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WO2017028759A1 - 芯片的制备方法、芯片及应用 - Google Patents

芯片的制备方法、芯片及应用 Download PDF

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Publication number
WO2017028759A1
WO2017028759A1 PCT/CN2016/095050 CN2016095050W WO2017028759A1 WO 2017028759 A1 WO2017028759 A1 WO 2017028759A1 CN 2016095050 W CN2016095050 W CN 2016095050W WO 2017028759 A1 WO2017028759 A1 WO 2017028759A1
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Prior art keywords
substrate
chip
transparent substrate
baking
layer
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PCT/CN2016/095050
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English (en)
French (fr)
Inventor
吴平
颜钦
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深圳市瀚海基因生物科技有限公司
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Publication of WO2017028759A1 publication Critical patent/WO2017028759A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • 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.
  • 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.
  • 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.
  • the second-generation sequencing chip usually has a large width of the chip channel.
  • 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.
  • 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 comprises the following steps:
  • 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;
  • PMGI polymethylglutarimide
  • (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.
  • NMP Methylpyrrolidone
  • the substrate comprises one of a silicon wafer, a glass, a metal or a ceramic.
  • the model glue comprises polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), ethylene-vinyl acetate (EVA) and polyurethane (PUA).
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • EVA ethylene-vinyl acetate
  • PUA polyurethane
  • the model glue is polydimethylsiloxane (PDMS).
  • the substrate is made of the same material as the model glue.
  • the polymethylglutarimide (PMGI) layer has a thickness of from 1 to 5 ⁇ m.
  • 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.
  • PMGI polymethylglutarimide
  • the product names are SF1, SF2, SF3, SF4, SF5, SF6, SF7.
  • the PMGI is polymethylglutarimide commercially available from MicroChem under the product name SF11.
  • the injection amount of N-methylpyrrolidone in each flow channel is from 100 to 500 ⁇ L.
  • 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.
  • 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.
  • 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.
  • 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.
  • the photolithography method comprises the following steps:
  • 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 ⁇ m;
  • 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;
  • 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;
  • 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;
  • 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.
  • the method 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.
  • the following processing is further included:
  • 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.
  • 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.
  • 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.
  • the negative photoresist is SU-8 2150.
  • the array of reaction cells comprises 15-25 flow channels.
  • the width of the spacer layer between the directions perpendicular to the longitudinal direction of the flow channel is 1-1.5 mm.
  • the spacing between adjacent flow channels is 1-1.5 mm.
  • the angle of the tapered end is 30-60°.
  • 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.
  • 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.
  • 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.
  • the fluid undergoes a biochemical reaction in the flow channel.
  • each flow channel has a length of 50 mm, a width of 1 mm, and a depth of 0.6 mm.
  • 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.
  • 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.
  • the fluid input aperture is coaxial with the fluid output aperture.
  • the fluid input aperture has a pore size of 300-500 ⁇ m.
  • the fluid output aperture has a pore size of 300-500 ⁇ m.
  • 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.
  • the fluid input hole and the fluid output hole are used for connecting fluid input and output devices, 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.
  • the material of the substrate comprises one or more of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), EVA (ethylene vinyl acetate) and PUA (polyurethane).
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • EVA ethylene vinyl acetate
  • PUA polyurethane
  • 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.
  • the spacer layer is made of polymethylglutarimide (PMGI).
  • PMGI polymethylglutarimide
  • 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.
  • 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 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.
  • the chip is subsequently applied to capture target regions, nucleic acid sequencing and the like.
  • 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.
  • the invention provides a chip made by the method of the first 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.
  • 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).
  • primers preferably targeting primers
  • 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.
  • 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.
  • CCD photoelectric coupling device
  • CMOS complementary metal oxide semiconductor
  • 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.
  • 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.
  • the reagents required may include immobilization reagents, extension reaction reagents, imaging reagents, and reagents for excising optical detection label molecules.
  • the kit also includes a buffer or other sequencing necessary reagent.
  • 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.
  • 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.
  • 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.
  • model gel such as PDMS, etc.
  • 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.
  • 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.
  • 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.
  • 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 are 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 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
  • 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.
  • 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.
  • 11 is a flow channel
  • 12 is a fluid input hole communicating with the second surface 1b of the substrate
  • the depth of the etching groove provided by the spacer layer 22 corresponding to the position of the flow channel is represented by h;
  • FIG. 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. ;
  • 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.
  • a method for preparing a single molecule sequencing chip includes 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 ⁇ m
  • the acceleration time of the homogenizer is set to 18 s
  • the time of uniform homogenization is 60 s
  • the speed of uniform homogenization is 1000 rpm.
  • 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;
  • 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;
  • 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
  • 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.
  • 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;
  • 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 ⁇ m;
  • 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 ⁇ L 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.
  • NMP N-methylpyrrolidone
  • 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.
  • 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.
  • 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 ⁇ m.
  • the angle of the tapered end 113 is 60°.
  • 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.
  • the length l of each flow path is 50 mm.
  • the width d of each flow path is 1 mm.
  • the depth d of each flow path is 0.6 mm.
  • 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.
  • the fluid input aperture 12 and the fluid output aperture 13 are coaxial.
  • the fluid input hole 12 and the fluid output hole 13 have a pore size of 300 ⁇ m.
  • 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 ⁇ m.
  • 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.
  • 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.
  • 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).
  • the fluid recirculation zone c indicated by the circle is hardly present in the tapered design B.
  • 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.
  • a method for preparing a single molecule sequencing chip includes 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 ⁇ m
  • the acceleration time of the homogenizer is set to 18 s
  • the time of uniform homogenization is 60 s
  • the speed of uniform homogenization is 1000 rpm.
  • 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;
  • 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;
  • 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;
  • 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.
  • 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.
  • 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 ⁇ m;
  • 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 ⁇ L 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.
  • the angle of the tapered end is 30°
  • the array of the reaction cell includes 25 flow paths
  • 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 ⁇ m.
  • 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 ⁇ m.
  • a method for preparing a single molecule sequencing chip includes the following steps:
  • 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;
  • 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;
  • 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;
  • 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.
  • 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.
  • 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 ⁇ m;
  • 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.
  • 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 ⁇ L 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.
  • 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.
  • the angle of the tapered end is 45°
  • 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 ⁇ m.
  • the transparent substrate is a transparent borosilicate glass with an epoxy group
  • the spacer layer is made of PMGI
  • the depth of the etching groove is 5 ⁇ m.
  • 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 (150-170 ⁇ m) 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.
  • 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 ⁇ L/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;
  • the deformation amount reaches about 28 ⁇ m under the line pressure drop of 15 kPa; for the chip of the third embodiment,
  • 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 5 ⁇ m, 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.
  • 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.
  • a method for preparing a single molecule sequencing chip includes the following steps:
  • 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 ⁇ m, 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;
  • 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;
  • 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;
  • 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
  • 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 ⁇ m;
  • 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.
  • 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 ⁇ L 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
  • 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.
  • the angle of the tapered end is 60°
  • 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 ⁇ m.
  • the transparent substrate is quartz having an aldehyde group on the surface
  • the spacer layer is made of PMGI
  • the depth of the etching groove is 3 ⁇ m.
  • 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.

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Abstract

一种芯片及其制备方法和应用。具体而言，涉及芯片的制备方法，方法包括阳膜的制备、基片的获得、基底修饰和封装芯片的步骤；根据该方法制备得到的芯片以及试剂盒，以及利用芯片或试剂盒在序列捕获和核酸测序中的用途；该芯片的制备方法工艺简单、制作成本低。

Description

芯片的制备方法、芯片及应用
本发明要求2015年08月14日向中国专利局递交的发明名称为“一种单分子测序芯片的制备方法”、申请号为201510500393.7的在先申请的优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及芯片的制备技术领域，具体涉及一种芯片的制备方法、芯片及应用。
背景技术
进入21世纪后，人类基因组计划的完成对当代的生物学研究和医学研究产生了巨大的影响。就基因序列分析而言，后基因组时代的重点已由单个物种的全基因组序列测定转移到了对某一物种在基因组DNA序列层次上对个体遗传差异及物种间遗传差异的比较。靶向基因重测序将会是未来临床基因检测的主流技术。单分子测序技术被誉为第三代测序技术，其显著特征是可以高保真地对DNA片段直接进行识别，单分子测序技术由于能识别到单个核酸分子，其具有比高通量测序技术(统称为二代测序技术)更高的检测灵敏度。
基因芯片是测序技术得以实现的关键部件。然而，二代测序芯片是目前市场上的主流产品，它们大多是采用半导体纳米加工工艺得到高密度的纳米阵列，加工工艺精细且复杂，成本非常高，且需要大型的高精度仪器和超高级别的洁净室来完成。另外，二代测序芯片为了实现高通量的测序目的，芯片通道的宽度通常较大，在负压抽液方式进样进行生化反应时，通常会存在流体的流场分布不均问题和盖玻片易变形的问题。流场分布不均问题会造成试剂切换不干净，并使生化反应受影响，盖玻片变形会影响芯片的质量，更会影响碱基光学信号的采集。
单分子测序技术由于在测序数据量上要求不高，无需像二代测序芯片要求超高密度的纳米阵列，因此，有必要提供一种适用于单分子测序的芯片的制备方法。
发明内容
鉴于此，本发明提供了一种芯片的制备方法，该方法制备工艺简单，成本低，采用该方法所制得的芯片的流场分布情况良好，芯片的变形率低，芯片内流体的冲刷切换彻底。
本发明提供的芯片的制备方法，包括以下步骤：
(1)取一基板，根据设计好的反应池阵列的图形模板，采用光刻法在基板的表面制作反应池阵列的阳膜；
(2)用模型胶浇注所述阳膜，经过真空除气后，在90-100℃下固化1-3h，使所述反应池阵列的阳膜转移在所述模型胶的底部，揭膜，得到带有多个流道的模型胶层，并在每个流道的两端各打一个孔，形成流体输入孔和输出孔，得到基片；
(3)基底修饰：取一透明基底，在所述透明基底的表面制备一聚甲基戊二酰亚胺(PMGI)层，得到表面修饰的透明基底；
(4)封装芯片：将上述基片与所述表面修饰的透明基底进行氧等离子体清洗，之后将所述基片与透明基底压合形成容纳流体的空间；然后往每个流道内注入N-甲基吡咯烷酮(简写为NMP)，反应时间为10min-15min，以清洗掉与各流道接触的聚甲基戊二酰亚胺层，得到位于所述透明基底表面的间隔层，完成单分子测序芯片的制备。
优选地，步骤(1)中，所述基板包括硅片、玻璃、金属或陶瓷中的一种。
优选地，步骤(2)中，所述模型胶包括聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、乙烯-醋酸乙烯(EVA)和聚胺脂(PUA)中的一种，但不限于此，只要是适用于软光刻法的模型胶即可。
更优选地，步骤(2)中，所述模型胶为聚二甲基硅氧烷(PDMS)。
如本发明所述的，所述基片的材质与模型胶相同。
优选地，步骤(3)中，所述聚甲基戊二酰亚胺(PMGI)层的厚度为1-5μm。
本发明中，所述聚甲基戊二酰亚胺，其英文全称为polymethylglutarimide(PMGI)，所述PMGI是购买自MicroChem公司的、产品名为SF1、SF2、SF3、SF4、SF5、SF6、SF7、SF7.5、SF8、SF9、SF10、SF11、SF12、SF13、SF14、SF15、SF17、SF19、SF23中的一种或多种。
优选地，所述PMGI是购买自MicroChem公司的、产品名为SF11的聚甲基戊二酰亚胺。
优选地，步骤(4)中，每个流道内N-甲基吡咯烷酮的注入量为100-500μL。
所述N-甲基吡咯烷酮的注入量可以将透明基底上的牺牲层全部或部分清洗掉，但只要保证与各流道接触的牺牲层清洗掉即可，使得注入流道的DNA流体可与基底表面的环氧基、氨基、羧基、巯基和醛基等官能团发生作用而被固定下来。
如本发明所述的，在将基片与表面修饰的透明基底进行氧等离子处理表面之前，制备 PMGI层的目的是为了保护透明基底表面的官能团(如环氧基、氨基、羧基、巯基和醛基中的一种)，避免其受等离子氧处理的影响，使得后续的基因样本固定到透明基底上。修饰后的透明基底在经过氧等离子清洗后，透明基底的表面由疏水性变成亲水性；再向每个流道注入N-甲基吡咯烷酮，该试剂可以将与各流道接触的PMGI腐蚀掉，使得透明基底表面的官能团再次暴露。
该单分子测序芯片的流道为亲水性，可以减少对待测DNA分子的非特异性吸附，同时基底表面的官能团不受影响。
优选地，步骤(4)中，所述基片与透明基底的压合具体为：先将基片与透明基底初步贴合，并放入温度为95-120℃的烤箱内，用重物压住烘烤1-3h。
优选地，步骤(1)中，所述光刻法包括以下步骤：
a、将负性光刻胶通过旋涂法制备到基板表面，得到匀胶后的基板，其中基板表面的光刻胶层厚度为600-650μm；
b、对匀胶后的基板进行前烘处理，所述前烘的温度控制为：缓慢加热到90-100℃并稳定15-20min，之后自然冷却至室温；
c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的基板表面，进行曝光处理，曝光时间为90-150s；
d、将曝光后的基板进行后烘处理，所述后烘的温度控制为：先缓慢加热到90-95℃并稳定5-10min，之后自然冷却至室温；
e、将后烘处理后的基板浸入显影液中，进行显影处理，清洗掉掩膜版以外的部分，得到反应池阵列的阳膜。
优选地，在进行所述光刻法的步骤a之前，还包括对基板进行以下预处理：
依次用无水乙醇、水清洗基板的表面，之后将清洁过的基板置于150℃的热板上加热10min，使表面水分彻底蒸发。
优选地，在所述光刻法的步骤e之后，还包括以下处理：
f、将步骤e得到的反应池阵列的阳膜进行硬烘处理，所述硬烘是在所述阳膜上加一玻璃板，并压一铁块，加热至130-150℃下烘烤45-60min。
所述硬烘是为了使阳膜的光刻胶层更牢固地粘附在基板表面，并增加光刻胶层的抗刻蚀能力。
优选地，步骤b中，所述前烘的温度控制为：先在65℃下烘烤6min，再按1℃/min 的速率逐渐升温至95℃，保持20min，自然冷却至室温。所述前烘的目的是将光刻胶中的有机溶剂蒸发掉，并使光刻胶凝固。
优选地，步骤e中，所述后烘的温度控制为：先以0.5℃/min的速率逐渐升温至95℃，保持5min，再以1-3℃/min的速率自然冷却至室温。所述后烘的目的是以使曝光部分的负性光刻胶的交联反应充分进行。
优选地，所述负性光刻胶为SU-8 2150。
优选地，所述反应池阵列包括15-25个流道。
优选地，所述间隔层沿与所述流道的长度方向垂直的方向之间的宽度为1-1.5mm。
如本发明所述的，相邻流道之间的间距为1-1.5mm。
优选地，所述锥形末端的夹角为30-60°。
优选地，每个所述流道相对设置的两个侧壁的交汇处的距离为每个流道的长度，每个所述流道的长度为50-75mm。
优选地，每个所述流道相对设置的两个侧壁之间的距离为每个流道的宽度，每个所述流道的宽度为1-2mm。
优选地，每个所述流道的深度为0.6-1mm。
将流道的深度优选为0.6-1mm，根据矩形流道流体力学阻力规律，厚度方向每增加一倍，流阻降低为原来的1/8，小的流阻，有利于流体的流动，便于单分子测序过程中，流体在流道内进行生化反应。
更优选地，每个流道的长度50mm，宽度为1mm，深度为0.6mm。
在流体上来说，宽度更窄的流道更有利于流体间的冲刷切换，将流道两端的纵截面设计成为三角形，当流体流过该流道内，可使流道内不存在回流现象。
优选地，所述基片具有与所述流道的长度方向垂直的第一边长，每个流道相对设置的的两个侧壁的交汇处距所述基片的第一边长的距离为0.5-1cm。
优选地，所述流体输入孔和流体输出孔同轴。
优选地，所述流体输入孔的孔径大小为300-500μm。
优选地，所述流体输出孔的孔径大小为300-500μm。
如发明所述的，所述基片第一表面间隔设置有多个流道组成单分子测序芯片的反应池阵列，每个流道的两个锥形末端表面开设有流体输入孔和输出孔，以供流体的流入与流出。
如本发明所述的，所述流体输入孔和流体输出孔用于连接流体的输入、输出装置，例 如，可以在所述流体输入孔和流体输出孔分别插上移液枪头、管接头等，以分散每个流道内流体的输入点、输出点，便于每个流道内流体的输入、输出不受干扰。
优选地，所述基片的材质包括聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、EVA(乙烯-醋酸乙烯)和PUA(聚胺脂)中一种或多种，但不限于此，只要能实现浇筑工艺即可。
优选地，所述透明基底包括表面带有官能团为环氧基、氨基、羧基、巯基和醛基中的一种的透明的玻璃、石英或有机聚合物材料。
优选地，所述间隔层的材质为聚甲基戊二酰亚胺(PMGI)。所述间隔层用于阻隔各流道内的样本之间的接触，保证每个流道内样本的单独控制。
优选地，所述间隔层是向每个流道内注入试剂以清洗掉与各流道接触的透明基底上的聚甲基戊二酰亚胺层，以使透明基底上表面带有的官能团(环氧基、氨基、羧基、巯基和醛基等)暴露出来。所述间隔层用于阻隔各流道内的样本之间的接触，保证每个流道内样本的单独控制。
优选地，所述芯片还包括探针，所述探针连接在所述芯片的透明基底的表面。
所述透明基底表面的官能团(如环氧基、氨基、羧基、巯基和醛基)，可与探针的官能团(如羧基、磷酸基、氨基等)发生作用，使得探针固定在芯片的透明基底上，从而后续改该芯片应用到捕获目标区域、核酸测序等领域。如透明基底上的环氧基可与修饰有-NH2的DNA探针发生化学反应，通过新的-CH2-NH-键将探针固定在修饰有环氧基团的基底表面。
所述探针可以包括引物(优选为靶向引物)。所述引物可以靶向或非靶向地捕获待测样品的核酸。
第二方面，本发明提供了采用第一方面的方法制得的芯片。
第三方面，本发明提供了第二方面所述的芯片在序列捕获和/或核酸测序中的应用。所述核酸测序包括DNA和/或RNA测序。
如无特殊说明，文中所称的“靶向引物”与“序列”、“引物”或“探针”可替换使用，指一段(寡)核苷酸序列。
所述序列捕获可以包括引物(优选为靶向引物)固定，以及采用固定的引物来捕获待测目标核酸(在核酸测序技术领域，也可称“模板核酸”)。“引物-待测核酸复合物”同“引物/待测核酸复合物”，表示由引物和待测核酸连接形成的复合物。若无特殊说明，本发明 所述的“待测核酸”与“模板核酸”可以互换。
所述芯片在应用时，在下层透明基底的外部设有荧光检测器，荧光检测器为光电耦合器件CCD或互补性氧化金属半导体CMOS中的一种。经过微流体通道中的生化反应，可用多种光学波长来检测固定在透明基底上的DNA分子中某一特定位置的碱基，从而确定固定在透明基底上的DNA序列。
第四方面，本发明提供了一种试剂盒，包括本发明第一方面所述的芯片和试剂。可以根据所述试剂盒的用途，来选择其所用的试剂。
第五方面，本发明提供了第四方面所述的试剂盒在在捕获目标区域和/或核酸测序中的用途。所述核酸测序包括DNA和/或RNA测序。
可以根据所述试剂盒的用途，来选择其所用的试剂，所述试剂可以包括一种或多种。例如，当所述芯片用于核酸测序(例如单分子测序)时，所需要的试剂可包括固定反应试剂、延伸反应试剂、成像试剂、切除光学检测标记分子的试剂。
可以理解的是，所述试剂盒还包括缓冲液或其他测序必要试剂。在本发明实施例中，所述固定反应试剂、延伸反应试剂、成像试剂、切除光学检测标记分子的试剂均没有特别限制。本领域现有普通常用的即可。比如，本领域技术人员根据需要分别配置针对的固定反应试剂、延伸反应试剂、切除光学检测标记分子等不同过程的缓冲液。
本发明通过光刻-浇注法将各流道制备在基片上，并与表面修饰的透明基底压合密封而成，得到所述单分子测序芯片，本发明提供的单分子测序芯片的制备方法简单，可操作性强，制造成本低，所述单分子测序芯片的制备可以摆脱昂贵、精细的半导体工艺的束缚。
采用该方法制得的芯片具有一定数目的流道，每个流道带有夹角的锥形末端，可使流道内流体的流场分布情况良好，流道内不存在回流现象，流场分布情况远好于二代测序芯片，同时集成化的多流道的设计增加了基底的支撑点，基底的变形问题几乎可以忽略。该单分子测序芯片可以实现每个流道内样本的单独控制，保证了样本间无交叉污染，同时可以简化后期数据处理工作。
本发明有益效果包括以下几个方面：
1、由于模型胶(如PDMS等)本身超强的疏水性，其容易非特异性地粘附DNA等生物大分子，使得其较少被报道应用于DNA测序芯片的基片。本发明基于软光刻-浇筑工艺制作出适合单分子DNA测序的芯片，在保护基底表面的官能团的前提下，采用等离子处理将基 片表面由疏水性改成亲水性，使其能符合DNA测序芯片的要求，该单分子测序芯片的流道既可以减少对待测DNA分子的非特异性吸附，同时基底表面是官能团不受影响。
2、由于光刻法制成的反应池阵列的阳膜可以重复多次浇筑使用，有利于单分子测序芯片的批量生产，并会进一步降低制造成本。
3、制得的芯片具有一定数目的流道，将各流道设计为较窄的流道，并且在流体进出口均设计成锥形，有利于形成流体缓冲带，可使流道内流体的冲刷切换彻底，不存在流体回流区，利于生化反应进行；同时流道的深度又较深，使流道内流体的流动阻力越小，可提高所述单分子测序芯片的相应时间。
4、制得的芯片中集成化的多流道，增加了基底的支撑面，有利于减小由于负压吸液造成的基底形变，克服了由于基底变形造成的芯片系统中流体的进样异常问题以及采集的图像效果不佳的问题。
5、制得的芯片，具有多条并行的流道，各流道之间相互独立，可以实现每个流道内样本的单独控制，保证了样本间无交叉污染，同时该单分子测序芯片无需像二代测序芯片中，在每个样本进样之前都要加入特定的一段序列“barcode”以识别出每个样本，简化基因样品准备的流程和后续的生物信息分析流程。
附图说明
图1为本发明实施例1中单分子测序芯片的一剖面结构示意图，1为基片，21为透明基底，22为透明基底21上的间隔层，2为21与22构成的基底层，1b为基片1的第二表面，11为流道，12为基片的第二表面1b连通的流体输入孔，间隔层22对应流道所在位置设置的腐蚀凹槽的深度用h表示；
图2是本发明实施例1中单分子测序芯片的基片的俯视结构示意图，1为基片，11为流道，12、13分别为流体输入孔和流体输出孔，111、112分别为流道相对设置的两个侧壁，113为流道的锥形末端，d表示的距离为每个流道的宽度，l表示的距离为每个流道的长度；
图3为本发明实施例的单分子测序芯片的制备方法的示意图，其中3为制作反应池阵列的阳膜的基底，1a为基片1的第一表面，1b为基片1的第二表面；
图4为二代测序芯片与本发明实施例1中单分子测序芯片的流体力学仿真模拟结果的对比；
图5为二代测序芯片与本发明实施例3中单分子测序芯片的透明基底的变形情况对比。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
实施例1
一种单分子测序芯片的制备方法(参见附图3的制备方法的示意图)，包括以下步骤：
(1)取一硅片作为基板，根据设计好的反应池阵列的图形模板，采用光刻法在硅片的表面制作反应池阵列的阳膜，具体包括以下步骤；
a、取一硅片，依次用无水乙醇、水清洗基板表面，之后将清洁过的硅片置于150℃的热板上加热10min，使表面水分彻底蒸发；将处理后的硅片置于匀胶机的旋转载物台上，旋涂负性光刻胶SU-8 2150，启动匀胶机，使光刻胶在硅片上均匀铺开，得到匀胶后的硅片，其中硅片表面的光刻胶层厚度为600μm，匀胶机的加速时间设定为18s，均匀匀胶的时间为60s，均匀匀胶的转速为1000转/分；
b、对匀胶后的硅片进行前烘处理，所述前烘的温度控制为：先在65℃时烘烤6min，再按1℃/min的速率逐渐升温至95℃，保持20min，自然冷却至室温；
c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的硅片表面，进行曝光处理，曝光时间为120s；
d、将曝光后的硅片进行后烘处理，所述后烘的温度控制为：先以0.5℃/min的速率逐渐升温至95℃，保持5min，再以2℃/min的速率自然冷却至室温；
e、将后烘处理后的硅片浸入SU-8显影液中，进行显影处理，清洗掉掩膜版以外的部分，使用异丙醇将残余的显影液洗去，最后使用去离子水将残留的异丙醇洗去，得到反应池阵列的阳膜；
f、将步骤e得到的反应池阵列的阳膜进行硬烘处理，以使阳膜的光刻胶层更牢固地粘附在硅片表面，所述硬烘是在所述阳膜上加一玻璃板，并压一铁块，加热至150℃烘烤60min，并自然冷却至室温，得到硬烘后的反应池阵列的阳膜，所述阳膜为凸起状；
(2)将陶氏化学公司的聚二甲基硅氧烷(PDMS)A、B胶按照质量比10:1混合，均匀搅拌后，倒入玻璃皿内，该玻璃皿内预先放置好上述制得的反应池阵列的阳膜，然后将该 玻璃皿放入真空设备内，抽真空1小时，待气泡抽干净后，放入烘箱内，保持底面水平，在95℃下固化1h，使所述反应池阵列的阳膜转移在所述模型胶的底部；从烘箱内取出玻璃皿，并冷却，用刀切下固化模型胶内的图案部分，之后揭膜，得到带有多个流道的凹槽的模型胶层，并在每个流道两端的流道底部各打一个孔，形成流体输入孔和输出孔，得到基片；
(3)基底修饰：取一表面带有环氧基的透明硼硅玻璃作为透明基底，在所述透明基底的表面制备一聚甲基戊二酰亚胺(PMGI，MicroChem公司的SF11系产品)层，得到表面修饰的透明基底，PMGI层的厚度为1μm；
(4)封装芯片：将上述基片与所述表面修饰的透明基底放入氧等离子清洗机进行清洗，之后将所述基片与透明基底压合形成容纳流体的空间；然后往每个流道内注入100μL的N-甲基吡咯烷酮(简写为NMP)，反应时间为10min，以清洗掉与各流道接触的PMGI层，以使硼硅玻璃表面带有的环氧团暴露出来，得到位于所述透明基底表面的间隔层，并在每个流道两端的流体输入孔和流体输出孔分别插上管接头，并用树脂密封胶合，完成单分子测序芯片的制作。
本实施例制得的的单分子测序芯片的结构示意图如图1所示，结合图1和基片的俯视结构示意图2一起来看，该单分子测序芯片包括基片1和所述基片压合设置的基底层2，所述基片1包括相对设置的第一表面(在图1中未画出，在图3中用1a表示)和第二表面1b，所述基片第一表面间隔设置有多个流道11形成的反应池阵列，每个所述流道11相对设置的两个侧壁111、112沿所述流道11的长度方向延伸并在所述流道的两端交汇形成两个带有夹角的锥形末端113，所述两个锥形末端113表面分别设置有与所述基片第二表面1b连通的流体输入孔12和流体输出孔13，所述基底层2包括透明基底21和设置在所述透明基底21表面的间隔层22，所述间隔层22与所述基片第一表面接触且所述间隔层11对应所述流道所在的位置设置有腐蚀凹槽，腐蚀凹槽的深度用h表示。
由图1可知，透明基底21上还有残留的聚甲基戊二酰亚胺(PMGI)层，但与各流道接触的对应透明基底上的PMGI层已经全部清除掉，即得到相间设置在透明基底21的间隔层22。所述间隔层22是向每个流道内注入清洗试剂以洗掉与各流道接触的透明基底上的PMGI层所形成，以使透明基底上表面带有的环氧基官能团暴露出来。所述间隔层22用于阻隔各流道内的样本之间的接触，保证每个流道内样本的单独控制。间隔层对应流道所在位置得到的腐蚀凹槽的深度为1μm。
在本实施例中，锥形末端113的夹角为60°。
在本实施例中，所述反应池阵列包括20个流道，相邻流道之间的间距为1mm，间隔层22沿与流道的长度方向垂直的方向之间的宽度为1mm。每个流道相对设置的两个侧壁的交汇处的距离为每个流道的长度，在图2中用l表示；每个流道相对设置的两个侧壁之间的距离为每个流道的宽度，在图2中用d表示。
在本实施例中，每个流道的长度l为50mm。
在本实施例中，每个流道的宽度d为1mm。
在本实施例中，每个流道的深度d为0.6mm。
在本实施例中，基片具有与流道的长度方向垂直的第一边长，每个流道相对设置的两个侧壁的交汇处与基片的第一边长的距离为0.5cm。
在本实施例中，流体输入孔12和流体输出孔13同轴。
在本实施例中，流体输入孔12和流体输出孔13的孔径大小为300μm。
在本实施例中，透明基底21为表面带有环氧基团的透明硼硅玻璃。间隔层的材质为PMGI，腐蚀凹槽的深度为1μm。
在本实施例中，基片1的材质为聚二甲基硅氧烷(PDMS)。
对实施例1制得的单分子测序芯片进行流体力学仿真模拟，其结果如图4所示，A是流道两端不带锥形设计的单分子测序芯片内的流场，B是实施例1带有锥形入口段的单分子测序芯片内的流场，图4中左列的颜色数据条表示流体的体积分数(底部为蓝色，顶部为红色)，蓝色代表初始存在于芯片内的空气，红色代表即将进入芯片的流体，流体从右往左流，流体从一个点进入(A、B中的红色点即为初始流入状态，分别用方框圈出，随着时间的进行，通道由蓝色逐渐变成红色，当通道变成全红色时，表面流体已经充满整个流道)。从图4可以明显看出，圆圈表示的流体回流区c几乎不存在于带锥形设计的B中。
以上对比说明，本发明制备的单分子测序芯片将流体进出口均设计成锥形，有利于形成流体缓冲带，可使流道内流体的冲刷切换彻底，不存在流体回流区，利于生化反应进行。
实施例2
一种单分子测序芯片的制备方法，包括以下步骤：
(1)取一硅片作为基板，根据设计好的反应池阵列的图形模板，采用光刻法在硅片的表面制作反应池阵列的阳膜，具体包括以下步骤；
a、取一硅片，依次用无水乙醇、水清洗基板表面，之后将清洁过的硅片置于150℃的热板上加热10min，使表面水分彻底蒸发；将处理后的硅片置于匀胶机的旋转载物台上，旋涂负性光刻胶SU-8 2150，启动匀胶机，使光刻胶在硅片上均匀铺开，得到匀胶后的硅片，其中硅片表面的光刻胶层厚度为650μm，匀胶机的加速时间设定为18s，均匀匀胶的时间为60s，均匀匀胶的转速为1000转/分；
b、对匀胶后的硅片进行前烘处理，所述前烘的温度控制为：缓慢加热到100℃并稳定15min，之后自然冷却至室温；
c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的硅片表面，进行曝光处理，曝光时间为150s；
d、将曝光后的硅片进行后烘处理，所述后烘的温度控制为：先缓慢加热到90℃并稳定10min，之后自然冷却至室温；
e、将后烘处理后的硅片浸入SU-8显影液中，进行显影处理，清洗掉掩膜版以外的部分，使用异丙醇将残余的显影液洗去，最后使用去离子水将残留的异丙醇洗去，得到反应池阵列的阳膜；
f、将步骤e得到的反应池阵列的阳膜进行硬烘处理，以使阳膜的光刻胶层更牢固地粘附在硅片表面，所述硬烘是在所述阳膜上加一玻璃板，并压一铁块，加热至130℃烘烤45min，并自然冷却至室温，得到硬烘后的反应池阵列的阳膜，所述阳膜为凸起状；
(2)用模型胶PDMS浇注所述阳膜，经过真空除气后，在90℃下固化3h，使所述反应池阵列的阳膜转移在所述模型胶的底部，揭膜，得到带有多个流道的模型胶层，并在每个流道的两端各打一个孔，形成流体输入孔和输出孔，得到基片；
(3)基底修饰：取一表面带有环氧基的透明硼硅玻璃作为透明基底，在所述透明硼硅玻璃的表面制备一PMGI层，得到表面修饰的透明基底，所述PMGI层的厚度为3μm；
(4)封装芯片：将上述基片与所述表面修饰的透明基底放入氧等离子清洗机进行清洗，之后取出，先将基片与透明基底初步贴合，并放入温度为120℃的烤箱内，用重物压住烘烤2h，完成所述基片与透明基底的压合，并形成容纳流体的空间；然后往每个流道内注入300μL的NMP，反应时间为12min，以清洗掉与各流道接触的PMGI层，以使硼硅玻璃表面带有的环氧团暴露出来，得到位于所述透明基底的表面的间隔层，并在每个流道两端的流体输入孔和流体输出孔分别插上管接头，并用树脂密封胶合，得到单分子测序芯片。
本实施例2中制得的单分子测序芯片，其包括基片和所述基片压合设置的基底层，所述 基片包括相对设置的第一表面和第二表面，所述基片第一表面间隔设置有多个流道形成的反应池阵列，每个所述流道相对设置的两个侧壁沿所述流道的长度方向延伸并在所述流道的两端交汇形成两个带有夹角的锥形末端，所述两个锥形末端表面分别设置有与所述基片第二表面连通的流体输入孔和流体输出孔，所述基底层包括透明基底和设置在所述透明基底表面的间隔层，所述间隔层与所述基片第一表面接触且所述间隔层对应所述流道所在的位置设置有腐蚀凹槽。
在本实施例2中，锥形末端的夹角为30°，所述反应池阵列包括25个流道，相邻流道之间的间距为1.2mm。每个流道的长度为75mm，每个流道的宽度为1.5mm，每个流道的深度d为0.8mm；基片具有与所述流道的长度方向垂直的第一边长，每个流道相对设置的两个侧壁的交汇处与基片的第一边长的距离为0.8cm；流体输入孔和流体输出孔同轴，其孔径大小均为400μm。在本实施例中，透明基底为表面带有环氧基团的透明硼硅玻璃，基片的材质为聚二甲基硅氧烷(PDMS)。间隔层的材质为PMGI，间隔层对应流道所在位置得到的腐蚀凹槽的深度为3μm。
实施例3
一种单分子测序芯片的制备方法，包括以下步骤：
(1)取一玻璃作为基板，根据设计好的反应池阵列的图形模板，采用光刻法在玻璃的表面制作反应池阵列的阳膜，具体包括以下步骤；
a、取一玻璃，依次用无水乙醇、水清洗基板表面，之后将清洁过的硅片置于150℃的热板上加热10min，使表面水分彻底蒸发；将处理后的硅片置于匀胶机的旋转载物台上，旋涂负性光刻胶SU-8 2150，启动匀胶机，使光刻胶在硅片上均匀铺开，得到匀胶后的硅片，其中硅片表面的光刻胶层厚度为620μm，匀胶机的加速时间设定为18s，均匀匀胶的时间为60s，均匀匀胶的转速为1000转/分；
b、对匀胶后的硅片进行前烘处理，所述前烘的温度控制为：缓慢加热到95℃并稳定18min，之后自然冷却至室温；
c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的硅片表面，进行曝光处理，曝光时间为150s；
d、将曝光后的硅片进行后烘处理，所述后烘的温度控制为：先缓慢加热到92℃并稳定8min，之后自然冷却至室温；
e、将后烘处理后的硅片浸入SU-8显影液中，进行显影处理，清洗掉掩膜版以外的部分，使用异丙醇将残余的显影液洗去，最后使用去离子水将残留的异丙醇洗去，得到反应池阵列的阳膜；
f、将步骤e得到的反应池阵列的阳膜进行硬烘处理，以使阳膜的光刻胶层更牢固地粘附在硅片表面，所述硬烘是在所述阳膜上加一玻璃板，并压一铁块，加热至140℃下烘烤50min，自然冷却至室温，得到硬烘后的反应池阵列的阳膜，所述阳膜为凸起状；
(2)用模型胶PDMS浇注所述阳膜，经过真空除气后，在100℃下固化2h，使所述反应池阵列的阳膜转移在所述模型胶的底部，揭膜，得到带有多个流道的凹槽的模型胶层，并在每个流道的两端各打一个孔，形成流体输入孔和输出孔，得到基片；
(3)基底修饰：取一表面带有环氧基的透明硼硅玻璃作为透明基底，在所述透明硼硅玻璃的表面制备一PMGI层，得到表面修饰的透明基底，所述PMGI层的厚度为5μm；
(4)封装芯片：将上述基片与所述表面修饰的透明基底放入氧等离子清洗机进行清洗，之后取出，先将基片与透明基底初步贴合，并放入温度为95℃的烤箱内，用重物压住烘烤1h，完成所述基片与透明基底的压合，并形成容纳流体的空间；然后往每个流道内注入500μL的NMP，反应时间为15min，以清洗掉与各流道接触的PMGI层，以使硼硅玻璃表面带有的环氧团暴露出来，得到位于所述透明基底的表面的间隔层，并在每个流道两端的流体输入孔和流体输出孔分别插上管接头，并用树脂密封胶合，得到单分子测序芯片，该单分子测序芯片包括基片和基片压合设置的基底层，基底层包括透明基底和设置在透明基底表面的间隔层。
本实施例3中制得的单分子测序芯片，包括基片和所述基片压合设置的基底层，所述基片包括相对设置的第一表面和第二表面，所述基片第一表面间隔设置有多个流道形成的反应池阵列，每个所述流道相对设置的两个侧壁沿所述流道的长度方向延伸并在所述流道的两端交汇形成两个带有夹角的锥形末端，所述两个锥形末端表面分别设置有与所述基片第二表面连通的流体输入孔和流体输出孔，所述基底层包括透明基底和设置在所述透明基底表面的间隔层，所述间隔层与所述基片第一表面接触且所述间隔层对应所述流道所在的位置设置有腐蚀凹槽。
在本实施例3中，锥形末端的夹角为45°，所述反应池阵列包括7个流道，相邻流道之间的间距为1.5mm。即间隔层沿与流道的长度方向垂直的方向之间的宽度为1.5mm。每个流道的长度为60mm，每个流道的宽度为2mm，每个流道的深度d为1mm；基片具有与 所述流道的长度方向垂直的第一边长，每个流道相对设置的两个侧壁的交汇处与基片的第一边长的距离为1cm；流体输入孔和流体输出孔同轴，其孔径大小均为500μm。
在本实施例3中，透明基底为带有环氧基团的透明硼硅玻璃，间隔层的材质为PMGI，腐蚀凹槽的深度为5μm。基片的材质为PDMS。
二代测序仪、单分子测序仪大多数采用注射泵负压吸液方式来实现试剂进样，由于芯片在键合时通常都使用很薄的玻璃(150-170μm)作为基底，管路中的总压降会使玻璃基底内外表面承受一定的压差，该压差会导致玻璃基底有一定的变形，这些变形不仅强烈影响整个流路系统中试剂的正常进样，还影响着芯片的采图效果。为了突出本发明的技术效果，本发明还对比了常见的二代测序芯片(两条流道)和本实施例3制得的芯片(7条流道)在相同负压作用下的玻璃基底的变形情况，对两种芯片施以相同的负压(15kpa)，假设玻璃基底的力学参数一致(杨氏模量72.9kN/mm2，泊松比0.2，密度2150g/cm3)，其结果如图5所示：
a为典型的二代芯片模型(中间一条支撑，两个流道)，b是其在典型流量10μL/s即15kpa压力作用下其玻璃基底的变形情况，c是其变形的侧视图；d是只有6个支撑面(7条流道)的芯片模型，e是有7条流道的芯片(实施例3)的基底的变形量。右边的颜色条表示形变量，底部为蓝色，顶部为红色，单位是m，其中，负号代表形变的方向向下；
从图5可以看出，由于二代测序仪芯片的流道宽度较大(约为5-10mm)，在15kpa的管路压降下变形量达到将近28μm；对于本实施例3所述芯片，当支撑的固面设置为6条时(形成7条流道)，玻璃基底在同样15kpa的压力作用下，其变形量小于5μm，可以预见，当芯片的流道数目增加到15-25条时，芯片的基底的形变量将减至更小。另外，通过计算，实施例3的芯片的流阻很小，基本不会达到15kpa的管路压降，实际上该芯片整体的管路压降不会超过5kpa。
实施例4
一种单分子测序芯片的制备方法，包括以下步骤：
(1)取一硅片作为基板，根据设计好的反应池阵列的图形模板，采用光刻法在硅片的表面制作反应池阵列的阳膜；
a、取一硅片，依次用无水乙醇、水清洗基板表面，之后将清洁过的硅片置于150℃的 热板上加热10min，使表面水分彻底蒸发；将处理后的硅片置于匀胶机的旋转载物台上，旋涂负性光刻胶SU-8 2150，启动匀胶机，使光刻胶在硅片上均匀铺开，得到匀胶后的硅片，其中硅片表面的光刻胶层厚度为650μm，匀胶机的加速时间设定为18s，均匀匀胶的时间为60s，均匀匀胶的转速为1000转/分；
b、对匀胶后的硅片进行前烘处理，所述前烘的温度控制为：缓慢加热到100℃并稳定15min，之后自然冷却至室温；
c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的硅片表面，进行曝光处理，曝光时间为150s；
d、将曝光后的硅片进行后烘处理，所述后烘的温度控制为：先以0.5℃/min的速率逐渐升温至95℃，保持5min，再以1℃/min的速率自然冷却至室温；
e、将后烘处理后的硅片浸入SU-8显影液中，进行显影处理，清洗掉掩膜版以外的部分，使用异丙醇将残余的显影液洗去，最后使用去离子水将残留的异丙醇洗去，得到反应池阵列的阳膜，所述阳膜为凸起状；
(2)用聚甲基丙烯酸甲酯(PMMA)作为模型胶，浇注所述阳膜，经过真空除气后，在95℃下固化1h，使所述反应池阵列的阳膜转移在所述模型胶的底部，揭膜，得到带有多个流道的模型胶层，并在每个流道的两端各打一个孔，形成流体输入孔和输出孔，得到基片；
(3)基底修饰：取表面带有醛基的石英作为透明基底，在所述透明基底的表面制备一PMGI层，得到表面修饰的透明基底，所述PMGI层的厚度为3μm；
(4)封装芯片：将上述基片与所述表面修饰的透明基底放入氧等离子清洗机进行清洗，将上述基片与所述表面修饰的透明基底放入氧等离子清洗机进行清洗，之后取出，先将基片与透明基底初步贴合，并放入温度为100℃的烤箱内，用重物压住烘烤3h，完成所述基片与基底的压合，形成容纳流体的空间；然后往每个流道内注入300μL的NMP，反应时间为10min，以清洗掉与各流道接触的PMGI层，得到位于所述透明基底表面的间隔层，完成得到单分子测序芯片的制作，该单分子测序芯片包括基片和基片压合设置的基底层，基底层包括透明基底和设置在透明基底表面的间隔层。
本实施例4制得的单分子测序芯片，包括基片和所述基片压合设置的基底层，所述基片包括相对设置的第一表面和第二表面，所述基片第一表面间隔设置有多个流道形成的反应池阵列，每个所述流道相对设置的两个侧壁沿所述流道的长度方向延伸并在所述流道的两 端交汇形成两个带有夹角的锥形末端，所述两个锥形末端表面分别设置有与所述基片第二表面连通的流体输入孔和流体输出孔，所述基底层包括透明基底和设置在所述透明基底表面的间隔层，所述间隔层与所述基片第一表面接触且所述间隔层对应所述流道所在的位置设置有腐蚀凹槽。
在本实施例4中，锥形末端的夹角为60°，所述反应池阵列包括15个流道，相邻流道之间的间距为1mm。即间隔层沿与流道的长度方向垂直的方向之间的宽度为1.5mm。每个流道的长度为50mm，每个流道的宽度为1mm，每个流道的深度d为0.6mm；基片具有与所述流道的长度方向垂直的第一边长，每个流道相对设置的两个侧壁的交汇处与基片的第一边长的距离为0.6cm；流体输入孔和流体输出孔同轴，其孔径大小均为300μm。
在本实施例4中，透明基底为表面带有醛基的石英，间隔层的材质为PMGI，腐蚀凹槽的深度为3μm。基片的材质为PMMA。修饰有-NH2的待测DNA分子可与本实施例芯片的透明基底上的醛基发生作用而被固定下来。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (20)

  1. 一种芯片的制备方法，其特征在于，包括以下步骤：
    (1)取一基板，根据设计好的反应池阵列的图形模板，采用光刻法在基板的表面制作反应池阵列的阳膜；
    (2)用模型胶浇注所述阳膜，经过真空除气后，在90-100℃下固化1-3h，使所述反应池阵列的阳膜转移在所述模型胶的底部，揭膜，得到带有多个流道的模型胶层，并在每个流道的两端各打一个孔，形成流体输入孔和输出孔，得到基片；
    (3)基底修饰：取一透明基底，在所述透明基底的表面制备一保护层，得到表面修饰的透明基底；
    (4)封装芯片：将上述基片与所述表面修饰的透明基底进行氧等离子体清洗，之后将所述基片与透明基底压合形成容纳流体的空间；然后往每个流道内注入腐蚀剂，以清洗掉与各流道接触的部分保护层，得到位于所述透明基底表面的间隔层，完成单分子测序芯片的制备。
  2. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(3)中，所述保护层的材质为聚甲基戊二酰亚胺。
  3. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(4)中，所述腐蚀剂为N-甲基吡咯烷酮。
  4. 如权利要求1所述的芯片的制备方法，其特征在于，所述间隔层为所述保护层经腐蚀剂清洗掉与各流道接触的部分得到，所述间隔层的材质为聚甲基戊二酰亚胺。
  5. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(3)中，所述保护层的厚度为1-5μm。
  6. 如权利要求3所述的芯片的制备方法，其特征在于，步骤(4)中，所述每个流道内腐蚀剂的注入量为100-500μL。
  7. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(4)中，所述反应池阵列包括15-25个流道，相邻流道之间的间距为1-1.5mm。
  8. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(4)中，每个所述流道相对设置的两个侧壁为每个流道的宽度，每个所述流道的宽度为1-2mm；每个所述流道的深度为0.6-1mm。
  9. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(2)中，所述基片的材质包括聚二甲基硅氧烷、聚甲基丙烯酸甲酯、乙烯-醋酸乙烯和聚胺脂中的一种。
  10. 如权利要求1所述的芯片的制备方法，其特征在于，所述透明基底包括表面带有官能团为环氧基、氨基、羧基、巯基和醛基中的一种的透明的玻璃、石英或有机聚合物材料。
  11. 如权利要求1所述的芯片的制备方法，其特征在于，步骤(1)中，所述光刻法包括以下步骤：
    a、将负性光刻胶通过旋涂法制备到基板表面，得到匀胶后的基板，其中基板表面的光刻胶层厚度为600-650μm；
    b、对匀胶后的基板进行前烘处理，所述前烘的温度控制为：缓慢加热到90-100℃并稳定15-20min，之后自然冷却至室温；
    c、将设计好的反应池阵列的图形模板作为掩膜版，并覆盖在前烘处理后的基板表面，进行曝光处理，曝光时间为90-150s；
    d、将曝光后的基板进行后烘处理，所述后烘的温度控制为：先缓慢加热到90-95℃并稳定5-10min，之后自然冷却至室温；
    e、将后烘处理后的基板浸入显影液中，进行显影处理，清洗掉掩膜版以外的部分，得到反应池阵列的阳膜。
  12. 如权利要求11所述的芯片的制备方法，其特征在于，在所述光刻法的步骤e之后，还包括以下处理：
    f、将步骤e得到的反应池阵列的阳膜进行硬烘处理，所述硬烘是在所述阳膜上加一玻璃板，并压一铁块，加热至130-150℃下烘烤45-60min。
  13. 如权利要求11所述的芯片的制备方法，其特征在于，步骤b中，所述前烘的温度控制为：先在65℃时烘烤6min，再按1℃/min的速率逐渐升温至95℃，保持20min，自然冷却至室温。
  14. 如权利要求11所述的芯片的制备方法，其特征在于，步骤e中，所述后烘的温度控制为：先以0.5℃/min的速率逐渐升温至95℃，保持5min，再以1-3℃/min的速率自然冷却至室温。
  15. 一种芯片，其特征在于，所述芯片利用权利要求1-14任一方法制备获得。
  16. 如权利要求15所述的芯片，所述芯片包括基片和所述基片压合设置的基底层，所述基片包括相对设置的第一表面和第二表面，所述基片的第一表面间隔设置有多个流道形成的反应池阵列，每个所述流道相对设置的两个侧壁沿所述流道的长度方向延伸并在所述流道的两端交汇形成两个带有夹角的锥形末端，所述两个锥形末端表面分别设置有与所述基片的第二表面连通的流体输入孔和流体输出孔，所述基底层包括透明基底和设置在所述透明基底表面的间隔层，所述间隔层与所述基片第一表面接触且所述间隔层对应所述流道所在的位置设置有腐蚀凹槽。
  17. 如权利要求16所述的芯片，其特征在于，所述芯片还包括探针，所述探针连接在所述芯片的透明基底的表面。
  18. 如权利要求15-17任一项所述的芯片在序列捕获和/或核酸测序中的应用。
  19. 一种试剂盒，其特征在于，包括如权利要求15-17任一项所述的芯片。
  20. 如权利要求19所述的试剂盒在序列捕获和/或核酸测序中的用途。
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