WO2022033453A1 - 氨基修饰的芯片及其制备方法和应用 - Google Patents

氨基修饰的芯片及其制备方法和应用 Download PDF

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WO2022033453A1
WO2022033453A1 PCT/CN2021/111672 CN2021111672W WO2022033453A1 WO 2022033453 A1 WO2022033453 A1 WO 2022033453A1 CN 2021111672 W CN2021111672 W CN 2021111672W WO 2022033453 A1 WO2022033453 A1 WO 2022033453A1
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group
high polymer
preparation
nhs
linking group
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PCT/CN2021/111672
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French (fr)
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冯叠文
林志峰
樊济才
刘磊
陈方
王�琦
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深圳市真迈生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/06—Coating with compositions not containing macromolecular substances
    • C08J7/065—Low-molecular-weight organic substances, e.g. absorption of additives in the surface of the article
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2463/00—Characterised by the use of epoxy resins; Derivatives of epoxy resins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2477/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
    • C08J2477/02—Polyamides derived from omega-amino carboxylic acids or from lactams thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2477/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
    • C08J2477/04—Polyamides derived from alpha-amino carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2479/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen, or carbon only, not provided for in groups C08J2461/00 - C08J2477/00
    • C08J2479/02—Polyamines

Definitions

  • the present application relates to the field of chip technology, in particular to an amino-modified chip and a preparation method and application thereof.
  • Gene chip also known as DNA chip, biochip
  • Gene chip technology is a product that complies with the requirements of this scientific development, and can be used to prepare and/or analyze biomolecules, specifically referring to the immobilization of probe molecules (not limited to nucleic acid sequences) on a support After hybridization with the labeled sample molecules, the quantity and sequence information of the sample molecules can be obtained by detecting the hybridization signal intensity of each probe molecule.
  • Chips are typically made from polymer-coated substrates, and molecular analysis and/or preparation, such as certain nucleic acid sequencing methods, relies on the binding of nucleic acid strands to the surface of the chip substrate, which is then determined by a number of different methods known in the art the sequence of the nucleic acid strand.
  • Existing substrates are typically flow cells, and current procedures for coating the flow cells include transferring the polymeric mixture into channels on the flow cell and incubating for a fixed period of time. The procedure is simple and produces reliable coatings that consistently support all downstream chemical processing steps including bridge amplification and sequencing.
  • the amino-modified chip can load probes at a higher density, meet the ever-evolving requirements for biomolecule preparation and/or analysis, and has good stability.
  • the embodiment of the present application provides an amino-modified chip, comprising:
  • the substrate is modified with an epoxy group
  • At least one structural unit of the high polymer contains an amino group, and the amino group is a primary amino group or/and a secondary amino group.
  • each of the structural units in the high polymer comprises 0.05 to 10 identical or different amino groups on average.
  • each of the structural units in the high polymer comprises 1 to 5 identical or different amino groups on average.
  • the polymer has a degree of polymerization ranging from 10 to 5,000.
  • the structural units in the polymer include at least one of them.
  • the high polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, and polyethyleneimine.
  • the chip further comprises probes grafted to the polymer.
  • the probe is bonded to the polymer 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 A linking group is bonded to the probe, and the second linking group is grafted to the high polymer through the amino group.
  • the second linking group is selected from at least one of a -NHS group, an epoxy group, and an isocyanate group.
  • the probe is modified with a group -DBCO or a group -N 3 ; the bonding mode is a covalent bond between the group -DBCO and the group -N 3 .
  • the embodiment of the present application also provides a method for preparing a chip, comprising the following steps:
  • At least one structural unit of the high polymer contains an amino group, and the amino group is a primary amino group or a secondary amino group.
  • each of the structural units in the high polymer comprises 0.05 to 10 identical or different amino groups on average.
  • each of the structural units in the high polymer comprises 1 to 5 identical or different amino groups on average.
  • the polymer has a degree of polymerization ranging from 10 to 5,000.
  • the structural units in the polymer include at least one of them.
  • the high polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers, and polyethyleneimine.
  • step (2) the reactive grafting is performed by contacting the high polymer with the substrate in an alkaline solution, the pH of the alkaline solution is 8.5 ⁇ 10.
  • step (2) the reaction grafting is carried out at a reaction temperature of 37-55° C. for 3-24 hours.
  • the preparation method further comprises step (3): grafting probes to the high polymer.
  • the probe is grafted with the polymer 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 A linking group is bonded to the probe, and the second linking group is grafted to the high polymer through the amino group.
  • the second linking group is selected from at least one of a -NHS group, an epoxy group, and an isocyanate group.
  • the probe is modified with a group -DBCO or a group -N 3 ; the bonding mode is a covalent bond between the group -DBCO and the group -N 3 .
  • the reactive grafting is performed by contacting the linking group with the high polymer in an alkaline solution, the pH of the alkaline solution being 7-9.
  • reaction grafting is carried out under the condition that the reaction temperature is room temperature for 30-90 min.
  • the covalent bonding is performed by contacting the probe with the linking group in an alkaline solution having a pH of 7-8.
  • the alkaline solution for the covalent bonding contains a surfactant, and the surfactant is selected from the group consisting of tetradecyltrimethylammonium bromide, cetyltrimethylammonium At least one of ammonium bromide and dodecyltrimethylammonium bromide.
  • the embodiments of the present application also provide the application of the chip as described above, or the chip prepared by the preparation method as described above, in biomolecule preparation or biomolecule analysis.
  • a substrate modified with a specific active group is reactively grafted with a high polymer containing primary amino groups and/or secondary amino groups.
  • the high polymer can form a high density and react with
  • the modified surface with high activity is used for grafting probes, which can load probes with higher density to meet the evolving needs of biomolecule preparation and/or analysis, and has good stability.
  • the method for preparing a chip disclosed in another embodiment of the present application can realize the grafting of probes without strict control of reaction conditions, so that the chip fabrication process is simple and easy to control, which is beneficial to the popularization and application of the chip.
  • Fig. 1 is a schematic diagram of the preparation process of the chip in the embodiment, X represents a polymer, and primer represents a primer/probe;
  • Fig. 2 is the probe density detection result graph of the chip prepared by embodiment 1-5;
  • Example 3 is a diagram showing the detection results of DNA clusters generated by the chip prepared in Example 1;
  • Fig. 4 is the DNA cluster detection result diagram generated by the chip prepared in Example 2;
  • FIG. 6 is a diagram showing the detection results of DNA clusters generated by the chip prepared in Example 4.
  • Example 7 is a diagram showing the detection results of DNA clusters generated by the chip prepared in Example 5;
  • FIG. 8 is a schematic diagram of the structure of the DNA library used in Example 7.
  • FIG. 8 is a schematic diagram of the structure of the DNA library used in Example 7.
  • amino-modified chip of the present application and its preparation method and application are described in further detail below with reference to specific examples.
  • the application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the present disclosure is provided.
  • the chip described herein may be a substrate grafted with the polymer, or a substrate further grafted with probes on the polymer.
  • the base material is not particularly limited, and the base is formed of at least one of glass, silicon wafer, plastic, gel and nylon film.
  • grafted on and modified with can mean directly grafted and modified on the object, or can further mean grafted and modified on the object through other transition groups.
  • Amino as used herein means, having the structural features of formula -N(X) 2 , wherein each "X" is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkane group, substituted or unsubstituted heterocyclic group, etc., it is understood that in the technical scheme of the present application, at least one X is H.
  • Non-limiting types of amino groups include -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. Suitable examples include, but are not limited to: methyl (Me, -CH3 ), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n - propyl, -CH2CH2CH ) 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(CH 3 ) )CH 2 CH 3 ), 2-methyl
  • cycloalkyl refers to a non-aromatic hydrocarbon containing ring carbon atoms, which may be monocycloalkyl, or spirocycloalkyl, or bridged cycloalkyl. Suitable examples include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Additionally, “cycloalkyl” may also contain one or more double bonds, and representative examples of cycloalkyl groups containing double bonds include cyclopentenyl, cyclohexenyl, cyclohexadienyl, and cyclobutadienyl .
  • Aryl refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing one hydrogen atom, which can be a monocyclic aryl group, a fused-ring aryl group, or a polycyclic aryl group.
  • polycyclic ring species at least One is an aromatic ring system. Suitable examples include, but are not limited to, benzene, biphenyl, naphthalene, anthracene, phenanthrene, perylene, triphenylene, and derivatives thereof.
  • Heterocyclyl means that at least one carbon atom is replaced by a non-carbon atom on the basis of a cycloalkyl group. Suitable examples include, but are not limited to: dihydropyridyl, tetrahydropyridyl (piperidinyl), tetrahydrothienyl, sulfur-oxidized tetrahydrothienyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinoline base, indoline.
  • Structural unit refers to the unit structure formed by one of the monomers in the polymer.
  • the high polymer may be formed by the polymerization of one monomer, or by the copolymerization of two or more different types of monomers, so the “structural units” may be the same or different.
  • degree of polymerization 2 ⁇ (algebra+1)-1, such as PamamDendrimer 3.0 generation, its degree of polymerization is 15.
  • the embodiment of the present application provides an amino-modified chip, comprising:
  • the substrate is modified with an epoxy group
  • At least one structural unit of the high polymer comprises an amino group, and the amino group is a primary amino group and/or a secondary amino group.
  • the chip can be a chip that can be used for gene detection, and can also be a chip that can be used to detect proteins, polypeptides, and the like.
  • each of the structural units in the high polymer contains 0.05 to 10 identical or different amino groups on average.
  • the structural unit may contain amino groups or may not contain amino groups, and the "average” refers to the average value obtained by dividing the total number of amino groups by the total number of the structural units.
  • the high polymer is a copolymer formed by polymerizing two or more monomers, wherein at least one of the monomers does not contain amino groups.
  • each of the structural units in the high polymer contains 0.5 amino groups on average, and the high polymer can be composed of 19 structural units without amino groups and 1 structural unit containing amino groups (containing 10 amino groups). The number of amino groups can be controlled by controlling the monomer combination during polymerization.
  • each of the structural units in the high polymer contains 1 to 5 amino groups on average.
  • the probe is connected to the surface of the chip through the linking group to the amino group of the polymer, so that each structural unit of the polymer contains 1-5 amino groups on average, which can better regulate the probe's Connecting density/quantity, so as to optimize the regulation of probe density, and at the same time, make it easier for probes to be grafted onto polymers through linking groups, and the density of probes is higher.
  • the polymerization degree of the high polymer ranges from 10 to 5000.
  • the molecular weight of the high polymer is 30KD-300KD. The higher the molecular weight, the higher the density of graftable nucleic acid chains, but at the same time, the effectiveness of the polymer to be immobilized on the surface of the substrate is increased. Therefore, in the embodiments of the present application, preferably, the molecular weight of the high polymer is 30KD-300KD.
  • the polyamide-amine dendrimer taking PamamDendrimer as an example, its generation number can be 3 to 11 generations.
  • the high polymer contains the same or different structural units, which can be commercially available or can be customized by a manufacturer, as long as it meets the requirements of the application on the number of amino groups contained in the polymer or/and the degree of polymerization of the polymer That's it.
  • the structural units in the high polymer include at least one of them.
  • the high polymer comprises the same structural unit, for example, the high polymer comprises the same structural unit the copolymer.
  • the high polymer comprises different structural units, eg, the high polymer comprises different structural units Copolymers, such as those disclosed in Kousaku Ohkawaa, Biodegradation of ornithine-containing polylysine hydrogels, Biomaterials 19 (1998) 1855-1860. include ornithine and lysine copolymers (Lys Orn) polymerized in different proportions , or polymers containing different structural units the copolymer.
  • the high polymer is selected from at least one of polylysine, polyornithine, chitosan, polyamidoamine dendrimers and polyethyleneimine.
  • the above-mentioned chip further comprises probes grafted on the high polymer.
  • the probe binds to the target molecule to capture the target molecule.
  • different probe types are selected, for example, oligonucleotide fragments, polypeptide sequences, fragments containing oligonucleotides, or fragments containing polypeptide sequences are selected.
  • the probes can selectively label fluorescent detection molecules, Detection molecules such as antigen, biotin or streptavidin.
  • the probe can be a nucleic acid chain and/or a polypeptide.
  • the probe is grafted with the high polymer through a linking group
  • the molecular structure of the linking group includes a first linking group and a second linking group; the One end of the first linking group is bonded to the nucleic acid strand probe, and the other end of the second linking group is grafted to the high polymer through the amino group.
  • the second linking group is selected from at least one of -NHS group, epoxy group and isocyanate group.
  • the probe is modified with a group -DBCO or a group -N 3 ; the bonding method is a covalent bond between the group -DBCO and the group -N 3 .
  • NHS is the abbreviation of succinimidyl ester
  • PEG is the abbreviation of polyethylene glycol
  • DBCO is the abbreviation of diphenylcyclooctyne
  • N3 is the abbreviation of azide.
  • the nucleic acid chain and the polymer are connected via the above-mentioned linking group.
  • NHS can be stably grafted with the amino group
  • DBCO or N can be modified with N or DBCO at room temperature.
  • the Click reaction is carried out without catalyst, the reaction conditions are mild, and the efficiency is high.
  • the embodiment of the present application also provides a method for preparing a chip, comprising the following steps:
  • At least one structural unit of the high polymer contains an amino group, and the amino group is a primary amino group or a secondary amino group.
  • step (1) is the step of obtaining a substrate, wherein the obtaining can be directly purchased, that is, directly purchased to obtain a modified epoxy-based substrate, or it can be self-made, and the self-made method can be by solution reaction, or by plasma
  • the coating method modifies the epoxy group-containing compound on the carrier.
  • the epoxy group-containing compound may be selected from epoxysilanes, such as 3-(2,3glycidoxy)propyltrimethoxysilane.
  • the material of the carrier is at least one of glass, silicon wafer, plastic, gel and nylon film.
  • the reaction conditions for modifying a compound containing an epoxy group on a carrier by a solution reaction include: the reaction temperature is room temperature, and the reaction time is 1-8 hours. Further, after the reaction is completed, drying is carried out under the condition of 80-150°C.
  • the carrier needs to be deeply cleaned and activated.
  • the alkali solution activation can be treated with 0.1M-2M NaOH aqueous solution for 1-20 minutes.
  • step (2) is a polymer grafting step.
  • the substrate modified is an epoxy group, and in step (2), the amino group is reacted and grafted with the epoxy group.
  • the reactive grafting is performed by contacting the high polymer with the substrate in an alkaline solution, and the pH of the alkaline solution is 8.5-10.
  • the pH values include, but are not limited to, the following values: 8.5, 8.8, 9, 9.16, 9.2, 9.5, 10.
  • the alkaline solution with a pH value of 8.5-10 is selected from at least one of phosphate buffer, carbonate buffer and borate buffer.
  • concentration of the buffer pair in the buffer with a pH value of 8.5-10 is 10-300 mM. Concentrations of the buffer pair include, but are not limited to, the following concentrations: 10 mM, 50 mM, 100 mM, 120 mM, 150 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 220 mM, 250 mM, 300 mM.
  • a carbonate buffer with a pH value of 9.16 is used as the reaction solvent, wherein the concentration of the buffer pair is 200 mM.
  • reaction grafting is carried out at a reaction temperature of 37-55° C. for 3-24 hours.
  • the combination of reaction temperature and reaction time includes but is not limited to the following combinations: 37°C*14h, 37°C*16h, 37°C*18h, 37°C*20h, 37°C*24h, 40°C*12h , 42°C*18h, 45°C*10h, 50°C*8h, 55°C*5h, 55°C*3h.
  • the reaction temperature is 37°C
  • the reaction time is 16h.
  • the preparation method of the chip further comprises step (3): grafting probes to the high polymer.
  • Step (3) is a probe grafting step.
  • the probe in step (3), is grafted with the high polymer through a linking group, and the molecular structure of the linking group includes a first linking group and a second linking group.
  • the second linking group is selected from at least one of -NHS group, epoxy group and isocyanate group.
  • step (3) can be divided into the following two steps to carry out:
  • the probe and the polymer are connected via the above-mentioned linking group.
  • -NHS can be relatively stably grafted with the amino group;
  • -DBCO or -N 3 can be modified with -N 3 or -DBCO.
  • the probe performs the Click reaction at room temperature without catalyst, with mild reaction conditions and high efficiency.
  • the active group-modified probe is modified with a group -DBCO or a group -N 3 ; the bonding method is a covalent bond between the group -DBCO and the group -N 3 . More specifically, the covalent bonding is a Click reaction.
  • the probe modified with the active group is a nucleic acid chain modified with the active group at the 5' end.
  • the linking group is prepared from NHS-PEGn-DBCO, that is, the group used for bonding is -DBCO, then the active group modified at the 5' end is -N 3 functional group; if the The linking group is prepared from NHS-PEGn-N 3 , that is, the group used for bonding is -N 3 , and the active group modified at the 5' end is -DBCO functional group.
  • the concentration of the compound providing the linking group in the reaction system is 50 ⁇ M to 5 mM.
  • concentrations include, but are not limited to, the following concentrations: 50 ⁇ M, 100 ⁇ M, 500 ⁇ M, 800 ⁇ M, 1 mM, 1.2 mM, 1.5 mM, 2 mM, 5 mM.
  • step (3-1) the reactive grafting is performed by contacting the linking group with the high polymer in an alkaline solution, the alkaline solution
  • the pH value is 7-9.
  • the pH value includes but is not limited to the following values: 7, 7.3, 7.5, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5.
  • the alkaline solution is selected from at least one of phosphate buffer and carbonate buffer.
  • the concentration of the buffer pair in the phosphate buffer or carbonate buffer is 10 mM to 300 mM.
  • Concentrations of the buffer pair include, but are not limited to, the following concentrations: 10 mM, 50 mM, 80 mM, 90 mM, 95 mM, 98 mM, 100 mM, 102 mM, 105 mM, 110 mM, 150 mM, 200 mM, 210 mM, 220 mM, 250 mM, 300 mM.
  • the reaction grafting uses a phosphate buffer with a pH value of 8 as a reaction solvent, wherein the concentration of the buffer pair is 100 mM.
  • the reaction grafting uses a carbonate buffer with a pH value of 8.3 as a reaction solvent, wherein the concentration of the buffer pair is 100 mM.
  • step (3-1) the reaction grafting is carried out under the condition that the reaction temperature is room temperature for 30-90 minutes.
  • the reaction time includes but is not limited to the following times: 30min, 40min, 50min, 55min, 58min, 60min, 62min, 65min, 70min, 80min, 90min.
  • step (3-2) the covalent bonding is performed by contacting the probe with the linking group in an alkaline solution, and the alkaline solution is The pH value is 7-8. Specifically, the pH value includes but is not limited to the following values: 7, 7.3, 7.5, 7.7, 7.8, 7.9, 8. More specifically, the alkaline solution is a citrate buffer. More specifically, the covalent bonding is carried out at room temperature.
  • the alkaline solution contains a surfactant, and the surfactant is selected from tetradecyl trimethyl ammonium bromide (TTAB), hexadecyl trimethyl bromide At least one of ammonium (CTAB) and dodecyltrimethylammonium bromide (DTAB).
  • TTAB tetradecyl trimethyl ammonium bromide
  • CTAB ammonium
  • DTAB dodecyltrimethylammonium bromide
  • the covalent bonding is performed by contacting the probe with the linking group in a citrate buffer supplemented with 1 mM TTAB at a reaction temperature of room temperature.
  • redundant functional groups can be selectively blocked before or after the probe is bonded, and the redundant functional groups can be, for example, -NH 2 , -DBCO, -N 3 and the like.
  • DIPEA Diethylamine
  • the embodiments of the present application also provide the application of the above chip, or the chip prepared by the above preparation method, in biomolecule preparation or biomolecule analysis.
  • the biomolecule preparation or biomolecule analysis can be, for example, nucleic acid sequencing, library hybridization, DNA cluster generation, etc., or protein detection, polypeptide detection, and the like.
  • Polyamide-amine dendrimer (PamamDendrimer), Pamam Dendrimer3.0 generation (abbreviation: PD3.0), molecular weight 6905.84, its molecular formula is as follows:
  • DMSO dimethyl sulfoxide
  • PEI Polyethyleneimine
  • Polylysine (PLL), molecular weight 150-300KD, its molecular formula is as follows:
  • PLO Polyornithine
  • Chitosan has a molecular weight of 100KD and the molecular formula is as follows:
  • Tetradecyltrimethylammonium bromide was purchased from sigma;
  • Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from sigma;
  • 20xSSC is purchased from Shenggong; Hepes is purchased from sigma; Tween20 is purchased from sigma;
  • SDS Sodium dodecyl sulfate
  • Washing solution 1 150mM Hepes+0.1%Tween20+0.1%SDS;
  • PLO polyornithine
  • step 2 Pass the solution configured in step 1 into the channel of the epoxy-modified substrate, pass 25 ⁇ l of the solution into each channel, seal the channel mouth (to prevent the solution from volatilizing), and react at 37° C. for 16 hours;
  • Functional groups modify the PLO-coated substrate.
  • Functional groups refer to groups that participate in subsequent reactions, which can be -N3, -DBCO and other groups. In this embodiment, the functional group is -N3 group.
  • the specific steps of functional group modification as follows:
  • step 4) Immediately pass the solution configured in step 3) into the substrate channel, 25 ⁇ l/channel; react at room temperature for 1 h;
  • the functional group-modified substrate can be immersed in RI02 solution and stored at 4°C for 1 year.
  • PLL polylysine
  • step 2 Pass the solution configured in step 1 into the channel of the epoxy-modified substrate, pass 25 ⁇ l of the solution into each channel, seal the channel mouth (to prevent the solution from volatilizing), and react at 37° C. for 16 hours;
  • Functional groups modify PLL-coated substrates.
  • Functional groups refer to groups that participate in subsequent reactions, which can be -N 3 , -DBCO and other groups. In this embodiment, the functional group is -N 3 group. Proceed as follows:
  • step 4) Immediately pass the solution configured in step 3) into the substrate channel, 25 ⁇ l/channel; react at room temperature for 1 h;
  • the functional group-modified substrate can be immersed in RI02 solution and stored at 4°C for 1 year.
  • step 2 Pass the solution configured in step 1 into the channel of the epoxy-modified substrate, pass 25 ⁇ l of the solution into each channel, seal the channel mouth (to prevent the solution from volatilizing), and react at 37° C. for 16 hours;
  • Functional groups modify the substrate coated with PamamDendrimer.
  • Functional groups refer to groups that participate in subsequent reactions, which can be -N 3 , -DBCO and other groups.
  • the functional group is -N 3 group.
  • step 4) Immediately pass the solution configured in step 3) into the substrate channel, 25 ⁇ l/channel; react at room temperature for 1 h;
  • the functional group-modified substrate can be immersed in RI02 solution and stored at 4°C for 1 year.
  • PEI polyethyleneimine
  • step 2 Pass the solution configured in step 1 into the channel of the epoxy-modified substrate, pass 25 ⁇ l of the solution into each channel, seal the channel mouth (to prevent the solution from volatilizing), and react at 37° C. for 16 hours;
  • Functional groups modify the PEI-coated substrate.
  • Functional groups refer to groups that participate in subsequent reactions, which can be -N3 , -DBCO and other groups.
  • the functional group is an N3 group.
  • the specific steps of functional group modification are as follows :
  • step 4) Immediately pass the solution configured in step 3) into the substrate channel, 25 ⁇ l/channel; react at room temperature for 1 h;
  • the functional group-modified substrate can be immersed in RI02 solution and stored at 4°C for 1 year.
  • step 2 Pass the solution configured in step 1 into the channel of the epoxy-modified substrate, pass 25 ⁇ l of the solution into each channel, seal the channel mouth (to prevent the solution from volatilizing), and react at 37° C. for 16 hours;
  • Functional groups modify Chitosan-coated substrates.
  • Functional groups refer to groups that participate in subsequent reactions, which can be -N3, -DBCO and other groups. In this embodiment, the functional group is an N3 group.
  • the specific steps of functional group modification are as follows:
  • step 4) Immediately pass the solution configured in step 3) into the substrate channel, 25 ⁇ l/channel; react at room temperature for 1 h;
  • the functional group-modified substrate can be immersed in RI02 solution and stored at 4°C for 1 year.
  • probe density detection is performed on the chips prepared in Examples 1-5, and the specific detection process is as follows:
  • T20-CY3 hybridization Take the chips prepared in Examples 1-5 respectively, drain the channel liquid on the fluid meter, and pass 20 ⁇ l of hybridization solution A (3xSSC (pH 7.2-7.4 containing 3uM T20-CY3) into each channel. )), hybridize at 55 °C for 15 min, and after cooling to room temperature, wash 1 ml with RI02 on a fluid meter.
  • the Cy3 of T20-CY3 is located at the 5' end of the T20 sequence.
  • Detection Use a fluorescence detection system with a 20x objective lens, use a wavelength of 532 nm, a laser power of 35 mW, and an exposure time of 60 ms, take pictures to collect data, and use ImageJ to analyze the image information.
  • the detection results are shown in Figure 2, the detection results of the chip probe density prepared in Examples 1-5, the ordinate is the fluorescence intensity (AU), and the unit dot/FOV is the average fluorescence intensity in the range of 110 ⁇ 110 ⁇ m in an observation area,
  • the abscissas PLL, PLO, PD3.0, CTS, and PEI correspond to the chips modified with polymer PLL, PLO, PD3.0 (polyamide-amine dendrimer), CTS (chitosan), and PEI, respectively. .
  • 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 3.
  • Insertion Insertion fragment, derived from phi-X174 standard strain; T20, Pe, and RD are SEQ Sequences shown in ID No. 2-4.
  • the DNA library was mixed with 52 ⁇ l of deionized water, and 18 ⁇ l of freshly prepared 0.2M NaOH solution was added to it. After mixing evenly, let stand for denaturation at room temperature for 8 minutes, and then add 20 ⁇ l of 400 mM Tris-HCl buffer pH 8.0 to stop the reaction. , to obtain a 100 pM denatured DNA library in a volume of 100 ⁇ l. 2.
  • 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, 3ug/ml Bst DNA polymerase, 200 ⁇ M dNTPs
  • extension buffer reagent 3ug/ml Bst DNA polymerase, 200 ⁇ M dNTPs

Abstract

本申请涉及一种氨基修饰的芯片及其制备方法和应用。所述氨基修饰的芯片，包括：基底，所述基底修饰有环氧基；经所述环氧基接枝于所述基底之上的高聚物，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或仲氨基。

Description

氨基修饰的芯片及其制备方法和应用 技术领域
本申请涉及芯片技术领域，特别是氨基修饰的芯片及其制备方法和应用。
背景技术
随着人类基因组(测序)计划(Human genome project)逐步实施以及分子生物学相关学科的迅猛发展，越来越多的动植物、微生物基因组序列得以测定，基因序列数据正在以前所未有的速度迅速增长。基因芯片(又称DNA芯片、生物芯片)技术就是顺应这一科学发展要求的产物，可用于制备和/或分析生物分子，具体是指将探针分子(不局限于核酸序列)固定于支持物上后与标记的样品分子进行杂交，通过检测每个探针分子的杂交信号强度进而获取样品分子的数量和序列信息。
芯片通常由聚合物包被基底制成，分子分析和/或制备，如某些核酸测序方法，依赖于核酸链与芯片基底表面的结合，然后通过本领域公知的许多不同的方法来确定所结合的核酸链的序列。现有的基底通常为流动池，当前的用于包覆流动池的程序包括，将聚合性混合物转移至流动池上的通道中，并孵育固定的时间。该程序简单，并且产生始终能够支持包括桥式扩增和测序在内的所有下游化学处理步骤的可靠包被。
然而，目前已有的芯片仍存在诸多缺陷或限制，限制了其应用，有待进一步改进。
申请内容
基于此，有必要提供一种氨基修饰的芯片。该氨基修饰的芯片能够更高密度地负载探针，满足不断发展的生物分子制备和/或分析需求，且稳定性好。
本申请的实施例提供一种氨基修饰的芯片，包括：
基底，所述基底修饰有环氧基；
经所述环氧基接枝于所述基底之上的高聚物，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或/和仲氨基。
在某些实施方式中，所述高聚物中平均每个所述结构单元包含0.05～10个相同或不同的氨基。
在某些实施方式中，所述高聚物中平均每个所述结构单元包含1～5个相同或不同的氨基。
在某些实施方式中，所述高聚物的聚合度范围为10～5000。
在某些实施方式中，所述高聚物中结构单元包括
Figure PCTCN2021111672-appb-000001
Figure PCTCN2021111672-appb-000002
中的至少一种。
在某些实施方式中，所述高聚物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子和聚乙烯亚胺中的至少一种。
在某些实施方式中，所述芯片还包括接枝于所述高聚物的探针。
在某些实施方式中，所述探针通过连接基团与所述高聚物键合，所述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；所述第一连接基团与所述探针键合，所述第二连接基团经所述氨基接枝于所述高聚物。
在某些实施方式中，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
在某些实施方式中，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n＝3～2000；所述NHS-PEGn-DBCO或NHS-PEGn-N 3中的基团-NHS与所述氨基进行反应接枝。
在某些实施方式中，所述探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3间的共价键合。
本申请的实施例还提供一种芯片的制备方法，包括如下步骤：
(1)获取基底，所述基底修饰有环氧基；
(2)将高聚物反应接枝至所述基底，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或仲氨基。
在某些实施方式中，所述高聚物中平均每个所述结构单元包含0.05～10个相同或不同的氨基。
在某些实施方式中，所述高聚物中平均每个所述结构单元包含1～5个相同或不同的氨基。
在某些实施方式中，所述高聚物的聚合度范围为10～5000。
在某些实施方式中，所述高聚物中结构单元包括
Figure PCTCN2021111672-appb-000003
Figure PCTCN2021111672-appb-000004
中的至少一种。
在某些实施方式中，所述高聚物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子和聚乙烯亚胺中的至少一种。
在某些实施方式中，步骤(2)中，所述反应接枝是通过使所述高聚物与所述基底在碱性溶液中接触进行的，所述碱性溶液的pH值为8.5～10。
在某些实施方式中，步骤(2)中，所述反应接枝是在反应温度为37～55℃的条件下反应3～24h。
在某些实施方式中，所述的制备方法还包括步骤(3)：将探针接枝至所述高聚物。
在某些实施方式中，所述探针通过连接基团与所述高聚物接枝，所述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；所述第一连接基团与所述探针键合，所述第二连接基团经所述氨基接枝于所述高聚物。
在某些实施方式中，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
在某些实施方式中，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n＝3～2000；所述NHS-PEGn-DBCO或NHS-PEGn-N 3中的基团-NHS与所述氨基进行反应接枝。
在某些实施方式中，所述探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3间的共价键合。
在某些实施方式中，所述反应接枝是通过使所述连接基团与所述高聚物在碱性溶液中接触进行的，所述碱性溶液的pH值为7～9。
在某些实施方式中，所述反应接枝是在反应温度为室温的条件下反应30～90min。
在某些实施方式中，所述共价键合是通过使所述探针与连接基团在碱性溶液中接触下进行的，所述碱性溶液的pH值为7～8。
在某些实施方式中，进行所述共价键合的所述碱性溶液中含有表面活性剂，所述表面活性剂选自十四烷基三甲基溴化铵、十六烷基三甲基溴化铵和十二烷基三甲基溴化铵中的至少一种。
本申请的实施的方式还提供如上所述的芯片、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。
本申请一实施方式公开的氨基修饰的芯片，其基底修饰有特定活性基团的基底反应接枝包含伯氨基和/或仲氨基的高聚物，该高聚物可以形成密度较高，且反应活性高的修饰表面，用于接枝探针，能够更高密度地负载探针，满足不断发展的生物分子制备和/或分析需求，且稳定性好。本申请另一实施方式公开的的制备芯片的方法，无需严格的反应条件控制，即可实现探针的接枝，使芯片的制作工艺简单易控，利于芯片的推广应用。
附图说明
图1为实施例中芯片的制备过程的示意图，X表示高聚物，primer表示引物/探针；
图2为实施例1-5制备的芯片的探针密度检测结果图；
图3为实施例1制备的芯片生成的DNA簇检测结果图；
图4为实施例2制备的芯片生成的DNA簇检测结果图；
图5为实施例3制备的芯片生成的DNA簇检测结果图；
图6为实施例4制备的芯片生成的DNA簇检测结果图；
图7为实施例5制备的芯片生成的DNA簇检测结果图；
图8为实施例7中采用的DNA文库的结构示意图。
具体实施方式
以下结合具体实施例对本申请的氨基修饰的芯片及其制备方法和应用作进一步详细的说 明。本申请可以以许多不同的形式来实现，并不限于本文所描述的实施方式。相反地，提供这些实施方式的目的是使对本申请公开内容理解更加透彻全面。
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申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。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。
本文所述芯片，可以为接枝有所述高聚物的基底，也可以为在所述高聚物上进一步接枝探针的基底。基底材料并不受特别限制，基底由玻璃、硅片、塑料、凝胶和尼龙膜中的至少之一形成。
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，也可以表示进一步经过其他的过渡基团接枝、修饰于对象之上。
本文所述的氨基是指，具有式-N(X) 2的结构特征，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，可以理解地，在本申请的技术方案中，至少一个X为H。氨基的非限制性类型包括-NH 2、-NH(烷基)、-NH(环烷基)、-NH(杂环基)、-NH(芳基)。
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。合适的实例包括但不限于：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)。
术语“环烷基”是指包含环碳原子的非芳香族烃，可以为单环烷基、或螺环烷基、或桥环烷基。合适的实例包括但不限于：环丙基、环丁基、环戊基、环己基和环庚基。另外，“环烷基”还可含有一个或多个双键，含有双键的环烷基的代表性实例包括环戊烯基、环己烯基、环己二烯基和环丁二烯基。
“芳基”是指在芳香环化合物的基础上除去一个氢原子衍生的芳族烃基，可以为单环芳基、或稠环芳基、或多环芳基，对于多环的环种，至少一个是芳族环系。合适的实例包括但不限于：苯、联苯、萘、蒽、菲、二萘嵌苯、三亚苯及其衍生物。
“杂环基”是指在环烷基的基础上至少一个碳原子被非碳原子所替代，非碳原子可以为N原子、O原子、S原子等，可以为饱和环或部分不饱和环。合适的实例包括但不限于：二氢吡啶基、四氢吡啶基(哌啶基)、四氢噻吩基、硫氧化的四氢噻吩基、四氢呋喃基、四氢喹啉基、四氢异喹啉基、二氢吲哚基。
“结构单元”是指高聚物中由其中一个单体形成的单元结构。所述高聚物可以由一种单体聚合而成，也可以由两个以上不同种类的单体共聚而成，因此所述“结构单元”可以相同，也可以不同。
聚酰胺-胺树枝状大分子聚合度与代数的关系：聚合度＝2^(代数+1)-1，如PamamDendrimer3.0代，其聚合度为15。
本申请的实施例提供一种氨基修饰的芯片，包括：
基底，所述基底修饰有环氧基；
经所述环氧基接枝于所述基底之上的高聚物，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基和/或仲氨基。
可以理解地，所述芯片为可以用于基因检测的芯片，也可以为用于蛋白、多肽等检测的芯片。
在其中一个具体的实施例中，所述高聚物中平均每个所述结构单元包含0.05～10个相同或不同的氨基。可以理解地，所述结构单元中可以包含氨基，也可以不包含氨基，所述“平均”是指以氨基的总数除以所述结构单元的总数所得的平均值，当平均每个所述结构单元包含氨基个数小于1时，所述高聚物为两个以上单体聚合而成的共聚物，其中至少一个单体不含氨基。举例可如，所述高聚物中平均每个所述结构单元包含0.5个氨基，所述高聚物可由19个无氨基结构单元和1个含氨基结构单元(含10个氨基)构成，通过控制聚合时的单体组合可控制氨基数量。
优选地，所述高聚物中平均每个所述结构单元包含1～5个氨基。测试发现，高聚物上的氨基的数量与氨基的反应活性相关，而氨基的反应活性直接影响氨基与连接到高聚物上的连接物例如探针的连接密度/数量。在一个示例中，探针通过连接基团与高聚物的氨基相连而连接在芯片表面，使高聚物的每个所述结构单元平均包含1～5个氨基能够较好地调控探针的连接密度/数量，从而实现探针密度调控的优化，同时，使探针更易于通过连接基团接枝到高聚物上且探针密度更高。
在其中一个具体的实施例中，所述高聚物的聚合度范围为10～5000。在另外一个具体的实施例中，所述高聚物的分子量为30KD～300KD。分子量越大，可接枝的核酸链密度越高，但是同时增加高聚物固定在基体表面的有效性，同时，分子量过大的分子还会对溶解度、反应效率等方便造成不良影响。故此在本申请的实施例中，优选所述高聚物的分子量为30KD～300KD。对于所述聚酰胺-胺树枝状大分子而言，以PamamDendrimer为例，其代数可以是3～11代。
优选地，所述高聚物包含相同或不同的结构单元，可以采用市售，也可以委托厂家定制，只需满足本申请对聚合物所包含的氨基数量或/和聚合物的聚合度的要求即可。
在其中一个具体的实施例中，所述高聚物中的结构单元包括
Figure PCTCN2021111672-appb-000005
Figure PCTCN2021111672-appb-000006
中的至少一种。
具体地，在一个示例中，所述高聚物包含相同的结构单元，如高聚物为包含相同结构单元的
Figure PCTCN2021111672-appb-000007
的共聚物。在另一个示例中，所述高聚物包含不同的结构单元，例如，高聚物为包含不同结构单元
Figure PCTCN2021111672-appb-000008
的共聚物，如参见Kousaku Ohkawaa,Biodegradation of ornithine-containing polylysine hydrogels,Biomaterials 19(1998)1855-1860.中所公开的内容包括以不同比例聚合的鸟氨酸和赖氨酸共聚物(Lys Orn)，或高聚物为包含不同结构单元
Figure PCTCN2021111672-appb-000009
Figure PCTCN2021111672-appb-000010
的共聚物。
优选地，所述高聚物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子和聚乙烯亚胺中的至少一种。
在其中一个具体的实施例中，上述芯片还包括接枝于所述高聚物的探针。其中探针通过与目标分子结合，从而捕获目标分子。基于不同的目的，选用不同的探针种类，如，选用寡核苷酸片段、多肽序列、包含寡核苷酸片段、或包含多肽序列的片段，同时，探针可选择性标记荧光检测分子、抗原、生物素或链霉亲和素等检测分子。
在其中一个具体的实施例中，所述探针可以为核酸链和/或多肽。
在其中一个具体的实施例中，所述探针通过连接基团与所述高聚物接枝，所述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；所述第一连接基团一端与所述核酸链探针键合，所述第二连接基团另一端经所述氨基接枝于所述高聚物。进一步地，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
在其中一个具体的实施例中，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n＝3～2000；所述NHS-PEGn-DBCO或NHS-PEGn-N 3中的基团-NHS与所述氨基进行反应接枝。
优选地，所述探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3 间的共价键合。其中，NHS为琥珀酰亚胺酯的缩写，PEG为聚乙二醇的缩写，DBCO为二苯基环辛炔的缩写，N 3为叠氮的缩写。经由上述连接基团连接所述核酸链和高聚物，一方面NHS能够在较为稳定地与所述氨基接枝，另一方面DBCO或N 3能够与修饰有N 3或DBCO的核酸链在常温、无催化剂的条件下进行Click反应，反应条件温和，效率高。
本申请的实施例还提供一种芯片的制备方法，包括如下步骤：
(1)获取基底，所述基底修饰有环氧基；
(2)将高聚物反应接枝至所述基底，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或仲氨基。
上述制备方法中，关于所述高聚物的进一步限定同上述芯片，在此不再赘述。
具体地，步骤(1)为获取基底步骤，其中所述获取可以是直接购买，即直接购买获得修饰有环氧基基底，也可以是自制，自制的方法可以为通过溶液反应，也可以通过等离子镀膜法在载体上修饰包含环氧基的化合物。
所述包含环氧基的化合物可以选自环氧硅烷，例如3-(2,3环氧丙氧)丙基三甲氧基硅烷。另外，在其中一个具体的实施例中，所述载体的材料为玻璃、硅片、塑料、凝胶和尼龙膜中的至少一种。
在其中一个具体的实施例中，采用溶液反应在载体上修饰包含环氧基的化合物的反应条件包括：反应温度为室温，反应时间为1～8小时。进一步地，反应结束后，于80～150℃条件下烘干。
可以理解地，在进行所述溶液反应修饰之前，需要对载体进行深度清洗和活化处理。例如，采用醇类溶剂、水在超声条件下交替清洗，使用碱液活化载体表面。所述碱液活化可采用如0.1M～2M的NaOH水溶液处理1～20分钟。
具体地，步骤(2)为高聚物接枝步骤。所述基底修饰的是环氧基，步骤(2)中，所述氨基与所述环氧基进行反应接枝。
优选地，所述反应接枝是通过所述高聚物与所述基底在碱性溶液中接触下进行的，所述碱性溶液的pH值为8.5～10。所述pH值包括但不限于以下数值：8.5、8.8、9、9.16、9.2、9.5、10。
优选地，所述pH值为8.5～10的碱性溶液选自磷酸盐缓冲液、碳酸盐缓冲液和硼酸盐缓冲液中的至少一种。更进一步地，所述pH值为8.5～10的缓冲液中缓冲对的浓度为10～300mM。所述缓冲对的浓度包括但不限于以下浓度：10mM、50mM、100mM、120mM、150mM、190mM、195mM、200mM、205mM、210mM、220mM、250mM、300mM。
优选地，以pH值为9.16的碳酸盐缓冲液作为反应溶剂，其中缓冲对的浓度为200mM。
另外，步骤(2)中，所述反应接枝是在反应温度为37～55℃的条件下反应3～24h。所述反应温度和反应时间的组合(温度*时间)包括但不限于以下组合：37℃*14h、37℃*16h、37℃*18h、37℃*20h、37℃*24h、40℃*12h、42℃*18h、45℃*10h、50℃*8h、55℃*5h、55℃*3h。
优选地，反应温度为37℃，反应时间为16h。
优选地，上述芯片的制备方法，还包括步骤(3)：将探针接枝至所述高聚物。步骤(3)为探针接枝步骤。
在其中一个具体的实施例中，步骤(3)中，所述探针通过连接基团与所述高聚物接枝，所述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；所述第一连接基团与所述探针键 合，所述第二连接基团经所述氨基接枝于所述高聚物。更为具体地，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
优选地，步骤(3)可以分为如下两步进行：
(3-1)将提供所述连接基团的化合物与所述高聚物的氨基进行接枝反应；
(3-2)将所述接枝反应的产物与活性基团修饰的探针进行键合。
在其中一个具体的实施例中，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n＝3～2000，所述NHS-PEG n-DBCO或NHS-PEGn-N 3中的基团-NHS与所述氨基进行反应接枝。经由上述连接基团连接所述探针和高聚物，一方面-NHS能够较为稳定地与所述氨基接枝，另一方面-DBCO或-N 3能够与修饰有-N 3或-DBCO的探针在常温、无催化剂的条件下进行Click反应，反应条件温和，效率高。
优选地，所述活性基团修饰的探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3间的共价键合。更为具体地，所述共价键合为Click反应。
在其中一个具体的实施例中，所述活性基团修饰的探针为在5’端进行所述活性基团修饰的核酸链。可以理解地，若所述连接基团由NHS-PEGn-DBCO制备而成，即用于键合的为基团-DBCO，那么5’端修饰的活性基团为-N 3官能团；若所述连接基团由NHS-PEGn-N 3制备而成，即用于键合的为基团-N 3，那么5’端修饰的活性基团为-DBCO官能团。
在其中一个具体的实施例中，步骤(3-1)中，所述提供连接基团的化合物在反应体系中的浓度为50μM～5mM。所述浓度包括但不限于以下浓度：50μM、100μM、500μM、800μM、1mM、1.2mM、1.5mM、2mM、5mM。
在其中一个具体的实施例中，步骤(3-1)中，所述反应接枝是通过使所述连接基团与所述高聚物在碱性溶液中接触进行的，所述碱性溶液的pH值为7～9。具体地，所述pH值包括但不限于以下数值：7、7.3、7.5、7.7、7.8、7.9、8、8.1、8.2、8.3、8.4、8.5。更为具体地，所述碱性溶液选自磷酸盐缓冲液和碳酸盐缓冲液中的至少一个。
在其中一个具体的实施例中，所述磷酸盐缓冲液或碳酸盐缓冲液中缓冲对的浓度为10mM～300mM。所述缓冲对的浓度包括但不限于以下浓度：10mM、50mM、80mM、90mM、95mM、98mM、100mM、102mM、105mM、110mM、150mM、200mM、210mM、220mM、250mM、300mM。
优选地，所述反应接枝是以pH值为8的磷酸盐缓冲液作为反应溶剂，其中缓冲对的浓度为100mM。在另一个具体的实施例中，所述反应接枝是以pH值为8.3的碳酸盐缓冲液作为反应溶剂，其中缓冲对的浓度为100mM。
在其中一个具体的实施例中，步骤(3-1)中，所述反应接枝是在反应温度为室温的条件下反应30～90min。所述反应时间包括但不限于以下时间：30min、40min、50min、55min、58min、60min、62min、65min、70min、80min、90min。
在其中一个具体的实施例中，步骤(3-2)中，所述共价键合是通过使所述探针与连接基团在碱性溶液中接触下进行的，所述碱性溶液的pH值为7～8。具体地，所述pH值包括但不限于以下数值：7、7.3、7.5、7.7、7.8、7.9、8。更为具体地，所述碱性溶液为柠檬酸盐缓冲液。更为具体地，所述共价键合是在室温条件下进行。
在其中一个具体的实施例中，所述碱性溶液中含有表面活性剂，所述表面活性剂选自十四 烷基三甲基溴化铵(TTAB)、十六烷基三甲基溴化铵(CTAB)和十二烷基三甲基溴化铵(DTAB)中的至少一种。采用所述表面活性剂有利于核酸链在表面沉降。
优选地，所述共价键合是在反应温度为室温的条件下，使所述探针与连接基团在添加有1mM TTAB的柠檬酸盐缓冲液中接触下进行的。
另外，在不同应用中，键合所述探针的之前或之后，可选择性地对多余的官能团进行封闭，多余的官能团可例如-NH 2、-DBCO、-N 3等。
具体地，在其中一个具体的实施例中，还包括封闭步骤：采用NHS-(PEG) n、乙酸酐、DBCO-苯甲酸、叠氮-苯甲酸中的至少一种进行封闭处理，其中n＝3～2000。更为具体地，-NH 2的封闭可选用NHS-(PEG) n(n可以为3～2000)、乙酸酐等；特殊地，例如使用1uL乙酸酐+1.75uL N,N-二异丙基乙胺(DIPEA)溶于甲酰胺对-NH 2进行封闭。-N 3的封闭可选用DBCO-苯甲酸进行封闭。-DBCO的封闭可选用叠氮苯甲酸进行封闭。
本申请的实施例还提供如上所述的芯片、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。具体地，所述生物分子制备或生物分子分析可如核酸测序、文库杂交、DNA簇的生成等，也可如蛋白检测、多肽检测等。
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(如阿拉丁、simga、生工等)或者根据实施例提供的结构式和分子量等信息自行制备或委托制备。
聚酰胺-胺树枝状大分子(PamamDendrimer)，Pamam Dendrimer3.0代(简称：PD3.0)，分子量6905.84，其分子式如下：
Figure PCTCN2021111672-appb-000011
NHS-PEG4-N 3和NHS-PEG4-DBCO，二者的分子式如下所示：
Figure PCTCN2021111672-appb-000012
Figure PCTCN2021111672-appb-000013
无水二甲基亚枫(DMSO)购买于sigma；
聚乙烯亚胺(PEI)，分子量为60KD，其分子式如下：
Figure PCTCN2021111672-appb-000014
聚赖氨酸(PLL)，分子量150-300KD，其分子式如下：
Figure PCTCN2021111672-appb-000015
聚鸟氨酸(PLO)，分子量30KD-70KD，其分子式如下：
Figure PCTCN2021111672-appb-000016
壳聚糖(Chitosan)，分子量为100KD，分子式如下：
Figure PCTCN2021111672-appb-000017
碳酸氢钠和碳酸钠购买于sigma；
十四烷基三甲基溴化铵(TTAB)购买于sigma；
磷酸氢二钠和磷酸二氢钠购买于sigma；
20xSSC购买于生工；Hepes购买于sigma；Tween20购买于sigma；
十二烷基硫酸钠(SDS)购买于阿拉丁；
SC：0.2M碳酸钠盐pH＝9.16(25℃)；
PC：0.1M磷酸钠盐pH＝8.0(25℃)；
固定液：3xSSC+1mM TTAB(十四烷基三甲基溴化铵)；
清洗液1：150mM Hepes+0.1％Tween20+0.1％SDS；
RI02：3xSSC pH＝7.2～7.4。
实施例1
PLO(聚鸟氨酸)包被环氧修饰的基底及官能团修饰PLO包被的基底的具体操作过程如下：
1.将0.1％PLO溶液室温平衡15min，取200μl PLO溶液，加入200ul SC缓冲液，涡旋混匀，短暂离心2s；
2.将步骤1中配置的溶液通入环氧修饰的基底的通道，每条通道通入25μl溶液，密封通道口(防止溶液挥发)，37℃反应16h；
3.反应结束后，待基底恢复至室温，在流体仪上依次使用清洗液1和RI02各1ml清洗，流体仪参数设置：试剂量(reagent amount)＝1000，速度(speed)＝10，循环(circle)＝1，延时＝0，次数＝1；
4.将PLO包被的基底浸泡在RI02溶液进行保存，4℃可保存1周；
5.官能团修饰PLO包被的基底，官能团指的是参与后续反应的基团，可为-N3、-DBCO等基团，在本实施例中官能团为-N 3基团，官能团修饰的具体步骤如下：
1)配置终浓度100mM的NHS-PEG4-N 3溶液，溶剂为无水DMSO(无水二甲基亚枫)；
2)将含有NHS-PEG4-N 3溶液通入PLO包被的基底的通道：在流体仪上将基底的通道液体抽干，使用1ml/通道的PC缓冲液润洗通道，润洗完后抽干通道液体；
3)将396μl PC缓冲液和4μl 100mM的NHS-PEG4-N 3溶液混合，涡旋混匀，短暂离心2s；
4)将步骤3)中配置的溶液立即通入基底通道，25μl/通道；室温反应1h；
5)反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6)在流体仪上将基底通道的液体抽干，取1个1.5离心管加入497.25μl甲酰胺，然后依次加入1.75μl DIPEA和1μl乙酸酐，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，室温反应15min；反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6.探针固定：
1)在流体仪上将基底通道的液体抽干，使用1ml/通道的固定液润洗通道，润洗完后抽干通道液体；
2)将360μl固定液与40μl 100μM A30(SEQ ID No.1)混合，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，37℃反应16h；
3)反应结束后，待芯片恢复至室温，在流体仪上依次使用清洗液1和RI02各1ml清洗；
官能团修饰后的基底可以浸泡在RI02溶液中进行保存，4℃保存1年。
实施例2
PLL(聚赖氨酸)包被环氧修饰的基底及官能团修饰PLL包被的基底的具体操作过程如下：
1.将0.1％PLL溶液室温平衡15min，取200μl PLL溶液，加入200μl SC缓冲液，涡旋混匀，短暂离心2s；
2.将步骤1中配置的溶液通入环氧修饰的基底的通道，每条通道通入25μl溶液，密封通道口(防止溶液挥发)，37℃反应16h；
3.反应结束后，待基底恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml，流体仪参数设置：试剂量(reagent amount)＝1000，速度(speed)＝10，循环(circle)＝1，延时＝0，次数＝1；
4.将PLL包被的基底浸泡在RI02溶液进行保存，4℃可保存1周；
5.官能团修饰PLL包被的基底，官能团指的是参与后续反应的基团，可为-N 3、-DBCO等基团，在本实施例中官能团为-N 3基团，官能团修饰的具体步骤如下：
1)配置终浓度100mM的NHS-PEG4-N 3溶液，溶剂为无水DMSO(无水二甲基亚枫)；
2)将含有NHS-PEG4-N 3溶液通入PLL包被的基底的通道：在流体仪上将基底的通道液体抽干，使用1ml/通道的PC缓冲液润洗通道，润洗完后抽干通道液体；
3)将396μl PC缓冲液和4μl 100mM的NHS-PEG4-N 3溶液混合，涡旋混匀，短暂离心2s；
4)将步骤3)中配置的溶液立即通入基底通道，25μl/通道；室温反应1h；
5)反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6)在流体仪上将基底通道的液体抽干，取1个1.5离心管加入497.25μl甲酰胺，然后依次加入1.75μl DIPEA和1μl乙酸酐，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，室温反应15min；反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6.探针固定：
1)在流体仪上将基底通道的液体抽干，使用1ml/通道的固定液润洗通道，润洗完后抽干通道液体；
2)将360μl固定液与40μl 100μM A30混合，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，37℃反应16h；
3)反应结束后，待芯片恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml；
官能团修饰后的基底可以浸泡在RI02溶液中进行保存，4℃保存1年。
实施例3
PamamDendrimer(聚酰胺-胺树枝状大分子)包被环氧修饰的基底及官能团修饰PamamDendrimer包被的基底的具体操作过程如下：
1.将5％PamamDendrimer室温平衡15min，取200μl PamamDendrimer溶液，加入380μl SC缓冲液，涡旋混匀，短暂离心2s；
2.将步骤1中配置的溶液通入环氧修饰的基底的通道，每条通道通入25μl溶液，密封通道口(防止溶液挥发)，37℃反应16h；
3.反应结束后，待基底恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml，流 体仪参数设置：试剂量(reagent amount)＝1000，速度(speed)＝10，循环(circle)＝1，延时＝0，次数＝1；
4.将PamamDendrimer包被的基底浸泡在RI02溶液进行保存，4℃可保存1周；
5.官能团修饰PamamDendrimer包被的基底，官能团指的是参与后续反应的基团，可为-N 3、-DBCO等基团，在本实施例中官能团为-N 3基团，官能团修饰的具体步骤如下：
1)配置终浓度100mM的NHS-PEG4-N 3溶液，溶剂为无水DMSO(无水二甲基亚枫)；
2)将含有NHS-PEG4-N 3溶液通入PLO包被的基底的通道：在流体仪上将基底的通道液体抽干，使用1ml/通道的PC缓冲液润洗通道，润洗完后抽干通道液体；
3)将396μl PC缓冲液和4μl 100mM的NHS-PEG4-N 3溶液混合，涡旋混匀，短暂离心2s；
4)将步骤3)中配置的溶液立即通入基底通道，25μl/通道；室温反应1h；
5)反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6.探针固定：
1)在流体仪上将基底通道的液体抽干，使用1ml/通道的固定液润洗通道，润洗完后抽干通道液体；
2)将360μl固定液与40μl 100uM A30混合，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，37℃反应16h；
3)反应结束后，待芯片恢复至室温，在流体仪上依次使用清洗液1和RI02各1ml清洗；
4)在流体仪上将基底通道的液体抽干，取1个1.5离心管加入497.25μl甲酰胺，然后依次加入1.75μl DIPEA和1μl乙酸酐，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，室温反应15min；反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
官能团修饰后的基底可以浸泡在RI02溶液中进行保存，4℃保存1年。
实施例4
PEI(聚乙烯亚胺)包被环氧修饰的基底及官能团修饰PEI包被的基底的具体操作过程如下：
1.将1％PEI溶液室温平衡15min，取200μl PEI溶液，加入380μl SC缓冲液，涡旋混匀，短暂离心2s；
2.将步骤1中配置的溶液通入环氧修饰的基底的通道，每条通道通入25μl溶液，密封通道口(防止溶液挥发)，37℃反应16h；
3.反应结束后，待基底恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml，流体仪参数设置：试剂量(reagent amount)＝1000，速度(speed)＝10，循环(circle)＝1，延时＝0，次数＝1；
4.将PEI包被的基底浸泡在RI02溶液进行保存，4℃可保存1周；
5.官能团修饰PEI包被的基底，官能团指的是参与后续反应的基团，可为-N3、-DBCO等基团，在本实施例中官能团为N 3基团，官能团修饰的具体步骤如下：
1)配置终浓度100mM的NHS-PEG4-N 3溶液，溶剂为无水DMSO(无水二甲基亚枫)；
2)将含有NHS-PEG4-N 3溶液通入PEI包被的基底的通道：在流体仪上将基底的通道液体抽干，使用1ml/通道的PC缓冲液润洗通道，润洗完后抽干通道液体；
3)将396μl PC缓冲液和4μl 100mM的NHS-PEG4-N 3溶液混合，涡旋混匀，短暂离心2s；
4)将步骤3)中配置的溶液立即通入基底通道，25μl/通道；室温反应1h；
5)反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6)在流体仪上将基底通道的液体抽干，取1个1.5离心管加入497.25μl甲酰胺，然后依次加入1.75ul DIPEA和1μl乙酸酐，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，室温反应15min；反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6.探针固定：
1)在流体仪上将基底通道的液体抽干，使用1ml/通道的固定液润洗通道，润洗完后抽干通道液体；
2)将360μl固定液与40μl 100μM A30混合，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，37℃反应16h；
3)反应结束后，待芯片恢复至室温，在流体仪上依次使用清洗液1和RI02各1ml清洗；
官能团修饰后的基底可以浸泡在RI02溶液中进行保存，4℃保存1年。
实施例5
Chitosan(壳聚糖)包被环氧修饰的基底及官能团修饰Chitosan包被的基底的具体操作过程如下：
1.将1％Chitosan溶液室温平衡15min，取20μl Chitosan溶液，加入380μl SC缓冲液，涡旋混匀，短暂离心2s；
2.将步骤1中配置的溶液通入环氧修饰的基底的通道，每条通道通入25μl溶液，密封通道口(防止溶液挥发)，37℃反应16h；
3.反应结束后，待基底恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml，流体仪参数设置：试剂量(reagent amount)＝1000，速度(speed)＝10，循环(circle)＝1，延时＝0，次数＝1；
4.将Chitosan包被的基底浸泡在RI02溶液进行保存，4℃可保存1周；
5.官能团修饰Chitosan包被的基底，官能团指的是参与后续反应的基团，可为-N3、-DBCO等基团，在本实施例中官能团为N3基团，官能团修饰的具体步骤如下：
1)配置终浓度100mM的NHS-PEG4-N 3溶液，溶剂为无水DMSO(无水二甲基亚枫)；
2)将含有NHS-PEG4-N 3溶液通入PLO包被的基底的通道：在流体仪上将基底的通道液体抽干，使用1ml/通道的PC缓冲液润洗通道，润洗完后抽干通道液体；
3)将396μl PC缓冲液和4μl 100mM的NHS-PEG4-N 3溶液混合，涡旋混匀，短暂离心2s；
4)将步骤3)中配置的溶液立即通入基底通道，25μl/通道；室温反应1h；
5)反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6)在流体仪上将基底通道的液体抽干，取1个1.5离心管加入497.25μl甲酰胺，然后依次加入1.75μl DIPEA和1μl乙酸酐，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，室温反应15min；反应结束后在流体仪上依次使用清洗液1、RI02和超纯水各1ml进行清洗；
6.探针固定：
1)在流体仪上将基底通道的液体抽干，使用1ml/通道的固定液润洗通道，润洗完后抽干通道液体；
2)将360μl固定液与40μl 100μM A30混合，涡旋混匀，短暂离心2s，立即通入基底通道，25μl/通道，37℃反应16h；
3)反应结束后，待芯片恢复至室温，在流体仪上依次使用清洗液1和RI02各清洗1ml；
官能团修饰后的基底可以浸泡在RI02溶液中进行保存，4℃保存1年。
实施例6
本实施例对实施例1-5制备的芯片进行探针密度检测，具体检测过程如下：
1.T20-CY3杂交：分别取实施例1-5中制备的芯片，在流体仪上抽干通道液体，每条通道通入20μl杂交液A(含3uM T20-CY3的3xSSC(pH 7.2-7.4))，于55℃杂交15min，待冷却至室温，在流体仪上使用RI02清洗1ml。其中T20-CY3的Cy3位于T20序列的5’端。
检测：利用20倍物镜的荧光检测系统，使用532nm的波长，激光功率为35mW，曝光时间为60ms，拍照收集数据，使用ImageJ分析图像信息。
检测结果如图2所示，实施例1-5中制备的芯片探针密度检测结果，纵坐标为荧光强度(A.U.)，单位dot/FOV即1观测区域为110×110μm范围的平均荧光强度，横坐标PLL、PLO、PD3.0、CTS、PEI分别对应的是修饰了高聚物PLL、PLO、PD3.0(聚酰胺-胺树枝状大分子)、CTS(壳聚糖)、PEI的芯片。
实施例7
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3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.2-4所示序列。
将DNA文库与52μl体积的去离子水混合，向其中加入18μl新鲜配制的0.2M 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的速度流通芯片通道160～260μl体积的延伸试剂(延伸缓冲试剂，3ug/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
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3-7以及表1，图4-8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
Figure PCTCN2021111672-appb-000018
从检测结果可看出，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。
以上所述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。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (29)

  1. 一种氨基修饰的芯片，其特征在于，包括：
    基底，所述基底修饰有环氧基；
    经所述环氧基接枝于所述基底之上的高聚物，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或/和仲氨基。
  2. 根据权利要求1所述的芯片，其特征在于，所述高聚物中平均每个所述结构单元包含0.05～10个相同或不同的氨基。
  3. 根据权利要求2所述的芯片，其特征在于，所述高聚物中平均每个所述结构单元包含1～5个相同或不同的氨基。
  4. 根据权利要求1-3任一项所述的芯片，其特征在于，所述高聚物的聚合度范围为10～5000。
  5. 根据权利要求4所述的芯片，其特征在于，所述高聚物中结构单元包括
    Figure PCTCN2021111672-appb-100001
    Figure PCTCN2021111672-appb-100002
    中的至少一种。
  6. 根据权利要求1-5任一项所述的芯片，其特征在于，所述高聚物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子和聚乙烯亚胺中的至少一种。
  7. 根据权利要求1-6任一项所述的芯片，其特征在于，所述芯片还包括接枝于所述高聚物的探针。
  8. 根据权利要求7所述的芯片，其特征在于，所述探针通过连接基团与所述高聚物接枝，所述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；所述第一连接基团与所述探针键合，所述第二连接基团经所述氨基接枝于所述高聚物。
  9. 根据权利要求8所述的芯片，其特征在于，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
  10. 根据权利要求9所述的芯片，其特征在于，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n为3～2000；所述NHS-PEG n-DBCO或NHS-PEG n-N 3中的基团-NHS与所述氨基进行反应接枝。
  11. 根据权利要求10所述的芯片，其特征在于，所述探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3间的共价键合。
  12. 一种芯片的制备方法，其特征在于，包括如下步骤：
    (1)获取基底，所述基底修饰有环氧基；
    (2)将高聚物反应接枝至所述基底，所述高聚物的至少一个结构单元包含氨基，所述氨基为伯氨基或仲氨基。
  13. 根据权利要求12所述的制备方法，其特征在于，所述高聚物中平均每个所述结构单元包含0.05～10个相同或不同的氨基。
  14. 根据权利要求13所述的制备方法，其特征在于，所述高聚物中平均每个所述结构单元包含1～5个相同或不同的氨基。
  15. 根据权利要求12-14任一项所述的制备方法，其特征在于，所述高聚物的聚合度范围 为10～5000。
  16. 根据权利要求15所述的制备方法，其特征在于，所述高聚物中结构单元包括
    Figure PCTCN2021111672-appb-100003
    中的至少一种。
  17. 根据权利要求12-16任一项所述的制备方法，其特征在于，所述高聚物选自聚赖氨酸、聚鸟氨酸、壳聚糖、聚酰胺-胺树枝状大分子和聚乙烯亚胺中的至少一种。
  18. 根据权利要求12-17任一项所述的制备方法，其特征在于，步骤(2)中，所述反应接枝是通过使所述高聚物与所述基底在碱性溶液中接触进行的，所述碱性溶液的pH值为8.5～10。
  19. 根据权利要求18所述的制备方法，其特征在于，步骤(2)中，所述反应接枝是在反应温度为37～55℃的条件下反应3～24h。
  20. 根据权利要求12-19任一项所述的制备方法，其特征在于，还包括步骤(3)：将探针接枝至所述高聚物。
  21. 根据权利要求20所述的制备方法，其特征在于，所述探针通过连接基团与所述高聚物接枝，所述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；所述第一连接基团与所述探针键合，所述第二连接基团经所述氨基接枝于所述高聚物。
  22. 根据权利要求21所述的制备方法，其特征在于，所述第二连接基团选自-NHS基团、环氧基团和异氰酸酯基团中的至少一种。
  23. 根据权利要求22所述的制备方法，其特征在于，所述连接基团由NHS-PEG n-DBCO或NHS-PEG n-N 3制成，其中，n为3～2000；所述NHS-PEG n-DBCO或NHS-PEG n-N 3中的基团-NHS与所述氨基进行反应接枝。
  24. 根据权利要求23所述的制备方法，其特征在于，所述探针修饰有基团-DBCO或基团-N 3；所述键合的方式为基团-DBCO与基团-N 3间的共价键合。
  25. 根据权利要求19-24任一项所述的制备方法，其特征在于，所述反应接枝是通过使所述连接基团与所述高聚物在碱性溶液中接触进行的，所述碱性溶液的pH值为7～9。
  26. 根据权利要求24所述的制备方法，其特征在于，所述反应接枝是在反应温度为室温的条件下反应30～90min。
  27. 根据权利要求19-25任一项所述的制备方法，其特征在于，所述共价键合是通过使所述探针与连接基团在碱性溶液中接触下进行的，所述碱性溶液的pH值为7～8。
  28. 根据权利要求27所述的制备方法，其特征在于，进行所述共价键合的所述碱性溶液中含有表面活性剂，所述表面活性剂选自十四烷基三甲基溴化铵、十六烷基三甲基溴化铵和十二烷基三甲基溴化铵中的至少一种。
  29. 权利要求1-11任一项所述的芯片、或权利要求12-28任一项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。
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