A high loading nucleic acid synthesis chip and a method for preparing the same

Abstract

The application provides a high-loading nucleic acid synthesis chip and a preparation method thereof. The chip comprises a chip substrate, a micropore array and microspheres which are layered on the inner wall of the micropores. The micropore array is a through hole which penetrates the chip substrate from top to bottom, and the inner wall of the through hole is self-assembled and fixed with the microspheres. The microspheres are uniformly and orderly arranged in a layered structure. The chip is prepared through five steps of cleaning the chip and a container, preparing a microsphere colloidal solution, self-assembling the microspheres by using a vertical deposition method, fixing the self-assembled structure by using a 'cold sintering' method and obtaining an expected structure by multiple self-assembling of the microspheres and fixing. By increasing the effective synthesis area of the reaction pool of the DNA synthesis chip, the DNA synthesis loading is improved, the high-loading chip is prepared, and the problem of low loading of the DNA chip is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of synthetic biology, and in particular to a high-loading nucleic acid synthesis chip and a preparation method thereof. BACKGROUND

[0002] DNA de novo synthesis is a key enabling technology of synthetic biology, and DNA synthesizer as its core equipment has experienced two stages. The first generation of column type synthesizer is mainly based on phosphoramidite method, and the synthesis technology using this method is mature and the instrument based on this method has a large market, but it has the disadvantages of low synthesis throughput, high synthesis cost and large consumption of reaction reagents. The second generation of chip type synthesizer takes high-throughput DNA synthesis chip as the core component, which can synthesize tens of thousands of oligonucleotide chains, realize high-throughput DNA synthesis, and reduce the synthesis cost and the amount of reagents consumed.

[0003] DNA synthesis chip is the core component of high-throughput DNA synthesis instrument. By adding A, G, C, T base monomer reagents and other chemical reagents to the reaction pool formed by the DNA chip microwells, chemical reactions are carried out in the DNA synthesis chip reaction pool to realize in-situ DNA synthesis. However, while improving the throughput of the DNA synthesis chip, the area of a single reaction pool is dramatically reduced, and the yield of a single nucleotide chain is very limited, which limits the subsequent detection and application.

[0004] At the same time, the method for fixing the self-assembled structure of the microspheres formed in the preparation process of the current DNA synthesis chip usually selects the high-temperature sintering method. This method requires a high temperature of 1000℃ or above for a long time to ensure that the self-assembled structure does not change. However, the high-temperature sintering condition is harsh, the processing time is long, and the high-temperature sintering will damage the chemical properties of the microsphere surface, resulting in low loading of the DNA synthesis chip. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a high-loading nucleic acid synthesis chip.

[0006] Another technical problem to be solved by the present application is to provide a preparation method of the high-loading nucleic acid synthesis chip.

[0007] To solve the above technical problems, the technical solution of the present application is as follows:

[0008] A high-loading nucleic acid synthesis chip, comprising a chip substrate, a microwell array and microspheres layered on the inner wall of the microwell, the microwell array is a plurality of through holes penetrating the chip substrate from top to bottom, and the inner wall of the through hole is self-assembled and fixed with microspheres, and the microspheres are uniformly and orderly arranged in a layered structure.

[0009] Preferably, the high-loading nucleic acid synthesis chip has a chip base made of pressure-resistant material, such as silicon, silicon-aluminum cast iron, high-aluminum cast iron, polystyrene or carbon-based material, and more preferably, the pressure-resistant material is silicon.

[0010] Preferably, the high-loading nucleic acid synthesis chip has microspheres with modified groups, such as amino, hydroxyl, aldehyde and other effective groups or molecules, and more preferably, the microspheres have amino modified groups.

[0011] Preferably, the high-loading nucleic acid synthesis chip is prepared by the following method: self-assembly of microspheres on the inner wall of the synthesis hole of the DNA synthesis chip is realized by vertical deposition method, the self-assembly structure of the microspheres is controlled by controlling the parameter variables in the self-assembly process, the self-assembly structure is fixed by using "cold sintering" method, and the expected assembly structure is obtained by increasing the self-assembly layers of the microspheres on the basis of the original self-assembly structure.

[0012] Preferably, the high-loading nucleic acid synthesis chip is fixed by alkali solution to have sufficient strength to resist the subsequent washing of the synthesis reagents.

[0013] The preparation method of the high-loading nucleic acid synthesis chip realizes the accurate control of the number of layers of the microspheres on the inner wall of the synthesis hole of the DNA synthesis chip, increases the effective synthesis area of the reaction pool by multiple self-assembly of the microspheres, and realizes the preparation of the high-loading DNA synthesis chip, which mainly includes the following five steps: cleaning the chip and container, preparing a microsphere colloidal solution, self-assembly of the microspheres by vertical deposition method, fixing the self-assembly structure by "cold sintering" method, and obtaining the expected structure by multiple self-assembly of the microspheres and fixing.

[0014] The preparation method of the high-loading nucleic acid synthesis chip realizes the accurate control of the number of layers of the microspheres on the inner wall of the synthesis hole of the DNA synthesis chip, increases the effective synthesis area of the reaction pool by multiple self-assembly of the microspheres, and realizes the preparation of the high-loading DNA synthesis chip, which mainly includes the following five steps: cleaning the chip and container, preparing a microsphere colloidal solution, self-assembly of the microspheres by vertical deposition method, fixing the self-assembly structure by "cold sintering" method, and obtaining the expected structure by multiple self-assembly of the microspheres and fixing.

[0015] Preferably, the preparation method of the high-loading nucleic acid synthesis chip has the following specific steps:

[0016] Step S1: cleaning the chip and container: ultrasonic cleaning of the chip and container with chemical reagents, drying of the container and chip with nitrogen, and obtaining clean chip and container;

[0017] Step S2: Preparation of microsphere colloidal solution: microspheres are added into solvent to obtain a microsphere colloidal solution with a mass fraction of 0.01%-2.5%; the microspheres can self-assemble on the inner wall of the chip hole, and the self-assembled structure formed thereby can be fixed to achieve the preparation of a high-loading chip; the solvent used does not chemically react with the microspheres, and can be quickly evaporated during the self-assembly of the microspheres using the vertical deposition method, so that the microspheres are deposited on the inner wall of the chip hole to form a self-assembled structure;

[0018] Step S3: Self-assembly of microspheres using the vertical deposition method: the microspheres are self-assembled on the inner wall of the hole of the clean chip using the vertical deposition method, and the microspheres are closely arranged on the inner wall of the hole to form a single-layer or multi-layer structure;

[0019] Step S4: Fixing of the self-assembled structure using the "cold sintering" method: to ensure that the structure can resist the subsequent washing of the reagents, the microspheres in the self-assembled structure in step S3 are treated with an alkaline solution at room temperature to fix the self-assembled structure; the self-assembled structure is treated using the "cold sintering" method, and the alkaline solution is added dropwise to the chip, thereby enhancing the binding force between the microspheres and between the microspheres and the inner wall of the chip hole under the premise that the shape of the microspheres and the self-assembled structure formed by the microspheres remain basically unchanged, so that the self-assembled structure formed during the subsequent self-assembly of the microspheres using the vertical deposition method remains stable;

[0020] Step S5: Multiple self-assembly of microspheres to obtain the desired structure and fixation: the self-assembled structure obtained in step S4 is again used to increase the number of layers of microspheres on the inner wall of the chip hole using the vertical deposition method until the number of layers of microspheres reaches the desired structure, and the assembled structure is fixed.

[0021] The microsphere structure obtained after step S5 has a sufficient number of layers (the maximum number of layers is the radius of the hole divided by the diameter of the microspheres), so that the reaction pool area of the synthesis hole of the chip is increased to meet the loading demand.

[0022] Preferably, in the above method for preparing a high-loading nucleic acid synthesis chip, to ensure that the chip and the container are clean and do not contain impurities that can affect the subsequent self-assembly process and the self-assembled structure of the microspheres, the chemical reagents used in step S1 are acetone, anhydrous ethanol, and deionized water, each with a mass fraction of greater than 99%.

[0023] Preferably, in the above method for preparing a high-loading nucleic acid synthesis chip, to ensure that the container can carry a certain mass fraction of the diluted microsphere colloidal solution and the chip, and to provide a good environment for the self-assembly of the microspheres, the container used for the preparation of the high-loading chip by the microspheres is a beaker, an evaporating dish, or a centrifuge tube, or any other container that meets the requirements.

[0024] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to ensure that the structure obtained by self-assembly of microspheres using the vertical deposition method is uniform and orderly, and the thickness of the chip is 200-1000 μm.

[0025] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to ensure that the DNA chip has a reaction pool and contains chemical reagents for reaction, so that the liquid such as microsphere colloidal solution can flow easily and no bubbles are generated, and the shape of the micropore is a circular, rectangular, oval or wedge-shaped pattern.

[0026] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to ensure that the dilution solvent does not chemically react with the microspheres, and in the process of self-assembly of microspheres using the vertical deposition method, the solvent can be quickly evaporated and the microspheres are deposited on the inner wall of the chip hole to form a self-assembly structure, and the solvent used in step S2 is anhydrous ethanol and / or deionized water.

[0027] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to ensure that the DNA synthesis loading is increased while the synthesis flux is not affected, and the chip hole diameter is 50-600 μm.

[0028] Preferably, the preparation method of the high-loading nucleic acid synthesis chip, the process of assembling the microsphere structure in steps S3 and S5 using the vertical deposition method mainly includes three steps of placing the chip and adding the microsphere colloidal solution, building the deposition device, and setting the temperature and evaporating the solvent, and the specific operation steps are as follows:

[0029] Step S31: placing the chip and adding the microsphere colloidal solution: placing the chip horizontally in the center of the deposition container so that the center line of the chip hole is perpendicular to the liquid surface, and adding the microsphere colloidal solution to the deposition container with a pipette until the chip surface is immersed;

[0030] Step S32: building the deposition device: placing the deposition container in a thermostat, and placing a large beaker upside down on the deposition container;

[0031] Step S33: temperature setting and solvent evaporation: setting the temperature of the thermostat for evaporation, and after the solvent in the solution in the deposition container is evaporated, taking out the chip from the deposition container with tweezers and placing it in a dry dish.

[0032] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to reduce the influence of gas flow on the self-assembly structure during the evaporation of the solvent in the process of self-assembly of microspheres using the vertical deposition method, and to realize uniform and orderly self-assembly of microspheres.

[0033] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to fix the self-assembled structure of the microspheres, enhance the binding force between the microspheres and the inner wall of the holes of the chip, and maintain the stability of the self-assembled structure formed in the subsequent self-assembly process of the microspheres by using the vertical deposition method, and resist the washing of the synthesis reagents in the subsequent DNA synthesis process. The self-assembled structure is fixed by using an alkaline solution, and the alkaline solution is a potassium hydroxide solution, a sodium hydroxide solution, or a sodium carbonate solution.

[0034] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to fix the self-assembled structure of the microspheres, enhance the binding force between the microspheres and the inner wall of the holes of the chip, and maintain the stability of the self-assembled structure formed in the subsequent self-assembly process of the microspheres by using the vertical deposition method, and resist the washing of the synthesis reagents in the subsequent DNA synthesis process. The self-assembled structure is fixed by using an alkaline solution, and the alkaline solution is a potassium hydroxide solution, a sodium hydroxide solution, or a sodium carbonate solution.

[0035] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to fix the self-assembled structure of the microspheres, enhance the binding force between the microspheres and the inner wall of the holes of the chip, and maintain the stability of the self-assembled structure formed in the subsequent self-assembly process of the microspheres by using the vertical deposition method, and resist the washing of the synthesis reagents in the subsequent DNA synthesis process. The self-assembled structure is fixed by using an alkaline solution, and the alkaline solution is a potassium hydroxide solution, a sodium hydroxide solution, or a sodium carbonate solution.

[0036] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to fix the self-assembled structure of the microspheres, enhance the binding force between the microspheres and the inner wall of the holes of the chip, and maintain the stability of the self-assembled structure formed in the subsequent self-assembly process of the microspheres by using the vertical deposition method, and resist the washing of the synthesis reagents in the subsequent DNA synthesis process. The self-assembled structure is fixed by using an alkaline solution, and the alkaline solution is a potassium hydroxide solution, a sodium hydroxide solution, or a sodium carbonate solution.

[0037] Preferably, the preparation method of the high-loading nucleic acid synthesis chip is to fix the self-assembled structure of the microspheres, enhance the binding force between the microspheres and the inner wall of the holes of the chip, and maintain the stability of the self-assembled structure formed in the subsequent self-assembly process of the microspheres by using the vertical deposition method, and resist the washing of the synthesis reagents in the subsequent DNA synthesis process. The self-assembled structure is fixed by using an alkaline solution, and the alkaline solution is a potassium hydroxide solution, a sodium hydroxide solution, or a sodium carbonate solution.

[0038] Beneficial effects:

[0039] The aforementioned high-capacity nucleic acid synthesis chip is a core component of a high-throughput DNA synthesizer. Utilizing arrayed micropores on the DNA chip, it achieves DNA synthesis throughput of tens of thousands. By increasing the effective synthesis area of ​​the reaction chamber of the DNA synthesis chip, the DNA synthesis capacity is increased, thus preparing a high-capacity chip and solving the problem of low DNA chip throughput. The developed method for preparing high-capacity chips through microsphere self-assembly utilizes a vertical deposition method to achieve the self-assembly of microspheres on the inner wall of the synthesis wells of the DNA synthesis chip. The self-assembly structure is controlled by adjusting parameters during the self-assembly process, and the formed self-assembly structure is fixed using a "cold sintering" method. The thickness of the microsphere self-assembly is increased multiple times using the vertical deposition method based on the original self-assembly structure, fixing the expected assembly structure and ensuring sufficient strength to resist the washing of synthesis reagents in subsequent processes. This method is mild, time-efficient, and does not affect the surface chemical properties of the microspheres. By fixing the self-assembly structure formed by the microspheres, the self-assembled microspheres increase the effective synthesis area of ​​the reaction chamber of the DNA synthesis chip, thereby increasing the DNA synthesis capacity and preparing a high-capacity chip, solving the problem of low DNA chip throughput. Specifically:

[0040] 1) The method of preparing high-capacity chips through microsphere self-assembly has the advantages of simple operation and inexpensive materials. The preparation of high-capacity chips does not require sophisticated instruments and equipment, and the materials are non-toxic and harmless. It only requires the self-assembly of microspheres on the inner wall of the synthesis well of the DNA synthesis chip to increase the effective reaction area of ​​the reaction cell, thereby increasing the synthesis capacity.

[0041] 2) A method for preparing high-capacity chips by microsphere self-assembly allows for control of the DNA synthesis load by controlling the number of microsphere layers on the inner wall of the chip wells. During the preparation process, the number of self-assembled layers of microspheres on the inner wall of the chip wells can be controlled by adjusting the concentration of the microsphere colloidal solution, thereby controlling the area of ​​the reaction chamber and ultimately the synthesis load.

[0042] 3) The method of preparing high-capacity chips by microsphere self-assembly can fix the self-assembled structure by "cold sintering". It can be carried out under mild conditions, requires a short time, and will not affect the self-assembled structure, especially the chemical properties of the microsphere surface. This ensures that the self-assembled structure can maintain its shape during the vertical deposition to increase the number of microsphere layers and can resist the washing of reagents in subsequent DNA synthesis.

[0043] 4) The method for preparing high-loading chip by microsphere self-assembly can meet the requirements of self-assembly layer number and synthesis loading of the chip by re-self-assembly of the microspheres assembled on the chip. For the chip with microspheres assembled on the inner wall of the hole, the self-assembly structure formed by vertical deposition can be fixed, and the microsphere self-assembly process can be performed again to increase the number of microspheres on the inner wall of the hole of the chip, realize the superposition of the self-assembly layer number, and finally meet the requirements of synthesis loading of the chip. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Figure 1 is a structural schematic diagram of a high-loading chip.

[0045] Figure 2 Figure 2 is an operation step block diagram of the method for preparing high-loading chip by microsphere self-assembly.

[0046] Figure 3 Figure 3 is an operation step block diagram of microsphere self-assembly by vertical deposition method.

[0047] Figure 4 Figure 4 is an experimental device diagram of microsphere self-assembly by vertical deposition method.

[0048] Figure 5 Figure 5 is an effect diagram of microsphere self-assembly structure on the inner wall of the hole of the chip.

[0049] In the figure, 101 is a chip substrate, 102 is a micro-hole array, and 103 is a microsphere.

[0050] 401 is a large beaker, 402 is a deposition container, 403 is a microsphere colloidal solution, and 404 is cotton. DETAILED DESCRIPTION

[0051] The microsphere self-assembly preparation of high-loading chip and the preparation method of the present application will be described in detail below in combination with examples and drawings. In the following embodiments, the present application can use microspheres and hole-bearing chips to prepare high-loading chips for DNA synthesis by microsphere self-assembly, thereby improving the DNA synthesis loading. In addition, the high-loading chip prepared by microsphere self-assembly can be used for micron-level, nanometer-level or picometer-level parallel manipulation of reaction reagents, without reducing the synthesis throughput of the chip.

[0052] Example 1

[0053] As Figure 1 , Figure 5As shown in the figure, the high-loading nucleic acid synthesis chip comprises a chip substrate 101, a micropore array 102, and microspheres 103 layered on the inner wall of the micropores, wherein the chip substrate is a silicon wafer, which is used as a pressure-resistant material to provide the arrangement of the micropore array and the assembly of the microspheres. The micropore array is a plurality of through holes penetrating the upper and lower surfaces of the substrate, and the inner wall of the through holes is self-assembled and fixed with the microspheres. The material of the microspheres is silica with an amino group on the surface, and the layered structure of the microspheres is uniformly and orderly arranged to increase the effective synthesis area of the reaction pool.

[0054] The size of the chip substrate required for preparing the high-loading nucleic acid synthesis chip is 1 mm x 1 mm;

[0055] The micropore of the micropore array required for preparing the high-loading nucleic acid synthesis chip is a circular hole;

[0056] The diameter of the micropore of the micropore array required for preparing the high-loading nucleic acid synthesis chip is 80 μm;

[0057] The pitch (distance between the center points of adjacent micropores) of the micropore of the micropore array required for preparing the high-loading nucleic acid synthesis chip is 80 μm;

[0058] The depth of the micropore of the micropore array required for preparing the high-loading nucleic acid synthesis chip is 300 μm;

[0059] The diameter of the microspheres required for preparing the high-loading nucleic acid synthesis chip is 300 nm.

[0060] As shown in the figure, the method for preparing the high-loading nucleic acid synthesis chip mainly comprises the following steps: Figure 2

[0061] (1) Cleaning the chip and the container: ultrasonic cleaning of the chip and the container with chemical reagents, the container is a beaker (also can be an evaporation dish or a centrifuge tube), drying the container and the chip with nitrogen, obtaining clean chip and container, wherein the chemical reagents used for cleaning the chip and the container are acetone, anhydrous ethanol and deionized water in turn, and the mass fraction of each is greater than 99%;

[0062] (2) Preparation of microsphere colloidal solution: adding microspheres into diluent to obtain a microsphere colloidal solution with a mass fraction of 0.2%, wherein the diluent is deionized water;

[0063] (3) Self-assembly of microspheres by vertical deposition method: self-assembly of microspheres on the inner wall of the holes of the clean chip by vertical deposition method, and obtaining a self-assembly structure with a certain number of layers on the inner wall of the holes, as shown in the figure, Figure 3 The main steps include: placement of the chip and dropwise addition of the microsphere colloidal solution, construction of the deposition device, and temperature setting and solvent evaporation.

[0064] As shown in the figure, Figure 4 ​The specific process of the chip placement and the dropping of the microsphere colloidal solution is as follows: the chip substrate 101 is placed horizontally in the center of the deposition container 402 so that the center line of the chip hole is perpendicular to the liquid level, and the microsphere colloidal solution 403 is dropped into the deposition container by using a pipette until the chip substrate surface is immersed;

[0065] The specific process of the construction of the deposition device is as follows: the deposition container is placed in a constant temperature box, a large beaker 401 is inverted on the deposition container, and the gap of the large beaker is sealed with cotton 404;

[0066] The specific process of temperature setting and solvent evaporation is as follows: the temperature of the constant temperature box is set for solvent evaporation, the temperature for temperature setting and solvent evaporation is 50℃, after the solvent in the solution in the deposition container is evaporated, the constant temperature box is taken out and closed, the chip is taken out from the deposition container with tweezers, and the chip substrate is placed in a drying dish for standing;

[0067] (4) "cold sintering" method for fixing the self-assembled structure: the microspheres in the self-assembled structure in step (3) are treated with an alkaline solution at room temperature to fix the self-assembled structure, and the alkaline solution used in the "cold sintering" method for fixing the self-assembled structure is 0.1 mol / L sodium hydroxide solution;

[0068] (5) multiple self-assembled microspheres to obtain the expected structure and fixation: the above self-assembled structure is again increased in the number of layers of microspheres 103 on the inner wall of the through hole of the chip micro-hole array 102 by using the vertical deposition method until the number of layers of microspheres reaches the expected structure of 3 layers, and the assembled structure is fixed.

[0069] Example 2

[0070] The structure and preparation method of the high-load nucleic acid synthesis chip are the same as those of Example 1, except that:

[0071] The material of the chip substrate required for preparing the high-load nucleic acid synthesis chip is aluminum-silicon cast iron, high-aluminum cast iron, polystyrene or carbon-based material;

[0072] The size of the chip substrate required for preparing the high-load nucleic acid synthesis chip is 1mm×0.5mm, 2mm×1mm or 2mm×2mm;

[0073] The micro-holes of the micro-hole array required for preparing the high-load nucleic acid synthesis chip are rectangular, elliptical or wedge-shaped holes;

[0074] The diameter of the micro-holes of the micro-hole array required for preparing the high-load nucleic acid synthesis chip is 100μm, 200μm or 300μm;

[0075] The pitch (distance between adjacent micro-holes) of the micro-holes of the micro-hole array required for preparing the high-load nucleic acid synthesis chip is 60μm, 100μm or 200μm;

[0076] The microwell depth of the microwell array required for preparing the high-loading nucleic acid synthesis chip is 200 μm, 400 μm or 500 μm;

[0077] The material of the microsphere required for preparing the high-loading nucleic acid synthesis chip is silica with hydroxyl or aldehyde groups on the surface;

[0078] The diameter of the microsphere required for preparing the high-loading nucleic acid synthesis chip is 700 nm, 1.5 μm or 2.5 μm;

[0079] In step (2) of the preparation method, the microspheres are added to a dilution solvent to obtain a microsphere colloidal solution with a mass fraction of 0.3%, 0.5% or 0.7%, and the dilution solvent is anhydrous ethanol or a mixed solution of deionized water and anhydrous ethanol; in step (3), the temperature is set to 35°C, 65°C or 80°C, which is the temperature used for solvent evaporation; in step (4), the alkali solution used for fixing the self-assembled structure by the "cold sintering" method is a 0.1 mol / L potassium hydroxide solution or a sodium carbonate solution.

[0080] Example 3

[0081] After the high-loading chip described in Example 1 is immersed in a base monomer acetonitrile solution for 12 hours, the excess monomers on the surface of the chip are cleaned with acetonitrile, the chip is treated with a deprotection solution to remove the base monomers connected to the chip, an equal amount of p-toluenesulfonic acid acetonitrile solution is added to the deprotection solution, and the peak value of the deprotection solution at a specific wavelength is measured using a UV spectrophotometer to reflect the synthesis loading of the nucleic acid synthesis chip. The same size chip (except for no self-assembled microsphere structure) is subjected to amino modification, and the subsequent treatment (connection of base monomers, deprotection of base monomers) is the same as that of the chip with the microspheres assembled. The peak value of the deprotection solution at a specific wavelength is measured using a UV spectrophotometer to reflect the synthesis loading of the conventional nucleic acid synthesis chip (without a self-assembled microsphere structure). Through experiments, the high-loading chip described in Example 1 can connect base monomers, indicating that the "cold sintering" fixing method does not damage the chemical properties of the microsphere surface. By comparing the peak values of the deprotection solutions of the two chips at a specific wavelength, the fold increase in the loading of the high-loading nucleic acid synthesis chip can be obtained. The experiments show that the deprotection solution corresponding to the high-loading chip described in Example 1 has a peak value at 500 nm, and the peak value is 0.43, indicating that the "cold sintering" fixing method does not damage the amino groups on the surface of the microspheres. The deprotection solution corresponding to the control chip of the same size (except for no self-assembled microsphere structure) also has a peak value at 500 nm, and the peak value is only 0.02. After comparison, the high-loading chip described in the present application has a loading that is more than 20 times higher than that of the conventional chip.

[0082] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing a high loading nucleic acid synthesis chip, characterized by: The application discloses a method for preparing a high-loading DNA synthesis chip by self-assembling microspheres on the inner wall of a chip substrate hole of a DNA synthesis chip, and precisely controlling the layer number of the microspheres on the inner wall, and increasing the effective synthesis area of a reaction pool by multiple self-assembling of the microspheres, and the method mainly comprises the following steps: cleaning a chip substrate and a deposition container, preparing a microsphere colloidal solution, self-assembling the microspheres by using a vertical deposition method, fixing the self-assembled structure by using a "cold sintering" method, and obtaining an expected microsphere layer number by multiple self-assembling of the microspheres and fixing.

2. The method for preparing a high loading nucleic acid synthesis chip according to claim 1, characterized by: The specific steps are as follows: Step S1: cleaning the chip substrate and the deposition container: the chip substrate and the deposition container are cleaned by using chemical reagents, and the deposition container and the chip substrate are dried by using nitrogen, so that a clean chip substrate and a clean deposition container are obtained; Step S2: preparing the microsphere colloidal solution: the microspheres are added into a solvent to obtain a microsphere colloidal solution with a mass fraction of 0.01%-2.5%; the solvent used does not react with the microspheres, and can be quickly evaporated in the process of self-assembling the microspheres by using the vertical deposition method, so that the microspheres are deposited on the inner wall of the chip substrate hole to form a self-assembled structure; Step S3: self-assembling the microspheres by using the vertical deposition method: the microspheres are self-assembled on the inner wall of the clean chip substrate hole by using the vertical deposition method, and the microspheres are closely arranged on the inner wall of the chip substrate hole to form a single-layer or multi-layer structure; Step S4: fixing the self-assembled structure by using the "cold sintering" method: in order to ensure that the structure can resist the subsequent washing of synthesis reagents, the microspheres in the self-assembled structure in step S3 are treated by using an alkaline solution at room temperature, so that the self-assembled structure is fixed; the self-assembled structure is treated by using the "cold sintering" method, the alkaline solution is added dropwise to the chip substrate, and under the premise that the shape of the microspheres and the self-assembled structure formed by the microspheres remain basically unchanged, the binding force between the microspheres and between the microspheres and the inner wall of the chip substrate hole is enhanced, so that the self-assembled structure formed in the subsequent self-assembling process of the microspheres by using the vertical deposition method is stable; Step S5: obtaining an expected structure by multiple self-assembling of the microspheres and fixing: the self-assembled structure obtained in step S4 is used to increase the layer number of the microspheres on the inner wall of the chip substrate hole by using the vertical deposition method again, until the layer number of the microspheres reaches the expected structure, and the self-assembled structure is fixed.

3. The method of claim 2, wherein the high loading nucleic acid synthesis chip is prepared by: The chemical reagents used in step S1 are acetone, anhydrous ethanol and deionized water in sequence, and the mass fraction of each is greater than 99%.

4. The method of claim 2, wherein the high loading nucleic acid synthesis chip is prepared by: The solvent used in step S2 is anhydrous ethanol and / or deionized water, and the hole diameter of the chip substrate hole is 50-600 μm.

5. The method for preparing a high-capacity nucleic acid synthesis chip according to claim 2, characterized in that: The process of self-assembling the microsphere structure by using the vertical deposition method in steps S3 and S5 mainly comprises three steps of placing the chip substrate and adding the microsphere colloidal solution, building a deposition device and setting the temperature and evaporating the solvent, and the specific operation steps are as follows: Step S31: placing the chip substrate and adding the microsphere colloidal solution: the chip substrate is placed horizontally in the center of the deposition container, so that the central axis of the chip substrate hole is perpendicular to the liquid surface, and the microsphere colloidal solution is added dropwise to the deposition container by using a pipette until the chip substrate surface is immersed; Step S32: setting up the deposition device: put the deposition container into the thermostat, and put a large beaker upside down on the deposition container; Step S33: temperature setting and solvent evaporation: set the temperature of the thermostat for evaporation, and after the solvent in the deposition container is evaporated, take out the deposition container from the thermostat, close the thermostat, take out the chip substrate from the deposition container with tweezers, and place the chip substrate in a dry dish.

6. The method of claim 5, wherein: The notch of the large beaker in step S32 is sealed with cotton, and the set value of the thermostat used in step S33 is 30-80℃.

7. The method of claim 2, wherein the high loading nucleic acid synthesis chip is prepared by: The basic solution in step S3 is potassium hydroxide solution, sodium hydroxide solution or sodium carbonate solution.

8. The high loading nucleic acid synthesis chip prepared according to the method of any one of claims 1 to 7, wherein: The chip substrate, the micropore array and the microspheres layered on the inner wall of the micropore, the micropore array is a plurality of chip substrate holes penetrating the chip substrate from top to bottom, and the inner wall of the chip substrate hole is self-assembled and fixed with microspheres, and the microspheres are uniformly and orderly arranged in a layered structure.

9. The high loading nucleic acid synthesis chip of claim 8, wherein: The thickness of the chip substrate is 200-1000μm, the chip substrate is made of pressure-resistant material, and the pressure-resistant material is silicon, aluminum-silicon cast iron, high-aluminum cast iron or carbon-based material; the shape of the micropore array is circular, rectangular, oval or wedge-shaped.

10. The high loading nucleic acid synthesis chip of claim 9, wherein: The carbon-based material is polystyrene.

11. The high loading nucleic acid synthesis chip of claim 8, wherein: The microspheres are silica, and the microspheres have modification groups of amino, hydroxyl or aldehyde group, and the average diameter of the microspheres is 150nm-3μm.

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