A portable instant muscle injury test strip and kit

Abstract

The invention belongs to the field of instant muscle injury detection, and specifically discloses a portable instant muscle injury detection test strip and a kit. The test strip consists of a nitrocellulose membrane, a conjugation pad, a sample pad, a water-absorbing pad and an adhesive-backed bottom plate. Antibody molecules 1 are fixed on the nitrocellulose membrane, and a fluorescent nano-microsphere-antibody molecule 2 complex is sprayed on the conjugation pad; the antibody molecule 1 is a skTnI capture antibody (Ab2); the fluorescent nano-microsphere-antibody molecule 2 is a europium fluorescent microsphere-antibody complex; and the present invention optimizes various parameters of a lateral flow immunochromatography test strip so that the test strip can detect a biomarker skTnI in serum at a minimum of 0.5 ng / mL, thereby realizing instant detection of muscle injury.

Description

Technical Field

[0001] The invention belongs to the field of instant muscle injury detection, and specifically discloses a portable instant muscle injury detection test paper and a reagent kit. Background Art

[0002] In the context of promoting quality education, timely detection and efficient management of muscle injuries are crucial to improving the physical quality of athletes or sports enthusiasts in college teams, reducing the frequency of sports injuries, and comprehensively improving the overall sports lifespan. However, in college sports teams, it is difficult to detect and prognose muscle injuries caused by sports in a timely and efficient manner, which makes muscle injuries affect the sports lifespan of college athletes or sports enthusiasts, becoming a current pain point. Traditional imaging detection equipment is bulky and expensive, and needs to be performed in large hospital laboratories. There are problems such as inconvenient medical treatment and long time. The user experience is poor, which often leads to low testing sentiment. Therefore, there is an urgent need for a convenient, fast, low-cost, and user-friendly instant muscle injury detection device for instant detection and long-term monitoring of muscle injuries after exercise, which is convenient for athletes to detect sports injuries in a timely manner, improve prognosis and efficacy, and provide guarantees for the improvement of overall sports lifespan. Summary of the invention

[0003] In view of the above problems, the present invention discloses a portable instant muscle injury detection test strip and a kit. In this paper, the skTnI concentration in the blood is detected by a lateral flow immunochromatography test strip, so as to realize the instant detection of human muscle injury.

[0004] The purpose of the present invention is achieved through the following technical solutions.

[0005] A portable instant muscle injury test strip, characterized in that the test strip is composed of a nitrocellulose membrane, a binding pad, a sample pad, a water-absorbing pad and an adhesive backing plate, the nitrocellulose membrane is fixed with an antibody molecule 1, and the binding pad is sprayed with a fluorescent nano-microsphere-antibody molecule 2 complex;

[0006] The antibody molecule 1 is: skTnI capture antibody (Ab2);

[0007] The fluorescent nanosphere-antibody molecule 2 is a europium fluorescent microsphere-antibody complex, and the antibody molecule 2 is a skTnI coated antibody (Ab1).

[0008] Furthermore, in the above-mentioned portable instant muscle injury detection test strip, the nitrocellulose membrane is selected from CN95.

[0009] Furthermore, in the above-mentioned portable instant muscle injury test strip, the fluorescent nano-microsphere-antibody molecule 2 complex is prepared by the following steps: the fluorescent nano-microsphere-antibody molecule 2 complex is prepared by the following steps: take 100 μL of europium fluorescent microspheres, wash four times with MES buffer; add 17.5 μL of EDC solution and 17.5 μL of sulfo-NHS solution, place the mixture on an oscillator and oscillate for 45 minutes to complete carboxyl activation, and then wash it with MES buffer for 3 times, and then add 500 μL of MES buffer and the optimal antibody of skTnI (skTnI coated antibody (Ab1)) coating mass to the activated mixture in sequence, and continue to shake for 2 hours; then wash it twice with ultrapure water, add 200 μL of blocking solution and continue to shake for 30 minutes; finally, wash it twice with ultrapure water, redisperse it with 200uL ultrapure water, complete the preparation of the europium fluorescent microsphere-antibody complex, and store it in a refrigerator at 4°C.

[0010] Furthermore, in the above-mentioned portable instant muscle injury detection test strip, the optimal antibody coating mass is 50-200 μg.

[0011] Furthermore, in the above-mentioned portable instant muscle injury detection test strip, the fluorescent nano-microspheres are selected from WD300+.

[0012] Furthermore, in the above-mentioned portable instant muscle injury detection test strip, the fluorescent nano-microspheres are coupled with the antibody molecules 2, and the pH value of the coupled solution is 5-7.

[0013] Furthermore, in the portable instant muscle injury test strip, the spraying concentration of the fluorescent nano-microsphere-antibody molecule 2 complex on the binding pad is 5-10%.

[0014] Furthermore, in the above-mentioned portable instant muscle injury test strip, the optimal coating concentration of the streaking antibody on the detection line is 1-3 mg / mL.

[0015] The invention also discloses a portable instant muscle injury detection kit, which is characterized by comprising the above-mentioned detection test paper.

[0016] Furthermore, the portable instant muscle injury detection kit further comprises a sample diluent, wherein the sample diluent contains 5wt% Tween-20 and 0.5wt% BSA.

[0017] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0018] The present invention prepares a lateral flow immunochromatographic test paper based on the principle of "sandwich method" to detect the muscle injury marker skTnI (skeletal muscle troponin) in human serum. The present invention achieves high-sensitivity detection of 0.5ng / mL skTnI within 10min by regulating the pretreatment method of the conjugate pad, screening of different types of NC membranes, screening of fluorescent nano-microspheres of different particle sizes, optimization of the pH of fluorescent nano-microsphere-coupled antibodies, optimization of the spraying concentration of fluorescent nano-microsphere-antibody complexes, and optimization of the concentration of sample diluent components (such as the content of Tween-20 and BSA). The sensitivity is about 10 times higher than that of traditional nanogold test strips. In addition, the present invention also successfully uses the test strip for the detection of skTnI clinical serum added samples, providing a new method for timely discovery, early detection and long-term monitoring of muscle damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : (a) Schematic diagram of the preparation and principle of the test paper, skTnI antibody 1 is fixed on the T line of the nitrocellulose membrane, and DNP-BSA is fixed on the C line; EuPSM-skTnI antibody 2 and EuPSM-DNP antibody are sprayed on the conjugate pad (b) Schematic diagram of the detection results of the test paper;

[0020] Figure 2 :A. Results of particle size screening of europium fluorescent microspheres; B. Results before and after pretreatment of sTnI conjugate pad; C. Results of antibody coating quality optimization; D. Results of pH optimization of EuPSM-coupled antibody; E. Results of gold spraying concentration optimization of EuPSM@sTnI-Ab; F. Results of NC membrane optimization; G. Results of Tween-20 content optimization; H. Results of BSA content regulation; I. Results of pH optimization of sample diluent; J. Results of membrane antibody concentration optimization;

[0021] Figure 3 : gradient photo;

[0022] Figure 4 : Quantitative standard curve of test strips. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] The experimental reagents and materials used in the present invention are shown in Table 1 below.

[0026] Table 1 Experimental reagents and materials

[0027]

[0028]

[0029]

[0030] Example 1 (Preparation Example)

[0031] Preparation of a portable instant muscle injury test strip.

[0032] like Figure 1 , a portable instant muscle injury test strip, the test strip is composed of a nitrocellulose membrane (NC membrane), a conjugation pad, a sample pad, a water-absorbing pad and a backing adhesive base plate, the nitrocellulose membrane is fixed with an antibody molecule 1, and the conjugation pad is sprayed with a fluorescent nano-microsphere-antibody molecule 2 complex;

[0033] The antibody molecule 1 is a skTnI capture antibody (Ab2);

[0034] The fluorescent nanosphere-antibody molecule 2 complex is prepared by the following steps: 100 μL of europium fluorescent microspheres (EuPSM) are taken and washed four times with MES buffer (50mM, pH=6.0). 17.5 μL of EDC solution (100 mg / mL) and 17.5 μL of sulfo-NHS solution (100 mg / mL) are added, and the mixture is placed in an oscillator (37°C, 220rpm) for 45 minutes to complete carboxyl activation. After washing with MES buffer for 3 times, 500 μL of MES buffer and the optimal antibody coating mass of skTnI coated antibody (Ab1) are added to the activated mixture in sequence, and the shaking is continued for 2 hours. Then wash twice with ultrapure water, and then add 200 μL of blocking solution (5 μl / mL ethanolamine and 20 mg / mL BSA) to continue shaking for 30 minutes. Finally, the mixture was washed twice with ultrapure water and re-dispersed with 200uL ultrapure water to complete the preparation of the europium fluorescent microsphere-antibody complex, which was then stored in a refrigerator at 4°C.

[0035] Among them, the NC membrane is the most important detection area of ​​the test paper. The antibody molecule 1 that recognizes the target to be tested is fixed on the membrane through electrostatic adsorption, hydrogen bonding and hydrophobic interaction, thus forming a detection line. The binding pad is pre-stored with a complex of fluorescent nano-microspheres-antibody molecules 2, which can track and display the target to be tested. The sample pad is used as the loading area, and the loading buffer is required to buffer and adjust the test sample to ensure the stability of the test. The absorbent pad is made of pure cotton wool containing high-density cellulose, and its strong capillary suction becomes the driving source of the entire test paper.

[0036] The above four parts are divided into Figure 1 As shown in a, stick them to the adhesive backing plate in sequence, with each adjacent part overlapping by 2mm, and the test paper is ready. Figure 1 As shown in FIG. 1 , when the sample contains the target to be tested, the target can be recognized by both the antibody molecule 2 connected with the fluorescent nanospheres and the antibody molecule 1 on the detection line, thereby forming a "sandwich" structure of antibody 1-target-antibody 2 on the detection line, and then fixing the fluorescent nanospheres on the detection line to form a color band; when the sample does not contain the target to be tested, the fluorescent nanospheres cannot be fixed on the detection line and will not form a color band. The intensity of the fluorescent signal on the detection line of the test paper is positively correlated with the concentration of the target to be tested in the sample. The higher the concentration of the target, the greater the signal intensity. Regardless of whether there is a target to be tested, the DNP antibody on the control line of the test paper can recognize the antibody molecule 2 connected with the fluorescent nanospheres, thereby forming a color band, indicating that the test paper is working normally.

[0037] Example 2

[0038] Optimization of test strip sensitivity

[0039] 1. Optimization of test strip sensitivity

[0040] This embodiment mainly optimizes the detection sensitivity of the test paper prepared in Example 1 by the following methods:

[0041] (1) Screening of europium fluorescent microspheres with the most suitable particle size. This study used seven types of europium fluorescent microspheres, WD200, WD300, WD300+, JK191, JK288, BY200, and BY300, and screened the europium fluorescent microspheres with the most suitable particle size through sensitivity comparison experiments;

[0042] (2) Regulating the pretreatment method of the test strip conjugate pad. This study pretreated the conjugate pad and performed a sensitivity optimization comparison experiment on the test strip before and after pretreatment;

[0043] (3) Optimize the optimal mass of the antibody coated on the europium fluorescent microspheres. In this study, the mass of the coated antibody was adjusted to 25, 50, 75, 100 and 150 μg, respectively, and the optimal antibody coating mass was screened out through sensitivity comparison experiments;

[0044] (4) Optimize the optimal pH for coupling europium fluorescent microspheres to antibodies. In this study, the pH of the europium fluorescent microsphere solution was adjusted to 5, 6, 7, 8, and 9, respectively, and the optimal coupling pH was screened through sensitivity comparison experiments;

[0045] (5) Optimize the optimal spraying concentration of europium fluorescent microsphere-antibody complex. In this study, the europium fluorescent microsphere-antibody complex was diluted to 2%, 4%, 6%, 8% and 10%, respectively, and sprayed onto the conjugate pad, and the optimal spraying concentration was screened through sensitivity comparison experiments;

[0046] (6) Screening of NC membranes with the most suitable pore size. This study used four commercially available NC membranes, CN95, CN110, CN140, and CN180, to conduct experiments and screen the most suitable NC membrane through sensitivity comparison experiments.

[0047] (7) Optimize the optimal component concentration of the sample diluent. This study optimized the content of Tween-20 and BSA in the diluent. Experiments were conducted on concentration gradients of 0.1%, 0.25%, 0.5%, 1%, 3%, and 5% Tween-20 and 0%, 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% BSA. The optimal content of Tween-20 and BSA was screened through sensitivity comparison experiments.

[0048] (8) Optimize the optimal pH of the sample diluent. In this study, the pH of the sample diluent was adjusted to 5, 6, 7, 8, 9, and 10 for experiments, and the optimal pH was screened through sensitivity comparison experiments;

[0049] (9) Optimize the concentration of the stripping antibody on the detection line. In this study, the concentration of the stripping antibody (skTnI capture antibody (Ab2)) on the detection line was adjusted to 0.5, 1, 1.5 and 2 mg / mL for experiments, and the optimal stripping concentration was screened through sensitivity comparison experiments.

[0050] The sensitivity comparison experiment was performed as follows: the skTnI antigen standard was gradiently diluted with sample diluent, and 100 μL was taken out with a pipette and added to the sample pads of the test paper in sequence; under the action of capillary suction, the sample flowed toward the absorbent pad; after waiting for about 15 minutes, the quantitative test results of the test paper were read using a fluorescence reader; the sensitivity of the portable test paper was optimized by analyzing and comparing the minimum detection signal intensity of the test results.

[0051] 2. Optimized test strips for detection of serum spiked samples

[0052] In order to verify the feasibility and accuracy of the high-sensitivity instant muscle injury detection test strip obtained by the above experiment optimization in clinical testing, negative human serum samples and sample diluents were selected as the detection matrix, and skTnI antigen standards were added to them in sequence, and serum-added samples with gradient concentrations of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0 ng / mL were prepared. Then 100 μL of each was taken with a pipette and added to the sample pad of the test strip in sequence; under the action of capillary suction, the sample flowed chromatographically in the direction of the absorbent pad; after waiting for about 15 minutes, the quantitative detection results of the test strip were read using a fluorescence reader.

[0053] 3. Evaluation of the test strips’ performance after optimization

[0054] By testing samples spiked with human serum of skTnI, the performance of the optimized test strips was evaluated in four aspects: detection linearity, minimum detection limit, detection accuracy, and detection precision.

[0055] 1) Evaluation of the linearity of the test strip after optimization

[0056] Human serum samples spiked with skTnI were prepared with gradient concentrations (100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0 ng / mL), and the samples were tested with the same batch of test strips. Each concentration sample was tested three times, and the quantitative test results of the test strip were read using a fluorescence reader. The average of the three test results was used as the detection signal value of the sample at that concentration. The quantitative relationship curve between the antigen concentration in the skTnI human serum spiked sample and the detection signal value of the test strip was drawn using Origin 2021 software. The linear fitting coefficient R2 of the curve was used as an evaluation index for the linearity of the test strip detection.

[0057] 2) Evaluation of the minimum detection limit of the test strip after optimization

[0058] Using the same batch of test strips, the skTnI serum spiked samples with zero concentration calibrators were tested 20 times, and the quantitative test results of the test strips were read using a fluorescence reader, and the mean (M) and standard deviation (SD) of the test results after 20 repeated measurements of the samples were calculated. According to the judgment index of the 95% confidence interval in statistics, the value obtained by M+3SD was used as the ordinate and substituted into the formula of the above quantitative relationship curve to calculate the corresponding concentration, which can be used as the evaluation index of the minimum detection limit of the test strip.

[0059] 3) Evaluation of the test strip detection accuracy after optimization

[0060] The recovery test method was used to evaluate the detection accuracy of the test strip. The skTnI antigen standard was added to the negative human serum sample, and the final concentration was calibrated to 90, 75, 60, 50, 40, 35, 30, 25, 12, 8, 6, 4, 2.5, 1.5, 0.8, 0.6, 0.4, and 0.25 ng / mL, respectively. The same batch of test strips were used for detection, and each concentration was repeated 3 times. The quantitative detection results of the test strips were read using a fluorescence reader. The average value of the test results after 3 repeated measurements of each sample was calculated, and the value was substituted into the above quantitative relationship curve formula to calculate the corresponding concentration. The ratio of the measured concentration to the calibrated concentration is the recovery rate of the test strip at this concentration, which is used to evaluate the detection accuracy of the test strip.

[0061] 4) Evaluation of the test strip detection precision after optimization

[0062] Based on the test results of 3), calculate the average value of the test results after each sample is measured three times and standard deviation (SD). Divide and multiply by the percentage to get the coefficient of variation (CV%), which is used to evaluate the detection precision of the test strip.

[0063] Example 3

[0064] Test strip sensitivity optimization results

[0065] (1) From the working principle of the test strip, it can be seen that the final test result is directly related to the presence and depth of the fluorescent signal on the test line, and the fluorescent signal is the signal that appears when the europium fluorescent microspheres gather on the test line. Therefore, the particle size of the europium fluorescent microspheres is crucial to the strength of the fluorescent signal. Figure 2 As shown in A, this study used seven types of europium fluorescent microspheres, including WD200, WD300, WD300+, JK191, JK288, BY200, and BY300, to conduct experiments. It was found that the europium fluorescent microspheres of WD300+ had a better detection signal-to-noise ratio than other microspheres regardless of high or low concentrations.

[0066] (2) The binding pad is a key component for storing the europium fluorescent microsphere-antibody 2 complex. The main raw material is glass fiber. The glass fiber has a small pore size, a thin diameter, and a tight vein, which can bind the europium fluorescent microsphere-antibody 2 complex very well. When the test strip is working, the above complex needs to be released from the binding pad. In order to release the complex comprehensively and thoroughly, this study pre-treated the binding pad. The binding pad treatment solution (200mM Tris-Hcl, 20% sucrose, 4% S9, pH=8) was absorbed into the gold spray film instrument and sprayed on the surface of the binding pad. It was placed in a constant temperature drying oven at 37°C for 2 hours and can be taken out for use. The sensitivity optimization comparison experiment of the test strips before and after pretreatment was carried out. Figure 2 As shown in B, it was found that the pretreated conjugate pad can play a sustained release role on the europium fluorescent microsphere-antibody 2 complex, thereby making the antigen-antibody binding more complete, thereby improving the overall detection signal;

[0067] (3) Figure 2 As shown in Figure C, this study optimized the antibody mass coated with europium fluorescent microspheres. The test results show that when the coated antibody mass is in the range of 25-100μg, the detection signal value increases with the increase of the coating mass. When the coating mass increases to 150μg, there is no significant difference between its detection signal value and the detection signal value at 100μg. For cost-saving considerations, this study selected 100μg as the optimal antibody mass for coating europium fluorescent microspheres;

[0068] (4) Figure 2 As shown in D, this study optimized the optimal pH for europium fluorescent microsphere-coupled antibodies. The test results show that when the pH is 6 and 7, the overall detection signal value of the test strip is significantly improved, but the discrimination for low concentrations is higher when the pH is 6, so it is selected as the optimal pH for europium fluorescent microsphere-coupled antibodies;

[0069] (5) By optimizing the antibody coating quality and coupling pH of the europium fluorescent microspheres, this study further optimized the optimal spray concentration of the europium fluorescent microsphere-antibody complex on the conjugate pad. Figure 2 As shown in Figure E, it was found that the overall detection signal value of the test paper increased with the increase of the spraying concentration, but when analyzing the test results of low concentrations, it was found that the spraying concentrations of 8% and 10% had poor discrimination and linearity for low concentration samples, while the spraying concentration of 6% had better linearity and discrimination, so 6% was selected as the optimal spraying concentration of the europium fluorescent microsphere-antibody complex;

[0070] (6) Due to the non-absolute uniformity of the drying and molding process during the production of NC membranes, the pore size of the membrane is also non-uniform and cannot be accurately defined. Therefore, in actual use, the pore size of NC membranes is generally indirectly expressed by the time of water chromatography per 4 cm of membrane, and the model is named after this time. Taking CN95 as an example, this model indicates that the time of water chromatography per 4 cm of membrane is 95 seconds. This study used four common NC membranes, namely CN95, CN110, CN140 and CN180, for experiments. The larger the NC membrane model, the slower the flow rate, the longer the reaction time with the detection line, and the higher the corresponding sensitivity. However, at the same time, the possibility of non-specific binding is also greater, resulting in false positives. Therefore, too long a reaction time does not necessarily mean a real sensitivity improvement. It is necessary to screen the appropriate balance point to find the best NC membrane suitable for this test paper. Figure 2F also shows that the larger the NC membrane model, the greater the detection signal value. However, by observing the fluorescence color development results of the test paper, it was found that CN110, CN140 and CN180 all had obvious fluorescence signals when the sample concentration was 0. Therefore, considering the problem of detection specificity, CN95 was selected as the most suitable NC membrane.

[0071] (7) This study optimized the content of Tween-20 and BSA in the sample diluent. Figure 2 As shown in G, when the Tween-20 content is 5%, the overall detection signal value of the test strip is significantly better than other contents; Figure 2 As shown in H, when the BSA content is 0.5%, the discrimination for low concentrations is higher, which helps to improve the detection sensitivity. Therefore, 5% Tween-20 and 0.5% BSA are selected as the optimal component content of the sample diluent;

[0072] (8) Figure 2 As shown in Figure 1, this study optimized the optimal pH of the sample diluent. The test results show that when the pH is 6, the overall detection signal value of the test strip is significantly better than other pH values, so it is selected as the optimal pH of the sample diluent;

[0073] (9) Figure 2 As shown in J, this study optimized the concentration of the streaking antibody on the test line. The test results show that with the increase in the concentration of the streaking antibody, the detection signal value of the test paper also increases, because more europium fluorescent microspheres-antibody complexes are captured at the test line, and the detection signal continues to increase, which increases the detection sensitivity of the test paper. However, when the concentration of the streaking antibody continues to increase, it is easy to increase the nonspecific adsorption at the test line, resulting in false positives. In addition, in order to save production costs, this study selected 1.5 mg / mL as the optimal coating concentration of the streaking antibody on the test line while meeting the sensitivity standard; Test Example 1

[0074] Optimized test strips for serum spiked samples

[0075] In order to verify the feasibility and accuracy of the high-sensitivity instant muscle injury test strip optimized in the above experiment in clinical detection, negative human serum samples were selected as the detection matrix, and skTnI antigen standards were added to them in sequence to prepare serum-added samples with gradient concentrations of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0 ng / mL. The optimized test strips were used to test the prepared skTnI serum-added samples, and the test results are shown in the figure. Figure 3As shown in the figure, it can be observed that when the concentration of skTnI serum added sample is 0 and 0.2 ng / mL, there is no significant difference in the detection signal intensity of the test strip (n=3, P>0.05); when the concentration of skTnI serum added sample is 0 and 0.5 ng / mL, there is a significant difference in the detection signal intensity of the test strip (n=3, P<0.05). This shows that when the optimized test strip is used for the detection of skTnI serum added sample, the detection sensitivity of 0.5 ng / mL can be achieved, and the skTnI concentration of 0.5 ng / mL is the exclusion threshold for clinical judgment of muscle damage. Therefore, the optimized test strip can be used to exclude whether the subject has muscle damage and realize the timely detection of muscle damage.

[0076] Test Example 2

[0077] Evaluation results of the test strips’ detection performance after optimization

[0078] 1) Evaluation results of the linearity of the optimized high-sensitivity portable test strips

[0079] The skTnI concentration in the serum-added sample (100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0 ng / mL) was used as the abscissa x, and the average of the detection signal intensity after three repeated measurements of each concentration was used as the ordinate y. The quantitative curve of the optimized high-sensitivity portable test strip for the detection of skTnI serum-added samples was calculated, as shown in FIG. Figure 4 The four-parameter fitting relationship of the quantitative curve is:

[0080] y=2.57374 / [1+(x / 71.53607)^(-1.0543)]+0.00694, indicating that the detection signal intensity of the test strip is positively correlated with the concentration of the target in the sample, that is, the detection signal intensity of the test strip increases with the increase of the concentration of the target in the sample. The linear correlation coefficient R2=0.999, indicating that the detection signal intensity of the test strip has a very good linear relationship with the concentration of the target contained in the serum added sample, that is, the optimized high-sensitivity portable test strip has a detection linearity that meets the standard.

[0081] 2) Evaluation results of the minimum detection limit of the optimized high-sensitivity portable test strips

[0082] After 20 repeated tests of zero-concentration skTnI serum with added sample calibrators, the mean and standard deviation of the signal intensity of the test strip were calculated, and the mean M = 0.012544, standard deviation SD = 0.002881, M + 3SD = 0.021187 were obtained. According to the 95% confidence interval judgment index in statistics, M + 3SD was substituted as y into the above quantitative curve y = 2.57374 / [1 + (x / 71.53607) ^ (-1.0543)]

[0083] +0.00694, we get x=0.520405, that is, the lowest detection limit (LOD) of the optimized high-sensitivity portable test strip is about 0.52 ng / mL.

[0084] 3) Evaluation results of the detection accuracy of the optimized high-sensitivity portable test strips

[0085] In this study, the detection accuracy of the highly sensitive portable test strips was evaluated by the recovery test method. The skTnI serum spiked samples with calibration concentrations of 30, 50, and 75 ng / mL were measured three times, and the average detection signal intensity of each test strip was 1.46231, 1.29458, and 0.90577, respectively. Substituting this average into the above quantitative curve y = 2.57374 / [1+(x / 71.53607)^(-1.0543)]

[0086] +0.00694, the corresponding concentration values ​​were calculated to be 91.838, 71.6171, and 39.6399 ng / mL, and the ratio of the measured concentration to the calibration concentration was the recovery rate (Table 2). The average recovery rate of the three concentrations was 122.45%, indicating that the optimized test strip has a good recovery rate and detection accuracy.

[0087] 4) Evaluation results of the detection precision of the optimized high-sensitivity portable test strips

[0088] In this study, the detection precision of the highly sensitive portable test strip was evaluated by the coefficient of variation (CV%). The skTnI human serum with a calibrated concentration of 30, 50, and 75 ng / mL was added to the sample and tested three times. The coefficient of variation of these three concentrations can be calculated according to the following formula:

[0089]

[0090] In the formula: CV%——coefficient of variation; SD——standard deviation; ——Average value. The coefficients of variation of the above 18 concentrations were calculated to be 2.99%, 7.16%, and 10.38%, respectively, as shown in Table 2. The average coefficient of variation of these three concentrations is 6.84%, indicating that the optimized high-sensitivity portable test strips have a low coefficient of variation and good detection precision, meeting the CV% <15% required by the detection standard.

[0091] Table 2 Recovery rate and coefficient of variation of test strips after optimization

[0092]

[0093] It can be seen from the above examples that the present invention prepares a lateral flow immunochromatographic test strip based on the principle of "sandwich method" to detect the muscle injury marker skTnI (skeletal muscle troponin) in human serum. The present invention achieves high-sensitivity detection of 0.5ng / mL skTnI within 10min by regulating the pretreatment method of the binding pad, screening of different types of NC membranes, screening of fluorescent nano-microspheres of different particle sizes, optimization of the pH of fluorescent nano-microsphere-coupled antibodies, optimization of the spraying concentration of fluorescent nano-microsphere-antibody complexes, and optimization of the concentration of sample diluent components (such as the content of Tween-20 and BSA). The sensitivity is about 10 times higher than that of traditional nanogold test strips. In addition, the present invention also successfully uses the test strip for the detection of skTnI clinical serum added samples, providing a new method for the timely discovery, early detection and long-term monitoring of muscle damage. And after a small test, the detection performance of the portable test strip was evaluated, and it was found that its detection linear R 2 It can reach 0.999, the minimum detection limit is as low as 0.52ng / mL, the average recovery rate is 122.45%, and the average coefficient of variation is 6.84%. This shows that the portable muscle injury instant detection test strip developed by this institute has high sensitivity and good detection performance, which can meet the needs of clinical detection and provide a new method for the timely discovery, early detection and long-term monitoring of muscle injury.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the specification and drawings of the present invention, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A portable instant muscle injury test strip, characterized in that: The test paper is composed of a nitrocellulose membrane, a conjugate pad, a sample pad, a water-absorbing pad and a backing adhesive base plate, wherein the nitrocellulose membrane is fixed with an antibody molecule 1, and the conjugate pad is sprayed with a fluorescent nano-microsphere-antibody molecule 2 complex; wherein the conjugate pad is pretreated: a conjugate pad treatment solution: 200mM Tris-Hcl, 20% sucrose, 4% S9, pH=8 is sucked into a gold spray film streaking instrument, and sprayed on the surface of the conjugate pad, and placed in a constant temperature drying oven at 37°C for 2 hours before being taken out for use; The antibody molecule 1 is: skTnI capture antibody Ab2; The fluorescent nanosphere-antibody molecule 2 is a europium fluorescent microsphere-antibody complex, and the antibody molecule 2 is a skTnI-coated antibody Ab1; The nitrocellulose membrane is selected from CN95, where CN95 means that the water chromatography time per 4 cm membrane is 95 seconds; The fluorescent nanosphere-antibody molecule 2 complex is prepared by the following steps: 100 μL of europium fluorescent microspheres are taken and washed four times with MES buffer; 17.5 μL of EDC solution and 17.5 μL of sulfo-NHS solution are added, and the mixture is placed on an oscillator for 45 minutes to complete carboxyl activation, and then washed three times with MES buffer, and 500 μL of MES buffer and skTnI coated antibody with the most suitable antibody coating mass are added to the activated mixture in sequence, and the shaking is continued for 2 hours; Then wash twice with ultrapure water, add 200 μL of blocking solution and continue shaking for 30 minutes; finally wash twice with ultrapure water, redisperse with 200 μL of ultrapure water to complete the preparation of europium fluorescent microsphere-antibody complex, and store in a refrigerator at 4°C; The optimal antibody coating mass is 100 μg; The fluorescent nanoparticles were selected from WD300+ and purchased from Suzhou Weidu Biotechnology Co., Ltd. The fluorescent nanospheres are coupled to the antibody molecule 2, and the pH value of the coupling solution is 6; The spraying concentration of the fluorescent nanosphere-antibody molecule 2 complex was 6%; The optimum coating concentration of the antibody molecule 1 on the detection line of the nitrocellulose membrane is 1.5 mg / mL.

2. A portable instant muscle injury detection kit, characterized in that: Including the test paper as claimed in claim 1.

3. A portable instant muscle injury detection kit according to claim 2, characterized in that: The method also includes a sample diluent, wherein the sample diluent contains 5 wt % Tween-20 and 0.5 wt % BSA, and the pH value is 6.

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