WO2017028759A1 - Chip, preparation method therefor, and application thereof - Google Patents

Abstract

A chip, preparation method therefor, and application thereof. Specifically, the present invention relates to a preparation method for a chip, the method comprising the steps of preparation of a film, obtaining of a substrate, modification of a base, and encapsulation of the chip; and the chip prepared according to the method, a kit, and application in sequence capturing and nucleic acid sequencing by utilizing the chip or the kit. The preparation method of the chip features a simple manufacturing process and low manufacturing costs.

Description

Chip preparation method, chip and application

The present invention claims the priority of the prior invention filed by the Chinese Patent Office on August 14, 2015, entitled "Preparation of a Single Molecule Sequencing Chip", Application No. 201510500393.7, the contents of which are incorporated herein by reference. The way is incorporated into this text.

Technical field

The present invention relates to the technical field of chip preparation, and in particular to a chip preparation method, a chip and an application thereof.

Background technique

After entering the 21st century, the completion of the Human Genome Project has had a tremendous impact on contemporary biological research and medical research. In terms of gene sequence analysis, the focus of the post-genome era has shifted from genome-wide sequence determination of individual species to the comparison of individual genetic differences and genetic differences between species at the genomic DNA sequence level of a species. Targeted gene resequencing will be the mainstream technology for future clinical genetic testing. Single-molecule sequencing technology is known as the third-generation sequencing technology. Its remarkable feature is that it can directly identify DNA fragments with high fidelity. Single-molecule sequencing technology has higher specificity than single-nucleotide sequencing technology. For second generation sequencing technology) higher detection sensitivity.

Gene chips are a key component of sequencing technology. However, the second-generation sequencing chips are currently the mainstream products on the market. Most of them use semiconductor nano-machining processes to obtain high-density nano-arrays. The processing technology is fine and complex, the cost is very high, and large-scale high-precision instruments and ultra-high-levels are required. The clean room to complete. In addition, in order to achieve high-throughput sequencing, the second-generation sequencing chip usually has a large width of the chip channel. When the biochemical reaction is carried out by vacuum pumping, there is usually a problem of uneven flow field distribution and cover glass. The piece is easy to deform. Uneven distribution of the flow field will cause the reagent to be switched uncleanly, and the biochemical reaction will be affected. The deformation of the cover glass will affect the quality of the chip, and will affect the collection of the base optical signal.

Since single-molecule sequencing technology does not require high amounts of sequencing data, there is no need for ultra-high-density nano-arrays like second-generation sequencing chips. Therefore, it is necessary to provide a method for preparing a chip suitable for single-molecule sequencing.

Summary of the invention

In view of this, the present invention provides a method for preparing a chip, which has a simple preparation process and low cost, and the flow field distribution of the chip prepared by the method is good, the deformation rate of the chip is low, and the fluid in the chip is switched. thorough.

The method for preparing a chip provided by the invention comprises the following steps:

(1) taking a substrate, according to the graphic template of the designed reaction cell array, using a photolithography method to form a positive film of the reaction cell array on the surface of the substrate;

(2) casting the positive film with a model glue, after vacuum degassing, curing at 90-100 ° C for 1-3 h, transferring the positive film of the reaction cell array to the bottom of the model glue, and peeling off the film. Obtaining a model glue layer with a plurality of flow channels, and making a hole at each end of each flow channel to form a fluid input hole and an output hole to obtain a substrate;

(3) substrate modification: taking a transparent substrate, preparing a polymethylglutarimide (PMGI) layer on the surface of the transparent substrate to obtain a surface-modified transparent substrate;

(4) encapsulating a chip: performing oxygen plasma cleaning on the substrate and the surface-modified transparent substrate, and then pressing the substrate and the transparent substrate to form a space for containing a fluid; and then injecting N- into each flow channel Methylpyrrolidone (abbreviated as NMP), the reaction time is 10 min-15 min, to wash away the polymethylglutarimide layer in contact with each channel, to obtain a spacer layer on the surface of the transparent substrate, to complete single molecule sequencing Preparation of the chip.

Preferably, in the step (1), the substrate comprises one of a silicon wafer, a glass, a metal or a ceramic.

Preferably, in the step (2), the model glue comprises polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), ethylene-vinyl acetate (EVA) and polyurethane (PUA). One, but not limited to, as long as it is a model glue suitable for soft lithography.

More preferably, in the step (2), the model glue is polydimethylsiloxane (PDMS).

As described in the present invention, the substrate is made of the same material as the model glue.

Preferably, in the step (3), the polymethylglutarimide (PMGI) layer has a thickness of from 1 to 5 μm.

In the present invention, the polymethylglutarimide is fully referred to as polymethylglutarimide (PMGI) in English, and the PMGI is purchased from MicroChem, and the product names are SF1, SF2, SF3, SF4, SF5, SF6, SF7. One or more of SF7.5, SF8, SF9, SF10, SF11, SF12, SF13, SF14, SF15, SF17, SF19, SF23.

Preferably, the PMGI is polymethylglutarimide commercially available from MicroChem under the product name SF11.

Preferably, in the step (4), the injection amount of N-methylpyrrolidone in each flow channel is from 100 to 500 μL.

The injection amount of the N-methylpyrrolidone may wash all or part of the sacrificial layer on the transparent substrate, but as long as the sacrificial layer in contact with each flow channel is cleaned, the DNA fluid injected into the flow channel may be combined with the substrate. Functional groups such as an epoxy group, an amino group, a carboxyl group, a thiol group, and an aldehyde group on the surface are fixed and fixed.

Preparation of a substrate and a surface modified transparent substrate prior to oxygen plasma treatment as described herein The purpose of the PMGI layer is to protect the functional groups on the surface of the transparent substrate (such as one of epoxy, amino, carboxyl, sulfhydryl and aldehyde groups) from being affected by plasma oxygen treatment, so that subsequent gene samples are fixed to the transparent substrate. on. After the modified transparent substrate is cleaned by oxygen plasma, the surface of the transparent substrate is changed from hydrophobic to hydrophilic; N-methylpyrrolidone is injected into each channel, and the reagent can corrode PMGI in contact with each channel. The functional group on the surface of the transparent substrate is exposed again.

The flow channel of the single-molecule sequencing chip is hydrophilic, which can reduce the non-specific adsorption of the DNA molecule to be tested, and the functional groups on the surface of the substrate are not affected.

Preferably, in step (4), the pressing of the substrate and the transparent substrate is: firstly, the substrate is initially adhered to the transparent substrate, and placed in an oven at a temperature of 95-120 ° C, and pressed with a heavy object. Stay baking for 1-3h.

Preferably, in the step (1), the photolithography method comprises the following steps:

a, the negative photoresist is prepared by spin coating to the surface of the substrate to obtain a uniform substrate, wherein the thickness of the photoresist layer on the surface of the substrate is 600-650 μm;

b. pre-baking the substrate after the homogenization, the temperature of the pre-baking is controlled to: slowly heat to 90-100 ° C and stabilize for 15-20 min, then naturally cool to room temperature;

c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the substrate after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 90-150 s;

d, the exposed substrate is post-baked, the temperature of the post-baking is controlled: first slowly heated to 90-95 ° C and stabilized for 5-10 min, then naturally cooled to room temperature;

e. The substrate after the post-baking treatment is immersed in a developing solution, developed, and the portion other than the mask is washed away to obtain a positive film of the cell array.

Preferably, before performing the step a of the photolithography method, the method further comprises: pre-treating the substrate:

The surface of the substrate was washed successively with absolute ethanol and water, and then the cleaned substrate was placed on a hot plate at 150 ° C for 10 minutes to completely evaporate the surface moisture.

Preferably, after the step e of the photolithography method, the following processing is further included:

f. The positive film of the reaction cell array obtained in step e is subjected to a hard baking treatment, wherein the hard baking is to add a glass plate to the positive film, press an iron block, and heat to 130-150 ° C for baking 45 -60min.

The hard baking is to make the photoresist layer of the anode film adhere more firmly to the surface of the substrate, and to increase the etching resistance of the photoresist layer.

Preferably, in step b, the temperature of the pre-baking is controlled by first baking at 65 ° C for 6 min, and then pressing 1 ° C / min. The rate was gradually increased to 95 ° C for 20 min and allowed to cool to room temperature. The purpose of the pre-baking is to evaporate the organic solvent in the photoresist and solidify the photoresist.

Preferably, in step e, the post-baking temperature is controlled by first gradually raising the temperature to 95 ° C at a rate of 0.5 ° C / min for 5 min, and then naturally cooling to room temperature at a rate of 1-3 ° C / min. The purpose of the post-baking is to sufficiently carry out the crosslinking reaction of the negative photoresist of the exposed portion.

Preferably, the negative photoresist is SU-8 2150.

Preferably, the array of reaction cells comprises 15-25 flow channels.

Preferably, the width of the spacer layer between the directions perpendicular to the longitudinal direction of the flow channel is 1-1.5 mm.

As described herein, the spacing between adjacent flow channels is 1-1.5 mm.

Preferably, the angle of the tapered end is 30-60°.

Preferably, the distance between the intersections of the two side walls of each of the flow passages is the length of each flow passage, and each of the flow passages has a length of 50-75 mm.

Preferably, the distance between the two side walls of each of the opposite flow channels is the width of each flow channel, and each of the flow channels has a width of 1-2 mm.

Preferably, each of the flow channels has a depth of from 0.6 to 1 mm.

The depth of the flow channel is preferably 0.6-1 mm. According to the hydrodynamic resistance law of the rectangular flow channel, the flow resistance is reduced to 1/8 of the original for each doubling of the thickness direction, and the flow resistance is small, which is favorable for the flow of the fluid, and is convenient for single flow. During the molecular sequencing process, the fluid undergoes a biochemical reaction in the flow channel.

More preferably, each flow channel has a length of 50 mm, a width of 1 mm, and a depth of 0.6 mm.

In terms of fluid, a narrower flow path is more advantageous for flushing between fluids, and the longitudinal section at both ends of the flow channel is designed as a triangle. When fluid flows through the flow channel, there is no backflow phenomenon in the flow channel.

Preferably, the substrate has a first side length perpendicular to a length direction of the flow channel, and a distance between two opposite side walls of each flow channel is a distance from a first side of the substrate It is 0.5-1 cm.

Preferably, the fluid input aperture is coaxial with the fluid output aperture.

Preferably, the fluid input aperture has a pore size of 300-500 μm.

Preferably, the fluid output aperture has a pore size of 300-500 μm.

According to the invention, the first surface of the substrate is provided with a plurality of flow channels to form a reaction cell array of single molecule sequencing chips, and the two tapered end surfaces of each flow channel are provided with a fluid input hole and an output hole. For the inflow and outflow of fluid.

According to the present invention, the fluid input hole and the fluid output hole are used for connecting fluid input and output devices, for example. For example, a pipette tip, a pipe joint, or the like may be inserted into the fluid input hole and the fluid output hole, respectively, to disperse the input point and the output point of the fluid in each channel, so that the input and output of the fluid in each channel are not allowed. Disturbed.

Preferably, the material of the substrate comprises one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), EVA (ethylene vinyl acetate) and PUA (polyurethane). Kind, but not limited to, as long as the pouring process can be realized.

Preferably, the transparent substrate comprises a transparent glass, quartz or organic polymeric material having a surface having a functional group of one of an epoxy group, an amino group, a carboxyl group, a thiol group and an aldehyde group.

Preferably, the spacer layer is made of polymethylglutarimide (PMGI). The spacer layer serves to block contact between samples within each flow channel, ensuring separate control of the sample within each flow channel.

Preferably, the spacer layer is a reagent which injects a reagent into each flow channel to wash away the polymethylglutarimide layer on the transparent substrate in contact with each flow channel, so as to have a functional group on the upper surface of the transparent substrate (ring The oxy group, the amino group, the carboxyl group, the thiol group and the aldehyde group are exposed. The spacer layer serves to block contact between samples within each flow channel, ensuring separate control of the sample within each flow channel.

Preferably, the chip further includes a probe attached to a surface of the transparent substrate of the chip.

The functional groups on the surface of the transparent substrate (such as epoxy group, amino group, carboxyl group, sulfhydryl group and aldehyde group) can interact with the functional groups of the probe (such as carboxyl group, phosphate group, amino group, etc.), so that the probe is fixed on the transparent of the chip. On the substrate, the chip is subsequently applied to capture target regions, nucleic acid sequencing and the like. For example, an epoxy group on a transparent substrate can be chemically reacted with a DNA probe modified with -NH ₂ , and the probe is immobilized on the surface of the substrate modified with an epoxy group by a new -CH ₂ -NH- bond.

The probe can include a primer (preferably a targeting primer). The primers can capture nucleic acids of the sample to be tested, either targeted or non-targeted.

In a second aspect, the invention provides a chip made by the method of the first aspect.

In a third aspect, the invention provides the use of the chip of the second aspect in sequence capture and/or nucleic acid sequencing. The nucleic acid sequencing includes DNA and/or RNA sequencing.

Unless otherwise specified, "targeting primers" and "sequences", "primers" or "probes" as used herein may be used interchangeably to refer to a (oligo)nucleotide sequence.

The sequence capture can include primers, preferably targeting primers, and immobilized primers to capture the target nucleic acid to be tested (also referred to as "template nucleic acid" in the field of nucleic acid sequencing technology). The "primer-test nucleic acid complex" is the same as the "primer/test nucleic acid complex", and represents a complex formed by ligation of a primer and a nucleic acid to be tested. Unless otherwise stated, the invention The "nucleic acid to be tested" and "template nucleic acid" are interchangeable.

When the chip is applied, a fluorescence detector is disposed outside the lower transparent substrate, and the fluorescence detector is one of a photoelectric coupling device CCD or a complementary metal oxide semiconductor CMOS. Through the biochemical reaction in the microfluidic channel, a plurality of optical wavelengths can be used to detect the base of a specific position in the DNA molecule immobilized on the transparent substrate, thereby determining the DNA sequence immobilized on the transparent substrate.

In a fourth aspect, the invention provides a kit comprising the chip and reagent of the first aspect of the invention. The reagents used can be selected depending on the use of the kit.

In a fifth aspect, the invention provides the use of the kit of the fourth aspect in capturing a target region and/or nucleic acid sequencing. The nucleic acid sequencing includes DNA and/or RNA sequencing.

The reagents used may be selected depending on the use of the kit, and the reagents may include one or more. For example, when the chip is used for nucleic acid sequencing (eg, single molecule sequencing), the reagents required may include immobilization reagents, extension reaction reagents, imaging reagents, and reagents for excising optical detection label molecules.

It will be appreciated that the kit also includes a buffer or other sequencing necessary reagent. In the embodiment of the present invention, the immobilization reagent, the extension reaction reagent, the imaging reagent, and the reagent for excising the optical detection label molecule are not particularly limited. It is generally used in the art. For example, a person skilled in the art separately configures a buffer solution for different processes such as a fixed reaction reagent, an extension reaction reagent, and an optical detection label molecule, as needed.

In the present invention, each flow channel is prepared on a substrate by a photolithography-casting method, and is pressed and sealed with a surface-modified transparent substrate to obtain the single-molecule sequencing chip. The preparation method of the single-molecule sequencing chip provided by the invention is simple. The operability is high and the manufacturing cost is low. The preparation of the single molecule sequencing chip can get rid of the limitation of expensive and fine semiconductor process.

The chip prepared by the method has a certain number of flow channels, and each flow channel has an angled tapered end, so that the flow field distribution of the fluid in the flow channel is good, there is no backflow phenomenon in the flow channel, and the flow field distribution is good. Far better than the second-generation sequencing chip, the integrated multi-channel design increases the support point of the substrate, and the deformation problem of the substrate is almost negligible. The single-molecule sequencing chip can realize separate control of samples in each channel, ensuring no cross-contamination between samples, and simplifying post-processing of data.

The beneficial effects of the invention include the following aspects:

1. Because the model gel (such as PDMS, etc.) itself is super hydrophobic, it is easy to non-specifically adhere to biological macromolecules such as DNA, making it less reported to be applied to substrates of DNA sequencing chips. The invention is based on a soft lithography-casting process to produce a chip suitable for single-molecule DNA sequencing, and under the premise of protecting the functional groups on the surface of the substrate, the substrate is treated by plasma treatment. The surface of the sheet is changed from hydrophobic to hydrophilic, so that it can meet the requirements of the DNA sequencing chip. The flow channel of the single-molecule sequencing chip can reduce the non-specific adsorption of the DNA molecule to be tested, and the surface of the substrate is not affected by the functional group.

2. The positive film of the reaction cell array made by photolithography can be repeatedly used for multiple times, which is beneficial to mass production of single molecule sequencing chips and further reduces manufacturing costs.

3. The obtained chip has a certain number of flow channels, and each flow channel is designed as a narrow flow channel, and the fluid inlet and outlet are designed to be tapered, which is favorable for forming a fluid buffer zone and can flush the fluid in the flow channel. The switching is complete, there is no fluid recirculation zone, which is beneficial to the biochemical reaction; at the same time, the depth of the flow channel is deeper, so that the smaller the flow resistance of the fluid in the flow channel, the corresponding time of the single molecule sequencing chip can be improved.

4. The integrated multi-channel in the obtained chip increases the supporting surface of the substrate, which is beneficial to reduce the deformation of the substrate caused by the suction of the negative pressure, and overcomes the abnormal injection of the fluid in the chip system due to the deformation of the substrate. The problem and the problem of poorly acquired images.

5. The obtained chip has a plurality of parallel flow channels, and each flow channel is independent of each other, which can realize individual control of samples in each flow channel, thereby ensuring no cross-contamination between samples, and the single-molecule sequencing chip does not need to be like In the second-generation sequencing chip, a specific sequence "barcode" is added before each sample is injected to identify each sample, simplifying the process of preparing the genetic sample and the subsequent bioinformatics analysis process.

DRAWINGS

1 is a schematic cross-sectional view showing a single molecule sequencing chip according to Embodiment 1 of the present invention, wherein 1 is a substrate, 21 is a transparent substrate, 22 is a spacer layer on the transparent substrate 21, and 2 is a base layer composed of 21 and 22, 1b. For the second surface of the substrate 1, 11 is a flow channel, 12 is a fluid input hole communicating with the second surface 1b of the substrate, and the depth of the etching groove provided by the spacer layer 22 corresponding to the position of the flow channel is represented by h;

2 is a top plan view showing a substrate of a single molecule sequencing chip according to Embodiment 1 of the present invention, wherein 1 is a substrate, 11 is a flow channel, and 12 and 13 are fluid input holes and fluid output holes, respectively, 111 and 112 are respectively flow. The two side walls of the opposite side of the road, 113 is the tapered end of the flow channel, d represents the distance of each flow channel, and l represents the distance of each flow channel;

3 is a schematic view showing a preparation method of a single molecule sequencing chip according to an embodiment of the present invention, wherein 3 is a substrate for forming a positive film of a reaction cell array, 1a is a first surface of the substrate 1, and 1b is a second surface of the substrate 1. ;

4 is a comparison of a hydrodynamic simulation simulation result of a second generation sequencing chip and a single molecule sequencing chip in Embodiment 1 of the present invention;

Figure 5 is a comparison of the deformation of the transparent substrate of the second generation sequencing chip and the single molecule sequencing chip of Example 3 of the present invention.

detailed description

The following is a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It is the scope of protection of the present invention.

Example 1

A method for preparing a single molecule sequencing chip (see the schematic diagram of the preparation method of FIG. 3) includes the following steps:

(1) taking a silicon wafer as a substrate, and forming a positive film of the array of the reaction cell on the surface of the silicon wafer by photolithography according to the graphic template of the designed reaction cell array, specifically including the following steps;

a. Take a silicon wafer, and then clean the surface of the substrate with anhydrous ethanol and water. Then, the cleaned silicon wafer is placed on a hot plate at 150 ° C for 10 minutes to completely evaporate the surface water; the treated silicon wafer is placed. On the rotating stage of the homogenizer, spin-coat the negative photoresist SU-8 2150, start the homogenizer, and spread the photoresist evenly on the silicon wafer to obtain the homogenized silicon wafer, wherein the silicon wafer The thickness of the photoresist layer on the surface is 600 μm, the acceleration time of the homogenizer is set to 18 s, the time of uniform homogenization is 60 s, and the speed of uniform homogenization is 1000 rpm.

b. pre-baking the homogenized silicon wafer, the temperature of the pre-baking is controlled by first baking at 65 ° C for 6 min, and then gradually heating to 95 ° C at a rate of 1 ° C / min for 20 min, naturally Cool to room temperature;

c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the silicon wafer after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 120 s;

d. The exposed silicon wafer is post-baked, and the temperature of the post-baking is controlled to: gradually increase the temperature to 95 ° C at a rate of 0.5 ° C / min, hold for 5 min, and then naturally cool to 2 ° C / min to Room temperature

e, immersing the post-baking silicon wafer in SU-8 developer, developing treatment, cleaning off the portion other than the mask, washing the residual developer with isopropyl alcohol, and finally using deionized water to leave the residue The isopropyl alcohol is washed away to obtain a positive film of the array of the reaction cell;

f. The anode film of the reaction cell array obtained in step e is subjected to a hard baking treatment so that the photoresist layer of the anode film is more firmly adhered to the surface of the silicon wafer, and the hard baking is to add a film to the anode film. Glass plate, and pressed an iron block, heated to 150 ° C for 60 min, and naturally cooled to room temperature, to obtain a positive film of the hard-baked reaction cell array, the positive film is convex;

(2) Mix Dow Chemical Company's polydimethylsiloxane (PDMS) A and B glue according to a mass ratio of 10:1, stir evenly, and pour into a glass dish. The above-mentioned system is pre-placed in the glass dish. The positive membrane of the array of reaction cells, and then The glass dish was placed in a vacuum apparatus, vacuumed for 1 hour, and after the air bubbles were cleaned, placed in an oven, the bottom surface was kept horizontal, and solidified at 95 ° C for 1 h to transfer the positive film of the reaction cell array to the model glue. The bottom of the glass; the glass dish is taken out from the oven and cooled, and the pattern portion in the cured model glue is cut with a knife, and then the film is peeled off to obtain a model glue layer with grooves of a plurality of flow paths, and in each flow path a hole is formed at the bottom of each of the flow channels at both ends to form a fluid input hole and an output hole to obtain a substrate;

(3) Substrate modification: A transparent borosilicate glass with an epoxy group on the surface was used as a transparent substrate, and a polymethylglutarimide (PMGI, SF11 series product of MicroChem) was prepared on the surface of the transparent substrate. a layer, a surface-modified transparent substrate, the PMGI layer having a thickness of 1 μm;

(4) packaging the chip: the substrate and the surface-modified transparent substrate are placed in an oxygen plasma cleaning machine for cleaning, and then the substrate and the transparent substrate are pressed together to form a space for containing fluid; and then into each flow channel 100 μL of N-methylpyrrolidone (abbreviated as NMP) was injected for 10 min to wash away the PMGI layer in contact with each channel to expose the epoxy group on the surface of the borosilicate glass. A spacer layer on the surface of the transparent substrate, and a fluid input hole and a fluid output hole at both ends of each flow channel are respectively inserted into the pipe joint, and sealed with a resin to complete the fabrication of the single molecule sequencing chip.

The schematic diagram of the structure of the single-molecule sequencing chip prepared in this embodiment is shown in FIG. 1. Referring to FIG. 1 together with the schematic structure diagram 2 of the substrate, the single-molecule sequencing chip includes the substrate 1 and the substrate pressure. a base layer 2 disposed, the substrate 1 including a first surface (not shown in FIG. 1, not shown in FIG. 3) and a second surface 1b, the first surface of the substrate is spaced apart A reaction cell array formed by a plurality of flow channels 11 is provided, and two opposite side walls 111, 112 of each of the flow channels 11 extend along the length direction of the flow channel 11 and meet at both ends of the flow channel Forming two tapered ends 113 having angles, the surfaces of the two tapered ends 113 are respectively provided with a fluid input hole 12 and a fluid output hole 13 communicating with the second surface 1b of the substrate, the base layer 2 includes a transparent substrate 21 and a spacer layer 22 disposed on a surface of the transparent substrate 21, the spacer layer 22 is in contact with the first surface of the substrate, and the spacer layer 11 is provided with corrosion corresponding to a position where the flow path is located The groove, the depth of the corrosion groove is denoted by h.

As can be seen from FIG. 1, there is a residual polymethylglutaimimide (PMGI) layer on the transparent substrate 21, but the PMGI layer on the corresponding transparent substrate that is in contact with each flow channel has been completely removed, that is, the phase is disposed between The spacer layer 22 of the transparent substrate 21. The spacer layer 22 is formed by injecting a cleaning agent into each of the flow channels to wash away the PMGI layer on the transparent substrate in contact with each flow path to expose the epoxy functional groups carried on the upper surface of the transparent substrate. The spacer layer 22 serves to block contact between samples within each flow channel, ensuring separate control of the samples within each flow channel. The depth of the etching groove obtained by the spacer layer corresponding to the position of the flow path is 1 μm.

In the present embodiment, the angle of the tapered end 113 is 60°.

In the present embodiment, the reaction cell array includes 20 flow paths, the spacing between adjacent flow channels is 1 mm, and the width of the spacer layer 22 in a direction perpendicular to the longitudinal direction of the flow channel is 1 mm. The distance between the intersections of the two side walls of each flow channel is the length of each flow channel, denoted by l in Fig. 2; the distance between the two side walls of each flow channel is set to each The width of the flow path is indicated by d in FIG.

In the present embodiment, the length l of each flow path is 50 mm.

In the present embodiment, the width d of each flow path is 1 mm.

In the present embodiment, the depth d of each flow path is 0.6 mm.

In the present embodiment, the substrate has a first side length perpendicular to the longitudinal direction of the flow path, and the intersection of the opposite side walls of each of the flow paths is 0.5 cm from the first side of the substrate.

In the present embodiment, the fluid input aperture 12 and the fluid output aperture 13 are coaxial.

In the present embodiment, the fluid input hole 12 and the fluid output hole 13 have a pore size of 300 μm.

In the present embodiment, the transparent substrate 21 is a transparent borosilicate glass having an epoxy group on its surface. The spacer layer is made of PMGI and the depth of the etched groove is 1 μm.

In the present embodiment, the material of the substrate 1 is polydimethylsiloxane (PDMS).

The single-molecule sequencing chip prepared in Example 1 was subjected to hydrodynamic simulation. The results are shown in Fig. 4. A is the flow field in the single-molecule sequencing chip without tapered design at both ends of the flow channel, and B is an example. 1 The flow field in a single-molecule sequencing chip with a tapered inlet section, the color data bar in the left column of Figure 4 represents the volume fraction of the fluid (blue at the bottom, red at the top), and blue represents the initial presence in the chip. The air, red represents the fluid that is about to enter the chip, the fluid flows from right to left, and the fluid enters from a point (the red point in A and B is the initial inflow state, which is circled by the box respectively, as time goes on, The channel gradually turns from blue to red, and when the channel becomes full red, the surface fluid has filled the entire flow path). As is apparent from Fig. 4, the fluid recirculation zone c indicated by the circle is hardly present in the tapered design B.

The above comparison shows that the single-molecule sequencing chip prepared by the invention designs the fluid inlet and outlet into a cone shape, which is favorable for forming a fluid buffer zone, and can completely switch the flushing of the fluid in the flow channel, and there is no fluid reflux zone, which is favorable for the biochemical reaction.

Example 2

A method for preparing a single molecule sequencing chip includes the following steps:

(1) taking a silicon wafer as a substrate, and forming a positive film of the array of the reaction cell on the surface of the silicon wafer by photolithography according to the graphic template of the designed reaction cell array, specifically including the following steps;

a. Take a silicon wafer, and then clean the surface of the substrate with anhydrous ethanol and water. Then, the cleaned silicon wafer is placed on a hot plate at 150 ° C for 10 minutes to completely evaporate the surface water; the treated silicon wafer is placed. On the rotating stage of the homogenizer, spin-coat the negative photoresist SU-8 2150, start the homogenizer, and spread the photoresist evenly on the silicon wafer to obtain the homogenized silicon wafer, wherein the silicon wafer The thickness of the photoresist layer on the surface is 650 μm, the acceleration time of the homogenizer is set to 18 s, the time of uniform homogenization is 60 s, and the speed of uniform homogenization is 1000 rpm.

b. pre-baking the homogenized silicon wafer, the temperature of the pre-baking is controlled to: slowly heat to 100 ° C and stabilize for 15 min, then naturally cool to room temperature;

c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the silicon wafer after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 150 s;

d, the exposed silicon wafer is post-baked, the post-baking temperature is controlled: first slowly heated to 90 ° C and stabilized for 10 min, then naturally cooled to room temperature;

e, immersing the post-baking silicon wafer in SU-8 developer, developing treatment, cleaning off the portion other than the mask, washing the residual developer with isopropyl alcohol, and finally using deionized water to leave the residue The isopropyl alcohol is washed away to obtain a positive film of the array of the reaction cell;

f. The anode film of the reaction cell array obtained in step e is subjected to a hard baking treatment so that the photoresist layer of the anode film is more firmly adhered to the surface of the silicon wafer, and the hard baking is to add a film to the anode film. Glass plate, and pressed an iron block, heated to 130 ° C for 45 min, and naturally cooled to room temperature, to obtain a positive film of the hard-baked reaction cell array, the positive film is convex;

(2) pouring the positive film with model glue PDMS, after vacuum degassing, curing at 90 ° C for 3 h, transferring the positive film of the reaction cell array to the bottom of the model glue, uncovering the film, and obtaining a plurality of flow path model glue layers, and a hole is formed at each end of each flow path to form a fluid input hole and an output hole to obtain a substrate;

(3) Substrate modification: a transparent borosilicate glass with an epoxy group on the surface is used as a transparent substrate, and a PMGI layer is prepared on the surface of the transparent borosilicate glass to obtain a surface-modified transparent substrate, and the thickness of the PMGI layer 3 μm;

(4) Encapsulating the chip: the substrate and the surface-modified transparent substrate are placed in an oxygen plasma cleaning machine for cleaning, and then taken out, and the substrate and the transparent substrate are initially laminated and placed in an oven at a temperature of 120 ° C. Inside, press and bake for 2h with heavy objects to complete the pressing of the substrate and the transparent substrate, and form a space for accommodating the fluid; then, inject 300 μL of NMP into each flow channel for 12 minutes to wash away The PMGI layer in contact with each flow channel exposes the epoxy group carried on the surface of the borosilicate glass to obtain a spacer layer on the surface of the transparent substrate, and a fluid input hole and a fluid output hole at both ends of each flow channel The tube joints were respectively inserted and sealed with a resin to obtain a single molecule sequencing chip.

The single-molecule sequencing chip prepared in the second embodiment, comprising a substrate and a substrate layer on which the substrate is press-fitted, The substrate includes a first surface and a second surface disposed opposite to each other, and the first surface of the substrate is spaced apart from the array of reaction cells formed by a plurality of flow channels, and the two sidewalls of each of the flow channels are disposed along the opposite side The flow path extends in the length direction and meets at both ends of the flow path to form two tapered ends with angles, and the two tapered end surfaces are respectively provided with fluids communicating with the second surface of the substrate An input aperture and a fluid output aperture, the base layer comprising a transparent substrate and a spacer layer disposed on a surface of the transparent substrate, the spacer layer being in contact with the first surface of the substrate and the spacer layer corresponding to the flow path The location is set with a corroded groove.

In the second embodiment, the angle of the tapered end is 30°, the array of the reaction cell includes 25 flow paths, and the spacing between adjacent flow channels is 1.2 mm. Each channel has a length of 75 mm, each channel has a width of 1.5 mm, and each channel has a depth d of 0.8 mm; the substrate has a first side length perpendicular to the length direction of the channel, each The intersection of the opposite side walls of the flow path is 0.8 cm from the first side of the substrate; the fluid input hole and the fluid output hole are coaxial, and the pore size is 400 μm. In this embodiment, the transparent substrate is a transparent borosilicate glass having an epoxy group on the surface, and the substrate is made of polydimethylsiloxane (PDMS). The spacer layer is made of PMGI, and the depth of the etching groove obtained by the spacer layer corresponding to the position of the runner is 3 μm.

Example 3

A method for preparing a single molecule sequencing chip includes the following steps:

(1) taking a glass as a substrate, according to the graphic template of the designed reaction cell array, using a photolithography method to form a positive film of the reaction cell array on the surface of the glass, specifically comprising the following steps;

a. Take a glass, and then clean the surface of the substrate with anhydrous ethanol and water. Then, the cleaned silicon wafer is placed on a hot plate at 150 ° C for 10 minutes to completely evaporate the surface water; On the rotating stage of the melter, spin-coat negative photoresist SU-8 2150, start the homogenizer, and spread the photoresist evenly on the silicon wafer to obtain the silicon wafer after homogenization, wherein the surface of the silicon wafer The thickness of the photoresist layer is 620 μm, the acceleration time of the homogenizer is set to 18 s, the time of uniform homogenization is 60 s, and the speed of uniform homogenization is 1000 rpm;

b. pre-baking the homogenized silicon wafer, the temperature of the pre-baking is controlled to: slowly heat to 95 ° C and stabilize for 18 min, then naturally cool to room temperature;

c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the silicon wafer after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 150 s;

d, the exposed silicon wafer is post-baked, the temperature of the post-baking is controlled: first slowly heated to 92 ° C and stabilized for 8 min, then naturally cooled to room temperature;

e, immersing the post-baking silicon wafer in SU-8 developer, developing treatment, cleaning off the portion other than the mask, washing the residual developer with isopropyl alcohol, and finally using deionized water to leave the residue The isopropyl alcohol is washed away to obtain a positive film of the array of the reaction cell;

f. The anode film of the reaction cell array obtained in step e is subjected to a hard baking treatment so that the photoresist layer of the anode film is more firmly adhered to the surface of the silicon wafer, and the hard baking is to add a film to the anode film. Glass plate, and pressed an iron block, heated to 140 ° C for 50 min, naturally cooled to room temperature, to obtain a positive film of the hard-baked reaction cell array, the positive film is convex;

(2) casting the positive film with model glue PDMS, after vacuum degassing, curing at 100 ° C for 2 h, transferring the positive film of the reaction cell array to the bottom of the model glue, uncovering the film, and obtaining a model glue layer of the grooves of the plurality of flow channels, and a hole is formed at each end of each flow path to form a fluid input hole and an output hole to obtain a substrate;

(3) Substrate modification: a transparent borosilicate glass with an epoxy group on the surface is used as a transparent substrate, and a PMGI layer is prepared on the surface of the transparent borosilicate glass to obtain a surface-modified transparent substrate, and the thickness of the PMGI layer 5 μm;

(4) Encapsulating the chip: the substrate and the surface-modified transparent substrate are placed in an oxygen plasma cleaning machine for cleaning, and then taken out, and the substrate is firstly bonded to the transparent substrate and placed in an oven at a temperature of 95 ° C. Inside, the workpiece was pressed and pressed for 1 hour to complete the pressing of the substrate and the transparent substrate, and a space for accommodating the fluid was formed; then 500 μL of NMP was injected into each flow channel, and the reaction time was 15 min to wash away The PMGI layer in contact with each flow channel exposes the epoxy group carried on the surface of the borosilicate glass to obtain a spacer layer on the surface of the transparent substrate, and a fluid input hole and a fluid output hole at both ends of each flow channel The tube joints are respectively inserted and sealed with a resin to obtain a single molecule sequencing chip comprising a substrate and a substrate layer provided with a substrate, and the substrate layer comprises a transparent substrate and a spacer layer disposed on the surface of the transparent substrate.

The single-molecule sequencing chip prepared in the third embodiment comprises a substrate and a substrate layer on which the substrate is press-fitted, the substrate comprising a first surface and a second surface disposed opposite to each other, the substrate being first The surface is spaced apart from the array of reaction cells formed by a plurality of flow channels, and the two opposite sidewalls of each of the flow channels extend along the length of the flow channel and meet at the two ends of the flow channel to form two bands. An angled tapered end, the two tapered end surfaces being respectively provided with a fluid input aperture and a fluid output aperture in communication with the second surface of the substrate, the base layer comprising a transparent substrate and disposed in the transparent a spacer layer on the surface of the substrate, the spacer layer is in contact with the first surface of the substrate, and the spacer layer is provided with a corrosion recess at a position corresponding to the flow path.

In the third embodiment, the angle of the tapered end is 45°, and the array of the reaction cell includes seven flow paths, and the interval between adjacent flow paths is 1.5 mm. That is, the width of the spacer layer between the directions perpendicular to the longitudinal direction of the flow channel is 1.5 mm. Each channel has a length of 60 mm, each channel has a width of 2 mm, and each channel has a depth d of 1 mm; the substrate has and The first side of the flow channel is perpendicular to the first side, and the intersection of the opposite side walls of each flow channel is 1 cm away from the first side of the substrate; the fluid input hole and the fluid output hole are coaxial The pore size is 500 μm.

In the third embodiment, the transparent substrate is a transparent borosilicate glass with an epoxy group, the spacer layer is made of PMGI, and the depth of the etching groove is 5 μm. The material of the substrate is PDMS.

The second-generation sequencer and single-molecule sequencer mostly use the syringe pump negative pressure aspiration method to achieve reagent injection. Since the chip is usually bonded with very thin glass (150-170μm) as the substrate, the pipeline The total pressure drop will cause a certain pressure difference between the inner and outer surfaces of the glass substrate. This pressure difference will cause a certain deformation of the glass substrate. These deformations not only strongly affect the normal injection of reagents in the entire flow path system, but also affect the acquisition of the chip. effect. In order to highlight the technical effects of the present invention, the present invention also compares the common second-generation sequencing chip (two flow channels) and the chip (7 flow channels) obtained in the third embodiment under the same negative pressure on the glass substrate. In the case of deformation, the same negative pressure (15 kPa) was applied to both chips, assuming that the mechanical parameters of the glass substrate were consistent (Young's modulus 72.9 kN/mm ² , Poisson's ratio 0.2, density 2150 g/cm ³ ), and the results were as follows. Figure 5 shows:

a is a typical second-generation chip model (one support in the middle, two flow paths), b is the deformation of its glass substrate under the pressure of 10 μL/s or 15 kPa typical flow, c is the side view of its deformation; d is There is only a chip model of six support faces (seven flow channels), and e is the amount of deformation of the base of the chip having three flow paths (Example 3). The color bar on the right represents the shape variable, the bottom is blue, the top is red, and the unit is m, where the negative sign represents the direction of deformation downward;

As can be seen from FIG. 5, since the flow path width of the second-generation sequencer chip is large (about 5-10 mm), the deformation amount reaches about 28 μm under the line pressure drop of 15 kPa; for the chip of the third embodiment, When the solid surface of the support is set to 6 (the formation of 7 flow channels), the deformation of the glass substrate under the pressure of 15kpa is less than 5μm, it is foreseen that when the number of flow channels of the chip is increased to 15-25 The shape variable of the base of the chip will be reduced to a smaller value. In addition, by calculation, the chip of the embodiment 3 has a small flow resistance and does not substantially reach a line pressure drop of 15 kPa. In fact, the overall line pressure drop of the chip does not exceed 5 kPa.

Example 4

A method for preparing a single molecule sequencing chip includes the following steps:

(1) taking a silicon wafer as a substrate, and forming a positive film of the array of the reaction cell on the surface of the silicon wafer by photolithography according to the graphic template of the designed reaction cell array;

a. Take a silicon wafer, wash the surface of the substrate with absolute ethanol and water in turn, and then place the cleaned silicon wafer at 150 ° C. The surface of the hot plate is heated for 10 minutes to completely evaporate the surface; the treated silicon wafer is placed on the rotating stage of the homogenizer, and the negative photoresist SU-8 2150 is spin-coated to start the homogenizing machine to enable photolithography. The glue is evenly spread on the silicon wafer to obtain the silicon wafer after the gelatinization, wherein the thickness of the photoresist layer on the surface of the silicon wafer is 650 μm, the acceleration time of the homogenizer is set to 18 s, and the uniform gel time is 60 s, uniform The speed of the glue is 1000 rpm;

b. pre-baking the homogenized silicon wafer, the temperature of the pre-baking is controlled to: slowly heat to 100 ° C and stabilize for 15 min, then naturally cool to room temperature;

c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the silicon wafer after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 150 s;

d. The exposed silicon wafer is post-baked, and the temperature of the post-baking is controlled to: gradually increase the temperature to 95 ° C at a rate of 0.5 ° C / min, hold for 5 min, and then naturally cool to 1 ° C / min to Room temperature

e, immersing the post-baking silicon wafer in SU-8 developer, developing treatment, cleaning off the portion other than the mask, washing the residual developer with isopropyl alcohol, and finally using deionized water to leave the residue The isopropyl alcohol is washed away to obtain a positive film of the reaction cell array, and the positive film is convex;

(2) using polymethyl methacrylate (PMMA) as a model glue, casting the positive film, after vacuum degassing, curing at 95 ° C for 1 h, transferring the positive film of the reaction cell array in the model The bottom of the glue, the film is peeled off, a model glue layer with a plurality of flow channels is obtained, and a hole is formed at each end of each flow channel to form a fluid input hole and an output hole to obtain a substrate;

(3) substrate modification: taking quartz with aldehyde groups on the surface as a transparent substrate, preparing a PMGI layer on the surface of the transparent substrate to obtain a surface-modified transparent substrate, the PMGI layer having a thickness of 3 μm;

(4) encapsulating the chip: the substrate and the surface-modified transparent substrate are placed in an oxygen plasma cleaning machine for cleaning, and the substrate and the surface-modified transparent substrate are placed in an oxygen plasma cleaning machine for cleaning, and then taken out. Firstly, the substrate is initially adhered to the transparent substrate, placed in an oven at a temperature of 100 ° C, and pressed with a weight for 3 hours to complete the pressing of the substrate and the substrate to form a space for containing the fluid; 300 μL of NMP was injected into each channel, and the reaction time was 10 min to wash away the PMGI layer in contact with each channel to obtain a spacer layer on the surface of the transparent substrate, thereby completing the fabrication of a single molecule sequencing chip. The sequencing chip includes a base layer in which the substrate and the substrate are press-fitted, and the base layer includes a transparent substrate and a spacer layer disposed on the surface of the transparent substrate.

The single-molecule sequencing chip prepared in the fourth embodiment comprises a substrate and a substrate layer on which the substrate is press-fitted, the substrate comprising a first surface and a second surface disposed opposite to each other, the first surface of the substrate An array of reaction cells formed by a plurality of flow channels is disposed at intervals, and two opposite sidewalls of each of the flow channels extend along a length direction of the flow channel and two of the flow channels The ends meet to form two tapered ends with angles, the two tapered end surfaces being respectively provided with a fluid input hole and a fluid output hole communicating with the second surface of the substrate, the base layer comprising a transparent substrate And a spacer layer disposed on the surface of the transparent substrate, the spacer layer is in contact with the first surface of the substrate, and the spacer layer is provided with a corrosion recess corresponding to a position where the flow path is located.

In the fourth embodiment, the angle of the tapered end is 60°, and the array of the reaction cell includes 15 flow paths, and the spacing between adjacent flow channels is 1 mm. That is, the width of the spacer layer between the directions perpendicular to the longitudinal direction of the flow channel is 1.5 mm. Each flow channel has a length of 50 mm, each flow channel has a width of 1 mm, and each flow channel has a depth d of 0.6 mm; the substrate has a first side length perpendicular to the longitudinal direction of the flow channel, and each flow The intersection of the two opposite sidewalls of the channel is 0.6 cm from the first side of the substrate; the fluid input aperture is coaxial with the fluid output aperture, and the aperture size is 300 μm.

In the fourth embodiment, the transparent substrate is quartz having an aldehyde group on the surface, the spacer layer is made of PMGI, and the depth of the etching groove is 3 μm. The material of the substrate is PMMA. The DNA molecule to be tested modified with -NH ₂ can be immobilized by acting on the aldehyde group on the transparent substrate of the chip of the present embodiment.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (20)

1. A method for preparing a chip, comprising the steps of:

   (1) taking a substrate, according to the graphic template of the designed reaction cell array, using a photolithography method to form a positive film of the reaction cell array on the surface of the substrate;

   (2) casting the positive film with a model glue, after vacuum degassing, curing at 90-100 ° C for 1-3 h, transferring the positive film of the reaction cell array to the bottom of the model glue, and peeling off the film. Obtaining a model glue layer with a plurality of flow channels, and making a hole at each end of each flow channel to form a fluid input hole and an output hole to obtain a substrate;

   (3) substrate modification: taking a transparent substrate, preparing a protective layer on the surface of the transparent substrate to obtain a surface-modified transparent substrate;

   (4) encapsulating a chip: performing oxygen plasma cleaning on the substrate and the surface-modified transparent substrate, and then pressing the substrate and the transparent substrate to form a space for containing a fluid; and then injecting an etchant into each of the channels. A portion of the protective layer in contact with each of the flow channels is washed to obtain a spacer layer on the surface of the transparent substrate, and the preparation of the single molecule sequencing chip is completed.
2. The method for preparing a chip according to claim 1, wherein in the step (3), the protective layer is made of polymethylglutarimide.
3. The method of producing a chip according to claim 1, wherein in the step (4), the etchant is N-methylpyrrolidone.
4. The method for preparing a chip according to claim 1, wherein the spacer layer is obtained by cleaning a portion of the protective layer that is in contact with each flow channel by an etchant, and the spacer layer is made of polymethylpentene. Imide.
5. The method of manufacturing a chip according to claim 1, wherein in the step (3), the protective layer has a thickness of 1-5 μm.
6. The method of manufacturing a chip according to claim 3, wherein in the step (4), the amount of the etchant injected into each of the channels is from 100 to 500 μL.
7. The method of preparing a chip according to claim 1, wherein in the step (4), the array of the reaction cells comprises 15-25 flow channels, and a spacing between adjacent flow channels is 1-1.5 mm.
8. The method of manufacturing a chip according to claim 1, wherein in the step (4), the two side walls of each of the flow channels are oppositely disposed, and the width of each of the flow channels is The width is 1-2 mm; the depth of each of the flow channels is 0.6-1 mm.
9. The method for preparing a chip according to claim 1, wherein in the step (2), the material of the substrate comprises polydimethylsiloxane, polymethyl methacrylate, ethylene-vinyl acetate and poly. One of the amines.
10. The method of preparing a chip according to claim 1, wherein the transparent substrate comprises transparent glass, quartz or organic having a functional group of one of an epoxy group, an amino group, a carboxyl group, a thiol group and an aldehyde group. Polymer material.
11. The method of manufacturing a chip according to claim 1, wherein in the step (1), the photolithography method comprises the following steps:

    a, the negative photoresist is prepared by spin coating to the surface of the substrate to obtain a uniform substrate, wherein the thickness of the photoresist layer on the surface of the substrate is 600-650 μm;

    b. pre-baking the substrate after the homogenization, the temperature of the pre-baking is controlled to: slowly heat to 90-100 ° C and stabilize for 15-20 min, then naturally cool to room temperature;

    c, the patterned template of the designed reaction cell array is used as a mask plate, and covers the surface of the substrate after the pre-baking treatment, and exposure treatment is performed, and the exposure time is 90-150 s;

    d, the exposed substrate is post-baked, the temperature of the post-baking is controlled: first slowly heated to 90-95 ° C and stabilized for 5-10 min, then naturally cooled to room temperature;

    e. The substrate after the post-baking treatment is immersed in a developing solution, developed, and the portion other than the mask is washed away to obtain a positive film of the cell array.
12. The method of fabricating a chip according to claim 11, wherein after the step e of the photolithography method, the following processing is further included:

    f. The positive film of the reaction cell array obtained in step e is subjected to a hard baking treatment, wherein the hard baking is to add a glass plate to the positive film, press an iron block, and heat to 130-150 ° C for baking 45 -60min.
13. The method for preparing a chip according to claim 11, wherein in the step b, the temperature of the pre-baking is controlled by first baking at 65 ° C for 6 min, and then gradually increasing the temperature to 95 at a rate of 1 ° C / min. °C, hold for 20min, and naturally cool to room temperature.
14. The method for preparing a chip according to claim 11, wherein in step e, the temperature of the post-baking is controlled to be gradually increased to 95 ° C at a rate of 0.5 ° C / min for 5 min, and then 1- The temperature was naturally cooled to room temperature at a rate of 3 ° C/min.
15. A chip characterized in that the chip is prepared by the method of any one of claims 1-14.
16. A chip according to claim 15, said chip comprising a substrate and a substrate layer press-bonded to said substrate, said substrate comprising oppositely disposed first and second surfaces, said first of said substrates The surface is spaced apart from the array of reaction cells formed by a plurality of flow channels, and the two opposite sidewalls of each of the flow channels extend along the length of the flow channel and meet at the two ends of the flow channel to form two bands. An angled tapered end, the two tapered end surfaces being respectively provided with a fluid input aperture and a fluid output aperture in communication with the second surface of the substrate, the base layer comprising a transparent substrate and disposed in the a spacer layer on the surface of the transparent substrate, the spacer layer is in contact with the first surface of the substrate, and the spacer layer is provided with a corrosion recess corresponding to a position where the flow path is located.
17. The chip of claim 16 wherein said chip further comprises a probe attached to a surface of said transparent substrate of said chip.
18. Use of the chip of any of claims 15-17 in sequence capture and/or nucleic acid sequencing.
19. A kit comprising the chip of any of claims 15-17.
20. Use of the kit of claim 19 for sequence capture and/or nucleic acid sequencing.

Images