Digital method for reducing non-specific adsorption based on PMMA ELISA plate and application

Abstract

The invention provides a digital method for reducing non-specific adsorption based on a PMMA ELISA plate and application, and belongs to the technical field of biological detection. According to the present invention, the PMMA is used for biomarker detection based on the convenience and the permeability of the PMMA, and the PMMA is subjected to surface activation so as to modify the surface of the PMMA into the active group capable of being combined with the antibody group, such that the non-specific adsorption can be reduced while the detection sensitivity and the detection accuracy are improved compared with the traditional physical adsorption elisa plate; therefore, the PMMA elisa plate has a wide application prospect in the field of biomarker detection, is expected to become an ideal substitute of a traditional material, provides new power for the development of a biomedical detection technology, and brings more convenience and possibility for human health and disease diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a digital method and application for reducing non-specific adsorption based on a PMMA microplate. Background Art

[0002] Biomarker detection is a method of evaluating the physiological or pathological state of an organism by measuring specific biochemical, immunological, or genetic indicators in biological fluids (such as blood, urine), cells, or tissues. These biomarkers can provide important information about disease risk, disease progression, treatment response, and biological age, etc., and contribute to the early diagnosis, prognosis assessment, and treatment monitoring of diseases.

[0003] Non-specific adsorption refers to the phenomenon of non-specific binding between the molecule to be detected and the solid-phase carrier during an experimental reaction. This phenomenon is usually caused by factors such as the physicochemical properties of the solid-phase carrier, the structural complexity of the molecule to be detected, and the unsuitability of the reaction conditions. The hydrophobicity, charge distribution on the surface of the solid-phase carrier, and the polarity of the molecule to be detected, etc., will all affect the degree of non-specific adsorption.

[0004] PMMA, commonly known as acrylic or plexiglass, is a thermoplastic plastic with extremely high transparency. Its light transmittance can reach more than 92%, it is light in weight and not easily broken, and is widely used in fields such as architecture, advertising, optics, and medical. The PMMA material not only has good weather resistance, can resist oxidation, weathering, and exposure to sunlight, rain, and wind, but also has good processing performance and can be formed by various methods such as injection molding and extrusion. In addition, the PMMA surface has relatively high hardness, is resistant to chemical corrosion, and is easy to dye and spray, being beautiful and durable. In the medical field, PMMA also has a wide range of applications, showing its excellent biocompatibility. Due to its unique advantages, this multifunctional material has become an ideal substitute for many traditional materials.

[0005] The digital fluorescence immunosorbent assay system (D-FLISA) is a technology applied in biomedical research and clinical diagnosis. It has high sensitivity and simplicity, and is a new method for detecting and quantitatively analyzing biomarkers, and has been used for the detection of various biomarkers. However, the problem of non-specific adsorption existing in the detection process of this technology often affects its accuracy and reliability.

[0006] Therefore, how to reduce the non-specific adsorption problem in the digital fluorescence immunosorbent assay process is crucial for the accuracy and reliability of biomarker detection. Summary of the Invention

[0007] The object of the present invention is to provide a digital method and application for reducing non-specific adsorption based on a PMMA microplate, which can reduce the non-specific adsorption problem in a digital fluorescence immunosorbent assay system and improve the accuracy and reliability of detection.

[0008] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a digital method for reducing non-specific adsorption based on a PMMA microplate, comprising the following steps:

[0010] (1) Treatment of the microplate: The polymethyl methacrylate microplate is subjected to plasma activation. After the plasma activation is completed, EDC solution and NHS solution with a concentration of 15 - 30 mg / mL are successively added and activation is continued for 1 - 2 h.

[0011] (2) Coating with the substrate antibody: The antibody is diluted with an antibody coupling buffer, and the diluted antibody is added to the activated polymethyl methacrylate microplate and coated for 1 - 4 h, then the microplate is washed.

[0012] (3) Adding a blocking solution to the carboxylated plate coated in step (2) to block for 1 - 3 h, washing the plate and patting it dry for standby.

[0013] Preferably, in step (1), the steps of the plasma activation are:

[0014] The polymethyl methacrylate microplate is placed in a plasma instrument for plasma surface activation for 0.5 - 10 min.

[0015] Preferably, in step (1), the temperature for continuing the activation with the EDC solution and the NHS solution is 35 - 38 °C, and the addition amount of the EDC solution and the NHS solution is 45 - 55 μL / well.

[0016] Preferably, in step (2), the concentration of the antibody is 1 - 5 μg / mL, and the addition amount is 100 μL / well.

[0017] Preferably, in step (2), the coating temperature is 35 - 38 °C.

[0018] Preferably, in step (3), the blocking solution is a PBS solution containing 1% BSA.

[0019] Preferably, in step (3), the blocking temperature is 35 - 38 °C.

[0020] The present invention also provides an application of the digital method for reducing non-specific adsorption based on a PMMA microplate in digital fluorescence immunoassay technology.

[0021] The beneficial effects of the present invention compared with the prior art are as follows:

[0022] (1) Based on the convenience and permeability of PMMA, the present invention uses it for biomarker detection. By surface activating PMMA, its surface is modified with active groups that can bind antibody groups. Compared with traditional physically adsorbed enzyme-linked immunosorbent assay (ELISA) plates, it can not only reduce non-specific adsorption, but also improve the sensitivity and accuracy of detection.

[0023] (2) The PMMA ELISA plate performs excellently in fluorescence immunoassay adsorption experiments. Its surface chemical properties are stable, which can effectively carry biomolecules while reducing the interference of background signals. In addition, the mechanical strength and durability of the PMMA ELISA plate have also been verified by experiments. Even after multiple uses and washings, it can still maintain good performance. These advantages make the PMMA ELISA plate have broad application prospects in the field of biomarker detection, and is expected to become an ideal substitute for traditional materials, providing new impetus for the development of biomedical detection technology and bringing more convenience and possibilities to human health and disease diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is a schematic diagram of the ELISA plate in Test Example 1 of the present invention;

[0026] Figure 2 It is the blank signal value of the PMMA ELISA plate and the polystyrene ELISA plate in Test Example 1 of the present invention;

[0027] Figure 3 It is the linear curve graph of the PMMA ELISA plate in Test Example 1 of the present invention;

[0028] Figure 4 It is the linear curve graph of the polystyrene ELISA plate in Test Example 1 of the present invention;

[0029] Figure 5 It is the repeatability test result graph of 10 batches of PMMA ELISA plates in Test Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] Example 1 of the present invention provides a digital method for reducing non-specific adsorption based on a PMMA microtiter plate, and the specific steps are as follows:

[0037] (1) Pretreatment of the microtiter plate: Put the polymethyl methacrylate (PMMA) microtiter plate into a plasma instrument for plasma surface activation for 10 min. Then, add 50 μL / well of EDC solution and NHS solution with a concentration of 20 mg / m each to the activated plate, and place it in an incubator at 37 °C for 1 h. After that, wash the plate with PBST solution;

[0038] (2) Coating of the substrate antibody: Dilute the antibody with MES solution to a concentration of 2 μg / mL, add 100 μL of the diluted antibody to each well of the microtiter plate, and place it in an incubator at 37 °C for 2 h. After that, wash the plate with PBST solution;

[0039] (3) Coating plate blocking: Add 150 μL of PBS solution containing 1% BSA, place it at 37°C for 2 h to block the coating plate. After completion, wash the plate with PBST solution and set aside.

[0040] Example 2

[0041] Example 2 of the present invention provides a digital method for reducing non-specific adsorption based on a PMMA enzyme-linked immunosorbent assay (ELISA) plate. The specific steps are as follows:

[0042] (1) Pretreatment of the ELISA plate: Place the polymethylmethacrylate (PMMA) ELISA plate into a plasma instrument for plasma surface activation for 5 min. After activation, add 50 μL / well of EDC solution and NHS solution with a concentration of 30 mg / m each to the plate, and place it in an incubator at 37°C for 2 h for activation. After completion, wash the plate with PBST solution;

[0043] (2) Coating of the substrate antibody: Dilute the antibody with MES solution to a concentration of 5 μg / mL, add 100 μL of the diluted antibody to each well of the well plate, and place it in an incubator at 35°C for 4 h for coating. After completion, wash the plate with PBST solution;

[0044] (3) Coating plate blocking: Add 150 μL of PBS solution containing 1% BSA, place it at 38°C for 1 h to block the coating plate. After completion, wash the plate with PBST solution and set aside.

[0045] Example 3

[0046] Example 3 of the present invention provides a digital method for reducing non-specific adsorption based on a PMMA ELISA plate. The specific steps are as follows:

[0047] (1) Pretreatment of the ELISA plate: Place the polymethylmethacrylate (PMMA) ELISA plate into a plasma instrument for plasma surface activation for 3 min. After activation, add 50 μL / well of EDC solution and NHS solution with a concentration of 30 mg / m each to the plate, and place it in an incubator at 37°C for 1 h for activation. After completion, wash the plate with PBST solution;

[0048] (2) Coating of the substrate antibody: Dilute the antibody with MES solution to a concentration of 1 μg / mL, add 100 μL of the diluted antibody to each well of the well plate, and place it in an incubator at 38°C for 2 h for coating. After completion, wash the plate with PBST solution;

[0049] (3) Coating plate blocking: Add 150 μL of PBS solution containing 1% BSA, place it at 35°C for 3 h to block the coating plate. After completion, wash the plate with PBST solution and set aside.

[0050] Test Example 1

[0051] In Test Example 1 of the present invention, a digital fluorescence immunoassay technique was used to detect interleukin 6 (both the LI-6 antibody and the antigen standard were purchased from Hangzhou Yiboxin Biotechnology Co., Ltd.). Using an enzyme-linked immunosorbent assay (ELISA) plate made of polystyrene (PS) as a control, the effect of the method for reducing non-specific adsorption of plate biomarkers in Example 1 was detected. The specific steps are as follows:

[0052] (1) ELISA plate made of polystyrene (PS):

[0053] Add 100 μL of the IL-6 basal antibody diluted with a pH 9.6 carbonate buffer solution, with an antibody concentration of 2 μg / mL, place it in an incubator at 37°C for 2 hours, and after completion, wash the plate with PBST solution;

[0054] Blocking of the excess sites on the coated plate: Add 150 μL of PBS solution containing 1% BSA, place it at 37°C for 2 hours to block the coated plate, and after completion, wash the plate with PBST solution.

[0055] (2) Verification of the adsorption of the plate:

[0056] Use the method in step (1) of Example 1 to prepare a PMMA ELISA plate for detecting IL-6. Add 50 μL of the antigen standard to both the PMMA ELISA plate for detecting IL-6 prepared by the method in step (1) of Example 1 and the ELISA plate made of polystyrene (PS) in step (1). The concentrations of the antigen standards are as follows: 1600, 800, 400, 200, 100, 50, 25, 0 pg / mL. Place it at 37°C for 1 hour, and after completion, wash the plate with PBST solution;

[0057] Add 100 μL of fluorescent microspheres containing the detection antibody, place it at 37°C for 1 hour, and after completion, wash the plate with PBST solution. Place the detection plate in a digital fluorescence immunoassay analyzer for reading and analysis. The results are shown in Tables 2, 3, and Figures 2 to 4 as shown.

[0058] All the ELISA plates used are 96-well plates. The schematic diagram of the ELISA plate is as shown in Figure 1 as shown. The sample loading layout is shown in Table 1, where S1-S8 are all standards, and T1-T10 are blank sample dilutions for testing:

[0059] Table 1 1 2 3 4 5 6 7 8 9 10 11 A S1 S1 T1 T9 B S2 S2 T2 T10 C S3 S3 T3 D S4 S4 T4 E S5 S5 T5 F S6 S6 T6 G S7 S7 T7 H S8 S8 T8

[0060] The detection results are as follows:

[0061] Table 2 ELISA plate made of polymethyl methacrylate (PMMA)

[0062]

[0063]

[0064] Table 3 ELISA plate made of polystyrene (PS)

[0065] 1 2 3 4 A 3724 3501 62 71 B 2719 2587 57 68 C 1598 1335 58 D 606 707 61 E 307 350 60 F 115 111 55 G 58 50 57 H 51 47 59

[0066] By comparing the test results of the above two forms of ELISA plates, it can be found that under the same test conditions, the ELISA plate made of polymethyl methacrylate (PMMA) shows a higher fluorescence signal value in the test, and the difference between the signal value of 0 and the signal value of the low value area is more obvious, indicating that it has better detection sensitivity and discrimination. This feature is of great significance for improving detection accuracy and reducing false negative results. On this basis, the physical properties of the two ELISA plates were further analyzed in detail, and it was found that PMMA material has better optical transparency and mechanical stability. These advantages may directly affect the accuracy and repeatability of the test results.

[0067] Test Example 2

[0068] Experimental Example 2 of the present invention compares the effects of multiple batches of PMMA ELISA plates, and the specific steps are as follows:

[0069] (1) Pretreatment of ELISA plates: 10 PMMA ELISA plates from different batches were taken and placed in a plasma instrument for plasma surface activation. After activation, 50 μL / well of EDC solution and NHS solution with a concentration of 20 mg / mL were added to the activated plates, respectively. The plates were placed in a 37°C incubator for activation for 1 h. After activation, the plates were washed with PBST solution.

[0070] (2) Coating with substrate antibody: Use MES solution to dilute IL-6 substrate to a concentration of 2 μg g / mL, add 100 μL of the diluted antibody to each well of the plate, place in a 37°C incubator for coating for 2 h, and wash the plate with PBST solution after completion;

[0071] (3) Blocking of coated plates: Add 150 μL of PBS solution containing 1% BSA and incubate at 37° C. for 2 h to block the coated plates. Afterwards, wash the plates with PBST solution and set aside.

[0072] (4) Take the above ELISA plates respectively, add 50 μL of antigen standard, the concentration of antigen standard is as follows: 1600, 800, 400, 200, 100, 50, 25, 0 pg / mL, place at 37°C for 1 h, and wash the plates with PBST solution;

[0073] (5) Add 100 μL of fluorescent microspheres containing detection antibodies, place at 37 °C for 1 h, after completion, wash the plate with PBST solution, place the detection plate on a digital fluorescence immunoassay analyzer for reading and analysis, and the results are shown in Table 5 and Figure 5 as follows.

[0074] The sample loading layouts of PMMA enzyme-linked immunosorbent assay (ELISA) plates from different batches are shown in the following table, where S1 - S8 are all standards, and columns 1 - 10 represent 10 batches of PMMA ELISA plates respectively:

[0075] Table 4 Sample Loading Layout of PMMA ELISA Plates

[0076] 1 2 3 4 5 6 7 8 9 10 11 12 A S1 S1 S1 S1 S1 S1 S1 S1 S1 S1 B S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 C S3 S3 S3 S3 S3 S3 S3 S3 S3 S3 D S4 S4 S4 S4 S4 S4 S4 S4 S4 S4 E S5 S5 S5 S5 S5 S5 S5 S5 S5 S5 F S6 S6 S6 S6 S6 S6 S6 S6 S6 S6 G S7 S7 S7 S7 S7 S7 S7 S7 S7 S7 H S8 S8 S8 S8 S8 S8 S8 S8 S8 S8

[0077] The test results are shown in Table 5

[0078] Table 5 Test Results of PMMA ELISA Plates from Different Batches

[0079]

[0080]

[0081] The results of 10 batches of tests show that the ELISA plates made of PMMA have good stability, with small fluctuations in background values in tests of different batches and can be controlled at a low level, indicating that this material has good application prospects in biomarker detection. It is found that its adsorption and detection sensitivity both meet expectations, indicating that the ELISA plates made of polymethyl methacrylate (PMMA) can effectively reduce non-specific adsorption and ensure the reliability and accuracy of experimental data. The specific experimental procedure is as follows:

[0082] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A digital method for reducing non-specific adsorption based on a PMMA enzyme-linked immunosorbent assay plate, characterized in that, It includes the following steps: (1) Treatment of the enzyme-labeled plate: Plasma activate the polymethyl methacrylate enzyme-labeled plate. After the plasma activation ends, sequentially add EDC solution and NHS solution with a concentration of 15 - 30 mg / mL each, and then continue the activation for 1 - 2 h; (2) Coating with the substrate antibody: Dilute the antibody with the antibody coupling buffer, and add the diluted antibody to the activated polymethyl methacrylate enzyme-labeled plate, coat for 1 - 4 h, and wash the plate; (3) Add the blocking solution to the carboxylated plate coated in step (2) to block for 1 - 3 h, wash the plate and pat it dry for standby.

2. The digital method for reducing non-specific adsorption based on a PMMA enzyme-labeled plate according to claim 1, wherein In step (1), the steps of the plasma activation are: Put the polymethyl methacrylate enzyme-labeled plate into a plasma instrument for plasma surface activation for 0.5 - 10 min.

3. The digital method for reducing non-specific adsorption based on a PMMA enzyme-labeled plate according to claim 1, wherein In step (1), the temperature for the continued activation of the EDC solution and NHS solution is 35 - 38 °C, and the addition amount of both the EDC solution and NHS solution is 45 - 55 μL / well.

4. The digital method for reducing non-specific adsorption based on a PMMA microplate according to claim 1, wherein In step (2), the concentration of the antibody is 1 - 5 μg / mL, and the addition amount is 100 μL / well.

5. The digital method for reducing non-specific adsorption based on a PMMA enzyme-labeled plate according to claim 1, wherein, In step (2), the coating temperature is 35 - 38 °C.

6. The digital method for reducing non-specific adsorption based on a PMMA microplate according to claim 1, wherein, In step (3), the blocking solution is a PBS solution containing 1% BSA.

7. The digital method for reducing non-specific adsorption based on a PMMA microplate according to claim 1, characterized in that, In step (3), the blocking temperature is 35 - 38 °C.

8. Application of the digital method for reducing non-specific adsorption based on the PMMA enzyme-labeled plate according to any one of claims 1 - 7 in the digital fluorescence immunoassay technology.

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