DE10113708A1 - Applying reagents to measurement bearer pits involves aligning stamp relative to bearer and bringing stamp into contact with bearer, whereby reagent remains on corresponding bearer pits - Google Patents

Abstract

According to the invention, one or more stamps are used to apply reagents to punctiform locations of a measurement carrier that are arranged similar to the pits of a CD. The stamps have pits, which correspond to the punctiform locations of the measurement carrier and which are wetted with different reagents by using a stamping pad. The reagents are transferred to the punctiform locations of the measurement carrier by pressing the stamp against the measurement carrier.

Description

The invention relates to methods and devices for Application of reagents to punctiform areas. Under punctiform points are points of a measuring carrier understand that on the order of pits from CDs or lie below.

An application where reagents are on punctiform spots analytical chemistry is to be applied. On a single-use carrier should be on exactly defined very small spots molecules are applied. It will very complex variations of different molecules different places applied. It's supposed to be a direct one and spatially reproducible addressing of the structures be enough. Such an arrangement could be used, among other things use for complex analytical detection methods the majority are based on the optical detection of molecules based on. Another application would be creation of molecular libraries on a support.  

Gene chips are generated either by direct synthesis from Oligonu Kleotiden on the carrier material or by systematic Application made using a pipetting robot. This Procedures are only applicable to a limited extent. The density and precision sion of the spots is limited, which affects the quality becomes.

The invention has for its object a possibility create a large number of different reagents to precisely addressable selectable punctiform digits of a measurement carrier.

The invention proposes a method for solving this problem with the features mentioned in claim 1. The invention also proposes a device for performing this Procedure as well as a stamp. Further training of the Er invention are the subject of subclaims, their wording as well as the wording of the summary by reference is made the content of the description.

Equally suitable as CD are their further developments, such as CD-ROM, CD-R and DVD as well as magneto-optical and mag netic carrier. In the further description the The term CD always synonymous with all such carriers and measurements carrier used.

The method proposed by the invention proceeds in such a way that at least one stamp is used, the punctiform Locations that are a subset of the punctiform Place the measuring carrier exactly. Under correspondence is to be understood as a mirror image. On the the reagents are in punctiform places on the stamp applied a thin layer. The stamp is opposite precisely aligned with the measuring carrier and placed on it  or pressed. This will ensure the reagents are exactly on the Places where they should be placed.

In a development of the invention it can be provided that the pits of the subset within a contiguous Be realm of a measurement carrier and thus also of the stamp be classified. Such a subset can, for example 100,000 punctiform digits contained within this Description can also be referred to as pits. It will be there assumed that the pits are similar to a CD are ordered. But there is no need for deepening or increases, but can also make such posts include that in a different way optically from the environment can be distinguished.

In a development of the invention it can be provided that a Stamp is used that has multiple subsets of the pits Measurement carrier addressed by appropriately structured pits. These different subsets can then be used with different Reagents are provided. This makes it possible to get the number to keep the required stamp small.

In further developments of the invention it can be provided that a stamp for applying several reagents to several Subsets of pits can be used.

To match all subsets of pits of the measurement carrier with the ent Providing speaking reagents can according to the invention be provided to use multiple stamps. these can then used sequentially to measure all of the pits to be provided with the appropriate materials.  

However, it is also possible and is based on the invention suggested using only a single stamp, the apply all reagents to all pits of the measurement carrier.

According to the invention it can be provided that the reagents in one monomolecular layer on the pits of the stamp bring it so that when stamping it in this layer be applied to the pits of the measurement carrier. In doing so the pits of the stamp are then free again. Such a stamp can be used immediately afterwards to create a second layer of the same reagent or another Take up reagent and stem again on the measuring carrier PelN. This can also be used to advantage sequentially molecular components of a polymer chemically to synthesize in the solid phase.

The invention also proposes to use molecules that are in serve as a reagent in a combination. These molecules can can be applied to the same pits one after the other. Therefore the invention proposes to open the pits of the stamp bring several different reagents to the same pits of the measurement carrier. So a stamp can do that serve the same subsets of the measurement in a row carrier with the same subsets but different Stamp reagents.

In connection with the above statements is a be particularly important application of the technique described to raise, which is also independent of the previously described Invention is important. This will be briefly below being represented.

Both oligonucleotides and oligopeptides are known with the help of the so-called solid phase technique on a solid phase  synthesized. For example, in oligonucleotide Solid-phase synthesis is the protective group for each synthesis step of the previously bound nucleotide monomer chemically with high Split efficiency. Then a new monomer in high concentration applied to a good saturation of the to reach free binding sites. Such a solid phase Synthesis is also used in the production of microarray genes chips applied, but with optically reactive protective groups with the help of lithographic masks and successive exposure must be split off. The application of the nucleotide Monomers then take place over the entire chip surface area. Light-activated protecting groups, however, face each other chemically cleaved protecting groups have the disadvantage that they on the one hand spontaneously split off in small numbers and on the other others are less completely removed by exposure can.

The application of the previously described invention Technology now makes it possible to use the ones described To avoid disadvantages. So the well-known conventional Solid phase synthesis (with chemically separated protective groups) both for oligonucleotides and for oligopeptides a corresponding array structure can be used. there the corresponding monomers or other polymer construction stones, preferably in a highly concentrated solution Stamp applied and with the help of these stamps exactly on applied the appropriate carrier. The remaining steps the synthesis of the oligonucleotide or oligopeptide can with With the help of conventional "chemical" solid phase technology respectively. Accordingly, complicated patterns can be created of oligonucleotides / oligopeptides on the array surface, d. H. for example on the surface of a CD polycar generate bonat slice without optically removable protection groups are needed. So this invention combines the  Advantages of (conventional) solid phase synthesis with those the so-called array technology.

This aspect will be explained in the following based on the structure of a Oligonucleotide are shown. It is obvious, that the corresponding technology by a specialist without white teres also on making connections from chemical or biochemical building blocks exist, such as for example oligopeptides can be transferred. In this case, for example, instead of the corresponding Nucleotide monomers inserted the corresponding amino acids puts.

According to the number of different nucleotides (A, C, G and T) for each position at which these nucleotides to be installed, a corresponding number of Stamping needed, d. H. usually four stamps. The Array surface is usually first with a linker, preferably a polycarbonate linker provided, however if appropriate also together with the first monomers can be brought. Then the stamp is selected, the to fits all places on the carrier where the syntheti based oligonucleotide, for example with monomer A. should start. A high con concentration of reactive monomers A (with protective group) introduced. The "first stamps" for the monomers C, G and T selected and coated. Then the appropriate places on the carrier are stamped and transfer the monomers to these sites. Selbstver the first stamps can also be used independently of one another be used. It is not imperative that everyone first First stamp coated with the monomers and then on the carrier be transferred.  

After the start nucleotides have been applied to the corresponding sites on the support, the unbound nucleotides are washed off the support and all protective groups are removed chemically (in one go). The stamps are then selected which are required in order to apply the second nucleotide monomers in a desired manner to the associated first monomers which have already been applied. These stamps are again coated with the corresponding monomers and these monomers (with protective group) are transferred / stamped onto the corresponding points. Then the washing step and the chemical removal of the protective groups take place again. This process is continued until the desired oligonucleotides have been synthesized. If, for example, oligonucleotides are to be produced from 20 nucleic acids at all locations on the support to be stamped, 20 .4, ie 80 stamps, are required for their synthesis.

For applying the reagents to the pits of the stamp or the stamp proposes the invention as a possibility to use an ink pad, for example with the help filled by Microfluidics or by a pipetting robot becomes.

To get the desired reagent on the correct pits of the stamp can apply if the ink pad several Contains reagents, be provided that the stamp against is aligned over the ink pad before using the Ink pad is brought into contact.

According to the invention it can be provided that for repeated Wetting the same pits with different reagents Stamps in several positions opposite an ink pad can be aligned.  

It is of course also possible for a stamp to use multiple ink pads.

According to the invention it can be provided that the pits of the Measurement carrier and thus also the ram along a curve or lines can be arranged. In particular, can be provided be that the pits are arranged along a spiral, if the measuring carrier is a rotating disc is.

It can also be provided that deviates from the spiral sections are provided in which the Curvature of the line along which the pits are arranged does not change or changes in the specified manner. For example the line in these areas is straight. While with a pure spiral, the curvature at every point the curve changes, areas can be created in which the arrangement of the pits is the same. Let it through stamps are used in several places on the measuring carrier.

It can be provided according to the invention that a plurality such sections with identical structure of the pits see is. For example, all can be addressed pits can be arranged in such areas.

The pits of the measuring carrier and / or the stamp can, like already mentioned briefly, as elevations and / or depressions be formed. You can also look in any other way are formed when their surface texture be visually distinguished from the surrounding areas can.

The device proposed by the invention for through implementation of the method contains a holder for the measuring carrier,  at least one stamp, at least one Subset of the pits of the measurement carrier corresponding pits has an alignment device for mutual alignment device of measuring carrier and stamp, as well as a device for Wetting the pits of the stamp.

According to the invention, it can be provided in a further development that the measuring carrier is circular disks, for example in the form and size of CDs or the like. Then you can read out the usual methods for reading CDs of reactions can be used.

With the device for wetting the pits of the stamp can it is advantageously an ink pad, given if more than one ink pad for one stamp.

The ink pad can have several departments have different reagents. These departments can be formed as coherent areas that are separated from each other by partitions.

The alignment of the stamp with respect to the measuring carrier can work mechanically, for example, with help of three pyramid-like depressions of the measuring carrier and three corresponding projections of the stamp. It is just if an optical alignment option is conceivable, with a or two crosshairs.

A similar alignment device can also be provided around the stamp or several stamps opposite the Align the ink pad, with several alignment posi tions can be provided.  

To the stamp pad in a reproducible way with the measurement A pressure device can bring the carrier into contact be provided.

The invention also proposes a measuring carrier that has a plurality of punctiform points that one predefined patterns are arranged accordingly. These pits are provided with reagents, the type and arrangement in a specified data stream are noted. The point digits are divided into subsets, whereby for the punctiform places of each subset a certain rea is provided that ver ver should be applied.

The subsets of pits are advantageously combined in one hanging surface arranged.

The invention also proposes a stamp for application of reagents on selected punctiform sites a multitude of measuring points carrier.

Further features, details and advantages of the invention are more preferred from the following description Embodiments of the invention, the claims and based on the drawing. Here show:

Figs. 1 to 6, the process steps for producing a measuring beam;

Fig. 7 to 9 process steps for producing a stamp;

Fig. 10 to 12 different process steps in a second embodiment;

FIG. 13 is a possibility of producing a submaster from the master structure;

Fig. 14 shows a further possibility of producing a submaster.

FIG. 15 is a plurality of ways in which the pits may be formed of the punch and the measuring beam;

Fig. 16 schematically illustrates the arrangement of different departments of an ink pad;

Fig. 17 shows the arrangement of several fields with identical arrangement of the dot-shaped sites.

The invention uses measuring carriers in which a variety of punctiform places according to a given pattern to be ordered. Therefore, one possibility is first wrote how such measurement carriers in precisely defined Way can be made.

Fig. 1 shows schematically and simplified a cross section through a glass master 1 , which is coated on one side with a photoresist 2 . This forms the output for the creation of a master. Layer 2 of the photoresist is exposed with the aid of a controlled laser, the laser beam exposing point-like locations which are arranged along a spiral line. This forms the original spiral shown schematically in FIG. 2.

A father 3 (see FIG. 3) is produced by nickel deposition, which thus represents a negative of the structure of FIG. 2. A further nickel deposition produces a mother structure 4 from the father 3, see FIG. 4. A son structure 5 is produced from the mother structure 4 by a further deposition process. Any number of son structures 5 can be produced. This son structure 5 is used to manufacture support structures 6 in the form of blank CDs on which all pits are present using injection molding processes. This is shown in Fig. 6 Darge. Any number of supports 6 can also be generated from any number of son structures 5 .

FIG. 7 shows how a glass master coated with negative photoresist is exposed from the glass master 1 shown in FIG. 2. This results in a negative substrate 7 , which is shown in FIG. 7 above the glass master 1 .

With the aid of a laser beam 8 , which is focused with optical devices 9 , the photoresist layer 10 of a further substrate 11 is partially exposed through the negative substrate 7 (see FIG. 8). From this further substrate 11 , which represents a first submaster, a negative for a silicone stamp is produced by depositing nickel stamps or by repeated electroforming. This stamp 12 is shown in FIG. 9. It contains are a subset of ridges 13, the animals at the Kontakt certain subsets of ridges 14 of the measuring beam 6 shown in FIG. 6 is determined. Detector molecules can be applied to certain elevations or pits of the measuring carrier by aligned contact of the stamps 12 with the measuring carriers 6 . By using a large number of stamps, which are produced with the help of many submasters, a wide variety of detector materials can be applied to the different subsets of pits.

A measuring carrier provided with the detector molecules can then be wetted with a sample to be examined, which then leads to various reactions. The result can then be read out using a CD device, whereby the assignment between the measurement results and the evaluator software.

FIGS. 10 and 11 show in which a nega tive substrate 15 is produced by coating with a negative photoresist and exposure by the Urmaster through an embodiment. A negative substrate 18, mirror-image spiral, is produced from the negative substrate 15 by exposure. This negative substrate 18 is used in a similar manner to produce a son substrate 16 , see FIG. 12, with the aid of which an inverse CD 17 can be produced by injection molding. The pits protrude from the side of the CD facing away from the laser. Starting from the substrate 18 , a further substrate with the original spiral can be produced by exposing a positive photoresist. This can be processed further according to FIGS. 8 and 9 into masters for silicone stamps, which subsets of the “negative” pits protruding from the CD can address.

In a further embodiment of the method, starting from a negative substrate 7, an etching mask is created by congruent mastering, which addresses defined subsets of lands. Applying a photo-resistive layer to a son and then exposing it through the congruent etching mask subsequently permits targeted etching of the son. This creates structures of a defined depth on the son, which after the injection molding on the CD represent elevations that protrude from the land plane from the side facing away from the laser. The height of these structures can differ from the depth of the pits. The increase allows greater tolerances both in the manufacture of the stamp and in the alignment of the CD and stamp.

FIG. 13 shows in more detail an alternative method step to the method shown in FIG. 8. A laser double head 20 contains two permanently connected laser heads. A laser head is used to generate a weak reading beam, represented by the double arrow 21 . This reading beam 21 reads out the negatively oriented CD master 22 . The laser head 20 contains a second strong laser that generates a write beam 23 controlled. This write beam 23 can be switched on and off by the logic of the double head 20 . A connected computer searches for the subset of the master's pits to be copied and controls the read-write logic. The fixed coupling of the two laser heads, in conjunction with a definition of a parallel orientation to the axis of rotation, ensures a spatial 1: 1 structure of the subset structure of the pits. The actual process for manipulating the stamp consists of curing or entry into the master stamp structure 24 , which is held on the underside of a stamp holder 25, for example by negative pressure. At the bottom of the stamp holder 25 existing solvent 26 is cured by exposure to the writing beam 23 . After the subset of pits is completely written, the excess solvent is rinsed out and removed. Tracking is only in the reading direction, since both heads are rigidly connected and the entire head is moved. The focus is adjusted in both arms.

FIG. 14 shows a device which is similar to the device according to FIG. 13. It is used here to create a positive sub-copy of a CD master. Connected by the axis 27 , the CD master 22 and the sub-copy 28 of the CD master that are to be produced rotate synchronously with one another. The side of the copy 28 facing the laser head 20 is coated with a layer 29 of photoresist which can be exposed by the write beam 23 . A connected computer searches for the subgroup of the master CD pits to be copied and controls the read-write logic. Attention is paid to an adapted focus size of the two beams, namely the reading beam 21 and the writing beam 23 . With the same size of the focus of the two beams, this results in a 1: 1 copy of the pits.

FIG. 15 now shows three different ways which can be designed in accordance to be brought punctate points of the stamp and the measuring beam together. For the sake of simplicity, it should be assumed that the respective measuring carrier 20 is arranged at the bottom, while the plunger 21 is arranged in the correct orientation above the measuring carrier 20 .

In the embodiment shown in FIG. 15 a, the punctiform points of the measuring carrier 20 are designed as elevations 22 , while at the same time the punctiform points of the stamp 21 are also designed as elevations 23 . If the ridges are provided with a monomolecular layer 23, this layer is at the contact between the stamp 21 and measurement beam 20 on the ridges 22 of the measuring beam 20 transmitted.

In FIG. 15b, the punctiform points of the measuring carrier 20 are again designed as elevations 22 . The punctiform points of the stamp 21 , on the other hand, are formed as depressions 24 . Such depressions 24 can serve to receive the reagent to be transferred as drops. This also allows the reagent to be transferred upon contact between the stamp 21 and the measuring carrier 20 .

Fig. 15c shows a third possibility, the ram 21 is formed with a smooth surface 25 in fact. The reagent to be transferred can also be applied to this smooth surface 25 as point-like spots. This also makes a transfer to the increases 22 of the measurement carrier 20 possible.

Of course, the punctiform points of the measuring carrier 20 can also be designed differently, namely also as depressions or smooth points on a smooth surface, so that the role of the stamp 21 and measuring carrier 20 can be reversed.

Fig. 16 shows a section of a stamp pad. This stamp pad contains arcuate sections 26 in which a particular reagent is present. The departments are arranged in concentric circles, along a spiral or linear. They are separated from one another by areas 27 , 28 without reagent. Each area 26 of the stamp pad corresponds to a specific area of punctiform points on the stamp and thus also on the measurement carrier. For example, 100,000 punctiform elevations can be arranged in a region of the stamp corresponding to a region 26 , which then stamp a corresponding number of pits of the measuring carrier with a specific material.

The invention uses circular disks in the form of conventional CDs or similar derivatives as measuring carriers. In this way, the usual methods for tracking the pits should be used with CD's. The pits on the CD's are arranged along a spiral that constantly changes their curvature. Deviations from a correct spiral shape can, however, be corrected by the usual reading devices. Here, the invention now proposes to consciously produce deviations from the spiral shape, for example in the shape as shown in FIG. 17. Here, in certain areas 29, the pits are arranged along a straight line, in which case the connection between the pits of one area and the pits of a next area is established by a line 31 , which ensures that the spiral shape is achieved again overall. The provision of regions 29 , 30 in which the curve has a constant curvature has the advantage that the regions 29 , 30 can be processed with the same stamp. It is also not necessary to deal with areas with linear arrangements of the pits. Concentric circles are also possible.

Claims (30)

1. A method for applying reagents to selected punctiform points on a measuring carrier ( 20 ), with the following steps:

• 1. 1.1 at least one stamp ( 21 ) is used, the
  • 1. 1.1.1 has pits corresponding to at least a subset of the pits of the measuring carrier ( 20 ),
• 2. 1.2 the pits of the stamp ( 21 ) are provided with a specific reagent,
• 3. 1.3 the stamp ( 21 ) is aligned with the measuring carrier ( 20 ),
• 4. 1.4 the stamp ( 21 ) is brought into contact with the measuring carrier ( 20 )
• 5. 1.5 the reagent remains on the pits of the subset of pits of the measuring carrier ( 20 ).

2. The method according to claim 1, wherein the pits of the subset of the measuring carrier ( 20 ) and the stamp ( 21 ) are arranged within a coherent area of the measuring carrier ( 20 ) and the stamp ( 21 ). 3. The method according to claim 1 or 2, in which a stamp ( 21 ) is used which addresses a plurality of subsets of pits of the measuring carrier ( 20 ) by appropriately structured pits. 4. The method of claim 3, wherein the different which subsets of pits with different Reagents are provided. 5. The method according to any one of the preceding claims, in which a stamp ( 21 ) for bringing up several reagents on several subsets of pits of the measuring carrier ( 20 ) is used. 6. The method according to any one of the preceding claims, in which a plurality of stamps ( 21 ) are used in succession. 7. The method according to any one of claims 1 to 5, in which a single stamp ( 21 ) for bringing up all reagents on all pits of the measuring carrier ( 20 ) is used. 8. The method according to any one of the preceding claims, in which reagents are applied in a monomolecular layer to the pits of the stamp ( 21 ) in such a way that the stamp surface is released again after stamping the measuring carrier ( 20 ). 9. The method according to any one of the preceding claims, in which the pits of the stamp ( 21 ) for applying several different reagents to the same pits of the measuring carrier ( 20 ) are used. 10. The method according to any one of the preceding claims, in which at least one stamp pad is used to apply the reagents to the pits of the stamp ( 21 ). 11. The method according to claim 10, wherein the stamp ( 21 ) is aligned with the ink pad. 12. The method according to claim 10 or 11, wherein for applying different reagents to the same pits, the stamp ( 21 ) is aligned in several positions with respect to the stamp pad. 13. The method according to any one of the preceding claims, wherein the pits of the measuring carrier ( 20 ) and / or the plunger ( 21 ) are arranged along a continuous curve. 14. The method according to claim 13, wherein the pits of the measuring carrier ( 20 ) and the pits of the stamp ( 21 ) are arranged along a spiral. 15. The method according to claim 13, wherein the curve deviates from a spiral in regions ( 29 , 30 ) in which the curvature of the curve along which the pits are arranged does not change. 16. The method according to claim 15, having a plurality of such sections ( 29 , 30 ). 17. The method according to any one of the preceding claims, in which as pits of the measuring carrier ( 20 ) and / or the stamp ( 21 ) elevations ( 22 , 23 ) and / or depressions ( 24 ) and / or in their surface quality from the rest of the measuring carrier ( 20 ) and / or the stamp ( 21 ) different places can be used. 18. Device for applying reagents to selected punctiform points of a measuring carrier ( 20 ) with

• 1. 18.1 a holder for the measuring carrier ( 20 ),
• 2. 18.2 at least one stamp ( 21 ) that
  • 1. 18.2.1 has pits corresponding to at least a subset of the pits of the measurement carrier ( 20 ),
• 3. 18.3 an alignment device for mutual alignment of the measuring carrier ( 20 ) and stamp ( 21 ), and with
• 4. 18.4 a device for applying the reagents to the pits of the stamp ( 21 ), in particular by wetting.

19. The apparatus of claim 18, wherein the measuring carrier ( 20 ) is a circular disc. 20. The apparatus of claim 18 or 19, wherein the pits of the subset are arranged within a contiguous area of the measurement carrier ( 20 ). 21. Device according to one of claims 18 to 20, wherein the device for applying the reagents to the pits of the stamp ( 21 ) comprises a stamp pad. 22. The apparatus of claim 21, wherein the stamp pad has a plurality of compartments ( 26 ) with different reagents. 23. The apparatus of claim 21 or 22, with an alignment device for aligning the stamp ( 21 ) with respect to the stamp pad, optionally in several positions. 24. Device according to one of claims 18 to 22, with a pressing device for pressing the stamp ( 21 ) onto the measuring carrier ( 20 ). 25. The device according to any one of claims 18 to 24, wherein the alignment device between the punch ( 21 ) and measuring carrier ( 20 ) works optically and / or mechanically. 26. measuring carrier ( 20 ) for carrying out a large number of reactions, with

• 1. 26.1 a variety of punctiform points (pits) that
• 2. 26.2 arranged according to a predetermined pattern and
• 3. 26.3 are provided with reagents, whereby
• 4. 26.4 the pits are divided into subsets and
• 5. 26.5 the pits of each subset are intended for a particular reaction.

27. Measuring carrier according to claim 26, in which the Subsets of pits in contiguous Areas are arranged. 28. Stamp for applying reagents to selected punctiform points (pits) of a multiplicity of punctiform points on pointing measuring carriers ( 20 )

• 1. 28.1 at least a subset of the pits of the measurement carrier ( 20 ) corresponding pits that
• 2. 28.2 are arranged in a coherent area.

29. Combination of measuring carrier ( 20 ) according to claim 26 or 27 and stamp ( 21 ) according to claim 28. 30. Use of the method according to one of claims 1 to 17, the device according to one of claims 18 to 25, the measuring support according to claim 26 or 27 and the stamp according to claim 28 in the field of

• 1. 30.1. routine diagnosis of genetic predispositions and defects,
• 2. 30.2. complete evaluation of the genome status with regard to DNA expression in healthy and diseased tissue,
• 3. 30.3. Characterization of the interaction in complex molecular mixtures,
• 4. 30.4. High throughput screening in pharmaceutical development and
• 5. 30.5. highly sensitive detection of pathogenic bacteria and viruses.