WO2021122984A1

WO2021122984A1 - Microarray

Info

Publication number: WO2021122984A1
Application number: PCT/EP2020/086757
Authority: WO
Inventors: Franz Laermer
Original assignee: Robert Bosch Gmbh
Priority date: 2019-12-18
Filing date: 2020-12-17
Publication date: 2021-06-24
Also published as: DE102019219948A1

Abstract

Microarray for performance of analyses on at least one sample which can be accommodated in array cells (16, 26) of the microarray (10, 20), wherein the array cells (16, 26) are in the form of a disk and the array cells (16, 26) are open on a front side of the disk for accommodation of the at least one sample and sealed on a back side of the disk, wherein the array cells (16, 26) are sealed with a hydrophobic and gas-permeable material in the region of the back side of the disk.

Description

description

title

Microarray

The invention relates to a microarray, a method for producing such a microarray and a method for using such a microarray.

State of the art

A microarray is understood here to mean an examination system, in particular a molecular biological examination system, which enables the parallel analysis of several, in particular several tens or several hundred to thousands, individual records in a small amount of biological sample material. There are different types of microarrays, which are also known as gene chips or biochips, as these can contain a lot of information in a very small space, comparable to a computer chip. The samples or sample materials are introduced into cells of the microarray, which are also referred to as trench cells.

Microarrays and in particular silicon microarrays are used, for example, for carrying out molecular biological reactions, such as, for example, PCR reactions (PCR: polymerase chain reaction), in a highly multiplexed manner, i. H. typically more than 100 individual features, which are amplified and tested in parallel, are used. It is known to functionalize such microarrays physically and / or biologically in various ways and to integrate them into a lab-on-chip environment.

A lab-on-chip system (LoC system) describes a microfluidic system that accommodates the entire functionality of a macroscopic laboratory on a plastic substrate the size of a plastic card. Leave with this technology Analyze the smallest sample quantities completely and automatically on a single chip. The sample liquid is transported between the various reaction and analysis chambers with the help of capillary forces or, particularly preferably, active pumping. Active pumping is made possible by elastic membranes, which can in turn provide valve functions and pumping functions when subjected to mechanical forces or pressures. The microarray according to the invention, which is also referred to as a microarray chip, is an essential component of such a lab-on-chip system.

A problem that arises particularly in connection with a lab-on-chip integration of microarray chips is the reliable and highly efficient filling of the microarrays, ie. H. the filling of the cells, for example with an eluate in a molecular biological process. An eluate is a discharged mixture of solvents and dissolved substances.

It should be noted here that a complete and gas bubble-free filling of microarrays in many cases requires a large excess of eluate, i.e. H. of sample-containing reaction liquid, with which the microarray must be slowly flushed in order to fill all array cells or trench cells with it reliably and without gas bubbles, with only a very small part of the reaction liquid, for example in the order of magnitude of nanoliters or tens of nanoliters per array cell, gets into the array cells and the rest, e.g. B. in the order of a few microliters or a few 10 microliters, must be discarded, which means a correspondingly large loss of sample material, such as. DNA or RNA strands, and thus a loss of sensitivity or sensitivity.

In this context, the term “efficient” or “highly efficient” means that only minimal amounts of sample supernatant are not filled into the array cell and are thus discarded, so that as large a portion of the sample as possible gets into the array cells and a high level of sensitivity or Sensitivity for the detection of the sample material can be achieved. At the same time, the proposed measures significantly improve the reliability of the filling. The filling properties of such microarrays, in particular silicon microarrays, can be improved, for example, by a series of surface functionalization measures in order to achieve, for example, targeted locally hydrophilic or hydrophobic surface properties in order to guide the sample liquid into the cells or microcavities in a targeted manner .

Disclosure of the invention

Against this background, a microarray having the features of claim 1, a method for producing a microarray according to claim 5 and a method for using such a microarray according to claim 9 are presented. Embodiments emerge from the dependent claims and from the description.

The presented microarray is used to carry out analyzes on at least one sample that is to be received in array cells of the microarray. A sample of one type or from a material or different samples can thus be examined. The array cells are formed in a disc, the array cells being open on a front side of the disc for receiving the at least one sample and being closed on a rear side of the disc. Furthermore, the array cells in the area of the rear side of the pane are sealed with a hydrophobic and gas-permeable material such as porous PTFE (polytetrafluoroethylene) or a PTFE sponge or a fabric with a PTFE coating.

It should be noted that the said disk can have any shape, in particular any outline. This disc can have a circular outline, but this is not necessary. The disk has a surface which is provided for the formation of the array cells.

The described method for producing a microarray comprises the following steps:

- Introduction of cavities into a disk from an upper side of the disk, - Causing that the cavities in the area of the underside of the pane are closed with a hydrophobic and gas-permeable material.

The presented microarray can be produced with a method for producing of the kind described herein.

Here, too, it should be noted that the disc has a circular shape in only one embodiment. One surface of the disk is provided for forming the array cells.

The presented method for using a microarray of the type described herein provides that at least one sample is first introduced into the array cells. The array cells are then sealed, if there is free space, for example with a perfluorinated medium.

Thus, structures of microarrays, in particular silicon microarrays, are presented here, which allow highly efficient filling with sample material. Corresponding manufacturing processes are described. Surface functionalization processes are combined in one embodiment in order to achieve the best possible result overall. A microfluidic workflow for using the presented hardware is also described.

Further advantages and configurations of the invention emerge from the description and the accompanying drawings.

It goes without saying that the features mentioned above and those yet to be explained below can be used not only in the respectively specified combination, but also in other combinations or on their own, without departing from the scope of the present invention.

Brief description of the drawings Figure 1 shows a first and second embodiment of the presented microarray.

FIG. 2 shows a third and fourth embodiment of the presented microarray.

FIGS. 3 to 9 show process steps for producing a microarray presented herein.

FIG. 10 shows a third and fourth embodiment of the presented microarray.

FIGS. 11 to 16 show process steps for producing a microarray presented herein.

Embodiments of the invention

The invention is shown schematically on the basis of embodiments in the drawings and is described in detail below with reference to the drawings.

Figures 1 to 9 describe designs and manufacturing processes for silicon microarrays with which the aforementioned advantageous properties can be achieved.

FIG. 1 shows a first embodiment of the presented microarray, which is designated as a whole by the reference number 10, and a second embodiment of the microarray, which is designated by the reference number 20.

The first microarray 10 comprises a silicon wafer 12, the surface 24 of which is formed by a layer made of a polytetrafluoroethylene-like material (PTFE). Array cells 16 and inlet areas 18 are also provided. The second microarray 20 comprises a silicon wafer 22, the surface 14 of which is formed by a layer of PTFE-like material. Array cells 26 and inlet areas 28 are also provided.

The two microarrays 10 and 20 can also be referred to as microarray chips.

In the embodiments shown, cavities, which later form the array cells or trench cells, are first introduced into the silicon wafer 12 or 22, for example, etched in, which are formed by the hydrophobic surface 14 or 24 and directly adjoining the hydrophilic inlet areas 18 or 28 are surrounded. The hydrophobic surface 14 or 24 is designed to be highly water-repellent by producing a PTFE-like layer on the surface, which is achieved, for example, by fluorinating a lacquer surface in a plasma using CF4 gas (tetrafluoromethane). This surface 14 or 24 is highly repellent for both aqueous and mineral oil media, but not for perfluorinated liquids.

An aqueous sample medium which washes over the microarry 10 or 20 is forced into the hydrophilic inlet areas 18 or 28 and drawn from there into the likewise hydrophilic array cells 16 and 26, respectively. A first arrow 30 denotes an upward flow of eluate, a second arrow 32 denotes a downward bisnonafluorotrifluoromethylamine flow.

The free space, which is also referred to as head space and determines the dead volume, above the microarray 10 or 20 can be kept to a minimum, for example designed to a value of 1 pm or even below 1 pm in order to reduce the loss To minimize sample material. After the array cells 16 and 26 have been filled, the free space can be filled with a perfluorinated medium and the array cells 16 and 26 sealed with it.

FIG. 2 shows a third embodiment of the presented microarray, which is designated as a whole by the reference number 50, and a fourth embodiment of the microarray, which is designated by the reference number 60. The third microarray 50 comprises a silicon wafer 52, the surface 54 of which is formed by a layer of PTFE-like material. Array cells 56 and inlet channels 58 are also provided.

The fourth microarray 60 comprises a silicon wafer 62, the surface 64 of which is formed by a layer of PTFE-like material. Array cells 66 and inlet channels 68 are also provided. A first arrow 70 denotes an upward flow of eluate, a second arrow 72 denotes a downward bisnonafluorotrifluoromethylamine flow.

In these two embodiments, cavities, which later form the array cells 56 and 66, are first introduced or etched into the silicon wafer 50 and 60, which are surrounded by the hydrophobic surface 54 and 64 and a network of hydrophilic inlet channels 58 and 68, respectively are. An aqueous sample medium is sucked in or pumped into the hydrophilic channel network formed by the additional channels 58 and 68 and thus reaches the hydrophilic array cells 56 and 66. If an explicit free space is provided above the microarray 50 and 60, respectively , this can then be filled up again with a perfluorinated medium and the array cells 56 and 66 are thus sealed.

If no free space is provided above the microarray 50 or 60, for example by closing it with a transparent cover or pressing it against a corresponding window of the lab-on-chip, this sealing with a perfluorinated medium on the top of the microarray chip is unnecessary there is no dead volume to be sealed above the microarray 50 or 60, respectively.

In the latter case, the sample liquid is then between the array cells in the network of filling channels, which are very flat, e.g. B. 1 .mu.m high or even less than 1 .mu.m high, can be designed in that a cover can be glued to the surface of the hydrophobic surface layer or mechanically clamped against it. The cover can also be implemented through a window in the lab-on-chip, for example by clamping the microarray chip against it. The following FIGS. 2 to 8 show process steps for producing a microarray presented herein.

FIG. 3 shows a silicon surface 100 of a disk 101 or a wafer, which in sections has a SiC ^ mask 102 with a thickness of 1 μm and a first photoresist 104 thereon. The illustration shows the intermediate step in the production after a first lithography step in which a window 106 was opened in the SiC> 2 mask 102 for an array cell.

FIG. 4 shows the silicon surface 100 which is covered in sections with a second photoresist 120. The illustration clarifies a second lithography step for the formation of a silicon channel area 122, which can also be configured as an inlet area.

FIG. 5 shows the intermediate step after etching through the wafer in the area of the window 106, so that a trench cell 130 is created.

FIG. 6 shows the next intermediate step after opening the SiC ^ mask 102 in the inlet areas or in the channel area 122.

FIG. 7 shows the intermediate step after fluorination of the residual lacquer by means of CF4 plasma and then hydrophilization of the silicon of the wafer, for example by means of H2O2 / H2O vapor, PEG, etc. Inflow areas are denoted by reference number 140 in the illustration. The second photoresist 120 forms a fluorinated, PTFE-like surface.

FIG. 8 shows the intermediate step after connecting the rear side of the pane or wafer with a floor mat 150, for example made of PTFE fabric or a PTFE sponge, a spotting of the biocontent and a single chip singulation.

FIG. 9 shows an alternative to the step from FIG. 8. In this step, a stopper 160, for example made of PTFE fabric or a PTFE sponge, is pressed into the trench cell from the back of the wafer. This is followed by a spotting of bio content and a single chip singulation. A decisive functional element is the venting of the trench cells, ie the controlled displacement of gas volumes in the array cells or trench cells by the penetrating sample liquid. For this purpose, a preferred embodiment provides for the trench cells to be etched through the wafer or the wafer thickness. The wafer thickness can e.g. B. 380 pm, which can be easily etched through with the so-called "Bosch process" for silicon deep structuring. To terminate the trench cells on the output side, a porous PTFE fabric material is applied to the back of the wafer, e.g. B. glued over the entire surface. It is also possible and, if necessary, expedient to press this porous PTFE fabric material into the trench cell. On the one hand, this can be done by means of a pick-and-place device, in which a porous PTFE fabric material plug can be manually and sequentially pressed into each trench cell. It is also possible to provide a mat of porous PTFE fabric material which is pre-structured, e.g. B. in the manner of an egg carton structure. The elevations of the mat exactly match the arrangement of the trench cell, so that when the back of the wafer is pressed against the egg-box-like structured PTFE fabric material mat, the elevations are pressed into the trench cells. The excess of PTFE fabric material can then be cut off, for example using a sharp blade, so that the PTFE fabric material bumps remain in the trench cells and the remaining mat is cut off. Alternatively, it is possible to provide so-called predetermined breaking points on the elevations, with which the mat can be torn off in a controlled manner by shearing off after the elevations have been pressed into the trench cells.

Suitable PTFE fabric materials are, for example, PTFE-coated textile fabrics or fiber materials used in the textile industry. Also suitable are sponges made of PTFE material or arrangements of extruded and thereby porosified PTFE films. All that is essential is a porous nature and perfluorinated inner surfaces of the structure in order to ensure water-repellent properties.

The function of these porous PTFE stoppers is as follows: When the trench cells of the microarray chips are filled, the penetrating or sucked in is displaced pumped-in aqueous sample liquid is the volume of gas enclosed in the trench cells. The hydrophilic surface interactions with the hydrophilized silicon intensify the effect of the penetration of the aqueous sample liquid and the displacement of the gas volume, which can easily penetrate through the porous PTFE structure and thus leave the trench cells below. It is one of the well-known properties of the above-mentioned PTFE fabrics that they are breathable, ie they allow gases and also water vapor to pass through, but repel aqueous media, such as rain, to a high degree and prevent them from passing through. As soon as the liquid column reaches the bottom of the PTFE fabric material, the further propagation of the liquid stops because the PTFE barrier represents an insurmountable obstacle for the aqueous solution. As limit values, one finds in the literature that, for example, a PTFE fabric can prevent the passage of water up to a pressure of 0.8 to 1 bar, which corresponds to a water column of 8 to 10 m in height.

Even when the microarray chips are filled with overpressure by active pumping, the penetration of the sample liquid at the interface with the PTFE fabric material can be reliably stopped, provided the pump pressure does not exceed 0.8 to 1 bar.

As soon as all the array cells are filled, the so-called "bottom space" can be filled with a perfluorinated liquid or a perfluorinated oil from below the microarray chip. These perfluorinated media fill the PTFE fabric material as liquid PTFE without pressure, which allows the perfluorinated liquids to penetrate without resistance. In this way, the array cells on the floor are now also closed gas-tight or water-vapor-tight and thus sealed during the further reactions. Thanks to its high thermal conductivity, the perfluorinated liquid also ensures excellent heat transport between the array cells and the environment if the array cells are to be heated and / or cooled alternately or continuously, for example to carry out a PCR. These perfluorinated liquids are therefore often used in technology as heat exchange media for the transfer of heat or cold, up to and including vapor phase soldering in electronics. This selective behavior of the PTFE fabric materials towards aqueous media on the one hand and perfluorinated media on the other hand not only allows the controlled filling and sealing of the array cells in the lab-on-chip during a practical molecular biological process sequence. In addition, the spotting of bio-reagents, such as primers and probes, etc., is made easier during bio-functionalization before the microarray chips are installed in the lab-on-chip environment. The aqueous spot with the mentioned bio-reagents simply flows down into each array cell after being spotted, displacing existing gas volumes and stops automatically when it reaches the bottom of the trench cell made of porous PTFE fabric material without penetrating it. This means that the entire amount of bio-reagents in each array cell is available for the subsequent molecular biological reactions in the lab-on-chip environment.

Furthermore, additional biological or physico-chemical functionalizations of the array cells can be carried out, for example by introducing polyethylene glycol or PEG-containing precursors, such as. B. PEG silanes, for growing PEG-ilated layers inside the trench cells or by covering the bio content or bio content with PEG, etc.

A molecular biological process in the lab-on-chip can proceed as follows:

- Filling the array cells in the lab-on-chip with an aqueous eluate or PCR master mix, which contains the sample material to be amplified and detected, either via a corresponding network of channels covered at the top or via a minimal free space up to a closure element or a combination of both until all the gas from the array cells has been displaced downwards through the PTFE fabric material. There the filling with the aqueous medium stops automatically.

- If necessary, overlaying the microarray chip with a perfluorinated medium if the microarray chip is open at the top, that is, uncovered, and thus a minimal free space is available, which is thus filled with the perfluorinated medium on the front side and thus sealed. If there is a cover This measure is not necessary because the chip is already sealed by the cover.

- Underlayers of the microarray chip with a perfluorinated medium that penetrates the PTFE fabric material from the back of the wafer and seals the array cells from below.

- Carrying out and analyzing the molecular biological analysis reaction, e.g. B. a qPCR or an isothermal PCR or a digital PCR. The excellent thermal conductivity of the perfluorinated medium can be an advantage here.

A possible first process flow for manufacturing the microarray is shown below:

1. 1000 nm thermal oxide is deposited over the entire surface of a silicon wafer,

2. Photolithography: photoresist mask 1 with trench cells or array cells,

3. Transferring the photomask 1 into thermal oxide: plasma etching oxide (CHF3 / Ar, CF4 + C4F / Ar) or wet etching with Buffered Oxide Etch (BOE, a hydrofluoric acid / ammonium fluoride mixture that is used in the semiconductor industry for etching S1O2), both Stop processes on silicon,

4.Remove photoresist mask 1,

5. Photolithography: photomask 2 (4 μm thick photoresist) with trench cells and feeding channel system or inlet areas,

6. Deep trenching of the silicon trench cells through the wafer: Masking by photoresist 2 and thermal oxide, whereby the photoresist 2 must not be used up: for example with the so-called BOSCH deep reactive ion etching process for anisotropic high-rate etching of silicon with S FB / CAFS alternating, lacquer mask and silicon oxide, channel areas are covered by the silicon oxide during the deep trench, 7. Transfer of the photo mask 2 (= feeding channel system) into the residual oxide by selective plasma etching of the oxide (CHF / Ar, CF4 + C4F / Ar) or wet etching (BOE) with a stop on silicon: Now the silicon is exposed in the channel area, if necessary briefly brushing the wafer with an _{O 2} plasma in an O2 plasma stripper for surface cleaning.

8. Fluorination of the remaining lacquer surface with CF4 plasma, fluorine termination of the lacquer surface 2 gives a hydrophobic PTFE-like highly stable surface, the silicon surface is provided with F-termination, which is subsequently converted into hydrophilic OH- by contact with water or suitable aqueous media. Can convert termination, see step 10,

9. Wet wafer cleaning with so-called SCl-clean, which is a mixture of ammonia (NH ₃ ), hydrogen peroxide (H2O2) and water, which z. B. can remove grease and organic contaminants from the wafer surface. The PTFE-like paint surface remains unaffected,

10. Final hydrophilization of the silicon with H ₂ _{O 2} vapor, formation of OH groups on all open silicon surfaces, ie any remaining Si-F or Si-H is completely converted into Si-OH. The PTFE-like paint surface remains unaffected,

11. Attaching a floor mat made of PTFE fabric or a PTFE sponge or a porous PTFE fabric material structure to the back of the wafer, e.g. B. by gluing onto the back of the wafer, or alternatively by pressing plugs into the trench cells from the back of the wafer, e.g. B. over the whole area by means of an egg carton structure and then cutting off the excess with a blade or tearing it off over predetermined breaking points,

12. Apply PEG if necessary as a self-assembled monolayer (SAM), e.g. using PEG-silanes (trichlorosilanes, trimethoxysilanes, triethoxsilanes, etc.) from the gas phase,

13. Mock biocontent in trench cells, 14. Mock PEG in trench cells about biocontent,

15. Cover microarray chips, if necessary with a transparent cover, e.g. cover glass or polymer cap,

16. Separation into individual chips, e.g. with sawing, breaking, Mahoh or stealth dicing, etc.,

17. Installation in LoC array chamber,

18. Filling with eluate in the direction against gravity using pressure and hydrophilic forces on the silicon surface. Fluorinated photoresist surface repels water and protects against cross-talk between neighboring array cells. Eluate stops at the bottom of the array cells on the hydrophobic PTFE fabric or PTFE sponge: Up to pressures of 0.8 to 1 bar or 8 to 10 meters of water column, such fabrics reject any penetration of aqueous media and only allow gases or perfluorinated liquids to pass through penetration,

19. Filling the PTFE fabric or PTFE sponge from or on the back of the wafer with perfluorinated material (liquid PTFE) that is readily absorbed by the PTFE fabric and seals the array cells on the underside and thermally connects them to the environment.

If the microarray chips are uncovered, i. H. without step 15:

20. Covering the free space with perfluorinated material by pumping in the direction of gravity, thereby the excess eluate is flushed away through the channel system without causing cross-talk to other neighboring array cells. Not applicable if there is no free space due to the cover.

It is also possible to dispense with the perfluorinated surface functionalization of the chips and, for example, to apply a silicon oxide, a silicon nitride or a sequence of layers of silicon oxide and silicon nitride there and to use them appropriately structure. The perfluorination of the surface via the process sequence described above merely represents an advantageous embodiment that does not necessarily have to be implemented in this way.

Embodiments and manufacturing methods for a silicon microarray chip and a molecular biological sequence, for example for a PCR in a lab-on-chip, are thus described above. It is shown that by introducing a porous, gas-permeable hydrophobic barrier, for example in the form of a PTFE fabric material, into the array cells of the microarray chip, a reliable, gas-bubble-free, highly efficient filling of the array cells with the sample solution can be achieved. H. the loss of sample material through dead volumes is minimized and the sensitivity in the detection of the sample material is maximized. After filling, the microarray chip is sealed with a perfluorinated sealing medium that effortlessly penetrates the PTFE fabric material from the back and seals it.

Embodiments of the method for using a microarray are presented below, which allow silicon microarray cells to be filled with eluate or aqueous sample material as efficiently as possible and without loss of sample material and then to be sealed. This sealing takes place, for example, with a perfluorinated sealing liquid. In the end, this is intended to prevent the need for pre-amplification of the sample material in the sense of an upstream PCR to enrich the eluate with DNA and RNA strands.

In microarray solutions of the prior art, there is the disadvantage that, as a rule, the loss of sample material is so significant that such a pre-amplification or upstream PCR is necessary. Such an upstream PCR means a high additional effort and high additional costs, entails quality risks in the implementation and subsequent dilution of the pre-amplification solution before filling the microarray chip and costs a considerable amount of additional process time.

In particular if, in the interest of accelerating the diagnostic process, rapid isothermal PCR processes are used in the microarray chips, How, for example, the rITA technology is to be used, there is a risk of at least partially losing the time advantage of the rITA due to the pre-amplification or even of doing less well overall than with a classic qPCR without rITA and microchips. The loss of sample material and the resulting limit-of-detection (DoL) that can be achieved in the best possible way can be estimated as indicated in the following example.

To illustrate this, it is assumed as an example:

100 trench cells each with 10 nl partial volumes on a chip area of 9 mm * 9 mm = 81 mm ² ;

Total sample volume in the trench cells: 100 * 10 nl = 1 ml;

For comparison, the dead volume with 1 pm of free space is (whereby the height of the free space can be increased or further reduced if necessary):

~ 81 mm ² * 0.001 mm = 0.081 mm ³ = 0.081 mI ~ 0.1 mI <<< ImI (factor 10).

LoD: a reliable start of a PCR or rITA reaction succeeds when about three DNA strands land in each trench cell => LoD about 300, namely three times the number of trench cells in the microarray chip.

For a free space of approx. 10 μm, the dead volume would be approx. 1 ml, that is to say of an order of magnitude comparable to the total sample volume in the trench cells, which can lead to a doubling of the above LoD.

In all cases in which sufficient sample material or DNA / RNA quantities are available for diagnostic detection, this is the case, for example, with RTI (respiratory infections), STI (sexually transmitted infections) or MSRA (methicillin resistant staph aureus) , one will advantageously refrain from the additional effort, the additional costs, the quality and process risks and the additional time required for pre-amplification and can be satisfied with LoD in the specified range without further ado, ie there is no pre-amplification or upstream PCR. A suitable microarray, a manufacturing process and a biotechnological sequence are now presented here with which the stated advantages can be achieved.

FIGS. 10 to 18 describe designs and manufacturing processes for microarrays, in particular silicon microarrays, with which the aforementioned properties can be achieved.

FIG. 10 shows a fifth embodiment of the presented microarray, which is designated as a whole by the reference number 200, and a sixth embodiment of the microarray, which is designated by the reference number 220.

The fifth microarray 200 comprises a silicon wafer 202, the surface 204 of which is covered by a PTFE-like layer, i. H. from a fluorinated polymer. Furthermore, array cells 206, inlet areas 208 and inlet channels 210 are provided.

The sixth microarray 220 comprises a silicon wafer 222, the surface 224 of which is formed by a PTFE-like layer. Array cells 226, inlet areas 228 and inlet channels 228 are also provided. Arrows 230 indicate an eluate flow and a flow of a perfluorinated liquid upward.

The following FIGS. 11 to 16 show process steps for producing a microarray presented herein.

FIG. 11 shows a silicon surface 300 of a disk 301 or a wafer, which in sections carries an SiO ₂ mask 302 with a thickness of 1 μm and a first photoresist 304 thereon. The illustration shows the intermediate step in the production after a first lithography step in which a window 306 in the SiO ₂ mask 302 was opened for an array cell.

FIG. 12 shows the silicon surface 100 which is covered in sections with a second photoresist 320. The illustration clarifies a second lithography step for the formation of a silicon channel area 322, which can also be configured as an inlet area. FIG. 13 shows the intermediate step after a deep trench in the area of the window 306, so that an array cell or trench cell 330 is created; the wafer is not etched through here.

FIG. 14 shows the next intermediate step after opening the SiC ^ mask 302 with a photoresist mask 2 in the inlet areas or in the channel area 322 with CF4 or C4F / Ar plasma.

FIG. 15 shows the intermediate step after fluorinating the residual paint in the CF4 plasma (hydrophobic OF) and then hydrophilizing the silicon, for example by means of H2O2 / H2O vapor, PEG, etc. In the illustration, a hydrophilized inlet area is designated by reference number 340. The second photoresist 320 forms a fluorinated, PTFE-like surface. The interior of the trench cell 330 is also hyd rophilized.

FIG. 16 shows the structure after the bio-content has been spotted, possibly with PEG, isolation and structure.

Cavities, which later form the trench cells or array cells, are thus introduced or etched into a silicon wafer, which are surrounded by a hydrophobic surface and directly adjoining hydrophilic inlet areas, together with a network of fine hydrophilic supply channels that supply the aqueous sample solution. The hydrophobic surface is made highly water-repellent through the production of a PTFE-like material on the surface, which is achieved, for example, by fluorinating a lacquer surface in a plasma using CF4 gas (tetrafluoromethane). This PTFE-like surface is highly repellent for both aqueous and mineral oil media, but not for perfluorinated liquids, which are also known as liquid PTFE.

As a result, there is no loss of sample material on these surfaces because DNA, RNA and proteins cannot bind to such surfaces. DNA and RNA have negative electrical charges and are repelled by PTFE-like surfaces, so there is no connection to them possible. This ensures that the range of hydrophilic, potentially DNA and RNA-binding surfaces remains limited to the reaction vessels, i.e. to the interior of the trench cells themselves, to the microchannels of the network of hydrophilic supply channels and to the hydrophilic inlet areas around the trench cells .

An aqueous sample medium that washes over the microarray chip is forced into the hydrophilic feed channels and via these into the hydrophilic inlet areas, which thus act as phase guides and direct the aqueous liquid to the hydrophilic array cells, into which they are subsequently drawn. The gas in the array cells is thereby displaced via the free space above the array cells. This free space or the dead volume over the microarray chips can and should be kept to a minimum in the sense of the lowest possible sample loss, for example at 10 pm, better to 1 pm, or even below 1 pm in order to prevent the loss of sample material minimize. This is possible simply by attaching appropriately dimensioned spacers between a transparent cover and the chip surface when the cover and the chip surface are glued. After filling the array cells with sample liquid, the free space should be filled with a perfluorinated medium and the array cells should be sealed with it. This perfluorinated medium, in conjunction with the PTFE-like surface of the array cell environment, also ensures their mutual isolation in order to keep a so-called cross-talk or material carryover between different array cells to a minimum.

In the latter case, the sealing liquid is then between the array cells and in the network of filling channels, which can be made very flat, for example 1 μm high or even less than 1 μm high, by placing a Cover can be glued or mechanically clamped against this. The cover can also be implemented through a window in the lab-on-chip, e.g. B. by the microarray chip is clamped against it with said spacer. Furthermore, additional biological or physico-chemical functionalizations of the array cells can be carried out, for example an introduction of polyethylene glycol or PEG-containing precursors, such as e.g. B. in the form of PEG-silanes, for the growth of PEG-ilated layers inside the array cells. After the bio-content has been introduced, the bio-content can be further covered with PEG, etc. A particular advantage of the PTFE-like surface layer is that it enables selective coating exclusively of the hydrophilic silicon surfaces, excluding the PTFE-like surfaces, for example in the form of Atomic Layer Deposition (ALD) processes or as a process for growing a self-built one (Monolayer) layer (= Seif Assembled (Mono-) Layer or SAM-Coating), since these processes only grow layers on the hydrophilic silicon surfaces, but not on the PTFE-like surfaces. Thus, for. B. a selective coating with permanently hydrophilic coatings or covers on the hydrophilic silicon surfaces in the sense of a self-built (monolayer) layer, while nothing grows on the PTFE-like surfaces and these consequently remain hydrophobic.

A molecular biological process in the lab-on-chip can thus proceed as follows:

- Filling the array cells in the lab-on-chip with an aqueous eluate or PCR master mix, which contains the sample material to be amplified and detected, via the hydrophilic channel network as so-called phase guides for the aqueous sample liquid, with a minimal head space between the chip surface and a closing element, cover or window, the gas volumes displaced from the array cells being "disposed of" via the free space.

- Overlaying the microarray chip with a perfluorinated medium, whereby the microarray chip or the array cells are sealed against one another.

- Carrying out and analyzing the molecular biological reaction, e.g. a qPCR, an isothermal PCR, an rITA or a digital PCR. A possible second process flow for manufacturing the microarray is shown below, the first nine steps corresponding to the first nine steps of the first process flow presented above, so that they are not listed here again:

10. PEG as a self-assembled layer (self-assembled monolayer = SAM coating), e.g. B. in the form of a PEG silane, the process selectively covering only hydrophilic silicon surfaces, but not the PTFE-like surfaces, on which no coating takes place,

11. Mock biocontent in array cells or trench cells,

12. Mock PEG in array cells about biocontent,

13. Cover chips, if necessary with a transparent cover, e.g. cover glass, polymer cap,

14. Separation into individual chips, e.g. by sawing, breaking, Mahoh dicing, etc.,

15. Installation in LoC array chamber,

16. Filling with eluate in the direction against gravity with the aid of pressure and hydrophilic forces on the silicon surface or in the network of supply channels and inlet areas. Fluorinated Foltolack surface repels water and protects against cross-talk between neighboring array cells,

17. Filling the free space with perfluorinated liquid also against gravity, thereby the excess eluate is flushed away through the channel system without causing cross-talk to other neighboring array cells.

Embodiments and manufacturing methods for a microarray or a microarray chip as well as a sequence, for example for a PCR, a qPCR, a digital PCR or an rITA in a lab-on-chip are thus described above described. A highly efficient filling of the array cells with the aqueous sample solution is achieved through minimal dead volumes, ie the loss of sample material due to dead volumes is minimized and the sensitivity of a detection of the sample material by the subsequent PCRs in the array cells is consequently improved to such an extent that a

Pre-amplification reaction or upstream PCR can be dispensed with. The microarray chip or the free space with minimal dead volume is sealed after filling with a perfluorinated sealing medium in order to suppress cross-talk and material carryover.

Claims

Expectations

1.Microarray for performing analyzes on at least one sample which is to be accommodated in array cells (16, 26, 56, 66, 130, 206, 226, 330) of the microarray (10, 20, 50, 60, 200, 220), wherein the array cells (16, 26, 56, 66, 130, 206, 226, 330) are formed in a disk (101, 301) and the array cells (16, 26, 56, 66, 130, 206, 226, 330) are open on a front side of the disc (101, 301) for receiving the at least one sample and closed on a rear side of the disc (101, 301), the array cells (16, 26, 56, 66, 130, 206, 226, 330 ) are closed in the area of the rear side of the pane (101, 301) with a hydrophobic and gas-permeable material.

2. Microarray according to claim 1, wherein the disc (101, 301) consists of silicon.

3. Microarray according to claim 1 or 2, in which the array cells (16, 26, 56, 66, 130, 206, 226, 330) on the back of the disc (101, 301) by plugs (160) made of a PTFE material , which is hydrophobic and gas-permeable, are closed.

4. Microarray according to claim 1 or 2, in which the array cells (16, 26, 56, 66, 130, 206, 226, 330) on the back of the disc (101, 301) by a mat (150) made of a PTFE Material that is hydrophobic and gas-permeable are closed.

5. Method for producing a microarray (10, 20, 50, 60, 200, 220) with the following steps:

- Introducing cavities into a disk (101, 301) from an upper side of the disk (101, 301),

- Causing that the cavities in the area of the underside of the disk (101, 301) are closed with a hydrophobic and gas-permeable material.

6. The method according to claim 5, in which the cavities are introduced continuously into the disk (101, 301) and the cavities in the area of the rear side of the disk (101, 301) are sealed with a PTFE-containing material.

7. The method according to claim 6, wherein the stopper (160) made of a PTFE material are pressed in.

8. The method according to claim 6, wherein a mat (150) made of a PTFE material is placed on the back.

9. The method for using a microarray (10, 20, 50, 60, 200, 220) according to one of claims 1 to 4, in which at least one sample is initially inserted into the array cells (16, 26, 56, 66, 130, 206, 226, 330) is introduced and then, if there is free space, the array cells (16, 26, 56, 66,

130, 206, 226, 330) are sealed on the front.

10. The method of claim 9, wherein the array cells (16, 26, 56, 66, 130, 206, 226, 330) are sealed by flushing a perfluorinated medium.