WO2018196265A1 - 基因检测芯片、其检测方法、制作方法及微流控芯片系统 - Google Patents

基因检测芯片、其检测方法、制作方法及微流控芯片系统 Download PDF

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WO2018196265A1
WO2018196265A1 PCT/CN2017/102514 CN2017102514W WO2018196265A1 WO 2018196265 A1 WO2018196265 A1 WO 2018196265A1 CN 2017102514 W CN2017102514 W CN 2017102514W WO 2018196265 A1 WO2018196265 A1 WO 2018196265A1
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reaction cell
aptamer
gene
upper substrate
fluorescence
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PCT/CN2017/102514
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English (en)
French (fr)
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古乐
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京东方科技集团股份有限公司
北京京东方光电科技有限公司
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Priority to US15/774,349 priority Critical patent/US20200164366A1/en
Priority to JP2018524215A priority patent/JP7051680B2/ja
Priority to EP17868484.1A priority patent/EP3617305A4/en
Publication of WO2018196265A1 publication Critical patent/WO2018196265A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/205—Aptamer
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/501—Detection characterised by immobilisation to a surface being an array of oligonucleotides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to the field of gene detection technologies, and in particular, to a gene detection chip, a detection method thereof, a manufacturing method thereof, and a microfluidic chip system.
  • the main methods of gene detection include direct sequencing, fluorescent polymerase chain reaction and gene chip.
  • the gene chip is widely favored in gene detection because of its small size, rapid and simple, and simultaneous detection of multiple genes.
  • the sequencing principle of the gene detection chip is hybridization sequencing, and the hybridization of the gene target sequence in the sample and the gene chip immobilized with the gene probe is detected.
  • gene chip detection requires labeling of the target gene, and the technique is cumbersome, and the fluorescence signal of the unhybridized gene molecule also interferes with the detection result.
  • An embodiment of the present disclosure provides a gene detection chip, including: an upper substrate and a lower substrate disposed opposite to each other; wherein
  • the upper substrate is provided with at least one pair of inlets and outlets; the inlets and the outlets are respectively located at opposite ends of the upper substrate;
  • reaction cell Providing at least one reaction cell and at least one microchannel on a side of the lower substrate facing the upper substrate; the reaction cell is respectively connected to the inlet and the outlet through the microchannel; wherein The surface of the reaction cell has an aptamer, wherein the aptamer is modified with a fluorescent label.
  • the method further includes: a first plastic hose connected to the inlet and a second plastic hose connected to the outlet;
  • the first plastic hose is used to introduce a solution through the inlet
  • the second plastic hose is used to direct the solution through the outlet.
  • the shape of the reaction cell is circular.
  • the material of the lower substrate is glass.
  • the upper substrate is a transparent substrate.
  • the embodiment of the present disclosure further provides a microfluidic chip system, including the above-mentioned gene detection chip and a power module provided by the embodiments of the present disclosure;
  • the power module is configured to provide power to the sample solution to introduce or derive the genetic detection chip.
  • the power module is a syringe pump.
  • the embodiment of the present disclosure further provides a method for detecting the above genetic detecting chip, comprising:
  • the sample solution to be tested is introduced into the reaction cell through the inlet;
  • the method before determining whether the sample solution recovers fluorescence after the hybridization reaction with the aptamer on the surface of the reaction cell, the method further includes:
  • the reaction cell is washed.
  • the cleaning of the reaction pool specifically includes:
  • the cleaning liquid is introduced into the reaction cell through the injection port, and the cleaning liquid is discharged through the sample outlet.
  • the embodiment of the present disclosure further provides a method for manufacturing a genetic detection chip, including:
  • the sample port is connected to the sample outlet.
  • the conjugated body modified with the fluorescent label is bonded to the inner surface of the reaction cell, and specifically includes:
  • the aptamer modified with fluorescent labeling was bonded to the inner surface of the reaction cell using a spotting machine.
  • the method further includes:
  • the reaction cell is washed.
  • a gene detection chip provided by an embodiment of the present disclosure includes: an upper substrate and a lower substrate disposed opposite to each other; wherein
  • the upper substrate is provided with at least one pair of inlets and outlets; the inlets and the outlets are respectively located at opposite ends of the upper substrate;
  • reaction cell Providing at least one reaction cell and at least one microchannel on a side of the lower substrate facing the upper substrate; the reaction cell is respectively connected to the inlet and the outlet through the microchannel; wherein The surface of the reaction cell has a complex of an aptamer and graphene oxide, wherein the aptamer is modified with a fluorescent label and the fluorescently labeled fluorescence is in a quenched state.
  • FIG. 1 is a schematic structural diagram of a gene detection chip according to an embodiment of the present disclosure
  • FIG. 2 is a flow chart of a gene detection method according to an embodiment of the present disclosure
  • FIG. 3 is a schematic diagram of a gene detection process according to an embodiment of the present disclosure.
  • FIG. 4 is a flow chart of a method for fabricating a gene detection chip according to an embodiment of the present disclosure.
  • the embodiment of the present disclosure provides a gene detecting chip, as shown in FIG. 1 , which may include: an upper substrate 01 and a lower substrate 02 disposed opposite to each other; wherein the upper substrate 01 is provided with at least one pair of inlets 03 and outlets 04; the inlet 03 and the outlet 04 are respectively located at opposite ends of the upper substrate 01; at least one reaction cell 05 and at least one microchannel 06 are disposed on one side of the lower substrate 02 facing the upper substrate 01; the reaction cell 05 passes through the microchannel 06 is connected to the inlet 03 and the outlet 04, respectively; wherein the surface of the reaction cell 05 has an aptamer, wherein the aptamer is modified with a fluorescent label.
  • a gene detection channel is formed through an inlet, a microchannel, a reaction cell, and a sample outlet, and the surface of the reaction cell has an aptamer, wherein the aptamer is modified with fluorescence. mark.
  • the fluorescently labeled fluorescence can be quenched first, and then the sample solution is introduced into the reaction cell through the inlet. If the target gene is present in the sample solution, the target gene is ligated with the aptamer on the surface of the reaction cell.
  • the hybridization reaction combines to restore fluorescence; if the target gene is not present in the sample solution, the fluorescence is still quenched.
  • the gene detection chip can realize the detection of the target gene without fluorescent labeling of the target gene, which not only simplifies the process of gene detection, but also prevents the influence of the fluorescent signal of the unhybridized target gene in the gene detection result on the hybrid gene;
  • the gene detection chip of the disclosure has the advantages of simple structure, convenient operation, high specificity of the aptamer and accurate detection result.
  • the gene detection chip of the present disclosure can set a plurality of detection channels, and simultaneously detect a plurality of different genes, and the detection efficiency is high.
  • the fluorescence in the fluorescent label Light can be quenched by graphene oxide.
  • graphene oxide is a derivative of graphene, and graphene oxide introduces hydroxyl and carboxyl groups on the basis of graphene, and has stronger hydrophilicity and biocompatibility than graphene.
  • As a quencher it can quench the fluorescence of most organic dyes and quantum dots.
  • the graphene oxide solution is passed through the reaction cell, a complex of the aptamer and the graphene oxide is formed on the surface of the reaction cell, and the graphene oxide can adsorb the single-stranded nucleic acid in the complex of the aptamer and the graphene oxide.
  • the aptamer then quenches the fluorescence of its label.
  • the target gene is present in the sample solution, the aptamer combines with the complementary DNA in the target gene to form a double-stranded DNA molecule.
  • the steric hindrance becomes larger, the adsorption capacity of the graphene oxide is weakened, and the fluorescence is recovered.
  • the above-mentioned gene detection chip provided by the embodiment of the present disclosure, as shown in FIG. 1 , may further include: a first plastic hose 031 connected to the inlet 03 and a second connected to the outlet 04
  • the plastic hose 041 is used to introduce the solution through the inlet 03; the second plastic hose 041 is used to guide the solution through the outlet 04.
  • the gene detection chip can also introduce a liquid into the gene detection chip and a solution in the gene chip through a plastic hose respectively connected to the inlet and the outlet, thereby facilitating the introduction and export of the solution.
  • the genetic test channel composed of the inlet, the microchannel, the reaction cell and the sample outlet can be cleaned by a plastic hose, that is, after the hybridization reaction, it is confirmed that the target gene is present, and the plastic tube can also be used.
  • the injection port and the sample outlet respectively inject and export the cleaning solution, thereby purifying the gene detection channel, thereby removing the unhybridized gene, facilitating observation of the hybridized gene, and improving the detection accuracy.
  • the upper substrate is a transparent substrate.
  • the shape of the reaction cell may be a circular shape or other shapes satisfying the design, which is not limited herein.
  • the shape of the reaction cell is designed to be circular, which can be conveniently observed under a microscope.
  • the material of the lower substrate is glass.
  • the material of the lower substrate is glass for silanization treatment, so that the fluorescent aptamer is bonded to the inner surface of the reaction cell, and then passes through the graphite oxide. The olefin quenches the fluorescence.
  • the sample solution with the target gene is The aptamer on the surface of the reaction cell is hybridized and bonded to the surface of the reaction cell.
  • the unhybridized gene can be separated from the hybridized gene, and the unhybridized gene in the detection result is prevented from affecting the hybrid gene, which is advantageous. Improve the accuracy of genetic testing.
  • the material of the upper substrate of the gene detecting chip may be glass or a polymer such as polydimethylsiloxane.
  • the upper substrate can form an inlet and a sample outlet through the through hole, and the lower substrate etches the microchannel and the reaction cell by photolithography, and the sample solution in the reaction cell can be hybridized with the aptamer on the surface of the reaction cell.
  • the lower substrate can be bonded by a low temperature adhesive.
  • the above-mentioned gene detection chip provided by the embodiment of the present disclosure needs to first quench the fluorescence in the fluorescent label when performing gene detection.
  • the fluorescence of the fluorescent label can be quenched before the gene detection chip is used.
  • an embodiment of the present disclosure further provides a gene detection chip, including: an upper substrate and a lower substrate disposed opposite to each other;
  • the upper substrate is provided with at least one pair of inlets and outlets; the inlet and the outlet are respectively located at opposite ends of the upper substrate;
  • the surface of the lower substrate facing the upper substrate is provided with at least one reaction cell and at least one microchannel; the reaction cell is respectively connected to the inlet and the outlet through the microchannel; wherein the surface of the reaction cell has an aptamer and graphene oxide A complex wherein the aptamer is modified with a fluorescent label and the fluorescently labeled fluorescence is in a quenched state.
  • the gene detecting chip provided by the embodiment of the present disclosure has an aptamer modified with a fluorescent label on the surface of the former reaction cell, and the surface of the reaction cell is an aptamer in the embodiment of the present disclosure.
  • a complex of graphene oxide wherein the aptamer is modified with a fluorescent label and the fluorescently labeled fluorescence is in a quenched state.
  • the target gene and the reaction cell are The aptamer of the surface is subjected to a hybridization reaction to bind, thereby recovering the fluorescence; if the target gene is not present in the sample solution, the fluorescence is still in a quenched state. Therefore, the gene detection chip can detect the target gene without fluorescent labeling of the target gene, which simplifies the process of gene detection and prevents the gene detection result.
  • the effect of the fluorescent signal of the unhybridized target gene on the hybrid gene; at the same time, the gene detection chip of the present disclosure has a simple structure, convenient operation, high specificity of the aptamer, and accurate detection result.
  • an embodiment of the present disclosure provides a microfluidic chip system, including: the above-mentioned gene detecting chip and a power module provided by the embodiments of the present disclosure; wherein the power module is used for introducing or deriving a gene detecting chip for a sample solution; Provide power. Since the principle of solving the problem of the microfluidic chip system is similar to that of the genetic detecting chip, the implementation of the microfluidic chip system can be referred to the implementation of the above genetic detecting chip, and the repetition will not be described again.
  • the power module may be a syringe pump.
  • the power of the gene detection chip for introducing and deriving the liquid can be powered by the syringe pump, and other powers can be used, which is not limited herein.
  • an embodiment of the present disclosure provides a method for detecting the above-mentioned gene detection chip according to an embodiment of the present disclosure. As shown in FIG. 2, the method may specifically include:
  • the sample solution to be tested is introduced into the reaction cell through the inlet;
  • the target gene exists in the sample solution, otherwise the target solution is not present in the sample solution.
  • the target gene is not required to be labeled, and only the graphene oxide solution is required to be introduced into the reaction cell to completely quench the fluorescently labeled fluorescence, and the fluorescently labeled fluorescence is completely quenched.
  • the sample is passed through the reaction cell, and the target sample is detected by performing hybridization reaction between the sample to be tested and the completely quenched modified fluorescent aptamer on the surface of the reaction cell.
  • the gene detection method is convenient to operate and the detection result is obtained. The accuracy rate is high.
  • graphene oxide is a derivative of graphene, and graphene oxide introduces a hydroxyl group and a carboxyl group on the basis of graphene, and has stronger hydrophilicity and biocompatibility than graphene.
  • As a quencher it can quench most organic dyes, fluorescence of quantum dots, and graphene oxide
  • the single-stranded nucleic acid aptamer is adsorbed and then the fluorescence of the label is quenched, so that the graphene oxide is added to the reaction cell and mixed with the fluorescently labeled aptamer to complete the quenching.
  • the aptamer When the aptamer binds to the complementary DNA in the binding target gene to form a double-stranded DNA molecule, the adsorption capacity of the graphene oxide is weakened due to the increase in steric hindrance, resulting in fluorescence recovery, thereby realizing detection of the target gene. Therefore, during the detection, the sample solution enters from the inlet and reacts with the aptamer on the surface of the reaction cell.
  • the target gene exists in the sample to be tested, the combination of the gene and the aptamer is detached from the graphene oxide, and the result is obtained.
  • the fluorescent signal Corresponding to the fluorescent signal, conversely, when the target gene is absent, all of the fluorescence is in a quenched state with no fluorescent signal.
  • the gene detection method of the embodiments of the present disclosure eliminates the cumbersome labeling of the target gene, simplifies the gene detection technology, and combines the microfluidic chip technology with the gene detection, and utilizes the microfluidic technology in the micron-scale chip.
  • the continuous microfluid is manipulated to realize gene detection, which has the characteristics of small reagent consumption, portable volume, fast and efficient reaction and easy integration.
  • the method before determining whether the sample solution and the aptamer on the surface of the reaction cell are subjected to a hybridization reaction, the method further comprises: cleaning the reaction cell.
  • the unhybridized fluorescent probe can be washed away by washing to prevent interference.
  • the specific cleaning process is to introduce the cleaning liquid into the reaction cell through the inlet, and the cleaning liquid is discharged through the sample outlet.
  • an embodiment of the present disclosure provides a method for fabricating a genetic detection chip, as shown in FIG. 4, including:
  • At least one pair of inlets and outlets are formed on the upper substrate; wherein the inlets and the outlets are respectively located at opposite ends of the upper substrate;
  • the step S201 and the step S202 are performed in a sequence, and the step S201 may be performed before the step S202 is performed; or the step S202 may be performed before the step S201 is performed; Step S201 is performed simultaneously with step S202, at This is not limited.
  • the conjugated body modified with the fluorescent label is bonded to the inner surface of the reaction cell, and specifically includes:
  • the fluorescently modified aptamer was bonded to the inner surface of the reaction cell using a spotting machine.
  • the silicide-labeled aptamer is bonded to the inner surface of the reaction cell by silanization treatment, and after the hybridization reaction, the sample solution with the target gene is hybridized with the aptamer and then bonded to On the surface of the reaction cell, the unhybridized gene can be separated from the hybridized gene during washing, and the unhybridized gene in the detection result is prevented from affecting the hybrid gene, which is advantageous for improving the accuracy of gene detection.
  • the aptamer modified with the fluorescent label is bonded to the step in step S202.
  • the method further includes:
  • the reaction cell is cleaned.
  • the graphene oxide solution may be passed into the reaction cell, and then the upper substrate and the lower substrate are bonded, and of course, the upper substrate and the lower substrate may be bonded. Then, the graphene oxide solution is introduced into the reaction cell through the inlet, which is not limited herein.
  • the upper substrate and the lower substrate are generally bonded by a low temperature adhesive.
  • the injection port and the sample port may be formed on the upper substrate by processing the through holes.
  • the microchannel and the reaction cell can be etched on the lower substrate by photolithography.
  • the reaction cell of the gene detection chip is combined with the aptamer modified with the fluorescent dye by chemical surface treatment, and then a 1 mg/ml graphene oxide solution is introduced into the reaction cell, and the quenching condition can be observed by a fluorescence confocal microscope. Until completely quenched.
  • a sample to be tested is added to the reaction cell of the chip, if the target gene is present, the target gene will bind to the aptamer on the surface of the glass to restore the fluorescence; if no target gene exists, it cannot be combined with the aptamer to maintain Quenched state.
  • the above-mentioned gene detection chip provided by the embodiments of the present disclosure can achieve the purpose of gene detection, and the specificity of the aptamer is high, and the detection result is accurate.
  • the gene detection chip of the present disclosure can have multiple parallel channels. For example, there are four parallel channels as shown in FIG. 1, which can simultaneously detect four different genes, and the detection efficiency is fast and efficient.
  • the embodiment of the present disclosure provides a gene detection chip, a detection method thereof, a manufacturing method thereof, and a microfluidic chip system.
  • the gene detection chip includes: an upper substrate and a lower substrate disposed opposite to each other; wherein the upper substrate is provided with at least one pair of a sample port and a sample outlet; the inlet port and the sample outlet are respectively located at opposite ends of the upper substrate; at least one reaction cell and at least one microchannel are disposed on one side of the lower substrate facing the upper substrate; and the reaction cells are respectively connected through the microchannel
  • the sample port is connected to the sample port.
  • a gene detection channel is formed through the inlet, the microchannel, the reaction cell, and the sample outlet.
  • the aptamer Since the surface of the reaction cell has an aptamer, the aptamer is modified with a fluorescent label. Thus, when performing the gene detection, the fluorescently labeled fluorescence can be quenched first, and then the sample solution is introduced into the reaction cell through the inlet. If the target gene is present in the sample solution, the target gene is ligated with the aptamer on the surface of the reaction cell. The hybridization reaction combines to restore fluorescence; if the target gene is not present in the sample solution, the fluorescence is still quenched.
  • the gene detection chip can realize the detection of the target gene without fluorescent labeling of the target gene, which not only simplifies the process of gene detection, but also prevents the influence of the fluorescent signal of the unhybridized target gene in the gene detection result on the hybrid gene;
  • the gene detection chip of the disclosure has the advantages of simple structure, convenient operation, high specificity of the aptamer and accurate detection result.

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Abstract

公开了一种基因检测芯片，其通过进样口、微通道、反应池、出样口组成基因检测通道，反应池的表面具有修饰有荧光标记的适配体，并公开了其检测方法、制作方法及微流控芯片系统。当进行基因检测时，先将荧光标记的荧光淬灭，后将样品溶液通过进样口通入反应池，如果存在目标基因，则目标基因与反应池表面的适配体杂交，使荧光恢复；如果不存在目标基因，则荧光仍处于淬灭状态。该基因检测芯片无需对目标基因进行荧光标记，简化了基因检测过程，也可以防止基因检测结果中未杂交的目标基因产生的荧光信号的干扰。

Description

基因检测芯片、其检测方法、制作方法及微流控芯片系统
本申请要求在2017年4月27日提交中国专利局、申请号为201710289072.6、发明名称为“一种基因检测芯片、其检测方法及微流控芯片系统”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及基因检测技术领域，尤其涉及一种基因检测芯片、其检测方法、制作方法及微流控芯片系统。
背景技术
现有技术中，基因检测的主要方法有直接测序法、荧光聚合酶链式反应法和基因芯片法。其中，基因芯片由于其体积小、快速简便并且可同时检测多种基因，在基因检测方面受到广泛青睐。基因检测芯片的测序原理是杂交测序，通过对样品中的基因靶序列和固定有基因探针的基因芯片进行杂交进行检测。然而，基因芯片检测需要对目标基因进行标记，技术繁琐，而且未杂交基因分子的荧光信号也会对检测结果造成干扰和影响。
因此，如何简化基因检测技术，且改善基因检测结果中未杂交的基因的荧光信号对杂交基因的影响，是本领域技术人员亟待解决的技术问题。
发明内容
本公开实施例提供了一种基因检测芯片，包括：相对设置的上基板和下基板；其中，
所述上基板设置有至少一对进样口和出样口；所述进样口和所述出样口分别位于所述上基板相对的两端；
所述下基板面向所述上基板的一面设置有至少一个反应池和至少一个微通道；所述反应池通过所述微通道分别与所述进样口和所述出样口相连；其中，所述反应池的表面具有适配体，其中所述适配体修饰有荧光标记。
在本公开实施例提供的上述基因检测芯片中，还包括：与所述进样口相连的第一塑胶软管和与所述出样口相连的第二塑胶软管；
所述第一塑胶软管用于将溶液通过所述进样口导入；
所述第二塑胶软管用于通过所述出样口将溶液导出。
在本公开实施例提供的上述基因检测芯片中，所述反应池的形状为圆形。
在本公开实施例提供的上述基因检测芯片中，所述下基板的材料为玻璃。
在本公开实施例提供的上述基因检测芯片中，所述上基板为透明基板。
本公开实施例还提供了一种微流控芯片系统，包括：本公开实施例提供的上述基因检测芯片以及动力模块；其中，
所述动力模块用于为所述样品溶液导入或导出所述基因检测芯片提供动力。
在本公开实施例提供的上述微流控芯片系统中，所述动力模块为注射泵。
本公开实施例还提供了一种上述基因检测芯片的检测方法，包括：
将氧化石墨烯溶液通入所述反应池以将所述荧光标记的荧光完全淬灭；
待所述荧光标记的荧光完全淬灭后，将待测样品溶液通过所述进样口导入所述反应池；
确定所述样品溶液与所述反应池表面的所述适配体发生杂交反应后是否恢复荧光，是则所述样品溶液存在目标基因，否则所述样品溶液不存在目标基因。
在本公开实施例提供的上述检测方法中，在确定所述样品溶液与所述反应池表面的所述适配体发生杂交反应后是否恢复荧光之前，还包括：
对所述反应池进行清洗。
在本公开实施例提供的上述检测方法中，对所述反应池进行清洗，具体包括：
将清洗液通过所述进样口导入所述反应池，并通过所述出样口将所述清洗液导出。
本公开实施例还提供了一种基因检测芯片的制作方法，包括：
在上基板上形成有至少一对进样口和出样口；其中，所述进样口和所述出样口分别位于所述上基板相对的两端；
在下基板上形成至少一个反应池和至少一个微通道，并将修饰有荧光标记的适配体键合到所述反应池的内表面；
以及，将所述上基板与所述下基板进行粘接，以使所述上基板覆盖所述下基板上的反应池和微通道，且所述反应池通过所述微通道分别与所述进样口和所述出样口相连。
在本公开实施例提供的上述制作方法中，将修饰有荧光标记的适配体键合到所述反应池的内表面，具体包括：
对所述反应池的内表面进行硅烷化处理；其中，所述下基板的材料为玻璃；
采用点样机将修饰有荧光标记的所述适配体键合到所述反应池的内表面。
在本公开实施例提供的上述制作方法中，在将修饰有荧光标记的适配体键合到所述反应池的内表面之后，还包括：
将氧化石墨烯溶液通入所述反应池以将所述荧光标记的荧光完全淬灭；
对所述反应池进行清洗。
本公开实施例提供的一种基因检测芯片，包括：相对设置的上基板和下基板；其中，
所述上基板设置有至少一对进样口和出样口；所述进样口和所述出样口分别位于所述上基板相对的两端；
所述下基板面向所述上基板的一面设置有至少一个反应池和至少一个微通道；所述反应池通过所述微通道分别与所述进样口和所述出样口相连；其中，所述反应池的表面具有适配体与氧化石墨烯的复合物，其中所述适配体修饰有荧光标记，且所述荧光标记的荧光处于淬灭状态。
附图说明
图1为本公开实施例提供的基因检测芯片的结构示意图；
图2为本公开实施例提供的基因检测方法流程图；
图3为本公开实施例提供的基因检测过程示意图；
图4为本公开实施例提供的基因检测芯片的制作方法的流程图。
具体实施方式
下面结合附图，对本公开实施例提供的基因检测芯片、其检测方法、制作方法及微流控芯片系统的具体实施方式进行详细的说明。
本公开实施例提供了一种基因检测芯片，如图1所示，可以包括：相对设置的上基板01和下基板02；其中，上基板01设置有至少一对进样口03和出样口04；进样口03和出样口04分别位于上基板01相对的两端；下基板02面向上基板01的一面设置有至少一个反应池05和至少一个微通道06；反应池05通过微通道06分别与进样口03和出样口04相连；其中，反应池05的表面具有适配体，其中，适配体修饰有荧光标记。
本公开实施例提供的上述基因检测芯片中，通过进样口、微通道、反应池、出样口组成一个基因检测通道，由于反应池的表面具有适配体，其中，适配体修饰有荧光标记。这样当进行基因检测时，可以先将荧光标记的荧光淬灭，然后将样品溶液通过进样口进入反应池，如果样品溶液中存在目标基因，则目标基因会与反应池表面的适配体进行杂交反应而结合，从而使荧光恢复；如果样品溶液中不存在目标基因，则荧光仍处于淬灭状态。因此该基因检测芯片无需对目标基因进行荧光标记，就可以实现目标基因的检测，既简化了基因检测的过程，也可以防止基因检测结果中未杂交的目标基因的荧光信号对杂交基因的影响；同时本公开的基因检测芯片结构简单、操作方便、适配体的专一性高、检测结果准确。
另外，本公开的基因检测芯片可以设置多个检测通道，同时检测多种不同基因，检测效率较高。
具体地，在本公开实施例提供的上述基因检测芯片中，荧光标记中的荧 光可以通过氧化石墨烯进行淬灭。其中，氧化石墨烯为石墨烯的衍生物，氧化石墨烯是在石墨烯的基础上引入了羟基和羧基基团，与石墨烯相比，有了更强的亲水性和生物相容性，作为一种淬灭剂能够淬灭大部分有机染料、量子点的荧光。因此，当将氧化石墨烯溶液通过反应池后，在反应池表面形成适配体与氧化石墨烯的复合物，在适配体与氧化石墨烯的复合物中，氧化石墨烯能够吸附单链核酸适配体，然后淬灭其标记物的荧光。这样当样品溶液中存在目标基因时，适配体与目标基因中的互补DNA相结合形成双链DNA分子，由于空间位阻变大，氧化石墨烯的吸附能力减弱，从而荧光恢复。
在具体实施时，本公开实施例提供的上述基因检测芯片中，如图1所示，还可以包括：与进样口03相连的第一塑胶软管031和与出样口04相连的第二塑胶软管041；第一塑胶软管031用于将溶液通过进样口03导入；第二塑胶软管041用于通过出样口04将溶液导出。具体地，基因检测芯片还可以通过分别与进样口和出样口相连的塑胶软管，向基因检测芯片内导入液体和将基因芯片内的溶液导出，从而方便溶液的导入和导出。另外，通过塑胶软管可以对进样口、微通道、反应池、出样口组成的基因检测通道进行清洗，即在进行杂交反应之后，确认是否存在目标基因之前，还可以通过塑胶软管从进样口和出样口分别注入和导出清洗液，从而实现基因检测通道的清洗，进而将未杂交的基因去除，方便观察杂交后的基因，提高检测准确率。
具体地，本公开实施例提供的上述基因检测芯片中，为了能够实现对反应池的观察，上基板为透明基板。
在具体实施时，本公开实施例提供的上述基因检测芯片中，反应池的形状可以为圆形，也可以为其他满足设计的形状，在此不做限定。优选地，将反应池的形状设计为圆形，可以方便在显微镜下进行观察。
在具体实施时，本公开实施例提供的上述基因检测芯片中，下基板的材料为玻璃。具体地，本公开实施例提供的上述基因检测芯片中，下基板的材料为玻璃，用于进行硅烷化处理，使得带有荧光的适配体键合到反应池的内表面，然后通过氧化石墨烯使荧光淬灭。从而当带有目标基因的样品溶液与 反应池表面的适配体进行杂交反应后键合到反应池的表面，在进行清洗时就可以将未杂交的基因与杂交的基因分离，避免检测结果中未杂交的基因影响杂交基因，有利于提高基因检测的准确率。另外，基因检测芯片的上基板的材料可以是玻璃或者聚合物例如聚二甲基硅氧烷。上基板通过加工通孔可以形成进样口和出样口，下基板通过光刻技术刻蚀出微通道和反应池，在反应池样品溶液可以与反应池表面的适配体进行杂交反应，上、下基板可以通过低温粘接剂粘接。
具体地，本公开实施例提供的上述基因检测芯片，在进行基因检测时需要先将荧光标记中的荧光淬灭。当然具体实施时，也可以使基因检测芯片在使用之前荧光标记中的荧光就是处于淬灭状态的。
因此，基于同一发明思想，本公开实施例还提供了一种基因检测芯片，包括：相对设置的上基板和下基板；其中，
上基板设置有至少一对进样口和出样口；进样口和出样口分别位于上基板相对的两端；
下基板面向上基板的一面设置有至少一个反应池和至少一个微通道；反应池通过微通道分别与进样口和出样口相连；其中，反应池的表面具有适配体与氧化石墨烯的复合物，其中适配体修饰有荧光标记，且荧光标记的荧光处于淬灭状态。
本公开实施例提供的该基因检测芯片，与前述的基因检测芯片相比，前者反应池的表面具有修饰有荧光标记的适配体，而本公开实施例中反应池的表面为适配体与氧化石墨烯的复合物，其中，适配体修饰有荧光标记，且荧光标记的荧光处于淬灭状态。这样在使用时由于荧光已经处于淬灭状态，因此不需要先将荧光淬灭，而是直接将样品溶液通过进样口进入反应池，如果样品溶液中存在目标基因，则目标基因会与反应池表面的适配体进行杂交反应而结合，从而使荧光恢复；如果样品溶液中不存在目标基因，则荧光仍处于淬灭状态。因此该基因检测芯片无需对目标基因进行荧光标记，就可以实现目标基因的检测，既简化了基因检测的过程，也可以防止基因检测结果中 未杂交的目标基因的荧光信号对杂交基因的影响；同时本公开的基因检测芯片结构简单、操作方便、适配体的专一性高、检测结果准确。
基于同一发明思想，本公开实施例提供了一种微流控芯片系统，包括：本公开实施例提供的上述基因检测芯片以及动力模块；其中，动力模块用于为样品溶液导入或导出基因检测芯片提供动力。由于该微流控芯片系统解决问题的原理与基因检测芯片相似，因此该微流控芯片系统的实施可以参见上述基因检测芯片的实施，重复之处不再赘述。
在具体实施时，本公开实施例提供的上述微流控芯片系统中，动力模块可以为注射泵。具体地，本公开实施例提供的上述微流控芯片系统中，可以通过注射泵为基因检测芯片导入和导出液体提供动力，当然也可以采用其他动力，在此不做限定。
基于同一发明思想，本公开实施例提供了一种本公开实施例提供的上述基因检测芯片的检测方法，如图2所示，可以具体包括：
S101、将氧化石墨烯溶液通入反应池以将荧光标记的荧光完全淬灭；
S102、待荧光标记的荧光完全淬灭后，将待测样品溶液通过进样口导入反应池；
S103、确定样品溶液与反应池表面的适配体发生杂交反应后是否恢复荧光；
是则样品溶液存在目标基因，否则样品溶液不存在目标基因。
本公开实施例提供的上述检测方法中，无需对目标基因进行标记，只需要将氧化石墨烯溶液通入反应池以将荧光标记的荧光完全淬灭，待荧光标记的荧光完全淬灭后将待测样品通过反应池，使待测样品与反应池表面的完全淬灭的修饰有荧光的适配体在反应池内进行杂交反应，就可以实现目标基因的检测，该基因检测方法操作方便、检测结果准确率较高。
具体地，氧化石墨烯是石墨烯的衍生物，氧化石墨烯在石墨烯的基础上引入了羟基和羧基基团，较石墨烯相比有了更强的亲水性和生物相容性，可以作为一种淬灭剂能够淬灭大部分有机染料、量子点的荧光，氧化石墨烯能 够吸附单链核酸适配体，然后淬灭其标记物的荧光，因此将氧化石墨烯加入到反应池与带有荧光标记的适配体进行混合完成淬灭。当适配体与键合目标基因中的互补DNA相结合形成双链DNA分子后，由于空间位阻变大，氧化石墨烯的吸附能力减弱，导致荧光恢复，从而实现目标基因的检测。因此，检测时将样品溶液从进样口进入，与反应池表面的适配体发生反应，当待测样品中目标基因存在时，基因和适配体结合从氧化石墨烯上脱落，就会得到相对应的荧光信号，相反，当目标基因不存在时，所有的荧光处于淬灭状态，没有荧光信号。综上，本公开实施例的基因检测方法免去了对目标基因的繁琐标记，简化了基因检测技术，且将微流控芯片技术与基因检测相结合，利用微流控技术在微米级的芯片上操控连续的微流体来实现基因检测，具有试剂消耗量小、体积便携、反应快速效率高且易于集成的特点。
在具体实施时，本公开实施例提供的上述检测方法中，在确定样品溶液与反应池表面的适配体发生杂交反应后是否恢复荧光之前，还可以包括：对反应池进行清洗。
具体地，通过清洗作用可以清洗掉未杂交的荧光探针，防止干扰。具体的清洗过程为将清洗液通过进样口导入反应池，并通过出样口将清洗液导出。
基于同一发明思想，本公开实施例提供了一种基因检测芯片的制作方法，如图4所示，包括：
S201、在上基板上形成有至少一对进样口和出样口；其中，进样口和出样口分别位于上基板相对的两端；
S202、在下基板上形成至少一个反应池和至少一个微通道，并将修饰有荧光标记的适配体键合到反应池的内表面；
S203、将上基板与下基板进行粘接，以使上基板覆盖下基板上的反应池和微通道，且反应池通过微通道分别与进样口和出样口相连。
具体地，在本公开实施例提供的上述制作方法中，步骤S201与步骤S202不分先后顺序，可以先执行步骤S201再执行步骤S202；或者，可以先执行步骤S202再执行步骤S201；当然也可以步骤S201与步骤S202同时执行，在 此不作限定。
具体地，在本公开实施例提供的上述制作方法中，将修饰有荧光标记的适配体键合到反应池的内表面，具体包括：
对反应池的内表面进行硅烷化处理；其中，下基板的材料为玻璃；
采用点样机将修饰有荧光的适配体键合到反应池的内表面。
具体地，通过硅烷化处理，使得带有荧光标记的适配体键合到反应池的内表面，从而进行杂交反应后，带有目标基因的样品溶液与适配体进行杂交反应后键合到反应池的表面，在进行清洗时就可以将未杂交的基因与杂交的基因分离，避免检测结果中未杂交的基因影响杂交基因，有利于提高基因检测的准确率。
具体地，为了避免在进行基因检测时需要将荧光标记的荧光完全淬灭，因此，在本公开实施例提供的上述制作方法中，在步骤S202将修饰有荧光标记的适配体键合到所述反应池的内表面之后，还包括：
将氧化石墨烯溶液通入反应池以将荧光标记的荧光完全淬灭；
对反应池进行清洗。
具体地，在本公开实施例提供的上述制作方法中，可以将氧化石墨烯溶液通过入反应池后再将上基板与下基板进行粘接，当然也可以将上基板与下基板进行粘接后，再通过进样口将氧化石墨烯溶液通入反应池，在此不作限定。
具体地，在本公开实施例提供的上述制作方法中，上基板与下基板一般通过低温粘接剂进行粘接。
具体地，在本公开实施例提供的上述制作方法中，可以通过加工通孔在上基板形成进样口和出样口。
具体地，在本公开实施例提供的上述制作方法中，可以通过光刻技术在下基板上刻蚀出微通道和反应池。
下面以一个具体实施例详细说明本公开实施例提供的上述基因检测芯片的检测原理，具体如下：
如图3所示，基因检测芯片的反应池内通过化学表面处理结合了修饰有荧光染料的适配体，之后向反应池内导入1mg/ml的氧化石墨烯溶液，可用荧光共聚焦显微镜观察淬灭情况直到完全淬灭。当向芯片的反应池内加入待测样品时，若有目标基因存在，则目标基因会与玻璃表面的适配体结合从而使荧光恢复；若无目标基因存在，则无法与适配体结合而保持淬灭状态。
本公开实施例提供的上述基因检测芯片可以达到基因检测的目的，并且适配体的专一性高，检测结果准确。同时本公开的基因检测芯片可以有多个平行通道，例如图1所示有四个平行通道，可以同时检测四种不同基因，检测效率快速高效。
本公开实施例提供了一种基因检测芯片、其检测方法、制作方法及微流控芯片系统，该基因检测芯片包括：相对设置的上基板和下基板；其中，上基板设置有至少一对进样口和出样口；进样口和出样口分别位于上基板相对的两端；下基板面向上基板的一面设置有至少一个反应池和至少一个微通道；反应池通过微通道分别与进样口和出样口相连。通过进样口、微通道、反应池、出样口组成一个基因检测通道，由于反应池的表面具有适配体，其中，适配体修饰有荧光标记。这样当进行基因检测时，可以先将荧光标记的荧光淬灭，然后将样品溶液通过进样口进入反应池，如果样品溶液中存在目标基因，则目标基因会与反应池表面的适配体进行杂交反应而结合，从而使荧光恢复；如果样品溶液中不存在目标基因，则荧光仍处于淬灭状态。因此该基因检测芯片无需对目标基因进行荧光标记，就可以实现目标基因的检测，既简化了基因检测的过程，也可以防止基因检测结果中未杂交的目标基因的荧光信号对杂交基因的影响；同时本公开的基因检测芯片结构简单、操作方便、适配体的专一性高、检测结果准确。
显然，本领域的技术人员可以对本公开实施例进行各种改动和变型而不脱离本公开实施例的精神和范围。这样，倘若本公开实施例的这些修改和变型属于本申请权利要求及其等同技术的范围之内，则本申请也意图包含这些改动和变型在内。

Claims (14)

  1. 一种基因检测芯片，其特征在于，包括：相对设置的上基板和下基板；其中，
    所述上基板设置有至少一对进样口和出样口；所述进样口和所述出样口分别位于所述上基板相对的两端；
    所述下基板面向所述上基板的一面设置有至少一个反应池和至少一个微通道；所述反应池通过所述微通道分别与所述进样口和所述出样口相连；其中，所述反应池的表面具有适配体，其中所述适配体修饰有荧光标记。
  2. 如权利要求1所述的基因检测芯片，其特征在于，还包括：与所述进样口相连的第一塑胶软管和与所述出样口相连的第二塑胶软管；
    所述第一塑胶软管用于将溶液通过所述进样口导入；
    所述第二塑胶软管用于通过所述出样口将溶液导出。
  3. 如权利要求1-2任一项所述的基因检测芯片，其特征在于，所述反应池的形状为圆形。
  4. 如权利要求1-3任一项所述的基因检测芯片，其特征在于，所述下基板的材料为玻璃。
  5. 如权利要求1-4任一项所述的基因检测芯片，其特征在于，所述上基板为透明基板。
  6. 一种微流控芯片系统，其特征在于，包括：如权利要求1-5任一项所述基因检测芯片以及动力模块；其中，
    所述动力模块用于为所述样品溶液导入或导出所述基因检测芯片提供动力。
  7. 如权利要求6所述的微流控芯片系统，其特征在于，所述动力模块为注射泵。
  8. 一种如权利要求1-5任一项所述基因检测芯片的检测方法，其特征在于，包括：
    将氧化石墨烯溶液通入所述反应池以将所述荧光标记的荧光完全淬灭；
    待所述荧光标记的荧光完全淬灭后，将待测样品溶液通过所述进样口导入所述反应池；
    确定所述样品溶液与所述反应池表面的所述适配体发生杂交反应后是否恢复荧光，是则所述样品溶液存在目标基因，否则所述样品溶液不存在目标基因。
  9. 如权利要求8所述的检测方法，其特征在于，在确定所述样品溶液与所述反应池表面的所述适配体发生杂交反应后是否恢复荧光之前，还包括：
    对所述反应池进行清洗。
  10. 如权利要求9所述的检测方法，其特征在于，对所述反应池进行清洗，具体包括：
    将清洗液通过所述进样口导入所述反应池，并通过所述出样口将所述清洗液导出。
  11. 一种基因检测芯片的制作方法，其特征在于，包括：
    在上基板上形成有至少一对进样口和出样口；其中，所述进样口和所述出样口分别位于所述上基板相对的两端；
    在下基板上形成至少一个反应池和至少一个微通道，并将修饰有荧光标记的适配体键合到所述反应池的内表面；
    以及，将所述上基板与所述下基板进行粘接，以使所述上基板覆盖所述下基板上的反应池和微通道，且所述反应池通过所述微通道分别与所述进样口和所述出样口相连。
  12. 如权利要求11所述的制作方法，其特征在于，将修饰有荧光标记的适配体键合到所述反应池的内表面，具体包括：
    对所述反应池的内表面进行硅烷化处理；其中，所述下基板的材料为玻璃；
    采用点样机将修饰有荧光标记的所述适配体键合到所述反应池的内表面。
  13. 如权利要求11所述的制作方法，其特征在于，在将修饰有荧光标记的适配体键合到所述反应池的内表面之后，还包括：
    将氧化石墨烯溶液通入所述反应池以将所述荧光标记的荧光完全淬灭；
    对所述反应池进行清洗。
  14. 一种基因检测芯片，其特征在于，包括：相对设置的上基板和下基板；其中，
    所述上基板设置有至少一对进样口和出样口；所述进样口和所述出样口分别位于所述上基板相对的两端；
    所述下基板面向所述上基板的一面设置有至少一个反应池和至少一个微通道；所述反应池通过所述微通道分别与所述进样口和所述出样口相连；其中，所述反应池的表面具有适配体与氧化石墨烯的复合物，其中所述适配体修饰有荧光标记，且所述荧光标记的荧光处于淬灭状态。
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