Screw rod in-hole type preparation method of microbead chip

Abstract

The invention provides a lead screw hole entering type preparation method of a microbead chip, which comprises the following steps: A, loading of silicon dioxide microspheres: injecting solid silicon dioxide microspheres into a buffer solution containing a dispersing agent, and preparing a monodisperse silicon dioxide microsphere solution with the concentration of 5mg / mL to 50mg / mL through oscillation dispersion; b, putting the microspheres into holes, namely putting a certain number of monocrystalline silicon plates with micropores on a coating table of a coating machine, adding a microsphere solution on the monocrystalline silicon plates, starting the coating machine, dispersing the silicon dioxide microspheres through the movement of a screw rod on the coating machine, and enabling the silicon dioxide microspheres to fall into the micropores of the silicon plates; and after coating is completed, cleaning the silicon plate to remove redundant silicon dioxide microspheres and the buffer solution on the surface to obtain the microsphere chip. By the adoption of the lead screw hole entering type, the hole entering rate of the microbeads reaches 97.6%-99.6%.

Description

Technical Field

[0001] The invention belongs to the technical field of chips, relates to the preparation of a microbead chip, and specifically relates to a screw-in-hole preparation method of a microbead chip. Background Art

[0002] As a low-cost, high-throughput, and high-accuracy detection method, chip detection has become a powerful detection tool for large-scale analysis, providing strong technical support for research in the fields of genetic research and analysis, personalized medicine, and agricultural breeding. The main methods for fixing materials in gene chip preparation include in situ synthesis, spotting, and microsphere loading. Among them, the microsphere loading method is the lowest cost and most efficient method for preparing high-density gene detection chips. The usual preparation method is to use a silicon substrate as the matrix, with hundreds of thousands, millions, or even hundreds of millions of small holes on the surface. Each small hole is used to fix a microsphere, and the surface of the microsphere is covalently coupled with a nucleic acid probe for detection.

[0003] The loading of microspheres on silicon substrates is a key step in the preparation of gene chips. Usually, hundreds of thousands of microspheres modified with different nucleic acid probes are mixed evenly in proportion and then randomly loaded into the small holes of the silicon substrate. How to improve the loading rate of microspheres and make them suitable for large-scale production is one of the key difficulties in the biochip manufacturing process.

[0004] In order to solve the above technical problems, many useful explorations have been made in the prior art. For example, Chinese invention patent No. CN112723303A discloses a method for preparing a microbead chip by spin coating; Chinese invention patent No. CN111282604A discloses a method for preparing a biochip by using bubbles generated by a foaming agent to standardize the range of microspheres that have entered a biochemical chip; Chinese invention patent No. CN112725417A discloses a method for preparing a microbead chip by electroplating. Summary of the invention

[0005] The present invention is based on the above research and continues to explore the preparation method of microbead chips, providing a screw-in-hole preparation method for microbead chips, which achieves the smooth falling of microspheres in the micropores of silicon plates by screw coating.

[0006] The main scheme of the screw-in-hole preparation method of the microbead chip provided by the present invention is as follows: a silicon wafer limiting device is set on the coating table of the coater, and then the microspheres are applied with external force by means of the back and forth movement of the screw of the coater to push them into the micropores of the silicon wafer.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a method for preparing a micro-bead chip by screw-in-hole method, comprising the following steps:

[0009] A. Silica microsphere loading

[0010] The solid silica microspheres are injected into a buffer solution containing a dispersant, and a monodisperse silica microsphere solution having a concentration of 5 mg / mL to 50 mg / mL is prepared by shaking and dispersing;

[0011] B. Microspheres entering the hole

[0012] A certain number of single-crystal silicon plates with micropores are placed on the coating table of the coater, and a microsphere solution is added thereon. The coater is turned on, and the silica microspheres are dispersed and dropped into the micropores of the silicon plate through the movement of the screw on the coater. After the coating is completed, the silicon plate is cleaned to remove excess silica microspheres and buffer solution on the surface to obtain a microbead chip.

[0013] The preferred technical scheme of these two steps is as follows:

[0014] 1. Step A:

[0015] (1) The solid silica microspheres are silica microspheres covalently bonded with oligonucleotide chains, and the preparation method thereof comprises the following steps ac:

[0016] a. Surface modification of silica microspheres with amino groups

[0017] Silica microspheres (CV<5.0%) with a particle size of 500nm to 5um (preferably 3um) are prepared into a 10mg / mL to 200mg / mL xylene suspension; a silanization agent (3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane) is added, and the volume concentration of the silanization agent in the mixed solution is less than 10%, and the reaction is fully carried out to modify the surface of the silica microspheres with amino groups;

[0018] b. Activated silica microspheres

[0019] The silica microspheres modified with amino groups obtained in step a are suspended in acetonitrile, wherein the mass concentration of the microspheres is 10 mg / mL to 200 mg / mL; diisopropylethylamine and cyanuric chloride are added, and the reaction is shaken; the excess reactants are washed with acetonitrile to remove them, and the silica microspheres are suspended in a sodium borate buffer solution, and the pH value is adjusted to 7.5 to 8.5;

[0020] c. Silica microspheres covalently linked to oligonucleotide chains

[0021] Dissolve 100 nmol of the oligonucleotide chain dry powder to be connected in 2M sodium chloride solution, mix with the silica microsphere sodium borate suspension obtained in step B, wherein the silica microsphere content is 10-100 mg, shake and react for 5-8 hours, centrifuge at 1000-3000 rpm, and retain the supernatant; wash and dry.

[0022] (2) The buffer containing a dispersant preferably contains a polymer, including but not limited to polyethylene glycol, polyvinyl alcohol, polyacrylamide, and mixtures thereof.

[0023] (3) After the solid silica microspheres are injected into the buffer solution containing the dispersant, the oscillation dispersion conditions are 30 min and 2500 rpm.

[0024] Step B

[0025] (1) About single crystal silicon panels with micropores

[0026] The preparation method belongs to the prior art, and micropores of a specified depth are set on a single crystal silicon plate by etching, such as: evenly spin-coating a photoresist on the surface of the single crystal silicon plate to form a uniform thin film, using a photolithography machine to transfer the pattern on the mask plate to the photoresist, and using plasma etching technology to etch densely arranged small holes with a diameter similar to that of silica microspheres on the surface of the silicon plate in the hollowed-out part of the photoresist, and the depth of the small holes is about 1μm to 2μm, and the photoresist on the surface of the single crystal silicon plate is removed by a chemical solvent. The surface of the silicon plate is washed three times with a large amount of deionized water to obtain a silicon plate with regularly arranged small holes.

[0027] (2) About coating machine

[0028] The coating machine structure is similar to the coating machine in the prior art. In order to limit the silicon wafer and prevent it from being pulled out of position by the lead screw, the present invention is provided with multiple silicon wafer placement frames on the coating table, and multiple silicon wafer frames are evenly arranged in each frame. The thickness of the silicon wafer frame is lower than the thickness of the silicon wafer to facilitate the coating operation.

[0029] Preferably, the number of silicon wafer placement frames is 5, and each frame is evenly provided with 12 silicon wafer frames, and the thickness of the silicon wafer frame is about 0.2 mm lower than the thickness of the silicon wafer. Specifically, the number of 75 mm*25 mm chips that can be placed on the coating table is 12, 24, 36, 48, or 60.

[0030] (3) Regarding dispersion coating operation

[0031] Fix the screw on the coater, place the silicon plates one by one on the homemade coater module, put the coater module in the clamp and tighten the two ends to fix it, lower the handle to drop the scraper, and add the silica microbead solution to the coater module. Rotate the fine tuner to make the scraper drop to the bottom (hear a click), press the zero key on the differential head to set the zero point, and after setting to zero, rotate the fine tuner in the opposite direction to set the coating thickness. Turn on the machine and click anywhere on the screen to enter the experimental interface. Click the input box behind "Set Speed", click the number to enter the speed required for the experiment, and press the "OK" key to save. Place the coating liquid in front of the scraper and click the "Start" button on the screen. After the coating is completed, lift the scraper lifting and lowering handle, click "Reset", clean the scraper, take out the coater module, take out the silicon plates one by one, and use buffer and water to wash them to obtain a group of silicon plates loaded with silica microspheres.

[0032] In terms of process parameters, 5000uL to 50000uL of silica microsphere solution is dripped into each silicon wafer placement frame; when dispersing the silica microspheres, the coating speed of the coater is 2-180mm / s, the screw specification accuracy of the coater is 0.003mm, the effective coating width is 30cm, and the coating thickness is 2-10μm.

[0033] The coating area of ​​the coater is 30 cm wide and 40 cm long.

[0034] The results show that the screw-type hole-entry method of the present invention can achieve a hole-entry rate of microbeads of 97.6% to 99.6%, and can achieve 60 silicon plates to be laid at a time, which is suitable for large-scale preparation of microbead chips.

[0035] The second aspect of the present invention provides a microbead chip prepared by the method described in the first aspect.

[0036] The third aspect of the present invention provides a use of the microbead chip as described above in the preparation of a biochip.

[0037] Functions and Effects of the Invention

[0038] In terms of effect, the screw-entry method of the present invention can achieve a micro-bead penetration rate of 97.6% to 99.6%.

[0039] In terms of production efficiency, the present invention can achieve the laying of 60 silicon plates at a time, greatly improving the production efficiency of micro-ball insertion into micro-bead chips.

[0040] In terms of input cost, the present invention can achieve corresponding operations and technical effects by improving the chip limiting position of the existing coating machine, and the production input cost is low, which is conducive to industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A schematic diagram of the structure of the coating station of the present invention is shown. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0043] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0044] Example 1: Preparation of 12 microbead chips simultaneously

[0045] 1. Preparation of Silica Microspheres Covalently Binding Oligonucleotide Chains

[0046] a. Surface modification of silica microspheres with amino groups

[0047] The raw material is solid silica microspheres with silanol groups on the surface, with a particle size of 3 μm, which are prepared into a 100 mg / mL xylene suspension. 3-aminopropyltrimethoxysilane, a silanization agent that can amino-aminolate the surface, is added, and the concentration of the silanization agent in the mixed solution is 1.0%. The mixed solution is shaken at room temperature for 1 hour. After the reaction is fully completed, the surface of the microspheres will be amino-grouped.

[0048] b. Activated silica microspheres

[0049] The microspheres with amino groups on the surface are suspended in acetonitrile, and the mass concentration of the microspheres is 100 mg / mL. Diisopropylethylamine and cyanuric chloride are added, and the concentration of diisopropylethylamine in the mixed solution is 0.3M, and the concentration of cyanuric chloride is 0.1M. The mixed solution is shaken at room temperature for 1 hour, and cyanuric chloride reacts with the amino groups on the surface of the microspheres to greatly enhance the activity of the surface groups of the microspheres. After washing with acetonitrile 3 to 5 times to remove excess reactants and products, rinse with 2M sodium borate buffer 3 to 5 times, and finally suspend the microspheres in sodium borate buffer, and add an appropriate amount of hydrochloric acid to adjust the pH value of the suspension to 7.5 to 8.5.

[0050] c. Silica microspheres covalently linked to oligonucleotide chains

[0051] Dissolve 100 nmol of the oligonucleotide chain powder to be connected in 2M sodium chloride solution (the length of the oligonucleotide chain is 15mer, and the terminal is modified with an amino group), mix it with an appropriate amount of sodium borate microsphere suspension, wherein the microsphere content is 50 mg, and shake the mixture at room temperature for 5 to 8 hours to allow the oligonucleotide chain to fully contact the microspheres and react with the active groups on the surface of the microspheres. After the connection reaction is completed, centrifuge the mixture at 1000 to 3000 rpm and retain the supernatant. Wash the microspheres with ultrapure water 3 to 5 times to remove excess reactants and products, dry them into dry powder form, and store them for use.

[0052] The microspheres coupled with the oligonucleotide chain were hybridized with the target (the target is complementary to the oligonucleotide chain and has a FAM fluorescent group at the end), incubated for 30 minutes at a temperature 20°C lower than the melting temperature of the corresponding oligonucleotide chain, and the fluorescence intensity was detected. The concentration of the microspheres during the detection was 2 mg / mL, and the excitation / emission filters used were 488 nm / 520 nm.

[0053] All fluorescence intensities mentioned in the present invention are measured under the above conditions. The conversion of fluorescence intensity and concentration uses a standard curve (obtained by measuring the fluorescence value of the prepared standard substance): C=8.797e-7*I-0.0055. Wherein C is the concentration of the fluorescent group, in nmol / mL, and I is the fluorescence intensity. The concentration of the fluorescent group can be obtained from the fluorescence intensity, and the amount of microspheres is known, so the number of fluorescent groups per unit surface area can be calculated. Thus, the amount of oligonucleotide chains involved in the connection reaction and the connection efficiency of the microspheres and the oligonucleotide chains are calculated. After multiple experimental verifications, the connection efficiency can reach about 9500 to 10500 oligonucleotide chains / square micron (microsphere particle size 3um, multiple measurements of fluorescence intensity of 40048, 42619, 39890).

[0054] 2. Preparation method of screw hole for microbead chip

[0055] A. Etching

[0056] The photoresist is evenly spin-coated onto the surface of the single crystal silicon plate to form a uniform thin film. The pattern on the mask is transferred to the photoresist using a photolithography machine. In the hollowed-out part of the photoresist, the plasma etching technology is used to etch densely arranged small holes with a diameter similar to that of the silicon dioxide microspheres on the surface of the silicon plate. The depth of the small holes is about 1um. The photoresist on the surface of the single crystal silicon plate is removed using a chemical solvent, and the surface of the silicon plate is cleaned 3 times with a large amount of deionized water.

[0057] B. Silica micro-ball screw-type hole entry

[0058] The coating machine is used, and the structure of the coating machine is similar to that of the coating machine in the prior art. In order to achieve silicon wafer limitation and prevent it from being pulled out of position by the screw rod, the coating station is improved, such as Figure 1 In the present invention, a plurality of silicon wafer placing frames 2 are arranged on a coating station 1, and a plurality of silicon wafer frames 21 are evenly arranged in each frame. The thickness of the silicon wafer frame is lower than the thickness of the silicon wafer to facilitate the coating operation.

[0059] Specifically, there are 5 silicon wafer placement frames, each of which is evenly equipped with 12 silicon wafer frames, and the thickness of the silicon wafer frame is 0.5mm, which is about 0.2mm lower than the thickness of the silicon wafer. Specifically, the number of 75mm*25mm chips that can be placed on the coating table is 12, 24, 36, 48, and 60.

[0060] In this embodiment, 12 silicon wafers are coated simultaneously to prepare microbead chips. The specific steps are as follows:

[0061] (1) A certain amount of monodisperse solid silica microspheres coupled with oligonucleotides were injected into a buffer containing a dispersant, and a monodisperse silica microsphere solution with a concentration of 5 mg / mL was prepared by oscillating and dispersing (30 min, 2500 rpm).

[0062] (2) Fix the screw (15 wires, total length 40cm, coating width 32cm) on the coater, place 12 silicon plates one by one on the homemade coater module, place the coater module in the clamp and tighten the two ends to fix it, lower the handle to drop the scraper, and add the silica microbead solution to the coater module. Rotate the fine tuner to make the scraper drop to the bottom (hear a click), press the zero key on the differential head to set the zero point, and after setting to zero, rotate the fine tuner in the opposite direction to set the coating thickness (3um). Turn on the machine and click anywhere on the screen to enter the experimental interface. Click the input box behind "Set Speed", click the number to enter the speed required for the test (5mm / s), and press the "OK" key to save. Place the coating liquid in front of the scraper and click the "Start" button on the screen. After coating is completed, lift the scraper lifting and lowering handle and click "Reset". Change the coating thickness (2um), and click anywhere on the screen again to enter the experimental interface. Click the input box behind "Set Speed", click the number to enter the speed required for the test (5mm / s), and press the "OK" button to save. Place the coating liquid in front of the scraper and click the "Start" button on the screen. After the coating is completed, lift the scraper lifting handle and click "Reset". Clean the scraper, take out the coater module, take out the silicon plates one by one, use buffer and water to clean, and get a set of 12 silicon plates loaded with silica microspheres.

[0063] The silicon plate obtained by the method has a microsphere penetration rate in the small pores on the silicon plate of 97.6% to 99.3%. The product results are summarized in Table 1.

[0064] Table 1 Summary of microsphere penetration rate of 12 silicon plates

[0065] Silicon board number 1 2 3 4 5 6 7 8 9 10 11 12 Porosity 97.8％ 98.3％ 98.0％ 98.5％ 99.2％ 99.3％ 99.1％ 99.2％ 98.7％ 98.8％ 98.4％ 97.6％

[0066] Example 2: Preparation of 60 microbead chips simultaneously

[0067] 1. Preparation of Silica Microspheres Covalently Binding Oligonucleotide Chains

[0068] The steps are the same as in Example 1.

[0069] 2. Preparation method of screw hole for microbead chip

[0070] A. Etching:

[0071] The photoresist is evenly spin-coated onto the surface of the single crystal silicon plate to form a uniform thin film. The pattern on the mask is transferred to the photoresist using a photolithography machine. In the hollowed-out part of the photoresist, the plasma etching technology is used to etch densely arranged small holes with a diameter similar to that of the silicon dioxide microspheres on the surface of the silicon plate. The depth of the small holes is about 1um. The photoresist on the surface of the single crystal silicon plate is removed using a chemical solvent, and the surface of the silicon plate is cleaned 3 times with a large amount of deionized water.

[0072] B. Loading silica microspheres:

[0073] A certain amount of monodisperse solid silica microspheres coupled with oligonucleotides were injected into the buffer containing dispersant, and after shaking and dispersing (30min, 2500rpm), a monodisperse silica microsphere solution with a concentration of 5mg / mL was prepared. The screw (15 wires, total length 40cm, coating width 32cm) was fixed on the coater, and 60 silicon plates were placed one by one on the homemade coater module. The coater module was placed in the clamp and tightened at both ends. The handle was lowered to drop the scraper, and the silica microbead solution was added to the coater module. Rotate the fine tuner to make the scraper drop to the bottom (hear a click), press the zero key on the differential head to set the zero point, and after zeroing, rotate the fine tuner in the opposite direction to set the coating thickness (3um). Turn on the machine and click anywhere on the screen to enter the experimental interface. Click the input box behind "Set Speed", click the number to enter the speed required for the experiment (5mm / s), and press the "OK" key to save. Place the coating liquid in front of the scraper and click the "Start" button on the screen. After the coating is completed, lift the scraper handle and click "Reset". Change the coating thickness (2um), click anywhere on the screen again to enter the experimental interface. Click the input box behind "Set Speed", click the number to enter the speed required for the test (5mm / s), and press the "OK" button to save. Place the coated liquid in front of the scraper and click the "Start" button on the screen. After the coating is completed, lift the scraper handle and click "Reset". Clean the scraper, take out the coater module, take out the silicon plates one by one, and wash them with buffer and water to obtain a group of 60 silicon plates loaded with silica microspheres.

[0074] The silicon plate obtained by the method has a microsphere penetration rate in the small holes on the silicon plate of 97.6% to 99.6%. The product results are summarized in Table 2.

[0075] Table 2 Summary of microsphere penetration rate of 60 silicon plates

[0076] Silicon board number 1 2 3 4 5 6 7 8 9 10 11 12 Porosity 97.8％ 98.3％ 98.0％ 98.5％ 99.2％ 99.3％ 99.1％ 99.2％ 98.7％ 98.8％ 98.4％ 97.6％ Silicon board number 13 14 15 16 17 18 19 20 21 22 23 24 Porosity 98.2％ 98.4％ 98.8％ 99.2％ 99.4％ 98.6％ 99.4％ 99.1％ 99.2％ 98.5％ 98.7％ 98.2％ Silicon board number 25 26 27 28 29 30 31 32 33 34 35 36 Porosity 97.7％ 98.5％ 98.6％ 98.4％ 99.3％ 99.3％ 99.4％ 99.4％ 99.3％ 98.9％ 98.7％ 98.0％ Silicon board number 37 38 39 40 41 42 43 44 45 46 47 48 Porosity 98.4％ 99.2％ 99.7％ 99.3％ 99.6％ 99.3％ 99.4％ 99.0％ 99.1％ 98.8％ 98.6％ 98.4％ Silicon board number 49 50 51 52 53 54 55 56 57 58 59 60 Porosity 98.2％ 98.8％ 99.3％ 99.2％ 99.4％ 99.5％ 99.3％ 99.2％ 99.0％ 98.4％ 98.3％ 97.8％

[0077] In summary, the screw hole entry method can meet the production needs of large-scale silicon wafer hole entry. If the output needs to be increased, it can be achieved by adding equipment. The requirements for personnel and operation are low, and batch production can be repeatable and operable, which greatly reduces production costs and greatly improves production efficiency.

[0078] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a micro-bead chip by screw-in-hole method, characterized in that: The steps include: A. Silica microsphere loading The solid silica microspheres are injected into a buffer solution containing a dispersant, and a monodisperse silica microsphere solution having a concentration of 5 mg / mL to 50 mg / mL is prepared by shaking and dispersing; B. Microspheres entering the hole A certain number of single crystal silicon plates with micropores are placed on the coating table of the coating machine, and the microsphere solution is added thereon. The coating machine is turned on, and the silicon dioxide microspheres are dispersed and dropped into the micropores of the silicon plate through the movement of the screw on the coating machine; After coating, the silicon plate is cleaned to remove excess silica microspheres and buffer on the surface to obtain a microbead chip.

2. The method for preparing a microbead chip by screw-in-hole method according to claim 1, characterized in that: in, In step A, the dispersant is selected from any one or more of polyethylene glycol, polyvinyl alcohol, and polyacrylamide.

3. The method for preparing a micro-bead chip by screw-in-hole method according to claim 1, characterized in that: in, In step A, the concentration of the monodispersed silica microsphere solution is 5 mg / mL.

4. The method for preparing a micro-bead chip by screw-in-hole method according to claim 1, characterized in that: in, In step B, the depth of the microholes on the single crystal silicon plate is 1 μm to 2 μm.

5. The method for preparing a micro-bead chip by screw-in-hole method according to claim 1, characterized in that: in, In step B, a plurality of silicon wafer placement frames are arranged on the coating table, and a plurality of silicon wafer frames are evenly arranged in each frame, and the thickness of the silicon wafer frame is lower than the thickness of the silicon wafer.

6. The method for preparing a micro-bead chip by screw-in-hole method according to claim 5, characterized in that: in, In step B, the number of the silicon wafer placement frames is 5, and 12 silicon wafer frames are evenly arranged in each frame. The thickness of the silicon wafer frame is about 0.2 mm lower than the thickness of the silicon wafer.

7. The method for preparing a micro-bead chip by screw-in-hole method according to claim 5, characterized in that: in, In step B, 5000uL to 50000uL of silica microsphere solution is dripped into each silicon wafer placement frame; when dispersing the silica microspheres, the coating speed of the coater is 2-180mm / s, the specification accuracy of the coating machine screw is 0.003mm, the effective coating width is 30cm, and the coating thickness is 2-10μm.

8. The method for preparing a micro-bead chip by screw-in-hole method according to claim 1, characterized in that: in, The coating area of ​​the coater is 30 cm wide and 40 cm long.

9. A microbead chip, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the microbead chip according to claim 9 in preparing a biochip.

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