Single-molecule array chip based on a functionalized epoxy resin substrate and its preparation method

Abstract

The present invention relates to the field of single-molecule immunoassay chips, and specifically discloses a preparation method of a single-molecule array chip based on a functionalized epoxy resin substrate, comprising the following steps: S1, pretreating a glass substrate to obtain a pretreated substrate; S2, heating a resin substrate material to melting and maintaining the temperature to keep the 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 20-60 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 on the premise of 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, the immunocomplex magnetic beads are distributed in the micro-wells, and then the fluorescence spots are counted with the aid of a high-resolution fluorescence microscope. 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) Capture the antigen in the sample with magnetic beads labeled with capture antibodies on the surface;

[0005] (2) Label the captured antigen with a detection antibody labeled with biotin;

[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 after the reaction, load them into the single molecule array chip, use a magnetic field to make the magnetic beads fall into the micro-wells that exactly 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 achieve the reduction of the manufacturing cost of single-molecule array chips and the improvement of chip flexibility while ensuring high-sensitivity detection, the present invention provides a single-molecule array chip based on a functionalized epoxy resin substrate and a preparation method thereof.

[0012] The technical solution adopted by the present invention is: a single-molecule array chip based on a functionalized epoxy resin substrate and a preparation method thereof. The raw materials for production include a glass substrate and a resin substrate material, and the resin substrate material includes epoxy resin.

[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 resin substrate material to melting and maintain the temperature to keep the resin substrate material in a molten state to obtain molten resin;

[0016] S3. Uniformly coat the molten resin on the surface of the pretreated substrate, and the coating thickness is 20 - 60 μm to obtain a coated substrate;

[0017] S4. Press a nanoimprint mold on the coating surface of the coated substrate, and 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 should be clear. When coating the molten resin, an offset press 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 and modification steps can be implemented as follows: completely immerse the imprinted substrate in the hexafluoroisobutyltrimethoxysilane solution for 3 to 20 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] As a further improvement of the present invention, the resin substrate material is compounded from epoxy resin and polymethyl methacrylate in a mass ratio of 1:0.1 to 0.8.

[0023] The pretreated substrate of the present invention can be prepared according to the following steps:

[0024] 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;

[0025] 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;

[0026] C. Dry the cleaned substrate in a nitrogen stream.

[0027] As a further improvement of the present invention, in step S4, the pressure application range is 2 to 5 MPa, the pressing temperature is 30 to 140°C, and the pressing time is 5 to 10 minutes.

[0028] The present invention also discloses a single-molecule array chip based on a functionalized epoxy resin substrate, which is prepared by the preparation method of the single-molecule array chip based on the functionalized epoxy resin substrate of the present invention.

[0029] 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

[0030] Figure 1 It is an imaging diagram of the single-molecule array chip in Example 1 under a fluorescence microscope (Zeiss Axio Observer). Detailed Description of the Invention

[0031] The present invention will be further described below in conjunction with the embodiments.

[0032] Example 1:

[0033] Prepare a single-molecule array chip according to the following steps:

[0034] (1) Clean the glass substrate using an ultrasonic cleaner, set the ultrasonic power to 100 W, and the cleaning time to 10 min;

[0035] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone with dust-free air to avoid water residue, and obtain a cleaned substrate;

[0036] (3) Dry the cleaned substrate in a nitrogen stream to ensure the surface is clean and dust-free, and obtain a pretreated substrate.

[0037] (4) Take epoxy resin as the resin substrate material, heat the resin substrate material to 200 °C to melt it, and maintain the temperature to keep the resin substrate material in a molten state to obtain molten resin;

[0038] (5) Use an offset printer 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;

[0039] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, 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 3 MPa, a pressing temperature of 80 °C, and a pressing time of 6 min; after cooling to 23 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0040] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.2% for 5 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.

[0041] 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 .

[0042] Example 2:

[0043] Prepare a single-molecule array chip according to the following steps:

[0044] (1) Clean the glass substrate using an ultrasonic cleaner, set the ultrasonic power to 80 W, and the cleaning time to 6 min;

[0045] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse the acetone with dust-free air to avoid water residue, and obtain a cleaned substrate;

[0046] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface, obtaining a pretreated substrate.

[0047] (4) Take epoxy resin as the resin substrate material, heat the resin substrate material to 210 °C to melt it, and maintain the temperature to keep the resin substrate material in a molten state, obtaining molten resin.

[0048] (5) Use an offset printing machine to evenly coat the molten resin on the surface of the pretreated substrate, with a coating thickness of 50 μm, obtaining a coated substrate.

[0049] (6) Press a nanoimprint mold of uniform specification (select silicon as the mold material, 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 5 MPa, a pressing temperature of 120 °C, and a pressing time of 5 min; after cooling to 25 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0050] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 0.5% for 8 min to obtain an immersed sample; then take out the immersed sample and dry it with dry nitrogen to remove the residual solvent, obtaining a single-molecule array chip.

[0051] Example 3:

[0052] This example is implemented under the same conditions and steps as Example 1, and the only difference is that: the resin substrate material in step (4) is compounded by epoxy resin and polymethyl methacrylate in a mass ratio of 1:0.5, while keeping the coating thickness unchanged. The specific steps are as follows:

[0053] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 100 W and the cleaning time to 10 min.

[0054] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then use dust-free air to rinse the acetone clean, avoiding water stain residues, obtaining a cleaned substrate.

[0055] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface, obtaining a pretreated substrate.

[0056] (4) Take a resin substrate material compounded by epoxy resin and polymethyl methacrylate in a mass ratio of 1:0.5, heat the resin substrate material to 200 °C to melt it, and maintain the temperature to keep the resin substrate material in a molten state, obtaining molten resin.

[0057] (5) Use an offset press 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;

[0058] (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) onto the coating surface of the coated substrate, with a pressure of 3 MPa, a pressing temperature of 80 °C, and a pressing time of 6 min; after cooling to 23 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0059] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.2% for 5 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.

[0060] Comparative Example 1:

[0061] 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 resin substrate material is all polymethyl methacrylate, and the coating thickness remains unchanged. The specific steps are as follows:

[0062] Prepare a single-molecule array chip according to the following steps:

[0063] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set to 100 W and the cleaning time to 10 min;

[0064] (2) Transfer the cleaned glass substrate to acetone for soaking to remove grease and organic contaminants; then rinse it clean with dust-free air to avoid water stain residue, to obtain a cleaned substrate;

[0065] (3) Dry the cleaned substrate in a nitrogen stream to ensure the surface is clean and dust-free, to obtain a pretreated substrate.

[0066] (4) Take polymethyl methacrylate as the resin substrate material, heat the resin substrate material to 200 °C to melt it, and maintain the temperature to keep the resin substrate material in a molten state, to obtain a molten resin;

[0067] (5) Use an offset press 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;

[0068] (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 microns and a height of 4 microns. 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 coated surface of the coated substrate, with a pressure of 3 MPa, a pressing temperature of 80 °C, and a pressing time of 6 min; after cooling to 23 °C, remove the nanoimprint mold to obtain an imprinted substrate.

[0069] (7) Immerse the imprinted substrate completely in a solution of hexafluoroisobutyltrimethoxysilane with acetonitrile as the solvent and a mass concentration of 1.2% for 5 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.

[0070] Comparative Example 2:

[0071] 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:

[0072] (1) Use an ultrasonic cleaner to clean the glass substrate, with the ultrasonic power set at 100 W and the cleaning time at 10 min;

[0073] (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, avoiding water stains remaining, to obtain a cleaned substrate;

[0074] (3) Dry the cleaned substrate in a nitrogen stream to ensure a clean and dust-free surface to obtain a pretreated substrate.

[0075] (4) Take epoxy resin as the resin substrate material, heat the resin substrate material to 200 °C to melt it, and maintain the temperature to keep the resin substrate material in a molten state to obtain molten resin;

[0076] (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, to obtain a coated substrate;

[0077] (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 microns and a height of 4 microns. 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 coated surface of the coated substrate, with a pressure of 3 MPa, a pressing temperature of 80 °C, and a pressing time of 6 min; after cooling to 23 °C, remove the nanoimprint mold to obtain an imprinted substrate;

[0078] (7) Dry the imprinted substrate with dry nitrogen to obtain a single-molecule array chip.

[0079] Bioanalysis experiment of low-abundance substances:

[0080] Use the single-molecule array chips obtained in the above examples and comparative examples for bioanalysis experiments of low-abundance substances to verify their accuracy and reliability in immunoassays. The specific experimental methods are as follows:

[0081] Modify magnetic beads with Tau217 primary antibody, modify Tau-217 secondary antibody with NHS-FITC, capture Tau-217 antigens at 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 methods are as follows:

[0082] I. Experimental materials:

[0083] Single-molecule array chips prepared in Examples 1 to 3 and Comparative Examples 1 and 2;

[0084] Tau217 primary antibody: Antibody used to modify magnetic beads;

[0085] NHS-FITC: Fluorescent dye used to modify Tau217 secondary antibody;

[0086] Tau217 antigen: Target molecule for capture;

[0087] Magnetic beads: Used to modify Tau217 primary antibody;

[0088] PBS buffer: Used for dilution and washing.

[0089] II. Experimental steps:

[0090] 1. Modification of magnetic beads:

[0091] (1) Dilute Tau217 primary antibody to a final concentration of 10 μg / mL;

[0092] (2) Add Tau217 primary antibody to magnetic beads to achieve an appropriate surface density, and the reaction temperature

[0093] is room temperature and the reaction time is 1 hour;

[0094] 2. Modification of Tau217 secondary antibody:

[0095] (1) Dissolve NHS-FITC in PBS buffer at a concentration of 1 mg / mL;

[0096] (2) Add NHS-FITC to the Tau217 secondary antibody to form a Tau217 secondary antibody-NHS-FITC complex. The reaction temperature is room temperature and the reaction time is 2 hours.

[0097] 3. Capture the target molecule:

[0098] 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

[0099] 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.

[0100] 4. Application of the complex:

[0101] Drop the captured Tau217 antigen complex onto the single-molecule chip on the high-entropy alloy chip substrate. Use 10 μL of the complex solution for each chip.

[0102] 5. Imaging analysis:

[0103] Perform fluorescence imaging on the single-molecule chip using a fluorescence microscope (Zeiss Axio Observer).

[0104] Detect and record the fluorescence signal using the FITC channel (excitation wavelength: 488 nm, emission wavelength: 515 nm).

[0105] 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.

[0106] Table 1 Statistical table of the number of single-molecule signals under the capture conditions of different concentrations of Tau217 antigen solutions

[0107]

[0108] 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 antigens.

[0109] From the comparison of Example 3, Example 1, and Comparative Example 1 in Table 1, it can be seen that on the premise that the resin coating thickness remains completely unchanged, the single-molecule signal intensities captured by the Tau217 antigen solutions with different concentrations of the composite resin substrate material composed of epoxy resin and polymethyl methacrylate in Example 3 are significantly higher than those in Example 1 using only epoxy resin and Comparative Example 1 using only polymethyl methacrylate. Especially at low antigen solution concentrations of 0.1 pg / mL, 0.5 pg / mL, and 1 pg / mL, extremely obvious sensitivity differences are shown. It can be seen that the components of the invented composite resin substrate material have an obvious synergistic effect in improving the single-molecule signal intensity.

[0110] From the comparison of Example 1 and Comparative Example 2 in Table 1, it can be seen that after the imprinted substrate in Example 1 is soaked in the solution of hexafluoroisobutyltrimethoxysilane, the single-molecule signal intensities captured by the Tau217 antigen solutions with different concentrations are significantly higher than those in Comparative Example 2 without soaking under the same conditions. It can be seen that the solution of hexafluoroisobutyltrimethoxysilane of the present invention has the technical effect of significantly improving the single-molecule signal intensity.

Claims

1. A method for preparing a single-molecule array chip based on a functionalized epoxy resin substrate, characterized in that: The production raw materials include a glass substrate and a resin base material. The resin base material is compounded from epoxy resin and polymethyl methacrylate in a mass ratio of 1:0.1 - 0.

8. The specific steps are as follows: S1. Pretreat the glass substrate to obtain a pretreated substrate; S2. Heat the resin base material to melting and maintain the temperature to keep the resin base material in a molten state to obtain molten resin; S3. Uniformly coat the molten resin on the surface of the pretreated substrate with a coating thickness of 20 - 60 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 based on the functionalized epoxy resin substrate according to claim 1, characterized in that: The mass concentration of the hexafluoroisobutyltrimethoxysilane solution is 0.2% - 2.0%.

3. The preparation method of the single-molecule array chip based on the functionalized epoxy 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 based on the functionalized epoxy resin substrate according to any one of claims 1 to 3, characterized in that: The specific step of the soaking and modification is: completely immerse the imprinted substrate in the hexafluoroisobutyltrimethoxysilane solution for an immersion time of 3 - 20 min 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 based on the functionalized epoxy 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 min; 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 based on the functionalized epoxy resin substrate according to any one of claims 1 to 3, characterized in that: In step S4, the pressure application range is 2 - 5 MPa, the pressing temperature is 30 - 140°C, and the pressing time is 5 - 10 min.

7. A single-molecule array chip based on a functionalized epoxy resin substrate prepared by the preparation method of the single-molecule array chip based on a functionalized epoxy resin substrate according to any one of claims 1 - 6.

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