Single-molecule array chip with composite resin substrate and preparation method thereof

Abstract

The present invention relates to the field of single-molecule immunoassay chips, and specifically discloses a method for preparing a single-molecule array chip with a composite resin substrate, comprising the following steps: S1, pretreating a glass substrate to obtain a pretreated substrate; S2, heating the composite resin substrate material to melting and maintaining the temperature to keep the composite resin substrate material in a molten state to obtain molten resin; S3, uniformly coating the molten resin on the surface of the pretreated substrate with a coating thickness of 30-75 μm to obtain a coated substrate; S4, pressing a nanoimprint mold onto the coating surface of the coated substrate, and after cooling to below 25°C, removing the nanoimprint mold to obtain an imprinted substrate. Its advantages are: it can reduce the manufacturing cost of the single-molecule array chip and improve the chip flexibility while ensuring high-sensitivity detection.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-detection chips, especially the production technology of single molecule immuno-detection chips. Background Art

[0002] Single Molecule Array (SiMoA) technology is a detection method that uses single molecule counting for trace biological markers. The detection limit of this technology can reach the femtogram (fg / mL) level, and it is currently the most representative single molecule immuno-detection technology. The system originated from a detection system developed by the teams of David Walt and David Duffy. Its detection principle is similar to that of digital PCR, both of which are achieved through single molecule isolation in micro-reactor units combined with single molecule level signal amplification.

[0003] SiMoA technology is a single molecule immuno-detection based on a microarray chip. Thousands of micron-sized micro-wells are engraved (or cast) on a millimeter-sized chip, and the volume of each micro-well is about 40 fl (i.e., the single molecule array chip). Subsequently, immunocomplex magnetic beads are distributed in the micro-wells, and then a high-resolution fluorescence microscope is used to count the fluorescence spots. According to the Poisson distribution theory, the ratio of the number of pores containing both beads and fluorescent products to the total number of pores containing beads is calculated to determine the concentration of the analyte in the test sample. Its operation process is roughly as follows:

[0004] (1) Use magnetic beads with capture antibodies on the surface to capture antigens in the sample;

[0005] (2) Use a biotin-labeled detection antibody to label the captured antigen;

[0006] (3) Add streptavidin-galactosidase complex to bind to the biotin on the detection antibody;

[0007] (4) Mix the washed magnetic beads with the substrate, load them into the single molecule array chip, use a magnetic field to make the magnetic beads fall into the micro-wells that perfectly match their size, and add an oil phase to physically isolate the micro-wells;

[0008] (5) The micro-wells containing galactosidase produce fluorescent products due to the catalytic action of enzyme molecules on the substrate;

[0009] (6) Perform fluorescence imaging on the micro-well array, and achieve quantitative detection by comparing the number of micro-wells emitting fluorescence signals with the standard curve.

[0010] Current SiMoA technology typically uses glass or silicon substrates as chip materials. Although these materials have good chemical stability and optical properties, they have high manufacturing costs, complex processing, and may limit detection sensitivity and the introduction of specific chemical functions in some cases. With the development of single-molecule detection technology, researchers have an urgent need for new chip materials that are more flexible, cost-effective, and capable of supporting high-sensitivity detection. Summary of the Invention

[0011] To reduce the manufacturing cost of single-molecule array chips and improve chip flexibility while ensuring high-sensitivity detection, the present invention provides a single-molecule array chip with a composite resin substrate and a preparation method therefor.

[0012] The technical solution adopted by the present invention is: a single-molecule array chip with a composite resin substrate and a preparation method therefor. The raw materials for production include a glass substrate and a composite resin substrate material. The composite resin substrate material is composed of a base resin material and a synergistic resin material in a mass ratio of 1:0.2 - 2. The base resin material is composed of an epoxy resin and a polycarbonate in a mass ratio of 1:0.1 - 1.5. The synergistic resin material includes polymethyl methacrylate.

[0013] The present invention can be specifically implemented according to the following steps:

[0014] S1. Pretreat the glass substrate to obtain a pretreated substrate.

[0015] S2. Heat the composite resin substrate material to melting and maintain the temperature to keep the composite resin substrate material in a molten state to obtain a molten resin.

[0016] S3. Uniformly coat the molten resin on the surface of the pretreated substrate with a coating thickness of 20 - 70 μm to obtain a coated substrate.

[0017] S4. Press a nanoimprint mold onto the coating surface of the coated substrate. After cooling to below 25°C, remove the nanoimprint mold to obtain an imprinted substrate.

[0018] Those skilled in the art can understand that the above nanoimprint mold is a nanoimprint mold with the required micron structure, and the surface of the mold should be flat and the structure clear. When coating the molten resin, an offset printing machine or a manual coater can be used.

[0019] As a further improvement of the present invention, it further includes a step of soaking and modifying the imprinted substrate with a solution of hexafluoroisobutyltrimethoxysilane. Preferably, the mass concentration of the hexafluoroisobutyltrimethoxysilane solution is 0.2% - 2.0%. Preferably, the solvent of the hexafluoroisobutyltrimethoxysilane solution is acetonitrile or acetone.

[0020] The above-mentioned soaking modification steps can be implemented as follows: completely immerse the imprinted substrate in the hexafluoroisobutyltrimethoxysilane solution for 5 to 30 minutes to obtain a soaked sample; then take out the soaked sample and remove the residual solvent.

[0021] Those skilled in the art can understand that the above method for removing the residual solvent can be to blow dry with dry nitrogen or place it in an oven at 70°C to 80°C to remove the remaining solvent.

[0022] The pretreated substrate of the present invention can be prepared according to the following steps:

[0023] A. Use an ultrasonic cleaner to clean the glass substrate, set the ultrasonic power to 50 to 100 W, and the cleaning time to 5 to 20 minutes;

[0024] B. Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone clean with dust-free air to obtain a cleaned substrate;

[0025] C. Dry the cleaned substrate in a nitrogen stream.

[0026] As a further improvement of the present invention, in step S4, the pressure application range is 5 to 20 MPa, the lamination temperature is 100 to 160°C, and the lamination time is 5 to 15 minutes.

[0027] The present invention also discloses a single-molecule array chip with a composite resin substrate, which is prepared by the preparation method of the single-molecule array chip with a composite resin substrate of the present invention.

[0028] The beneficial effects of the present invention: It can reduce the manufacturing cost of the single-molecule array chip and improve the chip flexibility while ensuring high-sensitivity detection. Description of the Drawings

[0029] Figure 1 It is an imaging diagram of the single-molecule array chip in Example 1 under a fluorescence microscope (Zeiss Axio Observer). Detailed Embodiments

[0030] The present invention will be further described below in conjunction with embodiments.

[0031] Example 1:

[0032] Prepare a single-molecule array chip according to the following steps:

[0033] (1) Use an ultrasonic cleaner to clean the glass substrate, set the ultrasonic power to 100 W, and the cleaning time to 6 minutes;

[0034] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse it thoroughly with dust-free air to avoid water residue, obtaining a cleaned substrate.

[0035] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface, obtaining a pretreated substrate.

[0036] (4) Prepare a composite resin base material, which is composed of a basic resin material and a synergistic resin material in a mass ratio of 1:0.8. The basic resin material is composed of epoxy resin and polycarbonate in a mass ratio of 1:1, and the synergistic resin material is polymethyl methacrylate. Heat the composite resin base material to 250 °C to melt it, and maintain the temperature to keep the composite resin base material in a molten state, obtaining a molten resin.

[0037] (5) Use an offset printing machine to uniformly coat the molten resin on the surface of the pretreated substrate, with a coating thickness of 45 μm, obtaining a coated substrate.

[0038] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, and use electron beam lithography to prepare a square array of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure excellent surface finish and high flatness of the mold, and guarantee the accuracy and reproducibility of the structure through precise manufacturing processes) on the coated surface of the coated substrate, with a pressure of 10 MPa, a pressing temperature of 150 °C, and a pressing time of 6 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0039] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.3% for 10 min to obtain an immersed sample; then take out the immersed sample and blow it dry with dry nitrogen to remove the residual solvent, obtaining a single-molecule array chip.

[0040] Use a fluorescence microscope (Zeiss Axio Observer) to perform fluorescence imaging on the single-molecule array chip of this example, and the results are shown in Figure 1 .

[0041] Example 2:

[0042] Prepare a single-molecule array chip according to the following steps:

[0043] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 90 W and the cleaning time to 10 min.

[0044] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone off with dust-free air to avoid water stain residue, obtaining a cleaned substrate.

[0045] (3) Dry the cleaned substrate in a nitrogen stream to ensure the surface is clean and dust-free, obtaining a pretreated substrate.

[0046] (4) Prepare a composite resin substrate material, which is composed of a base resin material and a synergistic resin material in a mass ratio of 1:0.5. The base resin material is composed of epoxy resin and polycarbonate in a mass ratio of 1:1.5, and the synergistic resin material is polymethyl methacrylate. Heat the composite resin substrate material to 260 °C to melt it, and maintain the temperature to keep the composite resin substrate material in a molten state, obtaining a molten resin.

[0047] (5) Use an offset printing machine to evenly coat the molten resin on the surface of the pretreated substrate, with a coating thickness of 60 μm, obtaining a coated substrate.

[0048] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, and use electron beam lithography to prepare a square array of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure that the surface finish of the mold is excellent and the flatness is high, and ensure the accuracy and reproducibility of the structure through precise manufacturing processes) on the coating surface of the coated substrate, with a pressure of 13 MPa, a pressing temperature of 110 °C, and a pressing time of 10 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0049] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 0.9% for 12 min to obtain a soaked sample; then take out the soaked sample and blow it dry with dry nitrogen to remove the residual solvent, obtaining a single-molecule array chip.

[0050] Example 3:

[0051] Prepare a single-molecule array chip according to the following steps:

[0052] (1) Clean the glass substrate using an ultrasonic cleaner, with the ultrasonic power set to 80 W and the cleaning time to 20 min.

[0053] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone off with dust-free air to avoid water stain residue, obtaining a cleaned substrate.

[0054] (3) Dry the cleaned substrate in a nitrogen stream to ensure the surface is clean and dust-free, obtaining a pretreated substrate.

[0055] (4) Prepare a composite resin base material, which is composed of a base resin material and a synergistic resin material in a mass ratio of 1:2. The base resin material is composed of epoxy resin and polycarbonate in a mass ratio of 1:0.2, and the synergistic resin material is polymethyl methacrylate. Heat the composite resin base material to 250 °C to melt it, and maintain the temperature to keep the composite resin base material in a molten state to obtain a molten resin;

[0056] (5) Use an offset printing machine to evenly coat the molten resin on the surface of the pretreated substrate, with a coating thickness of 30 μm, to obtain a coated substrate;

[0057] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, and use electron beam lithography to prepare a square array of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure that the surface finish of the mold is excellent and the flatness is high, and ensure the accuracy and reproducibility of the structure through precise manufacturing processes) on the coated surface of the coated substrate, with a pressure of 5 MPa, a pressing temperature of 100 °C, and a pressing time of 15 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0058] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 0.4% for 30 min to obtain an immersed sample; then take out the immersed sample and blow it dry with dry nitrogen to remove the residual solvent to obtain a single-molecule array chip.

[0059] Comparative Example 1:

[0060] This comparative example is a control experiment of Example 1, which is carried out under the same conditions and steps as Example 1, and the only difference is that: the composite resin base material does not include the synergistic resin material, and the coating thickness remains unchanged. The specific steps are as follows:

[0061] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 100 W and the cleaning time to 6 min;

[0062] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone clean with dust-free air to avoid water stain residue to obtain a cleaned substrate;

[0063] (3) Dry the cleaned substrate in a nitrogen stream to ensure that the surface is clean and dust-free to obtain a pretreated substrate.

[0064] (4) Prepare a composite resin base material, which only includes a base resin material composed of epoxy resin and polycarbonate in a mass ratio of 1:1. Heat the composite resin base material to 250 °C to melt it, and maintain the temperature to keep the composite resin base material in a molten state to obtain a molten resin;

[0065] (5) Use an offset printing machine to evenly coat the molten resin on the surface of the pretreated substrate, with a coating thickness of 45 μm, to obtain a coated substrate;

[0066] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, and use electron beam lithography to prepare a square array of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure excellent surface finish and high flatness of the mold, and ensure the accuracy and reproducibility of the structure through precise manufacturing processes) on the coating surface of the coated substrate, with a pressure of 10 MPa, a pressing temperature of 150 °C, and a pressing time of 6 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0067] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.3% for 10 min to obtain an immersed sample; then take out the immersed sample and dry it with dry nitrogen to remove the residual solvent to obtain a single-molecule array chip.

[0068] Comparative Example 2:

[0069] This comparative example is a control experiment of Example 1, which is carried out under the same conditions and steps as Example 1, and the only difference is that: the composite resin base material does not include the base resin material, and the coating thickness remains unchanged. The specific steps are as follows:

[0070] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 100 W and the cleaning time of 6 min;

[0071] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic pollutants; then rinse it with dust-free air to remove acetone completely, avoiding water stain residues, to obtain a cleaned substrate;

[0072] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface to obtain a pretreated substrate.

[0073] (4) Prepare a composite resin base material, which only includes a synergistic resin material, and the synergistic resin material is polymethyl methacrylate. Heat the composite resin base material to 250 °C to melt it, and maintain the temperature to keep the composite resin base material in a molten state to obtain a molten resin;

[0074] (5) Use an offset printing machine to uniformly coat the surface of the pretreated substrate with the molten resin, with a coating thickness of 45 μm, to obtain a coated substrate;

[0075] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, and use electron beam lithography to prepare an array arrangement of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure excellent surface finish and high flatness of the mold, and guarantee the accuracy and reproducibility of the structure through precise manufacturing processes) onto the coating surface of the coated substrate, with a pressure of 10 MPa, a pressing temperature of 150 °C, and a pressing time of 6 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0076] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.3% for 10 min to obtain an immersed sample; then take out the immersed sample and dry it with dry nitrogen to remove the residual solvent to obtain a single-molecule array chip.

[0077] Comparative Example 3:

[0078] This comparative example is a control experiment of Example 1, which is carried out under the same conditions and steps as Example 1, and the only difference is that: it does not include the step of soaking and modifying the imprinted substrate with a hexafluoroisobutyltrimethoxysilane solution. The specific steps are as follows:

[0079] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 100 W and the cleaning time of 6 min;

[0080] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse it with dust-free air to remove the acetone completely, avoiding water stain residues, to obtain a cleaned substrate;

[0081] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface to obtain a pretreated substrate.

[0082] (4) Prepare a composite resin base material, which is composed of a base resin material and a synergistic resin material in a mass ratio of 1:0.8. The base resin material is composed of epoxy resin and polycarbonate in a mass ratio of 1:1, and the synergistic resin material is polymethyl methacrylate. Heat the composite resin base material to 250 °C to melt it, and maintain the temperature to keep the composite resin base material in a molten state to obtain a molten resin;

[0083] (5) Use an offset printing machine to uniformly coat the surface of the pretreated substrate with the molten resin, with a coating thickness of 45 μm, to obtain a coated substrate;

[0084] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material and use electron beam lithography to prepare a square array of columnar structures with a diameter of about 5 μm and a height of 4 μm. Ensure excellent surface finish and high flatness of the mold, and guarantee the accuracy and reproducibility of the structure through precise manufacturing processes) on the coating surface of the coated substrate, with a pressure of 10 MPa, a pressing temperature of 150 °C, and a pressing time of 6 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0085] (7) Dry the imprinted substrate with dry nitrogen to obtain a single-molecule array chip.

[0086] Bioanalysis experiment of low-abundance substances:

[0087] Use the single-molecule array chips obtained in the above-mentioned examples and comparative examples for bioanalysis experiments of low-abundance substances to verify their accuracy and reliability in immunoassay. The specific experimental method is as follows:

[0088] Modify magnetic beads with Tau217 primary antibody, modify Tau-217 secondary antibody with NHS-FITC, capture Tau-217 antigens with different concentrations by one-step method and sandwich method, and after capture, drop the complex on the single-molecule chip for single-molecule signal number statistical analysis. The specific experimental method is as follows:

[0089] I. Experimental materials:

[0090] Single-molecule array chips prepared in Examples 1 to 3 and Comparative Examples 1 and 2;

[0091] Tau217 primary antibody: Antibody used to modify magnetic beads;

[0092] NHS-FITC: Fluorescent dye used to modify Tau217 secondary antibody;

[0093] Tau217 antigen: Target molecule for capture;

[0094] Magnetic beads: Used to modify Tau217 primary antibody;

[0095] PBS buffer: Used for dilution and washing.

[0096] II. Experimental steps:

[0097] 1. Modification of magnetic beads:

[0098] (1) Dilute Tau217 primary antibody to a final concentration of 10 μg / mL;

[0099] (2) Add Tau217 primary antibody to magnetic beads to achieve an appropriate surface density, and the reaction temperature

[0100] The temperature is room temperature and the reaction time is 1 hour;

[0101] 2. Modification of Tau217 secondary antibody:

[0102] (1) Dissolve NHS-FITC in PBS buffer at a concentration of 1 mg / mL;

[0103] (2) Add NHS-FITC to Tau217 secondary antibody to form Tau217 secondary antibody-NHS-FITC complex. The reaction temperature is room temperature and the reaction time is 2 hours;

[0104] 3. Capture of target molecules:

[0105] Use the one-step method to add different concentrations of Tau217 antigen solutions (0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 10

[0106] pg / mL, 100 pg / mL, 1 ng / mL) to the modified magnetic beads. The reaction temperature is 4°C and the reaction time is 12 hours;

[0107] 4. Application of the complex:

[0108] Drop the captured Tau217 antigen complex onto the single-molecule chip on the high-entropy alloy chip substrate. 10 μL of the complex solution is used for each chip;

[0109] 5. Imaging analysis:

[0110] Use a fluorescence microscope (Zeiss Axio Observer) to perform fluorescence imaging on the single-molecule chip;

[0111] Use the FITC channel (excitation wavelength: 488 nm, emission wavelength: 515 nm) to detect and record the fluorescence signal;

[0112] Count the number of single-molecule signals under the capture conditions of different concentrations of Tau217 antigen solutions. The results are shown in Table 1.

[0113] Table 1 Statistical table of the number of single-molecule signals under the capture conditions of different concentrations of Tau217 antigen solutions

[0114]

[0115] It can be seen from the detection results of Examples 1 to 3 in Table 1 that the single-molecule array chip prepared by the method of the present invention can generate stable single-molecule signals under the capture conditions of Tau217 antigen solutions with concentrations ranging from 0.1 pg / mL to 1 ng / mL, verifying the accuracy and reliability of the single-molecule chip of the present invention in capturing low-abundance Tau217 antigen.

[0116] From the comparison of Example 1, Comparative Example 1, and Comparative Example 2 in Table 1, it can be seen that on the premise that the thickness of the resin coating remains completely unchanged, the single-molecule signal intensities captured by different concentrations of Tau217 antigen solution on the composite resin substrate material composed of the base resin material and the synergistic resin material (polymethyl methacrylate) in Example 1 are significantly higher than those in Comparative Example 1 using only the base resin and Comparative Example 2 using only the synergistic resin. It can be seen that each component of the composite resin substrate material of the invention has an obvious synergistic effect in improving the single-molecule signal intensity.

[0117] From the comparison of Example 1 and Comparative Example 3 in Table 1, it can be seen that after the imprinted substrate of Example 1 is soaked in hexafluoroisobutyltrimethoxysilane solution, the single-molecule signal intensities captured by different concentrations of Tau217 antigen solution are significantly higher than those in Comparative Example 3 without soaking under the same conditions. It can be seen that the hexafluoroisobutyltrimethoxysilane solution of the present invention has the technical effect of significantly improving the single-molecule signal intensity.

Claims

1. Preparation method of single-molecule array chip with composite resin substrate, characterized in that: The production raw materials include a glass substrate and a composite resin base material. The composite resin base material is composed of a basic resin material and a synergistic resin material in a mass ratio of 1:0.2 - 2. The basic resin material is composed of epoxy resin and polycarbonate in a mass ratio of 1:0.1 - 1.

5. The synergistic resin material includes polymethyl methacrylate. Specifically, it includes the following steps: S1. Pretreat the glass substrate to obtain a pretreated substrate. S2. Heat the composite resin base material until it melts, and maintain the temperature to keep the composite resin base material in a molten state to obtain a molten resin. S3. Uniformly coat the molten resin on the surface of the pretreated substrate with a coating thickness of 20 - 70 μm to obtain a coated substrate. S4. Press a nanoimprint mold onto the coating surface of the coated substrate. After cooling to below 25°C, remove the nanoimprint mold to obtain an imprinted substrate. It also includes a step of soaking and modifying the imprinted substrate with a solution of hexafluoroisobutyltrimethoxysilane.

2. The preparation method of the single-molecule array chip of the composite resin substrate according to claim 1, wherein: The mass concentration of the hexafluoroisobutyltrimethoxysilane solution is 0.2% - 2.0%.

3. The preparation method of the single-molecule array chip with a composite resin substrate according to claim 2, characterized in that: The solvent of the hexafluoroisobutyltrimethoxysilane solution is acetonitrile or acetone.

4. The preparation method of the single-molecule array chip of the composite resin substrate according to any one of claims 1 to 3, characterized in that: The specific step of soaking and modification is: completely immerse the imprinted substrate in the hexafluoroisobutyltrimethoxysilane solution for 5 - 30 minutes to obtain a soaked sample; then take out the soaked sample and remove the residual solvent.

5. The preparation method of the single-molecule array chip of the composite resin substrate according to any one of claims 1 to 3, characterized in that, Step S1 is specifically: A. Clean the glass substrate using an ultrasonic cleaner with an ultrasonic power set to 50 - 100 W and a cleaning time of 5 - 20 minutes. B. Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants. Then rinse the acetone clean with dust-free air to obtain a cleaned substrate. C. Dry the cleaned substrate in a nitrogen stream.

6. The preparation method of the single-molecule array chip of the composite resin substrate according to any one of claims 1 to 3, characterized in that: In step S4, the pressure application range is 5 - 20 MPa, the pressing temperature is 100 - 160°C, and the pressing time is 5 - 15 minutes.

7. A single-molecule array chip of a composite resin substrate prepared by the method for preparing a single-molecule array chip of a composite resin substrate according to any one of claims 1 - 6.

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