DE102004031167A1 - Process for the production of biochips from porous substrates - Google Patents

Abstract

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.

Description

The The present invention relates to a process for the preparation of Biochips of porous Substrates in which in a corresponding assay as probes or Catcher molecules used Biochemical substances first on the pore surfaces of a relatively long, rod-shaped, porous substrate be applied, then in small finished biochips is isolated. By the method according to the invention, the homogeneity of the individual Chips are improved and the production speed is increased.

Porous substrates are suitable because of their advantageous optical and fluidic Properties very good as a substrate for DNA and protein microarrays. But technically complex is the application of a large number of biological substances (> 100), e.g. DNA, antibodies and Proteins. The biological substances are usually in one solved appropriate buffer and Spot for Spot in a sequential process on the surface of the porous Substrate applied by dispensing technique. By the sequential nature This process is very time-consuming. Especially for However, high number of spots arrays would be desirable to parallelize the dispensing process.

So far the biological-chemical substance is prepared by various "spotting processes" such as reservoir needle, Pin-and-ring or piezoprocessing, involving a biochemical Substance such as a previously synthesized oligonucleotide as a microdrop locally on the substrate surface is deposited, applied. On each chip will be consecutive various spots sold. Spotting can be done by using be parallelized by several such needles. This is but higher demands on the logistics of the substances to be mocked Another one Problem is the homogeneity of the Spots. For many needles can not be sure that the remote Liquid quantities at are identical to all needles.

One another approach, to higher Throughput is the use of a miniaturized Dispensierkopfes, which can settle up to 384 spots simultaneously (see w0 01/62377). Each dispensing nozzle is via a hose with a reservoir connected. The technical effort to, for example, substrate and Align dispensing head plane-parallel to one another (minimization the wedge error), but is extraordinary high. In case of insufficient adjustment are not reproducible and homogeneous spots available.

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

These The object is achieved by the embodiments characterized in the claims solved.

According to the present The invention provides methods which are characterized that the in a corresponding assay used as probes or capture molecules biochemical Substances first on the pore surfaces a rod-shaped porous Substrate are applied, then in small, finished biochips is isolated. By doing so, the homogeneity of the individual Biochips improved and the production speed increased.

• (i) Bereitstellen eines stabförmigen porösen Substrats, das Poren mit einem Durchmesser im Bereich von zwischen 1 μm und 100 μm und eine Querschnittsfläche aufweist, welche den Abmessungen eines zu fertigenden, einzelnen Biochips entspricht;
• (ii) Beschichten der Porenoberflächen des stabförmigen porösen Substrats mit als Fängermoleküle fungierenden chemischbiologischen Verbindungen, und
• (iii) scheibenweise Sägen des stabförmigen porösen Substrats derart, daß vereinzelte, fertige Biochips mit einer Dicke im Bereich von 100 bis 1.000 μm erhalten werden.

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 μm and a cross-sectional area corresponding 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 discrete, finished biochips having a thickness in the range of 100 to 1,000 μm.

The The macroporous substrate used in step (i) preferably has a pore diameter of 1 micron up to 50 μm, more preferably 1 to 20 μm. The distance from pore center to pore center, i. two to each other neighboring or

Adjacent pores is usually 1 to 100 microns, preferably 2 to 12 microns. 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, for example, the pores may be substantially round or elliptical be designed. The material of the rod-shaped porous substrate may in particular be selected from silicon oxide, aluminum oxide, glass or macroporous silicon, the latter being particularly preferred.

In Step (ii), the coating of the pore surfaces of the rod-shaped, porous Substrate with acting as catcher molecules chemical-biological compounds or biomolecules. 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 by pumping techniques supported. Thus, in step (ii), a solution of biomolecules taking advantage of the capillary force by means of at least one needle or needle assembly fed into the pores of the substrate used or filled become. For example, a surplus on liquid with a needle or an instrument which has a multiplicity of such needles and liquids has, over the pore opening be charged so that this pore by the capillary action with liquid crowded. After the desired Reaction time can through the liquid Applying overpressure removed to the pore opening become.

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 capture molecules such as in particular oligonucleotides or DNA molecules to the substrate material can be carried out via linker molecules according to the methods customary in the prior art, for example by treating the porous substrate material using epoxysilanes as linker molecules by subsequent reaction of the terminal epoxide groups Terminal primary amino groups or thiol groups of oligonucleotides or DNA molecules, which function in corresponding analysis methods as immobilized or fixed capture molecules for the present in the analyte target molecules. 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 functionalized such rod-shaped, porous substrate, as are a Mylar ^® film commonly used for such purposes arranged on a tensioned in a sawing frame sawing film for example, whereafter the sawing of the individual chips is carried out 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.

Of the Sawing process should be compatible with the biological coating. This can be, for example through the filling the pores before sawing can be achieved with substances that the surface is not attack, after sawing through a solvent completely again can be washed out of the pores, but still in the sawing process, the surface in the Pores in front of sawing water and protects particles. Suitable substances for this are, for example, long-chain polyalkylene glycols such as 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 × 1 cm, which corresponds to the later biochip dimensions, and a length of, for example, between 1 cm to 30 cm, in step (ii) be fluidly contacted by a fluidic mask at the two permeable end faces of the rod-shaped, porous substrate (see 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 in the pores of the The rod-shaped, porous substrate is then separated again in slices, and if relatively low spot densities are required, a large number of biochips can be produced in parallel, which reduces both costs and costs In a particularly advantageous manner, the rod-shaped, porous substrates can be connected directly to an oligo-synthesizer (eg provided for phosphoramidite synthesis chemistry).

A other embodiment The present invention provides, in the pores, the synthesis locally to prevent by the pore is closed, i. the pores local and defined to close, so that no synthesis reagent in the Pores can get. At the hidden place then finds no Synthesis takes place. For this purpose, the rod-shaped, porous substrate with pores in the area of between 1 μm and 100 μm and a cross-sectional area for example, 1 cm × 1 cm, which the later Biochip dimensions, and a length of, for example, between 1 cm to 30 cm, in step (ii) with a correspondingly configured Mask are covered. This mask locally prevents chemical Substances enter the pores. That's it, for example possible, by sequentially placing masks in locally different ones Areas to synthesize different oligonucleotides. For this can be a corresponding surface-structured Mask of elastomeric material in contact with the face of the rod-shaped, macroporous Substrate are brought. With one-sided application of a liquid drop a solution of biomolecules the liquid spreads by capillary forces in the pores determined by the mask structure.

The For example, a mask can be made of a flexible plastic such as a be elastomeric material that is compatible with synthetic chemistry (Deprotection, oxalic acid in water in the case of phosphoramidite chemistry). Preferably the elastomeric material of polydialkylsiloxanes, polyurethanes, Polyimides and crosslinked novolak resins selected. More preferably that is elastomeric material polydimethylsiloxane (PDMS). PDMS has one low surface energy and is chemically inert. In addition, PDMS is homogeneous, isotropic and up 300 nm optically transparent.

Various embodiments are applicable with variation of the mask area and the chip area. So the mask can contain holes (shadow mask) (see 2a ) or a relief structure (stamp) (see 2 B ), each of which 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.

Become namely for example, 100 chips are provided per rod-shaped substrate, so, so far the fluidic masks have the dimension of a chip, easily 100 Masks for the synthesis are necessary to be housed on a "mask wafer". The situation is different with planar substrates. To make 100 chips, based on a single 6 "wafer 100 pieces of 6 "masks are necessary. However, the production of such masks is expensive because each fluidic mask a lithographic mask is based.

One such stamp or mold or mask of elastomeric material having a relief structure can For example, be prepared by replica molds by the liquid polymer precursors of a Elastomers over a template (master) with a correspondingly predetermined surface relief structure is poured, as is known from soft lithography; please refer e.g. Xia et al., Angewandte Chemie, 1998, 110, pages 568-594. By applying such a stamp mask with relief structure may be a kind of closed capillary structure or at least parts of channels or capillaries that have at least one inlet and after passing at least a pore, preferably a region of pores, an outlet have generated. The mask can be structured that the formed channels while straight or meandering as well are formed continuously or comb-like.

It is possible, too, on the faces of the rod-shaped Substrates apply a structured seal, the adjacent Portions of the substrate fluidically decoupled. The mask then becomes Pressed on the seal and prevents chemical substances locally can not get into the pores.

The intended as catcher molecules Oligonucleotides are then returned directly to the now accessible Pores are synthesized, with synthesis in the pores of the whole Stabs and thus on many "chips" takes place simultaneously. Subsequently becomes the rod-shaped porous Substrate again separated by slices.

• (a) justiertes Aneinanderstapeln einer Vielzahl von porösen Substraten bzw. Chips mit einer Einzeldicke im Bereich von 100 bis 1.000 μm zu einem stabförmigen makroporösen Substrat;
• (b) reversibles Verbinden der einzelnen Substrate durch Tempern des erzeugten stabförmigen makroporösen Substrats bei einer Temperatur im Bereich von 80°C bis 200°C;
• (c) Beschichten der Porenoberflächen des in Schritt (b) erzeugten stabförmigen, porösen Substrats mit als Fängermoleküle fungierenden chemisch-biologischen Verbindungen, und
• (d) mechanisches Vereinzeln des stabförmigen, porösen Substrats derart, daß fertige Biochips mit einer Dicke im Bereich von 100 bis 1.000 μm erhalten werden.

In a second aspect of the present invention, a method of manufacturing a plurality of individual biochips from a porous one Substrate provided, comprising the steps:

• (a) aligned stacking of a plurality of porous substrates or chips having a single thickness in the range of 100 to 1000 microns to 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 chemical-biological compounds functioning as catcher molecules, and
• (d) mechanically dicing the rod-shaped porous substrate so as to obtain finished biochips having a thickness in the range of 100 to 1,000 μm.

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.

In Thus, in steps (a) and (b), polished single chips become reversible bonded to a rod. For this, the chips are stacked adjusted and connected by a tempering process. The holding forces the bond is low (only hydrogen bonds, Van the Waals forces), so that the connection without mechanical damage solved again can be. The achieved by the annealing process compound of single chips only has to fluidically seal to ensure that no fluid between the individual Chips are running.

To synthesis or coating with biomolecules, as described above, then the mechanical separation of the stack is done in single chips.

One Advantage of this method is that after the biological coating or the immobilization of the catcher molecules of the sawing process omitted can.

The Figures show:

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

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

In 1 It is shown how a rod-shaped porous substrate used according to the invention (US Pat. 10 ) with pores ( 11 ) with a fluidic mask ( 20 ) each at its two end faces ( 12 ) is fluidically contacted. On one of the fluidic masks ( 20 ) are separate supply lines ( 30 ) are provided for individual, determined pore areas through which different biomolecule solutions can be passed.

Each area of the face can be connected via the supply lines ( 30 ) are covered 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 supply lines ( 30 ) to an oligo-synthesizer (not shown).

In 2a ) is the placement of a shadow mask ( 50 shown while in 2 B ) arranging a stamp mask with relief structure ( 51 ) on a rod-shaped porous substrate used according to the invention ( 10 ) with pores ( 11 ) is shown. In both embodiments, pores or regions of pores are thereby locally closed as a function of the mask pattern, so that no synthesis reagent or biomolecules ( 40 ) can enter into these pores, while other pores or areas of pores are provided site-specifically for surface coating with biomolecules. Subsequently, the rod-shaped, porous substrate separated again by slice.

10

rod-shaped, porous substratum

11

pore

12

End faces of the substrate

20

fluidic mask

30

leads for the individual areas on the chip

40

biomolecules

50

shadow mask

51

stamp mask

Claims (13)

Method for producing a plurality of individual Biochips from a rod-shaped, porous substrate provided, comprising the steps: (i) provide a rod-shaped porous Substrate that has pores with a diameter ranging between 1 μm and 100 μm and a cross-sectional area having the dimensions of an individual to be manufactured Biochips corresponds; (ii) coating the pore surfaces of the rod-shaped porous Substrate with acting as catcher molecules chemical-biological compounds, and (iii) slice by slice Sawing the rod-shaped porous substrate such that isolated, finished biochips having a thickness in the range of 100 to 1,000 microns can be obtained. The method of claim 1, wherein the length of the rod-shaped porous Substrate is in the range of 1 cm to 30 cm. The method of claim 1 or 2, wherein the material of the rod-shaped porous Substrate of silicon oxide, alumina, glass or macroporous silicon selected is. Method according to one of claims 1 to 3, wherein in step (ii) coating the pore surfaces with the chemical-biological Connections by means of dispensing techniques, possibly by pumping techniques, he follows. Method according to one of claims 1 to 4, wherein before the Step (iii) the functionalized long-chain protection pores Polyalkylenglykolen and / or polyvinyl alcohols are filled. Method according to 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 fluidic mask is provided so that areas be sealed in array arrangement against each other and areas the face be covered with different chemical-biological compounds can. The method of claim 6, wherein the intended as catcher molecules Oligonucleotides are synthesized directly in the pores. The method of claim 6 or 7, wherein the rod-shaped, porous substrate on the at least one fluidic mask connected leads with an oligo synthesizer is connected. Method according to 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 stamp mask is provided so that regionally pore openings are covered so that no chemical-biological compounds get into these pores. Method for producing a plurality of individual Biochips from a porous Substrate, comprising the steps: (a) adjusted stacking a variety of porous Substrates with a single thickness in the range of 100 to 1,000 microns to one rod-shaped macroporous substrate; (b) reversibly connecting the individual substrates by Annealing the rod-shaped macroporous Substrate at a temperature in the range of 80 ° C to 200 ° C; (c) coating the pore surfaces the rod-shaped, porous substrate produced in step (b) with catcher molecules acting as catcher molecules chemical-biological compounds, and (d) mechanical singulation the rod-shaped, porous Substrate such that finished Biochips having a thickness in the range of 100 to 1,000 microns received become. The method of claim 10, wherein the length of the rod-shaped porous Substrate is in the range of 1 cm to 30 cm. A method according to claim 10 or 11, wherein the material of the rod-shaped porous Substrate of silicon oxide, alumina, glass or macroporous silicon selected is. A method according to any one of claims 10 to 12, wherein in step (c) coating the pore surfaces with the chemical-biological Connections by means of dispensing techniques, possibly by pumping techniques, he follows .