WO2021219073A1 - 一种芯片表面连接体及其制备方法和应用 - Google Patents

一种芯片表面连接体及其制备方法和应用 Download PDF

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WO2021219073A1
WO2021219073A1 PCT/CN2021/090905 CN2021090905W WO2021219073A1 WO 2021219073 A1 WO2021219073 A1 WO 2021219073A1 CN 2021090905 W CN2021090905 W CN 2021090905W WO 2021219073 A1 WO2021219073 A1 WO 2021219073A1
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chip
group
molecule
linker
acid
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PCT/CN2021/090905
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English (en)
French (fr)
Inventor
胡飞驰
吴政宪
王建鹏
范玉峰
王梦嘉
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南京金斯瑞生物科技有限公司
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Application filed by 南京金斯瑞生物科技有限公司 filed Critical 南京金斯瑞生物科技有限公司
Priority to EP21795393.4A priority Critical patent/EP4144860A4/en
Priority to JP2022564503A priority patent/JP7511025B2/ja
Priority to KR1020227040185A priority patent/KR20230002822A/ko
Priority to CN202180031788.0A priority patent/CN115461471A/zh
Priority to US17/922,182 priority patent/US20230174578A1/en
Publication of WO2021219073A1 publication Critical patent/WO2021219073A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • C07K9/003—Peptides being substituted by heterocyclic radicals, e.g. bleomycin, phleomycin
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/30—Characterised by physical treatment
    • C12Q2523/307—Denaturation or renaturation by electric current/voltage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to the technical field of biochip preparation, and in particular to a linker for synthesizing nucleic acid from an electric-assisted chip, and a preparation method and application thereof.
  • a stable linker on metal or semiconductor chips is very important. On the one hand, it can be used for surface in-situ synthesis or pre-preparation of a large number of oligo pools and DNA probes to ensure that nucleic acid molecules are synthesized It will not drop during the process to ensure the quality of synthesis; on the other hand, the linker can firmly connect the nucleic acid to the surface of the chip to achieve different applications, such as in-situ hybridization on the chip, chip screening and chip diagnosis, etc., through the detection of the sample hybridization signal Detection and analysis, and then qualitative or quantitative analysis of specific biomarkers in the sample, can play a huge application in the fields of disease diagnosis, drug screening, new drug development, and agricultural and environmental research.
  • the existing linkers used for chip synthesis of nucleic acids still have some problems, especially in terms of linker stability and preparation cost. These problems are related to the method used to prepare the linker.
  • the methods of preparing linker in the prior art mainly include small molecule adhesion method [1], metal-sulfhydryl reaction method [2], and polymer coating method [3,4].
  • the small molecule adhesion method uses the adhesion of high concentration of small molecules to stick to the chip surface to form a linker.
  • the disadvantage of this method is that the prepared linker has weak adhesion, especially during the nucleic acid synthesis process, which may cause synthesis errors. increase.
  • the linker prepared by this method is sensitive to water, and will fall off during subsequent applications, making it difficult to further meet the application requirements of our customers.
  • the metal-sulfhydryl reaction method uses sulfhydryl groups to react with metals to form a covalent bond as a linker. The problem with this method is that the sulfhydryl groups will be reduced and fall off from the metal surface when the nucleic acid is synthesized by electricity, resulting in the failure of nucleic acid synthesis and making subsequent applications more difficult. .
  • the polymer coating method uses polymer or nanomaterials on the surface of the chip and then modifies the corresponding molecules to form a linker, but the stability of the combination of the coating material and the metal surface is limited, and the thickness of each coating is difficult to control and difficult to achieve Highly uniform surface, and there are still insufficient stability under hot water or alkaline conditions, which needs to be solved urgently.
  • the present application provides a chip surface connector and a preparation method and application thereof.
  • the chip surface connector provided in the present application has good stability, is stable under hot water and alkaline conditions, and has good electrical conductivity. Electrical stability and resistance to organic solvents required for nucleic acid synthesis are conducive to applications such as nucleic acid synthesis.
  • the present invention provides a chip surface connector, which is prepared by the following steps: Step 1: In the presence of acid and nitrite, a direct current voltage is applied to make the aromatic amine bond molecules react with the chip surface to form a connection Bonding molecular groups on the surface of the chip; Step 2: Reaction modification with functionalized molecules to obtain a linker containing functionalized molecular groups, the functionalized molecular groups containing hydroxyl and ester groups.
  • the bonding molecule is an aniline substance. In other embodiments, the bonding molecule is selected from p-aminophenylacetic acid, p-aminophenethyl alcohol or p-aminophenylenediamine, preferably p-aminophenylacetic acid.
  • the direct current voltage is a constant direct current voltage
  • the applied direct current voltage is selected from 0.5V to 5.0V, preferably 2.5V to 3.0V, more preferably 2.5V.
  • Other preferred constant DC voltages are 0.5V, 0.6V, 0.7V, 0.8V, 0.9V, 1.0V, 1.1V, 1.2V, 1.3V, 1.4V, 1.5V, 1.6V, 1.7V, 1.8V, 1.9 V, 2.0V, 2.1V, 2.2V, 2.3V, 2.4V, 2.6V, 2.7V, 2.8V, 2.9V, 3.0V, 3.1V, 3.2V, 3.3V, 3.4V, 3.5V, 3.6V, 3.7V, 3.8V, 3.9V, 4.0V, 4.1V, 4.2V, 4.3V, 4.4V, 4.5V, 4.6V, 4.7V, 4.8V, 4.9V or 5.0V.
  • the applied DC voltage time is 10 minutes to 50 minutes, preferably the DC voltage application time is 10 minutes to 30 minutes, more preferably 20 minutes.
  • Other preferred DC voltage application time is 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes or 30 minutes.
  • the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite, preferably sodium nitrite.
  • the acid is selected from hydrochloric acid, nitric acid or sulfuric acid, preferably hydrochloric acid.
  • the functionalized molecule is a hydroxyl substance containing a long carbon chain and an ester group.
  • the functionalized molecule is selected from succinic anhydride modified base monomer, hydroxyethyl methacrylate, succinic acid modified base monomer, or oxalic acid modified base monomer , Preferably a base monomer modified by succinic anhydride.
  • the base monomer portion of the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine, and uracil.
  • the functionalized molecule is adenine, guanine, cytosine, thymine or uracil modified with succinic anhydride.
  • the bonding molecular group and the functionalized molecular group further include a linking arm molecular group.
  • the linker molecular group is connected by the reaction between the linker molecule and the bonding molecular group
  • the functionalized molecular group is connected by the reaction between the functionalized molecule and the linker molecular group.
  • the linker molecule is a diamine or glycol substance. In other embodiments, the linker molecule is selected from the group consisting of ethylene diamine, hexamethylene diamine, decane diamine, 1,8-octane diamine, ethylene glycol, hexanediol, decanediol or 1,8- Octanediol, preferably 1,8-octanediamine. In some embodiments, the linker molecule is a diamine substance. In other embodiments, the linker molecule is selected from ethylene diamine, hexamethylene diamine, decane diamine or 1,8-octane diamine, preferably 1,8-octane diamine.
  • the chip is a metal chip, preferably a gold, platinum or aluminum chip, more preferably a metal platinum chip.
  • the present invention also provides a method for preparing the chip surface connector, which includes the following steps:
  • Step 1 Mix the bonding molecules of aromatic amines with acid and nitrite to obtain a mixed solution
  • Step 2 Bring the mixed solution of step 1 into contact with the surface of the chip, and apply a direct current voltage to react to form bonding molecular groups connected to the surface of the chip;
  • Step 3 Reaction modification with a functionalized molecule to obtain a linker containing a functionalized molecular group, wherein the functionalized molecular group includes a hydroxyl group and an ester group.
  • step 3 the chip surface after the reaction in step 2 is contacted with the linker molecule to connect the linker molecule group.
  • the surface of the chip after the reaction is further contacted with the functionalized molecule in step 3 to connect functionalized molecular groups.
  • the bonding molecule is an aniline substance. In other embodiments, the bonding molecule is p-aminophenylacetic acid, p-aminophenethyl alcohol or p-aminophenylenediamine, preferably p-aminophenylacetic acid.
  • the functionalized molecule is a hydroxyl substance containing a long carbon chain and an ester group.
  • the functionalized molecule is selected from succinic anhydride modified base monomer, hydroxyethyl methacrylate, succinic acid modified base monomer, or oxalic acid modified base monomer , More preferably a monomer modified with succinic anhydride.
  • the base monomer part of the functionalized molecule is selected from one or more of adenine, guanine, cytosine, thymine, and uracil.
  • the DC voltage in step 2 is a constant DC voltage
  • the applied DC voltage is selected from 0.5V to 5.0V, preferably 0.5V to 3.0V, and more preferably 2.5V.
  • step 2 when the mixed solution obtained in step 1 is contacted with the chip surface and a constant DC voltage is applied to react, the temperature is 4°C to 40°C, preferably 20°C to 37°C, such as 20°C, 21°C ⁇ 22°C ⁇ 23°C ⁇ 24°C ⁇ 25°C ⁇ 26°C ⁇ 27°C ⁇ 28°C ⁇ 29°C ⁇ 30°C ⁇ 31°C ⁇ 32°C ⁇ 33°C ⁇ 34°C ⁇ 35°C ⁇ 36°C ⁇ 37°C, preferably It is room temperature; the reaction time is 10 minutes to 50 minutes, preferably 10 minutes to 30 minutes, such as 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes or 30 minutes, more preferably 20 minutes.
  • the reaction time is 10 minutes to 50 minutes, preferably 10 minutes to 30 minutes, such as 10 minutes, 12 minutes, 14 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes or 30 minutes, more preferably 20 minutes.
  • the nitrite is selected from sodium nitrite, potassium nitrite or calcium nitrite, preferably sodium nitrite.
  • the acid is selected from hydrochloric acid, nitric acid or sulfuric acid, preferably hydrochloric acid.
  • the linker molecule is a diamine or glycol substance selected from ethylene diamine, hexamethylene diamine, decane diamine, 1,8-octane diamine, ethylene glycol, hexanediol, Decanediol or 1,8-octanediol, preferably 1,8-octanediamine.
  • the linker molecule is a diamine substance selected from ethylene diamine, hexamethylene diamine, decane diamine or 1,8-octane diamine, preferably 1,8-octane diamine.
  • the chip is a metal chip selected from gold, platinum and aluminum chips, preferably a metal platinum chip.
  • the present invention also provides the application of the chip surface connector as described above in nucleic acid synthesis or preparation of a chip kit.
  • the present invention provides a chip surface linker.
  • the linker reacts with the surface of the chip by applying a constant DC voltage in the presence of acid and nitrite through the bonding molecules of aromatic amines to form a bond connected to the surface of the chip.
  • the functionalized molecule reacts with the linker molecular group to obtain a linker containing the functionalized molecular group, wherein
  • the functionalized molecular group includes a hydroxyl group and an ester group.
  • the bonding molecules described in the present invention are aromatic amines, such as anilines, which can be p-aminophenylacetic acid, p-aminophenethyl alcohol or p-aminophenylenediamine.
  • the bonding molecule is p-aminophenylacetic acid.
  • the bonding molecular group described in the present invention is a group covalently connected to the surface of the chip obtained after the bonding molecule and the surface of the chip undergo an electro-catalyzed reaction.
  • the bonding molecule group is a group formed by an aromatic amine-based bonding molecule that reacts with the surface of the chip by applying a constant DC voltage in the presence of acid and nitrite.
  • the bonding molecule can be p-aminophenylacetic acid, p-aminophenethyl alcohol or p-aminophenylenediamine.
  • the bonding molecules of aromatic amines are diazotized in the presence of acid and nitrite, and a constant DC voltage is applied to generate aromatic carbon radicals.
  • the aromatic carbon radicals react with chip electrode materials to form covalent bonds and bond. .
  • the linker molecules described in the present invention are diamines or glycols, which can be ethylenediamine, hexamethylenediamine, decanediamine, 1,8-octanediamine, ethylene glycol, hexanediol, decanediol Or 1,8-octanediol, in some embodiments of the invention 1,8-octanediamine.
  • the linker molecular group described in the present invention is a group formed by the reaction of the linker molecule with the bonding molecular group and the functionalized molecule successively.
  • the linker molecular group may be selected from the group consisting of ethylenediamine group, hexamethylenediamine group, decanediamino group, 1,8-octanediamino group, ethylene glycol group, hexanediol group, Decanediol or 1,8-octanediol.
  • the chip connected with the bonding molecular group is immersed in the linker molecule solution to modify the long-chain molecule, increase the distance between the functional group and the chip, and reduce the steric hindrance during subsequent application.
  • the functionalized molecule in the present invention is a hydroxyl substance containing a long carbon chain and containing an ester group, which can be a base monomer modified by succinic anhydride, hydroxyethyl methacrylate, a base monomer modified by succinic acid, or ethylene diacrylate.
  • the acid-modified base monomer uses a mixed solution of two or more of adenine, guanine, cytosine, or thymine modified with succinic anhydride in some embodiments of the present invention.
  • a solution of succinic anhydride modified adenine, succinic anhydride modified guanine, succinic anhydride modified cytosine, or succinic anhydride modified thymine is used alone.
  • the functionalized molecular group may be selected from succinic anhydride modified adenine, guanine, cytosine, thymine or uracil, hydroxyethyl methacrylate, succinic acid modified adenine , Guanine, cytosine, thymine or uracil, oxalic acid-modified adenine, guanine, cytosine, thymine, or uracil in which the carboxylic acid reacts with the amino group to remove the hydroxyl group.
  • the functional molecule is modified on the connecting arm, and the functionalized molecular group formed contains a hydroxyl group and an ester group, which is convenient for the subsequent electro-assisted DNA synthesis and cutting on the chip.
  • the chip surface connector of the present invention includes: (1) a bonding molecular group, which is covalently attached to the surface of the chip; (2) a functionalized molecular group, which contains a hydroxyl group A reactive group with an ester group; and (3) a linker molecular group that connects the bonding molecular group and the functionalized molecular group.
  • Figures 1a to 1d are schematic diagrams of preparing chip connectors and synthesizing DNA in accordance with an embodiment of the present invention. In Figure 1d, bonding molecules, linker molecules, and functional molecules are sequentially connected to the surface of the chip to obtain the chip surface connector.
  • the linker includes a bonding molecular group, a linking arm molecular group and a functionalized molecular group.
  • the bonding molecule sequentially connected to the chip surface connector described in the present invention is p-aminophenylacetic acid, the linker molecule is 1,8-octanediamine, and the functional molecule is adenine, guanine, and cytosine modified by succinic anhydride. Or thymine.
  • the metal is generally a sheet metal material, that is, a metal chip, and the metal component can be selected from gold, platinum or aluminum, and in some embodiments of the present invention is a metal platinum chip.
  • the chip is a CustomArray chip.
  • the metal sheet can be washed with water and alcohol in sequence, and then soaked with an acid solution, heated to a certain temperature, such as 40-70°C, placed for 5 to 30 minutes, rinsed with water, and dried to obtain a dry and clean metal surface.
  • the water is generally distilled water
  • the alcohol is ethanol
  • the present invention also provides a method for preparing a chip surface connector, which includes the following steps: mixing bonding molecules with hydrochloric acid and sodium nitrite solutions to obtain a mixed solution; then contacting the surface of the chip with the mixed solution and applying a constant DC voltage The reaction is carried out; the surface of the chip after the reaction is sequentially contacted with the linker molecule solution and the functionalized molecule solution for reaction modification, and the linker is prepared, as shown in Figure 1d.
  • the embodiment of the present invention first provides a clean metal chip, then prepares a mixed solution obtained by mixing the bonding molecules with the hydrochloric acid and sodium nitrite solutions, and finally contacts the metal chip with the mixed solution to react.
  • the bonding molecule is an aromatic amine substance, preferably an aniline substance, including but not limited to one or more of p-aminophenylacetic acid, p-aminophenethyl alcohol, and p-aminophenylenediamine. In some embodiments of the present invention In the scheme, it is p-aminophenylacetic acid.
  • 0.10-0.15 mM cold p-aminophenylacetic acid and 15 mM hydrochloric acid are mixed uniformly, and 0.07-0.10 mM sodium nitrite is added again, and the mixed solution is prepared by rapid shaking.
  • the cleaned metal chip is quickly immersed in the above mixed solution, that is, the metal surface is in contact with the mixed solution, and a fixed DC voltage is applied to stand still for reaction.
  • the applied fixed DC voltage is 0.5V to 3.0V, preferably 2.5V;
  • the reaction temperature is 20°C to 37°C, preferably room temperature; and the reaction time is 10 minutes to 30 minutes, preferably 20 minutes.
  • the constant DC voltage applied to the chip is 0.5V ⁇ 3.0V, and the reaction is promoted by standing at room temperature for 10 minutes to 30 minutes.
  • the constant DC voltage applied to the chip is 2.5V, and the reaction is promoted by standing at room temperature for 20 minutes.
  • the principle of this step is that in the presence of hydrochloric acid and sodium nitrite, the aromatic amine molecules are diazotized to form aromatic carbon radicals after a constant DC voltage is applied, and the aromatic carbon radicals react with the electrode material to form covalent bonds and combine.
  • the specific reaction As follows:
  • the chip After the reaction, the chip is taken out, and then washed with water and alcohol in turn, then the chip is washed with an acid solution, and finally with water, and dried.
  • the reacted chip utilizes the cross-linking acylation reaction of the carboxyl group and the amino group to modify the linker molecule to increase the distance between the functionalized group and the chip and reduce the steric hindrance during subsequent applications.
  • the linker molecule is a diamine substance, including but not limited to one or more of ethylene diamine, hexamethylene diamine, decane diamine, 1,8-octanediamine methanol, In some embodiments of the present invention, it is 1,8-octanediamine; the reacted chip is immersed in 1,8-octanediamine methanol solution, allowed to stand and react for 8-12h, preferably 8h, and then washed with water and blown dry .
  • Functionalized molecules are hydroxyl substances containing long carbon chains and ester groups, including but not limited to succinic anhydride modified base monomers, hydroxyethyl methacrylate, succinic acid modified base monomers or oxalic acid Modified base monomer.
  • succinic anhydride modified base monomers hydroxyethyl methacrylate
  • succinic acid modified base monomers oxalic acid Modified base monomer.
  • a solution of succinic anhydride modified adenine, guanine, cytosine, thymine, or uracil is used alone.
  • two or more mixed solutions of adenine, guanine, cytosine, thymine and uracil modified with succinic anhydride are used.
  • Functionalized molecules contain ester groups and hydroxyl groups, which can be used for DNA synthesis and cleavage.
  • the ester group is the cutting site, in order to cut the DNA synthesized on the chip, into a free solution-like oligo pool.
  • the hydroxyl group contained in the functionalized molecule is the initiation site of DNA synthesis.
  • the hydroxyl group provided by the base monomer portion is used for DNA synthesis.
  • the direct current of aniline substances (such as p-aminophenylacetic acid, p-phenylenediamine, etc.) is used to promote the grafting (for industrial production) to form a stable surface covalent on the surface of the metal chip, and then modify the connecting arms and functional molecules, thereby A stable connection is formed on the surface of the chip.
  • the formation principle includes the following steps: (1) Aromatic amine molecules are diazotized in the presence of hydrochloric acid and sodium nitrite, and a fixed DC voltage is applied to generate aromatic carbon radicals. The aromatic carbon radicals react with electrode materials to form covalent bonds and bond.
  • the invention also provides the application of the chip surface connector as described above in the synthesis of DNA or the preparation of a chip kit.
  • the chip surface connector of the present invention includes the following applications: DNA synthesis on the chip; for disease biomarker chip detection; for the development of POCT (Point-of-Care-Testing) chip kits; high-throughput chip screening Reagent test kit.
  • the chip surface connector of the present invention can also be used to synthesize DNA after hot water treatment; the synthesized DNA can be hybridized with oligonucleotides (such as DNA primers) and then subjected to alkali and thermal TE treatments, and can also be used to synthesize DNA with oligonucleosides. Acid (such as DNA primer) hybridization.
  • the DNA synthesized by the chip surface connector of the present invention is applied through multiple cycles of nucleic acid hybridization, elution and hybridization, and the chip surface connector can still maintain good stability.
  • the chip surface connector prepared in the embodiment of the present invention has good electrical conductivity and stable power on, and can be used for electrically assisting the synthesis of nucleic acid molecules such as DNA.
  • the chip surface connector of the present invention is resistant to organic solvents required for DNA synthesis, otherwise DNA synthesis cannot be performed.
  • the present invention can solve the problems of the existing connector being sensitive to water and unstable to heat, so the chip can be reused.
  • chip refers to a solid support formed of an inorganic substance such as a semiconductor or a metal such as gold, silver, platinum, etc., with a microarray formed by specific sites on the surface.
  • the sites are usually arranged in rows and columns. Can be used for a certain type of chemical or biochemical analysis, synthesis or method. These sites on the microarray are usually smaller than 100 microns.
  • the chip is a metal platinum chip.
  • linker refers to a molecule with one end connected or capable of being connected to a solid surface (such as a metal chip), and the other end having a reactive group, the reactive group and or capable of being related to chemical substances, such as small molecules, oligomers Or polymers are connected.
  • the linker can be bound to the solid surface and/or its reactive group has already been linked with related chemicals.
  • the reactive group of the linker may be connected with a protective group, wherein the protective group can be removed by chemical or electrochemical methods.
  • the linker may contain multiple molecular groups, wherein the molecules are covalently connected.
  • bonded molecular group refers to a chemical molecule located at the end of the linker, one end of the group can be covalently connected to a solid surface (such as a metal chip), and the other end has a reactive group, the reactive group and or can Connected to related chemical substances, such as small molecules, oligomers or polymers, can be connected to linker molecules or functionalized molecules in the present invention.
  • the bonding molecular group has been bound to the solid surface and/or its reactive group has been connected to a related chemical substance (such as a linking arm molecular group or a functionalized molecular group).
  • a protective group may be attached to the reactive group to which the molecular group is bonded, wherein the protective group can be removed by chemical or electrochemical methods.
  • linker molecular group refers to a chemical molecule located in the middle part of the linker, one end of the group can be connected to the bonding molecular group, and the other end has a reactive group, the reactive group and or can be related to the chemical molecule Substances, such as small molecules, oligomers or polymers, can be connected to functionalized molecules in the present invention.
  • the linker molecular group can be combined with the bonding molecular group and/or its reactive group has been connected to a related chemical substance (such as a bonding molecular group or a functionalized molecular group).
  • the reactive group linking the arm molecular group may be connected with a protective group, wherein the protective group can be removed by chemical or electrochemical methods.
  • the linker molecular group can be formed in situ on the bonded molecular group.
  • the linking arm molecular group can be formed first, and then connected to the bonding molecular group that has been connected to the solid surface.
  • the linker molecular group can be synthesized externally on the related chemical substance first, and then connected with the bonding molecular group that has been connected to the solid surface.
  • Related chemical substances can be connected to the linker molecular group connected to the bonding molecular group, and then the entire structure can be connected to the reaction site on the solid surface.
  • the purpose of the linking arm molecular group is to extend the distance between the related molecule and the solid surface of the chip and reduce the steric hindrance during subsequent applications.
  • the linker molecular group can be ethylenediamine group, hexamethylenediamine group, decanediamino group, 1,8-octanediamine group, ethylene glycol group, hexanediol group, decanediol group Or 1,8-octanediol group, etc.
  • the term "functionalized molecular group” refers to a chemical molecule located at the end of the linker, one end of the group can be connected to a linker molecular group or a bonding molecular group, and the other end has a chemical molecule with a reactive group that is connected to the Or it can be connected to related chemical substances, such as small molecules, oligomers or polymers, and can be connected to deoxyribonucleotide molecules in the present invention.
  • the functionalized molecular group can be connected to the linker molecular group and/or the reactive group to which the related chemical substance is connected.
  • a protective group may be attached to the reactive group of the functionalized molecular group, wherein the protective group can be removed by chemical or electrochemical methods.
  • the functionalized molecule can be adenine, guanine, cytosine, thymine or uracil modified with succinic anhydride, hydroxyethyl methacrylate, adenine modified with succinic anhydride, guanine, cytosine, Thymine or uracil, oxalic acid modified adenine, guanine, cytosine, thymine and uracil, etc.
  • base monomer means a molecule that can undergo polymerization to form a combined unit of the basic structure of a macromolecule, such as an oligomer, co-oligomer, polymer, or copolymer.
  • monomers include A, C, T, G, adenylic acid, guanylic acid, cytidine acid, uridine acid, amino acids, and other compounds.
  • aryl means an aromatic carbocyclic group having a monovalent value and about 4-20 carbon atoms.
  • aryl groups include, but are not limited to: phenyl, naphthyl, and anthracenyl.
  • One or more hydrogen atoms of the substituted aryl group may be substituted with other groups.
  • the aryl group used herein includes groups with multiple valences, so as to meet the requirement of substitution.
  • the aryl group may be part of a condensed ring structure, for example, N-hydroxysuccinimide is combined with phenyl (benzene) to form N-hydroxyphthalimide.
  • aromatic amine molecule refers to an amine with an aromatic substituent-that is -NH2, -NH- or a nitrogen-containing group connected to an aromatic ring, the structure of aromatic hydrocarbons usually contains one or more benzene ring.
  • Aniline is the simplest example of this class of compounds.
  • the aromatic amine molecule can be p-aminophenylacetic acid, p-aminophenethyl alcohol or p-aminophenylenediamine.
  • oligomer refers to molecules with intermediate relative molecular masses, the structure of which basically contains a small number of units derived from molecules of lower relative molecular mass in fact or concept. If after removing one or several units of a molecule, its properties are indeed significantly different, then the molecule can be regarded as having a medium relative molecular weight. If part or whole of the molecule has a medium relative molecular weight, and basically contains a small number of units actually or conceptually derived from a lower relative molecular weight molecule, it can be described as oligomeric or used as an adjective oligomeric Things to describe. Oligomers are usually composed of monomers.
  • the present invention utilizes aniline substances to effectively bond with the electrode surface through carbon free radicals under constant DC voltage conditions, thereby forming a metal-carbon covalent bond, stably bonding the molecules to the chip surface, and then proceeding
  • the linker molecules and functionalized molecules are modified to form a stable linker on the surface of the chip.
  • the invention obtains a chip surface connector with high adhesion, which can be stably bonded to the chip surface.
  • the chip surface connector of the present invention is stable under hot water and alkaline conditions, has good electrical conductivity, is stable when powered on, and resists organic solvents required for nucleic acid synthesis, and is extremely advantageous for subsequent nucleic acid synthesis and other applications.
  • Figure 1 is a schematic diagram of the preparation of a chip surface connector and its use for DNA synthesis in Example 1 of the present invention, wherein 1 is a metal chip, 2 is a linker prepared by this method, and 3 is a DNA synthesized on this linker. ;
  • Example 2 is a schematic diagram showing the comparison of the stability of the chip surface connector prepared by the method in Example 1 of the present invention and the traditional connector in hot water;
  • Figure 3 shows the stability of the chip surface connector prepared by the method in Example 1 of the present invention in the application of nucleic acid synthesis
  • Example 4 is a schematic diagram of a hybridization experiment of the chip surface connector prepared by the method in Example 1 of the present invention after being used for DNA synthesis;
  • FIG. 5 is a schematic diagram of the cleavability of DNA synthesized by the chip surface connector prepared by the method of Example 1 of the present invention.
  • FIG. 6 is a schematic diagram of DNA synthesized by the chip surface connector prepared by the method of Example 1 of the present invention.
  • Figure 7 is a diagram of DNA electrophoresis gel synthesized from the chip surface connector prepared by the method of Example 1 of the present invention.
  • the sample is the DNA (120nt) synthesized from the chip surface connector prepared in Example 1, and the reference is US20060105355A1 disclosed in Example 1.
  • DNA (120nt) synthesized by the original linker chip, ladder means standard product (after electrophoresis, it can display the nucleic acid position of 50nt, 75nt, 150nt, 200nt, 300nt).
  • Chip modification Take 18.15mg cold p-aminophenylacetic acid and 15mM hydrochloric acid (1820uL H 2 O, 180uL 0.5M HCl) and mix well, add 6.21mg sodium nitrite again, and shake well;
  • base monomer is adenine, thymine, guanine or cytosine 10.8mg and NHS 1.53mg, EDC 7.64mg dissolved in 100uL water
  • base monomer is adenine, thymine, guanine or cytosine 10.8mg and NHS 1.53mg, EDC 7.64mg dissolved in 100uL water
  • the schematic diagram of the linker prepared by the above operations is shown in Figure 1d.
  • the chip connects the bonding molecule, the linker molecule and the functionalized molecule in sequence to obtain the chip linker.
  • the linker includes the bond molecule group and the linker molecular group. Groups and functionalized molecular groups.
  • the prepared linker is then subjected to DNA synthesis (using CustomArray chip synthesizer), and the resultant effect diagram is shown in Figure 1c, and perfect DNA can be synthesized.
  • the original linker is to deposit small polyhydroxy molecules on the chip (see Example 1 in US20060105355A1), clean with distilled water and dry, then place it in distilled water at 80°C for 2 days, dry with argon, and then synthesize it on the chip with a CustomArray chip synthesizer. 33nt DNA, put the chip on the chip scanner (CustomArray, GenePix4000B) to scan the surface, the result is shown in Figure 2a, most of the area can not be synthesized by DNA.
  • the new linker is made according to the method of Example 1 of the present invention, placed in distilled water at 80°C for 2 days, dried with argon, and then synthesized 33nt DNA on the chip using the CustomArray chip synthesizer, and scanned on the chip scanner (CustomArray, GenePix4000B) As shown in Figure 2b, the DNA on the chip surface can still exist stably, indicating that the new linker has sufficient stability in hot water, which is of great value for subsequent high-temperature hybridization or other detection applications on the chip surface.
  • Example 3 The stability of the new linker in nucleic acid synthesis applications
  • the new linker was made according to the method of Example 1 of the present invention. Placed in distilled water at 80°C for 2 days, dried with argon, synthesized 33nt DNA on the chip with a CustomArray chip synthesizer, and scanned the surface on the chip scanner. The result is shown in the figure.
  • Example 4 The stability of the new linker in multiple hybridization-elution-hybridization applications
  • the new linker is made according to the method of Example 1 of the present invention.
  • Figure 4 after drying with argon, 33nt DNA is synthesized on the chip using the CustomArray chip synthesizer, and the surface is scanned on the chip scanner.
  • Figure 4b As shown; take two fluorescent DNA primers (100pM each), hybridize with the DNA synthesized on the chip (room temperature, hybridization reaction for 2 hours), wash with PBS buffer and scan on the chip scanner, the result is shown in Figure 4c, Shows different fluorescent spots; then rinse the chip with 100mM NaOH solution, remove the hybridization primers, dry it with argon, and scan the surface on the chip scanner.
  • Example 5 The cleavability of DNA from the chip after synthesis
  • the linker ( Figure 5a) is prepared using the method of Example 1 of the present invention, and then 120 nt DNA is synthesized on the chip with the CustomArray chip synthesizer ( Figure 5b).
  • the formed linker contains a cleavage group (ester group), which can be used in Cut the oligo from the chip under an alkaline (ammonia water) heating strip (65°C) (16 hours) to form free oligo. After washing the chip with water, scan it with a chip scanner ( Figure 5c), indicating that the DNA is almost completely cut. .
  • Example 6 The new linker is used for DNA quality characterization in DNA synthesis
  • the linker is prepared by the method in Example 1 of the present invention, and then 120 nt DNA is synthesized on the chip by the CustomArray chip synthesizer (as shown in Figure 6).
  • the formed linker contains a cleavage group (ester group), which can be used in alkaline (ammonia water).
  • oligos are cut (16h) from the chip to form a free oligo pool, which is measured by Thermo Scientific TM NanoDrop TM One Microvolume UV-Vis Spectrophotometers instrument
  • the concentration of the two chips is 21.4ng/uL and 19.4ng/uL, which meets the requirements.
  • perform PCR amplification and the resulting product is analyzed by electrophoresis gel image. As shown in Figure 7, it is similar in size to the standard product and is in the same position, indicating that the product synthesized by this linker is correct.

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Abstract

本发明涉及生物芯片领域，提供了一种芯片表面连接体及其制备方法和应用，所述芯片表面连接体通过芳香胺类的键合分子在酸和亚硝酸盐存在下，施加直流电压与芯片表面反应形成连接在芯片表面的键合分子基团，再用功能化分子反应修饰包含羟基和酯基的功能化分子基团制得。本发明获得的芯片表面连接体能更稳定地结合于芯片表面，在热水和碱性条件下稳定，具有较好的导电性，加电稳定性以及抗核酸合成所需有机溶剂，对于后续的核酸合成及其他应用极其有利。

Description

一种芯片表面连接体及其制备方法和应用 技术领域
本发明涉及生物芯片制备技术领域，尤其涉及一种用于电辅助芯片合成核酸的连接体及其制备方法和应用。
背景技术
金属或半导体芯片上稳定的连接体(linker)非常重要，一方面，其可用于表面原位合成或预制备大量的寡核苷酸聚合物(oligo pool)和DNA探针，确保核酸分子在合成过程中不会掉落，保证合成质量；另一方面，连接体能牢固连接核酸于芯片表面，以实现不同的应用，如芯片上原位杂交，芯片筛选和芯片诊断等，通过对样品杂交信号的检测分析，进而对样品中特定生物标志物(biomarker)实现定性或定量分析，可在疾病诊断、药物筛选、新药开发和农业与环境研究等领域发挥巨大的应用。然而，现有用于芯片合成核酸的linker依然存在一些问题，特别是在linker稳定性和制备成本上，这些问题和制备linker所使用的方法相关。现有技术中制备linker的方法主要有小分子黏附法[1]，金属-巯基反应法[2]，聚合物涂敷法[3,4]。其中小分子黏附法是利用高浓度小分子的粘附性粘于芯片表面形成linker，此方法的缺点在于制备的linker粘附性不牢固，特别是在核酸合成过程中可能存在脱落，导致合成错误增多。另外此方法制备的linker对水敏感，特别在后续应用时会脱落，难以进一步满足广大客户的应用需求。金属-巯基反应法是利用巯基与金属反应形成共价键来作为linker，此方法问题在于进行加电合成核酸时，巯基会被还原而从金属表面脱落，导致核酸合成失败，更难于进行后续应用。聚合物涂敷法是利用涂敷聚合物或纳米材料于芯片表面，再修饰相应分子来形成linker，但涂敷材料与金属表面结合的稳定性有限，每次涂层的厚度难于控制，难以实现高度均一表面，且在热水或碱性的条件下稳定性尚存在不足，急需解决。
发明内容
有鉴于此，本申请提供一种芯片表面连接体及其制备方法和应用，本申请提供的芯片表面连接体稳定性好，在热水和碱性条件下稳定，具有较好的导电性，加电稳定性以及抗核酸合成所需有机溶剂，有利于核酸合成等应用。
本发明提供一种芯片表面连接体，所述连接体通过以下步骤制得：步骤1：在酸和亚硝酸盐存在下，通过施加直流电压使得芳香胺类的键合分子与芯片表面反应形成连接在芯片表面的键合分子基团；步骤2：用功能化分子反应修饰，得到包含功能化分子基团的连接体，所述功能化分子基团包含羟基和酯基。
在一些实施方案中，所述键合分子为苯胺类物质。在另一些实施方案中，所述键合分子选自对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺，优选为对氨基苯乙酸。
在一些实施方案中，所述直流电压为恒定直流电压，所施加直流电压选自0.5V～5.0V，优选为2.5V～3.0V，更优选2.5V。其他优选的恒定直流电压为0.5V、0.6V、0.7V、0.8V、0.9V、1.0V、1.1V、1.2V、1.3V、1.4V、1.5V、1.6V、1.7V、1.8V、1.9V、2.0V、2.1V、2.2V、2.3V、2.4V、2.6V、2.7V、2.8V、2.9V、3.0V、3.1V、3.2V、3.3V、3.4V、3.5V、3.6V、3.7V、3.8V、3.9V、4.0V、4.1V、4.2V、4.3V、4.4V、4.5V、4.6V、4.7V、4.8V、4.9V或5.0V。
在一些实施方案中，所施加直流电压时间为10分钟～50分钟，优选施加直流电压时间为10分钟～30分钟，更优选为20分钟。其他优选施加直流电压时间为10分钟、12分钟、14分钟、16分钟、18分钟、20分钟、22分钟、24分钟、26分钟、28分钟或30分钟。
在一些实施方案中，所述亚硝酸盐选自亚硝酸钠、亚硝酸钾或亚硝酸钙，优选为亚硝酸钠。
在一些实施方案中，所述酸选自盐酸、硝酸或硫酸，优选盐酸。
在一些实施方案中，所述功能化分子为包含长碳链且含有酯基的羟基物质。在另一些实施方案中，所述功能化分子选自丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或乙二酸修饰的碱基单体，优选为丁二酸酐修饰的碱基单体。在一些实施方案中，所述功能化分子的碱基单体部分选自腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶中的一种或多种。在一个具体实施方案中，所述功能化分子为丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶。
在一些实施方案中，所述键合分子基团和所述功能化分子基团之间还包括连接臂分子基团。优选地，所述连接臂分子基团是通过连接臂分子与键合 分子基团反应连接的，所述功能化分子基团通过功能化分子与所述连接臂分子基团反应连接的。
在一些实施方案中，所述连接臂分子为二胺类或二醇类物质。在另一些实施方案中，所述连接臂分子选自乙二胺、己二胺、癸二胺、1,8-辛二胺、乙二醇、己二醇、癸二醇或1,8-辛二醇，优选为1,8-辛二胺。在一些实施方案中，所述连接臂分子为二胺类物质。在另一些实施方案中，所述连接臂分子选自乙二胺、己二胺、癸二胺或1,8-辛二胺，优选为1,8-辛二胺。
在一些实施方案中，所述芯片为金属芯片，优选为金、铂或铝芯片，更优选金属铂芯片。
本发明还提供一种芯片表面连接体的制备方法，包括以下步骤：
步骤1：将芳香胺类的键合分子与酸、亚硝酸盐混合得到混合溶液；
步骤2：将步骤1的混合溶液与芯片表面接触，施加直流电压进行反应形成连接在芯片表面的键合分子基团；
步骤3：用功能化分子反应修饰，得到包含功能化分子基团的连接体，其中所述功能化分子基团包含羟基和酯基。
在一些实施方案中，所述步骤3之前包括将步骤2反应后的芯片表面与连接臂分子接触反应连接上连接臂分子基团。反应后的芯片表面进一步与在步骤3中功能化分子接触反应连接上功能化分子基团。
在一些实施方案中，所述键合分子为苯胺类物质。在另一些实施方案中，所述键合分子为对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺，优选为对氨基苯乙酸。
在一些实施方案中，所述功能化分子为包含长碳链且含有酯基的羟基物质。在另一些实施方案中，所述功能化分子选自丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或乙二酸修饰的碱基单体，更优选为丁二酸酐修饰的单体。其中，所述功能化分子的碱基单体部分选自腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶、尿嘧啶中的一种或多种。
在一些实施方案中，步骤2中所述直流电压为恒定直流电压，所施加直流电压选自0.5V～5.0V，优选为0.5V～3.0V，更优选为2.5V。
在一些实施方案中，步骤2中使步骤1得到的混合溶液与所述芯片表面接触施加恒定直流电压反应时温度为4℃～40℃，优选为20℃～37℃，例如 20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃，优选为室温；反应时间为10分钟～50分钟，优选10分钟～30分钟，例如10分钟、12分钟、14分钟、16分钟、18分钟、20分钟、22分钟、24分钟、26分钟、28分钟或30分钟，更优选为20分钟。
在一些实施方案中，所述亚硝酸盐选自亚硝酸钠、亚硝酸钾或亚硝酸钙，优选为亚硝酸钠。
在一些实施方案中，所述酸选自盐酸、硝酸或硫酸，优选为盐酸。
在一些实施方案中，所述连接臂分子为二胺或二醇类物质，选自乙二胺、己二胺、癸二胺、1,8-辛二胺、乙二醇、己二醇、癸二醇或1,8-辛二醇，优选为1,8-辛二胺。在一些实施方案中，所述连接臂分子为二胺类物质，选自乙二胺、己二胺、癸二胺或1,8-辛二胺，优选为1,8-辛二胺。
在一些实施方案中，所述芯片为金属芯片，选自金、铂和铝芯片，优选金属铂芯片。
本发明还提供如前文所述的芯片表面连接体在核酸合成或制备芯片试剂盒中的应用。
发明详述
本发明提供了一种芯片表面连接体(linker)，所述连接体通过芳香胺类的键合分子在酸和亚硝酸盐存在下，施加恒定直流电压与芯片表面反应形成连接在芯片表面的键合分子基团，之后连接臂分子与键合分子基团进行反应连接形成连接臂分子基团，再用功能化分子与连接臂分子基团反应，得到包含功能化分子基团的连接体，其中所述功能化分子基团包含羟基和酯基。
本发明描述的键合分子为芳香胺类物质，如苯胺类物质，可以是对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺。在本发明的一些实施方案中键合分子为对氨基苯乙酸。本发明中描述的键合分子基团是键合分子与芯片表面发生电促反应后得到的共价连接在芯片表面的基团。在一些实施方案中键合分子基团是芳香胺类的键合分子在酸和亚硝酸盐存在条件下，施加恒定直流电压与芯片表面反应形成的基团。在一些实施方案中，所述的键合分子可以是对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺。本发明中芳香胺类的键合分 子在酸和亚硝酸盐存在的条件下重氮化，加恒定直流电压生成芳碳自由基，芳碳自由基与芯片类电极材料反应生成共价键而结合。
本发明描述的连接臂分子为二胺类或二醇类物质，可以是乙二胺、己二胺、癸二胺、1,8-辛二胺、乙二醇、己二醇、癸二醇或1,8-辛二醇，在本发明的一些实施方案中为1,8-辛二胺。本发明中描述的连接臂分子基团是连接臂分子先后与键合分子基团及功能化分子反应生成的基团。在一些实施方案中，所述连接臂分子基团可选自乙二胺基、己二胺基、癸二胺基、1,8-辛二胺基、乙二醇基、己二醇基、癸二醇基或1,8-辛二醇基。本发明中连接键合分子基团的芯片浸入连接臂分子溶液，修饰长链分子，增加功能基团与芯片间距离，减小后续应用时的空间位阻。
本发明中功能化分子为包含长碳链且含有酯基的羟基物质，可以是丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或乙二酸修饰的碱基单体，在本发明的一些实施方案中使用丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶或胸腺嘧啶中二种或多种混合溶液。在本发明的一些实施方案中单独使用丁二酸酐修饰的腺嘌呤、丁二酸酐修饰的鸟嘌呤、丁二酸酐修饰的胞嘧啶或丁二酸酐修饰的胸腺嘧啶溶液。在一些实施方案中，所述功能化分子基团可选自丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶，甲基丙烯酸羟乙酯，丁二酸修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶，乙二酸修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶中羧酸与氨基反应后脱除羟基的基团。本发明中将功能分子修饰于连接臂上，形成的功能化分子基团含有羟基和酯基，便于后续芯片上电促DNA的合成以及切割。
本发明的芯片表面连接体包括：(1)键合分子基团，所述键合分子基团共价连接在芯片表面；(2)功能化分子基团，所述功能化分子基团包含羟基和酯基的反应基团；和(3)连接键合分子基团和功能化分子基团的连接臂分子基团。图1a～图1d是本发明实施例芯片连接体制备及合成DNA的示意图，其中，图1d中芯片表面依次连接键合分子、连接臂分子以及功能化分子得到芯片表面连接体，所述芯片表面连接体包括键合分子基团、连接臂分子基团和功能化分子基团。本发明中描述的芯片表面连接体依次连接的键合分子为 对氨基苯乙酸，连接臂分子为1,8-辛二胺，功能化分子为丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶或胸腺嘧啶。
本发明描述的芯片表面连接体通过共价键稳定结合在芯片表面，可用于电辅助芯片合成核酸等应用。其中，所述的金属一般是片状的金属材料，也就是金属芯片，金属成分可选自金、铂或铝，在本发明的一些实施方案中为金属铂芯片。本发明的一个具体实施方案中，所述芯片为CustomArray芯片。本发明实施例可依次用水、醇冲洗，再用酸溶液浸泡金属片，并加热至一定温度例如40～70℃，放置5到30分钟，最后用水冲洗，干燥，即得干燥清洁的金属表面。其中，所述水一般为蒸馏水，醇为乙醇，而酸溶液主要采用prinaha溶液(H 2SO 4:H 2O 2＝3:1体积比)，主要去除污染物质如其他杂质金属粉尘、无机颗粒和有机小分子。
本发明还提供一种芯片表面连接体的制备方法，包括以下步骤：将键合分子与盐酸、亚硝酸钠溶液混合得到混合溶液；之后将芯片表面与所述混合溶液接触，并施加恒定直流电压进行反应；将反应后的芯片表面依次与连接臂分子溶液和功能化分子溶液接触反应修饰，制得连接体，如图1d所示。本发明实施例首先提供清洁的金属芯片，再配制键合分子与盐酸、亚硝酸钠溶液混合得到的混合溶液，最后将金属芯片与混合溶液接触反应。
在本发明的一些实施例中，优选以15mM盐酸为溶剂，将冷的键合分子与溶剂混合，再次加入亚硝酸钠，快速摇匀，制得混合溶液。所述键合分子为芳香胺类物质，优选为苯胺类物质，包括但不限于对氨基苯乙酸、对氨基苯乙醇、对氨基苯二胺中的一种或多种，在本发明的一些实施方案中为对氨基苯乙酸。在本发明一个实施方案中，将0.10～0.15mM冷的对氨基苯乙酸与15mM盐酸混合均匀，再次加入0.07～0.10mM亚硝酸钠，快速摇匀，制得混合溶液。将清洗后的金属芯片快速浸泡于上述混合溶液中，即金属表面与所述混合溶液接触，并施加固定直流电压静置进行反应。在本发明的一些实施方案中，施加的固定直流电压为0.5V～3.0V，优选2.5V；反应温度为20℃～37℃，优选室温；反应时间为10分钟～30分钟，优选20分钟。本发明的一些实施方案中，给芯片施加的恒定直流电压为0.5V～3.0V，室温下静置电促反应10分钟～30分钟。本发明的一个具体实施方案中，给芯片施加的恒定直流电压为2.5V，室温下静置电促反应20分钟。此步骤原理为芳香胺类分子在 盐酸和亚硝酸钠存在的条件下，加恒定直流电压后重氮化生成芳碳自由基，芳碳自由基与电极材料反应生成共价键而结合，具体反应如下所示：
Figure PCTCN2021090905-appb-000001
反应后，取出芯片，再依次用水、醇冲洗，再用酸溶液冲洗芯片，最后用水冲洗，吹干。
反应后的芯片利用羧基和氨基的交联酰化反应，进行连接臂分子修饰，增加功能化基团与芯片间的距离，减小后续应用时的空间位阻。在本发明的一些实施方案中，连接臂分子为二胺类物质，包括但不限于乙二胺、己二胺、癸二胺、1,8-辛二胺甲醇中的一种或多种，在本发明的一些实施方案中为1,8-辛二胺；将反应后的芯片浸泡于1,8-辛二胺甲醇溶液中，静置反应8～12h，优选8h，之后水洗，吹干。
反应后的芯片再进行功能化分子修饰。功能化分子为包含长碳链且含有酯基的羟基物质，包括但不限于丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或者乙二酸修饰的碱基单体。在本发明的一些实施方案中单独使用丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶的溶液。在本发明的另一些实施方案中使用丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶的两种或两种以上混合溶液。功能化分子中含有酯基和羟基，可用于DNA合成及切割。所述的酯基是切割位点，为了把芯片上合成的DNA切下来，成自由溶液状的oligo pool。功能化分子中的含有的羟基是DNA合成的启动位点。在本发明的一些实施方案中，碱基单体部分提供的羟基用于DNA合成。
综上，利用苯胺类物质(如，对氨基苯乙酸、对苯二胺等)的直流电促嫁接(便于工业化生产)于金属芯片表面形成表面稳定共价，再进行连接臂和功能分子修饰，从而在芯片表面形成稳定连接体。其形成原理包含如下步骤：①芳香胺类分子在盐酸和亚硝酸钠存在的条件下重氮化，加固定直流电 压生成芳碳自由基，芳碳自由基与电极材料反应生成共价键而结合；②再利用羧基氨基交联反应，将芯片浸入长链连接臂分子溶液中，修饰长链分子，增加功能基团与芯片间距离，减小后续应用是的空间位阻；③再将功能分子修饰与连接臂上，其含有羟基和酯基，便于后续芯片上电促DNA的合成以及切割。
本发明还提供如前文所述的芯片表面连接体在合成DNA或制备芯片试剂盒中的应用。本发明的芯片表面连接体包含如下应用：在芯片上进行DNA合成；用于疾病生物标志物芯片检测；用于开发POCT(Point-of-Care-Testing)的芯片试剂盒；高通量芯片筛选试剂盒。
本发明中的芯片表面连接体经热水处理，还能用于合成DNA；合成的DNA与寡核苷酸(如DNA引物)杂交后再经碱和热TE处理，还可用于与寡核苷酸(如DNA引物)杂交。本发明的芯片表面连接体合成的DNA经过多次核酸杂交、洗脱、杂交循环应用，该芯片表面连接体依然能保持较好的稳定性。本发明实施例制备的芯片表面连接体导电性好，加电稳定，可用于电辅助合成DNA等核酸分子，如连接体没有此性质会影响DNA合成。本发明的芯片表面连接体抗DNA合成所需的有机溶剂，否则无法进行DNA合成。在芯片连接体用于芯片杂交检测应用中，本发明可解决现有连接体对水敏感、对热不稳定等问题，因此芯片可以重复性利用。
术语“芯片”表示由半导体之类的无机物或金、银、铂等金属形成的固体支持物，其表面具有特定位点形成的微阵列，所述位点通常以行列形式排列，其中各位点可用于某种类型的化学或生物化学分析、合成或方法。微阵列上的这些位点通常小于100微米。在本发明中，所述芯片为金属铂芯片。
术语“连接体”表示一端与或能与固体表面(如金属芯片)相连，另一端具有反应基团的分子，所述反应基团与或能与相关的化学物质，例如小分子、寡聚物或聚合物相连。连接体可与固体表面结合和/或其反应基团已连接有相关的化学物质。连接体的反应基团可连接有保护基团，其中所述保护基团可用化学或电化学方法除去。连接体可含有多个分子基团，其中所述分子之间共价相连。
术语“键合分子基团”表示位于连接体端部，基团的一端能与固体表面(如金属芯片)共价相连，另一端具有反应基团的化学分子，所述反应基团与或能与相关的化学物质，例如小分子、寡聚物或聚合物相连，在本发明中能与连接臂分子或功能化分子相连。键合分子基团已经与固体表面结合和/或其反应基团已连接有相关的化学物质(如连接臂分子基团或功能化分子基团)。键合分子基团的反应基团可连接有保护基团，其中所述保护基团可用化学或电化学方法除去。
术语“连接臂分子基团”表示位于连接体中间部分，基团的一端能与键合分子基团相连，另一端具有反应基团的化学分子，所述反应基团与或能与相关的化学物质，例如小分子、寡聚物或聚合物相连，在本发明中能与功能化分子相连。连接臂分子基团可与键合分子基团结合和/或其反应基团已连接有相关的化学物质(如键合分子基团或功能化分子基团)。连接臂分子基团的反应基团可连接有保护基团，其中所述保护基团可用化学或电化学方法除去。连接臂分子基团可在键合分子基团上原位形成。可先形成连接臂分子基团，再与已连接在固体表面的键合分子基团相连。可先在相关的化学物质上外部合成连接臂分子基团，再与已连接在固体表面的键合分子基团相连。可将相关的化学物质连接在与键合分子基团相连的连接臂分子基团上，然后将整个结构连接于固体表面的反应位点。连接臂分子基团的目的是延长相关分子和芯片固体表面之间的距离，减小后续应用时的空间位阻。在本发明中，连接臂分子基团可以是乙二胺基、己二胺基、癸二胺基、1,8-辛二胺基、乙二醇基、己二醇基、癸二醇基或1,8-辛二醇基等。
术语“功能化分子基团”表示位于连接体末端部分，基团的一端能与连接臂分子基团或键合分子基团相连，另一端具有反应基团的化学分子，所述反应基团与或能与相关的化学物质，例如小分子、寡聚物或聚合物相连，在本发明中能与脱氧核糖核苷酸分子相连。功能化分子基团可与连接臂分子基团和/或通过其连接有相关化学物质的反应基团相连。功能化分子基团的反应基团可连接有保护基团，其中所述保护基团可用化学或电化学方法除去。在本发明中功能化分子可以是丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶，甲基丙烯酸羟乙酯，丁二酸酐修饰的腺嘌呤、鸟嘌呤、胞嘧 啶、胸腺嘧啶或尿嘧啶，乙二酸修饰的腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶等。
术语“碱基单体”表示能发生聚合从而构成大分子，例如寡聚物、共-寡聚物、聚合物或共聚物基本结构的组合单位的分子。单体的例子包括A、C、T、G、腺苷酸、鸟苷酸、胞苷酸、尿苷酸、氨基酸和其他化合物。
术语“芳基”表示具有单价和约4-20个碳原子的芳族碳环基团。芳基的例子包括但不限于：苯基、萘基和蒽基。取代的芳基的一个或多个氢原子可用其他基团取代。虽然按照芳基的定义是一价的，但本文所用的芳基包括具有多价的基团，从而符合取代的要求。芳基可以是稠环结构的一部分，例如N-羟基琥珀酰亚胺与苯基(苯)结合形成N-羟基苯邻二甲酰亚胺。
术语“芳胺类分子”是指具有一个芳香性取代基的胺——即-NH2、-NH-或含氮基团连接到一个芳香环上，芳香烃的结构中通常含有一个或多个苯环。苯胺是这类化合物最简单的实例。本发明中，芳胺类分子可以是对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺。
术语“寡聚物”表示具有中等相对分子质量(intermediate relative molecular mass)的分子，其结构基本上包含实际上或在概念上衍生自较低相对分子量分子的少量单位。如果除去某分子的一个或几个单位后，其特性确实显著不同，则该分子可视作具有中等相对分子量。如果该分子的部分或整体具有中等相对分子量，并且基本上包含实际上或在概念上衍生自较低相对分子量分子的少量单位，可将其描述为寡聚的，或用作为形容词使用的寡聚物来描述。寡聚物通常由单体构成。
有益效果
与现有技术相比，本发明利用苯胺类物质在恒定直流电压条件下，通过碳自由基有效与电极表面结合，从而形成金属-碳共价键，将分子稳定的结合于芯片表面，再进行连接臂分子和功能化分子修饰，从而在芯片表面形成稳定连接体。本发明获得粘附性高的芯片表面连接体，能稳定地结合于芯片表面。本发明所述芯片表面连接体在热水和碱性条件下稳定，具有较好的导电性，加电稳定，抗核酸合成所需有机溶剂，对于后续的核酸合成以及其他应用极其有利。
附图说明
图1为本发明实施例1芯片表面连接体制备及用于合成DNA的示意图，其中①是金属芯片，②是本方法制得的linker，③是在此linker上合成的DNA。；
图2为本发明实施例1方法制备的芯片表面连接体与传统连接体在热水中稳定性对比示意图；
图3为本发明实施例1方法制备的芯片表面连接体在核酸合成应用中的稳定性；
图4为本发明实施例1方法制备的芯片表面连接体在用于DNA合成后的杂交实验示意图；
图5为用本发明实施例1方法制备的芯片表面连接体合成的DNA可切割性示意图；
图6为用本发明实施例1方法制备的芯片表面连接体合成的DNA示意图；
图7为用本发明实施例1方法制备的芯片表面连接体合成的DNA电泳胶图，样品为实施例1制备的芯片表面连接体合成的DNA(120nt)，对照品为US20060105355A1实施例1中公开的原有linker芯片合成的DNA(120nt)，ladder表示标准品(电泳后可显示50nt、75nt、150nt、200nt、300nt的核酸位置)。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
为了进一步理解本申请，下面结合实施例对本申请提供的芯片表面连接体、其制备方法和应用进行具体地描述。但是应当理解，这些实施例是在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过 程，只是为进一步说明本发明的特征和优点，而不是对本发明权利要求的限制，本发明的保护范围也不限于下述的实施例。
实例1：芯片linker制备及用于DNA合成
具体实验步骤：
1.芯片清洗：金属铂芯片如图1a所示，用蒸馏水冲洗5次，乙醇冲洗5次，再以蒸馏水冲洗3次，然后用prinaha溶液(H 2SO 4:H 2O 2＝3:1,体积比)浸泡芯片于65℃烘箱放置30分钟，芯片用蒸馏水冲洗5次，氩气吹干；
2.芯片修饰：取18.15mg冷的对氨基苯乙酸与15mM盐酸(1820uL H 2O,180uL 0.5M HCl)混合均匀，再次加入6.21mg的亚硝酸钠，快速摇匀；
3.将此混合液快速加入芯片表面，给芯片加2.5v的直流电压，室温下静置电促反应20分钟，芯片修饰反应如下：
Figure PCTCN2021090905-appb-000002
4.取出芯片，蒸馏水冲洗芯片5次，乙醇冲洗5次，15mM盐酸冲洗5次，蒸馏水冲洗3次，氩气吹干；
5.将芯片浸泡于8.7mg的1,8-辛二胺的甲醇溶液中(1mL甲醇，NHS 1.53mg和EDC 7.64mg溶于100uL水，其中NHS是N-羟基琥珀酰亚胺，EDC是1-乙基-3-(3-二甲氨基丙基)碳化二亚胺)，静置反应8小时，蒸馏水冲洗5次，氮气吹干；
6.芯片浸泡于丁二酸酐修饰的碱基单体(碱基单体为腺嘌呤、胸腺嘧啶、鸟嘌呤或胞嘧啶10.8mg和NHS 1.53mg，EDC 7.64mg溶于100uL水)混合溶液，静置反应8小时；
7.芯片依次用乙醇，丙酮，乙醇，蒸馏水冲洗5次，氮气吹干，便得到此稳定的linker，如图1b所示；
8.放置于CustomArray芯片合成仪上，合成DNA，如图1c所示。
通过以上操作制得的linker(连接体)示意图如图1d所示，芯片依次连接键合分子、连接臂分子以及功能化分子得到芯片连接体，连接体包括键合分子基团、连接臂分子基团和功能化分子基团。制得的连接体再进行DNA合成 (应用CustomArray芯片合成仪)，所得效果示意图如图1c所示，可以合成出完美的DNA。
实例2：新旧linker在热水中稳定性对比
芯片上修饰linker后，80℃蒸馏水放置2天，再进行DNA合成。
原有linker是在芯片上沉积多羟基类小分子(参见US20060105355A1中实施例1)，蒸馏水清洗后干燥，再于80℃蒸馏水放置2天，氩气吹干后应用CustomArray芯片合成仪在芯片上合成33nt的DNA，放芯片于芯片扫描仪(CustomArray，GenePix4000B)上扫面，结果如图2a所示，大部分区域无法进行DNA的合成。
新linker是按本发明实施例1方法所做，于80℃蒸馏水放置2天，氩气吹干后应用CustomArray芯片合成仪在芯片上合成33nt的DNA，于芯片扫描仪(CustomArray，GenePix4000B)上扫面，结果如图2b所示，芯片表面DNA依然能稳定存在，说明此新linker在热水中有足够稳定性，这对后续芯片表面的高温杂交或其他检测应用有巨大的价值。
实例3：新linker在核酸合成应用中的稳定性
经热水处理以及合成DNA后再经热TE和碱液处理，再杂交应用。
新linker按本发明实施例1的方法所做，于80℃蒸馏水放置2天，氩气吹干后应用CustomArray芯片合成仪在芯片上合成33nt的DNA，于芯片扫描仪上扫面，结果如图3中a和b所示；取两种带荧光的DNA引物(各100pM)，与芯片上合成的DNA杂交(室温，杂交反应2小时)，PBS缓冲冲洗后于芯片扫描仪上扫描，结果如图3中c所示，显示出不同荧光点，说明两种DNA引物能与芯片上合成的DNA杂交；再用1MNaOH溶液冲洗芯片，去掉杂交引物，于80℃TE缓冲液(5mL pH 8.01M Tris-HCl Buffer与1mL pH 8.0 0.5M EDTA的混合溶液)中放置2天，氩气吹干后于芯片扫描仪上扫面，结果如图3d所示；再取两种带荧光的DNA引物(各100pM)，与芯片上合成的DNA杂交(室温，杂交反应2小时)，PBS缓冲冲洗后于芯片扫描仪上扫描，结果如图3e所示，显示出不同荧光点，说明两种DNA引物还能与芯片上合 成的DNA杂交。说明此新linker在热水中有足够稳定性，且耐碱和热TE，这对后续芯片表面的高温杂交或其他检测应用有巨大的价值。
实施例4：新linker在多次杂交-洗脱-杂交应用时的稳定性
新linker是按本发明实施例1的方法所做，如图4所示，氩气吹干后应用CustomArray芯片合成仪在芯片上合成33nt的DNA，于芯片扫描仪上扫面，结果如图4b所示；取两种带荧光的DNA引物(各100pM)，与芯片上合成的DNA杂交(室温，杂交反应2小时)，PBS缓冲冲洗后于芯片扫描仪上扫描，结果如图4c所示，显示出不同荧光点；再用100mM NaOH溶液冲洗芯片，去掉杂交引物，氩气吹干后于芯片扫描仪上扫面，结果如右图4d所示；取两种带荧光的DNA引物(各100pM)，与芯片上合成的DNA杂交(室温，杂交反应2小时)，PBS缓冲冲洗后于芯片扫描仪上扫描，结果如右图4e所示，如此循环多次，依然可用于杂交应用，且具备明显荧光信号，说明此linker足够稳定，可经受多次杂交和碱液洗脱。
实施例5：合成后DNA从芯片上的可切割性
应用本发明实施例1方法制得linker(图5a)，再应用CustomArray芯片合成仪在芯片上合成120nt的DNA(如图5b)，所形成的linker包含了切割基团(酯基)，可在碱性(氨水)加热条(65℃)件下将oligo从芯片上切割下(16小时)来形成自由oligo，水洗芯片后用芯片扫面仪扫描(图5c)，说明DNA基本全被切割下来。
实施例6：新linker用于DNA合成的DNA质量表征
应用本发明实施例1方法制得linker，再应用CustomArray芯片合成仪在芯片上合成120nt的DNA(如图6)，所形成的linker包含了切割基团(酯基)，可在碱性(氨水)加热条件下(65℃)将寡核苷酸(oligo)从芯片上切割(16h)下来形成自由寡核苷酸池(oligo pool)，用Thermo Scientific TMNanoDrop TM One Microvolume UV-Vis Spectrophotometers仪器测得两个芯片浓度为21.4ng/uL，19.4ng/uL，满足要求。再进行PCR扩增，所得产物进行电泳胶图分 析，如图7所示，与标准产物大小相近，且在同一位置，说明用此linker所合成的产物正确。
参考文献：
1.Sharma R.Small-molecule surfactant adsorption,polymer surfactant adsorption,and surface solubilization:An overview[M].1995.
2.Kokkin D L,Zhang R,Steimle T C,et al.Au–S bonding revealed from the characterization of diatomic gold sulfide,AuS[J].The Journal of Physical Chemistry A,2015,119(48):11659-11667.
3.Toma M,Tawa K.Polydopamine thin films as protein linker layer for sensitive detection ofinterleukin-6by surface plasmon enhanced fluorescence spectroscopy[J].ACS applied materials&interfaces,2016,8(34):22032-22038.
4.Saaem I,Ma K S,Marchi A N,et al.In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate[J].ACS applied materials&interfaces,2010,2(2):491-497

Claims (31)

  1. 一种芯片表面连接体，其特征在于，所述连接体通过以下步骤制得：
    步骤1：在酸和亚硝酸盐存在下，通过施加直流电压使得芳香胺类的键合分子与芯片表面反应形成连接在芯片表面的键合分子基团；
    步骤2：用功能化分子反应修饰，得到包含功能化分子基团的连接体，其中所述功能化分子基团包含羟基和酯基。
  2. 根据权利要求1所述的芯片表面连接体，所述键合分子为苯胺类物质。
  3. 根据权利要求1或2所述的芯片表面连接体，所述键合分子选自对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺，优选为对氨基苯乙酸。
  4. 根据权利要求1～3中任一项所述的芯片表面连接体，所述直流电压为恒定直流电压，所施加直流电压选自0.5V～5.0V，优选为0.5～3.0V。
  5. 根据权利要求1～4中任一项所述的芯片表面连接体，所施加直流电压时间为10分钟～50分钟，优选施加直流电压时间为10分钟～30分钟。
  6. 根据权利要求1～5中任一项所述的芯片表面连接体，所述亚硝酸盐选自亚硝酸钠、亚硝酸钾或亚硝酸钙，优选为亚硝酸钠。
  7. 根据权利要求1～6中任一项所述的芯片表面连接体，所述酸选自盐酸、硝酸或硫酸，优选为盐酸。
  8. 根据权利要求1～7中任一项所述的芯片表面连接体，所述功能化分子为包含长碳链且含有酯基的羟基物质。
  9. 根据权利要求8所述的芯片表面连接体，所述功能化分子选自丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或者乙二酸修饰的碱基单体，优选为丁二酸酐修饰的碱基单体。
  10. 根据权利要求9所述的芯片表面连接体，所述功能化分子中碱基单体部分选自腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶的一种或多种。
  11. 根据权利要求1～10中任一项所述的芯片表面连接体，所述键合分子基团和所述功能化分子基团之间还包括连接臂分子基团。
  12. 根据权利要求11所述的芯片表面连接体，所述连接臂分子基团是通过连接臂分子与键合分子基团反应连接的，所述功能化分子基团是通过功能化分子与所述连接臂分子基团反应连接的。
  13. 根据权利要求12所述的芯片表面连接体，所述连接臂分子为二胺类或二醇类物质。
  14. 根据权利要求12或13所述的芯片表面连接体，所述连接臂分子选自乙二胺、己二胺、癸二胺、1,8-辛二胺、乙二醇、己二醇、癸二醇或1,8-辛二醇，优选1,8-辛二胺。
  15. 根据权利要求1～14中任一项所述的芯片表面连接体，所述芯片为金属芯片，优选为金、铂或铝芯片。
  16. 根据权利要求15所述的芯片表面连接体，所述芯片为金属铂芯片。
  17. 一种芯片表面连接体的制备方法，其特征在于包括以下步骤：
    步骤1：将芳香胺类的键合分子与酸、亚硝酸盐混合得到混合溶液；
    步骤2：将步骤1的混合溶液与芯片表面接触，施加直流电压进行反应形成连接在芯片表面的键合分子基团；
    步骤3：将反应后的芯片表面与功能化分子反应，得到包含功能化分子基团的连接体，其中所述功能化分子基团包含羟基和酯基。
  18. 根据权利要求17所述的制备方法，所述步骤3之前包括将步骤2反应后的芯片表面与连接臂分子接触反应连接上连接臂分子基团。
  19. 根据权利要求17或18所述的制备方法，所述键合分子为苯胺类物质。
  20. 根据权利要求17-19中任一项所述的制备方法，所述键合分子选自对氨基苯乙酸、对氨基苯乙醇或对氨基苯二胺，优选为对氨基苯乙酸。
  21. 根据权利要求17～20中任一项所述的制备方法，所述直流电压为恒定直流电压，所施加直流电压选自0.5V～5.0V，优选为0.5V～3.0V。
  22. 根据权利要求17～21中任一项所述的制备方法，所施加直流电压时间为10分钟～50分钟，优选施加直流电压时间为10分钟～30分钟。
  23. 根据权利要求17～22中任一项所述的制备方法，所述亚硝酸盐选自亚硝酸钠、亚硝酸钾或亚硝酸钙，优选为亚硝酸钠。
  24. 根据权利要求17～23中任一项所述的制备方法，所述酸选自盐酸、硝酸或硫酸，优选为盐酸。
  25. 根据权利要求17～24中任一项所述的制备方法，所述功能化分子选自丁二酸酐修饰的碱基单体、甲基丙烯酸羟乙酯、丁二酸修饰的碱基单体或乙二酸修饰的碱基单体，优选为丁二酸酐修饰的碱基单体。
  26. 根据权利要求25所述的制备方法，所述功能化分子的碱基单体部分选自腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶的一种或多种。
  27. 根据权利要求17～26中任一项所述的制备方法，所述连接臂分子为二胺类或二醇类物质。
  28. 根据权利要求27所述的制备方法，所述连接臂分子选自乙二胺、己二胺、癸二胺、1,8-辛二胺、乙二醇、己二醇、癸二醇或1,8-辛二醇，优选为1,8-辛二胺。
  29. 根据权利要求17～28中任一项所述的制备方法，所述步骤2施加直流电压反应时温度为4℃～40℃，优选为20℃～37℃；反应时间为10分钟～50分钟，优选10分钟～30分钟。
  30. 根据权利要求17～29中任一项所述的制备方法，所述芯片为金属芯片，优选为金、铂和铝芯片。
  31. 如权利要求1～16任一项所述芯片表面连接体在核酸合成或制备芯片试剂盒中的应用。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515693A (zh) * 2003-01-02 2004-07-28 缪金明 利用数字微镜技术原位合成高密度dna芯片的方法
CN1553188A (zh) * 2003-06-06 2004-12-08 克 宋 微阵列信号放大方法
US20050202556A1 (en) * 2003-12-29 2005-09-15 Walter Gumbrecht Process and spotting solution for preparing microarrays
US20060105355A1 (en) 2004-11-18 2006-05-18 Karl Maurer Electrode array device having an adsorbed porous reaction layer having a linker moiety
CN107589254A (zh) * 2016-07-08 2018-01-16 奈尔公司 一种免疫脂质体复合物纳米粒子生物芯片的制造方法及应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020019A1 (en) * 1996-11-06 1998-05-14 Sequenom, Inc. Compositions and methods for immobilizing nucleic acids to solid supports
US7211654B2 (en) 2001-03-14 2007-05-01 Regents Of The University Of Michigan Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports
US20050019803A1 (en) * 2003-06-13 2005-01-27 Liu Timothy Z. Array electrode
KR101274854B1 (ko) * 2010-12-23 2013-06-13 한남대학교 산학협력단 감염성 호흡기 질환 진단용 전기화학 ｄｎａ 센서 및 이의 제조방법
CA2856163C (en) * 2011-10-28 2019-05-07 Illumina, Inc. Microarray fabrication system and method
KR101732706B1 (ko) 2015-06-05 2017-05-04 압타바이오 주식회사 신증 진단용 마커로서의 sh3yl1의 용도
WO2017117292A1 (en) 2015-12-28 2017-07-06 Vibrant Holdings, Llc Substrates, systems, and methods for nucleic acid array synthesis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1515693A (zh) * 2003-01-02 2004-07-28 缪金明 利用数字微镜技术原位合成高密度dna芯片的方法
CN1553188A (zh) * 2003-06-06 2004-12-08 克 宋 微阵列信号放大方法
US20050202556A1 (en) * 2003-12-29 2005-09-15 Walter Gumbrecht Process and spotting solution for preparing microarrays
US20060105355A1 (en) 2004-11-18 2006-05-18 Karl Maurer Electrode array device having an adsorbed porous reaction layer having a linker moiety
CN107589254A (zh) * 2016-07-08 2018-01-16 奈尔公司 一种免疫脂质体复合物纳米粒子生物芯片的制造方法及应用

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KOKKIN D LZHANG RSTEIMLE T C ET AL.: "Au-S Bonding Revealed From the Characterization of Diatomic Gold Sulfide, AuS[J", THE JOURNAL OF PHYSICAL CHEMISTRY A, vol. 119, no. 48, 2015, pages 11659 - 11667
SAAEM IMA K SMARCHI A N ET AL.: "In Situ Synthesis of DNA Microarray on Functional Cyclic Olefin Copolymer Substrate[J", ACS APPLIED MATERIALS & INTERFACES, vol. 2, no. 2, 2010, pages 491 - 497
See also references of EP4144860A4
SHARMA R, SMALL-MOLECULE SURFACTANT ADSORPTION, POLYMER SURFACTANT ADSORPTION, AND SURFACE SOLUBILIZATION: AN OVERVIEW [M, 1995
TOMA MTAWA K: "Polydopamine Thin Films as Protein Linker Layer for Sensitive Detection of Interleukin-6 by Surface Plasmon Enhanced Fluorescence Spectroscopy [J", ACS APPLIED MATERIALS & INTERFACES, vol. 8, no. 34, 2016, pages 22032 - 22038

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