Gene sequencing method adopting degradable hydrogel

Abstract

The invention discloses a depolymerization gene sequencing method, which is characterized in that a prepolymerization solution of a carrier is subjected to gas sealing or oil sealing in a gene sequencing chip, so that the carrier of a nucleic acid fragment to be detected is formed in a micro-pit of the gene sequencing chip. The degradable sites on the carrier can react through the degradation reagent and do not react with chemical modification on the inner surface of the gene sequencing chip, so that the aim of resetting the chip is fulfilled. According to the sequencing method disclosed by the invention, the chip can be efficiently utilized for repeated sequencing, and the gene sequencing chip does not need to be replaced.

Description

Technical Field

[0001] The invention relates to a sequencing method using a degradable hydrogel, and belongs to the field of gene sequencing. Background Art

[0002] Gene sequencing has played a significant role in the biological field. The basic principle of gene sequencing is the translation process of the gene to be tested. The detection of chemical reaction signals in this process is the core content. The first-generation dideoxy end-termination sequencing method is currently only suitable for small-scale sequencing due to its high cost and low throughput. Second-generation sequencing, namely the common SBS high-throughput sequencing method, has developed to a relatively mature stage. One of the core aspects of second-generation sequencing is to reduce costs while ensuring good sequencing signals. General sequencing is the process of collecting signals within an area of about 1 micron or even smaller. Usually, sequencing requires differential modification of DNA at specific locations; however, existing methods that can achieve differential DNA modification either have too many steps or are too complicated, and it is difficult to guarantee the stability of the final results.

[0003] While reuse is possible and has been demonstrated in the field of gene sequencing, the repetitive processes are often complex, and it is difficult to completely reset and recycle the chips directly on the sequencing machine. Therefore, if differential chips could be quickly modified on a sequencer or similar miniaturized machine, and the chips could be quickly and repeatedly reset after use, not only would the cost of sequencing be significantly reduced, but it would also significantly simplify and reduce the cost of resetting the instrument. Summary of the Invention

[0004] In order to solve the above problems, the inventors of this application have proposed the following solution. Instead of using conventional modified chips that are mainly hydrophilic, hydrophobic or functionalized hydrophobic chips are used. Then, by means of air sealing or oil sealing, RNA or DNA vectors are differentially modified in the micropits of the chip in one step, and a gene chip that can be used for subsequent biochemistry or sequencing is formed. This method can first perform hydrophobic or functionalized hydrophobic modification on the entire surface of a large-size wafer or other material substrate, and then cut and package it into multiple hydrophobic or functionalized hydrophobic chips; or the wafer or other material substrate can be cut first, packaged into multiple chips, and then a single chip is subjected to hydrophobic or functionalized hydrophobic treatment. Then, the RNA or DNA vector reaction solution is divided into micropits for separate reactions using air sealing or oil sealing, thereby forming a differential hydrophobic functionalized gene chip with only RNA / DNA vectors in the micropits. Compared with conventional chips, this type of gene chip has a higher load rate and better consistency, and can effectively improve sequencing performance.

[0005] The present invention aims to provide a novel method for reusing a gene chip and a sequencing chip based on hydrophobic modification, so as to solve the problem in the prior art of difficulty in rapid differential modification and rapid on-machine reuse.

[0006] According to one aspect of the present invention, a novel method for reusing a hydrophobic modified gene chip is provided. To achieve the above-mentioned purpose, the method mainly comprises the following four key steps: (1) performing a comprehensive hydrophobic modification or functional hydrophobic modification treatment on the chip; (2) using a gas seal or oil seal to differentially modify DNA or DNA carriers in situ within the chip micropits. The latter method requires utilizing a specific site on the carrier within the micropit to covalently connect DNA; (3) then using the chip for sequencing or other similar purposes; (4) using a special reagent to completely remove the hydrogel carrier of the chip without damaging the hydrophobic layer modification of the chip; repeating steps 2-4 can achieve the repeated use of the chip.

[0007] According to one aspect of the present invention, a gene sequencing method using a degradable hydrogel is provided, characterized in that the method comprises the following steps:

[0008] (1) adding a carrier polymerization reaction solution to a gene sequencing chip and sealing it in a micro-well of the gene sequencing chip; the inner surface of the gene sequencing chip is pre-hydrophobically chemically modified;

[0009] (2) The carrier to be polymerized in the micropits undergoes a cross-linking reaction to form a three-dimensional network polymer carrier, i.e., a degradable hydrogel;

[0010] (3) washing away the unreacted reaction solution to be polymerized;

[0011] (4) Immobilizing the nucleic acid molecules to be sequenced on a three-dimensional network polymer carrier;

[0012] (5) Sequencing;

[0013] (6) depolymerization, depolymerizing the hydrogel through the degradable sites of the three-dimensional network polymer carrier;

[0014] Repeat steps (1)-(6).

[0015] According to a preferred embodiment, the carrier to be polymerized in step (1) refers to a mixed solution of hydrogel functional monomers before polymerization; the carrier is a hydrogel;

[0016] According to a preferred embodiment, the hydrogel is a functionalized polysaccharide hydrogel, a functionalized polyethylene glycol and its derivative hydrogel, or a functionalized polyacrylamide and its derivative hydrogel; the functionalized polysaccharide hydrogel is selected from at least one of chitosan, hyaluronic acid, trehalose, and cellulose;

[0017] According to a preferred embodiment, a cross-linking reaction occurs in the solution to be polymerized to form the hydrogel, and the cross-linking is achieved by the mutual reaction of active functional groups obtained through functionalization on the prepolymer (PSA, PEG, PAM, etc.); further preferably, the cross-linking reaction type is: amino group with aldehyde group, epoxy group, thiol, succinimide, carboxyl group cross-linking, or azide with alkynyl group or norbornene-containing compound cross-linking, or free radical polymerization cross-linking;

[0018] According to a preferred embodiment, the polymerization initiation conditions of the reaction solution to be polymerized include heating or light irradiation;

[0019] According to a preferred embodiment, the reaction solution to be polymerized contains one or more of the following prepolymers or monomers: polysaccharides and their derivatives, polyethylene glycol and its derivatives, and polyacrylamide and its derivatives;

[0020] According to a preferred embodiment, the prepolymer or monomer includes chitosan, and the preparation of the reaction solution containing the prepolymer or monomer includes: dissolving chitosan in acetic acid solution, stirring and dissolving to obtain a chitosan solution; adding glutaraldehyde solution to the chitosan solution to obtain a reaction solution containing chitosan.

[0021] According to a preferred embodiment, in the carrier reaction solution to be polymerized, at least one component contains a degradable site adapted to acid hydrolysis, alkaline hydrolysis, fluoride decomposition or photolysis.

[0022] According to a preferred embodiment, the reaction solution to be polymerized in step (1) is a hydrogel precursor solution formed by dissolving a multi-arm polyethylene glycol backbone monomer with an active functional group FG1 and a cross-linking agent containing a degradable site in a solvent in a specific ratio; wherein the cross-linking agent has a linking group FG2; and the FG2 can undergo a coupling chemical reaction with FG1;

[0023] According to a preferred embodiment, the specific ratio refers to the mass ratio FG1:FG2 of the linking groups in the monomer and the cross-linking agent being between 10:1 and 1:1;

[0024] According to a preferred embodiment, the multi-arm polyethylene glycol is 2-8-arm polyethylene glycol, preferably, the multi-arm polyethylene glycol is four-arm polyethylene glycol, six-arm polyethylene glycol or eight-arm polyethylene glycol.

[0025] According to a preferred embodiment, the specific ratio refers to the mass ratio of the linking groups in the monomer and the cross-linking agent FG1:FG2 between 9:1-1:1, preferably between 8:1-1.5:1, more preferably between 7:1-1.8:1, and more preferably between 6:1-2:1.

[0026] According to a preferred embodiment, the hydrophobic chemical modification that does not damage the gene sequencing chip means that the hydrophobic chemical modification of the hydrophobic surface is not destroyed when steps (2) to (7) are repeated 1 to 50 times.

[0027] According to a preferred embodiment, the hydrophobic chemical modification that does not destroy the gene sequencing chip means that the loss of the hydrophobic chemical modification is within 10%; preferably within 9%; more preferably within 8%; more preferably within 7%; more preferably within 6%; more preferably within 5%; more preferably within 4%.

[0028] According to a preferred embodiment, the carrier to be polymerized reaction solution comprises the following structure:

[0029]

[0030] Wherein C and D are independently selected from C1-C6 alkyl, aryl, substituted aryl;

[0031] Preferably, the substituted aryl group refers to tolyl, ethylphenyl;

[0032] Preferably, the depolymerization refers to depolymerization by introducing a depolymerization solution, wherein the depolymerization solution is an aqueous solution containing soluble fluoride.

[0033] According to a preferred embodiment, the carrier to be polymerized reaction solution comprises the following structure:

[0034]

[0035] or

[0036]

[0037] Wherein, E and F are independently selected from C1-C3 alkyl groups; or E and F form a five-membered or six-membered cycloalkane.

[0038] wherein R1, R2, R3, and R4 are independently selected from hydrogen, C1-C4 alkyl, and halogenated C1-C4 alkyl;

[0039] Preferably, R1 is the same as R2, and R3 is the same as R4.

[0040] Preferably, the depolymerization and degradation method is washing with a solution having a pH value of less than or equal to 2.

[0041] Preferably, E and F are methyl groups.

[0042] According to a preferred embodiment, the carrier to be polymerized reaction solution comprises the following structure:

[0043] or

[0044] Preferably, the depolymerization and hydrolysis can be achieved by using an alkaline solution;

[0045] Preferably, the alkaline solution is selected from at least one of diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, lithium hydroxide, and potassium carbonate solution, and the concentration is 0.1M-3M;

[0046] Preferably, the concentration of the alkaline solution is 0.2, 0.3, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3; or a range consisting of any two numbers therein, and the concentration is a molar concentration.

[0047] According to a preferred embodiment, the carrier to be polymerized reaction solution comprises the following structure:

[0048]

[0049] Preferably, the depolymerization in step (7) is photodepolymerization, and the photodepolymerization is performed by irradiation with UV light in the wavelength range of 200-400 nm, for example, 365 nm or 254 nm.

[0050] According to one aspect of the present invention, a method for disaggregated gene sequencing is provided, comprising the following steps:

[0051] (1) adding a hydrogel prepolymer solution to a gene sequencing chip and sealing it in a micro-pit of the gene sequencing chip; the inner surface of the gene sequencing chip is pre-hydrophobically chemically modified;

[0052] (2) The hydrogel prepolymer solution in the micropits undergoes a cross-linking reaction to form a three-dimensional network hydrogel carrier;

[0053] (3) Immobilizing the nucleic acid molecules to be sequenced on a three-dimensional network hydrogel carrier;

[0054] (4) Sequencing;

[0055] (5) passing a cleaning solution into the hydrogel to react with the hydrogel to degrade the hydrogel without reacting with the hydrophobic chemical modification;

[0056] Repeat steps (1)-(5).

[0057] According to one aspect of the present invention, the present invention provides a gene sequencing kit, characterized in that it comprises:

[0058] hydrogel monomers,

[0059] hydrogel crosslinker,

[0060] Sequencing reagents,

[0061] Sealing gas or sealing reagent,

[0062] hydrogel cleanser;

[0063] Among them, the hydrogel monomer and the hydrogel cross-linker are used to form a hydrogel in the gene sequencing chip; the sequencing reagent is used for gene sequencing reaction; the sealing gas or sealing liquid is used to seal the mixed reagent formed by the hydrogel monomer and the hydrogel cross-linker in the micro-pits on the inner surface of the gene sequencing chip, thereby reacting to form a hydrogel in the micro-pits; the hydrogel cleaning liquid is used to react with the hydrogel to remove the hydrogel.

[0064] According to one aspect of the present invention, the present invention provides a gene sequencing system, characterized in that sequencing is performed using the aforementioned disaggregated gene sequencing method.

[0065] Among them, the applicant used a reducing agent method to remove the hydrogel in previous inventions, such as CN116555408A. This method only removes a special part of the hydrogel. However, the removal effect is not ideal. Later development data showed that the removal efficiency of reduction and other methods is about 95%. In this case, the quality of repeated sequencing data is difficult to guarantee. In addition, the method disclosed in this patent is to introduce external DNA carriers, such as hydrogel microspheres. This method cannot effectively achieve the repeated use of the chip. The reason is that in the process of introducing external hydrogel microspheres, centrifugation and other methods are often used, which requires the introduction of human operation. Under such circumstances, the reuse of gene sequencing chips cannot be achieved. The chip reuse method provided by the present invention can effectively remove the hydrogel according to the type of hydrogel, thereby completely removing the sequenced DNA in the chip.

[0066] The chip reuse method provided by the present invention not only has a high removal efficiency, for example, 99% or even higher, but is also applicable to a variety of nucleic acid carriers and carrier layers connected to nucleic acid carriers (for example, compatible with different hydrophobic surface chemical modifications). The method provided by the present invention can not only remove the hydrogel but also preserve the chemical modification on the chip surface.

[0067] The beneficial effects of the present invention include:

[0068] The present invention makes full use of the hydrophobic chip and uses oil or gas to independently divide the liquid in the flow channel into micropits, so that it can quickly generate DNA / DNA carrier hydrophobic chips only in the micropits. This process can greatly improve the efficiency of the differential modification process and greatly reduce the cost of modification, while also improving the stability and consistency of the modification.

[0069] Specific cleaning reagents are used. Specific cleaning reagents are selected for specific hydrogels, which are more specific and have a removal efficiency of over 99%. After multiple uses, the chip's functions remain intact.

[0070] Using an in situ hydrogel carrier synthesis method, hydrogels are synthesized directly within the microwells of a sequencing chip. This method allows for complete on-chip modification and reconfiguration, significantly simplifying chip reconfiguration and modification. The method of the present invention eliminates the need for additional coating techniques, effectively reducing the overall chip cost and facilitating ease of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 . Schematic diagram of the sequencing process, where 101 is an empty chip with an unmodified microarray structure; 102 is an empty chip with hydrophobic or functionalized hydrophobic modification; 103 is the sealing process: a method of dividing the liquid into separate micropits, which can be gas or oil, etc.; 104 is the reaction solution of DNA or DNA carriers that are divided into micropits after sealing and can be generated or reacted in the pits; 105 is a hydrophobic chip with DNA only in the micropits and no DNA in other areas.

[0072] Figure 2 .Reaction principle diagram.

[0073] Figure 3 .Mass spectrum of the compound in step 1 of Example 1.

[0074] Figure 4 .Mass spectrum of the compound in step 2 of Example 1.

[0075] Figure 5 .Actual picture of the hydrogel in Example 1.

[0076] Figure 6 .Microscope image of hydrogel in Example 1.

[0077] Figure 7 .Micrograph of the glue degradation effect in Example 1.

[0078] Figure 8 .Mass spectrum of the compound in step (1) of Example 2.

[0079] Figure 9 .Mass spectrum of the compound in step (ii) of Example 2.

[0080] Figure 10 . HPLC chart of step (three) of Example 2.

[0081] Figure 11 .Example 4 step (1) mass spectrum.

[0082] Figure 12.Mass spectrum of Example 4, step (ii).

[0083] Figure 13 .Mass spectrum of step (iii) of Example 4.

[0084] Figure 14 .Example 4 Hydrogel polymerization swelling diagram.

[0085] Figure 15 .Fluorescence microscope photograph of the hydrogel of Example 4.

[0086] Figure 16 .Screenshot of sequencing report.

[0087] Figure 17 . Fluorescence micrograph of the gel degradation effect after 3 repeats.

[0088] Figure 18 .Mass spectrum of step (iv) of Example 5.

[0089] Figure 19 .Actual picture of ammonium carbonate hydrogel.

[0090] Figure 20 .Example 5 Chip overall scanning puzzle.

[0091] Figure 21 .Microscopic photography of glue degradation.

[0092] Figure 22 .NMR image of step 1 of Example 6.

[0093] Figure 23 .Mass spectrum of step (5) of Example 6.

[0094] Figure 24 .Picture of hydrogel in Example 7. DETAILED DESCRIPTION

[0095] To make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments and accompanying drawings. However, those skilled in the art will appreciate that the present invention is not limited to the accompanying drawings and the following embodiments.

[0096] The present invention mainly involves the fluorescence switching sequencing method. However, it can be seen that different sequencing methods have no effect on the reuse of the chip. It can be a common second-generation sequencing method that uses an optical system to detect the signal of the nucleic acid cluster to be tested, or it can be a third or fourth-generation sequencing method that uses an electrical signal system to detect the nucleic acid fragment to be tested, or other similar methods. However, since the detection method of the sequencing signal is not the focus of the present invention, and the chip preparation itself is basically independent of the sequencing detection method, the focus of the present invention is: the preparation of the rapid in situ differential sequencing chip and its reuse method, which is basically unrelated to the choice of sequencing method.

[0097] Therefore, the two most critical parts of the technical solution of the present invention are the preparation of the discriminative DNA modification chip and the resetting of the chip after use.

[0098] The preparation of the in situ differential DNA-modified hydrophobic chip includes the following two steps:

[0099] a) Hydrophobic modification: including hydrophobicization or functionalized hydrophobic modification of microarray chip surface (see Appendix Figure 1 , 101 to 102), the purpose of which is to make the inside of the unmodified chip hydrophobic to ensure that the subsequent use of gas seal or oil seal to prepare for the in situ generation or connection of DNA in the micro-pits. Specifically:

[0100] The microarray chip structure is defined as a chip comprising a microreactor layer, inlets and outlets, and a base layer. The microreactor layer is also referred to as a microreactor chip. The microreactor layer contains a large number of separate microreactor structural units. The inlets and outlets of the chip serve as channels for fluids to enter and exit the chip. The base layer and other layers cooperate to form a reaction space, also known as a reaction chamber or fluid chamber. The reaction chamber is the space connected to the microreactor layer.

[0101] The microarray chip material can be glass, quartz, ceramic, metal, silicon-based, plastic (such as PMMA, PC, etc.); the micro-pit size can be 0.1μm-5μm.

[0102] The hydrophobicization or functional hydrophobicization modification of the inner surface of the microarray chip can be performed by using common trichlorofluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products) through CVD, liquid phase modification, or similar methods.

[0103] Considering the possibility of the chip being on-machine, it is preferred to use liquid phase hydrophobic modification and similar methods to perform hydrophobic and hydrophobic functional modification on the inside of the chip.

[0104] The main process of pure hydrophobic liquid phase modification is as follows: fluorosilane is dissolved in Novec 7200 (or other fluorinated oil) to prepare a hydrophobic reaction solution, which is introduced into the activated cavity. After a certain period of static reaction, it is rinsed with its solvent to remove excess unreacted fluorosilane. After drying, it is post-baked to ensure that all surfaces in the microcavity are evenly treated with fluorinated groups.

[0105] The fluorosilane used can be trichloroperfluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products), with a concentration of 0.01-1 wt%. The reaction time of the hydrophobic reaction solution can be 1-60 minutes, and the post-baking temperature can be 60-200°C for 1-300 minutes. Considering the convenience of on-machine modification, fluorochemical reagents that can quickly achieve surface hydrophobic silanization are preferred.

[0106] In order to achieve functionalized hydrophobic modification, silanes with different active functional groups can be directly mixed into the reaction reagents of the above fluorosilanes, such as common aminosilane coupling agents, silane coupling agents with carboxyl groups, silane coupling agents with mercapto groups, or silane coupling agents with olefin or alkyne groups at the end. The steps are the same as above and will not be repeated here.

[0107] b) Preparation of carrier hydrogels, etc.: On the hydrophobicized or functionalized hydrophobic modified chip, DNA is directly connected in situ only in the micro-pits or a support layer capable of subsequent DNA connection is connected in situ only in the micro-pits or a support layer is planted (see attached). Figure 1 (e.g., steps 102 to 105 in the above process). The support layer is preferably a hydrogel carrier. Embodiments of the present invention provide a method for achieving differential modification, including:

[0108] Solution: Hydrophobic chip + DNA carrier generation solution in micro-pits

[0109] Based on a modified hydrophobic chip (PF-chip for short), air or oil is used to divide the reaction solution to be polymerized, which can subsequently connect with DNA, into individual micropits (referred to as the seal process) for separate reactions. High-molecular-weight DNA carriers that can subsequently connect with DNA are directly generated in the micropits. The DNA with reaction sites on the carrier layer is then reacted to form a hydrophobic chip with DNA carriers only in the micropits.

[0110] The selection of DNA carriers takes into account their use in biological scenarios, and hydrogel polymer materials are preferred.

[0111] The main skeleton of the hydrogel can be a functionalized polysaccharide system (which can be referred to as PSA glue, such as chitosan, hyaluronic acid, trehalose, cellulose, etc.), a functionalized polyethylene glycol and its derivative glue (which can be referred to as PEG glue, which can be double-arm or multi-arm polyethylene glycol) system, or a functionalized polyacrylamide and its derivative glue (which can be referred to as PAM glue), etc., but the polymer skeleton is not limited to the above systems.

[0112] The cross-linking of hydrogels can be achieved through the mutual reaction of active functional groups on the functionalized prepolymers (PSA, PEG, PAM, etc.). For example, common reaction types are: amino groups reacting with aldehyde groups, epoxides, thiols, succinimide, and carboxyl groups for cross-linking; it can also be the reaction of azide with alkynyl groups or norbornene-containing compounds for cross-linking; it can also be other reaction systems such as free radical polymerization.

[0113] The method of separating the liquid into the micro-pits (seal) can be gas or oil.

[0114] The gas can be nitrogen, air, argon, etc.; the oil can be mineral oil, fluorocarbon oil, or other common non-polar oils, etc.

[0115] The triggering conditions for initiating polymerization are determined by the polymerization reaction system, and polymerization can be initiated by heating, light, etc.

[0116] The key step to achieve another key purpose of the present invention is to reset the chip after use to return it to its initial coating; and remove the state of the carrier such as hydrogel (see attached Figure 1-10 5 to 102). Specifically, a reagent that is specialized for removing specific hydrogels is used to reset the hydrophobic chip with discriminative DNA modification after use, so that the chip can return to its hydrophobic state for reuse.

[0117] Gene sequencing chips (also known as microarray chips) have a high-density array and surface microreactor structure at the micron scale. In order to adapt to different functions or usage scenarios, high molecular polymers are usually immobilized on the surface of the chip for coupling and loading biological molecules. Hydrogels are often used as carriers for biochemical reactions and are used to modify microarray chips due to their high water content, good biocompatibility (low surface protein and cell adhesion), variety of types and good adjustability. Traditional hydrogel materials usually meet the stability and functionality requirements of NGS processes such as DNA immobilization and amplification, but often only provide disposable chips. In order to improve the reusability and reuse times of the chip and reduce sequencing costs, it is necessary to develop hydrogel materials that can be controlled to degrade and can be completely removed so that the chip can be reset repeatedly.

[0118] Conventional hydrogel materials often have some shortcomings in achieving the goal of reconfigurable NGS chips, including:

[0119] 1) Structural issues: The hydrogel structure does not have a specific degradation control site design, so it is difficult to control the degradation rate, and it is impossible to provide a mild and controllable degradation mechanism and conditions that does not damage the chip; 2) Biocompatibility issues: Hydrogel materials are not suitable for biological reaction systems such as enzymes, DNA, and bases. In particular, degradation products may have adverse effects on biological reaction systems. In NGS sequencing applications, this effect may cause the sequencing signal to be unrecognizable or trigger negative biochemical reactions. 3) Degradation products are difficult to remove: Some hydrogels are difficult to completely degrade, resulting in the accumulation of residual fragments in the chip, which may affect the reuse of subsequent sequencing chips. 4) Stability and durability issues: Hydrogels may lose stability under specific environmental conditions, limiting long-term application and storage. 5) High preparation cost: The preparation process of some degradable hydrogels is complex and costly, which limits large-scale application. 6) Limited scope of application: The degradation mechanism of common medical degradable hydrogels often meets the purpose of sustained release in the body, but cannot meet the needs of in vitro fields such as those requiring rapid and controllable release.

[0120] To address these issues, the present invention has developed a novel hydrogel through molecular structure design and screening. The crosslinker of the hydrogel is designed with degradable functional groups. At the same time, the hydrogel monomer and crosslinker can quickly polymerize into a gel within the chip, providing carrier support performance for biomolecule immobilization and other biochemical reactions. The hydrogel material provided by this aspect can use inexpensive and easy-to-use degradation reagents to achieve controllable, rapid and complete degradation of the hydrogel, thereby being used for surface modification and re-clearance of materials such as NGS chips, enabling chip reuse.

[0121] Choice of carrier

[0122] The present invention relates to carriers within the microwells of a sequencing chip. These carriers are used to connect nucleic acid fragments to be sequenced. Carriers used in conventional sequencing can be microspheres, microparticles, or form a covering on the bottom or sidewalls of the microwells. Typically, these carriers are hydrogel carriers. Using different carriers requires different preliminary steps, which can impact sequencing. For example, when using hydrogel microspheres as carriers, centrifugation or other methods are required to load the hydrogel microspheres into the microwells of a gene sequencing chip. This method generally makes it difficult to load all microwells with microspheres, and some microwells may contain two microspheres. Areas without microspheres, or areas with multiple microspheres, will result in erroneous or invalid data in subsequent sequencing. The present invention uses an in situ synthesis method to prepare hydrogel carriers within the microwells. This method is uncommon. As far as is known in the sequencing field, this approach has not yet been used to synthesize hydrogels in situ within a chip for sequencing. Common methods for synthesizing hydrogels within sequencing chips involve externally synthesizing the hydrogel into microspheres or similar shapes; upon use, the microspheres are then loaded into the chip. There is a clear difference between these two approaches.

[0123] Sequencing methods

[0124] This invention primarily involves the fluorescence switching sequencing method. However, it can be seen that different sequencing methods have no effect on the reuse of the chip. The sequencing method is not the focus of this invention. The focus of this invention is the reuse of the sequencing chip, which is basically unrelated to the choice of sequencing method.

[0125] The sequencing methods involved in the embodiments of the present invention belong to the category of second-generation sequencing. Sequencing-by-synthesis, a common method within second-generation sequencing technology, is a widely used sequencing method. Typical examples include the Illumina sequencing method and the BGI sequencing method. Although each method has certain differences, they are all within the scope of the present invention. In the present invention, a hydrophobic modification method is used to create a hydrophobic chemical environment on the inner surface of the chip. Preferably, the hydrophobic modification is performed to create a hydrophobic environment covering 100% of the micro-pitted substrate area on the inner surface of the chip. This figure is adjustable, for example, to 50%, 60%, 70%, 80%, 90%, or 100%. Any two of these figures can also form a range, such as 50%-100%. This invention does not discuss intentionally reducing the effective modified area of the hydrophobic environment for other reasons. In the present invention, no special methods are used to maintain the hydrophobic modification ratio on the inner surface of the chip. It is conceivable that under unexpected conditions, the hydrophobic ratio may not reach 100%, and the hydrophobic reagent may not modify the entire inner surface, without affecting the function of other components. At the same time, because the carrier was synthesized using a sealed method, if the hydrophobic modification is not complete, the modified hydrogel will not be differentiated and is not within the scope of protection of this solution. The micro-pit area is the functional area of hydrophobic modification. If other areas are not properly hydrophobicized, it will not affect the overall results. It should be noted that in this invention, a full hydrophobic modification scheme is used. There is no intentional reduction in the modification ratio.

[0126] A hydrophobic chemical environment can provide the surface conditions required for sealing. Using the seal method, the in-situ formation of DNA carriers in micropits is an important method. Many of the applicant's previously disclosed patents describe the use of hydrophobic environments and oil seals in micropits for gene sequencing. However, in the present invention, the formation of DNA carriers also uses similar but essentially different means. It should be stated that this method has not appeared in the art, and the applicant believes that the method for forming the DNA carrier is very important for the reuse of the entire chip. For example, in patent CN 116333848 A, a complex photolithography process is used to prepare a gene sequencing chip that can be repeatedly sequenced, but this method has many impacts on the subsequent processing flow. When forming the DNA carrier for the second time, part of the process requires manual participation. In this case, it is not advisable in actual technology. The method described in the present invention can reuse the chip through the action of fluid, only in the gene sequencer.

[0127] Hydrophobic inner surface of the chip

[0128] The present invention describes hydrophobic modification of the inner surface. There are many common methods for hydrophobic modification. Generally, silane, fluorosilane, or other fluorinated compounds can be used for modification. It should be noted that in previous patents, the applicant has described a differentiated modification method. For example, after cleaning the chip surface, a hydrophobic compound is formed on the outer surface of the microwells using methods such as embossing, while the inner surface is hydrophilic using methods such as CVD. This inner surface can be used to attach specific groups, such as hydrogel microspheres. The present invention uses a single-step process to hydrophobically modify the inner surface of the entire chip. Reactants such as hydrogel prepolymers are enclosed in the microwells, and the reaction produces irregularly shaped hydrogel carriers. This method eliminates direct, pre-determined covalent bonds between the hydrogel and the interior of the microwells, but the hydrogel or other carrier remains firmly in the microwells and does not fall off during sequencing. The in-situ synthesis of hydrogels and other carriers using oil sealing, as employed in the present invention, has many advantages: it eliminates the need for additional modification of the substrate with compounds that covalently attach to the hydrogel.

[0129] Chip reset

[0130] In previous inventions of the applicant, such as CN116555408A, a reducing agent method was used to remove the hydrogel. This method only removed a specific part of the hydrogel. However, the removal effect was not good. Later developments showed that the removal efficiency of methods such as reduction was about 95%. In this case, the data quality of repeated sequencing is difficult to guarantee. The chip reuse method provided by the present invention not only has a relatively high removal efficiency, such as 99% or even higher, but is also applicable to a variety of hydrophobic surface chemical modifications. Although the present invention uses a similar method, in one aspect, the removal efficiency is higher, as high as 99% or more, and the hydrophobic modification of the chip is not damaged. The present invention provides a method for reusing chips on a machine, which does not require the chip to be disassembled from the chip, that is, it can be used multiple times, reducing the probability of contamination and reducing the process of manual participation.

[0131] Reuse of chips

[0132] In sequencing, the cost of the chip accounts for nearly half of the total cost. The reuse of chips is an important direction for the sequencing industry. The present invention provides a method for reusing sequencing chips. More importantly, the entire process is carried out in a sequencer. Only the liquid needs to be replaced to re-sequencing. Moreover, the steps are simple. For example, patent CN116333848 A uses a complex photolithography process. Other known sequencing methods have not yet provided a complete solution for chip reuse. The synthesis scheme of carriers such as hydrogels on the chip provided by the present invention is more suitable for chip reuse.

[0133] Chip hydrophobic modification, hydrophobic modification (PF-chip): includes hydrophobicization or functionalized hydrophobic modification of the microarray chip surface. Its purpose is to ensure that the subsequent use of gas or oil seals to prepare for in situ generation or ligation of DNA in the micro-pits. Specifically:

[0134] The hydrophobic modification is as follows: using a fluoroether reagent containing 0.1% wt of perfluoropolyether (HFPO) siloxane in the chip, reacting at room temperature for 10 minutes, then rinsing the chip with a fluoroether solvent and drying the chip, and aging at 85 degrees Celsius for 10 minutes to obtain a hydrophobic chip with fluorinated modification on the chip surface, which is subsequently labeled as a PF chip.

[0135] The hydrophobic modification of the chip can also be prepared using methods such as CVD, which provides conditions for hydrophobic micro-pit sealing. Optionally, the inner surface of the chip with micro-pits can be hydrophobically modified before chip preparation. For example, patent AU2016356395B2 also discloses that the chip needs to be hydrophobic modified. For example, patent CN207552330U discloses a hydrophobic sequencing system.

[0136] A gene sequencing method comprises the following steps:

[0137] (1) adding a carrier polymerization reaction solution to a gene sequencing chip and sealing it in a micro-well of a fluid chamber of the gene sequencing chip; the inner surface of the gene sequencing chip is pre-hydrophobically chemically modified;

[0138] (2) The carrier to be polymerized in the micropits undergoes a cross-linking reaction to form a three-dimensional network polymer carrier;

[0139] (3) washing away the unreacted reaction solution to be polymerized;

[0140] (4) Immobilizing the nucleic acid molecules to be sequenced on a three-dimensional network polymer carrier;

[0141] (5) Sequencing;

[0142] (6) depolymerization, depolymerizing the hydrogel through the degradable sites of the three-dimensional network polymer carrier;

[0143] Repeat steps (1)-(6).

[0144] According to a preferred embodiment, in the carrier reaction solution to be polymerized, at least one component contains a degradable site adapted to acid hydrolysis, alkaline hydrolysis, fluoride decomposition or photolysis.

[0145] According to a preferred embodiment, the carrier precursor solution refers to a mixed solution of hydrogel functional monomers before polymerization; the carrier is a hydrogel;

[0146] According to a preferred embodiment, the hydrogel is a functionalized polysaccharide hydrogel, a functionalized polyethylene glycol and its derivative hydrogel, or a functionalized polyacrylamide and its derivative hydrogel; the functionalized polysaccharide hydrogel is selected from at least one of chitosan, hyaluronic acid, trehalose, and cellulose;

[0147] Preferably, a cross-linking reaction occurs in the solution to be polymerized to form the hydrogel, and the cross-linking is achieved by the mutual reaction of active functional groups obtained through functionalization on the prepolymer (PSA, PEG, PAM, etc.); further preferably, the cross-linking reaction type is: amino group and aldehyde group, epoxy group, thiol, succinimide, carboxyl group reaction cross-linking, or azide and alkynyl group or norbornene group compound reaction cross-linking, or free radical polymerization cross-linking;

[0148] Preferably, the polymerization initiation conditions of the reaction solution to be polymerized include heating or light irradiation;

[0149] Preferably, the reaction solution to be polymerized contains one or more of the following prepolymers or monomers: polysaccharides and their derivatives, polyethylene glycol and its derivatives, and polyacrylamide and its derivatives;

[0150] Preferably, the prepolymer or monomer includes chitosan, and preparing the reaction solution containing the prepolymer or monomer includes: dissolving chitosan in acetic acid solution, stirring and dissolving to obtain a chitosan solution; adding glutaraldehyde solution to the chitosan solution to obtain a reaction solution containing chitosan.

[0151] According to a preferred embodiment, the reaction solution to be polymerized in step (1) is a hydrogel precursor solution formed by dissolving a multi-arm polyethylene glycol backbone monomer with an active functional group FG1 and a cross-linking agent containing a degradable site in a solvent in a specific ratio; wherein the cross-linking agent has a linking group FG2; and the FG2 can undergo a coupling chemical reaction with FG1;

[0152] The specific ratio refers to the mass ratio of the linking groups in the monomer and the cross-linking agent FG1:FG2 being between 10:1 and 1:1;

[0153] According to a preferred embodiment, the multi-arm polyethylene glycol is 2-8-arm polyethylene glycol, preferably, the multi-arm polyethylene glycol is four-arm polyethylene glycol, six-arm polyethylene glycol or eight-arm polyethylene glycol.

[0154] According to a preferred embodiment, the specific ratio refers to the mass ratio FG1:FG2 of the linking groups in the monomer and the cross-linker is between 9:1-1:1, preferably between 8:1-1.5:1, more preferably between 7:1-1.8:1, and more preferably between 6:1-2:1.

[0155] According to a preferred embodiment, the hydrophobic chemical modification that does not damage the gene sequencing chip means that the hydrophobic chemical modification of the hydrophobic surface is not destroyed when steps (2) to (7) are repeated 1 to 50 times.

[0156] According to a preferred embodiment, the hydrophobic chemical modification that does not destroy the gene sequencing chip means that the loss of the hydrophobic chemical modification is within 10%; preferably within 9%; more preferably within 8%; more preferably within 7%; more preferably within 6%; more preferably within 5%; more preferably within 4%.

[0157] According to a preferred embodiment, the hydrogel comprises the following structure:

[0158]

[0159] wherein C and D are independently selected from C1-C6 alkyl, aryl, and substituted aryl.

[0160] According to a preferred embodiment, the substituted aryl group refers to tolyl and ethylphenyl.

[0161] The depolymerization refers to introducing a depolymerization solution for depolymerization, wherein the depolymerization solution is an aqueous solution containing soluble fluoride.

[0162] According to a preferred embodiment, the hydrogel comprises the following structure:

[0163]

[0164] or

[0165]

[0166] Wherein, E and F are independently selected from C1-C3 alkyl groups; or E and F form a five-membered or six-membered cycloalkane.

[0167] Wherein, R1, R2, R3, and R4 are independently selected from hydrogen, C1-C4 alkyl, and halogenated C1-C4 alkyl. The C1-C4 refers to an alkyl group containing 1 to 4 carbon atoms.

[0168] According to a preferred embodiment, R1 is the same as R2, and R3 is the same as R4.

[0169] According to a preferred embodiment, the method for degrading the hydrogel is to wash it with a solution having a pH value less than or equal to 2.

[0170] According to a preferred embodiment, E and F are methyl groups.

[0171] According to a preferred embodiment, the hydrogel comprises the following structure:

[0172] or

[0173] According to a preferred embodiment, the hydrogel contains ammonium carbonate or carbonate structural groups. This can be hydrolyzed using an alkaline solution. The alkaline solution is selected from at least one of diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, lithium hydroxide, and potassium carbonate solutions, with a concentration of 0.1M to 3M. Preferably, the concentration is 0.2, 0.3, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, or a range consisting of any two of these numbers. The concentration is molar.

[0174] According to a preferred embodiment, the hydrogel comprises the following structure:

[0175]

[0176] Preferably, the depolymerization in step (8) is photodepolymerization, and the photodepolymerization is performed by irradiation with UV light in the wavelength range of 200-400 nm, for example, 365 nm or 254 nm.

[0177] Cross-linking agents are one of the key components in the preparation of hydrogels. Cross-linking agents react with hydrogel monomers with active groups to form three-dimensional hydrogels.

[0178] The hydrophobic chemical modification in step (2) includes: hydrophobic modification or functional hydrophobic modification of the microarray chip surface (see attached Figure 1 , 101 to 102), the purpose of which is to make the inside of the unmodified chip hydrophobic to ensure that the subsequent use of gas seal or oil seal to prepare for the in situ generation or connection of DNA in the micro-pits. Specifically:

[0179] The microarray chip structure is defined as a chip comprising a microreactor layer, inlets and outlets, and a base layer. The microreactor layer is also referred to as a microreactor chip. The microreactor layer contains a large number of separate microreactor structural units. The inlets and outlets of the chip serve as channels for fluids to enter and exit the chip. The base layer and other layers cooperate to form a reaction space, also known as a reaction chamber or fluid chamber. The reaction chamber is the space connected to the microreactor layer.

[0180] The microarray chip material can be glass, quartz, ceramic, metal, silicon-based, plastic (such as PMMA, PC, etc.); the micro-pit size can be 0.1μm-5μm.

[0181] The hydrophobicization or functional hydrophobicization modification of the inner surface of the microarray chip can be performed by using common trichlorofluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products) through CVD, liquid phase modification, or similar methods.

[0182] Considering the possibility of the chip being on-machine as a whole, it is preferred to use liquid phase hydrophobic modification and similar methods to perform hydrophobic and hydrophobic functional modification on the inside of the chip.

[0183] The main process of pure hydrophobic liquid phase modification is as follows: fluorosilane is dissolved in Novec 7200 (or other fluorinated oil) to prepare a hydrophobic reaction solution, which is introduced into the activated cavity. After a certain period of static reaction, it is rinsed with its solvent to remove excess unreacted fluorosilane. After drying, it is post-baked to ensure that all surfaces in the microcavity are evenly treated with fluorinated groups.

[0184] The fluorosilane used can be trichloroperfluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products), with a concentration of 0.01-1 wt%. The reaction time of the hydrophobic reaction solution can be 1-60 minutes, and the post-baking temperature can be 60-200°C for 1-300 minutes. Considering the convenience of on-machine modification, fluorochemical reagents that can quickly achieve surface hydrophobic silanization are preferred.

[0185] In order to achieve functionalized hydrophobic modification, silanes with different active functional groups can be directly mixed into the reaction reagents of the above fluorosilanes, such as common aminosilane coupling agents, silane coupling agents with carboxyl groups, silane coupling agents with mercapto groups, or silane coupling agents with olefin or alkyne groups at the end. The steps are the same as above and will not be repeated here.

[0186] The gene chip reuse method provided by the present invention is based on a degradable hydrogel, the starting material of which can quickly gel under specific conditions, and the functional groups it carries after gelling can continue to meet the performance requirements of the NGS sequencing process for RNA / DNA immobilization and amplification. At the same time, the hydrogel can be easily degraded, and its degraded components can be dissolved in a buffer solution, so that they can be completely removed from the NGS chip. Compared with traditional hydrogels, the hydrogel provided by the present invention has a specific degradable chemical structure and high biocompatibility. Its unique degradation characteristics make it an ideal material choice that can meet the needs of various application scenarios, including but not limited to gene sequencing chips, biomedical materials and other fields.

[0187] The degradable hydrogel of the present invention is mainly produced by the reaction of monomers and cross-linking agents.

[0188] Monomer: Multi-arm polyethylene glycol (Multi-Arm PEG) is used as the monomer skeleton to fully utilize the good biocompatibility of polyethylene glycol materials. Each polyethylene glycol branch side arm in the monomer has the same linking functional group (abbreviated as FG1). FG1 can be selected from carboxylic acid groups. Amine (-NH2), azide (-N3), terminal alkyne Activated alkynyl Acrylamide Non-polyethylene glycol monomers can also be selected, for example, one or more of linear polyacrylamide, polyacrylate, polyvinyl alcohol, polypyrrolidone, and linear polysaccharides.

[0189] Crosslinker: The crosslinker provided by the present invention has a degradable molecular structure, and the degradation conditions include acid degradation, alkali degradation, oxidation degradation, reduction degradation, and light degradation. In addition to the degradable site, the crosslinker structure also contains at least two functional groups (abbreviated as FG2) that can undergo chemical coupling reactions with the functional groups on the monomer side arms. FG2 is selected from carboxylic acid groups. Amine (-NH2), azide (-N3), terminal alkyne Activated alkynyl Acrylamide At least one of halogen (Cl, Br, I), sulfonic acid derivatives (such as Ts, Ms, Ns), carboxylic acid derivatives (such as Tf, C 1-6 acyl), and thiol derivatives.

[0190] In the present invention, the monomers and cross-linking agents may be polymerized into a gel on the chip surface, and the steps may include:

[0191] 1. A multi-arm polyethylene glycol backbone monomer and a cross-linker containing degradable sites are dissolved in a suitable solvent at a specific ratio to form a hydrogel precursor solution. The suitable solvent can be water or a mixture of water and an organic solvent such as DMF, DMSO, or acetonitrile. The specific ratio refers to a molar ratio of the linker groups in the monomer and cross-linker, FG1:FG2, between 10:1 and 1:1, with FG1 always in excess. 2. The hydrogel precursor solution is distributed within the microwells of the chip. Under specific reaction conditions (such as heating), the monomer and cross-linker undergo coupling polymerization to form a three-dimensional network hydrogel structure. 3. After polymerization is complete, the aqueous solution is replaced to allow the hydrogel to fully swell, and unreacted monomer and cross-linker are washed away. 4. The hydrogel structure contains excess FG1 functional groups that have not participated in cross-linking. These functional groups can undergo further coupling reactions with biomolecules bearing FG2 functional groups (such as DNA or protein), thereby achieving biomolecule immobilization on the chip.

[0192] Similarly, other types of hydrogels can also undergo cross-linking reactions if added in a manner similar to polyethylene glycol.

[0193] Gene sequencing chips

[0194] The gene sequencing chip described in the present invention refers to a gene sequencing chip with a micro-pit structure on the inner surface. In general gene sequencing chips, especially the common structure of second-generation gene sequencing chips, the inner surface contains a large number of micro-pits. The nucleic acid sequence fragments to be tested are fixed in the micro-pits, and the information of the nucleic acid sequence to be tested can be obtained by sequencing. The micro-pits of the gene sequencing chip can be cylindrical micro-pits, or quasi-cylindrical micro-pits, or truncated cone micro-pits, or micro-pits of irregular shapes. The size of the micro-pits can be characterized by the diameter of the micro-pit opening or a parameter similar to the diameter. Generally, the micro-pits can be 0.2-3 microns in size. The depth of the micro-pits can be 0.5-5 times the diameter of the micro-pit or a similar diameter. In the present invention, the micro-pits are not original structures or knowledge.

[0195] In summary, the characteristics of the on-chip degradable hydrogel of the present invention include:

[0196] Good rapid degradability: The use of degradable cross-linkers makes the hydrogel have excellent degradability. After degradation, the hydrogel decomposes into small molecular fragments at the monomer level, which can avoid problems such as large fragment residues caused by irregular depolymerization that may lead to adverse effects on subsequent biochemical reactions on the chip.

[0197] Good biocompatibility: The use of a polyethylene glycol monomer skeleton with high biocompatibility makes the hydrogel have good biocompatibility in the biochemical reaction system, avoiding problems such as nonspecific adsorption during the biochemical reaction process.

[0198] High hydrophilicity and porosity: The hydrogel of the present invention has excellent hydrophilicity, and the cross-linking degree and porosity can be conveniently controlled by adjusting the length of the hydrogel monomer polyethylene glycol backbone chain and the ratio of the cross-linker, thereby providing favorable support for the immobilization and diffusion of biomacromolecules on the chip surface.

[0199] In summary, the present invention provides an on-chip degradable hydrogel and a preparation method thereof. The invention can be applied to fields such as NGS and POCT and has broad application prospects.

[0200] The principle of hydrogel reaction is as follows Figure 2 As shown, polyethylene glycol monomers or other monomers react with cross-linking agents to form a three-dimensional hydrogel.

[0201] The multi-arm polyethylene glycol backbone monomer can be selected from Table 1.

[0202] Table 1 Multi-arm polyethylene glycol backbone monomers

[0203]

[0204]

[0205] exist Figure 2 The polymerization solution used in the reaction may also contain the following monomers:

[0206]

[0207] The functional unit can be selected from one or more of linear polyacrylamide, polyacrylate, polyvinyl alcohol, polypyrrolidone, and linear polysaccharide. The functional unit exists in the form of monomers in the reaction solution to be polymerized and reacts with a crosslinking agent to form a hydrogel.

[0208] exist Figure 2 In the example, the FG1 substituents and the corresponding FG2 substituents involved can be selected as shown in Table 2.

[0209] Table 2 Selection of substituents

[0210]

[0211] The following describes structures that are degradable under neutral conditions:

[0212] A hydrogel crosslinker containing a silyl ether structure, comprising a silyl ether structure. The silyl ether structure is sensitive to fluoride ions and degrades within a range of 1-10 (1-100) in terms of the molar ratio of the silyl ether structure to the fluoride ion, thereby causing hydrogel degradation.

[0213] A method for synthesizing a degradable hydrogel, characterized by comprising the following steps:

[0214] p-Toluenesulfonyl chloride reacts with polyethylene glycol in an alkaline dichloromethane solution to obtain a polyethylene glycol structure with a p-toluenesulfonyl group, i.e., compound A;

[0215] Compound A reacts with dimethylchlorosilane in an alkaline environment to obtain a crosslinker B;

[0216] The crosslinking agent B reacts with amino polyethylene glycol to remove the p-toluenesulfonic acid group, thereby obtaining a degradable hydrogel product with a silyl ether structure.

[0217] During the synthesis of the cross-linker, the p-toluenesulfonyl group, as a protecting group, can be removed and replaced under mild conditions. For example, sodium iodide can be reacted with compound A to obtain a cross-linker with an iodinated polyethylene glycol structure.

[0218] According to a preferred embodiment, the amino polyethylene glycol refers to one of four-arm amino PEG, six-arm amino PEG, and eight-arm amino PEG.

[0219] Wherein, the degradable hydrogel carrier under neutral conditions comprises the following structure:

[0220]

[0221] C and D are independently selected from C1-C6 alkyl, aryl, and substituted aryl.

[0222] According to a preferred embodiment, the substituted aryl group refers to tolyl, ethylphenyl

[0223] According to a preferred embodiment, the degradation liquid is an aqueous solution containing soluble fluoride.

[0224] According to a preferred embodiment, the concentration of fluoride in the degradation solution is adjusted according to different adaptation environments. When in a sealed environment, the amount of fluoride ions is 0.5-10 times the amount of silane ether in the hydrogel.

[0225] According to a preferred embodiment, the substituents C and D may also be present in the form of a five-membered ring or a six-membered ring.

[0226] The synthesis route of degradable hydrogels under neutral conditions is as follows:

[0227]

[0228] The hydrogel contains silyl ether structures, which are sensitive to fluoride ions, such as sodium fluoride, potassium fluoride, and ammonium fluoride. When the molar ratio of fluoride ions to silyl ether structures is in the range of 0.5-10, the hydrogel can be degraded. Preferably, F:silyl ether structures = 0.7-10, and more preferably, F:silyl ether structures = 1-5.

[0229] The following describes a hydrogel that can be degraded under acidic conditions for gene sequencing.

[0230] The structure that can be degraded under acidic conditions is preferably selected from the following structures:

[0231]

[0232] Or the following structure:

[0233]

[0234] A degradable hydrogel for gene sequencing, characterized by comprising the following structure:

[0235] or

[0236] Wherein, E and F are independently selected from C1-C3 alkyl groups; or E and F form a five-membered or six-membered cycloalkane.

[0237] Wherein, R1, R2, R3, and R4 are independently selected from hydrogen, C1-C4 alkyl, and halogenated C1-C4 alkyl. The C1-C4 refers to an alkyl group containing 1 to 4 carbon atoms.

[0238] According to a preferred embodiment, the method for degrading the hydrogel is to wash it with a solution having a pH value less than or equal to 2.

[0239] According to a preferred embodiment, E and F are methyl groups.

[0240] The acid for removing the hydrogel is selected from one or more of HCl, HBr, HI, CF3COOH, CF3SO3H, and H2SO4.

[0241] The concentration of the acid is 0.1M-10M, preferably 0.5M-5M; more preferably 1M-4M.

[0242] Synthesis method of hydrogel:

[0243] 1,4-cyclohexanedione reacts with glycerol in an alkaline environment and removes water molecules to form a cyclohexane derivative with a double five-membered ring structure, which is recorded as intermediate 1;

[0244] Intermediate 1 reacts with p-toluenesulfonyl chloride to produce intermediate 2 with a leaving group;

[0245] Intermediate 2 reacts with amino-modified hydrogel monomers to form hydrogel polymers.

[0246] Hydrogel synthesis method 2: 1,4-cyclohexanedione and glycerol are reacted in an alkaline environment to remove water molecules to form a cyclohexane derivative with a double five-membered ring structure, which is recorded as intermediate 1; intermediate 1 undergoes esterification reaction with TS-PEG-COOH to obtain intermediate 2;

[0247] Intermediate 2 reacts with amino-modified hydrogel monomers to form hydrogel polymers.

[0248] The synthesis route of degradable hydrogels under acidic conditions can be as follows:

[0249]

[0250] or

[0251]

[0252] The following describes the hydrogel structure that can be degraded under alkaline conditions.

[0253] The structure that can be degraded under alkaline conditions can be shown as follows:

[0254]

[0255] or as follows

[0256]

[0257] A degradable hydrogel for gene sequencing, characterized by comprising the following structure:

[0258] Either or both.

[0259] Example synthesis process of hydrogels that can be degraded under alkaline conditions:

[0260]

[0261] Hydrogels that degrade under alkaline conditions can be referred to as ammonium carbonate or carbonate hydrogels. These hydrogels contain ammonium carbonate or carbonate structural groups. They can be hydrolyzed using alkaline solutions. Alkaline solutions include 0.1M-3M solutions of diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, lithium hydroxide, potassium carbonate, and other alkaline solutions. Alternative concentrations include 0.2, 0.3, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, or any range consisting of any two of these numbers. The concentrations are molar.

[0262] The following describes a photodegradable hydrogel structure:

[0263] The photodegradable structure can be as follows:

[0264]

[0265] A photodegradable hydrogel comprising the following structure:

[0266]

[0267] Photodegradable hydrogels can be degraded by exposure to UV light in the 200-400nm wavelength range, for example, 365nm or 254nm. The decomposition time varies depending on the specific power.

[0268] In summary, the present invention describes hydrogels for gene sequencing that degrade under neutral conditions, acidic conditions, alkaline conditions, and light conditions. Further exemplary structures of the crosslinker portion of the hydrogel are shown in Table 3.

[0269] Table 3 Examples of hydrogel crosslinker structures and corresponding degradation modes

[0270]

[0271]

[0272] The present invention further relates to a method for degrading hydrogels to restore and reset a chip for reuse. Key steps include: adding a specific depolymerization agent (such as an acid, base, oxidant, or reducing agent) to a chip coated with the aforementioned degradable hydrogel, and applying external conditions such as light and heat to rapidly shear specific degradation sites on the hydrogel. After degradation is complete, a buffered cleaning solution is added to the chip to remove small molecular fragments formed by degradation, thereby resetting the chip.

[0273] A specific example of the hydrogel synthesis method is given below:

[0274] Embodiment 1:

[0275] Examples of degradable hydrogels under neutral conditions:

[0276]

[0277] The synthetic route of di-p-toluenesulfonic acid PEG silyl ether crosslinker is as follows:

[0278]

[0279] 1) Step 1: Synthesis of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0280] Compound sodium hydroxide (4.5 g, 116 mmol) and M2 (30 g, 154.5 mmol) were dissolved in 500 ml of dichloromethane solution. p-Toluenesulfonyl chloride (14.7 g, 77 mmol) was added under ice bath and reacted at room temperature for 6 hours. 200 ml of water was added for washing, the liquids were separated, dried, and the solvent was removed by spin drying. The oily compound 17 g (yield 42%) was separated and purified by DCM / MeOH (10:1-3:1) column chromatography.

[0281] LCMS: C15H24O7S, (M+H), 349.07. See the mass spectrum of step 1 compound for details. Figure 3 .

[0282] 2) Step 2: Synthesis of Compound 13,13-dimethyl-3,6,9,12,14,17,20,23-octaoxa-13-silapentacosane-1,25-diyl bis(4-methylbenzenesulfonate)

[0283] Compound M2 (7 g, 20.1 mmol) was dissolved in dry dichloromethane, pyridine (6.34 g, 81.4 mmol) was added to the reaction solution, and dimethylsilyl chloride (0.95 g, 10.05 mmol) was slowly added dropwise. The reaction was carried out at room temperature for 10 hours. The product was separated by silica gel (TEA-treated) column chromatography using DCM / MeOH (20:1-10:1) to obtain 1.1 g (yield 7.2%) of the oily compound.

[0284] LCMS: C 32 H 52 S2O 14 Si(M+H2O),770.37. The mass spectrum of step 2 is shown in Figure 4 .

[0285] Synthesis of di-p-toluenesulfonic acid PEG silyl ether hydrogel:

[0286] (1) In-bottle polymerization: 4Arm-PEG2K-NH2 15 mg, di-p-toluenesulfonic acid PEG silyl ether crosslinker 25 mg, DMF 60 μl, 100 ° C for 1 h (as Figure 5 Left), swelled after soaking in water (such as Figure 5 right).

[0287] Depend on Figure 5 It can be seen that after polymerization, a light yellow solid gel is formed, which swells after adding water and its volume increases several times, indicating that the gel has good water-swelling properties.

[0288] (2) In-chip polymerization:

[0289] The polymerization solution mix was prepared as follows: 700ul water + 200ul PEG (12.5mg / ml, 4Arm-PEG2K-NH2) + 100ul di-p-toluenesulfonic acid PEG silyl ether crosslinker (7.23mg / ml).

[0290] Cleaning: Pour 1000ul of ethanol / water / ethanol / water into the chip in sequence.

[0291] Air seal: Add 200ul of mix reagent to the cleaned chip, and then use a 1ml pipette to slowly inject air into the chip, trying to ensure that the air-liquid interface is as flat as possible, until all the liquid in the chip is pushed out of the chip.

[0292] Heat polymerization: Place the sealed chip on an 85°C hot plate. Heat the quartz surface for 30 seconds (with the chip exposed) (visible condensation vapor can be seen on the chip bottom). Use a 10ml pipette to pipette twice. The first shot should be very slow to push away the evaporated water vapor from the micro-pits. The second shot can be slightly faster to push away the water vapor from the edge of the glue. The chip is now completely dry. Continue heating and polymerization at 85°C for 30 minutes, then uncover. After polymerization, remove the chip and let it cool to room temperature.

[0293] Gel swelling: After the chip has cooled to room temperature, slowly drain 0.2 ml of ethanol followed by 1000 μl of water for rinsing. Heat at 60°C for 30 minutes to allow swelling, then seal. After swelling, rinse with 1000 μl of ethanol and water, respectively, and set aside.

[0294] Conversion of amino groups to azide:

[0295] Prepare the conversion solution: 492ul DMSO + 50ul FSO₂N₃ (400mM-MTBE) + 323ul water + 134ul NaHCO₃ (50mg / ml - freshly prepared). First add DMSO, then FSO₂N₃ solution and shake well. Then add water (the solution may become warm and slightly turbid). Finally, add the prepared NaHCO₃ solution and shake until the solution becomes clear.

[0296] 200 μl of amino conversion solution was added to each chip, and the reaction was allowed to proceed at 30°C for 20 minutes. The chip was then uncovered and rinsed with 1000 μl of water / ethanol / water in sequence.

[0297] Click mix preparation: Prepare 2uM DBCO-primer solution and inject 200ul.

[0298] Click temperature: 60℃ for 15min. Seal.

[0299] Wash with 1000ul pure water solution and hybridize with a 3'FAM probe that is complementary to the primer sequence;

[0300] Fluorescence microscope photography, whole chip scanning (see Figure 6 Left) and a partial enlarged view (see Figure 6 right).

[0301] Depend on Figure 6 It can be seen that the polymerization uniformity of the entire chip is good, with CV < 10%. The enlarged area of the chip shows that the glue is in a circular shape within the micro-pits and is evenly distributed.

[0302] (3) Sequencing: Cyna S201 sequencer. The technology of this sequencer has been described in the applicant’s previous patents (CN106755292B, AU2016356395B2). The sequencing sample was a λ phage genomic DNA fragment (about 300 bp). The sequencing report is shown in Table 4 below.

[0303] Table 4 Sequencing result report

[0304]

[0305]

[0306] (4) Glue degradation: 0.5M HF, RT℃-5min, repeat 3 times, take pictures to test the glue degradation effect. Figure 7 In the figure, the left picture is a microscopic picture before degradation, and the right picture is a microscopic picture after degradation.

[0307] It can be clearly seen that the glue in the chip micro-pits is evenly distributed before depolymerization and the statistical mean of brightness is 14902. After depolymerization, the statistical mean of brightness is 115, and the glue remains 0.7%.

[0308] (5) The results of repeating the above process 20 times are shown in Table 5:

[0309] Table 5 Repeat sequencing results

[0310]

[0311] As can be seen from Table 5, the results of 20 repetitions showed good reproducibility across all indicators, indicating that at least 20 repetitions of the polymerization-disaggregation-sequencing cycle can be achieved. It can be seen that when the cleaning and sequencing steps (number of cycles) are repeated multiple times, the overall sequencing flux loss is minimal. For example, when the number of cycles is 1, the sequencing flux is 652, and when the number of cycles is 5, the sequencing flux is 645, essentially unchanged. For example, when the number of cycles is 10, the flux is 684, showing no decrease. It should be noted that sequencing flux is not an absolute value. During sequencing, under the same conditions, sequencing flux is affected by conditions such as the sample, fluid, and surface. Therefore, when comparing flux under the same conditions, the only factor to consider is whether the variation is too great. In this example, the flux between multiple experiments did not vary much, and even increased slightly, indicating that there was no significant loss of sequenceable vectors. It can be seen that the values of flux, Q30, and Q40 did not vary significantly, demonstrating the repeatability of this sequencing method. The brightness statistics after depolymerization show that after depolymerization, the ratio of the brightness after depolymerization to the polymerization brightness is always in a relatively small numerical range and does not change much, which also proves that the hydrogel carrier does not change much.

[0312] Example 2

[0313] The synthetic route of diiodine PEG silyl ether crosslinker is as follows:

[0314]

[0315] The specific steps corresponding to the synthetic route are as follows:

[0316] Step 1: Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate

[0317] Compound M1 (20 g, 133 mmol) and sodium hydroxide (3.9 g, 99.8 mmol) were dissolved in 250 ml of dichloromethane solution. p-Toluenesulfonyl chloride (12.6 g, 66.2 mmol) was added under ice-cooling and the mixture was reacted at room temperature for 8 hours. 100 ml of water was added for washing, the mixture was separated, dried, and the solvent was removed by rotary evaporation. The mixture was separated and purified by DCM / MeOH (10:1-3:1) column chromatography to obtain 16.6 g of an oily compound (yield 41%). LCMS: C 13 H 20 SO6(M+H+)304.98. Mass spectrum see Figure 8 .

[0318] Step 2: Synthesis of compound 2-(2-(2-iodoethoxy)ethoxy)ethan-1-ol:

[0319] Compound M2 (10 g, 32.8 mmol) was dissolved in acetonitrile, sodium iodide (19.6 g, 131.4 mmol) was added, and the mixture was heated at 100°C for 8 hours. The mixture was cooled to room temperature and filtered to remove the precipitate. The solvent was then removed by swirl, and the mixture was washed with 100 ml of water, extracted with 200 ml of dichloromethane, and purified by column chromatography to obtain 5.5 g of an oily compound (yield 65%). LCMS: C6H 13 IO3(M+H+)260.98. Mass spectrum see Figure 9 .

[0320] Step 3: Synthesis of compound 1,19-diiodo-10,10-dimethyl-3,6,9,11,14,17-hexaoxa-10-silanonadecane

[0321] Compound M3 (5 g, 19.2 mmol) was dissolved in dry dichloromethane, pyridine (6.05 g, 77 mmol) was added to the reaction solution, and dimethylsilyl chloride (0.91 g, 9.6 mmol) was slowly added dropwise. The reaction was carried out at room temperature for 10 hours. The product was separated by silica gel (TEA-treated) column chromatography using DCM / MeOH (20:1-10:1) to obtain 2 g (yield 18%) of the following compound:

[0322] LCMS: C 14 H 30 I2O6Si, (M+NH4), 594.05. HPLC chart see Figure 10 .

[0323] The hydrogel sequencing involved in this embodiment showed no difference in specificity from that in Example 1, and will not be described in detail.

[0324] Example 3

[0325] In the present invention, the synthesis reaction of the acid-degraded hydrogel and the bisbromoketal crosslinker is as follows:

[0326]

[0327] Weigh 0.3 g of 2,2'-(propane-2,2-diylbis(oxy))diethylamine (0.3 g, 1.8 mmol, 1.0 eq.) into a 25 ml single-necked flask and dissolve it in 3 ml of DCM. Triethylamine (0.374 g, 3.6 mmol, 2.0 eq.) and bromoacetyl bromide (0.746 g, 3.6 mmol, 2.0 eq.) were then added under ice-cooling. Stir the mixture under ice-cooling for 30 min, then warm to room temperature and react for 16 h. MS: 404.9. Eq. refers to equivalents, or equimolar equivalents.

[0328] TLC showed that the reaction of the starting material was complete. The reaction solution was spin-dried and flash purified to obtain 350 mg of brown oil with a yield of 46.8%.

[0329] Example 4

[0330] The synthesis of Ts-977 acid-degradable crosslinker, see the steps below.

[0331]

[0332] Step 1: Add 1.0 eq. 1,4-cyclohexanedione, 2.0 eq. glycerol, 0.1 eq. p-toluenesulfonic acid and 10V (about 10 times the volume equivalent) of toluene to a glass reaction bottle. Start stirring, heat to reflux and separate the water. After reacting for 5 hours, cool to room temperature and add 1 eq. triethylamine. Vacuum concentrate to remove toluene. The concentrated product is purified by column to obtain compound 1. MS of compound 1: 261.20, see Figure 11 .

[0333] Step 2: Add 1.0 eq. of compound 1, 3.0 eq. of p-toluenesulfonyl chloride, 4.0 eq. of triethylamine, and 10 V of dichloromethane to a glass reaction flask. Stir and react at room temperature for 3 hours. Add 10 V of saturated sodium bicarbonate aqueous solution and separate the liquids. Wash the organic phase once with pure water. Concentrate the organic phase and purify it with a column. The p-toluenesulfonyl protected product is obtained and is designated as compound 2. The MS of compound 2 is 561.31, see Figure 12 .

[0334] Step 3: Add 1.0eq. of compound 1, 20V of dichloromethane and 2.0eq. of DMAP (4-dimethylaminopyridine) to the reaction flask and start stirring. Cool to 0°C, then add 2.0eq. of Ts-PEG4-COOH (a PEG derivative with COOH and p-toluenesulfonyl groups at both ends) and 4.0eq. of DCC (dicyclohexylcarbodiimide). After the addition is complete, slowly warm to room temperature and continue stirring for 20 hours. After the reaction is complete, add 20V of saturated sodium bicarbonate and wash once, then wash once with pure water. The organic phase is concentrated and dried, and then purified by reverse column. A cross-linker with p-toluenesulfonyl protection at the end is obtained, which is recorded as compound 3, that is, Ts-977 cross-linker. MS of compound 3: 977.90, see Figure 13 .

[0335] Synthesis of Ts-977 acid hydrolysis hydrogel:

[0336] (1) In-bottle polymerization: 4Arm-PEG2K-NH2 15 mg, TS-977 crosslinker (compound 3) 15 mg, DMF 30 μl, 100 °C for 1 h (as Figure 14 Left), swelled after soaking in water (such as Figure 14 right).

[0337] Depend on Figure 14 It can be seen that after polymerization, a yellow solid gel is formed, which swells after adding water and its volume increases several times, indicating that the gel has good water-swelling properties.

[0338] (2) In-chip polymerization:

[0339] Preparation of polymerization solution mix: 700ul water + 200ul PEG (12.5mg / ml, 4Arm-PEG2K-NH2) + 100ul TS-977 (9.39mg / ml).

[0340] Cleaning: Pour 500ul of ethanol / water / ethanol / water into the chip in sequence.

[0341] Air seal: Add 150ul of mix reagent to the cleaned chip, and then use a 1ml pipette to slowly inject air into the chip, trying to ensure that the air-liquid interface is flat, until all the liquid in the chip is pushed out of the chip.

[0342] Heat polymerization: Place the sealed chip on an 85°C hot plate. Heat the quartz surface for 30 seconds with the chip exposed (visible condensation vapor can be seen on the chip bottom). Use a 1ml pipette to pipette twice. The first shot is very slow, delivering 1ml of gas to dissipate the evaporated water vapor in the micro-pits. The second shot can be slightly faster to dissipate the water vapor around the edge of the glue. The chip is now completely dry. Continue heating and polymerization at 85°C for 30 minutes, then uncover. After polymerization, remove the chip and let it cool to room temperature.

[0343] Gel swelling: After the chip has cooled to room temperature, slowly drain 0.2 ml of ethanol and rinse with 500 μl of water. Heat at 60°C to allow swelling for 30 minutes, then seal. After swelling, rinse with 500 μl of ethanol and water sequentially, and set aside.

[0344] Conversion of amino groups to azide:

[0345] Prepare the conversion solution: 492ul DMSO + 50ul FSO₂N₃ (400mM-MTBE) + 323ul water + 134ul NaHCO₃ (50mg / ml - freshly prepared). First add DMSO, then FSO₂N₃ solution and shake well. Then add water (the solution may become warm and slightly turbid). Finally, add the prepared NaHCO₃ solution and shake until the solution becomes clear.

[0346] Add 150 μl of amino conversion solution to each chip, let it react at 30°C for 20 minutes, then uncover it. Rinse with 500 μl of water / ethanol / water in sequence.

[0347] Click mix preparation: Prepare 2uM DBCO-primer solution.

[0348] Click temperature: 60℃ for 15min. Seal.

[0349] Wash with 500ul of pure water and hybridize with a 3'FAM probe that is complementary to the primer sequence;

[0350] Fluorescence microscope photography, whole chip scanning (see Figure 15 Left) and partial enlarged image (see Figure 15 Middle figure), and the fluorescence intensity statistics of the local magnified image (see Figure 15 right).

[0351] exist Figure 15 The leftmost image is a full scan of the experimental chip. The middle section is the fluid flow area, which is also the sequencing area. It can be seen that the fluorescence intensity is relatively uniform across the entire area. The middle image is a fluorescence micrograph, in which each micropit has a period of 2 microns. It can be seen that each micropit is filled with hydrogel and is relatively uniform in size. The fluorescence intensity statistics in the right image show that its CV is 105.726 / 2313.736 = 4.5%. The uniformity of the hydrogel in the entire chip is maintained within 5%.

[0352] (3) Sequencing: Seina S201 sequencer, the sequencing sample is a λ phage genomic DNA fragment (about 300 bp), the sequencing report is as follows Figure 16 shown.

[0353] (4) Glue degradation: 3M HCl, 1 ml, 60℃-10min, repeat 3 times, take pictures to test the glue degradation effect. Figure 17 ( Figure 17 Left: before degradation, right: after degradation); it can be clearly seen that the glue in the chip micro-pits is evenly distributed before depolymerization and the statistical mean of the brightness is 2313.736. After depolymerization, the statistical mean of the brightness is 42.986, and the glue remains 1.8%.

[0354] (5) Repeat steps 2-4 above, using the same sequencing conditions. The results of multiple repetitions are shown in Table 6 below:

[0355] Table 6 Repeated sequencing results

[0356]

[0357] The results of 5 repetitions showed good reproducibility of various indicators, indicating that at least 5 repeated polymerization-disaggregation-sequencing cycles can be met.

[0358] Aggregate brightness is a relative value. All aggregate brightness values used in this article were obtained using uniform imaging conditions. Sequencing throughput is a value selected based on experimental conditions. Comparisons between experiments can provide insights into uniformity. Parameters such as Q30 and Q40 should be used for comparison between experiments. The brightness after deaggregation was obtained using the same imaging conditions as the aggregate brightness.

[0359] The present invention and examples may refer to descriptions of PEG. For example, 4Arm-PEG2K-NH2 refers to a 4-arm PEG with a molecular weight of approximately 2000 and amino-modified groups. This nomenclature is commonly used in the art.

[0360] According to the above experimental conditions, the hydrogel can be repeatedly polymerized and depolymerized more than 20 times under appropriate depolymerization conditions.

[0361] Example 5

[0362] The synthetic route of amine carbonate cross-linking agent is as follows:

[0363]

[0364] Step 1: Add 20g of PEG4 to a reaction flask, add 10V DCM (dichloromethane), and stir. Maintaining the temperature at 0°C, add 1.0eq. of sodium hydroxide and 1.0eq. of p-toluenesulfonyl chloride. Slowly warm to room temperature and continue stirring for 2 hours. Add water, separate the liquids, and concentrate the organic phase for column purification.

[0365] Step 2: Add 5g of mono-Ts-PEG4 to a reaction flask, add 10V DCM, and stir. Add 0.9eq. of CDI (carbonyldiimidazole, N,N-carbonyldiimidazole) at room temperature and stir for 1 hour. Then add 1.2eq. of ethanolamine and continue stirring for 1 hour. After adding water, separate the mixture. Wash the organic phase with 0.01M HCl aq. and concentrate to dryness for later use.

[0366] Step 3: Add 6 g of compound 2 to a reaction flask, add 10 V DCM, and stir. At room temperature, add 1.2 eq. of triethylamine. Then, add 1.2 eq. of p-toluenesulfonyl chloride and continue stirring for 16 hours. Add water, separate the liquids, and concentrate the organic phase for column purification.

[0367] Step 4: Add 7.4g of compound 3 to a reaction flask, add 15V of acetonitrile, and start stirring. At room temperature, add 5.0eq. of sodium iodide. Heat to 80-90°C and continue stirring for 4 hours. Cool to room temperature, filter, and concentrate the filtrate to dryness. Add water and extract once with dichloromethane. Concentrate the dichloromethane phase to dryness. See the mass spectrum for details. Figure 18 .

[0368] Ammonium carbonate hydrogel synthesis:

[0369] (1) In-bottle polymerization: weigh 4Arm-PEG2K-NH2 0.025mM, amine carbonate crosslinker 0.03mM, DMF 60ul, water 50ul, polymerize at 85℃ for 30min; after polymerization, the gel is as follows Figure 19As shown in the figure, it can be seen that the yellow solid gel is formed after polymerization (see Figure 19 Left), swells after adding water (see Figure 19 (right image), the volume increases several times, indicating that the gel has good water swelling properties. In the figure, the left side shows the solid hydrogel after polymerization, and the right side shows the hydrogel after adding water.

[0370] (2) In-chip polymerization:

[0371] Preparation of polymerization solution mix: 700ul water + 200ul PEG (12.5mg / ml, 4Arm-PEG2K-NH2) + 100ul carbonate crosslinker (4.39mg / ml);

[0372] Cleaning: Pour 1000ul of ethanol / water / ethanol / water into the chip in sequence.

[0373] Air seal: Add 200ul of mix reagent to the cleaned chip, and then use a 1ml pipette to slowly inject air into the chip, trying to ensure that the air-liquid interface is as flat as possible, until all the liquid in the chip is pushed out of the chip.

[0374] Heat polymerization: Place the sealed chip on an 85°C hot plate. Heat the quartz surface for 30 seconds (with the quartz surface exposed) until condensation is visible on the bottom of the chip. Use a 10ml pipette to slowly dispense 1ml of gas once to displace evaporated water vapor from the micro-pits. The second pipette can be slightly faster to displace water vapor from the edge of the adhesive. The chip is now completely dry. Continue heating and polymerization at 85°C for 30 minutes, then uncover. After polymerization, remove the chip and let it cool to room temperature.

[0375] Gel swelling: After the chip has cooled to room temperature, slowly drain 1ml of ethanol followed by 1000ul of water for rinsing. Heat at 60°C for 30 minutes to allow swelling, then seal. After swelling, rinse with 1000ul of ethanol and water, respectively, and set aside.

[0376] Conversion of amino groups to azide:

[0377] Prepare the conversion solution: 492ul DMSO + 50ul FSO₂N₃ (400mM-MTBE) + 323ul water + 134ul NaHCO₃ (50mg / ml - freshly prepared). First add DMSO, then FSO₂N₃ solution and shake well. Then add water (the solution may become warm and slightly turbid). Finally, add the prepared NaHCO₃ solution and shake until the solution becomes clear.

[0378] 200 μl of amino conversion solution was added to each chip, and the reaction was allowed to proceed at 30°C for 20 minutes. The chip was then uncovered and rinsed with 1000 μl of water / ethanol / water in sequence.

[0379] Click mix preparation: Prepare 2uM DBCO-primer solution.

[0380] Click temperature: 60℃ for 15min, seal.

[0381] Wash with 1000ul pure water solution and hybridize with a 3'FAM probe that is complementary to the primer sequence;

[0382] Fluorescence microscope photography, chip overall photography and scanning puzzle (see Figure 20 Left) and partial enlarged image (see Figure 20 right).

[0383] The images above show good uniformity across the entire chip's polymer mosaic. Zooming in on a specific area reveals evenly dispersed gel within the micropits. The left image shows a full scan of the chip, demonstrating relatively uniform fluorescence performance. Using software to measure the difference in values, see the middle image. Fluorescence is relatively uniform across the entire chip, with variations on either side likely due to reflections. Zooming in on a specific area, the fluorescence image reveals that hydrogel is present in virtually every micropit, with minimal variation in fluorescence intensity, with calculated CV differences within 5%.

[0384] (3) Sequencing: Seina S201 sequencer, sequencing sample is (same as Example 1), the sequencing report is as follows:

[0385] Table 7. Sequencing report

[0386] Number of sequencing fragments 782,535,421 / 786,452,563 Number of bases 804,225,658,487 / 806,325,598,265 Average read length 155.08 / 156.02 AQ30 98.56％ / 97.21％ AQ40 96.52％ / 96.34％

[0387] (4) Glue degradation: 0.1M NaOH, 60℃-10min, repeated 3 times, microscopic photography results as shown Figure 21 As shown (left before degradation, right after degradation);

[0388] It can be clearly seen that the glue in the chip micro-pits is evenly distributed before depolymerization and the statistical mean of brightness is 3673.909. After depolymerization, the statistical mean of brightness is 68.463, and the glue remaining is about 1.8%.

[0389] (5) The above process was repeated 20 times. The results are shown in Table 8. It can be seen that the reproducibility of various indicators of the 20 repeated results is good, indicating that at least 20 repeated polymerization-disaggregation-sequencing cycles can be met.

[0390] Table 8. Repeat sequencing results

[0391] Number of cycles Aggregate Brightness Sequencing throughput (M) AQ30 AQ40 Brightness after depolymerization 1 3673 782 98.43％ 97.25％ 68 2 3802 794 97.89％ 96.36％ 72 3 3725 750 97.98％ 96.32％ 75 4 3654 810 98.65％ 97.58％ 81 5 3698 790 98.15％ 96.26％ 80 15 3712 723 98.32％ 96.06％ 84 20 3665 769 98.07％ 96.25％ 87

[0392] Example 6

[0393] The synthesis route of the photodegradable crosslinker is as follows:

[0394]

[0395] Step 1: Take 20g SM (3-hydroxyacetophenone) and add it to the reaction bottle. Then add 5V glacial acetic acid and start stirring. Cool to 0 degrees Celsius and add 1.1eq. of concentrated nitric acid dropwise. After the addition is complete, slowly warm to room temperature and continue stirring for 5 hours. Add water and ethyl acetate to separate the liquid. The organic phase is concentrated and then purified by column. NMR spectrum see Figure 22 .

[0396] Step 2: 4 g of compound 1 was added to a reaction flask, 10 V of DMF was added, and stirring was started. 2.5 eq. of potassium carbonate was then added.

[0397] The temperature was raised to 80-90°C and stirring was continued for 5 hours. Water and ethyl acetate were added to separate the liquids. The organic phase was concentrated to dryness, slurried with methyl tert-butyl ether, and then filtered.

[0398] The filter cake is collected and dried, and the mother liquor is recovered through the column.

[0399] Step 3:

[0400] Add 3.5 g of compound 2 (comp. 2) to a reaction flask, add 10 V of anhydrous ethanol, and stir. Cool to 0°C, then add 4.0 eq. of sodium borohydride. Slowly warm to room temperature and continue stirring for 2 hours. Add water and ethyl acetate, and concentrate the organic phase to dryness for later use.

[0401] Step 4:

[0402] Add 3.8 g of compound 3 to a reaction flask, add 10 V DCM, and stir. Then, add 4.0 eq. of triethylamine. Cool to 0°C, and dropwise add 4.0 eq. of bromoacetyl bromide. Slowly warm to room temperature and continue stirring for 2 hours. Add water to the solution, concentrate the organic phase, and purify it on a column.

[0403] Step 5:

[0404] Add 0.5g of 2-Br compound to a reaction flask, add 10V acetone, and start stirring. Then add 10eq. of sodium iodide. Heat to 50°C and continue stirring for 5 hours. Add water and ethyl acetate to separate the liquids, and concentrate the organic phase to dryness for later use. For mass spectrum, see Figure 23 .

[0405] Example 7

[0406] Preparation and use examples of photodegradable hydrogels:

[0407] 1. Aggregation

[0408] In-bottle polymerization: weigh 4Arm-PEG2K-NH2 0.025mM, photodegradable crosslinker 0.03mM, DMF 60ul, water 50ul, polymerize at 85℃ for 30min; after polymerization, the gel is as follows Figure 24 As shown (left: after polymerization, middle: swelling after adding water, right: after λ = 365nm light exposure). The figure shows that after polymerization, a yellow solid gel is formed. After adding water, it swells and increases in volume several times, indicating that the gel has good water-swelling properties. After light exposure, the volume of the gel decreases significantly.

[0409] On-chip polymerization:

[0410] 1.1 Preparation of polymerization solution mix: 700ul water + 200ul PEG (12.5mg / ml) + 100ul photodegradable cross-linker (6.94mg / ml).

[0411] 1.2 Cleaning: Inject 1000ul of SR / MQ / SR / MQ into the chip in sequence.

[0412] 1.3 Air seal: Add 200ul of mix reagent to the cleaned chip, and then use a 200ul pipette to slowly inject air into the chip, trying to ensure that the air-liquid interface is flat, until all the liquid in the chip is pushed out of the chip.

[0413] 1.4 Heating and Polymerization: Place the sealed chip on an 85°C hot plate. Heat the quartz surface for 30 seconds with the chip exposed (clear condensation vapor can be seen on the chip bottom). Use a 10ml pipette to pipette twice. The first shot should be very slow to remove the evaporated water vapor from the micro-pits. The second shot can be slightly faster to remove the water vapor from the edge of the glue. At this point, the chip is completely dry. Continue heating and polymerizing at 85°C for 30 minutes, then uncover. After polymerization, remove the chip and cool it to room temperature.

[0414] 1.5 Gel Swelling: After the chip has cooled to room temperature, slowly drain 0.2 ml of SR and rinse with 1000 μl of MQ. Heat at 60°C to allow swelling for 30 min, then seal. After swelling, rinse with 1000 μl of SR and MQ sequentially, and set aside.

[0415] 2. Conversion of amino groups to azide

[0416] 2.1 Conversion solution preparation: 492ul DMSO + 50ul FSO₂N₃ (400mM-MTBE) + 323ul UW + 134ul NaHCO₃ (50mg / ml - freshly prepared). First add DMSO, then FSO₂N₃ solution and shake well. Then add UW (the solution may become warm and slightly turbid). Finally, add the prepared NaHCO₃ solution and shake until the solution becomes clear.

[0417] 2.2 Amino acid conversion: Add 200 μl of amino acid conversion solution to each chip, incubate at 30°C for 20 minutes, and then leave the chip uncovered. Then, add 1000 μl of MQ / SR / MQ solution sequentially for cleaning and use.

[0418] 3. Click DNA, hybridization probe;

[0419] 3.1Clean before click: Wash with 1000ul ethanol and water respectively.

[0420] 3.2Click mix preparation: Prepare 2uM DBCO-primer solution.

[0421] Click temperature: 60℃ for 15min, seal.

[0422] Wash with 1000ul pure water solution and hybridize with a 3'FAM probe that is complementary to the primer sequence;

[0423] 3.4 Clean with 500ul pure water cleaning solution.

[0424] 3.5 Take photos.

[0425] 4. Degradation of the hydrogel prepared above after sequencing is completed

[0426] 4.1 Degradation: Irradiate with λ = 365nm light for 30min, and the degradation is complete.

[0427] 4.3 After taking the photo, the empty chip value drops to the background value, and the hydrogel in the chip is removed.

[0428] 5. Resetting of the hydrogel.

[0429] Through experiments, it was found that the chip value after taking the photo dropped to the background value, and the difference from the background value was within 5%. It is believed that it does not affect further sequencing. The basic principles of the present disclosure are described above in conjunction with specific embodiments. However, it should be pointed out that the advantages, strengths, effects, etc. mentioned in the present disclosure are only examples and not limitations. It cannot be considered that these advantages, strengths, effects, etc. are required by each embodiment of the present disclosure. In addition, the specific details disclosed above are only for the purpose of illustration and ease of understanding, and are not limiting. The above details do not limit the present disclosure to the use of the above specific details to achieve it.

Claims

1. A gene sequencing method using a degradable hydrogel, characterized in that: The following steps are involved: (1) adding a carrier polymerization reaction solution to a gene sequencing chip and sealing it in a micro-well of the gene sequencing chip; the inner surface of the gene sequencing chip is pre-hydrophobically chemically modified; (2) The carrier to be polymerized in the micropits undergoes a cross-linking reaction to form a three-dimensional network polymer carrier; (3) washing away the unreacted support to be polymerized reaction solution; (4) Immobilizing the nucleic acid molecules to be sequenced on a three-dimensional network polymer carrier; (5) Sequencing; (6) depolymerization, depolymerizing the hydrogel through the degradable sites of the three-dimensional network polymer carrier; Repeat steps (1)-(6).

2. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: The carrier to be polymerized in step (1) is a mixed solution of hydrogel functional monomers before polymerization; the carrier is a hydrogel; Preferably, the hydrogel is selected from at least one of functionalized polysaccharide hydrogels, functionalized polyethylene glycol and its derivatives hydrogels, or functionalized polyacrylamide and its derivatives hydrogels; the functionalized polysaccharide hydrogel is selected from at least one of chitosan, hyaluronic acid, trehalose, and cellulose; Preferably, the carrier to be polymerized undergoes a cross-linking reaction to form the hydrogel, and the cross-linking is achieved by the mutual reaction of functional groups on the prepolymer (PSA, PEG, PAM, etc.) Further preferably, the cross-linking reaction type is: cross-linking by reaction of amino group with aldehyde group, epoxy group, thiol, succinimide, or carboxyl group, or cross-linking by reaction of azide with alkynyl group or norbornene-containing compound, or cross-linking by free radical polymerization; Preferably, the polymerization reaction of the carrier to be polymerized reaction solution is initiated by heating or light exposure; Preferably, the carrier reaction solution to be polymerized contains one or more of the following prepolymers or monomers: polysaccharides and their derivatives, polyethylene glycol and its derivatives, and polyacrylamide and its derivatives; Preferably, the prepolymer or monomer in the carrier to be polymerized reaction solution includes chitosan, and preparing the reaction solution containing the prepolymer or monomer includes: dissolving chitosan in acetic acid solution, stirring and dissolving to obtain a chitosan solution; adding glutaraldehyde solution to the chitosan solution to obtain a reaction solution containing chitosan.

3. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: In the carrier reaction solution to be polymerized, at least one component contains a degradable site adapted to acid hydrolysis, alkaline hydrolysis, fluoride decomposition or photolysis; Preferably, the carrier to be polymerized reaction solution in step (1) is prepared according to the following method: a multi-arm polyethylene glycol backbone monomer with an active functional group FG1 and a cross-linking agent containing a degradable site are dissolved in a solvent in a specific ratio to form a hydrogel precursor solution; wherein the cross-linking agent has a linking group FG2; and the FG2 can undergo a coupling chemical reaction with FG1; Preferably, the specific ratio refers to the mass ratio of the linking groups in the monomer and the cross-linking agent FG1:FG2 being between 10:1 and 1:1; Preferably, the multi-arm polyethylene glycol is 2-8-arm polyethylene glycol; preferably, the multi-arm polyethylene glycol is four-arm polyethylene glycol, six-arm polyethylene glycol, or eight-arm polyethylene glycol; Preferably, the specific ratio refers to the mass ratio of the linking groups in the monomer and the cross-linking agent FG1:FG2 between 9:1-1:1, preferably between 8:1-1.5:1, more preferably between 7:1-1.8:1, and more preferably between 6:1-2:1; Preferably, the hydrophobic chemical modification that does not damage the gene sequencing chip means that the hydrophobic chemical modification of the hydrophobic surface is not damaged when steps (2) to (7) are repeated 1 to 50 times; Preferably, the hydrophobic chemical modification that does not destroy the gene sequencing chip means that the loss of the hydrophobic chemical modification is within 10%; preferably within 9%; more preferably within 8%; more preferably within 7%; more preferably within 6%; more preferably within 5%; more preferably within 4%.

4. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: The carrier to be polymerized reaction solution comprises the following structure: Wherein C and D are independently selected from C1-C6 alkyl, aryl, substituted aryl; Preferably, the substituted aryl group includes tolyl, ethylphenyl; Preferably, the depolymerization is carried out by introducing a depolymerization solution, wherein the depolymerization solution is an aqueous solution containing soluble fluoride.

5. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: The carrier to be polymerized reaction solution comprises the following structure: or wherein E and F are independently selected from C1-C3 alkyl groups; or E and F form a five-membered or six-membered cycloalkane; wherein R1, R2, R3, and R4 are independently selected from hydrogen, C1-C4 alkyl, and halogenated C1-C4 alkyl; Preferably, R1 is the same as R2, and R3 is the same as R4; Preferably, the depolymerization and degradation method is washing with a solution having a pH value of less than or equal to 2; Preferably, E and F are methyl groups.

6. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: The carrier to be polymerized reaction solution comprises the following structure: or Preferably, the depolymerization is carried out by passing an alkaline solution to depolymerize and hydrolyze the product; Preferably, the alkaline solution is selected from at least one of diethylamine, triethylamine, sodium hydroxide, potassium hydroxide, lithium hydroxide, and potassium carbonate solution, and the concentration is 0.1M-3M; Preferably, the concentration of the alkaline solution is 0.2, 0.3, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3; or a range consisting of any two numbers therein, and the concentration unit is mole per liter.

7. The gene sequencing method using a degradable hydrogel according to claim 1, characterized in that: The carrier to be polymerized reaction solution comprises the following structure: Preferably, the depolymerization in step (7) is photodepolymerization, and the photodepolymerization is performed by irradiating UV light in the wavelength range of 200-400 nm, such as 365 nm or 254 nm.

8. A gene sequencing method using a degradable hydrogel, characterized in that: The following steps are involved: (1) adding a hydrogel prepolymer solution to a gene sequencing chip and sealing it in a micro-pit of the gene sequencing chip; the inner surface of the gene sequencing chip is pre-hydrophobically chemically modified; (2) The hydrogel prepolymer solution in the micropits undergoes a cross-linking reaction to form a three-dimensional network hydrogel carrier; (3) Immobilizing the nucleic acid molecules to be sequenced on a three-dimensional network hydrogel carrier; (4) Sequencing; (5) passing a cleaning solution into the hydrogel to react with the hydrogel to degrade the hydrogel without reacting with the hydrophobic chemical modification; Repeat steps (1)-(5).

9. A gene sequencing kit, characterized in that: The gene sequencing kit includes: hydrogel monomers, hydrogel crosslinker, Sequencing reagents, Sealing gas or sealing liquid; hydrogel cleanser; Among them, the hydrogel monomer and the hydrogel cross-linker are used to form a hydrogel in the gene sequencing chip; the sequencing reagent is used for gene sequencing reaction; the sealing gas or sealing liquid is used to seal the mixed reagent formed by the hydrogel monomer and the hydrogel cross-linker in the micro-pits on the inner surface of the gene sequencing chip, thereby reacting to form a hydrogel in the micro-pits; the hydrogel cleaning liquid is used to react with the hydrogel to remove the hydrogel.

10. A gene sequencing system, characterized in that: Sequencing is performed using the disaggregated gene sequencing method according to any one of claims 1 to 8.

Images