CN110628887A - Biomolecule microarray and preparation method and application thereof - Google Patents

Biomolecule microarray and preparation method and application thereof Download PDF

Info

Publication number
CN110628887A
CN110628887A CN201910915044.XA CN201910915044A CN110628887A CN 110628887 A CN110628887 A CN 110628887A CN 201910915044 A CN201910915044 A CN 201910915044A CN 110628887 A CN110628887 A CN 110628887A
Authority
CN
China
Prior art keywords
reaction
cleavable
reactive group
group
solid substrate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910915044.XA
Other languages
Chinese (zh)
Inventor
向国兵
张明航
吕华
施琦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Abduction Gene Technology Co Ltd
Original Assignee
Nanjing Abduction Gene Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Abduction Gene Technology Co Ltd filed Critical Nanjing Abduction Gene Technology Co Ltd
Priority to CN201910915044.XA priority Critical patent/CN110628887A/en
Publication of CN110628887A publication Critical patent/CN110628887A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Structural Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a biomolecule microarray and a preparation method and application thereof, belonging to the technical field of biological detection. According to the invention, a plurality of reaction areas which are arranged at intervals are formed on the surface of the solid substrate, and then biomolecules are respectively fixed in the reaction areas, so that a plurality of array points which are arranged in order are formed.

Description

Biomolecule microarray and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to a biomolecule microarray and a preparation method and application thereof.
Background
Biomolecule microarrays include various types such as DNA microarrays, protein microarrays, antibody microarrays, hapten microarrays, carbohydrate microarrays, etc., and are widely used in Point-of-care (POCT) technologies for qualitative and quantitative detection of a certain biomolecule in a biological sample. The DNA microarray is also called DNA array or DNA chip, the popular name is gene chip (genechip), which is a tool for genomics and genetics research, researchers can quantitatively analyze the expression level of a large amount of genes (thousands of genes) at the same time by using the gene chip, and the DNA microarray has the capability of quick, accurate and low-cost biological analysis and inspection.
DNA microarrays are widely used for gene expression and genotyping, and are also useful in high-throughput gene Sequencing technologies as flow cells for high-throughput Sequencing, as shown in fig. 1, in the conventional flow cells, a layer of macromolecules with reactive groups is spread on the surface of an activated slide, then, reactive primers (reactive primers) are covalently immobilized, then, in-situ bridge PCR amplification is performed in the flow cells to form an ultra-large number of monoclonal clusters of thousands or tens of thousands of the same DNA molecules and polyclonal clusters of two or more DNA molecules, and finally, Sequencing By Synthesis (SBS) is performed in the flow cells to obtain DNA Sequencing information. However, since only a single clone cluster can generate useful sequencing information, only about 30% of clusters in the existing flow cell are single clone clusters, and more than 60% of other clusters are multiple clone clusters, the generated sequencing information has no practical value, the signal-to-noise ratio of the generated sequencing information is low, the accuracy of the sequencing result is low, and the detection throughput of the sequencer is low.
Disclosure of Invention
The biomolecule microarray prepared by the invention can improve the proportion of monoclonal clusters when being applied to DNA sequencing, so that sequencing information with high signal-to-noise ratio is generated, the accuracy of a sequencing result is improved, and the detection flux of a sequencer is obviously improved.
To achieve the above object, the present invention first provides a method for preparing a biomolecule microarray, comprising:
step one, providing a solid substrate;
forming a plurality of reaction zones arranged at intervals and non-reaction zones positioned at the periphery of the reaction zones on the surface of the solid substrate, wherein the surfaces of the reaction zones are provided with reaction groups;
fixing a functional high molecular polymer by using a reaction group in the reaction zone;
and step four, fixing the biological molecules on the functional high molecular polymer to obtain the biological molecule microarray.
In some embodiments of the present invention, the surface of the solid substrate is provided with a plurality of features arranged at intervals, and the reaction region is formed at the positions of the features.
In some embodiments of the invention, the second step comprises:
forming a first reactive group on the surface of the solid substrate;
immobilizing a cleavable tether molecule with the first reactive group;
and carrying out patterned cracking on the cleavable linked chain molecules on the surface of the solid substrate to form a plurality of reaction zones arranged at intervals.
In some embodiments of the invention, the cleavable tether molecule does not itself carry a reactive group, and the cleavable tether molecule is cleaved to expose a first reactive group on the surface of the solid substrate;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are cracked, and the cleavable catenated molecules of the non-reaction zone are not cracked.
In some embodiments of the invention, the cleavable tether molecule itself carries a second reactive group that disappears upon cleavage of the cleavable tether molecule;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked.
In some embodiments of the invention, the cleavable tether molecule itself carries a protected second reactive group, which upon cleavage of the cleavable tether molecule disappears and produces a third reactive group;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked;
after the patterned cracking, a second reaction group of the cleavable linked chain molecule in the reaction zone is deprotected, so that the second reaction group recovers the reactivity; and protecting a third reactive group of the cleavable linked-chain molecule of the non-reactive zone, so that the third reactive group loses reactivity.
In some embodiments of the present invention, the plurality of reaction zones are arranged in a matrix.
In some embodiments of the invention, the distance between any two adjacent reaction zones of the plurality of reaction zones is between 0.5 μm and 20 μm.
The invention also provides a biomolecule microarray, which is prepared by the preparation method of the biomolecule microarray.
The invention also provides an application of the biomolecule microarray in DNA sequencing.
The invention has the beneficial effects that:
according to the invention, a plurality of reaction areas which are arranged at intervals are formed on the surface of the solid substrate, and then biomolecules are respectively fixed in the reaction areas, so that a plurality of array points which are arranged in order are formed.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of a conventional method for preparing a DNA microarray;
FIG. 2 is a schematic diagram of step 2 and step 3 in example 1 of the present invention;
FIGS. 3 and 4 are schematic views of step 4 of embodiment 1 of the present invention;
FIG. 5 is a schematic diagram of step 5 and step 6 in example 1 of the present invention;
FIG. 6 is a schematic view of step 3 in example 3 of the present invention;
FIGS. 7 and 8 are schematic views of step 4 of embodiment 3 of the present invention;
FIG. 9 is a schematic view of step 5 in example 3 of the present invention;
FIG. 10 shows the change of the reactive groups in the reaction region from COOH groups to NH groups in example 4 of the present invention2A schematic representation of a group;
FIGS. 11 and 12 are schematic diagrams of step 3 and step 4, respectively, according to embodiment 5 of the present invention;
FIG. 13 shows the features and locations of the reaction zones of the transparent solid substrate of example 6 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The invention provides a preparation method of a biomolecule microarray, which comprises the following steps:
step one, providing a solid substrate.
In particular, the material of the solid substrate may be a stable solid material that is insoluble in water. The solid substrate may be rigid or flexible. The solid substrate may be made in one geometry or a combination of geometries.
Specifically, the size and shape of the solid substrate are determined by the requirements of the detection method, and the material of the solid substrate can be one or a combination of several of inorganic crystals, inorganic glass, inorganic oxides and organic polymers. The material of the solid substrate may also be an inert high molecular polymer, such as polystyrene, polycarbonate, polyethersulfone, polyketone, poly aliphatic ether, polyetherketone, polyetheretherketone, polyarylether, polyamide-polyimide, polyester-polyacrylate, polymethacrylate, polyolefin, polycycloolefin, polyvinyl alcohol, cyclic olefin polymer, cyclic olefin copolymer, a mixture of various high molecular polymers, halogenated derivatives of high molecular polymers, crosslinked derivatives of high molecular polymers, and the like.
Preferably, the flow cell of the high-throughput gene sequencer based on fluorescence detection selects a material with high transparency as a solid substrate. The transparent solid substrate preferably has a transmittance of light of 250nm to 800nm of wavelength of more than 90% and low autofluorescence at a specific excitation wavelength. Meanwhile, the solid substrate needs to be hydrophilic enough to reduce non-specific adsorption, and to have strong resistance to organic solvents and detection solutions such as methanol, ethanol, propanol, acetone, acetonitrile, dimethylformamide, dimethylacetamide, dilute acids, dilute bases, synthetic surfactants, high salinity aqueous solutions, and the like.
The transparent solid substrate may be any of various transparent glass slides, for example, a transparent glass slide made of quartz, fused quartz, soda lime glass, borosilicate glass, or the like, and various transparent plastic sheets, transparent silicon wafers, transparent fiber sheets, and transparent nylon sheets. The transparent solid substrate can also be a structure formed by coating a layer of transparent metal oxide on the surface of an organic polymer.
And secondly, forming a plurality of reaction areas arranged at intervals on the surface of the solid substrate and non-reaction areas positioned at the periphery of the reaction areas, wherein the surfaces of the reaction areas are provided with reaction groups.
Specifically, the second step includes:
forming a first reactive group on the surface of the solid substrate;
immobilizing a cleavable tether molecule with the first reactive group;
and carrying out patterned cracking on the cleavable linked chain molecules on the surface of the solid substrate to form a plurality of reaction zones arranged at intervals.
Specifically, the reactive group may be a first reactive group formed on the surface of the solid substrate after the solid substrate is activated, or may be a second reactive group in a cleavable linked chain molecule covalently linked to the first reactive group. The reactive group is used for reacting with the functional high molecular polymer so as to fix the functional high molecular polymer on the surface of the solid substrate.
In particular, the reactive group is a functional group that can undergo a click reaction, such as-N3A group, -C ≡ CH (triple bond) group, -CH ≡ CH2(double bond) group, -CH (conjugated double bond) group, and the like.
Specifically, the method for forming the first reactive group on the surface of the solid substrate may be: the surface of the solid substrate is activated by a chemical method or/and a physical method, a layer of first reactive group with activity is formed on the surface of the activated solid substrate, the first reactive group is a functional group which can react with cleavable linked chain molecules to form covalent bond connection, and when the solid substrate is a high molecular polymer, the first reactive group can also be a reactive group carried by the high molecular polymer.
In particular, the second reactive group may be-OH, -NH2、-NHNH2、-ONH2CHO, -COOH and activated esters thereof (NHS, SO)3H-NHS, Pfp.), epoxy groups, halogen groups, maleimide groups, -N3A group, -C ≡ CH (triple bond) group, -CH ≡ CH2(double bond) group, -CH (conjugated double bond) group, -OSO3H、-SO3Cl、-SO3F、-SO3H、-SO2Cl、-SO2F. -SH, etc.
As shown in fig. 1, in the conventional biomolecule microarray, the reaction groups (R) formed on the surface of the solid substrate are disordered and dense, and thus, the biomolecule probes connected in the subsequent reaction are too close to each other to affect each other, thereby affecting the detection effect of the sample.
In order to prevent the mutual influence of too close distances between the biomolecule probes, the reaction groups formed on the surface of the formed solid substrate are subjected to patterning treatment, and are regularly arranged at intervals, namely, the reaction groups are limited in a plurality of reaction areas arranged in an array, the reaction areas can be in regular shapes such as square, rectangle and equilateral triangle arrangement, optionally, the distance between any two adjacent reaction areas is the same, so that the plurality of reaction areas are regularly arranged, and the reactive microarray is formed. The size of the reaction region (i.e., the microarray spot) can be adjusted according to the application, and for example, the diameter of the reaction region can be 0.5 μm to 20 μm (e.g., 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm) in case the reaction region is circular, but can be smaller than 0.5 μm or larger than 20 μm according to the application. Microarray spot flow cells with reaction zones around 1 μm in diameter can be used for high throughput DNA sequencing applications.
In some embodiments of the present invention, the plurality of reaction zones are arranged in a matrix form, and the distance between any two adjacent reaction zones is 0.5 μm to 20 μm (e.g., 0.5 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm).
Alternatively, the surface of the solid substrate may be smooth to facilitate detection by optical detection methods, although the surface of the solid substrate may also be patterned to have features.
In some embodiments of the present invention, the surface of the solid substrate is provided with a plurality of spaced features, and the reaction region is formed at the positions of the features.
In particular, the features may be pits, circles, barriers, protruding dots, pits, inlets, outlets, channels, grooves, diffraction gratings, etc. on a solid substrate. The features may be formed using techniques such as microcontact printing, photolithography, spotting, inkjet deposition, rubbing, etching, embossing, grooving, stretching, and oblique deposition of thin metal films.
Alternatively, the process of patterned cleavage may be: and (3) shielding the area, which is not required to be cracked, on the surface of the fixed substrate by using a MASK (MASK), and not shielding the area required to be cracked.
In some embodiments of the invention, the cleavable tether molecule does not itself carry a reactive group, and the cleavable tether molecule is cleaved to expose a first reactive group on the surface of the solid substrate;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are cracked, and the cleavable catenated molecules of the non-reaction zone are not cracked.
In other embodiments of the present invention, the cleavable tether molecule itself carries a second reactive group that disappears upon cleavage of the cleavable tether molecule;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked.
In other embodiments of the invention, the cleavable tether molecule itself carries a protected second reactive group, which disappears upon cleavage of the cleavable tether molecule and exposes a third reactive group;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked;
after the patterned cracking, a second reaction group of the cleavable linked chain molecule in the reaction zone is deprotected, so that the second reaction group recovers the reactivity; and protecting a third reactive group of the cleavable linked-chain molecule of the non-reactive zone, so that the third reactive group loses reactivity.
Specifically, the protection refers to the use of a protecting group to react with a group to be protected, so that the group to be protected is inactivated.
It will be appreciated that the cleavable tether molecules used in the present invention need to have a high cleavage efficiency when cleaved.
Specifically, the cleavage method used in the present invention may be photolysis or chemical cleavage, etc., according to the different cleavage requirements of different cleavable linked molecules.
When the cleavable linked chain molecule is cleaved by the photolysis method, the wavelength of the light used can be 254nm, 300nm, 308nm, 312nm, 315nm, 320nm, 333nm, 337nm, 342nm, 348nm, 350nm, 355nm, 334-365nm, 366nm, 400nm, 420nm or higher according to the different cleavage requirements of different cleavable linked chain molecules.
When the cleavable linked chain molecule is cleaved by a chemical cleavage method, the adopted chemical reagents can be enzymes, acids, bases, nucleophiles, electrophiles, organometallic or/and metal catalysts, reducing agents, oxidizing agents, F anions and the like according to different cleavage requirements of different cleavable linked chain molecules.
And step three, fixing the functional high molecular polymer by utilizing the reaction group of the reaction zone.
Specifically, the functional high molecular polymer is a macromolecule polymerized by monomers, and can be polymerized by one monomer or copolymerized by more than two monomers, wherein the high molecular polymer copolymerized by two monomers is a copolymer, and the high molecular polymer copolymerized by three monomers is a terpolymer.
High molecular polymers, which are typically copolymerized from two or more monomers, are also referred to as random polymers, such as random copolymers (which are copolymerized from two monomers) and random terpolymers (which are copolymerized from three monomers). The linking position between the monomers of the random polymer is random and random, and the molar ratio of the monomers of the random polymer can be randomly changed according to the application requirement. The random polymer has the general formula: - (M1)n1-(M2)n2-(M3)n3(with or without crosslinking between polymers of different monomers), wherein n1, n2 and n3 are any natural numbers, M1 is monomer 1, M2 is monomer 2, M3 is monomer 3, and the positions of M1, M2 and M3 in the random high-molecular polymer are arbitrary.
Specifically, examples of the monomer that can be polymerized into the functional polymer include monomers having a group such as allyl group, vinyl group, acryloyl group, methacryloyl group, etc., 2-vinyl-4, 4' -dimethyl azalide, glycidylcarboxylic acid, amine, acid anhydride, aldehyde, urea, thiourea, isocyanate, thioisocyanate, etc.
The high molecular polymerization requires the use of an initiator to initiate polymerization of the monomers, such as AIBN series initiators, benzophenone initiators, and the like, for free radical high molecular reactions. The residue of the initiator on the high molecular weight polymer is the portion of the initiator linked to the head and tail of the high molecular weight polymer by free radical reaction or other reaction mechanism.
The functional high molecular polymer with active reaction group is polymerized by monomer with active reaction group, usually by copolymerization of monomer with active reaction group and monomer without active reaction group according to specific proportion. The active reactive group of the functional polymer with the active reactive group has reactivity and can form covalent bond linkage with a specific other active reactive group (such as-OH, -NH) and the active reactive group carried by a molecule of the cleavable linking chain2、-NHR、-NH2OH、-NH2NH2、-N3-C.ident.CH, -CHO, -COOH, etc.
The functional high molecular polymer with only one reactive group can be polymerized by a monomer with the reactive group, or can be polymerized by a monomer with the reactive group and one or more than two monomers without the reactive group, and the functional high molecular polymer only has one reactive group.
The functional high molecular polymer with two active reaction groups can be formed by copolymerizing monomers with two active reaction groups, or can be formed by polymerizing monomers with two active reaction groups and another or more than two monomers without active reaction groups, and the functional high molecular polymer has two active reaction groups.
And step four, fixing the biological molecules on the functional high molecular polymer to obtain the biological molecule microarray.
In particular, when a biomolecule microarray is used for detection purposes, the biomolecule may be referred to as a biomolecule probe.
Specifically, the functional high molecular polymer fixed on the surface of the solid substrate is used as a support to covalently fix the biomolecule probes with active reaction groups to form a biomolecule microarray. For example, covalent bonds such as ester bond, amide bond, ether bond, amino bond, imino bond, oxime bond, acyloxime bond, hydrazone bond, hydrazine bond, sulfate bond, sulfamide bond, sulfoximine bond, thiohydrazide bond, sulfonate bond, sulfonamide bond, sulfonyloxime bond, sulfonylhydrazide bond, thioether bond, etc., covalent bonds formed by click chemistry (e.g., 1, 3-substituted triazolyl group), or covalent bonds formed by other conjugate addition chemistries (e.g., CH ═ CH)2Groups and CH ═ CH-CH ═ CH groups).
When the functional high molecular polymer with only one reactive group reacts with the solid substrate, only the reactive groups close enough to the solid substrate can covalently react with the reactive groups regularly arranged on the surface of the solid substrate to fix the functional high molecular polymer, and most of the unreacted reactive groups are used for reacting with the biomolecule probes with the reactive groups to covalently form the biomolecule microarray.
One active reactive group in the functional high molecular polymer with two active reactive groups is used for reacting with the reactive groups regularly arranged at intervals on the surface of the fixed substrate to fix the functional high molecular polymer, and the other active reactive group is used for reacting with the biomolecule probe with the active reactive group under a specific reaction condition to fix the biomolecule probe to form the biomolecule probe microarray.
The remaining reactive groups on the surface of the microarray (on the surface of the solid substrate and on the functional polymer) to which the biomolecule probes are covalently immobilized may result in the surface being hydrophobic, and a hydrophilic surface may be formed by covalently immobilizing reactive hydrophilic small molecules. The covalent bonds formed between the surface of the solid substrate and the functional high molecular polymer and the hydrophilic micromolecules are ester bonds, amido bonds, ether bonds, amino bonds, imino bonds, oxime bonds, acyl oxime bonds, hydrazone bonds, hydrazine bonds, sulfate bonds, sulfamide bonds, sulfuryl oxime bonds, thiohydrazide bonds, sulfonate bonds, sulfonic amide bonds, sulfonyl oxime bonds, sulfonyl hydrazide bonds, thioether bonds and the like.
Specifically, the biomolecule probe may include a capture probe for capturing an analyte to be detected and an analyte probe for performing an analysis detection on the analyte to be detected captured by the capture probe.
In particular, the capture probes and analyte probes may be peptides, proteins, enzymes, antibodies, antigens, hormones, glycosylated peptides, glycoconjugates, compatible oligomers, carbohydrates, polynucleotides, oligonucleotides, oligopeptides, DNA, and the like.
The analyte to be measured is an object to be analyzed, and the analyte to be measured is, for example, DNA, RNA, protein, carbohydrate, lipid, drug candidate, cofactor, metabolite, organic molecule, inorganic molecule, metal ion, or the like.
The analyte probe may be fluorescent or fluorescent quencher, the capture probe may or may not be fluorescent, the signal detected may be a fluorescent signal, and interaction of the analyte probe and the capture probe may result in an increase or decrease in the fluorescent signal.
The DNA microarray is characterized in that DNA probes are covalently immobilized on functional high molecular polymers which are covalently immobilized on the surface of a solid substrate in a flow cell, the DNA probes can be locally amplified in the flow cell to form DNA clusters to a sufficient amount for convenient detection, and the DNA clusters can be used for high-throughput DNA sequencing.
The invention also provides a biomolecule microarray, which is prepared by the preparation method of the biomolecule microarray.
The invention also provides an application of the biomolecule microarray in DNA sequencing.
Embodiments of the present invention will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
A method of preparing a biomolecule microarray, comprising:
step 1, carrying out activation treatment on the transparent solid substrate.
Introducing oxygen-bearing groups on the surface of a transparent solid substrate (25 mm. times.75 mm. times.0.5 mm slide) by using an oxygen plasma generator and an X-Y manipulator, placing a slide substrate sample on an aluminum scanning platform of the X-Y manipulator, and placing the surface to be treated to be upwards 4 mm from the ion source, setting the parameters of the plasma generator to be 60W, the helium gas flow rate to be 15 liters per minute, the oxygen gas flow rate to be 0.05 liters per minute, the plasma scanning source speed to be 20 mm per second, and the scanning times to be 1 to 10 times to adjust the hydroxyl group density on the surface of the transparent solid substrate, wherein the activated transparent solid substrate can be immediately used for forming a layer of NH2A group.
And 2, treating the surface of the activated transparent solid substrate by using 3-aminopropyl trimethoxy silane.
30 ml of absolute ethanol, 500. mu.l of 3-aminopropyltrimethoxysilane and 20. mu.l of triethylamine were added to a polypropylene chip tube with a screw cap, and then 5 glass slides which had been subjected to activation treatment were placed and shaken for 3 hours. Taking out the glass slide, cleaning the glass slide with enough 95% ethanol, drying the glass slide by using argon, putting the glass slide into a 110 ℃ oven for annealing treatment for 5 minutes, and forming a layer of NH on the surface of the activated glass slide2Group (i.e., first group), as shown in fig. 2. The prepared slide is immediately used for the next step of covalently immobilizing a layer of cleavable linker molecules on the slide surface.
Step 3, carrying NH2The 1- (bromomethyl) -2-nitrobenzene is covalently immobilized on the surface of the slide of the radical (i.e., the linker molecule can be cleaved).
In 2 ml of 5mMAdding 2.16mg 1- (bromomethyl) -2-nitrobenzene into absolute ethyl alcohol, adding 2 microliter 6mM Diisopropylethylamine (DIPEA), dissolving, mixing, and adding dropwise into the mixture with NH2Standing for 3 hours on the surface of the glass slide with the group surface, washing with ethanol, drying with argon, and carrying NH2The surface of the slide of groups is immobilized with cleavable linker molecules without reactive groups, as shown in FIG. 2.
Step 4, photolysis of NH formed on the surface of the slide obtained in step 32A microarray (i.e., a reaction area microarray).
As shown in FIG. 3, the NH band obtained in step 3 above was photolyzed using 365nm UV light2Cleavable linked-chain molecules without a reaction group are fixed on the surface of a group glass slide, a non-reaction area is shielded in the ultraviolet light irradiation process, the cleavable linked-chain molecules in the non-reaction area are prevented from being cleaved, only a reaction area (in a circle, the diameter of the reaction area is 1 mu m) is irradiated for 1 minute, the cleavable linked-chain molecules in the reaction area are cleaved after the irradiation, and NH on the surface of a solid substrate is exposed2A group (shown in FIG. 4), R in the reaction region represents NH2A group.
And 5, fixing functional high molecular polymers on the surfaces of the plurality of reaction areas obtained in the step 4 to form a functional high molecular microarray.
At room temperature, the carrier NH obtained in step 42The microarray substrate slide was prepared as a flow cell surface for high throughput sequencing, and 0.5 ml of anhydrous acetonitrile solution of N, N-dimethylacrylamide-2-vinyl-4, 4' -dimethylazalactone copolymer Poly (DMA-co-VAL) was added to the flow cell, and the formulation was: to a solution of 25 ml acetonitrile, 15. mu.l triethylamine and 87.0 mg of N, N-dimethylacrylamide-2-vinyl-4, 4' -dimethylazalactone copolymer (50% DMA and 50% VAL dipolymer) were added. After being gently rolled and shaken for 19 hours, the flow cell was taken out, washed with a sufficient amount of acetonitrile and dried by blowing to obtain a functional polymer microarray (shown in FIG. 5).
And 6, manufacturing an oligonucleotide primer microarray.
Mixing 25. mu.M of 5' H2Addition of N-labeled oligonucleotide primer (i.e., DNA) 5Adding 0mM sodium phosphate solution with pH8.5 into the functional polymer microarray flow cell obtained in the step 5. Then placing the microarray at 75% humidity for 4-18 hours, washing the microarray with distilled water, and drying the microarray to obtain the oligonucleotide primer microarray, wherein CL is a cleavable catenated chain molecule, and R is NH2Group (first reactive group), 10 is a functional high molecular polymer. The oligonucleotide primer microarray can be used for high-throughput sequencing of genes.
Example 2
Example 2 differs from example 1 in the manner in which the transparent solid substrate is subjected to the activation treatment in step 1. In example 2, the manner of performing the activation treatment on the transparent solid substrate was as follows: the slides were sonicated in 0.5 wt% SDS water for 20 minutes and then thoroughly rinsed with deionized water. Then, the mixture is subjected to ultrasonic treatment in a mixed solution of 29 wt% ammonia water, 30 wt% hydrogen peroxide and deionized water in a volume ratio of 1:1:5 for 20 minutes, and then the mixture is thoroughly washed with deionized water. Then carrying out ultrasonic treatment for 20 minutes in a mixed solution of 38 wt% HCl, 30 wt% hydrogen peroxide and deionized water in a volume ratio of 1:1:6, and then thoroughly cleaning with deionized water. The treated glass slide is placed in deionized water for storage, taken out for drying by using argon, and then baked at 110 ℃ for 5 minutes, wherein the water contact angle of the glass slide subjected to the pre-activation treatment is less than 10 degrees.
Example 3
Example 3 differs from example 1 in that in step 3, the strip NH is applied2The cleavable linker molecule covalently immobilized on the glass surface of the group is 4- (bromomethyl) -3-nitrobenzoic acid, which itself carries a COOH group (i.e. a second reactive group).
2.6mg of 4- (bromomethyl) -3-nitrobenzoic acid and 2. mu.l of 6mM diisopropylethylamine were added to 2 ml of absolute ethanol, and the mixed solution was dropwise added to the belt NH2The surface of the radical slide was left to stand for 3 hours, rinsed with ethanol, and blown dry with argon, as shown in FIG. 6, with NH2The solid substrate of the group immobilizes the cleavable tether molecule bearing the second reactive group.
In step 4, photolyzing the cleavable linked molecules on the surface of the solid substrate according to the method shown in fig. 7 to form a plurality of reaction regions arranged in an array, wherein during the illumination, the reaction regions are not irradiated by light, the cleavable linked molecules in the reaction regions (inside the circle) are not photolyzed and have COOH groups (i.e., second reaction groups), the COOH groups have reactivity, and the cleavable linked molecules in the non-reaction regions (outside the circle) are photolyzed to expose NH on the surface of the solid substrate2A group (i.e., a first reactive group) followed by NH of the non-reactive region2Protecting groups to make them inactive (as shown in FIG. 8), wherein the protection method is as follows: to 2 ml of DMF was added 5. mu.l of acetic anhydride (Ac)2O) and 2 microliter triethylamine are mixed evenly and then dripped on the surface of the glass slide after photolysis, and the glass slide is washed by DMF after standing for 2 hours and then dried by blowing after being washed by ethanol.
The COOH group of the cleavable linked chain molecule in the reaction region (in the circle) is a second reactive group, and is used for being connected with the functional high molecular polymer through a covalent bond in step 5, as shown in fig. 9, CL is the cleavable linked chain molecule, R is the COOH group (second reactive group), CL' is a photolysis product of the cleavable linked chain, and is a non-reactive group, and 10 is the functional high molecular polymer.
Example 4
Example 4 differs from example 3 in that, in step 3, the COOH microarray formed in example 3 is further converted into NH2Microarrays, i.e.by changing the reactive groups of the reaction area from COOH groups to NH2Group, as shown in figure 10.
Preparing a mixed solution: 120 microliter of water, 80 microliter of 5M NaCl, 1 milliliter of DMSO, 400 microliter of 1M NHS, 400 microliter of freshly prepared 1M EDC (EDC dissolved in 5mM imidazole hydrochloride solution at pH 5), 2 milliliters, mixed and left for 40 minutes, then 40 microliters of 2M solution of LiCl dissolved in DMSO and 400 microliters of 10mM H are added to the mixed solution2NCH2CH2NHBoc was further mixed, and then the further mixed solution was dropped on the surface of the slide glass with COOH microarray obtained in step 3 of example 3. After standing for 2 hours, the mixture is flushed by DMSOWashing, then dropping trifluoroacetic acid (TFA) on the surface of the slide, standing for 10 minutes, washing with water, washing with ethanol, and drying, thereby converting the COOH microarray formed in step 3 of example 3 into NH2A microarray.
Example 5
Example 5 differs from example 1 in that in step 3, NH is carried over2The covalently immobilized cleavable linker molecule on the glass surface of the group itself carries a protected second reactive group, as shown in fig. 11.
To 2 ml of 5mM absolute ethanol were added 3.59mg of t-butyl (4- (bromomethyl) -3-nitrophenylethyl) carbamate and 2. mu.l of 6mM diisopropylethylamine, followed by dropwise addition to the solution containing NH2The cleavable linking molecule with the protected second reactive group is fixed on the surface of the glass slide, and as shown in FIG. 11, the second reactive group carried by the cleavable linking molecule is NH2Radical, NH2The protecting group of the group was a Boc group.
In step 4, the cleavable linked chain molecules in the non-reaction zone are photolyzed, so that the cleavable linked chain molecules in the non-reaction zone are cleaved, and NH on the surface of the solid substrate is exposed2Group (i.e. first reactive group) followed by acetic anhydride (Ac)2O) NH to non-reaction zone2Protecting the group to render it inactive, and removing the Boc group of the NHBoc group from the cleavable linker molecule of the reaction zone to form NH in the reaction zone2Group (i.e. second reactive group) to form NH2Microarray, as shown in FIG. 12.
Example 6
Example 6 is different from examples 1 to 5 described above in that the surface of the transparent solid substrate is not smooth but has a plurality of grooves (features) arranged at intervals, the plurality of reaction regions are respectively located at the positions of the plurality of grooves, and the second reactive group is located at the bottom of the grooves, as shown in fig. 13.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the invention and form different embodiments. The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims (10)

1. A method of preparing a biomolecule microarray, comprising:
step one, providing a solid substrate;
forming a plurality of reaction zones arranged at intervals and non-reaction zones positioned at the periphery of the reaction zones on the surface of the solid substrate, wherein the surfaces of the reaction zones are provided with reaction groups;
fixing a functional high molecular polymer by using a reaction group in the reaction zone;
and step four, fixing the biological molecules on the functional high molecular polymer to obtain the biological molecule microarray.
2. The method of claim 1, wherein the solid substrate has a plurality of features spaced apart from each other, and the reaction region is formed at the positions of the features.
3. The method of preparing a biomolecule microarray according to claim 1, wherein the second step comprises:
forming a first reactive group on the surface of the solid substrate;
immobilizing a cleavable tether molecule with the first reactive group;
and carrying out patterned cracking on the cleavable linked chain molecules on the surface of the solid substrate to form a plurality of reaction zones arranged at intervals.
4. The method of claim 3, wherein the cleavable linker molecule does not itself carry a reactive group, and the cleavable linker molecule is cleaved to expose the first reactive group on the surface of the solid substrate;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are cracked, and the cleavable catenated molecules of the non-reaction zone are not cracked.
5. The method of claim 3, wherein the cleavable tether molecule itself carries a second reactive group, which disappears after the cleavable tether molecule is cleaved;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked.
6. The method of claim 3, wherein the cleavable tether molecule itself carries a protected second reactive group, which second reactive group disappears upon cleavage of the cleavable tether molecule and generates a third reactive group;
in the patterned cracking process, the cleavable catenated molecules of the reaction zone are not cracked, and the cleavable catenated molecules of the non-reaction zone are cracked;
after the patterned cracking, a second reaction group of the cleavable linked chain molecule in the reaction zone is deprotected, so that the second reaction group recovers the reactivity; and protecting a third reactive group of the cleavable linked-chain molecule of the non-reactive zone, so that the third reactive group loses reactivity.
7. The method of any one of claims 1 to 6, wherein the plurality of reaction regions are arranged in a matrix.
8. The method of claim 1 to 6, wherein the distance between any two adjacent reaction regions among the plurality of reaction regions is 0.5 μm to 20 μm.
9. A biomolecule microarray produced by the method for producing a biomolecule microarray according to any one of claims 1 to 8.
10. Use of a biomolecule microarray according to claim 9 for DNA sequencing.
CN201910915044.XA 2019-09-26 2019-09-26 Biomolecule microarray and preparation method and application thereof Pending CN110628887A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910915044.XA CN110628887A (en) 2019-09-26 2019-09-26 Biomolecule microarray and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910915044.XA CN110628887A (en) 2019-09-26 2019-09-26 Biomolecule microarray and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN110628887A true CN110628887A (en) 2019-12-31

Family

ID=68974531

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910915044.XA Pending CN110628887A (en) 2019-09-26 2019-09-26 Biomolecule microarray and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110628887A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1523354A (en) * 2003-02-17 2004-08-25 中国科学院力学研究所 Method and apparatus for bio-molecular chip minute quantity sample application and reaction
CN1932033A (en) * 2006-09-22 2007-03-21 东南大学 Nucleic acid sequencing process based on micro array chip
CN110055318A (en) * 2019-05-06 2019-07-26 南京拓远生物科技有限公司 A kind of preparation method of the three dimensional DNA microarray surface based on click chemistry
CN110066861A (en) * 2019-05-05 2019-07-30 南京拓远生物科技有限公司 A kind of patterning microarray flow cell, production method, detection method and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1523354A (en) * 2003-02-17 2004-08-25 中国科学院力学研究所 Method and apparatus for bio-molecular chip minute quantity sample application and reaction
CN1932033A (en) * 2006-09-22 2007-03-21 东南大学 Nucleic acid sequencing process based on micro array chip
CN110066861A (en) * 2019-05-05 2019-07-30 南京拓远生物科技有限公司 A kind of patterning microarray flow cell, production method, detection method and its application
CN110055318A (en) * 2019-05-06 2019-07-26 南京拓远生物科技有限公司 A kind of preparation method of the three dimensional DNA microarray surface based on click chemistry

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
钦传光等: "化学生物学中光敏分子微阵列的表面构建与应用", 《中国科学:化学》 *

Similar Documents

Publication Publication Date Title
US6664061B2 (en) Use and evaluation of a [2+2] photoaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix
JP4741499B2 (en) Binding of molecules to the surface
Rendl et al. Simple one-step process for immobilization of biomolecules on polymer substrates based on surface-attached polymer networks
JP2009522547A (en) Support for analyte assay and method for producing and using the same
US10100163B2 (en) Substrate independent copolymers for biofunctionalization
US20080206752A1 (en) Method For the Photochemical Attachment of Biomolecules to a Substrate
EP1307414A2 (en) The use and evaluation of a 2+2] photocycloaddition in immobilization of oligonucleotides on a three-dimensional hydrogel matrix
AU2019216624B2 (en) Polypeptide arrays and methods of attaching polypeptides to an array
US20030108917A1 (en) Method for manufacturing hydrogel biochip by using star-like polyethylene glycol derivative having epoxy group
US7879764B2 (en) Electrically active combinatorial chemical (EACC) chip for biochemical analyte detection
US7785769B2 (en) Immobilization of oligonucleotides and proteins in sugar-containing hydrogels
US11466321B2 (en) Array including sequencing primer and non-sequencing entity
US20230391997A1 (en) Flow cell including a heteropolymer
CN110628887A (en) Biomolecule microarray and preparation method and application thereof
US20060111517A1 (en) Recognition layers made of hydrogel based on polyacrylamide for use in biosensor technology
AU2018388360B2 (en) Catalytically active substances
JP5344438B2 (en) Substrate for fixing substance, substrate for fixing substance, and analysis method
CN114561455A (en) Chip surface chemical modification method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20191231