WO2001070400A1 - Multiblock micro-arrays or macro-arrays with lab-on-a-chip - Google Patents

Multiblock micro-arrays or macro-arrays with lab-on-a-chip Download PDF

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Publication number
WO2001070400A1
WO2001070400A1 PCT/FR2001/000881 FR0100881W WO0170400A1 WO 2001070400 A1 WO2001070400 A1 WO 2001070400A1 FR 0100881 W FR0100881 W FR 0100881W WO 0170400 A1 WO0170400 A1 WO 0170400A1
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micro
arrays
macro
array
flat
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PCT/FR2001/000881
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French (fr)
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WO2001070400A8 (en
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François Geli
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Geli Francois
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Priority to EP01921418A priority Critical patent/EP1268062A1/en
Priority to AU2001248414A priority patent/AU2001248414A1/en
Publication of WO2001070400A1 publication Critical patent/WO2001070400A1/en
Publication of WO2001070400A8 publication Critical patent/WO2001070400A8/en

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    • B82NANOTECHNOLOGY
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    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
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Definitions

  • the present invention relates to multi-block macro-arrays or three-dimensional multi-block micro-arrays integrated in a ready-to-use continuous ultra-compact chain of synthesis or chemical, biochemical or biological analysis.
  • These multi-block micro-arrays or macro-arrays incorporate Lab-on-a-Chip (labs on chips).
  • Biological analysis as well as chemical and biochemical analysis and synthesis increasingly use "micro-arrays" or "macro-arrays", the English term "array” designating in chemical analysis a dense or very flat grid pattern. dense of identical objects, generally wells or micro-locations for deposits or synthesis (see Figure IC).
  • the current densities of macro-arrays are of the order of 10 to 100 micro-locations per cm 2
  • the common densities of micro-arrays are of the order of 100 to 1000 micro-locations per cm 2 .
  • In these wells or on these micro-locations are deposited or synthesized small quantities of chemical molecules, peptides, nucleic acids or other biological or organic molecules.
  • Macro-arrays and micro-arrays are used for the research of new materials, for the screening of new synthetic polymers, for the discovery of new drugs, for the analysis of peptides and proteins, for the study of protein interactions , for Molecular Biology programs such as studies of genetic diseases, Genomics (mapping of genomes by molecular markers, study of genetic polymorphisms), of Functional Genomics (expressions of differential genes specific for physiopathological processes, organs or or tissues affected by diseases, search for new therapeutic targets), Pharmacogenomics (individual response to drugs, search for the mode of action of drugs).
  • Their interest lies in the economy of reagents, the simplification and improvement of analysis and synthesis processes, the increase in reaction speed, the preservation of contamination, the increase in the number of analyzes or synthesis by unit of volume and per unit of time, lower costs, portability of analysis media, etc.
  • the present invention provides the means of integrating Lab-on-a-chips into macro-arrays or micro-arrays by ensuring seamless connections in the analysis chain, which was not the case before, since in the previous processes, it was necessary to envisage a phase of exposure to the open air (such as for example pipetting) in order to pass from Lab-on-chips to macro-arrays or micro-arrays, or macro-arrays or micro- arrays with Lab-on-chips.
  • the multiblock configuration of macro-arrays and micro-arrays of the invention benefits from a compact architecture of the fluid connections between various parts of the analysis system or microfluidic process and makes it possible to handle very small quantities of fluids with waterproof connections to evaporation and contamination.
  • the invention also describes the micro-fabrication techniques used to manufacture the system of fluidic connections of these macro-arrays or microarrays.
  • the architecture and manufacturing design of the different modules that make up these micro-arrays or macro-arrays are available according to the analysis criteria requested and the different options selected in the mode of preparation of the reagents and in the detection mode.
  • nucleic acid analyzes is only an illustration of what can be achieved according to the invention in fields as diverse as for example combinatorial synthetic chemistry, combinatorial chemical analysis, immunoassays, in short any type of biological or biochemical or chemical analysis where very important research and analysis programs must be ensured, each elementary operation having to use the least possible of samples and reagents and before last as little time as possible.
  • the molecules are deposited on micro-arrays or macro-arrays using micro-arrayers (Tisone TC. Dispensing System for miniaturized diagnostics. IVD Technology, 1998, 4:40. _ Lemmo AV. Quantitative nanoliter dispensing. Broom Eng News, 1998, 14:30. _ Oldenburg KR, Zhang J, Chen T, Maffia A, Blom KF, Combs AP, Chung TDY. Assay miniarurization for ultra high througput screening of combinatorial and discrete compound libraries: a 9600 well (0.2 microliter) assay System. J. Biomol Screening, 1998, 3, 55-62. _ Papen R., Croker K, Kolb A. Nanoliter dispensing technology.
  • micro-needles Lebouitz KS, Pisano AP ,. Microneedles and microlancets fabricated using SOI wafers and isotropic etching. Electrochemical Society Proceedings, vol 98-14, 237-242) and miniature multipipettes (Papautsky I, Brazzle JD, Weiss RB, Ameel TA, Frazier AB. Parallel sample manipulation using micromachined pipette arrays. SPIE Proceedings 1998, Vol. 3515-09, 104-114. _ Brazzle JD, Papautsky I, Frazier AB. Fluid-coupled hollow metallic micromachined needle arrays.
  • micro-arrayers subject the analytes to exposure to the open air, where problems of evaporation and contamination are to be feared.
  • Each well of the micro-array or macro-array can be individually addressed by electrodes to direct an electric field, like the micro-arrays of CEA-LETI or of Nanogen (Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a micro-electronic chip array. Nature Biotechnology, 2000, 18, 199-204), or sensors, as for example in the patent "Microelectrochemical sensor and sensor array. US Patent 4874500 ".
  • Each well of the micro-array or macro-array can also be addressed individually by optical fibers (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal.
  • micro-arrays where ⁇ epoxes are made of its ⁇ can compete with in situ synthesis chips by using photolabile groups (Fodor SP Accessing genetic information with high-density DNA arrays. Science, 1996, 274, 610-613 _ Jacobs JW, Fodor SPA Combinatorial chemistry applications of light- directed chemical synthesis. TiBTECH, 1994, 12, 19-26. Lipshutz RJ, Morris D., Chee M ;, Hubbell E., Kozal MJ, Shah, ShenN ., Yang R., Fodor SPA Using oligonucleotide probe arrays to access genetic diversity.
  • the first very expensive reagent encountered is DNA polymerase used in various methods of Amplification of a nucleic acid fragment.
  • the preliminary phases to Amplification may without great economic consequences remain on a macro-scale.
  • the cost of DNA Polymerase makes it interesting to go to a micro-scale from this stage amplification.
  • the consequence of such an option is that the following phases (detection) must also happen on a micro-scale. Said detection phases also have a limit level of sensitivity and can therefore indicate the limit level of miniaturization and be a factor limiting the reduction in size.
  • miniaturized devices which could allow their manufacture at lower cost. Indeed, these miniaturized devices use less raw material, have fewer parts, use cheaper components, require less space, and aim to bring together several functions. At equal cost, it is to be expected that miniaturized systems will offer the possibility of carrying out more or more exhaustive analyzes.
  • miniaturization does not directly bring flexibility, on the other hand it can make flexible principles of analysis economically viable which would have remained too costly by remaining manufactured alongside a large format. For example, miniaturization can make DNA analysis systems economically relevant where the user decides his amplification sites on DNA and analyzes the amplified fragments at will. These systems are more flexible than those operating by hybridization where predetermined and non-interchangeable oligonucleotides are fixed on an analysis support.
  • Miniaturized devices using fewer reagents also represent a potential environmental advantage: fewer toxic reagents, less pollution.
  • FIG. 1A shows a microfluidic device which can be used in the current state of the art.
  • Said device has three stages consisting of flat modules (a), (b) and (c) provided with microchannels parallel to their surface. The three stages are connected by micro-channels perpendicular to the surface of said flat modules.
  • FIG. 1B shows a microfluidic device that can be used in the current state of the art.
  • Said device connects two flat modules provided with micro-channels parallel to their surface with connections provided by micro-channels perpendicular to the surface of said flat modules.
  • the following publications and patents illustrate these connection configurations:
  • Figure IC illustrates a configuration where a multipipette pipetting manifold (1776) dispenses the reagents on a micro-array (1777).
  • the current Lab-on-a-chip cannot directly feed micro-arrays or macro-arrays or be powered directly by them.
  • the different architectures adopted so far for articulations between the different parts of a microfluidic micro-system for chemical or biochemical or biological analysis are not able to provide the desired compactness, especially when certain components cannot, for the reasons described above, to be miniaturized and that they must therefore remain on a macro scale.
  • connections between miniaturized devices lack compactness also come from architectural constraints such as those of the dead volumes in the connections, and from difficulties of micro-positioning on dispersed architectures.
  • the insufficient compactness of the connections between miniaturized systems makes it difficult to reduce the cost of instrumentation, mass production of consumables with a view to reducing their cost, the design of integrated consumables where several stages of analysis are carried out on the same miniaturized support, the return to a compact system for the detection of analytes, useful to be adapted to a "biochip" type detection.
  • the conduits and components which guide or receive the fluids must be miniaturized, on the other hand that the components which manage the flow of fluids and reagents (micro-valves, micropumps, micro-sensors, micro-heaters, etc.) are also miniaturized, and finally that connections can be established at the inside and outside the device.
  • the components which manage the flow of fluids and reagents micro-valves, micropumps, micro-sensors, micro-heaters, etc.
  • the aspect ratio which represents the ability to respect the ribs of a three-dimensional plane, in particular to respect a profile from broken lines and not from curves
  • the manufacture of miniaturized systems at least in a first phase of manufacture, mostly starts from flat and flat supports (parallel to a plan and not very thick), called 2D, where most of the components are made from engravings, ablations and deposits on a flat surface.
  • sub-components which have a sufficient level of flatness and flatness to be accessible to machining techniques for a flat surface.
  • sub-components are made just enough planes and just enough dishes to be able to be micro-manufactured. Then they are superimposed and assembled by fusion or gluing after a possible interlocking or interlocking, this makes it possible to reconstitute the desired micro-system (US Patent ⁇ ° 5 932-315: Microfluidic structure assembly with mating microfeatures _ US Patent, N ° 5 611 214. Microcomponent sheet architecture _ US Patent 5252294. Micromechanical structure).
  • the desired microsystem can hold in a more or less flattened volume represented by the superposition of subcomponents themselves clearly flattened.
  • micro-fabrication techniques are, among others, wet chemical etching techniques of photolithography, dry etching with various photonic or particulate radiation, micro-shaping with micro-tools or lasers, cutting, ablation, d by fusion or anode assembly, bonding, welding, molding, hot stamping (hot-embossing in English), punching, drilling, electroplating, ceramic vapor deposition, manufacturing by progression by successive sheets (lamination in English).
  • wet etching has been applied for around forty years to silicon and its derivatives in the microelectronics industry. It may be isotropic. It can also be anisotropic when one seeks to take advantage of the orientation of the crystals and the properties of the gravitating substances to control its direction. (Sato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. of single crystal silicon: surface roughening as a function of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206).
  • the wet etching techniques both isotropic and anisotropic, have many variants.
  • New knowledge in surface treatment makes it possible to refine the qualities required for materials during manufacture or the qualities required for the finished product.
  • New knowledge in thermophysics and differential thermochemistry between two materials suggests new techniques of fusion, molding, stamping, punching, in particular plastics.
  • Microfabrication of polymers by stereolithography has enlarged the field of these techniques, in particular for rapid 3D prototyping.
  • micro-manufacturing techniques are applicable not only to the manufacture of the finished products, but also to those of the tools used to carry out these micro-manufacturing, as well as to the micro-molds and to the hot stamping micro-matrices used. to micro-replicate en masse a micro-object.
  • the other criteria which will help to select a manufacturing method and material are the intrinsic qualities of the materials making up the finished object, and the prospects for controlling manufacturing costs.
  • Certain techniques presuppose a unitary manufacturing method, today unsuitable for mass production: dry etching by photonic or particulate radiation (Bean. Anisotropic etching of silicon. 1978. vol ED-25 (10), pp 1185-1193.
  • micro-manufacturing replication masters for example micro-molds for injection molding or for reactive molding, or hot stamping micro-dies
  • two qualities are combined: a high aspect ratio and a surface compatible with the requirements of the replication process.
  • certain steps in replication are crucial, in particular the separation of the replication matrix from the newly replicated object.
  • the complexity of the process chosen to manufacture a replication matrix must be taken into account. For example, we can manufacture with very high precision an injection molding micromould or a hot stamping micro-matrix with the LIGA technique, where synchrotron radiation from very expensive, very rare and very heavy machinery, is used in the early stages.
  • PDMS poly (dimethylsiloxane) molding
  • wet etching is now applied to all types of silicon and quartz derivatives, as well as to different types of glass (for example pyrex, boro-phospho-silicate glasses, etc.).
  • microfluidics an important criterion is compatibility with the use of micro-electrophoresis, 2D micro-electrophoresis and micro-electro-chromatography to separate molecules.
  • Compatibility with electro-osmosis for moving fluids is also and above all important, this technique having the advantage of avoiding components such as microvalves and micropumps.
  • electro-osmosis as well as micro-electro-chromatography combined with electro-osmosis require large voltages. Consequently, they are incompatible in practice with the use of silicon.
  • thermocapillary force Bosset MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT. Microfabricated structures for integrated DNA analysis. PNAS 1996, vol. 93, pp5556-5561), or the forces coupled to alternations of surfaces or hydrophobic-surface lines or hydrophilic lines (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Selective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers.
  • Transparency a quality sought after in biological analysis, is a quality shared between glasses (Kricka L, Wilding P, et al. Micromachined Glass-Glass Microchips for In Vitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) and some plastics.
  • the glasses offer, among other things for biochemical analysis, compatibility with fluorescence detection and a good heat exchange coefficient. They are however etched only in an isotropic mode, which for example today limits the shape of the microchannels on glass to a circular shape.
  • Plastics even if they have less compatibility with fluorescence detection and a lower heat exchange coefficient than glasses, have many other qualities, including mainly the low cost price.
  • the very low cost of manufacturing plastic micro-manufactured objects comes from the low price of the raw material, the simplicity of the production processes that can be envisaged, among other things because of the ability to replicate by molding or stamping. hot, even for plastics photoresists to photolithography.
  • photoresists which can be machined, among other things, by photolithography, including for example PMMA for X-ray lithography, SU-8 (negative photoresist) and Novolac de Hoescht and
  • AZ 9260 photoresists positive for photolithography UN (Lorenz H, Despont M, Fahrni ⁇ , LaBianca ⁇ , Renaud P, SU-8: a low-cost negative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121 -124. _ .Lomül B, Maciossek A, Surface micro components fabricated by UN depth lithography and electroplating, SPIE vol 2639, 174- 184 _ Conédéra N, Le Goff B, Fabre Pot. Potentialities of a new positive photoresist for the realization of thick molds, J. Micromech, Microeng, 1999, 9, 173-175.
  • PA polyamides
  • PC polycarbonates
  • POM polyoxymethylenes
  • COC cyclopentadienenorbomen copolymer
  • PMMA polymethylmethacrylates
  • PE-ld low density polyethylene
  • PE-hd polyethylene high density
  • PP polypropylene
  • PS polystyrenes
  • COC polyetheretherketone
  • Still other plastics can be microfabricated: polybutylenete ⁇ htalate (PBT), polyphenylene ether (PPE), polysulfone (PSU), liquid crystal polymer (LCD), polyetherimide (PEI).
  • PBT polybutylenete ⁇ htalate
  • PPE polyphenylene ether
  • PSU polysulfone
  • LCD liquid crystal polymer
  • PEI polyetherimide
  • the biodegradable polyactide can also be microfabricated.
  • PMMA and PC are widely used in injection molding and hot stamping. COC is commonly cited in hot stamping.
  • the mass production processes for plastics are very varied.
  • the main processes can be mentioned:
  • the first photoresist layer is the bottom of said microchannel with a rectangular section.
  • the second photoresist layer forms the vertical walls of said micro-channel.
  • the third layer of photoresist terminates the capillary by constituting the cover part.
  • Mastrangelo CH An inexpensive plastic technology for microfabricated capillary electrophoresis chips, ⁇ -TAS 1998, 249-252), a technique which starts from parylene deposited on polycarbonate or silicon with subsequent use of sacrificial photoresist.
  • the advantage of this technique lies in the sealing of the microchannels naturally included in the method.
  • Microfabrication by laser of plastics is also possible, but as a unitary technique. It can be for example direct ablation in the mass or the cutting of a joint which one will slide in sandwich between two lids.
  • Plastic surface treatments depend on the application and the material used. For example, a hydrophobic surface must often be made hydrophilic. For a DNA analysis or protein analysis application, it is necessary to avoid bonds with the substrate by surface treatments specific to these two types of analysis and specific to the material chosen, respectively.
  • Figure 1A shows a microfluidic device which can be used in the current state of the art.
  • Figure 1B shows a microfluidic device that can be used in the current state of the art.
  • Figure IC shows a pipetting device (1776) dispensing reagents on a micro-array or a macro-array (1777) which can be used in the current state of the art.
  • Figure 2A shows a typical micro-well of a micro-array or a macro-array.
  • Figure 2B shows how a micro-channel (41) can be reconverted into a closable micro-well (42) that is both deep and of reduced section.
  • Figure 2C shows how a connection of a microfluidic element (4) is engaged from below and from above of said microwells (42).
  • Figure 2D shows a deep and narrow male microwell (42).
  • Figure 3A shows in section a microwell (42) obtained by reconversion of a micro-channel (41) passing right through a flat elementary module (1) and emerging in the thickness and on the edge of said flat elementary module (1).
  • FIG. 3B shows in perspective several parallel micro-channels (41) passing right through a flat elementary module (1) and emerging in the thickness and on the edge of said flat elementary module (1), so that obtains a line (2119) of microwells (42) from the micro-array or macro-array of the invention.
  • FIG. 3C shows the precise superposition of said flat elementary modules (1) which each carry a line (2119) of microwells (42) to form said micro-array or of the multiblock macro-array (3119) of the invention.
  • Figure 4A shows a flat elementary module (1) where the microchannels (41) are provided with enlarged portions.
  • Figure 4B shows in perspective a flat elementary module (1) where the microchannels (41) are provided with enlarged portions.
  • Figure 4C shows in perspective the micro-array (3119) formed by stacking flat elementary modules (1) whose microchannels (41) are provided with enlarged portions.
  • Figure 5 shows a top view of a micro-array or multi-block macro-array (319) of the invention constituted by the stack of flat elementary modules (110).
  • Figure 6 shows a front view of a flat elementary module (110) of the invention.
  • Figure 7 shows a back view of said flat elementary module (110) of the invention.
  • Figure 8 shows a bottom view of said micro-array or multi-block macro-array (319) of the invention.
  • Figure 9 shows a profile view of said micro-array or multi-block macro-array (319) of the invention.
  • Figure 10 shows the detachment of detachable parts (145) of said flat elementary modules (1 i0) ,. seen in profile.
  • Figure 11 shows a top view of said micro-array or macro-array with said detachable parts (145) connected to the sub-parts (155) of said flat elementary modules (110).
  • Figure 12 shows said detachable parts (145) alone, seen from above.
  • Figure 13 shows in side view a micro-array or multi-block macro-array (318) formed by stacking said detachable parts (145).
  • Figure 14 shows a top view of a multi-block micro-array or macro-array (318) formed by stacking said detachable parts (145).
  • Figure 15 shows in side view the face to face connection of a first multi-block micro-array or macro-array (1318) constituted by stacking of detachable parts (1145) of flat elementary modules with a second micro-array or macro- multiblock array (2318) formed by stacking other flat elementary modules (2145).
  • Figure 16 shows in front view the face to face connection of a first micro-array or multi-block macro-array (1318) constituted by stacking of detachable parts (1145) with with a second multi-block micro-array or macro-array (2318) formed by stacking other flat elementary modules (2145).
  • FIG. 17 shows an orthogonal connection of two flat elementary modules of the invention, said flat elementary modules being provided with detachable sub-parts.
  • FIG. 18 shows an orthogonal connection of two micro-arrays or multi-block macro-arrays formed respectively by stacking the flat elementary modules of FIG. 17.
  • Figure 19 first shows an orthogonal connection of two flat elementary modules of the invention, the first of the two said flat elementary modules being provided with detachable sub-parts. It then shows a direct connection of a first flat elementary module-second flat elementary module assembly with a third flat elementary module.
  • FIG. 20 shows an orthogonal connection of two multi-block micro-arrays or macro-arrays of the invention, the first micro-array or macro-array being constituted by stacking the assemblies of first flat elementary modules-second flat elementary modules of FIG. 19 , and the second micro-array or macro-array being formed by stacking the third flat elementary modules of FIG. 19.
  • Figure 21 first shows a direct connection of two flat elementary modules of the invention, the first of the two so-called flat elementary modules being provided with detachable sub-parts. It then shows an orthogonal connection of a first flat elementary module-second flat elementary module assembly with a third flat elementary module.
  • FIG. 22 shows the connections of micro-arrays of micro-arrays or multi-block macro-arrays of the invention constituted respectively by stacking of the flat elementary modules of FIG. 21.
  • FIG. 23 first shows a direct connection of two flat elementary modules of the invention, the first of the two so-called flat elementary modules being provided with detachable sub-parts. It then shows an orthogonal connection of an assembly of first flat elementary module-second flat elementary module with several juxtaposed flat elementary modules of a third type.
  • FIG. 24 shows the connections of the micro-arrays or multi-block macro-arrays of the invention constituted respectively by stacking the flat elementary modules of FIG. 23.
  • Figure 25 A shows a micro-array or multi-block macro-array (4319) of the invention is formed by stacking of flat elementary modules (4110) provided with microchannels (41) whose micro-mixers (4019) are located on the sub-parts (4155) of said flat elementary modules (4110).
  • Figure 25 B shows how a maximum density of the multiblock micro-arrays of the invention is obtained, with a multiblock micro-array (4318) formed by stacking detachable parts (4145) of said flat elementary modules (4110).
  • Figure 26A, Figure 26B and Figure 26C show that serial reagent transfer operations can be performed using micro-arrays or a multiblock macro-arrays (20317), "equivalent” to micro-arrays or multiblock macro-arrays (4319) used to prepare the reagents.
  • Figure 27 shows a high density multiblock micro-array or macro-array (5319) formed directly by stacking very thin flat elementary modules (5110),
  • Figure 28A, Figure 28B, Figure 28 C, Figure 28D show how a transverse intermediate piece (30339) provided with micro-channels can be connected between a stack of flat elementary modules (21110) and a stack of flat elementary modules (5110).
  • Figure 29 A shows in perspective a multi-block micro-array or macro-array (6319) of the invention with shutters making it possible to isolate a compartment of a flat elementary module (6110) from said multi-block micro-array or macro-array ( 6319).
  • Figure 29B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention with said shutters.
  • Figure 30A shows another perspective view of a micro-array or multi-block macro-array (6319) of the invention where the parallelism of the operations appears, with, for example, a washing circuit during the preparation of the reagents before sending to the surface of said micro-array or multi-block macro-array (6319).
  • FIG. 30B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention, with, for example, a washing circuit during the preparation of the reagents before sending to the surface of said micro-array or multi-block macro-array (6319).
  • Figure 31 shows a perspective view of a micro-array or multi-block macro-array (7319) of the invention where the parallelism of the operations appears, with, for example, the sending of a reaction mixture in surfacedudit micro-array or multi-block macro-array (6319).
  • Figure 32 shows a connection of a multiblock micro-array or macro-array of the invention with a multi-pipette block.
  • Figure 33A shows in perspective a multi-block micro-array or macro-array of the invention connected to a one-piece piece (555) of surface planarization.
  • Figure 33B shows the connection of a micro-array or multi-block macro-array of the invention connected to a one-piece piece (555) of surface planarization.
  • Figure 34 and Figure 35 show in perspective how a flat elementary module (6110) can be micro-fabricated by the assembly of two flat elementary hemi-modules (611012).
  • Figure 36A, Figure 36B, Figure 36C show a method of manufacturing all the flat elementary modules of the invention by superposition and fusion of complementary flat sub-parts, the upper flat sub-part serving as a cover.
  • Figure 36 A shows a first part
  • Figure 36 B shows its cover
  • Figure 36 C shows the micro-array or multi-block macro-array of the invention thus constituted.
  • Figure 37A shows a sub-part of a flat elementary module.
  • Figure 37B shows the assembly of a sub-part of a flat elementary module with its complementary part.
  • Figure 37C shows a multi-block micro-array or macro-array of the invention consisting of the stack of said flat elementary modules produced by assembling said complementary parts.
  • Figure 37 D shows by transparency the micro-array or macro-array thus formed.
  • Figure 38 shows that when basic flat modules (18110) do not have any components, the flat support which is used to make one of its complementary sub-parts (1811012) can be very thin and the micro-array or macro- constituted array (18319) can then be relatively dense.
  • Figure 39 shows that when flat elementary modules (19110) have components, the density of the micro-array or a multiblock macro-array (19319) which can be formed by stacking said flat elementary modules (19110) is limited.
  • Figure 40A shows an embodiment of the invention and resides in multi-block microarrays or macrbrarrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by chromatography by exchange anions then desalination on magnetic microparticles of silica gel.
  • Figure 40B and Figure 40C show the action of an electromagnet on said magnetic microparticles.
  • Figure 41 A shows another embodiment of the invention and resides in micro-arrays or multi-block macro-arrays (14319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by anion exchange chromatography then desalination on silica gel matrices.
  • Figure 41B shows another embodiment of the invention and resides in multi-block micro-arrays or macro-arrays (15319) dedicated to the analysis of DNA fragments with a metered preparation with extraction of DNA from matrices silica gel.
  • Figure 42 shows the orthogonal connection of a flat element for preparing samples and reagents with a flat element providing a specific reagent in one of the micro-wells of said flat element for preparing samples and reagents.
  • Figure 43 shows the orthogonal connection of a stack of flat elements for preparing samples and reagents with a stack of flat elements providing a specific reagent in each of the micro-wells of said flat elements for preparing samples and reagents.
  • Figure 44 shows the orthogonal connection of a flat element, in two assemblable parts, for preparing samples and reagents with a flat element, in two parts also assembled, and bringing a specific reagent in one of the micro-wells of said flat element preparation of samples and reagents.
  • Figure 45 shows the orthogonal connection of a stack of flat elements, in two assemblable parts, for preparing samples and reagents with a stack of flat elements, in two parts also assembled, and providing a specific reagent in each of the micro-wells of said flat elements for preparing the samples and reagents.
  • Figure 46 shows a flat sample preparation and reagent element capable of directing a line of first aliquots of said preparation towards macro-locations and capable of connecting to a stack of flat modules capable of taking each aliquot of the previous flat module to aliquot it again on micro-wells.
  • Figure 47 shows the connection of a flat element for preparing samples and reagents capable of directing a line of first aliquots of said preparation towards macro-locations with a stack of flat modules capable of taking up each aliquot of the previous flat module for the aliquot again on micro-wells.
  • Figure 48 shows the face to face between a first line, in the foreground, of n horizontal stacks of p flat modules individually identified specifically, with a second line, in the background, of n vertical stacks of p flat modules of reagents specific.
  • Figure 49 shows the orthogonal connection between a first line, in the foreground, of n horizontal stacks of p flat modules specifically identified individually, with a second line, in the background, of n vertical stacks of p flat modules of specific reagents , giving rise to n micro-arrays of np micro-wells, or in all n 2 p microwells, each of the nf ⁇ micro-wells having received one of the np specific reagents
  • Figure 50 shows the redistribution of the flat modules from the previous n works of flat modules marked differently in n new stacks grouping the flat modules with the same individual marking, each of said stacks with the same individual marking being dedicated to a single sample.
  • Figure 51 shows the connection of n flat modules each to a single sample with n new stacks grouping the flat modules with the same individual marking, each of the n samples may meet np specific reagents, the n stacks giving rise to n micro-arrays representing in total n'p micro-well.
  • Figure 52A shows a step of said integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) the introduction of said mixture of purified DNA and amplification buffer into the micro-channels of said micro -array or multiblock macro-array of the invention.
  • Figure 52B shows as a next step of said process of integrated DNA preparation on micro-arrays or multi-block macro-arrays (15319) a connection of said micro-array or macro-array (15319) with other stacks of flat elementary modules (9245).
  • Figure 52B shows as a next step of said integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) the disconnection of said micro-array or macro-array (15319) with other stacks of flat elementary modules (9245).
  • Figure 53 shows a bevel stack of flat elementary modules (40110) with subparts (40145) for compatibility with detection by microelectrophoresis with for example a connection to a bevel stack of flat elementary modules (40245) provided with micro -channels for micro-electrophoresis.
  • Figures 54 A, 54B, 54C, 54D show that one can synthesize in situ in the wells of a monoblock micro-array or macro-array (339) for example octamers from prefixed tetramers and tetramers brought by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • Figures 55A, 55B, 55C, 55D show that one can synthesize in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) octamers from prefixed tetramers and brought tetramers by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • Figures 56A, 56B, 56C, 56D show that octamers can be synthesized in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) from prefixed tetramers and tetramers brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • Figures 57A, 57B, 57C show that one can synthesize in situ in the micro-wells of a monoblock micro-array or macro-array (338) biochips in each micro-well of said micro-array or monoblock macro-array (338) through a connection with micro-arrays or multi-block macro-arrays of the invention.
  • Figures 58 A, 58B, 58C show that biochips can be synthesized in situ in each micro-well of a micro-array or multi-block macro-array (319)
  • Figure 59A shows how a multi-block micro-array or macro-array of the invention can be connected to a mono-block micro-array of plugs (340) which seal the microwells (42) of said micro-arrays or multi-block macro-arrays.
  • Figure 59B shows how a first multi-block micro-array or macro-array of the invention can be connected to another micro-array or macro-array of the invention formed by stacking flat elementary modules (1101), the micro- channels are provided with microvalves (396).
  • Figure 59C shows how a first multi-block micro-array or macro-array of the invention can be connected to another multi-block micro-array or macro-array of the invention formed by stacking flat elementary modules (1102) provided with devices (398) which create an electro-osmotic force by establishing an electrical potential between two places of the micro-channels of said flat elementary modules (1102)
  • Figure 59D shows how a first multi-block micro-array or macro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array or macro-array of the invention formed by stacking flat elementary modules (1103) whose micro-channels are provided with two micro-valves (397).
  • the classic micro-arrays or macro-arrays dedicated to chemical or biochemical or biological analysis are generally flat and flat, and made from a single block (monoblock).
  • micro-arrays or macro-arrays of the invention are planar, multi-block, thick, or even very thick, because they use the third dimension, the depth of the micro-array or of the macro-array.
  • the present invention relates to multi-block macro-arrays or three-dimensional multi-block micro-arrays integrated in a ready-to-use continuous ultra-compact chain of synthesis or chemical, biochemical or biological analysis.
  • Multibloc is opposed to the word “Monobloc”. It means that the "micro-arrays” or “macro-arrays” of the invention are, unlike conventional micro-arrays in Analysis Chemical, made up of several parts. More specifically, they consist of the assembly by superposition of a large number of identical or homologous elementary modules.
  • each elementary module of the micro-array or of the macro-array of the invention has inter alia for the function of constituting in itself either a column or a line of said micro-array.
  • the macro-arrays or microarrays designed according to the present invention use microwells obtained reconversion of micro-channels into microwells.
  • FIG. 2A shows a conventional micro-well of a micro-array or of a macro-array, the functionality of which suffers from exposure to the open air and therefore from contamination and evaporation problems.
  • FIG. 2B shows how a more or less long micro-channel (41) can be converted into a micro-well. It suffices that this micro-channel (41) passes through a more or less deep support right through, and that at one end of said micro-channel, that is to say at one of the orifices of said microchannel, any element is affixed obturator (4) or any device capable of opposing the passage of a fluid in said micro-channel (41).
  • the macro-arrays or micro-arrays designed according to the present invention use microwells (42) obtained by reconversion of microchannels (41) passing right through a flat elementary module (1 ) and opening into the thickness and on the edge of said flat elementary module (1) through orifices (42). With several parallel micro-channels (41) passing right through a flat elementary module (1) and opening into the thickness and on the edge of said flat elementary module (1), a line (2119) is obtained according to the invention.
  • microwells of the micro-array or macro-array of the invention as shown in Figure 3B
  • Figure 3C the precise superposition of these flat elementary modules (1) which each carry a line (2119) of the micro-array or of the macro-array, will form the multi-block micro-array or the multi-block macro-array (3119) of the invention.
  • the number of stacked flat elementary modules (1) will represent the number of lines of said micro-array or of the multiblock macro-array.
  • the number of wells (42) per elementary module that is to say the number of microchannels (41) per flat elementary module, will represent the number of columns of the micro-array or of the multiblock macro-array (3119) of the invention.
  • micro-wells of the micro-array or macro-array of the invention reduce the surface exposed to evaporation (see FIG. 2B).
  • a connection will be able to be engaged from below and from above of said microwells, which will seal the connection (see Figure 2C and Figure 2D).
  • the microwells of the microarray or of the multiblock macro-array of the invention can be closed on both sides, which suddenly transforms said micro-wells into micro-containers (see FIG. 2C).
  • the bottom of said microwell (42) is provided with a temporary closure means.
  • each flat elementary module which constitutes it will be able to be configured in "Lab-on-a-chip", that is to say in " laboratories on chips ", which will allow a supply of the micro-array or the macro-array with reaction products whose preparation has been integrated into said micro-array or multi-block macro-array without phase of exposure to the open air and without evaporation or contamination.
  • FIG. 4A shows flat elementary modules (1) where the microchannels (41) are, according to the invention, provided with micromixers (19) and enlarged portions. Enlarged portions (1701) of said micro-channels (41) may also include microcolumns (Cf Swedberg S, Kaltenbach-P, Witt K, Bek F, Mittlestadt LS. Fully integrated miniaturized planar liquid sample handling and analysis device. US Patent 5571410 _ He B, Tait N, Régnier F. Fabrication of nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797._. Pluskal MG, Microscale sample preparation, Nature Biotechnology, 2000, vol 18, 104-105) .
  • microcolumns Cf Swedberg S, Kaltenbach-P, Witt K, Bek F, Mittlestadt LS. Fully integrated miniaturized planar liquid sample handling and analysis device. US Patent 5571410 _ He B, Tait N, Régnier F. Fabrication of nanocolumns for liquid chromat
  • Enlarged portions (1706) can also receive passive or active microseparators provided with magnetic beads (Cf Ahn CH, Trimmer W, Jun YN, Erramilli S. A fully integrated micromachined magnetic particle separator. Journal of Microelectromechanical Systems, 1996, vol 5, No. 3, 151-158). Said enlarged portions (1706) for magnetic balls can be surrounded by housings (1767) for electromagnets (1768),
  • Figure 4B shows in perspective the same flat elementary module (1) as Figure 45 A. In this perspective view appears the line (2119) of micro-wells (42) in the thickness and on the edge of said flat elementary module (1).
  • Figure 4C shows in perspective the micro-array (3119) formed by stacking flat elementary modules (1) whose microchannels (41) are provided with micro-mixers (19), with enlarged portions (1701) provided with micro-columns, enlarged portions (1706) receiving magnetic balls and framed by housings (1767) for electromagnets (1768), said electromagnets (1768) passing right through said micro-array or multi-block macro-array (3119) .
  • said electromagnet (1768) is activated, the magnetic micro-particles are separated from the supernatant in said enlarged portion (1706) close to said electromagnet (1768).
  • a micro-array or a multiblock macro-array (319) is supplied from below with reagents or samples with compactness, integration and an optimized parallel architecture when all the elementary flat modules (110) which constitute it by stacking comprise a microfluidic circuit (55) provided with orifices for supplying and discharging samples and reagents located in the thickness and on the wafer said flat elementary modules (110).
  • These supply ports are such as (8), (10), (12), by way of example.
  • These discharge orifices are such as the orifice (11), by way of example.
  • said flat elementary modules (110) may consist of two parts (145) and (155), said part (145) supporting all or part of the microchannels (41) and possibly being detachable and very thin, of the order of 20 to 800 microns and said part (155) supporting all or part of the components of larger section.
  • Figure 5 shows a top view of said micro-array or multi-block macro-array (319) of the invention constituted by the stacking of said flat elementary modules (110), each of said flat elementary modules providing a line (219) of said micro -array or multi-block macro-array (319).
  • Figure 5 also shows by way of example the reagent supply orifices (10) located in the thickness and on the edge of one side of said flat elementary modules (110), and the reagent supply orifices ( 12) located in the thickness and on the edge of another side of said flat elementary modules (110).
  • FIG. 6 shows a front view of said flat elementary module (110) of the invention provided with microfluidic circuitry (55) capable of supplying said micro-channels (41) and finally said micro-array or multi-block macro-array (319 ).
  • Said microfluidic circuitry which may include a filtration zone (25) with filters (16), a purification zone (35) with micro-column (17), and a preparation zone (45) of the filtered and purified product before its introduction for a small part in said micro-channels (41) and finally, internally, from below, protected from any evaporation and from any contamination, in the micro-wells (42) of a line (219) of said micro-array or multiblock macro-array (319).
  • Figure 7 shows a back view of said flat elementary module (110) of the invention provided with electrical circuits (381) and (391) intended to activate the electrodes and components which are provided with said flat elementary modules (110) to control the movement of fluids.
  • Figure 8 shows a bottom view of said micro-array or multi-block macro-array (319) of the invention with supply ports for reagents or samples such as, for example, (7) and (8 ), and discharge orifices such as, for example, (11). According to the invention, said orifices are located in the thickness and on the edge of said flat elementary modules (110).
  • Figure 9 shows a profile view of said micro-array or multi-block macro-array (319) of the invention, Figure 9 also shows electrical connection pads (380) and (390) located, according to the invention, in the thickness and on the edge of said flat elementary modules (110)
  • Figures 5, 6, and 7 and 9 show the notches (71) and (72) which are optionally provided with the parts (155) of said flat elementary modules (110) to receive alignment guides such as (61), ( 62). They also show the notches (74) which are optionally provided with said detachable parts (145) of said flat elementary modules (110) to receive their own alignment guide in the event that they are detached and stacked separately.
  • the precision of the stacking can also be ensured by containers which conform to the contours of the shape of a micro-array or multi-block macro-array of the invention.
  • Figures 10, 11, 12, 13, 14 show how said detachable parts (145) of said flat elementary modules (110) can be detached and stacked to form a higher density micro-array or multi-block macro-array (318) as said multi-block micro-array or macro-array (319).
  • Figure 10 shows the detachment of said detachable parts (145), seen in profile, and Figures 11 and 12 show this detachment seen from above.
  • Figure 13 and Figure 14 show, respectively in side view and top view, a multi-block micro-array or macro-array (318) formed by stacking said detachable parts (145). Said multi-block micro-array or macro-array (318) is of higher density than said multi-block micro-array or macro-array (319).
  • the higher density multi-block micro-array or macro-array (318) constituted by the stacking of said detachable parts (145) can connect together face to face, as shown in FIG. 15 and the Figure 16.
  • FIGS. 15 and FIG. 16 respectively represent in side view and front view the face to face connection of a first multi-block micro-array or macro-array (2318) constituted by stacking of detachable parts (2145) of flat elementary modules with a second multi-block micro-array or macro-array (1318) formed by stacking detachable parts (1145).
  • a multi-block microarray or macro-array (3119) can be connected to another multi-block micro-array or macro-array (3119) orthogonally, provided that the stack of elementary flat modules (1) constituting the first micro-array or multi-block macro-array (3119) is offset by a rotation of 90 ° around the axis of the micro-channels (41) relative to the stack of flat elementary modules (1) of the second micro -array or multiblock macro-array (3119), so as to offer the possibility of a massive parallelism of reactions by configuration of XY matrices where lines of first reagents (xl x2 .. xn) in the first micro-array or macro -array multibloc (3119) cross with lines of second reagents (yl y2 ... yn) in the second micro-array or macroblock multibloc (3119).
  • Figures 17, 18, 19, 20, 21, 22, 23, 24 show how several micro-arrays or multi-block macro-arrays of the invention can connect face to face, before or after said detachable parts of said flat elementary modules are detached.
  • FIG 17 and Figure 18 show how a stack of flat elementary modules (910) with a single compartment (956) with an insertion opening (907) can connect directly to a stack of flat elementary modules (94610) provided with micro-channels (94641) with micro-mixers (94619), said micro-channels (94641) opening into the thickness and on the edge of said flat elementary modules (94610), said stack of said flat elementary modules (94610) being able to connect orthogonally to a stack of flat elementary modules (25110) constituting said micro-array or multi-block macro-array of the invention, said connection being able to be made before or after detaching the detachable parts (25145) from said flat elementary modules ( 25110) ..
  • Figure 19 and Figure 20 show how a stack of flat elementary modules (9010) provided with several compartments (9056) with insertion opening (9007) can connect directly to a stack of flat elementary modules (9046) provided with micro -channels (9041) with micro-mixers (9019), said micro-channels (9041) opening into the thickness and on the edge of said flat elementary modules (9046), said stack of said flat elementary modules (9046) being able to connect orthogonally to a stack of flat elementary modules (25110) constituting said micro-array or multiblock macro-array of the invention, said connection being made before or after detaching the detachable parts (25145) from said flat elementary modules (25110).
  • FIG. 21 and FIG. 22 show how a stack of flat elementary modules (1010) provided with a single compartment (1056) with insertion opening (1007) can be connected orthogonally to a stack of flat elementary modules (104610), said stack of said flat elementary modules (104610) being able to connect directly to a stack of flat elementary modules (25110) constituting said multi-block micro-array or macro-array of the invention, said connection being made before or after detaching the detachable parts (25145) of said flat elementary modules (25110).
  • Figure 23 and Figure 24 show how several juxtaposed stack of flat elementary modules (10010) provided with several compartments (10056) with insertion opening (10007) can connect orthogonally to a stack of flat elementary modules (1004610) stack of said flat elementary modules (1004610) which can be directly connected to a stack of flat elementary modules (25110) constituting said multi-block micro-array or macro-array of the invention, said connection being made before or after detaching the detachable parts ( 25145) of said flat elementary modules (25110).
  • Figure 25 A, Figure 25 B, Figure 25 C shows how a maximum density of the multiblock micro-arrays of the invention is obtained.
  • Figure 25A shows a micro-array (4319) formed by stacking flat elementary modules (4110) provided with two sub-parts (4i45) and (4155).
  • the micro-channels (41) open onto the micro-array (4319) in the thickness and on the edge of said sub-parts (4145).
  • micro-mixers 4019
  • said micro-channels (41) taking up a very small part of the fluids supplied by the micro-circuit (4055).
  • Said sub-part (4155) provides a large space between said micro-channels (41) and said sub-part (4145) receives said micro-channels (41) in a constricted configuration, so that the stacking of said flat elementary modules (4110) constitutes micro-arrays or multi-block macro-arrays (4319) of high density.
  • Figure 25B and Figure 25C show that after detachment, the stacking of said sub-parts (4145) constitutes micro-arrays or multi-block macro-arrays (4318) whose level of density is even higher compared to the micro- array (4319) is due to the lesser thickness of said detachable subparts (4145).
  • Figure 26 A, Figure 26B and Figure 26C show that one can choose not to detach the sub-parts (4145) of flat elementary modules (4110) forming by stacking a micro-array or a multi-block macro-array (4319 ) of the invention, but rather to create a micro-array or an equivalent multi-block macro-array (20317), then a micro-array or a multi-block macro-array (20316). of greater density.
  • Figure 26 A and Figure 26B show flat elementary modules (20046) traversed longitudinally right through by micro-channels emerging in the thickness and on the francKé on two of their sides, said sides being opposite, this which results in a stack of said flat elementary modules (20046) forming two multi-block micro-arrays or macro-arrays, each of said multi-block microarrays or macro-arrays constituting on an opposite face of the stack, said flat elementary modules ( 20046) which can therefore be used to reproduce identically by transfer of reagents said micro-array or macro-array (4319).
  • these “equivalent multi-block micro-arrays or macro-arrays” make it possible to work both in parallel, using a micro-array or multi-block macro-array (4319) with numerous micro-wells (42), but also in series by making successive different analyzes from the same preparation integrated on said micro-array or multi-block macro-array (4319), said integrated preparation being transferred into several micro-arrays or equivalent macro-arrays (20317 ) successive.
  • these configurations using "equivalent multi-block micro-arrays or macro-arrays” are suitable for chemical, biochemical or biological analysis or synthesis processes in series.
  • Figure 27 shows that a multiblock micro-array of high to very high density can also be directly constituted if it is made by stacking of flat elementary modules which, on the one hand are very thin, on the other hand have a configuration which allows their micro-channels to tighten on said flat elementary module.
  • FIG. 27 shows a high density multiblock micro-array or macro-array (5319) formed by stacking very thin flat elementary modules (5110) consisting of two parts (5145) and (5155) with alignment pads ( 71).
  • the micro-channels (41) lead to the multi-block micro-array (5319) in the thickness and on the edge of said sub-parts (5145).
  • micro-mixers 509
  • said micro-channels (41) taking up a very small part of the fluids supplied by the micro-circuit (5055).
  • Said sub-part (5155) provides a large space between said micro-channels (41) and said sub-part (5145) receives said micro-channels (41) in a constricted configuration, so that the stacking of said flat elementary modules (5110) directly constitutes very high density micro-arrays or multi-block macro-arrays (5319).
  • FIGS. 28A, 28B, Figure 28 C, Figure 28D show how a transverse intermediate piece (30339) provided with micro-channels can be connected between a stack of flat elementary modules (21110) and a stack of flat elementary modules (5110).
  • said flat elementary modules (21110) are traversed longitudinally right through by micro-channels opening into the thickness and on the edge of two of their sides, said sides being opposite, each of said multi-block microarrays or macro-arrays constituting on an opposite face of the stack, said stack being orthogonal to the stack of flat elementary modules (5110).
  • Said intermediate piece (30339) can be manufactured according to the mode of micro-arrays or multi-block macro-arrays of the invention, that is to say by stacking of flat elementary modules, said stack being parallel to the micro-channels, or else according to a different mode, for example with microchannels oriented perpendicular to the stack of flat elementary modules, that is to say perpendicular to a flat surface and not parallel to the length or the width of the flat surface as for the invention.
  • such a type of intermediate part (30339) can be used as a simple connector, or as a useful part in the chemical, biochemical or biological analysis or synthesis process, for example by being provided with molecules fixed and useful for process, or by being provided with micro-particles or micro-spheres fixing molecules useful to the process, or by being provided with micro-reservoirs of reagents in derivation of the micro-channels, or by being provided with micro-components such as micro-columns, microfilters, micromixers, micropumps, microvalves, micro-heaters, etc.
  • Figure 29A shows in perspective a micro-array or multi-block macro-array (6319) of the invention formed by stacking flat elementary modules (6110).
  • Said flat elementary modules (6110) are formed of sub-parts (6155) and sub-parts (6145).
  • Said sub-parts (6145) are provided with micro-channels (41) opening out in their thickness and on their edges to form the micro-wells (42) of said micro-array or multi-block macro-array (6319).
  • Said micro-channels (41) are provided with micro-mixers (19).
  • Said sub-parts (6145) are also provided with electrical connection pads (390) located in their thickness and on their edges.
  • said sub-parts (6155) are provided with a discharge orifice (11) and introduction orifices (7), (8), (10), (12) located in the thickness and on the edge of said flat elementary modules (6110), microfilters (16), and a microcolumn (17).
  • said micro-column (17) can be isolated by shutters (9) and (14) located in the thickness and on the edge of said flat elementary modules. (6110) and actuated from the outside of said flat elementary modules (6110)
  • Figure 29B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention.
  • Figure 30A and Figure 30B show respectively in perspective and in front, said micro-array or multi-block macro-array (6319) where the parallelism of operations appears, with, for example, a washing circuit (66) during preparation, before purified products (67) retained on the microlumn (17) are sent to the surface of said micro-array or multi-block macro-array (6319).
  • Figure 31 shows the parallelism of operations on a perspective view of a micro-array or multi-block macro-array (7319) of micro-wells (42) formed by stacking flat elementary modules (7110) provided with a zone of lysis-filtration (7025), a purification zone (7035) provided with a micro-column (7017), a zone for preparing the purified reagents (7045).
  • a reaction mixture (79) to the surface of said micro-array or multi-block macro-array (7319) is represented, via the micro-channels (41), said reaction mixture representing the product.
  • purified (77) added with a buffer (78).
  • Figure 32 shows a connection of a multi-pipette block (70211) with micro-array or multi-block macro-array of the invention.
  • Said multi-block micro-array or macro-array of the invention is formed by stacking flat elementary modules (70110) with sub-parts (70155) and (70145).
  • Said sub-parts (70155) are provided with microfluidic circuitry (70055), and said sub-parts (70145) are provided with microchannels (41) opening out in their thickness and on their edges.
  • Said multipipette block (70211) integrates a stack of flat elementary modules (70110) provided with subparts (70202) and subparts (70245).
  • Figure 33A shows in perspective how a multi-block micro-array or macro-array of the invention formed by stacking flat male elementary modules (8110) is connected to a monoblock surface planarization part (555).
  • Figure 33B shows the connection of the multi-block micro-array or macro-array with said one-piece surface flattening part (555).
  • Figures 33 A and Figure 33 B show a single piece (555) planarizing microarrays or multiblock macro-arrays consisting of a stack of 8110 male flat elementary modules.
  • Said planarization piece (555) is in one piece, has on the front a flat face, and has on the back its other face which fits into the recesses formed between the projecting parts of said flat male elementary modules 8110.
  • This part can be used in all cases where a micro-array or multi-block macro-array of the invention must have a flat surface, for example in cases where light reflections must be avoided for detections or photo-exhibitions .
  • Figure 42 shows the orthogonal connection of a flat element for preparing samples and reagents (27110) with a flat element (27210) providing a specific reagent in one of the micro-wells of said flat element for preparing samples and reagents (27110) .
  • Figure 43 shows the orthogonal connection of a stack of flat elements for sample and reagent preparation (27110) with a stack of flat elements (27210) providing a specific reagent in each of the micro-wells of said flat elements for preparation of samples and reagents (27210).
  • Figure 44 shows the orthogonal connection of a flat element, in two assemblable parts, for preparing samples and reagents, with a flat element, in two parts also assembled, and bringing a specific reagent in one of the micro-wells of said element sample preparation dish and reagents.
  • Figure 45 shows the orthogonal connection of a stack of flat elements, in two assemblable parts, for preparing samples and reagents with a stack of flat elements, in two parts also assembled, and providing a specific reagent in each of the micro-wells of said flat sample preparation elements and reagents.
  • Figure 46 shows a flat sample preparation and reagent element (47000) capable of directing a line of first aliquots of said preparation towards a line of macro-locations (47107) and capable of connecting to a stack of flat modules (37110 ) each provided with a reception room (37164).
  • the flat modules (37110) are able to take up by a line of orifices (37107) each aliquot of said module dish (47000) in said aliquot receiving chamber (37164) to re-aliquot it on micro-wells or micro-locations (37042) by means of microchannels (37041) passing through micro-mixers (47019).
  • Figure 47 shows the connection of a flat sample preparation and reagent element (47000) with a stack of flat modules (37110) capable of taking each aliquot of said flat module (47000) to re-label it on a micro- array of microwells or micro-locations (37042).
  • Figure 48 shows the face to face between a first line, in the foreground, of n horizontal stacks of p flat modules individually identified specifically, with a second line, in the background, of n vertical stacks of p flat modules of reagents specific.
  • the individual marking will later be used to assemble modules with the same marking to constitute a micro-array or multi-block macro-array of the invention dedicated to a sample corresponding to this marking, said micro-array or multi-block macro-array receiving np reagents specific in each of its micro-locations or micro-wells.
  • Figure 49 shows the orthogonal connection between a first line, in the foreground, of n horizontal stacks of p flat modules specifically identified individually, with a second line, in the background, of n vertical stacks of p flat modules of specific reagents , giving rise to n micro-arrays of np micro-wells, or in all n 2 p microwells, each of the nf ⁇ micro-wells having received one of the np specific reagents
  • Figure 50 shows the redistribution of the flat modules from the previous n works of flat modules marked differently in n new stacks grouping the flat modules with the same individual marking, each of said stacks with the same individual marking being dedicated to a single sample.
  • Figure 51 and Figure 52 show the connection of n flat modules each to a single sample with n new stacks grouping the flat modules with the same individual marking, each of the n samples may meet np specific reagents, the n stacks giving rise to n micro-arrays representing a total of micro-wells.
  • Figure 54A, Figure 54B, Figure 54C, Figure 54D show a solid piece of fixing and solid phase synthesis of molecules (339) plane, monoblock, connectable to a micro-array or a multi-block macro-array of l invention, provided with an array of micro-wells whose grid is modeled on that of said micro-array or multi-block macro-array of the invention.
  • Said one-piece attachment and solid phase synthesis of molecules (339) offers in each of its micro-wells a surface for attachment to a molecule to be synthesized in a solid phase synthesis process, or a surface for receiving microparticles fixing the first molecule of a molecular chain to be synthesized.
  • Figures 54 A, 54B, 54C, and 54D shows that one can synthesize in situ in the micro-wells of said part (339) octamers from prefixed tetramers and tetramers brought by the microchannels (41 ) of said multi-block micro-arrays or macro-arrays of the invention; - .
  • Figures 55A, 55B, 55C, 55D show that octamers can be synthesized in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) from tetramers prefixed in said micro- wells (42) and tetramers brought by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention. Said synthesis can possibly take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process.
  • the micro-channels (41) of said flat elementary modules (1) can be provided with enlarged portions and intended to fix active molecules in the analysis or synthesis process to which said flat elementary modules (1) are dedicated. ), said. fixing of active molecules which can take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process.
  • the precise example of Figures 56 A, 56 B, 56C, 56D shows that one can synthesize in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) of the octamers. from prefixed tetramers and tetramers brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • said enlarged portions of said microchannels (41) of said flat elementary modules (1) can also be intended to receive magnetic or passive microparticles for fixing molecules during the manufacturing phases to manufacture ready-to-use devices, or during the analysis or synthesis process. Said fixing of molecules can take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process.
  • a micro-array or a multi-block macro-array (3119) a part receiving deposits of microparticles, planar, monobloc, provided with an array of micro-wells whose grid is modeled on that of said micro-array or multi-block macro-array (3119), providing in each of its micro-wells a surface for depositing said microparticles provided with molecular link arms, which makes it possible to manufacture a micro-array or a macro-array of microparticles usable in chemical or biochemical analysis or synthesis.
  • said enlarged portions of said microchannels (41) of said flat elementary modules (1) can also be intended to be photoexposed, so as to be able to carry out syntheses with the use of photolabile groups instead of said portions of said microchannels (41 ), said syntheses having the aim of producing compounds on demand, or the aim of manufacturing a ready-to-use analysis device provided with molecules synthesized in situ.
  • Figures 57A, 57B, 57C show that biochips can be synthesized in situ in each micro-well of a solid piece of fixation and solid phase synthesis of molecules (338), using photolabile groups and micro- arrays or macro-arrays of micromasks (187) of photolithography.
  • Said monoblock fixing and solid phase synthesis part of molecules (338) is planar, monobloc, connectable to a micro-array or a multi-block macro-array (3119), provided with an array of micro-wells or spots including the grid is modeled on that of said micro-array or multi-block macro-array (3119).
  • the joint use of photolabile groupings and photolithography masks makes it possible to manufacture a micro-array or a macro-array of biochips having said part (338) as support.
  • the joint use of photolabile groupings and digital micro-mirrors also makes it possible to manufacture a micro-array or a macro-array of biochips having said part (338) as support.
  • Figures 58A, 58B, 58C show that biochips can be synthesized in situ in each micro-well (42) of a micro-array or multi-block macro-array (319) of the invention.
  • the joint use of photolabile groupings and photolithography masks makes it possible to manufacture a micro-array or a macro-array of biochips having for support said micro-array or multi-block macro-array (319).
  • the joint use of photolabile groupings and digital micro-mirrors also makes it possible to manufacture a micro-array or a macro-array of biochips having for support said micro-array or multi-block macro-array (319) of the invention.
  • Figure 59A shows how a micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to two monobloc and planar parts (340), each forming a monobloc micro-array of plugs .
  • Figure 59B shows how a first micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array of the invention formed by stacking flat elementary modules (1) 1101) whose micro-channels are provided with micro-valves (396).
  • a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and the so-called microvalve on the connected micro-array or multi-block macro-array.
  • Figure 59C shows how a first multi-block micro-array or micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another multi-block micro-array or macro-array of the invention formed by stacking of flat elementary modules (1102) provided with devices (398) which create an electro-osmotic force by establishing an electrical potential between two places of the micro-channels of said flat elementary modules (1102).
  • a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and said device (398).
  • Figure 59D shows how a first micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array of the invention formed by stacking flat elementary modules (1) 1103) whose micro-channels are provided with two micro-valves (397).
  • a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and the first micro valve (397) on the connected micro-array or multi-block macro-array.
  • Another volume is isolated between the two microvalves (397) of said second micro-array or multi-block macro-array of the invention.
  • Figure 59D also shows that upstream of an isolated volume between two volumes isolated by two microvalves, it can be considered the use of a micropump block (399) common to several channels.
  • Figure 34, Figure 35, Figure 36 A, Figure 36B, Figure 36C, Figure 37 A, Figure 37B, Figure 37C, Figure 37 D, Figure 38, Figure 39 show a method of manufacture of all the flat elementary modules of the invention in which any type of said flat elementary modules of the invention, micro-components not included, is manufactured with microfabrication techniques according to an assembly by superposition and fusion of complementary planar sub-parts .
  • Figure 34 and Figure 35 show in perspective how a flat elementary module (6110) to be provided with micro-channels (41), micro-mixers (19), micro-fluidic circuitry (55), housings for micro -filters (16), housings for microcolumns (17), orifices (7) and (11) for samples and reagents, perhaps micro-manufactured by the assembly of two flat elementary semi-modules (611012), each of its two flat elementary hemimodules (611012) having been micro-machined or micro-molded or micro-stamped so that a micro semi-circuitry is formed, inter alia -fluidic (5512), semi-microchannels (4112) opening onto the edge of said flat elementary semi-modules (611012) by semi-micro-wells (4212), semi-micro-mixers (1912), semi-housings (1612 ) for micro-filters (16), hemi-housings (1712) for micro-columns (17), hemi-orifices (712) and
  • Figure 36 A and Figure 36B show how a flat elementary module can be produced by assembling at least one lower flat sub-part (2) and one upper flat sub-part (3), said upper flat sub-part (3) acting as a cover.
  • Figure 36 C shows the multiblock micro-array (319) thus manufactured.
  • Figure 37A shows a sub-part of a flat elementary module
  • Figure 37B shows the assembly of a sub-part of a flat elementary module with its complementary part
  • Figure 37C shows a micro-array or macro- multiblock array consisting of the stack of said flat elementary modules manufactured by assembling said complementary parts
  • Figure 37 D shows by transparency the micro-array or macro-array thus formed.
  • Figure 38 shows that when elementary flat modules (18110) do not have any components, the flat support which is used to manufacture one of its complementary sub-parts (1811012) provided with its semi-micro-mixers (1801912) can be very thin, which makes it possible to directly constitute a micro-array or a multi-block macro-array (18319) of high density.
  • Figure 39 shows that when elementary flat modules (19110) have components activated by an electrical circuit (391) with connection pads (390), the flat support of one of its complementary sub-parts (1911012) must reserve housings such as 393, and 394 for components such as microvalves, sensors, micro-heaters, which limits the density of the micro-array or a multi-block macro-array (19319) that can be formed.
  • the manufacturing steps of any type of said flat elementary modules of the invention can be used to manufacture ready-to-use systems for synthesis or chemical, biochemical analysis. or biological, for example by constituting micro-reservoirs of reagents.
  • the surfaces of said micro-arrays or multi-block macro-arrays are micro-machined or subjected to a finishing work with the same microfabrication techniques as if it were a flat surface in one piece. , i.e. essentially techniques of cutting, dry etching or wet etching by photolithography, laser ablation, assembly or gluing by fusion or anodic assembly, drilling, stamping, welding, electroplating, electroless plating or chemical vapor deposition.
  • the configuration of said elementary flat modules of said micro-arrays or multi-block macro-arrays of the invention can be adapted to the type of detection (micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, optical fibers, microscopy confocal, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface, by measurement of evanescent wave, by electiochemistry, by radioactivity, by Ra an spectroscopy, etc.) in adequacy with the aim of the analysis.
  • detection micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, optical fibers, microscopy confocal, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface, by measurement of evanescent wave, by electiochemistry, by radioactivity, by Ra an
  • said integrated preparation includes cell lysis, filtration of debris, extraction and purification on silica matrices, then amplification with one of the many methods well known to those skilled in the art, such as, among others, PCR, RT-PCR, LCR, laNASBA, SDA, TMA, RCA.
  • Ion exchange chromatography on DNA aims to use adsorption surfaces of microparticles or hydrophilic supports with high density of positive charges and to bind the negative charges of phosphates from DNA under conditions of low salinity, which eliminate the possibilities of protein and carbohydrate adsorption.
  • the conditions for eluting the adsorbed DNA are, on the contrary, conditions for high salinity.
  • Anion exchange chromatography ends either with precipitation with an alcohol or with desalination on micro-particles or matrices of silica gels.
  • Adsorption on matrices or micro-particles of silica gel aims to adsorb DNA when high levels of chaotropic salts bind free water to aqueous solutions, while proteins and carbohydrates do not adsorb.
  • the elution of the DNA adsorbed on matrices or microparticles of silica gel takes place under conditions of low salinity, for example with water, and the purified DNA is ready for use without the need for precipitation.
  • DNA extraction-purification methods with anion exchange chromatography do not require centrifugation, because gravity is sufficient. They offer a high degree of purity. They are particularly useful in a DNA extraction protocol on Gram-Negative Bacteria, for which the precipitation with alcohol can be avoided by using in the second stage a passage over micro-particles or silica gel matrix with centrifugation, or with magnetic silica gel micro-particles without centrifugation.
  • DNA extraction methods on silica gel matrices are suitable for most samples, except for Gram-negative bacteria and plants where protocols using anion exchange chromatography are appropriate, should they constitute a first step followed by a second step on simple or magnetic silica gel microparticles or silica gel matrices
  • Figure 40 A shows an embodiment of the invention and resides in multi-block microarrays or macro-arrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by exchange chromatography anions and then desalination on magnetic microparticles of silica gel.
  • FIG. 1 shows an embodiment of the invention and resides in multi-block microarrays or macro-arrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by exchange chromatography anions and then desalination on magnetic microparticles of silica gel.
  • 40A represents an embodiment of the invention and resides in multi-block microarrays or macro-arrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation comprising cell lysis, filtration of debris, two successive extractions- purifications of DNA on two micro-columns of different nature, the upstream amplification of said micro-array or multi-block macro-array (13319) by one of the methods known to those skilled in the art, then the detection on or downstream of said multi-block micro-array or macro-array (13319) by one of the methods known to those skilled in the art.
  • Said multi-block micro-arrays or macro-arrays (13319) of micro-wells (42) are constituted by stacking of flat elementary modules (13110), provided with two sub-parts (13155) and (13145).
  • Said sub-part (13145) comprises the micro-channels (41) provided with micro-mixers (13019).
  • Said sub-part (13155) comprises a micro-fluidic circuit (13055), composed:
  • an extraction-purification-washing chamber with anion exchange chromatography 13061
  • an anion exchange chromatography micro-column 13117
  • orifice 13029
  • shutter making it possible to isolate said microcolumn (13117)
  • orifice for introducing the elution reagent (13113)
  • orifice (13038) for introducing the washing reagents from said micro-column (13117), orifice for discharging the washing reagents (13011), shutter (13028) for passage to the extraction-purification-washing chamber (13063) on magnetic micro-particles with iron and silica gel
  • a chamber 13063
  • a chamber 13063
  • an electromagnet 13723
  • an orifice for introducing the silica gel microparticles (13722)
  • an orifice 13010 for introducing the washing reagents
  • an orifice for introducing (13726) the reagent activating adsorption of l To said magnetic micro-particles
  • Figure 40C shows said free magnetic microparticles in said chamber (13063).
  • a chamber for the preparation of purified DNA 13064
  • the DNA sample is introduced into the cell lysis and filtration chamber through said orifice (13007).
  • the lysis reagents are introduced through said orifice (13008).
  • Cell lysis debris is filtered on microfilters (13016), and the lysate is introduced into the extraction-purification-washing chamber (13061) with anion exchange chromatography micro-column (13117).
  • anion exchange chromatography micro-column 13117
  • the washing reagents from said micro-column (13117) are introduced, which are discharged through the orifice (13011).
  • the said microcolumn (13117) is isolated, then the DNA is eluted by introducing the elution reagent through the orifice (13113).
  • the removal of the shutters (13028) and (13029) allows the eluate to pass into the extraction-purification-washing chamber (13063) on magnetic microparticles.
  • the magnetic micro-particles are introduced through the opening (13722), then washing is carried out with reagents introduced through the orifice (13010) and magnetized with the electromagnet (13723).
  • the elution reagent is introduced through the orifice (13013).
  • the withdrawal of the obturator (13024) allows the passage to the preparation chamber of the purified DNA (13064), so that in the process the DNA passes successively on the micro-column of chromatography by anion exchange (13117) in the purification chamber (13063) on magnetic beads with silica gel.
  • said purified DNA is mixed with the buffer suitable for the DNA amplification method chosen, said buffer being introduced through the orifice (13012) and being deprived of enzymes, primers and dNTPs necessary for amplification.
  • said mixture of purified DNA and amplification buffer is introduced into said micro-channels (41) with micro-mixers (13019) of said sub-part (13145).
  • a connection with other stacks of elementary flat modules allows the mixing of the DNA with the specific or expensive reagents of the desired amplification in each micro-channel (41) and finally in each microwell (42), said specific reagents or expensive being such as enzymes necessary for the chosen amplification method, deoxynucleotides and primers for amplification.
  • Said specific or expensive reagents can also be modified primers suitable for the chosen detection method, additional probes necessary for the chosen amplification or detection method, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNAs, etc.
  • Figure 40B and Figure 40C show the action of an electromagnet on said magnetic microparticles.
  • Figure 41 A shows another embodiment of the invention and resides in multi-block microarrays or macro-arrays (14319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by chromatography by anion exchange then desalination on silica gel matrices.
  • Figure 41 A shows micro-arrays or multi-block macro-arrays (14319) of micro-wells
  • (42) are constituted by stacking of flat elementary modules (14110), provided with two sub-parts (14155) and (14145).
  • Said sub-part (14145) comprises the micro-channels (41) provided with micro-mixers
  • Said sub-part (14155) comprises a micro-fluidic circuit (14055), composed:
  • an extraction-purification-washing chamber with anion exchange chromatography 14061
  • an anion exchange chromatography micro-column 14117
  • orifice 14029
  • the DNA sample is introduced into the cell lysis and filtration chamber through said orifice (14007).
  • the lysis reagents are introduced through said orifice (14008).
  • the cellular lysis debris is filtered on the microfilters (14016), and the lysate is introduced into the extraction-purification-washing chamber (14061) with anion exchange chromatography micro-column (14117).
  • the washing reagents from said micro-column (14117) are introduced, which are discharged through the orifice (14011).
  • the elution reagent is introduced through the orifice (14013). Then the withdrawal of the obturator (14024) allows the passage to the chamber (14064) of preparation of the eluate, so that in the process the DNA passes successively on the micro column of chromatography by anion exchange ( 14117) then on the silica gel micro-column (14017).
  • said purified DNA is mixed with the buffer suitable for the DNA amplification method chosen, said buffer being introduced through the orifice (14012) and being deprived of enzymes, primers and dNTPs necessary for amplification. Then said mixture of purified DNA and amplification buffer is introduced into said microchannels (41) with micro-mixers (14019) of said subpart (14145).
  • a connection with other stacks of elementary flat modules allows the mixing of the DNA with the specific or expensive reagents of the desired amplification in each micro-channel (41) and finally in each microwell (42), said specific reagents or expensive being such as enzymes necessary for the chosen amplification method, deoxynucleotides and primers for amplification.
  • Said specific or expensive reagents can also be modified primers adapted to the chosen detection method, additional probes necessary for the chosen amplification method, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNA, etc.
  • Figure 41 B, Figure 52A, Figure 52B, Figure 52C represent another embodiment of the invention and resides in micro-arrays or multi-block macro-arrays (15319) dedicated to the analysis of fragments of DNA with an integrated preparation including cell lysis, filtration of debris, DNA extraction-purification on a micro-column of silica gel, amplification upstream of said multi-block micro-array or macro-array (15319) by one of the methods known to those skilled in the art, then detection on or downstream of said micro-array or macro-array multiblock (15319) by one of the methods known to those skilled in the art.
  • Said multi-block micro-arrays or macro-arrays (15319) of micro-wells (42) are constituted
  • an extraction-purification-washing chamber (15061) provided with a micro-column (15017), with orifice (15029) for obturator making it possible to isolate the microcolumn (15017), orifice for introducing the reagent d elution (15013), orifice (15038) for introduction of the washing reagent, orifice for evacuation of the washing reagents (15011), obturator (15024) for passage to the preparation chamber of purified DNA, * of a chamber of preparation of purified DNA (15064) with insertion opening
  • Said sub-part (15145) comprises the micro-channels (41) provided with micro-mixers (15019).
  • Figure 42 shows that an integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) can take place in flat modules (15 110) where a already introduced into the chamber (15064) through the orifice (15 012) a buffer suitable for amplification, said buffer being deprived of the enzymes, primers and dNTPs necessary for the amplification.
  • the DNA sample is introduced into the cell lysis and filtration chamber (15015) through said orifice (15007).
  • Lysis reagents then take place through the orifice (15008), the cellular lysis debris being filtered on the microfilters (15016), then the passage of the lysate, by opening the obturator (15009), in the chamber (15061) extraction-purification-washing with micro-column on a silica gel matrix (15017).
  • the lysate overflow is discharged through the orifice (15011) while said microcolumn on a silica gel matrix (15017) is isolated by the action of the shutter (15029).
  • the isolation makes it possible to keep a sufficient amount of lysate which will continue to shed its DNA on said micro-column (15017) while any residual debris are evacuated. Centrifugation takes place to remove contaminants.
  • washing is carried out with reagents introduced through the orifice (15038), then the washing reagents are discharged through the orifice (15011), then the elution reagent is introduced through the orifice ( 15013), the withdrawal of the obturator (15024) allows the eluate to pass to the chamber (15064) for preparing the purified DNA and to mix with the buffer adequate for the chosen amplification method.
  • Figure 52A shows that said mixture of purified DNA and amplification buffer is introduced into said micro-channels (41) with micro-mixers (15019) of said sub-part (15145).
  • Figure 52B shows a connection of said micro-array or macro-array (15319) of the invention with other stacks of flat elementary modules (9245), said flat elementary modules (9245) being provided with microchannels (9241) with micromixers (92019), said microchannels (9241) opening into the thickness and on the edge of said flat elementary modules (9245) by orifices converted into microwells (9242).
  • Said flat elementary modules are connected at the other end to flat elementary modules (9255) provided with microreservoirs (9210).
  • Said micro-reservoirs (9210) contain the specific or expensive reagents of the desired amplification in each micro-channel (9241) and therefore in each microwell (9242).
  • Said specific or expensive reagents are the enzymes necessary for the chosen amplification method, the deoxynucleotides and the amplification primers. They can also be modified primers adapted to the chosen detection method, additional probes necessary for the amplification and detection method chosen, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNA , etc.
  • DNA fragments can be amplified in very small volumes, under certain conditions of passivation of the reaction support surfaces (Wilding P., Shoi ⁇ her MA, Kricka LJ.PCR in a silicon microstructure. Clin. Chem. 1994, 40 / 9, 1815-1818. _ Shoffher MA, Cheng J., Hvichia GE, Kricka LJ, Wilding P.Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Research, 1996, 24, 2, 375-379. _ Cheng J,. Shoffher MA, Hvichia GE, Kricka LJ, Wilding P. Chip PCR. 11. Investigation of different PCR amplification Systems in microfabricated silicon-glass chips.
  • DNA polymerase a buffer and deoxynucleotides are used.
  • copies of the two strands are produced directly by elongation from primers which have hybridized (paired) to the complementary sequence on a single strand of the DNA fragment to be amplified.
  • Elongation is the successive incorporation of deoxynucleotides on a newly formed strand thanks to the action of DNA Polymerase which progresses in the 5 '-> 3' direction.
  • the deoxynucleotide incorporated on the newly formed strand is that which is complementary to the nucleotide on the copied strand.
  • Tm Two strands of DNA pair below a temperature called Tm.
  • Tm Two strands of DNA pair below a temperature
  • the best specificities in amplification are obtained when the primers are paired at the highest possible temperature, so as to dissociate all non-specific pairings and to keep only the specific pairings, that is to say those of a primer and the strand to be amplified, those of the strand being elongated and the strand to be amplified, and those of the 2 complementary strands of the DNA fragment. to amplify.
  • annealing temperature generally fixed at Tm-5 ° C. Different amplifications on different DNA fragments differ in their Tm and therefore their Ta.
  • the annealing temperatures must not be too high, under penalty of not amplifying anything, nor too low, under penalty of not being specific enough.
  • concentration of MgC12 must be optimized: a too low concentration harms the yield, an excess induces a lack of specificity.
  • the specificity is also helped by the choice of certain DNA Polymerases, as well as by the experimental protocols.
  • the first cycle should start at an elevated temperature.
  • a complete DNA amplification reaction mixture should be stored at 4 ° C for the DNA polymerase to be inactive. If allowed to be active between 4 ° C and 60 ° C, nonspecific amplifications could occur.
  • the first cycle must start at a high temperature, for example with the denaturation temperature at 92 ° C - 96 ° C, so that the reaction mixture spends as little time as possible in a warming phase from 4 ° C to 60 ° C , where the risk of non-specific reaction exists.
  • the amplified DNA can also be labeled for example by incorporation of deoxynucleotides labeled during elongation by DNA Polymerase during the amplification.
  • a primer which can be linked in many ways, well known to those skilled in the art, to molecules which aid detection.
  • a primer can be linked to a fluorophore for fluorescence detection, or to affinity molecules such as biotin so that the amplified DNA strand binds to beads coated with streptavidin, or to labeling molecules such as labeling oligonucleotides.
  • the type of labeling will depend on the type of detection (micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, fiber optics, confocal microscopy, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface , by measurement of the evanescent wave, by electrochemistry, by radioactivity, etc) in adequacy with the aim of the analysis.
  • detection micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, fiber optics, confocal microscopy, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface , by measurement of the evanescent wave, by electrochemistry, by radioactivity, etc
  • each microchannel (41) of a micro-array or macro-array the purified DNA to be analyzed is mixed with primers specific for the DNA fragment to be amplified.
  • the four deoxynucleotides dATP, dTTP, dGTP, dCTP, and with the enzyme used for the amplification method chosen by connection with a second multi-block micro-array or macro-array formed by stacking flat elementary modules (9245).
  • Said flat elementary modules (9245) bring, from micro-reservoirs carried by said flat elementary modules (9255) to which they are connected, the desired mixture of said enzyme, deoxynucleotides, and said amplification primers so that a fragment precise DNA is amplified and presented in a precise microwell (42) of said mico-array or macro-array (15319).
  • primers used for the amplification depends on the chosen methodology. For example, these may be primers "beacons", with two sequences complementary to each other added at the ends, one linked to a fluorophore, the other linked to a repressor. These primers have the particularity of being detectable by a fluorescent signal only from the moment when they hybridize to a complementary strand of DNA, which removes the repressor from the fluorophore and allows the emission of a fluorescent signal.
  • Tyagi S. Kramer FR Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 1996, 14, 303-308.
  • Molecular beacon probes combined with amplification by NASBA enable homogenous, real-time detection of RNA. Nucleic Acids Research, 1998, 26, 9, 2150-2155. ).
  • the fluorescence of molecular beacons can be detected in real time by measuring the evanescent wave from optical fibers (Liu X, Tan W. A Fiber-Optic Evanescent Wave DNA biosensor based on novel molecular beacons. Anal. Chem. 1999, 71, 5054-5059)
  • an amplification detected by the exonuclease activity of a DNA polymerase requires the use of a third probe complementary to a sequence inside the DNA fragment to be amplified, and linked to a fluorophore and to its repressor .
  • This third probe can be added to the reaction mixture kept cold with primers and deoxynucleotides , and DNA Polymerase.
  • Other real-time amplification detection methods can be implemented.
  • each micro-well can be electronically addressed to exercise individualized control over amplification and hybridization reactions (Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M.
  • each micro-well of a micro-array or multi-block macro-array of the invention can be addressed individually by electrodes to control the reaction in progress in said micro-well.
  • the device according to the invention can be used for its own manufacture in the factory if one wishes to manufacture ready-to-use flat functional units for DNA amplification.
  • a micro-array 1519
  • flat module units 15110
  • the amplified DNA can be detected by one of the detection methods known in the current state of the art, such as detection of the amplification directly in real time with a measurable marking during the amplification, or detection by mass spectrometry. , or detection by micro-electrophoresis, or detection by chromatography, or detection by hybridization on probes. Fluorescence, bioluminescence, chemiluminescence, colorimetry, measurement of the evanescent wave, the plasmon resonance surface, elecfrochemistry, radioactivity, densimetry can be used.
  • the labeling authorizing one of these detections can be by fluorescence, bioluminescence, chentiluminescence, or else carried out with molecules having a redox potential differential, or with label molecules such as a short oligonucleotide sequence, or with molecules affinity, or a combination of said previous labeling methods.
  • said hybridization can be detected by numerous methods, including methods with labeling, methods without labeling, methods of dynamic observation of the evolution of hybridization.
  • the colorimetric methods by fluorescence, by radioactivity, by plasmon resonance surface, by mass spectrometry, etc.
  • double strand on magnetic beads coated with streptavidin This separation can take place in the microwells of a second multi-block micro-array or macro-array filled with said beads coated with streptavidin, or else in a micro-array or macro-array provided with microchannels with enlarged portions and filled with said coated beads. with streptavidin.
  • This method of separation of the two strands makes it possible to separate on the one hand the strand of amplified DNA biotynilized with the magnetic beads, and on the other hand the strand of free amplified DNA in the supernatant.
  • each microwell (42) of a micro-array or multi-block macro-array (15319) It is also possible, in each microwell (42) of a micro-array or multi-block macro-array (15319), to detect the amplification by mass spectrometry by one of the methods known in the present state of the art. Art.
  • a DNA preparation for one of the detection methods by MALDI-TOF mass spectrometry (described by Little DP, Cornish TJ, O'Donnell MJ, Braun A., Cotter RJ, Kôster H.. MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantifies of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezo-electric pipet.
  • DNA can be amplified in the micro-channels (41) of a micro-array or multi-block macro-array (15319) with one of the two biotynilized primers, then the two strands of the amplified double strand DNA can be separated by means of beads magnetic housed in the enlarged portions of said microchannels (41).
  • the supernatant containing the amplified single-stranded DNA can be presented in the microwells (42) of said micro-array or macro-array (15319) and mixed with a derivative of hydroxysuccinic or hydroxypiccolinic acid, either directly on said micro-array or macro -array multibloc (15319), either in the micro-wells of another connected macro-array or multi-block micro-array.
  • a laser shot in each micro-well then makes it possible to ionize the DNA fragment and to accelerate it in an electric field for detection by mass spectrometry., Where the comparison of the expected mass of said DNA fragment and the mass measured confirms the conformity of the fragment to the expected mass
  • Figure 53 shows a stack of flat modules (40110) provided with sub-parts (40145) supporting all or part of the micro-channels (41).
  • the stacking of said flat elementary modules (40110) is carried out in an offset manner in order to obtain a bevel on a lateral face.
  • the spacing between said sub-parts (40145) allows the intercalation of electrical insulating flat modules (40195) in the stack.
  • This bevel multi-block microarray or macroarray can be connected to a second bevel stack of flat elementary modules (40245) alternated with insulating flat modules.
  • Said flat elementary modules (40245) are provided at their end with a reading range (40250), as well as a device for discharging the analytes.
  • microelectrophoresis can be envisaged by applying a voltage to the two ends of the microchannels of the flat elementary modules (40245) connected last.
  • the elongation fragments of the Sanger reaction are read on the reading range (40250) according to the method controlled by the type of labeling (fluorescence, electro-chemical, etc.).
  • microelectrophoresis can also take place by mass spectrometry, as is done for protein fragments (Minarik M, Foret F, Karger BL. Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing, Electrophoresis 2000, 21, 247-254).
  • the micro-arrays or macro-arrays of the invention are suitable for these sequential processes.
  • the multi-block micro-arrays or macro-arrays of the invention are also suitable for sequential processes, such as for example separation by chromatography followed by separation by micro-electrophoresis (Cf Tragas C , Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interace. Electrophoresis 2000, 21, 227-237).
  • Hybridization can take place either on beads that can be transported from one place to another (Fan ZH, Mangru S, Granzow R, Heaney P, Ho W, Dong Q, Kumar R. Dynamic DNA hybridization on a chip using paramagnetic beads. Anal. Chem., 1999, 71, 4851-4859), or on a surface dedicated to this use.
  • Oligonucleotide probes are generally synthesized according to the phospharamidite method in automated systems.
  • eaucage SL Caruthers MH Deoxynucleoside phospharamidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, vol 22, 70, 1859-1862. 1981.
  • Mcbride LJ Caruthers, MH An investigation of several oligonucleotide phospharamidites usefùynuucleotides Tetrahedron Letters 24 (3): 245-248, 1983.
  • Polynucleotide synthesizing apparatus ⁇ iina A., Ohira T., Miyamoto S., Nippon Zeon Co Ltd, US Patent 4 671 941. _ Multiple polymer synthesizer. Judd AK. SRI International. US 5,053,454. Polynucleotide synthesizer. Whitehouse CM., Whitehouse GP, Sesholtz DA, Norman D., Eastman Kodak Company, Rochester. . US patent 5,112,575. _ Automated synthesis of oligonucleotides. McGraw RA, Grosse WM, University of Georgia Research Foundation, Inc. US Patent 5368823.
  • WO Patent 9925724 Nery large scale immobilized polymer synthesis using mechanically directed flow paths. Winkler JL, Fodor SA, Buchko CJ, Ross DA, Aldwin L. Affymetrix, Inc Santa Clara. US Patent 5 885 837. _ General purpose gene synthesizer., Zelinka RJ, Itakura K., Sims CW, Kaplan BE, Systec Inc., US Patent 4 598 049. Remotely programmable matrices with memories. Nova MP, Senyei AE, IRORI, US 5 874214. Method and apparatus for producing position addressable combinatorial libraries. Dehlinger PJ, Palo Alto, CA. US Patent 5,763,263.
  • the photoexposure used to select the polymers to be deprotected is usually done by photomasks. But it can also be done through the use of digital micro-mirrors (Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide micro-arrays using a digital micromirror array. Nature Biotechnology. 1999. Vol 17.974-978).
  • the oligonucleotide probes can also be synthesized outside and deposited on said micro-wells (42) of said multi-block micro-array or macro-array (15319) or by a multi-array block or by means of a micro-array or macro. connected multiblock array of the invention. Adequate surface treatments of said micro-wells (42) can allow adsorption of the probes. Some methodologies recommend, on the contrary, their covalent fixation.
  • the synthesis can be envisaged either nucleotide by nucleotide, or by groups of several nucleotides.
  • the multi-block micro-arrays or macro-arrays of the invention allow, for example an in situ synthesis of octamers from a library of tetramers. Two octamers can then be linked to form a 16-sea.
  • Figures 54 A, 54B, 54C, 54D show that one can synthesize in situ in the wells of a monoblock micro-array or macro-array (339) octa-nucleotides from prefixed tetra-nucleotides and tetranucleotides brought by microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • Figures 55A, 55B, 55C, 55D show that one can synthesize in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) of the octa-nucleotides from prefixed tetra-nucleotides and tetranucleotides brought by the microchannels (41) of said microarrays or multiblock macro-arrays (319) of the invention.
  • Figures 5A, 56B, 56C, 56D show that octa nucleotides from tetra can be synthesized in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) -fixed nucleotides and tetranucleotides brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
  • the same operation can also be carried out by connection with another micro-array or multi-block macro-array of the invention.
  • Figures 57A, 57B, 57C show that one can synthesize in situ in the wells of a monoarray microarray or macro-array (338) DNA chips in each micro-well of said monoblock micro-array or macro-array (338 ) using said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention to supply the reagents and using photolabile groups and micromasks for specific photoexposure of each micro-well of said micro-array or monoblock macro-array (338).
  • the same result can be obtained by phot ⁇ exposing each of said micro-wells using micro-mirrors, and by bringing the reagents with micro-arrays or macro-arrays of the invention connected.
  • Figures 58A, 58B, 58C show that DNA chips can be synthesized in situ in the wells of a multi-block microarray or macro-array (319) in each micro-well (42) of said micro-array or macro-array monoblock (338) using said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention to supply the reagents and using photolabile groups and micromasks (187) for specific photoexposure of each micro- well (42) of said micro-array or multi-block macro-array (319).
  • the same result can be obtained by photoexposing each of said micro-wells using micro-mirrors, and by bringing the reagents with micro-arrays or macro-arrays of the invention connected.
  • Each well of a multi-block micro-array or macro-array of the invention can be addressed individually by optical fibers for the detection of reaction products (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal. Biochem., 1997, 251, 270-279), or to control electro- kinetics or the aggregation of microparticles or molecules in said microwell (WO9740385A1 - Light-controUed electrokinetic assembly of microparticles near surfaces), or to direct a photoexposure in said microwell.
  • (3119) is ensured by the use of polymers, either by the use of polymers for mass production of the flat modules constituting the stack of said micro-arrays or multi-block macro-arrays (3119), or by coating with a polymer of the edge of each of said flat modules constituting the stack, either by the use of a monobloc intermediate piece of polymer acting as a joint between two of said micro-arrays or multi-block macro-arrays (3119).
  • a barcode, color or electromagnetic code marking of each flat module to be included in the constitution of a micro-array of the invention dedicated to a sample or to a specific operation allows the recomposition of stacks of flat modules having " received specific reagents in other stacks

Abstract

The invention concerns multiblock micro-arrays or macro-arrays incorporating laboratories on chips, for use in chemical, biochemical or biological analysis or chemical or biochemical synthesis. An inventive multiblock micro-array or macro-array consists of a stack of flat elementary modules provided with parallel microchannels at their surface which emerge into the thickness and on the edge of their sides, each flat elementary module providing a line to said multiblock micro-array or macro-array. The microchannels can be provided with micro-mixers and enlarged portions, provided with molecule-fixing surface and can receive micro-columns or micro-particles or micro-spheres. The juxtaposition of the lines first set of reagents enables to perform the parallel reactions on very small volumes. Two inventive multiblock micro-arrays or macro-arrays can be orthogonally connected to cross a first set of reagents a with a second, and form a sealed chain of analysis or synthesis.

Description

Micro-arrays ou macro-arrays multiblocs avec laboratoires sur puces intégrésMulti-block micro-arrays or macro-arrays with laboratories on integrated chips
La présente invention concerne des macro-arrays multiblocs ou micro-arrays multi-blocs en trois dimensions intégrés dans une chaîne prête à l'emploi continue ultra compacte de synthèse ou d'analyse chimique, biochimique ou biologique. Ces micro-arrays ou macro- arrays multi-blocs intègrent des Lab-on-a-Chip (laboratoires sur puces). L'analyse biologique ainsi que l'analyse et la synthèse chimique et biochimique utilisent de plus en plus des "micro-arrays" ou des "macro-arrays", le terme anglais «array» désignant en analyse chimique un quadrillage plan dense ou très dense d'objets identiques, généralement des puits ou des micro-emplacements pour dépôts ou synthèse (voir Figure IC). Les densités courantes des macro-arrays sont de l'ordre de 10 à 100 micro-emplacements au cm2, les densités courantes des micro-arrays sont de l'ordre de 100 à 1000 micro-emplacements au cm2. Dans ces puits ou sur ces micro-emplacements sont déposées ou synthétisées de petites quantités de molécules chimiques, de peptides, d'acides nucléiques ou d'autres molécules biologiques ou organiques. Les macro-arrays et les micro-arrays sont utilisés pour la recherche de nouveaux matériaux, pour le screening de nouveaux polymères synthétiques, pour la découverte de nouveaux médicaments, pour l' analyse de peptides et de protéines, pour l'étude des interactions protéiques, pour des programmes de Biologie Moléculaire tels qu'études de maladies génétiques, Génomique (cartographie des génomes par marqueurs moléculaires, étude des polymorphismes génétiques), de Génomique Fonctionnelle (expressions de gènes différentielles spécifiques de processus physio-pathologiques, d'organes ou de ou de tissus affectés par des maladies, recherche de nouvelles cibles thérapeutiques), de Pharmacogénomique (réponse individuelle aux médicaments, recherche du mode d'action de médicaments). Le terme anglais Lab-on-a-Chip, laboratoire sur puce, désigne des supports miniaturisés d'analyse, fabriqués avec des techniques de micro-fabrication issues de la micro-électronique, où les fluides sont convoyés dans des micro-canaux, classiquement de 10 à 500 microns de diamètre. Leur intérêt réside dans l'économie de réactifs, la simplification et l'amélioration des procédés d'analyse et de synthèse, l'augmentation de la vitesse réactionnelle, la préservation des contaminations, l'augmentation du nombre d'analyses ou de synthèse par unité de volume et par unité de temps, la baisse des coûts, la portabilité des supports d'analyse, etc.The present invention relates to multi-block macro-arrays or three-dimensional multi-block micro-arrays integrated in a ready-to-use continuous ultra-compact chain of synthesis or chemical, biochemical or biological analysis. These multi-block micro-arrays or macro-arrays incorporate Lab-on-a-Chip (labs on chips). Biological analysis as well as chemical and biochemical analysis and synthesis increasingly use "micro-arrays" or "macro-arrays", the English term "array" designating in chemical analysis a dense or very flat grid pattern. dense of identical objects, generally wells or micro-locations for deposits or synthesis (see Figure IC). The current densities of macro-arrays are of the order of 10 to 100 micro-locations per cm 2 , the common densities of micro-arrays are of the order of 100 to 1000 micro-locations per cm 2 . In these wells or on these micro-locations are deposited or synthesized small quantities of chemical molecules, peptides, nucleic acids or other biological or organic molecules. Macro-arrays and micro-arrays are used for the research of new materials, for the screening of new synthetic polymers, for the discovery of new drugs, for the analysis of peptides and proteins, for the study of protein interactions , for Molecular Biology programs such as studies of genetic diseases, Genomics (mapping of genomes by molecular markers, study of genetic polymorphisms), of Functional Genomics (expressions of differential genes specific for physiopathological processes, organs or or tissues affected by diseases, search for new therapeutic targets), Pharmacogenomics (individual response to drugs, search for the mode of action of drugs). The English term Lab-on-a-Chip, laboratory on a chip, designates miniaturized analysis supports, manufactured with micro-manufacturing techniques from microelectronics, where the fluids are conveyed in micro-channels, conventionally from 10 to 500 microns in diameter. Their interest lies in the economy of reagents, the simplification and improvement of analysis and synthesis processes, the increase in reaction speed, the preservation of contamination, the increase in the number of analyzes or synthesis by unit of volume and per unit of time, lower costs, portability of analysis media, etc.
La présente invention apporte le moyen d'intégrer des Lab-on-a-chip aux macro-arrays ou aux micro-arrays en assurant des connexions sans rupture dans la chaîne d'analyse, ce qui n'était pas le cas auparavant, puisque dans les procédés antérieurs il fallait envisager une phase d'exposition à l'air libre (telle que par exemple un pipetage ) pour passer des Lab-on- chips aux macro-arrays ou micro-arrays, ou des macro-arrays ou micro-arrays aux Lab-on- chips. En effet, la configuration multiblocs de macro-arrays et de micro-arrays de l'invention bénéficie d'une architecture compacte des connexions fluidiques entre diverses parties du système d'analyse ou de process microfluidique et permet de manipuler de très petites quantités de fluides avec des connexions étanches à l'évaporation et aux contaminations.The present invention provides the means of integrating Lab-on-a-chips into macro-arrays or micro-arrays by ensuring seamless connections in the analysis chain, which was not the case before, since in the previous processes, it was necessary to envisage a phase of exposure to the open air (such as for example pipetting) in order to pass from Lab-on-chips to macro-arrays or micro-arrays, or macro-arrays or micro- arrays with Lab-on-chips. Indeed, the multiblock configuration of macro-arrays and micro-arrays of the invention benefits from a compact architecture of the fluid connections between various parts of the analysis system or microfluidic process and makes it possible to handle very small quantities of fluids with waterproof connections to evaporation and contamination.
L'invention décrit aussi les techniques de micro-fabrication utilisées pour fabriquer le système de connexions fluidiques de ces macro-arrays ou microarrays.The invention also describes the micro-fabrication techniques used to manufacture the system of fluidic connections of these macro-arrays or microarrays.
A titre d'exemple, il est fait une description d'une application dans le domaine de l'analyse d'acides nucléiques en Biologie Moléculaire qui permet l 'analyse et la synthèse d'acides nucléiques sur des macro-arrays ou des micro-arrays multiblocs de l'invention intégrant des laboratoires d'analyse sur puces (Lab- on- a -Chip) assurant au moins la préparation des échantillons et des réactifs.By way of example, a description is given of an application in the field of nucleic acid analysis in Molecular Biology which allows the analysis and synthesis of nucleic acids on macro-arrays or micro- multiblock arrays of the invention integrating analysis laboratories on chips (Lab-on-a -Chip) ensuring at least the preparation of samples and reagents.
Il est montré le type de configuration générale du systèmes d'analyse ou de synthèse intégré et miniaturisé que permet l'invention.The type of general configuration of the integrated and miniaturized analysis or synthesis systems shown in the invention is shown.
L'architecture et la conception de fabrication des différents modules qui composent ces micro-arrays ou macro-arrays sont déclinés selon les critères d'analyse demandés et les différentes options retenues dans le mode de préparation des réactifs et dans le mode de détection .The architecture and manufacturing design of the different modules that make up these micro-arrays or macro-arrays are available according to the analysis criteria requested and the different options selected in the mode of preparation of the reagents and in the detection mode.
L'exemple retenu en matière d'analyses d'acides nucléiques n'est qu'une illustration de ce qui peut être réalisé selon l'invention dans des domaines aussi divers que par exemple la chimie de synthèse combinatoire, l'analyse chimique combinatoire, les immuno-analyses, bref tout type d'analyse biologique ou biochimique ou chimique où l'on doit assurer de très importants de programmes de recherche et d'analyse, chaque opération élémentaire devant utiliser le moins possible d'échantillons et de réactifs et devant durer le moins de temps possible. The example chosen for nucleic acid analyzes is only an illustration of what can be achieved according to the invention in fields as diverse as for example combinatorial synthetic chemistry, combinatorial chemical analysis, immunoassays, in short any type of biological or biochemical or chemical analysis where very important research and analysis programs must be ensured, each elementary operation having to use the least possible of samples and reagents and before last as little time as possible.
Etat Actuel de l'Art dans les micro-arrays ou macro-arrays en Analyse Chimique, Biochimique ou Biologique:Current State of the Art in micro-arrays or macro-arrays in Chemical, Biochemical or Biological Analysis:
55
On peut se reporter à un certain nombre de références pour l'utilisation des micro-arrays ou macro-arrays en analyse et en synthèse chimique et biochimique, et en analyse biologique:We can refer to a certain number of references for the use of micro-arrays or macro-arrays in chemical and biochemical analysis and synthesis, and in biological analysis:
* pour la recherche de nouveaux matériaux (Xiang XD, Sun X, Briceno G, Lou Y, Wang* for research on new materials (Xiang XD, Sun X, Briceno G, Lou Y, Wang
10 KA,Chang H, Wallace-Freedman WD, Chen SW, Schultz PG. A combinatorial approach to material discovery. Science 1994, 268, 1738.)10 KA, Chang H, Wallace-Freedman WD, Chen SW, Schultz PG. A combinatorial approach to material discovery. Science 1994, 268, 1738.)
* pour l'analyse de molécules organiques.(Braun RM, Beyder A, Xu J, Wood MC, Ewing AG, Winograd N. Spatially resolved détection of attomole quantifies of organic molécules localized in picoliter vials using Time-of-Flight secondary ion mass spectrometry.* for the analysis of organic molecules. (Braun RM, Beyder A, Xu J, Wood MC, Ewing AG, Winograd N. Spatially resolved detection of attomole quantifies of organic molecules localized in picoliter vials using Time-of-Flight secondary ion mass spectrometry.
15 AnaLChem., 1999, 71, 3318-3324).15 AnaLChem., 1999, 71, 3318-3324).
* pour le screening de nouveaux polymères synthétiques (Reynolds CH. Designing diverse and focused combinatorial libraries of synthetic polymers. Journal;of Combinatorial Chemistry, 1999, 1, 297-306).* for the screening of new synthetic polymers (Reynolds CH. Designing diverse and focused combinatorial libraries of synthetic polymers. Journal; of Combinatorial Chemistry, 1999, 1, 297-306).
* pour la découverte de nouveaux médicaments (Hogan J. Combinatorial Chemistry in Drug 20. Discovery. Nature Biotechnology, 1997. 15, 4.)* for the discovery of new drugs (Hogan J. Combinatorial Chemistry in Drug 20. Discovery. Nature Biotechnology, 1997. 15, 4.)
* pour l' analyse de peptides et de protéines (Dawies H, Lomas L, Austen B. Profiling of Amyloïd beta peptide variants using SELDI ProteinChip arrays.. Biotechniques. 1999, 27, 1258-1261. _ Kaur S, Me Guire L, Tang D, Dollinger G, Huebner V. Affinity sélection and mass-spectrometry-based stratégies to identify lead compounds in combinatorial libraries.* for the analysis of peptides and proteins (Dawies H, Lomas L, Austen B. Profiling of Amyloid beta peptide variants using SELDI ProteinChip arrays .. Biotechniques. 1999, 27, 1258-1261. _ Kaur S, Me Guire L, Tang D, Dollinger G, Huebner V. Affinity selection and mass-spectrometry-based strategies to identify lead compounds in combinatorial libraries.
25 Journal of Protein Chemistry, 1997, Vol 16, N°5, 505-511. _ Tartar A, La chimie combinatoire. Biofutur, 1997, 168, 26-31 _ Michelet D, Hélène C. La synthèse combinatoire. Pour la science. 1997. 241. 50-5425 Journal of Protein Chemistry, 1997, Vol 16, No. 5, 505-511. _ Tartar A, Combinatorial chemistry. Biofutur, 1997, 168, 26-31 _ Michelet D, Hélène C. Combinatorial synthesis. For science. 1997. 241. 50-54
* pour l'étude des interactions protéiques (Colas P. Micro-arrays pour l'étude des interactions protéiques à l'échelle génomique. Médecine Sciences. 2000, 506, 50- 55.)* for the study of protein interactions (Colas P. Micro-arrays for the study of protein interactions on a genomic scale. Médecine Sciences. 2000, 506, 50-55.)
30 * pour des programmes de Biologie Moléculaire tels qu'études de maladies génétiques, de génomique (cartographie des génomes par marqueurs moléculaires, étude des polymorphismes génétiques), de génomique fonctionnelle (expressions différentielles spécifiques d'organes ou de processus physiopathologiques ou de tissus affectés par des maladies, recherche de nouvelles cibles thérapeutiques), de pharmacogénomique (réponse30 * for Molecular Biology programs such as studies of genetic diseases, genomics (mapping of genomes by molecular markers, study of genetic polymorphisms), functional genomics (specific differential expressions of organs or pathophysiological processes or affected tissues by diseases, search for new therapeutic targets), pharmacogenomics (response
35 individuelle aux médicaments, recherche du mode d'action de médicaments), etc. (Southern E.M., Case-Green S.C, Elder J.K., Johnson M., Mir K.U., Wang L., Williams J.C. Arrays of complementary oligonucleotides for analysing the hybridization behaviour of nucleic acids. Nucleic Acids Research, 1994, Vol 22, No8, 1368-1373. _ Maskos U., Southern E.M. A study of oligonucleotide reassociation using large arrays of oligonucleotides synthetised on a glass support. Nucleic Acids Research, 1993, Nol21, Νo20, 4663-4669. _ Schena M., Shalon D., Davis R.W., Brown P.O._ Quantitative monitoring of gène expression patterns with a complementary DNA microarray. Science, 1995, 270, 467-4 70. _ Yang GP, Ross DT, Kuang WW, Brown PO, Weigel RJ.. Combining SSH and cDNA microarrays for rapid identification of differentially expressed gènes. Nucleic Acids Research, 1999, 27, 6, 1517- 1523. _ Lashkari D.A., McCusker J.H., Davis R.W.Whole génome analysis: expérimental access to ail génome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR. Proc. Natl. Acad. Sci. USA. 1997, 94, 8945-8947. _ Livache T., Fouque B., Roget A., Marchand J., Bidan G., Téoule R., Mathis G. Polypyrrole DNA chip on a silicon device: example of Hepatitis C virus genotyping. Analytical Biochemistry, 1998, 255, 188-194. _ Guo Z., Guilfoyle R.A., Thiel A.J., Wang R., Smith L.M. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Research, 1994, Nol.22, Νo24, 5456-5465. _ O'Donnell- Maloney M.J., Smith C.L., Cantor C.R.. The development of microfabricated arrays for DNA sequencing and analysis. TiBTECH, 1996, 14, 69-73. _ Little D.P., Braun A., O'Donnell M.J., Kôster H.. Mass spectrometry from miniaturized arrays for full comparative DNA analysis. Nat. Med., 1996, 13, Nol2, 1413-16. _ Little D.P., Cornish T.J., O'Donnell M.J., Braun A., Cotter R.J., Koster H. __ MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantities of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezo-electric pipet. Anal. Chem. 1997, 69, 4540-46. _ Hacia J.G., Edgemon K., Sun B., Stern D.., Fodor S.P.A., Collins FS.Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.Nucleic Acids Research, 1998, 26, 16, 3865-3866 _ Weiler J, Hoheisel JD. Combining the préparation of oligonucleotide arrays and synthesis of high-quality primers. Analytical Chemistry, 1996, 243, 218-227 _ Cheng J, Sheldon EL, Wu L, Uribe A, Gerrue LO, Carrino J, Heller MJ, O'Connell JP. Préparation and hybridization analysis of DNA/RNA from E.Coli on microfabricated bioelectronic chips. Nature Biotechnology. 1998, 16,. 541-546. _ Mirzabekov et al.. DNA analysis and diagnostics on oligonucleotide microchips. PNAS, 1996, 93, pp4913-4918 __ Mirzabekov et al. Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology. Applied and Environmental Microbiology, 1997, 63, 6, 2397-2402 -35 individual drug, research mode of action of drugs), etc. (Southern EM, Case-Green SC, Elder JK, Johnson M., Mir KU, Wang L., Williams JC Arrays of complementary oligonucleotides for analyzing the hybridization behavior of nucleic acids. Nucleic Acids Research, 1994, Vol 22, No8, 1368 -1373. _ Maskos U., Southern EM A study of oligonucleotide reassociation using large arrays of oligonucleotides synthetized on a glass support. Nucleic Acids Research, 1993, Nol21, Νo20, 4663-4669. _ Schena M., Shalon D., Davis RW, Brown PO_ Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995, 270, 467-4 70. _ Yang GP, Ross DT, Kuang WW, Brown PO, Weigel RJ .. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Research, 1999, 27, 6, 1517-1523. _ Lashkari DA, McCusker JH, Davis RWWhole genome analysis: experimental access to ail genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR. Proc. Natl. Acad. Sci. USA. 1997, 94, 8945-8947. _ Livache T., Fouque B., Roget A., Marchand J., Bidan G., Téoule R., Mathis G. Polypyrrole DNA chip on a silicon device: example of Hepatitis C virus genotyping. Analytical Biochemistry, 1998, 255, 188-194. _ Guo Z., Guilfoyle RA, Thiel AJ, Wang R., Smith LM Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Research, 1994, Nol. 22, Νo24, 5456-5465. _ O'Donnell- Maloney MJ, Smith CL, Cantor CR. The development of microfabricated arrays for DNA sequencing and analysis. TiBTECH, 1996, 14, 69-73. _ Little DP, Braun A., O'Donnell MJ, Kôster H .. Mass spectrometry from miniaturized arrays for full comparative DNA analysis. Nat. Med., 1996, 13, Nol2, 1413-16. _ Little DP, Cornish TJ, O'Donnell MJ, Braun A., Cotter RJ, Koster H. __ MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantities of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezo- electric pipet. Anal. Chem. 1997, 69, 4540-46. _ Hacia JG, Edgemon K., Sun B., Stern D .., Fodor SPA, Collins FS.Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.Nucleic Acids Research, 1998, 26, 16, 3865-3866 _ Weiler J, Hoheisel JD. Combining the preparation of oligonucleotide arrays and synthesis of high-quality primers. Analytical Chemistry, 1996, 243, 218-227 _ Cheng J, Sheldon EL, Wu L, Uribe A, Gerrue LO, Carrino J, Heller MJ, O'Connell JP. Preparation and hybridization analysis of DNA / RNA from E.Coli on microfabricated bioelectronic chips. Nature Biotechnology. 1998, 16 ,. 541-546. _ Mirzabekov et al .. DNA analysis and diagnostics on oligonucleotide microchips. PNAS, 1996, 93, pp4913-4918 __ Mirzabekov et al. Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology. Applied and Environmental Microbiology, 1997, 63, 6, 2397-2402 -
Mirzabekov et al. DNA séquence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5-mers contiguously stacked to microchip oligonucleotides. Nucleic Acids research, 2000, 28, 5, 1193-1198 _ Rehman FN, Audeh M, Abrams ES, Hammond PW, Kenney M, Boles TC. Immobilization of acrylamide-modified oligonucleotides by co-polymerization. Nucleic Acids research, 1999, 27, 2, 649-655).Mirzabekov et al. DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5-mers contiguously stacked to microchip oligonucleotides. Nucleic Acids research, 2000, 28, 5, 1193-1198 _ Rehman FN, Audeh M, Abrams ES, Hammond PW, Kenney M, Boles TC. Immobilization of acrylamide-modified oligonucleotides by co-polymerization. Nucleic Acids research, 1999, 27, 2, 649-655).
Le dépôt des molécules sur les micro-arrays ou macro-arrays s'effectue grâce à des micro- arrayers (Tisone TC. Dispensing System for miniaturized diagnostics. IVD Technology, 1998, 4:40. _ Lemmo AV. Quantitative nanoliter dispensing. Genêt Eng News, 1998, 14:30. _ Oldenburg KR, Zhang J, Chen T, Maffia A, Blom KF, Combs AP, Chung TDY. Assay miniarurization for ultra high througput screening of combinatorial and discrète compound libraries: a 9600 well (0.2 microliter) assay System. J. Biomol Screening, 1998, 3, 55-62. _ Papen R., Croker K, Kolb A. Nanoliter dispensing technology. Genêt. Eng. Neews. 1998, 9:16-17. Lemmo AV, Rose DJ, Tisdone T. InkJet dispensing technology: applications in Drug Discovery. Current Opinion in Biotechnology. 1998, 9, 615-617._ Graves DJ, Su HJ, McKenzie SE, Surrey S, Fortina P. System for preparing microhybridization arrays on glass slides. Anal. Chem, 1998, 5085-5092 _ Me Bride SE, Moroney RM, Chiang W. Electrohydrodynamic pumps for high density microf uidic arrays. μ-TAS 1998, Canada, pp45-50.)The molecules are deposited on micro-arrays or macro-arrays using micro-arrayers (Tisone TC. Dispensing System for miniaturized diagnostics. IVD Technology, 1998, 4:40. _ Lemmo AV. Quantitative nanoliter dispensing. Broom Eng News, 1998, 14:30. _ Oldenburg KR, Zhang J, Chen T, Maffia A, Blom KF, Combs AP, Chung TDY. Assay miniarurization for ultra high througput screening of combinatorial and discrete compound libraries: a 9600 well (0.2 microliter) assay System. J. Biomol Screening, 1998, 3, 55-62. _ Papen R., Croker K, Kolb A. Nanoliter dispensing technology. Broom. Eng. Neews. 1998, 9: 16-17. Lemmo AV, Rose DJ, Tisdone T. InkJet dispensing technology: applications in Drug Discovery. Current Opinion in Biotechnology. 1998, 9, 615-617._ Graves DJ, Su HJ, McKenzie SE, Surrey S, Fortina P. System for preparing microhybridization arrays on glass slides. Anal. Chem, 1998, 5085-5092 _ Me Bride SE, Moroney RM, Chiang W. Electrohydrodynamic pumps for high density microf uidic arrays. μ-TAS 1998, Canada, pp45-50.)
Des développements existent aujourd'hui pour fabriquer des micro-aiguilles (Lebouitz KS, Pisano AP,. Microneedles and microlancets fabricated using SOI wafers and isotropic etching. Electrochemical Society Proceedings, vol 98-14, 237-242) et des multipipettes miniaturisées (Papautsky I, Brazzle JD, Weiss RB, Ameel TA, Frazier AB. Parallel sample manipulation using micromachined pipette arrays. SPIE Proceedings 1998, Vol. 3515-09, 104-114. _ Brazzle JD, Papautsky I, Frazier AB. Fluid-coupled hollow metallic micromachined needle arrays. SPIE Proceedings 1998, 3515-13, ppl 16-124 _ Frazier AB, Methods for preparing devices having metallic hollow microchannels on planar substrate surfaces. US Patent 5876582 _ Szita N, Buser R. A pneumatically actuated micropipetting device. SPIE 3258. 156 - 160.).Developments exist today to manufacture micro-needles (Lebouitz KS, Pisano AP ,. Microneedles and microlancets fabricated using SOI wafers and isotropic etching. Electrochemical Society Proceedings, vol 98-14, 237-242) and miniature multipipettes (Papautsky I, Brazzle JD, Weiss RB, Ameel TA, Frazier AB. Parallel sample manipulation using micromachined pipette arrays. SPIE Proceedings 1998, Vol. 3515-09, 104-114. _ Brazzle JD, Papautsky I, Frazier AB. Fluid-coupled hollow metallic micromachined needle arrays. SPIE Proceedings 1998, 3515-13, ppl 16-124 _ Frazier AB, Methods for preparing devices having metallic hollow microchannels on planar substrate surfaces. US Patent 5876582 _ Szita N, Buser R. A pneumatically actuated micropipetting device. SPIE 3258. 156 - 160.).
Ces développerments sont illustrés par la Figure IC où un dispositif multipipette (1776) dispense les réactifs sur un micro-array (1777).These developments are illustrated in Figure IC where a multipipette device (1776) dispenses the reagents on a micro-array (1777).
Tels qu'ils sont conçus aujourd'hui, les micro-arrayers font subir aux analytes une exposition à l'air libre, où des problèmes d'évaporation et de contamination sont à craindre.As they are designed today, micro-arrayers subject the analytes to exposure to the open air, where problems of evaporation and contamination are to be feared.
Chaque puits du micro-array ou macro-array peut être adressé individuellement par des électrodes pour diriger un champ électrique, comme les micro-arrays du CEA-LETI ou de la société Nanogen ( Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a micro-electronic chip array. Nature Biotechnology, 2000, 18, 199-204)., ou bien des capteurs, comme par exemple dans le brevet "Microelectrochemical sensor and sensor array. US Patent 4874500". Chaque puits du micro-array ou macro-array peut aussi être adressé individuellement par des fibres optiques (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal. Biochem., 1997, 251, 270-279). En Analyse Biologique, les micro-arrays où sont faits des αepoxs αe sonαes peuvent e e eu concurrence avec des puces de synthèse in situ par utilisation de groupements photolabiles (Fodor S.P. Accessing genetic information with high-density DNA arrays. Science, 1996, 274, 610-613 _ Jacobs J.W., Fodor S.P.A. Combinatorial chemistry applications of light- directed chemical synthesis. TiBTECH, 1994, 12, 19-26. Lipshutz R.J., Morris D., Chee M;, Hubbell E., Kozal M.J., Shah , ShenN., Yang R., Fodor S.P.A. Using oligonucleotide probe arrays to access genetic diversity. Biotechniques, 1995, 19, No3, 442-447. _ Singh- Gasson S; Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide micro-arrays using a digital micromirror array. Nature Biotechnology.1999, 17, 974-978). C'est sur la synthèse par groupements photolabiles que repose en partie les systèmes d'Affymetrix pour la Biologie Moléculaire et ceux d'Affymax pour l'étude d'affinité de peptides (Brevet International WO9210092A1 d'Affymax Technologies). Il s'effectue alors un changement d'échelle dans la densité d'intégration (jusqu'à 400 000 sondes au cm2), mais par contre ces systèmes sont dédiés à l'analyse exhaustive d'un seul échantillon. Des procédés sont envisagés pour les intégrer dans une chaîne de préparation et d'analyse, où un seul échantillon est totalement pris en charge dans une micro-circuiterie fermée (Anderson R.C., Lipshutz R. J., Rava R.P., Fodor S.P. Integratéd nucleic acid diagnostic device. US Patent WO 97/02357). Parallèlement à cela, afin de parer au fait que ces dispositifs de synthèse in situ sont dédiés à l'analyse exhaustive d'un seul échantillon, il est envisagé de fabriquer des micro-arrays ou macro-arrays de puces (Affymax Technologies. Biόarray chip reaction apparatus and its manufacture. WO 9533846 _ Methods for concurrently processing multiple biological chip assays. Affymetrix. US Patent 5874219 _ Affymax Technologies. Very large scale immobilized polymer synthesis, WO9210092 ).Each well of the micro-array or macro-array can be individually addressed by electrodes to direct an electric field, like the micro-arrays of CEA-LETI or of Nanogen (Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a micro-electronic chip array. Nature Biotechnology, 2000, 18, 199-204), or sensors, as for example in the patent "Microelectrochemical sensor and sensor array. US Patent 4874500 ". Each well of the micro-array or macro-array can also be addressed individually by optical fibers (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal. Biochem., 1997, 251, 270- 279). In Biological Analysis, micro-arrays where αepoxes are made of its αα can compete with in situ synthesis chips by using photolabile groups (Fodor SP Accessing genetic information with high-density DNA arrays. Science, 1996, 274, 610-613 _ Jacobs JW, Fodor SPA Combinatorial chemistry applications of light- directed chemical synthesis. TiBTECH, 1994, 12, 19-26. Lipshutz RJ, Morris D., Chee M ;, Hubbell E., Kozal MJ, Shah, ShenN ., Yang R., Fodor SPA Using oligonucleotide probe arrays to access genetic diversity. Biotechniques, 1995, 19, No3, 442-447. _ Singh- Gasson S; Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide micro-arrays using a digital micromirror array. Nature Biotechnology. 1999, 17, 974-978). It is on the synthesis by photolabile groups that partially rests the systems of Affymetrix for Molecular Biology and those of Affymax for the study of affinity of peptides (International Patent WO9210092A1 of Affymax Technologies). There is then a change of scale in the integration density (up to 400,000 probes per cm 2 ), but on the other hand these systems are dedicated to the exhaustive analysis of a single sample. Methods are envisaged to integrate them into a preparation and analysis chain, where a single sample is fully supported in a closed micro-circuit (Anderson RC, Lipshutz RJ, Rava RP, Fodor SP Integrate nucleic acid diagnostic device. US Patent WO 97/02357). At the same time, in order to counter the fact that these in situ synthesis devices are dedicated to the exhaustive analysis of a single sample, it is envisaged to manufacture micro-arrays or macro-arrays of chips (Affymax Technologies. Biόarray chip reaction apparatus and its manufacture. WO 9533846 _ Methods for concurrently processing multiple biological chip assays. Affymetrix. US Patent 5874219 _ Affymax Technologies. Very large scale immobilized polymer synthesis, WO9210092).
Avantages de la miniaturisation et des systèmes micro-fluidiques:Advantages of miniaturization and microfluidic systems:
De nombreux laboratoires essaient d'ajuster la consommation de réactifs au minimum permis par la sensibilité du système d'analyse. Le but est l'économie en réactifs, cruciale lorsque certains d'entre eux sont particulièrement chers. La miniaturisation est une piste particulièrement intéressante à suivre, dès lors que se présentent les premiers réactifs onéreux dans une chaîne d'analyse.Many laboratories try to adjust the consumption of reagents to the minimum allowed by the sensitivity of the analysis system. The goal is the economy in reagents, crucial when some of them are particularly expensive. Miniaturization is a particularly interesting avenue to follow, as soon as the first expensive reagents appear in an analysis chain.
Par exemple, en matière d'analyse d'acides nucléiques, le premier réactif rencontré très onéreux est l'ADN polymérase utilisée dans diverses méthodes d'Amplification d'un fragment d'acides nucléiques. Dans cet exemple, les phases préliminaires à l'Amplification (Prélèvement, Lyse cellulaire, Extraction, Concentration, Purification) peuvent sans grande conséquence économique rester à une macro-échelle. Par contre, le coût de l' ADN Polymérase fait qu'il est intéressant de passer à une micro-échelle dès cette étape d'amplification. La conséquence d'une telle option est que les phases qui suivent (détection) doivent elles aussi se passer à une micro-échelle. Lesdites phases de détection possèdent par ailleurs un niveau limite de sensibilité et peuvent par conséquent indiquer le niveau limite de miniaturisation et se situer en facteur limitant de la réduction de taille.For example, in nucleic acid analysis, the first very expensive reagent encountered is DNA polymerase used in various methods of Amplification of a nucleic acid fragment. In this example, the preliminary phases to Amplification (Sampling, Cell Lysis, Extraction, Concentration, Purification) may without great economic consequences remain on a macro-scale. On the other hand, the cost of DNA Polymerase makes it interesting to go to a micro-scale from this stage amplification. The consequence of such an option is that the following phases (detection) must also happen on a micro-scale. Said detection phases also have a limit level of sensitivity and can therefore indicate the limit level of miniaturization and be a factor limiting the reduction in size.
Les impératifs de qualité exigent une minimisation des faux-positifs et des faux-négatifs. Aux causes internes (bruits de fond dûs aux limites de l'instrumentation et aux défauts de purification, réactions croisées avec des analytes normalement présents dans l'échantillon) peuvent s'ajouter en effet des causes externes, généralement des contaminations des échantillons par les molécules présentes dans l'environnement.Quality imperatives require minimization of false positives and false negatives. Internal causes (background noises due to instrumentation limits and lack of purification, cross-reactions with analytes normally present in the sample) may be added to external causes, generally contamination of samples by molecules present in the environment.
Des circuits d'analyse étanches doivent permettre de s'affranchir de ce risque externe, généralement en réduisant le plus possible des opérations de pipetage.Sealed analysis circuits must overcome this external risk, generally by reducing pipetting operations as much as possible.
Dès le moment où les volumes d'analyses sont très réduits, la manipulation des échantillons et des analytes peut se faire sur des supports haute densité qui non seulement bénéficient d'une cinétique de réaction grandement améliorée par les faibles volumes mis enjeu, mais permettent de réduire le nombre et la longueur de déplacements des organes des robots de manipulation des échantillons et des réactifs, ce qui améliore les temps demandés pour ces opérations. De plus ces hautes densités favorisent les architectures massivement parallèles, ce qui permet de réduire considérablement le temps total d'analyses d'un lot d'échantillons. Enfin, la réduction de taille permet d'envisager plusieurs étapes de l'analyse sur le même consommable, facilitant et réduisant les manipulations et menant aux concept de consommables « intégrés », dont l'utilisation permet là-aussi d'accélérer l'analyse.From the moment when the volumes of analyzes are very reduced, the handling of samples and analytes can be done on high density supports which not only benefit from a kinetics of reaction greatly improved by the low volumes involved, but allow to reduce the number and length of movements of the organs of the robots for handling the samples and reagents, which improves the times required for these operations. In addition, these high densities favor massively parallel architectures, which considerably reduces the total analysis time for a batch of samples. Finally, the reduction in size makes it possible to envisage several stages of the analysis on the same consumable, facilitating and reducing the manipulations and leading to the concept of “integrated” consumables, the use of which also makes it possible to speed up the analysis. .
Les hautes cadences d'analyse doivent être recherchées en minimisant les investissements de robotisation. Là aussi, la piste suivie est la miniaturisation des appareils qui pourrait autoriser leur fabrication à moindre coût. En effet, ces appareils miniaturisés utilisent moins de matière première, ont moins de pièces, utilisent des composants meilleur marché, demandent moins de place, et ont pour ambition de rassembler plusieurs fonctions. A coût égal, il est à prévoir que les systèmes miniaturisés offriront la possibilité de faire des analyses plus nombreuses ou plus exhaustives.High rates of analysis must be sought while minimizing investment in robotization. There too, the track followed is the miniaturization of the devices which could allow their manufacture at lower cost. Indeed, these miniaturized devices use less raw material, have fewer parts, use cheaper components, require less space, and aim to bring together several functions. At equal cost, it is to be expected that miniaturized systems will offer the possibility of carrying out more or more exhaustive analyzes.
La réduction de taille permet d'envisager des consommables « intégrés », qui sont conçus en fonction d'une fabrication potentielle à moindre coût, par exemple en étant adaptés à une fabrication de masse ou en utilisant des matériaux peu onéreux. Ces matériaux peu onéreux sont représentés non seulement par le silicium et le verre et leurs dérivés, mais aussi par les plastiques Par ailleurs, la miniaturisation n'apporte pas directement la flexibilité, par contre elle peut rendre économiquement viables des principes d'analyse flexibles qui seraient restés trop coûteux en restant fabriqués aux côtes d'un grand format. Par exemple, la miniaturisation peut rendre économiquement pertinents des systèmes d'analyse d'ADN où l'utilisateur décide de ses sites d'amplification sur l'ADN et analyse à volonté les fragments amplifiés. Ces systèmes-là sont plus flexibles que ceux fonctionnant par hybridation où des oligonucleotides prédéterminés et non interchangerables sont fixés sur un support d'analyse.The reduction in size makes it possible to envisage “integrated” consumables, which are designed as a function of potential manufacture at lower cost, for example by being suitable for mass production or by using inexpensive materials. These inexpensive materials are represented not only by silicon and glass and their derivatives, but also by plastics On the other hand, miniaturization does not directly bring flexibility, on the other hand it can make flexible principles of analysis economically viable which would have remained too costly by remaining manufactured alongside a large format. For example, miniaturization can make DNA analysis systems economically relevant where the user decides his amplification sites on DNA and analyzes the amplified fragments at will. These systems are more flexible than those operating by hybridization where predetermined and non-interchangeable oligonucleotides are fixed on an analysis support.
Les appareils miniaturisés utilisant moins de réactifs représentent aussi un avantage potentiel sur le plan environnemental : moins de réactifs toxiques, moins de pollution.Miniaturized devices using fewer reagents also represent a potential environmental advantage: fewer toxic reagents, less pollution.
Etat Actuel de l'Art dans les connexions micro-fïuidiques:Current state of the art in micro-fluid connections:
Lorsqu'on envisage des analyses miniaturisées, il est important pour les connexions fluidiques entre les diverses parties du système:When considering miniaturized analyzes, it is important for fluid connections between the various parts of the system:
* d'opter pour des options qui permettent d'éviter l'évaporation (Mayer G, Kôhler M, Michromechanical compartments for biotechnological applications: fabrication and investigation of liquid evaporation. Sensors and Actuators A , 1997, 202 - 207). Des solutions pour lutter contre l'évaporation existent comme des systèmes étanches, ou en atmosphère saturée d'humidité, ou avec utilisation de ménisques d'huiles en surface des chambres de réaction. Mais aucune d'entre elles ne résout la difficulté de conserver l'étanchéité des connections entre deux parties d'un système d'analyse *d'opter pour des options qui permettent d'éviter les contaminations.* opt for options that avoid evaporation (Mayer G, Kôhler M, Michromechanical compartments for biotechnological applications: fabrication and investigation of liquid evaporation. Sensors and Actuators A, 1997, 202 - 207). Solutions to combat evaporation exist as sealed systems, or in an atmosphere saturated with humidity, or with the use of menisci of oils on the surface of the reaction chambers. But none of them solves the difficulty of maintaining the tightness of the connections between two parts of an analysis system * of opting for options which make it possible to avoid contamination.
Des développements ont amené à proposer certaines parties du système d'analyse avec un degré élevé de miniaturisation . Les configurations actuelles de connexion font appel à des juxtapositions et à des superpositions de composants aplatis connectés de proche en proche par des connecteurs placés sur les surfaces supérieures ou inférieures desdits composants aplatis. La Figure 1A montre un dispositif microfluidique pouvant être utilisé en l'Etat Actuel de l'Art. Ledit dispositif est à trois étages constitués des modules plats (a), (b) et (c) pourvus de microcanaux parallèles à leur surface. Les trois étages sont connectés par des micro-canaux perpendiculaires à la surface desdits modules plats.Developments have led to proposing certain parts of the analysis system with a high degree of miniaturization. Current connection configurations use juxtapositions and overlays of flattened components connected step by step by connectors placed on the upper or lower surfaces of said flattened components. Figure 1A shows a microfluidic device which can be used in the current state of the art. Said device has three stages consisting of flat modules (a), (b) and (c) provided with microchannels parallel to their surface. The three stages are connected by micro-channels perpendicular to the surface of said flat modules.
La Figure 1 B montre un dispositif microfluidique pouvant être utilisé en l'Etat Actuel de l'Art. Ledit dispositif connecte deux modules plats pourvus de micro-canaux parallèles à leur surface avec des connexions assurées par des micro-canaux perpendiculaires à la surface desdits modules plats. Les publications et brevets suivants illustrent ces configurations de connexions:Figure 1B shows a microfluidic device that can be used in the current state of the art. Said device connects two flat modules provided with micro-channels parallel to their surface with connections provided by micro-channels perpendicular to the surface of said flat modules. The following publications and patents illustrate these connection configurations:
* Flexible packaging and interconnect scheme for microfluidic Systems, SPIE Proceedings, 3606-16, ppl 11-118.* Flexible packaging and interconnect scheme for microfluidic Systems, SPIE Proceedings, 3606-16, ppl 11-118.
*Kovacs et al.; Novel interconnection technologies for integrated microfluidics Systems. Proceedings of the Solid-State sensor and Actuator Workshop, Hilton Head, South Carolina, 1998, June 8-1 l, pp 112-115. *NerLee D, Alcock A^ Clarl G et al.. Fluid circuit technology: integrated interconnect technology for miniature fluidic devices. Proceedings of Solid State Sensors and Actuator Workshop, Hilton Head Island, SC, pp 9-14, 1996. * Gonzalez C, Pan JY, Collins SD, Smith RL. Packaging technology for miniature IND instrumentation. MD§DI April 1998.* Kovacs et al .; Novel interconnection technologies for integrated microfluidics Systems. Proceedings of the Solid-State sensor and Actuator Workshop, Hilton Head, South Carolina, 1998, June 8-1 l, pp 112-115. * NerLee D, Alcock A ^ Clarl G et al .. Fluid circuit technology: integrated interconnect technology for miniature fluidic devices. Proceedings of Solid State Sensors and Actuator Workshop, Hilton Head Island, SC, pp 9-14, 1996. * Gonzalez C, Pan JY, Collins SD, Smith RL. Packaging technology for miniature IND instrumentation. MD§DI April 1998.
* Gonzalez C, Smith RL, Collins SD. Fluidic interconnects for modular assembly of chemical microsystems. Proceedings of 1997 International Conférence on Solid State sensors and Actuators, Chicago, Institute of Electrical and Electronics Engineers, 1997, pp527-530.* Gonzalez C, Smith RL, Collins SD. Fluidic interconnects for modular assembly of chemical microsystems. Proceedings of 1997 International Conférence on Solid State sensors and Actuators, Chicago, Institute of Electrical and Electronics Engineers, 1997, pp527-530.
* Man PF, Jones KD, Mastrangelo CH. Microfluidic plastics interconnects for multi -bioanal sis chip modules. SPIE vol 3224, ppl 96- 200.* Man PF, Jones KD, Mastrangelo CH. Microfluidic plastics interconnects for multi -bioanal sis chip modules. SPIE vol 3224, ppl 96-200.
* Karger et al., Microscale fluid handling System, US Patent 5 872 010, *.Kovacs GTA, Micromachined fluidic coupler, US Patent 5 890 745 * Zanzucchi PJ, Cherukuri SC, Me Bride SE. Partioned microelectronic aiid fluidic device array for clinical diagnostics and chemical synthesis. US Patent 5585069.* Karger et al., Microscale fluid handling System, US Patent 5,872,010, * .Kovacs GTA, Micromachined fluidic coupler, US Patent 5,890,745 * Zanzucchi PJ, Cherukuri SC, Me Bride SE. Partioned microelectronic aiid fluidic device array for clinical diagnostics and chemical synthesis. US Patent 5585069.
* Cherukuri SC, Demers RR, Fan ZH, Levine AW, Me Bride SE, Zanzucchi PJ. Method and System inhibiting cross-contaminatioi in fluids of combinatorial chemistry device.* Cherukuri SC, Demers RR, Fan ZH, Levine AW, Me Bride SE, Zanzucchi PJ. Method and System inhibiting cross-contaminatioi in fluids of combinatorial chemistry device.
US Patent 5603351. * Bings ΝH, Wang C, Skinner CD, Colyer CL, Thibault P, Harrison DJ. Microfluidic devices connected to fused -silica capillaries with minimal dead volume. Anal. Chem. 1999, 71, 3292-3296.US Patent 5603351. * Bings ΝH, Wang C, Skinner CD, Colyer CL, Thibault P, Harrison DJ. Microfluidic devices connected to fused -silica capillaries with minimal dead volume. Anal. Chem. 1999, 71, 3292-3296.
* Elwenspoek M, Lammerink TS J, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245.* Elwenspoek M, Lammerink TS J, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245.
Les configurations actuelles des connexions des dispositifs miniaturisés ne permettent pas de connecter des micro-arrays ou des macro-arrays avec des Lab-on-a-chip (laboratoires sur puce) avec compacité, et souffrent de ruptures dans les connexions fluidiques, matérialisées par des phases de pipetage avec exposition des analytes à l'air libre, ce qui entraîne des problèmes d'évaporation et de contamination. La Figure IC illustre une configuration où une rampe de pipetage multipipette (1776) dispense les réactifs sur un micro-array (1777).The current configurations of the connections of miniaturized devices do not allow micro-arrays or macro-arrays to be connected with Lab-on-a-chip (laboratories on chip) with compactness, and suffer from breaks in the fluidic connections, materialized by pipetting phases with exposure of the analytes to the open air, which causes problems of evaporation and contamination. Figure IC illustrates a configuration where a multipipette pipetting manifold (1776) dispenses the reagents on a micro-array (1777).
Parallèllement à cela, les Lab-on-a-chip actuels ne peuvent pas directement alimenter des micro-arrays ou des macro-arrays ou être alimentés directement par eux. Les différentes architectures adoptées jusqu'à présent pour des articulations entre les différentes parties d'un micro- système microfluidique d'analyse chimique ou biochimique ou biologique ne sont pas en mesure d'apporter la compacité souhaitée, surtout lorsque certains composants ne peuvent pas, pour les raisons décrites précédemment, être miniaturisés et qu'ils doivent par conséquent rester à une macro-échelle.At the same time, the current Lab-on-a-chip cannot directly feed micro-arrays or macro-arrays or be powered directly by them. The different architectures adopted so far for articulations between the different parts of a microfluidic micro-system for chemical or biochemical or biological analysis are not able to provide the desired compactness, especially when certain components cannot, for the reasons described above, to be miniaturized and that they must therefore remain on a macro scale.
En l'Etat Actuel de l'Art , aucune solution proposée ne résout la difficulté de conserver une architecture compacte des connexions tout en gardant l'étanchéité desdits connexions entre deux parties d'un système d'analyse ou de process microfluidique.In the current state of the art, no solution proposed solves the difficulty of maintaining a compact architecture of the connections while keeping the tightness of the said connections between two parts of an analysis system or microfluidic process.
Pourtant, le manque de compacité des connections entre dispositifs πiiniaturisés rend difficile l' analyse de macro volumes d'échantillons prélevés aune macroéchelle avec des réactifs que l'on souhaite utiliser en très petites quantités. L'insuffisance de compacité des connexions entre dispositifs miniaturisés rend également difficiles les configurations massivement parallèles recherchées pour des hautes cadences d'analyse, en particulier sur un grand nombre d'échantillons. Elle rend difficiles l'isolement et la manipulation de microvolumes ou de nanovolumes, ainsi que la précision de l'analyse, à cause des fortes contraintes physico- chimiques comme la diffusion et les phénomènes non désirés d' électro-osmose, alors que dans le même temps la miniaturisation rehausse l'attention qu'on doit apporter aux forces de capillarité et de l'hydrophobicité des contenants. Les difficultés constatées lorsque des connexions entre dispositifs miniaturisés manquent de compacité viennent aussi des contraintes architecturales comme celles des volumes morts dans les connexions, et des difficultés de micropositionnement sur des architectures dispersées. L'insuffisance de compacité des connexions entres systèmes miniaturisés rend difficile la diminution du coût de l'instrumentation, la fabrication de masse des consommables en vue de la diminution de leur coût, la conception de consommables intégrés où plusieurs étapes de l'analyse sont effectués sur le même support miniaturisé, le retour à un système compact pour la détection des analytes, utile pour être adapté à une détection de type "biopuces".However, the lack of compactness of the connections between miniature devices makes it difficult to analyze macro volumes of samples taken at a macro scale with reagents which one wishes to use in very small quantities. The insufficient compactness of the connections between miniaturized devices also makes the massively parallel configurations sought for high analysis rates difficult, in particular on a large number of samples. It makes it difficult to isolate and manipulate microvolumes or nanovolumes, as well as the precision of the analysis, because of the strong physicochemical constraints such as diffusion and unwanted electro-osmosis phenomena, while in the at the same time, miniaturization enhances the attention that must be given to the capillary forces and the hydrophobicity of the containers. The difficulties observed when connections between miniaturized devices lack compactness also come from architectural constraints such as those of the dead volumes in the connections, and from difficulties of micro-positioning on dispersed architectures. The insufficient compactness of the connections between miniaturized systems makes it difficult to reduce the cost of instrumentation, mass production of consumables with a view to reducing their cost, the design of integrated consumables where several stages of analysis are carried out on the same miniaturized support, the return to a compact system for the detection of analytes, useful to be adapted to a "biochip" type detection.
En l'Etat actuel de l'Art, les différentes architectures adoptées jusqu'à présent des articulations entre les différentes parties d'un microsystème microfluidique d'analyse chimique ou biochimique ou biologique montrent un manque d'adaptation à la vie du consommable: i)fabrication,ii) utilisation suite aux prélèvements des échantillons iii) analyse des échantillons.In the current state of the art, the different architectures adopted up to now for articulations between the different parts of a microfluidic microsystem for chemical or biochemical or biological analysis show a lack of adaptation to the life of the consumable: i ) manufacturing, ii) use following the taking of samples iii) analysis of samples.
En effet, des assemblages très compacts de consommables destinés à l'analyse de très nombreux échantillons peuvent être mis à profit pour la fabrication à l'usine et lors des phases d'analyses massivement parallèles. Par contre, ces assemblages compacts de consommables n'ont pas d'intérêt lors des phases de prélèvement sur de très nombreux échantillons. Ces phases de prélèvement ont lieu soit chez le malade ou dans l'environnement dans lequel est prélevé l'échantillon, soit au dispensaire au laboratoire d'analyse et sont par nature des étapes dispersées dans le temps et dans l'espace.Indeed, very compact assemblies of consumables intended for the analysis of a large number of samples can be used for manufacturing in the factory and during massively parallel analysis phases. On the other hand, these compact assemblies of consumables have no interest during the sampling phases from very many samples. These sampling phases take place either from the patient or in the environment in which the sample is taken, either at the dispensary at the analysis laboratory and are by nature stages dispersed in time and space.
En d'autres termes , en l'Etat Actuel de l'Art , rien dans la conception des consommables intégrés ne s'adapte véritablement au fait que dans la vie dudit consommable, la fabrication à l'usine est une étape de resserrement dans le temps et de concentration dans l'espace, l'étape de prélèvement est une étape de dispersion spatio-temporelle, l'étape d'analyse au laboratoire peut être à nouveau une phase de resserrement spatio-temporel.In other words, in the current state of the art, nothing in the design of integrated consumables truly adapts to the fact that in the life of said consumable, manufacturing at the factory is a step of tightening in the time and concentration in space, the sampling step is a spatio-temporal dispersion step, the laboratory analysis step can again be a spatio-temporal tightening phase.
En l'Etat actuel de l'Art, les différentes architectures adoptées jusqu'à présent des articulations entre les différentes parties d'un microsystème microfluidique d'analyse chimique ou biochimique ou biologique montrent un décalage entre le potentiel d'utilisation de très petits volumes qui seraient suffisants pour effectuer des détections (certaines techniques de détection permettraient d'utliser quelques nanolitres seulement, voire moins) et les quantités réellement utilisées pour les mélanges réactionnels (souvent des microlitres). Ceci est dû à un déficit de savoir-faire dans les connexions pour manipuler et transporter les très petits volumes.In the current state of the art, the different architectures adopted up to now for articulations between the different parts of a microfluidic microsystem for chemical or biochemical or biological analysis show a gap between the potential for using very small volumes which would be sufficient to carry out detections (certain detection techniques would make it possible to use only a few nanoliters, or even less) and the quantities actually used for the reaction mixtures (often microliters). This is due to a lack of know-how in the connections for handling and transporting very small volumes.
Finalement, dans les configurations proposées jusqu'ici, certaines possibilités techniques ne sont pas assez exploitées, comme par exemple celles montrées par le publications comme : -"A new fabrication method for borosilicate glass capillary tubes with latéral Mets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij NF. Sensors and Actuators A 60, 1997, 219-222 ". Cette publication montre qu'il est possible de fabriquer des microcanaux débouchant dans l'épaissseur et sur la tranche de micro-dispositifs plats. - " Zanzucchi PJ, Cherukuri SC, Me Bride SE.. Etching to form cross-over, non intersecting channel networks for use in partitioned microelectronic and fluidic device arrays for clinical diagnostics and chemical synthesis. US Patent 5681484". Ce brevet montre qu'il est possible de fabriquer des micro-canaux qui se croisent en s'évitant. Finally, in the configurations proposed so far, certain technical possibilities are not exploited enough, such as for example those shown by the publications like: - "A new fabrication method for borosilicate glass capillary tubes with lateral Mets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij NF. Sensors and Actuators A 60, 1997, 219-222 ". This publication shows that it is possible to manufacture microchannels opening into the thickness and on the edge of flat micro-devices. - "Zanzucchi PJ, Cherukuri SC, Me Bride SE .. Etching to form cross-over, non intersecting channel networks for use in partitioned microelectronic and fluidic device arrays for clinical diagnostics and chemical synthesis. US Patent 5681484". This patent shows that it is possible to manufacture micro-channels which cross each other while avoiding each other.
Etat Actuel de l'Art dans les techniques de microfabrication des systèmes miniaturisés d'analyse chimique, biochimique et biologique:Current state of the art in microfabrication techniques for miniaturized systems of chemical, biochemical and biological analysis:
Pour qu'un système d'analyse chimique ou biochimique puisse être miniaturisé, il faut d'une part que les conduits et composants qui guident ou reçoivent les fluides (microcanaux, microréservoirs, micro-mixers, micro-colonnes, etc) soient miniaturisés, d'autre part que les composants qui gèrent le parcours des fluides et des réactifs (micro-vannes, micropompes, micro-capteurs, micro-chauffeurs, etc) soient eux-aussi miniaturisés, et enfin que des connexions puissent s'établir à l'intérieur et vers l'extérieur du dispositif. (Elwenspoek M, Lammerink TSJ, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245. __ Nerpoorte EMJ, van der Schoot BH, Jeanneret S, Manz A, Widmer HM, de Rooij ΝF. Three-dimensional micro flow manifolds for miniaturized chemical anlysis Systems . J. Micromech. Microeng.1994, 4, 246-256, 1994 _ Schabmueller CGJ, Koch M, Evans AGR, Brunnschweiler A. . Design and fabrication of a microfluidic circuitboard. J. Micromech.Microeng. 1999, 9, 176-179._ Lammerink TSJ, Spiering NL, Elwenspoek M, van den Berg A, Modular concept for fluid handling System. Proc. IEEE Micro Electro Mechanical Systems, 1996, San Diego pp389-384 _ Richter M, Prak A, Eberl M, Leeuwis H, Woias P, Steckenborn A. 1997. A chemical microanalysis System as a microfluid demonstrator. Proc. Transducers 97, IEEE Chicago, pp303-306._ Kovacs GTA, Petersen K, Albin M. Silicon micromachining: sensors to Systems. Analytical Chemistry, 1996, 407A - 412A _ Gravesen P, Branebjerg J, Jensen OS.. .Microfluidics. A review.J. Micromech. Microeng. 1993. 3. 168-182. _ Shoji S, Esahi M. Microflow devices and Systems. J. Micromech. Microeng. 1994.4. 157-171. _ Bϋttgenbach S., Robohm C. Microflow devices for miniaturized chemical analysis Systems. SPIE 1998, vol 3539, 51-61 _ Urban G, Jobst G, Moser I. Chemo-and biosensor microsystems for clinical applications. SPIE 1998. Vol 3539, 46-50).In order for a chemical or biochemical analysis system to be miniaturized, on the one hand, the conduits and components which guide or receive the fluids (microchannels, microreservoirs, micro-mixers, micro-columns, etc.) must be miniaturized, on the other hand that the components which manage the flow of fluids and reagents (micro-valves, micropumps, micro-sensors, micro-heaters, etc.) are also miniaturized, and finally that connections can be established at the inside and outside the device. (Elwenspoek M, Lammerink TSJ, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245. __ Nerpoorte EMJ, van der Schoot BH, Jeanneret S, Manz A, Widmer HM , de Rooij ΝF. Three-dimensional micro flow manifolds for miniaturized chemical anlysis Systems. J. Micromech. Microeng. 1994, 4, 246-256, 1994 _ Schabmueller CGJ, Koch M, Evans AGR, Brunnschweiler A.. Design and fabrication of a microfluidic circuitboard, J. Micromech.Microeng. 1999, 9, 176-179._ Lammerink TSJ, Spiering NL, Elwenspoek M, van den Berg A, Modular concept for fluid handling System. Proc. IEEE Micro Electro Mechanical Systems, 1996, San Diego pp389-384 _ Richter M, Prak A, Eberl M, Leeuwis H, Woias P, Steckenborn A. 1997. A chemical microanalysis System as a microfluid demonstrator. Proc. Transducers 97, IEEE Chicago, pp303-306._ Kovacs GTA , Petersen K, Albin M. Silicon micromachining: sensors to Systems. Analytical Chemistry, 1996, 407A - 412A _ Gravesen P, Br anebjerg J, Jensen OS ... Microfluidics. A review.J. Micromech. Microeng. 1993. 3. 168-182. _ Shoji S, Esahi M. Microflow devices and Systems. J. Micromech. Microeng. 1994.4. 157-171. _ Bϋttgenbach S., Robohm C. Microflow devices for miniaturized chemical analysis Systems. SPIE 1998, vol 3539, 51-61 _ Urban G, Jobst G, Moser I. Chemo-and biosensor microsystems for clinical applications. SPIE 1998. Vol 3539, 46-50).
Il pourra être envisagé des techniques et des fabrications séparées d'une part pour les supports avec micro-conduits et microcanaux, d'autre part pour les micro-composants, quitte à procéder à un montage, de préférence automatisé, en fin de fabrication.It will be possible to envisage separate techniques and manufacturing on the one hand for the supports with micro-conduits and microchannels, on the other hand for the micro-components, even if it means mounting, preferably automated, at the end of manufacture.
Parmi les critères qui aident à orienter le choix d'un mode de fabrication pour une pièce donnée d'un dispositif miniaturisé d'analyse chimique ou biochimique figure le ratio d'aspect (aspect ratio en anglais) qui représente l'aptitude à respecter les côtes d'un plan en trois dimensions, en particulier à respecter un profil à partir de lignes brisées et non pas à partir de courbesAmong the criteria that help guide the choice of a manufacturing method for a given part of a miniaturized chemical or biochemical analysis device is the aspect ratio which represents the ability to respect the ribs of a three-dimensional plane, in particular to respect a profile from broken lines and not from curves
La fabrication des systèmes miniaturisés, au moins dans une première phase de fabrication, part la plupart du temps de supports plans et plats (parallèles à un plan et peu épais), dits en 2D, où l'essentiel des composants est fabriqué à partir de gravures, d'ablations et de dépôts sur surface plane.The manufacture of miniaturized systems, at least in a first phase of manufacture, mostly starts from flat and flat supports (parallel to a plan and not very thick), called 2D, where most of the components are made from engravings, ablations and deposits on a flat surface.
La possibilité existe aussi maintenant de faire des composants de moins en moins plans et plats tout en respectant de plus en plus finement un profil de fabrication . Ceci est obtenu entre autres grâce à l'amélioration des techniques substractives (gravure chimique, ablation physique), à l'amélioration des techniques additives (dépôts tels que électrodéposition (electioplating^-electroless plating, par vapeur chimique (CND et PENCD)) et enfin à l'amélioration dès techniques de moulage et d'estampage. Très souvent cependant, les techniques de micro-usinage d'une surface plane n'ont pas assez progressé soit dans la profondeur limite d'usinage, soit dans les techniques de dépôt ou de moulage pour sculpter des dispositifs où des formes en 3D avec un rapport élevé hauteur/ surface de base sont requises. Une solution possible consiste en des assemblages de plusieurs pièces, que l'ont peut appeler «sous-composants», qui ont un degré de planéité et de platitude suffisant pour être accessibles aux techniques d'usinage d'une surface plane. Dans certaines configurations, on fabrique des sous-composants juste assez plans et juste assez plats pour pouvoir être micro- fabriqués. Puis on les superpose et assemble par fusion ou collage après un éventuel emboîtement ou enclenchement, ce permet de reconstituer le micro-système voulu (US Patent Ν° 5 932-315 : Microfluidic structure assembly with mating microfeatures _ US Patent, N° 5 611 214. Microcomponent sheet architecture _ US Patent 5252294. Micromechanical structure).The possibility also exists now of making less and less planar and flat components while more and more respecting a manufacturing profile. This is achieved inter alia thanks to the improvement of substractive techniques (chemical etching, physical ablation), to the improvement of additive techniques (deposits such as electrodeposition (electioplating ^ -electroless plating, by chemical vapor (CND and PENCD)) and Finally, improvement in molding and stamping techniques. Very often, however, micro-machining techniques for a flat surface have not progressed enough either in the limit machining depth or in deposition techniques. or molding to sculpt devices where 3D shapes with a high height / base area ratio are required. One possible solution consists of assemblies of several parts, which we can call "sub-components", which have a sufficient level of flatness and flatness to be accessible to machining techniques for a flat surface. In some configurations, sub-components are made just enough planes and just enough dishes to be able to be micro-manufactured. Then they are superimposed and assembled by fusion or gluing after a possible interlocking or interlocking, this makes it possible to reconstitute the desired micro-system (US Patent Ν ° 5 932-315: Microfluidic structure assembly with mating microfeatures _ US Patent, N ° 5 611 214. Microcomponent sheet architecture _ US Patent 5252294. Micromechanical structure).
Pour que cette solution soit applicable, il faut entre autres choses que le microsystème voulu puisse tenir dans volume plus ou moins aplati représenté par la superposition de sous- composants eux-mêmes nettement aplatis.For this solution to be applicable, it is necessary, among other things, that the desired microsystem can hold in a more or less flattened volume represented by the superposition of subcomponents themselves clearly flattened.
Mais il arrive aussi que la fabrication de certains composants ne puisse pas faire appel à des techniques de micro-usinage des surfaces planes. Ce peut être le cas parce que les formes sont trop sophistiquées pour être techniquement réalisables ou pour être fabriquées par ces techniques à un coût raisonnable. Ce peut aussi être le cas parce que la fonction demandée à ces composants s'accomode mal de la miniaturisation elle-même ou des techniques de miniaturisation. La conséquence d'une telle situation est que ces composants vont rester à une macro-échelle, et que le packaging du microsystème devra être conçu assembler ou connecter des macro-pièces avec des micro-pièces.( van der Schoot BH, Interfacing micro and macro mechanical worlds. J. Micromech. Microeng.1995, 5, 72-73 ).However, it also happens that the manufacture of certain components cannot use micromachining techniques for flat surfaces. This may be the case because the shapes are too sophisticated to be technically feasible or to be manufactured by these techniques at a reasonable cost. This may also be the case because the function required of these components does not go well with the miniaturization itself or with the miniaturization techniques. The consequence of such a situation is that these components will remain on a macro-scale, and that the packaging of the microsystem will have to be designed to assemble or connect macro-parts with micro-parts. (Van der Schoot BH, Interfacing micro and macro mechanical worlds. J. Micromech. Microeng. 1995, 5, 72-73).
Parmi les très nombreuses techniques de micro-fabrication figurent entre autres des techniques de gravure chimique humide de photolithographie, de gravure sèche avec divers rayonnements photoniques ou particulaires, de micro-façonnage avec micro-outillages ou lasers, de découpage, d'ablation, d'assemblage par fusion ou d'assemblage anodique, de collage, de soudure, de moulage, d'estampage à chaud (hot-embossing en anglais), de poinçonnage, de forage, d'électrodéposition, de dépôt de vapeur ctamique, de fabrication par progression par feuilles successives (lamination en anglais).Among the many micro-fabrication techniques are, among others, wet chemical etching techniques of photolithography, dry etching with various photonic or particulate radiation, micro-shaping with micro-tools or lasers, cutting, ablation, d by fusion or anode assembly, bonding, welding, molding, hot stamping (hot-embossing in English), punching, drilling, electroplating, ceramic vapor deposition, manufacturing by progression by successive sheets (lamination in English).
La gravure humide est appliquée depuis une quarantaine d'années au silicium et à ses dérivés dans l'industrie de la micro-électronique. Elle est peut être isotrope. Elle peut aussi être anisotrope lorsqu'on cherche à profiter de l'orientation des cristaux et des propriétés des substances gravantes pour maîtriser sa direction.(Sato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. Characterization of anisotropic etching properties of single crystal silicon: surface roughening as a function of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206). Les techniques de gravure humide tant isotropes qu'anisotropes possèdent de nombreuses variantes. En effet, les nouvelles connaissances en physique des matériaux, chimie orbitale, physique des rayonnements, dopage des matériaux, permettent de tirer profit de la structure atomique de différents matériaux utilisés, aident à concevoir des méthodes de contrôle de la direction, de la profondeur et de l'arrêt des gravures sur différentes couches et suggèrent de nouvelles pistes pour de nouvelles techniques .Wet etching has been applied for around forty years to silicon and its derivatives in the microelectronics industry. It may be isotropic. It can also be anisotropic when one seeks to take advantage of the orientation of the crystals and the properties of the gravitating substances to control its direction. (Sato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. of single crystal silicon: surface roughening as a function of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206). The wet etching techniques, both isotropic and anisotropic, have many variants. Indeed, new knowledge in materials physics, orbital chemistry, radiation physics, doping of materials, allow to take advantage of the atomic structure of different materials used, help to design methods of control of the direction, the depth and of the etchings on different layers and suggest new tracks for new techniques.
Chacune des techniques citées possède de nombreuses variantes dont l'éventail ne cesse de se développer à un rythme soutenu.Each of the techniques cited has many variations, the range of which is constantly developing at a sustained pace.
Les nouvelles connaissances en traitement de surface permettent d'affiner les qualités demandées aux matériaux en cours de fabrication ou les qualités demandées au produit fini. Les nouvelles connaissances en thermophysique et thermochimie différentielle entre deux matériaux suggèrent des nouvelles techniques de fusion, de moulage, d'estampillage, de poinçonnage, en particulier des plastiques.New knowledge in surface treatment makes it possible to refine the qualities required for materials during manufacture or the qualities required for the finished product. New knowledge in thermophysics and differential thermochemistry between two materials suggests new techniques of fusion, molding, stamping, punching, in particular plastics.
La microfabrication des polymères par stéréolithographie est venue grossir le champ de ces techniques, en particulier pour le prototypage rapide en 3D.Microfabrication of polymers by stereolithography has enlarged the field of these techniques, in particular for rapid 3D prototyping.
Selon que l'on grave un matériau dans sa masse ou que l'on grave seulement des couches superficielles, on parle respectivement de "bulk micromachining" et de "surface micromachining".Depending on whether one engraves a material in its mass or whether one engraves only surface layers, one speaks respectively of "bulk micromachining" and "surface micromachining".
Toutes ces techniques de micro-fabrication sont applicables non seulement à la fabrication des produits finis, mais aussi à celles des outils utilisés pour effectuer ces micro-fabrications, ainsi qu'aux micro-moules et aux micro-matrices d'estampage à chaud utilisés pour micro- répliquer en masse un micro-objet. Parmi les autres critères qui vont aider à sélectionner un mode et un matériau de fabrication figurent les qualités intrinsèques des matériaux composant l'objet fini, et les perspectives de maîtrise des coûts de fabrication. Certaines techniques supposent un mode de fabrication unitaire, aujourd'hui peu adapté à la fabrication de masse: gravure sèche par rayonnements photoniques ou particulaires (Bean. Anisotropic etching of silicon. 1978. vol ED-25(10), pp 1185-1193. IEEE Transactions of Electron devices.), ablation lasers, gravure avec micro-pointe. Mais ces techniques à vocation unitaire peuvent être utilisées comme première étape dans un process de fabrication de masse d'objets en plastique ou en céramique ou en métal selon des procédés appelés "par réplication" (Niggemann M., Ehrfeld W., Weber L.. Fabrication of miniaturized biotechnical devices. SPIE Conférence on Micromachining and Microfabrication Process Technology IV, Santa Clara, California, Sept 1998, vol 3511, pp 204 - 213 _ Ruprecht R, Bâcher W, Hausselt JH, Piotter N. Injection Molding of LIGA and LIGA-similar microstructures using filled and unfilled thermoplastics. SPIE, vol 2639, ppl 46-158 _ Fleming JG, Barron CC, Νovel silicon fabrication process for high aspect ratio micromachined parts, SPIE vol 2639, 185-190 _ Keller CG, Howe RT. Νickel-filled HEXSIL thermally actuated tweezers, 8a International Conférence on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, June 25-29, pp 376-379. _ Selvakumar A, Νajafi K, High density vertical comb array microactuators fabricated using a novel bulk/polysilicon trench refill technology, Solid State Sensor and Actuator Workshop, Hilton head , 1994, SC June 13-16, pp 138-141 _ Becker H., Dietz W.. Microfluidic devices for μ-TAS applications fabricated by polymer hot embossing. Proceedings of SPIE. Microfluidic Devices and Systems. 21-22 sept 1998, Santa Clara, ρρl77-182 _ Grzybowski BA, Haag R, Bowden Ν, Whitesides GM. Génération of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645- 4652 _ Martynova L, Locascio E, Gaitan G, Kramer W, Christensen RG, MacCrehan WA.. Fabrication of plastic microfluid channels by imprinting methods. Anal. Chem. 1997, 69, 4763-4789) .All these micro-manufacturing techniques are applicable not only to the manufacture of the finished products, but also to those of the tools used to carry out these micro-manufacturing, as well as to the micro-molds and to the hot stamping micro-matrices used. to micro-replicate en masse a micro-object. Among the other criteria which will help to select a manufacturing method and material are the intrinsic qualities of the materials making up the finished object, and the prospects for controlling manufacturing costs. Certain techniques presuppose a unitary manufacturing method, today unsuitable for mass production: dry etching by photonic or particulate radiation (Bean. Anisotropic etching of silicon. 1978. vol ED-25 (10), pp 1185-1193. IEEE Transactions of Electron devices.), Laser ablation, micro-tip engraving. However, these unitary techniques can be used as a first step in a process for the mass production of plastic or ceramic or metal objects according to processes called "by replication" (Niggemann M., Ehrfeld W., Weber L. . Fabrication of miniaturized biotechnical devices. SPIE Conference on Micromachining and Microfabrication Process Technology IV, Santa Clara, California, Sept 1998, vol 3511, pp 204 - 213 _ Ruprecht R, Bâcher W, Hausselt JH, Piotter N. Injection Molding of LIGA and LIGA-similar microstructures using filled and unfilled thermoplastics. SPIE, vol 2639, ppl 46-158 _ Fleming JG, Barron CC, Νovel silicon fabrication process for high aspect ratio micromachined parts, SPIE vol 2639, 185-190 _ Keller CG, Howe RT . Νickel-filled HEXSIL thermally actuated tweezers, 8 a International Conférence on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, June 25-29, pp 376-379. _ Selvakumar A, Νajafi K, High density vertical com b array microactuators fabricated using a novel bulk / polysilicon trench refill technology, Solid State Sensor and Actuator Workshop, Hilton head, 1994, SC June 13-16, pp 138-141 _ Becker H., Dietz W .. Microfluidic devices for μ- TAS applications fabricated by polymer hot embossing. Proceedings of SPIE. Microfluidic Devices and Systems. 21-22 Sept 1998, Santa Clara, ρρl77-182 _ Grzybowski BA, Haag R, Bowden Ν, Whitesides GM. Generation of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645- 4652 _ Martynova L, Locascio E, Gaitan G, Kramer W, Christensen RG, MacCrehan WA .. Fabrication of plastic microfluid channels by imprinting methods. Anal. Chem. 1997, 69, 4763-4789).
On peut en effet tirer de ces techniques à vocation unitaire un profit considérable pour micro- fabriquer des masters de réplication (par exemple des micro-moules pour moulage par injection ou pour moulage réactif, ou des micro-matrices d'estampage à chaud), à condition de réunir deux qualités: un ratio d'aspect élevé et une surface compatible avec les exigences du process de réplication. En effet, certaines étapes dans la réplication sont cruciales, en particulier la séparation de la matrice de réplication de l'objet nouvellement répliqué. Cependant, la complexité du process choisi pour fabriquer une matrice de réplication est à prendre en compte. Par exemple, on peut fabriquer avec une très grande précision un micromoule de moulage par injection ou une micro-matrice d'estampage à chaud avec la technique LIGA, où un rayonnement synchrotron issu d'une machinerie très onéreuse, très rare et très lourde, est utilisé dans les premières étapes. Mais de nouvelles techniques de gravure sèche et surtout de gravure humide avec des performances accrues peuvent se révéler plus souples avec des ratios d'aspect qui se rapprochent de plus en plus de la technique LIGA. Ainsi le gravure humide anisotrope a beaucoup progressé (Holke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57). D'autres résultats montrent aussi un progrès dans les performances de la gravure humide isotrope (Wet chemical isotropic etching procédures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A.. SPIE vol 3223, p 72-79). Quant aux techniques à vocation unitaire, certaines d'entre elles pourront être adaptées à la fabrication en masse lorsque les instruments de fabrication eux-mêmes qui servent à les mettre en oeuvre seront miniaturisés et pourront être utilisés de manière massivement parallèles. C'est une perspective proche pour l'ablation laser (grâce à la fabrication de micro- lasers) et la gravure par micropointe, plus lointaine pour certaines techniques de gravure sèche.One can indeed derive from these unitary techniques a considerable profit for micro-manufacturing replication masters (for example micro-molds for injection molding or for reactive molding, or hot stamping micro-dies), provided that two qualities are combined: a high aspect ratio and a surface compatible with the requirements of the replication process. Indeed, certain steps in replication are crucial, in particular the separation of the replication matrix from the newly replicated object. However, the complexity of the process chosen to manufacture a replication matrix must be taken into account. For example, we can manufacture with very high precision an injection molding micromould or a hot stamping micro-matrix with the LIGA technique, where synchrotron radiation from very expensive, very rare and very heavy machinery, is used in the early stages. But new techniques of dry etching and especially of wet etching with increased performances can prove to be more flexible with aspect ratios which approach more and more the LIGA technique. Thus anisotropic wet etching has progressed a lot (Holke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57). Other results also show progress in the performance of wet chemical isotropic etching procedures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A .. SPIE vol 3223, p 72-79 ). As for unitary techniques, some of them can be adapted to mass production when the manufacturing instruments themselves which are used to implement them are miniaturized and can be used in a massively parallel manner. This is a close prospect for laser ablation (thanks to the manufacture of micro-lasers) and microtip etching, more distant for certain dry etching techniques.
Aujourd'hui la fabrication de masse est plutôt réservée aux techniques qui ont naturellement cette vocation, dont entre autres: gravure humide sur silicium et dérivés, et sur verres, photolithographie UN sur photorésists, fabrication par progression par couches successives de polymères avec utilisation de couches sacrificielles selon Webster et Mastrangelo cités ci- après en référence, moulage de poly(dimethylsiloxane) (PDMS), moulage de plastiques par injection avec micro-moule, moulage de céramiques et de métaux, estampage à chaud de polymères avec micro-matrice d'estampage.Today mass production is rather reserved for techniques which naturally have this vocation, including among others: wet etching on silicon and derivatives, and on glasses, UN photolithography on photoresists, production by progression by successive layers of polymers with the use of layers sacrificial according to Webster and Mastrangelo cited below with reference, poly (dimethylsiloxane) molding (PDMS), injection molding of plastics with micro-mold, molding of ceramics and metals, hot stamping of polymers with micro-matrix of stamping.
Comptant parmi ces dernières, la gravure humide est désormais appliquée à tous types de dérivés du silicium et au quartz, ainsi qu' aux différents types de verre (par exemple pyrex, verres boro-phospho-silicatés, etc).One of the latter, wet etching is now applied to all types of silicon and quartz derivatives, as well as to different types of glass (for example pyrex, boro-phospho-silicate glasses, etc.).
En matière de microfluidique, un critère important est la compatibilité avec l'utilisation de la micro-électrophorèse, de la micro-électrophorèse 2D et de la micro-électro-chromatographie pour séparer les molécules. Est importante aussi et surtout la compatibilité avec l'électro- osmose pour mouvoir des fluides, cette technique ayant l'avantage d'éviter des composants telles que microvannes et micropompes. Comme la micro-électrophorèse et la micro- électrophorèse 2D, l'électro-osmose ainsi que la micro-électro-chromatographie alliée à l'électro-osmose nécessitent de gros voltages. En conséquence, elles sont incompatibles en pratique avec l'utilisation du silicium.In microfluidics, an important criterion is compatibility with the use of micro-electrophoresis, 2D micro-electrophoresis and micro-electro-chromatography to separate molecules. Compatibility with electro-osmosis for moving fluids is also and above all important, this technique having the advantage of avoiding components such as microvalves and micropumps. Like micro-electrophoresis and 2D micro-electrophoresis, electro-osmosis as well as micro-electro-chromatography combined with electro-osmosis require large voltages. Consequently, they are incompatible in practice with the use of silicon.
Par contre, elle sont compatibles avec les verres et les plastiques. (Manz A., Effenhauser CS, Burggraf Ν, Harrison DJ, Seiler K, Fluri K. Electroosmotic pumping and electrophoretic séparations for miniaturized chemical analysis Systems. J. Micromech. Microeng., 1994, 4, 257-265. - Mac Cormick RM, Nelson JR, Alonso-Amigo MG, Benvegnu DJ, Hooper HH. Microchannel electrophoretic séparations of DNA in injection-molded plastic substrates. Anal. Chem., 1997, 69, 2626-2630 _ Jacobson SJ, Kutter JP, Culbertson CT, Ramsey JM. .Rapid electrophoretic and chromatographic analysis on microchips, μ-TAS 1998, Banff, Canada, 315-318. _ Microfabricated liquid chromatography columns based on collocated monolith support structures, μ-TAS 1998, Banff, Canada, 451-455. _Paulus A., Williams SJ, Sassi AP, Kao PH, Tan H, Hooper HH . Integrated capillary electrophoresis using glass and plastic chips for multiplexed DNA analysis, pp 94-103. SPIE Proceedings Vol 3515 #3515- 08. _ PM Martin, DW Matson, Bennett WD, Hammerstrom DJ. Fabrication of plastic microfluidic components. Polymer-based microfluidic analytical devices. SPIE Proceedings Vol 3515 # 3515-19).On the other hand, they are compatible with glasses and plastics. (Manz A., Effenhauser CS, Burggraf Ν, Harrison DJ, Seiler K, Fluri K. Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis Systems. J. Micromech. Microeng., 1994, 4, 257-265. - Mac Cormick RM , Nelson JR, Alonso-Amigo MG, Benvegnu DJ, Hooper HH. Microchannel electrophoretic separations of DNA in injection-molded plastic substrates. Anal. Chem., 1997, 69, 2626-2630 _ Jacobson SJ, Kutter JP, Culbertson CT, Ramsey JM.. Rapid electrophoretic and chromatographic analysis on microchips, μ-TAS 1998, Banff, Canada, 315-318. _ Microfabricated liquid chromatography columns based on collocated monolith support structures, μ-TAS 1998, Banff, Canada, 451-455. _Paulus A., Williams SJ, Sassi AP, Kao PH, Tan H, Hooper HH. Integrated capillary electrophoresis using glass and plastic chips for multiplexed DNA analysis, pp 94-103. SPIE Proceedings Vol 3515 # 3515- 08. _ PM Martin, DW Matson, Bennett WD, Hammerstrom DJ. Fabrication of plastic microfluidic components. Polymer-based microfluidic analytical devices. SPIE Proceedings Vol 3515 # 3515-19).
Toutefois on peut envisager d'utiliser d'autres forces que la force électro-osmotique pour mouvoir les liquides où l'utilisation de micro-vannes et micropompes peut être minimisée, comme la gravité, la force centrifuge (Madou MJ, Kellogg GJ: The LabCD: a centrifuge- based microfluidic platform for diagnostics. SPIE vol 3259, pp 80-93).However we can consider using other forces than electro-osmotic force to move liquids where the use of micro-valves and micropumps can be minimized, like gravity, centrifugal force (Madou MJ, Kellogg GJ: The LabCD: a centrifuge-based microfluidic platform for diagnostics. SPIE vol 3259, pp 80-93).
D'autres modçs de propulsion de liquides peuvent être envisagés comme la force thermocapillaire ( Burns MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT. Microfabricated structures for integrated DNA analysis. P.N.A.S. 1996, vol. 93, pp5556-5561), ou les forces couplées à des alternances surfaces ou raies hydrophobes-surfaces ou raies hydrophiles (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Sélective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers. SPIE vol 3515, 136- 143 _ Eastman Kodak» Device for fluid sύpply of a micro-metering device; US Patent N° 5805189 _ Beckton Dickinson. DNA microwell device and method.US Patent N° 5795748).Other liquid propulsion models can be considered such as thermocapillary force (Burns MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT. Microfabricated structures for integrated DNA analysis. PNAS 1996, vol. 93, pp5556-5561), or the forces coupled to alternations of surfaces or hydrophobic-surface lines or hydrophilic lines (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Selective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers. SPIE vol 3515, 136- 143 _ Eastman Kodak »Device for fluid sύpply of a micro-metering device; US Patent N ° 5805189 _ Beckton Dickinson. DNA microwell device and method.US Patent N ° 5795748).
Par ailleurs, une publication récente (Characterization of silicon-based insulated channels for capillary electrophoresis, Van den Berg et al., μ-TAS 98, Canada, pp-327-330) montre que des travaux sont entrepris pour faire acquérir au silicium une compatibilité avec de gros voltages, mais la technique de fabrication employée est une gravure sèche (deep reactive ion etchnig), donc à vocation unitaire.Furthermore, a recent publication (Characterization of silicon-based insulated channels for capillary electrophoresis, Van den Berg et al., Μ-TAS 98, Canada, pp-327-330) shows that work is being done to acquire silicon a compatibility with large voltages, but the manufacturing technique used is a dry etching (deep reactive ion etchnig), therefore with a unitary vocation.
La transparence, qualité recherchée en analyse biologique, est une qualité partagée entre les verres (Kricka L, Wilding P, et al.. Micromachined Glass-Glass Microchips for In Vitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) et certains plastiques.Transparency, a quality sought after in biological analysis, is a quality shared between glasses (Kricka L, Wilding P, et al. Micromachined Glass-Glass Microchips for In Vitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) and some plastics.
Certains verres ayant les bons compromis de conditions de dopage et d'expansion thermique, ont la qualité de pouvoir s'assembler facilement au silicium (Albaugh KB, Rasmussen DH, "Mechanisms of anodic bonding of silicon to pyrex glass. Proc IEEE Solid State Sensors and Actuators Workshop. 1988. 109-110).Certain glasses having the good compromises of conditions of doping and thermal expansion, have the quality of being able to assemble easily with silicon (Albaugh KB, Rasmussen DH, "Mechanisms of anodic bonding of silicon to pyrex glass. Proc IEEE Solid State Sensors and Actuators Workshop. 1988. 109-110).
La gravure humide sur verre, par nature isotrope, est parfaitement maîtrisée (A new fabrication method for borosilicate glass capillary tubes with latéral inlets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij NF. Sensors and Actuators A 60, 1997, 219-222. _ Corman T, Enoksson P, Stemme G. Deep wet etching of borosilicate glass using an anodically bonded silicon substrate as mask J. Micromech. Microeng., 1998, 8, 84-87.)Wet etching on glass, by nature isotropic, is perfectly mastered (A new fabrication method for borosilicate glass capillary tubes with lateral inlets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij NF. Sensors and Actuators A 60, 1997, 219-222. _ Corman T, Enoksson P, Stemme G. Deep wet etching of borosilicate glass using an anodically bonded silicon substrate as mask J. Micromech. Microeng., 1998, 8, 84-87.)
Comparés aux plastiques, les verres offrent entre autres pour l'analyse biochimique la compatibilité avec la détection par fluorescence et un bon coefficient d'échange thermique. Ils sont gravés cependant uniquement selon un mode isotrope, ce qui par exemple limite aujourd'hui la forme des microcanaux sur verre à une forme circulaire.Compared to plastics, the glasses offer, among other things for biochemical analysis, compatibility with fluorescence detection and a good heat exchange coefficient. They are however etched only in an isotropic mode, which for example today limits the shape of the microchannels on glass to a circular shape.
Les plastiques, même s'ils ont une moindre compatibilité avec la détection par fluorescence et un moindre coefficient d'échange thermique que les verres, ont de nombreuses autres qualités, dont principalement le faible prix de revient.Plastics, even if they have less compatibility with fluorescence detection and a lower heat exchange coefficient than glasses, have many other qualities, including mainly the low cost price.
Des travaux qui portent sur l'amélioration de la détection par fluorescence avec les plastiques et l'élimination des bruits de fond (en modulant la vitesse de migration des analytes et en utilisant une source liraiineuse LED) ont été rapportés par Wang Shau-Chun et Michael D. Morris de l'Université du Michigan à la 10 ème "Frederick Conférence on Capillary Electrophoresis " en Octobre 1999.Studies that focus on improving fluorescence detection with plastics and eliminating background noise (by modulating the migration speed of the analytes and using a LED light source) have been reported by Wang Shau-Chun and Michael D. Morris of the University of Michigan at the 10th "Frederick Conference on Capillary Electrophoresis" in October 1999.
Le très faible coût de fabrication des objets micro-fabriqués en plastique vient du faible prix de la matière première, de la simplicité des process de production qui peuvent être envisagés, entre autres du fait de l'aptitude à la réplication par moulage ou par estampage à chaud, voire même pour les plastiques photoresists à la photolithographie .The very low cost of manufacturing plastic micro-manufactured objects comes from the low price of the raw material, the simplicity of the production processes that can be envisaged, among other things because of the ability to replicate by molding or stamping. hot, even for plastics photoresists to photolithography.
Un inconvénient des plastiques est en voie d'être surmonté: il était difficile de déposer le métal de la circuiterie électrique une fois le produit fini. Mais des solutions voient le jour, comme le marquage avec une encre conductrice.A disadvantage of plastics is being overcome: it was difficult to remove the metal from the electrical circuit once the product was finished. But solutions are emerging, such as marking with a conductive ink.
Parmi les plastiques, il est intéressant de faire la classification suivante:Among plastics, it is interesting to make the following classification:
- les photoresists, usinables entre autres par photolithographie, dont par exemple le PMMA pour la lithographie aux rayons X, SU-8 ( photorésist négatif) et Novolac de Hoescht et- photoresists, which can be machined, among other things, by photolithography, including for example PMMA for X-ray lithography, SU-8 (negative photoresist) and Novolac de Hoescht and
AZ 9260 (photoresists positif) pour la photolithographie UN (Lorenz H, Despont M, Fahrni Ν, LaBianca Ν, Renaud P, SU-8: a low-cost négative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121-124. _ .Loechtel B, Maciossek A, Surface micro components fabricated by UN depth lithography and electroplating, SPIE vol 2639, 174- 184 _ Conédéra N, Le Goff B, Fabre Ν. Potentialities of a new positive photorésist for the realization of thick moulds, J. Micromech. Microeng, 1999, 9, 173-175. _ Guérin LJ, Bossel M, Demierre M, Calmes S, Renaud P. Simple and low cost fabrication of embedded microchannels by using a new thick-film photoplastic. Proceedings of Transducers, Chicago, USA, 1997, ppl419-1422.) - les élastomères siliconés, dont le poly(dimethylsiloxane) (PDMS), utilisables entre autres par moulage simple, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly(dimethylsiloxane), Electrophoresis 2000, 21, 27-40. _ Ocvirk G, Munroe M; TangAZ 9260 (photoresists positive) for photolithography UN (Lorenz H, Despont M, Fahrni Ν, LaBianca Ν, Renaud P, SU-8: a low-cost negative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121 -124. _ .Loechtel B, Maciossek A, Surface micro components fabricated by UN depth lithography and electroplating, SPIE vol 2639, 174- 184 _ Conédéra N, Le Goff B, Fabre Pot. Potentialities of a new positive photoresist for the realization of thick molds, J. Micromech, Microeng, 1999, 9, 173-175. _ Guérin LJ, Bossel M, Demierre M, Calmes S, Renaud P. Simple and low cost fabrication of embedded microchannels by using a new thick-film photoplastic. Proceedings of Transducers, Chicago, USA, 1997, ppl419-1422.) - silicone elastomers, including poly (dimethylsiloxane) (PDMS), usable among others by simple molding, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly (dimethylsiloxane), Electrophoresis 2000, 21, 27-40. _ Ocvirk G, Munroe M; Tang
T, Oleschuk R, Westra K, Harrison DJ, Electrokinetic control of fluid flow in native poly(dimethysiloxane) capillary electrophoretic devices, Electrophoresis 2000, 21, 107- 115.)T, Oleschuk R, Westra K, Harrison DJ, Electrokinetic control of fluid flow in native poly (dimethysiloxane) capillary electrophoretic devices, Electrophoresis 2000, 21, 107-115.)
- un ensemble de plus en plus vaste usinable entre autres par moulage par injection et par emboutissage à chaud. Parmi ces derniers, on peut citer des polyamides (PA), des polycarbonates (PC), des polyoxyméthylènes (POM), le cyclopentadienenorbomen copolymer (COC), des polyméthylmethacrylates (PMMA), le polyéthylène basse densité (PE-ld), le polyéthylène haute densité (PE-hd), le polypropylène (PP), des polystyrènes (PS), le cycloolefin copolymère (COC), le polyetheretherketone (PEEK). (Niggemann- an increasingly large assembly which can be machined, inter alia, by injection molding and hot stamping. Among these, polyamides (PA), polycarbonates (PC), polyoxymethylenes (POM), cyclopentadienenorbomen copolymer (COC), polymethylmethacrylates (PMMA), low density polyethylene (PE-ld), polyethylene high density (PE-hd), polypropylene (PP), polystyrenes (PS), cycloolefin copolymer (COC), polyetheretherketone (PEEK). (Niggemann
M., Ehrfeld W., Weber L.; Fabrication of miniaturized biotechnical devices, SPIE , vol 3511, pp 204 - 213 _ Becker H, Gartner C, Polymer microfabrication methods for microfluidic analytical applications, Electrophoresis 2000, 21, 12-26).M., Ehrfeld W., Weber L .; Fabrication of miniaturized biotechnical devices, SPIE, vol 3511, pp 204 - 213 _ Becker H, Gartner C, Polymer microfabrication methods for microfluidic analytical applications, Electrophoresis 2000, 21, 12-26).
D'autres plastiques encore peuvent être microfabriqués: le polybutylèneteφhtalate (PBT), le polyphenylène ether (PPE), le polysulfone (PSU), le liquid crystal polymer (LCD), le polyetherimide (PEI). Le polyactide biodégradable peut aussi être microfabriqué.Still other plastics can be microfabricated: polybutyleneteφhtalate (PBT), polyphenylene ether (PPE), polysulfone (PSU), liquid crystal polymer (LCD), polyetherimide (PEI). The biodegradable polyactide can also be microfabricated.
Le PMMA et le PC sont couramment employés dans le moulage par injection et l'estampage à chaud. Le COC est couramment cité dans l'estampage à chaud.PMMA and PC are widely used in injection molding and hot stamping. COC is commonly cited in hot stamping.
Les procédés de fabrication de masse des plastiques sont très variés. On peut citer comme principaux procédés:The mass production processes for plastics are very varied. The main processes can be mentioned:
- l'impression par matrices filiformes (wire imprinting) ( Locascio LF, Gitan M, Hong J, Eldefrawi M, Plastic microfluidic devices for clinical measurements, μ-TAS 1998, 367- 370 _ Chen YH, Chen SH, Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire-imprinting method, Electrophoresis 2000, 21, 165-170)- printing using wire imprinting (Locascio LF, Gitan M, Hong J, Eldefrawi M, Plastic microfluidic devices for clinical measurements, μ-TAS 1998, 367- 370 _ Chen YH, Chen SH, Analysis of DNA fragments by microchip electrophoresis fabricated on poly (methyl methacrylate) substrates using a wire-imprinting method, Electrophoresis 2000, 21, 165-170)
l'estampage à chaud (Hot embossing) (Becker H., Dietz W, Dannberg P. Microfluidic manifolds by polymer hot embossing for μ-TAS applications. μ-TAS 1998, Banff, Canada, 253-256. _ Kempen LU, Kunz RE, Gale MT. Micromolded structures for integrated optical sensors. SPIE vol 2639, 278-285.). - le moulage par injection (Hagmann P, Ehrfeld W. Fabrication of microstructures of extrême structural heights by reaction injection molding, International Polymer Processing, 1989, Vol IN, Ν°3, pp 188-195. _ Weber L, Ehrfeld W, Freimuth H, Lâcher M, Lehr H, Pech B. . Micro-moulding - a powerful tool for the large scale production of précise microstructures. Proc. SPIE Symp. Micromacliining and Microfabrication, 1996, vol 2879, pp 156- 167.).hot embossing (Becker H., Dietz W, Dannberg P. Microfluidic manifolds by polymer hot embossing for μ-TAS applications. μ-TAS 1998, Banff, Canada, 253-256. _ Kempen LU, Kunz RE, Gale MT. Micromolded structures for integrated optical sensors. SPIE vol 2639, 278-285.). - injection molding (Hagmann P, Ehrfeld W. Fabrication of microstructures of extreme structural heights by reaction injection molding, International Polymer Processing, 1989, Vol IN, Ν ° 3, pp 188-195. _ Weber L, Ehrfeld W, Freimuth H, Lâcher M, Lehr H, Pech B.. Micro-molding - a powerful tool for the large scale production of precise microstructures. Proc. SPIE Symp. Micromacliining and Microfabrication, 1996, vol 2879, pp 156- 167.).
- le moulage simple pour les élastomères siliconés (Kumar A, Whitesides GM. Appl. Phys. Lett, 1993, 63, 2002-2004 _ Wilbur JL, Kumar A, kim E, Whitesides GM, Adv. Mat.- simple molding for silicone elastomers (Kumar A, Whitesides GM. Appl. Phys. Lett, 1993, 63, 2002-2004 _ Wilbur JL, Kumar A, kim E, Whitesides GM, Adv. Mat.
1994, 7, 600-604.).1994, 7, 600-604.).
- la photolithographie des photoresists, dont par exemple la lithographie aux rayons X pour le PMMA, la photolithographie UN pour le photopolymère Epson SU-8. Dans cette dernière, trois procédés sont couramment utilisés (Renaud P., Nan Lintel H,- photolithography of photoresists, including for example X-ray lithography for PMMA, UN photolithography for the Epson SU-8 photopolymer. In the latter, three methods are commonly used (Renaud P., Nan Lintel H,
Heusckel M, Guérin L.. Photo-polymer microchannel technologies and applications, μ- TAS 1998, Banff. Canada, ppl7-22.). Ils commencent toutes trois par le dépôt d'une première couche de SU-8 qu'on expose aux UN. Pour la fabrication dfun microcanal, la première couche de photorésist fait le fond dudit micro-canal à section rectangulaire. La deuxième couche de photorésist fait les parois verticales dudit micro-canal. La troisième couche de photorésist termine le capillaire en constituant la partie couvercle.Heusckel M, Guérin L .. Photo-polymer microchannel technologies and applications, μ- TAS 1998, Banff. Canada, ppl7-22.). They all start with the deposition of a first layer of SU-8 which is exposed to the UN. For the manufacture of a microchannel f, the first photoresist layer is the bottom of said microchannel with a rectangular section. The second photoresist layer forms the vertical walls of said micro-channel. The third layer of photoresist terminates the capillary by constituting the cover part.
* le "fill process" . On procède par remplissage avec une couche sacrificielle, comme par exemple l'Araldite GT6063 de Ciba-Geigy entre la deuxième et la troisième couche de photorésist. En fin de process, la couche sacrificielle est dissoute. * le "mask process" . On interpose une couche de métal sur la deuxième couche de photorésist qu'on ne développe pas. Cette deuxième couche de métal masque le microcanal. Une troisième couche de photorésist est déposée puis illuminée. Puis le photorésist est développé à l'intérieur et à l'extérieur dudit micro-canal.* the "fill process". This is done by filling with a sacrificial layer, such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer. At the end of the process, the sacrificial layer is dissolved. * the "mask process". A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel. A third layer of photoresist is deposited and then illuminated. Then the photoresist is developed inside and outside of said micro-channel.
* le "lamination process", un procédé sans dissolution, où l'on déroule une couche de film sec de SU-8 sur la construction faite à partir de la première couche de photorésist pour la sceller.* the "lamination process", a non-dissolving process, where a layer of dry film of SU-8 is unwound on the construction made from the first layer of photoresist to seal it.
- la fabrication par progression par couches successives de polymères , avec utilisation de couches sacrificielles, dont par exemple le process utilisé par Webster JR, Burns MA, Mastrangelo CH, Man PF, Jones DK, Burke DT., (Webster JR, Burns MA, Burke DT.,- the production by progression by successive layers of polymers, with the use of sacrificial layers, including for example the process used by Webster JR, Burns MA, Mastrangelo CH, Man PF, Jones DK, Burke DT., (Webster JR, Burns MA, Burke DT.,
Mastrangelo CH , An inexpensive plastic technology for microfabricated capillary electrophoresis chips, μ-TAS 1998, 249-252), une technique qui part de parylène déposé sur du polycarbonate ou du silicium avec utilisation ultérieure de photorésist sacrificiel. L'avantage de cette technique réside dans le scellement des microcanaux naturellement inclus dans la méthode.Mastrangelo CH, An inexpensive plastic technology for microfabricated capillary electrophoresis chips, μ-TAS 1998, 249-252), a technique which starts from parylene deposited on polycarbonate or silicon with subsequent use of sacrificial photoresist. The advantage of this technique lies in the sealing of the microchannels naturally included in the method.
La microfabrication par laser des plastiques est aussi possible, mais en tant technique unitaire. Ce peut être par exemple l'ablation directe dans la masse ou la découpe d'un joint que l'on glissera en sandwich entre deux couvercles.Microfabrication by laser of plastics is also possible, but as a unitary technique. It can be for example direct ablation in the mass or the cutting of a joint which one will slide in sandwich between two lids.
Les traitements de surface de plastiques dépendent de l'application et du matériau utilisé. Par exemple, il faut souvent rendre hydrophile une surface hydrophobe. Pour une application d'analyse d'ADN, ou d'analyse de protéines, il faut éviter des liaisons avec le substrat par des traitements de surfaces spécifiques respectivement à ces deux types d'analyse et spécifiques du matériau choisi.Plastic surface treatments depend on the application and the material used. For example, a hydrophobic surface must often be made hydrophilic. For a DNA analysis or protein analysis application, it is necessary to avoid bonds with the substrate by surface treatments specific to these two types of analysis and specific to the material chosen, respectively.
Pour assembler et sceller d'un couvercle des microfabrications en plastique, plusieurs procédés existent. On peut citer entre autres; - le scellement par déroulement à chaud d'une feuille recouvrante de PET d'environ 30 microns coatée avec une couche d'un matériau, le plus souvent un polymère que l'on porte à son point de fusion pour qu'elle se mélange avec le substrat, - le scellement d'un couvercle ou l'assemblage d'une partie complémentaire par collage, ou par pression à chaud, ou par soudure au laser, ou par l'emploi d'ultrasons, ou par l'emploi de plasmas, etc. Several methods exist for assembling and sealing plastic microfabrications with a cover. One can cite among others; - the sealing by hot unwinding of a PET covering sheet of about 30 microns coated with a layer of a material, most often a polymer which is brought to its melting point so that it mixes with the substrate, - the sealing of a cover or the assembly of a complementary part by gluing, or by hot pressing, or by laser welding, or by the use of ultrasound, or by the use of plasmas , etc.
FIGURES:FIGURES
La Figure 1 A montre un dispositif microfluidique pouvant être utilisé en l'Etat Actuel de l'Art.Figure 1A shows a microfluidic device which can be used in the current state of the art.
La Figure 1 B montre un dispositif microfluidique pouvant être utilisé en l'Etat Actuel de l'Art.Figure 1B shows a microfluidic device that can be used in the current state of the art.
La Figure IC montre un dispositif de pipetage (1776) dispensant des réactifs sur un micro- array ou un macro-array (1777) pouvant être utilisé en l'Etat Actuel de l'Art.Figure IC shows a pipetting device (1776) dispensing reagents on a micro-array or a macro-array (1777) which can be used in the current state of the art.
La Figure 2A montre un micro-puits classique d'un micro-array ou d'un macro-array.Figure 2A shows a typical micro-well of a micro-array or a macro-array.
La Figure 2B montre comment on peut reconvertir un micro-canal (41) en micro-puits (42) obturable à la fois profond et à section réduite.Figure 2B shows how a micro-channel (41) can be reconverted into a closable micro-well (42) that is both deep and of reduced section.
La Figure 2C montre comment une connexion d'un élément microfluidique (4) est engagée par le dessous et par le dessus desdits micropuits (42).Figure 2C shows how a connection of a microfluidic element (4) is engaged from below and from above of said microwells (42).
La Figure 2D montre un micropuits (42) mâle profond et étroit.Figure 2D shows a deep and narrow male microwell (42).
La Figure 3 A montre en coupe un micropuits (42) obtenu par reconversion d'un micro-canal (41) traversant de part en part un module élémentaire plat (1) et débouchant dans l'épaisseur et sur la tranche dudit module élémentaire plat (1).Figure 3A shows in section a microwell (42) obtained by reconversion of a micro-channel (41) passing right through a flat elementary module (1) and emerging in the thickness and on the edge of said flat elementary module (1).
La Figure 3B montre en perspective plusieurs micro-canaux (41) parallèles traversant de part en part un module élémentaire plat (1) et débouchant dans l'épaisseur et sur la tranche dudit module élémentaire plat (1), de telle sorte qu'on obtient une ligne (2119) de micropuits (42) du micro-array ou macro-array de l'invention.FIG. 3B shows in perspective several parallel micro-channels (41) passing right through a flat elementary module (1) and emerging in the thickness and on the edge of said flat elementary module (1), so that obtains a line (2119) of microwells (42) from the micro-array or macro-array of the invention.
La Figure 3C montre la superposition précise desdits modules élémentaires plats (1) qui portent chacun une ligne (2119) de micropuits (42) pour former ledit micro-array ou du macro-array multibloc (3119) de l'invention.FIG. 3C shows the precise superposition of said flat elementary modules (1) which each carry a line (2119) of microwells (42) to form said micro-array or of the multiblock macro-array (3119) of the invention.
La Figure 4 A montre un module élémentaire plat (1) où les microcanaux (41) sont pourvus de portions élargies.Figure 4A shows a flat elementary module (1) where the microchannels (41) are provided with enlarged portions.
La Figure 4B montre en perspective un module élémentaire plat (1) où les microcanaux (41) sont pourvus de portions élargies. La Figure 4C montre en perspective le micro-array (3119) formé par empilement des modules élémentaires plats (1) dont les microcanaux (41) sont pourvus de portions élargies.Figure 4B shows in perspective a flat elementary module (1) where the microchannels (41) are provided with enlarged portions. Figure 4C shows in perspective the micro-array (3119) formed by stacking flat elementary modules (1) whose microchannels (41) are provided with enlarged portions.
La Figure 5 montre une vue de dessus d'un micro-array ou macro-array multibloc (319) de l'invention constitué par l'empilement de modules élémentaires plats (110).Figure 5 shows a top view of a micro-array or multi-block macro-array (319) of the invention constituted by the stack of flat elementary modules (110).
La Figure 6 montre une vue de face d'un module élémentaire plat (110) de l'invention.Figure 6 shows a front view of a flat elementary module (110) of the invention.
La Figure 7 montre une vue de dos dudit module élémentaire plat (110) de l'invention.Figure 7 shows a back view of said flat elementary module (110) of the invention.
La Figure 8 montre une vue de dessous dudit micro-array ou macro-array multibloc (319) de l'invention.Figure 8 shows a bottom view of said micro-array or multi-block macro-array (319) of the invention.
La Figure 9 montre une vue de profil dudit micro-array ou macro-array multibloc (319) de l'invention.Figure 9 shows a profile view of said micro-array or multi-block macro-array (319) of the invention.
La Figure 10 représente le détachement de parties détachables (145) desdits modules élémentaires plats (1 i0),. vu de profil.Figure 10 shows the detachment of detachable parts (145) of said flat elementary modules (1 i0) ,. seen in profile.
La Figure 11 représente une vue de dessus dudit micro-array ou macro-array avec lesdites parties détachables (145) connectées aux sous parties (155) desdits modules élémentaires plats (110).Figure 11 shows a top view of said micro-array or macro-array with said detachable parts (145) connected to the sub-parts (155) of said flat elementary modules (110).
La Figure 12 représente lesdites parties détachables (145) seules, vues de dessus.Figure 12 shows said detachable parts (145) alone, seen from above.
La Figure 13 représente en vue de profil un micro-array ou macro-array multibloc (318) formé par empilement desdites parties détachables (145).Figure 13 shows in side view a micro-array or multi-block macro-array (318) formed by stacking said detachable parts (145).
La Figure 14 représente en vue de dessus un micro-array ou macro-array multibloc (318) formé par empilement desdites parties détachables (145).Figure 14 shows a top view of a multi-block micro-array or macro-array (318) formed by stacking said detachable parts (145).
La Figure 15 représente en vue de profil la connexion face à face d'un premier micro-array ou macro-array multibloc (1318) constitué par empilement de parties détachables (1145) de modules élémentaires plats avec un deuxième micro-array ou macro-array multibloc (2318) constitué par empilement d'autres modules élémentaires plats (2145).Figure 15 shows in side view the face to face connection of a first multi-block micro-array or macro-array (1318) constituted by stacking of detachable parts (1145) of flat elementary modules with a second micro-array or macro- multiblock array (2318) formed by stacking other flat elementary modules (2145).
La Figure 16 représente en vue de face la connexion face à face d'un premier micro-array ou macro-array multibloc (1318) constitué par empilement de parties détachables (1145) avec avec un deuxième micro-array ou macro-array multibloc (2318) constitué par empilement d'autres modules élémentaires plats (2145).Figure 16 shows in front view the face to face connection of a first micro-array or multi-block macro-array (1318) constituted by stacking of detachable parts (1145) with with a second multi-block micro-array or macro-array (2318) formed by stacking other flat elementary modules (2145).
La Figure 17 montre une connexion orthogonale de deux modules élémentaires plats de l'invention, lesdits modules élémentaires plats étant pourvus de sous-parties détachables. La Figure 18 montre une connexion orthogonale de deux micro-arrays ou macro-arrays multiblocs constitués respectivement par empilement des modules élémentaires plats de la figure 17.Figure 17 shows an orthogonal connection of two flat elementary modules of the invention, said flat elementary modules being provided with detachable sub-parts. FIG. 18 shows an orthogonal connection of two micro-arrays or multi-block macro-arrays formed respectively by stacking the flat elementary modules of FIG. 17.
La Figure 19 montre d'abord une connexion orthogonale de deux modules élémentaires plats de l'invention, le premier des deux dits modules élémentaires plats étant pourvus de sous- parties détachables. Elle montre ensuite une connexion directe d'un assemblage premier module élémentaire plat-deuxième module élémentaire plat avec un troisième module élémentaire plat. La Figure 20 montre une connexion orthogonale de deux micro-arrays ou macro-arrays multiblocs de l'invention, le premier micro-array ou macro-array étant constitué par empilement des assemblages premiers modules élémentaires plats-deuxième modules élémentaires plats de la figure 19, et le deuxième micro-array ou macro-array étant constitué par empilement des troisièmes modules élémentaires plats de la figure 19.Figure 19 first shows an orthogonal connection of two flat elementary modules of the invention, the first of the two said flat elementary modules being provided with detachable sub-parts. It then shows a direct connection of a first flat elementary module-second flat elementary module assembly with a third flat elementary module. FIG. 20 shows an orthogonal connection of two multi-block micro-arrays or macro-arrays of the invention, the first micro-array or macro-array being constituted by stacking the assemblies of first flat elementary modules-second flat elementary modules of FIG. 19 , and the second micro-array or macro-array being formed by stacking the third flat elementary modules of FIG. 19.
La Figure 21 montre d'abord une connexion directe de deux modules élémentaires plats de l'invention, le premier des deux dits modules élémentaires plats étant pourvus de sous-parties détachables. Elle montre ensuite une connexion orthogonale d'un assemblage premier module élémentaire plat-deuxième module élémentaire plat avec un troisième module élémentaire plat.Figure 21 first shows a direct connection of two flat elementary modules of the invention, the first of the two so-called flat elementary modules being provided with detachable sub-parts. It then shows an orthogonal connection of a first flat elementary module-second flat elementary module assembly with a third flat elementary module.
La Figure 22 montre les connexions de micro-arrays des micro-arrays ou macro-arrays multiblocs de l'invention constitués respectivement par empilement des modules élémentaires plats de la figure 21.FIG. 22 shows the connections of micro-arrays of micro-arrays or multi-block macro-arrays of the invention constituted respectively by stacking of the flat elementary modules of FIG. 21.
La Figure 23 montre d'abord une connexion directe de deux modules élémentaires plats de l'invention, le premier des deux dits modules élémentaires plats étant pourvus de sous-parties détachables. Elle montre ensuite une connexion orthogonale d'un assemblage premier module élémentaire plat-deuxième module élémentaire plat avec plusieurs modules élémentaire plats juxtaposés d'un troisième type. La Figure 24 montre les connexions des micro-arrays ou macro-arrays multiblocs de l'invention constitués respectivement par empilement des modules élémentaires plats de la figure 23. La Figure 25 A montre un micro-array ou macro-array multibloc (4319) de l'invention est formé par empilement de modules élémentaires plats (4110) pourvus de microcanaux (41) dont les micro-mélangeurs (4019) sont situés sur les sous-parties (4155) desdits modules élémentaires plats (4110).Figure 23 first shows a direct connection of two flat elementary modules of the invention, the first of the two so-called flat elementary modules being provided with detachable sub-parts. It then shows an orthogonal connection of an assembly of first flat elementary module-second flat elementary module with several juxtaposed flat elementary modules of a third type. FIG. 24 shows the connections of the micro-arrays or multi-block macro-arrays of the invention constituted respectively by stacking the flat elementary modules of FIG. 23. Figure 25 A shows a micro-array or multi-block macro-array (4319) of the invention is formed by stacking of flat elementary modules (4110) provided with microchannels (41) whose micro-mixers (4019) are located on the sub-parts (4155) of said flat elementary modules (4110).
La Figure 25 B, puis la Figure 25 C montre comment s'obtient une densité maximale des micro-arrays multiblocs de l'invention, avec un micro-array multibloc (4318) formé par empilement des parties détachables (4145) desdits modules élémentaires plats (4110).Figure 25 B, then Figure 25 C shows how a maximum density of the multiblock micro-arrays of the invention is obtained, with a multiblock micro-array (4318) formed by stacking detachable parts (4145) of said flat elementary modules (4110).
La Figure 26 A, la Figure 26B et la Figure 26C montrent qu'on peut faire des opérations de transfert de réactifs en série grâce à des micro-arrays ou un macro-arrays multibloc (20317), "équivalents " aux micro-arrays ou macro-arrays multiblocs (4319) ayant servi à la préparation des réactifs.Figure 26A, Figure 26B and Figure 26C show that serial reagent transfer operations can be performed using micro-arrays or a multiblock macro-arrays (20317), "equivalent" to micro-arrays or multiblock macro-arrays (4319) used to prepare the reagents.
La Figure 27 montre un micro-array ou macro-array multibloc de haute densité (5319) formé directement par empilement de modules élémentaires plats (5110) très peu épais,Figure 27 shows a high density multiblock micro-array or macro-array (5319) formed directly by stacking very thin flat elementary modules (5110),
La Figure 28A, la Figure 28B, la Figure 28 C, la Figure 28D montrent comment une pièce intermédiaire transversale (30339) pourvue de micro-canaux peut être connectée entre un empilement de modules élémentaires plats (21110) et un empilement de modules élémentaires plats (5110).Figure 28A, Figure 28B, Figure 28 C, Figure 28D show how a transverse intermediate piece (30339) provided with micro-channels can be connected between a stack of flat elementary modules (21110) and a stack of flat elementary modules (5110).
La Figure 29 A montre en perspective un micro-array ou macro-array multibloc (6319) de l'invention avec obturateurs permettant d'isoler un compartiment d'un module élémentaire plat (6110) dudit micro-array ou macro-array multibloc (6319).Figure 29 A shows in perspective a multi-block micro-array or macro-array (6319) of the invention with shutters making it possible to isolate a compartment of a flat elementary module (6110) from said multi-block micro-array or macro-array ( 6319).
La Figure 29B montre une vue de face d'un module élémentaire plat (6110) dudit micro-array ou macro-array multibloc (6319) de l'invention avec lesdits obturateurs.Figure 29B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention with said shutters.
La Figure 30A montre une autre vue en perspective d'un micro-array ou macro-array multibloc (6319) de l'invention où apparaît le parallélisme des opérations, avec, à titre d'exemple, un circuit de lavage pendant la préparation des réactifs avant leur envoi en surface dudit micro-array ou macro-array multibloc (6319).Figure 30A shows another perspective view of a micro-array or multi-block macro-array (6319) of the invention where the parallelism of the operations appears, with, for example, a washing circuit during the preparation of the reagents before sending to the surface of said micro-array or multi-block macro-array (6319).
La Figure 30B montre une vue de face d'un module élémentaire plat (6110) dudit micro-array ou macro-array multibloc (6319) de l'invention, avec, à titre d'exemple, un circuit de lavage pendant la préparation des réactifs avant leur envoi en surface dudit micro-array ou macro- array multibloc (6319). La Figure 31 montre une vue en perspective d'un micro-array ou macro-array multibloc (7319) de l'invention où apparaît le parallélisme des opérations, avec, à titre d'exemple, l'envoi d'un mélange réactionnel en surfacedudit micro-array ou macro-array multibloc (6319)..FIG. 30B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention, with, for example, a washing circuit during the preparation of the reagents before sending to the surface of said micro-array or multi-block macro-array (6319). Figure 31 shows a perspective view of a micro-array or multi-block macro-array (7319) of the invention where the parallelism of the operations appears, with, for example, the sending of a reaction mixture in surfacedudit micro-array or multi-block macro-array (6319).
La Figure 32 montre une connexion d'un micro-array ou macro-array multibloc de l'invention avec un bloc multi-pipette.Figure 32 shows a connection of a multiblock micro-array or macro-array of the invention with a multi-pipette block.
La Figure 33A montre en perspective un micro-array ou macro-array multibloc de l'invention connecté à une une pièce monobloc (555) d'aplanissement de surface.Figure 33A shows in perspective a multi-block micro-array or macro-array of the invention connected to a one-piece piece (555) of surface planarization.
La Figure 33B en coupe la connexion d'un micro-array ou macro-array multibloc de l'invention connecté à une une pièce monobloc (555) d'aplanissement de surface.Figure 33B shows the connection of a micro-array or multi-block macro-array of the invention connected to a one-piece piece (555) of surface planarization.
La Figure 34 et la Figure 35 montrent en perspective comment un module élémentaire plat (6110) peut-être micro-fàbriqué par l'assemblage de deux hémi-modules élémentaires plats (611012).Figure 34 and Figure 35 show in perspective how a flat elementary module (6110) can be micro-fabricated by the assembly of two flat elementary hemi-modules (611012).
La Figure 36A, la Figure 36B, la Figure 36C montrent une méthode de fabrication de tous les modules élémentaires plats de l'invention par superposition et fusion de sous-parties planes complémentaires, la sous partie plane supérieure faisant office de couvercle. . La Figure 36 A montre une première pièce, la Figure 36 B montre son couvercle, la Figure 36 C montre le micro-array ou macro-array multibloc de l'invention ainsi constitué.Figure 36A, Figure 36B, Figure 36C show a method of manufacturing all the flat elementary modules of the invention by superposition and fusion of complementary flat sub-parts, the upper flat sub-part serving as a cover. . Figure 36 A shows a first part, Figure 36 B shows its cover, Figure 36 C shows the micro-array or multi-block macro-array of the invention thus constituted.
La Figure 37A montre une sous-partie d'un module élémentaire plat.Figure 37A shows a sub-part of a flat elementary module.
La Figure 37B montre l'assemblage d'une sous-partie d'un module élémentaire plat avec sa partie complémentaire.Figure 37B shows the assembly of a sub-part of a flat elementary module with its complementary part.
La Figure 37C montre un micro-array ou macro-array multibloc de l'invention constitué de l'empilement desdits modules élémentaires plats fabriqués par assemblage desdites parties complémentaires. La Figure 37 D représente par transparence le micro-array ou macro-array ainsi constitué.Figure 37C shows a multi-block micro-array or macro-array of the invention consisting of the stack of said flat elementary modules produced by assembling said complementary parts. Figure 37 D shows by transparency the micro-array or macro-array thus formed.
La Figure 38 montre que lorsque des modules élémentaires plats (18110) ne présentent pas de composants, le support plat qui sert à fabriquer l'une de ses sous-parties complémentaires (1811012) peut être très mince et le micro-array ou macro-array constitué (18319) peut alors être relativement dense. La Figure 39 montre que lorsque des modules élémentaires plats (19110) présentent des composants, la densité du micro-array ou un macro-array multibloc (19319) pouvant être constitué par empilement desdits modules élémentaires plats (19110) est limitée.Figure 38 shows that when basic flat modules (18110) do not have any components, the flat support which is used to make one of its complementary sub-parts (1811012) can be very thin and the micro-array or macro- constituted array (18319) can then be relatively dense. Figure 39 shows that when flat elementary modules (19110) have components, the density of the micro-array or a multiblock macro-array (19319) which can be formed by stacking said flat elementary modules (19110) is limited.
La Figure 40A représente un mode de réalisation de l'invention et réside dans des micro- arrays ou macrbrarrays multiblocs (13319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée avec extraction de l'ADN par chromatographie par échange d'anions puis dessalement suif microparticules magnétiques de gel de silice.Figure 40A shows an embodiment of the invention and resides in multi-block microarrays or macrbrarrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by chromatography by exchange anions then desalination on magnetic microparticles of silica gel.
La Figure 40B et la Figure 40C montrent l'action d'un électro-aimant sur lesdites microparticules magnétiques.Figure 40B and Figure 40C show the action of an electromagnet on said magnetic microparticles.
La Figure 41 À-montre un autre mode de réalisation de l'invention et réside dans des micro- arrays ou macro-arrays multiblocs (14319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée avec extraction de l'ADN par chromatographie par échange d'anions puis dessalement sur matrices de gel de silice.Figure 41 A shows another embodiment of the invention and resides in micro-arrays or multi-block macro-arrays (14319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by anion exchange chromatography then desalination on silica gel matrices.
La Figure 41B montre un autre mode de réalisation de l'invention et réside dans des micro- arrays ou macro-arrays multiblocs (15319) dédiés à l'analyse de fragments d'ADN avec Une préparation mtégrée avec extraction de l'ADN sur matrices de gel de silice.Figure 41B shows another embodiment of the invention and resides in multi-block micro-arrays or macro-arrays (15319) dedicated to the analysis of DNA fragments with a metered preparation with extraction of DNA from matrices silica gel.
La Figure 42 montre la connexion orthogonale d'un élément plat de préparation des échantillons et réactifs avec un élément plat apportant un réactif spécifique dans un des micro-puits dudit élément plat de préparation des échantillons et réactifs.Figure 42 shows the orthogonal connection of a flat element for preparing samples and reagents with a flat element providing a specific reagent in one of the micro-wells of said flat element for preparing samples and reagents.
La Figure 43 montre la connexion orthogonale d'un empilement d'éléments plats de préparation des échantillons et réactifs avec un empilement d'éléments plats apportant un réactif spécifique dans chacun des micro-puits desdits éléments plats de préparation des échantillons et réactifs.Figure 43 shows the orthogonal connection of a stack of flat elements for preparing samples and reagents with a stack of flat elements providing a specific reagent in each of the micro-wells of said flat elements for preparing samples and reagents.
La Figure 44 montre la connexion orthogonale d'un élément plat, en deux parties assemblables, de préparation des échantillons et réactifs avec un élément plat, en deux parties assemblables lui aussi, et apportant un réactif spécifique dans un des micro-puits dudit élément plat de préparation des échantillons et réactifs.Figure 44 shows the orthogonal connection of a flat element, in two assemblable parts, for preparing samples and reagents with a flat element, in two parts also assembled, and bringing a specific reagent in one of the micro-wells of said flat element preparation of samples and reagents.
La Figure 45 montre la connexion orthogonale d'un empilement d'éléments plats , en deux parties assemblables, de préparation des échantillons et réactifs avec un empilement d'éléments plats, en deux parties assemblables eux aussi, et apportant un réactif spécifique dans chacun des micro-puits desdits éléments plats de préparation des échantillons et réactifs.Figure 45 shows the orthogonal connection of a stack of flat elements, in two assemblable parts, for preparing samples and reagents with a stack of flat elements, in two parts also assembled, and providing a specific reagent in each of the micro-wells of said flat elements for preparing the samples and reagents.
La Figure 46 montre un élément plat de préparation des échantillons et réactifs capable de diriger une ligne de premiers aliquots de ladite préparation vers des macro-emplacements et capable de se connecter à un empilement de modules plats capables de reprendre chaque aliquot du module plat précédent pour l'aliquoter à nouveau sur des micro-puits.Figure 46 shows a flat sample preparation and reagent element capable of directing a line of first aliquots of said preparation towards macro-locations and capable of connecting to a stack of flat modules capable of taking each aliquot of the previous flat module to aliquot it again on micro-wells.
La Figure 47 montre la connexion d'un élément plat de préparation des échantillons et réactifs capable de diriger une ligne de premiers aliquots de ladite préparation vers des macro-emplacements avec un empilement de modules plats capables de reprendre chaque aliquot du module plat précédent pour l'aliquoter à nouveau sur des micro-puits.Figure 47 shows the connection of a flat element for preparing samples and reagents capable of directing a line of first aliquots of said preparation towards macro-locations with a stack of flat modules capable of taking up each aliquot of the previous flat module for the aliquot again on micro-wells.
La Figure 48 montre le face à face entre une première ligne, au premier plan, de n empilements horizontaux de p modules plats identifiés individuellement de manière spécifique, avec une deuxième ligne, en arrière plan, de n empilements verticaux de p modules plats de réactifs spécifiques.Figure 48 shows the face to face between a first line, in the foreground, of n horizontal stacks of p flat modules individually identified specifically, with a second line, in the background, of n vertical stacks of p flat modules of reagents specific.
La Figure 49 montre la connexion orthogonale entre une première ligne, au premier plan, de n empilements horizontaux de p modules plats identifiés individuellement de manière spécifique, avec une deuxième ligne, en arrière plan, de n empilements verticaux de p modules plats de réactifs spécifiques, donnant lieu à n micro-arrays de np micro-puits, soit en tout n2 p micropuits, chacun des nfγ micro-puits ayant reçu l'un des np réactifs spécifiquesFigure 49 shows the orthogonal connection between a first line, in the foreground, of n horizontal stacks of p flat modules specifically identified individually, with a second line, in the background, of n vertical stacks of p flat modules of specific reagents , giving rise to n micro-arrays of np micro-wells, or in all n 2 p microwells, each of the nfγ micro-wells having received one of the np specific reagents
La Figure 50 montre la redistribution des modules plats des n empliments précédents de modules plats marqués différemment dans n nouveaux empilements regroupant les modules plats avec le même marquage individuel, chacun desdits empilement avec le même marquage individuel étant dédié à un seul échantillon.Figure 50 shows the redistribution of the flat modules from the previous n works of flat modules marked differently in n new stacks grouping the flat modules with the same individual marking, each of said stacks with the same individual marking being dedicated to a single sample.
La Figure 51 montre la connexion de n modules plats chacun à un seul échantillon avec n nouveaux empilements regroupant les modules plats avec le même marquage individuel, chcun des n échantillons pouvant rencontrer np réactifs spécifiques, les n empilements donnant lieu à n micro-arrays représentant au total n'p micro-puits.Figure 51 shows the connection of n flat modules each to a single sample with n new stacks grouping the flat modules with the same individual marking, each of the n samples may meet np specific reagents, the n stacks giving rise to n micro-arrays representing in total n'p micro-well.
La Figure 52A montre une étape dudit process de préparation intégrée de l'ADN sur micro- arrays ou macro-arrays multiblocs (15319) l'introduction dudit mélange de l'ADN purifié et du tampon d'amplification dans les micro-canaux dudit micro-array ou macro-array multibloc de l'invention. La Figure 52B montre comme étape suivante dudit process de préparation intégrée de l'ADN sur micro-arrays ou macro-arrays multiblocs (15319) une connexion dudit micro-array ou macro-array (15319) avec d'autres empilements de modules élémentaires plats (9245).Figure 52A shows a step of said integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) the introduction of said mixture of purified DNA and amplification buffer into the micro-channels of said micro -array or multiblock macro-array of the invention. Figure 52B shows as a next step of said process of integrated DNA preparation on micro-arrays or multi-block macro-arrays (15319) a connection of said micro-array or macro-array (15319) with other stacks of flat elementary modules (9245).
La Figure 52B montre comme étape suivante dudit process de préparation intégrée de l'ADN sur micro-arrays ou macro-arrays multiblocs (15319) la déconnexion dudit micro-array ou macro-array (15319) avec d'autres empilements de modules élémentaires plats (9245).Figure 52B shows as a next step of said integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) the disconnection of said micro-array or macro-array (15319) with other stacks of flat elementary modules (9245).
La Figure 53 montre un empilement en biseau de modules élémentaires plats (40110) avec sous-parties (40145) pour unne compatibilité avec une détection par microélectrophorèse avec par exemple une connexion à un empilement en biseau de modules élémentaires plats (40245) pourvus de micro-canaux pour micro-électrophorèse.Figure 53 shows a bevel stack of flat elementary modules (40110) with subparts (40145) for compatibility with detection by microelectrophoresis with for example a connection to a bevel stack of flat elementary modules (40245) provided with micro -channels for micro-electrophoresis.
Les Figures 54 A, 54B, 54C, 54D montrent qu'on peut synthésiser in situ dans les puits d'un micro-array ou macro-array monobloc (339) par exemple des octamers à partir de tetramers préfixés et de tetramers amenés par les microcanaux (41) desdits micro-arrays ou macro- arrays multiblocs (319) de l'invention.Figures 54 A, 54B, 54C, 54D show that one can synthesize in situ in the wells of a monoblock micro-array or macro-array (339) for example octamers from prefixed tetramers and tetramers brought by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
Les Figures 55A, 55B, 55C, 55D montrent qu'on peut synthésiser in situ dans les micro-puits (42) d'un micro-array ou macro-array multibloc (319) des octamers à partir de tetramers préfixés et de tetramers amenés par les microcanaux (41) desdits micro-arrays ou macro- arrays multiblocs (319) de l'invention.Figures 55A, 55B, 55C, 55D show that one can synthesize in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) octamers from prefixed tetramers and brought tetramers by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
Les Figures 56A, 56B, 56C, 56D montrent qu'on peut synthésiser in situ dans des portions élargies des micro-canaux (41) d'un micro-array ou macro-array multibloc (319) des octamers à partir de tetramers préfixés et de tetramers amenés par lesdits microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention.Figures 56A, 56B, 56C, 56D show that octamers can be synthesized in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) from prefixed tetramers and tetramers brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
Les Figures 57A, 57B, 57C montrent qu'on peut synthésiser in situ dans les micro-puits d'un micro-array ou macro-array monobloc (338) des biopuces dans chaque micro-puits dudit micro-array ou macro-array monobloc (338) grâce à une connexion avec des micro-arrays ou macro-arrays multiblocs de l'invention.Figures 57A, 57B, 57C show that one can synthesize in situ in the micro-wells of a monoblock micro-array or macro-array (338) biochips in each micro-well of said micro-array or monoblock macro-array (338) through a connection with micro-arrays or multi-block macro-arrays of the invention.
Les Figures 58 A, 58B, 58C montrent qu'on peut synthésiser des biopuces in situ dans chaque micro-puits d'un micro-array ou macro-array multibloc (319) La Figure 59A montre comment un micro-array ou macro-array multibloc de l'invention peut être connecté à un micro-array monobloc de bouchons (340) qui viennent obturer les micropuits (42) desdits micro-arrays ou macro-arrays multiblocs.Figures 58 A, 58B, 58C show that biochips can be synthesized in situ in each micro-well of a micro-array or multi-block macro-array (319) Figure 59A shows how a multi-block micro-array or macro-array of the invention can be connected to a mono-block micro-array of plugs (340) which seal the microwells (42) of said micro-arrays or multi-block macro-arrays.
La Figure 59B montre comment un premier micro-array ou macro-array multibloc de l'invention peut être connecté à un autre micro-array ou macro-array de l'invention formé par empilement de modules élémentaires plats (1101) dont les micro-canaux sont pourvus de micro- vannes (396).Figure 59B shows how a first multi-block micro-array or macro-array of the invention can be connected to another micro-array or macro-array of the invention formed by stacking flat elementary modules (1101), the micro- channels are provided with microvalves (396).
La Figure 59C montre comment un premier micro-array ou macro-array multibloc de l'invention peut être connecté à un autre micro-array ou macro-array multibloc de l'invention formé par empilement de modules élémentaires plats (1102) pourvus de dispositifs (398) qui créent une force électro-osmotique en établissant un potentiel électrique entre deux endroits des micro-canaux desdits de modules élémentaires plats (1102)Figure 59C shows how a first multi-block micro-array or macro-array of the invention can be connected to another multi-block micro-array or macro-array of the invention formed by stacking flat elementary modules (1102) provided with devices (398) which create an electro-osmotic force by establishing an electrical potential between two places of the micro-channels of said flat elementary modules (1102)
La Figure 59D montre comment un premier micro-array ou macro-array multibloc de l'invention formé par emplilement de modules élémentaires plats (1) peut être connecté à un autre micro-array ou macro-array de l'invention formé par empilement de modules élémentaires plats (1103) dont les micro-canaux sont pourvus de deux micro-vannes (397).Figure 59D shows how a first multi-block micro-array or macro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array or macro-array of the invention formed by stacking flat elementary modules (1103) whose micro-channels are provided with two micro-valves (397).
DESCRIPTION DE L'INVENTION:DESCRIPTION OF THE INVENTION:
Les micro-arrays ou macro-arrays classiques dédiés à l'analyse chimique ou biochimique ou biologique sont généralement plans et plats, et fabriqués d'un seul bloc (monobloc).The classic micro-arrays or macro-arrays dedicated to chemical or biochemical or biological analysis are generally flat and flat, and made from a single block (monoblock).
Les micro-arrays ou macro-arrays de l'invention sont plans, multiblocs, épais, voire très épais, car ils utilisent la troisième dimension, la profondeur du micro-array ou du macro- array.The micro-arrays or macro-arrays of the invention are planar, multi-block, thick, or even very thick, because they use the third dimension, the depth of the micro-array or of the macro-array.
La présente invention concerne des macro-arrays multiblocs ou micro-arrays multi-blocs en trois dimensions intégrés dans une chaîne prête à l'emploi continue ultra compacte de synthèse ou d'analyse chimique, biochimique ou biologique.The present invention relates to multi-block macro-arrays or three-dimensional multi-block micro-arrays integrated in a ready-to-use continuous ultra-compact chain of synthesis or chemical, biochemical or biological analysis.
Ces micro-arrays ou macro-arrays multi-blocs de l'invention intègrent dans leur architecture des Lab-on-a-Chip (laboratoires sur puces), ou bien, selon les configurations décrites dans l'invention, sont directement reliés ou connectables à ces Lab-on-a-Chip.These multi-block micro-arrays or macro-arrays of the invention integrate in their architecture Lab-on-a-Chip (laboratories on chips), or, depending on the configurations described in the invention, are directly connected or connectable to these Lab-on-a-Chip.
Le mot "Multibloc" s'oppose au mot "Monobloc". Il signifie que les "micro-arrays "ou "macro-arrays" de l'invention sont, à l'inverse des micro-arrays classiques en Analyse Chimique, constitués de plusieurs pièces. Plus précisément, ils sont constitués de l'assemblage par superposition d'un nombre élevé de modules élémentaires identiques ou homologues.The word "Multibloc" is opposed to the word "Monobloc". It means that the "micro-arrays" or "macro-arrays" of the invention are, unlike conventional micro-arrays in Analysis Chemical, made up of several parts. More specifically, they consist of the assembly by superposition of a large number of identical or homologous elementary modules.
Si l'on considère qu'un micro-array ou un macro-array est un quadrillage de puits ou de spots identiques, avec x lignes et y colonnes, chaque module élémentaire du micro-array ou du macro-array de l'invention a entre autres pour fonction de constituer en soi soit une colonne, soit une ligne dudit micro-array.If we consider that a micro-array or a macro-array is a grid of identical wells or spots, with x rows and y columns, each elementary module of the micro-array or of the macro-array of the invention has inter alia for the function of constituting in itself either a column or a line of said micro-array.
Les macro-arrays ou microarrays conçus selon la présente invention utilisent des micropuits obtenus reconversion de micro-canaux en micropuits.The macro-arrays or microarrays designed according to the present invention use microwells obtained reconversion of micro-channels into microwells.
La Figure 2A montre un micro-puits classique d'un micro-array ou d'un macro-array, dont la fonctionnalité souffre d'une exposition à l'air libre et donc de problèmes de contamination et d'évaporation.FIG. 2A shows a conventional micro-well of a micro-array or of a macro-array, the functionality of which suffers from exposure to the open air and therefore from contamination and evaporation problems.
La Figure 2B montre comment on peut reconvertir en micro-puit un micro-canal (41) plus ou moins long. Il suffit que ce micro-canal (41) traverse un support plus ou moins profond de part en part, et qu'à une extrémité dudit micro-canal, c'est à dire à un des orifices dudit microcanal, on appose un élément quelconque obturateur (4) ou un dispositif quelconque pouvant s'opposer au passage d'un fluide dans ledit micro-canal (41).FIG. 2B shows how a more or less long micro-channel (41) can be converted into a micro-well. It suffices that this micro-channel (41) passes through a more or less deep support right through, and that at one end of said micro-channel, that is to say at one of the orifices of said microchannel, any element is affixed obturator (4) or any device capable of opposing the passage of a fluid in said micro-channel (41).
Ainsi que le montrent la Figure 3A et la Figure 3B, les macro-arrays ou micro-arrays conçus selon la présente invention utilisent des micropuits (42) obtenus par reconversion de microcanaux (41) traversant de part en part un module élémentaire plat (1) et débouchant dans l'épaisseur et sur la tranche dudit module élémentaire plat (1) par des orifices (42). Avec plusieurs micro-canaux (41) parallèles traversant de part en part un module élémentaire plat (1) et débouchant dans l'épaisseur et sur la tranche dudit module élémentaire plat (1), on obtient selon l'invention une ligne (2119) de micropuits du micro-array ou macro-array de l'invention, ainsi que le montre la Figure 3B, Selon l'invention, ainsi que le montre la Figure 3C, la superposition précise de ces modules élémentaires plats (1) qui portent chacun une ligne (2119) du micro-array ou du macro-array, va former le micro-array multibloc ou le macro-array multibloc (3119) de l'invention. Le nombre de modules élémentaires plats (1) empilés va représenter le nombre de lignes dudit micro-array ou du macro-array multibloc. Le nombre de puits (42) par module élémentaire, c'est à dire le nombre de microcanaux (41) par module élémentaire plat, va représenter le nombre de colonnes du micro-array ou du macro-array multibloc (3119) de l'invention.As shown in Figure 3A and Figure 3B, the macro-arrays or micro-arrays designed according to the present invention use microwells (42) obtained by reconversion of microchannels (41) passing right through a flat elementary module (1 ) and opening into the thickness and on the edge of said flat elementary module (1) through orifices (42). With several parallel micro-channels (41) passing right through a flat elementary module (1) and opening into the thickness and on the edge of said flat elementary module (1), a line (2119) is obtained according to the invention. microwells of the micro-array or macro-array of the invention, as shown in Figure 3B, According to the invention, as shown in Figure 3C, the precise superposition of these flat elementary modules (1) which each carry a line (2119) of the micro-array or of the macro-array, will form the multi-block micro-array or the multi-block macro-array (3119) of the invention. The number of stacked flat elementary modules (1) will represent the number of lines of said micro-array or of the multiblock macro-array. The number of wells (42) per elementary module, that is to say the number of microchannels (41) per flat elementary module, will represent the number of columns of the micro-array or of the multiblock macro-array (3119) of the invention.
Les micro-puits obturables du micro-array ou macro-array de l'invention, à la fois profonds et à section réduite, diminuent la surface exposée à l'évaporation (voir Figure 2B). Une connexion va pouvoir être engagée par le dessous et par le dessus desdits micropuits, ce qui va rendre la connexion étanche.(voir Figure 2C et Figure 2D). De plus, les micropuits du microarray ou du macro-array multibloc de l'invention peuvent être obturés des deux côtés , ce qui du coup transforme lesdits micro-puits en micro-récipients (voir Figure 2C). Ainsi qu'il a été dit , pour reconvertir un microcanal (41) en micropuits (42) étroit et profond, le fonds dudit micropuits (42) est pourvu d'un moyen d'obturation provisoire. Lorsque cette configuration. est alternée avec la configuration opposée à 180°C, on peut alimenter le micropuits dύfmcro-array ou du macro-array de l'invention indifféremment par le dessous ou le dessus. Un des avantages des micro-arrays ou macro-arrays multiblocs de l'invention réside dans le fait que chaque module élémentaire plat qui le constitue va pouvoir être configuré en "Lab- on-a-chip", c'est à dire en "laboratoires sur puces", ce qui permettra une alimentation du micro-array ou du macro-array avec des produits de réaction dont la préparation a été intégrée audit micro-array ou macro-array multibloc sans phase d'exposition à l'air libre et sans évaporation ni contamination.The closable micro-wells of the micro-array or macro-array of the invention, both deep and of reduced section, reduce the surface exposed to evaporation (see FIG. 2B). A connection will be able to be engaged from below and from above of said microwells, which will seal the connection (see Figure 2C and Figure 2D). In addition, the microwells of the microarray or of the multiblock macro-array of the invention can be closed on both sides, which suddenly transforms said micro-wells into micro-containers (see FIG. 2C). As has been said, to convert a microchannel (41) into microwells (42) narrow and deep, the bottom of said microwell (42) is provided with a temporary closure means. When this configuration. is alternated with the opposite configuration at 180 ° C, one can feed the microwell dύfmcro-array or the macro-array of the invention either from below or from above. One of the advantages of the multi-block micro-arrays or macro-arrays of the invention lies in the fact that each flat elementary module which constitutes it will be able to be configured in "Lab-on-a-chip", that is to say in " laboratories on chips ", which will allow a supply of the micro-array or the macro-array with reaction products whose preparation has been integrated into said micro-array or multi-block macro-array without phase of exposure to the open air and without evaporation or contamination.
La Figure 4A montre des modules élémentaires plats (1) où les microcanaux (41) sont, selon l'invention, pourvus de micromélangeurs (19) et de portions élargies. Des portions élargies (1701) desdits micro-canaux (41) peuvent aussi comporter des microcolonnes (Cf Swedberg S, Kaltenbach-P, Witt K, Bek F, Mittlestadt LS. Fully integrated miniaturized planar liquid sample handling and analysis device. US Patent 5571410 _ He B, Tait N, Régnier F. Fabrication of nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797._. Pluskal MG, Microscale sample préparation, Nature Biotechnology, 2000, vol 18, 104-105). Des portions élargies (1706) peuvent aussi recevoir des microséparateurs passifs ou actifs pourvus de billes magnétiques (Cf Ahn CH, Trimmer W, Jun YN, Erramilli S. A fully integrated micromachined magnetic particle separator. Journal of Microelectromechanical Systems, 1996, vol 5, N° 3, 151-158). Lesdites portions élargies (1706) pour billes magnétiques peuvent être encadrées par des logements (1767) pour électro-aimants (1768),Figure 4A shows flat elementary modules (1) where the microchannels (41) are, according to the invention, provided with micromixers (19) and enlarged portions. Enlarged portions (1701) of said micro-channels (41) may also include microcolumns (Cf Swedberg S, Kaltenbach-P, Witt K, Bek F, Mittlestadt LS. Fully integrated miniaturized planar liquid sample handling and analysis device. US Patent 5571410 _ He B, Tait N, Régnier F. Fabrication of nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797._. Pluskal MG, Microscale sample preparation, Nature Biotechnology, 2000, vol 18, 104-105) . Enlarged portions (1706) can also receive passive or active microseparators provided with magnetic beads (Cf Ahn CH, Trimmer W, Jun YN, Erramilli S. A fully integrated micromachined magnetic particle separator. Journal of Microelectromechanical Systems, 1996, vol 5, No. 3, 151-158). Said enlarged portions (1706) for magnetic balls can be surrounded by housings (1767) for electromagnets (1768),
La Figure 4B montre en perspective le même module élémentaires plat (1) que la Figure 45 A. Dans cette vue en perspective apparaît la ligne (2119) de micro-puits (42) dans l'épaisseur et sur la tranche dudit module élémentaire plat (1).Figure 4B shows in perspective the same flat elementary module (1) as Figure 45 A. In this perspective view appears the line (2119) of micro-wells (42) in the thickness and on the edge of said flat elementary module (1).
La Figure 4C montre en perspective le micro-array (3119) formé par empilement des modules élémentaires plats (1) dont les microcanaux (41) sont pourvus de micromélangeurs (19), de portions élargies (1701) pourvues de micro-colonnes, de portions élargies (1706) recevant des billes magnétiques et encadrées par des logements (1767) pour électro-aimants (1768), lesdits électro-aimants (1768) traversant de part en part transversalement ledit micro- array ou macro-array multibloc (3119). Lorsque ledit électro-aimant (1768) est activé, les micro-particules magnétiques sont séparées du surnagenant dans lesdites portion élargies (1706) proches dudit électro-aimant (1768).Figure 4C shows in perspective the micro-array (3119) formed by stacking flat elementary modules (1) whose microchannels (41) are provided with micro-mixers (19), with enlarged portions (1701) provided with micro-columns, enlarged portions (1706) receiving magnetic balls and framed by housings (1767) for electromagnets (1768), said electromagnets (1768) passing right through said micro-array or multi-block macro-array (3119) . When said electromagnet (1768) is activated, the magnetic micro-particles are separated from the supernatant in said enlarged portion (1706) close to said electromagnet (1768).
Selon l'invention, ainsi que le montrent les Figures 5, 6, 7, 8, 9, un micro-array ou un macro- array multibloc (319) est alimenté par le dessous en réactifs ou en échantillons avec une compacité, une intégration et une architecture parallèle optimisées lorsque tous les modules élémentaires plats (110) qui le constituent par empilement comportent une circuiterie microfluidique (55) pourvue d'orifices d'alimentation et d'évacuation en échantillons et réactifs situés dans l'épaisseur et sur la tranche desdits modules élémentaires plats (110). Ces orifices d'alimentation sont tels que (8), (10), (12), à titre d'exemple. Ces orifices d'évacuation sont tels que l'orifice (11), à titre d'exemple.According to the invention, as shown in Figures 5, 6, 7, 8, 9, a micro-array or a multiblock macro-array (319) is supplied from below with reagents or samples with compactness, integration and an optimized parallel architecture when all the elementary flat modules (110) which constitute it by stacking comprise a microfluidic circuit (55) provided with orifices for supplying and discharging samples and reagents located in the thickness and on the wafer said flat elementary modules (110). These supply ports are such as (8), (10), (12), by way of example. These discharge orifices are such as the orifice (11), by way of example.
Selon l'invention, , ainsi que le montrent les Figures 5, 6, 7, 8, 9, lesdits modules élémentaires plats (110) peuvent être constitués de deux parties (145) et (155), ladite partie (145) supportant tout ou partie des microcanaux (41) et pouvant être éventuellement détachables et de très faible épaisseur, de l'ordre de 20 à 800 microns et ladite partie (155) supportant tout ou partie des composants de plus grande section.According to the invention, as shown in Figures 5, 6, 7, 8, 9, said flat elementary modules (110) may consist of two parts (145) and (155), said part (145) supporting all or part of the microchannels (41) and possibly being detachable and very thin, of the order of 20 to 800 microns and said part (155) supporting all or part of the components of larger section.
La Figure 5 montre une vue de dessus dudit micro-array ou macro-array multibloc (319) de l'invention constitué par l'empilement desdits modules élémentaires plats (110), chacun desdits modules élémentaires plats apportant une ligne (219) dudit micro-array ou macro- array multibloc (319). La Figure 5 montre aussi à titre d'exemple les orifices d'alimentation en réactifs (10) situés dans l'épaisseur et sur la tranche d'un côté desdits modules élémentaires plats (110), et des orifice d'alimentation en réactifs (12) situés dans l'épaisseur et sur la tranche d'un autre côté desdits modules élémentaires plats (110).Figure 5 shows a top view of said micro-array or multi-block macro-array (319) of the invention constituted by the stacking of said flat elementary modules (110), each of said flat elementary modules providing a line (219) of said micro -array or multi-block macro-array (319). Figure 5 also shows by way of example the reagent supply orifices (10) located in the thickness and on the edge of one side of said flat elementary modules (110), and the reagent supply orifices ( 12) located in the thickness and on the edge of another side of said flat elementary modules (110).
La Figure 6 montre une vue de face dudit module élémentaire plat (110) de l'invention pourvue d'une circuiterie microfluidique (55) pouvant alimenter lesdits micro-canaux (41) et finalement ledit micro-array ou macro-array multibloc (319). Ladite circuiterie microfluidique pouvant comprendre une zone de filtration (25) avec filtres (16), une zone de purification (35) avec miocrocolonne (17), et une zone de préparation (45) du produit filtré et purifié avant son introduction pour faible partie dans lesdits micro-canaux (41) et finalement, par voie interne, par le dessous, à l'abri de toute évaporation et de toute contamination, dans les micro-puits (42) d'une ligne (219) dudit micro-array ou macro-array multibloc (319).Figure 6 shows a front view of said flat elementary module (110) of the invention provided with microfluidic circuitry (55) capable of supplying said micro-channels (41) and finally said micro-array or multi-block macro-array (319 ). Said microfluidic circuitry which may include a filtration zone (25) with filters (16), a purification zone (35) with micro-column (17), and a preparation zone (45) of the filtered and purified product before its introduction for a small part in said micro-channels (41) and finally, internally, from below, protected from any evaporation and from any contamination, in the micro-wells (42) of a line (219) of said micro-array or multiblock macro-array (319).
La Figure 7 montre une vue de dos dudit module élémentaire plat (110) de l'invention pourvu de circuiteries électriques (381) et (391) destinée à activer les électrodes et composants dont sont pourvus lesdits modules élémentaires plats (110) pour commander le mouvement des fluides. La Figure 8 montre une vue de dessous dudit micro-array ou macro-array multibloc (319) de l'invention avec des orifices d'alimentation en réactifs ou en échantillons tels que, à titre d'exemple, (7) et (8), et des orifices d'évacuation tels que, à titre d'exemple, (11). Selon l'invention, lesdits orifices sont situés dans l'épaisseur et sur la tranche desdits modules élémentaires plats (110).Figure 7 shows a back view of said flat elementary module (110) of the invention provided with electrical circuits (381) and (391) intended to activate the electrodes and components which are provided with said flat elementary modules (110) to control the movement of fluids. Figure 8 shows a bottom view of said micro-array or multi-block macro-array (319) of the invention with supply ports for reagents or samples such as, for example, (7) and (8 ), and discharge orifices such as, for example, (11). According to the invention, said orifices are located in the thickness and on the edge of said flat elementary modules (110).
La Figure 9 montre une vue de profil dudit micro-array ou macro-array multibloc (319) de l'invention, La Figure 9 montre également des plots de connexion électrique (380) et (390) situés, selon l'invention, dans l'épaisseur et sur la tranche desdits modules élémentaires plats (110)Figure 9 shows a profile view of said micro-array or multi-block macro-array (319) of the invention, Figure 9 also shows electrical connection pads (380) and (390) located, according to the invention, in the thickness and on the edge of said flat elementary modules (110)
Les Figures 5, 6, et 7 et 9 montrent les encoches (71) et (72) dont sont éventuellement pourvus les parties (155) desdits modules élémentaires plats (110) pour recevoir des guides d'alignement tels que (61), (62). Elles montrent aussi les encoches (74) dont sont éventuellement pourvues lesdites parties détachables (145) desdits modules élémentaires plats (110) pour recevoir leur propre guide d'alignement au cas où elles seraient détachées et empilées séparément.Figures 5, 6, and 7 and 9 show the notches (71) and (72) which are optionally provided with the parts (155) of said flat elementary modules (110) to receive alignment guides such as (61), ( 62). They also show the notches (74) which are optionally provided with said detachable parts (145) of said flat elementary modules (110) to receive their own alignment guide in the event that they are detached and stacked separately.
Selon l'invention, la précision de l'empilement peut aussi être assurée par des contenants qui épousent les contours de la forme d'un micro-array ou macro-array multibloc de l'invention.According to the invention, the precision of the stacking can also be ensured by containers which conform to the contours of the shape of a micro-array or multi-block macro-array of the invention.
Les Figures 10, 11, 12, 13, 14 montrent comment lesdites parties détachables (145) desdits modules élémentaires plats (110) peuvent être détachées et être empilées pour former un micro-array ou macro-array multibloc (318) de plus grande densité que ledit micro-array ou macro-array multibloc (319).Figures 10, 11, 12, 13, 14 show how said detachable parts (145) of said flat elementary modules (110) can be detached and stacked to form a higher density micro-array or multi-block macro-array (318) as said multi-block micro-array or macro-array (319).
La Figure 10 représente le détachement desdites parties détachables (145), vu de profil, et les Figures 11 et 12 représentent ce détachement vu de dessus.Figure 10 shows the detachment of said detachable parts (145), seen in profile, and Figures 11 and 12 show this detachment seen from above.
La Figure 13 et la Figure 14 représentent, respectivement en vue de profil et vue de dessus, un micro-array ou macro-array multibloc (318) formé par empilement desdites parties détachables (145). Ledit micro-array ou macro-array multibloc (318) est de plus grande densité que ledit micro-array ou macro-array multibloc (319).Figure 13 and Figure 14 show, respectively in side view and top view, a multi-block micro-array or macro-array (318) formed by stacking said detachable parts (145). Said multi-block micro-array or macro-array (318) is of higher density than said multi-block micro-array or macro-array (319).
Selon l'invention, les micro-array ou macro-array multibloc de plus grande densité (318) constitués par l'empilement desdites parties détachables (145) peuvent se connecter entre eux face à face, ainsi que le montre la Figure 15 et la Figure 16. Les Figure 15 et Figure 16 représentent respectivement en vue de profil et vue de face la connexion face à face d'un premier micro-array ou macro-array multibloc (2318) constitué par empilement de parties détachables (2145) de modules élémentaires plats avec un deuxième micro-array ou macro-array multibloc (1318) constitué par empilement de parties détachables (1145).According to the invention, the higher density multi-block micro-array or macro-array (318) constituted by the stacking of said detachable parts (145) can connect together face to face, as shown in FIG. 15 and the Figure 16. FIGS. 15 and FIG. 16 respectively represent in side view and front view the face to face connection of a first multi-block micro-array or macro-array (2318) constituted by stacking of detachable parts (2145) of flat elementary modules with a second multi-block micro-array or macro-array (1318) formed by stacking detachable parts (1145).
Selon l'invention, un microarray ou macro-array multibloc (3119) peut se connecter à un autre micro-array ou macro-array multibloc (3119) de façon orthogonale, dès lors que que l'empilement de modules plats élémentaires (1) constituant le premier micro-array ou macro- array multibloc (3119) est décalé par une rotation de 90° autour de l'axe des micro-canaux (41) par rapport à l'empilement des modules élémentaires plats (1) du deuxième micro-array ou macro-array multibloc (3119), de manière à offrir la possibilité d'un parallélisme massif des réactions par configuration de matrices XY où des lignes de premiers réactifs (xl x2 .. xn) dans le premier micro-array ou macro-array multibloc (3119) se croisent avec des lignes de deuxièmes réactifs (yl y2 ... yn) dans le deuxième micro-array ou macro-array multibloc (3119).According to the invention, a multi-block microarray or macro-array (3119) can be connected to another multi-block micro-array or macro-array (3119) orthogonally, provided that the stack of elementary flat modules (1) constituting the first micro-array or multi-block macro-array (3119) is offset by a rotation of 90 ° around the axis of the micro-channels (41) relative to the stack of flat elementary modules (1) of the second micro -array or multiblock macro-array (3119), so as to offer the possibility of a massive parallelism of reactions by configuration of XY matrices where lines of first reagents (xl x2 .. xn) in the first micro-array or macro -array multibloc (3119) cross with lines of second reagents (yl y2 ... yn) in the second micro-array or macroblock multibloc (3119).
Les Figures 17, 18,19, 20, 21, 22, 23, 24 représentent comment plusieurs micro-arrays ou macro-arrays multiblocs de l'invention peuvent se connecter face à face, avant ou après que lesdites parties détachables desdits modules élémentaires plats soient détachées.Figures 17, 18, 19, 20, 21, 22, 23, 24 show how several micro-arrays or multi-block macro-arrays of the invention can connect face to face, before or after said detachable parts of said flat elementary modules are detached.
La Figure 17 et la Figure 18 représentent comment un empilement de modules élémentaires plats (910) pourvus d'un seul compartiment (956) avec un orifice d'introduction (907) peut se connecter directement à un empilement des modules élémentaires plats (94610) pourvus de micro-canaux (94641) avec micro-mélangeurs (94619), lesdits micro-canaux (94641) débouchant dans l'épaisseur et sur la tranche desdits modules élémentaires plats (94610), ledit empilement desdits modules élémentaires plats (94610) pouvant se connecter orthogonalement à un empilement de modules élémentaires plats (25110) constituant ledit micro-array ou macro-array multibloc de l'invention, ladite connexion se pouvant se faire avant ou après le détachement des parties détachables (25145) desdits modules élémentaires plats (25110)..Figure 17 and Figure 18 show how a stack of flat elementary modules (910) with a single compartment (956) with an insertion opening (907) can connect directly to a stack of flat elementary modules (94610) provided with micro-channels (94641) with micro-mixers (94619), said micro-channels (94641) opening into the thickness and on the edge of said flat elementary modules (94610), said stack of said flat elementary modules (94610) being able to connect orthogonally to a stack of flat elementary modules (25110) constituting said micro-array or multi-block macro-array of the invention, said connection being able to be made before or after detaching the detachable parts (25145) from said flat elementary modules ( 25110) ..
La Figure 19 et la Figure 20 représentent comment un empilement de modules élémentaires plats (9010) pourvu de plusieurs compartiments (9056) avec orifice d'introduction (9007) peut se connecter directement à un empilement des modules élémentaires plats (9046) pourvus de micro-canaux (9041 ) avec micro-mélangeurs (9019), lesdits micro-canaux (9041 ) débouchant dans l'épaisseur et sur la tranche desdits modules élémentaires plats (9046), ledit empilement desdits modules élémentaires plats (9046) pouvant se connecter orthogonalement à un empilement de modules élémentaires plats (25110) constituant ledit micro-array ou macro-array multibloc de l'invention, ladite connexion se faisant avant ou après le détachement des parties détachables (25145) desdits modules élémentaires plats (25110).Figure 19 and Figure 20 show how a stack of flat elementary modules (9010) provided with several compartments (9056) with insertion opening (9007) can connect directly to a stack of flat elementary modules (9046) provided with micro -channels (9041) with micro-mixers (9019), said micro-channels (9041) opening into the thickness and on the edge of said flat elementary modules (9046), said stack of said flat elementary modules (9046) being able to connect orthogonally to a stack of flat elementary modules (25110) constituting said micro-array or multiblock macro-array of the invention, said connection being made before or after detaching the detachable parts (25145) from said flat elementary modules (25110).
La Figure 21 et la Figure 22 représentent comment un empilement de modules élémentaires plats (1010) pourvu d'un seul compartiment (1056) avec orifice d'introduction (1007) peut se connecter orthogonalement à un empilement des modules élémentaires plats (104610), ledit empilement desdits modules élémentaires plats (104610) pouvant se connecter directement à un empilement de modules élémentaires plats (25110) constituant ledit micro-array ou macro-array multibloc de l'invention, ladite connexion se faisant avant ou après le détachement des parties détachables (25145) desdits modules élémentaires plats (25110).FIG. 21 and FIG. 22 show how a stack of flat elementary modules (1010) provided with a single compartment (1056) with insertion opening (1007) can be connected orthogonally to a stack of flat elementary modules (104610), said stack of said flat elementary modules (104610) being able to connect directly to a stack of flat elementary modules (25110) constituting said multi-block micro-array or macro-array of the invention, said connection being made before or after detaching the detachable parts (25145) of said flat elementary modules (25110).
La Figure 23 et la Figure 24 représentent comment plusieurs empilement juxtaposés de modules élémentaires plats (10010) pourvus de plusieurs compartiments (10056) avec orifice d'introduction (10007) peuvent se connecter orthogonalement à un empilement des modules élémentaires plats (1004610), ledit empilement desdits modules élémentaires plats (1004610) pouvant se connecter directement à un empilement de modules élémentaires plats (25110) constituant ledit micro-array ou macro-array multibloc de l'invention, ladite connexion se faisant avant ou après le détachement des parties détachables (25145) desdits modules élémentaires plats (25110). La Figure 25 A, la Figure 25 B, la Figure 25 C montre comment s'obtient une densité maximale des micro-arrays multiblocs de l'invention.Figure 23 and Figure 24 show how several juxtaposed stack of flat elementary modules (10010) provided with several compartments (10056) with insertion opening (10007) can connect orthogonally to a stack of flat elementary modules (1004610) stack of said flat elementary modules (1004610) which can be directly connected to a stack of flat elementary modules (25110) constituting said multi-block micro-array or macro-array of the invention, said connection being made before or after detaching the detachable parts ( 25145) of said flat elementary modules (25110). Figure 25 A, Figure 25 B, Figure 25 C shows how a maximum density of the multiblock micro-arrays of the invention is obtained.
En effet, la Figure 25 A, montre un micro-array (4319) constitué par empilement de modules élémentaires plats (4110) pourvus de deux sous- parties (4i45) et (4155). Les micro-canaux (41) débouchent sur le micro-array (4319) dans l'épaisseur et sur la tranche desdites sous-parties (4145).In fact, Figure 25A shows a micro-array (4319) formed by stacking flat elementary modules (4110) provided with two sub-parts (4i45) and (4155). The micro-channels (41) open onto the micro-array (4319) in the thickness and on the edge of said sub-parts (4145).
La portion desdits micro-canaux (41) sur ladite sous-partie (4155) est pourvue de micromélangeurs (4019), lesdits micro-canaux (41) reprenant une très petite partie des fluides amenés par la micro-circuiterie (4055). Ladite sous-partie (4155) ménage un espace important entre lesdits micro-canaux (41) et ladite sous-partie (4145) reçoit lesdits micro-canaux (41) dans une configuration resserrée, de manière que l'empilement desdits modules élémentaires plats (4110) constitue des microarrays ou macro-arrays multiblocs (4319) de forte densité.The portion of said micro-channels (41) on said sub-part (4155) is provided with micro-mixers (4019), said micro-channels (41) taking up a very small part of the fluids supplied by the micro-circuit (4055). Said sub-part (4155) provides a large space between said micro-channels (41) and said sub-part (4145) receives said micro-channels (41) in a constricted configuration, so that the stacking of said flat elementary modules (4110) constitutes micro-arrays or multi-block macro-arrays (4319) of high density.
La Figure 25 B et la Figure 25 C montrent qu'après détachement, l'empilement desdites sous- parties (4145) constitue des micro-arrays ou macro-arrays multiblocs (4318) dont le niveau encore supérieur de densité par rapport au micro-array (4319) est dû à la moindre épaisseur desdites sous-parties détachables (4145). La Figure 26 A, la Figure 26B et la Figure 26C montrent qu'on peut choisir de ne pas détacher les sous parties (4145) de modules élémentaires plats (4110) formant par empilement un micro-array ou un macro-array multibloc (4319) de l'invention, mais plutôt de créer un micro-array ou un macro-array multibloc équivalent (20317), puis un micro-array ou un macro-array multibloc (20316).de plus grande densité.Figure 25B and Figure 25C show that after detachment, the stacking of said sub-parts (4145) constitutes micro-arrays or multi-block macro-arrays (4318) whose level of density is even higher compared to the micro- array (4319) is due to the lesser thickness of said detachable subparts (4145). Figure 26 A, Figure 26B and Figure 26C show that one can choose not to detach the sub-parts (4145) of flat elementary modules (4110) forming by stacking a micro-array or a multi-block macro-array (4319 ) of the invention, but rather to create a micro-array or an equivalent multi-block macro-array (20317), then a micro-array or a multi-block macro-array (20316). of greater density.
En effet, la Figure 26 Aet la Figure 26B montrent des modules élémentaires plats (20046) traversés longitùdinalement de part en part par des micro-canaux débouchant dans l'épaisseur et sur la francKè de deux de leurs côtés, lesdits côtés étant opposés, ce qui a pour conséquence qu'un empilement desdits modules élémentaires plats (20046) forme deux micro-arrays ou macro-arrays multiblocs, chacun desdits microarrays ou macro-arrays multiblocs se constituant sur une face opposée de l'empilement, lesdits modules élémentaires plats (20046) pouvant dès lors être utilisés pour reproduire à l'identique par transfert de réactifs ledit micro- array ou macro-array (4319). Ainsi l'empilement desdits modules élémentaires plats (20046) peut former le micro-array ou macro-array multibloc (20317) puis le microarray (20316) équivalent à celui qui aurait été constitué en détachant puis en empilant les parties (4145) desdits modules élémentaires plats (4110) dudit micro-array ou macro-array multibloc (4319).Indeed, Figure 26 A and Figure 26B show flat elementary modules (20046) traversed longitudinally right through by micro-channels emerging in the thickness and on the francKè on two of their sides, said sides being opposite, this which results in a stack of said flat elementary modules (20046) forming two multi-block micro-arrays or macro-arrays, each of said multi-block microarrays or macro-arrays constituting on an opposite face of the stack, said flat elementary modules ( 20046) which can therefore be used to reproduce identically by transfer of reagents said micro-array or macro-array (4319). Thus the stacking of said flat elementary modules (20046) can form the micro-array or multi-block macro-array (20317) then the microarray (20316) equivalent to that which would have been formed by detaching and then stacking the parts (4145) of said modules. flat elementaries (4110) of said micro-array or multi-block macro-array (4319).
Selon l'invention, ces "micro-arrays ou macro-arrays multiblocs équivalents" permettent de travailler à la fois en parallèle, en utilisant un micro-array ou macro-array multibloc (4319) avec de nombreux micro-puits (42), mais aussi en série en faisant se succéder des analyses différentes à partir d'une même préparation intégrée sur ledit micro-array ou macro-array multibloc (4319), ladite préparation intégrée étant transférée dans plusieurs micro-arrays ou macro-arrays équivalents (20317) successifs. De même, ces configurations utilisant des "micro-arrays ou macro-arrays multiblocs équivalents" sont adaptées à des process d'analyse ou de synthèse chimique, biochimique ou biologique en série.According to the invention, these “equivalent multi-block micro-arrays or macro-arrays” make it possible to work both in parallel, using a micro-array or multi-block macro-array (4319) with numerous micro-wells (42), but also in series by making successive different analyzes from the same preparation integrated on said micro-array or multi-block macro-array (4319), said integrated preparation being transferred into several micro-arrays or equivalent macro-arrays (20317 ) successive. Likewise, these configurations using "equivalent multi-block micro-arrays or macro-arrays" are suitable for chemical, biochemical or biological analysis or synthesis processes in series.
La Figure 27 montre qu'un micro-array multibloc de haute à très haute densité peut aussi être directement constitué s'il est fait par empilement de modules élémentaires plats qui, d'une part sont de très faible épaisseur, d'autre part ont une configuration qui autorise leurs micro- canaux à resserrer sur lesdits module élémentaire plat.Figure 27 shows that a multiblock micro-array of high to very high density can also be directly constituted if it is made by stacking of flat elementary modules which, on the one hand are very thin, on the other hand have a configuration which allows their micro-channels to tighten on said flat elementary module.
La Figure 27 montre un micro-array ou macro-array multibloc de haute densité (5319) formé par empilement de modules élémentaires plats (5110) de très faible épaisseur constitué de deux parties (5145) et (5155) avec plots d'alignement (71). Les micro-canaux (41) débouchent sur le micro-array multibloc (5319) dans l'épaisseur et sur la tranche desdites sous-parties (5145).FIG. 27 shows a high density multiblock micro-array or macro-array (5319) formed by stacking very thin flat elementary modules (5110) consisting of two parts (5145) and (5155) with alignment pads ( 71). The micro-channels (41) lead to the multi-block micro-array (5319) in the thickness and on the edge of said sub-parts (5145).
La portion desdits micro-canaux (41) sur ladite sous-partie (5155) est pourvue de micromélangeurs (5019), lesdits micro-canaux (41) reprenant une très petite partie des fluides amenés par la micro-circuiterie (5055). Ladite sous-partie (5155) ménage un espace important entre lesdits micro-canaux (41) et ladite sous-partie (5145) reçoit lesdits micro-canaux (41) dans une configuration resserrée, de manière que l'empilement desdits modules élémentaires plats (5110) constitue directement des micro-arrays ou macro-arrays multiblocs (5319) de très haute densité.The portion of said micro-channels (41) on said sub-part (5155) is provided with micro-mixers (5019), said micro-channels (41) taking up a very small part of the fluids supplied by the micro-circuit (5055). Said sub-part (5155) provides a large space between said micro-channels (41) and said sub-part (5145) receives said micro-channels (41) in a constricted configuration, so that the stacking of said flat elementary modules (5110) directly constitutes very high density micro-arrays or multi-block macro-arrays (5319).
La Figure 28A, la Figure 28B, la Figure 28 C, la Figure 28D montrent comment une pièce intermédiaire transversale (30339) pourvue de micro-canaux peut être connectée entre un empilement de modules élémentaires plats (21110) et un empilement de modules élémentaires plats (5110). Dans l'exemple relaté sur les Figures 28A, 28B, 28C, 28D, lesdits modules élémentaires plats (21110) sont traversés longitudinalement de part en part par des micro-canaux débouchant dans l'épaisseur et sur la tranche de deux de leurs côtés, lesdits côtés étant opposés, chacun desdits microarrays ou macro-arrays multiblocs se constituant sur une face opposée de l'empilement, ledit empilement étant orthogonal par rapport à l'empilement des modules élémentaires plats (5110).Figure 28A, Figure 28B, Figure 28 C, Figure 28D show how a transverse intermediate piece (30339) provided with micro-channels can be connected between a stack of flat elementary modules (21110) and a stack of flat elementary modules (5110). In the example described in FIGS. 28A, 28B, 28C, 28D, said flat elementary modules (21110) are traversed longitudinally right through by micro-channels opening into the thickness and on the edge of two of their sides, said sides being opposite, each of said multi-block microarrays or macro-arrays constituting on an opposite face of the stack, said stack being orthogonal to the stack of flat elementary modules (5110).
La dite pièce intermédiaire (30339) peut être fabriquée selon le mode des micro-arrays ou macro-arrays multiblocs de l'invention, c'est à dire par empilement de modules élémentaires plats, ledit empilement étant parallèle aux micro-canaux, ou bien selon un mode différent, par exemple avec des micro-canaux orientés perpendiculairement à l'empilement de modules élémentaires plats, c'est à dire perpendiculairement à une surface plane et non pas parallèllement à la longueur ou à la largeur de la surface plane comme pour l'invention. Selon l'invention, un tel type de pièce intermédiare (30339) peut être utilisée comme simple connecteur,ou comme pièce utile au process d'analyse ou de synthèse chimique, biochimique ou biologique, par exemple en étant pourvus de molécules fixées et utiles au process, ou en étant pourvue de micro-particules ou micro-sphères fixant des molécules utiles au process, ou bien en étant pourvues de micro-réservoirs de réactifs en dérivation des micro-canaux, ou bien en étant pourvues de micro-composants tels que micro-colonnes, microfiltres, micromélangeurs, micropompes, microvannes, micro-réchauffeurs, etc. La Figure 29 A montre en perspective un micro-array ou macro-array multibloc (6319) de l'invention formé par empilement de modules élémentaires plats (6110). Lesdits modules élémentaires plats (6110) sont formés de sous-parties (6155) et de sous-parties (6145). Lesdites sous-parties (6145) sont pourvues de micro-canaux (41) débouchant dans leur épaisseur et sur leur tranche pour former les micro-puits (42) dudit micro-array ou macro- array multibloc (6319). Lesdits micro-canaux (41) sont pourvus de micro-mélangeurs (19). Lesdites sous-parties (6145) sont également pourvues de plots de connexion électrique (390) situés dans leur épaisseur et sur leur tranche.Said intermediate piece (30339) can be manufactured according to the mode of micro-arrays or multi-block macro-arrays of the invention, that is to say by stacking of flat elementary modules, said stack being parallel to the micro-channels, or else according to a different mode, for example with microchannels oriented perpendicular to the stack of flat elementary modules, that is to say perpendicular to a flat surface and not parallel to the length or the width of the flat surface as for the invention. According to the invention, such a type of intermediate part (30339) can be used as a simple connector, or as a useful part in the chemical, biochemical or biological analysis or synthesis process, for example by being provided with molecules fixed and useful for process, or by being provided with micro-particles or micro-spheres fixing molecules useful to the process, or by being provided with micro-reservoirs of reagents in derivation of the micro-channels, or by being provided with micro-components such as micro-columns, microfilters, micromixers, micropumps, microvalves, micro-heaters, etc. Figure 29A shows in perspective a micro-array or multi-block macro-array (6319) of the invention formed by stacking flat elementary modules (6110). Said flat elementary modules (6110) are formed of sub-parts (6155) and sub-parts (6145). Said sub-parts (6145) are provided with micro-channels (41) opening out in their thickness and on their edges to form the micro-wells (42) of said micro-array or multi-block macro-array (6319). Said micro-channels (41) are provided with micro-mixers (19). Said sub-parts (6145) are also provided with electrical connection pads (390) located in their thickness and on their edges.
Selon l'invention, les dites sous-parties (6155) sont pourvues d'un orifice d'évacuation (11) et d'orifices d'introduction (7), (8), (10) , (12) situés dans l'épaisseur et sur la tranche desdits modules élémentaires plats (6110), de microfiltres (16), et d'une microcolonne (17). Selon l'invention, ladite micro-colonne (17), comme d'autres micro-composants du système microfluidique, peut être isolée par des obturateurs (9) et (14) situés dans l'épaisseur et sur la tranche desdits modules élémentaires plats (6110) et actionnés par l'extérieur desdits modules élémentaires plats (6110) La Figure 29B montre une vue de face d'un module élémentaire plat (6110) dudit micro-array ou macro-array multibloc (6319) de l'invention .According to the invention, said sub-parts (6155) are provided with a discharge orifice (11) and introduction orifices (7), (8), (10), (12) located in the thickness and on the edge of said flat elementary modules (6110), microfilters (16), and a microcolumn (17). According to the invention, said micro-column (17), like other micro-components of the microfluidic system, can be isolated by shutters (9) and (14) located in the thickness and on the edge of said flat elementary modules. (6110) and actuated from the outside of said flat elementary modules (6110) Figure 29B shows a front view of a flat elementary module (6110) of said micro-array or multi-block macro-array (6319) of the invention.
La Figure 30A et la Figure 30 B montrent respectivement en perpective et de face, ledit micro-array ou macro-array multibloc (6319) où apparaît le parallélisme des opérations, avec, à titre d'exemple, un circuit de lavage (66) pendant la préparation, avant que des produits purifiés (67) retenus sur la microlonne (17) soient envoyés en surface dudit micro-array ou macro-array multibloc (6319). Dans cet exemple, alors que les orifices d'introduction sont bouchés par des bouchons (351), (352), (353), et alors que ladite microcolonne (17) est isolée par le double obturateur (354), un produit de lavage introduit par l'orifice (10) est évacué par l'orifice (11) dans tous lesdits modules élémentaires (6110) en même temps, de sorte qu'une opération de préparation de réactifs ou d'échantillons a lieu en même temps pour tous lesdits micro-puits (42) desdits micro-arrays ou macro-arrays multiblocs (6319).Figure 30A and Figure 30B show respectively in perspective and in front, said micro-array or multi-block macro-array (6319) where the parallelism of operations appears, with, for example, a washing circuit (66) during preparation, before purified products (67) retained on the microlumn (17) are sent to the surface of said micro-array or multi-block macro-array (6319). In this example, while the introduction orifices are blocked by plugs (351), (352), (353), and while said microcolumn (17) is isolated by the double shutter (354), a washing product introduced through the orifice (10) is discharged through the orifice (11) in all of said elementary modules (6110) at the same time, so that an operation for preparing reagents or samples takes place at the same time for all said micro-wells (42) of said multi-block micro-arrays or macro-arrays (6319).
La Figure 31 montre le parallélisme des opérations sur une vue en perspective d'un micro- array ou macro-array multibloc (7319) de micro-puits (42) formé par empilement de modules élémentaires plats (7110) pourvus d'une zone de lyse-filtration (7025), d'une zone de purification (7035) munie d'une micro-colonne (7017), d'une zone de préparation des réactifs purifiés (7045). A titre d'exemple, il est représenté l'envoi d'un mélange réactionnel (79) en surface dudit micro-array ou macro-array multibloc (7319), via les micro-canaux (41), ledit mélange réactionnel représentant le produit purifié (77) additionné d'un tampon (78) .Figure 31 shows the parallelism of operations on a perspective view of a micro-array or multi-block macro-array (7319) of micro-wells (42) formed by stacking flat elementary modules (7110) provided with a zone of lysis-filtration (7025), a purification zone (7035) provided with a micro-column (7017), a zone for preparing the purified reagents (7045). By way of example, the sending of a reaction mixture (79) to the surface of said micro-array or multi-block macro-array (7319) is represented, via the micro-channels (41), said reaction mixture representing the product. purified (77) added with a buffer (78).
La Figure 32 montre une connexion d'un un bloc multi-pipette (70211) avec micro-array ou macro-array multibloc de l'invention. Ledit micro-array ou macro-array multibloc de l'invention est formé par empilement de modules élémentaires plats (70110) avec sous-parties (70155) et (70145) . Lesdites sous-parties (70155) sont pourvues d'une circuiterie microfluidique (70055), et lesdites sous parties (70145) sont pourvues de microcanaux (41) débouchant dans leur épaissseur et sur leur tranche. Ledit bloc multipipette (70211) intègre un empilement de modules élémentaires plats (70110) pourvus de sous -parties (70202) et de sous-parties (70245).Figure 32 shows a connection of a multi-pipette block (70211) with micro-array or multi-block macro-array of the invention. Said multi-block micro-array or macro-array of the invention is formed by stacking flat elementary modules (70110) with sub-parts (70155) and (70145). Said sub-parts (70155) are provided with microfluidic circuitry (70055), and said sub-parts (70145) are provided with microchannels (41) opening out in their thickness and on their edges. Said multipipette block (70211) integrates a stack of flat elementary modules (70110) provided with subparts (70202) and subparts (70245).
La Figure 33A montre en perspective comment un micro-array ou macro-array multibloc de l'invention formé par empilement de modules élémentaires plats mâles (8110) est connecté à une pièce monobloc d'aplanissement de surface (555). La Figure 33B en coupe la connexion du micro-array ou macro-array multibloc avec ladite pièce monobloc d'aplanissement de surface (555).Figure 33A shows in perspective how a multi-block micro-array or macro-array of the invention formed by stacking flat male elementary modules (8110) is connected to a monoblock surface planarization part (555). Figure 33B shows the connection of the multi-block micro-array or macro-array with said one-piece surface flattening part (555).
Les Figures 33 A et la Figure 33 B montrent une pièce monobloc (555) d'aplanissement de microarrays ou macro-arrays multiblocs constitué par un empilement de modules élémentaires plats 8110 mâles. Ladite pièce d'aplanissement (555) est monobloc, présente au recto une face plane, et présente au verso son autre face qui s'emboite dans les creux ménagés entre les parties saillantes desdits modules élémentaires plats 8110 mâles . Cette pièce est utilisable dans tous les cas où un micro-array ou macro-array multibloc de l'invention doit présenter une surface plane, par exemple dans les cas où l'on doit éviter des réflexions lumineuses pour des détections ou des photo-expositions.Figures 33 A and Figure 33 B show a single piece (555) planarizing microarrays or multiblock macro-arrays consisting of a stack of 8110 male flat elementary modules. Said planarization piece (555) is in one piece, has on the front a flat face, and has on the back its other face which fits into the recesses formed between the projecting parts of said flat male elementary modules 8110. This part can be used in all cases where a micro-array or multi-block macro-array of the invention must have a flat surface, for example in cases where light reflections must be avoided for detections or photo-exhibitions .
La Figure 42 montre la connexion orthogonale d'un élément plat de préparation des échantillons et réactifs (27110) avec un élément plat (27210) apportant un réactif spécifique dans un des micro-puits dudit élément plat de préparation des échantillons et réactifs (27110).Figure 42 shows the orthogonal connection of a flat element for preparing samples and reagents (27110) with a flat element (27210) providing a specific reagent in one of the micro-wells of said flat element for preparing samples and reagents (27110) .
La Figure 43 montre la connexion orthogonale d'un empilement d'éléments plats de préparation des échantillons et réactifs (27110) avec un empilement d'éléments plats (27210) apportant un réactif spécifique dans chacun des micro-puits desdits éléments plats de préparation des échantillons et réactifs (27210).Figure 43 shows the orthogonal connection of a stack of flat elements for sample and reagent preparation (27110) with a stack of flat elements (27210) providing a specific reagent in each of the micro-wells of said flat elements for preparation of samples and reagents (27210).
La Figure 44 montre la connexion orthogonale d'un élément plat, en deux parties assemblables, de préparation des échantillons et réactifs, avec un élément plat, en deux parties assemblables lui aussi, et apportant un réactif spécifique dans un des micro-puits dudit élément plat de préparation des échantillons et réactifs.Figure 44 shows the orthogonal connection of a flat element, in two assemblable parts, for preparing samples and reagents, with a flat element, in two parts also assembled, and bringing a specific reagent in one of the micro-wells of said element sample preparation dish and reagents.
La Figure 45 montre la connexion orthogonale d'un empilement d'éléments plats , en deux parties assemblables, de préparation des échantillons et réactifs avec un empilement d'éléments plats, en deux parties assemblables eux aussi, et apportant un réactif spécifique dans chacun des micro-puits desdits éléments plats de préparation des échantillons et réactifs.Figure 45 shows the orthogonal connection of a stack of flat elements, in two assemblable parts, for preparing samples and reagents with a stack of flat elements, in two parts also assembled, and providing a specific reagent in each of the micro-wells of said flat sample preparation elements and reagents.
La Figure 46 montre un élément plat de préparation des échantillons et réactifs (47000) capable de diriger une ligne de premiers aliquots de ladite préparation vers une ligne de macro-emplacements (47107) et capable de se connecter à un empilement de modules plats (37110) pourvus chacun d'une chambre de réception (37164). Les modules plats (37110) sont eux capables de reprendre par une ligne d'orifices (37107) chaque aliquot dudit module plat (47000) dans ladite chambre de réception des aliquots (37164) pour l'aliquoter à nouveau sur des micro-puits ou de micro-emplacements (37042) par le biais de microcanaux (37041) traversant des micromélangeurs (47019).Figure 46 shows a flat sample preparation and reagent element (47000) capable of directing a line of first aliquots of said preparation towards a line of macro-locations (47107) and capable of connecting to a stack of flat modules (37110 ) each provided with a reception room (37164). The flat modules (37110) are able to take up by a line of orifices (37107) each aliquot of said module dish (47000) in said aliquot receiving chamber (37164) to re-aliquot it on micro-wells or micro-locations (37042) by means of microchannels (37041) passing through micro-mixers (47019).
La Figure 47 montre la connexion d'un élément plat de préparation des échantillons et réactifs (47000) avec un empilement de modules plats (37110) capables de reprendre chaque aliquot dudit module plat (47000) pour l'aliquoter à nouveau sur un micro-array des micropuits ou de micro-emplacements (37042).Figure 47 shows the connection of a flat sample preparation and reagent element (47000) with a stack of flat modules (37110) capable of taking each aliquot of said flat module (47000) to re-label it on a micro- array of microwells or micro-locations (37042).
La Figure 48 montre le face à face entre une première ligne, au premier plan, de n empilements horizontaux de p modules plats identifiés individuellement de manière spécifique, avec une deuxième ligne, en arrière plan, de n empilements verticaux de p modules plats de réactifs spécifiques. Le marquage individuel servira plus tard à rassembler des modules avec le même marquage pour consituer un micro-array nou macro-array multibloc de l'invention dédié à un échantillon correspondant à ce marquage, ledit micro-array ou macro-array multibloc recevant np réactifs spécifiques dans chacun de ses micro-emplacements ou micro-puits.Figure 48 shows the face to face between a first line, in the foreground, of n horizontal stacks of p flat modules individually identified specifically, with a second line, in the background, of n vertical stacks of p flat modules of reagents specific. The individual marking will later be used to assemble modules with the same marking to constitute a micro-array or multi-block macro-array of the invention dedicated to a sample corresponding to this marking, said micro-array or multi-block macro-array receiving np reagents specific in each of its micro-locations or micro-wells.
La Figure 49 montre la connexion orthogonale entre une première ligne, au premier plan, de n empilements horizontaux de p modules plats identifiés individuellement de manière spécifique, avec une deuxième ligne, en arrière plan, de n empilements verticaux de p modules plats de réactifs spécifiques, donnant lieu à n micro-arrays de np micro-puits, soit en tout n2 p micropuits, chacun des nfγ micro-puits ayant reçu l'un des np réactifs spécifiquesFigure 49 shows the orthogonal connection between a first line, in the foreground, of n horizontal stacks of p flat modules specifically identified individually, with a second line, in the background, of n vertical stacks of p flat modules of specific reagents , giving rise to n micro-arrays of np micro-wells, or in all n 2 p microwells, each of the nfγ micro-wells having received one of the np specific reagents
La Figure 50 montre la redistribution des modules plats des n empliments précédents de modules plats marqués différemment dans n nouveaux empilements regroupant les modules plats avec le même marquage individuel, chacun desdits empilement avec le même marquage individuel étant dédié à un seul échantillon.Figure 50 shows the redistribution of the flat modules from the previous n works of flat modules marked differently in n new stacks grouping the flat modules with the same individual marking, each of said stacks with the same individual marking being dedicated to a single sample.
La Figure 51 et la Figure 52 montrent la connexion de n modules plats chacun à un seul échantillon avec n nouveaux empilements regroupant les modules plats avec le même marquage individuel, chcun des n échantillons pouvant rencontrer np réactifs spécifiques, les n empilements donnant lieu à n micro-arrays représentant au total micro-puits.Figure 51 and Figure 52 show the connection of n flat modules each to a single sample with n new stacks grouping the flat modules with the same individual marking, each of the n samples may meet np specific reagents, the n stacks giving rise to n micro-arrays representing a total of micro-wells.
La Figure 54A, la Figure 54B, la Figure 54C, la Figure 54D montrent une pièce monobloc de fixation et de synthèse en phase solide de molécules (339) plane, monobloc, connectable à un micro-array ou un macro-array multibloc de l'invention, pourvue d'un array de micro-puits dont le quadrillage est calqué sur celui dudit micro-array ou macro-array multibloc de l'invention. Ladite pièce monobloc de fixation et de synthèse en phase solide de molécules (339) offre dans chacun de ses micro-puits une surface de fixation à une molécule à synthétiser dans un process de synthèse en phase solide, ou une surface de réception de microparticules fixant la première molécule d'une chaîne moléculaire à synthétiser. L'exemple sur les Figures 54 A, 54B, 54C, et 54D montre qu'on peut synthétiser in situ dans les micro-puits de ladite pièce (339) des octamers à partir de tetramers préfixés et de tetramers amenés par les microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs de l'invention;-. Figure 54A, Figure 54B, Figure 54C, Figure 54D show a solid piece of fixing and solid phase synthesis of molecules (339) plane, monoblock, connectable to a micro-array or a multi-block macro-array of l invention, provided with an array of micro-wells whose grid is modeled on that of said micro-array or multi-block macro-array of the invention. Said one-piece attachment and solid phase synthesis of molecules (339) offers in each of its micro-wells a surface for attachment to a molecule to be synthesized in a solid phase synthesis process, or a surface for receiving microparticles fixing the first molecule of a molecular chain to be synthesized. The example in Figures 54 A, 54B, 54C, and 54D shows that one can synthesize in situ in the micro-wells of said part (339) octamers from prefixed tetramers and tetramers brought by the microchannels (41 ) of said multi-block micro-arrays or macro-arrays of the invention; - .
Les Figures 55A, 55B, 55C, 55D montrent qu'on peut synthésiser in situ dans les micro-puits (42) d'un micro-array ou macro-array multibloc (319) des octamers à partir de tetramers préfixés dans lesdits micro-puits (42) et de tetramers amenés par les microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention. Ladite synthèse peut éventuellement avoir lieu pendant les phases de fabrication desdits modules élémentaires plats (1) pour fabriquer des dispositifs prêts à l'emploi, ou pendant le process de l'analyse ou de la synthèse.Figures 55A, 55B, 55C, 55D show that octamers can be synthesized in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) from tetramers prefixed in said micro- wells (42) and tetramers brought by the microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention. Said synthesis can possibly take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process.
Selon l'invention, les micro-canaux (41) desdits modules élémentaires plats (1) peuvent être pourvus de portions élargies et destinées à fixer des molécules actives dans le process d'analyse ou de synthèse auquel sont dédiés lesdits modules élémentaires plats (1), ladite . fixation de molécules actives pouvant avoir lieu pendant les phases de fabrication desdits modules élémentaires plats (1) pour fabriquer des dispositifs prêts à l'emploi, ou pendant le process de l'analyse ou de la synthèse. L'exemple précis des Figures 56 A, 56 B, 56C, 56D montre qu'on peut synthésiser in situ dans des portions élargies des micro-canaux (41) d'un micro-array ou macro-array multibloc (319) des octamers à partir de tetramers préfixés et de tetramers amenés par lesdits microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention.According to the invention, the micro-channels (41) of said flat elementary modules (1) can be provided with enlarged portions and intended to fix active molecules in the analysis or synthesis process to which said flat elementary modules (1) are dedicated. ), said. fixing of active molecules which can take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process. The precise example of Figures 56 A, 56 B, 56C, 56D shows that one can synthesize in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) of the octamers. from prefixed tetramers and tetramers brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention.
Selon l'invention, lesdites portions élargies desdits microcanaux (41) desdits modules élémentaires plats (1) peuvent aussi être destinées à recevoir des microparticules magnétiques ou passives de fixation de molécules pendant les phases de fabrication pour fabriquer des dispositifs prêts à l'emploi, ou pendant le process de l'analyse ou de la synthèse . Ladite fixation de molécules peut avoir lieu pendant les phases de fabrication desdits modules élémentaires plats (1) pour fabriquer des dispositifs prêts à l'emploi, ou pendant le process de l'analyse ou de la synthèseAccording to the invention, said enlarged portions of said microchannels (41) of said flat elementary modules (1) can also be intended to receive magnetic or passive microparticles for fixing molecules during the manufacturing phases to manufacture ready-to-use devices, or during the analysis or synthesis process. Said fixing of molecules can take place during the manufacturing phases of said flat elementary modules (1) for manufacturing ready-to-use devices, or during the analysis or synthesis process.
Selon l'invention, on peut connecter à un micro-array ou un macro-array multibloc (3119) une pièce recevant des dépôts de microparticules, plane, monobloc, pourvue d'un array de micro-puits dont le quadrillage est calqué sur celui dudit micro-array ou macro-array multibloc (3119), offrant dans chacun de ses micro-puits une surface de dépôt à desdites microparticules pourvues de bras moléculaires de liaison, ce qui permet de fabriquer un micro-array ou un macro-array de microparticules utilisable en analyse ou en synthèse chimique ou biochimique. Une modalité est représentée par une application du brevet "WO9740385A1 - Light-controlled electrokinetic assembly of microparticules near surfaces", où, sur un subtrat en silicium, les micropuits sont adressés par des électrodes en oxide de silicium et baignés par une solution électrolyte, et où lesdites microparticules peuvent être assemblées sous le contrôle de la lumièreAccording to the invention, it is possible to connect to a micro-array or a multi-block macro-array (3119) a part receiving deposits of microparticles, planar, monobloc, provided with an array of micro-wells whose grid is modeled on that of said micro-array or multi-block macro-array (3119), providing in each of its micro-wells a surface for depositing said microparticles provided with molecular link arms, which makes it possible to manufacture a micro-array or a macro-array of microparticles usable in chemical or biochemical analysis or synthesis. One method is represented by an application of the patent "WO9740385A1 - Light-controlled electrokinetic assembly of microparticles near surfaces", where, on a silicon substrate, the microwells are addressed by electrodes in silicon oxide and bathed in an electrolyte solution, and where said microparticles can be assembled under the control of light
Selon l'invention, lesdites portions élargies desdits microcanaux (41) desdits modules élémentaires plats (1) peuvent aussi être destinées à être photoexposées, de manière à pouvoir procéder à des synthèses avec utilisation de groupements photolabiles au lieu même desdites portions desdits microcanaux (41), lesdites synthèses ayant pour but de produire des composés à la demande, ou pour but de fabriquer un dispositif d'analyse prêt à l'emploi pourvu de molécules synthétisées in situ. Les Figures 57A, 57B, 57C montrent qu'on peut synthésiser in situ des biopuces dans chaque micro-puits d'une pièce monobloc de fixation et de synthèse en phase solide de molécules (338), en utilisant des groupements photolabiles et des micro-arrays ou macro-arrays de micromasques (187) de photolithographie. Ladite pièce monobloc de fixation et de synthèse en phase solide de molécules (338) est plane, monobloc, connectable à un micro-array ou un macro-array multibloc (3119), pourvue d'un array de micro-puits ou de spots dont le quadrillage est calqué sur celui dudit micro-array ou macro-array multibloc (3119).According to the invention, said enlarged portions of said microchannels (41) of said flat elementary modules (1) can also be intended to be photoexposed, so as to be able to carry out syntheses with the use of photolabile groups instead of said portions of said microchannels (41 ), said syntheses having the aim of producing compounds on demand, or the aim of manufacturing a ready-to-use analysis device provided with molecules synthesized in situ. Figures 57A, 57B, 57C show that biochips can be synthesized in situ in each micro-well of a solid piece of fixation and solid phase synthesis of molecules (338), using photolabile groups and micro- arrays or macro-arrays of micromasks (187) of photolithography. Said monoblock fixing and solid phase synthesis part of molecules (338) is planar, monobloc, connectable to a micro-array or a multi-block macro-array (3119), provided with an array of micro-wells or spots including the grid is modeled on that of said micro-array or multi-block macro-array (3119).
L'utilisation conjointe des groupements photolabiles et des masques de photolithographie, permet de fabriquer un micro-array ou un macro-array de biopuces ayant pour support ladite pièce (338). L'utilisation conjointe de groupements photolabiles et de micro-mirroirs digitaux permet aussi de fabriquer un micro-array ou un macro-array de biopuces ayant pour support ladite pièce (338).The joint use of photolabile groupings and photolithography masks makes it possible to manufacture a micro-array or a macro-array of biochips having said part (338) as support. The joint use of photolabile groupings and digital micro-mirrors also makes it possible to manufacture a micro-array or a macro-array of biochips having said part (338) as support.
Les Figures 58A, 58B, 58C montrent qu' on peut synthésiser in situ des biopuces dans chaque micro-puits (42) d'un micro-array ou macro-array multibloc (319) de l'invention. L'utilisation conjointe des groupements photolabiles et des masques de photolithographie, permet de fabriquer un micro-array ou un macro-array de biopuces ayant pour support ledit micro-array ou macro-array multibloc (319). L'utilisation conjointe de groupements photolabiles et de micro-mirroirs digitaux permet aussi de fabriquer un micro-array ou un macro-array de biopuces ayant pour support ledit micro-array ou macro-array multibloc (319) de l'invention.Figures 58A, 58B, 58C show that biochips can be synthesized in situ in each micro-well (42) of a micro-array or multi-block macro-array (319) of the invention. The joint use of photolabile groupings and photolithography masks makes it possible to manufacture a micro-array or a macro-array of biochips having for support said micro-array or multi-block macro-array (319). The joint use of photolabile groupings and digital micro-mirrors also makes it possible to manufacture a micro-array or a macro-array of biochips having for support said micro-array or multi-block macro-array (319) of the invention.
La Figure 59A montre comment un micro-array ou micro-array multibloc de l'invention formé par emplilement de modules élémentaires plats (1) peut être connecté à deux pièces (340) monobloc et planes , chacune formant un micro-array monobloc de bouchons. La Figure 59B montre comment un premier micro-array ou micro-array multibloc de l'invention formé par empilement de modules élémentaires plats (1) peut être connecté à un autre micro-array de l'invention formé par empilement de modules élémentaires plats (1101) dont les micro-canaux sont pourvus de micro-vannes (396). Dans cette configuration, un micro-puits dudit premier micro-array ou micro-array multibloc de l'invention isole un volume compris entre l'orifice du micro-canal à la surface dudit premier micro-array ou macro-array multibloc de l'invention et la dite microvanne sur le micro-array ou macro-array multibloc connecté.Figure 59A shows how a micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to two monobloc and planar parts (340), each forming a monobloc micro-array of plugs . Figure 59B shows how a first micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array of the invention formed by stacking flat elementary modules (1) 1101) whose micro-channels are provided with micro-valves (396). In this configuration, a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and the so-called microvalve on the connected micro-array or multi-block macro-array.
La Figure 59C montre comment un premier micro-array ou micro-array multibloc de l'invention formé par empilement de modules élémentaires plats (1) peut être connecté à un autre micro-array ou macro-array multibloc de l'invention formé par empilement de modules élémentaires plats (1102) pourvus de dispositifs (398) qui créent une force électro-osmotique en établissant un potentiel électrique entre deux endroits des micro-canaux desdits de modules élémentaires plats (1102). Dans cette configuration, un micro-puits dudit premier micro-array ou micro-array multibloc de l'invention isole un volume compris entre l'orifice du micro-canal à la surface dudit premier micro-array ou macro-array multibloc de l'invention et ledit dispositif (398).Figure 59C shows how a first multi-block micro-array or micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another multi-block micro-array or macro-array of the invention formed by stacking of flat elementary modules (1102) provided with devices (398) which create an electro-osmotic force by establishing an electrical potential between two places of the micro-channels of said flat elementary modules (1102). In this configuration, a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and said device (398).
La Figure 59D montre comment un premier micro-array ou micro-array multibloc de l'invention formé par empilement de modules élémentaires plats (1) peut être connecté à un autre micro-array de l'invention formé par empilement de modules élémentaires plats (1103) dont les micro-canaux sont pourvus de deux micro-vannes (397). Dans cette configuration, un micro-puits dudit premier micro-array ou micro-array multibloc de l'invention isole un volume compris entre l'orifice du micro-canal à la surface dudit premier micro-array ou macro-array multibloc de l'invention et la première micro vanne (397) sur le micro-array ou macro-array multibloc connecté. Un autre volume est isolé entre les deux microvannes (397) dudit deuxième micro-array ou macro-array multibloc de l'invention . La Figure 59 D montre aussi qu'en amont d'un volume isolé entre deux volumes isolés par deux microvannes, il peut être envisagé l'utilisation d'un bloc micropompe (399) commun à plusieurs canaux.Figure 59D shows how a first micro-array or multi-block micro-array of the invention formed by stacking flat elementary modules (1) can be connected to another micro-array of the invention formed by stacking flat elementary modules (1) 1103) whose micro-channels are provided with two micro-valves (397). In this configuration, a micro-well of said first micro-array or multi-block micro-array of the invention isolates a volume comprised between the orifice of the micro-channel on the surface of said first micro-array or multi-block macro-array of the invention and the first micro valve (397) on the connected micro-array or multi-block macro-array. Another volume is isolated between the two microvalves (397) of said second micro-array or multi-block macro-array of the invention. Figure 59D also shows that upstream of an isolated volume between two volumes isolated by two microvalves, it can be considered the use of a micropump block (399) common to several channels.
La Figure 34, la Figure 35, la Figure 36 A, la Figure 36B, la Figure 36C, la Figure 37 A, la Figure 37B, la Figure 37C, la Figure 37 D, la Figure 38, la Figure 39 montrent une méthode de fabrication de tous les modules élémentaires plats de l'invention dans laquelle tout type desdits modules élémentaires plats de l'invention, micro-composants non compris, est fabriqué avec les techniques de microfabrication selon un assemblage par superposition et fusion de sous-parties planes complémentaires.Figure 34, Figure 35, Figure 36 A, Figure 36B, Figure 36C, Figure 37 A, Figure 37B, Figure 37C, Figure 37 D, Figure 38, Figure 39 show a method of manufacture of all the flat elementary modules of the invention in which any type of said flat elementary modules of the invention, micro-components not included, is manufactured with microfabrication techniques according to an assembly by superposition and fusion of complementary planar sub-parts .
La Figure 34 et la Figure 35 montrent en perspective comment un module élémentaire plat (6110) devant être pourvu de micro-canaux (41), de micromélangeurs (19), d'une circuiterie micro-fluidique (55), de logements pour micro-filtres (16), de logements pour microcolonnes (17), d'orifices (7) et (11) pour échantillons et réactifs, peut-être micro-fabriqué par l'assemblage de deux hémi-modules élémentaires plats (611012), chacun de ses deux hémimodules élémentaires plats (611012) ayant été micro-usiné ou micro-moulé ou micro- estampé pour que soient formés, entre autres, une hémi-circuiterie micro-fluidique (5512), des hémi-microcanaux (4112) débouchant sur la tranche desdits hémi-modules élémentaires plats (611012) par les hémi-micro-puits (4212), des hémi-micromélangeurs (1912), des hémi logements (1612) pour micro-filtres (16), des hemi-logements (1712) pour micro- colonnes(17), des hémi-orifices (712) et (1112) pour échantillons et réactifs .Figure 34 and Figure 35 show in perspective how a flat elementary module (6110) to be provided with micro-channels (41), micro-mixers (19), micro-fluidic circuitry (55), housings for micro -filters (16), housings for microcolumns (17), orifices (7) and (11) for samples and reagents, perhaps micro-manufactured by the assembly of two flat elementary semi-modules (611012), each of its two flat elementary hemimodules (611012) having been micro-machined or micro-molded or micro-stamped so that a micro semi-circuitry is formed, inter alia -fluidic (5512), semi-microchannels (4112) opening onto the edge of said flat elementary semi-modules (611012) by semi-micro-wells (4212), semi-micro-mixers (1912), semi-housings (1612 ) for micro-filters (16), hemi-housings (1712) for micro-columns (17), hemi-orifices (712) and (1112) for samples and reagents.
La Figure 36 A et la Figure 36B montrent comment un module élémentaire plat peut-être fabriqué par l'assemblage d'au moins une sous-partie plane inférieure (2) et une sous partie plane supérieure (3), ladite sous partie plane supérieure (3) faisant office de couvercle.Figure 36 A and Figure 36B show how a flat elementary module can be produced by assembling at least one lower flat sub-part (2) and one upper flat sub-part (3), said upper flat sub-part (3) acting as a cover.
La Figure 36 C montre le micro-array multibloc (319) ainsi fabriqué.Figure 36 C shows the multiblock micro-array (319) thus manufactured.
La Figure 37A montre une sous-partie d'un module élémentaire plat, la Figure 37B montre l'assemblage d'une sous-partie d'un module élémentaire plat avec sa partie complémentaire, la Figure 37C montre un micro-array ou macro-array multibloc constitué de l'empilement desdits modules élémentaires plats fabriqués par assemblage desdites parties complémentaires, la Figure 37 D représente par transparence le micro-array ou macro-array ainsi constitué.Figure 37A shows a sub-part of a flat elementary module, Figure 37B shows the assembly of a sub-part of a flat elementary module with its complementary part, Figure 37C shows a micro-array or macro- multiblock array consisting of the stack of said flat elementary modules manufactured by assembling said complementary parts, Figure 37 D shows by transparency the micro-array or macro-array thus formed.
La Figure 38 montre que lorsque des modules élémentaires plats (18110) ne présentent pas de composants, le support plat qui sert à fabriquer l'une de ses sous-parties complémentaires (1811012) muni de ses hémi-micro-mélangeurs (1801912) peut être très mince, ce qui permet de constituer directement un micro-array ou un macro-array multibloc (18319) de densité élevée. La Figure 39 montre que lorsque des modules élémentaires plats (19110) présentent des composants activés par une circuiterie électrique (391) avec des plots de connexion (390), le support plat d'une de ses sous-parties complémentaires (1911012) doit réserver des logements tels que 393, et 394 pour composants tels que micro- vannes, capteurs, micro-réchauffeurs, ce qui limite la densité du micro-array ou un macro-array multibloc (19319) pouvant être constitué.Figure 38 shows that when elementary flat modules (18110) do not have any components, the flat support which is used to manufacture one of its complementary sub-parts (1811012) provided with its semi-micro-mixers (1801912) can be very thin, which makes it possible to directly constitute a micro-array or a multi-block macro-array (18319) of high density. Figure 39 shows that when elementary flat modules (19110) have components activated by an electrical circuit (391) with connection pads (390), the flat support of one of its complementary sub-parts (1911012) must reserve housings such as 393, and 394 for components such as microvalves, sensors, micro-heaters, which limits the density of the micro-array or a multi-block macro-array (19319) that can be formed.
Selon l'invention, à l'usine de fabrication, les étapes de fabrication de tout type desdits modules élémentaires plats de l'invention peuvent être mises à profit pour fabriquer des systèmes prêts à l'emploi de synthèse ou d'analyse chimique, biochimique ou biologique, par exemple par constitution de micro-réservoirs de réactifs.According to the invention, at the manufacturing plant, the manufacturing steps of any type of said flat elementary modules of the invention can be used to manufacture ready-to-use systems for synthesis or chemical, biochemical analysis. or biological, for example by constituting micro-reservoirs of reagents.
Selon l'invention, les surfaces desdits micro-arrays ou macro-arrays multiblocs sont micro- usinées ou soumises à un travail de finition avec les mêmes techniques de microfabrication que s'il s'agissait d'une surface plane d'un seul tenant, c'est à dire essentiellement des techniques de découpage, de gravure sèche ou gravure humide par photolithographie, d'ablation laser, d'assemblage ou collage par fusion ou d'assemblage anodique, de forage, d'estampage, de soudure, d' électrodéposition, d'electroless plating ou de dépôt de vapeur chimique.According to the invention, the surfaces of said micro-arrays or multi-block macro-arrays are micro-machined or subjected to a finishing work with the same microfabrication techniques as if it were a flat surface in one piece. , i.e. essentially techniques of cutting, dry etching or wet etching by photolithography, laser ablation, assembly or gluing by fusion or anodic assembly, drilling, stamping, welding, electroplating, electroless plating or chemical vapor deposition.
Selon un version de l'invention, on peut utiliser pour l'analyse chimique, biochimique, et biologique, des empilements décalés de modules élémentaires plats dont un côté au moins a une forme en arc de cercle, lesdits modules élémentaires plats étant pourvus de micro-canaux débouchant dans l'épaissseur et sur la tranche dudit côté en arc de cercle. L'empilement ainsi constitué est adapté à une connexion avec des éléments homologues pour une détection par détecteur rotatif. Un exemple de ce type de détecteur rotatif est décrit pour la détection après micro-électrophorèse dans le brevet " Rotating scanning apparatus WO 96/13716".According to a version of the invention, it is possible to use for chemical, biochemical, and biological analysis, staggered stacks of flat elementary modules of which at least one side has a shape in an arc of a circle, said flat elementary modules being provided with micro -channels opening into the thickness and on the edge of said side in an arc. The stack thus formed is suitable for connection with homologous elements for detection by a rotary detector. An example of this type of rotary detector is described for detection after micro-electrophoresis in the patent "Rotating scanning apparatus WO 96/13716".
La configuration desdits modules élémentaires plats desdits micro-arrays ou macro-arrays multiblocs de l'invention peut être adaptée au type de détection (micro-électrophorèse, hybridation, spectrométrie de masse, chromatographie, micro-électrochromatographie, caméras CCD, fibres optiques, microscopie confocale, détection avec fluorescence, chimiluminescence, bioluminescence, colorimétrie, par surface plasmon résonance, par mesure de l'onde évanescente, par électiochimie, par radioactivité, par spectroscopie Ra an, etc) en adéquation avec le but de l'analyse.The configuration of said elementary flat modules of said micro-arrays or multi-block macro-arrays of the invention can be adapted to the type of detection (micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, optical fibers, microscopy confocal, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface, by measurement of evanescent wave, by electiochemistry, by radioactivity, by Ra an spectroscopy, etc.) in adequacy with the aim of the analysis.
APPLICATION AUX MICRO-ARRAYS OU MACRO-ARRAYS DE L'INVENTION DEDIES A L'ANALYSE DE L'ADN:APPLICATION TO THE MICRO-ARRAYS OR MACRO-ARRAYS OF THE INVENTION DEDICATED TO DNA ANALYSIS:
Pour des micro-arrays ou macro-arrays dédiés à l'analyse d'ADN, ladite préparation intégrée comprend la lyse cellulaire, la filtration des débris, l'extraction et la purification sur matrices de silice, puis l'amplification avec une des nombreuses méthodes bien connues de l'Homme de l'Art, telles que, parmi d'autres, la PCR, la RT-PCR, la LCR, laNASBA, la SDA, la TMA, la RCA.For micro-arrays or macro-arrays dedicated to DNA analysis, said integrated preparation includes cell lysis, filtration of debris, extraction and purification on silica matrices, then amplification with one of the many methods well known to those skilled in the art, such as, among others, PCR, RT-PCR, LCR, laNASBA, SDA, TMA, RCA.
De nombreux protocoles existent pour la lyse cellulaire et rextraction-purification de l'ADN, selon qu'on à affaire à des prélèvements de sang total, de plasma, de sérum, de tissus, de cheveux, de suspension cellulaire, de moelle osseuse, de liquide cérébro-spinal, de prélèvement buccaux, de liquides corporels, de déchets corporels, de biposies, de biopsies protèges par de la paraffine, de bactéries Gram-positive, de bactéries Gram-Négative, de mossissures, de plantes., de gels d'agarose ou de polyacrylamide, ou bien que l'analyse vise de virus à ARN ou des virus à ADN. Les procédés d'extraction au phénol-chloroforme et par filtration tendent à laisser la place à des procédés comme la chromatographie par échange d'ions et l'extraction sur matrices de gel de silice. La chromatographie par échange d'ions sur l'ADN vise à utiliser des surfaces d'adsorption de microparticules ou de supports hydrophiles à haute densité de charges positives et à lier les charges négatives des phosphates des ADN dans des conditions de salinité faibles, qui éliminent les possibilités d'adsorption des protéines et des carbohydrates. Les conditions d'élution de l'ADN adsorbé sont au contraire des conditions de forte salinité. La chromatographie par échange d'anions se termine soit par une précipitation avec un alcool, soit par un dessalement sur micro-particules ou matrices de gels de silice.Many protocols exist for cell lysis and DNA extraction-purification, depending on whether we are dealing with whole blood, plasma, serum, tissue, hair, cell suspension, bone marrow, cerebrospinal fluid, oral swabs, body fluids, body wastes, biposia, paraffin-protected biopsies, Gram-positive bacteria, Gram-Negative bacteria, mosses, plants., gels agarose or polyacrylamide, or that the analysis is aimed at RNA viruses or DNA viruses. Phenol-chloroform and filtration extraction processes tend to give way to processes such as ion exchange chromatography and extraction on silica gel matrices. Ion exchange chromatography on DNA aims to use adsorption surfaces of microparticles or hydrophilic supports with high density of positive charges and to bind the negative charges of phosphates from DNA under conditions of low salinity, which eliminate the possibilities of protein and carbohydrate adsorption. The conditions for eluting the adsorbed DNA are, on the contrary, conditions for high salinity. Anion exchange chromatography ends either with precipitation with an alcohol or with desalination on micro-particles or matrices of silica gels.
L'adsorption sur matrices ou micro-particules de gel de silice vise à adsorber l'ADN lorsque de fortes teneurs en sels chaotropiques lient l'eau libre des solutions aqueuses, tandis que les protéines et les carbohydrates ne s'adsorbent pas. L'élution de l'ADN adsorbé sur matrices ou micro-particules de gel de silice se fait dans des conditions de faible salinité, par exemple à l'eau, et l'ADN purifié est prêt à l'emploi sans nécessité de précipitation.Adsorption on matrices or micro-particles of silica gel aims to adsorb DNA when high levels of chaotropic salts bind free water to aqueous solutions, while proteins and carbohydrates do not adsorb. The elution of the DNA adsorbed on matrices or microparticles of silica gel takes place under conditions of low salinity, for example with water, and the purified DNA is ready for use without the need for precipitation.
Lorsqu'on utilise des matrices de gel de silice simples ou des micro-particules sans de gel de silices simples, ladite méthode d'extraction-purification sur matrice de gels de silices nécessite des centrifugations. Lorsque par contre on utilise des micropartiçules magnétiques de gel de silice et de fer, on peut se passer-de centrifugations.When using simple silica gel matrices or microparticles without simple silica gel, said extraction-purification method on a matrix of silica gels requires centrifugations. When, on the other hand, magnetic microparticles of silica gel and iron are used, centrifugations can be dispensed with.
Les méthodes d'extraction-purification de l'ADN avec chromatographie par échange d'anions ne nécessitent pas de centrifugation, car la gravité suffit. Elles offrent un grand degré de pureté. Elles sont particulièrement utiles dans un protocole d'extraction d'ADN sur Bactéries Gram- Négatives, pour laquelle on peut éviter la précipitation à l'alcool en utilisant en deuxième étape un passage sur micro-particules ou matrice de gel de silice avec centrifugation, ou bien avec micro-particules magnétiques de gel de silice sans centrifugation.DNA extraction-purification methods with anion exchange chromatography do not require centrifugation, because gravity is sufficient. They offer a high degree of purity. They are particularly useful in a DNA extraction protocol on Gram-Negative Bacteria, for which the precipitation with alcohol can be avoided by using in the second stage a passage over micro-particles or silica gel matrix with centrifugation, or with magnetic silica gel micro-particles without centrifugation.
Les méthodes d'extraction d'ADN sur matrices de gel de silice conviennent pour la plupart des prélèvements, excepté pour les Bactéries Gram-Négatives et les plantes où des protocoles utilisant la chromatographie par échanghe d'anions sont appropriées, dussent-elles constituer une première étape suivie dune deuxième étape sur micro-particules simples ou magnétiques de gel de silice ou matrices de gel de siliceDNA extraction methods on silica gel matrices are suitable for most samples, except for Gram-negative bacteria and plants where protocols using anion exchange chromatography are appropriate, should they constitute a first step followed by a second step on simple or magnetic silica gel microparticles or silica gel matrices
La Figure 40 A représente un mode de réalisation de l'invention et réside dans des microarrays ou macro-arrays multiblocs (13319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée avec extraction de l'ADN par chromatographie par échanges d'anions puis dessalement sur microparticules magnétiques de gel de silice. La Figure 40A représente un mode de réalisation de l'invention et réside dans des microarrays ou macro-arrays multiblocs (13319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée comprenant la lyse cellulaire, la filtration des débris, deux extractions- purifications successives de l'ADN sur deux micro-colonnes de nature différente, l'amplification en amont dudit micro-array ou macro-array multibloc (13319) par une des méthodes connues de l'Homme de l'Art, puis la détection sur ou en aval dudit micro-array ou macro-array multibloc (13319) par une des méthodes connues de l'Homme de l'Art..Figure 40 A shows an embodiment of the invention and resides in multi-block microarrays or macro-arrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by exchange chromatography anions and then desalination on magnetic microparticles of silica gel. FIG. 40A represents an embodiment of the invention and resides in multi-block microarrays or macro-arrays (13319) dedicated to the analysis of DNA fragments with an integrated preparation comprising cell lysis, filtration of debris, two successive extractions- purifications of DNA on two micro-columns of different nature, the upstream amplification of said micro-array or multi-block macro-array (13319) by one of the methods known to those skilled in the art, then the detection on or downstream of said multi-block micro-array or macro-array (13319) by one of the methods known to those skilled in the art.
Lesdits micro-arrays ou macro-arrays multiblocs (13319) de micro-puits (42) sont constitués par empilement de modules élémentaires plats (13110), pourvus de deux sous-parties (13155) et (13145).Said multi-block micro-arrays or macro-arrays (13319) of micro-wells (42) are constituted by stacking of flat elementary modules (13110), provided with two sub-parts (13155) and (13145).
Ladite sous-partie (13145) comporte les micro-canaux (41) pourvus de micromélangeurs (13019). Ladite sous-partie (13155) comporte une circuiterie micro-fluidique (13055), composée:Said sub-part (13145) comprises the micro-channels (41) provided with micro-mixers (13019). Said sub-part (13155) comprises a micro-fluidic circuit (13055), composed:
*d'une chambre de lyse cellulaire et de filtration (13015) pourvue de microfiltres (13016), avec orifice d'introduction de l'échantillon (13007), orifice d'introduction des réactifs de lyse (13008), orifice pour obturateur (13009) de passage à la chambre d'extraction-purification-lavage avec chromatographie par échange d'anion(13061)* a cell lysis and filtration chamber (13015) provided with microfilters (13016), with orifice for introducing the sample (13007), orifice for introducing the lysis reagents (13008), orifice for obturator ( 13009) passage to the extraction-purification-washing chamber with anion exchange chromatography (13061)
*. d'une chambre d'extraction-purification-lavage avec chromatographie par échange d'anions (13061) pourvue d'une micro-colonne de chromatographie par échange d'anions (13117), avec orifice (13029) pour obturateur permettant d'isoler ladite microcolonne (13117), orifice d'introduction du réactif d'élution (13113), orifice (13038) d'introduction des réactifs de lavage de ladite micro-colonne (13117), orifice pour évacuation des réactifs de lavage (13011), obturateur (13028) de passage à la chambre (13063) d'extraction-purification-lavage sur micro-particules magnétiques avec fer et gel de silice, * d'une chambre (13063) pourvue d'un électroaimant (13723), d'un orifice d'introduction des micro-particules de gel de silice (13722), d'un orifice (13010) d'introduction des réactifs de lavage, d'un orifice d'introduction (13726) du réactif activateur d'adsorption de l'ADN auxdites micro-particules magnétiques, d'un orifice d'introduction du réactif d'élution (13013), d'un obturateur (13024) de passage à la chambre de préparation de l'ADN purifié. La Figure 40B montre les microparticules magnétisées par l'électro-aimant (13723) activé dans ladite chambre (13063), et la*. an extraction-purification-washing chamber with anion exchange chromatography (13061) provided with an anion exchange chromatography micro-column (13117), with orifice (13029) for shutter making it possible to isolate said microcolumn (13117), orifice for introducing the elution reagent (13113), orifice (13038) for introducing the washing reagents from said micro-column (13117), orifice for discharging the washing reagents (13011), shutter (13028) for passage to the extraction-purification-washing chamber (13063) on magnetic micro-particles with iron and silica gel, * of a chamber (13063) provided with an electromagnet (13723), an orifice for introducing the silica gel microparticles (13722), an orifice (13010) for introducing the washing reagents, an orifice (for introducing (13726) the reagent activating adsorption of l To said magnetic micro-particles, an orifice for introducing the elution reagent (13013), an obturate ur (13024) passing through the preparation chamber of the purified DNA. Figure 40B shows the microparticles magnetized by the electromagnet (13723) activated in said chamber (13063), and the
Figure 40C montre lesdites microparticules magnétiques libres dans ladite chambre (13063). *d' une chambre de préparation de l'ADN purifié (13064) avec orifice d'introduction (13012) du tampon adéquat pour la méthode d'amplification de l'ADN choisie et donnant accès auxdits micro-canaux (41) de ladite sous partie (13145), ledit tampon étant privé des enzymes, des primers et des dNTP nécessaires à l'amplificationFigure 40C shows said free magnetic microparticles in said chamber (13063). * a chamber for the preparation of purified DNA (13064) with an opening for introduction (13012) of the buffer suitable for the DNA amplification method chosen and giving access to said micro-channels (41) of said sub part (13145), said buffer being deprived of the enzymes, primers and dNTPs necessary for the amplification
Dans ce mode de réalisation de l'invention, l'échantillon d'ADN est introduit dans la chambre de lyse cellulaire et de filtration par ledit orifice (13007). Les réactifs de lyse sont introduits par ledit orifice (13008). Les débris cellulaires de lyse sont filtrés sur les microfiltres (13016), et le lysat est introduit dans la chambre (13061) d'extraction-purification-lavage avec micro-colonne de chromatographie par échange d'anions (13117). Après l'adsorption de l'ADN sur ladite micro-colonne (13117), on procède à l'introduction des réactifs de lavage de ladite micro-colonne (13117) qui sont évacués par l' orifice (13011). Ensuite on isole grâce à un obturateur introduit par l'orifice (13029) ladite microcolonne (13117), puis on procède à l'élution de l'ADN en introduisant le réactif d'élution par l'orifice (13113). Le retrait des obturateurs (13028) et (13029)permet à l'éluat de passer dans la chambre (13063) d'extraction-purification-lavage sur microparticules magnétiques. Après introduction par l'orifice (13726) d'un tampon adéquat pour la méthode puis du réactif activateur d'adsorption de l'ADN auxdites micro-particules magnétiques, les micro-particules magnétiques sont introduites par l'orifice (13722), puis on procède à un lavage avec des réactifs introduits par l'orifice (13010) et on magnétise avec l'électro-aimant (13723). Après élimination du réactif de lavage par l'orifice (13011), le réactif d'élution est introduit par l'orifice (13013). Après une nouvelle magnétisation, le retrait de l'obturateur (13024) permet le passage à la chambre de préparation de l'ADN purifié (13064), si bien que dans le process l'ADN passe successivement sur la micro-colonne de chromatographie par échange d'anions (13117) à la chambre (13063) de purification sur billes magnétiques avec gel de silice. Dans ladite chambre de préparation de l'ADN purifié (13064), on mélange ledit ADN purifié avec le tampon adéquat pour la méthode d'amplification de l'ADN choisie, ledit tampon étant introduit par l'orifice (13012) et étant privé des enzymes, des primers et des dNTP nécessaires à l'amplification. Ensuite ledit mélange de l'ADN purifié et du tampon d'amplification est introduit dans lesdits micro-canaux (41) avec micro-mélangeurs (13019) de ladite sous partie (13145). Une connexion avec d'autres empilements de modules élémentaires plats permet le mélange de l'ADN avec les réactifs spécifiques ou onéreux de l'amplification souhaitée dans chaque micro-canal (41) et finalement dans chaque micropuits (42), lesdits réactifs spécifiques ou onéreux étant tels qu'enzymes nécessaires à la méthode d'amplification choisie, déoxynucléotides et primers d'amplification. Lesdits réactifs spécifiques ou onéreux peuvent être aussi des primers modifiés adaptés à la méthode de détection choisie, des sondes supplémentaires nécessaires à la méthode d'amplification ou de détection choisie, des déoxynucléotides modifiés, des didéoxynucléotides, des didéoxynucléotides modifiés, des analogues peptidiques de sondes tels que des PNA, etc. La Figure 40B et la Figure 40C montrent l'action d'un électro-aimant sur lesdites micro- particules magnétiques.In this embodiment of the invention, the DNA sample is introduced into the cell lysis and filtration chamber through said orifice (13007). The lysis reagents are introduced through said orifice (13008). Cell lysis debris is filtered on microfilters (13016), and the lysate is introduced into the extraction-purification-washing chamber (13061) with anion exchange chromatography micro-column (13117). After the adsorption of DNA on said micro-column (13117), the washing reagents from said micro-column (13117) are introduced, which are discharged through the orifice (13011). Then, using a shutter inserted through the orifice (13029), the said microcolumn (13117) is isolated, then the DNA is eluted by introducing the elution reagent through the orifice (13113). The removal of the shutters (13028) and (13029) allows the eluate to pass into the extraction-purification-washing chamber (13063) on magnetic microparticles. After introduction via the opening (13726) of a buffer suitable for the method and then of the reagent activating adsorption of DNA to said magnetic micro-particles, the magnetic micro-particles are introduced through the opening (13722), then washing is carried out with reagents introduced through the orifice (13010) and magnetized with the electromagnet (13723). After removal of the washing reagent through the orifice (13011), the elution reagent is introduced through the orifice (13013). After a new magnetization, the withdrawal of the obturator (13024) allows the passage to the preparation chamber of the purified DNA (13064), so that in the process the DNA passes successively on the micro-column of chromatography by anion exchange (13117) in the purification chamber (13063) on magnetic beads with silica gel. In said chamber for the preparation of purified DNA (13064), said purified DNA is mixed with the buffer suitable for the DNA amplification method chosen, said buffer being introduced through the orifice (13012) and being deprived of enzymes, primers and dNTPs necessary for amplification. Then said mixture of purified DNA and amplification buffer is introduced into said micro-channels (41) with micro-mixers (13019) of said sub-part (13145). A connection with other stacks of elementary flat modules allows the mixing of the DNA with the specific or expensive reagents of the desired amplification in each micro-channel (41) and finally in each microwell (42), said specific reagents or expensive being such as enzymes necessary for the chosen amplification method, deoxynucleotides and primers for amplification. Said specific or expensive reagents can also be modified primers suitable for the chosen detection method, additional probes necessary for the chosen amplification or detection method, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNAs, etc. Figure 40B and Figure 40C show the action of an electromagnet on said magnetic microparticles.
La Figure 41 A montre un autre mode de réalisation de l'invention et réside dans des microarrays ou macro-arrays multiblocs (14319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée avec extraction de l'ADN par chromatgraphie par échange d'anions puis dessalement sur matrices de gel de silice.Figure 41 A shows another embodiment of the invention and resides in multi-block microarrays or macro-arrays (14319) dedicated to the analysis of DNA fragments with an integrated preparation with DNA extraction by chromatography by anion exchange then desalination on silica gel matrices.
La Figure 41 A montre des micro-arrays ou macro-arrays multiblocs (14319) de micro-puitsFigure 41 A shows micro-arrays or multi-block macro-arrays (14319) of micro-wells
(42) sont constitués par empilement de modules élémentaires plats (14110), pourvus de deux sous-parties (14155) et (14145).(42) are constituted by stacking of flat elementary modules (14110), provided with two sub-parts (14155) and (14145).
Ladite sous-partie (14145) comporte les micro-canaux (41) pourvus de micromélangeursSaid sub-part (14145) comprises the micro-channels (41) provided with micro-mixers
(14019). Ladite sous-partie (14155) comporte une circuiterie micro-fluidique (14055), composée:(14019). Said sub-part (14155) comprises a micro-fluidic circuit (14055), composed:
*d'une chambre de lyse cellulaire et de filtration (14015) pourvue de microfiltres (14016), avec orifice d'introduction de l'échantillon (14007), orifice d'introduction des réactifs de lyse (14008), orifice pour obturateur (14009) de passage à la chambre d'extraction-purification-lavage avec chromatographie par échange d'anion(14061)* a cell lysis and filtration chamber (14015) provided with microfilters (14016), with orifice for introducing the sample (14007), orifice for introducing the lysis reagents (14008), orifice for obturator ( 14009) passage to the extraction-purification-washing chamber with anion exchange chromatography (14061)
*. d'une chambre d'extraction-purification-lavage avec chromatographie par échange d'anions (14061) pourvue d'une micro-colonne de chromatographie par échange d'anions (14117), avec orifice (14029) pour obturateur permettant d'isoler ladite microcolonne (14117), orifice d'introduction du réactif d'élution (14113), orifice*. an extraction-purification-washing chamber with anion exchange chromatography (14061) provided with an anion exchange chromatography micro-column (14117), with orifice (14029) for shutter making it possible to isolate said microcolumn (14117), orifice for introducing the elution reagent (14113), orifice
(14038) d'introduction des réactifs de lavage de ladite micro-colonne (14117), orifice pour évacuation des réactifs de lavage (14011), obturateur (14028) de passage de l'ADN purifié par chromatographie par échange d'anions à la chambre (14063) d'extraction-purification-lavage sur matrice de gel de silice.(14038) for introducing the washing reagents from said micro-column (14117), orifice for evacuation of the washing reagents (14011), stopper (14028) for passage of the purified DNA by anion exchange chromatography at the extraction-purification-washing chamber (14063) on a silica gel matrix.
* d'une chambre d'extraction-purification-lavage sur matrice de gel de silice (14063) pourvue d'une micro-colonne sur matrice de gel de silice (14017), avec orifice (14010) d'introduction des réactifs de lavage de la microcolonne sur matrice de gel de silice (14017), orifice d'introduction du réactif d'élution (14013), obturateur (14026) pour isoler ladite microcolonne (14017) sur matrice de gel de silice, obturateur* an extraction-purification-washing chamber on a silica gel matrix (14063) provided with a micro-column on a silica gel matrix (14017), with orifice (14010) for introducing the washing reagents microcolumn on silica gel matrix (14017), orifice for introducing the elution reagent (14013), obturator (14026) for isolating said microcolumn (14017) on silica gel matrix, obturator
(14024) de passage à la chambre de préparation de l'ADN purifié,(14024) passing through the preparation chamber for the purified DNA,
*d' une chambre de préparation de l'ADN purifié (14064) avec orifice d'introduction (14012) du tampon adéquat pour la méthode d'amplification de l'ADN choisie et donnant accès auxdits micro-canaux (41) de ladite sous partie (14145), ledit tampon étant privé des enzymes, des primers et des dNTP nécessaires à l'amplification* a chamber for the preparation of purified DNA (14064) with an opening for the introduction (14012) of the buffer suitable for the DNA amplification method chosen and giving access to said micro-channels (41) of said subpart (14145), said buffer being deprived of the enzymes, primers and dNTPs necessary for the amplification
Dans ce mode de réalisation de l'invention, l'échantillon d'ADN est introduit dans la chambre de lyse cellulaire et de filtration par le dit orifice (14007). Les réactifs de lyse sont introduits par ledit orifice (14008). Les débris cellulaires de lyse sont filtrés sur les microfîltres (14016), et le lysat est introduit dans la chambre (14061) d'extraction-purification-lavage avec micro-colonne de chromatographie par échange d'anions (14117). Après l'adsorption de l'ADN sur ladite micro-colonne (14117), on procède à l'introduction des réactifs de lavage de ladite micro-colonne (14117) qui sont évacués par l' orifice (14011). Ensuite on isole grâce à un obturateur introduit par l'orifice (14029) ladite microcolonne (14117), puis on procède à l'élution de l'ADN en introduisant le réactif d'élution par l'orifice (14113). Le retrait des obturateurs (14028) et (14029)permet à l'éluat de passer dans la chambre d'extraction- purification-lavage sur matrice de gel de silice (14063). L'ADN subit une deuxième purification sur la microcolonne (14017) de gel de silice. Après un lavage avec des réactifs de lavage introduits par l' orifice (14010), centrifugation et isolement de ladite microcolonne sur matrice de gel de silice (14017) par l'action de l'obturateur (14026), le réactif d'élution est introduit par l'orifice (14013). Puis le retrait de l' obturateur (14024) permet le passage à la chambre (14064) de préparation de l'éluat, si bien que dans le process l'ADN passe successivement sur la micro-colonne de chromatographie par échange d'anions (14117) puis sur la micro-colonne de gel de silice (14017). Dans ladite chambre de préparation de l'ADN purifié (14064), on mélange ledit ADN purifié avec le tampon adéquat pour la méthode d'amplification de l'ADN choisie, ledit tampon étant introduit par l'orifice (14012) et étant privé des enzymes, des primers et des dNTP nécessaires à l'amplification. Ensuite ledit mélange de l'ADN purifié et du tampon d'amplification est introduit dans lesdits microcanaux (41) avec micro-mélangeurs (14019) de ladite sous partie (14145). Une connexion avec d'autres empilements de modules élémentaires plats permet le mélange de l'ADN avec les réactifs spécifiques ou onéreux de l'amplification souhaitée dans chaque micro-canal (41) et finalement dans chaque micropuits (42), lesdits réactifs spécifiques ou onéreux étant tels qu'enzymes nécessaires à la méthode d'amplification choisie, déoxynucléotides et primers d'amplification. Lesdits réactifs spécifiques ou onéreux peuvent être aussi des primers modifiés adaptés à la méthode de détection choisie, des sondes supplémentaires nécessaires à la méthode d'amplification choisie, des déoxynucléotides modifiés, des didéoxynucléotides, des didéoxynucléotides modifiés, des analogues peptidiques de sondes tels que des PNA, etc.In this embodiment of the invention, the DNA sample is introduced into the cell lysis and filtration chamber through said orifice (14007). The lysis reagents are introduced through said orifice (14008). The cellular lysis debris is filtered on the microfilters (14016), and the lysate is introduced into the extraction-purification-washing chamber (14061) with anion exchange chromatography micro-column (14117). After the adsorption of DNA on said micro-column (14117), the washing reagents from said micro-column (14117) are introduced, which are discharged through the orifice (14011). Then, using a shutter inserted through the orifice (14029), said microcolumn (14117) is isolated, then the DNA is eluted by introducing the elution reagent through the orifice (14113). The removal of the obturators (14028) and (14029) allows the eluate to pass into the extraction-purification-washing chamber on a silica gel matrix (14063). The DNA undergoes a second purification on the microcolumn (14017) of silica gel. After washing with washing reagents introduced through the orifice (14010), centrifugation and isolation of said microcolumn on a silica gel matrix (14017) by the action of the shutter (14026), the elution reagent is introduced through the orifice (14013). Then the withdrawal of the obturator (14024) allows the passage to the chamber (14064) of preparation of the eluate, so that in the process the DNA passes successively on the micro column of chromatography by anion exchange ( 14117) then on the silica gel micro-column (14017). In said preparation chamber for purified DNA (14064), said purified DNA is mixed with the buffer suitable for the DNA amplification method chosen, said buffer being introduced through the orifice (14012) and being deprived of enzymes, primers and dNTPs necessary for amplification. Then said mixture of purified DNA and amplification buffer is introduced into said microchannels (41) with micro-mixers (14019) of said subpart (14145). A connection with other stacks of elementary flat modules allows the mixing of the DNA with the specific or expensive reagents of the desired amplification in each micro-channel (41) and finally in each microwell (42), said specific reagents or expensive being such as enzymes necessary for the chosen amplification method, deoxynucleotides and primers for amplification. Said specific or expensive reagents can also be modified primers adapted to the chosen detection method, additional probes necessary for the chosen amplification method, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNA, etc.
La Figure 41 B, la Figure 52A, la Figure 52B, la Figure 52C représentent un autre mode de réalisation de l'invention et réside dans des micro-arrays ou macro-arrays multiblocs (15319) dédiés à l'analyse de fragments d'ADN avec une préparation intégrée comprenant la lyse cellulaire, la filtration des débris, rextraction-purification de l'ADN sur une micro-colonne de gel de silice, l'amplification en amont dudit micro-array ou macro-array multibloc (15319) par une des méthodes connues de l'Homme de l'Art, puis la détection sur ou en aval dudit micro-array ou macro-array multibloc (15319) par une des méthodes connues de l'Homme de l'Art.. Lesdits micro-arrays ou macro-arrays multiblocs (15319) de micro-puits (42) sont constituésFigure 41 B, Figure 52A, Figure 52B, Figure 52C represent another embodiment of the invention and resides in micro-arrays or multi-block macro-arrays (15319) dedicated to the analysis of fragments of DNA with an integrated preparation including cell lysis, filtration of debris, DNA extraction-purification on a micro-column of silica gel, amplification upstream of said multi-block micro-array or macro-array (15319) by one of the methods known to those skilled in the art, then detection on or downstream of said micro-array or macro-array multiblock (15319) by one of the methods known to those skilled in the art. Said multi-block micro-arrays or macro-arrays (15319) of micro-wells (42) are constituted
Figure imgf000054_0001
fl idi ( *d' une chambre de lyse cellulaire et de filtration (15015) pourvue de microfiltres
Figure imgf000054_0001
fl idi (* of a cell lysis and filtration chamber (15015) provided with microfilters
(15016), avec orifice d'introduction de l'échantillon (15007), orifice d'introduction des réactifs de lyse (15008), orifice pour obturateur (15009) de passage à la chambre d'extraçtion-purification-lavage (15061 ),(15016), with hole for introducing the sample (15007), hole for introducing lysis reagents (15008), hole for obturator (15009) for passage to the extraction-purification-washing chamber (15061) ,
*d'une chambre d'extraction-purification-lavage (15061) pourvue d'une micro- colonne (15017), avec orifice (15029) pour obturateur permettant d'isoler la microcolonne (15017), orifice d'introduction du réactif d'élution (15013), orifice (15038) pour introduction du réactif de lavage, orifice pour évacuation des réactifs de lavage (15011), obturateur (15024) de passage à la chambre de préparation de l'ADN purifié, *d' unexhambre de préparation de l'ADN purifié (15064) avec orifice d'introduction* an extraction-purification-washing chamber (15061) provided with a micro-column (15017), with orifice (15029) for obturator making it possible to isolate the microcolumn (15017), orifice for introducing the reagent d elution (15013), orifice (15038) for introduction of the washing reagent, orifice for evacuation of the washing reagents (15011), obturator (15024) for passage to the preparation chamber of purified DNA, * of a chamber of preparation of purified DNA (15064) with insertion opening
(15012) du tampon adéquat pour la méthode d'amplification de l'ADN choisie et donnant accès auxdits micro-canaux (41) de ladite sous partie (15145), ledit tampon étant privé des enzymes, des primers et des dNTP nécessaires à l'amplification(15012) of the buffer suitable for the DNA amplification method chosen and giving access to said micro-channels (41) of said subpart (15145), said buffer being deprived of the enzymes, primers and dNTPs necessary for the 'amplification
Ladite sous-partie (15145) comporte les micro-canaux (41) pourvus de micromélangeurs (15019).Said sub-part (15145) comprises the micro-channels (41) provided with micro-mixers (15019).
Pour ce mode de réalisation de l'invention, la Figure 42 montre qu'un process de préparation intégrée de l'ADN sur micro-arrays ou macro-arrays multiblocs (15319) peut avoir lieu dans des modules plats (15 110) où a déjà été introduit dans la chambre (15064) par l'orifice (15 012) un tampon adéquat pour l'amplification, ledit tampon étant privé des enzymes, des primers et des dNTP nécessaires à l'amplification. L'échantillon d'ADN est introduit dans la chambre de lyse cellulaire et de filtration(15015) par le dit orifice (15007). Ont ensuite lieu des réactifs de lyse par l'orifice (15008), les débris cellulaires de lyse étant filtrés sur les microfiltres (15016), puis le passage du lysat, en ouvrant l'obturateur (15009), dans la chambre (15061) d'extraction-purification-lavage avec micro-colonne sur matrice de gel de silice (15017). Le trop-plein de lysat est évacué par l'orifice (15011) tandis que ladite microcolonne sur matrice de gel de silice (15017) est isolée par l'action de l'obturateur (15029). L'isolement permet de garder une quantité suffisante de lysat qui va continuer de délester son ADN sur ladite micro-colonne (15017) pendant que d'éventuels débris résiduels sont évacués. Une centrifugation a lieu pour évacuer des contaminants. On procède ensuite à un lavage avec des réactifs introduits par l' orifice (15038), puis à l'évacuation des réactifs de lavage par l'orifice (15011), puis à l'introduction du réactif d'élution par l'orifice (15013), le retrait de l' obturateur (15024) permet à l'éluat de passer à la chambre (15064) de préparation de l'ADN purifié et de se mélanger avec le tampon adéquat pour la méthode d"amplification choisie.For this embodiment of the invention, Figure 42 shows that an integrated DNA preparation process on micro-arrays or multi-block macro-arrays (15319) can take place in flat modules (15 110) where a already introduced into the chamber (15064) through the orifice (15 012) a buffer suitable for amplification, said buffer being deprived of the enzymes, primers and dNTPs necessary for the amplification. The DNA sample is introduced into the cell lysis and filtration chamber (15015) through said orifice (15007). Lysis reagents then take place through the orifice (15008), the cellular lysis debris being filtered on the microfilters (15016), then the passage of the lysate, by opening the obturator (15009), in the chamber (15061) extraction-purification-washing with micro-column on a silica gel matrix (15017). The lysate overflow is discharged through the orifice (15011) while said microcolumn on a silica gel matrix (15017) is isolated by the action of the shutter (15029). The isolation makes it possible to keep a sufficient amount of lysate which will continue to shed its DNA on said micro-column (15017) while any residual debris are evacuated. Centrifugation takes place to remove contaminants. Next, washing is carried out with reagents introduced through the orifice (15038), then the washing reagents are discharged through the orifice (15011), then the elution reagent is introduced through the orifice ( 15013), the withdrawal of the obturator (15024) allows the eluate to pass to the chamber (15064) for preparing the purified DNA and to mix with the buffer adequate for the chosen amplification method.
La Figure 52A montre que ledit mélange de l'ADN purifié et du tampon d'amplification est introduit dans lesdits micro-canaux (41) avec micro-mélangeurs (15019) de ladite sous partie (15145).Figure 52A shows that said mixture of purified DNA and amplification buffer is introduced into said micro-channels (41) with micro-mixers (15019) of said sub-part (15145).
La Figure 52B montre une connexion dudit micro-array ou macro-array (15319) de l'invention avec d'autres empilements de modules élémentaires plats (9245) , lesdits modules élémentaires plats (9245) étant pourvus de microcanaux (9241) avec micromélangeurs (92019), lesdits microcanaux (9241) débouchant dans l'épaisseur et sur la tranche desdits modules élémentaires plats (9245) par des orifices convertis en micropuits (9242) . Lesdits modules élémentaires plats sont connectés à l'autre extrémité à des modules élémentaires plats (9255) pourvus de microréservoirs (9210). Lesdits micro-réservoirs (9210) contiennent les réactifs spécifiques ou onéreux de l'ampHfication souhaitée dans chaque micro-canal (9241) et par conséquent dans chaque micropuits (9242).Figure 52B shows a connection of said micro-array or macro-array (15319) of the invention with other stacks of flat elementary modules (9245), said flat elementary modules (9245) being provided with microchannels (9241) with micromixers (92019), said microchannels (9241) opening into the thickness and on the edge of said flat elementary modules (9245) by orifices converted into microwells (9242). Said flat elementary modules are connected at the other end to flat elementary modules (9255) provided with microreservoirs (9210). Said micro-reservoirs (9210) contain the specific or expensive reagents of the desired amplification in each micro-channel (9241) and therefore in each microwell (9242).
Cette connexion permet le mélange de l'ADN avec les réactifs spécifiques ou onéreux de l'amplification souhaitée dans chaque micro-canal (41) et par conséquent dans chaque micropuits (42). Lesdits réactifs spécifiques ou onéreux sont les enzymes nécessaires à la méthode d'amplification choisie, les déoxynucléotides et les primers d'amplification. Ils peuvent être aussi des primers modifiés adaptés à la méthode de détection choisie, des sondes supplémentaires nécessaires à la méthode d'amplification et de détection choisie, des déoxynucléotides modifiés, des didéoxynucléotides, des didéoxynucléotides modifiés, des analogues peptidiques de sondes tels que des PNA, etc.This connection allows the DNA to be mixed with the specific or expensive reagents of the desired amplification in each micro-channel (41) and therefore in each microwell (42). Said specific or expensive reagents are the enzymes necessary for the chosen amplification method, the deoxynucleotides and the amplification primers. They can also be modified primers adapted to the chosen detection method, additional probes necessary for the amplification and detection method chosen, modified deoxynucleotides, dideoxynucleotides, modified dideoxynucleotides, peptide analogs of probes such as PNA , etc.
Des fragments d'ADN peuvent être amplifiés dans de très petits volumes, sous certaines conditions de passivations des surfaces de support de la réaction (Wilding P. , Shoiïher M.A., Kricka LJ.PCR in a silicon microstructure. Clin. Chem. 1994, 40/9, 1815-1818. _ Shoffher M.A., Cheng J., Hvichia G.E., Kricka L.J., Wilding P.Chip PCR. I . Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Research, 1996, 24, 2, 375-379. _ Cheng J, .Shoffher M.A., Hvichia G.E., Kricka L.J., Wilding P. Chip PCR. 11. Investigation of différent PCR amplification Systems in microfabricated silicon-glass chips. Nucleic Acids Research, 1996, 24, 2, 380-385.). Il existe de nombreuses méthodes d'amplification de l'ADN bien connues de l'Homme de l'Art. Ce sont par exemple, pour les plus connues de ces méthodes, la PCR, la RT-PCR, la LCR, la NASBA, la SDA, la TMA, la RCA,etc. Dans la méthode d'amplification par PCR, comme dans beaucoup d'autres méthodes d'amplification de l'ADN ou de l'ARN bien connues de l' Hommes de l'Art, on utilise un couple de primers spécifiques du fragment à amplifier. Les deux primers sont deux oligonucleotides spécifiques. Le premier oligonucleotide spécifique est complémentaire d'une séquence située sur le brin «sens» à une extrémité du fragment d'ADN double brin à amplifier. Le deuxième oligonucleotide spécifique est complémentaire d'une séquence située sur le brin «anti-sens» à l'autre extrémité de ce même fragment d'ADN double brin à amplifier.DNA fragments can be amplified in very small volumes, under certain conditions of passivation of the reaction support surfaces (Wilding P., Shoiïher MA, Kricka LJ.PCR in a silicon microstructure. Clin. Chem. 1994, 40 / 9, 1815-1818. _ Shoffher MA, Cheng J., Hvichia GE, Kricka LJ, Wilding P.Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Research, 1996, 24, 2, 375-379. _ Cheng J,. Shoffher MA, Hvichia GE, Kricka LJ, Wilding P. Chip PCR. 11. Investigation of different PCR amplification Systems in microfabricated silicon-glass chips. Nucleic Acids Research, 1996, 24, 2, 380 -385.). There are many methods of amplifying DNA well known to those skilled in the art. For example, for the best known of these methods, PCR, RT-PCR, LCR, NASBA, SDA, TMA, RCA, etc. In the PCR amplification method, as in many other DNA or RNA amplification methods well known to those skilled in the art, a pair of primers specific for the fragment to be amplified is used. . The two primers are two specific oligonucleotides. The first specific oligonucleotide is complementary to a sequence located on the “sense” strand at one end of the double stranded DNA fragment to be amplified. The second specific oligonucleotide is complementary to a sequence located on the “antisense” strand at the other end of this same double stranded DNA fragment to be amplified.
De même, dans la méthode d'amplification par PCR, comme dans beaucoup d'autres méthodes d'amplification de l'ADN bien connues de l' Hommes de l'Art, on utilise une ADN polymérase, un tampon et des déoxynucléotides. Dans la PCR, on fabrique directement des copies des deux brins par élongation à partir de primers qui se sont hybrides (appariés) à la séquence complémentaire sur un monobrin du fragment d'ADN à amplifier. L' élongation est l'incorporation successive des déoxynucléotides sur un brin néoformé grâce à l'action de l'ADN Polymérase qui progresse dans le sens 5'->3'. Le déoxynucléotide incorporé sur le brin néoformé est celui qui est complémentaire du nucléotide sur le brin copié. Une alternance de températures, soit dans le temps, soit dans l'espacé, permet de commander un appariement ou hybridation entre les brins copiés, ou au contraire la dénaturation desdits brins copiés. Les températures élevées favorisent la dénaturation, les températures modérées favorisent le réappariement. Tout brin copié peut être réapparié avec le brin copié complémentaire, le nouveau fragment d'ADN double brin ainsi formé peut lui-même servir de matrice et être copié par le même procédé. A chaque alternance, dans le temps ou l'espace, de température modérée-température élevée-température modérée, tous les fragments d'ADN sont copiés en un exemplaire, ce qui représente un doublement du nombre de ces fragments. Le nombre d'alternances de températures que subissent les fragments d'ADN est donc le nombre de doublements du nombre de fragments d'ADN cible.Likewise, in the PCR amplification method, as in many other DNA amplification methods well known to those skilled in the art, DNA polymerase, a buffer and deoxynucleotides are used. In PCR, copies of the two strands are produced directly by elongation from primers which have hybridized (paired) to the complementary sequence on a single strand of the DNA fragment to be amplified. Elongation is the successive incorporation of deoxynucleotides on a newly formed strand thanks to the action of DNA Polymerase which progresses in the 5 '-> 3' direction. The deoxynucleotide incorporated on the newly formed strand is that which is complementary to the nucleotide on the copied strand. An alternation of temperatures, either in time or in space, makes it possible to control a pairing or hybridization between the copied strands, or on the contrary the denaturation of said copied strands. High temperatures favor denaturation, moderate temperatures favor re-pairing. Any copied strand can be re-paired with the complementary copied strand, the new double stranded DNA fragment thus formed can itself serve as a template and be copied by the same process. At each alternation, in time or space, of moderate temperature-high temperature-moderate temperature, all the DNA fragments are copied in a copy, which represents a doubling of the number of these fragments. The number of alternations of temperatures that the DNA fragments undergo is therefore the number of doublings of the number of target DNA fragments.
Deux brins d'ADN s'apparient au-dessous d'une température dite Tm. Les meilleures spécificités dans l'amplification sont obtenues lorsqu'on apparie les primers à la température la plus haute possible, de manière à désapparier tous les appariements non spécifiques et à ne conserver que les appariements spécifiques, c'est à dire ceux d'un primer et du brin à amplifier, ceux du brin en cours d' élongation et du brin à amplifier, et ceux des 2 brins complémentaires du fragment d'ADN à amplifier. On va donc hybrider les primers à des température les plus proches possibles de leurs température Tm, à une température Ta dite température d'annealing (généralement fixée à Tm-5°C). Différentes amplifications sur différents fragments d'ADN diffèrent par leur Tm et donc leur Ta.Two strands of DNA pair below a temperature called Tm. The best specificities in amplification are obtained when the primers are paired at the highest possible temperature, so as to dissociate all non-specific pairings and to keep only the specific pairings, that is to say those of a primer and the strand to be amplified, those of the strand being elongated and the strand to be amplified, and those of the 2 complementary strands of the DNA fragment. to amplify. We will therefore hybridize the primers at temperatures as close as possible to their temperature Tm, at a temperature Ta called annealing temperature (generally fixed at Tm-5 ° C). Different amplifications on different DNA fragments differ in their Tm and therefore their Ta.
Les températures d'annealing ne doivent être ni trop élevées, sous peine de ne rien amplifier, ni trop basses, sous peine de ne pas être assez spécifiques. De même, la concentration en MgC12 doit être optimisée: une concentration trop faible nuit au rendement, un excès induit un manque de spécificité. La spécificité est aussi aidée par le choix de certaines ADN Polymérase, ainsi que par les protocoles expérimentaux. Le premier cycle doit commencer à une température élevée Un mélange réactionnel complet d'amplification de l'ADN doit être conservé à 4°C, pour que l'ADN polymérase soit inactive. Si on la laissait être active entre 4° C et 60°C, des amplifications non spécifiques pourraient se produire.The annealing temperatures must not be too high, under penalty of not amplifying anything, nor too low, under penalty of not being specific enough. Likewise, the concentration of MgC12 must be optimized: a too low concentration harms the yield, an excess induces a lack of specificity. The specificity is also helped by the choice of certain DNA Polymerases, as well as by the experimental protocols. The first cycle should start at an elevated temperature. A complete DNA amplification reaction mixture should be stored at 4 ° C for the DNA polymerase to be inactive. If allowed to be active between 4 ° C and 60 ° C, nonspecific amplifications could occur.
Le premier cycle doit commencer à une température élevée, par exemple par la température de dénaturation à 92°C - 96 °C, pour que le mélange réactionnel passe le moins de temps possible dans une phase de réchauffement de 4°C à 60 °C, où le risque de réaction non spécifique existe.The first cycle must start at a high temperature, for example with the denaturation temperature at 92 ° C - 96 ° C, so that the reaction mixture spends as little time as possible in a warming phase from 4 ° C to 60 ° C , where the risk of non-specific reaction exists.
Si on veut éliminer totalement le risque de réaction non spécifique du mélange réactionnel entre 4°C et 65°C, il faut le séparer en 2 par une couche de paraffine, de telle manière que cette séparation rende impossible la réaction. Seule la fusion de la paraffine à 65° C reconstituera le mélange réactionnel..If the risk of non-specific reaction of the reaction mixture between 4 ° C and 65 ° C is to be completely eliminated, it must be separated in 2 by a layer of paraffin, in such a way that this separation makes the reaction impossible. Only the melting of the paraffin at 65 ° C. will reconstitute the reaction mixture.
Lorsque plusieurs amplifications différentes sont effectuées en même temps avec le souhait de les optimiser toutes, une solution est de fixer des températures d'annealing spécifiques pour chaque amplificationWhen several different amplifications are carried out at the same time with the desire to optimize them all, one solution is to set specific annealing temperatures for each amplification
Si par contre l'on est seulement désireux d'obtenir une amplification pour chaque fragment optimisée sur le plan de la spécificité, mais pas forcément sur celui du rendement, on peut tenter des températures d'annealing identiques et élevées pour toutes les amplifications, autour de 60°C pour des primers de 20 mers, et un peu moins pour des primers plus courts (pour les primers de 20 mers, 60 °c entraînera une meilleure spécificité que 55 °C, mais pour certaines amplifications, le rendement sera un peu faible).If on the other hand we are only eager to obtain an amplification for each fragment optimized in terms of specificity, but not necessarily in terms of yield, we can attempt identical and high annealing temperatures for all the amplifications, around of 60 ° C for primers of 20 seas, and a little less for shorter primers (for primers of 20 seas, 60 ° c will lead to better specificity than 55 ° C, but for certain amplifications, the yield will be a little low).
Enfin, certains travaux ( par exemple Thuong et al, NucleidcAcids Research, 1998, 26, 18, 4249-4258 ; Nucleic Acids Research, 1997, 25, 15, 3059-3065), décrivent des modifications de bases qui rendent les Tm de brins d'ADN indépendants de leur composition en bases. Ceci fait envisager l'emploi de ce type de méthodes pour permettre une homogénéité des températures d'annealing pour un grand nombre d'amplifications effectuées en même temps.Finally, certain works (for example Thuong et al, NucleidcAcids Research, 1998, 26, 18, 4249-4258; Nucleic Acids Research, 1997, 25, 15, 3059-3065), describe basic modifications which make the Tm of strands of DNA independent of their base composition. This makes it possible to consider the use of this type of method to allow homogeneity of the annealing temperatures for a large number of amplifications carried out at the same time.
En ce qui concerne le marquage pour la détection après l'amplification, plusieurs stratégies existent. On peut utiliser le fragment d'ADN amplifié que l'on va lier à une autre molécule, en utilisant ou en combinant différentes propriétés chimiques, bien connues de V Homme de l'Art, comme les réactions enzymatiques et la fluorescence. On peut aussi utiliser un marquage radioactif. L'ADN amplifié peut aussi être marqué par exemple par incorporation de déoxynucléotides marqués lors de l'élongation par l'ADN Polymérase pendant l'amplification.With regard to labeling for detection after amplification, several strategies exist. We can use the amplified DNA fragment that we will link to another molecule, using or combining different chemical properties, well known to those skilled in the art, such as enzymatic reactions and fluorescence. Radioactive labeling can also be used. The amplified DNA can also be labeled for example by incorporation of deoxynucleotides labeled during elongation by DNA Polymerase during the amplification.
Mais ce peut être aussi un primer qui peut être lié selon de nombreuses façons, bien connues de l' Homme de l'Art, à des molécules aidant la détection. Parmi de nombreux exemples, un primer peut être lié à un fluorophore pour la détection par fluorescence, ou à des molécules d'affinité comme la biotine pour que le brin d'ADN amplifié se lie à des billes coatées à la streptavidine, ou à des molécules d'étiquetage comme des oligonucleotides d'étiquetage. Le type de marquage va dépendre du type de détection (micro-électrophorèse, hybridation, spectrométrie de masse, chromatographie, micro-électrochromatographie, caméras CCD, fibres optiques, microscopie confocale, détection avec fluorescence, chimiluminescence, bioluminescence, colorimétrie, par surface plasmon résonance, par mesure de l'onde évanescente, par électrochimie, par radioactivité, etc) en adéquation avec le but de l'analyse.But it can also be a primer which can be linked in many ways, well known to those skilled in the art, to molecules which aid detection. Among many examples, a primer can be linked to a fluorophore for fluorescence detection, or to affinity molecules such as biotin so that the amplified DNA strand binds to beads coated with streptavidin, or to labeling molecules such as labeling oligonucleotides. The type of labeling will depend on the type of detection (micro-electrophoresis, hybridization, mass spectrometry, chromatography, micro-electrochromatography, CCD cameras, fiber optics, confocal microscopy, detection with fluorescence, chemiluminescence, bioluminescence, colorimetry, by plasmon resonance surface , by measurement of the evanescent wave, by electrochemistry, by radioactivity, etc) in adequacy with the aim of the analysis.
On recherche parfois à sophistiquer la réaction d'amplification . Par exemple, dans les process combinant l'amplification et le séquençage selon Sanger, on peut vouloir introduire un biais en incorporant non seulement des déoxynucléotides, mais aussi un certain pourcentage de didéoxynucléotides. Comme ces didéoxynucléotides sont correctement incorporés par l'ADN polymérase mais que par contre ils empêchent l'élongation de se poursuivre au-delà de leur propre incorporation, ils provoquent un arrêt dans la polymérisation de l'ADN et génèrent un certain pourcentage de fragments d'ADN représentant des copies partielles du fragment d'ADN à amplifier, dans la mesure où la copie commence bien mais se termine prématurément. Lorsqu'on hiérachise ces fragments selon une variété de protocoles, on peut déterminer quel est le didéoxynucléotide qui a provoqué l'arrêt d'un fragment d'une longueur donnée, ce qui permet de découvrir la séquence de l'ADN amplifié. Une des méthodes pour hiérarchiser les fragments est la mobilité électrophorétique sur gel sur une seule ligne, mais avec des didéoxynucléotides marqués différemment, une autre est la mobilité électrophorétique sur gel sur 4 lignes, chaque ligne correspondant à une réaction où un seul des quatre didéoxy a été incorporé, une autre est la spectrométrie de masse qui mesure directement la masse des fragments, généralement dans quatre puits où la réaction de Sanger diffère par le didéoxynucléotide incorporé.We sometimes try to improve the amplification reaction. For example, in processes combining amplification and sequencing according to Sanger, one may want to introduce a bias by incorporating not only deoxynucleotides, but also a certain percentage of dideoxynucleotides. As these dideoxynucleotides are correctly incorporated by DNA polymerase but on the other hand prevent the elongation from continuing beyond their own incorporation, they cause a stop in the polymerization of DNA and generate a certain percentage of fragments of DNA representing partial copies of the DNA fragment to be amplified, insofar as the copy starts well but ends prematurely. When hierarchizing these fragments according to a variety of protocols, one can determine which is the dideoxynucleotide which caused the arrest of a fragment of a given length, which makes it possible to discover the sequence of the amplified DNA. One of the methods for prioritizing the fragments is electrophoretic mobility on gel on a single line, but with dideoxynucleotides labeled differently, another is electrophoretic mobility on gel on 4 lines, each line corresponding to a reaction where only one of the four dideoxy has another was mass spectrometry which directly measures the mass of the fragments, generally in four wells where the Sanger reaction differs by the incorporated dideoxynucleotide.
Selon une version de l'invention, dans chaque microcanal (41) d'un micro-array ou macro- array (15319), on procède au mélange de l'ADN purifié à analyser avec des primers spécifiques du fragment d'ADN à amplifier, les quatre déoxynucléotides dATP, dTTP, dGTP, dCTP, et avec l'enzyme utilisée pour la méthode d'amplification choisie, par connexion avec un deuxième micro-array ou macro-array multibloc formé par empilement de modules élémentaires plats (9245). Lesdits modules élémentaires plats (9245) amènent, à partir de micro-réservoirs portés par lesdits modules élémentaires plats (9255) auxquels ils sont connectés, le mélange voulu de ladite enzyme, des déoxynucléotides, et desdits primers d'amplification pour qu'un fragment précis de l'ADN soit amplifié et présenté dans un micropuits précis (42) dudit mico-array ou macro-array (15319). Ceci permet, par le biais des jonctions étanches ainsi réalisées, de préparer l'ADN pour des amplifications puis des détections sur de très faibles quantités dans une configuration de grande compacité dans des conditions étanches et sans rupture de chaîne, avec comme corollaires d'éviter l'évaporation et les contaminations.According to a version of the invention, in each microchannel (41) of a micro-array or macro-array (15319), the purified DNA to be analyzed is mixed with primers specific for the DNA fragment to be amplified. , the four deoxynucleotides dATP, dTTP, dGTP, dCTP, and with the enzyme used for the amplification method chosen, by connection with a second multi-block micro-array or macro-array formed by stacking flat elementary modules (9245). Said flat elementary modules (9245) bring, from micro-reservoirs carried by said flat elementary modules (9255) to which they are connected, the desired mixture of said enzyme, deoxynucleotides, and said amplification primers so that a fragment precise DNA is amplified and presented in a precise microwell (42) of said mico-array or macro-array (15319). This makes it possible, by means of the tight junctions thus produced, to prepare the DNA for amplifications then detections on very small quantities in a configuration of great compactness under tight conditions and without chain break, with as corollaries to avoid evaporation and contamination.
La construction des primers utilisés pour l'amplification dépend de la méthodologie choisie. Par exemple, ce peuvent être des primers "beacons", avec deux séquences complémentaires l'une de l'autre rajoutées aux extrémités, l'une liée à un fluorophore, l'autre liée à un répresseur. Ces primers ont la particularité de n'être détectables par un signal fluorescent qu' à partir du moment où ils s'hybrident à un brin complémentaire d'ADN, ce qui éloigne le répresseur du fluorophore et permet l'émission d'un signal fluorescent^ Tyagi S., Kramer F.R. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 1996, 14, 303-308. _ Tyagi S., Bratu D.P., Kramer F.R. Multicolor beacons for allele discrimination. Nature Biotechnology, 1998, 16, 49^53. _ _Leone G., van Sçhijndel H., van Gemen B., Kramer F.R., Schoe CD. Molecular beacon probes combined with amplification by NASBA enable homogenéous, real-time détection of RNA. Nucleic Acids Research, 1998, 26, 9, 2150-2155. ). La détection de fluorescence des beacons moléculaires peut se faire en temps réel par mesure de l'onde évanescente à partir de fibres optiques (Liu X, Tan W. A Fiber-Optic Evanescent Wave DNA biosensor based on novel molecular beacons. Anal. Chem. 1999, 71, 5054-5059)The construction of the primers used for the amplification depends on the chosen methodology. For example, these may be primers "beacons", with two sequences complementary to each other added at the ends, one linked to a fluorophore, the other linked to a repressor. These primers have the particularity of being detectable by a fluorescent signal only from the moment when they hybridize to a complementary strand of DNA, which removes the repressor from the fluorophore and allows the emission of a fluorescent signal. ^ Tyagi S., Kramer FR Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnology, 1996, 14, 303-308. _ Tyagi S., Bratu DP, Kramer FR Multicolor beacons for allele discrimination. Nature Biotechnology, 1998, 16, 49 ^ 53. _ _Leone G., van Sçhijndel H., van Gemen B., Kramer FR, Schoe CD. Molecular beacon probes combined with amplification by NASBA enable homogenous, real-time detection of RNA. Nucleic Acids Research, 1998, 26, 9, 2150-2155. ). The fluorescence of molecular beacons can be detected in real time by measuring the evanescent wave from optical fibers (Liu X, Tan W. A Fiber-Optic Evanescent Wave DNA biosensor based on novel molecular beacons. Anal. Chem. 1999, 71, 5054-5059)
Dans le mélange réactionnel, il peut être rajouté à partir desdits modules élémentaires plats (9245) des réactifs spécifiques d'autres méthodologies de détection de l'amplifiation en temps réel comme celles décrites par Kalinina O., Lebedeva L, Brown J.,Silver J. Nanoliter scale PCR with Taqman détection. Nucleic Acids Research, 1997, Vol. 25, No 10, 1999-2004. _ Holland P. M., Abramson R.D., Watson R., Gelfand D.H. Détection of spécifie polymérase chain reaction product by utilizing the 5 ' 3 ' exonuclease activity of Thermus aquaticus DNA polymérase. Proc. Natl. Acad. Sci. USA. 1991, 88, 7276-7280. _ Lee L.G., Connell, C.R., Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids research, 1993, 21, 16, 3761-3766. __ Livak K, Flood, S.J.A., Marmaro J. Giusti W., Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe System useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications. 357- 362, 1995. _ Mullah B., Livak K, Andrus A., Kenney P. Efficient synthesis of double-dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay. Nucleic Acids Research, 1998, Vol 26, No4. _ Gibson U.E.M., Heid C.A., Williams P.M. A novel method for real time quantitative RT-PCR. Génome Research, 1996, 995-1001. _ Heid C.A., Stevens J; Livak K.J., Williams P.M. Real time Quantitative PCR .Génome Research, 1996, 986-994. Ainsi, une amplification détectée par l'activité exonucléasique d'une ADN polymérase nécessite l'emploi d'une troisième sonde complémentaire d'une séquence à l'intérieur du fragment d'ADN à amplifier, et liée à un fluorophore et à son répresseur. Lors de l'élongation, l'activité exonucléasique de l'ADN Polymérase va séparer le fluorophore de son répresseur et permettre l'émission d'un signal.Cette troisième sonde peut être rajoutée au mélange réactionnel conservé au froid avec les primers et les déoxynucléotides, et l'ADN Polymérase. D'autres méthodes de détection de l'amplification en temps réel peuvent être mises en œuvre.In the reaction mixture, it is possible to add, from said flat elementary modules (9245), specific reagents from other real-time amplification detection methodologies such as those described by Kalinina O., Lebedeva L, Brown J., Silver J. Nanoliter scale PCR with Taqman detection. Nucleic Acids Research, 1997, Vol. 25, No 10, 1999-2004. _ Holland PM, Abramson RD, Watson R., Gelfand DH Detection of specifies polymerase chain reaction product by utilizing the 5 '3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA. 1991, 88, 7276-7280. _ Lee LG, Connell, CR, Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids research, 1993, 21, 16, 3761-3766. __ Livak K, Flood, SJA, Marmaro J. Giusti W., Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe System useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications. 357-362, 1995. _ Mullah B., Livak K, Andrus A., Kenney P. Efficient synthesis of double-dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay. Nucleic Acids Research, 1998, Vol 26, No4. _ Gibson UEM, Heid CA, Williams PM A novel method for real time quantitative RT-PCR. Genome Research, 1996, 995-1001. _ Heid CA, Stevens J; Livak KJ, Williams PM Real time Quantitative PCR. Génome Research, 1996, 986-994. Thus, an amplification detected by the exonuclease activity of a DNA polymerase requires the use of a third probe complementary to a sequence inside the DNA fragment to be amplified, and linked to a fluorophore and to its repressor . During elongation, the exonucleosic activity of DNA Polymerase will separate the fluorophore from its repressor and allow the emission of a signal. This third probe can be added to the reaction mixture kept cold with primers and deoxynucleotides , and DNA Polymerase. Other real-time amplification detection methods can be implemented.
Puis on procède à l'amplification spécifique d'un fragment d'ADN par une des méthodes d'amplification connues en l'Etat Actuel de l'Art en pourvoyant le dispositif de réchauffeurs chargés de tempérer tout ou partie desdits microcanaux (41) desdites unités fonctionnelles planes (15145) et tout ou partie desdits microcanaux (9241) desdites unités fonctionnelles planes (9245), de manière à ce que la température desdits microcanaux (41) et micro-canaux (9241) convienne à tout moment au protocole de la méthode d'amplification d'ADN choisie. Certaines méthodes d'amplifications sont isothermes, comme la SDA. De plus, on peut adresser électroniquement chaque micro-puits pour exercer un contrôle individualisé sur les réactions d'amplification et d'hybridation ( Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a micro-electronic chip array. Nature Biotechnology, 2000, 18, 199-204). De même, chaque micro-puits d'un micro-array ou macro-array multibloc de l'invention peut être adressé individuellement par des électrodes pour contrôler la réaction en cours dans ledit-micro-puits.Then one proceeds to the specific amplification of a DNA fragment by one of the amplification methods known in the current state of the art by providing the device with heaters responsible for tempering all or part of said microchannels (41) of said planar functional units (15145) and all or part of said microchannels (9241) of said planar functional units (9245), so that the temperature of said microchannels (41) and microchannels (9241) is suitable at all times for the protocol of the DNA amplification method chosen. Some amplification methods are isothermal, such as SDA. In addition, each micro-well can be electronically addressed to exercise individualized control over amplification and hybridization reactions (Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a micro-electronic chip array. Nature Biotechnology, 2000, 18, 199-204). Similarly, each micro-well of a micro-array or multi-block macro-array of the invention can be addressed individually by electrodes to control the reaction in progress in said micro-well.
Le dispositif selon l'invention est utilisable pour sa propre fabrication à l'usine si l'on veut fabriquer des unités fonctionnelles planes prêtes à l'emploi pour l'amplification d'ADN. A l'usine de fabrication, dans chaque microcanal (41) d'un micro-array (15319), on peut procéder à l'introduction de primers spécifiques, des déoxynucléotides, de l'enzyme d'amplification pendant les phases terminales de fabrication des unités modules plats (15110), de manière à ce que, lors d'une utilisation sur le terrain pour amplifier un fragment d'ADN, l'opération d'ajouter lesdits primers spécifiques au mélange réactionnel ne soit plus nécessaire pour mener à bien pour ladite réaction d'amplification. De cette façon l'on peut disposer d'un micro-array ou macro-array de l'invention "prêt-à-1'emploi". Cette opération effectuée en usine peut éventuellement se faire grâce à une connexion orthogonale à un micro-array ou macro-array de l'invention, soit en fin de fabrication, soit en cours de fabrication. L'ADN amplifié peut être détecté par une des méthodes de détection connues en l'Etat Actuel de l'Art, telle que détection de l'amplification directement en temps réel avec un marquage mesurable pendant l'amplification, ou détection par spectrométrie de masse, ou détection par micro-électrophorèse, ou détection par chromatographie, ou détection par hybridation sur sondes. La fluorescence, la bioluminescence, la chimiluminescence, la colorimétrie, la mesure de l'onde évanescente, la surface plasmon résonance, l'élecfrochimie, le radioactivité, la densimétrie peuvent être utilisées. Le marquage autorisant l'une de ces détections peut être par fluorescence, bioluminescence, chentiluminescence, ou bien effectué avec des molécules ayant un différentiel de potentiel d'oxydo-réduction, ou avec molécules étiquette tels qu'une courte séquence oligonucléotidique, ou avec molécules d'affinité, ou une combinaison desdites méthodes de marquage précédentes. Dans le cas de détection par hybridation sur sondes, ladite hybridation peut être détectée par de nombreux procédés, dont des procédés avec marquage, des procédés sans marquage, des procédés d'observation dynamique de l'évolution de l'hybridation.. Parmi les très nombreuses méthodes de détection de l'hybridation, citons entre autres les méthodes colorimétriques, par fluorescence, par radioactivité, par surface plasmon résonance, par spectrométrie de masse, etc.The device according to the invention can be used for its own manufacture in the factory if one wishes to manufacture ready-to-use flat functional units for DNA amplification. At the manufacturing plant, in each microchannel (41) of a micro-array (15319), it is possible to introduce specific primers, deoxynucleotides, the amplification enzyme during the final manufacturing phases flat module units (15110), so that, when used in the field to amplify a DNA fragment, the operation of adding said specific primers to the reaction mixture is no longer necessary to carry out successfully for said amplification reaction. In this way one can have a micro-array or macro-array of the invention "ready-to-use". This operation carried out in the factory can possibly be done by means of an orthogonal connection to a micro-array or macro-array of the invention, either at the end of manufacture, or during manufacture. The amplified DNA can be detected by one of the detection methods known in the current state of the art, such as detection of the amplification directly in real time with a measurable marking during the amplification, or detection by mass spectrometry. , or detection by micro-electrophoresis, or detection by chromatography, or detection by hybridization on probes. Fluorescence, bioluminescence, chemiluminescence, colorimetry, measurement of the evanescent wave, the plasmon resonance surface, elecfrochemistry, radioactivity, densimetry can be used. The labeling authorizing one of these detections can be by fluorescence, bioluminescence, chentiluminescence, or else carried out with molecules having a redox potential differential, or with label molecules such as a short oligonucleotide sequence, or with molecules affinity, or a combination of said previous labeling methods. In the case of detection by hybridization on probes, said hybridization can be detected by numerous methods, including methods with labeling, methods without labeling, methods of dynamic observation of the evolution of hybridization. Among the very Numerous methods of detection of hybridization, let us quote inter alia the colorimetric methods, by fluorescence, by radioactivity, by plasmon resonance surface, by mass spectrometry, etc.
On peut procéder, dans chaque micrôpuits (42) d'un micro-array ou macro-arry multibloc (15319) à la détection de l'amplification en temps réel par fluorescence par une des méthodes connues en l'Etat Actuel de l'Art, comme par exemple la méthode dite de primers sous forme de «beacons moléculaires» avec fluorophores et répresseurs, ou la méthode dite «Taqman» de détection d'une activité exonucléasique de l'ADN polymérase grâce à un fluorophore et un répresseur pendant les élongations ayant cours lors des méthodes d'amplification . Cette détection peut être effectuée ou non à travers un couvercle transparent.One can proceed, in each microwell (42) of a micro-array or multi-block macro-arry (15319), to detect the amplification in real time by fluorescence by one of the methods known in the current state of the art. , such as the so-called primers method in the form of “molecular beacons” with fluorophores and repressors, or the so-called “Taqman” method for detecting an exonuclease activity of DNA polymerase using a fluorophore and a repressor during elongation used during amplification methods. This detection may or may not be carried out through a transparent cover.
On peut aussi transférer les ADN amplifiés présentés dans ledit micro-array ou macro-array multibloc (15319) sur un micro-array monobloc (340) et procéder à la détection dans ledit micro-array ou macro-array monobloc (340), ce dernier pouvant être pourvu d'un couvercle transparent.It is also possible to transfer the amplified DNAs presented in said micro-array or multi-block macro-array (15319) to a mono-block micro-array (340) and to carry out the detection in said micro-block or monoblock macro-array (340), this the latter can be provided with a transparent cover.
On peut également conditionner les ADN amplifiés présents dans ledit micro-array ou macro-array multibloc (15319) en utilisant des transferts en série sur d'autres micro-array ou macro-array multiblocs pour procéder à des rajouts de réactifs accompagnés ou non de lavages, de séparations, de fixations et de purifications.It is also possible to condition the amplified DNAs present in said micro-array or multi-block macro-array (15319) by using transfers in series on other micro-array or multi-block macro-array to add reagents with or without washes, separations, fixations and purifications.
Par exemple, on peut commencer par amplifier les ADN avec un des deux primers biotynilés dans un premier micro-array ou macro-array multibloc (15319), ce qui permet, dans des conditions dénaturantes, de séparer les deux brins de l'ADN amplifié double brin sur des billes magnétiques coatées à la streptavidine. Cette séparation peut avoir lieu dans les micropuits d'un deuxième micro-array ou macro-array multibloc remplis desdites billes coatées à la streptavidine, ou bien dans un micro-array ou macro-array pourvu de microcanaux avec portions élargies et remplies desdites billes coatées à la streptavidine. Cette méthode de séparation des deux brins permet de séparer d'une part le brin d'ADN amplifié biotynilé avec les billes magnétiques, et d'autre part le brin d'ADN amplifié libre dans le surnageant.For example, we can start by amplifying DNA with one of the two biotynilized primers in a first micro-array or multi-block macro-array (15319), which allows, under denaturing conditions, to separate the two strands of the amplified DNA. double strand on magnetic beads coated with streptavidin. This separation can take place in the microwells of a second multi-block micro-array or macro-array filled with said beads coated with streptavidin, or else in a micro-array or macro-array provided with microchannels with enlarged portions and filled with said coated beads. with streptavidin. This method of separation of the two strands makes it possible to separate on the one hand the strand of amplified DNA biotynilized with the magnetic beads, and on the other hand the strand of free amplified DNA in the supernatant.
On peut en outre procéder, dans chaque micropuits (42) d'un micro-array ou macro-array multibloc (15319) à la détection de l'amplification par spectrométrie de masse par une des méthodes connues en l'Etat Actuel de l'Art.It is also possible, in each microwell (42) of a micro-array or multi-block macro-array (15319), to detect the amplification by mass spectrometry by one of the methods known in the present state of the art. Art.
Par exemple, pour suivre selon l'invention une préparation de l'ADN pour une des méthodes de détection par spectrométrie de masse MALDI-TOF (décrite par Little D.P., Cornish T.J., O'Donnell M.J., Braun A., Cotter R.J., Kôster H. . MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantifies of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezo-electric pipet. Anal. Chem. 1997, 69, 4540-46.) , l'ADN peut être amplifié dans les micro-canaux (41) d'un micro-array ou macro-array multibloc (15319) avec un des deux primers biotynilés, puis les deux brins de l'ADN amplifié double brin peuvent être séparés grâce à des billes magnétiques logées dans les portions élargies desdits microcanaux (41). Le surnageant contenant l'ADN amplifié monobrin peut être présenté dans les micropuits (42) dudit micro-array ou macro-array (15319) et mélangé à un dérivé de l'acide hydroxysuccinique ou hydroxypiccolinique, soit directement sur ledit micro-array ou macro-array multibloc (15319), soit dans les micro-puits d'un autre macro- array ou micro-array multibloc connecté. Un tir au laser dans chaque micro-puits permet alors d'ioniser le fragment d'ADN et de l'accélérer dans une champ électrique pour une détection par spectrométrie de masse., où la comparaison de la masse attendue dudit fragment d'ADN et la masse mesurée permet de confirmer la conformité du fragment, à la masse attendueFor example, to follow, according to the invention, a DNA preparation for one of the detection methods by MALDI-TOF mass spectrometry (described by Little DP, Cornish TJ, O'Donnell MJ, Braun A., Cotter RJ, Kôster H.. MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantifies of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezo-electric pipet. Anal. Chem. 1997, 69, 4540-46.), DNA can be amplified in the micro-channels (41) of a micro-array or multi-block macro-array (15319) with one of the two biotynilized primers, then the two strands of the amplified double strand DNA can be separated by means of beads magnetic housed in the enlarged portions of said microchannels (41). The supernatant containing the amplified single-stranded DNA can be presented in the microwells (42) of said micro-array or macro-array (15319) and mixed with a derivative of hydroxysuccinic or hydroxypiccolinic acid, either directly on said micro-array or macro -array multibloc (15319), either in the micro-wells of another connected macro-array or multi-block micro-array. A laser shot in each micro-well then makes it possible to ionize the DNA fragment and to accelerate it in an electric field for detection by mass spectrometry., Where the comparison of the expected mass of said DNA fragment and the mass measured confirms the conformity of the fragment to the expected mass
Cet exemple de process de détection par spectrométrie de masse MALDI-TOF est cité pour montrer la polyvalence et la grande adaptabilité des micro-arrays ou macro-arrays multiblocs de l'invention. En spectrométrie de masse, par exemple, d'autres méthodes ne nécessitent pas la séparation des deux brins sur billes de streptavidine. Dans les autres possibilités de détection, de très nombreuses variantes existent également, variantes auxquelles peuvent s'adapter les micro-arrays ou macro-arrays multiblocs de l'invention.This example of the MALDI-TOF mass spectrometry detection process is cited to show the versatility and the great adaptability of the multi-block micro-arrays or macro-arrays of the invention. In mass spectrometry, for example, other methods do not require the separation of the two strands on streptavidin beads. In the other detection possibilities, numerous variants also exist, variants to which the micro-arrays or multi-block macro-arrays of the invention can be adapted.
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Functional intégration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem., 1996, 68, 4081-4086. _ Kheterpal L, Scherer J.R., Clark S.M., Radhakrishnan A., Ju J., Ginther CL., Sensabaugh G.F., Mathies R.A. DNA sequencing using a four-color confocal fluorescence capillary array scanner. Electrophoresis, 1996, 17, 1852-1859. _ Wang Y., Wallin J.W., JU J., Sensabaugh G.F., Mathies R.A. High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers. Electrophoresis 1996, 17, 1485-1490. _ Woolley A.T., Sensabaugh G.F., Mathies R.A. High-speed DNA genotyping using microfabricated capillary array electrophoresis chips. Anal. Chem. 1997, 69, 2181- 2186. _ Woolley A.T., Lao K., Glazer A_N., Mathies R.A. Capillary electrophoresis chips with integrated electrochemical détection. Anal. Chem., 1998, 70, 684-688. _ Woolley A.T., Hadley D., Landre P., de Mello A.J., Mathies R.A., Northrup M.A. Functional intégration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem., 1996, 68, 4081-4086. _Mansfield E.S. et al. Versatile low-viscosity sieving matrices for non-denaturing DNA séparations using capillary array electrophoresis. Electrophoresis, 1997, 18, 104-111. _ Simpson P.C., Roach D., Wooley A.T., Thorsen T., Johnston R., Sensabaugh G.F., Mathies R.A. High throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates. Pr.Natl.Acad.Sci. USA, 1998, 95, 2256-2261. _ Jacobson SC, Hergenrôder R, Koutny LB, Warmack RJ, Ramsey JM. Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices. Anal. Chem, 1994, 66, 1107-1113).Integrated cell isolation and polymerase chain reaction analysis using silicon microfilter chambers. Analytical Biochemistry, 1998, 257, 95-100. _ Cheng J., Waters LC, Fortina P., Hvichia G., Jacobson SC, Ramsey JM, Kricka LJ, Wilding P. Degenerate oligonucleotide primed polymerase chain reaction and capillary electrophoresis analysis of human DNA on microchip-based devices. Analytical Biochemistry, 1998, 257, 101-106. _ Ueno K ;, Yeung ES Capillary electrophoresis and sample injection Systems integrated on a planar glass chiρ.Anal. Chem., 1992, 64, 1926-1932. _ Manz A., Harrison DJ, Verpoorte EMJ, Fettinger JC, Paulus A., Ludi H., Widmer HM Planar chips technology for miniaturization and integration of separation techniques into monitoring Systems. Capillary electrophoresis on a chip. J. of Chromatography, 1992, 593, 253-258. _ Harrison DJ, Fluri K., Seiler K., Fan Z., Effenhauser CS, Manz A. Micromachninig a miniaturized capillary electrophoresis- based chemical analysis System on a chip. Science, 1993, 261, 895-897 _ Liang Z., Chiem N., Ocvirrk G., Tang T., Fluri K., Harrison DJ. Microfabrication of a planar absorbance and fluorescence cell for integrated capillary electrophoresis devices. Anal. Chem., 1996, 1040-1046. _ Coyler CL., Tang T., Chiem N., Harrison D. Clinical potential of microchip capillary electrophoresis Systems. Electrophoresis, 1997, 18, 1733-1741. _ Li PCH, Harrison JD Transport, manipulation, and reaction of biological cells On-chip using electrokinetic effects. Anal. Chem., 1997, 69, 1564-1568. _ Effenhauser CS, Manz A., Widmer HM Glass chips for high-speed capillary electrophoresis separations with submicrometer plate heights. Anal. Chem. 1993, 65, 2637-2642. _ Effenhauser CS, Paulus A., Manz A., Widmer HM High-speed separation of antisense oligonucletides on a micromachined capillary electrophoresis device. Anal. Chem. 1994, 66, 2949-2953. _ Effenhauser CS, Bruin GJM, Paulus A. Integrated chip-based capillary electrophoresis. Electrophoresis, 1997, 18, 2203-2213. _ Effenhauser CS, Bruin GJM, Paulus A., Ehrat M. Integrated capillary electrophoresis on flexible silicone microdevices: analysis of DNA restriction fragments and detection of single DNA molecules on microchips. Anal. Chem. 1998, 69, 3451-3457. _ Jacobson SC, Koutny LB, Hergenrôder R., Moore AW, Ramsey JM. Microchip capillary electrophoresis with an integrated postcolumn reactor. Anal. Chem. 1994,, 3472-3476. _ Jacobson JC, Ramsey JM. Integrated microdevice for DNA restriction fragment analysis. Anal. Chem., 1996, 68, 720-723. _ Waters LC, Jacobson SC, KroutclïiîώnaN., Khandurina J., Foote RS, Ramsey JM Microchip device for cell lysis, multiplexr ÇR amplification, and electrophoretic sizing. Anal. Chem., 1998, 70, 158-162. _ Fister JC, Jacobson SC, Davis LM, Ramsey JM Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices.Anal. Chem., 1998, 70, 431-437. _Hurst GB, Weaver K., Doktycz. Simultaneous monitoring of DNA fragments separated by electrophoresis in a multiplexed array of 100 capillaries. Anal. Chem. , 1994, 66, 1424-1431. _ Me Cormick RM, Nelson RJ, Alonso-Amigo MG, Benvegnu DJ, Hooper HH Microchannel electrophoretic separations of DNA in injection-molded plastic substrates. Anal. Chem, 1997, 69, 2626-2630. Burns MA, Mastrangelo CH, Sammarco TS, Man FP, Webster JR, Johnson BN, Foerster B., Jones D., Fields Y., Kaiser A., Burke D. Microfabricated structures for integrated DNA analysis. Proc. Natl. Acad. Sci. USA, 1996, 93, 5556-5561. __ Huang XC, Quesada MA, Mathies RA Capillary array electrophoresis using laser-excited confocal fluorescence detection. Anal. chem. 1992, 64, 967-972. _-Ηuang X. C, Quesada MA, Mathies R.À. DNA sequencing using capillary array electrophoresis. Anal. Chem. , 1992, 64, 2149-2154. _ Woolley AT, Mathies RA Ultra high-speed DNA fragment separations using microfabricated capillary array electrophoresis. Proc. Natl. acad. SDEI. USA, 1994, 91, 11348-11352 _ Haab BB, Mathies RA A single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis. Anal. Chem., 1995, 67, 3253-3260. _ Zhu H., Clark SM, Benson SC, Rye HS, Glazer AN, Mathies RA High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes. Anal. Chem., 1994, 66, 1941-1948. _ Wang, Ju J., Carpenter BA, Atherton JM, Sensabaugh GF, Mathies RA Rapid sizing of short tandem repeat alleles using capillary array electrophoresis and energy transfer fluorecent primers. Anal. Chem., 1995, 67, 1197-1203. _ Woolley AT, Hadley D., Landre P., de Mello AJ, Mathies RA, Norfhrup MA Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem., 1996, 68, 4081-4086. _ Kheterpal L, Scherer JR, Clark SM, Radhakrishnan A., Ju J., Ginther CL., Sensabaugh GF, Mathies RA DNA sequencing using a four-color confocal fluorescence capillary array scanner. Electrophoresis, 1996, 17, 1852-1859. _ Wang Y., Wallin JW, JU J., Sensabaugh GF, Mathies RA High-resolution capillary array electrophoretic sizing of multiplexed short tandem repeat loci using energy-transfer fluorescent primers. Electrophoresis 1996, 17, 1485-1490. _ Woolley AT, Sensabaugh GF, Mathies RA High-speed DNA genotyping using microfabricated capillary array electrophoresis chips. Anal. Chem. 1997, 69, 2181-2166. _ Woolley AT, Lao K., Glazer A_N., Mathies RA Capillary electrophoresis chips with integrated electrochemical detection. Anal. Chem., 1998, 70, 684-688. _ Woolley AT, Hadley D., Landre P., de Mello AJ, Mathies RA, Northrup MA Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem., 1996, 68, 4081-4086. _Mansfield ES et al. Versatile low-viscosity sieving matrices for non-denaturing DNA separations using capillary array electrophoresis. Electrophoresis, 1997, 18, 104-111. _ Simpson PC, Roach D., Wooley AT, Thorsen T., Johnston R., Sensabaugh GF, Mathies RA High throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates. Pr.Natl.Acad.Sci. USA, 1998, 95, 2256-2261. _ Jacobson SC, Hergenrôder R, Koutny LB, Warmack RJ, Ramsey JM. Effects of injection schemes and column geometry on the performance of microchip electrophoresis devices. Anal. Chem, 1994, 66, 1107-1113).
La Figure 53 montre un empilement de modules plats (40110) pourvus de sous-parties (40145) supportant tout ou partie des micro-canaux (41). L'empilement desdits modules élémentaires plats (40110) est effectué de manière décalée afin d'obtenir sur une face latérale un biseau. L'espacement entre lesdites sous-parties (40145) permet l'intercalation de modules plats isolants électriques (40195) dans l'empilement.Figure 53 shows a stack of flat modules (40110) provided with sub-parts (40145) supporting all or part of the micro-channels (41). The stacking of said flat elementary modules (40110) is carried out in an offset manner in order to obtain a bevel on a lateral face. The spacing between said sub-parts (40145) allows the intercalation of electrical insulating flat modules (40195) in the stack.
Ce microarray- ou macroarray multibloc en biseau peut être connecté à un deuxième empilement en biseau de modules élémentaires plats (40245) alternés avec des modules plats isolants.This bevel multi-block microarray or macroarray can be connected to a second bevel stack of flat elementary modules (40245) alternated with insulating flat modules.
Lesdits modules élémentaires plats (40245) sont pourvus à leur extrémité d'un plage de lecture (40250), ainsi que d'un dispositif d'évacuation des analytes.Said flat elementary modules (40245) are provided at their end with a reading range (40250), as well as a device for discharging the analytes.
Après avoir soumis l'ADN amplifié à une réaction de Sanger dans les micro-canaux (41) desdits micro-array ou macro-arrays multiblocs (15319), ou alternativement sur un autre micro-array ou macro-array multibloc de l'invention provisoirement connecté, une microélectrophorèse peut être envisagée en apposant une tension aux deux extrémités des microcanaux des modules élémentaires plats (40245) connectés en dernier lieu. La lecture des fragments d'élongation de la réaction de Sanger se fait sur la plage de lecture (40250) selon la méthode commandée par le type de marquage (fluorescence, élecfrochimie, etc). La détection d'analytes séparés par micro-éléctrophorèse peut aussi avoir lieu par spectrométrie de masse, ainsi qu'il est pratiqué pour des fragments de protéines ( Minarik M, Foret F, Karger BL. Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing. Electrophoresis 2000, 21, 247-254). Les micro-arrays ou macro-arrays de l'invention sont adaptés à ces process séquentiels. Ainsi qu'il a été dit précédemment, les micro-arrays ou macro-arrays multiblocs de l'invention sont aussi adaptés à des process séquentiels, comme par exemple une séparation par chromatographie suivie d'une séparation par micro-électrophorèse (Cf Tragas C, Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interace. Electrophoresis 2000, 21, 227-237).After having subjected the amplified DNA to a Sanger reaction in the micro-channels (41) of said micro-array or multi-block macro-arrays (15319), or alternatively on another micro-array or multi-block macro-array of the invention provisionally connected, microelectrophoresis can be envisaged by applying a voltage to the two ends of the microchannels of the flat elementary modules (40245) connected last. The elongation fragments of the Sanger reaction are read on the reading range (40250) according to the method controlled by the type of labeling (fluorescence, electro-chemical, etc.). Detection of separate analytes by microelectrophoresis can also take place by mass spectrometry, as is done for protein fragments (Minarik M, Foret F, Karger BL. Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing, Electrophoresis 2000, 21, 247-254). The micro-arrays or macro-arrays of the invention are suitable for these sequential processes. As mentioned above, the multi-block micro-arrays or macro-arrays of the invention are also suitable for sequential processes, such as for example separation by chromatography followed by separation by micro-electrophoresis (Cf Tragas C , Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interace. Electrophoresis 2000, 21, 227-237).
On peut aussi détecter dans chaque micropuits (42) d'un micro-array ou macro-array multibloc (15319) l'hybridation d'un fragment d'ADN sur sondes telles que des oligonucleotides ou des fragments d'ADN.One can also detect in each microwell (42) of a micro-array or multi-block macro-array (15319) the hybridization of a DNA fragment on probes such as oligonucleotides or DNA fragments.
L'hybridation peut avoir lieu soit sur des billes qu'on peut transporter d'un endroit à un autre ( Fan ZH, Mangru S, Granzow R, Heaney P, Ho W, Dong Q, Kumar R. Dynamic DNA hybridization on a chip using paramagnetic beads. Anal. Chem., 1999, 71, 4851-4859), soit sur une surface dédiée à cet usage.Hybridization can take place either on beads that can be transported from one place to another (Fan ZH, Mangru S, Granzow R, Heaney P, Ho W, Dong Q, Kumar R. Dynamic DNA hybridization on a chip using paramagnetic beads. Anal. Chem., 1999, 71, 4851-4859), or on a surface dedicated to this use.
Les sondes oligonucléotidiques sont généralement synthétsisées selon la méthode phospharamidite dans des systèmes automatisés. (Beaucage SL, Caruthers M.H. Deoxynucleoside phospharamidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, vol 22, 70, 1859-1862. 1981. _ Mcbride L.J., Caruthers, M.H. An investigation of several oligonucleotide phospharamidites usefùl for synthesizing déoxynucléotides Tetrahedron Letters 24 (3) :245-248, 1983. _ Sinha N.D. et al. Polymer Support oligonucleotide synthesis XVIII . Nucl. Acid Res. 12 (11) -.4539-4556, 1984. _ Andrus A., Wright P., Wang J., Mullah B., Baier J., Mason G., Kaufman J. Synthesis and purification in a single column on a high-throughput automated oligonucleotide production System. Nucleic Acids Symposium Séries. N° 34, 183 - 184, 1995. _ Weiler J., Hoheisel J.D. Combining the préparation of oligonucleotide arrays and synthesis of high quality primers. Analytical Biochemistry. 1996. 243, 218 - 227. _ Beier M. , Hoheisel JD. Versatile derivatisation of solid support média for covalent bonding on DNA microchips. Nue. Ac. Res. 1999, 27, 9, 1970 - 1977 . _ Supports useful in solid phase synthesis of oligonucleotides. Buendia J., Nierat J. Roussel Uclaf. US Patent 4 780 504. _ Multifunctional controlled pore glass reagent for solid phase oligonucleotide synthesis. Clontech Laboratories, Inc, Palo Alto, CA. US patent 5 141 813 _ Reusable solid support for oligonucleotide synthesis, process for production thereof and process for use thereof. Pon R., Yu S., Univ. Techn. Int.WO 9723496. _ Disposable reagent storage and delivery cartridge. Kaplan B.E., Swiderski P.M.. City of Hope, Duarfe, CA. US Patent 5 766 550. __ Singh R.K. et al. Protecting groups used in oligonucleotide synthesis : a current survey . J. Sci. § Ind. Res.49 :441-448, 1990. _ Vu H et al. Fast oligonucleotide deprotection phosporamidite chemistry for DNA synthesis. Tetrahedron Letters 31(50) :7269-7272, 1990. _ Base protecting groups and process of oligonucleotide synthesis. Yu D., Agrawal S., Habus L, Iyer R.P., Hybridon Inc, WO Patent 9849183. _ Methods and reagents for cleaving and deprotecting oligonucleotides. Beckman Instruments, Inc., Fullerton, CA. US Patent 5 518 651 - Sindelar L.E., Jaklevic J.M. High throughput DNA synthesis in a multichannel format. Nucleic Acid Research, 1995, vol 23, n°6, 982 - 987. _ Rapid, large scale automatable high pressure peptide synthesis. Nerlander M.S., Fuller W.D., Goodman M. Bio Research Inc. US Patent 4 192 798. _ Apparatus for high pressure peptide synthesis . Nerlander M.S., Fuller W.D., Goodman M. Bio Research Inc. US Patent 4 362 699 _ Chemical synthesis apparatus for préparation of polynucleotides. Bender R., Duck P.D. Ens Bio Logicals US Patent 4 353 989. Multiple reactor System and method for polynucleoti.de synthesis. Urdea M.S., Warner .O. Chiron Corporation. US Patent 4 517338. _Oligonucleotide probes are generally synthesized according to the phospharamidite method in automated systems. (Beaucage SL, Caruthers MH Deoxynucleoside phospharamidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, vol 22, 70, 1859-1862. 1981. _ Mcbride LJ, Caruthers, MH An investigation of several oligonucleotide phospharamidites usefùynuucleotides Tetrahedron Letters 24 (3): 245-248, 1983. _ Sinha ND et al. Polymer Support oligonucleotide synthesis XVIII. Nucl. Acid Res. 12 (11) -.4539-4556, 1984. _ Andrus A., Wright P. , Wang J., Mullah B., Baier J., Mason G., Kaufman J. Synthesis and purification in a single column on a high-throughput automated oligonucleotide production System. Nucleic Acids Symposium Series. N ° 34, 183 - 184, 1995. _ Weiler J., Hoheisel JD Combining the preparation of oligonucleotide arrays and synthesis of high quality primers. Analytical Biochemistry. 1996. 243, 218 - 227. _ Beier M., Hoheisel JD. Versatile derivatisation of solid support media for covalent bonding on DNA microchips. Naked. Ac. R es. 1999, 27, 9, 1970 - 1977. _ Supports useful in solid phase synthesis of oligonucleotides. Buendia J., Nierat J. Roussel Uclaf. US Patent 4,780,504. _ Multifunctional controlled pore glass reagent for solid phase oligonucleotide synthesis. Clontech Laboratories, Inc, Palo Alto, CA. US patent 5,141,813 _ Reusable solid support for oligonucleotide synthesis, process for production thereof and process for use thereof. Pon R., Yu S., Univ. Techn. Int.WO 9723496. _ Disposable reagent storage and delivery cartridge. Kaplan BE, Swiderski PM. City of Hope, Duarfe, CA. US Patent 5,766,550. __ Singh RK et al. Protecting groups used in oligonucleotide synthesis: a current survey. J. Sci. § Ind. Res. 49: 441-448, 1990. _ Vu H et al. Fast oligonucleotide of protection phosporamidite chemistry for DNA synthesis. Tetrahedron Letters 31 (50): 7269-7272, 1990. _ Base protecting groups and process of oligonucleotide synthesis. Yu D., Agrawal S., Habus L, Iyer RP, Hybridon Inc, WO Patent 9849183. _ Methods and reagents for cleaving and deprotecting oligonucleotides. Beckman Instruments, Inc., Fullerton, CA. US Patent 5,518,651 - Sindelar LE, Jaklevic JM High throughput DNA synthesis in a multichannel format. Nucleic Acid Research, 1995, vol 23, n ° 6, 982 - 987. _ Rapid, large scale automatable high pressure peptide synthesis. Nerlander MS, Fuller WD, Goodman M. Bio Research Inc. US Patent 4 192 798. _ Apparatus for high pressure peptide synthesis. Nerlander MS, Fuller WD, Goodman M. Bio Research Inc. US Patent 4,362,699 _ Chemical synthesis apparatus for preparation of polynucleotides. Bender R., Duck PD Ens Bio Logicals US Patent 4 353 989. Multiple reactor System and method for polynucleoti.de synthesis. Urdea MS, Warner .O. Chiron Corporation. US Patent 4 517338. _
Polynucleotide synthesizing apparatus. Νiina A., Ohira T., Miyamoto S., Nippon Zeon Co Ltd, US Patent 4 671 941. _ Multiple polymer synthesizer. Judd AK. SRI International. US 5 053 454. _ Polynucleotide synthesizer. Whitehouse CM., Whitehouse G.P., Sesholtz D.A., Norman D., Eastman Kodak Company, Rochester. . US patent 5 112 575. _ Automated synthesis of oligonucleotides. McGraw R.A., Grosse W.M., University of Georgia Research Foundation , Inc. US Patent 5368823. _ Apparatus for multiple simultaneous synthesis, Cody D.R., De Witt S.H.H., Hodges J.C, Kiely J.S., Moos W.H., Pavia M.R., Roth B.D., Schroeder M.C, Stankovic C.J., Warner-Lambert Company, US Patent 5 324483 _ Method and apparatus for biopolymer synthesis. Zuckermann R.N., Heubner V.D., Santi D.V., Sianti M.A.. Chiron Corporation. US Patent 5 705 610. _ Method and apparatus for biopolymer synthesis. Zuckermann R.N., Heubner V.D., Santi D.N., Sianti M.A.. Chiron Corporation. US Patent 5 705 610. _ Microscale fluid handling Systems. Karger B.L., Foret F., Zavracky, P.M., McGruer E.Ν., Xue Q., Dunayeskiy, Y.M., Northeastern University, Boston, MA,USA. US Patent 5 872 010. _ Methods and solvent vehicles for reagent delivery in oligonucleotide synthesis using automated puise jetting devices. Gamble R.C, Theriault T.P., Winder S.C, Incyte Phaπnarceutical , Inc, Palo Alto, CA. US Patent 5 874 554. _ Method and composition for oligonucleotide synthesis on an open environment support surface using high boiling point organic solvents to control evaporation. Frauendorf A. W., Brennan T.M., Protogene Lab Inc. WO Patent 9925724. _ Nery large scale immobilized polymer synthesis using mechanically directed flow paths. Winkler JL, Fodor SA, Buchko CJ, Ross DA, Aldwin L. Affymetrix, Inc Santa Clara. US Patent 5 885 837. _ General purpose gène synthesizer., Zelinka R.J., Itakura K., Sims C.W., Kaplan B.E., Systec Inc., US Patent 4 598 049. Remotely programmable matrices with memories. Nova M.P., Senyei A.E., IRORI, US 5 874214. Method and apparatus for producing position addressable combinatorial libraries. Dehlinger PJ, Palo Alto, CA. US Patent 5 763 263 . Photoelectric synthesis of DNA or protein probe arrays, Krihak M., Lee H-C, Shieh CL. Motorola . US 54810989 . Weiler J., Hoheisel J.D. Combining the préparation of oligonucleotide arrays and synthesis of high quality primers. Analytical Biochemistry. 1996. 243, 218 - 227. Beier M. , Hoheisel JD. Versatile derivatisation of solid support média for covalent bonding on DNA microchips. Nue. Ac. Res. 1999, 27, 9, 1970 - 1977. _ Process for combining parallel, oligonucleotide synthesis and préparation of oligomer chips. Weiler J., Hoheisel JD. WO Patent 9749714.Polynucleotide synthesizing apparatus. Νiina A., Ohira T., Miyamoto S., Nippon Zeon Co Ltd, US Patent 4 671 941. _ Multiple polymer synthesizer. Judd AK. SRI International. US 5,053,454. Polynucleotide synthesizer. Whitehouse CM., Whitehouse GP, Sesholtz DA, Norman D., Eastman Kodak Company, Rochester. . US patent 5,112,575. _ Automated synthesis of oligonucleotides. McGraw RA, Grosse WM, University of Georgia Research Foundation, Inc. US Patent 5368823. _ Apparatus for multiple simultaneous synthesis, Cody DR, De Witt SHH, Hodges JC, Kiely JS, Moos WH, Pavia MR, Roth BD, Schroeder MC, Stankovic CJ, Warner-Lambert Company, US Patent 5 324 483 _ Method and apparatus for biopolymer synthesis. Zuckermann RN, Heubner VD, Santi DV, Sianti MA. Chiron Corporation. US Patent 5,705,610. _ Method and apparatus for biopolymer synthesis. Zuckermann RN, Heubner VD, Santi DN, Sianti MA. Chiron Corporation. US Patent 5,705,610. _ Microscale fluid handling Systems. Karger BL, Foret F., Zavracky, PM, McGruer E.Ν., Xue Q., Dunayeskiy, YM, Northeastern University, Boston, MA, USA. US Patent 5,872,010. _ Methods and solvent vehicles for reagent delivery in oligonucleotide synthesis using automated puise jetting devices. Gamble RC, Theriault TP, Winder SC, Incyte Phaπnarceutical, Inc, Palo Alto, CA. US Patent 5,874,554. _ Method and composition for oligonucleotide synthesis on an open environment support surface using high boiling point organic solvents to control evaporation. Frauendorf AW, Brennan TM, Protogene Lab Inc. WO Patent 9925724. _ Nery large scale immobilized polymer synthesis using mechanically directed flow paths. Winkler JL, Fodor SA, Buchko CJ, Ross DA, Aldwin L. Affymetrix, Inc Santa Clara. US Patent 5 885 837. _ General purpose gene synthesizer., Zelinka RJ, Itakura K., Sims CW, Kaplan BE, Systec Inc., US Patent 4 598 049. Remotely programmable matrices with memories. Nova MP, Senyei AE, IRORI, US 5 874214. Method and apparatus for producing position addressable combinatorial libraries. Dehlinger PJ, Palo Alto, CA. US Patent 5,763,263. Photoelectric synthesis of DNA or protein probe arrays, Krihak M., Lee HC, Shieh CL. Motorola. US 54810989. Weiler J., Hoheisel JD Combining the preparation of oligonucleotide arrays and synthesis of high quality primers. Analytical Biochemistry. 1996. 243, 218 - 227. Beier M., Hoheisel JD. Versatile derivatisation of solid media support for covalent bonding on DNA microchips. Naked. Ac. Res. 1999, 27, 9, 1970 - 1977. _ Process for combining parallel, oligonucleotide synthesis and preparation of oligomer chips. Weiler J., Hoheisel JD. WO Patent 9749714.
Les sondes oligonucléotidiques peuvent être synthétisées in situ dans lesdits micro-puits (42) d'un micro-array (15319) grâce à l'utilisation de groupements protecteurs photolabiles. (Photolabile reagents for incorporation into oligonucleotide chains. Urdea M.S., Horn Thomas . Chiron Corporation . US Patent 5 258 506 . . _ Large scale photolithographic solid phase synthesis of an array of polymers. Pirrung M.C, Read J.L., Fodor S., Stryer L. . Affymax Technologies. US 5 405 783 . Very large scale immobilized polymer synthesis . Pirrung M.C, Read J.L., Fodor S., Stryer L. . Affymax Technologies. US 5 424 186 . _ Photolabile nucleoside and peptide protecting groups. Fodor S., Stryer R., Winkler JM , Holmes CP, Solas DW. Affymax Technologies. US Patent 5 489 678 . Spatially addressable immobilization of oligonucleotides and other biological polymers on surfaces. McGall. G.H., Fodor S., Sheldon E.L. Affymax Technologies. US 5 412 087.The oligonucleotide probes can be synthesized in situ in said micro-wells (42) of a micro-array (15319) thanks to the use of photolabile protective groups. (Photolabile reagents for incorporation into oligonucleotide chains. Urdea MS, Horn Thomas. Chiron Corporation. US Patent 5 258 506.. _ Large scale photolithographic solid phase synthesis of an array of polymers. Pirrung MC, Read JL, Fodor S., Stryer L Affymax Technologies. US 5,405,783. Very large scale immobilized polymer synthesis. Pirrung MC, Read JL, Fodor S., Stryer L.. Affymax Technologies. US 5,424,186. _ Photolabile nucleoside and peptide protecting groups. Fodor S. , Stryer R., Winkler JM, Holmes CP, Solas DW. Affymax Technologies. US Patent 5,489,678. Spatially addressable immobilization of oligonucleotides and other biological polymers on surfaces. McGall. GH, Fodor S., Sheldon EL Affymax Technologies. US 5 412,087.
La photoexposition pratiquée pour sélectionner les polymères à déprotéger se fait habituellement par photomasques. Mais elle peut aussi se faire grâce à l'utilisation de micro- mirroirs digitaux ( Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide micro-arrays using a digital micromirror array . Nature Biotechnology.1999. Vol 17.974-978).The photoexposure used to select the polymers to be deprotected is usually done by photomasks. But it can also be done through the use of digital micro-mirrors (Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide micro-arrays using a digital micromirror array. Nature Biotechnology. 1999. Vol 17.974-978).
Les sondes oligonucléotidiques peuvent aussi être synthétisées à l'extérieur et déposées sur lesdits micro-puits (42) dudit micro-array ou macro-array multibloc (15319) ou par un bloc multi-arrayer ou grâce à un micro-array ou macro-array multibloc connecté de l'invention. Des traitements de surface adéquats desdits micro-puits (42) peuvent autoriser une adsorption des sondes . Certaines méthodologies préconisent au contraire leur fixation covalente .The oligonucleotide probes can also be synthesized outside and deposited on said micro-wells (42) of said multi-block micro-array or macro-array (15319) or by a multi-array block or by means of a micro-array or macro. connected multiblock array of the invention. Adequate surface treatments of said micro-wells (42) can allow adsorption of the probes. Some methodologies recommend, on the contrary, their covalent fixation.
Dans un procédé de synthèse de sondes oligonucléotidiques, la synthèse peut être envisagée soit nucléotide par nucléotide, soit par groupes de plusieurs nucléotides. Les micro-arrays ou macro-arrays multiblocs de l'invention permettent, par exemple un synthèse in situ d'octamers à partir d'une bibliothèque de tetramers. Deux octamers peuvent ensuite être liés pour constituer un 16-mers.In a process for the synthesis of oligonucleotide probes, the synthesis can be envisaged either nucleotide by nucleotide, or by groups of several nucleotides. The multi-block micro-arrays or macro-arrays of the invention allow, for example an in situ synthesis of octamers from a library of tetramers. Two octamers can then be linked to form a 16-sea.
Les Figures 54 A, 54B, 54C, 54D montrent qu'on peut synthésiser in situ dans les puits d'un micro-array ou macro-array monobloc (339) des octa-nucléotides à partir de tetra-nucléotides préfixés et de tétranucléotides amenés par microcanaux (41) desdits micro-arrays ou macro- arrays multiblocs (319) de l'invention. Les Figures 55A, 55B, 55C, 55D montrent qu'on peut synthésiser in situ dans les micro-puits (42) d'un micro-array ou macro-array multibloc (319) des octa-nucléotides à partir de tetra- nucléotides préfixés et de tétranucléotides amenés par les microcanaux (41) desdits microarrays ou macro-arrays multiblocs (319) de l'invention.Figures 54 A, 54B, 54C, 54D show that one can synthesize in situ in the wells of a monoblock micro-array or macro-array (339) octa-nucleotides from prefixed tetra-nucleotides and tetranucleotides brought by microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention. Figures 55A, 55B, 55C, 55D show that one can synthesize in situ in the micro-wells (42) of a micro-array or multi-block macro-array (319) of the octa-nucleotides from prefixed tetra-nucleotides and tetranucleotides brought by the microchannels (41) of said microarrays or multiblock macro-arrays (319) of the invention.
Les Figures 5 Â, 56B, 56C, 56D montrent qu'on peut synthésiser in situ dans des portions élargies des miçro-canaux (41) d'un micro-array ou macro-array multibloc (319) des octa- nucléotides àpartir de tetra-nucléotides préfixés et de tétranucléotides amenés par lesdits microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention. La même opération peut aussi être effectuée par connexion avec un autre micro-array ou macro- array multibloc de l'invention..Figures 5A, 56B, 56C, 56D show that octa nucleotides from tetra can be synthesized in situ in enlarged portions of the micro-channels (41) of a micro-array or multi-block macro-array (319) -fixed nucleotides and tetranucleotides brought by said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention. The same operation can also be carried out by connection with another micro-array or multi-block macro-array of the invention.
Les Figures 57A, 57B, 57C montrent qu'on peut synthésiser in situ dans les puits d'un microarray ou macro-array monobloc (338) des puces à ADN dans chaque micro-puits dudit micro-array ou macro-array monobloc (338) en utilisant lesdits microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention pour amener les réactifs et en utilisant des groupements photolabiles et des micromasques pour photoexposition spécifique de chaque micro-puits dudit micro-array ou macro-array monobloc (338). Le même résultat peut être obtenu en photόexposant chacun desdits micro-puits à l'aide de micro-mirroirs, et en amenant les réactifs avec des micro-arrays ou des macro-arrays de l'invention connectés.Figures 57A, 57B, 57C show that one can synthesize in situ in the wells of a monoarray microarray or macro-array (338) DNA chips in each micro-well of said monoblock micro-array or macro-array (338 ) using said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention to supply the reagents and using photolabile groups and micromasks for specific photoexposure of each micro-well of said micro-array or monoblock macro-array (338). The same result can be obtained by photόexposing each of said micro-wells using micro-mirrors, and by bringing the reagents with micro-arrays or macro-arrays of the invention connected.
Les Figures 58A, 58B, 58C montrent qu'on peut synthésiser in situ dans les puits d'un microarray ou macro-array multibloc (319) des puces à ADN dans chaque micro-puits (42) dudit micro-array ou macro-array monobloc (338) en utilisant lesdits microcanaux (41) desdits micro-arrays ou macro-arrays multiblocs (319) de l'invention pour amener les réactifs et en utilisant des groupements photolabiles et des micromasques (187) pour photoexposition spécifique de chaque micro-puits (42) dudit micro-array ou macro-array multibloc (319). Le même résultat peut être obtenu en photoexposant chacun desdits micro-puits à l'aide de micro-mirroirs, et en amenant les réactifs avec des micro-arrays ou des macro-arrays de l'invention connectés..Figures 58A, 58B, 58C show that DNA chips can be synthesized in situ in the wells of a multi-block microarray or macro-array (319) in each micro-well (42) of said micro-array or macro-array monoblock (338) using said microchannels (41) of said micro-arrays or multi-block macro-arrays (319) of the invention to supply the reagents and using photolabile groups and micromasks (187) for specific photoexposure of each micro- well (42) of said micro-array or multi-block macro-array (319). The same result can be obtained by photoexposing each of said micro-wells using micro-mirrors, and by bringing the reagents with micro-arrays or macro-arrays of the invention connected.
Chaque puits d'un micro-array ou macro-array multiblocs de l'invention peut être adressé individuellement par des fibres optiques pour la détection des produits de réaction (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal. Biochem., 1997, 251, 270-279), ou pour contrôler des réactions électro- cinétiques ou l'aggrégation de micro-particules ou de molécules dans ledit micropuits (WO9740385A1 - Light-controUed electrokinetic assembly of microparticules near surfaces), ou pour diriger une photoexposition dans ledit micro-puits.Each well of a multi-block micro-array or macro-array of the invention can be addressed individually by optical fibers for the detection of reaction products (Healey BG, Matson RS, Walt DR, Fiberoptic DNA sensor array capable of detecting point mutations, Anal. Biochem., 1997, 251, 270-279), or to control electro- kinetics or the aggregation of microparticles or molecules in said microwell (WO9740385A1 - Light-controUed electrokinetic assembly of microparticles near surfaces), or to direct a photoexposure in said microwell.
L'étanchéité de connexion entre deux desdits micro-arrays ou macro-arrays multiblocsThe tightness of connection between two of said micro-arrays or multi-block macro-arrays
(3119) est assurée par l'emploi de polymères, soit par l'emploi de polymères pour fabriquer dans la masse les modules plats constituant l'empilement desdits micro-arrays ou macro- arrays multiblocs (3119), soit par revêtement avec un polymère de la tranche de chacun desdits modules plats constituant l'empilement , soit par l'usage d'une pièce intermédiaire monobloc en polymère faisant office de joint entre deux desdits micro-arrays ou macro- arrays multiblocs (3119).(3119) is ensured by the use of polymers, either by the use of polymers for mass production of the flat modules constituting the stack of said micro-arrays or multi-block macro-arrays (3119), or by coating with a polymer of the edge of each of said flat modules constituting the stack, either by the use of a monobloc intermediate piece of polymer acting as a joint between two of said micro-arrays or multi-block macro-arrays (3119).
Un marquage de type code barre ou code couleur ou électromagnétique de chaque module plat devant être inclus dans la constitution d'un micro-array de l'invention dédié à un échantillon ou à une opération spécifique permet la recomposition d'empilements de modules plats ayant "reçu des réactifs spécifiques dans d'autres empilements A barcode, color or electromagnetic code marking of each flat module to be included in the constitution of a micro-array of the invention dedicated to a sample or to a specific operation allows the recomposition of stacks of flat modules having " received specific reagents in other stacks

Claims

REVENDICATIONS - "Micro-arrays" ou "macro-arrays" (3119) au sens de micro-réseaux quadrillés plans denses ou très denses de puits ou de micro-emplacements où sont déposées ou synthétisées de petites quantités de molécules chimiques, biologiques ou organiques, caractérisés en ce qu'ils sont multi-blocs et micro-fabriqués par superposition et empilement de modules élémentaires plats (1) typiquement de forme aplatie, lesdits modules élémentaires plats (1) étant pourvus d'une part d'orifices d'introduction et d'évacuation des échantillons et des réactifs situés uniquement dans leur épaisseur et sur leur tranche, lesdits modules élémentaires plats (1) étant pourvus d'autre part de connexions électriques situées uniquement dans leur épaisseur et sur leur tranche, lesdits modules élémentaires plats (1) étant pourvus aussi d'un circuit fluidique avec obturateurs actionnables par l'extérieur et situés uniquement dans leur épaisseur et sur leur tranche, lesdits modules élémentaires plats (1) étant pourvus enfin de microcanaux (41) orientés parallèllement à leur surface, lesdits microcanaux (41) débouchant dans l'épaisseur et sur la tranche d'un côté au moins desdits modules élémentaires plats (1) et pouvant de ce fait être reconvertis en micro-puits (42) profonds et étroits, de sorte qu'une ligne (2119) desdits micro-puits (42) est constituée sur la tranche dudit côté dudit module élémentaire plat (1), et que l'empilement de plusieurs desdits modules élémentaires plats (1) crée une juxtaposition de lignes (2119) desdits micropuits (42) et crée par conséquent ledit microarray ou macro-array multibloc (3119) tout entier, le nombre desdits modules élémentaires plats (1) empilés constituant le nombre de lignes dudit micro-array ou macro-array (3119), et le nombre desdits micro-canaux (41) par module élémentaire plat (1) constituant le nombre de colonnes dudit micro-array ou macro-array multibloc (3119), lesdits micro-arrays ou macroarrays multiblocs (3119) pouvant être interconnectés orthogonalement deux à deux , de manière à ce que l'empilement de modules plats élémentaires (1) constituant le premier micro-array ou macro-array multibloc soit décalé par une rotation de 90° autour de l'axe des micro-canaux (41) par rapport à l'empilement des modules élémentaires plats (1) du deuxième micro-array ou macro-array multibloc, de manière à offrir la possibilité d'un parallélisme massif des réactions par configuration de matrices XY où des lignes de premiers réactifs (xl x2 .. xn) dans le premier microarray ou macro-array multibloc se croisent avec des lignes de deuxièmes réactifs spécifiques (y 1 y2 ... yn) dans le deuxième micro-array ou macro-array multibloc. - "Micro-arrays" ou "macro-arrays" (3119) selon la revendication 1, caractérisés en ce que les modules plats (1) ont des microcanaux (41) d'une longueur de quelques millimètres à quelques centimètres, et une section de très petit diamètre, de l'ordre de 5 à 500 microns, de forme arrondie ou rectangulaire ou en ligne brisée. - "Micro-arrays" ou "macro-arrays" (3119) selon la revendication 1, caractérisés en ce que leur géométrie est assurée par des encoches portées par lesdits modules élémentaires plats (1) et par des contenants épousant leur forme. - "Micro-arrays" ou "macro-arrays" (3119) selon la revendication 1, caractérisés en ce que les modules élémentaires plats (1) comportent des microcanaux (41) pourvus de micro- micro-array ou macro-array- multibloc (3119) avec un autre liquide arrivant de manière interne par le dessous dudit micro-array ou macro-array multibloc (3119). - "Micro-arrays" ou "macro-arrays" (3119) mâles (femelles) selon la revendication 1, caractérisés en ce que les micro-canaux (41) peuvent déboucher dans l'épaisseur et sur la tranche d'au moins un côté desdits modules élémentaires plats (1) à l'endroit de zones convexes (cocaves), pouvant s'accoupler avec la forme femelle (mâle) complémentaire. - "Micro-arrays" ou "macro-arrays" (37119) femelles selon la revendication 1, caractérisés en ce qu'ils sont constitués par un empilement de modules plats (37100) formant un parallélépipède dont l'une des faces constitue ledit micro-array ou macro-array (37119) et dont une autre face se connecte à un élément plat (47000) de préparation des échantillons et réactifs puis de répartition d' aliquots sur des micro-emplacements (47107), chacun desdits aliquots étant repris par un desdits modules plats (37110) à partir d'une chambre de réception (37164), puis à nouveau aliquoté et dirigé vers les micro-puits ou microemplacements (37042) dudit micro-array ou macro-array multibloc (37119).CLAIMS - "Micro-arrays" or "macro-arrays" (3119) in the sense of dense or very dense planar grid micro-networks of wells or micro-locations where small quantities of chemical, biological or organic molecules are deposited or synthesized , characterized in that they are multi-block and micro-manufactured by superposition and stacking of flat elementary modules (1) typically of flat shape, said flat elementary modules (1) being provided on the one hand with introduction orifices and for discharging samples and reagents situated only in their thickness and on their edges, said flat elementary modules (1) being provided on the other hand with electrical connections located only in their thickness and on their edges, said flat elementary modules (1) 1) being also provided with a fluid circuit with shutters operable from the outside and located only in their thickness and on their edges, said flat elementary modules (1) being finally provided with microchannels (41) oriented parallel to their surface, said microchannels (41) opening out in the thickness and on the edge of at least one side of said flat elementary modules (1) and being able to this fact be reconverted into micro-wells (42) deep and narrow, so that a line (2119) of said micro-wells (42) is formed on the edge of said side of said flat elementary module (1), and that the stacking of several of said flat elementary modules (1) creates a juxtaposition of lines (2119) of said microwells (42) and consequently creates said entire microarray or multi-block macro-array (3119), the number of said flat elementary modules (1) stacked constituting the number of lines of said micro-array or macro-array (3119), and the number of said micro-channels (41) per flat elementary module (1) constituting the number of columns of said micro-array or multi-block macro-array (3119 ), said micro -arrays or multi-block macroarrays (3119) which can be orthogonally interconnected two by two, so that the stack of elementary flat modules (1) constituting the first multi-block micro-array or macro-array is offset by a rotation of 90 ° around the axis of the micro-channels (41) relative to the stacking of the flat elementary modules (1) of the second micro-array or multi-block macro-array, so as to offer the possibility of a massive parallelism of the reactions by configuration of XY matrices where lines of first reagents (xl x2 .. xn) in the first microarray or multiblock macro-array intersect with lines of second specific reagents (y 1 y2 ... yn) in the second micro-array or multiblock macro-array. - "Micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that the flat modules (1) have microchannels (41) with a length of a few millimeters to a few centimeters, and a section of very small diameter, of the order of 5 to 500 microns, of rounded or rectangular shape or in broken line. - "Micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that their geometry is ensured by notches carried by said flat elementary modules (1) and by containers conforming to their shape. - "Micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that the flat elementary modules (1) comprise microchannels (41) provided with micro- micro-array or multi-block macro-array (3119) with another liquid arriving internally from below said multi-block micro-array or macro-array (3119). - "Micro-arrays" or "macro-arrays" (3119) male (female) according to claim 1, characterized in that the micro-channels (41) can lead into the thickness and on the edge of at least one side of said flat elementary modules (1) at the place of convex zones (cocaves), capable of coupling with the complementary female (male) shape. - "Micro-arrays" or "macro-arrays" (37119) females according to claim 1, characterized in that they consist of a stack of flat modules (37100) forming a parallelepiped of which one of the faces constitutes said micro -array or macro-array (37119) and of which another face is connected to a flat element (47000) for preparing the samples and reagents then for distributing aliquots on micro-locations (47107), each of said aliquots being taken up by one of said flat modules (37110) from a receiving chamber (37164), then again aliquoted and directed towards the micro-wells or micro-locations (37042) of said micro-array or multi-block macro-array (37119).
- "Micro-arrays" ou "macro-arrays" multiblocs (319) selon la revendication 1, caractérisés en ce que les modules élémentaires plats (110) sont pourvus d'une zone de préparation des réactifs et des échantillons avant leur envoi dans les microcanaux (41), ladite zone étant intégrée dans une circuiterie microfluidique (55) et pouvant comprendre une zone de filtration (25) avec filtres (16), une zone de purification (35) comportant une ou plusieurs microcolonnes (17), et une zone de traitement d'un réactif ou d'un échantillon filtré et purifié (45), lesdites zones étant sous le contrôle d'obturateurs positionnés dans l'épaisseur et sur la tranche desdits modules élémentaires plats (110) , lesdits obturateurs pouvant être actionnés par l'extérieur . - "Micro-arrays" ou "macro-arrays" multiblocs (319) selon la revendication 1, caractérisés en ce que les modules élémentaires plats (110) sont constitués de deux au moins sous- parties détachables (145) et (155), ladite sous-partie détachable (145) supportant tout ou partie des microcanaux (41) et pouvant être de très faible épaisseur, de l'ordre de 20 à 800 microns et ladite sous-partie détachable (155) supportant tout ou partie des composants de plus grande section. - "Micro-arrays" ou "macro-arrays" multiblocs (319) selon les revendications 1, 7, et 8, caractérisés en ce que les modules élémentaires plats (110) sont d'une part pourvus d'une circuiterie électrique (391) chargée d'activer des micro-électrodes ou des microcomposants utilisés dans ladite circuiterie microfluidique (55) et dans les micro-canaux (41), d'autre part pourvus de plots de connexion électrique (390) et (380) situés dans l'épaisseur et sur la tranche respectivement desdites sous-parties (155) et (145) desdits modules élémentaires plats (110) de manière à connecter lesdites sous- parties desdits modules élémentaires plats à une alimentation électrique. 0- "Micro-arrays" ou "macro-arrays" multiblocs (319) selon la revendication 1, caractérisés en ce que les sous-parties détachables (145) desdits modules élémentaires plats (110) peuvent être séparées des sous-parties (155) puis empilées, de manière à former un micro-array ou un macro-array multibloc (318) de densité supérieure, du fait que lesdites sous-parties détachables (145) ont une plus faible épaisseur que les sous-parties (155). - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en ce que deux "micro-arrays" ou "macro-arrays" multiblocs (3119) peuvent se connecter face à face, de telle manière que soit assuré de manière étanche et à l'abri des contaminations le mélange de fluides et de réactifs d'un premier micro-array ou macro- array multibloc (3119) avec les fluides et réactifs d' un deuxième micro-array ou macro- array multibloc (3119), ledit mélange ayant lieu en partie dans les micromélangeurs (19) , et de telle manière que soient assurées les réactions chimiques, biochimiques ou biologiques désirées sur de très petits volumes contenus dans les portions de microcanaux (41) dans lesquelles s'effectuent les réactions. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 11, caractérisés en ce que deux "micro-arrays" ou "macro-arrays" multiblocs (3119) ayant été connectés face à face peuvent être déconnectés après que les réactions chimiques désirées aient eu lieu, de telle manière qu'on puisse disposer de l'un ou l'autre ou des deux microarrays ou macro-arrays multiblocs (3119), chacun des deux ayant tout ou partie des produits de réactions désirés. - "Micro-arrays" ou "macro-arrays" multiblocs (319) selon la revendication 1, caractérisés en ce qu'ils peuvent se connecter orthogonalement à un empilement de modules élémentaires plats (946, 9046), eux-mêmes connectés directement à un empilement de modules élémentaires plats (910, 9010) munis d'un ou plusieurs compartiments (956, 9056). - "Micro-arrays" ou "macro-arrays" multiblocs (319) selon la revendication 1, caractérisés en ce qu'ils peuvent se connecter directement à un empilement de modules élémentaires plats (1046, 10046) ), eux-mêmes connectés orthogonalement à un empilement de modules élémentaires plats (1010, 10010) munis d'un ou plusieurs compartiments (1056,- "Multi-block micro-arrays" or "macro-arrays" (319) according to claim 1, characterized in that the flat elementary modules (110) are provided with a zone for preparing the reagents and samples before being sent to the microchannels (41), said zone being integrated into microfluidic circuitry (55) and may include a filtration zone (25) with filters (16), a purification zone (35) comprising one or more microcolumns (17), and a zone for treatment of a reagent or a filtered and purified sample (45), said zones being under the control of obturators positioned in the thickness and on the edge of said flat elementary modules (110), said obturators being able to be actuated from the outside. - "Multi-block" micro-arrays "or" macro-arrays "(319) according to claim 1, characterized in that the flat elementary modules (110) consist of at least two detachable sub-parts (145) and (155), said detachable sub-part (145) supporting all or part of the microchannels (41) and being able to be very thin, of the order of 20 to 800 microns and said detachable sub-part (155) supporting all or part of the components of larger section. - "Multi-block" micro-arrays "or" macro-arrays "(319) according to claims 1, 7, and 8, characterized in that the flat elementary modules (110) are on the one hand provided with electrical circuitry (391 ) responsible for activating micro-electrodes or microcomponents used in said microfluidic circuitry (55) and in micro-channels (41), on the other hand provided with electrical connection pads (390) and (380) located in the thickness and on the edge respectively of said sub-parts (155) and (145) of said flat elementary modules (110) so as to connect said sub-parts of said flat elementary modules to a power supply. 0- "Multi-block" micro-arrays "or" macro-arrays "(319) according to claim 1, characterized in that the detachable sub-parts (145) of said flat elementary modules (110) can be separated from the sub-parts (155 ) then stacked, so as to form a micro-array or a multi-block macro-array (318) of higher density, because said detachable sub-parts (145) have a smaller thickness than the sub-parts (155). - "Multi-block" micro-arrays "or" macro-arrays "(3119) according to claim 1, characterized in that two" multi-block "micro-arrays" or "macro-arrays" (3119) can be connected face to face, such so that the mixture of fluids and reagents of a first multi-block micro-array or macro-array (3119) with the fluids and reagents of a second micro-array or macro is ensured in a sealed and protected from contamination manner - multiblock array (3119), said mixing taking place partly in micromixers (19), and in such a way that the desired chemical, biochemical or biological reactions are ensured on very small volumes contained in the portions of microchannels (41) in which the reactions take place. - "Multi-block" micro-arrays "or" macro-arrays "(3119) according to claim 11, characterized in that two" multi-block "micro-arrays" or "macro-arrays" (3119) having been connected face to face can be disconnected after the desired chemical reactions have taken place, so that one or the other or both microarrays or multi-block macro-arrays (3119) are available, each having all or some of the reaction products desired. - "Multi-block" micro-arrays "or" macro-arrays "(319) according to claim 1, characterized in that they can be connected orthogonally to a stack of flat elementary modules (946, 9046), themselves connected directly to a stack of flat elementary modules (910, 9010) provided with one or more compartments (956, 9056). - "Multi-block" micro-arrays "or" macro-arrays "(319) according to claim 1, characterized in that they can be connected directly to a stack of flat elementary modules (1046, 10046)), themselves orthogonally connected to a stack of flat elementary modules (1010, 10010) provided with one or more compartments (1056,
10056). - "Micro-arrays" ou "macro-arrays" multiblocs (4319) selon la revendication 8, caractérisés en ce que les modules élémentaires plats (4110) sont pourvus de deux sous- parties (4145) et (4155), ladite sous-partie (4155) ménageant un espace important entre lesdits micro-canaux (41), ladite sous-partie (4145) recevant lesdits micro-canaux (41) dans une configuration resserrée, l'empilement desdits modules élémentaires plats (4110) constituant des micro-arrays ou macro-arrays multiblocs (4319) dont le niveau de densité est dû au resserrement desdits micro-canaux (41) sur ladite sous partie (4145), et l'empilement desdites sous-parties (4145) constituant des micro-arrays ou macro-arrays multiblocs (4318) dont le niveau encore supérieur de densité est dû à la moindre épaisseur desdites sous-parties détachables (4145). - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en qu'ils se connectent à une pièce d'aplanissement monobloc (555) desdits10056). - "Multi-block micro-arrays" or "macro-arrays" (4319) according to claim 8, characterized in that the flat elementary modules (4110) are provided with two sub-parts (4145) and (4155), said sub- part (4155) providing a large space between said micro-channels (41), said sub-part (4145) receiving said micro-channels (41) in a constricted configuration, the stacking of said flat elementary modules (4110) constituting micro multi-block arrays or macro-arrays (4319) the density level of which is due to the tightening of said micro-channels (41) on said sub-part (4145), and the stacking of said sub-parts (4145) constituting micro-arrays or multi-block macro-arrays (4318) whose even higher level of density is due to the lesser thickness of said detachable sub-parts (4145). - "Multi-block micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that they connect to a one-piece planarization part (555) of said
"microarrays" ou "macro-arrays multiblocs" (3119) mâles, ladite pièce d'aplanissement présentant au recto une face plane, et au verso son autre face qui s'emboite dans ledit "micro-array" ou "macro-array" (3119) mâle. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en ce qu'ils se connectent à une pièce (340) monobloc et plane formant un microarray de bouchons qui viennent obturer les micro-puits (42) desdits micro-arrays ou macro-arrays multiblocs (3119). - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractériserai ce qu'ils se connectent à une pièce monobloc de fixation de molécules pourvue d'un "array" de micro-puits ou de spots dont le quadrillage est calqué sur celui dudit "micro-array" ou "macro-array" multibloc (3119) offrant dans chacun de ses micro- puits ou sur chacun de ses spots une surface de fixation à une molécule utile dans un process d'analyse chimique ou biochimique ou biologique. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1 , caractérisés en ce qu'une pièce de fixation et de synthèse en phase solide de molécules est plane, monβbloc, connectable à un "micro-array " ou un " macro-array" multibloc (3119), pourvue d'un "array" de micro-puits dont le quadrillage est calqué sur celui dudit "micro- array" ou "macro-array" multibloc (3119), offrant dans chacun de ses micro-puits une surface de fixation à une molécule à synthétiser dans un process de synthèse en phase solide . - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon l'une quelconque des . revendications 1 à 19, caractérisés en ce que. tous les modules élémentaires plats, microcomposants compris ou non, sont fabriqués avec les techniques de microfabrication selon un assemblage par superposition et fusion de sous-parties planes complémentaires. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon l'une quelconque des revendications 1 à 19, caractérisés en ce que les surfaces desdits "micro-arrays" ou"microarrays" or "multi-block macro-arrays" (3119) male, said planarization part having on the front a flat face, and on the back its other face which fits into said "micro-array" or "macro-array" (3119) male. - "Micro-arrays" or "macro-arrays" multiblocks (3119) according to claim 1, characterized in that they connect to a piece (340) monobloc and plane forming a microarray of plugs which seal the micro-wells (42) of said micro-arrays or multi-block macro-arrays (3119). - "Multi-block micro-arrays" or "macro-arrays" (3119) according to claim 1, will characterize that they are connected to a monobloc piece for fixing molecules provided with an "array" of micro-wells or spots the grid of which is modeled on that of said multi-block "micro-array" or "macro-array" (3119) offering in each of its micro-wells or on each of its spots a surface for attachment to a molecule useful in a process of chemical or biochemical or biological analysis. - "Multi-block" micro-arrays "or" macro-arrays "(3119) according to claim 1, characterized in that a fixing and solid phase synthesis part of molecules is plane, monβblock, connectable to a" micro-array "or a multiblock macro-array (3119), provided with a micro-well" array "whose grid is modeled on that of said multiblock" micro-array "or" macro-array "(3119), offering in each of its micro-wells has a surface for attachment to a molecule to be synthesized in a solid phase synthesis process. - "Micro-arrays" or "macro-arrays" multiblocks (3119) according to any one of. claims 1 to 19, characterized in that. all the elementary flat modules, microcomponents included or not, are manufactured with microfabrication techniques according to an assembly by superposition and fusion of complementary flat sub-parts. - "Multi-block" micro-arrays "or" macro-arrays "(3119) according to any one of claims 1 to 19, characterized in that the surfaces of said" micro-arrays "or
"macro-arrays" multiblocs sont micro-usinées ou soumises à un travail de finition avec les mêmes techniques de microfabrication que s'il s'agissait d'une surface plane d'un seul tenant, c'est à dire essentiellement des techniques de découpage, de gravure sèche ou gravure humide par photolithographie, d'ablation laser, d'assemblage ou collage par fusion ou d'assemblage anodique, de forage, d'estampage, de soudure, d'électrodéposition, d'electroless plating ou de dépôt de vapeur chimique. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon l'une quelconque des revendications 1 à 19, caractérisés en ce que les surfaces desdits "micro-arrays" ou "macro-arrays" multiblocs, pourvus d'une pièce d'aplanissement (555) sont micro-usinées ou soumises à un travail de finition avec les mêmes techniques que s'il s'agissait d'une surface plane d'un seul tenant. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en ce que les modules élémentaires plats ont un côté au moins en forme d'arc de cercle, lesdits modules élémentaires plats étant pourvus de micro-canaux débouchant dans l'épaissseur et sur la tranche dudit côté en arc de cercle, l'empilement des modules élémentaires plats ainsi constitué ayant une face dont la surface épouse la forme d'un cylindre et étant adapté à une connexion avec des éléments homologues pour une détection à partir d'un détecteur rotatif - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 23, caractérisés en ce qu'ils sont constitués d'empilements décalés de modules élémentaires plats dont un côté au moins a une forme en arc de cercle, lesdits modules élémentaires plats étant pourvus de micro-canaux débouchant dans l'épaissseur et sur la tranche dudit côté en arc de cercle, ledit empilement décalé ainsi étant adapté à une connexion avec des éléments homologues pour une détection à partir d'un détecteur rotatif - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en ce que chaque micro-puits est adressé individuellement par des fibres optiques, lesdites fibres optiques assurant la détection des produits de réaction, ou contrôlant des réactions électro-cinétiques ou l'aggrégation de micro-particules ou de molécules dans ledit micropuits, ou dirigeant une photoexposition dans ledit micro-puits dudit micro-array ou macro-array multibloc. - "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon la revendication 1, caractérisés en ce que chaque micro-puits est adressé individuellement par des électrodes, lesdites électrodes devant contrôler la réaction en cours dans chaque micro-puits dudit micro-array ou macro-array multibloc.multiblock "macro-arrays" are micro-machined or subjected to a finishing work with the same microfabrication techniques as if it were a flat surface in one piece, ie essentially cutting, dry etching or wet etching by photolithography, laser ablation, assembly or gluing by fusion or anodic assembly, drilling, stamping, welding, electroplating, electroless plating or deposition chemical vapor. - "Multi-block" micro-arrays "or" macro-arrays "(3119) according to any one of claims 1 to 19, characterized in that the surfaces of said multi-block" micro-arrays "or" macro-arrays ", provided with a flattening part (555) are micro-machined or subjected to a finishing work with the same techniques as if it were a flat surface in one piece. - Multi-block "micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that the flat elementary modules have one side at least in the form of an arc of a circle, said flat elementary modules being provided with micro -channels opening into the thickness and on the edge of said side in an arc, the stacking flat elementary modules thus constituted having a face whose surface takes the shape of a cylinder and being suitable for connection with homologous elements for detection from a rotary detector - "Micro-arrays" or "macro-arrays "multiblock (3119) according to claim 23, characterized in that they consist of staggered stacks of flat elementary modules at least one side of which has an arcuate shape, said flat elementary modules being provided with micro-channels opening out in the thickness and on the edge of said side in an arc, said offset stack thus being suitable for connection with homologous elements for detection from a rotary detector - "Micro-arrays" or "macro-arrays" multiblock (3119) according to claim 1, characterized in that each micro-well is addressed individually by optical fibers, said optical fibers ensuring the detection of products ts of reaction, or controlling electro-kinetic reactions or the aggregation of micro-particles or molecules in said microwell, or directing a photoexposure in said micro-well of said micro-array or multi-block macro-array. - Multiblock "micro-arrays" or "macro-arrays" (3119) according to claim 1, characterized in that each micro-well is addressed individually by electrodes, said electrodes having to control the reaction in progress in each micro-well of said multi-block micro-array or macro-array.
- "Micro-arrays" ou "macro-arrays" multiblocs (3119) selon les revendications 1, 5, 8, 11, 12, 13, 14, 15, 23, 24, caractérisés en ce que l'étanchéité de connexion entre deux desdits micro-arrays ou macro-arrays multiblocs (3119) est assurée par l'emploi de polymères, soit par l'emploi de polymères pour fabriquer dans la masse les modules plats constituant l'empilement desdits micro-arrays ou macro-arrays multiblocs (3119), soit par revêtement avec un polymère de la tranche de chacun desdits modules plats constituant l'empilement , soit par l'usage d'une pièce intermédiaire monobloc en polymère faisant office de joint entre deux desdits micro-arrays ou macro-arrays multiblocs (3119).- "Multi-block" micro-arrays "or" macro-arrays "(3119) according to claims 1, 5, 8, 11, 12, 13, 14, 15, 23, 24, characterized in that the connection seal between two said multi-block micro-arrays or macro-arrays (3119) is ensured by the use of polymers, or by the use of polymers for mass production of the flat modules constituting the stack of said multi-block micro-arrays or macro-arrays ( 3119), either by coating with a polymer the edge of each of said flat modules constituting the stack, or by the use of a monobloc intermediate piece of polymer acting as a joint between two of said micro-arrays or multi-block macro-arrays (3119).
- "Micro-arrays" ou "macro-arrays" multiblocs selon la revendication 1, caractérisés en ce qu'un marquage de chaque module plat devant être inclus dans la constitution d'un micro-array de l'invention dédié à un échantillon ou à une opération spécifique permet la recomposition d'empilements de modules plats ayant reçu des réactifs spécifiques dans d'autres empilements. - "Multi-block" micro-arrays "or" macro-arrays "according to claim 1, characterized in that a marking of each flat module to be included in the constitution of a micro-array of the invention dedicated to a sample or to a specific operation allows the recomposition of stacks of flat modules having received specific reagents in other stacks.
PCT/FR2001/000881 2000-03-22 2001-03-22 Multiblock micro-arrays or macro-arrays with lab-on-a-chip WO2001070400A1 (en)

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