CN118226046B - Troponin I detection kit and preparation method thereof - Google Patents
Troponin I detection kit and preparation method thereof Download PDFInfo
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- CN118226046B CN118226046B CN202410650767.2A CN202410650767A CN118226046B CN 118226046 B CN118226046 B CN 118226046B CN 202410650767 A CN202410650767 A CN 202410650767A CN 118226046 B CN118226046 B CN 118226046B
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Abstract
The invention discloses a troponin I detection kit and a preparation method thereof, belonging to the technical field of biological detection. The kit comprises a detection card, a data disc and a quality control product, wherein the detection card comprises an anti-cTnI antibody, an anti-sheep anti-rabbit IgG antibody, a fluorescent microsphere coupled anti-cTnI antibody, a fluorescent microsphere coupled anti-rabbit IgG antibody, a bottom plate, absorbent paper, a nitrocellulose membrane and a release pad. The fluorescent microsphere is a quantum dot-RBITC fluorescent microsphere, is mainly prepared by wrapping oil-soluble CdTe/CdSe/ZnS quantum dot fluorescent microsphere by a polymer and then carrying out electrostatic interaction with tetraethyl rhodamine fluorescein, and has high fluorescence and light stability. The minimum detection limit of the kit is 0.1 ng/mL, the linear range is 0.1-20ng/mL, the detection sensitivity is high, the linear range is wide, the accuracy is good, and the development of cTnI detection is facilitated.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a troponin I detection kit and a preparation method thereof.
Background
Myocardial ischemia causes myocardial cell injury and necrosis, and releases intracellular cardiac markers such as myoglobin, myocardial creatine kinase, myocardial adenosine deaminase, troponin I, etc., wherein cardiac troponin I (Cardiac Troponin I, CTnI) is widely recognized as one of the best biomarkers of myocardial injury due to its high sensitivity and specificity to myocardial injury. CTnI are specific proteins in myocardial cells, which are released into the blood during ischemia or necrosis of myocardial cells. Therefore, CTnI detection can provide important information on myocardial damage, and plays a key role in early diagnosis of myocardial infarction, patient management and treatment effect evaluation.
CTnI the detection method mainly comprises a colloidal gold method, an enzyme-linked immunosorbent method, a chemiluminescence method and the like, wherein the colloidal gold method can only carry out preliminary qualitative analysis, cannot carry out quantitative analysis, has poor repeatability and is easy to cause misreading and misjudgment; the ELISA method has long measurement time and low sensitivity; the chemiluminescent method has poor precision and insufficient sensitivity. Most of the methods are limited to be used in a central laboratory, cannot meet the requirements of the market on CTnI for rapid detection and accurate results, and limit the clinical application of the methods, so that development of a novel product which is simple, convenient, easy to use and rapid in diagnosis is urgently needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention designs a troponin I detection kit and a preparation method thereof, wherein the kit has high accuracy and high sensitivity.
In order to achieve the above purpose, the technical solution of the present invention is as follows:
in one aspect, the invention provides a troponin I detection kit comprising a detection card comprising an anti-cTnI antibody, an anti-sheep anti-rabbit IgG antibody, a fluorescent microsphere coupled anti-cTnI antibody, a fluorescent microsphere coupled anti-rabbit IgG antibody, a bottom plate, absorbent paper, a nitrocellulose membrane, and a release pad;
The release pad is treated by a pretreatment liquid, and the pretreatment liquid comprises, by mass, 0.5-2% of Triton-100, 2-7% of sucrose, 1-3% of sodium citrate, 1-4% of PEG-20000, 0.1-2% of ethanol and 0.1-0.3% of NaCl.
Further preferably, the pretreatment liquid comprises 1% Triton-100, 5% sucrose, 2% sodium citrate, 2.5% PEG-20000, 1% ethanol, 0.2% NaCl.
Preferably, the fluorescent microsphere is a quantum dot-RBITC fluorescent microsphere, and the preparation of the fluorescent microsphere comprises the following steps:
S1: dissolving polymaleic anhydride stearyl ester, polyethyl methacrylate and CdTe/CdSe/ZnS quantum dots in a solvent, carrying out ultrasonic dispersion, standing for 5-10 h, evaporating the solvent at 55-65 ℃, centrifuging 8000-14000 rpm for 20-40 min, taking precipitate, activating in 0.01 mol/L alkali liquor for 8-12h, washing, and adding the solvent to prepare solution A;
s2: and (3) mixing the solution A obtained in the step (S1), tetraethylrhodamine fluorescein and a buffer solution with the pH value of 4-6.5 for reaction to obtain the fluorescent microsphere.
Preferably, the mass ratio of the poly (stearyl maleate), the poly (ethyl methacrylate) and the CdTe/CdSe/ZnS quantum dots is (2-5): (3-7): 2.
Further preferably, the mass ratio of the poly (stearyl maleate), the poly (ethyl methacrylate) and the CdTe/CdSe/ZnS quantum dots is 3:4:2.
Preferably, the nitrocellulose membrane comprises a detection line and a quality control line, wherein the detection line is coated with an anti-cTnI antibody, and the quality control line is coated with an anti-sheep anti-rabbit IgG antibody.
Preferably, the anti-cTnI antibody coupled with the fluorescent microsphere and the anti-rabbit IgG antibody coupled with the fluorescent microsphere are respectively prepared by reacting the anti-cTnI antibody and the anti-rabbit IgG antibody with the quantum dot-RBITC fluorescent microsphere for 1-4 hours under the action of a coupling agent; the fluorescent microsphere-coupled anti-cTnI antibody and the fluorescent microsphere-coupled anti-rabbit IgG antibody are as follows (10-20): (0.5-1.5) and spraying on the release pad in an amount of 2-6 mu L/cm.
Further preferably, the fluorescent microsphere-conjugated anti-cTnI antibody and fluorescent microsphere-conjugated anti-rabbit IgG antibody are prepared according to a ratio of 15:1 and sprayed onto the release pad in an amount of 2-6 μl/cm.
Preferably, the release pad is selected from one of a glass fiber film and a polyester cellulose film.
Preferably, the coupling agent is one or more of carbodiimide hydrochloride, 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide, N-hydroxysuccinimide, suberic acid imine, N-hydroxy inositol, succinic anhydride, and N, N' -dicyano-N, N- (dimethylformamide) guanidine.
On the other hand, the invention also provides a method for detecting troponin I, wherein the troponin I detection kit is used for detecting, 50-100 mu L of a sample to be detected is added into a sample adding hole of a detection card, and the sample is added for 15min to interpret the result.
On the other hand, the invention also provides application of the troponin I detection kit in detection of troponin I.
Compared with the prior art, the invention has the following beneficial effects:
The invention discloses a troponin I detection kit which comprises a detection card, a data disc and a quality control product, wherein the detection card comprises an anti-cTnI antibody, an anti-goat anti-rabbit IgG antibody, a fluorescent microsphere coupled anti-cTnI antibody, a fluorescent microsphere coupled anti-rabbit IgG antibody, a bottom plate, water absorbing paper, a nitrocellulose membrane and a release pad. The fluorescent microsphere is a quantum dot-RBITC fluorescent microsphere, is mainly prepared by wrapping oil-soluble CdTe/CdSe/ZnS quantum dot fluorescent microsphere by a polymer and then reacting with tetraethyl rhodamine fluorescein by an electrostatic effect, and has high fluorescence and light stability; and then respectively coupling with the anti-cTnI antibody and the anti-rabbit IgG antibody to obtain the fluorescent microsphere coupled anti-cTnI antibody and the fluorescent microsphere coupled anti-rabbit IgG antibody. The minimum detection limit of the kit is 0.05ng/mL, the detection sensitivity is high, the linear range is wide, the accuracy is high, and the development of troponin I detection is facilitated.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard graph of the troponin I assay kit of example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Principle of: the polymer is used for wrapping the oil-soluble CdTe/CdSe/ZnS quantum dots to prepare the fluorescent microsphere, the carboxyl group of the fluorescent microsphere is negatively charged, the ethyl group of the tetraethyl rhodamine fluorescein (Rhodamine B isothiocyanate, RBITC) is positively charged, and therefore the quantum dot-RBITC fluorescent microsphere with high fluorescence performance and light stability is obtained through combination of positive and negative charges, and the fluorescence signal intensity is enhanced, and the detection sensitivity is improved.
Example 1
The preparation method of the troponin I detection kit specifically comprises the following steps:
1. preparation of fluorescent microspheres
1. Adding 4 mg oil-soluble CdTe/CdSe/ZnS quantum dots, 6mg of polymaleic anhydride stearyl ester and 8mg poly ethyl methacrylate into 50mL chloroform, performing ultrasonic dispersion, standing for 8 h, performing rotary evaporation on chloroform at 60 ℃, centrifuging 30min at 10000 rpm after the evaporation of chloroform is finished to remove supernatant, activating in 0.01mol/L sodium hydroxide for 10 hours, washing with pure water for three times, dispersing precipitate into 10mL of pure water after washing, and preserving to obtain the quantum dot fluorescent microsphere solution.
2. 1 ML of 1% tetraethyl rhodamine fluorescein aqueous solution, 5mL quantum dot fluorescent microsphere solution and 7 mL HAc-NaAc buffer solution (pH is 5.0) are mixed, and ultrasonic treatment is carried out at room temperature for 25 min, thus obtaining the quantum dot-RBITC fluorescent microsphere.
2. Quantum dot-RBITC fluorescent microsphere coupled antibody
1. 200. Mu.L of quantum dot-RBITC fluorescent microspheres were centrifuged at 12000 rpm for 10. min, the microspheres were allowed to settle and the supernatant removed, 200. Mu.L of phosphate buffer (50 mM pH: 6.0) was added to the pellet and mixed well, and 15. Mu.L of carbodiimide hydrochloride (EDC, 1 mg/mL), 0.1 mg anti-cTnI antibody (or anti-rabbit IgG antibody) were added to 15. Mu.L of hydroxysuccinimide (NHS, 1 mg/mL) and reacted at room temperature for 2 h, centrifuged at 12000 rpm for 20 min, the microspheres were allowed to settle and the supernatant removed.
2. The reaction was continued by adding 100. Mu.L of 1% BSA in water for 30min, centrifuging at 12000 rpm for 20min, allowing the microspheres to settle and removing the supernatant.
3. 200. Mu.L of preservation solution (prepared from 0.5% BSA, 2% glucose, 3% trehalose, tirs-HCl 1% polyethylene glycol PEG-15000, 1% ethanol and the balance water, pH 6.5) was added and mixed.
3. Assembly of immunochromatographic test strips
The immunochromatography test strip comprises a sample pad (SB 06), a PVC bottom plate, absorbent paper, a nitrocellulose membrane and a release pad; the release pad is made of glass fiber film.
1. An anti-cTnI antibody and an anti-goat anti-rabbit IgG antibody are respectively fixed on a nitrocellulose membrane to form a detection region (T line) and a quality control region (C line): the anti-cTnI antibody recognizing a single epitope was diluted to a concentration of 1. 1 mg/mL with a phosphate buffer solution of pH 7.0 containing 2% sucrose and 1% anti-erythrocyte antibody at a concentration of 0.1 mol/L, respectively, and the anti-goat anti-mouse IgG antibody was diluted to a concentration of 2 mg/mL, and both were sprayed on nitrocellulose membrane at an interval of 0.5 cm using a quantitative film spraying apparatus, and dried at room temperature for use.
2. Firstly, soaking a release liner in a pretreatment liquid for 15 min percent, wherein the pretreatment liquid comprises 1 percent Triton-100, 5 percent sucrose, 2 percent sodium citrate, 2.5 percent PEG-20000, 1 percent ethanol and 0.2 percent NaCl; after drying at room temperature, the fluorescent microsphere-coupled anti-cTnI antibody and the fluorescent microsphere-coupled anti-rabbit IgG antibody were mixed according to a ratio of 15:1 and spraying the mixture on a release liner (5 cm X5 cm) in an amount of 4. Mu.L/cm using a quantitative film spraying apparatus, and drying at room temperature in the absence of light.
3. Sequentially sticking absorbent paper, a sample pad, a prepared release pad and a nitrocellulose membrane on a PVC bottom plate, chopping into a test strip with the width of 4mm, loading into a card shell, namely a detection card, and storing in a sealed environment with the humidity not higher than 20%.
Example 2
The preparation method of the troponin I detection kit specifically comprises the following steps:
1. preparation of fluorescent microspheres
1. Adding 4mg oil-soluble CdTe/CdSe/ZnS quantum dots, 10 mg polymaleic anhydride stearyl ester and 6mg polyethyl methacrylate into 50 mL chloroform, performing ultrasonic dispersion, standing for 5h, performing rotary evaporation on chloroform at 65 ℃, centrifuging 40min at 8000 rpm after the chloroform evaporation is finished to remove supernatant, activating 8 h in 0.01 mol/L sodium hydroxide, washing with pure water for three times, dispersing the precipitate in 10 mL pure water after washing, and storing to obtain the quantum dot fluorescent microsphere solution.
2. Mixing 2 mL of 1% tetraethyl rhodamine fluorescein aqueous solution, 7 mL quantum dot fluorescent microsphere solution and 9 mL HAc-NaAc buffer solution (pH is 4.5), and performing ultrasonic treatment at room temperature for 25 min to obtain the quantum dot-RBITC fluorescent microsphere.
2. Quantum dot-RBITC fluorescent microsphere coupled antibody
1. 200. Mu.L of quantum dot-RBITC fluorescent microspheres were centrifuged at 12000 rpm for 10. min, the microspheres were allowed to settle and the supernatant removed, 200. Mu.L of phosphate buffer (50 mM pH: 6.0) was added to the pellet and mixed well, and 15. Mu.L of carbodiimide hydrochloride (EDC, 1 mg/mL), 0.1 mg anti-cTnI antibody (or anti-rabbit IgG antibody) were added to 15. Mu.L of hydroxysuccinimide (NHS, 1 mg/mL) and reacted at room temperature for 2 h, centrifuged at 12000 rpm for 20min, the microspheres were allowed to settle and the supernatant removed.
2. The reaction was continued by adding 100. Mu.L of 1% BSA in water for 30 min, centrifuging 20 at 12000 rpm for min, allowing the microspheres to settle and removing the supernatant.
3. 200. Mu.L of preservation solution (prepared from 0.5% BSA, 2% glucose, 3% trehalose, tirs-HCl 1% polyethylene glycol PEG-15000, 1% ethanol and the balance water, pH 6.5) was added and mixed.
The pretreatment liquid comprises 0.5% Triton-100, 2% sucrose, 3% sodium citrate, 4% PEG-20000, 0.1% ethanol and 0.3% NaCl. Step three of preparation of troponin I assay kit the remaining steps are the same as in example 1.
Example 3
The preparation method of the troponin I detection kit specifically comprises the following steps:
1. preparation of fluorescent microspheres
1. Adding 4 mg oil-soluble CdTe/CdSe/ZnS quantum dots, 4 mg polymaleic anhydride stearyl ester and 14 mg poly ethyl methacrylate into 50 mL chloroform, performing ultrasonic dispersion, standing for 10 h, performing rotary evaporation on chloroform at 55 ℃, centrifuging 20min at 14000 rpm after the evaporation of chloroform is finished to remove supernatant, activating 12 h in 0.01 mol/L sodium hydroxide, washing with pure water for three times, dispersing precipitate in 10 mL pure water after washing, and storing to obtain the quantum dot fluorescent microsphere solution.
2. 1 ML of 1% tetraethyl rhodamine fluorescein aqueous solution, 3mL quantum dot fluorescent microsphere solution and 5mL HAc-NaAc buffer solution (pH is 6.5) are mixed, and ultrasonic treatment is carried out at room temperature for 25 min, thus obtaining the quantum dot-RBITC fluorescent microsphere.
2. Quantum dot-RBITC fluorescent microsphere coupled antibody
1. 200. Mu.L of quantum dot-RBITC fluorescent microspheres were centrifuged at 12000 rpm for 10 min, the microspheres were allowed to settle and the supernatant removed, 200. Mu.L of phosphate buffer (50 mM pH: 6.0) was added to the pellet and mixed well, and 15. Mu.L of carbodiimide hydrochloride (EDC, 1 mg/mL), 0.1 mg anti-cTnI antibody (or anti-rabbit IgG antibody) was added to 15. Mu.L of hydroxysuccinimide (NHS, 1 mg/mL) and reacted at room temperature for 2h, centrifuged at 12000 rpm for 20 min, the microspheres were allowed to settle and the supernatant removed.
2. The reaction was continued by adding 100. Mu.L of 1% BSA in water for 30 min, centrifuging 20 at 12000 rpm for min, allowing the microspheres to settle and removing the supernatant.
3. 200. Mu.L of preservation solution (prepared from 0.5% BSA, 2% glucose, 3% trehalose, tirs-HCl 1% polyethylene glycol PEG-15000, 1% ethanol and the balance water, pH 6.5) was added and mixed.
The pretreatment liquid comprises 2% Triton-100, 7% sucrose, 1% sodium citrate, 1% PEG-20000, 2% ethanol and 0.1% NaCl; step three of preparation of troponin I assay kit the remaining steps are the same as in example 1.
Comparative example 1
Comparative example 1 differs from example 1 in that the fluorescent microspheres were prepared differently, all else being identical.
Preparing fluorescent microspheres:
1. Adding 12 mg oil-soluble CdTe/CdSe/ZnS quantum dots, 3mg polymaleic anhydride stearyl ester and 14mg of polyethyl methacrylate into 5mL chloroform, performing ultrasonic dispersion, standing for 4h, performing rotary evaporation on chloroform at 50 ℃, centrifuging 50 min at 15000 rpm after the chloroform evaporation is finished to remove supernatant, activating 6h in 0.01 mol/L sodium hydroxide, washing with pure water for three times, dispersing the precipitate into 10 mL pure water after washing, and storing to obtain the quantum dot fluorescent microsphere solution.
2. Mixing 2 mL of 1% tetraethyl rhodamine fluorescein aqueous solution, 3mL quantum dot fluorescent microsphere solution and 5mL HAc-NaAc buffer solution (pH is 6.5), and performing ultrasonic treatment at room temperature for 25 min to obtain the quantum dot-RBITC fluorescent microsphere.
Comparative example 2
Comparative example 2 differs from example 1 in that the quantum dots are oil-soluble CdSe/ZnS quantum dots, all of which are identical.
Comparative example 3
Comparative example 3 differs from example 1 in that the quantum dots are water-soluble CdTe/CdSe/ZnS quantum dots, all other things being equal.
Comparative example 4
Comparative example 4 is different from example 1 in that the release pad was not subjected to pretreatment liquid, and the other are the same.
Comparative example 5
Comparative example 5 is different from example 1in that the pretreatment liquid of the release pad is different and the other are the same.
The pretreatment liquid comprises 3% Triton-100, 1% sucrose, 5% PEG-20000 and 0.4% NaCl.
Comparative example 6
Comparative example 6 is different from example 1in that the pretreatment liquid of the release pad is different and the other are the same.
The pretreatment liquid comprises 0.5-2% Triton-100, 1-3% sodium citrate and 0.1-0.3% NaCl.
Example 4
The troponin I antigen was measured using the troponin I detection kit prepared in example 1, and the sample to be measured was serum.
1. Preparation before testing
(1) Starting a detector (a dry fluorescence immunoassay analyzer AFS-1000 type), preheating for 5min, taking out the detection kit and the sample from a low-temperature environment (4 ℃) and recovering to room temperature (20-25 ℃).
(2) After the kit is unsealed, the data disc is taken out and inserted into the detector, the data disc contains standard curve and lot number information, and the information in the data disc is led into the detector.
(3) Under the condition that the information of the data disc is correctly read, the current batch number kit can be used for the data disc in a common mode, and the data discs with different batch numbers cannot be used in a mixed mode.
2. And (3) sucking 80 μl of the sample to be detected by using a pipette with a proper measuring range, and adding the sample to be detected into a sample adding hole of the detection card, wherein obvious bubbles are not required to be generated during sucking and sample adding.
3. And after the sample is added to the detection card for 15min, the detection card is immediately inserted into the detection groove to start detection, the detection card is automatically scanned together, and the detection result is read/printed from the detector after the detection is finished.
4. The test card is removed and disposed of as potentially biohazardous waste.
Test example 1
1. Linear experiments
The cTnI standard was diluted to 0.05, 0.1, 0.5, 1, 5, 10, 20 ng/mL with phosphate buffer containing 5% bovine serum albumin (pH 7.2), and 80 μl was added to the kit of example 1 and incubated 15 min, and 3 replicates were set for each concentration as detected by a dry fluorescent immunoassay, and an average was taken, and a fitted standard curve was established with the cTnI standard concentration as the abscissa and the fluorescent signal intensity test value as the ordinate.
As shown in FIG. 1, the kit has a good linear relationship between 0.05 and 20 ng/mL, and meanwhile, the linear equation fitted by the kit is as follows: y= 275.13 x+382.7, r 2 = 0.9908, no HOOK effect occurs. The minimum detection limit is 0.05 ng/mL.
2. Repeatability experiments
20 Test cards were prepared in examples 1 to 3 and comparative examples 1 to 6, respectively, the experiment was repeated three times, the test was performed by 0.1 ng/mL of serum containing the cTnI standard, the average value and the standard value were calculated from the test results, and the coefficient of variation CV was calculated from the average value and the standard value, and the formula was as follows:
；
The results show that the coefficient of variation CV of comparative examples 1-6 is > 15%, while the kits CV of examples 1-3 are less than or equal to 15%, indicating good intra-and inter-batch reproducibility of the kits of the present invention.
3. Stability test
The kits of examples 1-3 and comparative examples 1-6 were tested for cTnI quality control at concentrations of 0.5 and 10 ng/mL for 3 months, 6 months, 12 months, and 20 months, respectively, and each concentration was repeated 5 times, and the detection results were expressed as mean ± SD. The results showed that the relative deviation of the test results of comparative examples 1-6 exceeded.+ -. 15%, while the relative deviation of the test results of examples 1-3 did not exceed.+ -. 15%, indicating good stability of the kit.
4. Sensitivity experiment
The cTnI standard was diluted to 0.06, 0.15, 0.55, 1.3, 5.2, 10.3, 19 ng/mL with a phosphate buffer solution (pH 7.2) containing 5% bovine serum albumin, and 80. Mu.L was measured using the kits of examples 1 to 3 and comparative examples 1 to 6, respectively, and the measurement values were expressed as a ratio T/C. The experimental results are shown in table 1 below.
Table 1 sensitivity test results table
The results showed that the sensitivity of the kits of examples 1-3 was better than the kits of comparative examples 1-6.
5. Specificity experiments
The kits of examples 1-3 were used to detect 5 ng/mL CK-MB, cTnI, cTnC, myoglobin, tropin I, etc. markers, each of which was repeated three times, the detection results are shown in Table 2 below, and the kits of examples 1-3 were all negative, indicating that the kits of examples 1-3 were excellent in specificity.
Table 2 results of specificity experiments
In the table, "+" indicates that the T line has a fluorescent band; "-" indicates that no fluorescent band appears on the T line.
In conclusion, the kit quantitatively detects the content of troponin I (cTnI) in human serum and plasma samples by using an immunochromatography technology according to the principle of a double-antibody sandwich method. Troponin I (cTnI) in the sample was combined with a fluorescently labeled anti-troponin I (cTnI) antibody to form an immune complex. The immune complex flows upwards from capillary action to react with the anti-cTnI antibody in the test area, and a fluorescent signal is generated in the reaction area after the immune complex is excited by the excitation light source with specific wavelength. The intensity of the signal is in direct proportion to the content of troponin I (cTnI) in the sample, the detection sensitivity is high, the linear range is wide, the accuracy is high, and the development of troponin I detection technology is facilitated.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (7)
1. The troponin I detection kit is characterized by comprising a detection card, wherein the detection card comprises an anti-cTnI antibody, an anti-sheep anti-rabbit IgG antibody, a fluorescent microsphere coupled anti-cTnI antibody, a fluorescent microsphere coupled anti-rabbit IgG antibody, a bottom plate, absorbent paper, a nitrocellulose membrane and a release pad;
The release pad is treated by a pretreatment liquid, and the pretreatment liquid comprises, by mass, 0.5-2% of Triton-100, 2-7% of sucrose, 1-3% of sodium citrate, 1-4% of PEG-20000, 0.1-2% of ethanol and 0.1-0.3% of NaCl;
The fluorescent microsphere is a quantum dot-RBITC fluorescent microsphere, and the preparation of the fluorescent microsphere comprises the following steps:
s1: dissolving polymaleic anhydride stearyl ester, polyethyl methacrylate and oil-soluble CdTe/CdSe/ZnS quantum dots in a solvent, carrying out ultrasonic dispersion, standing for 5-10 h, evaporating the solvent at 55-65 ℃, centrifuging 8000-14000 rpm for 20-40 min, taking precipitate, activating 8-12 h in 0.01 mol/L alkali liquor, washing, and adding the solvent to prepare a solution A;
s2: and (3) mixing the solution A obtained in the step (S1), tetraethylrhodamine fluorescein and a buffer solution with the pH value of 4-6.5 for reaction to obtain the fluorescent microsphere.
2. The troponin I detection kit according to claim 1, wherein the mass ratio of poly (stearyl maleate), poly (ethyl methacrylate), cdTe/CdSe/ZnS quantum dots is (2-5): (3-7): 2.
3. The troponin I detection kit according to claim 2, wherein the mass ratio of poly (stearyl maleate), poly (ethyl methacrylate), cdTe/CdSe/ZnS) quantum dots is 3:4:2.
4. The troponin I detection kit of claim 1, wherein the nitrocellulose membrane comprises a detection line and a quality control line, wherein the detection line is coated with an anti-cTnI antibody, and the quality control line is coated with an anti-sheep anti-rabbit IgG antibody.
5. The troponin I detection kit according to claim 1, wherein the fluorescent microsphere-conjugated anti-cTnI antibody and the fluorescent microsphere-conjugated anti-rabbit IgG antibody are prepared by reacting the anti-cTnI antibody and the anti-rabbit IgG antibody with quantum dot-RBITC fluorescent microspheres for 1-4 hours, respectively, under the action of a coupling agent; the fluorescent microsphere-coupled anti-cTnI antibody and the fluorescent microsphere-coupled anti-rabbit IgG antibody are as follows (10-20): (0.5-1.5) and spraying on the release pad in an amount of 2-6 mu L/cm.
6. The troponin I assay kit according to claim 1, wherein the release liner is selected from one of a glass fiber membrane, a polyester cellulose membrane.
7. The troponin I assay kit of claim 5, wherein the coupling agent is one or more of carbodiimide hydrochloride, 1- [3- (dimethylamino) propyl ] -3-ethylcarbodiimide, N-hydroxysuccinimide, suberic acid imine, N-hydroxystearinositols, succinic anhydride, and N, N' -dicyano-N, N- (dimethylformamide) guanidine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105092852A (en) * | 2015-08-05 | 2015-11-25 | 上海交通大学 | Fluorescence immunoassay test strip used for detecting tumor marker CA72-4 and preparation method of fluorescence immunoassay test strip |
| CN112111268A (en) * | 2020-09-27 | 2020-12-22 | 上海交通大学 | Fluorescent coding microsphere and array and preparation method thereof |
| CN220690951U (en) * | 2023-08-21 | 2024-03-29 | 深圳市迈科龙生物技术有限公司 | Kit for detecting cardiac troponin I |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105092852A (en) * | 2015-08-05 | 2015-11-25 | 上海交通大学 | Fluorescence immunoassay test strip used for detecting tumor marker CA72-4 and preparation method of fluorescence immunoassay test strip |
| CN112111268A (en) * | 2020-09-27 | 2020-12-22 | 上海交通大学 | Fluorescent coding microsphere and array and preparation method thereof |
| CN220690951U (en) * | 2023-08-21 | 2024-03-29 | 深圳市迈科龙生物技术有限公司 | Kit for detecting cardiac troponin I |
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