A cascade signal amplification detection method based on platinum nanozymes and its application in ovarian cancer biomarkers

Abstract

The present invention discloses a cascade signal amplification detection method based on platinum nanozymes, comprising the following steps: (1) constructing a magnetic nanoprobe; (2) constructing a platinum nanoprobe; (3) constructing a sandwich immune sandwich structure; (4) inducing a cascade signal amplification reaction; (5) visual quantitative detection of analytes. On the one hand, the present invention utilizes the specific recognition principle of antibodies to antigens to construct a sandwich immune sandwich structure, and on the other hand, utilizes platinum nanozymes to induce a cascade signal amplification reaction, thereby realizing the conversion of analyte recognition signals to visual ink advance distances, and at the same time realizing ultrasensitive quantitative detection of analytes, and applying this to the detection of ovarian cancer biomarkers. The detection method is low in cost, simple in operation, short in reaction time, high in sensitivity and strong in specificity, providing a new analytical means for the instant diagnosis of cancer biomarkers, and can be extended to the detection of more biomarkers.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a cascade signal amplification detection method based on platinum nanozymes and its application in ovarian cancer biomarkers. Background Art

[0002] Ovarian cancer is a common gynecological malignancy, and its mortality rate ranks first among various gynecological tumors. Since the clinical symptoms of ovarian cancer are not obvious, it is easy to be missed and misdiagnosed. Clinical statistics show that more than 70% of ovarian cancer patients are in the late stage of cancer when diagnosed, and the cancer has spread to other organs, missing the best time for treatment. In the past 20 years, the 5-year survival rate of ovarian cancer has been at a low level, and the recurrence rate within 5 years is as high as 80%, and the recurrence is mainly concentrated in the first 3 years of treatment. If ovarian cancer can be diagnosed as early as possible, the cancer cells only exist in the ovaries, and the patient's survival rate can be increased to 75%-95%, so early diagnosis of ovarian cancer is very important. Currently, there are two main detection methods for diagnosing ovarian cancer. One method is transvaginal ultrasonography (TUV). This imaging method can be used to examine the female reproductive organs, including the uterus, ovaries, cervix and vagina. Although it is widely used, this method cannot accurately detect whether the mass is benign or malignant. In addition, this method also relies on experienced clinical technicians to interpret the test results. Another conventional detection method is to detect the biomarkers CA125 carbohydrate antigen 125 and HE4 human epididymis protein 4 in the patient's blood. Among them, CA125 carbohydrate antigen 125 has low specificity and sensitivity, and is prone to false negatives or false positives. Compared with CA125 carbohydrate antigen 125, HE4 human epididymis protein 4 has higher sensitivity and stronger specificity, especially in the early stage of the disease when there are no symptoms. The sensitivity of HE4 human epididymis protein 4 in the early stage of the disease is 82.7%, while CA125 carbohydrate antigen 125 is only 45.9%. Compared with the specificity of CA125 carbohydrate antigen 125 of only 20%, the specificity of HE4 human epididymis protein 4 is as high as 99%. HE4 human epididymis protein 4 and CA125 carbohydrate antigen 125 are complementary markers. The combined use of the two can increase the sensitivity to 92%, reduce false negative results by 30%, and greatly increase the accuracy of ovarian cancer diagnosis. Therefore, the combined use of the two can avoid the missed diagnosis of disease recurrence monitoring caused by the negative results of a single application. The development of low-cost, accurate and sensitive biomarker quantitative detection methods is of great significance for the early diagnosis of ovarian cancer, and can monitor the progression of the disease in real time, thereby assisting the early treatment process. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a cascade signal amplification detection method based on platinum nanozymes and its application in ovarian cancer biomarkers. The present invention utilizes the specific recognition of antibodies and antigens to construct a sandwich immune sandwich structure of magnetic nanoprobe-analyte-platinum nanoprobe, utilizes the horseradish peroxidase of the platinum nanoprobe to ultra-fast catalyze dopamine to polydopamine under hydrogen peroxide conditions, and deposits it on the surface of the composite probe, thereby cross-linking a large number of platinum nanoparticles, and then utilizes the catalase activity of the platinum nanoparticles for visual detection.

[0004] To achieve the above object, the present invention adopts the following technical scheme: A cascade signal amplification detection method based on platinum nanozyme of the present invention comprises the following steps:

[0005] (1) constructing a magnetic nanoprobe: diluting amino group-modified magnetic silica nanoparticles with PBS phosphate buffered saline solution, adding glutaraldehyde to the modified magnetic silica nanoparticles for activation, separating the magnetic silica nanoparticles with a magnet and adding PBS phosphate buffered saline solution; adding a capture antibody thereto, incubating overnight, and discarding the supernatant to prepare a magnetic nanoprobe;

[0006] Then, BSA bovine serum albumin solution was added for blocking, adsorbed by magnet, washed and resuspended with PBS phosphate buffered saline solution, and stored in dark place for future use;

[0007] (2) constructing a platinum nanoprobe: diluting platinum nanoparticles with a Tris-HCl buffer solution, adding a detection antibody thereto, incubating overnight, and centrifuging the mixed solution to obtain a platinum nanoprobe;

[0008] Then, add BSA bovine serum albumin solution to make the final concentration 0.1-1%, and continue the reaction; centrifuge again, and finally disperse the blocked platinum nanoprobes in PBS phosphate buffered saline solution, and store in the dark for later use;

[0009] (3) constructing a sandwich immune structure: adding different concentrations of analytes to the magnetic nanoprobe solution prepared in step (1), incubating, adsorbing with a magnet and washing with a PBS phosphate buffer solution; then adding the platinum nanoprobe constructed in step (2), incubating to form a sandwich immune structure, adsorbing with a magnet and washing with a Tris-HCl tris-hydroxymethylaminomethane-hydrochloric acid buffer solution;

[0010] (4) Induced cascade signal amplification reaction:

[0011] The composite probe with sandwich immune sandwich structure constructed in step (3) is added to the dopamine solution, and reacted at room temperature. The platinum nanoparticles ultra-fast catalyze dopamine to form polydopamine and deposit on the surface of magnetic silica nanoparticles blocked by BSA bovine serum albumin, separate with a magnet and resuspend with PBS phosphate buffered saline solution; then, the platinum nanoparticles modified with mercaptophenylboronic acid are added, and the reaction is slowly shaken at room temperature. The supernatant is separated with a magnet and dispersed in the aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles;

[0012] (5) Visual quantitative detection of analytes:

[0013] Use a pipette to add the ink, H2O2, and the sample solution in step (4) into the corresponding channels in the multi-volume column chip, slide the chip, and the platinum nanoparticles react with H2O2 to produce O2, thereby pushing the ink forward. The distance the ink column moves forward is recorded, thereby achieving visual quantitative detection of the analyte.

[0014] Furthermore, a cascade signal amplification detection method based on platinum nanozymes of the present invention comprises the following steps:

[0015] In step (1), 1-5 mg / ml of 200-500 nm amino group-modified magnetic silica nanoparticles are diluted with 0.01 M PBS phosphate buffered saline with a pH value of 7.4, 20-100 μL of 25% glutaraldehyde is added to 2 ml of the modified magnetic silica nanoparticles, activated for 0.5-2 h under slow shaking conditions, the magnetic silica nanoparticles are separated by a magnet and 1 ml of 0.01 M PBS phosphate buffered saline with a pH value of 7.4 is added; 10 μL of 0.01-1 mg / ml capture antibody is added thereto, incubated overnight at 4° C., the magnetic silica nanoparticles are adsorbed by a magnet and the supernatant is discarded to remove unreacted capture antibody, thereby obtaining a magnetic nanoprobe; then a BSA bovine serum albumin solution is added for blocking for 0.5-2 h, the final concentration is 0.1-1%, the magnetic nanoprobe is adsorbed by a magnet, washed with 0.01 M PBS phosphate buffered saline with a pH value of 7.4 and the magnetic nanoprobe is resuspended, and stored at 4° C. in the dark for later use;

[0016] In step (2), 0.5-3 mg / ml of 10-50 nm platinum nanoparticles are diluted with 0.01 M Tris-HCl buffer solution with a pH value of 7.4, 10 μL of 0.01-1 mg / ml detection antibody is added thereto, and the mixture is incubated overnight at 4° C. The mixed solution is centrifuged at 5000-10000 r / min for 10 min to obtain a platinum nanoprobe; then a BSA bovine serum albumin solution is added to make a final concentration of 0.1-1%, and the reaction is continued for 0.5-2 h; the mixture is centrifuged again at 5000-10000 r / min for 10 min, and finally the blocked platinum nanoprobe is dispersed in 0.01 M PBS phosphate buffer solution with a pH value of 7.4, and stored at 4° C. in the dark for later use;

[0017] In step (3), different concentrations of analytes are added to 100 μL of the magnetic nanoprobe solution prepared in step (1), incubated at 37° C. for 0.5 to 5 h, adsorbed by a magnet, and washed with a 0.01 M PBS phosphate buffer solution with a pH value of 7.4; then, 100 μL of the platinum nanoprobe constructed in step (2) is added, incubated at 37° C. for 0.5 to 5 h to form a sandwich immune sandwich structure, adsorbed by a magnet, and washed with a 0.01 M Tris-HCl buffer solution with a pH value of 8.5;

[0018] In step (4), the sandwich immune sandwich structure composite probe constructed in step (3) is added to the dopamine solution, and the reaction is carried out at room temperature for 10 to 60 minutes. The platinum nanoparticles ultra-fast catalyze dopamine to form polydopamine and deposit on the surface of the magnetic silica nanoparticles blocked by BSA bovine serum albumin. The particles are separated by a magnet and resuspended in a 0.01M PBS phosphate buffer solution with a pH value of 7.4. Then, 5 to 30 μL of mercaptophenylboronic acid-modified platinum nanoparticles are added, and the reaction is slowly shaken at room temperature for 10 to 60 minutes. The supernatant is removed by a magnet and dispersed in 50 μL of an aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles.

[0019] In step (5), the ink, 0.5-5M H2O2, and the sample solution in step (4) are added to the corresponding channels in the multi-volume column chip using a pipette, and the chip is slid. The platinum nanoparticles react with H2O2 and produce O2, thereby pushing the ink forward. The distance the ink column moves forward within 5 minutes is recorded, thereby achieving visual quantitative detection of the analyte.

[0020] Furthermore, in step (1), the concentration of the capture antibody and the detection antibody in step (2) are both 0.5-1 mg / ml.

[0021] Furthermore, in step (1), the PBS phosphate buffered saline solution used to resuspend the magnetic nanoprobes and in step (2) used to resuspend the platinum nanoprobes contain 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin.

[0022] Furthermore, in step (3), the incubation time between the magnetic nanoprobe and the analyte, and between the platinum nanoprobe and the magnetic nanoprobe-analyte is 0.5 to 2 hours; the sandwich immune structure is formed by the specific recognition of the antibody to the antigen.

[0023] Further, in step (3), the analyte is the ovarian cancer biomarker CA125 carbohydrate antigen 125 or HE4 human epididymis protein 4.

[0024] Furthermore, in step (4), the dopamine solution contains 1-5M H2O2, 0.002-0.01g dopamine hydrochloride and 0.01M Tris-HCl buffer solution with a pH value of 8.5.

[0025] Furthermore, in step (4), the ultra-fast formation of polydopamine from dopamine utilizes the peroxidase activity of platinum nanoparticles; the deposition of polydopamine utilizes the superior reactivity of polydopamine to BSA bovine serum albumin containing amino groups.

[0026] Furthermore, in step (4), polydopamine and mercaptophenylboronic acid-modified platinum nanoparticles are combined by covalent reaction; the reaction of platinum nanoparticles with H2O2 and production of O2 utilizes the catalase activity of platinum nanoparticles.

[0027] Application of the platinum nanoparticles of the present invention in ovarian cancer biomarkers.

[0028] Beneficial effects: The detection method of the present invention has low cost, simple operation, short reaction time, high sensitivity and strong specificity, and provides a new analytical means for the instant diagnosis of cancer biomarkers, and can be extended to the detection of more biomarkers.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The present invention utilizes the principle of specific antigen recognition by antibodies to form a composite probe of a sandwich immune sandwich structure, namely, a magnetic nanoprobe-analyte-platinum nanoprobe, in the presence of a specific analyte, and then utilizes the horseradish peroxidase activity of the platinum nanoprobe to ultra-rapidly catalyze dopamine to polydopamine under H2O2 conditions, and deposit it on the surface of the composite probe, thereby cross-linking a large number of platinum nanoparticles containing mercaptophenylboronic acid functional groups. The entire process has a short reaction time, strong specificity, and can easily achieve detection signal enhancement.

[0031] (2) The present invention realizes the conversion of analyte recognition signals into visualized ink advancement distances, and also realizes ultrasensitive quantitative detection of analytes.

[0032] (3) The present invention requires a small amount of sample and reagents, and the detection cost is low. It can be used for low-cost quantitative detection, does not require the help of professional operators and complex equipment, and is easy to operate.

[0033] (4) The present invention is universal and can be extended to more biomarkers with high sensitivity and specificity by replacing antibodies that specifically recognize different biomarkers. The present invention provides a new analytical method for the instant diagnosis of cancer biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of the principle of a cascade signal amplification detection method based on platinum nanozymes provided by the present invention.

[0035] Figure 2 This is a comparison diagram of the ink advancement distance of the composite probe described in the present invention before and after the cascade signal amplification reaction.

[0036] Figure 3 This is a standard curve diagram of the ovarian cancer biomarker CA125 carbohydrate antigen 125 of the present invention.

[0037] Figure 4 This is a standard curve diagram of the ovarian cancer biomarker HE4 human epididymis protein 4 of the present invention. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0039] Example 1

[0040] Note: All proteins described in the present invention were purchased from Shanghai Lingchao Biotechnology Co., Ltd.

[0041] In this example, the ovarian cancer biomarker CA125 carbohydrate antigen 125 was used as the analyte, and a quantitative detection of the signal amplification was performed on it.

[0042] The present invention provides a cascade signal amplification detection method based on platinum nanozymes, comprising the following steps: (1) constructing a magnetic nanoprobe

[0043] 2mg / ml 200nm amino group modified magnetic silica nanoparticles were diluted with 0.01M PBS phosphate buffered saline with a pH value of 7.4, 90μLμL 25% glutaraldehyde was added to 2ml of the modified magnetic silica nanoparticles, activated under slow shaking for 1h, magnetic silica nanoparticles were separated by magnet and 1ml of 0.01M PBS phosphate buffered saline with a pH value of 7.4 was added; 10μL 1mg / ml CA125 carbohydrate antigen 125-capture antibody was added thereto, incubated overnight at 4°C, magnetic silica nanoparticles were adsorbed by magnet and the supernatant was discarded to remove unreacted capture antibody, and magnetic nanoprobes were prepared; then BSA bovine serum albumin solution was added for blocking for 0.5h, the final concentration was 1%, magnet adsorption, and 0.01M containing 0.1% Triton X-100, 5% sucrose, 1% BSA bovine serum albumin was used. The magnetic nanoprobes were washed and resuspended in PBS phosphate buffered saline with a pH value of 7.4 and stored at 4°C in the dark until use.

[0044] (2) Construction of platinum nanoprobes

[0045] 1 mg / ml of 50 nm platinum nanoparticles were diluted with 0.01 M Tris-HCl buffer solution with a pH value of 7.4, and 10 μL of 1 mg / ml carbohydrate antigen 125-detection antibody was added thereto, incubated overnight at 4°C, and the mixed solution was centrifuged at 8000 r / min for 10 min to obtain platinum nanoprobes; then BSA bovine serum albumin solution was added to make a final concentration of 0.5%, and the reaction was continued for 0.5 h; centrifuged again at 8000 r / min for 10 min, and finally the blocked platinum nanoprobes were dispersed in 0.01 M PBS phosphate buffer solution with a pH value of 7.4 containing 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin, and stored at 4°C in the dark for future use.

[0046] (3) Construction of sandwich immune structure

[0047] Different concentrations of CA125 carbohydrate antigen 125 were added to 100 μL of the magnetic nanoprobe solution prepared in step (1), incubated at 37°C for 1 hour, adsorbed by a magnet and washed with a 0.01M PBS phosphate buffer solution with a pH value of 7.4; then 100 μL of the platinum nanoprobe constructed in step (2) was added, incubated at 37°C for 1 hour to form a sandwich immune sandwich structure, adsorbed by a magnet and washed with a 0.01M Tris-HCl buffer solution with a pH value of 8.5. The sandwich immune sandwich structure is formed by the specific recognition of the antibody to the antigen. The formation of the sandwich immune sandwich structure is based on the principle of specific recognition of the antibody to the antigen.

[0048] (4) Induced cascade signal amplification reaction

[0049] The sandwich immune composite probe constructed in step (3) is added to the dopamine solution and reacted at room temperature for 30 minutes. The platinum nanoparticles use peroxidase activity to ultra-rapidly catalyze dopamine to form polydopamine and deposit on the surface of the magnetic silica nanoparticles blocked by BSA bovine serum albumin. The particles are separated by a magnet and resuspended in a 0.01M PBS phosphate buffer solution with a pH value of 7.4. Then, 30 μL of mercaptophenylboronic acid-modified platinum nanoparticles are added and the reaction is slowly shaken at room temperature for 20 minutes. The supernatant is removed by a magnet and dispersed in 50 μL of an aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles.

[0050] The dopamine solution is a 0.01M Tris-HCl tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution containing 5M H2O2, 0.002g dopamine hydrochloride and a pH value of 8.5.

[0051] The deposition of polydopamine utilizes the superior reactivity of polydopamine to BSA bovine serum albumin containing amino groups.

[0052] The polydopamine and the platinum nanoparticles modified with mercaptophenylboronic acid are combined through a covalent reaction.

[0053] (5) Visual quantitative detection of biomarkers

[0054] The ink, 5M H2O2, and the sample solution in step (4) were added to the corresponding channels in the multivariate volume column chip using a pipette. The chip was slid and the platinum nanoparticles reacted with H2O2 and produced O2 using catalase activity, thereby pushing the ink forward. The distance the ink column advanced within 5 minutes was recorded to achieve visual quantitative detection of biomarkers.

[0055] Application of the platinum nanoparticles of the present invention in ovarian cancer biomarkers.

[0056] Experimental Results

[0057] Figure 2 The figure shows the ink advancement distance of the composite probe before and after the cascade signal amplification reaction. It can be seen from the figure that, through comparison, it is proved that the cascade signal amplification reaction based on platinum nanozymes significantly improves the sensitivity of detection.

[0058] Figure 3 The standard curve of the ovarian cancer biomarker CA125 carbohydrate antigen 125 is shown. As can be seen from the figure, the signal of the detection system increases with the increase of CA125 carbohydrate antigen 125 concentration.

[0059] pass Figure 1 Briefly describe the principle of a cascade signal amplification detection method based on platinum nanozymes provided by the present invention:

[0060] Firstly, a magnetic nanoprobe is constructed by modifying a capture antibody on magnetic silica nanoparticles, and a platinum nanoprobe is constructed by modifying a detection antibody on platinum nanoparticles. In the presence of a specific analyte, the principle of antibody-antigen specific recognition is used to form a composite probe with a sandwich immune structure, namely, a magnetic nanoprobe-analyte-platinum nanoprobe. Then, the horseradish peroxidase activity of the platinum nanoprobe is used to ultra-rapidly catalyze dopamine into polydopamine under H2O2 conditions, and deposit it on the surface of the composite probe, thereby cross-linking a large number of platinum nanoparticles containing mercaptophenylboronic acid functional groups to achieve amplification of the detection signal. Finally, the catalase activity of the platinum nanoparticles is used to catalyze H2O2 to produce O2, and the analyte recognition signal is converted into a visualized ink advancement distance, thereby achieving quantitative analysis of the analyte.

[0061] Example 2

[0062] The difference between Example 2 and Example 1 is that the cascade signal amplification detection method based on platinum nanozymes of the present invention comprises the following steps:

[0063] In step (1), a magnetic nanoprobe is constructed:

[0064] 5 mg / ml of 400 nm amino group-modified magnetic silica nanoparticles were diluted with 0.01M PBS phosphate buffered saline with a pH value of 7.4, 20 μL of 25% glutaraldehyde was added to 2 ml of the modified magnetic silica nanoparticles, activated for 0.5 h under slow shaking conditions, separated the magnetic silica nanoparticles with a magnet and added with 1 ml of 0.01M PBS phosphate buffered saline with a pH value of 7.4; 10 μL of 0.5 mg / ml capture antibody was added thereto, incubated overnight at 4°C, the magnetic silica nanoparticles were adsorbed by a magnet and the supernatant was discarded to remove the unreacted capture antibody, thereby obtaining a magnetic nanoprobe; then a BSA bovine serum albumin solution was added for blocking for 1.5 h, the final concentration was 0.1%, and the magnetic nanoprobe was adsorbed; the magnetic nanoprobe was washed and resuspended with a 0.01M PBS phosphate buffered saline with a pH value of 7.4 containing 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin, and stored at 4°C in the dark for later use.

[0065] In step (2), construct a platinum nanoprobe:

[0066] 0.5 mg / ml of 40 nm platinum nanoparticles were diluted with 0.01 M Tris-HCl buffer solution with a pH value of 7.4, and 10 μL of 0.5 mg / ml detection antibody was added thereto, incubated overnight at 4°C, and the mixed solution was centrifuged at 5000 r / min for 10 min to obtain platinum nanoprobes; then BSA bovine serum albumin solution was added to make a final concentration of 1%, and the reaction was continued for 1.5 h; centrifuged again at 10000 r / min for 10 min; finally, the blocked platinum nanoprobes were dispersed in 0.01 M PBS phosphate buffer solution with a pH value of 7.4 containing 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin (, and stored at 4°C in the dark for future use.

[0067] In step (3), a sandwich immune sandwich structure is constructed:

[0068] Analytes of different concentrations were added to 100 μL of the magnetic nanoprobe solution prepared in step (1), incubated at 37°C for 0.5 h, adsorbed by a magnet and washed with a 0.01 M PBS phosphate buffer solution with a pH value of 7.4; then 100 μL of the platinum nanoprobe constructed in step (2) was added, incubated at 37°C for 0.5 h to form a sandwich immune structure, adsorbed by a magnet and washed with a 0.01 M Tris-HCl buffer solution with a pH value of 8.5; the sandwich immune structure was formed by the specific recognition of the antibody to the antigen.

[0069] In step (4), a cascade signal amplification reaction is induced: the sandwich immune sandwich structure composite probe constructed in step (3) is added to the dopamine solution, and the reaction is carried out at room temperature for 10 minutes. The platinum nanoparticles ultra-rapidly catalyze dopamine to form polydopamine and deposit on the surface of the magnetic silica nanoparticles blocked by BSA bovine serum albumin, and the particles are separated by a magnet and resuspended in a 0.01M PBS phosphate buffer solution with a pH value of 7.4; then, 5 μL of mercaptophenylboronic acid-modified platinum nanoparticles are added, and the reaction is slowly shaken at room temperature for 10 minutes. The supernatant is removed by magnetic separation and dispersed in 50 μL of aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles;

[0070] The dopamine solution contains 3M H2O2, 0.008g dopamine hydrochloride and 0.01M Tris-HCl buffer solution with a pH value of 8.5. The ultra-fast formation of dopamine from polydopamine utilizes the peroxidase activity of platinum nanoparticles; the deposition of polydopamine utilizes the superior reactivity of polydopamine to BSA bovine serum albumin containing amino groups. Polydopamine is bound to mercaptophenylboronic acid-modified platinum nanoparticles through a covalent reaction.

[0071] In step (5), visual quantitative detection of the analyte:

[0072] Use a pipette to add ink, 0.5M H2O2, and the sample solution in step (4) into the corresponding channels of the multi-volume column chip. Slide the chip, and the platinum nanoparticles react with H2O2 to produce O2, thereby pushing the ink forward. The distance the ink column moves within 5 minutes is recorded to achieve visual quantitative detection of the analyte.

[0073] Example 3

[0074] The difference between Example 3 and Example 1: A cascade signal amplification detection method based on platinum nanozymes of the present invention comprises the following steps:

[0075] In step (1), a magnetic nanoprobe is constructed:

[0076] 5 mg / ml of 400 nm amino group-modified magnetic silica nanoparticles were diluted with 0.01M PBS phosphate buffered saline with a pH value of 7.4, 20 μL of 25% glutaraldehyde was added to 2 ml of the modified magnetic silica nanoparticles, activated for 0.5 h under slow shaking conditions, separated the magnetic silica nanoparticles with a magnet and added with 1 ml of 0.01M PBS phosphate buffered saline with a pH value of 7.4; 10 μL of 0.8 mg / ml capture antibody was added thereto, incubated overnight at 4°C, the magnetic silica nanoparticles were adsorbed by a magnet and the supernatant was discarded to remove the unreacted capture antibody, thereby obtaining a magnetic nanoprobe; then a BSA bovine serum albumin solution was added for blocking for 1.5 h, the final concentration was 0.1%, and the magnetic nanoprobe was adsorbed; the magnetic nanoprobe was washed and resuspended with a 0.01M PBS phosphate buffered saline with a pH value of 7.4 containing 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin, and stored at 4°C in the dark for later use.

[0077] In step (2), construct a platinum nanoprobe:

[0078] 0.5 mg / ml of 40 nm platinum nanoparticles were diluted with 0.01 M Tris-HCl buffer solution with a pH value of 7.4, and 10 μL of 0.8 mg / ml detection antibody was added thereto, incubated overnight at 4°C, and the mixed solution was centrifuged at 5000 r / min for 10 min to obtain a platinum nanoprobe; then BSA bovine serum albumin solution was added to make a final concentration of 1%, and the reaction was continued for 1.5 h; centrifuged again at 10000 r / min for 10 min; finally, the blocked platinum nanoprobe was dispersed in 0.01 M PBS phosphate buffer solution with a pH value of 7.4 containing 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin, and stored at 4°C in the dark for future use.

[0079] In step (3), a sandwich immune sandwich structure is constructed:

[0080] Analytes of different concentrations were added to 100 μL of the magnetic nanoprobe solution prepared in step (1), incubated at 37°C for 2 h, adsorbed by a magnet and washed with a 0.01 M PBS phosphate buffer solution with a pH value of 7.4; then 100 μL of the platinum nanoprobe constructed in step (2) was added, incubated at 37°C for 2 h to form a sandwich immune structure, adsorbed by a magnet and washed with a 0.01 M Tris-HCl buffer solution with a pH value of 8.5; the sandwich immune structure was formed by the specific recognition of the antibody to the antigen.

[0081] In step (4), a cascade signal amplification reaction is induced:

[0082] The composite probe with sandwich immune sandwich structure constructed in step (3) is added to the dopamine solution, and the mixture is reacted at room temperature for 60 minutes. The platinum nanoparticles ultra-rapidly catalyze dopamine to form polydopamine and deposit on the surface of magnetic silica nanoparticles blocked by BSA bovine serum albumin. The mixture is separated by a magnet and resuspended in a 0.01M PBS phosphate buffer solution with a pH value of 7.4. Then, 20 μL of platinum nanoparticles modified with mercaptophenylboronic acid are added, and the mixture is slowly shaken and reacted at room temperature for 60 minutes. The supernatant is removed by a magnet and dispersed in 50 μL of an aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles.

[0083] The dopamine solution contains 1M H2O2, 0.01g dopamine hydrochloride and 0.01M Tris-HCl buffer solution with a pH value of 8.5. The ultrafast formation of dopamine into polydopamine utilizes the peroxidase activity of platinum nanoparticles.

[0084] In step (5), visual quantitative detection of the analyte:

[0085] Use a pipette to add the ink, 4M H2O2, and the sample solution in step (4) into the corresponding channels in the multi-volume column chip.

[0086] Example 4

[0087] In this example, HE4 human epididymis protein 4, a biomarker for ovarian cancer, was used as an analyte, and a quantitative detection was performed on it by signal amplification.

[0088] The difference from Example 1 is that the CA125 carbohydrate antigen 125-capture antibody is replaced by HE4 human epididymis protein 4-capture antibody, and the CA125 carbohydrate antigen 125-detection antibody is replaced by HE4 human epididymis protein 4-detection antibody.

[0089] Figure 4 The standard curve of the ovarian cancer biomarker HE4 human epididymis protein 4 is shown. As can be seen from the figure, the signal of the detection system increases with the increase of HE4 human epididymis protein 4 concentration.

[0090] The above embodiments are only preferred implementation modes of the present invention and do not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, the technical solutions obtained by equivalent replacement or equivalent transformation, or several improvements and modifications made without departing from the principles of the present invention, are all within the protection scope of the present invention.

Claims

1. A cascade signal amplification detection method based on platinum nanozymes, characterized in that The steps include: (1) Construction of magnetic nanoprobes: dilute amino group-modified magnetic silica nanoparticles with PBS phosphate buffered saline solution, add glutaraldehyde to the modified magnetic silica nanoparticles for activation, separate the magnetic silica nanoparticles with a magnet and add PBS phosphate buffered saline solution; add capture antibodies, incubate overnight, discard the supernatant, and prepare a magnetic nanoprobe solution; Then, BSA bovine serum albumin solution was added for blocking, adsorbed by magnet, washed and resuspended with PBS phosphate buffered saline solution, and stored in dark place for future use; (2) Constructing platinum nanoprobes: diluting platinum nanoparticles with Tris-HCl buffer solution, adding detection antibodies thereto, incubating overnight, and centrifuging the mixed solution to obtain platinum nanoprobes; Then add BSA bovine serum albumin solution to make the final concentration of 0.1-1%, and continue the reaction; centrifuge again, and finally disperse the blocked platinum nanoprobes in PBS phosphate buffered saline solution and store in the dark for later use; (3) Constructing a sandwich immune structure: adding different concentrations of analytes to the magnetic nanoprobe solution prepared in step (1), incubating, adsorbing with a magnet and washing with a PBS phosphate buffer solution; then adding the platinum nanoprobe constructed in step (2), incubating to form a sandwich immune structure, adsorbing with a magnet and washing with a Tris-HCl tris-hydroxymethylaminomethane-hydrochloric acid buffer solution; (4) Induced cascade signal amplification reaction: The sandwich immune sandwich structure constructed in step (3) is added to the dopamine solution and reacted at room temperature. The platinum nanoparticles ultra-rapidly catalyze dopamine to form polydopamine and deposit on the surface of the magnetic silica nanoparticles blocked by BSA bovine serum albumin. The mixture is separated by a magnet and resuspended in PBS phosphate buffered saline solution. Then, the platinum nanoparticles modified with mercaptophenylboronic acid are added, and the reaction is slowly shaken at room temperature. The supernatant is separated by a magnet and dispersed in the aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles. (5) Visual quantitative detection of analytes: Use a pipette to add the ink, H2O2, and the sample solution in step (4) into the corresponding channels of the multi-volume column chip. Slide the chip, and the platinum nanoparticles react with H2O2 to produce O2, thereby pushing the ink forward. The distance the ink column moves forward is recorded, thereby achieving visual quantitative detection of the analyte.

2. The cascade signal amplification detection method based on platinum nanozymes according to claim 1, characterized in that: In step (1), 1-5 mg / ml of 200-500 nm amino group-modified magnetic silica nanoparticles are diluted with 0.01 M PBS phosphate buffered saline with a pH value of 7.4, 20-100 µL of 25% glutaraldehyde is added to 2 ml of the modified magnetic silica nanoparticles, activated under slow shaking for 0.5-2 h, separated by a magnet and 1 ml of 0.01 M PBS phosphate buffered saline with a pH value of 7.4 is added; 10 µL of 0.01-1 mg / ml capture antibody is added thereto, incubated overnight at 4°C, the magnetic silica nanoparticles are adsorbed by a magnet and the supernatant is discarded to remove unreacted capture antibody, thereby obtaining a magnetic nanoprobe; then a BSA bovine serum albumin solution is added for blocking for 0.5-2 h, the final concentration is 0.1-1%, adsorbed by a magnet, washed and resuspended with 0.01 M PBS phosphate buffered saline with a pH value of 7.4, and stored at 4°C in the dark for later use; In step (2), 0.5-3 mg / ml of 10-50 nm platinum nanoparticles are diluted with 0.01 M Tris-HCl buffer solution with a pH value of 7.4, 10 µL of 0.01-1 mg / ml detection antibody is added thereto, and the mixture is incubated overnight at 4°C. The mixed solution is centrifuged at 5000-10000 r / min for 10 min to prepare platinum nanoprobes; then, a BSA bovine serum albumin solution is added to make a final concentration of 0.1-1%, and the reaction is continued for 0.5-2 h; the mixture is centrifuged again at 5000-10000 r / min for 10 min, and finally the blocked platinum nanoprobes are dispersed in 0.01 M PBS phosphate buffer solution with a pH value of 7.4, and stored at 4°C in the dark for later use; In step (3), different concentrations of analytes are added to 100 μL of the magnetic nanoprobe solution prepared in step (1), incubated at 37°C for 0.5-5 h, adsorbed by a magnet and washed with a 0.01 M PBS phosphate buffer solution with a pH value of 7.4; then, 100 μL of the platinum nanoprobe constructed in step (2) is added, incubated at 37°C for 0.5-5 h to form a sandwich immune sandwich structure, adsorbed by a magnet and washed with a 0.01 M Tris-HCl buffer solution with a pH value of 8.5; In step (4), the sandwich immune sandwich structure constructed in step (3) is added to the dopamine solution, and the reaction is carried out at room temperature for 10 to 60 minutes. The platinum nanoparticles ultra-rapidly catalyze dopamine to form polydopamine and deposit on the surface of the magnetic silica nanoparticles blocked by BSA bovine serum albumin. The particles are separated by a magnet and resuspended in a 0.01M PBS phosphate buffer solution with a pH value of 7.

4. Then, 5 to 30 μL of platinum nanoparticles modified with mercaptophenylboronic acid are added, and the reaction is slowly shaken at room temperature for 10 to 60 minutes. The supernatant is removed by a magnet and dispersed in 50 μL of an aqueous phase, thereby preparing a sample solution containing a large amount of platinum nanoparticles. In step (5), the ink, 0.5-5 M H2O2, and the sample solution in step (4) are added to the corresponding channels in the multi-volume column chip using a pipette. The chip is slid, and the platinum nanoparticles react with H2O2 to produce O2, thereby pushing the ink forward. The distance the ink column moves within 5 minutes is recorded to achieve visual quantitative detection of the analyte.

3. The cascade signal amplification detection method based on platinum nanozymes according to claim 2, characterized in that: In step (1), the concentration of the capture antibody and the detection antibody in step (2) are both 0.5-1 mg / ml.

4. The cascade signal amplification detection method based on platinum nanozymes according to claim 3, characterized in that: The PBS phosphate buffered saline solution used for resuspending the magnetic nanoprobes in step (1) and for the platinum nanoprobes in step (2) contains 0.1% Triton X-100, 5% sucrose, and 1% BSA bovine serum albumin.

5. The cascade signal amplification detection method based on platinum nanozymes according to claim 3, characterized in that: In step (3), the incubation time between the magnetic nanoprobe and the analyte, and between the platinum nanoprobe and the magnetic nanoprobe-analyte is 0.5 to 2 h; the sandwich immune structure is formed by the specific recognition of the antibody to the antigen.

6. The cascade signal amplification detection method based on platinum nanozymes according to claim 5, characterized in that: In step (3), the analyte is the ovarian cancer biomarker CA125 carbohydrate antigen 125 or HE4 human epididymis protein 4.

7. The cascade signal amplification detection method based on platinum nanozymes according to claim 1, characterized in that: In step (4), the dopamine solution contains 1-5 M H2O2, 0.002-0.01 g dopamine hydrochloride and 0.01 M Tris-HCl buffer solution with a pH value of 8.

5.

8. The cascade signal amplification detection method based on platinum nanozymes according to claim 3, characterized in that: In step (4), the ultra-fast formation of polydopamine from dopamine utilizes the peroxidase activity of platinum nanoparticles.

9. The cascade signal amplification detection method based on platinum nanozymes according to claim 8, characterized in that: In step (4), the polydopamine and mercaptophenylboronic acid-modified platinum nanoparticles are combined by covalent reaction; the platinum nanoparticles react with H2O2 to produce O2 by utilizing the catalase activity of the platinum nanoparticles.

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