Photothermal sensor for detecting cardiac troponin I based on copper sulfide

Abstract

The invention provides a photo-thermal sensor for detecting cardiac troponin I based on copper sulphide, and belongs to the field of instant detection.The photo-thermal sensor comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper, the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are arranged on one face of the bottom plate, and the sample pad contains a cardiac troponin I monoclonal antibody 1 marked by copper sulphide; and a cardiac troponin I monoclonal antibody 2 and a cardiac troponin I monoclonal antibody 3 are fixed on a detection line of the nitrocellulose membrane. According to the invention, dual-mode detection of colorimetric and photo-thermal sensing is successfully applied to cTnI detection for the first time, and the obtained scheme has good anti-interference capability, good specificity, accuracy and stability, can meet the detection requirements of various scenes, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of point-of-care testing (POCT), and in particular to a photothermal sensor based on copper sulfide for cardiac troponin I, and preparation and application thereof. Background Art

[0002] Cardiac troponin I (cTnI) is a regulatory protein unique to myocardial tissue that inhibits the binding of myosin to actin and plays an important role in myocardial contraction. A large number of studies have shown that in the early stages of acute myocardial infarction (AMI), cTnI levels vary widely and have a high sensitivity. Moreover, cTnI is not expressed in any type of skeletal muscle and has a high specificity for myocardial cells. The average concentration of cTnI in normal human serum is less than 0.06 ng / ml. When the myocardium is slightly damaged, its concentration increases significantly and can reach 100-1300 ng / ml. cTnI is the most specific and sensitive marker of myocardial tissue damage that can be detected in the blood today. It is the best marker for diagnosing acute myocardial infarction and risk stratification of heart disease. The specificity of cTnI in diagnosing myocardial infarction is 96% and the sensitivity is 97%. Therefore, cTnI is one of the most sensitive and specific serum markers of myocardial cell injury. In addition, cTnI has the advantages of clear threshold, wide window period and rapid detection. It has gradually become the main biochemical indicator for judging myocardial cell injury in AMI patients.

[0003] At present, the commonly used methods for detecting cardiac troponin I include fluorescent PCR, chemiluminescence, colloidal gold method, etc. Compared with other methods limited by instruments, sites, reagents and transportation, the colloidal gold method has the advantages of rapid detection results, convenient testing, convenient transportation, and no dependence on instrument testing. However, the colloidal gold method has low sensitivity and cannot be quantitatively analyzed, which is not conducive to the judgment of the disease. Compared with colorimetric visual detection, photothermal sensing has higher sensitivity and can perform quantitative analysis, but it requires the assistance of instruments and equipment. There is no report on the use of photothermal sensors to detect cardiac troponin I. Summary of the invention

[0004] In view of this, the present invention provides a photothermal sensor for cardiac troponin I based on copper sulfide and a preparation method and application thereof, aiming to solve the problem that the colloidal gold method cannot perform quantitative detection, and the scheme can achieve semi-quantitative detection of cTnI in special circumstances where no instrument is available.

[0005] The technical solution of the present invention is as follows: In the first aspect, the present invention provides a photothermal sensor based on copper sulfide for cardiac troponin I, the photothermal sensor comprising a base plate and a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper arranged on one side of the base plate, wherein the sample pad contains a cardiac troponin I monoclonal antibody 1 labeled with copper sulfide, and the detection line of the nitrocellulose membrane is fixed with a cardiac troponin I monoclonal antibody 2 and a monoclonal antibody 3.

[0006] It can be understood that in the above-mentioned photothermal sensor, cardiac troponin I monoclonal antibody 1, monoclonal antibody 2 and monoclonal antibody 3 represent three different cardiac troponin I monoclonal antibodies. In some embodiments of the present invention, cardiac troponin I monoclonal antibody 1, monoclonal antibody 2 and monoclonal antibody 3 are selected from the following three types: An antibody that specifically binds to the amino acid fragment 24-40 of cardiac troponin I; An antibody that specifically binds to the amino acid fragment 41-49 of cardiac troponin I; An antibody that specifically binds to the 83rd to 93rd amino acid fragment of cardiac troponin I.

[0007] Preferably, in the above-mentioned photothermal sensor, the particle size of copper sulfide is 3-4 nm, and the mass ratio of copper sulfide to cardiac troponin I monoclonal antibody 1 is (1.80-2.20):(0.85-1.15).

[0008] In a second aspect, the present invention provides the use of the above-mentioned photothermal sensor based on copper sulfide for cardiac troponin I in the preparation of cardiac troponin I detection products, such as reagent cards or kits. For example, in some specific embodiments, the reagent card includes the above-mentioned optical sensor and a card body, the optical sensor is arranged in the card body, and the card body is provided with a sample addition hole at a position corresponding to the sample pad, and a detection window is provided at the detection line of the nitrocellulose membrane.

[0009] In a third aspect, the present invention provides a method for preparing the above-mentioned photothermal sensor based on copper sulfide for cardiac troponin I, which comprises the following steps: (1) synthesizing copper sulfide with a carboxyl group and cardiac troponin I monoclonal antibody 1 with an amino group, obtaining copper sulfide-labeled cardiac troponin I monoclonal antibody 1 through dehydration condensation of the carboxyl group and the amino group, and then spraying the copper sulfide-labeled cardiac troponin I monoclonal antibody 1 on the pretreated binding pad and drying; (2) preparing a mixed solution of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3, streaking the solution onto a nitrocellulose membrane, and drying the solution to obtain a nitrocellulose membrane with a detection line; (3) Fix the nitrocellulose membrane on the base plate, stick absorbent paper on the upper end of the nitrocellulose membrane, stick the binding pad on the lower end of the nitrocellulose membrane, and finally stick the sample pad on the bottom.

[0010] Preferably, in the above preparation method, the sample pad is obtained by impregnating glass fiber with a sample pad treatment solution and then drying the sample pad treatment solution, wherein the sample pad treatment solution consists of 9-12 mM Tri-HCl, 4-6% trehalose and 0.85-1.15% NaCl.

[0011] Preferably, in the above preparation method, the pretreatment of the conjugate pad is: soaking the glass fiber in a conjugate pad treatment solution and then drying it, wherein the conjugate pad treatment solution consists of 9-12 mM Tri-HCl and 0.2-0.4% Tween.

[0012] In a fourth aspect, the present invention provides a method for semi-quantitative or quantitative detection of cardiac troponin I using the above-mentioned photothermal sensor, comprising the following operations: The sample to be tested is added dropwise to the sample pad, reacted for 10-20 minutes, the nitrocellulose membrane detection area is irradiated with 808nm laser, and the temperature change is recorded with an infrared thermal imager; the concentration of cardiac troponin I in the sample to be tested is calculated using the temperature difference.

[0013] For example, in some embodiments of the present invention, the concentration of cardiac troponin I is calculated specifically by the following formula: ; Wherein, ΔT is the temperature value increased compared with the initial temperature, and C is the concentration of cardiac troponin I in the sample to be tested.

[0014] In the detection method of the present invention, the reaction process and detection principle are specifically as follows: when the sample to be tested is dripped onto the sample pad, the cardiac troponin I antigen in the sample to be tested and the cardiac troponin I monoclonal antibody 1 labeled with copper sulfide in the binding pad combine to form an antigen-antibody complex, which diffuses forward along the nitrocellulose membrane under capillary action, and the complex is captured by the cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3 fixed on the detection line. The more cardiac troponin I in the sample to be tested, the more complexes captured on the detection line, and the more corresponding copper sulfide. Through the presence and depth of color changes, cTnI can be semi-quantitatively analyzed. Under 808nm laser irradiation, the aggregated copper sulfide increases the temperature of the detection area, and the temperature of the fixed detection area can be quantified by a thermal imager; the increased temperature is positively correlated with the concentration of cardiac troponin I in the sample, and the concentration of cardiac troponin I in the sample can be obtained by bringing the increased temperature into the standard curve for calculation.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully applies the dual-mode detection of colorimetry and photothermal sensing to the detection of cTnI for the first time, and experimental data show that the photothermal sensor constructed by the present invention has good anti-interference ability, good specificity, accuracy and stability, and can realize reliable detection of cTnI in blood samples.

[0016] The present invention can realize quantitative detection using a portable thermal imager, and can also perform semi-quantitative detection of cTnI in special circumstances where no instrument is available. It is simple to operate and low in cost, can meet the diagnostic needs of various scenarios, and has great potential in the fields of disease diagnosis and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 Schematic diagram of the structure of the photothermal sensor in Example 1 of the present invention; Figure 2 This is a graph showing the specific detection results of cTnI, cTnT (cardiac troponin T) and cTnC (cardiac troponin C) by the photothermal sensor in Example 4 of the present invention; Figure 3 This is a linear interval result diagram measured by the photothermal sensor in Example 4 of the present invention. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the specification and claims of the present invention and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0021] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0022] Example 1 This example provides a photothermal sensor based on copper sulfide for cardiac troponin I, and its structure is as follows Figure 1 As shown, it includes a PVC base plate, a nitrocellulose membrane, absorbent paper, a conjugation pad and a sample pad, the nitrocellulose membrane is fixed on the PVC base plate, one end of the nitrocellulose membrane is overlapped with the absorbent paper, and the other end is overlapped with the conjugation pad, and the end of the conjugation pad away from the nitrocellulose membrane is overlapped with the sample pad; the nitrocellulose membrane is provided with a detection line, and the detection line is fixed with a cardiac troponin I monoclonal antibody 2 and a cardiac troponin I monoclonal antibody 3; the conjugation pad uniformly contains a copper sulfide-labeled cardiac troponin I monoclonal antibody 1.

[0023] The information of the three monoclonal antibodies used in this example is shown in Table 1.

[0024] Table 1 Information on cardiac troponin I monoclonal antibodies

[0025] Example 2 This example provides a method for preparing the photothermal sensor shown in Example 1, comprising the following steps: (1) Preparation of copper sulfide with carboxyl group.

[0026] 15 μL of thioglycolic acid was added to 10 mL of 2 mM Cu(NO3)2 solution, and the pH was adjusted to 9 with 0.5 M NaOH solution. After stirring for 30 min, Na2S solution (10 mL, 6.7 mM) was added dropwise to the mixture, and stirring was continued until the solution turned green. After the reaction was completed, the solution was transferred to an ultrafiltration tube (Millipore-10KD) and centrifuged and washed. The final quantification was 2.5 mL and stored at 4 °C for future use.

[0027] (2) Copper sulfide-conjugated cTnI monoclonal antibody 1.

[0028] Take 100 μL of the copper sulfide solution prepared in step (1) and add it to 900 μL of MES (10 mM, pH 6.0) and mix well. Weigh 5 mg of EDC and 5 mg of NHS, dissolve them in ultrapure water to make 5 mg / mL solutions, then add 100 μL of NHS solution to the above solution and mix well, then add 200 μL of EDC solution and mix well, and react in a shaker at 30°C and 220 rpm for 30 min.

[0029] The product was washed twice by centrifugation with BB (10mM, pH8.0) and then redispersed in 1mL BB (10mM, pH8.0). cTnI monoclonal antibody 1 was diluted with BB (10mM, pH8.0) to a 1mg / mL solution, 100μL was added to the above solution, mixed evenly, and reacted in a shaker at 30℃ and 220rpm for 1h. After the reaction was completed, the product was washed twice by centrifugation with BB (10mM, pH8.0), redispersed in 1mL BB (10mM, pH8.0), 100μL 10% BSA solution was added, and reacted in a shaker at 30℃ and 220rpm for 1h. The product copper sulfide-cTnI monoclonal antibody 1 complex was centrifuged to remove the supernatant, and the precipitate was redispersed in 1 mL of drying solution (10 mM BB, 0.5% NaCl, 0.5% trehalose, 0.1% BSA, 0.01% NaN3) and placed at 4°C for use.

[0030] (3) Pretreatment of sample pad and conjugate pad.

[0031] Place the glass fiber on the sample loading rack. Before loading, mix the sample pad treatment solution (10mM Tri-HCl, 5% trehalose, 1% NaCl) thoroughly. Use a sample pipette to evenly apply the sample pad treatment solution to the glass fiber so that the glass fiber is fully soaked with the sample pad treatment solution and reaches saturation. Spread the treated glass fiber flat on the grid and put it into an oven at 45℃ for 16 hours.

[0032] Place the glass fiber on the sample rack, mix the binding pad treatment solution (10mMTri-HCl, 0.3% Tween-20) thoroughly before adding the sample, and use a pipette to evenly add the binding pad treatment solution to the glass fiber to make the glass fiber fully infiltrated with the binding pad treatment solution to reach saturation. Spread the treated glass fiber flat on the grid and put it into the oven at 45℃ for 16 hours.

[0033] (4) Preparation of bonding pad.

[0034] Cut the pretreated conjugate pad into 1.00 cm × 30 cm size for later use. Dilute the solution prepared in step (2) 5 times, spray it evenly on the cut pretreated conjugate pad with a gold label sprayer at a spray volume of 10 μL / cm, and place it in an oven at 45°C for 16 hours.

[0035] (5) Immobilization of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3.

[0036] Cut the nitrocellulose membrane into 2.5cm×30cm / sheet, take the PVC base plate, remove the 2.5cm wide protective paper in the middle area of ​​the base plate, and align the cut nitrocellulose membrane along the edge of the middle area and paste it in the middle. Prepare the cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3 into a mixed solution of 1.0mg / mL and 0.5mg / mL using the coating solution (10mMPB, 1% trehalose), and use a continuous film drawing instrument to draw the antibody on the membrane at a spray volume of 1μL / cm, and place it in an oven at 45℃ for 16h to fix the cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3 on the NC membrane.

[0037] (6) Construction of photothermal sensor.

[0038] Remove the 2 cm wide protective layer of paper on the upper area of ​​the PVC base plate after drying in step (5), align the absorbent paper along the 300 mm side of the base plate, press 2 mm on the 30 cm side of the nitrocellulose membrane, and stick it tightly. Glue the binding pad on the lower end of the nitrocellulose membrane, requiring the binding pad to press the nitrocellulose membrane 2 mm, and finally glue the sample pad to the bottom end, ensuring that the sample pad presses the binding pad 2 mm. Cut the pasted large plate into 4 mm wide test strips, which are the constructed photothermal sensors.

[0039] Example 3 This example provides a method for detection using the photothermal sensor prepared in Example 2, comprising the following steps: (1) Construction of standard curve.

[0040] 100 μL of a solution containing a certain concentration of cTnI antigen was dripped onto the sample pad. After 15 minutes of reaction, the NC membrane detection area was irradiated with an 808 nm laser and the temperature change was recorded with a portable infrared thermal imager. The four-parameter growth model was fitted based on the antigen concentration and the increased temperature value to obtain the following formula: ; Where ΔT is the temperature increase compared to the initial temperature, and C is the concentration of the target.

[0041] (2) Testing of samples to be tested.

[0042] The sample to be tested is added to the sample pad, and after 15 minutes of reaction, the NC membrane detection area is irradiated with 808nm laser, and the temperature change is recorded with a portable infrared thermal imager. The increased temperature value is positively correlated with the concentration of cardiac troponin I in the sample to be tested. The increased temperature value is brought into the above standard curve for calculation, and the concentration of cardiac troponin I in the sample to be tested can be obtained.

[0043] Example 4 This example examines the performance of the photothermal sensor of the present invention from aspects such as anti-interference ability, specificity, and precision, and specifically includes the following experiments: (1) Interference experiment

[0044] The low value solution (0.50 ng / mL) and high value solution (20.00 ng / mL) of cTnI were selected as the basic solution, and the addition of hemoglobin, triglyceride and bilirubin interferors was studied. The final concentrations of hemoglobin 5.0 g / L, triglyceride 10 g / L and bilirubin 0.2 g / L were added to the sample, and the detection conditions were the same as in Example 3.

[0045] The results are shown in Table 2. When the hemoglobin is 5.0 g / L, the triglyceride is 10 g / L, and the bilirubin is 0.2 g / L, the relative deviation is within ±5%, indicating that the sensor of the present invention has a certain anti-interference ability. It should be noted that the mean values ​​corresponding to hemoglobin and bilirubin in Table 2 are both 0.51, but the reason for the inconsistency of the relative deviation is that the calculation is performed according to the actual specific concentration value.

[0046] Table 2 Test results of the anti-interference ability of the photothermal sensor

[0047] (2) Specificity experiments

[0048] The specificity of the photothermal sensor was verified using a blank group, cTnI, cTnT, and cTnC, wherein the concentrations of cTnI, cTnT, and cTnC were all 20 ng / mL. The detection conditions were the same as those in Example 3.

[0049] The results are as follows Figure 2 As shown, the temperature will increase significantly only in the presence of cTnI, indicating that the sensor of the present invention has good specificity.

[0050] (3) Determination of linear interval.

[0051] The cTnI antigen solution close to the upper limit of the linear range was gradiently diluted to 5 concentrations. According to the conditions in Example 3, the above solutions were tested using the photothermal sensor of the present invention, and each concentration was tested three times to calculate the average value.

[0052] Take the dilution concentration as the independent variable and the mean of the test results as the dependent variable to find the linear regression equation and calculate the correlation coefficient R of the linear regression. 2 =0.9953( Figure 3 ), indicating that the sensor of the present invention has good linearity between 0-50ng / mL.

[0053] (4) Precision experiment.

[0054] The cTnI antigen solutions at two different concentration levels (0.50 ng / mL and 20.00 ng / mL) were measured 10 times respectively, and the mean and standard deviation of the 10 measurement results were calculated. The coefficient of variation was calculated.

[0055] The test results are shown in Table 3. The CVs of the solutions at the high and low concentration levels are both less than 10%, indicating that the sensor of the present invention has good precision.

[0056] Table 3 Test results of photothermal sensor precision

[0057] Example 5 Using the photothermal sensor of the present invention, this example tested the serum sample, as follows: Different concentrations of antigens were added to negative serum, including 0.50 ng / mL, 5.00 ng / mL, 10.00 ng / mL, 20.00 ng / mL, and 50.00 ng / mL. The detection conditions were the same as those in Example 3, and the content of the target in the sample to be tested was calculated based on the temperature change measured in the detection zone and the constructed standard curve.

[0058] The results are shown in Table 4. For serum samples containing different concentrations of cTnI, the accuracy of the detection results of the detection method established by the present invention in serum is between 98% and 106.7%, and the relative deviation between multiple detection results is less than or equal to 6.7%, indicating that the sensing method established by the present invention has good accuracy and stability.

[0059] Table 4 Detection results of target substances in serum samples

[0060] In summary, the photothermal sensor provided by the present invention can be used for the quantitative detection of cTnI in blood samples, and has good anti-interference ability, good specificity, accuracy and stability, which is of great significance for clinical instant detection.

[0061] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and embodiments having substantially the same structure as the technical idea and exerting the same effects within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, within the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other methods of combining some of the constituent elements in the embodiments to construct are also included in the scope of the present invention.

Claims

1. A photothermal sensor based on copper sulfide for cardiac troponin I, characterized in that: The invention comprises a bottom plate and a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper arranged on one side of the bottom plate. The sample pad contains a copper sulfide-labeled cardiac troponin I monoclonal antibody 1, and a cardiac troponin I monoclonal antibody 2 and a monoclonal antibody 3 are fixed on the detection line of the nitrocellulose membrane.

2. The photothermal sensor according to claim 1, characterized in that: The cardiac troponin I monoclonal antibody 1, monoclonal antibody 2 and monoclonal antibody 3 are selected from the following three types: An antibody that specifically binds to the amino acid fragment 24-40 of cardiac troponin I; An antibody that specifically binds to the amino acid fragment 41-49 of cardiac troponin I; An antibody that specifically binds to the 83rd to 93rd amino acid fragment of cardiac troponin I.

3. The photothermal sensor according to claim 1, characterized in that: The particle size of the copper sulfide is 3-4 nm, and the mass ratio of the copper sulfide to the cardiac troponin I monoclonal antibody 1 is (1.80-2.20):(0.85-1.15).

4. Use of the photothermal sensor according to any one of claims 1 to 3 in preparing a cardiac troponin I detection reagent card or kit.

5. A method for preparing a photothermal sensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) synthesizing copper sulfide with a carboxyl group and cardiac troponin I monoclonal antibody 1 with an amino group, obtaining copper sulfide-labeled cardiac troponin I monoclonal antibody 1 through dehydration condensation of the carboxyl group and the amino group, and then spraying the copper sulfide-labeled cardiac troponin I monoclonal antibody 1 on the pretreated binding pad and drying; (2) preparing a mixed solution of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3, streaking the solution onto a nitrocellulose membrane, and drying the solution to obtain a nitrocellulose membrane with a detection line; (3) Fix the nitrocellulose membrane on the base plate, stick absorbent paper on the upper end of the nitrocellulose membrane, stick the binding pad on the lower end of the nitrocellulose membrane, and finally stick the sample pad on the bottom.

6. The preparation method according to claim 5, characterized in that The sample pad is obtained by soaking glass fiber in sample pad treatment solution and then drying it, wherein the sample pad treatment solution consists of 9-12 mM Tri-HCl, 4-6% trehalose and 0.85-1.15% NaCl.

7. The preparation method according to claim 5, characterized in that The pretreatment of the conjugate pad is as follows: the glass fiber is soaked in a conjugate pad treatment solution and then dried, wherein the conjugate pad treatment solution is composed of 9-12 mM Tri-HCl and 0.2-0.4% Tween.

8. A method for semi-quantitative or quantitative detection of cardiac troponin I, characterized in that: Detection is performed using the photothermal sensor described in any one of claims 1 to 3.

9. The method according to claim 8, characterized in that The sample to be tested is added dropwise to the sample pad, reacted for 10-20 minutes, the nitrocellulose membrane detection area is irradiated with 808nm laser, and the temperature change is recorded with an infrared thermal imager; the concentration of cardiac troponin I in the sample to be tested is calculated using the temperature difference.

10. The method according to claim 9, characterized in that The concentration of cardiac troponin I was calculated by the following formula: ； Wherein, ΔT is the temperature value increased compared with the initial temperature, and C is the concentration of cardiac troponin I in the sample to be tested.

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