Micro whole blood heart failure marker immunochromatographic test strip and preparation method thereof

Abstract

The present invention discloses a micro-whole blood heart failure marker immunochromatographic test strip and its preparation method, which relates to the field of heart failure detection technology. The micro-whole blood heart failure marker immunochromatographic test strip comprises a blood filter pad, a binding pad, a nitrocellulose membrane, a blotting paper and a polystyrene base plate. The blood filter pad, the binding pad, the nitrocellulose membrane and the blotting paper are fixed on the polystyrene base plate in sequence. A detection line T line and a quality control line C line are respectively provided on both sides of the nitrocellulose membrane. The detection line T line is adjacent to the binding pad, and the quality control line C line is adjacent to the blotting paper. The present invention also provides a preparation method and a method for using the micro-whole blood heart failure marker immunochromatographic test strip, which has high detection sensitivity and simple operation, and effectively solves the problem of large interference of endogenous peroxidase in fingertip blood / micro whole blood / plasma under acidic conditions in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of heart failure detection, and in particular to a trace whole blood heart failure marker immunochromatographic detection test strip and a preparation method thereof. Background Art

[0002] Heart failure (HF) is a clinical syndrome caused by structural and / or functional abnormalities of the heart, resulting in reduced cardiac output and / or increased intracardiac pressure at rest or during stress. It is reported that approximately 64.3 million people worldwide suffer from HF, with approximately 8.9 million in my country. The prevalence of HF in people aged 35 and over is 1.3%. This prevalence is increasing due to an aging population, poorly controlled diabetes, and hypertension. Despite advances in HF treatment and care, the prognosis for patients remains poor, with a five-year survival rate of approximately 50%. Furthermore, HF limits patients' physical, psychological, and social functioning, severely impacting their quality of life and placing a significant burden on individuals, their families, and the healthcare system. Therefore, it is crucial to develop point-of-care (POC) biomarkers for HF to accurately monitor disease progression and treatment efficacy in patients with HF and to adjust treatment plans.

[0003] Nanozyme immunochromatographic technology, an emerging detection method, has achieved breakthroughs in rapid diagnostics thanks to its high sensitivity and specificity, providing innovative solutions for clinical point-of-care testing. Currently, approximately 90% of peroxidase nanozymes (PODs) are optimally catalyzed in acidic conditions. However, endogenous peroxidases in fingertip blood, trace whole blood, or plasma significantly interfere with these conditions, hindering their potential biomedical applications. Currently, there are no comparable neutral nanozyme-catalyzed test strips on the market for detecting heart failure markers in fingertip blood, trace whole blood, or plasma. Summary of the Invention

[0004] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing a trace whole blood heart failure marker immunochromatographic test strip, which effectively solves the problem in the prior art of large interference from endogenous peroxidase in fingertip blood / trace whole blood / plasma under acidic conditions.

[0005] To achieve the above object, the present invention solves the technical problem by adopting a technical solution: providing a method for preparing a micro whole blood heart failure marker immunochromatographic test strip, comprising the following steps:

[0006] S1. Dispersing the Au1Os2 bimetallic nanozyme in a phosphate buffer solution, then adding a heart failure marker detection antibody and mixing, centrifuging and discarding the supernatant, adding a phosphate buffer solution to terminate the reaction, and then ultrasonically dispersing the mixture, adding a bovine serum albumin solution to obtain an Au1Os2 nanozyme-heart failure marker detection antibody probe, and diluting the Au1Os2 nanozyme-heart failure marker detection antibody probe twice with a diluent for later use;

[0007] S2, soaking the conjugate pad in the conjugate pad pretreatment solution, soaking the blood filter pad in the blood filter pad pretreatment solution, and after drying, spraying the Au1Os2 nanozyme-heart failure marker detection antibody probe on the conjugate pad and drying;

[0008] S3. Draw the quality control line C and the detection line T on both sides of the nitrocellulose membrane, and then stick the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene base plate in sequence for assembly to obtain a trace whole blood heart failure marker immunochromatographic test strip.

[0009] Furthermore, in step S1, the Au1Os2 bimetallic nanozyme is prepared by the following method: tetrachloroauric acid, potassium hexachloroosmate and polyvinylpyrrolidone solution are mixed and stirred, and then sodium borohydride solution is added dropwise and stirred again, centrifuged, washed with water, and resuspended with ultrapure water to obtain the Au1Os2 bimetallic nanozyme.

[0010] Furthermore, in step S1, 50-500 μL of 40 mM tetrachloroauric acid, 100-1000 μL of 40 mM potassium hexachloroosmate and 4-40 μL of 20 mg / mL polyvinylpyrrolidone solution were mixed, stirred at 500 rpm at room temperature for 5 min, and then 500-1000 μL of 100 mM sodium borohydride solution was added dropwise and stirred at 20-100°C for 10 min. The mixture was centrifuged at 9000-14000 rpm for 5-15 min, washed with water three times, and resuspended with ultrapure water to obtain Au1Os2 bimetallic nanozyme.

[0011] Furthermore, in step S1, the heart failure marker is at least one of B-type natriuretic peptide, N-terminal pro-brain natriuretic peptide and growth-stimulated gene 2 protein.

[0012] Furthermore, in step S1, the volume ratio of Au1Os2 bimetallic nanozyme, phosphate buffer and heart failure marker detection antibody is 20-160 μL:200-1500 μL:10-80 μL.

[0013] Furthermore, in step S1, the concentration of Au1Os2 bimetallic nanozyme is 5-6 mg / mL.

[0014] Furthermore, in step S1, the concentration of Au1Os2 bimetallic nanozyme was 5 mg / mL.

[0015] The beneficial effect of adopting the above further scheme is that the Au1Os2 bimetallic nanozyme is a peroxidase and has high peroxidase-like activity under neutral conditions.

[0016] Furthermore, in step S1, the concentration of the phosphate buffer is 0.01-0.02 M, and the pH value is 7.3-7.5.

[0017] Furthermore, in step S1, the concentration of the phosphate buffer is 0.01 M and the pH value is 7.4.

[0018] Furthermore, in step S1, the concentration of the heart failure marker detection antibody is 1-2 mg / mL.

[0019] Furthermore, in step S1, the concentration of the heart failure marker detection antibody is 1 mg / mL.

[0020] Furthermore, in step S1, the mixture is mixed by inversion at room temperature for 1-1.5 h.

[0021] Furthermore, in step S1, the mixture was mixed by inversion at room temperature for 1 h.

[0022] Furthermore, in step S1, centrifugation is performed at 13,000-15,000 r / min for 9-11 min.

[0023] Furthermore, in step S1, centrifugation is performed at 14000 r / min for 10 min.

[0024] Furthermore, in step S1, 20-160 μL of phosphate buffer was added to terminate the reaction.

[0025] Furthermore, in step S1, ultrasonic dispersion is performed at 20-100 W for 1-3 min.

[0026] Furthermore, in step S1, the volume ratio of bovine serum albumin solution to Au1Os2 bimetallic nanozyme is 20-90 μL:20-160 μL.

[0027] Furthermore, in step S1, the concentration of the bovine serum albumin solution is 10-11%.

[0028] Furthermore, in step S1, the concentration of the bovine serum albumin solution is 10%.

[0029] Furthermore, in step S1, a bovine serum albumin solution is added and blocked for 0.5-1 h.

[0030] Furthermore, in step S1, bovine serum albumin solution was added and blocked for 0.5 h.

[0031] Furthermore, in step S1, the diluent includes the following raw materials in percentage by mass: 1-2% bovine serum albumin, 4-6% sucrose, 0.1-0.5% Tween-20, and the remainder is phosphate buffer.

[0032] Furthermore, in step S2, the conjugate pad pretreatment solution includes the following raw materials in percentage by mass: 0.1-0.2% polyvinyl pyrrolidone, 0.2-0.3% Tween-20, 5-6% sucrose and 1-2% bovine serum albumin, with the remainder being phosphate buffer.

[0033] Furthermore, in step S2, the conjugate pad pretreatment solution includes the following raw materials in percentage by mass: 0.1% polyvinyl pyrrolidone, 0.2% Tween-20, 5% sucrose, and 1% bovine serum albumin, with the remainder being phosphate buffer.

[0034] Furthermore, in step S2, the concentration of the phosphate buffer is 0.01-0.02 M, and the pH value is 7.3-7.5.

[0035] Furthermore, in step S2, the concentration of the phosphate buffer is 0.01 M and the pH value is 7.4.

[0036] Furthermore, in step S2, the blood filter pad pretreatment liquid is prepared by the following method: rabbit anti-human red blood cell antibody is added to the above-mentioned conjugate pad pretreatment liquid to obtain the blood filter pad pretreatment liquid.

[0037] Furthermore, the concentration of rabbit anti-human red blood cell antibody in the blood filter pad pretreatment solution is 0.4-0.6 mg / mL.

[0038] Furthermore, the concentration of rabbit anti-human red blood cell antibody in the blood filter pad pretreatment solution was 0.5 mg / mL.

[0039] Further, in step S2, soaking is performed for 15-20 minutes.

[0040] Furthermore, in step S2, soaking is performed for 15 minutes.

[0041] Furthermore, in step S2, the mixture is placed together with absorbent paper in a constant temperature and humidity chamber and dried for 2-24 hours at 37-50°C and a humidity of 15-30%.

[0042] Furthermore, in step S2, the Au1Os2 nanozyme-heart failure marker detection antibody probe is sprayed at a rate of 4-10 μL / cm using a film gold spraying apparatus.

[0043] Furthermore, in step S2, the mixture is placed in a constant temperature incubator and dried for 9-11 hours at 39-41° C. and a humidity of 19-21%.

[0044] Furthermore, in step S2, the sample was placed in a constant temperature incubator and dried for 10 h at 40° C. and 20% humidity.

[0045] Furthermore, in step S3, the capture antibody corresponding to the heart failure marker is sprayed on the nitrocellulose membrane as the detection line T line, and the goat anti-mouse monoclonal antibody is sprayed on the nitrocellulose membrane as the quality control line C line.

[0046] Furthermore, in step S3, the heart failure marker capture antibody is diluted to 2-3 mg / mL using a membrane diluent.

[0047] Furthermore, in step S3, the heart failure marker capture antibody is diluted to 2 mg / mL using a membrane diluent.

[0048] Furthermore, the stripping diluent includes the following raw materials in mass percentage: 0.5%-2% sodium chloride, 1%-3% sucrose, and the balance is phosphate buffer.

[0049] Furthermore, in step S3, a heart failure marker capture antibody is sprayed onto one side of the nitrocellulose membrane using a film gold spraying apparatus as a detection line T line.

[0050] Furthermore, in step S3, the goat anti-mouse monoclonal antibody is diluted to 0.8-0.9 mg / mL using a membrane diluent.

[0051] Furthermore, in step S3, the goat anti-mouse monoclonal antibody is diluted to 0.8 mg / mL using a membrane diluent.

[0052] Furthermore, in step S3, goat anti-mouse monoclonal antibody is sprayed on the other side of the nitrocellulose membrane using a film gold spraying apparatus as a detection line C line.

[0053] Furthermore, in step S3, during assembly, two adjacent parts overlap by 1-2 mm.

[0054] Furthermore, in step S3, the micro whole blood heart failure marker immunochromatographic test strip is cut into 4 mm width and stored in a sealed aluminum foil bag at 24-26°C.

[0055] The micro whole blood heart failure marker immunochromatographic detection test strip is prepared by the preparation method of the micro whole blood heart failure marker immunochromatographic detection test strip.

[0056] The above-mentioned trace whole blood heart failure marker immunochromatographic test strip includes a blood filter pad, a conjugation pad, a nitrocellulose membrane, absorbent paper and a polystyrene base. The blood filter pad, conjugation pad, nitrocellulose membrane and absorbent paper are arranged in parallel from left to right on the surface of the polystyrene base, and the two adjacent parts overlap by 1-2 mm. The detection line T line and the quality control line C line are respectively arranged on both sides of the nitrocellulose membrane. The detection line T line is adjacent to the conjugation pad, and the quality control line C line is adjacent to the absorbent paper.

[0057] The method for using the above-mentioned trace whole blood heart failure marker immunochromatographic test strip includes the following steps: diluting the sample to be tested and adding it dropwise to the blood filter pad, starting the chromatographic test, applying a color developing solution on the T line of the nitrocellulose membrane after completion, observing the color signals of the T line and C line before and after color development, determining the test results, and then analyzing the grayscale values of the T line and C line, establishing a standard curve based on the T / C signal ratio and the corresponding heart failure marker concentration value, and substituting the T / C signal value of the test strip to be tested into the standard curve for quantitative detection.

[0058] Furthermore, the sample to be tested is fingertip blood and / or a trace amount of whole blood.

[0059] Further, the sample to be tested is diluted with a sample diluent.

[0060] Furthermore, the sample diluent was phosphate buffered saline containing 1-2% Tween-20.

[0061] Furthermore, the sample diluent was phosphate buffered saline containing 1.5% Tween-20.

[0062] Furthermore, the volume of the sample to be tested is 5-35 μL.

[0063] Furthermore, the volume ratio of the sample to be tested to the sample diluent is 5-35 μL:50-100 μL.

[0064] Furthermore, the volume ratio of the sample to be tested to the sample diluent is 15 μL:60 μL.

[0065] Furthermore, the chromatography detection time is 5-30 min.

[0066] Furthermore, the volume ratio of the sample to be tested to the color developing solution is 5-35 μL:0.6-0.8 μL.

[0067] Furthermore, the volume ratio of the sample to be tested to the color developing solution is 15 μL:0.7 μL.

[0068] Furthermore, the color developing solution includes the following components: 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide solution and phosphate buffer.

[0069] Furthermore, the concentration of the 3,3',5,5'-tetramethylbenzidine solution in the color developing solution is 8-9 mM.

[0070] Furthermore, the concentration of the 3,3',5,5'-tetramethylbenzidine solution in the color developing solution is 8 mM.

[0071] Furthermore, the concentration of the hydrogen peroxide solution in the color developing solution is 0.1-0.2 mM.

[0072] Furthermore, the concentration of the hydrogen peroxide solution in the color developing solution is 0.1 mM.

[0073] Furthermore, the concentration of phosphate buffer in the color developing solution is 0.01-0.2 M.

[0074] Furthermore, the concentration of phosphate buffer in the color developing solution is 0.01 M.

[0075] Furthermore, the color development time is 15-60 s.

[0076] Furthermore, the color development time was 30 s.

[0077] Furthermore, the test result is determined as follows: if only the quality control line C appears, the test result is determined to be negative; if both the quality control line C and the test line T appear, the test result is determined to be positive.

[0078] The present invention has the following beneficial effects:

[0079] 1. This invention, for the first time, utilizes a liquid-phase reduction method to prepare Au1Os2 bimetallic nanozymes, resulting in a particle size of approximately 3.78 nm. The synthesis method is simple, and the dispersibility and stability are excellent. The Au1Os2 bimetallic nanozymes are peroxidase nanozymes with high peroxidase-like activity under neutral conditions, effectively addressing the existing problem of significant interference from endogenous peroxidases in fingertip blood, trace whole blood, or plasma under acidic conditions.

[0080] 2. The immunochromatographic test strip for micro-whole-blood heart failure markers prepared by the present invention has a real sample detection sensitivity of up to 0.058 ng / mL, strong anti-interference ability, good reliability, and strong specificity. It combines the simplicity, rapidity, and field application of colloidal gold technology with high sensitivity and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 This is a comparison diagram before and after catalytic color development of Example 1;

[0082] Figure 2 This is a standard curve diagram of the T / C ratio before and after catalysis and the corresponding N-terminal pro-brain natriuretic peptide concentration in Example 1;

[0083] Figure 3 This is the test result diagram of colloidal gold test strips in Comparative Example 1;

[0084] Figure 4 is a standard curve diagram of the T / C ratio and the corresponding N-terminal pro-brain natriuretic peptide concentration in the case of colloidal gold test strip detection in Comparative Example 1;

[0085] Figure 5 Transmission electron microscopy image of Au1Os2 bimetallic nanozyme;

[0086] Figure 6 Element distribution map of Au1Os2 bimetallic nanozyme;

[0087] Figure 7 Calculation diagram of the peroxidase specific activity of Au1Os2 bimetallic nanozyme under acidic and neutral conditions;

[0088] Figure 8 This is an analysis diagram of the specific detection results described in an embodiment of the present invention;

[0089] Figure 9 This is a box plot of the precision experiment described in the embodiment of the present invention. DETAILED DESCRIPTION

[0090] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0091] Example 1

[0092] The micro whole blood heart failure marker immunochromatographic test strip includes a blood filter pad, a conjugation pad, a nitrocellulose membrane, absorbent paper and a polystyrene base. The blood filter pad, conjugation pad, nitrocellulose membrane and absorbent paper are arranged in parallel from left to right on the surface of the polystyrene base, and the two adjacent parts overlap by 1-2 mm. The detection line T line and the quality control line C line are respectively arranged on both sides of the nitrocellulose membrane. The detection line T line is adjacent to the conjugation pad, and the quality control line C line is adjacent to the absorbent paper.

[0093] The method for preparing the above-mentioned micro whole blood heart failure marker immunochromatographic test strip comprises the following steps:

[0094] S1. Disperse 100 μL of 5 mg / mL Au1Os2 bimetallic nanozyme in 1000 μL of 0.01 M phosphate buffer at a pH of 7.4, then add 50 μL of 1 mg / mL N-terminal brain natriuretic peptide precursor detection antibody and mix well. Centrifuge at 14000 r / min for 10 min and discard the supernatant. Add 100 μL of phosphate buffer to terminate the reaction, then perform ultrasonic dispersion, add 10% bovine serum albumin solution to obtain Au1Os2 nanozyme-heart failure marker detection antibody probe, and dilute the Au1Os2 nanozyme-heart failure marker detection antibody probe twice with diluent for standby use. The diluent includes the following percentages of raw materials: 1% bovine serum albumin, 5% sucrose, 0.3% Tween-20, and the remainder is phosphate buffer;

[0095] S2. Soak the conjugate pad in the conjugate pad pretreatment solution for 15 min, soak the blood filter pad in the blood filter pad pretreatment solution for 15 min, place it in a constant temperature and humidity chamber together with absorbent paper, dry it at 40°C and 20% humidity for 10 h, spray the Au1Os2 nanozyme-heart failure marker detection antibody probe on the conjugate pad with a film spray gold instrument at a rate of 8 μL / cm and place it in a constant temperature incubator, dry it at 40°C and 20% humidity for 10 h, the above-mentioned conjugate pad pretreatment solution includes the following raw materials by mass percentage: 0.1% polyvinyl pyrrolidone, 0.2% Tween-20, 5% sucrose and 1% bovine serum albumin, and the balance is phosphate buffer solution, the above-mentioned blood filter pad pretreatment solution includes the following raw materials by mass percentage: 0.1% polyvinyl pyrrolidone, 0.2% Tween-20, 5% sucrose, 1% bovine serum albumin and 0.5 mg / mL rabbit anti-human erythrocyte antibody, the remainder being phosphate buffer, the concentration of phosphate buffer being 0.01 M, and the pH being 7.4;

[0096] S3. Dilute the N-terminal pro-brain natriuretic peptide capture antibody to 2 mg / mL with a membrane diluent, spray the N-terminal pro-brain natriuretic peptide capture antibody on one side of the nitrocellulose membrane using a membrane gold sprayer as the detection line T line, dilute the goat anti-mouse monoclonal antibody to 0.8 mg / mL with a membrane diluent, spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane using a membrane gold sprayer as the detection line C line, the above-mentioned membrane diluent includes the following raw materials in mass percentage: 1% sodium chloride, 2% sucrose, and the remainder is phosphate buffer, and then stick the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene base plate in sequence for assembly. During assembly, the two adjacent parts overlap each other by 1-2 mm to obtain a micro whole blood heart failure marker immunochromatographic test strip, cut the micro whole blood heart failure marker immunochromatographic test strip into 4 mm width, and store it in a sealed aluminum foil bag at 25°C.

[0097] The method for using the above-mentioned micro-whole blood heart failure marker immunochromatographic test strip includes the following steps: taking a total of 15 μL of the subject's micro-whole blood as the test sample, diluting the test sample with 60 μL of phosphate buffer containing 1-2% Tween-20, and then dropping it onto the blood filter pad to start the chromatographic test. After 15 minutes, apply a color developing solution (8mM 3,3',5,5'-tetramethylbenzidine solution (TMB), 0.1 M hydrogen peroxide solution (H2O2), 0.01 M phosphate buffer (PBS), pH 7) on the T line of the nitrocellulose membrane, and take a photo after 30 seconds of color development to record the results. Figure 1 As shown, Figure 1 In the figure, the eye pattern is marked as the test strip test results corresponding to the concentration of the detection limit. The upper figure shows Au1Os2 before color development, and the lower figure shows Au1Os2+TMB+H2O2, pH 7.0 after color development. Image J software was used to analyze the grayscale values of the T line and C line, and a standard curve was established based on the T / C signal ratio and the corresponding N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration value. The results showed that the detection limit of the trace whole blood heart failure marker immunochromatographic test strip was 5.76 ng / mL before catalytic color development and 0.058 ng / mL after catalytic color development. The linear correlation coefficient R was 5.76-115.2 ng / mL before catalytic color development. 2 The linear correlation coefficient R was 97.47% in the range of 0.058-115.2 ng / mL after catalytic color development. 2 The standard curve established by the T / C ratio before and after catalysis and the corresponding N-terminal pro-brain natriuretic peptide concentration is as follows Figure 2 As shown, Figure 2 In the figure, Au1Os2 is the standard curve before catalysis, and Au1Os2+TMB+H2O2, pH 7.0 is the standard curve after catalysis.

[0098] Example 2

[0099] The immunochromatographic test strip for micro whole blood heart failure markers is similar to that in Example 1.

[0100] The method for preparing the above-mentioned micro whole blood heart failure marker immunochromatographic test strip comprises the following steps:

[0101] S1. Disperse 20 μL of 5 mg / mL Au1Os2 bimetallic nanozyme in 200 μL of 0.01 M phosphate buffer at a pH of 7.3, then add 10 μL of 1 mg / mL B-type natriuretic peptide detection antibody and mix well. Centrifuge at 13,000 r / min for 9 min and discard the supernatant. Add 20 μL of phosphate buffer to terminate the reaction, then perform ultrasonic dispersion, add 10% bovine serum albumin solution to obtain Au1Os2 nanozyme-heart failure marker detection antibody probe, and dilute the Au1Os2 nanozyme-heart failure marker detection antibody probe twice with diluent for standby use. The diluent includes the following percentages of raw materials: 1% bovine serum albumin, 4% sucrose, 0.1% Tween-20, and the remainder is phosphate buffer;

[0102] S2. Soak the conjugate pad in the conjugate pad pretreatment solution for 15 min, soak the blood filter pad in the blood filter pad pretreatment solution for 15 min, place it in a constant temperature and humidity chamber together with absorbent paper, dry it at 37°C and 15% humidity for 2 h, spray the Au1Os2 nanozyme-heart failure marker detection antibody probe on the conjugate pad with a film spray gold instrument at a dosage of 4 μL / cm and place it in a constant temperature incubator, dry it at 39°C and 19% humidity for 9 h, the above-mentioned conjugate pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinyl pyrrolidone, 0.2% Tween-20, 5% sucrose and 1% bovine serum albumin, and the balance is phosphate buffer solution, the above-mentioned blood filter pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinyl pyrrolidone, 0.2% Tween-20, 5% sucrose, 1% bovine serum albumin and 0.5 mg / mL rabbit anti-human erythrocyte antibody, the remainder being phosphate buffer, the concentration of phosphate buffer being 0.01 M, and the pH being 7.3;

[0103] S3. Dilute the B-type natriuretic peptide capture antibody to 2 mg / mL with a membrane diluent, spray the B-type natriuretic peptide capture antibody on one side of the nitrocellulose membrane using a membrane gold sprayer as the detection line T line, dilute the goat anti-mouse monoclonal antibody to 0.8 mg / mL with a membrane diluent, the above-mentioned membrane diluent includes the following raw materials in mass percentage: 0.5% sodium chloride, 1% sucrose, and the remainder is phosphate buffer, spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane using a membrane gold sprayer as the detection line C line, and then stick the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene base plate in sequence for assembly. During assembly, the two adjacent parts overlap each other by 1-2 mm to obtain a micro whole blood heart failure marker immunochromatographic test strip, cut the micro whole blood heart failure marker immunochromatographic test strip into 4 mm width, and store it in a sealed aluminum foil bag at 24°C.

[0104] Example 3

[0105] The immunochromatographic test strip for micro whole blood heart failure markers is similar to that in Example 1.

[0106] The method for preparing the above-mentioned micro whole blood heart failure marker immunochromatographic test strip comprises the following steps:

[0107] S1. Disperse 160 μL of 6 mg / mL Au1Os2 bimetallic nanozyme in 1500 μL of 0.02 M phosphate buffer at a pH of 7.5, then add 80 μL of 1 mg / mL growth stimulation expression gene 2 protein detection antibody and mix well. Centrifuge at 15000 r / min for 11 min and discard the supernatant. Add 160 μL of phosphate buffer to terminate the reaction, then perform ultrasonic dispersion, add 11% bovine serum albumin solution to obtain Au1Os2 nanozyme-heart failure marker detection antibody probe, and dilute the Au1Os2 nanozyme-heart failure marker detection antibody probe twice with diluent for standby use. The diluent includes the following percentages of raw materials: 2% bovine serum albumin, 6% sucrose, 0.5% Tween-20, and the remainder is phosphate buffer;

[0108] S2, soaking the conjugate pad in the conjugate pad pretreatment solution for 20 min, soaking the blood filter pad in the blood filter pad pretreatment solution for 20 min, placing it in a constant temperature and humidity chamber together with absorbent paper, drying it at 50°C and 30% humidity for 24 h, spraying the Au1Os2 nanozyme-heart failure marker detection antibody probe on the conjugate pad with a film spray gold instrument at a rate of 10 μL / cm and placing it in a constant temperature incubator, drying it at 41°C and 21% humidity for 11 h, the above-mentioned conjugate pad pretreatment solution includes the following raw materials by mass percentage: 0.2% polyvinyl pyrrolidone, 0.3% Tween-20, 6% sucrose and 2% bovine serum albumin, and the balance is phosphate buffer. The above-mentioned blood filter pad pretreatment solution includes the following raw materials by mass percentage: 0.2% polyvinyl pyrrolidone, 0.3% Tween-20, 6% sucrose, 2% bovine serum albumin and 0.5 mg / mL rabbit anti-human red blood cell antibody, and the balance is phosphate buffer;

[0109] S3. Dilute the growth stimulation expression gene 2 protein capture antibody to 3 mg / mL with a membrane diluent, spray the growth stimulation expression gene 2 protein capture antibody on one side of the nitrocellulose membrane using a membrane gold sprayer as the detection line T line, dilute the goat anti-mouse monoclonal antibody to 0.9 mg / mL with a membrane diluent, spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane using a membrane gold sprayer as the detection line C line, the above-mentioned membrane diluent includes the following raw materials in mass percentage: 2% sodium chloride, 3% sucrose, and the remainder is phosphate buffer, and then stick the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene base in sequence for assembly. During assembly, the two adjacent parts overlap each other by 1-2 mm to obtain a micro whole blood heart failure marker immunochromatographic test strip, cut the micro whole blood heart failure marker immunochromatographic test strip into 4 mm width, and store it in a sealed aluminum foil bag at 26°C.

[0110] Comparative Example 1

[0111] The method of use shown in Example 1 was used to replace the micro whole blood heart failure marker immunochromatographic test strips with colloidal gold test strips to conduct the experiment. The test results of the colloidal gold test strips were as follows: Figure 3 As shown, Figure 3 The eye sample is marked as the test strip test result corresponding to the concentration of the lower limit of detection. The standard curve established by T / C ratio and corresponding N-terminal pro-brain natriuretic peptide concentration is as follows Figure 4 As shown, Figure 4 In the figure, Au NPs are the results of colloidal gold test strips.

[0112] Depend on Figure 3 and Figure 4 It can be seen that the sensitivity of the colloidal gold test strip is only 1 ng / mL, and the linear correlation coefficient R is within the range of 1-100 ng / mL. 2 It is 95.37%.

[0113] Test Example 1

[0114] The transmission electron microscopy image of the Au1Os2 bimetallic nanozyme in Example 1 is as follows: Figure 5 shown.

[0115] Depend on Figure 5 It can be seen that the particles have a spherical structure with an average particle size of about 3.78 nm, which is consistent with the morphology of some alloy nanozymes synthesized by liquid phase reduction method.

[0116] Test Example 2

[0117] The element distribution diagram of the Au1Os2 bimetallic nanozyme with peroxidase activity in Example 1 is as follows: Figure 6 shown. Figure 6In the figure, A is the energy dispersive spectrometer line scan of Au1Os2, B is the surface scan of element distribution of Au1Os2, C is the surface scan of Au element in Au1Os2, and D is the surface scan of Os element in Au1Os2.

[0118] Depend on Figure 6 It can be seen that the synthesized Au1Os2 elements are evenly distributed and the synthesis quality is good.

[0119] Test Example 3

[0120] The absorption intensity of the colorimetric reaction catalyzed by the Au1Os2 bimetallic nanozyme was quantitatively measured to determine the specific activity. The specific activity calculation diagram of the Au1Os2 bimetallic nanozyme with peroxidase at pH 4 and pH 7 is shown in the figure. Figure 7 shown.

[0121] Depend on Figure 7 It can be seen that the specific activity of Au1Os2 bimetallic nanozyme at pH 4.0 and pH 7.0 is 580.89 U / mg and 722.57 U / mg, respectively, far exceeding the existing technology.

[0122] Test Example 4

[0123] Add 1 μg / mL of interfering proteins to the sample diluent, including heart-type fatty acid binding protein (H-FABP), myoglobin (MYO), matrix metalloproteinase 9 (MMP9), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). At the same time, prepare a sample solution with 10 ng / mL of N-terminal pro-brain natriuretic peptide (NT-proBNP). Add 75 μL to the test strip, react for 15 minutes, take a picture and record, then apply 0.7 μL of color development solution on the T line, take a picture and record after 30 seconds of color development. The results are as follows: Figure 8 shown.

[0124] Depend on Figure 8 It can be seen that the nanozyme test strips can effectively distinguish NT-proBNP from other interfering proteins.

[0125] Test Example 5

[0126] Three sample solutions containing different concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) protein were tested using nanozyme test strips. Each sample was tested six times and a box plot was drawn. The CV values were between 6.70% and 9.77%. The results are as follows: Figure 9 shown.

[0127] Depend on Figure 9 It can be seen that the nanoenzyme test strips have high accuracy in detecting heart failure markers.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a micro whole blood heart failure marker immunochromatographic test strip, characterized in that: The following steps are involved: S1. Dispersing the Au1Os2 bimetallic nanozyme in a phosphate buffer solution, then adding a heart failure marker detection antibody and mixing, centrifuging and discarding the supernatant, adding a phosphate buffer solution to terminate the reaction, and then ultrasonically dispersing the mixture, adding a bovine serum albumin solution to obtain an Au1Os2 nanozyme-heart failure marker detection antibody probe, and diluting the Au1Os2 nanozyme-heart failure marker detection antibody probe twice with a diluent for later use; S2, soaking the conjugate pad in the conjugate pad pretreatment solution, soaking the blood filter pad in the blood filter pad pretreatment solution, and after drying, spraying the Au1Os2 nanozyme-heart failure marker detection antibody probe obtained in step S1 on the conjugate pad and drying; S3. Draw the quality control line C and the detection line T on both sides of the nitrocellulose membrane, and then stick the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene base plate in sequence for assembly to obtain a trace whole blood heart failure marker immunochromatographic test strip.

2. The method for preparing the micro whole blood heart failure marker immunochromatographic test strip according to claim 1, wherein: In step S1, the Au1Os2 bimetallic nanozyme is prepared by the following method: tetrachloroauric acid, potassium hexachloroosmate and polyvinylpyrrolidone solution are mixed and stirred, and then sodium borohydride solution is added dropwise and stirred again, centrifuged, washed with water, and resuspended with ultrapure water to obtain Au1Os2 bimetallic nanozyme.

3. The method for preparing the micro whole blood heart failure marker immunochromatographic test strip according to claim 1, wherein: In step S1, the heart failure marker is at least one of B-type natriuretic peptide, N-terminal pro-brain natriuretic peptide and growth-stimulated gene 2 protein.

4. The method for preparing the micro whole blood heart failure marker immunochromatographic test strip according to claim 1, wherein: In step S2, the conjugate pad pretreatment solution comprises the following raw materials in percentage by weight: 0.1-0.2% polyvinyl pyrrolidone, 0.2-0.3% Tween-20, 5-6% sucrose, and 1-2% bovine serum albumin, with the remainder being phosphate buffer.

5. The method for preparing the micro whole blood heart failure marker immunochromatographic test strip according to claim 1, wherein: In step S3, the capture antibody corresponding to the heart failure marker is sprayed on one side of the nitrocellulose membrane as the detection line T line, and the goat anti-mouse monoclonal antibody is sprayed on the other side of the nitrocellulose membrane as the quality control line C line. The distance between the T line and the C line is 7-9 mm.

6. A micro whole blood heart failure marker immunochromatographic test strip prepared by the method for preparing a micro whole blood heart failure marker immunochromatographic test strip according to any one of claims 1 to 5.

7. The immunochromatographic test strip for micro whole blood heart failure markers according to claim 6, wherein: The apparatus comprises a blood filter pad, a conjugation pad, a nitrocellulose membrane, absorbent paper and a polystyrene base plate. The blood filter pad, conjugation pad, nitrocellulose membrane and absorbent paper are arranged in parallel from left to right on the surface of the polystyrene base plate, and adjacent parts overlap by 1-2 mm. A detection line T line and a quality control line C line are respectively arranged on both sides of the nitrocellulose membrane. The detection line T line is adjacent to the conjugation pad, and the quality control line C line is adjacent to the absorbent paper.

8. The method for using the micro whole blood heart failure marker immunochromatographic test strip according to claim 6 or 7, characterized in that: The following steps are involved: The sample to be tested is added dropwise to the blood filter pad to start the chromatographic test. After completion, the color developing solution is applied to the T line of the nitrocellulose membrane. The color signals of the T line and C line before and after color development are observed to determine the test results. The grayscale values of the T line and C line are then analyzed. A standard curve is established based on the T / C signal ratio and the corresponding heart failure marker concentration value. The T / C signal value of the test strip to be tested is substituted into the standard curve for quantitative detection.

9. The method for using the micro whole blood heart failure marker immunochromatographic test strip according to claim 8, wherein: The sample to be tested is fingertip blood and / or a trace amount of whole blood.

10. The method for using the micro whole blood heart failure marker immunochromatographic test strip according to claim 8, wherein: The test result determination criteria are: if only the quality control line C line appears, the test result is determined to be negative; if both the quality control line C line and the test line T line appear at the same time, the test result is determined to be positive.

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