Nuclear track membrane-based protein biomarker detection method and application

Abstract

The invention provides a detection method and application of a nuclear track membrane-based protein biomarker, and relates to the technical field of electrochemical detection.The detection method comprises the steps that amination modified gold nanoparticles are connected to the inner wall of a nuclear track membrane pore channel with carboxyl, the conductivity of the pore channel is improved, then a first antibody is added to be combined with the gold nanoparticles, and the first antibody is obtained; then incubating with a protein marker and non-conductive nanoparticles combined with a second antibody, and after incubation is finished and cleaning is performed, detecting a difference value of current passing through a pore channel through an electrochemical workstation so as to realize quantitative detection of the concentration of the marker. The non-conductive nanoparticles connected with the second antibody can block the pore channel and obviously reduce the current, so that the current difference is larger, the change is more obvious, and the detection range is larger. The detection method disclosed by the invention has the characteristics of rapidness, accuracy, high sensitivity, simplicity and convenience in operation and low cost, and has very important theoretical and practical significance in detection of clinical protein markers.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to a nuclear pore membrane-based protein biomarker detection method and application. Background Art

[0002] Protein biomarkers are specific proteins present in body fluids or tissues that can reflect disease states, physiological processes, or treatment effects. For example, PSA (prostate-specific antigen) is used for prostate cancer monitoring, and AFP (carcinoembryonic antigen) assists in colorectal cancer management. Quantitative testing provides objective data for clinicians by measuring their concentrations, supporting precision medical decision-making.

[0003] Common methods for protein biomarker detection include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), fluorescent immunoassay (FIA), immunoblotting, immunoturbidimetry / immunoscattering, mass spectrometry, and electrochemical detection. Among them, electrochemical detection quantifies the target protein through the changes in electrical signals (current, potential, impedance, etc.) caused by biorecognition events (such as antigen-antibody binding) occurring on the electrode surface. Its advantages are high sensitivity, rapid detection, and low cost. Disadvantages are matrix interference in complex samples (such as blood), long-term stability and repeatability of electrodes, and cross-reactions during multi-marker joint testing.

[0004] Nuclear pore membrane, also known as heavy ion microporous filter membrane, is a membrane material with regular geometric shape and uniform pore size. Its pore size is basically a cylindrical straight hole, the surface is flat and smooth, the filtration speed is large and the retention characteristics are good. In addition, the nuclear pore membrane also has high mechanical strength and flexibility, light weight and good weight consistency, low absorption, no adsorption of the effective ingredients of the drug solution, and is not easily affected by moisture and deterioration. These characteristics make the nuclear pore membrane perform well in biological detection. In the prior art, the detection method of the nuclear pore membrane for protein antigens has the problems of poor sensitivity and narrow detection range due to its unclear current change, which makes it difficult to meet the accurate detection of markers.

[0005] Therefore, there is an urgent need to develop a nuclear pore membrane-based detection method that is fast, accurate, highly sensitive, simple and convenient to operate, low-cost, and has a wide detection range to meet the needs of clinical testing. Summary of the invention

[0006] One of the purposes of the present invention is to provide a method for detecting nuclear pore membrane-based protein biomarkers to solve the technical problems of existing nuclear pore membrane detection methods such as long time consumption, poor sensitivity and narrow detection range.

[0007] The present invention provides a method for detecting a nuclear pore membrane-based protein biomarker, which comprises the following steps: (1) connecting amino-modified gold nanoparticles to the inner wall of a nuclear pore membrane channel having a carboxyl group, then adding a first antibody to bind the gold nanoparticles, and then drying the membrane after sealing and washing to obtain a nuclear pore membrane modified with the first antibody; (2) placing the nuclear pore membrane modified with the first antibody in an electrolytic cell and detecting the membrane using an electrochemical workstation to obtain an initial current value I0, and then incubating the membrane with a protein biomarker and a non-conductive nanomaterial combined with a second antibody for 10-90 minutes, and after the incubation is completed and the membrane is washed, placing the membrane in an electrolytic cell and detecting the membrane using an electrochemical workstation to obtain a detection current value I1; and (3) establishing a standard curve by detecting the change in the current difference (I0-I1) corresponding to markers of different concentrations, thereby achieving quantitative detection of the marker concentration.

[0008] Furthermore, the methods for modifying the gold nanoparticles by amino modification include but are not limited to 3-aminopropyltrimethoxysilane (APTMS) modification, polyethylene glycol (PEG) modification and aminophenol (AP) modification; And / or, the particle size of the amino-modified gold nanoparticles is 20-200 nm.

[0009] Furthermore, the method for connecting the amino-modified gold nanoparticles to the inner wall of the nuclear pore membrane channel having carboxyl groups comprises the following steps: immersing the nuclear pore membrane in an MES buffer solution containing EDC and NHS for oscillation reaction for 0.5-1 h to activate the carboxyl groups (-COOH) on the inner wall of the nuclear pore membrane channel, then taking out and immersing in a solution containing amino-modified gold nanoparticles for oscillation reaction for 3-5 h, and finally washing with pure water 2-3 times and drying to complete the preparation.

[0010] Furthermore, the method for combining the first antibody with gold nanoparticles is as follows: adding the first antibody to a MES buffer solution containing EDC and NHS and oscillating the reaction for 0.5-1 h for carboxyl activation, then immersing the nuclear pore membrane modified with gold nanoparticles in the activated first antibody solution and oscillating the reaction for 3-5 h, adding it to a PBS buffer solution containing BSA and blocking it for 0.5-3 h, and finally washing it with pure water 2-3 times and drying it to complete the preparation.

[0011] Furthermore, the preparation method of the non-conductive nanoparticles bound to the second antibody is: first, the non-conductive nanoparticles are amino-modified, and then the second antibody is carboxyl-activated using an MES buffer containing EDC and NHS, and then the amino-modified non-conductive nanoparticles are added for oscillation reaction for 3-5 hours, and finally the preparation is completed after sealing and centrifugal washing 2-3 times.

[0012] Furthermore, the concentration of EDC is 8-12 mg / mL, the concentration of NHS is 8-12 mg / mL, the concentration of MES buffer is 0.05-0.2 M, and the pH of MES buffer is 4-6.

[0013] Further, the non-conductive nanoparticles include but are not limited to silicon dioxide (SiO2) nanoparticles, magnetic nanoparticles, polystyrene nanoparticles, polylactic acid nanoparticles, aluminum oxide (Al2O3) nanoparticles and titanium dioxide (TiO2) nanoparticles; And / or, the particle size of the non-conductive nanoparticles is 0.2-2 μm.

[0014] The second object of the present invention is to provide a method for detecting a nuclear pore membrane-based protein biomarker and its application in detecting protein markers by sandwich method.

[0015] The nuclear pore membrane-based protein biomarker detection method provided by the present invention can realize the detection of protein biomarkers with rapid accuracy, high sensitivity, simple and convenient operation, low cost and wide detection range by combining nuclear pore membrane technology, immunoassay technology and nanotechnology. First, the nuclear pore membrane technology provides a nuclear pore membrane with carboxyl groups, and then combines with amino gold nanoparticles (nanotechnology) through amide bonds to enhance the initial current signal of the nuclear pore membrane, so that the current variation range during detection is also larger, and a larger detection range can be achieved. Subsequently, the first antibody and the second antibody are specifically combined with the marker (immunotechnology) through immune reaction, making the detection more accurate. At the same time, the non-conductive nanoparticles (nanotechnology) connected to the second antibody are enriched in large quantities in the pores of the nuclear pore membrane, blocking the pores. Due to its non-conductive characteristics, the current signal in the pores can be significantly reduced, thereby making the current change more significant, achieving a detection effect of high sensitivity and a larger detection range. The present invention achieves the technical effect of higher sensitivity and a larger detection range through this "open source and throttling" design scheme (gold nanoparticles increase the initial current signal as open source, and non-conductive nanoparticles block the pores as throttling).

[0016] The nuclear pore membrane-based protein biomarker detection method provided by the present invention has a wide range of applications and is conducive to clinical detection application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Schematic diagram of the method for detecting nuclear pore membrane-based protein biomarkers; Figure 2 This is an electron micrograph of the nuclear pore membrane; Figure 3 Transmission electron microscopy images of gold nanoparticles before and after amino modification; Figure 4 This is the Zeta potential change diagram of gold nanoparticles before and after amino modification; Figure 5 Transmission electron microscopy images of MSNs before modification and after antibody binding with AFP secondary antibody; Figure 6 The Zeta potential change diagram of MSNs before modification, after amino modification and after antibody binding to AFP secondary antibody; Figure 7 A schematic diagram of a detection device for a nuclear pore membrane-based protein biomarker detection method; Figure 8 The standard curve is the current change (I0-I1) in response to different concentrations of AFP. DETAILED DESCRIPTION

[0019] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0020] According to one aspect of the present invention, the present invention provides a method for detecting nuclear pore membrane-based protein biomarkers, such as Figure 1 As shown in the figure, the specific steps of the detection method are as follows: (1) connecting amino-modified gold nanoparticles to the inner wall of the nuclear pore membrane channel with carboxyl groups, and then adding the first antibody to make it bind to the gold nanoparticles. After sealing and washing, it is dried to obtain the nuclear pore membrane modified with the first antibody; (2) placing the nuclear pore membrane modified with the first antibody in an electrolytic cell and detecting it through an electrochemical workstation to obtain an initial current value I0, and then incubating it with protein biomarkers and non-conductive nanomaterials bound to the second antibody for 10-90 minutes. After the incubation is completed and washed, it is placed in an electrolytic cell and detected through an electrochemical workstation to obtain a detection current value I1; (3) By detecting the change in the current difference (I0-I1) corresponding to different concentrations of the marker, a standard curve is established to achieve quantitative detection of the marker concentration.

[0021] In typical but non-limiting embodiments of the invention, the incubation time is, for example, 10, 20, 30, 40, 50, 60, 70, 80 or 90 minutes.

[0022] In a preferred embodiment of the present invention, the methods for modifying the gold nanoparticles by amino modification include but are not limited to 3-aminopropyltrimethoxysilane (APTMS) modification, polyethylene glycol (PEG) modification and aminophenol (AP) modification.

[0023] In a preferred embodiment of the present invention, the particle size of the amino-modified gold nanoparticles is 20-200 nm.

[0024] In typical but non-limiting embodiments of the invention, the particle size of the amino-modified gold nanoparticles is 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nm.

[0025] In a preferred embodiment of the present invention, the method for connecting the amino-modified gold nanoparticles to the inner wall of the nuclear pore membrane pore channel having carboxyl groups comprises the following steps: immersing the nuclear pore membrane in an MES buffer solution containing EDC and NHS for oscillation reaction for 0.5-1 h to activate the carboxyl groups (-COOH) on the inner wall of the nuclear pore membrane pore channel, then taking out and immersing in a solution containing amino-modified gold nanoparticles for oscillation reaction for 3-5 h, and finally washing with pure water 2-3 times and drying to complete the preparation.

[0026] In a preferred embodiment of the present invention, the method for binding the first antibody to the gold nanoparticles is as follows: the first antibody is added to a MES buffer solution containing EDC and NHS and oscillated for 0.5-1 h for carboxyl activation, and then the nuclear pore membrane modified with the gold nanoparticles is immersed in the activated first antibody solution and oscillated for 3-5 h, and then blocked with a PBS buffer solution containing BSA for 0.5-3 h, and finally washed with pure water 2-3 times and dried to complete the preparation.

[0027] In a preferred embodiment of the present invention, the preparation method of the non-conductive nanomaterial bound to the second antibody is as follows: first, the non-conductive nanomaterial is amino-modified, and then the second antibody is carboxyl-activated using an MES buffer containing EDC and NHS, followed by adding the amino-modified non-conductive nanoparticles for oscillation reaction for 3-5 hours, and finally the preparation is completed after sealing and centrifugal washing 2-3 times.

[0028] In a preferred embodiment of the present invention, the concentration of EDC is 8-12 mg / mL, the concentration of NHS is 8-12 mg / mL, the concentration of MES buffer is 0.05-0.2 M, and the pH of MES buffer is 4-6.

[0029] In a typical but non-limiting embodiment of the present invention, the concentration of EDC is 8, 9, 10, 11 or 12 mg / mL; the concentration of NHS is 8, 9, 10, 11 or 12 mg / mL; the concentration of MES buffer is 0.05, 0.1, 0.15 or 0.2 M; and the pH of MES buffer is 4, 5 or 6.

[0030] In a preferred embodiment of the present invention, the non-conductive nanomaterials include but are not limited to silicon dioxide (SiO2) nanoparticles, magnetic nanoparticles, polystyrene nanoparticles, polylactic acid nanoparticles, aluminum oxide (Al2O3) nanoparticles and titanium dioxide (TiO2) nanoparticles.

[0031] In a preferred embodiment of the present invention, the particle size of the non-conductive nanomaterial is 0.2-2 μm.

[0032] In typical but non-limiting embodiments of the present invention, the particle size of the non-conductive nanomaterial is, for example, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 or 2 μm.

[0033] According to a second aspect of the present invention, the present invention provides an application of a method for detecting a nuclear pore membrane-based protein biomarker in detecting protein markers using a sandwich method.

[0034] Example 1 Preparation and characterization of nuclear pore membrane modified with AFP primary antibody

[0035] 1. Preparation of nuclear pore membrane with carboxyl groups (the pore size of the nuclear pore membrane is about 5 μm, see Figure 2 ): (1) A polyethylene terephthalate (PET) film was used as the base film. The base film was irradiated with heavy ions from a linear accelerator and then sensitized under ultraviolet light for 2 h. (2) The base film was chemically etched. After the etching was completed, the carboxyl groups were exposed in the pores. The etching solution was 8 M sodium hydroxide solution, the blocking solution was 3 M potassium chloride and 3 M formic acid, and the applied voltage was 1.5 V.

[0036] 2. Amino modification of gold nanoparticles: (1) Use gold nanoparticles with a particle size of 50 nm and disperse them in 0.01 M PBS buffer (pH=9); (2) Dissolve HS-PEG-NH2 in 0.01 M PBS buffer (pH=9), add it to the gold nanoparticles at a molar ratio of PEG to gold atoms on the surface of AuNPs of 5000:1, and vortex mix at room temperature for 24 h; (3) Centrifuge (12000 rpm, 10-90 minutes) to remove free PEG, resuspend the precipitate in buffer, and repeat 3 times. Resuspend in PBS and store at 4°C in the dark.

[0037] 3. Modification of gold nanoparticles in the pores of nuclear pore membranes: The nuclear pore membranes were immersed in MES buffer (0.1 M) containing EDC (10 mg / mL) and NHS (10 mg / mL) and oscillated for 0.5 h to activate the carboxyl groups (-COOH) on the inner wall of the pores of the nuclear pore membranes. The membranes were then taken out and immersed in a solution of amino-modified gold nanoparticles and oscillated for 4 h. Finally, the membranes were washed with pure water three times and air-dried to complete the preparation.

[0038] 4. Connection of AFP primary antibody in the nuclear pore membrane pore: Add AFP primary antibody to MES buffer (0.1 M) containing EDC (10 mg / mL) and NHS (10 mg / mL) and oscillate for 0.5 h to activate the carboxyl group. Then, immerse the nuclear pore membrane modified with gold nanoparticles in the activated AFP primary antibody solution and oscillate for 4 h. Add PBS buffer (0.05 M, pH=7.4) containing BSA (0.1%) to block for 1 h. Finally, wash with pure water 3 times and dry it to complete the preparation.

[0039] 5. Characterization of aminated gold nanoparticles: After the preparation of aminated gold nanoparticles, the gold nanoparticles before and after preparation were first characterized by transmission electron microscopy. The results are as follows Figure 3 As shown in the figure, the particle size of the gold nanoparticles is about 50 nm. Before amino modification, they are evenly dispersed and have a smooth surface. However, after modification, they aggregate to a certain extent and the surface is not smooth, indicating that the amino modification is successful. The Zeta potential of the gold nanoparticles was then characterized. The results are shown in Figure 4 As shown in the figure, the Zeta potential of the gold nanoparticles before and after modification changed significantly from -25 mV to 22 Mv, indicating that the amino modification was successful.

[0040] 6. Characterization of the nuclear pore membrane modified with the first AFP antibody: During the step-by-step modification of the nuclear pore membrane until the antibody modification is completed, the current signal of the nuclear pore membrane will change significantly. By detecting the current signal of the nuclear pore membrane at different stages, it can be verified whether the antibody modification is successful, and it can also be verified that the current signal of the nuclear pore membrane will become larger after the modification of the gold nanoparticles. In this embodiment, an electrochemical workstation is used to detect the change of the current signal during the modification of the nuclear pore membrane. The chronopotentiometry is used, the applied voltage is 2 V, and the duration is 5 seconds. The results show that the current signals of the nuclear pore membrane before modification, after gold nanoparticle modification, and after antibody modification are 6 μA, 55 μA, and 50 μA, respectively, which have changed significantly, indicating that the first AFP antibody has been successfully modified into the pore of the nuclear pore membrane. The current signal of the nuclear pore membrane after the gold nanoparticle modification is significantly larger, indicating that the gold nanoparticle modification of the nuclear pore membrane can achieve the purpose of increasing the initial current signal, which provides support for the high sensitivity and larger detection range of the detection method provided by the present invention.

[0041] Example 2 Preparation and Characterization of Non-Conductive Nanoparticles (Mesoporous Silica Nanoparticles) Binding to AFP Secondary Antibody

[0042] 1. Preparation of mesoporous silica nanoparticles bound to AFP secondary antibody: The non-conductive nanomaterial selected in this example is mesoporous silica nanoparticles (MSNs) with a particle size of 500 nm. The non-conductive nanoparticles are amino-modified as follows: 10 mg of MSNs and 6 mg of 3-aminopropyltriethoxysilane (APTES) are added to a mixed solution of 20 mL of ethanol and water, wherein ethanol: water = 95:5 (v / v), and then acetic acid is added to adjust the pH to 4, and the reaction is stirred at room temperature (25°C) for 24 h at a stirring speed of 300 rpm. After the reaction is completed, the amino-modified MSNs are obtained by centrifugation and washing with ethanol and deionized water at 10,000 rpm for 10 minutes for 3 times. 20 μg of AFP secondary antibody was added to MES buffer (0.1 M) containing EDC (10 mg / mL) and NHS (10 mg / mL) and oscillated for 0.5 h to activate the carboxyl group. Then, the amino-modified MSNs were mixed with the activated AFP secondary antibody solution and oscillated for 4 h. The mixture was blocked in PBS buffer (0.05 M, pH=7.4) containing BSA (0.1%) for 1 h. Finally, the mixture was centrifuged (10,000 rpm, 10 min) and washed three times to complete the preparation.

[0043] 2. Characterization of MSNs bound to AFP secondary antibody: In this example, transmission electron microscopy was performed on MSNs bound to AFP secondary antibody before modification and after antibody binding. The results are as follows: Figure 5 As shown in the figure, the surface of MSNs before modification is smooth and well dispersed, with obvious mesoporous structure. After antibody binding, the surface is not smooth, the dispersion is characterized by local aggregation, and the mesopores disappear, indicating that the antibody modification is successful. Subsequently, the Zeta potential of MSNs bound to the AFP secondary antibody was characterized before modification, after amino modification, and after antibody binding. The results are shown in the figure. Figure 6 As shown, the Zeta potential of MSNs at each stage was -21 mV, 32 mV and 15 mV, respectively, which changed significantly during the modification process, indicating that the antibody modification was successful.

[0044] Example 3 Detection of Tumor Marker AFP

[0045] 1. Preparation of standard solutions: Prepare AFP standards S1-S6 at the following concentrations: 1, 5, 20, 100, 500 and 1000 ng / mL.

[0046] 2. Preparation of standard curve: Before testing, place the nuclear pore membrane in Example 1 into the electrolytic cell (see Figure 7 ), 0.5×PBS phosphate buffer solution was injected into the electrolytic cell as an electrolyte, and Ag / AgCl electrodes were inserted at both ends of the electrolytic cell and connected to an electrochemical workstation. The chronopotentiometry was used with a voltage of 2V and a duration of 5 seconds. The electrochemical workstation automatically recorded the corresponding microcurrent value on the connected computer to obtain the initial current value I0. Subsequently, the nuclear pore membrane in Example 1 was immersed in PBS buffer (0.05 M, pH=7.4), and then 5 μL of AFP antigen standards of different concentrations and MSNs bound to the AFP secondary antibody in Example 2 were added and incubated with shaking for 30 min. After the incubation, it was washed three times with deionized water and finally placed in an electrolytic cell. The electrochemical workstation was used to use the chronopotentiometry with a voltage of 2V and a duration of 5 seconds for detection to obtain the detection current value I1. The changes in the current difference (I0- I1) corresponding to the markers of different concentrations were detected, and a standard curve was established with the concentration of the AFP standard as the horizontal axis and the corresponding current difference (I0- I1) as the vertical axis (see Figure 8 ).

[0047] 3. Concentration detection: The AFP sample to be detected is detected according to the method used to establish the standard curve, and then the obtained current difference is compared with the standard curve prepared above to obtain the concentration of the AFP to be detected.

[0048] 4. Determination of detection limit: Use zero concentration calibrator as sample for detection, repeat the measurement 20 times, obtain the current change rate (I0- I1) of 20 measurement results, calculate its mean value (M) and standard deviation (SD), obtain the (I0-I1) value corresponding to M+2SD, perform two-point regression fitting based on the concentration-(I0- I1) value between zero concentration calibrator and adjacent calibrator to obtain a linear equation, substitute the (I0- I1) value corresponding to M+2SD into the above equation, and obtain the corresponding concentration value, which is the detection limit. The results are shown in Table 1: The detection limit is 0.04398 ng / mL.

[0049] Table 1 Detection limit determination

[0050] 5. Accuracy test: Prepare the AFP national standard into samples with concentrations of 16 ng / mL and 330 ng / mL (allowable deviation is ±10%). Test each concentration three times and calculate the relative deviation according to formula (1). If all three results meet the requirement of relative deviation within ±10%, the test is considered qualified. If two or more results fail to meet the requirement, the test is considered unqualified. If one result fails to meet the requirement, the test should be repeated 20 times in a row and the relative deviation should be calculated. If the results meet the requirement for 19 or more tests, the test meets the accuracy requirement.

[0051] B = (C t -T) / T×100%…………………………………………(1)

[0052] In the formula: B——relative deviation; C t ——determine the concentration; T——determine the target value of the sample.

[0053] Table 2 Determination of accuracy

[0054] 6. Intra-batch variation test: The same batch of nuclear pore membranes provided in Example 1 and MSNs bound to the AFP secondary antibody provided in Example 2 were used to test C1 (20 ng / mL) and C2 (400 ng / mL) 10 times, respectively. The results are shown in Table 3. The intra-batch variations of C1 and C2 were 3.4% and 1.7%, respectively, meeting the test requirements.

[0055] Table 3 Intra-batch difference test

[0056] 7. Stability test: The same batch of nuclear pore membranes provided in Example 1 and MSNs bound to the AFP secondary antibody provided in Example 2 were placed at 4°C for 0, 1, 2, 6, and 12 months, respectively. The concentration of the standard S5 was tested three times, and the relative deviations from 0 month were calculated. The results are shown in Table 4. When placed for 12 months, the relative deviation was -3.53%, which met the test requirements.

[0057] Table 4 Stability test

[0058] 8. Specificity test: AFP (500 ng / mL), CA125 (500 U / mL) and CA199 (500 U / mL) were detected using the nuclear pore membrane provided in Example 1 and the MSNs bound to the AFP secondary antibody provided in Example 2. The results are shown in Table 5. The concentration values ​​of the three detected were all less than 1 ng / mL, indicating that the detection method provided by the present invention has good specificity for AFP detection.

[0059] Table 5 Specificity test

[0060] In summary, the detection method provided by the present invention has an extremely low detection limit, excellent intra-batch variation, stability and specificity, and a short detection time. It is fast and accurate, highly sensitive, simple and convenient to operate, and low cost. It can meet the detection of protein biomarkers and is very worthy of promotion and application.

Claims

1. A method for detecting nuclear pore membrane-based protein biomarkers, characterized in that: The specific steps of the detection method are as follows: (1) connecting amino-modified gold nanoparticles to the inner wall of the nuclear pore membrane channel with carboxyl groups, then adding a first antibody to bind to the gold nanoparticles, and then drying after sealing and washing to obtain a nuclear pore membrane modified with the first antibody; (2) placing the nuclear pore membrane modified with the first antibody in an electrolytic cell and detecting it through an electrochemical workstation to obtain an initial current value I0, and then incubating it with a protein biomarker and a non-conductive nanomaterial bound to a second antibody for 10-90 minutes. After the incubation is completed and washed, placing it in an electrolytic cell and detecting it through an electrochemical workstation to obtain a detection current value I1; (3) by detecting the change in the current difference (I0-I1) corresponding to different concentrations of the marker, a standard curve is established to achieve quantitative detection of the marker concentration.

2. The method for detecting nuclear pore membrane-based protein biomarkers according to claim 1, characterized in that: The methods for modifying the gold nanoparticles by amino modification include but are not limited to 3-aminopropyltrimethoxysilane (APTMS) modification, polyethylene glycol (PEG) modification and aminophenol (AP) modification; And / or, the particle size of the amino-modified gold nanoparticles is 20-200 nm.

3. The method for detecting nuclear pore membrane-based protein biomarkers according to claim 1, characterized in that: The method for connecting amino-modified gold nanoparticles to the inner wall of the pore of a nuclear pore membrane modified with carboxyl groups comprises the following steps: immersing the nuclear pore membrane in an MES buffer solution containing EDC and NHS and oscillating the reaction for 0.5-1 h to activate the carboxyl groups (-COOH) on the inner wall of the pore of the nuclear pore membrane; then taking out the membrane and immersing it in a solution containing amino-modified gold nanoparticles and oscillating the reaction for 3-5 h; and finally washing it with pure water 2-3 times and drying it to complete the preparation.

4. The method for detecting nuclear pore membrane-based protein biomarkers according to claim 1, characterized in that: The method for combining the first antibody with gold nanoparticles is as follows: adding the first antibody to a MES buffer solution containing EDC and NHS and oscillating the reaction for 0.5-1 h to activate the carboxyl group, then immersing the nuclear pore membrane prepared in claim 3 in the activated first antibody solution and oscillating the reaction for 3-5 h, adding it to a PBS buffer solution containing BSA for blocking for 0.5-3 h, and finally washing it with pure water 2-3 times and drying it to complete the preparation.

5. The method for detecting nuclear pore membrane-based protein biomarkers according to claim 1, characterized in that: The preparation method of the non-conductive nanoparticles bound to the second antibody is as follows: first, the non-conductive nanoparticles are amino-modified, and then the second antibody is carboxyl-activated using an MES buffer containing EDC and NHS, and then the amino-modified non-conductive nanoparticles are added for oscillation reaction for 3-5 hours, and finally the preparation is completed after sealing and centrifugal washing for 2-3 times.

6. The method for detecting nuclear pore membrane-based protein biomarkers according to any one of claims 3 to 5, characterized in that: The concentration of EDC is 8-12 mg / mL, the concentration of NHS is 8-12 mg / mL, the concentration of MES buffer is 0.05-0.2 M, and the pH of MES buffer is 4-6.

7. The method for detecting nuclear pore membrane-based protein biomarkers according to claim 1 or 5, characterized in that: The non-conductive nanoparticles include, but are not limited to, silicon dioxide (SiO2) nanoparticles, magnetic nanoparticles, polystyrene nanoparticles, polylactic acid nanoparticles, aluminum oxide (Al2O3) nanoparticles, and titanium dioxide (TiO2) nanoparticles; And / or, the particle size of the non-conductive nanoparticles is 0.2-2 μm.

8. Application of the nuclear pore membrane-based protein biomarker detection method according to claim 1 in sandwich method detection of protein markers.

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