WO2019091207A1 - 芯片及其制备方法 - Google Patents

芯片及其制备方法 Download PDF

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Publication number
WO2019091207A1
WO2019091207A1 PCT/CN2018/105472 CN2018105472W WO2019091207A1 WO 2019091207 A1 WO2019091207 A1 WO 2019091207A1 CN 2018105472 W CN2018105472 W CN 2018105472W WO 2019091207 A1 WO2019091207 A1 WO 2019091207A1
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silane
group
molecule
nucleic acid
epoxy
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PCT/CN2018/105472
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English (en)
French (fr)
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赵�智
黄天逊
赵陆洋
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 WO2019091207A1 publication Critical patent/WO2019091207A1/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
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/28—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C17/00—Surface treatment of glass, not in the form of fibres or filaments, by coating
    • C03C17/28—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material
    • C03C17/30—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material with silicon-containing compounds
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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"
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Definitions

  • the present application relates to the field of biotechnology, and in particular, the present application relates to a chip, a capture chip, and a method of fabricating the same.
  • chips for nucleic acid detection including the use of chips for target nucleic acid capture, nucleic acid sequencing on the chip, etc.
  • chip performance including surface affinity / hydrophobic properties, non-specific adsorption of nucleic acid properties, probe / primer amount, probe / Primer fixation density, etc., plays a key role in the detection or measurement results.
  • the chip still needs to be further developed or improved, especially for specific situations or chips that can meet specific detection requirements.
  • a first aspect of the present application provides a chip.
  • a chip includes a solid phase substrate, a plurality of graft molecules, and a plurality of first silane molecules; one end of the plurality of graft molecules and the solid phase substrate Connected, a plurality of graft molecules have an epoxy group; one end of the first silane molecule is connected to the solid phase substrate, the first silane molecule has a silane group end and at least one hydrophilic group end, the epoxy group and the hydrophilic group The molar ratio of the end of the group is 1:1 to 1:1000.
  • the chip of the present application has superior surface performance, low non-specific adsorption to nucleic acid, facilitates immobilization and uniform distribution of a large number of probes, and is applied to nucleic acid capture detection, which is advantageous for obtaining a large amount of data and high quality detection results, and is particularly suitable for Highly accurate nucleic acid detection, such as nucleic acid detection at the single molecule level.
  • a second aspect of the present application provides another chip.
  • the chip includes a solid phase substrate, a plurality of graft molecules, a nucleic acid probe molecule, and a plurality of first silane molecules; One end of the molecule is connected to the solid phase substrate; the nucleic acid probe molecule is connected to at least a portion of the plurality of graft molecules via an -O- bond; one end of the plurality of first silane molecules is connected to the solid phase substrate, and the first silane molecule has a silane group The ends of the cluster and at least one end of the hydrophilic group.
  • the second chip of the present application also has superior surface performance, low non-specific adsorption to nucleic acid, facilitates immobilization and uniform distribution of a large number of probes, and is applied to nucleic acid capture detection, which is advantageous for obtaining a large amount of data and high quality detection results. It is especially suitable for nucleic acid detection with high precision requirements, such as nucleic acid detection at the single molecule level.
  • solid phase substrate may be any solid support that can be used for immobilizing a nucleic acid sequence, such as a nylon membrane, a glass sheet, a plastic, a silicon wafer, a magnetic bead, etc., unless otherwise specified, the surface of the chip and The substrate surfaces are used interchangeably.
  • the molar ratio of the -O- bond to the end of the hydrophilic group of the chip is 1:1 to 1:1000.
  • the molar ratio of the -O- bond to the end of the hydrophilic group of the chip is positively correlated with the density of the nucleic acid probe molecule, and the positive correlation includes: when the -O- bond and the end of the hydrophilic group The molar ratio is 1:1000, the density of the nucleic acid probe molecule is 0.6 / ⁇ M 2 ; when the molar ratio of the -O- bond to the end of the hydrophilic group is 1:500, the density of the nucleic acid probe molecule is 1.2 / ⁇ M 2 ; when the molar ratio of the -O- bond to the end of the hydrophilic group is 1:250, the density of the nucleic acid probe molecule is 2.4 / ⁇ M 2 ; when the molar ratio of the -O- bond to the end of the hydrophilic group is 1 : 1 to 1:250, the density of the nucleic acid probe molecules is greater than 2.4 / ⁇ M 2 .
  • any numerical value expressed in an accurate manner represents a range, that is, an interval including plus or minus 10% of the numerical value, unless otherwise specified. The description will not be repeated below.
  • the solid phase substrate is selected from at least one of ordinary glass, quartz glass, ordinary glass containing a metal plating layer, and quartz glass containing a metal plating layer.
  • the graft molecule is an epoxy silane molecule having a silane group end and at least one epoxy group end.
  • the graft molecule and/or the first silane molecule have a silane group, and the silane group is selected from the group consisting of monomethylsilane, dimethylsilane, trimethylsilane, monomethyloxysilane, and dimethyl At least one of oxysilane, trimethoxysilane, monoethoxysilane, diethoxysilane, and triethoxysilane.
  • the above silane groups are suitable for attachment to the surface of the glass.
  • the graft molecule and/or the first silane molecule have a linking group, the linking group is bonded to the silane group, and the linking group is 1 to 12 alkane chains or 1 to 8 ethoxylated chains.
  • the above-mentioned linking group is advantageous for forming a monolayer having a relatively uniform surface height on the surface of the solid phase substrate and functioning to isolate the surface of the substrate.
  • the hydrophilic group end of the first silane molecule is at least one selected from the group consisting of a hydroxyl group, an amino group, a trifluoropropyl group, and a methacryloxy group.
  • the graft molecule is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, glycidyloxypropylethoxysilane, glycidyloxypropylmethyldiethoxylate Silane, glycidyloxypropylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane and 2-(3,4-epoxy ring At least one of alk)-ethyltriethoxysilane.
  • the first silane molecule is selected from the group consisting of hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxylate Silane, 3-ureidopropyltrimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropyl)methyldiethoxy At least one of a silane, a 3-methacryloxypropyltrimethoxysilane, and a methacryloxypropyltris(trimethylsiloxy)silane.
  • a third aspect of the present application provides a method for preparing two chips of the present application.
  • the method includes: surface modifying a solid phase substrate, and modifying the surface comprises using a mixture to surface the solid phase substrate.
  • the mixture contains a 1:1 to 1:1000 molar ratio of epoxy groups and hydrophilic group ends.
  • the method of the present application can conveniently adjust the surface epoxy group (reactive group, which can be used for reaction with the probe) and the hydrophilic group by controlling the molar ratio of the epoxy group and the hydrophilic group according to actual needs.
  • the density, so as to achieve the probe load capacity of the chip and the surface properties can be adjusted, which is beneficial to reduce the non-specific adsorption of the chip surface and improve the detection performance of the obtained chip.
  • the method of the present application further comprises: immobilizing the nucleic acid probe molecule on the surface modified solid phase substrate.
  • the epoxy group is derived from an epoxy silane having a silane group end and at least one epoxy group end.
  • the hydrophilic group end is derived from a first silane, and the first silane has a silane group end and at least one hydrophilic group end.
  • the silane group of the epoxy silane and/or the first silane is selected from the group consisting of monomethylsilane, dimethylsilane, trimethylsilane, monomethyloxysilane, dimethoxysilane, and trimethyl At least one of oxysilanes.
  • the epoxy silane and/or the first silane have a linking group, the linking group and the silane group are bonded, and the linking group is 1 to 12 alkane chains or 1 to 8 ethoxy chains.
  • the epoxy silane is selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, glycidyloxypropylethoxysilane, glycidyloxypropylmethyldiethoxylate Silane, glycidyloxypropylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane and 2-(3,4-epoxy ring At least one of alk)-ethyltriethoxysilane.
  • the first silane is selected from the group consisting of hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxylate Silane, 3-ureidopropyltrimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropyl)methyldiethoxy At least one of silane, 3-methacryloxypropyltrimethoxysilane, and methacryloxypropyltris(trimethylsiloxy)silane.
  • the surface modification is carried out in the liquid phase and/or the gas phase.
  • the total content of epoxy silane and first silane in the mixture is from 1% to 3%. Further, the silanization efficiency is further improved.
  • the method of the present application further comprises: surface activation of the solid phase substrate prior to surface modification to impart a hydroxyl group to the surface of the solid phase substrate.
  • the nucleic acid probe molecule is a nucleic acid molecule modified by terminal amination.
  • the amino group is attached to the modified solid phase substrate via a 1-8 alkyl chain or a 1-4 ethoxy chain.
  • the fixed density of the nucleic acid probe molecule has a controllable correspondence with the molar ratio of the epoxy group to the hydrophilic group, and the controllable correspondence includes: an epoxy group and a hydrophilic group.
  • the molar ratio of the group is 1:1000, the fixed density of the nucleic acid probe molecule is 0.6 / ⁇ M 2 ; the molar ratio of the epoxy group to the hydrophilic group is 1:500, and the fixed density of the nucleic acid probe molecule is 1.2 / ⁇ M 2 ; the molar ratio of the epoxy group to the hydrophilic group is 1:250, the fixed density of the nucleic acid probe molecule is 2.4 / ⁇ M 2 ; and the molar ratio of the epoxy group to the hydrophilic group is 1: 1 to 1:250, the nucleic acid probe molecule has a fixed density of more than 2.4 / ⁇ M 2 .
  • the fixed density of the nucleic acid probe molecules in the prepared chip increases as the molar ratio of the epoxy group to the hydrophilic group increases.
  • the activated solid phase substrate is obtained by contacting a solid phase substrate with ethanol or isopropanol and a Piranha solution. This results in a sufficient amount of reactive hydroxyl groups on the surface of the solid phase substrate.
  • the epoxy silane is 3-glycidoxypropyltrimethoxysilane. Further, the silanization efficiency is further improved.
  • the hydroxysilane is hydroxypropyltrimethoxysilane. Further, the silanization efficiency is further improved.
  • the amino group is modified at the 5' end of the nucleic acid probe molecule.
  • the nucleic acid probe molecule is a sequence of polyT.
  • the length of the nucleic acid probe molecule is 10 to 30 bp.
  • the epoxy ring opening reaction is carried out in a phosphate solution. Further, the reaction pH is effectively controlled to 8 to 10.
  • the phosphate is a 0.1 M to 2 M K 2 HPO 4 solution. Further, the efficiency of the epoxy ring opening reaction is further improved.
  • the epoxy ring-opening reaction is carried out at 25 to 60 ° C for 4 to 24 hours. Further, the epoxy ring-opening reaction is sufficient, and the nucleic acid probe molecules can be maximally immobilized on the silanized solid phase substrate.
  • the method further comprises: performing a first washing on the silanized solid phase substrate.
  • the excess epoxy silane and the first silane are washed away from the surface of the solid phase substrate to further improve the non-specific adsorption capacity of the chip.
  • the immobilizing the nucleic acid probe molecule further comprises: performing a second washing on the solid phase substrate to which the nucleic acid probe molecule is immobilized. The excess unfixed nucleic acid probe molecule or non-specific binding is washed away from the surface of the solid phase substrate, further improving the non-specific adsorption capacity of the chip.
  • the first wash is alternately washed with ethanol or isopropanol, water.
  • the second wash is followed by washing with a mixture of 3 x SSC and 0.1% Triton, 0.1 M to 2 M K 2 HPO 4 , 150 mM HEPES and 150 mM NaCl solution and ultrapure water.
  • FIG. 1 is a schematic view showing a process of a sequencing chip in an embodiment of the present application
  • Fig. 4 is a graph showing the results of changes in the number of surface non-specifically adsorbed fluorescent dots at different mixing ratios in the examples of the present application.
  • the surface resistance of the chip to non-specific adsorption and the like can not meet the requirements of chip-based single molecule sequencing.
  • the test found that the surface properties of the chip include non-specific adsorption, etc., which have an important influence on the immobilization, immobilization and distribution of the surface primer/probe, the capture of the template nucleic acid and the biochemical reaction of the subsequent sequencing process. Directly affect the final sequencing results.
  • the present application is intended to solve at least one of the technical problems in the related art at least to some extent; therefore, the chip of the present application and a method of preparing the same are creatively proposed.
  • CY3 and CY5 are nucleic acid labeling molecules, and both Cy 5 and Cy 3 are water-soluble 3H-phthalocyanine type bioluminescent labeling dyes, which are capable of emitting red color fluorescence and green fluorescence under laser irradiation of 650 nm and 550 nm, respectively. .
  • the base chip is an empty chip and does not contain a probe.
  • the epoxy silane may be 3-glycidoxypropyltrimethoxysilane, glycidoxypropyl ethoxysilane, glycidyloxypropylmethyldiethoxysilane, glycidyl ether oxygen Propylmethyldimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltrimethoxysilane, 2-(3,4-epoxycyclohexane)-ethyltriethyl Oxysilane or the like.
  • 3-glycidoxypropyltrimethoxysilane is exemplified.
  • the first silane may be hydroxypropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-ureidopropyltriethoxysilane, 3-ureidopropyl Trimethoxysilane, (3,3,3-trifluoropropyl)methyldimethoxysilane, (3,3,3-trifluoropropyl)methyldiethoxysilane, 3-methylpropene Acyloxypropyltrimethoxysilane, methacryloxypropyltris(trimethylsiloxy)silane, and the like.
  • hydroxypropyltrimethoxysilane is exemplified.
  • step (2) the same as step (2) of the embodiment 1;
  • the surface grafting is a 1:100 mixture of 3-glycidoxypropyltrimethoxysilane and hydroxypropyltrimethoxysilane, and the mixture is separately adjusted in this example.
  • the ratio of 3-glycidoxypropyltrimethoxysilane and hydroxypropyltrimethoxysilane was 1:1, 1:10, 1:100, 1:1000, and different ratios were prepared according to the method of Example 2.
  • Three sheets of the substrate chip were each mixed with PolyA-CY3 at the same concentration of 0.6 nM, and the average number of fluorescent dots per square micrometer of field of view was counted under a fluorescence microscope. The results are shown in FIG.
  • the results in Figure 3 show that: 1) surface primers are successfully immobilized on the base glass at different ratios; 2) the surface fixation density can be effectively adjusted by adjusting the proportion of the silane mixture.
  • the substrate chips of different proportions in Example 4 were immersed in a solution containing 5% C-base containing CY5, reacted at room temperature for 5 minutes, and then washed with 2 ⁇ PBS solution and ultrapure water in order to obtain a certain adsorption.
  • the average number of fluorescent dots per square micrometer of field of view was counted under a microscope, and the results are shown in FIG.
  • the results in Figure 4 show that: 1) the base wafer with a mixing ratio of 1:1000 has the least number of fluorescent dots, indicating that the anti-base non-specific adsorption of this surface is strong; 2) as the ratio becomes smaller, the number of fluorescent dots decreases. It is indicated that the anti-base non-specific adsorption of the substrate chip can be achieved by adjusting the mixing ratio.

Abstract

一种芯片及其制备方法。芯片包括：固相基底；以及多个接枝分子，多个接枝分子的一端与固相基底相连，多个接枝分子具有环氧基团；多个第一硅烷分子，第一硅烷分子的一端与固相基底相连，第一硅烷分子具有硅烷基团末端和至少一个亲水基团末端，其中，环氧基团与亲水基团末端的摩尔比例是1:1～1:1000。芯片具有较优的表面性能，对核酸的非特异性吸附低、利于大量探针固定上以及均匀分布，应用于核酸捕获检测，利于获得数据量大、质量高的检测结果，特别适用于高精确要求的核酸检测，例如单分子水平的核酸检测。

Description

芯片及其制备方法 技术领域
本申请涉及生物技术领域，具体地，本申请涉及芯片、捕获芯片及其制备方法。
背景技术
近年来，主要发达国家包括美国，英国，法国等都陆续将基因技术作为国家战略，基因检测技术的发展引人关注。
利用芯片进行核酸检测，包括利用芯片进行目标核酸捕获、在芯片上进行核酸序列测定等，芯片的性能，包括表面亲/疏水特性、非特异性吸附核酸性能、探针/引物的量、探针/引物固定密度等，对检测或测定结果起到关键作用。
芯片仍有待进一步开发或改进，特别是适用于特定情形或者能够满足特定检测要求的芯片。
发明内容
本申请的第一方面提供了一种芯片，本申请的一个实施例中，芯片包括固相基底、多个接枝分子和多个第一硅烷分子；多个接枝分子的一端与固相基底相连，多个接枝分子具有环氧基团；第一硅烷分子的一端与固相基底相连，第一硅烷分子具有硅烷基团末端和至少一个亲水基团末端，环氧基团与亲水基团末端的摩尔比例是1:1～1:1000。
本申请的芯片具有较优的表面性能，对核酸的非特异性吸附低、利于大量探针固定上以及均匀分布，应用于核酸捕获检测，利于获得数据量大、质量高的检测结果，特别适用于高精确要求的核酸检测，例如单分子水平的核酸检测。
本申请的第二方面提供了另一种芯片，本申请的一个实施例中，该芯片包括固相基底、多个接枝分子、核酸探针分子和多个第一硅烷分子；多个接枝分子的一端与固相基底相连；核酸探针分子与多个接枝分子的至少一部分通过-O-键相连；多个第一硅烷分子的一端与固 相基底相连，第一硅烷分子具有硅烷基团末端和至少一个亲水基团末端。
本申请的第二种芯片同样具有较优的表面性能，对核酸的非特异性吸附低、利于大量探针固定上以及均匀分布，应用于核酸捕获检测，利于获得数据量大、质量高的检测结果，特别适用于高精确要求的核酸检测，例如单分子水平的核酸检测。
需要说明的是，本申请的“固相基底”可以是任何可用于固定核酸序列的固体支持物，例如尼龙膜、玻璃片、塑料、硅片、磁珠等，如无特殊说明，芯片表面与基底表面可互换使用。
本申请的实施例中，芯片的-O-键和亲水基团末端的摩尔比为1:1～1:1000。
本申请的实施例中，芯片的-O-键和亲水基团末端的摩尔比与核酸探针分子的密度呈正相关关系，正相关关系包括：当-O-键和亲水基团末端的摩尔比为1:1000，核酸探针分子的密度为0.6个/μM 2；当-O-键和亲水基团末端的摩尔比为1:500，核酸探针分子的密度为1.2个/μM 2；当-O-键和亲水基团末端的摩尔比为1:250，核酸探针分子的密度为2.4个/μM 2；当-O-键和亲水基团末端的摩尔比为1:1～1:250，核酸探针分子的密度大于2.4个/μM 2。
需要说明的是，由于本申请实施例中涉及的具体数据大多具有统计意义，因此，如无特殊说明，任意以精确方式表达的数值均代表一个范围，即包含该数值正负10％的区间，以下不再重复说明。
本申请的实施例中，固相基底选自普通玻璃、石英玻璃、含有金属镀层的普通玻璃和含有金属镀层的石英玻璃中的至少一种。
本申请的实施例中，接枝分子为环氧硅烷分子，环氧硅烷分子具有硅烷基团末端和至少一个环氧基团末端。
本申请的实施例中，接枝分子和/或第一硅烷分子具有硅烷基团，硅烷基团选自一甲基硅烷、二甲基硅烷、三甲基硅烷、一甲基氧硅烷、二甲氧基硅烷、三甲氧基硅烷、一乙氧基硅烷、二乙氧基硅烷和三乙氧基硅烷中的至少之一种。上述硅烷基团适于与玻璃表面连接。
本申请的实施例中，接枝分子和/或第一硅烷分子具有连接基团，连接基团和硅烷基团相连，连接基团为1～12个烷烃链或者1～8乙氧 基链。上述连接基团有利于在固相基底表面形成表面高低较一致的单分子层，且起到隔离基底表面的作用。
本申请的实施例中，第一硅烷分子的亲水基团末端选自羟基、氨基、三氟丙基和甲基丙烯酰氧基中的至少一种。
本申请的实施例中，接枝分子选自3-缩水甘油醚氧基丙基三甲氧基硅烷、缩水甘油醚氧基丙基乙氧基硅烷、缩水甘油醚氧基丙基甲基二乙氧基硅烷、缩水甘油醚氧基丙基甲基二甲氧基硅烷、2-(3,4-环氧环已烷)-乙基三甲氧基硅烷和2-(3,4-环氧环已烷)-乙基三乙氧基硅烷中的至少一种。
本申请的实施例中，第一硅烷分子选自羟基丙基三甲氧基硅烷、3-氨丙基三乙氧基硅烷、3-氨丙基三甲氧基硅烷、3-脲丙基三乙氧基硅烷、3-脲丙基三甲氧基硅烷、(3,3,3-三氟丙基)甲基二甲氧基硅烷、(3,3,3-三氟丙基)甲基二乙氧基硅烷、3-甲基丙烯酰氧基丙基三甲氧基硅烷和甲基丙烯酰氧基丙基三(三甲基硅氧基)硅烷中的至少一种。
本申请的第三方面提供了本申请的两种芯片的制备方法，本申请的一个实施例中，该方法包括：对固相基底进行表面修饰，表面修饰包括利用混合物对固相基底的表面进行处理，混合物包含1:1～1:1000摩尔比的环氧基团和亲水基团末端。
本申请的方法，能够通过控制环氧基和亲水基团的摩尔比例，根据实际需求，方便地调节表面环氧基团(反应基团，可用于与探针反应)和亲水基团的密度，从而达到芯片的探针承载量以及表面性能可调控，利于降低芯片表面非特异吸附，提高获得的芯片的检测性能。
本申请的实施例中，本申请的方法进一步包括：将核酸探针分子固定在表面修饰后的固相基底上。
本申请的实施例中，环氧基团来自环氧硅烷，环氧硅烷具有硅烷基团末端和至少一个环氧基团末端。
本申请的实施例中，亲水基团末端来自第一硅烷，第一硅烷具有硅烷基团末端和至少一个亲水基团末端。
本申请的实施例中，环氧硅烷和/或第一硅烷的硅烷基团选自一甲基硅烷、二甲基硅烷、三甲基硅烷、一甲基氧硅烷、二甲氧基硅烷和三甲氧基硅烷中的至少之一种。
本申请的实施例中，环氧硅烷和/或第一硅烷具有连接基团，连接基团和硅烷基团连接，连接基团为1～12个烷烃链或者1～8乙氧基链。
本申请的实施例中，环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、缩水甘油醚氧基丙基乙氧基硅烷、缩水甘油醚氧基丙基甲基二乙氧基硅烷、缩水甘油醚氧基丙基甲基二甲氧基硅烷、2-(3,4-环氧环已烷)-乙基三甲氧基硅烷和2-(3,4-环氧环已烷)-乙基三乙氧基硅烷中的至少一种。
本申请的实施例中，第一硅烷选自羟基丙基三甲氧基硅烷、3-氨丙基三乙氧基硅烷、3-氨丙基三甲氧基硅烷、3-脲丙基三乙氧基硅烷，3-脲丙基三甲氧基硅烷、(3,3,3-三氟丙基)甲基二甲氧基硅烷、(3,3,3-三氟丙基)甲基二乙氧基硅烷、3-甲基丙烯酰氧基丙基三甲氧基硅烷和甲基丙烯酰氧基丙基三(三甲基硅氧基)硅烷中的至少一种。
本申请的实施例中，表面修饰在液相和/或气相中进行。
本申请的实施例中，混合物中环氧硅烷和第一硅烷的总含量为1％-3％。进而硅烷化效率进一步提高。
本申请的实施例中，本申请的方法还包括：进行表面修饰之前，对固相基底进行表面活化，以使固相基底的表面带有羟基。
本申请的实施例中，核酸探针分子为经过末端氨基化修饰的核酸分子。氨基通过1～8烷基链或1～4乙氧基链与修饰处理的固相基体连接。
本申请的实施例中，核酸探针分子的固定密度与环氧基团和亲水基团的摩尔比具有可控的对应关系，该可控的对应关系包括：环氧基团和亲水基团的摩尔比为1:1000，核酸探针分子的固定密度为0.6个/μM 2；环氧基团和亲水基团的摩尔比为1:500，核酸探针分子的固定密度为1.2个/μM 2；环氧基团和亲水基团的摩尔比为1:250，核酸探 针分子的固定密度为2.4个/μM 2；环氧基团和亲水基团的摩尔比为1:1～1:250，核酸探针分子的固定密度大于2.4个/μM 2。
本申请的实施例中，制备的芯片中的核酸探针分子的固定密度随着环氧基团和亲水基团的摩尔比的增大而增大。
本申请的实施例中，活化固相基底是通过将固相基底与乙醇或异丙醇以及Piranha溶液进行接触获得的。进而使固相基底表面产生足够多的活性羟基基团。
本申请的实施例中，环氧硅烷为3-缩水甘油醚氧基丙基三甲氧基硅烷。进而硅烷化效率进一步提高。
本申请的实施例中，羟基硅烷为羟基丙基三甲氧基硅烷。进而硅烷化效率进一步提高。
本申请的实施例中，氨基修饰在核酸探针分子的5’端。
本申请的实施例中，核酸探针分子为polyT的序列。
本申请的实施例中，核酸探针分子的长度为10～30bp。
本申请的实施例中，环氧开环反应是在磷酸盐溶液中进行的。进而将反应pH有效控制在8～10。
本申请的实施例中，磷酸盐为0.1M～2M的K 2HPO 4溶液。进而环氧开环反应的效率进一步提高。
本申请的实施例中，环氧开环反应是在25～60℃的条件下进行4～24小时。进而环氧开环反应充分，核酸探针分子可最大化固定在经过硅烷化处理的固相基体上。
本申请的实施例中，表面修饰之后、固定核酸探针分子之前进一步包括：对经过硅烷化处理的固相基体进行第一洗涤。将多余的环氧硅烷和第一硅烷从固相基体表面洗掉，进一步提高芯片的抗非特异性吸附能力。
本申请的实施例中，固定核酸探针分子之后进一步包括：对固定有核酸探针分子的固相基体进行第二洗涤。将多余的没有固定的核酸探针分子或非特异性结合从固相基体表面洗掉，进一步提高芯片的抗非特异性吸附能力。
本申请的实施例中，第一洗涤是用乙醇或异丙醇，水交替清洗。
本申请的实施例中，第二洗涤是依次使用3×SSC与0.1％的Triton组成的混合液，0.1M～2M K 2HPO 4，150mM HEPES与150mM NaCl溶液和超纯水来清洗。
附图说明
图1是本申请实施例中测序芯片的工艺示意图；
图2是本申请实施例中不同杂交浓度下的表面杂交荧光点数变化的结果图；
图3是本申请实施例中不同混合比例下的表面杂交荧光点数变化的结果图；
图4是本申请实施例中不同混合比例下的表面非特异吸附荧光点数变化的结果图。
具体实施方式
本申请是基于发明人对以下事实和问题的发现和认识作出的：
目前市售的芯片或者利用已知方法制备得的芯片，芯片表面抗非特异吸附等性能并不能满足基于芯片的单分子测序的要求。在芯片上进行测序时，测试发现，芯片的表面性能包括非特异吸附等，对表面引物/探针的固定、固定量及分布，模板核酸的捕获及后续测序过程的生化反应都有重要影响，直接影响最终测序结果。
本申请旨在至少在一定程度上解决以上相关技术中的技术问题之一；因此，创造性的提出了本申请的芯片及其制备方法。
本申请要求2017年11月10日提交中国专利局的申请号为201711105069.0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。
本申请中，CY3和CY5为核酸标记分子，Cy 5和Cy 3都属于水溶性3H-吲哚菁型生物荧光标示染料，分别能够在650nm和550nm的激光照射下发出红光色荧光和绿色荧光。
基底芯片为空载芯片，不含探针。
环氧硅烷可以采用3-缩水甘油醚氧基丙基三甲氧基硅烷、缩水甘油醚氧基丙基乙氧基硅烷、缩水甘油醚氧基丙基甲基二乙氧基硅烷、 缩水甘油醚氧基丙基甲基二甲氧基硅烷、2-(3,4-环氧环已烷)-乙基三甲氧基硅烷、2-(3,4-环氧环已烷)-乙基三乙氧基硅烷等。本申请实施例中以3-缩水甘油醚氧基丙基三甲氧基硅烷为例。
第一硅烷可以采用羟基丙基三甲氧基硅烷、3-氨丙基三乙氧基硅烷、3-氨丙基三甲氧基硅烷、3-脲丙基三乙氧基硅烷、3-脲丙基三甲氧基硅烷、(3,3,3-三氟丙基)甲基二甲氧基硅烷、(3,3,3-三氟丙基)甲基二乙氧基硅烷、3-甲基丙烯酰氧基丙基三甲氧基硅烷、甲基丙烯酰氧基丙基三(三甲基硅氧基)硅烷等。本申请实施例中以羟基丙基三甲氧基硅烷为例。
下面通过具体实施例对本申请作进一步详细说明。以下实施例仅对本申请进行进一步说明，不应理解为对本申请的限制。实施例中的试剂、检测仪器等，如无特殊说明，可自配或者通过市售途径获取。
实施例1
(1)清洗，活化玻璃表面：用乙醇或异丙醇，水交替清洗直径为40mm玻璃表面，使用Piranha溶液活化玻璃，使表面产生足够多活性羟基基团；
(2)表面接枝：配制1:100的3-缩水甘油醚氧基丙基三甲氧基硅烷和羟基丙基三甲氧基硅烷混合水溶液，调整PH＝5.5，将玻璃片浸泡在该溶液中，室温下反应12小时，反应完，用乙醇或异丙醇，水交替清洗三次。
实施例2
(1)同实施例1的步骤(1)；
(2)同实施例1的步骤(2)；
(3)引物固定：将接枝后的玻璃片吹干后，置于浓度为1.0M的氨基修饰的PolyT核酸引物的K 2HPO 4溶液中，K 2HPO 4溶液的浓度为150mM，反应1小时，依次用3×SSC溶液(含0.1％的Triton)，0.2M K 2HPO 4溶液，150mM HEPES与150mM NaCl混合溶液，及超 纯水清洗后，含有PolyT引物(探针)的基底芯片制作完成。本例的制备流程简单表述如图1所示。
实施例3
吹干实施例2制备的基底芯片后，分别杂交不同浓度的PolyA-CY3于上述基底芯片，杂交浓度为0.2nM、0.4nM、0.6nM、0.8nM，重复三次，在显微镜下统计每平方微米视野内的平均荧光点数。不同杂交浓度统计的荧光点数结果如图2所示。由图2可知：1)不同杂交浓度下均有荧光点，说明引物成功固定在基底玻璃表面；2)0.6nM后，提高杂交浓度，杂交点数均没有明显变化，这说明表面固定引物数量一定，杂交点数在一定浓度后不随点数变化。
实施例4
实施例2的步骤(2)中，表面接枝采用的是1:100的3-缩水甘油醚氧基丙基三甲氧基硅烷和羟基丙基三甲氧基硅烷混合水溶液，本例分别调整混合液中3-缩水甘油醚氧基丙基三甲氧基硅烷和羟基丙基三甲氧基硅烷比例为1:1、1:10、1:100、1:1000，按照实施例2的方法制作不同比例的基底芯片各3张，然后用同一浓度0.6nM的PolyA-CY3杂交，在荧光显微镜下统计每平方微米视野内的平均荧光点数，结果如图3所示。图3的结果显示：1)不同比例下，表面引物均成功固定在基底玻璃上；2)通过调整硅烷混合液比例，可以有效调整表面固定密度。
实施例5
将实施例4中不同比例的基底芯片，一起浸泡在含有5％的含CY5的C碱基溶液中，室温反应5分钟后，依次用2×PBS溶液和超纯水清洗后，得到吸附了一定碱基的芯片，在显微镜下统计每平方微米视野内的平均荧光点数，结果如图4所示。图4的结果显示：1)混合比例为1:1000的基底芯片具有最少的荧光点数，说明此表面的抗碱基非特异吸附较强；2)随着比例变小，荧光点数呈递减趋势，说明基底芯片的抗碱基非特异吸附可以通过调整混合比例来实现。
在本申请说明书的描述中，参考术语“一个实施例”、“实施例”、等的描述意指结合实施例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。
以上应用了具体个例对本申请进行阐述，只是用于帮助理解本申请，并不用以限制本申请。对于本领域的一般技术人员，依据本申请的思想，可以对上述具体实施方式进行变化。

Claims (25)

  1. 一种芯片，其特征在于，包括：
    固相基底；以及
    多个接枝分子，所述多个接枝分子的一端与所述固相基底相连，所述多个接枝分子具有环氧基团；
    多个第一硅烷分子，所述第一硅烷分子的一端与所述固相基底相连，所述第一硅烷分子具有硅烷基团末端和至少一个亲水基团末端，
    其中，所述环氧基团与所述亲水基团末端的摩尔比例是1:1～1:1000。
  2. 一种芯片，其特征在于，包括：
    固相基底；以及
    多个接枝分子，所述多个接枝分子的一端与所述固相基底相连；
    核酸探针分子，所述核酸探针分子与所述多个接枝分子的至少一部分通过-O-键相连；
    多个第一硅烷分子，所述多个第一硅烷分子的一端与所述固相基底相连，所述第一硅烷分子具有硅烷基团末端和至少一个亲水基团末端。
  3. 根据权利要求2所述的芯片，其特征在于，所述-O-键和亲水基团末端的摩尔比为1:1～1:1000。
  4. 根据权利要求3所述的芯片，其特征在于，所述-O-键和亲水基团末端的摩尔比与所述核酸探针分子的密度呈正相关关系，所述正相关关系包括：
    当所述-O-键和亲水基团末端的摩尔比为1:1000，所述核酸探针分子的密度为0.6个/μM 2；
    当所述-O-键和亲水基团末端的摩尔比为1:500，所述核酸探针分子的密度为1.2个/μM 2；
    当所述-O-键和亲水基团末端的摩尔比为1:250，所述核酸探针分子的密度为2.4个/μM 2；
    当所述-O-键和亲水基团末端的摩尔比为1:1～1:250，所述核酸探针分子的密度大于2.4个/μM 2。
  5. 根据权利要求1或2所述的芯片，其特征在于，所述固相基底选自普通玻璃、石英玻璃、含有金属镀层的普通玻璃和含有金属镀层的石英玻璃中的至少一种。
  6. 根据权利要求1或2所述的芯片，其特征在于，所述接枝分子为环氧硅烷分子，所述环氧硅烷分子具有硅烷基团末端和至少一个环氧基团末端。
  7. 根据权利要求1或2所述的芯片，其特征在于，所述接枝分子和/或第一硅烷分子具有硅烷基团，所述硅烷基团选自一甲基硅烷、二甲基硅烷、三甲基 硅烷、一甲基氧硅烷、二甲氧基硅烷、三甲氧基硅烷、一乙氧基硅烷、二乙氧基硅烷和三乙氧基硅烷中的至少之一种。
  8. 根据权利要求7所述的芯片，其特征在于，所述接枝分子和/或第一硅烷分子具有连接基团，所述连接基团和所述硅烷基团相连，所述连接基团为1～12个烷烃链或者1～8乙氧基链。
  9. 根据权利要求7或8所述的芯片，其特征在于，所述第一硅烷分子的亲水基团末端选自羟基、氨基、三氟丙基和甲基丙烯酰氧基中的至少一种。
  10. 根据权利要求7或8所述的芯片，其特征在于，所述接枝分子选自3-缩水甘油醚氧基丙基三甲氧基硅烷、缩水甘油醚氧基丙基乙氧基硅烷、缩水甘油醚氧基丙基甲基二乙氧基硅烷、缩水甘油醚氧基丙基甲基二甲氧基硅烷、2-(3，4-环氧环已烷)-乙基三甲氧基硅烷和2-(3，4-环氧环已烷)-乙基三乙氧基硅烷中的至少一种。
  11. 根据权利要求9所述的芯片，其特征在于，所述第一硅烷分子选自羟基丙基三甲氧基硅烷、3-氨丙基三乙氧基硅烷、3-氨丙基三甲氧基硅烷、3-脲丙基三乙氧基硅烷、3-脲丙基三甲氧基硅烷、(3，3，3-三氟丙基)甲基二甲氧基硅烷、(3，3，3-三氟丙基)甲基二乙氧基硅烷、3-甲基丙烯酰氧基丙基三甲氧基硅烷和甲基丙烯酰氧基丙基三(三甲基硅氧基)硅烷中的至少一种。
  12. 一种制备权利要求1-11任一所述芯片的方法，其特征在于，包括：
    对固相基底进行表面修饰，所述表面修饰包括利用混合物对所述固相基底的表面进行处理，所述混合物包含1:1～1:1000摩尔比的环氧基团和亲水基团末端。
  13. 根据权利要求12所述的方法，其特征在于，所述方法进一步包括：
    将核酸探针分子固定在表面修饰后的固相基底上。
  14. 根据权利要求12所述的方法，其特征在于，所述环氧基团来自环氧硅烷，所述环氧硅烷具有硅烷基团末端和至少一个环氧基团末端；
    所述亲水基团末端来自第一硅烷，所述第一硅烷具有硅烷基团末端和至少一个亲水基团末端。
  15. 根据权利要求14所述的方法，其特征在于，所述环氧硅烷和/或所述第一硅烷的硅烷基团选自一甲基硅烷、二甲基硅烷、三甲基硅烷、一甲基氧硅烷、二甲氧基硅烷和三甲氧基硅烷中的至少之一种。
  16. 根据权利要求14或15所述的方法，其特征在于，所述环氧硅烷和/或所述第一硅烷具有连接基团，所述连接基团和所述硅烷基团连接，所述连接基团为1～12个烷烃链或者1～8乙氧基链。
  17. 根据权利要求14或15所述的方法，其特征在于，所述环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、缩水甘油醚氧基丙基乙氧基硅烷、缩水甘油醚氧基丙基甲基二乙氧基硅烷、缩水甘油醚氧基丙基甲基二甲氧基硅烷、2-(3，4-环氧环已烷)-乙基三甲氧基硅烷和2-(3，4-环氧环已烷)-乙基三乙氧基硅烷中的至少一种。
  18. 根据权利要求14或15所述的方法，其特征在于，所述第一硅烷选自羟基丙基三甲氧基硅烷、3-氨丙基三乙氧基硅烷、3-氨丙基三甲氧基硅烷、3-脲丙基三乙氧基硅烷、3-脲丙基三甲氧基硅烷、(3，3，3-三氟丙基)甲基二甲氧基硅烷、(3，3，3-三氟丙基)甲基二乙氧基硅烷、3-甲基丙烯酰氧基丙基三甲氧基硅烷和甲基丙烯酰氧基丙基三(三甲基硅氧基)硅烷中的至少一种。
  19. 根据权利要求12所述的方法，其特征在于所述表面修饰在液相和/或气相中进行。
  20. 根据权利要求12所述的方法，其特征在于，所述环氧基团来自环氧硅烷，所述亲水基团末端来自第一硅烷，所述混合物中环氧硅烷和第一硅烷的总含量为1％-3％。
  21. 根据权利要求12所述的方法，其特征在于，进行所述表面修饰之前，对所述固相基底进行表面活化，以使所述固相基底的表面带有羟基。
  22. 根据权利要求13所述的方法，其特征在于，所述核酸探针分子为经过末端氨基化修饰的核酸分子。
  23. 根据权利要求14-22任一所述的方法，其特征在于，所述环氧硅烷为3-缩水甘油醚氧基丙基三甲氧基硅烷，所述第一硅烷为羟基丙基三甲氧基硅烷。
  24. 根据权利要求23所述的方法，其特征在于，表面修饰之后、固定核酸探针分子之前进一步包括：对经过表面修饰的固相基底进行第一洗涤。
  25. 根据权利要求24所述的方法，其特征在于，固定核酸探针分子之后进一步包括：对固定有核酸探针分子的固相基底进行第二洗涤。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114682309A (zh) * 2020-12-29 2022-07-01 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101643321A (zh) * 2009-09-01 2010-02-10 博奥生物有限公司 高分子聚合物三维氨基基片及其制备方法与应用
CN102168146A (zh) * 2011-05-18 2011-08-31 蔡伟文 高特异性高灵敏度的基因芯片及其制备方法与应用
CN105112408A (zh) * 2015-08-14 2015-12-02 深圳市瀚海基因生物科技有限公司 一种单分子靶向测序中靶向引物的固定方法、单分子靶向测序试剂盒及应用
CN105154323A (zh) * 2015-08-14 2015-12-16 深圳市瀚海基因生物科技有限公司 一种单分子测序芯片

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528264B1 (en) * 2000-11-01 2003-03-04 Corning Incorporated Polymer support for DNA immobilization
US20070275411A1 (en) * 2006-05-25 2007-11-29 Mcgall Glenn H Silane mixtures
US20080261825A1 (en) * 2004-04-02 2008-10-23 Brennan John D Method of immobilizing membrane-associated molecules
FR2870143B1 (fr) * 2004-05-12 2006-07-14 Commissariat Energie Atomique Procede sol-gel de fonctionnalisation d'une surface d'un substrat solide.
SG150405A1 (en) * 2007-08-29 2009-03-30 Agency Science Tech & Res Method of coating a particle
US8501406B1 (en) * 2009-07-14 2013-08-06 Pacific Biosciences Of California, Inc. Selectively functionalized arrays

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101643321A (zh) * 2009-09-01 2010-02-10 博奥生物有限公司 高分子聚合物三维氨基基片及其制备方法与应用
CN102168146A (zh) * 2011-05-18 2011-08-31 蔡伟文 高特异性高灵敏度的基因芯片及其制备方法与应用
CN105112408A (zh) * 2015-08-14 2015-12-02 深圳市瀚海基因生物科技有限公司 一种单分子靶向测序中靶向引物的固定方法、单分子靶向测序试剂盒及应用
CN105154323A (zh) * 2015-08-14 2015-12-16 深圳市瀚海基因生物科技有限公司 一种单分子测序芯片

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAI, MENGJUN ET AL.: "Study of Surface Modification on Polymer Microfluidic Cell Chip", ACTA ACADEMIAE MEDICINAE MILITARIS TERTIAE, vol. 33, no. 24, 30 December 2011 (2011-12-30), pages 2571 - 2574 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114682309A (zh) * 2020-12-29 2022-07-01 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途
CN114682309B (zh) * 2020-12-29 2024-04-09 深圳华大生命科学研究院 芯片、制备芯片的方法及芯片的用途

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