Gene chip and reuse method thereof

Abstract

The repeated use method of the gene chip comprises the following steps: (1) resetting the gene chip; (2) carrying out overall hydrophobic treatment on the reset gene chip; and (3) generating the RNA or DNA carrier in the micro-pit of the gene chip in a gas sealing or oil sealing manner. The liquid in the flow channel is independently divided into each micro-pit of the hydrophobic chip by oil or gas, so that the RNA / DNA carrier can be quickly generated only in the micro-pits, the efficiency of a distinguishing modification process can be greatly improved, the modification cost can be greatly reduced, and the stability and consistency of modification can be improved. The liquid-phase free radical oxidation organic layer is used for resetting the chip, so that the chip can be quickly cleaned and activated, the resetting process is greatly simplified, and the resetting cost is greatly reduced. According to the method, the chip can be completely modified and reset on the machine, and the chip resetting and modifying process is greatly simplified.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic chips, and in particular relates to a gene chip and a method for preparing and reusing the same. Background Art

[0002] In the field of gene sequencing, to ensure sequencing quality and improve the signal-to-noise ratio, the most common way to implement chip modification is to differentially modify DNA at specific locations. However, existing methods for achieving differential DNA modification either have too many steps or are too complex, 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, they use whole-surface hydrophobic or functionalized hydrophobic chips, and then use air sealing or oil sealing to differentially modify RNA or DNA vectors in the micropits of the chip in one step, and form a gene chip that can be used for subsequent biochemistry or sequencing. 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 it can first cut the wafer or other material substrate, package it into multiple chips, and then perform hydrophobic or functionalized hydrophobic treatment on a single chip. Then, use air sealing or oil sealing to divide the RNA or DNA vector reaction solution into micropits for separate reactions, 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 in situ differentially modify RNA or DNA carriers within the chip micropits. The latter method requires utilizing a special site on the carrier within the micropit to covalently link RNA or DNA; (3) then using the chip for sequencing or other similar purposes; (4) using an oxidizing substance that can generate a large number of free radicals to remove the hydrophobic layer and other organic matter of the chip, returning the chip to its original state; repeating steps 1-4 can achieve the repeated use of the chip.

[0007] 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.

[0008] 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.

[0009] The preparation of the in situ differential DNA-modified hydrophobic chip includes the following two steps:

[0010] a) Overall hydrophobic modification: including overall hydrophobic modification or overall functional hydrophobic modification of the 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:

[0011] The microarray chip structure is defined as a chip comprising a microreactor layer, an inlet and outlet, and a base layer. The microreactor layer is also referred to as a microreactor chip. The microreactor layer comprises a large number of separate microreactor structural units. The outlet and inlet of the chip are channels for fluid to enter and exit the chip. The base layer and other layers cooperate to form a reaction space. This reaction space is also referred to as a reaction chamber or fluid chamber. The reaction chamber is the space connected to the microreactor layer. The so-called overall hydrophobic modification refers to the hydrophobic modification of all surfaces within the chip that can be reached by fluid.

[0012] The microarray chip can be made of glass, quartz, ceramic, metal, silicon-based, or plastic (such as PMMA, PC, etc.); the micropit (reaction chamber) size can be 0.1 μm-5 μm.

[0013] The gene chip or gene sequencing chip described herein refers to a chip used for gene sequencing. Common gene sequencing chips include fluid inlets and outlets, reaction chambers, and may or may not include micropits on the inner surface of the reaction chamber. The size of the micropits can be 0.1-5 microns. A gene sequencing chip can also be called a microarray chip, which refers to a gene sequencing chip containing micropits (microreaction chambers).

[0014] The hydrophobicization or functional hydrophobicization modification of the inner surface of the microarray chip can be performed by using trichlorofluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products) through CVD, liquid phase modification, or similar methods.

[0015] 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.

[0016] 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.

[0017] The fluorosilane used can be trichloroperfluorosilane, trimethylfluorosilane, or perfluoropolyether silane (1720 or 2202 or similar products), with a concentration of 0.01-1 wt% (mass concentration). 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, fluorinated agents that can quickly achieve surface hydrophobic silanization, such as perfluorohalosilanes, are preferred.

[0018] 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 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.

[0019] b) Synthesis of hydrogel: 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 1-102 to 105). An embodiment of the present invention provides a solution for generating a fully hydrophobic chip + a DNA carrier in a micro-pit.

[0020] Based on the above-mentioned modified hydrophobic chip (PF-chip for short), air or oil is used to divide the reaction solution to be polymerized with the subsequent ability to connect with DNA into individual micropits (referred to as the seal process) for separate reactions. High-molecular DNA carriers that can subsequently connect with DNA are directly generated in the micropits. Then, the DNA with reaction sites on the carrier layer is reacted to form a hydrophobic chip with DNA carriers only in the micropits.

[0021] The selection of DNA carriers takes into account their use in biological scenarios, and hydrogel polymer materials are preferred.

[0022] The main skeleton of the hydrogel can be a functionalized polysaccharide glue system (which can be referred to as PSA glue, such as chitosan, hyaluronic acid, trehalose, cellulose, etc.), or 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 polymeric skeleton is not limited to the above systems.

[0023] The cross-linking method of the hydrogel can be obtained by the mutual reaction of the functional groups obtained by functionalization on the prepolymer (PSA, PEG, PAM, etc.), such as the reaction type: amino group and aldehyde group, epoxy group, thiol, succinimide, carboxyl group reaction cross-linking; it can also be the reaction cross-linking of azide and alkynyl or norbornene-containing compounds; it can also be other reaction systems such as free radical polymerization.

[0024] The method of separating the liquid into the micro-pits (seal) can be gas or oil.

[0025] The gas can be nitrogen, air, argon, etc.; the oil can be mineral oil, fluorocarbon oil, or other non-polar oils, etc.

[0026] The triggering conditions for initiating polymerization are determined by the polymerization reaction system, and polymerization can be initiated by heating, light, etc.

[0027] The key step to achieve another key purpose of the present invention is to reset the chip after use to return it to its original state without coating, glue and other organic modifications (see attached Figure 1-10 5 to 101). Specifically, a reagent that can generate strong oxidative free radicals is used to reset the hydrophobic chip with discriminative DNA modification after use, so that it can be returned to its original clean state for reuse.

[0028] In previous inventions by the applicant, such as CN116555408A, a reducing agent was used to remove the hydrogel. This method only removed a specific portion of the hydrogel. However, its removal effect was less than ideal. Subsequent data showed that the removal efficiency of reduction methods was approximately 95%. In this case, the quality of the data from repeated sequencing was difficult to guarantee. The chip reuse method provided by the present invention not only has a relatively high removal efficiency, for example, 99% or even higher, but is also applicable to a variety of nucleic acid carriers and receiving layers for connecting nucleic acid carriers (for example, compatible with different hydrophobic surface chemical modifications). The method provided by the present invention not only removes the chemical modification on the chip surface while removing the hydrogel, but also acts as an activator for the next surface modification, activating the surface and facilitating the connection of the newly modified coating. The present invention is a more effective chip resetting method. However, patent CN116555408A only discloses a non-on-chip, reusable gene sequencing method that uses pre-synthesized carriers such as hydrogels to be loaded into the chip through centrifugation or other methods. Its loading efficiency is not 100%, and external carriers such as microspheres cannot be loaded on-chip.

[0029] The cleaning agents used include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; acidic hydrogen peroxide, or peroxides such as Fenton's oxidation reagent (Fenton) can also be used, or strong oxidizing compounds such as sodium hypochlorite and potassium permanganate can be used.

[0030] There are subtle differences between these cleaning agents. Persulfide can remove nucleic acid organic carriers and hydrophobic coatings by generating highly oxidizing sulfate radicals; while peroxide can remove nucleic acid organic carriers and hydrophobic coatings by generating highly oxidizing hydroxyl radicals. However, hydroxyl radicals generally have a short existence time and poor stability. Therefore, considering the removal efficiency, convenience, economy and environmental protection, persulfate is preferred.

[0031] The beneficial effects of the present invention include:

[0032] 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 RNA / 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.

[0033] The use of liquid-phase free radical oxidation of the organic layer can quickly reset and activate the chip, greatly simplifying the reset process and reset cost.

[0034] This method allows the chip to be modified and reset completely on-chip, greatly simplifying the chip reset and modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the steps of the gene chip reuse method provided in an embodiment of the present invention, wherein: 101 is a blank gene chip with an unmodified microarray structure; 102 is a chip obtained by modifying the blank gene chip to be hydrophobic or functionalized; 103 is the sealing process: using oil or gas to divide the liquid into micropits; 104 is the reaction solution divided into the micropits after sealing and capable of generating RNA or DNA vectors in the micropits; 105 is a chip with DNA only in the micropits and no DNA in the remaining areas.

[0036] Figure 2 This is a picture of the PF-PAM-DNA chip prepared in an example of the present invention, where the left is a single-view image and the right is an enlarged image. It can be seen from the figure that only in the micropits are there very regular and consistent DNA units with a small CV (coefficient of variation).

[0037] Figure 3 This is a picture of the chip after resetting after PF-PAM-DNA sequencing. The lower left corner is an enlarged picture after improving the contrast. It can be seen from the picture that there is almost no fluorescence brightness, indicating that the resetting method can effectively remove the RNA / DNA carrier in the pit.

[0038] Figure 4 Comparative microscope images of the chip interior wetted with pure water before and after APS removes the chip's hydrophobic layer. The left image shows the chip interior being non-wettable by water before removal, while the right image shows the chip interior being completely wetted by water after APS treatment.

[0039] Figure 5 This is a microscopic microscope image of the first F-PEG-DNA chip prepared in an embodiment of the present invention. From the image, it can be seen that the DNA fluorescence signal in each micro-pit prepared for the first time is uniform and the CV is small.

[0040] Figure 6 This is an image of the PF-PEG-DNA chip after KPS treatment. As can be seen from the image, there is almost no fluorescence, indicating that the reset method can effectively remove the RNA / DNA carrier in the pit;

[0041] Figure 7 This is an image of replanting the DNA vector and clickDNA in the microwells after sequencing was repeated 20 times using the method provided in an embodiment of the present invention.

[0042] Figure 8 This is a microscopic image of the FPF-PEG-DNA chip prepared for the first time in an embodiment of the present invention. From the image, it can be seen that the distribution of DNA in different micropits is uniform and consistent, and the CV is small.

[0043] Figure 9This is an image of the FPF-PEG-DNA modified chip after reset. As can be seen from the image, there is almost no fluorescence, indicating that the reset method can effectively remove the RNA / DNA carrier in the pit.

[0044] Figure 10 This is a characterization diagram of the modified FPF-PEG-DNA chip after repeated resetting 20 times using the method provided in an embodiment of the present invention.

[0045] Figure 11 This is a microscopic microscope image of the first FPF-DNA chip prepared in an embodiment of the present invention. From the image, it can be seen that the DNA fluorescence signal in each micro-pit prepared for the first time is uniform and the CV is small.

[0046] Figure 12 This is a comparison before and after resetting the PF-PEG-DNA chip using m-chloroperbenzoic acid.

[0047] Figure 13 This is a comparison of the PF-PEG-DNA chip before and after treatment with 30% hydrogen peroxide. DETAILED DESCRIPTION

[0048] 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.

[0049] As disclosed, the applicant's previous patents, such as CN202010087598.8, CN202010986795.3, and CN202010063461.9, describe methods and steps for synthesizing microparticles such as hydrogels. The hydrogels described in this patent can be synthesized using the same methods and steps, differing in the specific ratios of the components. The contents of the aforementioned inventions may be incorporated herein by reference.

[0050] Choice of carrier

[0051] The present invention relates to carriers within the microwells of a sequencing chip. These carriers are used to attach nucleic acid fragments to be sequenced. The carriers can be microspheres, microparticles, or form a covering on the bottom or sidewalls of the microwells. Typically, the 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 is generally difficult to load microspheres into all microwells, 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. Alternatively, hydrogel carriers can be prepared within the microwells using in situ synthesis. This method is uncommon, and 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 are significant differences between these two methods. Another method involves introducing a hydrogel reaction solution throughout the chip, which then reacts with a surface pre-modified with linker groups to form a hydrogel attached to the surface. In this method, the hydrogel is non-discriminatory, with hydrogel present both inside and outside the micropits, which is detrimental to sequencing. The degradable hydrogel of the present invention is primarily composed of the following components:

[0052] 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

[0053] Cross-linking agent: The cross-linking agent provided by the present invention is designed with a degradable molecular structure, and the degradation conditions include acid degradation, alkaline degradation, oxidation degradation, reduction degradation, and light degradation. In addition to the degradable site, the cross-linking agent 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 can be selected from carboxylic acid groups. Amine (-NH2), azide (-N3), terminal alkyne Activated alkynyl Acrylamide Halogen (Cl, Br, I), sulfonic acid derivatives (such as Ts, Ms, Ns), carboxylic acid derivatives (such as Tf, C 1-6 acyl), thiol derivatives, etc.

[0054] Multi-arm polyethylene glycol refers to polyethylene glycol with 4 to 8 arms, preferably polyethylene glycol with 4 arms, 6 arms, or 8 arms.

[0055] Sequencing methods

[0056] This invention primarily involves the fluorescence switching sequencing method. However, it can be seen that different sequencing methods have no effect on chip reuse. 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.

[0057] The sequencing methods involved in the embodiments of the present invention belong to the category of second-generation sequencing. Sequencing-by-synthesis, a common sequencing method within second-generation sequencing technology, is widely used. Typical examples include the Illumina sequencing method and the BGI sequencing method. Although each sequencing 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 entire 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 chip's inner surface. 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. Furthermore, since the carrier was synthesized using a sealed method, if the hydrophobic modification is not complete, the hydrogel will not be differentiated and will not be protected by this scheme. The micro-pit area is the functional area for hydrophobic modification, and if other areas are not properly hydrophobicized, it will not affect the overall results.

[0058] A hydrophobic chemical environment can provide the surface conditions required for sealing. Using the seal method, it is an important method to form DNA carriers in situ in micro-pits. Many of the patents previously disclosed by the applicant describe the use of hydrophobic environments and oil seals in micro-pits 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, patent CN 116333848A uses a complex photolithography process 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.

[0059] Hydrophobic inner surface of the chip

[0060] The present invention describes hydrophobic modification of the inner surface. Many common methods for hydrophobic modification exist. Generally, silane, fluorosilane, or other fluorinated compounds can be used for modification. It should be noted that the applicant has previously described a differential modification method in a previous patent. 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 made hydrophilic using methods such as CVD. This inner surface can be used to attach specific groups, such as hydrogel microspheres. The present invention utilizes a single-step process to hydrophobically modify the inner surface of the entire chip. By enclosing reactants such as hydrogel prepolymers within the microwells, the reaction produces irregularly shaped hydrogel carriers. The inventors of this application surprisingly discovered that, using this method, while there is no direct, pre-determined covalent bond between the hydrogel and the interior of the microwells, the hydrogel or other carrier remains well within the microwells and does not fall off during sequencing. The in-situ synthesis of hydrogels and other carriers using an oil seal employed in the present invention offers numerous advantages: it eliminates the need for additional modification of the substrate with the compound covalently attached to the hydrogel.

[0061] Chip reset

[0062] In previous inventions of the applicant, such as CN116555408A, a reducing agent method was used to remove the hydrogel. This method only removes a specific part of the hydrogel. However, its removal effect is not good. Later developments show that the removal efficiency of methods such as reduction is 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. The method provided by the present invention can remove the chemical modification on the chip surface while removing the hydrogel. It is a more effective method for resetting the chip on the machine. The main cleaning agents used include persulfates such as potassium persulfate and sodium persulfate; acidic hydrogen peroxide or peroxides such as Fenton oxidation reagent (Fenton) can also be used.

[0063] Reuse of chips

[0064] 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. And the steps are simple. For example, in patent CN116333848 A, a complex photolithography process is used. 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 the reuse of chips. The present invention provides usage data of multiple reuses, for example 20 times, and the sequencing quality has not been significantly reduced or changed.

[0065] The pictures of the various embodiments involved in this invention are all taken under an optical microscope, wherein the period of a single micro-pit in the chip is about 2 microns.

[0066] Implementation process (chip modification process)

[0067] 1. Example of Fully Hydrophobic-Free Radical Oil-Seal Polymerization PAM Chip Modification (PF-PAM-DNA Chip). A key benefit of this invention is to fully utilize the advantages of the hydrophobic chip to rapidly generate RNA / DNA carrier hydrophobicity-differentiated modified chips in situ within micropits. To achieve this goal, the process involves two main steps:

[0068] (1) Overall hydrophobic modification (PF-chip): This includes overall hydrophobicization or overall 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 connection of DNA in the micropits. Specifically:

[0069] The hydrophobic modification is as follows: a fluoroether reagent containing 0.1% wt of perfluoropolyether (HFPO) siloxane is introduced into the chip after activation with APS (ammonium persulfate), and the reaction is carried out at room temperature for 10 minutes. The chip is then rinsed with a fluoroether solvent and dried, and aged at 85 degrees Celsius for 10 minutes. This results in a hydrophobic chip with the entire chip surface being fluorinated and subsequently labeled as a PF chip.

[0070] (2) In situ generation of DNA-linked polyacrylamide hydrogel in micropits (PF-PAM-chip), which includes first in situ generation of polyacrylamide hydrogel with DNA-linkable sites in micropits of a hydrophobic chip, and then linking DNA to the polyacrylamide hydrogel in the micropits. Specifically:

[0071] The preparation process of PF-PAM is as follows: 4.43 g of acrylamide (AM) is dissolved in ultrapure water to prepare a 2.5 mol / L acrylamide solution; 3.85 g of N'N-methylenebisacrylamide (bis-AM) is dissolved in ultrapure water to prepare a 1 mol / L bis-AM solution; 0.30 g of acrylamide polyethylene glycol (200) azide (N3-PEG200-AM) is dissolved in ultrapure water to prepare a 0.2 mol / L solution; and the prepared 2.5 mol / L AM solution and 1 mol / L Prepare a bis-AM solution (5ml) in a 20:1 volume ratio, then add 0.5g potassium persulfate and 5ml ultrapure water to create a mixed solution (herein referred to as "water mix"). Take a PF chip (a microarray chip with a microwell diameter of 1.9µm) and rinse the cavity with 1000µL of isopropyl alcohol and 1000µL of ultrapure water. Then, inject 400µL of the prepared water mix solution (the specific volume depends on the chip cavity size). Finally, inject 200µL of fluoro oil FC40 solution to partition the hydrogel monomer solution within the microwells for separate reactions. Seal the inlet and outlet ports tightly and heat at 50°C for 1 hour on a PCR instrument or a dedicated hotplate. Finally, rinse thoroughly with 2mL of isopropyl alcohol and 1mL of wash buffer.

[0072] The PF-PAM-DNA chip preparation process is as follows: 200 μl of DNA primer reagent that reacts with the hydrogel is injected into the polymerized microarray chip. The chip entrance and exit are sealed, and the chip is placed in a PCR instrument and heated to 55°C. After reacting for 10 minutes, the chip is removed and rinsed with 2 ml of buffer solution. Then, 200 μl of the fluorescent probe 3'fam-P7rc is added to the chip. The chip is placed in a PCR instrument and heated to 60°C for 10 minutes. The chip is then cooled to 40°C for 5 minutes, removed, and rinsed with 2 ml of buffer solution. After rinsing, fluorescence is observed on an inverted fluorescence microscope using a 488 / 520 nm filter. Figure 2As can be seen from the figure, the left one is a single tile image and the right one is its enlarged image. It can be seen from the figure that not only are there DNA carriers in all the micro-pits, but the morphology and DNA brightness are very consistent. From the statistical chart in the upper right corner of the left figure, it can be seen that the CV of the DNA brightness of different micro-pits is less than 5%. The DNA unit with a small CV well demonstrates the beneficial effect of this method.

[0073] 2. Example of fully hydrophobic-cross-linked polymerized PEG chip modification (PF-PEG-DNA)

[0074] Using this method, the in situ generation of DNA carriers in micropits can be diverse. In addition to the above-mentioned in situ polymerization method, long-chain prepolymers can be used to seal into the micropits and cross-link into hydrogel DNA carriers in one step. The hydrogel cross-linking method can be achieved by the mutual reaction of functional groups on the prepolymer (PSA, PEG, PAM, etc.). The reaction types include: amino groups with aldehyde groups, epoxy groups, thiols, succinimide, and carboxyl groups. Taking the preparation of PEG-type hydrogel as an example, the details are as follows:

[0075] The method of making the chip fully hydrophobic is the same as above and will not be repeated here;

[0076] The preparation process of the PF-PEG-DNA chip is as follows: take 1 mg of 4-arm-PEG5K-NH2, add 40 ul of DMF solvent respectively, then add pure water to 400 ul, then add 0.5 eq of PEG prepolymer with I groups at both ends, shake and mix, and set aside (polymerization mix); then take a chip (PF-chip) with a micro-pit size period of 2.6 um that has been hydrophobicized, rinse the chip with 1 ml of ethanol and pure water respectively, and then inject 200 ul of the prepared polymerization mix solution into the rinsed chip respectively, and then use 200 ul of air (the specific gas volume can be adjusted according to the size of the chip cavity volume) to remove the remaining aqueous phase mix in the chip flow channel except for the micro-pits at a speed of 5 ul / s to complete the gas sealing process; then place the sealed chip on a heater and heat it at 70 degrees for 30 minutes of polymerization; after polymerization, rinse it with SR and pure water, and soak it in pure water for 30 minutes for standby use;

[0077] The PF-PEG-DNA chip preparation process is as follows: 200 μl of DNA primer reagent that reacts with the hydrogel is injected into the polymerized microarray chip. The chip entrance and exit are sealed, and the chip is placed in a PCR instrument and heated to 55°C. After reacting for 10 minutes, the chip is removed and rinsed with 2 ml of buffer solution. Then, 200 μl of the fluorescent probe 3'fam-P7rc is added to the chip. The chip is placed in a PCR instrument and heated to 60°C for 10 minutes. The chip is then cooled to 40°C for 5 minutes, removed, and rinsed with 2 ml of buffer solution. After rinsing, fluorescence is observed on an inverted fluorescence microscope using a 488 / 520 nm filter. Figure 5 As can be seen from the figure, the left one is a single tile image, and the right one is its enlarged image. It can be seen from the figure that the PEG hydrogel generated in situ in the micropit is also very regular and consistent, and the DNA brightness CV after click is very small, which also well illustrates the universal effectiveness and simplicity of this type of differential modification DNA carrier method.

[0078] According to a typical embodiment of the present invention, a sequencing chip is provided. The sequencing chip is a reused chip obtained by any of the above methods.

[0079] The beneficial effects of the present invention will be further illustrated below with reference to the examples.

[0080] General implementation process (reset process)

[0081] 1. Use APS (ammonium persulfate) to reset the surface of the PF-PAM-DNA chip

[0082] First, a PF-PAM-DNA chip was prepared according to the above example and subjected to a single round of sequencing. A 1M APS solution was then injected into the chip, reacted at 95°C for 5 minutes, and then washed with pure water and ethanol. This washing step was repeated two to three times to restore the chip to its original clean state. The PF-PAM-DNA chip was then re-prepared according to the above example and subjected to a new round of sequencing using a Cyna S100 sequencer. The chip was then reset, differentially modified, and sequenced. This cycle was repeated dozens of times. Data statistics are shown in Table 1 below. The amplification rate represents the proportion of micropits on the chip that generated valid data. The table shows that the proportion of micropits that generated valid data was very similar for each reset sequencing under the same conditions, demonstrating the consistency of the reset method. The throughput represents the amount of valid data output from the final sequencing run. Comparing the throughput of the repeated sequencing after multiple resets with the initial sequencing data shows little difference in throughput. Comparing key sequencing parameters such as decay and lead times shows that this method allows for repeated resets of the chip with very similar sequencing quality. In Table 1, seq refers to the number of repetitions of sequencing, for example, seq10 refers to the result of resetting sequencing for the 10th time. Other parameters have conventional meanings in the art.

[0083] Table 1 List of repeated sequencing results

[0084]

[0085] As can be seen in Table 1, the data for sequencing runs 1, 5, 10, 15, and 20 showed no significant changes. Sequencing throughput remained stable, with a slight decrease in amplification rate, but the decline was not linear. The applicant's extensive sequencing tests also showed a slight decrease in amplification rate, but this was not a significant defect. Data quality (AQ30) showed no regular changes. More specifically, the decay coefficient and lead coefficient remained largely unchanged, remaining within the required range for accurate data.

[0086] in Figure 2 The example is a prepared PF-PAM-DNA image. Figure 3 This is the comparison image after APS is cleared. Figure 3 It can be seen that the chip has almost no fluorescence brightness, indicating that the original PAM and DNA in the micropits have been completely removed; Figure 4 The example is a hydrophilic test diagram of the hydrophobic surface compared to the surface after APS treatment. From the figure, it can be seen that the cavity of the chip after hydrophobic treatment is difficult to be wetted by water, so water will not enter the pit. After APS treatment, the chip is drained and the hydrophilicity test is performed again. It can be seen that the chip has become completely hydrophilic, indicating that the chip coating is also removed during APS cleaning and restored to its original state (see Figure 1 105 to 101 process).

[0087] 2. Using KPS to Reset the PF-PEG-DNA Chip Surface

[0088] First, prepare the PF-PEG-DNA chip according to the above example ( Figure 5 ), perform one round of sequencing first. Then use 1MKPS solution to inject into the chip, react at 95 degrees for 20 minutes, then wash with pure water and ethanol, and repeat the washing steps 2 to 3 times to restore the original clean chip state ( Figure 6 Then, the PF-PAM-DNA chip was re-prepared according to the above example ( Figure 7 ), control Figure 5 It can be seen that whether it is the brightness of DNA in the micro-pit or the CV between different pits, the effect is almost completely consistent after resetting 20 times.

[0089] 3. Resetting the FPF-PEG-DNA Chip Surface Using APS

[0090] First, prepare the PFP-PEG-DNA chip according to the above example ( Figure 8 ), perform one round of sequencing first. Then use 2MAPS solution to inject into the chip, react at 95 degrees for 20 minutes, then wash with pure water and ethanol, and repeat the washing steps 2 to 3 times to restore the original clean chip state ( Figure 9 Then, the chip is repeatedly reset, differentially modified, and characterized according to the above example. After repeated cycles of dozens of times, the repeatedly modified PFP-PEG-DNA reset chip is re-characterized ( Figure 10 ), control Figure 5 , the effect after reset is almost exactly the same.

[0091] 4. Using NaPS (sodium persulfate) to reset the FPF-DNA chip surface

[0092] First, prepare the FPF-DNA chip according to the above example ( Figure 11 ), perform one round of sequencing first. Then use 1.5M NaPS solution to inject into the chip, react at 95 degrees for 10 minutes, then wash with pure water and ethanol, and repeat the washing steps 2 to 3 times to restore the original clean chip state ( Figure 12 ).

[0093] 5. Resetting the PF-PEG-DNA Chip Surface Using m-Chloroperoxybenzoic Acid

[0094] First, prepare the FP-PEG-DNA chip according to the above example ( Figure 12Left), first perform a round of sequencing. Then inject 40% m-chloroperoxybenzoic acid into the chip, react at 30 degrees for 10 minutes, then wash with pure water and ethanol, and repeat the washing steps 2 to 3 times. The residue is characterized by the following diagram ( Figure 12 (right picture), it can be seen that there are still a lot of DNA carriers remaining in the micropits, indicating that the actual removal ability of this type is relatively weak, or further optimization of conditions is needed.

[0095] 6. Reset the FPF-PEG-DNA chip surface using hydrogen peroxide

[0096] First, prepare the PFP-PEG-DNA chip according to the above example ( Figure 13 Left), a round of sequencing was performed first. Then, a hydrogen peroxide solution (35%) was injected into the chip, reacted at 30 degrees for 120 minutes, and then washed with pure water and ethanol. The washing steps were repeated 2 to 3 times. The residue was characterized by fluorescence microscopy (Figure 1). Figure 13 (right picture), it can be seen that there are still a lot of DNA carriers remaining in the micropits, indicating that the actual removal ability of this type is relatively weak, or further optimization of conditions is needed.

Claims

1. A method for reusing a gene chip, characterized in that: The method comprises: (1) Reset the gene chip; (2) performing overall hydrophobic treatment on the reset gene chip; (3) Generate RNA or DNA vectors in the micropits of the gene chip using gas seals or oil seals.

2. The method for reusing a gene chip according to claim 1, wherein: The step (1) resetting the gene chip is to clean the gene chip with a cleaning agent to return the gene chip to its initial state without organic modifications; Preferably, the cleaning agent comprises persulfate, peroxide and strong oxidizing compound; Preferably, the persulfate includes potassium persulfate, sodium persulfate and ammonium persulfate; the peroxide includes acidic hydrogen peroxide and Fenton oxidation reagent; and the strong oxidizing compound includes sodium hypochlorite and potassium permanganate.

3. The method for reusing a gene chip according to claim 1, wherein: The step (2) of performing overall hydrophobic treatment on the chip comprises: using fluorosilane or fluorochlorosilane silane to perform hydrophobic treatment on the inner surface of the chip by gas phase or liquid phase modification; Preferably, the step (2) of performing hydrophobic treatment on the chip as a whole comprises: performing hydrophobic treatment on the inside of the chip using a liquid phase hydrophobic modification method; Preferably, the liquid-phase hydrophobic modification comprises: dissolving a hydrophobic reagent in fluorinated oil (e.g., Novec 7200) to prepare a hydrophobic reaction solution, introducing the solution into the activated gene chip cavity, allowing the solution to react for a certain period of time, then washing the solution with a solvent to remove unreacted fluorosilane, and drying the solution and then performing post-baking. Preferably, the hydrophobic agent includes fluorosilane, and the fluorosilane is trichloroperfluorosilane, trimethylfluorosilane or perfluoropolyether silane (such as 1720 or 2202 or similar products), with a concentration of 0.01-1wt%; the reaction time of the static reaction is 1-60min; the post-baking temperature is 60-200°C, and the post-baking time is 1-300min; Preferably, the fluorosilane is a perfluorohalosilane.

4. The method for reusing a gene chip according to claim 1, wherein: The step (3) of directly connecting RNA / DNA in the micropits of the gene chip using a gas seal or oil seal or generating a polymer carrier capable of connecting RNA / DNA in the micropits comprises: using gas or oil to separate the carriers that can subsequently connect with RNA or DNA into individual micropits (referred to as the sealing process), reacting to generate a polymer carrier that can subsequently connect with RNA or DNA in the micropits, and then reacting the RNA or DNA with the reaction sites of the carrier layer to form a hydrophobic chip with RNA or DNA only in the micropits; Preferably, the RNA or DNA carrier is a hydrogel; Preferably, the hydrogel is a functionalized polysaccharide hydrogel, a functionalized polyethylene glycol and its derivatives hydrogel, or a functionalized polyacrylamide and its derivatives hydrogel; further preferably, the functionalized polysaccharide hydrogel is at least one selected from chitosan, hyaluronic acid, trehalose, and cellulose; Preferably, a cross-linking reaction occurs in the reaction 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 group compound cross-linking, or free radical polymerization cross-linking; The gas includes nitrogen, air, and argon; the oil includes mineral oil, fluorocarbon oil or non-polar oil; Preferably, the polymerization initiation conditions of the reaction solution to be polymerized include heating or light irradiation; 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; Preferably, the concentration of the prepolymer or monomer in the reaction solution to be polymerized is 0.025 mg / ml to 25 mg / ml; 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.

5. The method for reusing a gene chip according to claim 1, wherein: The gene chip in step (1) is prepared as follows: (1.1) Perform overall hydrophobic treatment on the blank gene chip; (1.2) Generate RNA or DNA vectors in the chip micro-pits obtained in the previous step using a gas seal or oil seal.

6. The method for reusing a gene chip according to claim 1, wherein: The following steps are also included: (4) cleaning the reactants from the unreacted carrier; (5) Connecting the nucleic acid fragment to be sequenced to the vector; (6) Sequencing; (7) Repeat steps (1)-(6).

7. The method for reusing a gene sequencing chip according to any one of claims 1 to 6, characterized in that: The method of generating RNA or DNA carriers in the micropits of the gene chip using a gas seal or oil seal refers to passing a mixed solution of a multi-arm polyethylene glycol backbone monomer and a cross-linking agent into the gene chip, and then sealing the mixed solution in each micropit of the gene chip by a gas seal or oil seal, and forming RNA or DNA hydrogel carriers in the micropits after the reaction.

8. The method for reusing a gene sequencing chip according to claim 7, characterized in that: The multi-arm polyethylene glycol skeleton monomer is connected to an active group FG1, and the cross-linking agent is connected to an active group FG2, wherein the FG1 is selected from: Carboxylic acid group Amine (-NH2), azide (-N3), terminal alkyne Activated alkynyl Acrylamide One of the following; The FG2 is selected from: Carboxylic acid group Amine (-NH2), azide (-N3), terminal alkyne Activated alkynyl Acrylamide 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.

9. A gene sequencing system, characterized in that: The gene sequencing system comprises: Gene chips, fluid systems and optical systems; Among them, the inner surface of the gene chip has pre-processed micro-pits; The fluid system includes a supply system for multiple reagents, wherein the multiple reagents include: gene sequencing reagents, hydrophobic modification reagents, carrier preparation reagents, and cleaning reagents, wherein the hydrophobic modification reagents are used for hydrophobic chemical modification of the inner surface of the gene chip; the carrier preparation reagents are used for preparing carriers independently existing in the micropits of the gene sequencing chip; and the cleaning reagents are used for resetting the chip.

10. A gene sequencing kit, characterized in that: The gene sequencing kit includes: Gene sequencing reagents, hydrophobic modification reagents, hydrogel preparation reagents, cleaning reagents; Among them, the gene testing reagent is used for sequencing; the hydrophobic modification reagent is used for hydrophobic chemical modification of the inner surface of the gene chip; the hydrogel preparation reagent is used to prepare hydrogel carriers independently existing in the micropits of the gene sequencing chip; and the cleaning reagent is used to reset the chip.

Images