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WO2022111613A1 - 基底及其制备方法以及基底的用途 - Google Patents

基底及其制备方法以及基底的用途 Download PDF

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WO2022111613A1
WO2022111613A1 PCT/CN2021/133358 CN2021133358W WO2022111613A1 WO 2022111613 A1 WO2022111613 A1 WO 2022111613A1 CN 2021133358 W CN2021133358 W CN 2021133358W WO 2022111613 A1 WO2022111613 A1 WO 2022111613A1
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Prior art keywords
polymer
peg
alkyl
substrate
independently selected
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PCT/CN2021/133358
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English (en)
French (fr)
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WO2022111613A9 (zh
Inventor
林志峰
孙雷
王�琦
冯叠文
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深圳市真迈生物科技有限公司
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Priority to EP21897113.3A priority Critical patent/EP4253434A4/en
Priority to US18/037,423 priority patent/US20230406983A1/en
Publication of WO2022111613A1 publication Critical patent/WO2022111613A1/zh
Publication of WO2022111613A9 publication Critical patent/WO2022111613A9/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
    • C08F283/06—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals
    • C08F283/065—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals on to unsaturated polyethers, polyoxymethylenes or polyacetals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3234—Inorganic material layers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3278—Polymers being grafted on the carrier
    • 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
    • B01L3/502707—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 manufacture of the container or its components
    • 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
    • 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
    • C03C17/32—Surface treatment of glass, not in the form of fibres or filaments, by coating with organic material with synthetic or natural resins
    • 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
    • C03C17/34—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions
    • C03C17/3405—Surface treatment of glass, not in the form of fibres or filaments, by coating with at least two coatings having different compositions with at least two coatings of organic materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals
    • C08F220/325—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals containing glycidyl radical, e.g. glycidyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/56—Acrylamide; Methacrylamide
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/52—Amides or imides
    • C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D133/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Coating compositions based on derivatives of such polymers
    • C09D133/04—Homopolymers or copolymers of esters
    • C09D133/06—Homopolymers or copolymers of esters of esters containing only carbon, hydrogen and oxygen, the oxygen atom being present only as part of the carboxyl radical
    • C09D133/062—Copolymers with monomers not covered by C09D133/06
    • C09D133/068—Copolymers with monomers not covered by C09D133/06 containing glycidyl groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D133/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Coating compositions based on derivatives of such polymers
    • C09D133/24—Homopolymers or copolymers of amides or imides
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • B01L2200/12—Specific details about manufacturing devices
    • 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
    • B01L2300/12—Specific details about materials

Definitions

  • the present invention relates to the field of surface treatment and biomolecule detection, in particular to a substrate with a polymer coating on the surface, a method for preparing a substrate with a polymer coating on the surface, and the use of the substrate.
  • Biochips/arrays can be made by modifying the surface of substrates and incorporating polymer coatings.
  • Surface modification technology from two-dimensional modification, to 2.5-dimensional modification, to three-dimensional modification, can greatly increase the density of functional groups contained on the surface.
  • the increased density of functional groups can make the biochip suitable for high-throughput testing, such as hybridization, amplification, and sequencing on the chip surface.
  • the present invention aims to solve at least one of the above technical problems at least to a certain extent or to provide an optional technical solution.
  • embodiments of the present invention provide a substrate having a surface comprising a polymer coating covalently attached thereto, the polymer coating comprising a polymer comprising a polymer as described in Formula I Repeating unit A shown and repeating unit B as shown in formula II or formula III:
  • X is selected from -O- or -NH-
  • R 01 ', R 01 " and R 01 "' are each independently selected from -H or C1-C3 alkyl
  • R 0 is selected from C1-C10 alkyl or -(C1-C5 alkyl)-NH-NHS-PEG4, and R 0 contains at least one R 02 substitution, and at least one R 02 substitution is independently selected from epoxy, amino or azido,
  • R 03 ', R 03 “, R 03 “', R 03 “”, R 04 ', R 04 “, R 04 “', R 05 ', R 05 “ and R 05 “' are each independently selected from -H , C1-C3 alkyl, amide or ester group,
  • L 1 is selected from C1-C3 alkylene or -C(O)-R 06 -C(O)-,
  • R 06 is selected from PEG or alkyldiamine, and when R 06 is selected from PEG, the molecular weight of PEG is 200-2000.
  • the polymer-coated surface has high density and uniformly distributed functional groups, high functional group reactivity, stable performance and controllable reaction characteristics, which can meet the needs of a large number of loaded biomolecules and high-pass biomolecules. Quantitatively detect the needs of application scenarios.
  • Embodiments of the present invention also provide a method of making a substrate having a surface coated with a polymer, comprising: contacting a polymer with the surface to attach the polymer to the surface, the polymer comprising, for example, The repeating unit A shown in formula I and the repeating unit B shown in formula II or formula III:
  • X is selected from -O- or -NH-
  • R 01 ', R 01 " and R 01 "' are each independently selected from -H or C1-C3 alkyl
  • R 0 is selected from C1-C10 alkyl or -(C1-C5 alkyl)-NH-NHS-PEG4, and R 0 contains at least one R 02 substitution, and at least one R 02 substitution is independently selected from epoxy, amino or azide, where PEG4 refers to HO(CH 2 CH 2 O) nH where n is 4;
  • R 03 ', R 03 “, R 03 “', R 03 “”, R 04 ', R 04 “, R 04 “', R 05 ', R 05 “ and R 05 “' are each independently selected from -H , C1-C3 alkyl, amide or ester group,
  • L 1 is selected from C1-C3 alkylene or -C(O)-R 06 -C(O)-,
  • R 06 is selected from PEG or alkyldiamine, and when R 06 is selected from PEG, the molecular weight of PEG is 200-2000.
  • Embodiments of the present invention also provide the use of the substrate of any of the above embodiments or the substrate prepared by the method of any of the above embodiments in the capture and/or detection of biomolecules.
  • the surface provided by the above embodiment includes a polymer coating, the polymer is composed of a specific repeating unit A and a repeating unit B, and the polymer-coated surface has the characteristics of high functional group density, uniform distribution and high functional group reactivity , thereby enabling the surface to be loaded with biocomponents/biomolecules at a high density, meeting the evolving demands of biomolecule preparation and/or analysis.
  • the polymer on the surface has good stability and is relatively insensitive to air, which facilitates the fabrication of the substrate or the surface treatment, and has strong industrial practicability.
  • the chip of any of the above-mentioned embodiments or the chip prepared by the method of any of the above-mentioned embodiments has a surface with high biochemical reactivity and uniform modification, which is beneficial to control the subsequently loaded oligonucleotides directly or indirectly connected to the polymer. Sequence (primer or probe) and/or amount and/or density of nucleic acid molecules to be detected.
  • the chip is especially suitable for applications with high throughput requirements and stable and controllable surface properties.
  • the substrate with the above-mentioned surface properties is suitable for a sequencing platform based on chip detection and using the principle of Sequencing by Synthesis (SBS) to realize sequencing, such as a single molecule sequencing platform or a high-throughput sequencing platform, specifically, for example, to be tested.
  • Nucleic acid molecules are connected to the surface for direct single-molecule detection without amplification.
  • nucleic acid molecules to be tested are connected to the surface and amplified into clusters (amplified signals) on the surface before detection; the substrate/chip is suitable for detection. Equipped with the current mainstream sequencing platforms based on the SBS principle, such as ILLUMINA, BGI and other sequencing platforms.
  • chips with stable and consistent surface properties can be easily and controllably prepared in batches, which has strong industrial practicability.
  • FIG. 1 is a schematic flow chart of preparing a chip according to an embodiment of the present invention
  • Fig. 2 is the GPC molecular weight test chart of the polymer prepared in Example 1 of the present invention.
  • Fig. 3 is the light intensity diagram of the surface quality detection of the chip prepared in Example 1 of the present invention.
  • Fig. 4 is the light intensity diagram of the biological application of the chip prepared in Example 1 of the present invention.
  • Fig. 5 is the GPC molecular weight test chart of the polymer prepared in Example 2 of the present invention.
  • Fig. 6 is the light intensity diagram of the surface quality detection of the chip prepared in Example 2 of the present invention.
  • Fig. 7 is the light intensity diagram of the biological application of the chip prepared in Example 2 of the present invention.
  • Fig. 8 is the GPC molecular weight test chart of the polymer prepared in Example 3 of the present invention.
  • Example 9 is a schematic diagram of nuclear magnetic detection of azide-based macromolecules in Example 3 of the present invention.
  • Fig. 10 is the light intensity diagram of the surface quality detection of the chip prepared in Example 3 of the present invention.
  • Example 11 is a light intensity diagram of the biological application of the chip prepared in Example 3 of the present invention.
  • Fig. 12 is the GPC molecular weight test chart of the polymer prepared in Example 4 of the present invention.
  • Example 13 is a light intensity diagram of the surface quality detection of the chip prepared in Example 4 of the present invention.
  • Example 14 is a light intensity diagram of the biological application of the chip prepared in Example 4 of the present invention.
  • Fig. 15 is the GPC molecular weight test chart of the polymer prepared in Example 5 of the present invention.
  • Example 16 is a light intensity diagram of the surface quality detection of the chip prepared in Example 5 of the present invention.
  • Example 17 is a light intensity diagram of the biological application of the chip prepared in Example 5 of the present invention.
  • Figure 18 is a molecular structure diagram of the library in Example 6 of the present invention.
  • 19 to 23 are the fluorescence intensity detection diagrams of the chips prepared in Examples 1 to 5 of the present invention, wherein the left picture is the original picture, and the right picture is the picture processed by ImageJ binarization.
  • the chip referred to herein includes a substrate with a surface comprising a polymer coating, and also includes a substrate with biomolecules attached to the polymer coating.
  • the material of the substrate is not particularly limited, for example, at least one selected from glass, silicon wafer, plastic, gel, and nylon film. Substrates, chips and biochips can be used interchangeably unless otherwise stated.
  • connection should be understood in a broad sense, for example, it may be directly connected, or indirectly connected through an intermediate medium, and may be chemically connected or physically connected. Unless otherwise expressly defined, in the descriptions of connection relationships involving compounds, biomolecules, functional groups, groups, etc. herein, connection generally refers to chemical connection, such as binding by covalent bonds, based on van der Waals forces, or electrostatic interactions adsorption, etc. For those skilled in the art, the specific meanings of the above terms in this document can be understood according to specific situations.
  • the polymer referred to herein refers to a high molecular compound, the molecular weight is usually greater than 1000, and sometimes it is also called a high polymer.
  • a polymer as used herein is the formation of a plurality of monomer molecules through a polymerization reaction such as addition polymerization.
  • the functional groups, functional groups, and reactive groups referred to herein can be used interchangeably, and represent groups that make the compound have certain or certain characteristics.
  • grafted on and “modified with" described herein may refer to direct grafting and modification on the object, or may refer to indirect grafting or modification, such as through other transition groups or structures. on the object.
  • attachment herein includes grafting, immobilization, binding, etc.; and, herein, grafting, immobilization, binding and covalent attachment/covalent attachment (covalent attachment) are used interchangeably herein.
  • alkyl refers to a saturated hydrocarbon containing primary (normal) carbon atoms, or secondary carbon atoms, or tertiary carbon atoms, or quaternary carbon atoms, or a combination thereof.
  • C 1 -C 10 alkyl refers to an alkyl group containing 1 to 10 carbon atoms, and each occurrence may independently be C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, C 9 alkyl, C 10 alkyl.
  • alkyl groups include but are not limited to: methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH) 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH ( CH3 ) CH2CH3 ), 2 -methyl-2-propyl (t-Bu, t-butyl, -C( CH3 ) 3 ), 1-pentyl (n-pentyl, -CH2 CH2CH2CH2CH3 ), 2 -pentyl (n
  • Epoxy refers to a group comprising the structure -X-CH(O)CH-X, where each X is independently H or an alkyl group; epoxy groups include but are not limited to -CH(O)CH 2 , -CH(O)CH(alkyl).
  • Amino refers to a derivative of ammonia, which is a group comprising the structure -N(X) 2 , where each X is independently H or an alkyl group.
  • Amino groups include but are not limited to -NH2 , -N(alkyl) 2 , -NH(alkyl), -N(cycloalkyl) 2 , -NH(cycloalkyl), -N(heterocyclyl) 2 , -NH(heterocyclyl), -N(aryl) 2 , -NH(aryl), -N(alkyl)(aryl), -N(alkyl)(heterocyclyl), -N(ring alkyl)(heterocyclyl), -N(aryl)(heteroaryl), and -N(alkyl)(heteroaryl).
  • Ester group refers to a group comprising -C(O)OX structure, wherein X is an alkyl group; ester group includes but is not limited to -C(O)OCH 3 , -C(O)OCH 2 CH 3 .
  • “Amido” refers to a group comprising -C(O)N(X) 2 structure, wherein each X is independently H or alkyl; amide groups include but are not limited to -C(O)NH 2 , - C(O)NH(alkyl), -C(O)N(alkyl) 2 .
  • Biomolecules or biological components referred to herein include nucleic acids and/or proteins.
  • Nucleic acid can be DNA, cDNA, RNA or RNA-DNA complex, and can be double-stranded or single-stranded.
  • Probes/primers as referred to herein are nucleic acid molecules of known sequence that can be used to capture target nucleic acid sequences, or as primers to amplify target nucleic acid sequences. It can be DNA and/or RNA, etc., and is usually an oligonucleotide chain with a length of less than 150 nt. By structurally and/or chemically treating the substrate surface, the probes attached to the surface can be randomly or regularly distributed.
  • a specific embodiment of the present invention provides a substrate, which has a surface, the surface comprises a polymer coating covalently linked thereto, the polymer coating comprises a polymer, and the polymer comprises a repeating unit A as shown in formula I and a Repeating unit B represented by formula II or formula III:
  • X is selected from -O- or -NH-;
  • R 01 ', R 01 " and R 01 "' are independently selected from -H or C1-C3 alkyl;
  • R 0 is selected from C1-C10 alkyl or - (C1-C5 alkyl)-NH-NHS-PEG4, and
  • R 0 contains at least one R 02 substitution, and at least one R 02 substitution is each independently selected from epoxy, amino or azide, where PEG4 refers to HO ( CH 2 CH 2 O) n in H is 4;
  • R 03 ', R 03 '', R 03 '', R 03 '', R 03 '', R 04 ', R 04 '', R 04 '', R 05 ', R 05 " and R 05 "' are independently selected from -H, C1-C3 alkyl, amide or ester group;
  • L 1 is selected from C1-C3 alkylene or -C(O)-R 06
  • R 06 is selected from PEG, and the molecular weight of PEG is 200-2000, preferably 500-1000.
  • the polymer comprises a structure of Formula III, Formula IV, or Formula V:
  • the polymer coating comprises a polymer of Formula VI or Formula VII:
  • n is selected from an integer in the range of 1-2000
  • n is selected from an integer in the range of 1-3000
  • m1 and m2 are independently selected from an integer in the range of 1-2000
  • n1 or n2 is selected from an integer in the range of 1-1500 and n1 is equal to n2
  • R 06 is selected from PEG
  • the molecular weight of PEG is less than 1000, preferably the molecular weight of PEG is 500-1000.
  • the polymer coating comprises the polymer of formula VI, and the ratio of m to n is 1:1 to 1:30; preferably, 1:2 to 1:30; more preferably, 1:1: 10 ⁇ 1:20.
  • the polymer coating comprises a polymer represented by formula VII, and the ratio of the sum of m1 and m2 to n1 or n2 is 1:1 to 1:30; preferably, 1:2 to 1:30; More preferably, 1:10 ⁇ 1:20.
  • the molecular weight of the polymer is 1-200,000. Polymers with molecular weights in this range can be attached to the surface so that the surface has the desired and stable and controllable properties. Regarding the effect of polymer molecular weight on surface properties, for polymers in this molecular weight range formed by polymerization of the same proportion of monomers, based on the knowledge of the polymer, the morphology/structure of the polymer on the surface and according to the test Experience, the inventors guessed that a polymer with a relatively high molecular weight such as 180,000 would give the surface more superior properties than a polymer with a relatively low molecular weight such as 20,000, such as a larger amount of functional groups, a more uniform distribution, etc. However, unexpectedly, , the same probes were connected to the two surfaces coated with polymers with these two molecular weights, and it was found that the properties of the surfaces coated with polymers with these two molecular weights were equivalent, and there was no significant difference.
  • the polymer coating is attached to the surface by covalent bonding with reactive groups on the surface selected from the group consisting of amino, epoxy, alkynyl, cyano, vinyl, and propenyl. at least one.
  • the reactive groups are amino groups and the polymers contain epoxy groups. At least a portion of the amino groups react with at least a portion of the epoxy groups on the polymer to covalently bond the polymer to the surface through reactive groups.
  • the reactive group is an epoxy group and the polymer contains -NH2 . At least a portion of the epoxy groups react with at least a portion of -NH2 on the polymer to covalently bond the polymer to the surface through reactive groups.
  • the reactive group is one of alkynyl, cyano, vinyl, and propenyl
  • the polymer contains -N3 . At least a portion of the designated reactive groups react with at least a portion of the N3 on the polymer such that the polymer is covalently bound to the surface through the reactive groups.
  • X is selected from -O-.
  • R 01 ', R 01 “ and R 01 “' are each independently selected from -H or methyl, and R 0 is selected from C1-C3 alkyl; and/or R 03 ', R 03 “, R 03 “', R 03 “", R 04 ', R 04 “, R 04 “', R 05 ', R 05 “, R 05 “' are each independently selected from -H, methyl, -C (O)NH 2 or -C(O)OCH 3 , L 1 is selected from -C(O)-PEG-C(O)- or -C(O)-NH-CH 2 -NH-C(O)- .
  • repeating unit A is selected from one of the following structures:
  • repeating unit B is selected from one of the following structures:
  • the molecular weight of the PEG is 200-2000, preferably 500-1000.
  • the polymer comprises one of the following structures:
  • the polymer coating comprises one of the following polymers:
  • p is selected from an integer in the range of 1 to 3000
  • p1 and p2 are independently selected from an integer in the range of 1 to 1300 and p1 is equal to p2
  • q, q1 and q2 are each independently selected from an integer in the range of 1 to 2000 .
  • X is selected from -NH-.
  • repeating unit B is shown in formula III.
  • R 01 ', R 01 “ and R 01 “' are each independently selected from -H or methyl, and R 0 is selected from C1-C3 alkyl or -(C1-C5 alkyl)-NH-NHS -PEG4 ; and/or R04 ', R04 “, R04 “', R05', R05 “ and R05 “' are each independently selected from -H, methyl, -C(O) NH2 or -C(O)OCH 3 , L 1 is selected from -C(O)-PEG-C(O)- or -C(O)-NH-CH 2 -NH-C(O)-, L 1 is selected from - In the case of C(O)-PEG-C(O)-, the molecular weight of PEG is 200-2000, preferably 500-1000.
  • repeating unit A is selected from one of the following structures:
  • PEG4 means that n in HO(CH 2 CH 2 O)nH is 4.
  • repeating unit B is selected from one of the following structures:
  • the polymer comprises one of the following structures:
  • the polymer coating comprises one of the following polymers:
  • p3 and p4 are independently selected from integers in the range of 1 to 1300 and p3 is equal to p4, q3 and q4 are independently selected from integers in the range of 1 to 500, and q5 and q6 are independently selected from the range of 1 to 1500. Integer inside.
  • repeating unit B is as shown in formula II.
  • the repeating unit B is selected from one of the following structures:
  • R 0 is selected from -(C1-C5 alkyl)-NH-NHS-PEG4-N 3 .
  • PEG4 means that n in HO(CH 2 CH 2 O)nH is 4.
  • repeating unit A is
  • the polymer includes one of the following structures:
  • the polymer coating includes one of the following polymers:
  • p7 is selected from the integer in the range of 1-3000, and q7 is selected from the integer in the range of 1-500.
  • monomer A of formula VIII corresponding to repeating unit A and monomer B of formula IX or formula X corresponding to repeating unit B are polymerized to obtain a polymer, polymerized
  • the reaction is carried out in the presence of an initiator,
  • X is selected from -O- or -NH-
  • R 01 ', R 01 " and R 01 "' are independently selected from -H or C1-C3 alkyl
  • R 0 is selected from C1-C10 alkyl or -(C1 -C5 alkyl)-NH-NHS-PEG4, and R 0 contains at least one R 02 substitution, and at least one R 02 substitution is each independently selected from epoxy, amino or azide, where PEG4 refers to HO(CH 2 n in CH2O)nH is 4 ;
  • R03 ', R03 ", R03 "', R03 "", R04 ', R04 “, R04 “', R05 ', R05 “ and R 05 “' is independently selected from -H, C1-C3 alkyl group, amide group or ester group
  • L 1 is selected from C1-C3 alkylene group or -C(O)-R 06 -C(O)-
  • R 06 is selected from P
  • R 06 is PEG
  • the molecular weight of PEG is 200-2000, preferably the molecular weight of PEG is 500-1000.
  • the molar ratio of monomer A and monomer B in the polymerization reaction is selected from a value in the range of 1:1 to 1:30. Preferably, it is selected from a value within the range of 1:2 to 1:30; more preferably, it is selected from a value within the range of 1:10 to 1:20.
  • the surface also has biomolecules attached to the surface that are covalently bound to the polymer.
  • the biomolecule is selected from at least one of proteins and nucleic acids.
  • the biomolecule is, for example, a probe, and the end of the probe is immobilized on the surface with a polymer-binding group; further, the probe can hybridize to a target nucleic acid molecule, and the biomolecule can also include a target nucleic acid Molecules or probe-target nucleic acid molecule complexes.
  • Another specific embodiment of the present invention provides a method for preparing a substrate with a polymer coating on the surface, and the method can be used for the preparation of the substrate in any of the above embodiments or examples.
  • the method includes contacting a polymer with a surface to attach the polymer to the surface, the polymer comprising a repeating unit A as shown in Formula I and a repeating unit B as shown in Formula II or Formula III:
  • X is selected from -O- or -NH-
  • R 01 ', R 01 " and R 01 "' are independently selected from -H or C1-C3 alkyl
  • R 0 is selected from C1-C10 alkyl or - (C1-C5 alkyl)-NH-NHS-PEG4, and R 0 comprises at least one R 02 substitution, and at least one R 02 substitution is each independently selected from epoxy, amino or azide
  • R 03 ', R 03 ", R 03 “', R 03 “", R 04 ', R 04 “, R 04 “', R 05 ', R 05 “ and R 05 “' are each independently selected from -H, C1-C3 alkyl , amide group or ester group
  • L 1 is selected from C1-C3 alkylene or -C(O)-R 06 -C(O)-
  • R 06 is selected from PEG or alkyldiamine.
  • the polymer comprises a structure of Formula III, Formula IV, or Formula V:
  • the polymer coating comprises a polymer of Formula VI or Formula VII:
  • n is selected from an integer in the range of 1-2000
  • n is selected from an integer in the range of 1-3000
  • m1 and m2 are independently selected from an integer in the range of 1-2000
  • n1 or n2 is selected from an integer in the range of 1-1500 and n1 is equal to n2
  • R 06 is selected from PEG
  • the molecular weight of PEG is 200-2000, preferably 500-1000.
  • the polymer coating comprises the polymer of formula VI, and the ratio of m to n is 1:1 to 1:30; preferably, 1:2 to 1:30; more preferably, 1:1: 10 ⁇ 1:20.
  • the polymer coating comprises a polymer represented by formula VII, and the ratio of the sum of m1 and m2 to n1 or n2 is 1:1 to 1:30; preferably, 1:2 to 1:30; More preferably, 1:10 ⁇ 1:20.
  • the molecular weight of the polymer is 1-200,000.
  • X is selected from -O-.
  • R 01 ', R 01 “ and R 01 “' are each independently selected from -H or methyl, and R 0 is selected from C1-C3 alkyl; and/or R 03 ', R 03 “, R 03 “', R 03 “", R 04 ', R 04 “, R 04 “', R 05 ', R 05 “, R 05 “' are each independently selected from -H, methyl, -C(O) NH2 or -C(O) OCH3 , L1 is selected from -C(O) -PEG -C(O)- or -C(O)-NH- CH2 -NH-C(O)-.
  • repeating unit A is selected from one of the following structures:
  • repeating unit B is selected from one of the following structures:
  • the molecular weight of PEG is 200-2000, for example, not more than 1000, preferably 500-1000.
  • the polymer comprises one of the following structures:
  • the polymer comprises one of the following polymers:
  • p is selected from an integer in the range of 1 to 3000
  • p1 and p2 are independently selected from an integer in the range of 1 to 1300 and p1 is equal to p2
  • q, q1 and q2 are each independently selected from an integer in the range of 1 to 2000 .
  • X is selected from -NH-.
  • the repeating unit B is shown in formula III.
  • repeating unit A is selected from one of the following structures:
  • repeating unit B is selected from one of the following structures:
  • the polymer comprises one of the following structures:
  • the polymer comprises one of the following polymers:
  • p3 and p4 are independently selected from integers in the range of 1 to 1300 and p3 is equal to p4, q3 and q4 are independently selected from integers in the range of 1 to 500, and q5 and q6 are independently selected from the range of 1 to 1500. Integer inside.
  • repeating unit B is shown in formula II.
  • repeating unit B is selected from one of the following structures:
  • R 0 is selected from -(C1-C5 alkyl)-NH-NHS-PEG4-N 3 .
  • repeating unit A is
  • the polymer includes one of the following structures:
  • the polymer includes one of the following polymers: Wherein, p7 is selected from the integer in the range of 1-3000, and q7 is selected from the integer in the range of 1-500.
  • the method includes: modifying the surface to have reactive groups on the surface, the reactive groups being selected from at least one of amino, epoxy, alkynyl, cyano, vinyl, and propenyl; The polymer is brought into contact with the surface to covalently bind the polymer to the reactive groups for attachment to the surface.
  • the reactive group is an amino group
  • the polymer contains epoxy groups, and at least a portion of the epoxy groups react with at least a portion of the amino groups to attach the polymer to the surface.
  • the reactive groups are epoxy groups
  • the polymer contains amino groups
  • at least a portion of the amino groups react with at least a portion of the epoxy groups to attach the polymer to the surface.
  • the reactive group is selected from one of alkynyl, cyano, vinyl, and propenyl
  • the polymer comprises -N3 , and at least a portion of the -N3 reacts with at least a portion of the reactive group to polymerize attached to the surface.
  • the surface is treated with a silane coupling agent selected from the group consisting of 3-aminopropyltrimethoxysiloxane, 3-(triethoxysilyl )propyl]carbamate 2-propynyl, 4-(triethoxy)silylbutyronitrile and gamma-ylbutyronitrile and at least one of alkylglycidoxy)propyltrimethoxysilane.
  • a silane coupling agent selected from the group consisting of 3-aminopropyltrimethoxysiloxane, 3-(triethoxysilyl )propyl]carbamate 2-propynyl, 4-(triethoxy)silylbutyronitrile and gamma-ylbutyronitrile and at least one of alkylglycidoxy)propyltrimethoxysilane.
  • the reaction is performed at 40°C to 50°C for 1 h to 8 h to covalently bind the polymer to the reactive group for attachment to the surface.
  • it also includes covalently binding the biomolecule to the polymer for attachment to the surface.
  • the biomolecule is selected from at least one of proteins and nucleic acids.
  • the reaction is performed at 50°C to 60°C for 0.5 to 5 h to covalently bind the biomolecule to the polymer.
  • the polymer contains an epoxy group and the biomolecule is modified with an amino group at at least one end.
  • the polymer contains an amino group and the biomolecule has an NHS on at least one end.
  • the polymer comprises -N3 and the biomolecule bears DBCO on at least one end.
  • Particular embodiments of the present invention also provide the use of the substrate of any of the above embodiments or examples for biomolecule capture and/or detection.
  • An embodiment of the present invention provides a polymer; the polymer includes a repeating unit A and a repeating unit B; the repeating unit A has the following structural features:
  • X is selected from -O- or -NH-, R 01 ', R 01 ", R 01 “' are independently selected from: -H or C1-C3 alkyl; R 0 is selected from C1-C10 alkyl or -C1 -C5 alkyl-NH-NHS-PEG4, and R 0 is substituted by at least one R 02 ; R 02 is independently selected from: epoxy, amino or azide; the repeating unit B has the following structural features : R 03 ', R 03 “, R 03 “', R 03 “”, R 04 ', R 04 “, R 04 “', R 05 ', R 05 “, R 05 “' are each independently selected from:- H, C1-C3 alkyl, amide or ester group, L 1 is selected from C1-C3 alkylene or -C(O)-R 06 -C(O)-; R 06 is selected from PEG or alkyldiamine .
  • X is selected from -O-.
  • R 01 ', R 01 ", R 01 “' are each independently selected from: -H or methyl; R 0 is selected from C1-C3 alkyl.
  • R 03 ', R 03 “, R 03 “', R 03 “”, R 04 ', R 04 “, R 04 “', R 05 ', R 05 “, R 05 “' are independently selected from : -H, methyl, -C(O)NH 2 or -C(O)OCH 3
  • L 1 is selected from -C(O)-PEG-C(O)- or -C(O)-NH-CH 2 -NH-C(O)-.
  • repeating unit A is selected from one of the repeating units shown below:
  • repeating unit B is selected from one of the repeating units shown below:
  • X is selected from -NH-.
  • R 04 ', R 04 ", R 04 “', R 05 ', R 05 “, R 05 “' are each independently selected from: -H, C1-C3 alkyl group, amide group or ester group, L 1 is selected from C1-C3 alkylene or -C(O)-R 06 -C(O)-; R 06 is selected from PEG or alkyldiamine.
  • R 01 ', R 01 ", R 01 “' are each independently selected from: -H or methyl; R 0 is selected from C1-C3 alkyl or -(C1-C5 alkyl)-NH-NHS- PEG4.
  • R 04 ', R 04 “, R 04 “', R 05 ', R 05 “, R 05 “' are each independently selected from: -H, methyl, -C(O)NH 2 or -C (O) OCH3
  • L1 is selected from -C(O) -PEG -C(O)- or -C(O)-NH- CH2 -NH-C(O)-.
  • repeating unit A is selected from one of the repeating units shown below:
  • repeating unit B is selected from one of the repeating units shown below:
  • repeating unit B has the following structural features: R 03 ', R 03 “, R 03 “', R 03 “” are each independently selected from: -H, C1-C3 alkyl, amide or ester.
  • repeating unit B is one of the repeating units shown below:
  • R 0 is selected from -(C1-C5 alkyl)-NH-NHS-PEG4-N 3 .
  • the repeating unit A is
  • the molecular weight of the polymer is 10,000 to 120,000.
  • Embodiments of the present invention also provide a method for preparing the above polymer, which includes the following steps: performing a copolymerization reaction with monomer A and monomer B to prepare the polymer; the monomer A forms the repeating unit A, The monomer B forms the repeating unit B.
  • the molar ratio of the monomer A and the monomer B is 1:(1-30). Specifically, the molar ratio of the monomer A and the monomer B is 1:1, 1:3, 1:5, 1:8, 1:10, 1:12, 1:15, 1:20, 1:1 25, 1:30.
  • the copolymerization reaction refers to the polymerization reaction of the monomer A and the monomer B under the initiation of an initiator at 30-60° C. (reaction temperature).
  • reaction temperature can adopt the following specific temperature values: 30°C, 35°C, 36°C, 37°C, 38°C, 40°C, 41°C, 42°C, 43°C, 45°C, 50°C, 52°C , 54°C, 55°C, 56°C, 58°C, 60°C.
  • the initiator is selected from at least one of azobisisobutyronitrile (AIBN) and potassium persulfate (KPS).
  • AIBN azobisisobutyronitrile
  • KPS potassium persulfate
  • the polymerization reaction is terminated with oxygen.
  • the product obtained from the polymerization reaction is extracted with methanol, and then dried to prepare the polymer.
  • An embodiment of the present invention further provides a chip, the chip includes a substrate, and a polymer grafted on the surface of the substrate; the polymer is the polymer as described above.
  • the substrate is modified with active groups, and the polymer is grafted on the surface of the substrate through the active groups; the active groups are selected from amino groups, epoxy groups, At least one of alkynyl, cyano, vinyl and propenyl.
  • the biochip further includes a biocomponent, and the biocomponent is grafted to the polymer.
  • the biological component is selected from at least one of amino acid sequence and nucleotide sequence.
  • amino acid sequences include proteins, oligopeptides, polypeptides, etc.
  • nucleotide sequences include oligonucleotide sequences, polynucleotide sequences, and the like.
  • An embodiment of the present invention also provides a method for preparing a chip, comprising the following steps: obtaining a substrate, and grafting a polymer onto the surface of the substrate; the polymer is selected from the polymers in any of the above examples.
  • the preparation method of the chip does not require strict control of reaction conditions, and the process is simple and easy to control, which is beneficial to the popularization and application of the chip.
  • the active group is grafted on the surface of the substrate first, and then the polymer is grafted on the surface of the substrate through the active group; the active group is selected from At least one of amino, epoxy, alkynyl, cyano, vinyl and propenyl.
  • grafting the reactive group refers to using a silane coupling agent to react with the substrate;
  • the silane coupling agent is selected from 3-aminopropyltrimethoxysiloxane, 3-(Triethoxysilyl)propyl]carbamate, 2-propynyl, 4-(triethoxy)silylbutyronitrile and gamma butyronitrile and alkylglycidoxy)propyltrimethoxy at least one of the base silanes.
  • the grafting of the polymer on the surface of the substrate through the active group refers to the reaction at 40-50° C. for 1-8 hours (reaction temperature*time).
  • reaction temperature*time may adopt the following combinations: 40°C*4h, 43°C*4h, 44°C*4h, 45°C*4h, 46°C*4h, 47°C*4h, 50°C*4h, 45°C °C*2h, 45°C*6h, 45°C*1h, 45°C*8h.
  • grafting the reactive group means that the silane coupling agent reacts with the substrate at 20-30° C. for 1-5 hours.
  • the preparation method may further include the following step: grafting a biological component to the high polymer.
  • the biological component is selected from at least one of amino acid sequence and nucleotide sequence.
  • amino acid sequences include proteins, oligopeptides, polypeptides, etc.
  • nucleotide sequences include oligonucleotide sequences, polynucleotide sequences, and the like.
  • the grafting of the biological component to the high polymer refers to a reaction at 50° C. ⁇ 60° C. for 0.5 ⁇ 5 h (reaction temperature*time).
  • reaction temperature*time may adopt the following combinations: 55°C*0.5h, 55°C*1h, 55°C*2h, 55°C*3h, 55°C*5h, 54°C*1h, 56°C*1h, 52°C*1h, 50°C*1h, 58°C*1h, 60°C*1h.
  • the embodiments of the present invention provide applications of the above polymers, or the above chips, or the chips prepared by the above preparation methods in biomolecule preparation or biomolecule analysis.
  • T20 ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt (SEQ ID NO: 1); Pe: caacaacaacaacaacaacaacaacaacaacaacaacaacaacaa (SEQ ID NO: 2); RD: ctgccccgggttcctcattc tat (SEQ ID NO: 3).
  • the substrate is made of glass, and the glass is subjected to surface amination coating by solution method.
  • the coating method is as follows:
  • APTMS aminopropyltrimethoxysilane
  • the glass after the amination coating was placed in a 0.1 wt % isopropanol solution of epoxy macromolecules, and reacted at 45° C. for 4 hours to obtain a surface grafted with epoxy macromolecules.
  • NH 2 -CY3 (purchased from Xi'an Kaixin Biotechnology Co., Ltd.) was used to detect the glass surface quality of modified epoxy macromolecules.
  • the specific detection process is as follows:
  • the NH2-CY3 solution with a concentration of 20 ⁇ M was poured into the surface, and after reacting at 55 °C for 1 h, under a fluorescence microscope, a 532 nm band laser was used to take pictures according to the parameters of 35 mW and 60 ms to detect the light intensity.
  • the light intensity detection results are shown in Figure 3.
  • the average brightness of the obtained surface is 21919a.u. through ImageJ signal statistics.
  • Oligonucleotide connection (a) The substrate modified with macromolecules adopts microchannel packaging technology, such as surface modification packaging, thermocompression bonding packaging or anodic bonding microchannel packaging technology.
  • the surface forms microfluidic channels.
  • a 5 ⁇ M solution of NH 2 -oligo(NH 2- A 30 ) was added to the microfluidic channel, and the reaction was carried out at 37° C. for 24 hours, so that the surface was grafted with a high-density oligo (oligonucleotide) sequence, where A 30 represented 30 adenines Oligonucleotide chains formed from nucleotides.
  • the substrate is made of glass, and the glass is subjected to surface amination coating by solution method.
  • the coating method is as follows:
  • the glass was placed in a 5% APTMS (aminopropyltrimethoxysilane) ethanol solution, reacted at 25° C. for 2 hours, then taken out and baked in an oven at 130° C. for 2 hours to complete the amination of the glass surface.
  • APTMS aminopropyltrimethoxysilane
  • the glass after amination coating was placed in a 0.1 wt % isopropanol solution of epoxy macromolecules, and reacted at 45° C. for 4 hours to obtain a surface modified with epoxy macromolecules.
  • NH 2 -CY3 (purchased from Xi'an Kaixin Biotechnology Co., Ltd.) was used to detect the glass surface quality of modified epoxy macromolecules.
  • the specific detection process is as follows:
  • the NH 2 -CY3 solution with a concentration of 20 uM was poured into the surface and reacted at 55 °C for 1 h.
  • the 532 nm band laser was used to take pictures according to the parameters of 35 mW and 60 ms to detect the light intensity.
  • the light intensity detection results are shown in Figure 6, and the average signal intensity obtained by ImageJ software analysis is 13768a.u.
  • the substrate modified with macromolecules is used to form microfluidic channels on the surface of the substrate by microfluidic packaging technology.
  • 5uM NH 2 -oligo (NH 2 -T 35 ) solution was added to the microfluidic channel, and the reaction was carried out at 37°C for 24 hours, so that the surface was grafted with a high-density oligo (oligonucleotide) sequence, where T 35 represented 35 thymus Oligonucleotide chains formed from pyrimidine nucleotides.
  • the NMR test is shown in Figure 9.
  • the H absorption peaks of saturated chain scission after olefin polymerization appear at 1.43, 1.55, and 2.09, and the H absorption peaks on CH 2 in the intramolecular diamine appear in the range of 3.04-3.41, around 3.93.
  • the formation of azide macromolecules is evidenced by the appearance of H absorption peaks within the PEG chain scission adjacent to the azide.
  • the substrate is made of glass, and the glass is coated by solution method on the surface.
  • the coating method is as follows:
  • the glass was placed in a 1% solution of 2-propynyl 3-(triethoxysilyl)propyl]carbamate in toluene. After reacting at 25°C for 2 hours, the glass was taken out and baked in an oven at 130°C for 2 hours. Alkynylation of glass surfaces.
  • the alkynyl-coated glass was placed in an isopropanol solution of 0.1 wt % azide macromolecules, and reacted at 45° C. for 4 hours to obtain a surface grafted with azide macromolecules.
  • the DBCO-CY3 solution with a concentration of 20 ⁇ M was poured into the surface, reacted at 55°C for 1 h, and then irradiated with a 532 nm band laser under a fluorescence microscope, and photographed and detected the light intensity according to the parameters of 35 mW and 60 ms.
  • the light intensity detection results are shown in Figure 10, and were analyzed in ImageJ software to obtain a surface with an average signal intensity of 19464a.u.
  • the substrate modified with macromolecules is used to form microfluidic channels on the surface of the substrate by microfluidic packaging technology.
  • the substrate is made of glass, and the glass is coated with cyanide by solution method.
  • the coating method is as follows:
  • the glass was placed in a 1% solution of 4-(triethoxy)silylbutyronitrile in toluene, reacted at 25° C. for 2 hours, and then taken out of the glass and baked at 130° C. for 2 hours in an oven. Complete cyanation of glass surfaces.
  • the cyanide-coated glass was placed in an isopropanol solution of 0.1 wt % azide macromolecules and reacted at 45° C. for 4 hours to obtain a surface grafted with azide macromolecules.
  • DBCO-CY3 purchased from Xi'an Kaixin Biotechnology Co., Ltd. detects the glass surface quality of modified azide macromolecules. The specific detection process is as follows:
  • the DBCO-CY3 solution with a concentration of 20 ⁇ M was poured into the surface and reacted at 55°C for 1 h.
  • a 532 nm band laser was used to take pictures according to the parameters of 35 mW and 60 ms to detect the light intensity.
  • the light intensity detection results are shown in Figure 13, and the average signal intensity obtained by ImageJ software analysis is 28531a.u.
  • the substrate modified with macromolecules is used to form microfluidic channels on the surface of the substrate by microfluidic packaging technology.
  • a 5uM solution of DBCO-oligo (DBCO-T 35 ) was added to the microfluidic channel, and the reaction was carried out at 37° C. for 24 hours, so that the surface was grafted with a high-density oligo (oligonucleotide) sequence, where T 35 represented 35 thymine nuclei. Oligonucleotide chains formed from nucleotides.
  • the substrate is made of glass, and the surface of the glass is epoxidized by solution method.
  • the coating method is as follows:
  • the glass after epoxidation coating was placed in a 0.1 wt % solution of amino macromolecules in isopropanol, and reacted at 45° C. for 4 hours to obtain a surface grafted with azide macromolecules.
  • the NHS-CY3 solution with a concentration of 20 ⁇ M was poured into the surface, and after reacting at 55 °C for 1 h, under a fluorescence microscope, irradiated with a 532 nm band laser, and photographed according to the parameters of 35 mW and 60 ms to detect the light intensity.
  • the light intensity detection results are shown in Fig. 16, which were analyzed in ImageJ software, and the average signal intensity obtained was 25437a.u.
  • DNA library a DNA library with a fragment length of 150-300bp containing known sequences at both ends.
  • the molecular structure of the library is shown in Figure 18.
  • Insertion Insertion fragment, derived from phi-X174 standard strain; T20, Pe, and RD are SEQ respectively Sequences shown in ID NO: 1-3.
  • the DNA library was mixed with 52 ⁇ l volume of deionized water, 18 ⁇ l of 0.2M NaOH solution was added to it, mixed evenly, left to stand for denaturation at room temperature for 8 minutes, and then 20 ⁇ l volume of 400 mM Tris-HCl buffer pH 8.0 was added to terminate the reaction to obtain 100 ⁇ l volume of 100 pM denatured DNA library.
  • the denatured DNA library was diluted to 5 pM using pH 7.3 containing 3 ⁇ SSC (20 ⁇ SSC buffer diluted with nuclease-free water (Rnase-free water)) hybridization solution, and then passed into the chip channel at 42
  • the hybridization reaction was performed at °C for 30 minutes, and then a volume of 160-260 ⁇ l of washing reagent (5 ⁇ SSC, 0.05% Tween 20, pH 7.0) was circulated at a speed of 250 ⁇ l/min to complete the hybridization reaction.
  • extension buffer reagent (20 mM Tris, 10 mM ammonium sulfate, 2 mM magnesium sulfate, 1.5 M betaine, 1.3% dimethylmethylene) was circulated through the chip channel at a speed of 500 ⁇ l/min. Sulfone, 0.45M N-methylformamide, 1.5M carboxamide, 0.1% TritonX-100, pH 9.0);
  • extension reagent extension buffer reagent, 3 ⁇ g/ml Bst DNA polymerase, 200 ⁇ M dNTPs
  • extension buffer reagent 3 ⁇ g/ml Bst DNA polymerase, 200 ⁇ M dNTPs
  • the ILLUMINA sequencing platform operating manual can be used for amplification and clustering; the article Isothermal amplification method for next-generation sequencing (Zhaochun Ma, et.al, PNAS August 27, 2013 110(35)14320-14323, https:// doi.org/10.1073/pnas.1311334110) for amplification to generate DNA clusters.
  • the detection results of DNA clusters are shown in Figures and Table 1.
  • the chips corresponding to Figures 19 to 23 are the detection results of DNA clusters generated by the chips prepared in Examples 1-5, respectively.
  • the chip containing DNA clusters on the surface is loaded into a sequencing platform, such as the sequencing platform of ILLUMINA Company, for sequencing, and sequencing data of quality meeting specific application requirements can be obtained.
  • a sequencing platform such as the sequencing platform of ILLUMINA Company

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Abstract

本发明涉及一种基底及其制备方法及其应用。所称的基底，具有表面，该表面包含与之共价连接的聚合物包被，聚合物包被包含聚合物，聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：(I), (II), (III) 其中，X选自-O-或-NH-， R 01'、R 01"和R 01"'各自独立地选自-H或C1～C3烷基，R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，R 03'、R 03"、R 03"'、R 03""、R 04'、R 04"、R 04" '、R 05'、R 05"和R 05"'各自独立地选自-H、C1～C3烷基、酰胺基或酯基，L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，R 06选自PEG或烷基二胺。该表面能够更高密度地负载生物分子，满足不断发展的生物分子制备和/或分析需求，且稳定性好。

Description

基底及其制备方法以及基底的用途 技术领域
本发明涉及表面处理和生物分子检测领域，特别涉及一种表面具有聚合物包被的基底、一种制备表面具有聚合物包被的基底的方法以及基底的用途。
背景技术
本部分中讨论的主题不应仅因为是在本部分提及就被认为是现有技术。
生物芯片/阵列(array)可在基底的表面上进行修饰以及结合聚合物包被来制成。
表面修饰技术，从二维修饰，到2.5维度的修饰，再到三维修饰，可使得表面上包含的功能性基团的密度得到大幅度的提升。功能性基团的密度提升后，可使得该生物芯片适用于高通量测试，比如在芯片表面上进行杂交、扩增、测序等。
如何处理表面以使表面获得期望的参数或者使表面满足检测应用的要求，而且使表面性能稳定可控、易再现，是值得关注的问题。
申请内容
本发明旨在至少一定程度上解决上述技术问题至少之一或者提供一种可选择的技术方案。
为此，本发明的实施方式提供一种基底，其具有表面，该表面包含与之共价连接的聚合物包被，所述聚合物包被包含聚合物，所述聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
Figure PCTCN2021133358-appb-000001
其中，
X选自-O-或-NH-，
R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，
R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，
R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，
L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，
R 06选自PEG或烷基二胺，在R 06选自PEG的情况下，其中的PEG的分子量为200～2000。
具有该聚合物包被的表面，具有较高密度且均匀分布的官能团、且官能团反应活性高，还具有性能稳定和反应可控的特点，能够满足需要大量负载生物分子、对生物分子进行高通量检测应用情景的需求。
本发明的实施方式还提供一种制备表面具有聚合物包被的基底的方法，包括：使聚合物与所述表面接触，以使所述聚合物连接至所述表面，所述聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
Figure PCTCN2021133358-appb-000002
其中，
X选自-O-或-NH-，
R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，
R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，这里的PEG4指HO(CH 2CH 2O)nH中的n为4；
R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，
L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，
R 06选自PEG或烷基二胺，在R 06选自PEG的情况下，其中的PEG的分子量为200～2000。
本发明的实施方式还提供上述任一实施方式的基底或者利用上述任一实施方式的方法所制备得的基 底在生物分子捕获和/或检测中的用途。
上述实施方式提供的包含聚合物包被的表面，聚合物由特定的重复单元A和重复单元B组成，具有该聚合物包被的表面具有官能团密度较高、分布均匀且官能团反应活性高的特点，由此使得该表面能够高密度地负载生物组分/生物分子，满足不断发展的生物分子制备和/或分析的需求。另外，该表面上的聚合物的稳定性好，对空气相对不敏感，使该基底的制作或者表面的处理易于进行，具有较强的工业实用性。
上述任一实施方式的芯片或者通过上述任一实施方式的方法制备得的芯片，具有生化反应活性高且均匀修饰的表面，利于控制后续载入的、与该聚合物直接或间接连接的寡核酸序列(引物或探针)和/或待测核酸分子的量和/或密度。该芯片特别适用于通量要求高且需要表面性能稳定可控的应用情景。
具有上述表面特性的基底适用于基于芯片检测、利用边合成边测序(Sequencing by synthesis，SBS)原理实现测序的测序平台，如单分子测序平台或高通量测序平台，具体地，例如使待测核酸分子连接到该表面不经扩增直接进行单分子检测，又例如使待测核酸分子连接到该表面并在该表面上扩增成簇(放大信号)后再进行检测；该基底/芯片适配于当前市面上主流的基于SBS原理实现测序的平台，如ILLUMINA、华大基因等的测序平台。此外，利用上述实施方式的方法，容易可控地批量制备得表面性能稳定且较一致的芯片，具有较强的工业实用性。
附图说明
本发明实施方式的上述和/或附加的方面和优点从结合下面附图对实施方式的描述中将变得明显和容易理解，其中：
图1为本发明一实施例中的制备芯片的流程示意图；
图2为本发明实施例1制备的聚物的GPC分子量测试图；
图3为本发明实施例1制备的芯片的表面质量检测的光强图；
图4为本发明实施例1制备的芯片的生物应用的光强图；
图5为本发明实施例2制备的聚物的GPC分子量测试图；
图6为本发明实施例2制备的芯片的表面质量检测的光强图；
图7为本发明实施例2制备的芯片的生物应用的光强图；
图8为本发明实施例3制备的聚物的GPC分子量测试图；
图9为本发明实施例3中叠氮基大分子核磁检测示意图；
图10为本发明实施例3制备的芯片的表面质量检测的光强图；
图11为本发明实施例3制备的芯片的生物应用的光强图；
图12为本发明实施例4制备的聚物的GPC分子量测试图；
图13为本发明实施例4制备的芯片的表面质量检测的光强图；
图14为本发明实施例4制备的芯片的生物应用的光强图；
图15为本发明实施例5制备的聚物的GPC分子量测试图；
图16为本发明实施例5制备的芯片的表面质量检测的光强图；
图17为本发明实施例5制备的芯片的生物应用的光强图；
图18为本发明实施例6中文库的分子结构图；
图19～23为本发明实施例1～5制备的芯片的的荧光强度检测图，其中，左图为原始图片，右图为使用ImageJ二值化处理后的图片。
具体实施方式
以下结合具体实施例对本发明的基底/芯片及其制备方法和应用作进一步详细的说明。下面通过参考附图描述的实施方式是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。示例中的试剂、检测仪器等，如无特殊说明，可自配或者通过市售途径获取。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。此外，由于本文涉及的具体数据大多具有统计意义或者难以精确测定，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，如包含该数值正负10％的区间，以下不再重复说明。
本文所称的芯片包括表面包含聚合物包被的基底，也包括在聚合物包被上连接有生物分子的基底。基底的材料并不受特别限制，例如选自玻璃、硅片、塑料、凝胶和尼龙膜中的至少之一。若无另外说明，基底、芯片和生物芯片可替换使用。
本文所称的“连接”应做广义理解，例如，可以是直接相连，也可以通过中间媒介间接相连，可以是通过化学连接也可以是物理连接。除非另有明确的限定，在本文中，在涉及化合物、生物分子、官能团、基团等的连接关系的描述中，连接一般指化学连接，如通过共价键的结合、基于范德华力或静电作用的吸附等。对于本领域的技术人员而言，可以根据具体情况理解上述术语在本文中的具体含义。
本文所称的聚合物指高分子化合物，分子量通常大于1000，有时也称高聚物。本文中的聚合物是多个 单体分子经过聚合反应例如加成聚合反应而形成。
本文所称的官能团、功能性基团、活性基团可以替换使用，代表使得化合物具有某种或某类特性的基团。
本文所述的“经……接枝于”、“修饰有……”可以表示直接接枝、修饰于对象之上，也可以表示非直接地比如经过其他的过渡基团或结构接枝、修饰于对象之上。除非另有明确的说明，本文中的连接包括接枝、固定、结合等；并且，本文中的接枝、固定、结合与共价连接/共价结合(共价键连接)均可互换使用。
术语“烷基”是指包含伯(正)碳原子、或仲碳原子、或叔碳原子、或季碳原子、或其组合的饱和烃。例如，“C 1～C 10烷基”是指包含1～10个碳原子的烷基，每次出现时，可以互相独立地为C 1烷基、C 2烷基、C 3烷基、C 4烷基、C 5烷基、C 6烷基、C 7烷基、C 8烷基、C 9烷基、C 10烷基。所称的烷基包括但不限于：甲基(Me、-CH 3)、乙基(Et、-CH 2CH 3)、1-丙基(n-Pr、n-丙基、-CH 2CH 2CH 3)、2-丙基(i-Pr、i-丙基、-CH(CH 3) 2)、1-丁基(n-Bu、n-丁基、-CH 2CH 2CH 2CH 3)、2-甲基-1-丙基(i-Bu、i-丁基、-CH 2CH(CH 3) 2)、2-丁基(s-Bu、s-丁基、-CH(CH 3)CH 2CH 3)、2-甲基-2-丙基(t-Bu、t-丁基、-C(CH 3) 3)、1-戊基(n-戊基、-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH3)CH2CH2CH3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、1-己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3和辛基(-(CH 2) 7CH 3)。
“环氧基”是指包含-X-CH(O)CH-X结构的基团，其中的每个X独立地是H或烷基；环氧基包括但不限于-CH(O)CH 2、-CH(O)CH(烷基)。
“氨基”是指氨的衍生物，为包含-N(X) 2结构的基团，其中的每个X独立地是H或烷基。氨基包括但不限于-NH 2、-N(烷基) 2、-NH(烷基)、-N(环烷基) 2、-NH(环烷基)、-N(杂环基) 2、-NH(杂环基)、-N(芳基) 2、-NH(芳基)、-N(烷基)(芳基)、-N(烷基)(杂环基)、-N(环烷基)(杂环基)、-N(芳基)(杂芳基)和-N(烷基)(杂芳基)。
“叠氮基”或者“叠氮”指-N 3。
“酯基”是指包含-C(O)O-X结构的基团，其中的X是烷基；酯基包括但不限于-C(O)OCH 3、-C(O)OCH 2CH 3。
“酰胺基”是指包含-C(O)N(X) 2结构的基团，其中的每个X独立地是H或烷基；酰胺基包括但不限于-C(O)NH 2、-C(O)NH(烷基)、-C(O)N(烷基) 2。
本文所称的生物分子或生物组分，包括核酸和/或蛋白。核酸可以是DNA，也可以是cDNA或RNA或者RNA-DNA复合物，可以是双链、也可以是单链。
本文所称的探针/引物是序列已知的核酸分子，可用以捕获目标核酸序列，或作为引物用于扩增目标核酸序列。可以是DNA和/或RNA等，通常是长度小于150nt的寡核苷酸链。通过对基底表面进行结构性和/或化学处理，可以使连接在该表面上的探针呈随机或规则分布。
本发明具体实施方式提供的一种基底，其具有表面，表面包含与之共价连接的聚合物包被，聚合物包被包含聚合物，聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
Figure PCTCN2021133358-appb-000003
其中，X选自-O-或-NH-；R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基；R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，这里的PEG4指HO(CH 2CH 2O)nH中的n为4；R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基；L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-；R 06选自PEG或烷基二胺。
在某个示例中，R 06选自PEG，PEG的分子量为200～2000，较佳地500～1000。
在某些示例中，聚合物包含式III、式IV或者式V所示的结构：
Figure PCTCN2021133358-appb-000004
在某些示例中，聚合物包被包含式VI或式VII所示的聚合物：
Figure PCTCN2021133358-appb-000005
其中，m选自1～2000范围内的整数，n选自1～3000范围内的整数，m1和m2分别独立的选自1～2000范围内的整数，n1或n2选自1～1500范围内的整数且n1等于n2，并且，在R 06选自PEG的情况下，PEG的分子量小于1000，较佳地PEG的分子量为500～1000。
在某些示例中，聚合物包被包含式VI所示聚合物，m和n的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
在某些示例中，聚合物包被包含式VII所示聚合物，m1与m2之和与n1或n2的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
在某些示例中，聚合物的分子量为1-20万。分子量在此范围的聚合物连接于表面都能使得表面具有预期的且稳定可控的性能。关于聚合物分子量对表面性能的影响，对于该分子量范围中的、由相同单体比例聚合形成的高聚物，基于对高聚物、高聚物在表面上的形态/结构的认识以及据测试经验，发明人猜测分子量相对高的如18万的聚合物会比分子量相对低的如2万的聚合物会使表面具有更优越的性能，例如官能团更大量、分布更均匀等，然而，意外地，在具有这两种分子量的聚合物包被的两表面上分别连接相同的探针测试发现，具有这两种分子量聚合物包被的表面的性能相当，无明显差别。
在某些示例中，聚合物包被与表面上的活性基团共价结合而连接于该表面，活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种。
在某些示例中，活性基团为氨基，聚合物包含环氧基团。至少一部分氨基和聚合物上的至少一部分环氧基团反应而使得聚合物通过活性基团共价结合至表面。
在某些示例中，活性基团为环氧基，聚合物包含-NH 2。至少一部分环氧基和聚合物上的至少一部分-NH 2反应而使得聚合物通过活性基团共价结合至表面。
在某些示例中，活性基团为炔基、氰基、乙烯基和丙烯基中的一种，聚合物包含-N 3。至少一部分指定活性基团与聚合物上的至少一部分N 3发生反应而使得聚合物通过活性基团共价结合至表面。
在某些示例中，X选自-O-。
具体地，在一些示例中，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基；和/或R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
在某些示例中，重复单元A选自如下结构中的一种：
Figure PCTCN2021133358-appb-000006
在一些示例中，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000007
其中的PEG的分子量为200～2000，较佳地500-1000。
在一些示例中，聚合物包含如下结构中的一种：
Figure PCTCN2021133358-appb-000008
在一些示例中，聚合物包被包含如下聚合物中的一种：
Figure PCTCN2021133358-appb-000009
Figure PCTCN2021133358-appb-000010
其中，p选自1～3000范围内的整数，p1和p2分别独立地选自1～1300范围内的整数且p1等于p2，q、q1和q2分别独立地选自1～2000范围内的整数。
在另外某些示例中，X选自-NH-。
具体地，在一些示例中，重复单元B如式III所示。
在一些示例中，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基或-(C1-C5烷基)-NH-NHS-PEG4；和/或R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-，L 1选自-C(O)-PEG-C(O)-的情况下，其中的PEG的分子量为200～2000，较佳地500～1000。
在某些示例中，重复单元A选自如下结构中的一种：
Figure PCTCN2021133358-appb-000011
这里的PEG4表示HO(CH 2CH 2O)nH中的n为4。
在一些示例中，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000012
在一些示例中，聚合物包含如下结构中的一种：
Figure PCTCN2021133358-appb-000013
在一些示例中，聚合物包被包含如下聚合物中的一种：
Figure PCTCN2021133358-appb-000014
其中，p3和p4分别独立地选自1～1300范围内的整数且p3等于p4，q3和q4分别独立地选自1～500范围内的整数，q5和q6分别独立地选自1～1500范围内的整数。
在另一些示例中，重复单元B如式II所示。
具体地，在一些示例中，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000015
在一些示例中，R 0选自-(C1-C5烷基)-NH-NHS-PEG4-N 3。这里的PEG4表示HO(CH 2CH 2O)nH中的n为4。
在一些示例中，重复单元A为
Figure PCTCN2021133358-appb-000016
在一些示例中，聚合物包括如下结构中的一种：
Figure PCTCN2021133358-appb-000017
在一些示例中，聚合物包被包括如下聚合物中的一种：
Figure PCTCN2021133358-appb-000018
其中，p7选自1～3000范围内的整数，q7选自1～500范围内的整数。
在某些示例中，使对应于重复单元A的如式VIII所示的单体A以及对应于重复单元B的如式IX或式X所示的单体B进行聚合反应以获得聚合物，聚合反应在引发剂存在的情况下进行，
Figure PCTCN2021133358-appb-000019
其中，
X选自-O-或-NH-，R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，这里的PEG4指HO(CH 2CH 2O)nH中的n为4；R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，R 06选自PEG或烷基二胺。
在某些具体示例中，R 06为PEG的情况下，PEG分子量为200～2000，较佳地PEG分子量为500～1000。
在某些示例中，聚合反应中单体A和单体B的摩尔比例选自1:1至1:30范围内的数值。较佳地，选自1:2～1:30范围内的数值；更佳地，选自1:10～1:20范围内的数值。
在某些示例中，表面还连接有生物分子，生物分子与聚合物共价结合而连接于表面。
在某些示例中，生物分子选自蛋白和核酸中的至少一种。在某个示例中，生物分子例如是探针，探针末端带有能与聚合物结合的基团而固定至表面；进一步地，探针可与目标核酸分子杂交，生物分子还可以包括目标核酸分子或是探针-目标核酸分子复合物。
本发明另一具体实施方式提供的一种制备表面具有聚合物包被的基底的方法，该方法可以用于上述任一实施方式或示例中的基底的制备。该方法包括：使聚合物与表面接触，以使聚合物连接至表面，聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
Figure PCTCN2021133358-appb-000020
其中，X选自-O-或-NH-，R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，R 06选自PEG或烷基二胺。
可以理解地，上述任一示例中的基底具有技术特征和优点，同样也适用于本实施方式中的方法。
例如，在某些示例中，聚合物包含式III、式IV或者式V所示的结构：
Figure PCTCN2021133358-appb-000021
在某些示例中，聚合物包被包含式VI或式VII所示的聚合物：
Figure PCTCN2021133358-appb-000022
其中，m选自1～2000范围内的整数，n选自1～3000范围内的整数，m1和m2分别独立的选自1～2000范围内的整数，n1或n2选自1～1500范围内的整数且n1等于n2，并且，在R 06选自PEG的情况下，PEG的分子量为200～2000，较佳地为500～1000。
在某些示例中，聚合物包被包含式VI所示聚合物，m和n的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
在某些示例中，聚合物包被包含式VII所示聚合物，m1与m2之和与n1或n2的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
在某些示例中，聚合物的分子量为1-20万。
具体地，在一些示例中，X选自-O-。
在一些示例中，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基；和/或R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1 选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
在一些示例中，重复单元A选自如下结构中的一种：
Figure PCTCN2021133358-appb-000023
在一些示例中，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000024
其中的PEG的分子量为200～2000例如不大于1000，较佳地500～1000。
在一些示例中，聚合物包含如下结构中的一种：
Figure PCTCN2021133358-appb-000025
Figure PCTCN2021133358-appb-000026
在一些示例中，聚合物包含如下聚合物中的一种：
Figure PCTCN2021133358-appb-000027
Figure PCTCN2021133358-appb-000028
其中，p选自1～3000范围内的整数，p1和p2分别独立地选自1～1300范围内的整数且p1等于p2，q、q1和q2分别独立地选自1～2000范围内的整数。
在另一些示例中，X选自-NH-。具体地，重复单元B如式III所示。
更具体地，在某些示例中，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基或-(C1-C5烷基)-NH-NHS-PEG4；和/或R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、甲基、-C(O)NH 2或 -C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
在某些示例中，重复单元A选自如下结构中的一种：
Figure PCTCN2021133358-appb-000029
在某些示例中，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000030
在某些示例中，聚合物包含如下结构中的一种：
Figure PCTCN2021133358-appb-000031
在某些示例中，聚合物包含如下聚合物中的一种：
Figure PCTCN2021133358-appb-000032
其中，p3和p4分别独立地选自1～1300范围内的整数且p3等于p4，q3和q4分别独立地选自1～500范围内的整数，q5和q6分别独立地选自1～1500范围内的整数。
具体地，在另一些示例中，重复单元B如式II所示。
例如，重复单元B选自如下结构中的一种：
Figure PCTCN2021133358-appb-000033
在某些示例中，R 0选自-(C1-C5烷基)-NH-NHS-PEG4-N 3。
在某些示例中，重复单元A为
Figure PCTCN2021133358-appb-000034
在某些示例中，聚合物包括如下结构中的一种：
Figure PCTCN2021133358-appb-000035
在某些示例中，聚合物包括如下聚合物中的一种：
Figure PCTCN2021133358-appb-000036
其中，p7选自1～3000范围内的整数，q7选自1～500范围内的整数。
在又一些示例中，该方法包括：修饰表面，以使表面带有活性基团，活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种；使聚合物与表面接触，以使聚合物与活性基团共价结合以连接至表面。
具体地，在一个示例中，活性基团为氨基，聚合物包含环氧基，至少一部分环氧基与至少一部分氨基发生反应以使聚合物连接至表面。
在另一示例中，活性基团为环氧基，聚合物包含氨基，至少一部分氨基与至少一部分环氧基发生反应以使聚合物连接至表面。
在再一些示例中，活性基团选自炔基、氰基、乙烯基和丙烯基中的一种，聚合物包含-N 3，至少一部分-N 3与至少一部分活性基团发生反应以使聚合物连接至表面。
在某些示例中，使用硅烷偶联剂处理表面，以使表面带有活性基团，硅烷偶联剂选自3-氨丙基三甲氧基硅氧烷、3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯、4-(三乙氧基)硅基丁腈和γ基丁腈和烷基环氧丙氧)丙基三甲氧基硅烷中的至少一种。
在某些示例中，于40℃～50℃反应1h～8h以使聚合物与活性基团共价结合以连接至表面。
在某些示例中，还包括：使生物分子与聚合物共价结合以连接至表面。
在某些示例中，生物分子选自蛋白和核酸中的至少一种。
在某些示例中，于50℃～60℃反应0.5～5h以使生物分子与聚合物共价结合。
在某些示例中，聚合物包含环氧基，生物分子的至少一个末端带有氨基修饰。
在某些示例中，聚合物包含氨基，生物分子的至少一个末端带有NHS。
在某些示例中，聚合物包含-N 3，生物分子的至少一个末端带有DBCO。
本发明的具体实施方式还提供上述任一实施方式或示例中的基底在生物分子捕获和/或检测中的用途。应用或用途例如核酸检测，更具体地，例如测序。
本发明一实施例提供一种聚合物；所述聚合物包括重复单元A和重复单元B；所述重复单元A具有如下所示结构特征：
Figure PCTCN2021133358-appb-000037
X选自-O-或-NH-，R 01’、R 01”、R 01”’各自独立地选自：-H或C1～C3烷基；R 0选自C1～C10烷基或-C1-C5烷基-NH-NHS-PEG4，且R 0被至少一个R 02取代；R 02各自独立地选自：环氧基、氨基或叠氮基；所述重复单元B具有如下所示结构特征：
Figure PCTCN2021133358-appb-000038
R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自：-H、C1～C3烷基、酰胺基或酯基，L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-；R 06选自PEG或烷基二胺。
在其中一个具体的实施例中，X选自-O-。
进一步地，R 01’、R 01”、R 01”’各自独立选自：-H或甲基；R 0选自C1～C3烷基。
进一步地，R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立选自：-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
具体地，所述重复单元A选自如下所示重复单元中的一种：
Figure PCTCN2021133358-appb-000039
具体地，所述重复单元B选自如下所示重复单元中的一种：
Figure PCTCN2021133358-appb-000040
在另外一个具体的实施例中，X选自-NH-。
进一步地，所述重复单元B具有如下所示结构特征：
Figure PCTCN2021133358-appb-000041
R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自：-H、C1～C3烷基、酰胺基或酯基，L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-；R 06选自PEG或烷基二胺。
更进一步地，R 01’、R 01”、R 01”’各自独立选自：-H或甲基；R 0选自C1～C3烷基或-(C1-C5烷基)-NH-NHS-PEG4。
更进一步地，R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立选自：-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
具体地，所述重复单元A选自如下所示重复单元中的一种：
Figure PCTCN2021133358-appb-000042
具体地，所述重复单元B选自如下所示重复单元中的一种：
Figure PCTCN2021133358-appb-000043
另进一步地，所述重复单元B具有如下所示结构特征：
Figure PCTCN2021133358-appb-000044
R 03’、R 03”、R 03”’、R 03””各自独立地选自：-H、C1～C3烷基、酰胺基或酯基。
具体地，所述重复单元B自如下所示重复单元中的一种：
Figure PCTCN2021133358-appb-000045
更进一步地，R 0选自-(C1-C5烷基)-NH-NHS-PEG4-N 3。
具体地，所述重复单元A为
Figure PCTCN2021133358-appb-000046
另外，在其中一个具体的实施例中，所述聚合物的分子量为1万～12万。
本发明的实施例还提供如上所述聚合物的制备方法，包括如下步骤：以单体A和单体B进行共聚反应，制备所述聚合物；所述单体A形成所述重复单元A，所述单体B形成所述重复单元B。
在其中一个具体的实施例中，所述单体A和所述单体B的摩尔比为1:(1～30)。具体地，单体A和所述单体B的摩尔比为1:1、1:3、1:5、1:8、1:10、1:12、1:15、1:20、1:25、1:30。
在其中一个具体的实施例中，所述共聚反应是指所述单体A和所述单体B在引发剂的引发下，于30～60℃(反应温度)进行聚合反应。具体地，所述反应温度可采用如下具体的温度值：30℃、35℃、36℃、37℃、38℃、40℃、41℃、42℃、43℃、45℃、50℃、52℃、54℃、55℃、56℃、58℃、60℃。
在其中一个具体的实施例中，所述引发剂选自偶氮二异丁腈(AIBN)、过硫酸钾(KPS)中的至少一种。
在其中一个具体的实施例中，所述聚合反应以氧气终止。
在其中一个具体的实施例中，所述聚合反应所得产物以甲醇提取，再干燥，制备所述聚合物。
本发明的实施例还提供一种芯片，所述芯片包括基底，以及接枝于所述基底表面的聚合物；所述聚合物为如上所述的聚合物。
在其中一个具体的实施例中，所述基底修饰有活性基团，所述聚合物通过所述活性基团接枝于所述基底的表面；所述活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种。
在其中一个具体的实施例中，所述生物芯片还包括生物组分，所述生物组分接枝于所述聚合物。由此用于进一步的生物反应和应用。具体地，所述生物组分选自氨基酸序列和核苷酸序列中的至少一种。进一步地，氨基酸序列包含蛋白质、寡肽、多肽等；核苷酸序列包含寡核苷酸序列、多核苷酸序列等。
本发明的实施例还提供一种芯片的制备方法，包括如下步骤：获取基底，将聚合物接枝至所述基底的表面；所述聚合物选自上述任一示例中的聚合物。该芯片的制备方法，无需严格的反应条件控制，工艺简单易控，利于芯片的推广应用。
在其中一个具体的实施例中，先于所述基底的表面接枝活性基团，再将所述聚合物经所述活性基团接枝于所述基底的表面；所述活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种。
在其中一个具体的实施例中，接枝所述活性基团是指采用硅烷偶联剂与所述基底进行反应；所述硅烷偶联剂选自3-氨丙基三甲氧基硅氧烷、3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯、4-(三乙氧基)硅基丁腈和γ基丁腈和烷基环氧丙氧)丙基三甲氧基硅烷中的至少一种。
在其中一个具体的实施例中，所述聚合物经所述活性基团接枝于所述基底的表面是指于40～50℃反应1～8h(反应温度*时间)。具体地，所述反应温度*时间可采用如下组合：40℃*4h、43℃*4h、44℃*4h、45℃*4h、46℃*4h、47℃*4h、50℃*4h、45℃*2h、45℃*6h、45℃*1h、45℃*8h。
在其中一个具体的实施例中，接枝所述活性基团是指所述硅烷偶联剂与所述基底在20～30℃反应1～5h。
另外，在其中一个具体的实施例中，所述制备方法还可以包括如下步骤：将生物组分接枝至所述高聚 物。由此用于进一步的生物反应和应用。具体地，所述生物组分选自氨基酸序列和核苷酸序列中的至少一种。进一步地，氨基酸序列包含蛋白质、寡肽、多肽等；核苷酸序列包含寡核苷酸序列、多核苷酸序列等。
在其中一个具体的实施例中，将所述生物组分接枝至所述高聚物是指于50℃～60℃反应0.5～5h(反应温度*时间)。具体地，所述反应温度*时间可采用如下组合：55℃*0.5h、55℃*1h、55℃*2h、55℃*3h、55℃*5h、54℃*1h、56℃*1h、52℃*1h、50℃*1h、58℃*1h、60℃*1h。
另外，本发明的实施例提供如上所述的聚合物，或如上所述的芯片，或如上所述的制备方法制备得到的芯片在生物分子制备或生物分子分析中的应用。
如下为具体的实施例，如无特别说明，实施例中采用的原料均为市售获得，比例若无另外说明均指质量百分比。
T20：tttttttttttttttttttttttttt(SEQ ID NO:1)；Pe：caacaacaacaacaacaacaacaacaa(SEQ ID NO:2)；RD：ctgccccgggttcctcattc tat(SEQ ID NO:3)。
实施例1
请参图1。
(1)聚合物制备
以甲苯为溶剂，按照体积比，体系中加入5wt％的GMA(甲基丙烯酸缩水甘油酯)，0.1wt％的分子量为1000g/mol的PEGDA(聚乙二醇(二醇)二丙烯酸酯)，0.1wt％的AIBN(azodiisobutyronitrile；azobisisobutyronitrile，中文名称为偶氮二异丁腈)引发剂，通氮除氧后，在55℃下发生聚合反应。反应进行3h后，注入氧气终止反应，使用甲醇提取聚合物后，真空干燥获得体型的环氧基大分子，使用GPC检测(凝胶渗透色谱)，检测结果如图2所示，获得峰位分子量为98215的环氧基大分子。
Figure PCTCN2021133358-appb-000047
(2)基底表面修饰
基底采用玻璃，玻璃使用溶液法进行表面氨基化镀膜，镀膜方法如下：
将玻璃置于5％的APTMS(氨丙基三甲氧基硅烷)乙醇溶液中25℃反应2h后，取出玻璃与烘箱中130℃烘烤2h，完成玻璃表面的氨基化。
将氨基化镀膜后的玻璃置于0.1wt％的环氧基大分子的异丙醇溶液中，45℃反应4h后，得到接枝有环氧大分子的表面。
利用NH 2-CY3(采购自西安凯新生物科技有限公司)检测修饰环氧大分子的玻璃表面质量，具体检测过程如下：
将20μM浓度的NH2-CY3溶液通入到表面，55℃反应1h后，在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图3所示，经ImageJ信号统计，获得的表面平均亮度为21919a.u.。
(3)寡核苷酸连接(a)将修饰有大分子的基底采用微流道封装技术，例如采用表面改性封装方式、热压键合封装或阳极键合微流道封装技术，在基底表面形成微流道。在微流道中加入NH 2-oligo(NH 2-A 30)5μM溶液，在37℃下反应24h，使表面接枝有高密度oligo(寡核苷酸)序列，其中A 30表示30个腺嘌呤核苷酸形成的寡核苷酸链。
(b)用纯水将微流道表面冲洗干净后，加入5uM T 20-CY3的溶液，在55℃下杂交30min。
(c)在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图4所示，获得的平均亮度值为28267a.u.。
实施例2
(1)聚合物制备
以水和DMF(二甲基甲酰胺)为溶剂，其中水和DMF的体积比未4:1。按照质量比，体系中加入2％的丙烯酰胺AM、0.2％的GMA单体、0.1％的KPS(过硫酸钾)引发剂和0.01％的TEMED(四甲基乙二胺)凝胶促进剂，通氮除氧后，在42℃下发生聚合反应。反应进行3h后，注入氧气终止反应，使用甲醇提取聚合物后，真空干燥获得线性的环氧基大分子，经GPC检测(凝胶渗透色谱)，检测结果如图5所示，获得峰位分子量为191619的环氧基大分子。
Figure PCTCN2021133358-appb-000048
(2)基底表面修饰
基底采用玻璃，玻璃使用溶液法进行表面氨基化镀膜，镀膜方法如下：
将玻璃置于5％的APTMS(氨丙基三甲氧基硅烷)乙醇溶液中，25℃反应2h后，取出玻璃与烘箱中130℃烘烤2h，完成玻璃表面的氨基化。
将氨基化镀膜后的玻璃置于0.1wt％的环氧基大分子的异丙醇溶液中，45℃反应4h后，得到修饰有环氧大分子的表面。
利用NH 2-CY3(采购自西安凯新生物科技有限公司)检测修饰环氧大分子的玻璃表面质量，具体检测过程如下：
将20uM浓度的NH 2-CY3溶液通入到表面，55℃反应1h后，在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图6所示，经ImageJ软件分析，获得的平均信号强度为13768a.u.。
(3)寡核苷酸连接
(a)将修饰有大分子的基底采用微流道封装技术在基底表面形成微流道。在微流道中加入5uM的NH 2-oligo(NH 2-T 35)溶液，在37℃下反应24h，使表面接枝有高密度oligo(寡核苷酸)序列，其中T 35表示35个胸腺嘧啶核苷酸形成的寡核苷酸链。
(b)用纯水将微流道表面冲洗干净后，加入5μM的A 30-CY3溶液，在55℃下杂交30min，其中A 30表示30个腺嘌呤核苷酸形成的寡核苷酸链。
(c)在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强图如图7所示，获得的平均信号强度为13039a.u.。
实施例3
(1)聚合物制备
以水和DMF(二甲基甲酰胺)为溶剂，其中水和DMF的体积比为4:1。按照质量比，体系中加入2％的丙烯酰胺(AM)，1％的烯基叠氮单体(采购自西安凯新生物科技有限公司)，0.1％的KPS引发剂，0.01％的TEMED凝胶促进剂，通氮除氧后，在37℃下发生聚合反应。反应进行3h后，注入氧气终止反应，使用乙醇提取聚合物后，真空干燥获得线性的叠氮基大分子，经GPC检测(凝胶渗透色谱)，检测结果如图8所示，获得峰位分子量为15167的叠氮基大分子。
核磁测试如图9所示，在1.43、1.55、2.09处出现烯烃聚合后形成饱和链断的H吸收峰，在3.04-3.41范围出现分子内二胺内CH 2上的H吸收峰，在3.93附近出现叠氮临近的PEG链断内的H吸收峰，证明了叠氮大分子的形成。
Figure PCTCN2021133358-appb-000049
(2)基底表面修饰
基底采用玻璃，玻璃使用溶液法进行表面炔基化镀膜，镀膜方法如下：
将玻璃置于1％的3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯的甲苯溶液中，25℃反应2h后，取出玻璃于烘箱中130℃烘烤2h，完成玻璃表面的炔基化。
将炔基化镀膜后的玻璃置于0.1wt％的叠氮基大分子的异丙醇溶液中，45℃反应4h后，得到接枝有叠氮大分子的表面。
利用DBCO(二苯并环辛炔)-CY3(采购自西安凯新生物科技有限公司)检测修饰叠氮大分子的玻璃表面质量，具体检测过程如下：
将20μM浓度的DBCO-CY3溶液通入到表面，55℃反应1h后，在荧光显微镜下，使用532nm波段激光照射，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图10所示，在ImageJ软件中进行分析，获得平均信号强度为19464a.u.的表面。
(3)寡核苷酸连接
(a)将修饰有大分子的基底采用微流道封装技术在基底表面形成微流道。在微流道中加入5μM的DBCO-oligo(DBCO-T 35)溶液，在37℃下反应24h，使表面接枝有高密度oligo(寡核苷酸)序列，其中T 35表示35个胸腺嘧啶核苷酸。
(b)将表面用纯水冲洗干净后，使用A 30-cy3的5uM溶液在55℃下杂交30min，其中A 30表示30个腺嘌呤核苷酸形成的寡核苷酸链。
(c)在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图11所示，获得平均信号强度为14535a.u.。
实施例4
(1)聚合物制备
以水为溶剂，按照质量比，体系中加入2％的烯基叠氮单体，0.1％的分子量为1000g/mol或者575g/mol的PEGDA单体，0.1％的KPS引发剂，0.01％的TEMED凝胶促进剂，通氮除氧后，在37℃下发生聚合反应。反应进行3h后，停止反应，使用乙醇提取聚合物后，真空干燥后获得体型的的叠氮基大分子，经GPC检测，检测结果如图12所示，获得其峰位分子量为129976的叠氮基大分子；
Figure PCTCN2021133358-appb-000050
(2)基底表面修饰
基底采用玻璃，玻璃使用溶液法进行表面氰基化镀膜，镀膜方法如下：
将玻璃置于1％的4-(三乙氧基)硅基丁腈的甲苯溶液中，25℃反应2h后，取出玻璃于烘箱中130℃烘烤2h。完成玻璃表面的氰基化。
将氰基化镀膜后的玻璃置于0.1wt％的叠氮基大分子的异丙醇溶液中，45℃反应4h后，得到接枝有叠氮大分子的表面。DBCO-CY3(采购自西安凯新生物科技有限公司)检测修饰叠氮大分子的玻璃表面质量，具体检测过程如下：
将20μM浓度的DBCO-CY3溶液通入到表面，55℃反应1h后，在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图13所示，经ImageJ软件分析，获得的平均信号强度为28531a.u.。
(3)寡核苷酸连接
(a)将修饰有大分子的基底采用微流道封装技术在基底表面形成微流道。在微流道中加入5uM的DBCO-oligo(DBCO-T 35)溶液，在37℃下反应24h，使表面接枝有高密度oligo(寡核苷酸)序列，其中T 35表示35个胸腺嘧啶核苷酸形成的寡核苷酸链。
(b)将表面用纯水冲洗干净后，使用5μM的A 30-CY3溶液在55℃下杂交30min。
(c)在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图14所示，获得的平均信号强度为32108a.u.。
实施例5
(1)聚合物制备
以甲苯为溶剂，按照体积比，体系中加入5％的N-(3-氨基丙基)甲基丙烯酸盐盐酸盐单体，0.1％的分子量为1000g/mol或者575g/mol的PEGDA单体，0.1％的AIBN引发剂，通氮除氧后，在55℃下发生聚合反应。反应进行3h后，注入氧气终止反应，使用甲醇提取聚合物后，真空干燥获得体型的氨基大分子，经GPC检测(凝胶渗透色谱)，检测结果如图15，所示获得峰位分子量为99396的氨基大分子。
Figure PCTCN2021133358-appb-000051
(2)基底表面修饰
基底采用玻璃，玻璃使用溶液法进行表面环氧化镀膜，镀膜方法如下：
将玻璃置于1％的γ％璃置于璃，环氧丙氧)丙基三甲氧基硅烷的甲苯溶液中，25℃反应2h后，取出玻璃与烘箱中130℃烘烤2h，完成玻璃表面的环氧化。
将环氧化镀膜后的玻璃置于0.1wt％的氨基大分子的异丙醇溶液中，45℃反应4h后，得到接枝有叠氮大分子的表面。
利用NHS-CY3(定制/采购自西安凯新生物科技有限公司)检测修饰叠氮大分子的玻璃表面质量，具体检测过程如下：
将20μM浓度的NHS-CY3溶液通入到表面，55℃反应1h后，在荧光显微镜下，使用532nm波段激光照射，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图16所示，在ImageJ软件中进行分析，获得的平均信号强度为25437a.u.。
(3)制备得到的生物芯片的生物应用：
(a)将修饰有大分子的玻璃封装成带有微流道能够容纳一定液体的芯片(流动池)。往封装后芯片的微流道中加入NHS-oligo(NHS-T35)5μM溶液，在37℃下反应24h，使表面接枝有高密度引物；
(b)将表面用纯水冲洗干净后，使用A30-CY3的5μM溶液在55℃下杂交30min；
(c)在荧光显微镜下，使用532nm波段激光，按照35mW，60ms参数进行拍照检测光强。光强检测结果如图17所示，获得的平均信号强度为33155a.u.。
实施例6
利用实施例1-5中制备的芯片进行文库杂交及检测，具体流程如下：
1.DNA杂交文库准备
DNA文库：片段长度为150-300bp双端含有已知序列的DNA文库，文库的分子结构如图18所示，Insertion：插入片段，来源于phi-X174标准菌株；T20、Pe、RD分别为SEQ ID NO:1-3所示序列。
将DNA文库与52μl体积的去离子水混合，向其中加入18μl0.2M NaOH溶液，混合均匀后于室温静置变性8分钟，然后加入20μl体积pH8.0的400mMTris-HCl缓冲液终止反应，获得100μl体积的100pM变性DNA文库。
2.DNA变性文库与芯片探针杂交
使用pH 7.3含有3×SSC(是将20×SSC缓冲液用无核酸酶水(Rnase-free水)稀释而成)杂交液将变性好的DNA文库稀释至5pM，然后通入芯片通道中于42℃杂交反应30分钟，接着以250μl/min的速度流通160～260μl体积的清洗试剂(5×SSC，0.05％吐温20，pH 7.0)，完成杂交反应。
3.模板初始延伸
1)以500μl/min的速度流通芯片通道160～260μl体积的延伸缓冲试剂(20mM三羟甲基氨基甲烷(Tris)，10mM硫酸铵，2mM硫酸镁，1.5M甜菜碱，1.3％二甲基亚砜，0.45M N-甲基甲酰胺，1.5M甲酰胺(carboxamide)，0.1％TritonX-100，pH 9.0)；
2)以500μl/min的速度流入芯片通道160～260μl体积的延伸试剂(延伸缓冲试剂，3μg/ml Bst DNA聚合酶，200μM dNTPs)，然后于50～60℃反应5min，完成模板初始延伸。
4.DNA簇生成
可以利用ILLUMINA测序平台操作手册进行扩增成簇；也可以利用文章Isothermal amplification method for next-generation sequencing(Zhaochun Ma，et.al,PNAS August 27,2013 110(35)14320-14323,https://doi.org/10.1073/pnas.1311334110)公开的template walking技术进行扩增，生成DNA簇。
5.检测DNA簇(cluster)
1)设置热循环温度为50～60℃；
2)以500μl/min的速度流通芯片通道160～260μl体积的变性试剂甲酰胺，变性5分钟打开DNA双螺旋结构；
3)以500μl/min的速度流通芯片通道160～260μl体积的质控试剂(0.5μM RD-Cy3，3×SSC)，其中RD-CY3中的CY3位于RD序列的5’端；
4)设置热循环温度为25℃，反应15～30分钟；
5)以500μl/min的速度流通芯片通道160～260μl体积的清洗试剂；
6)利用20倍物镜的荧光检测系统，设置532nm波长激光功率为300mW，曝光时间为20ms，进行拍照观察。
DNA簇检测结果见图以及表1，图19～23对应的芯片分别为利用实施例1-5制备的芯片生成的DNA簇检测结果。
表1
生物芯片 密度(cluster/μm^2) 密度(cluster/mm^2) 平均每个cluster亮度(cts)
实施例1 0.0692875 69288 1906
实施例2 0.1271716 127172 1145
实施例3 0.1742481 174248 1730
实施例4 0.1519920 151992 5116
实施例5 0.0860361 86036 399
由表1可知，利用实施例1～5制备的芯片可成功用于文库的杂交、DNA簇的生成。
此外，进一步地，将表面包含DNA簇的芯片载入测序平台例如ILLUMINA公司的测序平台进行测序，能获得质量满足特定应用要求的测序数据。
在本说明书的描述中，参考术语“一个实施方式”、“某些实施方式”、“示意性实施方式”、“示例”、“具体示例”、或“一些示例”等的描述意指结合所述实施方式或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施方式或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施方式或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施方式或示例中以合适的方式结合。
尽管上面已经示出和描述了本发明的实施方式，可以理解的是，上述实施方式是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施方式进行变化、修改。

Claims (71)

  1. 一种基底，其具有表面，其特征在于，所述表面包含与之共价连接的聚合物包被，所述聚合物包被包含聚合物，所述聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
    Figure PCTCN2021133358-appb-100001
    其中，
    X选自-O-或-NH-，
    R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，
    R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，
    R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，
    L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，
    R 06选自PEG或烷基二胺，在R 06选自PEG的情况下，PEG分子量为200～2000。
  2. 根据权利要求1所述的基底，其特征在于，所述聚合物包含式III、式IV或者式V所示的结构：
    Figure PCTCN2021133358-appb-100002
  3. 根据权利要求1或2所述的基底，其特征在于，所述聚合物包被包含式VI或式VII所示的聚合物：
    Figure PCTCN2021133358-appb-100003
    其中，
    m选自1～2000范围内的整数，
    n选自1～3000范围内的整数，
    m1和m2分别独立的选自1～2000范围内的整数，
    n1或n2选自1～1500范围内的整数且n1等于n2，并且，在R 06选自PEG的情况下，PEG的分子量选自200～2000。
  4. 根据权利要求3所述的基底，其特征在于，所述聚合物包被包含式VI所示聚合物，m和n的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
  5. 根据权利要求3所述的基底，其特征在于，所述聚合物包被包含式VII所示聚合物，m1与m2之和与n1或n2的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
  6. 根据权利要求1～5任一项所述的基底，其特征在于，所述聚合物的分子量为1-20万。
  7. 根据权利要求1～6任一项所述的基底，其特征在于，所述聚合物包被与所述表面上的活性基团共价结合而连接于该表面，所述活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种。
  8. 根据权利要求7所述的基底，其特征在于，所述活性基团为氨基，所述聚合物包含环氧基团。
  9. 根据权利要求7所述的基底，其特征在于，所述活性基团为环氧基，所述聚合物包含-NH 2。
  10. 根据权利要求7所述的基底，其特征在于，所述活性基团为炔基、氰基、乙烯基和丙烯基中的一种，所述聚合物包含-N 3。
  11. 根据权利要求1～10任一项所述的基底，其特征在于，X选自-O-。
  12. 根据权利要求11所述的基底，其特征在于，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基；和/或
    R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
  13. 根据权利要求12所述的基底，其特征在于，所述重复单元A选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100004
  14. 根据权利要求11～13任一项所述的基底，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100005
    其中的PEG的分子量为200～2000，较佳地为500-1000。
  15. 根据权利要求14所述的基底，其特征在于，所述聚合物包含如下结构中的一种：
    Figure PCTCN2021133358-appb-100006
    Figure PCTCN2021133358-appb-100007
    其中的PEG的分子量为200～2000，较佳地为500～1000。
  16. 根据权利要求14所述的基底，其特征在于，所述聚合物包被包含如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100008
    Figure PCTCN2021133358-appb-100009
    其中，
    p选自1～3000范围内的整数，
    p1和p2分别独立地选自1～1300范围内的整数且p1等于p2，
    q、q1和q2分别独立地选自1～2000范围内的整数，
    PEG的分子量为200～2000，较佳地为500～1000。
  17. 根据权利要求1～10任一项所述的基底，其特征在于，X选自-NH-。
  18. 根据权利要求17所述的基底，其特征在于，所述重复单元B如式III所示。
  19. 根据权利要求18所述的基底，其特征在于，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基或-(C1-C5烷基)-NH-NHS-PEG4；和/或
    R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-。
  20. 根据权利要求19所述的基底，其特征在于，所述重复单元A选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100010
  21. 根据权利要求18～20任一项所述的基底，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100011
    其中，PEG的分子量为200～2000，较佳地为500～1000。
  22. 根据权利要求21所述的基底，其特征在于，所述聚合物包含如下结构中的一种：
    Figure PCTCN2021133358-appb-100012
  23. 根据权利要求21所述的基底，其特征在于，所述聚合物包被包含如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100013
    其中，
    p3和p4分别独立地选自1～1300范围内的整数且p3等于p4，
    q3和q4分别独立地选自1～500范围内的整数，
    q5和q6分别独立地选自1～1500范围内的整数。
  24. 根据权利要求17所述的基底，其特征在于，所述重复单元B如式II所示。
  25. 根据权利要求24所述的基底，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100014
  26. 根据权利要求25所述的基底，其特征在于，R 0选自-(C1-C5烷基)-NH-NHS-PEG4-N 3。
  27. 根据权利要求24～26任一项所述的基底，其特征在于，所述重复单元A为
    Figure PCTCN2021133358-appb-100015
  28. 根据权利要求27所述的基底，其特征在于，所述聚合物包括如下结构中的一种：
    Figure PCTCN2021133358-appb-100016
  29. 根据权利要求27所述的基底，其特征在于，所述聚合物包被包括如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100017
    其中，
    p7选自1～3000范围内的整数，
    q7选自1～500范围内的整数。
  30. 根据权利要求1～29所述的基底，其特征在于，使对应于所述重复单元A的如式VIII所示的单体A以及对应于所述重复单元B的如式IX或式X所示的单体B进行聚合反应以获得所述聚合物，所述聚合反应在引发剂存在的情况下进行，
    Figure PCTCN2021133358-appb-100018
    其中，
    X选自-O-或-NH-，
    R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，
    R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，
    R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，
    L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，
    R 06选自PEG或烷基二胺，在R 06选自PEG的情况下，PEG的分子量为200～2000，较佳地为500～1000。
  31. 根据权利要求30所述的基底，其特征在于，所述聚合反应中单体A和所述单体B的摩尔比例选自1:1至1:30范围内的数值。
  32. 根据权利要求1～31任一项所述的基底，其特征在于，所述表面还连接有生物分子，所述生物分子接枝于所述聚合物而连接于所述表面。
  33. 根据权利要求33所述的基底，其特征在于，所述生物分子选自蛋白和核酸中的至少一种。
  34. 一种制备表面具有聚合物包被的基底的方法，其特征在于，包括：
    使聚合物与所述表面接触，以使所述聚合物连接至所述表面，所述聚合物包含如式I所示的重复单元A以及如式II或式III所示的重复单元B：
    Figure PCTCN2021133358-appb-100019
    其中，
    X选自-O-或-NH-，
    R 01’、R 01”和R 01”’各自独立地选自-H或C1～C3烷基，
    R 0选自C1～C10烷基或-(C1-C5烷基)-NH-NHS-PEG4，且R 0包含至少一个R 02取代，至少一个R 02取代各自独立地选自环氧基、氨基或叠氮基，
    R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、C1～C3烷基、酰胺基或酯基，
    L 1选自C1～C3亚烷基或-C(O)-R 06-C(O)-，
    R 06选自PEG或烷基二胺，在R 06选自PEG的情况下，PEG的分子量为200～2000。
  35. 根据权利要求34所述的方法，其特征在于，所述聚合物包含式III、式IV或者式V所示的结构：
    Figure PCTCN2021133358-appb-100020
  36. 根据权利要求34或35所述的方法，其特征在于，所述聚合物包被包含式VI或式VII所示的聚合物：
    Figure PCTCN2021133358-appb-100021
    其中，
    m选自1～2000范围内的整数，
    n选自1～3000范围内的整数，
    m1和m2分别独立的选自1～2000范围内的整数，
    n1或n2选自1～1500范围内的整数且n1等于n2，并且，在R 06选自PEG的情况下，PEG的分子量为200～2000。
  37. 根据权利要求36所述的方法，其特征在于，所述聚合物包被包含式VI所示聚合物，m和n的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
  38. 根据权利要求36所述的方法，其特征在于，所述聚合物包被包含式VII所示聚合物，m1与m2之和与n1或n2的比值为1:1～1:30；较佳地，1:2～1:30；更佳地，1:10～1:20。
  39. 根据权利要求34～38任一项所述的方法，其特征在于，所述聚合物的分子量为1-20万。
  40. 根据权利要求34～39任一项所述的方法，其特征在于，X选自-O-。
  41. 根据权利要求40所述的方法，其特征在于，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基；和/或
    R 03’、R 03”、R 03”’、R 03””、R 04’、R 04”、R 04”’、R 05’、R 05”、R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-，在L 1选自-C(O)-PEG-C(O)-的情况下，其中的PEG的分子量为500～1000。
  42. 根据权利要求41所述的方法，其特征在于，所述重复单元A选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100022
  43. 根据权利要求40～42任一项所述的方法，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100023
    其中的PEG的分子量为200～2000，较佳地 500～1000。
  44. 根据权利要求43所述的方法，其特征在于，所述聚合物包含如下结构中的一种：
    Figure PCTCN2021133358-appb-100024
  45. 根据权利要求43所述的方法，其特征在于，所述聚合物包含如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100025
    Figure PCTCN2021133358-appb-100026
    其中，
    p选自1～3000范围内的整数，
    p1和p2分别独立地选自1～1300范围内的整数且p1等于p2，
    q、q1和q2分别独立地选自1～2000范围内的整数。
  46. 根据权利要求34～39任一项所述的方法，其特征在于，X选自-NH-。
  47. 根据权利要求46所述的方法，其特征在于，所述重复单元B如式III所示。
  48. 根据权利要求47所述的方法，其特征在于，R 01’、R 01”和R 01”’各自独立地选自-H或甲基，R 0选自C1～C3烷基或-(C1-C5烷基)-NH-NHS-PEG4；和/或
    R 04’、R 04”、R 04”’、R 05’、R 05”和R 05”’各自独立地选自-H、甲基、-C(O)NH 2或-C(O)OCH 3，L 1选自-C(O)-PEG-C(O)-或-C(O)-NH-CH 2-NH-C(O)-，在L 1选自-C(O)-PEG-C(O)-的情况下，其中的PEG的分子量为200～2000，较佳地500～1000。
  49. 根据权利要求48所述的方法，其特征在于，所述重复单元A选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100027
  50. 根据权利要求46～49任一项所述的方法，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100028
    其中的PEG的分子量为200～2000，较佳地500～1000。
  51. 根据权利要求50所述的方法，其特征在于，所述聚合物包含如下结构中的一种：
    Figure PCTCN2021133358-appb-100029
  52. 根据权利要求50所述的方法，其特征在于，所述聚合物包含如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100030
    其中，
    p3和p4分别独立地选自1～1300范围内的整数且p3等于p4，
    q3和q4分别独立地选自1～500范围内的整数，
    q5和q6分别独立地选自1～1500范围内的整数。
  53. 根据权利要求46所述的方法，其特征在于，所述重复单元B如式II所示。
  54. 根据权利要求53所述的方法，其特征在于，所述重复单元B选自如下结构中的一种：
    Figure PCTCN2021133358-appb-100031
  55. 根据权利要求53所述的方法，其特征在于，R 0选自-(C1-C5烷基)-NH-NHS-PEG4-N 3。
  56. 根据权利要求53～55任一项所述的方法，其特征在于，所述重复单元A为
    Figure PCTCN2021133358-appb-100032
  57. 根据权利要求56所述的方法，其特征在于，所述聚合物包括如下结构中的一种：
    Figure PCTCN2021133358-appb-100033
  58. 根据权利要求56所述的方法，其特征在于，所述聚合物包括如下聚合物中的一种：
    Figure PCTCN2021133358-appb-100034
    其中，
    p7选自1～3000范围内的整数，
    q7选自1～500范围内的整数。
  59. 根据权利要求34～58任一项所述的方法，其特征在于，包括：
    修饰所述表面，以使所述表面带有活性基团，所述活性基团选自氨基、环氧基、炔基、氰基、乙烯基和丙烯基中的至少一种；
    使所述聚合物与所述表面接触，以使所述聚合物与所述活性基团共价结合以连接至所述表面。
  60. 根据权利要求59所述的方法，其特征在于，所述活性基团为氨基，所述聚合物包含环氧基，至少一部分所述环氧基与至少一部分所述氨基发生反应以使所述聚合物连接至所述表面。
  61. 根据权利要求59所述的方法，其特征在于，所述活性基团为环氧基，所述聚合物包含氨基，至少一部分所述氨基与至少一部分所述环氧基发生反应以使所述聚合物连接至所述表面。
  62. 根据权利要求59所述的方法，其特征在于，所述活性基团选自炔基、氰基、乙烯基和丙烯基中的一种，所述聚合物包含-N 3，至少一部分所述-N 3与至少一部分所述活性基团发生反应以使所述聚合物连接至所述表面。
  63. 根据权利要求59所述的方法，其特征在于，使用硅烷偶联剂处理所述表面，以使所述表面带有所述活性基团，
    所述硅烷偶联剂选自3-氨丙基三甲氧基硅氧烷、3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯、4-(三乙氧基)硅基丁腈和γ基丁腈和烷基环氧丙氧)丙基三甲氧基硅烷中的至少一种。
  64. 根据权利要求63所述的方法，其特征在于，于40℃～50℃反应1h～8h以使所述聚合物与所述活性 基团共价结合以连接至所述表面。
  65. 根据权利要求34～64任一项所述的方法，其特征在于，还包括：
    使生物分子与所述聚合物共价结合以连接至所述表面。
  66. 根据权利要求65所述的方法，其特征在于，所述生物分子选自蛋白和核酸中的至少一种。
  67. 根据权利要求66所述的方法，其特征在于，于50℃～60℃反应0.5～5h以使所述生物分子与所述聚合物共价结合。
  68. 根据权利要求65所述的方法，其特征在于，所述聚合物包含环氧基，所述生物分子的至少一个末端带有氨基修饰。
  69. 根据权利要求65所述的方法，其特征在于，所述聚合物包含氨基，所述生物分子的至少一个末端带有NHS。
  70. 根据权利要求65所述的方法，其特征在于，所述聚合物包含-N 3，所述生物分子的至少一个末端带有DBCO。
  71. 权利要求1～33任一项所述的基底在生物分子捕获和/或检测中的用途。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101560061A (zh) * 2009-05-27 2009-10-21 上海交通大学 图型化聚合物刷的制备方法
CN104761745A (zh) * 2015-03-16 2015-07-08 北京化工大学 一种三维生物芯片基片制备方法
CN111171229A (zh) * 2020-02-12 2020-05-19 赛纳生物科技(北京)有限公司 一种核酸固载微球的制备方法
CN111426841A (zh) * 2020-03-31 2020-07-17 中科康磁医疗科技（苏州）有限公司 一种具有防污微凝胶芯片的制备方法、芯片，以及传感器

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2335377C (en) * 2001-03-05 2010-12-14 Bioneer Corporation Bio-molecular microchip and manufacturing process thereof
KR100487167B1 (ko) * 2002-10-30 2005-05-03 (주)바이오니아 바이오칩 및 이의 제조방법
EP2386061A4 (en) * 2009-01-12 2012-07-18 Univ California WOVEN POLYMERNANOPARTICLES
CN103233274B (zh) * 2013-05-06 2014-12-31 北京化工大学 一种聚合物基三维生物芯片的制备方法
EP3431614B1 (en) * 2013-07-01 2021-09-08 Illumina, Inc. Catalyst-free surface functionalization and polymer grafting
SI3212684T1 (sl) * 2014-10-31 2020-04-30 Illumina Cambridge Limited Polimeri in DNK kopolimer premazi

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101560061A (zh) * 2009-05-27 2009-10-21 上海交通大学 图型化聚合物刷的制备方法
CN104761745A (zh) * 2015-03-16 2015-07-08 北京化工大学 一种三维生物芯片基片制备方法
CN111171229A (zh) * 2020-02-12 2020-05-19 赛纳生物科技(北京)有限公司 一种核酸固载微球的制备方法
CN111426841A (zh) * 2020-03-31 2020-07-17 中科康磁医疗科技（苏州）有限公司 一种具有防污微凝胶芯片的制备方法、芯片，以及传感器

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP4253434A4
ZHAOCHUN MA ET AL., PNAS, vol. 110, no. 35, 27 August 2013 (2013-08-27), pages 14320 - 14323, Retrieved from the Internet <URL:https://doi.org/10.1073/pnas.1311334110>

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