Polymer, gene sequencing chip, and preparation methods

Abstract

The present application relates to a polymer, a gene sequencing chip, preparation methods, and a gene sequencing method. The polymer has the following structure: formula 1, wherein R1 is H or formula 2, R2 is formula 3, formula 4, formula 5 or formula 6, and R3 is formula 7, formula 8, formula 9 or formula 10; x is selected from an integer from about 1 to about 10000, y is selected from an integer from about 1 to about 1000, z is selected from an integer from about 1 to about 1000, and the ratio of x, y, and z is about 1:(0.015-0.05):(0.045-0.10); and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each independently selected from an integer from about 1 to about 10.

Description

Polymer, gene sequencing chip and preparation method

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 2023109405248, filed on July 28, 2023, entitled “Polymers and methods for preparing same, applications in gene sequencing chips, and sequencing chips,” the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present application relates to the field of gene sequencing technology, and in particular to a polymer, a gene sequencing chip, and a preparation method. Background Art

[0004] Polymer-coated substrates have applications in many technological fields. For example, implantable medical devices can be coated with highly biocompatible polymers, or they can be used in gene sequencing. In recent years, gene sequencing technology has garnered increasing attention. Through gene sequencing, it is possible to analyze and determine gene sequences from blood or human appendages, predicting the likelihood of developing various diseases, such as cancer or leukemia. Polymer coatings play a key role in genetic testing methods. For example, some sequencing methods rely on the attachment of nucleic acid chains to a surface polymer coating. In another example, one or more surfaces of a flow cell are coated with a polymer that can be grafted with nucleic acids. By coating the polymer in the flow cell channels and incubating for a fixed period of time, a stable and reliable coating is formed that supports all downstream biochemical steps, including amplification and sequencing. Current polymers used for nucleic acid attachment in gene sequencing have several limitations. For example, they rely on functionalization of the substrate surface with silane coupling agents before covalent attachment, resulting in structural instability. 2. Some polymers are air-sensitive, and prolonged exposure to air can lead to performance degradation. 3. Polymers are expensive to use, require high coating concentrations, and are generally not reusable.

[0005] Summary of the Invention

[0006] According to various embodiments of the present application, the present application provides a polymer and a preparation method thereof.

[0007] According to various embodiments of the present application, the present application also provides a gene sequencing chip and a method for preparing the same.

[0008] An embodiment of the present application provides a polymer having the following structure:

[0009] R1 is H or

[0010] R2 is

[0011] R3 is

[0012] x is selected from an integer of about 1 to about 10,000, y is selected from an integer of about 1 to about 1,000, and z is selected from an integer of about 1 to about 1,000, and the ratio of x, y, and z is about 1:(0.015-0.05):(0.045-0.10);

[0013] n1, n2, n3, n4, n5, n6, n7, n8 and n9 are each independently selected from integers of about 1 to about 10.

[0014] In one embodiment, the weight average molecular weight of the polymer is about 1,000 to 1,000,000.

[0015] In one embodiment, the R1 is H, the R2 is The R3 is

[0016] In one embodiment, the n3 is approximately 5-7, and the n9 is approximately 3-5.

[0017] In one embodiment, the structure of the polymer is:

[0018] An embodiment of the present application further provides a method for preparing the polymer as described in any of the above embodiments, comprising the following steps:

[0019] Mixing the main monomer, the phosphonic acid functional monomer and the azide functional monomer, and performing a polymerization reaction;

[0020] The structural formula of the main monomer is:

[0021] The structural formula of the phosphonic acid function is:

[0022] The structural formula of the azide functional monomer is:

[0023] In one embodiment, the main monomer includes acrylamide, the phosphonic acid functional monomer includes (6-acrylamidohexyl)phosphonic acid, and the azide functional monomer includes N-(14-azido-3,6,9,12-tetraoxetane)acrylamide.

[0024] In one embodiment, the step of performing the polymerization reaction comprises:

[0025] Mixing the main monomer, the phosphonic acid functional monomer, and the azide functional monomer in a solvent to form a mixed solution; and

[0026] Under a protective gas atmosphere, the mixed solution is heated, and a catalyst and an initiator are added to carry out free radical polymerization.

[0027] An embodiment of the present application further provides a method for preparing a gene sequencing chip, which is prepared using the polymer described in any of the above embodiments.

[0028] In one embodiment, the steps include:

[0029] Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate;

[0030] Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkyne group;

[0031] Immersing the transition metal modified chip substrate in a solution containing the polymer, so that the polymer bonds to the transition metal atoms in the transition metal oxide via the phosphonic acid groups, thereby preparing a chip substrate in which the polymer is grafted onto the surface of the transition metal oxide; and

[0032] The chip substrate with the polymer grafted onto the surface of the transition metal oxide is immersed in a solution containing the nucleic acid load. The chip substrate with the polymer grafted onto the surface of the transition metal oxide is connected to the alkyne group in the modification group of the nucleic acid load via the azide group.

[0033] In one embodiment, the steps include:

[0034] Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate;

[0035] Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkyne group;

[0036] mixing the nucleic acid loading substance with the polymer, allowing the alkyne group in the modified group of the nucleic acid loading substance to react with the azide group on the polymer to prepare a complex in which the nucleic acid loading substance is grafted onto the polymer; and

[0037] The complex of the nucleic acid load grafted onto the polymer is coated on the surface of the transition metal modified chip substrate, and the complex of the nucleic acid load grafted onto the polymer is bonded to the transition metal atom in the transition metal oxide through the phosphonic acid group.

[0038] In one embodiment, the modifying group is introduced by at least one compound selected from the group consisting of alkyne, dibenzocyclooctyne, and bicyclo[6.1.0]nonyne.

[0039] In one embodiment, the transition metal oxide is one or more of titanium dioxide, zirconium dioxide and hafnium dioxide.

[0040] In one embodiment, the nucleic acid cargo is a primer nucleic acid.

[0041] An embodiment of the present application further provides a gene sequencing chip, which is prepared by the preparation method described in any of the above embodiments.

[0042] An embodiment of the present application further provides a gene sequencing method, which uses the above-mentioned gene sequencing chip for sequencing.

[0043] The details of one or more embodiments of the present application are set forth in the following drawings and description. Other features, objects, and advantages of the present application will become apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the conventional technology, the following briefly introduces the drawings required for use in the embodiments or the conventional technology descriptions. Obviously, the drawings described below are merely embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the disclosed drawings without any creative work.

[0045] FIG1 is a schematic diagram of the reaction process between a polymer and a nucleic acid loading substance containing an alkyne-modified group in one embodiment;

[0046] FIG2 is a schematic diagram of the chemical structure of the polymer prepared in Example 1;

[0047] FIG3 is a graph showing the aqueous phase GPC characterization results of the polymer prepared in Example 1;

[0048] FIG4 is a photograph of the rolling circle amplification (RCA) reaction performed in Example 2, stained with SYBR Gold and observed under an inverted fluorescence microscope at 200x using 488 nm excitation light;

[0049] FIG5 is a photograph of the sequencer after running 10 cycles of the rolling circle amplification (RCA) reaction in Example 2 using a Gendow sequencer;

[0050] FIG6 is a photograph of the rolling circle amplification (RCA) reaction performed in Example 3, stained with SYBR Gold and observed under an inverted fluorescence microscope at 200x using 488 nm excitation light;

[0051] FIG7 is a photograph of the sequencer after running 10 cycles using a Gendow sequencer after bridge PCR in Example 4;

[0052] FIG8 is a graph showing the intensity changes of Cy3 complementary fluorescence test after primers of different concentrations are fixed on the chip surface in Example 5;

[0053] FIG9 shows a photograph of the sequencer after bridge PCR was performed on a gene sequencing chip obtained in Example 5 using primers with a final concentration of 10 μM and running 10 cycles on a Gendow sequencer;

[0054] FIG10 is a graph showing the intensity changes of Cy3 complementary fluorescence test of a gene sequencing chip prepared after the polymer of Example 6 was reused for different times. DETAILED DESCRIPTION

[0055] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0057] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0058] An embodiment of the present application provides a polymer having the following structure:

[0059] R1 is H or

[0060] R2 is

[0061] R3 is

[0062] x is selected from an integer of about 1 to about 10,000, y is selected from an integer of about 1 to about 1,000, and z is selected from an integer of about 1 to about 1,000, and the ratio of x, y, and z is about 1:(0.015-0.05):(0.045-0.10);

[0063] n1, n2, n3, n4, n5, n6, n7, n8 and n9 are each independently selected from integers of about 1 to about 10.

[0064] The polymer provided in this application is a polymer containing phosphonic acid groups and azide groups. It has stable properties and is not easy to deteriorate. The specific modification of transition metals by phosphonic acid groups can achieve chemical treatment of the chip substrate surface to form a stable and reliable polymer coating. At the same time, after chemical connection between the azide group and the nucleic acid containing the modification group, the nucleic acid can be successfully fixed on the surface of the chip substrate to form a stable surface structure, thereby forming a gene sequencing chip. The polymer can be reused multiple times and can perform various amplification reactions and support sequencing. It has good stability and lower usage cost.

[0065] In one embodiment, x is selected from an integer of about 2000 to about 4000. In one embodiment, x is selected from about 3000.

[0066] In one embodiment, y is selected from an integer of about 1 to about 100. In one embodiment, y is selected from about 60.

[0067] In one embodiment, z is selected from an integer of about 100 to about 200. In one embodiment, z is selected from about 150.

[0068] In one embodiment, the weight average molecular weight of the polymer is about 1,000 to 1,000,000. In one embodiment, the weight average molecular weight of the polymer is about 100,000 to 200,000.

[0069] In one embodiment, R1 is H, R2 is R3 is

[0070] In one embodiment, n3 is approximately 5-7, and n9 is approximately 3-5.

[0071] In one embodiment, the polymer has the structure:

[0072] An embodiment of the present application further provides a method for preparing a polymer as in any of the above embodiments, comprising the following steps:

[0073] Mixing the main monomer, the phosphonic acid functional monomer and the azide functional monomer, and performing a polymerization reaction;

[0074] The structural formula of the main monomer is:

[0075] The structural formula of the phosphonic acid function is:

[0076] The structural formula of the azide functional monomer is:

[0077] In the preparation method of the polymer provided by the present application, the main monomer contains an acrylamide group, which serves as the backbone of the polymer and can provide the polymer with good biocompatibility and antifouling properties; one end of the phosphonic acid functional monomer contains an unsaturated group, and the other end contains a phosphonic acid group, which serves to introduce a phosphonic acid group into the structure of the polymer, and utilize the bonding effect of the phosphonic acid group with the transition metal on the surface of the chip substrate to fix the polymer to the surface of the chip substrate to form a stable and reliable polymer coating; one end of the azide functional monomer contains an unsaturated group, and the other end contains an azide group, which serves to introduce an azide group into the structure of the polymer, and utilize the mechanism of the click chemistry reaction between the azide group and the alkyne group to realize the connection between the polymer and the nucleic acid load, so that the nucleic acid load can be stably fixed to the surface of the chip substrate to form a gene sequencing chip with a stable structure, which can perform a variety of amplification reactions and support sequencing. There are many types of polymers prepared by the above method, and monomers of various structures can be introduced by adjusting the structural combination of R1, R2, and R3 to achieve regulation of the molecular weight of the polymer.

[0078] It can be understood that, depending on the R1 group, the main monomer may contain a PEG-modified or unmodified acrylamide group; depending on the R2 group, the unsaturated group contained at one end of the phosphonic acid functional monomer may be an acrylamide group or a vinyl group; depending on the R3 group, the unsaturated group contained at one end of the azide functional monomer may also be an acrylamide group or a vinyl group.

[0079] In one embodiment, the main monomer includes acrylamide, the phosphonic acid functional monomer includes (6-acrylamidohexyl)phosphonic acid, and the azide functional monomer includes N-(14-azido-3,6,9,12-tetraoxetane)acrylamide.

[0080] In one embodiment, the molar ratio of the main monomer, the phosphonic acid functional monomer, and the azide functional monomer is about 1:(0.015-0.05):(0.045-0.10).

[0081] In one embodiment, the step of performing the polymerization reaction comprises:

[0082] Mixing the main monomer, the phosphonic acid functional monomer, and the azide functional monomer in a solvent to form a mixed solution; and

[0083] Under a protective gas atmosphere, the mixed solution is heated, and a catalyst and an initiator are added to carry out free radical polymerization.

[0084] In one embodiment, the solvent is water.

[0085] In one embodiment, the step of forming the mixed solution comprises:

[0086] dissolving the main monomer in the first part of the solvent to form a main monomer solution;

[0087] dissolving the phosphonic acid functional monomer in the second portion of the solvent to form a phosphonic acid functional monomer solution;

[0088] dissolving the azide functional monomer in the third portion of the solvent to form an azide functional monomer solution; and

[0089] The main monomer solution, the phosphonic acid functional monomer solution and the azide functional monomer solution are mixed and fixed to volume to form a mixed solution.

[0090] In one embodiment, the protective gas is nitrogen.

[0091] In one embodiment, the catalyst for free radical polymerization is one or more of N,N,N',N'-tetramethylethylenediamine (TEMED), sodium sulfite, and sodium thiosulfate.

[0092] In one embodiment, the initiator for free radical polymerization is one or more of potassium persulfate (KPS), ammonium sulfate (APS), and an azo initiator. Alternatively, the azo initiator may be, but is not limited to, 2,2'-azobisisobutylamidine dihydrochloride (AAPH).

[0093] In one embodiment, the temperature for performing the free radical polymerization is about 4°C to 60°C.

[0094] In one embodiment, the reaction time of the free radical polymerization is about 1 hour to 3 hours.

[0095] In one embodiment, the method for preparing the polymer further comprises the step of terminating the polymerization after the reaction is completed:

[0096] The reaction product is exposed to air and stirred to quench free radicals.

[0097] In one embodiment, the stirring time is about 30 min to 60 min.

[0098] In one embodiment, the method for preparing the polymer further comprises the step of collecting the product:

[0099] The product after the reaction is precipitated in a precipitant, and the precipitate is collected and dried.

[0100] In one embodiment, the precipitant may be, for example but not limited to, isopropyl alcohol.

[0101] In one embodiment, the polymer prepared by the above method can be dissolved in water to prepare a solution for easy use.

[0102] An embodiment of the present application further provides a method for preparing a gene sequencing chip, which is prepared using the polymer described in any of the above embodiments.

[0103] The polymer provided in this application contains phosphonic acid groups and azide groups, wherein the phosphonic acid groups can coordinate with transition metals, thereby achieving specific modification of chip substrates covered with transition metals. The bonding effect of phosphonic acid on transition metals can form monodentate, bidentate, or tridentate bonding structures, thereby fixing the polymer to the surface of the chip substrate; the azide groups can be connected to nucleic acid payloads containing alkynyl modification groups introduced by compounds such as alkynes, dibenzocyclooctyne (DBCO), and bicyclo[6.1.0]nonyne (BCN) through a click chemistry reaction, thereby achieving the purpose of fixing the nucleic acid payload to the surface of the chip substrate.

[0104] Based on the structural characteristics of the polymers provided in this application, this application provides two methods for preparing gene sequencing chips using the above polymers:

[0105] The first method is: first use phosphonic acid groups to specifically modify the transition metal covering the surface of the chip substrate, and then graft the polymer to the surface of the transition metal to make the surface of the chip substrate covered with azide groups. The azide groups can undergo click chemistry reactions with alkynyl-containing modification groups introduced by compounds such as alkynes, dibenzocyclooctyne (DBCO), and bicyclo[6.1.0]nonyne (BCN), thereby achieving the purpose of fixing the nucleic acid load containing alkynyl modification groups to the surface of the chip substrate, and preparing a gene sequencing chip.

[0106] The second method is: first, use the alkyne-containing modification group introduced by compounds such as alkynes, dibenzocyclooctyne (DBCO), and bicyclo[6.1.0]nonyne (BCN) on the nucleic acid load to conduct a click chemistry reaction with the azide group on the polymer to prepare a complex of the nucleic acid load grafted on the polymer, so that the complex contains phosphonic acid groups, and then bond the phosphonic acid groups to the transition metal on the surface of the transition metal-modified chip substrate, thereby fixing the nucleic acid load on the chip substrate surface to prepare a gene sequencing chip.

[0107] The above two methods can be used to obtain a gene sequencing chip with high-density nucleic acid molecules fixed on the surface of a transition metal-modified chip substrate, and the gene sequencing chip can perform various amplification reactions.

[0108] Specifically:

[0109] Corresponding to the first method above, in one embodiment, the preparation of the gene sequencing chip includes the following steps:

[0110] Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate;

[0111] Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkynyl group; and

[0112] The transition metal modified chip substrate is immersed in a solution containing a polymer, so that the polymer is bonded to the transition metal atoms in the transition metal oxide through the phosphonic acid group, thereby preparing a chip substrate with the polymer grafted on the surface of the transition metal oxide;

[0113] The chip substrate with the polymer grafted on the surface of the transition metal oxide is immersed in a solution containing a nucleic acid load. The chip substrate with the polymer grafted on the surface of the transition metal oxide is connected to the alkyne group in the modification group of the nucleic acid load through the azide group.

[0114] Corresponding to the second method described above, in another embodiment, the preparation of the gene sequencing chip includes the following steps:

[0115] Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate;

[0116] Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkyne group;

[0117] mixing the nucleic acid loading substance with the polymer, allowing the alkyne group in the modification group of the nucleic acid loading substance to react with the azide group on the polymer to prepare a complex in which the nucleic acid loading substance is grafted onto the polymer; and

[0118] The nucleic acid loading substance grafted onto the polymer complex is coated on the surface of the transition metal modified chip substrate, and the nucleic acid loading substance grafted onto the polymer complex is bonded to the transition metal atom in the transition metal oxide through the phosphonic acid group.

[0119] In one embodiment, the modifying group is introduced by at least one compound selected from the group consisting of alkyne, dibenzocyclooctyne, and bicyclo[6.1.0]nonyne.

[0120] In one embodiment, the transition metal oxide is one or more of titanium dioxide, zirconium dioxide, and hafnium dioxide.

[0121] In one embodiment, the nucleic acid cargo is a primer nucleic acid.

[0122] It is understandable that the chip substrate may be, for example, but not limited to, one or more materials including plastic, silicon, silicon dioxide, silicon nitride, quartz, ceramic, resin, polymer, copolymer, and the like.

[0123] It is understood that the transition metal-modified chip substrate, in which the transition metal oxide is coated on the surface of the chip substrate, can be produced in-house or commercially available. It is understood that the method for producing the transition metal-modified chip substrate in-house can be conventional methods in the art, such as coating methods such as atomic layer deposition and magnetron sputtering, or chemical synthesis methods such as in-situ generation, and is not particularly limited herein.

[0124] It is understandable that the nucleic acid cargo containing a modification group such as an alkyne group can be homemade or purchased commercially.

[0125] Figure 1 shows a schematic diagram of a reaction for preparing a complex of a nucleic acid loading substance grafted onto a polymer, in one embodiment, when the nucleic acid loading substance is a primer and the alkynyl group in the modifying group is introduced by an alkyne. It is understood that when the nucleic acid loading substance is another loading substance and / or the alkynyl group in the modifying group is introduced by another compound such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN), the reaction schematic diagram is similar and will not be repeated here.

[0126] In one embodiment, a catalyst is added during the preparation of a complex of a nucleic acid payload grafted onto a polymer. In one embodiment, the catalyst is a monovalent copper ion. In one embodiment, the monovalent copper ion can be obtained by: forming a ligand with CuSO4 and THPTA, which is then reduced to monovalent copper in the presence of sodium ascorbate.

[0127] It is understood that in the two preparation methods described above, the solvent in the solution involved can be water, ethanol, methanol, tetrahydrofuran, or mixtures thereof. The specific solvent and concentration used can be selected based on the polymer solubility. The lower the solvent dielectric constant, the fewer defects the phosphonic acid polymer has in transition metal modification. The higher the phosphonic acid polymer concentration and the longer the reaction time, the higher the density of the polymer grafted onto the transition metal-modified chip substrate surface.

[0128] An embodiment of the present application further provides a gene sequencing chip, which is prepared by the preparation method in any of the above embodiments.

[0129] An embodiment of the present application further provides a gene sequencing method, which uses the above-mentioned gene sequencing chip for sequencing.

[0130] The present application is further described in detail below through specific examples. The following examples are relatively specific, and it is understood that in other embodiments, they are not limited thereto. In the following specific examples, the instruments, reagents, and materials involved, unless otherwise specified, are all conventional instruments, reagents, and materials already in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already in the prior art.

[0131] Nucleic acid loading material: purchased from Sangon Biotech (Shanghai) Co., Ltd., with the following primer nucleic acid sequence:

[0132] First primer:

[0133] SEQ 1: 5`CHCH-Spacer18-ATCTCGTATGCCGTCTTCTGCTTG.

[0134] Second primer:

[0135] SEQ 2: 5`CHCH-Spacer18-CAAGCAGAAGACGGCATACGA.

[0136] Third primer:

[0137] SEQ 3: 5`CHCH-Spacer18-AATGATACGGCGACCACCGATCTACAC.

[0138] Transition metal modified chip substrate with transition metal oxide covering the surface of the chip substrate: purchased from Titanium Technology Co., Ltd., the chip substrate is made of silicon dioxide and the transition metal oxide is TiO2.

[0139] Example 1

[0140] Preparation of polymer:

[0141] 1. Weigh the main monomer acrylamide (about 500 mg, about 7.03 mmol), dissolve it in about 2 mL of ultrapure water, and mix thoroughly;

[0142] 2. Weigh the phosphonic acid functional monomer (6-acrylamidohexyl)phosphonic acid (about 33 mg, about 0.14 mmol, about 2% of the amount of the main monomer substance) and dissolve it in about 2 mL of ultrapure water. Dissolve thoroughly and mix well.

[0143] 3. Weigh the azide-functional monomer N-(14-azido-3,6,9,12-tetraoxetane) acrylamide (N3-PEG-Acrylamide, about 111 mg, about 0.35 mmol, about 5% of the amount of the main monomer substance) and dissolve it in about 2 mL of ultrapure water. Dissolve thoroughly and mix well.

[0144] 4. Mix the solutions prepared in step 1, step 2, and step 3 and dilute to about 10 mL. Filter using a 0.22 μm filter membrane and continue to flow nitrogen for about 30 minutes.

[0145] 5. Preheat a water bath to about 40°C, transfer the mixed solution obtained in step 4 to the three-necked flask in the water bath at a speed of about 200 rpm, and continuously flow nitrogen;

[0146] 6. Add N,N,N',N'-tetramethylethylenediamine (TEMED, about 18 μL, about 0.15 mmol) to the mixed solution obtained in step 5 and mix thoroughly;

[0147] 7. Add potassium persulfate (KPS, about 5 mg, about 0.02 mmol) to the mixed solution obtained in step 6 to start the reaction;

[0148] 8. The reaction mixture of step 7 was continued to react at about 40° C. for about 2 hours;

[0149] 9. Expose the reacted material from step 8 to air and stir for about 30 minutes to quench free radicals and terminate the reaction;

[0150] 10. The reaction product obtained in step 9 was precipitated with a large amount of isopropanol, stirred for about 30 minutes, the precipitate was collected, and vacuum-dried to obtain a polymer;

[0151] 11. The polymer obtained in step 10 was quantified and prepared into a 10% (w / v) polymer solution using purified water at a polymer mass to water volume ratio of approximately 0.1 g:1 mL, and stored at approximately 4°C.

[0152] Among them, the chemical structure of the main monomer acrylamide is:

[0153] The chemical formula of the phosphonic acid functional monomer (6-acrylamidohexyl)phosphonic acid is:

[0154] The chemical formula of the azide functional monomer N-(14-azido-3,6,9,12-tetraoxetane) acrylamide is:

[0155] The polymerization process for preparing polymers is:

[0156] The polymer prepared in Example 1 was structurally characterized:

[0157] The polymer prepared in Example 1 was 1 HNMR (D2O) characterization was performed. Combined with Figure 2, the characterization results are as follows:

[0158] The characteristic absorption peak of -CH2- at the D, E, F, and G label positions appears at approximately 1.28 ppm; the characteristic absorption peak of -CH2- at the B and H label positions appears at approximately 1.43 ppm to 1.66 ppm; the characteristic absorption peak of -CH2- at the A label position and the characteristic absorption peak of -CH2- at the M label position appear at approximately 2.08 ppm to 2.21 ppm; the characteristic absorption peak of -CH2- at the I and C label positions appears at approximately 3.53 ppm to 3.60 ppm; and the characteristic absorption peak of -CH2- at the K, J, and L label positions appears at approximately 3.60 ppm to 3.96 ppm.

[0159] Figure 3 shows the aqueous phase GPC characterization results of the polymer prepared in Example 1. GPC testing showed that the polymer prepared in Example 1 had a number average molecular weight Mn of approximately 59,169, a weight average molecular weight Mw of approximately 171,873, and a molecular weight distribution D of approximately 2.90478.

[0160] Example 2

[0161] The primers are fixed to the surface of the chip substrate to prepare a gene sequencing chip, and RCA amplification is performed;

[0162] The preparation method of the gene sequencing chip is: first preparing a chip substrate with a polymer grafted on the surface of a transition metal oxide, and then connecting the primer to the polymer.

[0163] Step 1: providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate.

[0164] Step 2: Preparation of chip substrate with polymer grafted onto transition metal oxide surface

[0165] 1. Take about 100 μL of the approximately 10% polymer solution prepared in Example 1, add about 9.9 mL of purified water, and mix thoroughly to prepare a polymer working solution;

[0166] 2. Soak the transition metal modified chip substrate provided in step 1 in isopropanol solution for about 2 minutes, take it out and blow it dry with nitrogen, and then clean it with plasma for about 3 minutes;

[0167] 3. Soak the cleaned transition metal modified chip substrate in the polymer working solution prepared in step 1 and place it at room temperature for about 15 hours;

[0168] 4. Take out the chip substrate treated in step 3, rinse it repeatedly with purified water, and blow dry it with nitrogen.

[0169] Step 3: Connect the primer to the polymer

[0170] 1. Prepare the following solution:

[0171] About 200 mM tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA): Weigh about 8.69 mg of tris-(3-hydroxypropyltriazolylmethyl)amine and add purified water to make a solution of about 100 μL;

[0172] About 100 mM copper sulfate (CuSO4): Weigh about 159.6 mg of copper sulfate and add purified water to make about 10 mL of solution;

[0173] About 100 mM sodium ascorbate: Weigh about 19.81 mg of ascorbic acid and add purified water to make about 1 mL of solution;

[0174] THPTA / CuSO4 ligand: Mix about 200 mM THPTA and about 100 mM CuSO4 solution in a volume ratio of about 1:1;

[0175] About 100 μM of the first primer (SEQ 1): Dissolve and prepare by adding NF water.

[0176] 2. Primers are linked to polymers and RCA amplified

[0177] (1) Take about 50 μL of about 100 μM first primer, add about 50 μL of NF water, and mix thoroughly;

[0178] (2) Add about 2 μL of THPTA / CuSO4 ligand to the solution in step (1) and mix thoroughly;

[0179] (3) adding about 3 μL of about 100 mM sodium ascorbate to the solution of step (2) and mixing thoroughly to prepare a reaction solution;

[0180] (4) injecting the reaction solution of step (3) into the chip substrate treated in step 2 and reacting at room temperature for about 1 hour;

[0181] (5) Rinse the chip with purified water and store at approximately 4°C;

[0182] (6) Perform rolling circle amplification (RCA) reaction using the chip obtained in step (5).

[0183] Example 3

[0184] The primers are fixed to the surface of the chip substrate to prepare a gene chip, and RCA amplification is performed;

[0185] The preparation method of the gene sequencing chip is: firstly, the primer and the polymer are mixed and reacted to prepare a complex of the primer grafted on the polymer, and then the complex is fixed on the surface of the transition metal modified chip substrate.

[0186] Step 1: providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate.

[0187] Step 2: Preparation of primer-grafted polymer complex

[0188] 1. Prepare the following solution:

[0189] About 200 mM tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA): Weigh about 8.69 mg of tris-(3-hydroxypropyltriazolylmethyl)amine and add purified water to make a solution of about 100 μL;

[0190] About 100 mM copper sulfate (CuSO4): Weigh about 159.6 mg of copper sulfate and add purified water to make about 10 mL of solution;

[0191] About 100 mM sodium ascorbate: Weigh about 19.81 mg of ascorbic acid and add purified water to make about 1 mL of solution;

[0192] THPTA / CuSO4 ligand: Mix about 200 mM THPTA and about 100 mM CuSO4 solution in a volume ratio of about 1:1;

[0193] About 100 μM of the first primer (SEQ 1): Dissolve and prepare in NF water.

[0194] 2. Preparation of primer-grafted polymer complexes

[0195] (1) Take about 50 μL of the approximately 10% polymer solution prepared in Example 1, add about 800 μL of purified water, and mix well to obtain a polymer working solution;

[0196] (2) adding about 100 μL of about 100 μM first primer to the polymer working solution of step (1) and mixing well;

[0197] (3) Add about 20 μL of THPTA / CuSO4 ligand to the solution in step (2) and mix well;

[0198] (4) adding about 30 μL of about 100 mM sodium ascorbate to the solution of step (3) and mixing uniformly to obtain a reaction solution;

[0199] (5) The reaction solution of step (4) is reacted at room temperature for about 1 hour to obtain a primer-grafted polymer complex, which is stored at about 4°C;

[0200] Step 3: Fix the complex on the surface of the transition metal modified chip substrate

[0201] 1. Coat the primer-grafted polymer complex prepared in step 2 on the surface of the transition metal-modified chip substrate provided in step 1, and react at room temperature for about 15 hours;

[0202] 2. After the reaction is completed, wash the gene chip three times with purified water and store at about 4°C;

[0203] 3. Use the gene sequencing chip obtained in step 2 to perform rolling circle amplification (RCA) amplification.

[0204] Example 4

[0205] The primers are fixed to the surface of the chip substrate to prepare a gene sequencing chip, and bridge PCR amplification is performed;

[0206] The preparation method of the gene sequencing chip is: first preparing a chip substrate with a polymer grafted on the surface of a transition metal oxide, and then connecting the primer to the polymer.

[0207] Step 1: providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate.

[0208] Step 2: Preparation of chip substrate with polymer grafted onto transition metal oxide surface

[0209] 1. Take about 100 μL of the approximately 10% polymer solution prepared in Example 1, add about 9.9 mL of purified water, and mix thoroughly to prepare a polymer working solution;

[0210] 2. Soak the transition metal modified chip substrate obtained in step 1 in isopropanol solution for about 2 minutes, take it out and blow it dry with nitrogen, and then clean it with plasma for about 3 minutes;

[0211] 3. Soak the cleaned transition metal modified chip substrate in the polymer working solution prepared in step 1 and place it at room temperature for about 15 hours;

[0212] 4. Take out the chip substrate treated in step 3, rinse it repeatedly with purified water, and blow dry it with nitrogen.

[0213] Step 3: Connect the primer to the polymer

[0214] 1. Prepare the following solution:

[0215] About 200 mM tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA): Weigh about 8.69 mg of tris-(3-hydroxypropyltriazolylmethyl)amine and add purified water to make a solution of about 100 μL;

[0216] About 100 mM copper sulfate (CuSO4): Weigh about 159.6 mg of copper sulfate and add purified water to make about 10 mL of solution;

[0217] About 100 mM sodium ascorbate: Weigh about 19.81 mg of ascorbic acid and add purified water to make about 1 mL of solution;

[0218] THPTA / CuSO4 ligand: Mix approximately 200 mM THPTA and 100 mM CuSO4 solution in a volume ratio of approximately 1:1;

[0219] About 100 μM of the second primer (SEQ 2): Dissolve and prepare in NF water;

[0220] About 100 μM of the third primer (SEQ 3): Dissolve and prepare by adding NF water.

[0221] 2. Primers are linked to polymers and bridge PCR amplification is performed

[0222] (1) Take about 50 μL of about 100 μM second primer, add about 50 μL of about 100 μM third primer, and mix thoroughly;

[0223] (2) Add about 2 μL of THPTA / CuSO4 ligand to the solution in step (1) and mix thoroughly;

[0224] (3) adding about 3 μL of about 100 mM sodium ascorbate to the solution of step (2) and mixing thoroughly to prepare a reaction solution;

[0225] (4) injecting the reaction solution of step (3) into the chip substrate treated in step 2 and reacting at room temperature for 1 hour;

[0226] (5) Rinse the chip with purified water and store at approximately 4°C.

[0227] (6) Using the chip of step (5), bridge PCR reagents are added to generate clusters.

[0228] Figure 4 shows the results of photographing the gene sequencing chip prepared by the method of Example 2 and performing a rolling circle amplification (RCA) reaction, using SYBR Gold staining, and observing the results of photographing under an inverted fluorescence microscope with a 200x field of view using 488nm excitation light; Figure 5 shows the results of photographing the gene sequencing chip prepared by the method of Example 2 and performing a rolling circle amplification (RCA) reaction, using a Gendow sequencer after running 10 cycles; Figure 6 shows the results of photographing the gene sequencing chip prepared by the method of Example 3 and performing a rolling circle amplification (RCA) reaction, using SYBR Gold staining, and observing the results of photographing under an inverted fluorescence microscope with a 200x field of view using 488nm excitation light; Figure 7 shows the results of photographing the gene sequencing chip prepared by the method of Example 4 and performing bridge PCR, using a Gendow sequencer after running 10 cycles. As can be seen from Figures 4 to 7, the methods of Examples 2 to 4 can all stably fix high-density primers to the chip substrate surface and perform amplification reactions.

[0229] Example 5

[0230] Step 1: Same as Example 4.

[0231] Step 2: Same as Example 4.

[0232] Step 3:

[0233] 1. Prepare the following solution: same as in Example 4.

[0234] 2. Primer and polymer connection:

[0235] (1) Prepare a reaction solution according to Example 4, wherein the final concentrations of the second primer and the third primer in the reaction solution are about 1 μM, about 10 μM, about 20 μM, about 30 μM, and about 40 μM, respectively;

[0236] in:

[0237] When the final concentration of the second primer and the third primer in the reaction solution is about 1 μM, the amount of each reagent added includes: about 1 μL of about 100 μM second primer + about 1 μL of about 100 μM third primer + about 93 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL 100 mM sodium ascorbate;

[0238] When the final concentration of the second primer and the third primer in the reaction solution is about 10 μM, the amount of each reagent added includes: about 10 μL of about 100 μM second primer + about 10 μL of about 100 μM third primer + about 75 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL of about 100 mM sodium ascorbate;

[0239] When the final concentration of the second primer and the third primer in the reaction solution is about 20 μM, the amount of each reagent added includes: about 20 μL of about 100 μM second primer + about 20 μL of about 100 μM third primer + about 55 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL of about 100 mM sodium ascorbate;

[0240] When the final concentration of the second primer and the third primer in the reaction solution is about 30 μM, the amount of each reagent added includes: about 30 μL of about 100 μM second primer + about 30 μL of about 100 μM third primer + about 35 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL of about 100 mM sodium ascorbate;

[0241] When the final concentration of the second primer and the third primer in the reaction solution is about 40 μM, the amount of each reagent added includes: about 40 μL of about 100 μM second primer + about 40 μL of about 100 μM third primer + about 15 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL of about 100 mM sodium ascorbate;

[0242] (2) injecting the reaction solutions of the second primer and the third primer prepared in step (1) with final concentrations of about 1 μM, about 10 μM, about 20 μM, about 30 μM, and about 40 μM into the chip substrate treated in step 2, respectively, and reacting at room temperature for about 1 hour;

[0243] (3) Rinse the chip with purified water and store at approximately 4°C.

[0244] The final concentrations of the second primer and the third primer in the reaction solution are about 1 μM, about 10 μM, about 20 μM, about 30 μM, and about 40 μM, respectively. The surface nucleic acid density of the chip formed in step (3) is measured by fluorescence. The test method includes the following steps:

[0245] (1) Approximately 3 μM Cy3-labeled oligonucleotides were added to each chip in 2x SSC buffer. The oligonucleotides were reverse complementary to the primer nucleic acid immobilized on the chip surface. The chip was placed in an oven at approximately 40°C for a hybridization reaction of approximately 30 minutes. After the reaction, the chip was allowed to cool to room temperature and each chip channel was rinsed twice with approximately 1 mL of 2x SSC buffer.

[0246] (2) The surface nucleic acid density of each chip was detected using the Gendow sequencing platform. The nucleic acid density on the surface of each chip was evaluated by detecting the Cy3 fluorescence on the complementary strand and taking the average brightness of the photographed field of view. The average fluorescence intensity in each photographed field of view with an area of ​​approximately 110 × 110 μm was calculated. The average brightness of the flow channel was obtained by taking the average of the fluorescence values ​​in 50 fields of view in the middle area.

[0247] As shown in Figure 8, as the final concentration of the primer nucleic acid in the reaction solution increases, the fluorescence intensity also increases continuously, indicating that the density of the nucleic acid fixed and grafted on the chip surface increases with the increase of the final concentration of the primer nucleic acid in the reaction solution. The fluorescence intensity is positively correlated with the density of the nucleic acid fixed and grafted on the chip surface, and the grafting status of the nucleic acid on the chip surface can be reflected by the change in fluorescence intensity.

[0248] After the fluorescence test, formamide was used to remove the fluorescent hybrid chains, and the final concentration of the second primer and the third primer in the reaction solution was about 10 μM. The chip formed in step (3) was added with a bridge PCR reagent for cluster generation. The detection results obtained by taking a picture of one of the flow channels on the Gendow sequencer are shown in Figure 9. As can be seen from Figure 9, this embodiment successfully and stably fixed the high-density primers on the surface of the chip substrate and supported amplification.

[0249] Example 6

[0250] Step 1: Same as Example 4.

[0251] Step 2: Same as Example 4.

[0252] Step 3:

[0253] 1. Prepare the following solution: same as in Example 4.

[0254] 2. Primer and polymer connection:

[0255] (1) A reaction solution was prepared with reference to Example 4. The final concentrations of the second primer and the third primer in the reaction solution were respectively about 10 μM. The amount of each reagent added included: about 10 μL of about 100 μM second primer + about 10 μL of about 100 μM third primer + about 75 μL NF water + about 2 μL THPTA / CuSO4 ligand + about 3 μL of about 100 mM sodium ascorbate.

[0256] (2) Inject the reaction solution prepared in step (1) into the chip substrate treated in step 2 and react at room temperature for about 1 hour.

[0257] (3) Rinse the chip with purified water and store at approximately 4°C.

[0258] (4) Recover the used polymer working solution and use it for the preparation of the next gene sequencing chip.

[0259] The surface nucleic acid density on the gene sequencing chip prepared using the polymer working solution after 0, 5, 10, 15, 20, 25, and 30 cycles of recovery was tested. The surface nucleic acid density was evaluated by the change in fluorescence intensity with reference to Example 5. The test results are shown in FIG10 .

[0260] As shown in Figure 10, when the polymer that has been recycled 25 times is used to prepare a gene sequencing chip, the fluorescence intensity of the obtained sequencing chip can still reach more than about 90% of the fluorescence intensity of the chip prepared from the polymer used for the first time, indicating that the polymer is stable and reusable.

[0261] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0262] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A polymer, characterized in that It has the following structure: R1 is H or R2 is R3 is x is selected from an integer of about 1 to about 10,000, y is selected from an integer of about 1 to about 1,000, z is selected from an integer of about 1 to about 1,000, and the ratio of x, y and z is about 1:(0.015-0.05):(0.045-0.10); n1, n2, n3, n4, n5, n6, n7, n8 and n9 are each independently selected from integers of about 1 to about 10.

2. The polymer according to claim 1, characterized in that The weight average molecular weight of the polymer is about 1,000 to 1,000,000.

3. The polymer according to claim 1 or 2, characterized in that R1 is H, and R2 is The R3 is 4. The polymer according to any one of claims 1 to 3, characterized in that The n3 is approximately 5-7, and the n9 is approximately 3-5.

5. The polymer according to any one of claims 1 to 4, characterized in that The structure of the polymer is:

6. A method for preparing a polymer according to any one of claims 1 to 5, characterized in that: The steps include: Mixing the main monomer, the phosphonic acid functional monomer and the azide functional monomer, and performing a polymerization reaction; The structural formula of the main monomer is: The structural formula of the phosphonic acid function is: The structural formula of the azide functional monomer is:

7. The method for preparing a polymer according to claim 6, characterized in that: The main monomer includes acrylamide, the phosphonic acid functional monomer includes (6-acrylamidohexyl)phosphonic acid, and the azide functional monomer includes N-(14-azido-3,6,9,12-tetraoxetane)acrylamide.

8. The method for preparing a polymer according to claim 6 or 7, characterized in that: The steps of carrying out the polymerization reaction include: Mixing the main monomer, the phosphonic acid functional monomer and the azide functional monomer in a solvent to form a mixed solution; and Under a protective gas atmosphere, the mixed solution is heated, and a catalyst and an initiator are added to carry out free radical polymerization.

9. A method for preparing a gene sequencing chip, characterized in that: The method is prepared by using the polymer described in any one of claims 1 to 5.

10. The preparation method according to claim 9, characterized in that: The steps include: Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate; Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkynyl group; Immersing the transition metal modified chip substrate in a solution containing the polymer, so that the polymer is bonded to the transition metal atom in the transition metal oxide through the phosphonic acid group, to prepare a chip substrate with the polymer grafted on the surface of the transition metal oxide; as well as The chip substrate with the polymer grafted on the surface of the transition metal oxide is immersed in a solution containing the nucleic acid load, and the chip substrate with the polymer grafted on the surface of the transition metal oxide is connected to the alkyne group in the modification group of the nucleic acid load through the azide group.

11. The preparation method according to claim 9, characterized in that: The steps include: Providing a transition metal modified chip substrate in which a transition metal oxide is covered on the surface of the chip substrate; Providing a nucleic acid cargo, wherein the nucleic acid cargo contains a modification group, wherein the modification group includes an alkynyl group; The nucleic acid loading substance is mixed with the polymer, and the alkynyl group in the modification group of the nucleic acid loading substance is fused with the stacked group on the polymer. nitrogen group reaction to prepare a complex in which a nucleic acid load is grafted onto a polymer; and The complex of the nucleic acid load grafted onto the polymer is coated on the surface of the transition metal modified chip substrate, and the complex of the nucleic acid load grafted onto the polymer is bonded to the transition metal atom in the transition metal oxide through the phosphonic acid group.

12. The preparation method according to claim 10 or 11, characterized in that: The modifying group is introduced from at least one compound selected from the group consisting of alkyne, dibenzocyclooctyne and bicyclo[6.1.0]nonyne.

13. The preparation method according to any one of claims 10 to 12, characterized in that: The transition metal oxide is one or more of titanium dioxide, zirconium dioxide and hafnium dioxide.

14. The preparation method according to any one of claims 10 to 13, characterized in that: The nucleic acid loading substance is a primer nucleic acid.

15. A gene sequencing chip, characterized in that: The compound is prepared by the preparation method according to any one of claims 10 to 14.

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