阅读说明：本技术 生物分子微阵列及其制备方法与应用 (Biomolecule microarray and preparation method and application thereof ) 是由 向国兵 张明航 吕华 施琦 于 2019-09-26 设计创作，主要内容包括：本发明提供一种生物分子微阵列及其制备方法与应用,属于生物检测技术领域。本发明通过在固体基底表面形成间隔排列的数个反应区,之后分别在数个反应区固定生物分子,从而形成排列有序的数个阵点,本发明制得的生物分子微阵列应用于DNA测序时能够提高单克隆簇比例,进而产生高信噪比的测序信息,提高测序结果的准确性,同时显著提高测序仪的检测通量。(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.)

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.

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.