Microscale whole blood heart failure marker immunochromatography test strip and preparation method thereof

Abstract

The invention discloses a trace whole-blood heart failure marker immunochromatography detection test strip and a preparation method thereof, and relates to the technical field of heart failure detection. The microscale whole blood heart failure marker immunochromatography detection test strip comprises a blood filtering pad, a combination pad, a nitrocellulose membrane, absorbent paper and a polystyrene bottom plate, the blood filtering pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially fixed to the polystyrene bottom plate, and a detection line T and a quality control line C are arranged on the two sides of the nitrocellulose membrane respectively. The detection line T is adjacent to the combination pad, and the quality control line C is adjacent to the absorbent paper. The invention also provides a preparation method and a use method of the microscale whole blood heart failure marker immunochromatography detection test strip, the detection sensitivity is high, the operation is simple, and the problem of large interference of endogenous peroxidase in fingertip blood / microscale whole blood / plasma under an acidic condition in the prior art is effectively solved.

Description

Technical Field

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

[0002] Heart failure (HF) is a group of clinical syndromes caused by abnormal heart structure and / or function, resulting in reduced cardiac output and / or increased intracardiac pressure at rest or under stress. It is reported that approximately 64.3 million people worldwide suffer from heart failure. In China, the number of heart failure patients is about 8.9 million, and the prevalence of heart failure in people over 35 years old is 1.3%. Due to problems such as population aging, diabetes, and poor control of hypertension, the prevalence of heart failure is continuously increasing. Although new progress has been made in the treatment and care of heart failure, the prognosis of patients is still poor, and the five-year survival rate is about 50%. In addition, heart failure limits the physiological, psychological, and social functions of patients, seriously affecting their quality of life and bringing a huge burden to patients, their families, and the healthcare system. Therefore, it is particularly important to establish a point-of-care testing (POCT) method for heart failure-related biomarkers to timely and accurately monitor the disease progression and treatment effect of heart failure patients and adjust the treatment plan.

[0003] As an emerging detection method, nanozyme immunochromatography technology has achieved breakthrough progress in the field of rapid diagnosis with its advantages of high sensitivity and specificity, providing an innovative solution for clinical point-of-care testing. At present, about 90% of peroxidase (POD) nanozymes have optimal catalytic conditions in acidic environments, but the interference of endogenous peroxidases in fingertip blood / trace whole blood / plasma is extremely large under acidic conditions, hindering their potential biomedical applications. There are currently no similar products on the market for test strips catalyzed by neutral nanozymes for detecting heart failure markers in fingertip blood / trace whole blood / plasma. Summary of the Invention

[0004] Aiming at the above deficiencies in the prior art, the present invention provides a preparation method for an immunochromatographic test strip for detecting heart failure markers in trace whole blood, effectively solving the problem of large interference of endogenous peroxidases in fingertip blood / trace whole blood / plasma under acidic conditions in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: providing a preparation method for an immunochromatographic test strip for detecting heart failure markers in trace whole blood, comprising the following steps: S1. Disperse Au1Os2 bimetallic nanozyme in phosphate buffer solution, then add the heart failure biomarker detection antibody and mix well. Centrifuge and discard the supernatant. Add phosphate buffer solution to terminate the reaction, then perform ultrasonic dispersion, and add bovine serum albumin solution to obtain the Au1Os2 nanozyme-heart failure biomarker detection antibody probe. Dilute the Au1Os2 nanozyme-heart failure biomarker detection antibody probe two-fold with diluent and set aside for later use; S2. Immerse the conjugate pad in the conjugate pad pretreatment solution, and immerse the blood filter pad in the blood filter pad pretreatment solution. After drying, spray the Au1Os2 nanozyme-heart failure biomarker detection antibody probe on the conjugate pad and dry it; S3. Draw the quality control line C and the test line T on both sides of the nitrocellulose membrane respectively. Then paste the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene bottom plate in sequence for assembly to obtain the micro whole blood heart failure biomarker immunochromatographic test strip.

[0006] Furthermore, in step S1, the Au1Os2 bimetallic nanozyme is prepared by the following method: Mix chloroauric acid, potassium hexachloroiridate and polyvinylpyrrolidone solution, stir, then dropwise add sodium borohydride solution and stir again, centrifuge, wash with water, and resuspend with ultrapure water to obtain the Au1Os2 bimetallic nanozyme.

[0007] Furthermore, in step S1, mix 50 - 500 μL of 40 mM chloroauric acid, 100 - 1000 μL of 40 mM potassium hexachloroiridate and 4 - 40 μL of 20 mg / mL polyvinylpyrrolidone solution, stir at 500 rpm for 5 min at room temperature, then dropwise add 500 - 1000 μL of 100 mM sodium borohydride solution and stir at 20 - 100 °C for 10 min, centrifuge at 9000 - 14000 rpm for 5 - 15 min, wash with water three times, and resuspend with ultrapure water to obtain the Au1Os2 bimetallic nanozyme.

[0008] Furthermore, in step S1, the heart failure biomarker is at least one of B-type natriuretic peptide, N-terminal pro-brain natriuretic peptide and growth stimulating expressed gene 2 protein.

[0009] Furthermore, in step S1, the volume ratio of Au1Os2 bimetallic nanozyme, phosphate buffer solution and heart failure biomarker detection antibody is 20 - 160 μL:200 - 1500 μL:10 - 80 μL.

[0010] Furthermore, in step S1, the concentration of Au1Os2 bimetallic nanozyme is 5 - 6 mg / mL.

[0011] Furthermore, in step S1, the concentration of Au1Os2 bimetallic nanozyme is 5 mg / mL.

[0012] The beneficial effects of adopting the above further scheme are as follows: The Au1Os2 bimetallic nanozyme is a peroxidase and has high peroxidase-like activity under neutral conditions.

[0013] Further, in step S1, the concentration of the phosphate buffer solution is 0.01 - 0.02 M, and the pH value is 7.3 - 7.5.

[0014] Further, in step S1, the concentration of the phosphate buffer solution is 0.01 M, and the pH value is 7.4.

[0015] Further, in step S1, the concentration of the heart failure biomarker detection antibody is 1 - 2 mg / mL.

[0016] Further, in step S1, the concentration of the heart failure biomarker detection antibody is 1 mg / mL.

[0017] Further, in step S1, invert and mix evenly at room temperature for 1 - 1.5 h.

[0018] Further, in step S1, invert and mix evenly at room temperature for 1 h.

[0019] Further, in step S1, centrifuge at 13000 - 15000 r / min for 9 - 11 min.

[0020] Further, in step S1, centrifuge at 14000 r / min for 10 min.

[0021] Further, in step S1, add 20 - 160 μL of phosphate buffer solution to terminate the reaction.

[0022] Further, in step S1, ultrasonically disperse at 20 - 100 W for 1 - 3 min.

[0023] Further, in step S1, the volume ratio of the bovine serum albumin solution to the Au1Os2 bimetallic nanozyme is 20 - 90 μL:20 - 160 μL.

[0024] Further, in step S1, the concentration of the bovine serum albumin solution is 10 - 11%.

[0025] Further, in step S1, the concentration of the bovine serum albumin solution is 10%.

[0026] Further, in step S1, after adding the bovine serum albumin solution, seal for 0.5 - 1 h.

[0027] Further, in step S1, after adding the bovine serum albumin solution, seal for 0.5 h.

[0028] Further, in step S1, the diluent comprises the following raw materials by mass percentage: 1-2% bovine serum albumin, 4-6% sucrose, 0.1-0.5% Tween-20, and the balance is phosphate buffer solution.

[0029] Further, in step S2, the binding pad pretreatment solution comprises the following raw materials by mass percentage: 0.1-0.2% polyvinylpyrrolidone, 0.2-0.3% Tween-20, 5-6% sucrose, and 1-2% bovine serum albumin, and the balance is phosphate buffer solution.

[0030] Further, in step S2, the binding pad pretreatment solution comprises the following raw materials by mass percentage: 0.1% polyvinylpyrrolidone, 0.2% Tween-20, 5% sucrose, and 1% bovine serum albumin, and the balance is phosphate buffer solution.

[0031] Further, in step S2, the concentration of the phosphate buffer solution is 0.01-0.02 M, and the pH value is 7.3-7.5.

[0032] Further, in step S2, the concentration of the phosphate buffer solution is 0.01 M, and the pH value is 7.4.

[0033] Further, in step S2, the filter blood pad pretreatment solution is prepared by the following method: adding rabbit anti-human red blood cell antibody to the above binding pad pretreatment solution to obtain the filter blood pad pretreatment solution.

[0034] Further, the concentration of the rabbit anti-human red blood cell antibody in the filter blood pad pretreatment solution is 0.4-0.6 mg / mL.

[0035] Further, the concentration of the rabbit anti-human red blood cell antibody in the filter blood pad pretreatment solution is 0.5 mg / mL.

[0036] Further, in step S2, soak for 15-20 min.

[0037] Further, in step S2, soak for 15 min.

[0038] Further, in step S2, put it into a thermostatic and humidified box together with absorbent paper, and dry it at 37-50 °C and a humidity of 15-30% for 2-24 h.

[0039] Further, in step S2, use a membrane scribing and gold spraying instrument to spray the Au1Os2 nanozyme-heart failure biomarker detection antibody probe at a dosage of 4-10 μL / cm.

[0040] Further, in step S2, put it into a thermostatic incubator, and dry it at 39-41 °C and a humidity of 19-21% for 9-11 h.

[0041] Further, in step S2, it is placed in an incubator and dried at 40°C and a humidity of 20% for 10 h.

[0042] Further, in step S3, the capture antibody corresponding to the heart failure marker is sprayed on the nitrocellulose membrane as the test line T line, and the goat anti-mouse monoclonal antibody is sprayed on the nitrocellulose membrane as the quality control line C line.

[0043] Further, in step S3, the capture antibody for the heart failure marker is diluted to 2 - 3 mg / mL with a membrane scribing diluent.

[0044] Further, in step S3, the capture antibody for the heart failure marker is diluted to 2 mg / mL with a membrane scribing diluent.

[0045] Further, the membrane scribing diluent includes the following raw materials by mass percentage: 0.5% - 2% sodium chloride, 1% - 3% sucrose, and the balance is phosphate buffer solution.

[0046] Further, in step S3, the capture antibody for the heart failure marker is sprayed on one side of the nitrocellulose membrane as the test line T line with a membrane scribing and gold spraying instrument.

[0047] Further, in step S3, the goat anti-mouse monoclonal antibody is diluted to 0.8 - 0.9 mg / mL with a membrane scribing diluent.

[0048] Further, in step S3, the goat anti-mouse monoclonal antibody is diluted to 0.8 mg / mL with a membrane scribing diluent.

[0049] Further, in step S3, the goat anti-mouse monoclonal antibody is sprayed on the other side of the nitrocellulose membrane as the quality control line C line with a membrane scribing and gold spraying instrument.

[0050] Further, in step S3, during assembly, the adjacent two parts overlap by 1 - 2 mm.

[0051] Further, in step S3, the immunochromatographic test strip for the heart failure marker in trace whole blood is cut to a width of 4 mm and stored in a sealed aluminum foil bag at 24 - 26°C.

[0052] An immunochromatographic test strip for the heart failure marker in trace whole blood prepared by the above method for preparing an immunochromatographic test strip for the heart failure marker in trace whole blood.

[0053] The above-mentioned immunochromatographic test strip for detecting heart failure markers in trace whole blood includes a blood filtration pad, a conjugate pad, a nitrocellulose membrane, a blotting paper, and a polystyrene bottom plate. The blood filtration pad, the conjugate pad, the nitrocellulose membrane, and the blotting paper are arranged in parallel from left to right on the surface of the polystyrene bottom plate, with an overlap of 1-2 mm between adjacent two parts. A test line T line and a control line C line are respectively arranged on both sides of the nitrocellulose membrane. The test line T line is adjacent to the conjugate pad, and the control line C line is adjacent to the blotting paper.

[0054] The method for using the above-mentioned immunochromatographic test strip for detecting heart failure markers in trace whole blood includes the following steps: Dilute the sample to be tested and then drop it onto the blood filtration pad to start chromatographic detection. After the detection is completed, apply a chromogenic solution to the T line on the nitrocellulose membrane, observe the color signals of the T line and the C line before and after chromogenesis to determine the test result, and then analyze the gray values of the T line and the C line. Establish a standard curve based on the T / C signal ratio and the corresponding heart failure marker concentration value, and substitute the T / C signal value of the test strip to be detected into the standard curve for quantitative detection.

[0055] Furthermore, the sample to be tested is fingertip blood or / and trace whole blood.

[0056] Furthermore, dilute the sample to be tested with a sample diluent.

[0057] Furthermore, the sample diluent is a phosphate buffer solution containing 1-2% Tween-20.

[0058] Furthermore, the sample diluent is a phosphate buffer solution containing 1.5% Tween-20.

[0059] Furthermore, the volume of the sample to be tested is 5-35 μL.

[0060] Furthermore, the volume ratio of the sample to be tested to the sample diluent is 5-35 μL:50-100 μL.

[0061] Furthermore, the volume ratio of the sample to be tested to the sample diluent is 15 μL:60 μL.

[0062] Furthermore, the time for chromatographic detection is 5-30 min.

[0063] Furthermore, the volume ratio of the sample to be tested to the chromogenic solution is 5-35 μL:0.6-0.8 μL.

[0064] Furthermore, the volume ratio of the sample to be tested to the chromogenic solution is 15 μL:0.7 μL.

[0065] Furthermore, the chromogenic solution includes the following components: 3,3',5,5'-tetramethylbenzidine, hydrogen peroxide solution, and phosphate buffer solution.

[0066] Further, the concentration of 3,3',5,5'-tetramethylbenzidine solution in the chromogenic solution is 8 - 9 mM.

[0067] Further, the concentration of 3,3',5,5'-tetramethylbenzidine solution in the chromogenic solution is 8 mM.

[0068] Further, the concentration of hydrogen peroxide solution in the chromogenic solution is 0.1 - 0.2 mM.

[0069] Further, the concentration of hydrogen peroxide solution in the chromogenic solution is 0.1 mM.

[0070] Further, the concentration of phosphate buffer solution in the chromogenic solution is 0.01 - 0.2 M.

[0071] Further, the concentration of phosphate buffer solution in the chromogenic solution is 0.01 M.

[0072] Further, the chromogenic time is 15 - 60 s.

[0073] Further, the chromogenic time is 30 s.

[0074] Further, the criterion for judging the test result is as follows: if only the control line C appears, the test result is judged to be negative; if both the control line C and the test line T appear simultaneously, the test result is judged to be positive.

[0075] The present invention has the following beneficial effects: 1. In the present invention, the preparation of Au1Os2 bimetallic nanozyme is realized for the first time by the liquid-phase reduction method. The particle size is about 3.78 nm. The synthesis method is simple, and the dispersibility and stability are good. The Au1Os2 bimetallic nanozyme is a peroxidase nanozyme, which has high peroxidase-like activity under neutral conditions, effectively solving the problem of large interference of endogenous peroxidase in fingertip blood / micro whole blood / plasma under acidic conditions in the prior art.

[0076] 2. The immunochromatographic test strip for detecting heart failure markers in micro whole blood prepared by the present invention has a detection sensitivity for real samples that can reach 0.058 ng / mL. It has strong anti-interference ability, good reliability, and strong specificity. It combines the simplicity, rapidity, and on-site applicability of colloidal gold technology, and at the same time has high sensitivity, with broad market prospects. Description of the Drawings

[0077] Figure 1 It is a comparison diagram before and after catalytic chromogenesis for Example 1; Figure 2 It is a standard curve diagram of the T / C ratio and the corresponding concentration of N-terminal pro-brain natriuretic peptide before and after catalysis for Example 1; Figure 3 It is a test result diagram of the colloidal gold test strip for Comparative Example 1; Figure 4 The standard curve graph of the T / C ratio and the corresponding concentration of N-terminal pro-brain natriuretic peptide under the detection of the colloidal gold test strip in Comparative Example 1; Figure 5 The transmission electron microscope image of Au1Os2 bimetallic nanozyme; Figure 6 The elemental distribution map of Au1Os2 bimetallic nanozyme; Figure 7 The calculation graph of the peroxidase specific activity of Au1Os2 bimetallic nanozyme under acidic and neutral conditions; Figure 8 The analysis graph of the specific detection results described in the embodiments of the present invention; Figure 9 The box plot of the precision experiment described in the embodiments of the present invention. Detailed implementation manners

[0078] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0079] Example 1 The immunochromatographic test strip for detecting heart failure markers in trace whole blood includes a blood filter pad, a conjugate pad, a nitrocellulose membrane, a water-absorbing paper and a polystyrene bottom plate. The blood filter pad, the conjugate pad, the nitrocellulose membrane and the water-absorbing paper are arranged in parallel from left to right on the surface of the polystyrene bottom plate. The adjacent two parts overlap by 1-2 mm. A test line T line and a quality control line C line are respectively arranged on both sides of the nitrocellulose membrane. The test line T line is adjacent to the conjugate pad, and the quality control line C line is adjacent to the water-absorbing paper.

[0080] The preparation method of the above immunochromatographic test strip for detecting heart failure markers in trace whole blood includes the following steps: S1. Disperse 100 μL of Au1Os2 bimetallic nanozyme with a concentration of 5 mg / mL in 1000 μL of phosphate buffer with a concentration of 0.01 M and a pH value of 7.4. Then add 50 μL of N-terminal pro-brain natriuretic peptide detection antibody with a concentration of 1 mg / mL 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, and then perform ultrasonic dispersion. Add 10% bovine serum albumin solution to obtain the Au1Os2 nanozyme-heart failure biomarker detection antibody probe. Dilute the Au1Os2 nanozyme-heart failure biomarker detection antibody probe two-fold with a diluent. The above diluent includes the following raw materials in percentage: 1% bovine serum albumin, 5% sucrose, 0.3% Tween-20, and the balance is phosphate buffer; S2. Immerse the conjugate pad in the conjugate pad pretreatment solution for 15 min, immerse the blood filter pad in the blood filter pad pretreatment solution for 15 min, and put them into a thermostatic and humidified incubator together with the absorbent paper. Dry at 40 °C and a humidity of 20% for 10 h. Spray the Au1Os2 nanozyme-heart failure biomarker detection antibody probe on the conjugate pad with a dosage of 8 μL / cm using a membrane scribing and gold spraying instrument and put it into a thermostatic incubator. Dry at 40 °C and a humidity of 20% for 10 h. The above conjugate pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinylpyrrolidone, 0.2% Tween-20, 5% sucrose, and 1% bovine serum albumin, and the balance is phosphate buffer. The above blood filter pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinylpyrrolidone, 0.2% Tween-20, 5% sucrose, 1% bovine serum albumin, and 0.5 mg / mL rabbit anti-human red blood cell antibody, and the balance is phosphate buffer. The concentration of the phosphate buffer is 0.01 M and the pH is 7.4; S3. Dilute the N-terminal pro-brain natriuretic peptide capture antibody to 2 mg / mL with a membrane scribing diluent. Spray the N-terminal pro-brain natriuretic peptide capture antibody on one side of the nitrocellulose membrane as the test line T line using a membrane scribing and gold spraying instrument. Dilute the goat anti-mouse monoclonal antibody to 0.8 mg / mL with a membrane scribing diluent. Spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane as the control line C line. The above membrane scribing diluent includes the following raw materials in mass percentage: 1% sodium chloride, 2% sucrose, and the balance is phosphate buffer. Then paste the blood filter pad, conjugate pad, nitrocellulose membrane, and absorbent paper on the polystyrene bottom plate in sequence for assembly. During assembly, the adjacent two parts overlap by 1-2 mm to obtain a micro whole blood heart failure biomarker immunochromatographic test strip. Cut the micro whole blood heart failure biomarker immunochromatographic test strip into 4 mm wide and store it in a sealed aluminum foil bag at 25 °C.

[0081] The method for using the above-mentioned immunochromatographic test strip for detecting heart failure markers in trace whole blood includes the following steps: Take 15 μL of the test subject's trace whole blood as the test sample. After diluting the test sample with 60 μL of phosphate buffer containing 1-2% Tween-20, drop it onto the blood filter pad and start chromatographic detection. After 15 minutes, apply a chromogenic solution (8 mM 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. After 30 seconds of color development, take a photo to record the result as Figure 1 shown, Figure 1 In, the eye pattern marks the test strip detection result corresponding to the concentration of the lower limit of detection. The upper picture Au1Os2 is the picture before color development, and the lower picture Au1Os2+TMB+H2O2, pH 7.0 is the picture after color development. Use Image J software to analyze the gray values of the T line and the C line, and establish a standard curve based on the T / C signal ratio and the corresponding N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration value. The results prove that the lower limit of detection of the immunochromatographic test strip for heart failure markers in trace whole blood is 5.76 ng / mL before catalytic color development, and 0.058 ng / mL after catalytic color development. The linear correlation coefficient R 2 is 97.47% in the range of 5.76 - 115.2 ng / mL before catalytic color development, and the linear correlation coefficient R 2 is 99.32% in the range of 0.058 - 115.2 ng / mL after catalytic color development. The standard curve established based on the T / C ratio before and after catalysis and the corresponding N-terminal pro-brain natriuretic peptide concentration is as Figure 2 shown, Figure 2 In, Au1Os2 is the standard curve before catalysis, and Au1Os2+TMB+H2O2, pH 7.0 is the standard curve after catalysis.

[0082] Example 2 The immunochromatographic test strip for detecting heart failure markers in trace whole blood is similar to that in Example 1.

[0083] The preparation method of the above-mentioned immunochromatographic test strip for detecting heart failure markers in trace whole blood includes the following steps: S1. Disperse 20 μL of Au1Os2 bimetallic nanozyme with a concentration of 5 mg / mL in 200 μL of phosphate buffer with a concentration of 0.01 M and a pH value of 7.3. Then add 10 μL of B-type natriuretic peptide detection antibody with a concentration of 1 mg / mL 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, and add 10% bovine serum albumin solution to obtain the Au1Os2 nanozyme-heart failure biomarker detection antibody probe. Dilute the Au1Os2 nanozyme-heart failure biomarker detection antibody probe two-fold with a diluent. The above diluent includes the following raw materials in percentage: 1% bovine serum albumin, 4% sucrose, 0.1% Tween-20, and the balance is phosphate buffer; S2. Immerse the conjugate pad in the conjugate pad pretreatment solution for 15 min, immerse the blood filter pad in the blood filter pad pretreatment solution for 15 min, and put them into a thermostatic and humidified incubator together with the absorbent paper. Dry at 37°C and a humidity of 15% for 2 h. Spray the Au1Os2 nanozyme-heart failure biomarker detection antibody probe on the conjugate pad with a dosage of 4 μL / cm using a membrane scribing and gold spraying instrument and put it into a thermostatic incubator. Dry at 39°C and a humidity of 19% for 9 h. The above conjugate pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinylpyrrolidone, 0.2% Tween-20, 5% sucrose, and 1% bovine serum albumin, and the balance is phosphate buffer. The above blood filter pad pretreatment solution includes the following raw materials in mass percentage: 0.1% polyvinylpyrrolidone, 0.2% Tween-20, 5% sucrose, 1% bovine serum albumin, and 0.5 mg / mL rabbit anti-human red blood cell antibody, and the balance is phosphate buffer. The concentration of the phosphate buffer is 0.01 M and the pH is 7.3; S3. Dilute the B-type natriuretic peptide capture antibody to 2 mg / mL with a membrane scribing diluent. Spray the B-type natriuretic peptide capture antibody on one side of the nitrocellulose membrane as the test line T line using a membrane scribing and gold spraying instrument. Dilute the goat anti-mouse monoclonal antibody to 0.8 mg / mL with a membrane scribing diluent. The above membrane scribing diluent includes the following raw materials in mass percentage: 0.5% sodium chloride, 1% sucrose, and the balance is phosphate buffer. Spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane as the control line C line using a membrane scribing and gold spraying instrument. Then paste the blood filter pad, conjugate pad, nitrocellulose membrane, and absorbent paper on the polystyrene bottom plate in sequence for assembly. During assembly, the adjacent two parts overlap by 1-2 mm to obtain a micro whole blood heart failure biomarker immunochromatographic test strip. Cut the micro whole blood heart failure biomarker immunochromatographic test strip into 4 mm wide and store it in a sealed aluminum foil bag at 24°C.

[0084] Example 3 The micro whole blood heart failure biomarker immunochromatographic test strip is similar to Example 1.

[0085] The preparation method of the above-mentioned immunochromatographic test strip for detecting heart failure markers in trace whole blood includes the following steps: S1. Disperse 160 μL of Au1Os2 bimetallic nanozyme with a concentration of 6 mg / mL in 1500 μL of phosphate buffer with a concentration of 0.02 M and a pH value of 7.5. Then add 80 μL of the detection antibody for growth stimulation expressed gene 2 protein with a concentration of 1 mg / mL, mix well, centrifuge at 15000 r / min for 11 min, 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 the Au1Os2 nanozyme-heart failure marker detection antibody probe. Dilute the Au1Os2 nanozyme-heart failure marker detection antibody probe two times with a diluent. The above diluent includes the following raw materials in percentage: 2% bovine serum albumin, 6% sucrose, 0.5% Tween-20, and the balance is phosphate buffer; S2. Immerse the conjugate pad in the conjugate pad pretreatment solution for 20 min, immerse the blood filter pad in the blood filter pad pretreatment solution for 20 min, put them together with the absorbent paper into a thermostatic and humidity-controlled box, and dry at 50 °C and a humidity of 30% for 24 h. Spray the Au1Os2 nanozyme-heart failure marker detection antibody probe on the conjugate pad with a dosage of 10 μL / cm using a membrane scribing and gold spraying instrument, and then put it into an incubator, and dry at 41 °C and a humidity of 21% for 11 h. The above conjugate pad pretreatment solution includes the following raw materials in mass percentage: 0.2% polyvinylpyrrolidone, 0.3% Tween-20, 6% sucrose, and 2% bovine serum albumin, and the balance is phosphate buffer. The above blood filter pad pretreatment solution includes the following raw materials in mass percentage: 0.2% polyvinylpyrrolidone, 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; S3. Dilute the capture antibody for growth stimulation expressed gene 2 protein to 3 mg / mL with a membrane scribing diluent, spray the capture antibody for growth stimulation expressed gene 2 protein on one side of the nitrocellulose membrane as the test line T line using a membrane scribing and gold spraying instrument, dilute the goat anti-mouse monoclonal antibody to 0.9 mg / mL with a membrane scribing diluent, and spray the goat anti-mouse monoclonal antibody on the other side of the nitrocellulose membrane as the control line C line. The above membrane scribing diluent includes the following raw materials in mass percentage: 2% sodium chloride, 3% sucrose, and the balance is phosphate buffer. Then paste the blood filter pad, conjugate pad, nitrocellulose membrane, and absorbent paper on the polystyrene bottom plate in sequence for assembly. During assembly, the adjacent two parts overlap by 1-2 mm to obtain the immunochromatographic test strip for detecting heart failure markers in trace whole blood. Cut the immunochromatographic test strip for detecting heart failure markers in trace whole blood into 4 mm wide and store it at 26 °C in a sealed aluminum foil bag.

[0086] Comparative Example 1 Using the usage method shown in Example 1, an experiment was carried out by replacing the immunochromatographic test strip for trace whole blood heart failure markers with a colloidal gold test strip. The test results of the colloidal gold test strip are as Figure 3 shown, Figure 3 in which the test strip test result corresponding to the concentration marked as the lower limit of detection in the eye pattern. The standard curve established by the T / C ratio and the corresponding N-terminal pro-brain natriuretic peptide concentration is as Figure 4 shown, Figure 4 in which Au NPs are the results of the colloidal gold test strip.

[0087] From 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 2 is 95.37% in the range of 1 - 100 ng / mL.

[0088] Test Example 1 The transmission electron micrograph of the Au1Os2 bimetallic nanozyme in Example 1 is as Figure 5 shown.

[0089] From Figure 5 it can be seen that the particles show a spherical structure, and the average particle size is about 3.78 nm, which is consistent with the morphology of some alloy nanozymes synthesized by the liquid-phase reduction method.

[0090] Test Example 2 The elemental distribution map of the Au1Os2 bimetallic nanozyme with peroxidase activity in Example 1 is as Figure 6 shown. Figure 6 in which A is the line scan diagram of the energy dispersive spectrometer of Au1Os2, B is the surface scan diagram of the elemental distribution of Au1Os2, C is the surface scan diagram of the Au element in Au1Os2, and D is the surface scan diagram of the Os element in Au1Os2.

[0091] From Figure 6 it can be seen that the synthesized Au1Os2 has a uniform elemental distribution and better synthesis quality.

[0092] Test Example 3 The absorption intensity of the colorimetric reaction catalyzed by the Au1Os2 bimetallic nanozyme was quantitatively measured to determine the specific activity. The calculation diagram of the specific activity of the Au1Os2 bimetallic nanozyme with peroxidase at pH 4 and pH 7 is as Figure 7 shown.

[0093] From Figure 7It can be seen that the specific activities of the Au1Os2 bimetallic nanozyme are 580.89 U / mg and 722.57 U / mg under the conditions of pH 4.0 and pH 7.0 respectively, far exceeding the prior art.

[0094] Test Example 4 Add 1 μg / mL of interfering proteins, including heart-type fatty acid-binding protein (H-FABP), myoglobin (MYO), matrix metalloproteinase 9 (MMP9), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), to the sample diluent. At the same time, prepare a sample solution added with 10 ng / mL of N-terminal pro-brain natriuretic peptide (NT-proBNP), drop 75 μL onto the test strip, take a photo record after reacting for 15 min, then apply 0.7 μL of the chromogenic solution on the T line, and take a photo record after chromogenic reaction for 30 s. The results are as Figure 8 shown.

[0095] It can be seen from Figure 8 that the nanozyme test strip can well distinguish NT-proBNP from other interfering proteins.

[0096] Test Example 5 Use the nanozyme test strip to detect 3 sample solutions containing different concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) protein. Each sample is measured six times, and a box plot is drawn. The CV value is between 6.70% and 9.77%. The results are as Figure 9 shown.

[0097] It can be seen from Figure 9 that the nanozyme test strip has high detection accuracy for heart failure markers.

[0098] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of an immunochromatographic test strip for detecting heart failure markers in trace whole blood, characterized in that, It includes the following steps: S1. Disperse Au1Os2 bimetallic nanozyme in phosphate buffer solution, then add the heart failure biomarker detection antibody and mix well. Centrifuge and discard the supernatant. Add phosphate buffer solution to terminate the reaction, then perform ultrasonic dispersion, and add bovine serum albumin solution to obtain the Au1Os2 nanozyme - heart failure biomarker detection antibody probe. Dilute the Au1Os2 nanozyme - heart failure biomarker detection antibody probe two - fold with dilution solution and reserve it for use. S2. Immerse the conjugate pad in the conjugate pad pretreatment solution, and immerse the blood filter pad in the blood filter pad pretreatment solution. After drying, spray the Au1Os2 nanozyme - heart failure biomarker detection antibody probe obtained in step S1 on the conjugate pad and dry it. S3. Draw the quality control line C and the test line T on both sides of the nitrocellulose membrane respectively. Then paste the blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper on the polystyrene bottom plate in sequence for assembly to obtain the immunochromatographic test strip for detecting heart failure biomarkers in micro - whole blood.

2. The preparation method of the micro whole blood heart failure biomarker immunochromatographic test strip according to claim 1, characterized in that, In step S1, the Au1Os2 bimetallic nanozyme is prepared by the following method: Mix chloroauric acid, potassium hexachloroosmate and polyvinylpyrrolidone solution, stir, then dropwise add sodium borohydride solution and stir again. Centrifuge, wash with water, and resuspend with ultrapure water to obtain Au1Os2 bimetallic nanozyme.

3. The preparation method of the immunochromatographic test strip for trace whole blood heart failure markers according to claim 1, characterized in that, In step S1, the heart failure biomarker is at least one of B - type natriuretic peptide, N - terminal pro - brain natriuretic peptide and growth - stimulating expressed gene 2 protein.

4. The preparation method of the micro whole blood heart failure biomarker immunochromatographic test strip according to claim 1, characterized in that, In step S2, the conjugate pad pretreatment solution includes the following raw materials by mass percentage: 0.1 - 0.2% polyvinylpyrrolidone, 0.2 - 0.3% Tween - 20, 5 - 6% sucrose and 1 - 2% bovine serum albumin, and the balance is phosphate buffer solution.

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

6. The immunochromatographic test strip for detecting heart failure biomarkers in micro - whole blood prepared by the method for preparing the immunochromatographic test strip for detecting heart failure biomarkers in micro - whole blood according to any one of claims 1 - 5.

7. The immunochromatographic test strip for detecting heart failure markers in trace whole blood according to claim 6, wherein It includes a blood filter pad, a conjugate pad, a nitrocellulose membrane, an absorbent paper and a polystyrene bottom plate. The blood filter pad, conjugate pad, nitrocellulose membrane and absorbent paper are arranged in parallel from left to right on the surface of the polystyrene bottom plate, and the adjacent two parts overlap by 1 - 2 mm. The test line T and the quality control line C are respectively arranged on both sides of the nitrocellulose membrane. The test line T is adjacent to the conjugate pad, and the quality control line C is adjacent to the absorbent paper.

8. Method for using the immunochromatographic test strip for detecting heart failure markers in trace whole blood according to claim 6 or 7, characterized in that, It includes the following steps: Drop the test sample onto the blood filter pad, start the chromatographic detection. After completion, apply the chromogenic solution on the T line of the nitrocellulose membrane, observe the color signals of the T line and the C line before and after color development to determine the test result. Then analyze the gray - scale values of the T line and the C line, establish a standard curve based on the T / C signal ratio and the corresponding heart failure biomarker concentration value, and substitute the T / C signal value of the test strip to be detected into the standard curve for quantitative detection.

9. The method for using the immunochromatographic test strip for detecting trace whole blood heart failure markers as described in claim 8, characterized in that, The test sample is fingertip blood or / and micro - whole blood.

10. The method for using the immunochromatographic test strip for detecting heart failure markers in trace whole blood according to claim 8, characterized in that, The judgment criteria for the detection results are as follows: if only the control line C appears, the detection result is judged to be negative; if both the control line C and the test line T appear simultaneously, the detection result is judged to be positive.

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