WO2006000275A1

WO2006000275A1 - Method for producing biochips from porous substrates

Info

Publication number: WO2006000275A1
Application number: PCT/EP2005/005056
Authority: WO
Inventors: Stephan Dertinger; Thomas Haneder; Alfred Martin
Original assignee: Infineon Technologies Ag
Priority date: 2004-06-28
Filing date: 2005-05-10
Publication date: 2006-01-05
Also published as: DE102004031167A1

Abstract

The invention relates to a method for the production of biochips from porous substrates, wherein the biological-chemical substances used in a corresponding assay as probes and/or catcher molecules are initially applied to the porous surface of a relatively long, bar-shaped, porous substrate which is subsequently divided into small, prepared biochips. According to the inventive method, the homogeneity of the individual chips is improved and the production rate is increased.

Description

Process for the production of biochips from porous substrates

The present invention relates to a method for the production of biochips from porous substrates, wherein the biological-chemical substances used in a corresponding assay as probes or capture molecules are first applied to the pore surfaces of a relatively long, rod-shaped, porous substrate, which subsequently into small finished Biochips is isolated. By the method according to the invention, the homogeneity of the individual chips can be improved and the production speed can be increased.

Porous substrates are due to their advantageous optical and fluid properties very well as a substrate for DNA and protein microarrays. However, the application of a large number of biochemical substances (> 100), such as, for example, is technically complicated. DNA, antibodies and proteins. The biological substances are usually dissolved in a suitable buffer and applied spot by spot in a sequential process on the surface of the porous substrate by means of dispensing technique. Due to the sequential nature of this process is very time consuming. However, especially for high spot count arrays, it is desirable to parallelize the dispensing process.

So far, the biological-chemical substance by various "spotting ^u such as reservoir needle, pin-and-ring or Piezoverfahren in which a biochemical substance such as a previously synthesized oligonucleotide as a microdroplets deposited locally on the substrate surface is, applied. On each chip will be consecutive various spots sold. The spotting can be parallelized by the use of several such needles. However, this places higher demands on the logistics of the substances to be mocked. Another problem is the homogeneity of the spots. For many needles, it can not be ensured that the amounts of liquid deposited are identical for all needles.

Another approach to achieving higher throughput is to use a miniaturized dispensing head that can deposit up to 384 spots simultaneously (see WO 01/62377). Each dispensing nozzle is connected to a reservoir via a hose. The technical effort, for example, to align the substrate and Dispensierkopf plan-parallel to each other (minimizing the wedge error), however, is extremely high. In case of insufficient adjustment, no reproducible and homogeneous spots are available.

The present invention is therefore based on the object to enable a method which overcomes the above problems.

This object is achieved by the embodiments characterized in the claims.

According to the present invention, methods are provided, which are characterized in that the biological-chemical substances used in a corresponding assay as probes or catcher molecules are first applied to the pore surfaces of a rod-shaped porous substrate, which is then singulated into small, finished biochips. By doing so, the homogeneity of the individual biochips can be improved and the production speed can be increased. In a first aspect of the present invention there is provided a method of manufacturing a plurality of discrete biochips from a rod-shaped, porous substrate, comprising the steps of: (i) providing a rod-shaped porous substrate having pores with a diameter in the range of between 1 μm and 100 microns and has a cross-sectional area which corresponds to the dimensions of a single biochip to be manufactured; (ii) coating the pore surfaces of the rod-shaped porous substrate with chemical molecules acting as catcher molecules, and (iii) slicing the rod-shaped porous substrate so as to obtain singulated, finished biochips having a thickness in the range of 100 to 1,000 μm.

The macroporous substrate used in step (i) preferably has a pore diameter of 1 μm to 50 μm, more preferably 1 to 20 μm. The distance from the center of the pore to the center of the pore (pitch), ie, two adjacent or adjacent pores, is usually 1 to 100 μm, preferably 2 to 12 μm. The pore density is usually in the range of 10 ⁴ to 10 ⁸ / cm ² . The length of the rod-shaped porous substrate is not subject to any specific limitation, but is usually in the range of about 1 cm to 30 cm for process reasons. The cross-sectional area of the rod-shaped, porous substrate corresponds to the dimensions of a single biochip to be manufactured and is therefore usually 1 cm × 1 cm. The pores may be in hexagonal or square arrangement. Further, the pores may be designed, for example, substantially round or elliptical. The material of the rod-shaped porous substrate may in particular be selected from silica, alumina, glass or macroporous silicon, the latter being particularly preferred.

In step (ii), the coating of the pore surfaces of the rod-shaped, porous substrate takes place with chemical-biological compounds or biomolecules acting as catcher molecules. The site-specific attachment of such chemical-biological compounds or biomolecules to the pore surfaces is usually carried out by means of dispensing techniques, possibly assisted by pumping techniques. Thus, in step (ii), a solution of biomolecules can be charged or filled by utilizing at least one needle or needle assembly into the pores of the substrate employed by utilizing the capillary force. For example, an excess of liquid may be charged through the pore opening with a needle or instrument having a multiplicity of such needles and liquids so that this pore fills with liquid through the capillary action. After the desired reaction time, the liquid can be removed by applying overpressure to the pore opening.

As biomolecules which are coupled or bound site-specifically to the pore surfaces in step (ii), in particular DNA, RNA, PNA (in the case of nucleic acids and their chemical derivatives, eg single strands, triplex structures or combinations thereof can be present), saccharides, peptides , Proteins (eg antibodies, antigens, receptors), derivatives of combinatorial chemistry (eg organic molecules), cell components (eg organelles), cells, multicellular cells and cell aggregates. If the finished biochip is to be used in the context of an EIA or ELISA, in particular specific antibodies are used as biomolecules. The binding or coupling of the catcher molecules as In particular, oligonucleotides or DNA molecules to the substrate material can be via linker molecules according to the methods commonly used in the art, for example by treating the porous substrate material when using epoxy silanes as linker molecules by subsequent reaction of the terminal epoxy groups with terminal primary amino groups or thiol groups of oligonucleotides or DNA molecules which act as immobilized or fixed capture molecules for the target molecules present in the analyte of interest in corresponding analysis methods. In this case, for example, the oligonucleotides which can be used as catcher molecules can be synthesized using the synthesis strategy as described in Tet. Let. 22, 1981, pages 1859-1862. The oligonucleotides can be derivatized during the preparation process either at the 5 or the 3-terminal position with terminal amino groups. Another way of attaching such capture molecules to the inner wall surfaces of the pores can be carried out by first exposing the substrate to a chlorine source such as Cl ₂ , SOCl ₂ , COCl ₂ or (COCl) ₂ , optionally using a free radical initiator such as peroxides, azo compounds or Bu ₃ SnH, and then with a corresponding nucleophilic compound, in particular with oligonucleotides or DNA molecules having terminal primary amino groups or thiol groups are reacted (see WO 00/33976).

In step (iii), the then finished biochips are separated by sawing the rod-shaped, porous substrate so that individual, finished biochips having a thickness in the range from 100 to 1000 .mu.m, preferably 250 to 450 .mu.m are obtained. The thus functionalized, rod-shaped, porous substrate can be placed on a sawing foil stretched in a sawing frame, such as, for example, a MylarO foil commonly used for such purposes, after which the Sawing out the individual chips from the rod-shaped substrate according to conventional techniques. The slicing sawing has the consequence that advantageously no biological molecules are immobilized on the cut edges (top and bottom of the chips) in contrast to "spotted" porous substrates.

The sawing process should be compatible with the biological coating. This can be achieved, for example, by filling the pores before sawing with substances which do not attack the surface, can be washed out completely again from the pores after sawing by a solvent, but nevertheless in the sawing process, the surface in the pores before Sägewasser and particles protects. Suitable substances for this purpose are, for example, long-chain polyalkylene glycols, in particular polyethylene glycol (PEG) and polyvinyl alcohols.

In one embodiment of the present invention, the rod-shaped porous substrate having pores in the range of between 1 .mu.m and 100 .mu.m and a cross-sectional area of, for example, 1 cm x 1 cm, which corresponds to the later biochip dimensions, and a length of, for example, between 1 cm to 30 cm, in step (ii) are fluidly contacted by a fluidic mask at the two permeable end faces of the rod-shaped, porous substrate (see Figure 1). The fluidic mask ensures that areas in array array are sealed against each other. Each area of the face can be covered with different substances. The oligonucleotides provided as catcher molecules are then synthesized directly in the pores, whereby the synthesis takes place simultaneously in the pores of the entire rod and thus on many "chips." The rod-shaped, porous substrate is then separated again in slices, if only relatively small Spot densities are required, a large number of biochips can be made in parallel with this method, which improves both the cost and the homogeneity of the biochip. In a particularly advantageous manner, the 5 rod-shaped, porous substrates can be connected directly to an oligosynthesizer (eg provided for phosphoramidite synthesis chemistry).

Another embodiment of the present invention provides for LO to locally prevent synthesis in the pores by occluding the pore, i. to close the pores locally and in a defined manner, so that no synthesis reagent can enter the pores. At the hidden place then no synthesis takes place. For this purpose, the rod-shaped, porous substrate having pores L5 in the range of between 1 .mu.m and 100 .mu.m and a cross-sectional area of, for example, 1 cm x 1 cm, which corresponds to the later biochip dimensions, and a length of for example between 1 cm to 30 cm, in step (ii) are covered with a suitably configured mask. This -0 mask locally prevents chemical substances from entering the pores. Thus, for example, it is also possible to synthesize different oligonucleotides by sequentially placing masks in locally different regions. For this purpose, a corresponding surface-structured mask 25 made of elastomeric material can be brought into contact with the end face of the rod-shaped, macroporous substrate. When one-sided application of a liquid drop of a solution of biomolecules occurs, the liquid spreads by capillary forces into the i0 pores determined by the mask structure.

For example, the mask may be made of a flexible plastic such as an elastomeric material compatible with synthetic chemistry (deprotecting agent, oxalic acid in water in the Case of phosphoroaraidite chemistry). Preferably, the elastomeric material is selected from polydialkylsiloxanes, polyurethanes, polyimides and crosslinked novolak resins. More preferably, the elastomeric material is polydimethylsiloxane (PDMS). PDMS has a low surface energy and is chemically inert. In addition, PDMS is homogeneous, isotropic and optically transparent up to 300 nm.

Various embodiments are applicable with variation of the mask area and the chip area. Thus, the mask can contain holes (shadow mask) (see FIG. 2 a) or have a relief structure (stamp) (see FIG. 2 b), which respectively covers specific pores or pore areas. In the hole variant, the substrate-inclined surface of the mask should have a flexible layer that is biocompatible and fluid-tight, which can be achieved, for example, with the use of PDMS as the mask material. The layer must cover the surface in conformity, but must not close the holes. One advantage is that the mask area is relatively small, and thus a complete mask set for a 30mer (about 100 different masks) can be accommodated on a single 150 mm substrate.

If, for example, 100 chips per rod-shaped substrate are provided, since the fluidic masks have the dimension of one chip, it is easy to place 100 masks, which are necessary for the synthesis, on a "mask wafer." The situation is different for planar substrates Making 100 chips on a single 6 "wafer requires 100 pieces of 6" masks, but producing such masks is expensive because each fluidic mask is based on a lithographic mask.

Such a stamp or mold or mask made of elastomeric material having a relief structure, for example, by Replica molds can be prepared by casting the liquid polymer precursor of an elastomer over a template (Master) with a correspondingly predetermined surface relief structure, as is known from soft lithography; See, for example, Xia et al., Angewandte Chemie, 1998, 110, pages 568 to 594. By applying such a stamp mask with relief structure can be a kind of closed capillary structure or at least parts of channels or capillaries, the at least one inlet and after passing at least a pore, preferably a portion of pores, having an outlet. The mask can be structured such that the channels formed are straight or meander-shaped and continuous or comb-shaped.

It is also possible to apply a structured seal to the end faces of the rod-shaped substrate, which fluidically decouples adjacent regions of the substrate. The mask is then pressed onto the seal and prevents chemical substances from entering the pores locally.

The oligonucleotides provided as catcher molecules are then synthesized again directly in the now accessible pores, whereby the synthesis takes place simultaneously in the pores of the entire rod and thus on many "chips." Subsequently, the rod-shaped porous substrate is again separated in slices.

In a second aspect of the present invention, there is provided a method of making a plurality of discrete biochips from a porous substrate, comprising the steps of: (a) adjusting stacking a plurality of porous substrates in the range of 100 to 1000 microns to a rod-shaped macroporous substrate; (b) reversibly connecting the individual substrates by Annealing the rod-shaped macroporous substrate produced at a temperature in the range of 80 ⁰ C to 200 ⁰ C; (c) coating the pore surfaces of the rod-shaped porous substrate formed in step (b) with chemical-biological compounds functioning as catcher molecules, and (d) mechanically dicing the rod-shaped porous substrate such that finished biochips having a thickness in the range of 100 to 1,000 microns are obtained.

The substrates or individual chips used in step (a) preferably have a pore diameter of from 1 .mu.m to 50 .mu.m, more preferably from 1 to 20 .mu.m. The distance from the center of the pore to the center of the pore (pitch), ie, two adjacent or adjacent pores, is usually 1 to 100 μm, preferably 2 to 12 μm. The pore density is usually in the range of 10 ⁴ to 10 ⁸ / cm ² . The length of the rod-shaped porous substrate produced therefrom is not specifically limited, but is usually in the range of about 1 cm to 30 cm for process reasons. The cross-sectional area of the individual substrates or chips, and thus also of the rod-shaped porous substrate, corresponds to the dimensions of a single biochip to be manufactured and is therefore usually 1 cm × 1 cm. The pores may be in hexagonal or square arrangement. Further, the pores may be designed, for example, substantially round or elliptical. The material may for example be selected from silica, alumina, glass or macroporous silicon.

Thus, in steps (a) and (b), polished single chips are reversibly bonded to a bar. For this, the chips are stacked adjusted and connected by a tempering process. The holding forces of the bond connection are low (only Hydrogen bonds, Van der Waals forces), so that the connection can be released again without mechanical damage. The connection of the individual chips achieved by the annealing process must only fluidically seal to ensure that no liquid runs between the individual chips.

After the synthesis or coating with biomolecules, as described above, the mechanical separation of the LO stack then takes place in individual chips.

An advantage of this method is that after the biological coating or the immobilization of the catcher molecules of the sawing process can be omitted. L5 The figures show:

Fig. 1 shows schematically the contacting of a rod-shaped substrate used according to the invention with a fluidic mask, by which it is achieved that areas of the chip can be addressed separately fluidly.

Fig. 2 shows schematically the embodiment according to the invention, wherein the rod-shaped substrate is covered with a mask, 5 in order to achieve that the catcher molecule compounds get locally into the pores determined by the mask, wherein Fig. 2a, the provision of a mask with through holes (shadow mask ) and Fig. 2b show the provision of a stamp mask.

FIG. 1 shows how a rod-like porous substrate (10) with pores (11) used according to the invention is in fluidic contact with a fluidic mask (20) at its two end faces (12). Disposed on one of the fluidic masks (20) are separate supply lines (30) for individual, 5 determined pore areas, through which Different biomolecule solutions can be directed. Each region of the end face can be covered by the supply lines (30) with different substances or biomolecules. In addition, the fluidic mask ensures that regions in array arrangement are sealed against each other. For example, with such an arrangement, the oligonucleotides provided as catcher molecules can also be synthesized directly in the pores, whereby the synthesis takes place simultaneously in the pores of the entire rod and thus on many "chips." The rod-shaped, porous substrate is then separated again in slices. In a particularly advantageous manner, the rod-shaped, porous substrate (10) via leads (30) to an oligo-synthesizer (not shown) are connected.

FIG. 2 a) shows the arrangement of a shadow mask (50), while FIG. 2 b) shows the arrangement of a stamp mask with a relief structure (51) on a rod-shaped porous substrate (10) with pores (11) used in accordance with the invention. In both embodiments, depending on the mask pattern thereby locally pores or areas of pores are closed, so that no synthesis reagent or biomolecules (40) can get into these pores or, while other pores or areas of pores site-specific with the surface coating Biomolecules are provided. Subsequently, the rod-shaped, porous substrate is again separated by slices.

reference numeral

10 rod-shaped, porous substrate 11 pore 12 faces of the substrate fluidic mask leads for each area on the chip biomolecules perforated mask stamp mask

Claims

claims

A method for producing a plurality of individual biochips from a rod-shaped, porous substrate, comprising the steps of: (i) providing a rod-shaped porous substrate having pores with a diameter in the range of between 1 μm and 100 μm and a cross-sectional area, which corresponds to the dimensions of a single biochip to be manufactured; (ii) coating the pore surfaces of the rod-shaped porous substrate with chemical-biological compounds acting as catcher molecules, and (iii) slicing the rod-shaped porous substrate so as to obtain singulated, finished biochips having a thickness in the range of 100 to 1,000 μm.

2. The method of claim 1, wherein the length of the rod-shaped porous substrate is in the range of 1 cm to 30 cm.

3. The method of claim 1 or 2, wherein the material of the rod-shaped porous substrate of silicon oxide, alumina, glass or macroporous silicon is selected.

4. The method according to any one of claims 1 to 3, wherein in step (ii) the coating of the pore surfaces with the chemical-biological compounds by means of dispensing techniques, optionally by pumping techniques, takes place.

5. The method according to any one of claims 1 to 4, wherein prior to step (iii) the functionalized pores for protection with long-chain polyalkylene glycols and / or polyvinyl alcohols are filled.

6. The method according to any one of claims 1 to 5, wherein in step (ii) at least one of the two end faces of the rod-shaped, porous substrate, a fluidic mask is provided so that areas in array arrangement are sealed against each other and areas of the end face with different chemical-biological compounds can be occupied.

7. The method according to claim 6, wherein the oligonucleotides provided as catcher molecules are synthesized directly in the pores.

8. The method of claim 6 or 7, wherein the rod-shaped, porous substrate is connected via connected to the at least one fluid mask feed lines with an oligo-synthesizer.

9. The method according to any one of claims 1 to 5, wherein in step (ii) on at least one of the two end faces of the rod-shaped porous substrate, a shadow mask or a punch mask is provided so that partially pore openings are covered such that no chemisch¬ biological compounds get into these pores.

A method of making a plurality of discrete biochips from a porous substrate, comprising the steps of: (a) adjusting a plurality of porous substrates having a single thickness ranging from 100 to 1000 microns into a rod-shaped macroporous substrate; (b) reversibly bonding the individual substrates by annealing the formed rod-shaped macroporous substrate at a temperature in the range of 80 ^° C to 200 ^° C; (c) coating the pore surfaces of the rod-shaped porous substrate produced in step (b) with as And (d) mechanically dicing the rod-shaped, porous substrate so as to obtain finished biochips having a thickness in the range of 100 to 1000 μm.

11. The method of claim 10, wherein the length of the rod-shaped porous substrate is in the range of 1 cm to 30 cm.

12. The method according to claim 10 or 11, wherein the material of the rod-shaped porous substrate made of silicon oxide,. Alumina, glass or macroporous silicon is selected.

13. The method according to any one of claims 10 to 12, wherein in step (c) the coating of the pore surfaces with the chemical-biological compounds by means of dispensing techniques, optionally by pumping techniques, takes place.