A photothermal sensor based on copper sulfide for detecting cardiac troponin I

Abstract

This invention provides a photothermal sensor for detecting cardiac troponin I based on copper sulfide, belonging to the field of point-of-care testing. The photothermal sensor includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper disposed on one side of the base plate. The sample pad contains a copper sulfide-labeled cardiac troponin I monoclonal antibody 1, and cardiac troponin I monoclonal antibodies 2 and 3 are immobilized on the detection lines of the nitrocellulose membrane. This invention is the first to successfully apply dual-mode detection of colorimetry and photothermal sensing to the detection of cTnI. Furthermore, the resulting scheme exhibits good anti-interference ability, good specificity, accuracy, and stability, meeting the detection needs of various scenarios and showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of point-of-care testing (POCT) technology, specifically to a photothermal sensor based on copper sulfide for cardiac troponin I, and its preparation and application. Background Technology

[0002] Cardiac troponin I (cTnI) is a regulatory protein specific to myocardial tissue. It inhibits the binding of myosin to actin and plays an important role in myocardial contraction. Numerous studies have shown that cTnI levels fluctuate significantly in the early stages of acute myocardial infarction (AMI), exhibiting high sensitivity. Furthermore, cTnI is not expressed in any type of skeletal muscle, demonstrating high cardiomyocyte specificity. The average serum concentration of cTnI in normal individuals is <0.06 ng / ml, but its concentration significantly increases to 100–1300 ng / ml in cases of minor myocardial injury. cTnI is currently the most specific and sensitive biomarker for detecting myocardial tissue damage in blood, making it the best biomarker for diagnosing acute myocardial infarction and for 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 biomarkers for cardiomyocyte damage. In addition, cTnI has advantages such as a clear threshold, a wide window period, and rapid detection, and has gradually become the main biochemical indicator for judging cardiomyocyte damage in AMI patients.

[0003] Currently, common methods for detecting cardiac troponin I include fluorescent PCR, chemiluminescence, and colloidal gold methods. Compared to other methods limited by instruments, facilities, reagents, and transportation, the colloidal gold method offers advantages such as rapid results, convenient testing, easy transport, and independence from instrumentation. However, the colloidal gold method has low sensitivity and cannot provide quantitative analysis, which is detrimental to disease diagnosis. Compared to colorimetric detection, photothermal sensing offers higher sensitivity and can perform quantitative analysis, but it requires auxiliary equipment, and there are currently no reports of using 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, its preparation method and application, aiming to solve the problem that colloidal gold method cannot quantitatively detect it, and this solution can achieve semi-quantitative detection of cTnI in special circumstances where no instruments are available.

[0005] The specific technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a photothermal sensor based on copper sulfide for cardiac troponin I. The photothermal sensor includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper disposed on one side of the base plate. The sample pad contains a copper sulfide-labeled cardiac troponin I monoclonal antibody 1, and the detection lines of the nitrocellulose membrane are fixed with cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3.

[0007] It is understood that in the aforementioned 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:

[0008] An antibody that specifically binds to amino acid fragments 24-40 of cardiac troponin I;

[0009] An antibody that specifically binds to amino acid fragments 41-49 of cardiac troponin I;

[0010] An antibody that specifically binds to the 83-93 amino acid fragment of cardiac troponin I.

[0011] 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).

[0012] Secondly, this invention provides the application of the aforementioned 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. In some specific embodiments, the reagent card includes the aforementioned optical sensor and a card body. The optical sensor is disposed within the card body, which has a sample application hole at the corresponding location on the sample pad and a detection window at the detection line of the nitrocellulose membrane.

[0013] Thirdly, the present invention provides a method for preparing the above-mentioned photothermal sensor based on copper sulfide for cardiac troponin I, which includes the following steps:

[0014] (1) Synthesize copper sulfide with carboxyl group and cardiac troponin I monoclonal antibody 1 with amino group. Copper sulfide labeled cardiac troponin I monoclonal antibody 1 is obtained by dehydration condensation of carboxyl group and amino group. Then, copper sulfide labeled cardiac troponin I monoclonal antibody 1 is sprayed onto the pretreated binding pad and dried.

[0015] (2) Prepare a mixed solution of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3, apply it to a nitrocellulose membrane, and dry it to obtain a nitrocellulose membrane with detection lines;

[0016] (3) Fix the nitrocellulose membrane to the base plate, attach absorbent paper to the upper end of the nitrocellulose membrane, attach the binding pad to the lower end of the nitrocellulose membrane, and finally attach the sample pad to the bottom.

[0017] Preferably, in the above preparation method, the sample pad is obtained by wetting glass fiber with a sample pad treatment solution and then drying it, wherein the sample pad treatment solution is composed of 9-12mM Tri-HCl, 4-6% trehalose and 0.85-1.15% NaCl.

[0018] Preferably, in the above preparation method, the pretreatment of the conjugate pad is as follows: the glass fiber is soaked in the conjugate pad treatment solution and then dried, wherein the conjugate pad treatment solution is composed of 9-12mM Tri-HCl and 0.2-0.4% Tween.

[0019] Fourthly, 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:

[0020] The sample to be tested is dropped onto the sample pad and reacted for 10-20 minutes. The detection area of ​​the nitrocellulose membrane is irradiated with an 808nm laser, and the temperature change is recorded with an infrared thermal imager. The concentration of cardiac troponin I in the sample is calculated using the temperature difference.

[0021] In some embodiments of the present invention, the concentration of cardiac troponin I is specifically calculated using the following formula:

[0022] ;

[0023] In the formula, ΔT is the temperature increase compared to the initial temperature, and C is the concentration of cardiac troponin I in the sample to be tested.

[0024] In the detection method of this invention, the reaction process and detection principle are as follows: When the sample to be tested is added to the sample pad, the cardiac troponin I antigen in the sample and the copper sulfide-labeled cardiac troponin I monoclonal antibody 1 in the conjugate pad combine to form an antigen-antibody complex. This complex diffuses forward along the nitrocellulose membrane under capillary action. The complex is captured by cardiac troponin I monoclonal antibodies 2 and 3 fixed on the detection line. The more cardiac troponin I in the sample, the more complex is captured on the detection line, and consequently, the more copper sulfide is present. Semi-quantitative analysis of cTnI can be performed by observing the presence and intensity of color changes. Under 808nm laser irradiation, the accumulated copper sulfide raises the temperature of the detection area. The temperature of the fixed detection area can be quantified using a thermal imager. The increased temperature is positively correlated with the concentration of cardiac troponin I in the sample. The concentration of cardiac troponin I in the sample can be calculated by substituting the increased temperature into a standard curve.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] This invention is the first to successfully apply dual-mode detection of colorimetry and photothermal sensing to the detection of cTnI. Moreover, experimental data show that the photothermal sensor constructed in this invention has good anti-interference ability, good specificity, accuracy and stability, and can realize reliable detection of cTnI in blood samples.

[0027] This invention enables quantitative detection using a portable thermal imager, and can also perform semi-quantitative detection of cTnI in special circumstances where no instruments are available. It is simple to operate and low in cost, and can meet the diagnostic needs of various scenarios, showing great potential in the fields of disease diagnosis and clinical application. Attached Figure Description

[0028] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0029] Figure 1 This is a schematic diagram of the photothermal sensor in Embodiment 1 of the present invention;

[0030] Figure 2 This is a graph showing the specific detection results of the photothermal sensor for cTnI, cTnT (cardiac troponin T) and cTnC (cardiac troponin C) in Embodiment 4 of the present invention.

[0031] Figure 3 This is a graph showing the linear interval results measured by the photothermal sensor in Embodiment 4 of the present invention. Detailed Implementation

[0032] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the invention, are intended to cover non-exclusive inclusion.

[0034] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0035] Example 1

[0036] This example provides a photothermal sensor based on copper sulfide for cardiac troponin I, the structure of which is as follows: Figure 1 As shown, the apparatus includes a PVC base plate, a nitrocellulose membrane, absorbent paper, a conjugate pad, and a sample pad. The nitrocellulose membrane is fixed to the PVC base plate. One end of the nitrocellulose membrane overlaps with absorbent paper, and the other end overlaps with the conjugate pad. The end of the conjugate pad away from the nitrocellulose membrane overlaps with the sample pad. The nitrocellulose membrane has a detection line, and cardiac troponin I monoclonal antibody 2 and cardiac troponin I monoclonal antibody 3 are fixed on the detection line. The conjugate pad uniformly contains copper sulfide-labeled cardiac troponin I monoclonal antibody 1.

[0037] The information on the three monoclonal antibodies used in this embodiment is shown in Table 1.

[0038] Table 1 Information on cardiac troponin I monoclonal antibodies

[0039]

[0040] Example 2

[0041] This example provides a method for preparing the photothermal sensor shown in Example 1, including the following steps:

[0042] (1) Preparation of copper sulfide with carboxyl groups.

[0043] Add 15 μL of mercaptoacetic acid to 10 mL of 2 mM Cu(NO3)2 solution, and adjust the pH to 9 with 0.5 M NaOH solution. After stirring for 30 min, add 10 mL of 6.7 mM Na2S solution dropwise to the mixture, and continue stirring until the solution turns green. After the reaction is complete, transfer the solution to an ultrafiltration tube (Millipore-10KD), centrifuge and wash, and finally measure 2.5 mL. Store at 4 °C for later use.

[0044] (2) Copper sulfide coupled with cTnI monoclonal antibody 1.

[0045] 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, and dissolve them in ultrapure water to make a solution of 5 mg / mL. Then, add 100 μL of NHS solution to the above solution and mix well, then add 200 μL of EDC solution and mix well. React at 30 °C and 220 rpm in a shaker for 30 min.

[0046] The product was washed twice with BB (10mM, pH 8.0) by centrifugation, and then redispersed in 1 mL of BB (10mM, pH 8.0). cTnI monoclonal antibody 1 was diluted to a 1 mg / mL solution with BB (10mM, pH 8.0), and 100 μL was added to the diluted solution. The mixture was thoroughly mixed and reacted at 30°C and 220 rpm for 1 h in a shaker. After the reaction was complete, the product was washed twice with BB (10mM, pH 8.0) by centrifugation, and then redispersed in 1 mL of BB (10mM, pH 8.0). 100 μL of 10% BSA solution was added, and the mixture was reacted at 30°C and 220 rpm for 1 h in a shaker. The product, copper sulfide-cTnI monoclonal antibody 1 complex, was centrifuged to remove the supernatant. The precipitate was then redispersed in 1 mL of drying buffer (10 mM BB, 0.5% NaCl, 0.5% trehalose, 0.1% BSA, 0.01% NaN3) and stored at 4 °C for later use.

[0047] (3) Pretreatment of sample pads and conjugate pads.

[0048] Place the glass fiber on the sample rack. Before adding the sample, thoroughly mix the sample pad treatment solution (10mM Tri-HCl, 5% trehalose, 1% NaCl). Use a sampler to evenly apply the sample pad treatment solution onto the glass fiber, ensuring that the glass fiber is fully soaked and saturated. Spread the treated glass fiber flat on the mesh rack and place it in an oven at 45℃ for 16 hours to dry.

[0049] Place the glass fiber on the sample rack. Before adding the sample, thoroughly mix the conjugate pad treatment solution (10mM Tri-HCl, 0.3% Tween-20). Use a sampler to evenly add the conjugate pad treatment solution onto the glass fiber, ensuring that the glass fiber is fully soaked and saturated. Spread the treated glass fiber flat on the mesh rack and place it in an oven at 45℃ for 16 hours to dry.

[0050] (4) Preparation of binding pad.

[0051] Cut the pretreated conjugate pad into 1.00cm×30cm pieces for later use. Dilute the solution prepared in step (2) 5 times and spray it evenly onto the cut pretreated conjugate pad using a gold sprayer at a spray rate of 10μL / cm. Place it in an oven at 45℃ and dry for 16h.

[0052] (5) Fixation of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3.

[0053] Cut nitrocellulose membranes into 2.5cm × 30cm sheets. Take a PVC base plate, remove the 2.5cm protective paper in the middle area, and align the cut nitrocellulose membranes with the edges of the middle area and paste them in the center. Prepare a mixed solution of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3 with coating solution (10mMPB, 1% trehalose) at concentrations of 1.0mg / mL and 0.5mg / mL, respectively. Apply the antibodies to the membrane using a continuous streaking device at a spray rate of 1μL / cm. Place the membrane in an oven at 45℃ for 16 hours to fix the cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3 onto the NC membrane.

[0054] (6) Construction of photothermal sensor.

[0055] Remove the 2cm wide protective paper from the upper part of the PVC base plate after drying in step (5). Align the absorbent paper with the 300mm side of the base plate and press it 2mm onto the 30cm side of the nitrocellulose membrane, ensuring a tight seal. Attach the binding pad to the lower end of the nitrocellulose membrane, ensuring the binding pad overlaps the nitrocellulose membrane by 2mm. Finally, attach the sample pad to the bottom, ensuring the sample pad overlaps the binding pad by 2mm. Cut the finished plate into 4mm wide test strips; these test strips constitute the constructed photothermal sensor.

[0056] Example 3

[0057] This example provides a method for detection using the photothermal sensor prepared in Example 2, including the following steps:

[0058] (1) Construction of standard curve.

[0059] 100 μL of a solution containing a certain concentration of cTnI antigen was added to the sample pad. After reacting for 15 min, the NC membrane detection area was irradiated with an 808 nm laser, and the temperature change was recorded using a portable infrared thermal imager. Based on the antigen concentration and the temperature rise, a four-parameter growth model was fitted to obtain the following formula:

[0060] ;

[0061] In the formula, ΔT is the temperature increase compared to the initial temperature, and C is the concentration of the target substance.

[0062] (2) Detection of the sample to be tested.

[0063] The sample to be tested was dropped onto the sample pad and reacted for 15 minutes. Then, the detection area of ​​the NC membrane was irradiated with an 808nm laser, and the temperature change was recorded using a portable infrared thermal imager. The increased temperature value was positively correlated with the concentration of cardiac troponin I in the sample. By substituting the increased temperature value into the standard curve mentioned above, the concentration of cardiac troponin I in the sample could be obtained.

[0064] Example 4

[0065] This example examines the performance of the photothermal sensor of this invention in terms of anti-interference capability, specificity, and precision, specifically including the following experiments:

[0066] (1) Interference experiment.

[0067] Low-value (0.50 ng / mL) and high-value (20.00 ng / mL) cTnI solutions were selected as basal solutions to investigate the addition of hemoglobin, triglycerides, and bilirubin interfering substances. The solutions were added to the samples at final concentrations of 5.0 g / L hemoglobin, 10 g / L triglycerides, and 0.2 g / L bilirubin, and the detection conditions were the same as in Example 3.

[0068] The results are shown in Table 2. At hemoglobin levels of 5.0 g / L, triglycerides of 10 g / L, and bilirubin of 0.2 g / L, the relative deviations were all within ±5%, indicating that the sensor of this invention has a certain anti-interference capability. It should be noted that the reason why the mean values ​​for hemoglobin and bilirubin in Table 2 are both 0.51 but the relative deviations are inconsistent is that the calculations were performed based on actual specific concentration values.

[0069] Table 2. Detection results of the anti-interference capability of the photothermal sensor

[0070]

[0071] (2) Specificity test.

[0072] The specificity of the photothermal sensor was verified using a blank control group, cTnI, cTnT, and cTnC, with the concentrations of cTnI, cTnT, and cTnC all at 20 ng / mL. The detection conditions were the same as in Example 3.

[0073] The results are as follows Figure 2 As shown, the temperature only increases significantly in the presence of cTnI, indicating that the sensor of the present invention has good specificity.

[0074] (3) Determination of linear intervals.

[0075] A cTnI antigen solution close to the upper limit of the linear range was serially diluted to five concentrations. Following the conditions in Example 3, the solutions were tested using the photothermal sensor of this invention, with each concentration tested three times, and the mean value was calculated.

[0076] Using dilution concentration as the independent variable and the mean of the test results as the dependent variable, a linear regression equation was derived, and the correlation coefficient R of the linear regression was calculated. 2 =0.9953 ( Figure 3 This indicates that the sensor of the present invention has good linearity in the range of 0-50 ng / mL.

[0077] (4) Precision test.

[0078] Ten replicate measurements were performed on cTnI antigen solutions at two different concentration levels (0.50 ng / mL and 20.00 ng / mL). The mean and standard deviation of the ten measurements were calculated, and the results were then analyzed using the formula... The coefficient of variation was calculated.

[0079] The test results are shown in Table 3. The CV of the solutions at both high and low concentration levels is less than 10%, indicating that the sensor of the present invention has good precision.

[0080] Table 3. Detection results of the precision of the photothermal sensor

[0081]

[0082] Example 5

[0083] Using the photothermal sensor of this invention, a serum sample was tested in this example, as follows:

[0084] Different concentrations of antigen were added to the 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 in Example 3. The content of the target analyte in the sample was calculated based on the temperature change measured in the detection zone and the constructed standard curve.

[0085] The results are shown in Table 4. For serum samples containing different concentrations of cTnI, the detection method established in this invention has an accuracy of 98%-106.7% in serum, and the relative deviation between multiple detection results is less than or equal to 6.7%, indicating that the sensing method established in this invention has good accuracy and stability.

[0086] Table 4. Detection results of target analytes in serum samples

[0087]

[0088] In summary, the photothermal sensor provided by this 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 point-of-care testing.

[0089] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A photothermal sensor based on copper sulfide and cardiac troponin I, characterized in that, The sample pad includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper disposed on one side of the base plate. The sample pad contains a copper sulfide-labeled cardiac troponin I monoclonal antibody 1, and the detection lines of the nitrocellulose membrane are fixed with cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3. The copper sulfide has a particle size of 3-4 nm, and the mass ratio of copper sulfide to monoclonal antibody 1 is (1.80-2.20):(0.85-1.15). The copper sulfide labels monoclonal antibody 1 by dehydration condensation of carboxyl and amino groups.

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: An antibody that specifically binds to amino acid fragments 24-40 of cardiac troponin I; An antibody that specifically binds to amino acid fragments 41-49 of cardiac troponin I; An antibody that specifically binds to the 83-93 amino acid fragment of cardiac troponin I.

3. The application of the photothermal sensor as described in claim 1 or 2 in the preparation of cardiac troponin I detection reagent cards or kits.

4. A method for fabricating a photothermal sensor as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesize copper sulfide with carboxyl group and cardiac troponin I monoclonal antibody 1 with amino group. Copper sulfide labeled cardiac troponin I monoclonal antibody 1 is obtained by dehydration condensation of carboxyl group and amino group. Then, copper sulfide labeled cardiac troponin I monoclonal antibody 1 is sprayed onto the pretreated binding pad and dried. (2) Prepare a mixed solution of cardiac troponin I monoclonal antibody 2 and monoclonal antibody 3, apply it to a nitrocellulose membrane, and dry it to obtain a nitrocellulose membrane with detection lines; (3) Fix the nitrocellulose membrane to the base plate, attach absorbent paper to the upper end of the nitrocellulose membrane, attach the binding pad to the lower end of the nitrocellulose membrane, and finally attach the sample pad to the bottom.

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

6. The preparation method according to claim 4, 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 consists of 9-12mM Tri-HCl and 0.2-0.4% Tween.

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