DE10053472B4 - Device and method for the combinatorial immobilization of sample molecules on fiber elements - Google Patents

Abstract

Device for immobilizing molecules on the outer surface of each of an endless fiber or a fiber element,
with a substantially tubular reactor unit (1),
with at least one insertion opening for an endless fiber or a fiber element,
with a supply line (2) for supplying a fluid which contains the desired sample molecules or sample molecule species or sample molecule species group for immobilization
characterized,
the molecules are sample molecules which are selected from the group consisting of nucleic acids, proteins, peptides, saccharides and mixtures of these sample molecules
in that the device comprises a plurality of reactor units (1) forming a reactor, and
in that the supply line (2) comprises a mask (5) which can be attached to the reactor units (1) and is exchangeable on the line side.

Description

The The invention relates to a device according to the preamble of the claim 1 and a method thus for the immobilization of sample molecules on fiber elements.

components of the above construction are for the rapid analysis of samples on the presence or absence of target molecules. It is essentially a parallel process, as a sample simultaneously contacted with several or all elements of the component is and target molecules with react to those elements which are specific for the target molecule sample molecules wear.

Biochips are known in various embodiments. For example, the US 5,744,305 A describes a biochip with a planar structure, on the surface of which in selected areas, the so-called spots, respectively selected and assigned sample molecules are applied. Such planar biochips are very expensive to manufacture, since each spot must be sequentially equipped with the respectively assigned sample molecules. In addition, each component is as it were a single piece, ie the production of a second biochip same structure, even the same assembly with sample molecules, again requires the same effort as the production of the first biochip. Mass production is therefore not possible and the known biochips are therefore extremely expensive.

One Component of other construction is known from US-6037186 A. Accordingly, become a plurality of porous ones rods each prepared with a solution containing a selected sample molecule species soaked, as it were become. After bundling the soaked Become a staff from the bundle in a plane orthogonal to the longitudinal extent of the bunch Slices cut off, which form the component. The cut surfaces form while the spots. Although this technology allows mass production a Biochiptyps, but has the significant disadvantage that contamination of the Spots with the sample molecules each adjacent spots take place and consequently disturbing "crosstalking" in the reading takes place. Various usual Detection methods are also not applicable to porous bodies. Finally, porous bodies have worse kinetic properties due to diffusion - controlled transport processes in the Pores inside.

Both The components described above have the common disadvantage that only one low effective sensor area with sample molecules to disposal stands. In addition, that to Reading a suitable detector above the surface, but this does not have to be positioned exactly contacting. With increasing miniaturization strengthen themselves Positioning problems and the risk of misallocation between Signals and spots increase considerably.

A component of the input structure is known from US 5837196 A known. In the case of the known component, this is a bundle of. formed optical fiber elements, whose arranged in a plane faces carry the sample molecules. Readout is by, decrease of optical signals at the opposite ends of the fiber elements carrying the sample molecules. In this structure, the problem of positioning the detector is no longer, however, must be made for producing a biochip functioning component each time a fitting of the generated faces with sample molecules with the result that a little expensive mass production of a Biochiptyps is just as possible, as in the case of a biochip according to the above-described reference US 5,744,305 A ,

From the DE-publications DE 100 53 394 C2 and DE 100 53 473 A1 a component is known as an internal state of the art having a plurality of fiber elements and with sample molecules immobilized on the fiber elements of selected sample molecule species or selected sample molecule species groups, wherein each fiber element is assigned a specific sample molecule species or sample molecule species group, the sample molecules are immobilized on shell surfaces of the fiber elements, and wherein the fiber elements by means of a support member in the radial direction, relative to the fiber elements, fixed spaced from each other or bundled with line contact to each other. Although it is not excluded that sample molecules are also bound in the volume of the fiber elements, it is essential that the interaction with target molecules takes place on or via the lateral surfaces.

Accordingly, the prior art discloses a method for producing such a component with the following process stages: a) at least one continuous fiber is produced, b) the endless fiber is passed through a fluid containing a selected sample molecule species or a selected sample molecule species group, c) the sample molecules of the D) optionally the continuous fiber is fed to at least one washing process stage; e) the endless fiber is assigned directly or indirectly to the sample sample or sample molecule group immobilized on the fiber in step c); f) of different continuous fibers or from different A fiber element is cut off in each case in regions of an endless fiber, and the fiber elements are connected or bundled with a support element. In principle, a plurality of continuous fibers may each be passed through different sample molecule species or fluid containing sample molecule species groups. Alternatively, the fluid is changed in sections in a single continuous fiber.

in the In connection with such components, it is desirable to have a particular develop efficient technology of immoilization.

From the publication DE 689 08 107 T2 For example, a method for producing a carbon-coated optical fiber is known. The optical fiber is fed to a chemical vapor deposition reactor at a predetermined speed so as to traverse it in its axis. The reactor tube is preheated by a heater to a predetermined temperature, and the raw materials for the carbon layer are introduced into the reactor tube through an upper tube. The heater can serve for the thermal decomposition of the feed material. Typical feed compositions include halogenated hydrocarbon compositions and can be used in the gaseous state or with inert gases such as argon or the like. be supplied diluted. The hydrocarbon composition is decomposed to achieve formation of a carbon coating on the surface of the optical fiber.

In the publication US 5,679,220 describes the application of calcium carbonate or equivalent fillers to papermaking fibers. The application of the filler takes place in the pulp, a first vessel having the paper pulp with the fibers of a certain strength, a second vessel having a slurry of granular calcium hydroxide and both pulps react in a reactor with a gaseous precipitant such as gaseous carbon dioxide. In this case, the application of the filler takes place in the flowing stream of the reactants.

Of the Invention is based on the object, a method and an apparatus specify by means of which in a particularly efficient way fiber elements with sample molecules can be provided In particular, a combinatorial synthesis of sample molecules to the Fiber elements feasible and the sample molecules selected from the group consisting from nucleic acids, DNA, RNA, PNA, aptamers, proteins, peptides, saccharides and mixtures of these sample molecules are.

to solution this object the invention teaches a device for immobilization of sample molecules selected from the group consisting of nucleic acids, DNA, RNA, PNA, aptamers, Proteins, peptides, saccharides and mixtures of these sample molecules on the Outer surface respectively an endless fiber or a fiber element, with a substantially tubular reactor unit, with at least one, preferably at one end face of the Reactor unit mounted insertion opening for an endless fiber or a fiber element, with a feed line for feeding a Fluids, which the desired sample molecules or sample molecule species or sample molecule species group for immobilization contains. Essentially tubular means that Any other cross-sectional areas are possible, provided the largest diameter small opposite the length is. The reactor can but must not, on the opposite face a vent for fibers exhibit.

In the supply line may be a preferably electrically controllable shut-off valve or flow control valve be introduced.

The Device may have a plurality of preferably parallel to each other, relative to a reactor center axis, running reactors have. Then Several endless fibers or fiber elements can be processed in parallel.

Of the Reactor can be reversible from two mutually mirror symmetrical reactor halves together be. This allows, for example, an endless fiber or a Fiber element first in a reactor half insert and then by attaching the second half of the reactor to close this. In this case, such a device with a plurality of reactors from two half-blocks, containing mutually complementary reactor halves, reversibly joined together. Znächst then all the fibers are inserted in the one half block and then becomes the second half block as it were pushed over.

Two or more of such constructs can be reversible as it were stacked reversibly connected to each other, wherein all reactors preferably run parallel to one another. The above described equipment with fibers then takes place for Plane, referring to the half-blocks.

The feed line can be connected to the cylinder jacket surface of a reactor. It will be appreciated that when a plurality of reactors are set up, one supply line can feed all the reactors, only a portion of the reactors, or only one reactor at a time. The feed line is connected to an end face of the reactor. The same applies. Fluid may be transferred from the reactor via either the supply line or via separate, possibly closed via valves discharge lines are deducted.

at Establishment of a plurality of reactors and reactor front side feed the fluid can be the supply line comprise a mask attachable to the reactor end face of the device. Such a mask has for certain reactors passages for a fluid, while other reactors over that Fluid are separated. By replacing the mask and the supplied Fluids or the Probenmolekülspezies can be the fibers with a plurality of sample molecule species occupy; it can be done even perform a combinatorial synthesis. Likewise, for example also a sequencing of nucleic acids possible. A mask set becomes in accordance with the desired Combination composed of a plurality of masks. It is but also possible to make a mask of a material in which in the reactor mounted state openings attach and also reseal, for example thermally by means of a laser. It is also possible a slider, which as it were a perforated tape coded successively on the slide contains different masks in front of the faces the reactors in discrete steps, a mask dimension unit, pass by. While the "forwarding" can on the slider a washing fluid should be abandoned to prevent cross-contamination.

The Invention further teaches a method for the immobilization of sample molecules selected from the group consisting of "nucleic acids, DNA, RNA, PNA, aptamers, proteins, peptides, saccharides and mixtures of these sample molecules "on the outer surface of a Fiber element or an endless fiber, wherein the fiber element or the continuous fiber is introduced into a reactor unit, wherein via a feed a fluid containing the desired sample molecules or sample molecule species or sample molecule species group fed to the reactor unit is and then wherein in the reactor unit, the immobilization of the Sample molecule species or the sample molecules or the sample molecule species group carried out becomes.

The Supply of sample molecules, possibly also the immobilization, can containing different fluids different sample molecule species be repeated. If in repetition cycles there is no immobilization, but a chain extension reaction carried out In particular, a combinatorial synthesis is possible if the respective sample molecule species the respective fluids in chain extension reaction to each other react. In the case of nucleic acids, the sample molecule species are then individual bases, from which then, for example, an oligonucleotide is built up in each reactor and therefore on each Fiber another.

insofar comprises the concept of sample molecule species also building blocks of a sample molecule structure created from this (finished sample molecule species). The combinatorial synthesis is an independent aspect of the invention.

Conveniently, In this case, a device according to one of claims 1 to 9 is used.

definitions:

• - When Fiber elements are designated by an endless fiber cut pieces. As a rule, the cut in a plane becomes orthogonal to the longitudinal extent the endless fiber is executed but it is self-evident also a contrast less than 90 ° angled Cutting plane possible.
• An endless fiber is a rod-like or thread-like structure, opposite to the length of long-length fiber elements, which can typically be made by means of drawing technologies, blowing technologies and / or extrusion technologies and wound and stored on drums or the like. An endless fiber and / or a fiber element can have the most varied cross-sectional shapes in a cross-sectional plane that is orthogonal to the longitudinal extent. Only preferred is a substantially circular cross-section. In this respect, the term of the lateral surface in the context of the invention comprises not only cylinder jacket surfaces, but also lateral surfaces in the case of non-circular cross-sections. In this respect, the term radial direction in the context of the invention denotes all directions in a cross-sectional plane. In this respect, finally, the diameter in the context of the invention designates d = 2 * (F / 2π) ^0.5 , where F is the cross-sectional area (of any shape). The end face of a fiber element is formed by a section through an endless fiber.
• - spacing the fiber elements means that the lateral surfaces adjacent Fiber elements do not touch each other. It is then a bundling the fiber elements without line contact between individual fiber elements of the bunch created. Line contact means that the (mechanical) contact not in areas of mutually parallel surfaces of adjacent fiber elements consists. A spacing of the fiber elements and / or a line contact between equivalent fiber elements is equivalent to the device of radially extending noses in the region of lateral surface a fiber element, whereby adjacent fiber elements except for the Point, line or surface contact be kept spaced apart in the region of the noses.
• - One Fiber elements Buffer usually has mutually coplanar faces of the bundled Fiber elements on. However, it is also possible to have groups within a fiber element bundle of fiber elements with coplanar faces, the strin areas of fiber elements of different groups to each other are not coplanar.
• - are sample molecules molecules which with target molecules can enter into a specific interaction. Examples of such Interactions are: antibody antigen, Lectin carbohydrate, protein aptamer, Nucleic acid nucleic acid, nucleic acid ribozyme, biotin-avidin, etc.
• - are target molecules molecules on which a sample to be analyzed, which abandoned the component is being investigated. But target molecules can also be molecules specifically from one (for example with other methods or with the same method to be analyzed) sample should be removed.
• - One Sample molecule species contains sample molecules exclusively a structure, for example a sequence in the case of nucleic acids or Proteins or peptides. Alternatively, the notion of sample molecule species in the context of combinatorial synthesis but also building blocks for a sample molecule species in the above sense, for example nucleotides, amino acids, sugar units and the same.
• - One Sample molecule species group contains as group elements at least two sample molecule species. This may be the case with the sample molecule species to deal with similar or different sample molecule types. As sample molecule types for example, nucleic acids, Called peptides, proteins and saccharides.
• - When functional groups of a polymer material are such chemical Groups of polymer backbone denotes a non-specific binding between target molecules and enable the polymer material. In the case of nucleic acids as target molecules would be functional Groups, for example, amino groups, hydroxyl groups, thiol groups or carboxyl groups. It goes without saying that what is said on the Polymer material optionally added excipients applies.
• - Cooperative Effects between molecules several sample molecule species and a target molecule species are characterized in that the energetic gain through simultaneous interaction between each the molecules the different sample molecule species on the one hand and between the different sample molecule species and the target molecule overall, on the other hand, is larger as the sum of the energetic gains of the interactions of a molecule each of a sample molecule species with a molecule the target molecule species. In the case of nucleic acids as sample molecules and target molecules are examples of stacking effects. The stacking effect is an energy gain through interactions, namely delocalization of n-electrons the hydrophobic ring structures of adjacent bases in double-stranded nucleic acids. in the Trap of proteins can cooperative effects from special secondary structures interacting with each other Proteins result. In general, cooperative effects become one higher Specificity and Binding energy of a bond between sample molecules and a target molecule reached.

It is preferred if the fiber elements are optical fiber elements. With this embodiment Signals, for example, fluorescence signals from corresponding Stimulate (optically) marker groups of fibers bound to the fibers and to Detection by means of optical sensors to optically contactable Guide the points of the fiber elements. But also luminescence can be detected. Such bodies can in particular the faces be the fiber elements. For example, optical fibers can at least partly made of glass. It is preferred, however, if the Fiber elements are formed from a polymer material, which preferably selected is from the group consist of "polyethylene (PE), Polycarbonate (PC), Polyvinylchloride (PVC), Polystyrene (PS), Polytherephthalate (PETP), Polyethersulfone (PES), Polyetheretherketone (PEEK), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polybuthylene terephthalate (PBT, Polyoxymethylene (POM), polysulfone (PSU), polyetherimide (PEI), polyamide (PA) and mixtures and copolymers of the monomers of such polymers ", in particular selected from the group consisting of "polycarbonate (PC), polyvinyl chloride (PVC), polystyrene and mixtures as well as copolymers the monomers of such polymers ". Essential in the selection of materials is only that the material is sufficient (permanently) temperature resistant against in the course of processing or use of the biochips occurring temperatures. As in this sense, be resistant to high temperatures all polymer materials, which under exposure to at least 90 ° C, preferably at least 120 ° C, in particular at least 140 ° C, over 1000 hours as resistant prove. Typically, this is satisfied when the glass transition temperature is above half the said temperature limit is.

Especially temperature resistant is for example polycarbonate with a glass transition temperature of 150 ° C. It understands that the Polymer material in plastic technology contain conventional excipients can, such as plasticizers, light stabilizers, in particular UV stabilizers, and the like. Also, the (wavelength dependent) dielectric constant influencing additives be incorporated for the purpose of optimizing the optical properties in a wavelength range of interest.

One Fiber element can also be made of several materials in a composite be. In particular, you can in the area of the lateral surface be different materials used by the material of the core, for example, the reflectivity of running in the fiber element Light at the interface solid / liquid or solid / gas optionally wavelength-selective to modify. Also, for example, the core of a mechanical rigid material, for example metal, glass or polycarbonate, during the the core surrounding optically transparent material then less rigid can be.

In geometric terms, the fiber elements may be formed and arranged as follows. The fiber elements preferably have a diameter in the range of 0.01 μm to 1000 μm and a length in the range of 0.1 μm to 100 mm. The diameter to length ratio can range from 100 to 10 ^-9 . Furthermore, it is preferred if the fiber elements are packed in a density of 1 to 10 ⁷ fibers / cm ² , based on a radial cross-sectional plane of the fiber elements.

The Sample molecule species or sample molecule species group can be selected be from the group consisting of "nucleic acids, DNA, RNA, PNA, aptamers, Proteins, peptides, saccharides and mixtures of these sample molecules. "In the case of nucleic acids it is single-stranded or double-stranded nucleic acids is. The bases of the nucleic acids can naturally occurring bases, they can but also chemically derivatized, for example by incorporation of marker groups. The same applies in the case of the other classes of compounds the aforementioned group. Likewise, the sequences can be natural or not natural be. In the case of nucleic acids In principle, the number of bases of a hybridizable region can be arbitrary To be long. However, it is recommended that the number of bases possible keep small, for example, to a maximum of 25, better 17, limit, thus a mismatch between a sample molecule and a target molecule in only a base leads to a sufficient destabilization.

The sample molecules can directly or via Spacer compounds to be bonded to the fiber elements. The latter is preferred. As spacer compounds are all customary Connections in question. For example, the spacer compound from a chain (for example, the length 5 to 80, preferably 25 to 40 bases) of (identical) nucleic acid bases, for example thymidine, be formed. Then it is advantageous if the number of bases greater than in the spacer compound is the number of bases in a hybridizable region of the sample molecule. It is also possible, to use synthetic organic oligomers or polymers in whose Polymer chain, for example carbamate groups can be incorporated. It is possible, too two or more sample molecules over one To connect spacer compound and the spacer compound via a Binding site of the spacer compound to bind to the fiber element.

each Fiberelement can be a sample molecules each selected, different sample molecule species wear. In other words, each fiber element carries sample molecules one single structure, where the structures of the sample molecules different Fiber elements differ. But it is also possible that each Fiber element sample molecules a respective selected, different Probenmolekülspeziesgruppe carries, wherein the group elements of each sample molecule species group together under Training of cooperative effects, such as stacking, to a defined target molecule tie. Then wear each fiber element sample molecules with two (or more) different structures (group elements), wherein different fiber elements have different combinations on sample molecules bear different structures. In terms of used Cooperative effects will be recommended, the molar amounts the sample molecules different structures on a fiber element within Deviations up to + 50% the same size. It is advisable, that the Sample molecules different Structure on a fiber element in terms of spatial Arrangement of the binding sites on the fiber element stochastically distributed should.

It is possible, too, within one or more of the fiber elements each discrete Fields set up which different sample molecule species or sample molecule species groups wear.

In With regard to a reduction of non-specific binding of target molecules directly on the surfaces the fiber elements, it is advantageous if the polymer material carries no functional groups on its surface, and if the sample molecules are nucleic acids, that the nucleic acids from a preferably aqueous solution below Irradiation with UV light to the surface of the polymer material are bound.

Of course it is possible an inventive component not only analytically for the detection of target molecules, but also preparative, For example, for the targeted separation of substances from complex Mixtures, such as blood.

A erfindunggemäßer reactor can be constructed of a variety of materials. So it is possible, for example, the reactor or Bautei le from an optically transparent material, in particular for UV transparent to make. Then it is possible to carry out immobilization processes under UV irradiation, simultaneously in all the reactors of a structural unit.

in the Below, the invention is based on merely embodiments illustrative examples closer explained. It shows:

1 A device according to the invention with a plurality of reactor units 1 , built from a total of four reactor halves 6 .

2 : Detail view of a reactor half with feed lines connected to cylinder jacket surfaces of the reactor units 6 and

3 A device according to the invention with reactor-end surface-side supply of fluids via one or more masks 5 ,

The figures speak for themselves, so that may be referred to. In such a reactor, for example, the following reaction can be carried out. An oligonucleotide of the sequence T (20) -AGT CTA ATC TGA TCT AGA is introduced into a crosslink solution containing 1 M NaCl, 0.2 M MgCl ₂ and 0.3 M Tris, pH 8, at a concentration of 10 nM. In a reactor unit half 3 a device according to the invention a PS endless fiber of 80 microns in diameter, which was extruded in a conventional manner inserted. Then the second half of the reactor is put on. The feed line 2 is over a valve 4 with a peristaltic pump communicating with a reservoir in which the above oligonucleotide solution is stored. The oligonucleotide solution is introduced into the reactor and the UV-transparent reactor is irradiated with an overlying UV lamp with a main emission at 254 nm. Thereafter, the endless fiber, if necessary after a washing step, again removed and supplied for further processing in the context of a 3D biochip.

1

reactor

2

retort half

3

Valve

4

feed

5

electrical control lines

6

pass socket

7

dowel

8th

mask

Claims (5)

Device for immobilizing molecules on the outer surface of each of an endless fiber or a fiber element, with a substantially tubular reactor unit ( 1 ), with at least one insertion opening for an endless fiber or a fiber element, with a supply line ( 2 ) for supplying a fluid which contains the desired sample molecules or sample molecule species or immobilized speciation group, characterized in that the molecules are sample molecules selected from the group consisting of nucleic acids, proteins, peptides, saccharides and mixtures of these sample molecules from a reactor forming reactor units ( 1 ) and that the supply line ( 2 ) a strinseitig the reactor units ( 1 ) attachable and replaceable mask ( 5 ). Apparatus according to claim 1, characterized in that in the supply line ( 2 ) a preferably electrically controllable shut-off valve or flow control valve ( 4 ) is introduced. Device according to one of claims 1 or 2, characterized in that the reactor consists of two mutually mirror-symmetrical reactor halves ( 6 ) is reversibly joined together. Method for immobilizing sample molecules selected from the group consisting of nucleic acids, proteins, peptides, saccharides, and mixtures of these sample molecules on the outer surface of each of a fiber element or an endless fiber, wherein the fiber element or the endless fibers in a reactor unit ( 1 ) is introduced into a device according to one of claims 1 to 3, wherein via a supply line ( 2 ) a fluid containing the desired sample molecules or sample molecule species or sample molecule species group is supplied to the reactor unit and then in the reactor unit the immobilization of the sample molecules or sample molecule species or sample molecule species group is performed. The method of claim 4, wherein the supply and the Immobilization with different fluids containing different sample molecules or sample molecule species or sample molecule species groups is repeated.