WO2022100637A1 - 芯片、芯片的制备方法及应用 - Google Patents

芯片、芯片的制备方法及应用 Download PDF

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WO2022100637A1
WO2022100637A1 PCT/CN2021/129931 CN2021129931W WO2022100637A1 WO 2022100637 A1 WO2022100637 A1 WO 2022100637A1 CN 2021129931 W CN2021129931 W CN 2021129931W WO 2022100637 A1 WO2022100637 A1 WO 2022100637A1
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chip
silane
group
nucleic acid
amino group
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PCT/CN2021/129931
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English (en)
French (fr)
Inventor
王�琦
孙雷
冯叠文
高锦鸿
林志峰
刘磊
陈方
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深圳市真迈生物科技有限公司
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Priority to EP21891157.6A priority Critical patent/EP4245864A1/en
Priority to US18/037,056 priority patent/US20240035084A1/en
Publication of WO2022100637A1 publication Critical patent/WO2022100637A1/zh

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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • C09D143/00—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 containing boron, silicon, phosphorus, selenium, tellurium, or a metal; Coating compositions based on derivatives of such polymers
    • C09D143/04—Homopolymers or copolymers of monomers containing silicon
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • 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

Definitions

  • the present application relates to the technical field of nucleic acid detection, and in particular, to a chip, a method for preparing a chip, and an application of the chip.
  • nucleic acid detection chips and related technologies including capturing target nucleic acids on the chip surface, performing specific molecular biological reactions, and determining nucleic acid sequences, are the key factors that determine the performance and application scope of nucleic acid detection technology.
  • An embodiment of the present application provides a chip, comprising: a substrate with an amino group grafted on the surface, the amino group is a primary amino group or a secondary amino group; and a first compound grafted on the surface through the amino group, the first The compound contains the structure shown below:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy, and n is 1 or 2.
  • An embodiment of the present application also provides a method for preparing a chip, the method can be used to prepare the chip in any of the above embodiments, the method includes: obtaining a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group ; and attaching a first compound to the surface through the amino group, the first compound comprising the structure:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy, and n is 1 or 2.
  • the embodiments of the present application also provide the application of the chip of any of the above embodiments or the chip prepared by the method of any of the above embodiments in nucleic acid detection.
  • nucleic acid detection is, for example, the detection of designated site variation, sequencing, and the like.
  • 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 oligomers directly or indirectly connected to the first compound.
  • Nucleic acid sequences primers or probes
  • the chip is especially suitable for applications that require high surface properties and require stable and controllable surface properties.
  • the chip with the above-mentioned surface characteristics is suitable for a sequencing platform based on chip detection and using the Sequencing by Synthesis (SBS) principle to realize sequencing, such as a single-molecule sequencing platform or a high-throughput sequencing platform.
  • Nucleic acid molecules are connected to the surface of the chip for direct single-molecule detection without amplification.
  • nucleic acid molecules to be tested are connected to the surface of the chip and amplified into clusters (amplified signals) on the surface before detection; the 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 top view of a chip according to an embodiment of the present application.
  • FIG. 2 is an image of a certain field of view on the chip surface according to an embodiment of the application
  • FIG. 4 is an image of a certain field of view on the chip surface according to an embodiment of the application.
  • FIG. 6 is an image of a certain field of view on the chip surface according to an embodiment of the present application.
  • first and second herein are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature. As used herein, “plurality” means two or more, unless expressly defined otherwise.
  • 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 description of connection relationships involving compounds, biomolecules, groups, etc. herein, connection generally refers to chemical connection, such as binding by covalent bonds, adsorption based on van der Waals forces or electrostatic interactions Wait. For those skilled in the art, the specific meanings of the above terms in this document can be understood according to specific situations.
  • grafted on and “modified with" described herein can mean directly grafted or modified on the object, or can mean indirectly, such as grafted or modified through other groups or structures, on the object. on the object.
  • attachment herein includes grafting, immobilization, binding, etc., and, unless expressly stated otherwise, grafting, immobilization, binding and covalent attachment/covalent attachment (covalent bond attachment) herein ) can be used interchangeably.
  • Amino is a group comprising the structure -N(X) 2 , wherein each "X" is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, Substituted or unsubstituted heterocyclyl, etc.; unless expressly stated otherwise, in embodiments of the present application, at least one X is H.
  • the so-called amino group includes, but is not limited to, -NH2 , -NH(alkyl), -NH(cycloalkyl), -NH(heterocyclyl), -NH(aryl).
  • 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. Including but 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, -CH2CH( CH3 ) 2 ), 2 -butyl (s-Bu, s-butyl, -CH( CH3 ) CH2 CH 3 ), 2-methyl-2-propyl (t
  • alkoxy refers to an alkyl group containing an -O- group, such as the alkyl group exemplified above linked to an oxygen atom.
  • C1-C5 alkoxy means that the alkyl moiety contains 1-5 carbon atoms, and each time it appears, it can be independently C1 alkoxy, C2 alkoxy, C3 alkoxy, C4 Alkoxy, C5 alkoxy. Suitable examples include, but are not limited to: methoxy (-O- CH3 or -OMe), ethoxy (-O- CH2CH3 or -OEt) and tert-butoxy (-OC( CH3 ) 3 or -OtBu).
  • silanes referred to herein refer to silicon hydrocarbons; for example, referred to as aminosilanes, alkenylsilanes, alkynylsilanes, etc., are silicon hydrocarbons containing corresponding groups or bonds, for example, aminosilane refers to containing at least one amino group Silane, alkenyl silane refers to a silane containing at least one double bond, and alkynyl silane refers to a silane containing at least one triple bond.
  • a "structural unit” as referred to herein refers to a repeating unit typically contained in a polymer/polymer, sometimes also referred to as a monomer (monomer unit), repeating unit or chain segment.
  • a polymer may contain one or more structural units. Polymers can be polymerized from one monomer or from multiple monomers.
  • the chip referred to herein is a solid-phase support or solid-phase substrate, which can be a substrate grafted with silane, polymer or squaraine compound, or a nucleic acid sequence further grafted on polymer or squaraine compound the base.
  • the material for making the substrate is not particularly limited, for example, at least one selected from glass, silicon wafer, plastic, gel and nylon film. Unless explicitly stated otherwise, chip surface and substrate surface are used interchangeably.
  • the nucleic acid sequence referred to herein can be DNA and/or RNA.
  • the so-called “probe” is a nucleic acid sequence with a known sequence, which can be DNA and/or RNA, etc., also called “primer” in some embodiments, and is usually an oligonucleotide chain with a length of less than 150 nt.
  • the probes attached to the surface can be randomly or regularly distributed.
  • the chip provided by the embodiment of the present application includes a first chip, including: a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; and a first compound grafted on the surface through the amino group, the The first compound contains the structure shown below:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy, and n is 1 or 2.
  • the chip through the modified layer comprising the first compound with the above-mentioned structural features, has a surface with high biochemical reactivity and uniform modification, which is convenient for controlling the oligonucleotide sequences (primers) loaded subsequently and directly or indirectly connected to the first compound. or probe) and/or the amount and/or density of nucleic acid molecules to be detected.
  • the chip with this surface characteristic is suitable for single-molecule sequencing platforms and next-generation sequencing platforms, for example, the nucleic acid molecules to be tested are connected to the surface of the chip for direct single-molecule detection without amplification, for example, the nucleic acid molecules to be tested are connected to the chip.
  • the surface is amplified into clusters (signal amplification) and then detected, which is suitable for commercially available mainstream second-generation sequencing platforms such as ILLUMINA's sequencing-by-synthesis platform.
  • the chip is easy to prepare, including easy and stable preparation of a surface with relatively consistent reproducibility, and has strong industrial applicability.
  • R 1 , R 2 are each independently selected from -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound comprises one of the following structures:
  • the first compound comprising any one of the above structures can make the surface have the characteristics of high reactivity, and can make the surface capable of reacting with various compounds/groups containing amino groups.
  • the squaraine in the above four first compounds can continue to react with other compounds containing amino groups, such as nucleic acid containing amino groups, polylysine, etc., so that it can participate in the substrate
  • Surface-reactive compounds have more and more flexible options, potentially enabling the resulting surfaces to have richer or more controllable properties.
  • different amino group-containing compounds can be selected to react with the first compound according to the requirements of the amount/density of probes on the surface for various applications, such as the selection of polymer macromolecular polylysine with The squaraine reaction fixes the polymer macromolecules on the surface of the chip substrate, which can increase the density of active groups on the chip surface related to the immobilization of probes.
  • the amino group is derived from at least one of aminosilane, polylysine, polyornithine, chitosan, polyamide-amine dendrimers, polyacrylamide, and polyethyleneimine compounds. In this way, it is advantageous to form a surface having the above-mentioned characteristics.
  • the amino groups are derived from polymers such as polylysine, polyornithine, chitosan, polyamidoamine dendrimers, or polyacrylamide, thus providing greater/more A high density of amino groups is used to connect the first compound, which is beneficial to control or increase the amount/density of the first compound on the surface, and further facilitates to control or increase the amount/density of molecules further connected to the first compound.
  • the amino group is from an aminosilane.
  • the aminosilane may be selected from (3-aminopropyl)triethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldiethoxysilane, and aminoethylaminopropyl At least one of triethoxysilane.
  • the surface-grafted amino group comprises a silane attached to the surface and a polymer attached to the surface via the silane, the silane being selected from epoxy silanes, alkenyl silanes, and At least one of the alkynyl silanes, the polymer comprising a plurality of structural units, at least one of the structural units comprising the amino group.
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide, and polyethyleneimine compounds.
  • the silane comprises an epoxysilane selected from the group consisting of 3-glycidyloxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, At least one of 3-glycidyloxypropylmethyldiethoxysilane and 3-glycidyloxypropylmethyldimethoxysilane.
  • the silane comprises an alkenyl silane selected from the group consisting of vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane and at least one of vinyltriisopropoxysilane.
  • the silane comprises an alkynyl silane selected from propynyloxytrimethoxysilane and 2-propyne [3-(triethoxysilyl)propyl]carbamate at least one of esters.
  • the chip further includes a nucleic acid sequence with an amino modification at at least one end linked to the first compound.
  • the chip further includes a polymer linked to the first compound, the polymer comprising a plurality of structural units, at least one of the structural units comprising an amino group.
  • the chip further comprises nucleic acid sequences linked to the polymer.
  • the polymer and the nucleic acid sequence are linked by a linking group comprising a first terminus capable of bonding to the nucleic acid sequence and the amino group capable of bonding to the polymer the second end of the ligation.
  • the second terminus comprises at least one of -NHS, an epoxy group, and an isocyanate group.
  • the nucleic acid sequence is modified with -DBCO or -N3 and the first terminus comprises -DBCO or -N3 .
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide, and polyethyleneimine compounds.
  • the chip provided by the embodiment of the present application includes a second chip, including: a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; and a first compound grafted on the surface of the substrate through the amino group;
  • the first compound has the following structural features:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy; n is 1 or 2; the substrate with amino groups grafted on the surface comprises a substrate layer, which is grafted on the substrate layer.
  • silane and a polymer grafted on the silane the silane is selected from at least one of epoxy silane, alkenyl silane and alkynyl silane; at least one structural unit of the polymer comprises the amino.
  • R 1 and R 2 are each independently selected from: -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide and polyethyleneimine compounds.
  • the silane comprises an epoxy silane, which can be selected from 3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropyltriethoxysilane , at least one of 3-glycidyloxypropylmethyldiethoxysilane and 3-glycidyloxypropylmethyldimethoxysilane.
  • the silane comprises an alkenyl silane, which can be selected from vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxy)silane, and At least one of vinyltriisopropoxysilane.
  • the silane comprises an alkynyl silane, which can be selected from propynyloxytrimethoxysilane and 2-propynyl [3-(triethoxysilyl)propyl]carbamate at least one of them.
  • the second chip of this embodiment may also have additional technical features and technical effects of the chip in any of the above-mentioned embodiments or the first chip, which will not be repeated here.
  • the chip provided by the embodiment of the present application includes a third chip, including: a substrate with an amino group grafted on the surface, the amino group being a primary amino group or a secondary amino group; a first compound grafted on the surface through the amino group; and, A second compound grafted to the first compound, the second compound comprising an amino group; the first compound has the following structural features:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy; n is 1 or 2.
  • R 1 and R 2 are each independently selected from: -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the second compound is a nucleic acid sequence with amino modifications.
  • the nucleic acid sequence is a nucleic acid sequence with a known sequence, and the nucleic acid sequence can be used as a probe or primer for capturing the target sequence and/or performing amplification or sequencing detection.
  • the first compound reacts with the amino group modified by the nucleic acid sequence, and then the nucleic acid sequence is connected to the chip surface; it is understood that the amount of the active group involved in the reaction contained in the first compound directly affects the nucleic acid sequence connected to the surface. amount.
  • the second compound is a polymer in which at least one structural unit includes an amino group.
  • the chip further comprises a nucleic acid sequence linked to the second compound through a linking group.
  • the molecular structure of the linking group includes a first linking group and a second linking group, the first linking group is bonded to the nucleic acid sequence, and the second linking group is grafted on the polymer the polymer.
  • the second linking group is selected from at least one of a -NHS group, an epoxy group, and an isocyanate group.
  • the second linking group is -NHS
  • the reaction conditions of -NHS and amino group are simple and easy to control, and the reaction can be carried out at room temperature of pH 7-9.
  • At least one end of the nucleic acid sequence is modified with -DBCO or -N 3
  • the first linking group is selected from the group -DBCO or the group -N 3
  • the first linking group is connected to the nucleic acid sequence through the group -DBCO is covalently bonded to the group -N3 . It can be understood that when the group modified by the nucleic acid sequence is -DBCO, the first linking group is -N 3 ; when the group modified by the nucleic acid sequence is -N 3 , the first linking group is -DBCO.
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide, and polyethyleneimine compounds.
  • the second compound is a polymer whose at least one structural unit contains an amino group, such as polylysine, polyornithine, chitosan, polyamide-amine dendrimers, or polyacrylamide , because its own structure includes the spatial structure formed on the surface, the polymer can provide a larger number/higher density of amino groups on the surface of the substrate, thereby increasing the density of immobilized nucleic acid sequences.
  • an amino group such as polylysine, polyornithine, chitosan, polyamide-amine dendrimers, or polyacrylamide
  • the third chip of any of the foregoing embodiments may also have additional technical features and technical effects of the chips of any of the foregoing embodiments, the first chip, and the second chip, which will not be repeated here.
  • a method for preparing a chip provided by an embodiment of the present application can be used to prepare the chip in any of the above embodiments, and the method includes: obtaining a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; and
  • the first compound comprises the following structure:
  • R 1 and R 2 are independently selected from -OH or C1-C5 alkoxy, and n is 1 or 2.
  • R 1 , R 2 are each independently selected from -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the amino group is derived from at least one of aminosilane, polylysine, polyornithine, chitosan, polyamide-amine dendrimers, polyacrylamide, and polyethyleneimine compounds .
  • the reaction is carried out at room temperature for 3 h to 5 h to attach the first compound to the amino group.
  • the amino group is from an aminosilane.
  • the aminosilane is selected from (3-aminopropyl)triethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldiethoxysilane and aminoethylaminopropyl at least one of triethoxysilane.
  • the aminosilane is reacted at room temperature for 1 h to 8 h to attach the amino silane to the surface, so as to obtain the amino group-grafted substrate on the surface.
  • the surface-grafted amino group comprises a silane attached to the surface and a polymer attached to the surface via the silane, the silane being selected from epoxy silanes, alkenyl silanes, and At least one of the alkynyl silanes, the polymer comprising a plurality of structural units, at least one of the structural units comprising the amino group.
  • the polymer is selected from at least the group consisting of polylysine, polyornithine, chitosan, polyamide-amine dendrimers, polyacrylamide and polyethyleneimine compounds A sort of.
  • the silane comprises the epoxy silane selected from the group consisting of 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropyltriethoxy At least one of silane, 3-glycidyloxypropylmethyldiethoxysilane, and 3-glycidyloxypropylmethyldimethoxysilane.
  • the silane comprises the alkenyl silane selected from the group consisting of vinyltrimethoxysilane, vinyltriethoxysilane, vinyltris(2-methoxyethoxysilane) ) silane and at least one of vinyltriisopropoxysilane.
  • the silane comprises the alkynyl silane selected from propynyloxytrimethoxysilane and [3-(triethoxysilyl)propyl]carbamic acid 2- At least one of propargyl esters.
  • the reaction is performed at room temperature for 1-8 h to attach the silane to the surface.
  • the reaction is carried out at a temperature of 30°C to 65°C for 1 h to 48 h to attach the polymer to the silane.
  • the method further comprises: linking a nucleic acid sequence having an amino modification at at least one terminus to the first compound nucleic acid sequence via the amino modification.
  • the method further comprises: treating the surface with a blocking reagent selected from at least one of NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3, and acetic anhydride kind.
  • a blocking reagent selected from at least one of NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3, and acetic anhydride kind.
  • the nucleic acid sequence is reacted at 30°C to 80°C for 1 h to 48 h so that the nucleic acid sequence is linked to the first compound.
  • the method further includes linking a polymer to the first compound, the polymer comprising a plurality of structural units, at least one of the structural units comprising an amino group.
  • the method also includes: connecting the nucleic acid sequence to the polymer through a linking group, the linking group comprising a first end capable of being linked to the nucleic acid sequence, and a linker capable of being linked to the polymer Amino-linked second terminus.
  • it also includes: treating the surface with a blocking agent, the blocking agent is selected from at least one of NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3 and acetic anhydride kind.
  • a blocking agent is selected from at least one of NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3 and acetic anhydride kind.
  • the nucleic acid sequence is modified with -DBCO or -N3 and the first terminus comprises -DBCO or -N3 .
  • the second terminus comprises at least one of a -NHS group, an epoxy group, and an isocyanate group.
  • the reaction is carried out at a temperature of 30°C to 65°C for 1 to 48 h to connect the first end of the linking group to the nucleic acid sequence.
  • the reaction is carried out at room temperature for 0.5 h to 5 h to attach the second end of the linking group to the amino group of the polymer.
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide, and polyethyleneimine compounds.
  • reaction is carried out at 30°C to 40°C for 1 h to 5 h to link the polymer with the first compound.
  • the preparation method provided by the embodiment of the present application includes a first preparation method, and the method includes the following steps: (1) obtaining a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; (2) grafting the first compound branched from the amino group; the first compound has the following structural features:
  • R 1 and R 2 are independently selected from: -OH or C1-C5 alkoxy, and n is 1 or 2.
  • the method is simple and easy to operate, and can prepare a chip whose surface includes the first compound modified layer with the above-mentioned specific structural features, and the surface of the prepared chip has high biochemical reaction activity, good repeatability and stability of the modified layer.
  • the prepared chip is suitable for nucleic acid detection based on chip detection, and can regulate and control the oligonucleotide sequence (primer or probe) and/or the nucleic acid molecule to be tested subsequently loaded on the chip and directly or indirectly connected to the first compound quantity and density.
  • the chip with the above-mentioned surface properties prepared by this method is suitable for single-molecule sequencing platforms and next-generation sequencing platforms, for example, the nucleic acid molecules to be tested are connected to the surface of the chip for direct detection without amplification, for example, the nucleic acid molecules to be tested are directly detected. It is connected to the surface of the chip and amplified into clusters (amplified signal) on the surface before detection, which is adapted to the mainstream second-generation sequencing platforms available in the market, such as ILLUMINA and BGI's sequencing platform.
  • R 1 and R 2 are each independently selected from: -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the first compound has the characteristics of high reactivity, and can be used as a linker of various amino group-containing compounds.
  • the squaraine in the above-mentioned four first compounds is grafted to aminosilane, the squaraine can continue to react with the compound containing amino group, such as nucleic acid containing amino group, polylysine and the like.
  • the compound containing amino group such as nucleic acid containing amino group, polylysine and the like.
  • using the first compound to fabricate the chip increases the flexibility of selecting other compounds needed to fabricate the substrate surface of the chip.
  • different compounds containing amino groups can be selected to react with the first compound.
  • the macromolecules are immobilized on the surface of the chip substrate, which can increase the density of active groups related to the immobilization of probes on the chip surface.
  • the above-mentioned grafting is carried out at room temperature for 3 h to 5 h.
  • the amino group is derived from at least one of aminosilane, polylysine, polyornithine, chitosan, polyamide-amine dendrimers, polyacrylamide and polyethyleneimine compounds kind.
  • the amino group is derived from aminosilane
  • the amino group-grafted substrate includes a substrate layer and the aminosilane grafted on the substrate layer.
  • the above aminosilane is selected from the group consisting of (3-aminopropyl)triethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropylmethyldiethoxysilane, and ammonia At least one of ethylaminopropyltriethoxysilane.
  • the above-mentioned aminosilane is grafted onto the base material layer by reacting at room temperature for 1 h to 8 h.
  • the preparation method provided by the embodiment of the present application includes a second preparation method, and the method includes the following steps: including the following steps: (1) obtaining a substrate with an amino group grafted, and the amino group is a primary amino group or a secondary amino group; (2) preparing the first amino group A compound is grafted to the amino group; the amino group-grafted substrate includes a silane grafted to the surface and a polymer grafted to the silane; the silane is selected from epoxy silanes, alkenyl silanes, and alkynes At least one of the base silanes; at least one structural unit of the polymer comprises the amino group, and the first compound has the following structural features:
  • R 1 and R 2 are independently selected from: -OH or C1-C5 alkoxy, and n is 1 or 2.
  • the first compound is selected from one of the following compounds:
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide and polyethyleneimine compounds.
  • the steric structure of the amino group-containing polymer can provide a higher density of amino groups, thereby increasing the density of the first compound that is graft-reacted with the amino groups.
  • the epoxysilane is selected from the group consisting of 3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-glycidyloxypropyl At least one of propylmethyldiethoxysilane and 3-glycidyloxypropylmethyldimethoxysilane; alkenyl silane is selected from vinyltrimethoxysilane, vinyltriethoxysilane at least one of silane, vinyltris(2-methoxyethoxy)silane and vinyltriisopropoxysilane; alkynylsilane propynyloxytrimethoxysilane and [3-(triethoxysilane) at least one of 2-propynyl silyl)propyl]carbamate.
  • the silane is grafted to the surface by reacting at room temperature for 1 h to 8 h.
  • the polymer is grafted on the silane by reacting at a temperature of 30° C. ⁇ 65° C. for 1 h ⁇ 48 h.
  • the method provided by the embodiment of the present application includes a third preparation method, including the following steps: (1) obtaining a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; (2) grafting the first compound on the surface of the substrate; (3) grafting a second compound on the first compound, the second compound comprising an amino group; the first compound has the following structural features:
  • R 1 and R 2 are independently selected from: -OH or C1-C5 alkoxy, and n is 1 or 2.
  • R 1 and R 2 are each independently selected from: -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the second compound is an amino-modified nucleic acid sequence.
  • the method further includes step (4): blocking the amino groups on the surface of the chip with a blocking reagent.
  • blocking reagents to block unreacted amino groups or incompletely reacted amino groups on the chip surface can reduce the non-specific adsorption of protein and/or nucleic acid sequences by amino groups on the chip surface, such as non-specific adsorption of DNA polymerase and template nucleic acid sequences. , thereby reducing the impact on experimental procedures such as sequencing or nucleic acid hybridization.
  • Reagents that can theoretically remove amino activity without affecting the subsequent experimental process can be used as blocking reagents, for example, blocking reagents can be selected from but not limited to NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3 and acetic anhydride at least one of the reagents.
  • the blocking reagent is NHS-PEG4.
  • the nucleic acid sequence is grafted on the first compound by reacting at a temperature of 30°C to 80°C for 1 h to 48 h.
  • the method provided by the embodiment of the present application includes a fourth preparation method, including the following steps: (1) obtaining a substrate with an amino group grafted on the surface, and the amino group is a primary amino group or a secondary amino group; (2) grafting the first compound on the surface of the substrate; (3) grafting a second compound on the first compound; the second compound is a polymer whose at least one structural unit contains an amino group; the first compound has the following structural features:
  • R 1 and R 2 are independently selected from: -OH or C1-C5 alkoxy, and n is 1 or 2.
  • R 1 and R 2 are each independently selected from: -OH, -OCH 3 or -OCH 2 CH 3 .
  • the first compound is selected from one of the following compounds:
  • the method further includes step (4): grafting the nucleic acid sequence to the second compound through a linking group, and the molecular structure of the linking group includes a first linking group and a second linking group group, the first linking group is bonded to the nucleic acid sequence, and the second linking group is grafted to the polymer through the amino group contained in the polymer.
  • the method further includes step (5): blocking the amino groups on the surface of the chip with a blocking reagent.
  • blocking reagents to block unreacted amino groups or incompletely reacted amino groups on the chip surface can reduce the non-specific adsorption of protein and/or nucleic acid sequences by amino groups on the chip surface, such as non-specific adsorption of DNA polymerase and template nucleic acid sequences. , thereby reducing the impact on experimental procedures such as sequencing or nucleic acid hybridization.
  • Reagents that can theoretically remove amino activity without affecting the subsequent experimental process can be used as blocking reagents, for example, blocking reagents can be selected from but not limited to NHS-PEG4, NHS-PEG4-NHS, NHS-PEG-N3 and acetic anhydride at least one of the reagents. In a specific embodiment, the blocking reagent is preferably acetic anhydride.
  • the nucleic acid sequence is modified with a group-DBCO or a group-N 3
  • the first linking group is selected from a group-DBCO or a group-N 3
  • the first linking group and the nucleic acid sequence Covalent bonding is carried out through the group -DBCO to the group -N3 .
  • the first linking group is -N 3
  • the first linking group is -DBCO.
  • the second linking group is grafted to the second compound by reacting at room temperature for 0.5 h to 5 h.
  • reaction conditions for the covalent bonding of the first linking group to the nucleic acid sequence are the reaction at a temperature of 30°C to 65°C for 1 to 48 hours.
  • the polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, polyacrylamide and polyethyleneimine compounds.
  • the second compound is a polymer whose at least one structural unit contains an amino group, such as polylysine, polyornithine, chitosan, polyamide-amine dendrimers or polyacrylamide, the polymer itself
  • the space structure can provide a higher density of amino groups on the surface of the chip substrate, thereby increasing the density of immobilized nucleic acid sequences.
  • the polymer is grafted on the first compound by reacting at a temperature of 30° C. ⁇ 40° C. for 1 h ⁇ 5 h.
  • the embodiments of the present application also provide the chip of any of the above embodiments, or the application/use of the chip prepared by the method of any of the above embodiments in nucleic acid detection.
  • the so-called application/use may include applications for the purpose of diagnosis and treatment of diseases, and applications not for the purpose of diagnosis and treatment of diseases, such as the detection of microorganisms in the air and the detection of microorganisms in sewage. , Pathological examination of dead animals, etc.
  • the nucleic acid detection described above is sequencing, such as single-molecule sequencing or next-generation sequencing.
  • Detection of probe density the use of nucleic acid chains with fluorescent groups to hybridize with probes on the surface of the chip to detect the density of probes;
  • Chip surface amplification for next-generation sequencing: use bridge PCR (bridgePCR), recombinase polymerase amplification (RPA), template walking (Templatewalking) and other methods to perform nucleic acid amplification on the chip surface, or directly Emulsion PCR beads were loaded on the chip surface.
  • bridgePCR bridge PCR
  • RPA recombinase polymerase amplification
  • Tempolatewalking template walking
  • the single-molecule sequencing platforms mentioned herein are commercial automations based on the Sequencing by Synthesis (SBS) principle, using virtual terminators and TIRF optical detection systems to determine the nucleotide or base sequence of nucleic acid molecules
  • SBS Sequencing by Synthesis
  • TIRF optical detection systems to determine the nucleotide or base sequence of nucleic acid molecules
  • For sequencing platforms please refer to the article [Single molecule targeted sequencing for cancer gene mutation detection, Scientific RepoRts
  • Genocare1600 can perform sequencing by multiple rounds of single-color SBS (1-channel), two-color (2-channel), or four-color (4-channel) reactions, for example, four bases by sequentially adding four nucleotides
  • An extension reaction is used to determine the nucleotide or base type (1-channel) at a position on the nucleic acid molecule to be tested.
  • nucleotide composition of the nucleic acid molecule/nucleic acid fragment for example, four nucleotides have the same fluorescence
  • two nucleotides with different fluorescent labels are added in each round of reaction for reaction and signal detection; for another example, four nucleotides with four different fluorescent labels are respectively added, and four nucleosides are added in each round of reaction acid for reaction and signal detection.
  • 3 ⁇ SSC buffer made up of 20 ⁇ SSC buffer (Sigma, #S6639-1L) diluted with nuclease-free water (Rnase-free water).
  • Polylysine (PLL) The degree of branching can be controlled between 0.35-0.45.
  • This example describes the process and method for preparing chips based on squaraine and polylysine (PLL).
  • the chip prepared by this method was used for single-molecule fluorescence sequencing after fluorescence densitometry.
  • the sequence of compound modification on the chip surface is as follows: aminosilane is plated on the glass substrate by solution reaction, then a layer of squaraine is connected by reaction with amino group, then a layer of PLL is connected by squaraine molecule, and then a layer of PLL is connected by reaction with amino group in PLL.
  • the prepared chip can be used for fluorescence density determination using the GenoCare1600 single-molecule fluorescence sequencing platform, or the nucleic acid can be sequenced on this platform.
  • Aminosilane modification add 0.1% volume of glacial acetic acid to a water-ethanol mixed solution with a volume ratio of 1:20, and then add 2.5% volume of (3-aminopropyl)triethoxysilane to prepare into aminosilane modification solution.
  • the activated glass sheet was soaked in the aminosilane modification solution, reacted at room temperature for 2 hours, after the reaction was completed, washed alternately with ethanol and water, and then placed in a vacuum oven to dry at 110 degrees Celsius.
  • Packaged chip a chip having a chamber for accommodating liquid and a liquid inlet and outlet can be prepared with reference to the contents disclosed in WO2017205876A1, EP3590603A1, etc. For example, bonding a glass sheet and a base layer etched with channels to encapsulate to form a chip containing multiple independent channels and a designated surface with aminosilane modification, each channel is physically isolated, and can independently accommodate solutions and control liquid in and out. and subsequent reactions.
  • 1 is a schematic top view of a packaged chip with 16 channels. The specifications of the channels are, for example, 90 mm long x 1.8 mm wide x 0.1 mm high, and each channel can independently control liquid in and out or reaction control. The channel size and/or number can be varied depending on the detection throughput requirements.
  • liquid control equipment can be used to pass various reaction reagents, washing reagents, etc. into the encapsulated channels respectively, so as to carry out further chemical modification/connection on the designated surface.
  • the surface performance parameters of the chips are more consistent.
  • Probe ligation 5nM N 3 -D9 solution (solvent is 3xSSC buffer, N 3 is the 5' end of D9 connected/modified azide, D9 is the probe sequence (SEQ ID No. 1)) according to A volume of 25 ⁇ L per channel was passed into the channel and placed in a 37°C oven for reaction for about 16 hours. After the reaction, each channel was washed one or more times with 1 mL of a buffer containing 150 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) and 150 mM NaCl, optionally with 1 mL of 3 ⁇ SSC buffer. channel one or more times.
  • solvent is 3xSSC buffer
  • N 3 is the 5' end of D9 connected/modified azide
  • D9 is the probe sequence (SEQ ID No. 1)) according to A volume of 25 ⁇ L per channel was passed into the channel and placed in a 37°C oven for reaction for about 16 hours. After the reaction, each channel was washed one
  • Probe quantity/density detection Pass 2nM D9'-Cy3 solution (solvent is 3 ⁇ SSC buffer, D9' is the reverse complementary sequence of D9) into the chip channel in a volume of 25 ⁇ L per channel, Place in a 40°C oven for hybridization reaction for about 30 minutes, and wash each channel with 1 mL of 3 ⁇ SSC buffer after the reaction.
  • the surface probe density of the chip prepared in this example was detected using the GenoCare1600 single-molecule fluorescence platform.
  • the average number of fluorescent spots (the number of bright spots) in the photographed field of view was used to evaluate the probe density on the chip surface.
  • the number of fluorescent dots in each photographing field of view (FOV) with an area of 110 ⁇ 110 ⁇ m is counted.
  • FOV photographing field of view
  • the average number of fluorescent points in a FOV field of view range is about 32,000 to 38,000.
  • the average of 16 channels is about 35,500.
  • the probe density on the surface of the chip prepared by this method can meet the requirements of the GenoCare1600 single-molecule sequencing platform.
  • This example describes the process and method for preparing chips based on squaraine and polylysine (PLL).
  • the chip prepared by this method is used for nucleic acid amplification and/or next-generation sequencing on the chip surface.
  • the nucleic acid on the chip surface can be measured by fluorescence microscope, or sequenced on a next-generation sequencing platform (such as Illumina NextSeq 500 sequencing platform).
  • each channel was washed with 1 mL of buffer containing 150 mM HEPES and 150 mM NaCl. Then 25 ⁇ L of 3uM P1'-Cy3 (solvent is 3 ⁇ SSC buffer, P1' is the reverse complement of P1) and 25 ⁇ L of P2'-Cy3 (P2' is the reverse complement of P2)) solution was passed into the channel respectively
  • P1'-Cy3 solvent is 3 ⁇ SSC buffer, P1' is the reverse complement of P1
  • 25 ⁇ L of P2'-Cy3 P2' is the reverse complement of P2
  • the average fluorescence brightness in a FOV field of view is about 28000-30000 , as shown in Figure 3, by photographing the number of fluorescent spots in 100 fields of view in the middle area of each chip channel, the average brightness of the entire channel is about 28500 after taking the average.
  • the measured average brightness is about 25200. Therefore, under this measurement system, the fluorescence brightness corresponding to the average density of probes in the entire channel (the sum of the average brightness detected by P1'-Cy3 and P2'-Cy3) is 53700.
  • This example describes the process and method for the fabrication of chips based on squaraine and polylysine (PLL).
  • the chip prepared by this method is used for isothermal amplification of the chip surface or and/or for next-generation sequencing.
  • the chip was prepared using the same steps as steps (1)-(3) in Example 20, and then the DNA library was used as a template, using The reagents in the Basic kit (TwistDx, Cambridge UK) were amplified on the chip surface to obtain amplified clusters.
  • the operation steps are as follows: add 18 ⁇ L of DNA library denatured to single-stranded 29.5 ⁇ L of buffer and de-nucleic acid water in the Basic kit make the total volume 50 ⁇ L, and the final concentration of the library is 10 pM. After shaking and briefly centrifuging, add 2.5 ⁇ L of magnesium acetate reagent, and mix well, use a pipette to mix The good reaction solution is poured into the chip channel, the chip is incubated at 37 to 42 degrees Celsius for 15 to 30 minutes, and then the channel is washed with 1 mL of 3 ⁇ SSC buffer. The amplified clusters can be sequenced and detected using the Illumina NextSeq 500 sequencing platform. .
  • This example describes the process and method for preparing chips based on squaraine and chitosan.
  • the chip prepared by this method is used for single-molecule fluorescence sequencing, solid-phase amplification on the chip surface or for next-generation sequencing.
  • the modification sequence of the surface of the chip substrate is as follows: epoxy silane is plated on the surface of the substrate by solution reaction, and then a layer of chitosan is modified by the reaction between epoxy groups and amino groups in chitosan, Then, a layer of squaraine molecule was connected by the amino group reaction in chitosan, and the squaraine molecule was used as a linker to connect the DNA molecule with NH 2 functional group.
  • the prepared chip uses the Illumina NextSeq 500 fluorescent sequencing platform to measure the fluorescence density, that is, the density of nucleic acid molecules on the chip surface, and then uses the template walking method to perform nucleic acid amplification, and the obtained nucleic acid amplification clusters (clusters) are used for subsequent use. Sequencing.
  • Packaged chip The surface is treated with a known packaging process to encapsulate the glass sheet with epoxy silane modification and the substrate to form a chip containing a plurality of independent channels and a designated surface with epoxy silane modification. Channels can independently carry out subsequent reactions.
  • 1 is a schematic top view of a packaged chip with 16 channels. The specifications of the channels are, for example, 90 mm long x 1.8 mm wide x 0.1 mm high, and each channel can independently control liquid in and out or reaction control. The channel size and/or number can be varied depending on the detection throughput requirements.
  • liquid control equipment can be used to pass various reaction reagents, washing reagents, etc. into the encapsulated channels respectively, so as to carry out further chemical modification/connection on the designated surface.
  • the chips with relatively consistent surface performance parameters are beneficial to the industrialization of chip preparation.
  • Probe ligation 25 ⁇ L of 10uM NH 2 -A50 solution (wherein NH 2 is the amino group modified/linked at the 5' end of A50) prepared by using 3 ⁇ SSC and ethanol in a volume ratio of 1:4 as a solvent, respectively.
  • the functional group, A50 is the nucleic acid single strand formed by 50 adenine nucleotides) into each channel, placed in a 37 °C oven for about 16 hours, after the reaction, each channel was washed with ethanol, and then used 1mL containing Buffer wash with 150 mM HEPES and 150 mM NaCl.
  • Probe density detection 3uM T35-Cy3 solution (solvent is 3 ⁇ SSC buffer, Cy3 is the modified Cy3 fluorophore at the 3' end of T35, T35 is the nucleic acid mononucleotide formed by 35 thymine nucleotides) chain) into channel 1 and channel 2 according to the volume of 25 ⁇ L of each channel, and placed in a 40 °C oven for hybridization reaction for about 30 minutes. After the reaction, the chip was placed at room temperature, and then each channel was washed with 1 mL of 3xSSC buffer. . The surface probe density of the chip prepared in this example was detected using the Illumina NextSeq 500 sequencing platform.
  • the immobilization density of the DNA chip was evaluated by the average fluorescence brightness in the photographed field of view.
  • the average fluorescence brightness in each field of view (FOV) with an area of 110 ⁇ 110 ⁇ m is counted.
  • FOV field of view
  • the average value in a FOV field of view is obtained.
  • the fluorescence brightness range is 46000-52000.
  • the average brightness of the channel is about 47500, as shown in Figure 5. Therefore, under this measurement system, the probe density of the entire channel corresponds to a fluorescence brightness of 47500.
  • Removal of fluorescent hybridized strand Use formamide to remove fluorescent hybridized strand T35-Cy3.
  • Nucleic acid amplification and sequencing Using the DNA library as a template, template walking solid-phase amplification is performed on the chip surface to obtain amplified clusters, and the amplified clusters are labeled with fluorescently-labeled hybrid strands. Detected on the NextSeq 500 sequencing platform, and the obtained results are shown in Figure 6. The amplified clusters can then be sequenced on the Illumina NextSeq 500 sequencing platform.
  • Chips prepared by this method are used for single-molecule fluorescence sequencing.
  • the sequence of compound modification on the chip surface is: plating aminosilane on the glass substrate by solution reaction, then connecting a layer of squaraine molecules by reacting with amino groups, then connecting DNA molecules with NH 2 through squaraine molecules, and finally using NHS -PEG4 blocks amino groups on glass substrates that may not be involved in the reaction.
  • the prepared chip can use GenoCare1600 single-molecule fluorescence sequencing platform for fluorescence density measurement, or perform sequencing application on this platform.
  • Aminosilane modification add 0.1% volume of glacial acetic acid to a water-ethanol mixed solution with a volume ratio of 1:20, and then add 2.5% volume of (3-aminopropyl)triethoxysilane to prepare into aminosilane modification solution.
  • the activated glass sheet was soaked in the aminosilane modification solution and reacted at room temperature for 2 hours. After the reaction, it was washed alternately with ethanol and water, and then placed in a vacuum oven and dried at 110 degrees Celsius for several hours.
  • Packaged chip a chip having a chamber for accommodating liquid and a liquid inlet and outlet can be prepared with reference to the contents disclosed in WO2017205876A1, EP3590603A1, etc.
  • a glass sheet with an aminosilane-modified surface on a specified surface is bonded to another substrate layer with channels etched on its surface by using a viscous substance such as glue to form a chip containing a plurality of channels.
  • Figure 1 is a top view of a packaged chip with 16 channels.
  • the specifications of the channels are, for example, 90 mm long, 1.8 mm wide, and 0.1 mm high, and each channel can independently control liquid in and out or reaction control.
  • the channel size and/or number can be varied depending on the detection throughput requirements.
  • liquid control equipment can be used to pass various reaction reagents, washing reagents, etc. into the encapsulated channels respectively, so as to realize the chemical reaction on the surface of the chip.
  • Probe ligation 25 ⁇ L of 10 nM NH2-D9 solution (wherein NH2 is the amino functional group modified/linked at the 5' end of D9) prepared by using 3 ⁇ SSC and ethanol in a volume ratio of 1:4 as solvent, respectively. , probe sequence) into each chip channel, seal the channel opening, and place it in a 37 °C oven for about 16 hours. After the reaction, each channel was washed with ethanol, and then 1 mL of buffer containing 150mM HEPES and 150mM NaCl was used. cleaning.
  • Probe density detection Pass 2nM D9'-Cy3 solution (solvent is 3 ⁇ SSC buffer) into each chip channel in a volume of 25 ⁇ L per channel, and place it in a 40°C oven for hybridization reaction for about 30 minutes. After the reaction, wash the channel with 1 mL of 3 ⁇ SSC buffer.
  • the GenoCare1600 single-molecule fluorescence platform was used to detect the probe density on the chip surface prepared in this example. Specifically, by detecting the Cy3 fluorescence on the D9'-Cy3 chain, the immobilization density of the DNA chip was evaluated by the number of fluorescent spots in the photographic field of view. In this example, the number of fluorescent spots in each photographing field of view (FOV) with an area of 110 ⁇ 110 ⁇ m is counted.
  • the number of fluorescent spots in a FOV field of view interval is about 39,000.
  • the probe density on the surface of the chip prepared by this method can meet the requirements of the GenoCare1600 single-molecule sequencing platform.

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Abstract

本申请涉及一种芯片、芯片的制备方法和应用。本申请实施方式的芯片包括：表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及经所述氨基连接于所述表面的第一化合物；该第一化合物包含如下结构，式（1）或式（2），其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。该芯片可以用于核酸检测，例如单分子测序、二代测序等。

Description

芯片、芯片的制备方法及应用 技术领域
本申请涉及核酸检测技术领域，特别是涉及一种芯片、一种芯片的制备方法和芯片应用。
背景技术
近年来，随着大健康和精准医疗等领域的快速发展，核酸检测技术的迭代和进步受到越来越多的关注。其中，核酸检测芯片及其相关技术，包括在芯片表面上进行目标核酸的捕获、进行特定的分子生物学反应、进行核酸序列的测定等，是决定核酸检测技术的性能和应用范围的关键因素。
在基于芯片检测实现核酸序列测定(测序)的技术的发展迭代过程中，发展形成了一代Sanger测序，二代测序，三代或四代的单分子测序。
基于芯片检测实现测序的多种测序技术或测序平台对芯片表面的性能、其上化合物或序列的连接/固定量或分布情况等均有着各自的要求，因此，对于每一种测序技术或测序平台，一般地，都需要开发和设计特定的符合指定要求的或者说能与相应的测序平台或测序技术相适配的芯片。
申请内容
本申请的实施方式提供一种芯片，包括：表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及经所述氨基接枝于所述表面的第一化合物，所述第一化合物包含下示结构：
Figure PCTCN2021129931-appb-000001
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
本申请的实施方式还提供一种制备芯片的方法，该方法可用于制备上述任一实施方式中的芯片，该方法包括：获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及使第一化合物通过所述氨基连接于所述表面，所述第一化合物包含如下结构：
Figure PCTCN2021129931-appb-000002
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
本申请的实施方式还提供上述任一实施方式的芯片或者上述任一实施方式的方法制备得的芯片在核酸检测中的应用。所称的核酸检测例如指定位点变异检测、测序等。
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、与该第一化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。该芯片特别适用于对表面性能要求高、需要表面性能稳定可控的应用。
具有上述表面特性的芯片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芯片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芯片表面并在该表面上扩增成簇（放大信号）后再进行检测；该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
本申请实施方式的上述和/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为本申请实施方式的芯片俯视示意图；
图2为本申请实施方式的芯片表面某个视野的图像；
图3为本申请实施方式的芯片表面某个视野的图像；
图4为本申请实施方式的芯片表面某个视野的图像；
图5为本申请实施方式的芯片表面某个视野的图像；
图6为本申请实施方式的芯片表面某个视野的图像。
具体实施方式
以下结合具体实施例对本申请的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，仅用于解释本申请，而不能理解为对本申请的限制。
除非另有定义，本文所使用的所有的技术和科学术语与本申请所属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。
本文中的术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本文中，“多个”的含义是两个或两个以上，除非另有明确的限定。
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、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，而且，除非另有明确的说明，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。
本文所称的氨基为包含-N(X) 2结构的基团，其中每个“X”独立地是H、取代的或未被取代的烷基、取代的或未被取代的环烷基、取代的或未被取代的杂环基等；除非另有明确的说明，在本申请的实施方式中，至少一个X为H。所称的氨基包括但不限于-NH 2、-NH(烷基)、-NH(环烷基)、-NH(杂环基)、-NH(芳基)。
所称的“烷基”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。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(CH 3)CH 2CH 2CH 3)、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)。
所称的“烷氧基”指包含-O-基团的烷基，例如上面所示例的烷基与氧原子连接形成的基团。所称的“C1～C5烷氧基”是指烷基部分包含1～5个碳原子，每次出现时，可以互相独立地为C1烷氧基、C2烷氧基、C3烷氧基、C4烷氧基、C5烷氧基。合适的实例包括但不限于：甲氧基(-O-CH 3或-OMe)、乙氧基(-O-CH 2CH 3或-OEt)和叔丁氧基(-O-C(CH 3) 3或-OtBu)。
本文所称的各种类型的硅烷指硅烃；例如，所称的氨基硅烷、烯基硅烷、炔基硅烷等，为包含相应基团或键的硅烃，例如，氨基硅烷指包含至少一个氨基的硅烃，烯基硅烷指包含至少一个双键的硅烃，炔基硅烷指包含至少一个三键的硅烃。
本文所称的“结构单元”指聚合物/高聚物中一般包含的重复单元，有时也称为单体(单体单元)、重复单元或链节。一种聚合物可以包含一种或多种结构单元。高聚物可以由一种单体聚合而成，也可以由多种单体聚合而成。
本文所称的芯片，属于固相支持物或固相基底，可以为接枝有硅烷、聚合物或方酸类化合物的基底，也可以为在聚合物或方酸类化合物上进一步接枝核酸序列的基底。制作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。除非另有明确的说明，芯片表面与基底表面可互换使用。
本文所称的核酸序列，可以是DNA和/或RNA。所称的“探针”是序列已知的核酸序列，可以是DNA和/或RNA等，在一些实施方式中也称为“引物”，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。
本申请实施方式提供的芯片包括第一芯片，包括：表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及经所述氨基接枝于所述表面的第一化合物，所述第一化合物包含下示结构：
Figure PCTCN2021129931-appb-000003
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
该芯片，通过包含具有上述结构特征的第一化合物的修饰层，具有生化反应活性高和均匀修饰的表面，便于控制后续载入的、与该第一化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。具有该表面特性的芯片适配于单分子测序平台和二代测序平台，例如使待测核酸分子连接到该芯片表面不经扩增直接进行单分子检测，例如使待测核酸分子连接到该芯片表面并在该表面上扩增成簇(放大信号)后再进行检测，适配于市售的主流的二代测序平台如ILLUMINA的边合成边测序平台。此外，该芯片易于制备，包括易于稳定地制备再现性能较一致的表面，具有较强的工业实用性。
这里的烷氧基为直链和/或具有支链的含氧烷基，例如可为-OCH 3、-OCH 2CH 3、-O(CH 2) mCH 3(m＝2～4)、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、-OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。
在某些实施例中，R 1、R 2分别独立地选自-OH、-OCH 3或-OCH 2CH 3。
在某些实施例中，所述第一化合物包含如下结构中的一种：
Figure PCTCN2021129931-appb-000004
包含上述任一种结构的第一化合物可使表面具有反应活性高的特性，可使得表面能够与多种含有氨基的化合物/基团发生反应。如上述四种第一化合物中的方酸接枝于氨基硅烷的氨基后，方酸还可以继续与含有氨基的其他化合物反应，如含有氨基的核酸、聚赖氨酸等，从而使能够参与基底表面反应的化合物具有更多的、更灵活的选择，可能可以使形成的表面具有更丰富或更利于控制的特性。利用该第一化合物的特性，可以根据各种应用对表面上探针的量/密度的需求，选择不同的含有氨基的化合物与第一化合物进行反应，如选择聚合物大分子聚赖氨酸与方酸反应，将聚合物大分子固定在芯片基底表面，可提高芯片表面与探针固定相关的活性基团的密度。
在某些示例中，氨基来自氨基硅烷、聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。如此，利于形成具有上述特性的表面。
具体地，在某些示例中，氨基来自聚合物，如聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子或聚丙烯酰胺，如此，可提供更大量/更高密度的氨基用于连接第一化合物，利于控制或提高第一化合物在表面的量/密度，进而利于控制或提高进一步连接于第一化合物的分子的量/密度。
在某些示例中，氨基来自氨基硅烷。具体地，氨基硅烷可选自(3-氨基丙基)三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨基丙基甲基二乙氧基硅烷和氨乙基氨丙基三乙氧基硅烷中的至少一种。
在某些示例中，所述表面接枝有氨基的基底包括连接于所述表面的硅烷和经所述硅烷连接于所述表面的聚合物，所述硅烷选自环氧硅烷、烯基硅烷和炔基硅烷中的至少一种，所述聚合物包含多个结构单元，至少一个所述结构单元包含所述氨基。
在某些示例中，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某些示例中，所述硅烷包含环氧硅烷，所述环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含烯基硅烷，所述烯基硅烷选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含炔基硅烷，所述炔基硅烷选自丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
在某些示例中，所述芯片还包括与所述第一化合物连接的、至少一个末端具有氨基修饰的核酸序列。
在某些示例中，所述芯片还包括与所述第一化合物连接的聚合物，所述聚合物包含多个结构单元，至少一个所述结构单元包含氨基。
在某些示例中，所述芯片还包含连接于所述聚合物的核酸序列。
在某些示例中，所述聚合物和所述核酸序列通过连接基团连接，所述连接基团包含能够与所述核酸序列键合的第一末端以及能够与所述聚合物的所述氨基连接的第二末端。
在某些示例中，所述第二末端包含-NHS、环氧基团和异氰酸酯基团中的至少一种。
在某些示例中，所述核酸序列带有-DBCO或-N 3修饰，所述第一末端包含-DBCO或-N 3。
在某些示例中，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚 丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
本申请实施方式提供的芯片包括第二芯片，包括：表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及经所述氨基接枝于所述基底表面的第一化合物；所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000005
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基；n为1或2；所述表面接枝有氨基的基底包括基材层、接枝于所述基材层之上的硅烷和接枝于所述硅烷的聚合物，所述硅烷选自环氧基硅烷、烯基硅烷和炔基硅烷中的至少一种；所述聚合物的至少一个结构单元包含所述氨基。
具体地，在某些示例中，烷氧基中的烷基可为直连烷基，也可为含有支链的烷基，例如可为-OCH 3、-OCH 2CH 3、-O(CH 2) mCH 3(m＝2～4)、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、-OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。在某个具体实施方式中，R 1、R 2分别独立选自：-OH、-OCH 3或-OCH 2CH 3。
在某个具体实施方式中，第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000006
在某个具体实施方式中，聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某个具体实施方式中，所述硅烷包含环氧硅烷，环氧硅烷可选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含烯基硅烷，烯基硅烷可选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含炔基硅烷，炔基硅烷可选自丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
可以理解地，该实施方式的第二芯片还可以具有上述任一实施方式中的芯片或第一芯片的附加技术特征和技术效果，在此不再赘述。
本申请的实施方式提供的芯片包括第三芯片，包括：表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；经所述氨基接枝于所述表面的第一化合物；以及，接枝于所述第一化合物的第二化合物，所述第二化合物包含氨基；所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000007
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基；n为1或2。
具体地，在某些示例中，烷氧基中的烷基可为直连烷基，也可为含有支链的烷基，例如可为-OCH 3、-OCH 2CH 3、-O（CH 2） mCH 3（m=2～4）、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、 -OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。在某个具体实施方式中，R 1、R 2分别独立选自：-OH、-OCH 3或-OCH 2CH 3。
在某些示例中，第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000008
在某些示例中，第二化合物为带有氨基修饰的核酸序列。
具体地，在某个示例中，核酸序列为序列已知的核酸序列，该核酸序列可作为探针或引物使用，用于捕获目标序列和/或进行扩增或测序检测等。第一化合物与该核酸序列修饰的氨基进行反应，进而将该核酸序列连接至芯片表面；可以理解地，第一化合物包含的参与该反应的活性基团的量直接影响连接到该表面的核酸序列的量。
在某些示例中，第二化合物为至少一个结构单元包含氨基的聚合物。
进一步地，在某些示例中，该芯片还包含通过连接基团与所述第二化合物连接的核酸序列。
在某些示例中，连接基团的分子结构中包含第一连接基团和第二连接基团，第一连接基团与核酸序列键合，第二连接基团经聚合物包含的接枝于该聚合物。
在某些示例中，第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。较佳地，第二连接基团为-NHS，-NHS与氨基反应条件操作简单、易控制，可在pH7～9室温下反应。
在某些示例中，核酸序列的至少一个末端修饰有-DBCO或-N 3，第一连接基团选自基团-DBCO或基团-N 3，第一连接基团与核酸序列通过基团-DBCO与基团-N 3进行共价键合。可以理解，当核酸序列修饰的基团为-DBCO，第一连接基团为-N 3；当核酸序列修饰的基团为-N 3，第一连接基团为-DBCO。
在某些示例中，聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
具体地，在某些示例中，第二化合物为至少一个结构单元含有氨基的聚合物，如聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子或聚丙烯酰胺，因其本身的结构包括在表面上形成的空间结构，聚合物可在基底表面提供更大量/更高密度的氨基，从而可提高固定的核酸序列的密度。
可以理解地，上述任一实施方式的第三芯片还可以具有上述任一实施方式的芯片、第一芯片、第二芯片的附加技术特征和技术效果，在此不再赘述。
本申请实施方式提供的一种制备芯片的方法，可用于制备上述任一实施方式中的芯片，该方法包括：获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及
使第一化合物通过所述氨基连接至所述表面，
所述第一化合物包含如下结构：
Figure PCTCN2021129931-appb-000009
其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
在某些示例中，R 1、R 2分别独立地选自-OH、-OCH 3或-OCH 2CH 3。
在某些示例中，所述第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000010
Figure PCTCN2021129931-appb-000011
在某些示例中，所述氨基来自氨基硅烷、聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某些示例中，在室温条件下反应3h～5h以使所述第一化合物与所述氨基连接。
在某些示例中，所述氨基来自氨基硅烷。具体地，所述氨基硅烷选自(3-氨基丙基)三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨基丙基甲基二乙氧基硅烷和氨乙基氨丙基三乙氧基硅烷中的至少一种。
在某些示例中，在室温条件下反应1h～8h使所述氨基硅烷连接于所述表面，以获得所述表面接枝有氨基的基底。
在某些示例中，所述表面接枝有氨基的基底包括连接于所述表面的硅烷和经所述硅烷连接于所述表面的聚合物，所述硅烷选自环氧硅烷、烯基硅烷和炔基硅烷中的至少一种，所述聚合物包含多个结构单元，至少一个所述结构单元包含所述氨基。
具体地，在某些示例中，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某些示例中，所述硅烷包含所述环氧硅烷，所述环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含所述烯基硅烷，所述烯基硅烷选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种。
在某些示例中，所述硅烷包含所述炔基硅烷，所述炔基硅烷选自丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
在某些示例中，在室温下反应1h～8h以将所述硅烷连接至所述表面。
在某些示例中，在30℃～65℃温度下反应1h～48h以使所述聚合物连接至所述硅烷。
在某些示例中，该方法还包括：使至少一个末端具有氨基修饰的核酸序列经所述氨基修饰连接于所述第一化合物核酸序列。
具体地，在某些示例中，该方法进一步包括：利用封闭试剂处理所述表面，所述封闭试剂选自NHS-PEG4、NHS-PEG4-NHS、NHS-PEG-N3和乙酸酐中的至少一种。
在某些示例中，核酸序列在30℃～80℃下反应1h～48h以使所述核酸序列连接于所述第一化合物。
在某些示例中，该方法还包括：使聚合物与所述第一化合物相连，所述聚合物包括多个结构单元，至少一个所述结构单元包含氨基。
进一步地，该方法还包括：核酸序列通过连接基团使核酸序列连接于所述聚合物，所述连接基团包含能够与所述核酸序列连接的第一末端，以及能够与所述聚合物的氨基连接的第二末端。
具体地，在某些示例中，还包括：利用封闭试剂对所述表面进行处理，所述封闭试剂选自 NHS-PEG4、NHS-PEG4-NHS、NHS-PEG-N3和乙酸酐中的至少一种。
在某些示例中，所述核酸序列带有-DBCO或-N 3修饰，所述第一末端包含-DBCO或-N 3。
在某些示例中，所述第二末端包含-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
在某些示例中，于30℃～65℃温度下反应1～48h以使所述连接基团的第一末端与所述核酸序列连接。
在某些示例中，于室温下反应0.5h～5h以使所述连接基团的第二末端与所述聚合物的氨基连接。
在某些示例中，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某些示例中，于30℃～40℃下反应1h～5h以使所述聚合物与所述第一化合物连接。
本申请实施方式提供的制备方法包括第一制备方法，该方法包括如下步骤：(1)获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；(2)使第一化合物接枝于所述氨基；所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000012
其中，R 1、R 2分别独立选自：-OH或C1～C5烷氧基，n为1或2。
该方法操作简便，能够制备出表面包含上述特定结构特征的第一化合物修饰层的芯片，而且，制备得的芯片的表面该修饰层的生化反应活性高、重复性和稳定性均较好。制备得的该芯片适用于基于芯片检测的核酸检测，并且能够调控后续载至该芯片的、与该第一化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密度。通过该方法制备得的具有上述表面特性的芯片适配于单分子测序平台和二代测序平台，例如使待测核酸分子连接到该芯片表面不经扩增直接进行检测，例如使待测核酸分子连接到该芯片表面并在该表面上扩增成簇(放大信号)后再进行检测，适配于市售的主流的二代测序平台如ILLUMINA和华大基因的测序平台。
上述烷氧基中的烷基可为直连烷基，也可为含有支链的烷基，例如可为-OCH 3、-OCH 2CH 3、-O(CH 2) mCH 3(m＝2～4)、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、-OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。在某个具体实施方式中，R 1、R 2分别独立选自：-OH、-OCH 3或-OCH 2CH 3。
在某个具体实施方式中，所述第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000013
第一化合物具有反应活性高的特性，可作为多种含有氨基的化合物的连接物。如上述四种第一化合物中的方酸接枝于氨基硅烷后，方酸还可以继续与含有氨基的化合物反应，如含有氨基的核酸、聚赖氨酸等。从而，利用第一化合物制备芯片增加选择用于制备芯片基底表面所需的其它化合物的灵活性。利用此类化合物制备芯片，可以根据对芯片探针密度的不同需求，选择不同的含有氨基的化合物与第一化合物进行反应，如选择聚合物大分子聚赖氨酸与方酸反应，将聚合物大分子固定在芯片基底表面，可提高芯片表面与探针固定相关的活性基团的密度。
在某个具体实施方式中，上述接枝是在室温条件下反应3h～5h。
在某个具体实施方式中，上述氨基来自氨基硅烷、聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺 树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
在某个具体实施方式中，上述氨基来自氨基硅烷，所述接枝有氨基的基底包括基材层以及接枝于所述基材层之上的所述氨基硅烷。
在某个具体实施方式中，上述氨基硅烷选自(3-氨基丙基)三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨基丙基甲基二乙氧基硅烷和氨乙基氨丙基三乙氧基硅烷中的至少一种。
在某个具体实施方式中，上述氨基硅烷通过在室温条件下反应1h～8h接枝于所述基材层之上。
本申请实施方式提供的制备方法包括第二制备方法，该方法包括如下步骤：包括如下步骤：(1)获取接枝有氨基的基底，所述氨基为伯氨基或仲氨基；(2)将第一化合物接枝于所述氨基；所述接枝有氨基的基底包括接枝于表面的硅烷和接枝于所述硅烷的聚合物；所述硅烷选自环氧基硅烷、烯基硅烷和炔基硅烷中的至少一种；所述聚合物的至少一个结构单元包含所述氨基，所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000014
其中，R 1、R 2分别独立选自：-OH或C1～C5烷氧基，n为1或2。
在某个具体实施方式中，所述第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000015
在某个具体实施方式中，聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。含有氨基的聚合物的空间结构可提供更高密度的氨基，从而可提高与氨基接枝反应的第一化合物的密度。
在某个具体实施方式中，环氧基硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种；烯基硅烷选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种；炔基硅烷丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
在某个具体实施方式中，硅烷是通过在室温条件下反应1h～8h接枝于所述表面。
在某个具体实施方式中，聚合物是在30℃～65℃温度下反应1h～48h接枝于所述硅烷上。
可以理解地，上述任一实施方式的第二制备方法还可以具有上述任一实施方式的方法的附加技术特征和技术效果，在此不再赘述。
本申请实施方式提供的方法包括第三制备方法，包括如下步骤：(1)获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；(2)使第一化合物接枝于所述氨基；(3)于所述第一化合物上接枝第二化合物，所述第二化合物包含氨基；所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000016
其中，R 1、R 2分别独立选自：-OH或C1～C5烷氧基，n为1或2。
在某些示例中，烷氧基中的烷基可为直连烷基，也可为含有支链的烷基，例如可为-OCH 3、-OCH 2CH 3、-O(CH 2) mCH 3(m＝2～4)、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、-OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。在某个具体实施方式中，R 1、R 2分别独立选自：-OH、-OCH 3或-OCH 2CH 3。
在某个具体实施方式中，所述第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000017
在某个具体实施方式中，第二化合物为氨基修饰的核酸序列。
在某个具体实施方式中，该方法还包括步骤(4)：利用封闭试剂对所述芯片的表面的氨基进行封闭处理。
采用封闭试剂封闭芯片表面的未参与反应的氨基或反应不完全的氨基，可降低芯片表面的氨基对蛋白和/或核酸序列的非特异性吸附，如对DNA聚合酶、模板核酸序列的非特异性吸附，从而降低对测序或核酸杂交等实验流程的的影响。理论上可以去除氨基活性且不影响后续实验过程的试剂都可以用作封闭试剂，例如，封闭试剂可选自但不局限于NHS-PEG4、NHS-PEG4-NHS、NHS-PEG-N3和乙酸酐中的至少一种试剂。
较佳地，在某个具体实施方式中，封闭试剂为NHS-PEG4。
在某个具体实施方式中，核酸序列是在30℃～80℃温度下反应1h～48h接枝于所述第一化合物上。
可以理解，该第三制备方法还可以具有上述任一实施方式中的方法的附加技术特征和优势，在此不再赘述。
本申请实施方式提供的方法包括第四制备方法，包括如下步骤：(1)获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；(2)使第一化合物接枝于所述氨基；(3)于所述第一化合物上接枝第二化合物；所述第二化合物为至少一个结构单元包含氨基的聚合物；所述第一化合物具有如下所示结构特征：
Figure PCTCN2021129931-appb-000018
其中，R 1、R 2分别独立选自：-OH或C1～C5烷氧基，n为1或2。
在第四制备方法的某些示例中，烷氧基中的烷基可为直连烷基，也可为含有支链的烷基，例如可为-OCH 3、-OCH 2CH 3、-O(CH 2) mCH 3(m＝2～4)、-OCH(CH 3) 2、-OC(CH 3) 3、-OCH(CH 3)CH 2CH 3、-OCH(CH 3)CH 2CH 2CH 3、-OCH(CH 3)CH 2CH 3、-OC(CH 3) 2CH 2(CH 3) 2或-OCH 2CH(CH 3) 2等。在某个具体实施方式中，R 1、R 2分别独立选自：-OH、-OCH 3或-OCH 2CH 3。
在某个具体实施方式中，所述第一化合物选自如下化合物中的一种：
Figure PCTCN2021129931-appb-000019
在某个具体实施方式中，该方法还包括步骤(4)：将核酸序列通过连接基团接枝于所述第二化 合物，连接基团的分子结构中包含第一连接基团和第二连接基团，第一连接基团与核酸序列键合，第二连接基团经聚合物包含的氨基接枝于该聚合物。
在某个具体实施方式中，该方法还包括步骤(5)：利用封闭试剂对所述芯片的表面的氨基进行封闭处理。
采用封闭试剂封闭芯片表面的未参与反应的氨基或反应不完全的氨基，可降低芯片表面的氨基对蛋白和/或核酸序列的非特异性吸附，如对DNA聚合酶、模板核酸序列的非特异性吸附，从而降低对测序或核酸杂交等实验流程的的影响。理论上可以去除氨基活性且不影响后续实验过程的试剂都可以用作封闭试剂，例如，封闭试剂可选自但不局限于NHS-PEG4、NHS-PEG4-NHS、NHS-PEG-N3和乙酸酐中的至少一种试剂。在某个具体实施方式中，封闭试剂优先选择乙酸酐。
在某个具体实施方式中，上核酸序列修饰有基团-DBCO或基团-N 3，第一连接基团选自基团-DBCO或基团-N 3，第一连接基团与核酸序列通过基团-DBCO与基团-N 3进行共价键合。可以理解，当核酸序列修饰的基团为-DBCO，第一连接基团为-N 3；当核酸序列修饰的基团为-N 3，第一连接基团为-DBCO。
在某个具体实施方式中，第二连接基团是在室温条件下反应0.5h～5h接枝于所述第二化合物。
在某个具体实施方式中，第一连接基团与核酸序列共价键合的反应条件为30℃～65℃温度下反应1～48h。
在某个具体实施方式中，聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。当第二化合物为至少一个结构单元含有氨基的聚合物时，如聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子或聚丙烯酰胺时，聚合物因其本身的空间结构可在芯片基底表面提供更高密度的氨基，从而可提高固定的核酸序列的密度。
在某个具体实施方式中，聚合物是在30℃～40℃温度下反应1h～5h接枝于所述第一化合物上。
可以理解地，上述任一实施方式中的第四制备方法还可以具有上述任一实施方式中的方法附加技术特征和优势，在此不再赘述。
本申请的实施方式还提供上述任一实施方式的芯片，或上述任一实施方式的方法制备得的芯片在核酸检测中的应用/用途。可以理解的，所称的应用/用途可以包括以疾病的诊断和治疗为目的的应用，也可以包括非以疾病的诊断和治疗为目的的应用，例如空气中微生物的检测、污水中微生物的检测、已死亡动物体的病理检测等。
在某个具体实施方式中，上述核酸检测为测序，例如单分子测序或二代测序。
在实际应用时，根据需要，还可以对芯片进行如下程序中的一种或多种：
(1)检测探针密度：利用带荧光基团的核酸链与芯片表面的探针杂交，用于检测探针的密度；
(2)拍照并计算表面荧光分子数量/密度：在高倍荧光显微镜下，拍摄芯片表面多个视野，用图像软件如ImageJ软件统计图像中的亮斑(亮点)数目，计算一个视野的平均(亮)点数，从而推出荧光分子的密度；或者在普通荧光显微镜下，拍摄芯片表面多个视野，通过图片局部或整体亮度，来计算表面荧光分子的相对密度。以测量得到的表面探针的量/密度情况，来指导后续应用流程中的处理条件；
(3)测量杂交之后的单分子比例(对于单分子测序芯片)：通过在在单分子荧光显微镜联系曝光拍照(movie)，检测杂交之后的所有亮点中有多少亮点是一个荧光分子发出的荧光；
(4)芯片表面扩增(用于二代测序)：使用桥式PCR(bridgePCR)，重组酶聚合酶扩增(RPA)，模板步行(Templatewalking)等方法在芯片表面进行核酸扩增，或者直接将乳液PCR的微珠(beads)加载(loading)在芯片表面。
如下为具体的实施例，除非有特殊说明，所有的实验材料均来自商业供应商或者委托合成/制备(如阿拉丁、西格玛(Sigma)、生工等)。
本文中提及的单分子测序平台，例如Genocare TM1600，是基于边合成边测序(SBS)原理、利用虚拟终止子和TIRF光学检测系统测定核酸分子的核苷酸或碱基排列次序的商业自动化测序平台，可参见文章【Single molecule targeted sequencing for cancer gene mutation detection，Scientific RepoRts|6:26110|DOI:10.1038/srep26110】、已公开的专利申请如CN201680047468.3、CN201910907555.7、CN201880077576.4和/或CN201911331502.1等披露的技术方案进行平台/功能模块的搭建和适配试剂/耗材的制备。Genocare1600可以通过单色SBS(1-channel)、双色(2-channel)或者四色(4-channel)等进行多轮反应来实现测序，例如，通过依次加入四种核苷酸进行四次碱基延伸反应来确定待测核酸分子上的一个位置的核苷酸或碱基类型(1-channel)。具体地，例如，利用四种带有相同荧光标记的核苷酸进行四轮反应，一轮反应加入一种核苷酸并采集信号，进行四轮反应获得四种核苷酸的聚合反应信号，以此来确定模板上的一个位置的核苷酸，然后，重复多轮测序，测得核酸分子/核酸片段的核苷酸组成；再例如，使四种核苷酸两两带有相同的荧光标记，每轮反应加入两种带有不同荧光标记的核苷酸进行反应和信号检测；又例如，使四种核苷酸分别带有四种不同的荧光标记，每轮反应加入四种核苷酸进行反应和信号检测。
实施例中涉及的部分试剂的来源、具体结构或组成如下：
3×SSC缓冲液，由20×SSC缓冲液(西格玛，#S6639-1L)用无核酸酶水(Rnase-free水)稀释而成。
方酸二甲酯：
Figure PCTCN2021129931-appb-000020
聚赖氨酸(PLL)：
Figure PCTCN2021129931-appb-000021
支化度可控制在0.35-0.45之间。
DBCO-PEG4-NHS：
Figure PCTCN2021129931-appb-000022
壳聚糖：
Figure PCTCN2021129931-appb-000023
方酸二乙酯：
Figure PCTCN2021129931-appb-000024
NHS-PEG4：
Figure PCTCN2021129931-appb-000025
D9：tttttttttttccttgatacctgcgaccatccagttccactcagatgtgtataagagacag(SEQ ID No.1)
P1：ttttttttttaatgatacggcgaccaccga(SEQ ID No.2)
P2：ttttttttttcaagcagaagacggcatacga(SEQ ID No.3)
实施例10
本实施例描述了基于方酸和聚赖氨酸(PLL)制备芯片的过程和方法。利用此方法制备的芯片经过荧光密度测定后用于单分子荧光测序。在芯片表面的化合物修饰顺序为：在玻璃基底上通过溶液反应镀上氨基硅烷，再通过与氨基反应连接一层方酸分子，再通过方酸分子连接一层PLL，再通过与PLL中的氨基反应连接上NHS-DBCO，然后使用乙酸酐封闭未参与反应的氨基，最后通过二苯基环辛炔(DBCO)官能团连接上带有叠氮官能团的DNA分子。制备好的芯片可以使用GenoCare1600单分子荧光测序平台进行荧光密度测定，或者在该平台上对核酸进行测序。
具体步骤如下：
(1)清洗、活化玻璃表面(基底表面)：使用乙醇、水交替清洗玻璃表面(如肖特D263M系列玻璃)，并同时进行超声处理，超声时间约10分钟，超声水浴温度37摄氏度。清洗结束后，使用0.5M的NaOH溶液活化玻璃表面2分钟，使玻璃表面产生活性羟基基团。
(2)氨基硅烷修饰：在体积比为1：20的水乙醇混合溶液中加入0.1％体积比的冰乙酸，再加入2.5％体积比的(3-氨基丙基)三乙氧基硅烷后配制成氨基硅烷修饰溶液。将活化后的玻璃片浸泡在氨基硅烷修饰溶液中，室温下反应2小时，反应结束后，用乙醇、水交替清洗，再放置于真空烘箱中110摄氏度干燥。
(3)封装芯片：可参照如WO2017205876A1、EP3590603A1等公开的内容制备具有容纳液体的腔室、进出液口的芯片。例如，粘合玻璃片和蚀刻有通道的基底层以封装形成包含多个独立通道、且指定表面带有氨基硅烷修饰的芯片，各通道是物理隔离的，可以独立的容纳溶液、进行进出液控制和后续反应。图1为具有16条通道的封装后的芯片的俯视示意图，通道的规格例如为长90mm×宽1.8mm×高0.1mm，各通道可以独立地进行进出液控制或反应控制。据检测通量要求，可改变通道尺寸和/或数量。
后续步骤，可采用液体控制设备将各类反应试剂、洗涤试剂等分别通入到封装后的通道内，以在指定表面进行进一步的化学修饰/连接，如此，利于芯片的批量制备，利于获得大量的表面性能参数较为一致的芯片。
(4)方酸修饰：分别将25μL 1mM方酸二甲酯的乙醇溶液通入每个通道中，室温下反应约3小时，之后用乙醇清洗。取200μL 0.1％PLL(分子量150～300K)水溶液，加入200μL乙醇，涡旋混匀，按照每条通道25μL的体积通入通道中，可密封通道口(防止溶液挥发)，放置于37℃烘箱反应3小时，反应结束后。随后分别将25μL的使用0.1M NaHCO 3(pH约为8.3)缓冲液配制20mM的DBCO-PEG4-NHS溶液通入每个通道中，在室温下反应约1小时，反应结束后，可使用1mL3×SSC 缓冲液清洗。再配制乙酸酐反应液(例如497.25μL甲酰胺中加入1.75μL DIPEA(N,N-二异丙基乙胺)和1μL乙酸酐)，涡旋混匀之后，立即通入通道，每个通道25μL，室温反应15min，反应结束后，每个通道使用1mL3×SSC缓冲液清洗。
(5)探针连接：将5nM的N 3-D9溶液(溶剂为3xSSC缓冲液，N 3为D9的5’端连接/修饰叠氮，D9为探针序列(SEQ ID No.1))按照每条通道25μL的体积通入通道中，放置于37℃烘箱反应约16小时。反应结束后，每个通道使用1mL的含有150mM HEPES(4-羟乙基哌嗪乙磺酸)与150mMNaCl的缓冲液清洗一次或多次，可选地，再使用1mL3×SSC缓冲液清洗每个通道一次或多次。
(6)探针量/密度检测：将2nM的D9’-Cy3溶液(溶剂为3×SSC缓冲液，D9’为D9的反向互补序列)按照每条通道25μL的体积通入芯片通道中，放置于40℃烘箱进行杂交反应约30分钟，反应结束后使用1mL 3×SSC缓冲液对每个通道进行清洗。使用GenoCare1600单分子荧光平台检测本实施例中制备的芯片的表面探针密度。
具体的，通过检测D9’-Cy3链上的Cy3荧光，以拍摄视野中的平均荧光点数(亮点数)来评价芯片表面的探针密度。本实施例中统计的是面积为110×110μm的每个拍摄视野(FOV)中的荧光点数。通过拍摄每个芯片通道中间区域的100个视野的荧光点数，取平均值后得到一个FOV视野区间的荧光点数约为32000～38000，如图2所示，16个通道的平均值约为35500。
利用此方法制备的芯片表面的探针密度可以满足GenoCare1600单分子测序平台的要求。
实施例20
本实施例描述了基于方酸和聚赖氨酸(PLL)制备芯片过程和方法。利用此方法制备的芯片用于芯片表面的核酸扩增和/或二代测序。芯片表面的核酸可以使用荧光显微镜进行荧光强度的测定，或者在二代测序平台(如Illumina NextSeq 500测序平台)上进行测序。
具体步骤如下：
(1)利用与实施例10中步骤(1)-(4)相同步骤制备获得指定表面带有PLL修饰的芯片，然后将芯片在室温静置半小时。随后按照体积比为1：1混合10uM的N 3-P1溶液和10uM的N 3-P2溶液(溶剂为3×SSC缓冲液，N 3为P1的5’端连接/修饰叠氮官能团，P1(SEQ ID No.2)和P2(SEQ ID No.3)分别为探针序列)，并按照每条通道25μL的体积通入芯片通道中，密封通道进出液口，放置于37℃烘箱反应约16小时。反应结束后，每个通道使用1mL的含有150mM HEPES与150mMNaCl的缓冲液清洗。随后将25μL3uM的P1’-Cy3(溶剂为3×SSC缓冲液，P1’为P1的反向互补序列)和25μL P2’-Cy3(P2’为P2的反向互补序列))溶液分别通入通道1和通道2中，将芯片放置于40℃烘箱进行杂交反应约30分钟，反应结束后放置芯片至室温后分别使用1mL3×SSC缓冲液清洗。将芯片的进出液口封住保存或进行下一步使用及检测。
(2)使用Illumina NextSeq 500测序平台检测本例中制备的芯片表面的探针密度。具体的，通过检测P1’-Cy3或P2’-Cy3链上的Cy3荧光，以拍照视野中整体的平均荧光亮度来评价芯片表面的探针密度。本例中统计的面积为110×110μm的每个拍摄视野(FOV)中的平均荧光亮度，对于通道1，即P1’-Cy3杂交的通道，一个FOV视野内的平均荧光亮度约为28000～30000，如图3所示，通过拍摄每个芯片通道中间区域的100个视野的荧光点数，取平均值后得到整个通道的平均亮度约为28500。对于通道2，即P2’-Cy3杂交的通道，测得的平均亮度约为25200。所以在此测量体系下，整个通道的探针平均密度对应的荧光亮度为(P1’-Cy3和P2’-Cy3检测的平均亮度之和)为53700。
(3)去除带荧光的杂交链：使用甲酰胺去除带荧光的杂交链。
(4)核酸扩增及测序：以DNA文库作为模板，在芯片表面进行桥式PCR扩增获得扩增簇， 利用带有荧光标记的杂交链标记扩增得到的扩增簇，然后在Illumina NextSeq 500测序平台进行检测。检测结果如图4所示，图4示意的是其中一个通道经拍照后获得的检测结果。之后扩增簇可以在Illumina NextSeq 500测序平台上进行测序检测。
实施例30
本实施例描述了基于方酸和聚赖氨酸(PLL)制备的芯片的过程和方法。利用此方法制备的芯片进行芯片表面的等温扩增或者和/或用于二代测序。
具体步骤如下：
使用与实施例20中步骤(1)-(3)相同步骤制备获得芯片，然后以DNA文库作为模板，使用
Figure PCTCN2021129931-appb-000026
Basic试剂盒(TwistDx,Cambridge UK)中的试剂在芯片表面进行扩增获得扩增簇。
操作步骤如下：将18μL变性为单链的DNA文库中加入
Figure PCTCN2021129931-appb-000027
Basic试剂盒中的29.5μL缓冲液和去核酸水，使得总体积为50μL，文库终浓度为10pM，震荡并短暂离心后，加入2.5μL乙酸镁试剂，充分混匀后，使用移液器将混合好的反应液打入芯片通道，将芯片至于37至42摄氏度孵育15至30分钟，然后使用1mL 3×SSC缓冲液清洗通道，扩增得到的扩增簇可以使用Illumina NextSeq 500测序平台进行测序检测。
实施例40
本实施例描述了基于方酸和壳聚糖(chitosan)制备芯片的过程和方法。利用此方法制备的芯片用于单分子荧光测序、芯片表面的固相扩增或用于二代测序。芯片基底表面(本实施例芯片基底为玻璃)修饰顺序为：通过溶液反应在基地表面镀上环氧硅烷，再通过环氧基团与壳聚糖中氨基的反应修饰上一层壳聚糖，随后再通过壳聚糖中氨基反应连接一层方酸分子，方酸分子作为连接剂连接上带有NH 2官能团的DNA分子，最后使用NHS-PEG4封闭未参与反应的氨基。制备好的芯片使用Illumina NextSeq 500荧光测序平台测定荧光密度，即测定芯片表面核酸分子的密度，再使用模板行走(Tamplatewalking)的方法进行核酸扩增，得到的核酸扩增簇(clusters)用于后续测序。
具体步骤如下：
(1)清洗、活化玻璃表面(基底表面)：使用乙醇、水交替清洗玻璃表面(如肖特D263M系列玻璃)，并同时进行超声处理，超声时间约10分钟，超声水浴温度37摄氏度。清洗结束后，使用0.5M的NaOH溶液活化玻璃表面2分钟，使玻璃表面产生活性羟基基团。
(2)环氧硅烷修饰：将表面活化后的玻璃片浸泡在pH＝5.5，体积比为1：100的3-缩水甘油醚氧基丙基和羟基丙基三甲氧基硅烷混合水溶液中，室温下反应8小时，反应结束后，用乙醇、水交替清洗，再放置于真空烘箱中100摄氏度干燥。
(3)封装芯片：采用已知的封装工艺对表面进行处理以将带环氧硅烷修饰的玻璃片和基底封装形成包含多个独立通道且指定表面具有环氧硅烷修饰的芯片，芯片中的各通道可以独立的进行后续反应。图1为具有16条通道的封装后的芯片的俯视示意图，通道的规格例如为长90mm×宽1.8mm×高0.1mm，各通道可以独立地进行进出液控制或反应控制。据检测通量要求，可改变通道尺寸和/或数量。
后续步骤，可采用液体控制设备将各类反应试剂、洗涤试剂等分别通入到封装后的通道内，以在指定表面进行进一步的化学修饰/连接，如此，利于芯片的自动化制备，利于获得大量的、表面性能参数较为一致的芯片，利于芯片制备的工业化。
(4)方酸修饰：分别将25μL利用0.2M pH＝9.2的碳酸盐缓冲液配置的0.1％的壳聚糖(分子量160K)溶液通入每个芯片通道中，密封通道口(防止溶液挥发)，放置于37℃烘箱反应16小时。反应结束后，每个通道使用1mL3×SSC缓冲液清洗。随后每条通道通入25μL 1mM方酸二乙酯的 乙醇溶液，室温下反应约4小时，之后用乙醇清洗。
(5)探针连接：分别将25μL的使用体积比为1：4的3×SSC和乙醇作为溶剂配制的10uM的NH 2-A50溶液(其中NH 2为A50的5’端修饰/连接的氨基官能团，A50为50个腺嘌呤核苷酸形成的核酸单链)通入每个通道中，放置于37℃烘箱反应约16小时，反应结束后，每个通道用乙醇清洗，再使用1mL的含有150mM HEPES与150mMNaCl的缓冲液清洗。随后分别将25μL的使用0.1M NaHCO 3(pH＝8.3)缓冲液配制的20mM的NHS-PEG4溶液通入每个芯片通道中，在室温下反应约1小时，用于封闭未参与反应的氨基，反应结束后，使用1mL3×SSC缓冲液清洗通道。
(6)探针密度检测：将3uM的T35-Cy3溶液(溶剂为3×SSC缓冲液，Cy3为T35的3’端修饰Cy3荧光基团，T35为35个胸腺嘧啶核苷酸形成的核酸单链)按照每条通道25μL的体积分别通入通道1和通道2中，放置于40℃烘箱进行杂交反应约30分钟，反应结束后将芯片放置至室温，然后使用1mL 3xSSC缓冲液清洗每个通道。使用Illumina NextSeq 500测序平台检测本实施例中制备的芯片的表面探针密密度。
具体的，通过检测通道1和通道2中T35-Cy3链上的Cy3荧光，以拍照视野中的平均荧光亮度来评价DNA芯片的固定密度。本实施例中统计的是面积为110×110μm的每个拍摄视野(FOV)中的平均荧光亮度，通过拍摄每个芯片通道1和通道2中间区域的100个视野，得到一个FOV视野内的平均荧光亮度区间为46000～52000，对所有FOV取平均值后，通道的平均亮度约为47500，如图5所示。所以在此测量体系下，整个通道的探针密度对应47500的荧光亮度。
(7)去除带荧光的杂交链：使用甲酰胺去除带荧光的杂交链T35-Cy3。
(8)核酸扩增及测序：以DNA文库作为模板，在芯片表面进行template walking固相扩增获得扩增簇，利用带有荧光标记的杂交链标记扩增得到的扩增簇，然后在Illumina NextSeq 500测序平台上检测，得到的结果如图6所示。之后扩增簇可以在Illumina NextSeq 500测序平台上进行测序。
实施例50
本实施例描述了基于方酸和氨基基底制备芯片的过程和方法。利用此方法制备的芯片用于单分子荧光测序。在芯片表面的化合修饰顺序为：在玻璃基底上通过溶液反应镀上氨基硅烷，再通过与氨基反应连接一层方酸分子，再通过方酸分子连接带有NH 2的DNA分子，最后使用NHS-PEG4对玻璃基底上可能未参与反应的氨基进行封闭。制备好的芯片可以使用GenoCare1600单分子荧光测序平台进行荧光密度测定，或者在该平台上进行测序应用。
具体步骤如下：
(1)清洗、活化玻璃表面(基底表面)：使用乙醇、水交替清洗玻璃表面(如肖特D263M系列玻璃)，并同时进行超声处理，超声时间约10分钟，超声水浴温度37摄氏度。清洗结束后，使用0.5M的NaOH溶液活化玻璃表面2分钟，使玻璃表面产生活性羟基基团。
(2)氨基硅烷修饰：在体积比为1：20的水乙醇混合溶液中加入0.1％体积比的冰乙酸，再加入2.5％体积比的(3-氨基丙基)三乙氧基硅烷后配制成氨基硅烷修饰溶液。将活化后的玻璃片浸泡在氨基硅烷修饰溶液中，室温下反应2小时，反应结束后，用乙醇、水交替清洗，再放置于真空烘箱中，110摄氏度干燥数小时。
(3)封装芯片：可参照如WO2017205876A1、EP3590603A1等公开的内容制备具有容纳液体的腔室、进出液口的芯片。例如，利用胶等粘性物质将指定表面带有氨基硅烷修饰的玻璃片与另一表面蚀刻有通道的基底层粘合以形成包含多个通道的芯片。图1为具有16条通道的封装后的芯片的俯视图，通道的规格例如为长90mm×宽1.8mm×高0.1mm，各通道可以独立地进行进出液控制 或反应控制。据检测通量要求，可改变通道尺寸和/或数量。
后续步骤，可采用液体控制设备将各类反应试剂、洗涤试剂等分别通入到封装后的通道内，实现芯片表面的化学反应。
(4)方酸修饰：分别将25μL1mM方酸二甲酯的乙醇溶液通入每个芯片通道中，室温下反应约3小时，之后用乙醇清洗2-4次。
(5)探针连接：分别将25μL的使用体积比为1：4的3×SSC和乙醇作为溶剂配制的10nM的NH2-D9溶液(其中NH 2为D9的5’端修饰/连接的氨基官能团，探针序列)通入每个芯片通道中，密封通道口，放置于37℃烘箱反应约16小时，反应结束后，每个通道用乙醇清洗，再使用1mL的含有150mM HEPES与150mMNaCl的缓冲液清洗。随后分别将25μL的使用0.1M NaHCO 3(pH＝8.3)缓冲液配制的20mM的NHS-PEG4溶液通入每个芯片通道中，在室温下反应约1小时，封闭之前未参与反应的氨基，反应结束后，可使用1mL3×SSC缓冲液清洗每个通道。
(6)探针密度检测：将2nM的D9’-Cy3溶液(溶剂为3×SSC缓冲液)按照每条通道25μL的体积通入每个芯片通道中，放置于40℃烘箱进行杂交反应约30分钟，反应结束后使用1mL 3×SSC缓冲液清洗通道。使用GenoCare1600单分子荧光平台检测本实施例中制备的芯片表面探针密度。具体的，通过检测D9’-Cy3链上的Cy3荧光，以拍照视野中的荧光点数来评价DNA芯片的固定密度。本实施例中统计的是面积为110×110μm的每个拍摄视野(FOV)中的荧光点数。通过拍摄每个芯片通道中间区域的100个视野，取平均值后得到一个FOV视野区间的荧光点数约为39000。利用此方法制备的芯片表面的探针密度可以满足GenoCare1600单分子测序平台的要求。
以上实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上实施例详细表述了本申请申请的几种实施方式，但并不能因此而理解为对申请范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请请求的保护范围。

Claims (47)

  1. 一种芯片，其特征在于，包括：
    表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及
    经所述氨基接枝于所述表面的第一化合物，所述第一化合物包含如下结构：
    Figure PCTCN2021129931-appb-100001
    其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
  2. 根据权利要求1所述的芯片，其特征在于，R 1、R 2分别独立地选自-OH、-OCH 3或-OCH 2CH 3。
  3. 根据权利要求1所述的芯片，其特征在于，所述第一化合物包含如下结构中的一种：
    Figure PCTCN2021129931-appb-100002
  4. 根据权利要求1～3任一项所述的芯片，其特征在于，所述氨基来自氨基硅烷、聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  5. 根据权利要求4所述的芯片，其特征在于，所述氨基来自氨基硅烷。
  6. 根据权利要求5所述的芯片，其特征在于，所述氨基硅烷选自(3-氨基丙基)三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨基丙基甲基二乙氧基硅烷和氨乙基氨丙基三乙氧基硅烷中的至少一种。
  7. 根据权利要求1～3任一项所述的芯片，其特征在于，所述表面接枝有氨基的基底包括连接于所述表面的硅烷和经所述硅烷连接于所述表面的聚合物，
    所述硅烷选自环氧硅烷、烯基硅烷和炔基硅烷中的至少一种，
    所述聚合物包含多个结构单元，至少一个所述结构单元包含所述氨基。
  8. 根据权利要求7所述的芯片，其特征在于，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  9. 根据权利要求7所述的芯片，其特征在于，所述硅烷包含环氧硅烷，所述环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种。
  10. 根据权利要求7所述的芯片，其特征在于，所述硅烷包含烯基硅烷，所述烯基硅烷选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种。
  11. 根据权利要求7所述的芯片，其特征在于，所述硅烷包含炔基硅烷，所述炔基硅烷选自丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
  12. 根据权利要求1～11任一项所述的芯片，其特征在于，所述芯片还包括与所述第一化合物连接的、至少一个末端具有氨基修饰的核酸序列。
  13. 根据权利要求1～3任一项所述的芯片，其特征在于，所述芯片还包括与所述第一化合物连接的聚合物，所述聚合物包含多个结构单元，至少一个所述结构单元包含氨基。
  14. 根据权利要求13所述的芯片，其特征在于，所述芯片还包含连接于所述聚合物的核酸序列。
  15. 根据权利要求14所述的芯片，其特征在于，所述聚合物和所述核酸序列通过连接基团连接，所述连接基团包含能够与所述核酸序列键合的第一末端以及能够与所述聚合物的所述氨基连接的第二末端。
  16. 根据权利要求15所述的芯片，其特征在于，所述第二末端包含-NHS、环氧基团和异氰酸酯基团中的至少一种。
  17. 根据权利要求15所述的芯片，其特征在于，所述核酸序列带有-DBCO或-N 3修饰，所述第一末端包含-DBCO或-N 3。
  18. 根据权利要求13～17任一项所述的芯片，其特征在于，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  19. 一种制备芯片的方法，其特征在于，包括：
    获取表面接枝有氨基的基底，所述氨基为伯氨基或仲氨基；以及
    使第一化合物通过所述氨基连接至所述表面，所述第一化合物包含如下结构：
    Figure PCTCN2021129931-appb-100003
    其中，R 1、R 2分别独立地选自-OH或C1～C5烷氧基，n为1或2。
  20. 根据权利要求19所述的方法，其特征在于，R 1、R 2分别独立地选自-OH、-OCH 3或-OCH 2CH 3。
  21. 根据权利要求19所述的方法，其特征在于，所述第一化合物选自如下化合物中的一种：
    Figure PCTCN2021129931-appb-100004
  22. 根据权利要求19～21任一项所述的方法，其特征在于，所述氨基来自氨基硅烷、聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  23. 根据权利要求19～21任一项所述的方法，其特征在于，在室温条件下反应3h～5h以使所述第一化合物与所述氨基连接。
  24. 根据权利要求22所述的方法，其特征在于，所述氨基来自氨基硅烷。
  25. 根据权利要求24所述的方法，其特征在于，所述氨基硅烷选自(3-氨基丙基)三乙氧基硅烷、3-氨基丙基三甲氧基硅烷、3-氨基丙基甲基二乙氧基硅烷和氨乙基氨丙基三乙氧基硅 烷中的至少一种。
  26. 根据权利要求24或25所述的方法，其特征在于，在室温条件下反应1h～8h使所述氨基硅烷连接于所述表面，以获得所述表面接枝有氨基的基底。
  27. 根据权利要求19～21任一项所述的方法，其特征在于，所述表面接枝有氨基的基底包括连接于所述表面的硅烷和经所述硅烷连接于所述表面的聚合物，
    所述硅烷选自环氧硅烷、烯基硅烷和炔基硅烷中的至少一种，
    所述聚合物包含多个结构单元，至少一个所述结构单元包含所述氨基。
  28. 根据权利要求27所述的方法，其特征在于，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  29. 根据权利要求27所述的方法，其特征在于，所述硅烷包含所述环氧硅烷，所述环氧硅烷选自3-缩水甘油醚氧基丙基三甲氧基硅烷、3-缩水甘油醚氧基丙基三乙氧基硅烷、3-缩水甘油醚氧基丙基甲基二乙氧基硅烷和3-缩水甘油醚氧基丙基甲基二甲氧基硅烷中的至少一种。
  30. 根据权利要求27所述的方法，其特征在于，所述硅烷包含所述烯基硅烷，所述烯基硅烷选自乙烯基三甲氧基硅烷、乙烯基三乙氧基硅烷、乙烯基三(2-甲氧基乙氧基)硅烷和乙烯基三异丙氧基硅烷中的至少一种。
  31. 根据权利要求27所述的方法，其特征在于，所述硅烷包含所述炔基硅烷，所述炔基硅烷选自丙炔氧基三甲氧基硅烷和[3-(三乙氧基硅烷基)丙基]氨基甲酸2-丙炔酯中的至少一种。
  32. 根据权利要求27～31任一项所述的方法，其特征在于，在室温下反应1h～8h以将所述硅烷连接至所述表面。
  33. 根据权利要求27～31任一项所述的方法，其特征在于，在30℃～65℃温度下反应1h～48h以使所述聚合物连接至所述硅烷。
  34. 根据权利要求19～21任一项所述的方法，其特征在于，还包括：
    使至少一个末端具有氨基修饰的核酸序列经所述氨基修饰连接于所述第一化合物核酸序列。
  35. 根据权利要求34所述的方法，其特征在于，还包括：
    利用封闭试剂处理所述表面，所述封闭试剂选自NHS-PEG4、NHS-PEG4-NHS、NHS-PEG-N3和乙酸酐中的至少一种。
  36. 根据权利要求34或35所述的方法，其特征在于，核酸序列在30℃～80℃下反应1h～48h以使所述核酸序列连接于所述第一化合物。
  37. 根据权利要求19～21任一项所述的方法，其特征在于，还包括：
    使聚合物与所述第一化合物相连，所述聚合物包括多个结构单元，至少一个所述结构单元包含氨基。
  38. 根据权利要求37所述的方法，其特征在于，还包括：
    核酸序列通过连接基团使核酸序列连接于所述聚合物，所述连接基团包含能够与所述核酸序列连接的第一末端，以及能够与所述聚合物的氨基连接的第二末端。
  39. 根据权利要求37所述的方法，其特征在于，还包括：
    利用封闭试剂对所述表面进行处理，所述封闭试剂选自NHS-PEG4、NHS-PEG4-NHS、 NHS-PEG-N3和乙酸酐中的至少一种。
  40. 根据权利要求38所述的方法，其特征在于，所述核酸序列带有-DBCO或-N 3修饰，所述第一末端包含-DBCO或-N 3。
  41. 根据权利要求38所述的方法，其特征在于，所述第二末端包含-NHS基团、环氧基团和异氰酸酯基团中的至少一种。核酸序列
  42. 根据权利要求40所述的方法，其特征在于，于30℃～65℃温度下反应1～48h以使所述连接基团的第一末端与所述核酸序列连接。
  43. 根据权利要求41所述的方法，其特征在于，于室温下反应0.5h～5h以使所述连接基团的第二末端与所述聚合物的氨基连接。
  44. 根据权利要求37～43任一项所述的方法，其特征在于，所述聚合物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子、聚丙烯酰胺和聚乙烯亚胺化合物中的至少一种。
  45. 根据权利要求44所述的方法，其特征在于，于30℃～40℃下反应1h～5h以使所述聚合物与所述第一化合物连接。
  46. 权利要求1～18任一项所述的芯片或利用权利要求19～44任一项所述的方法制备得的芯片在核酸检测中的用途。
  47. 根据权利要求46所述的用途，其特征在于，所述核酸检测为测序。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4908404A (en) * 1988-08-22 1990-03-13 Biopolymers, Inc. Synthetic amino acid-and/or peptide-containing graft copolymers
US20010010902A1 (en) * 1995-03-04 2001-08-02 Jorg Kleiber Sequence-specific detection of nucleic acids
US20060068204A1 (en) * 2004-09-30 2006-03-30 3M Innovative Properties Company Substrate with attached dendrimers
WO2017202917A1 (en) * 2016-05-27 2017-11-30 F. Hoffmann-La Roche Ag Tagged multi-nucleotides useful for nucleic acid sequencing
WO2017205876A1 (en) 2016-05-27 2017-11-30 Idex Health & Science Llc Methods and apparatus for coated flowcells
CN110511973A (zh) * 2019-07-16 2019-11-29 艾吉泰康生物科技(北京)有限公司 一种用于核酸原位合成的固相载体及其制备方法
EP3590603A1 (en) 2018-07-03 2020-01-08 Illumina, Inc. Interposer with first and second adhesive layers

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4341524C2 (de) * 1993-12-06 1997-01-16 Gluesenkamp Karl Heinz Dr Verfahren zur Immobilisierung von Biomolekülen und Affinitätsliganden an polymere Träger
ES2272099T3 (es) * 1998-10-16 2007-04-16 Ge Healthcare Bio-Sciences Ab Composicion estable en el almacenamiento que comprende un soporte activado por acido escuarico utilizable para la inmovilizacion de compuestos que contienen grupos de amina.
DE19850680A1 (de) * 1998-11-03 2000-05-04 Peter Nehls Verfahren zur Bestimmung der Reparaturkapazität von DNS-Reparaturenzyme enthaltenden Lösungen und zum Nachweis von DNS-Strukturmodifikationen und Basenfehlpaarungen
EP1476753B1 (en) * 2001-11-14 2013-08-14 Luminex Corporation Functionalized compositions for improved immobilization

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4908404A (en) * 1988-08-22 1990-03-13 Biopolymers, Inc. Synthetic amino acid-and/or peptide-containing graft copolymers
US20010010902A1 (en) * 1995-03-04 2001-08-02 Jorg Kleiber Sequence-specific detection of nucleic acids
US20060068204A1 (en) * 2004-09-30 2006-03-30 3M Innovative Properties Company Substrate with attached dendrimers
WO2017202917A1 (en) * 2016-05-27 2017-11-30 F. Hoffmann-La Roche Ag Tagged multi-nucleotides useful for nucleic acid sequencing
WO2017205876A1 (en) 2016-05-27 2017-11-30 Idex Health & Science Llc Methods and apparatus for coated flowcells
EP3590603A1 (en) 2018-07-03 2020-01-08 Illumina, Inc. Interposer with first and second adhesive layers
CN110511973A (zh) * 2019-07-16 2019-11-29 艾吉泰康生物科技(北京)有限公司 一种用于核酸原位合成的固相载体及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCIENTIFIC REPORTS, vol. 6, pages 26110

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