CN118330235A - Immunochromatography chest pain detection kit capable of rapidly distinguishing acute myocardial infarction from acute aortic dissection, and preparation method and application thereof - Google Patents

Abstract

The invention particularly relates to a multichannel immunochromatography chest pain detection kit for rapidly distinguishing acute myocardial infarction and acute aortic dissection, which consists of three multichannel immunochromatography kits for respectively detecting cardiac troponin (cTnI) and soluble growth stimulation expression gene 2 protein (sST 2), cTnI and heart type fatty acid binding protein (h-FABP), sST2 and high mobility group protein (HMGB 1), and the main components of the kit are four immune probes based on ZrO ₂ @PEI-QDs fluorescent composite microspheres. The invention also discloses four preparation methods of the ZrO ₂ @PEI-QDs fluorescent composite microsphere, which are based on the detection principle of a double-antibody sandwich method. The invention can realize the rapid detection of acute myocardial infarction and acute aortic dissection, has simple and rapid operation, and can be used for distinguishing and monitoring the progress of acute chest pain for the purposes of diagnosis and treatment of non-diseases.

Description

Immunochromatography chest pain detection kit capable of rapidly distinguishing acute myocardial infarction from acute aortic dissection, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to an immunochromatography chest pain kit capable of rapidly distinguishing acute myocardial infarction and acute aortic dissection, and a preparation method and application thereof.

Background

Chest pain is a common cause of medical visits in emergency departments and can be caused by various diseases, wherein Acute Myocardial Infarction (AMI) and Acute Aortic Dissection (AAD) have similar typical symptoms, clinically have the characteristics of acute onset and high death rate, and the death risk of patients is greatly increased due to diagnosis delay and misdiagnosis. Acute myocardial infarction is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery due to acute occlusion of coronary artery, and most patients with acute myocardial infarction have sudden angina, about 10% of patients who visit emergency departments for chest pain are diagnosed as AMI each year, and the acute myocardial infarction is a main cause of death and morbidity of cardiovascular diseases worldwide, and accounts for about 15% of the worldwide deaths (DK Arnett et al, circulation,2019, 140 (11) 596-646). Acute aortic dissection is the most dangerous type of cardiovascular disease, and clinically manifests itself mainly as sudden, severe chest and back pain, with incidence rates of 5 to 30 out of 100 tens of thousands per year (J Golledge et al, THE LANCET,2008, 372 (9632) 55-66); although unusual, the mortality rate is extremely high, with a total mortality rate of between 15% and 25% (A EVANGELISTA et al, circulation,2018, 137, 1846-1860). AMI and AAD have high misdiagnosis rates in clinical diagnosis, and a foreign investigation about hospitalization of AMI patients in emergency department finds that the misdiagnosis rate in emergency department can reach 29% at most (MJ Schull et al ANN EMERG MED, 2006, 48, 647-655), while the probability of clinically correct diagnosis of AAD is only 15% to 43% (U Sampson et al, global Heart,2014,9 (1), 171-180). Especially for AAD patients, the mortality rate of untreated AAD patients in 24 hours is up to 50% with an increase of 1% to 2% per hour (A EVANGELISTA et al, circulation,2018, 137, 1846-1860) (U Sampson et al, global Heart,2014,9 (1), 171-180). Given the similarity of typical symptoms of AMI and ADD, doctors may not be able to accurately diagnose the patient's condition nor perform effective treatment, which not only greatly delays treatment time, but even endangers the patient's life. In clinical diagnosis, imaging examination such as coronary angiography, pulmonary artery CTA, electrocardiogram and the like can be used for diagnosing AMI and AAD, but for some areas with insufficient medical equipment or special working environments, accurate diagnosis and subsequent effective treatment of patients cannot be ensured. Therefore, rapid, effective, accurate diagnosis of AMI and ADD is a critical clinical problem. Besides the method, the diagnosis of the patient can be performed by identifying and quantifying the biomarker, and the correct and rapid diagnosis of the disease condition of the patient can be realized by qualitatively and quantitatively detecting the chest pain specific biomarker.

Cardiac troponin (cardiac troponin, cTn) is a marker of myocardial injury and is responsible for regulating the contraction of cardiomyocytes. Mainly consists of three separately encoded subunits, cardiac troponin T (cardiac troponin T, cTnT), cardiac troponin I (cardiac troponin I, cTnI) and troponin C (troponin C, tnC), playing a specific role in regulating the interaction and cooperation of myosin with key actin and tropomyosin binding domains. The content of cTnI in normal blood of a human body is extremely low, but when ischemia or hypoxia occurs in the body, the integrity of myocardial cell membranes is damaged, the free cTnI can quickly pass through the damaged cell membranes to directly enter the blood circulation, and the concentration of the cTnI is quickly increased; has become the most ideal marker for diagnosing AMI at present. When the AMI patient is ill for 3 to 6 hours, the concentration of the AMI patient can reach 50 ng/mL, and the AMI patient can reach peak 550-ng/mL in 10 to 24 hours; the AMI has the specificity of 97%, the sensitivity of 87% and a longer detection window period (ZP Yuan et al, analysis, 2021, 146, 5474). The concentration of cTnI in normal human serum is very low, about 0 to 0.03 ng/mL; clinically, cTnI concentrations of 0.5 to 2.0 ng/mL are considered as a boundary between normal and AMI patients, and the risk of developing AMI is usually determined with a threshold of 0.5 ng/mL (Lakshmanakumar et al, sci. Rep,2019,9 (1), 1-7). Therefore, detecting the level of cTnI in human serum is of great importance for the correct diagnosis of AMI.

Cardiac fatty acid binding protein (h-FABP) is a relatively small (15 kDa) protein found in the cytoplasm and, due to its small molecular weight, can be released early in myocardial ischemia or infarction. h-FABP has a high degree of cardiac specificity, 80% specificity and 81% sensitivity, and is expressed at a concentration 10 times higher in the heart than skeletal muscle (C Carroll et al, emerg Med J,2013, 30, 280-286). When myocardial ischemia is damaged, myocardial cells mobilize fatty acid for energy, h-FABP can be rapidly released into blood, the h-FABP concentration begins to rise within 1 to 3 hours, and peaks after about 4 hours; it is superior to traditional myocardial markers in early diagnosis (MH Bruins Slot et al, heart,2010, 96, 1957-63). When the concentration of h-FABP in blood exceeds 5.8 ng/mL, the possibility of acute myocardial ischemic injury is considered. The combination diagnosis of cTnI and h-FABP can enhance the sensitivity of AMI detection, and the combination of two biomarkers can show higher AUC (0.881; P < 0.001) and higher combined sensitivity (0.91; P < 0.001) than the cTnI alone, thereby having higher diagnostic value (G Lippi et al Clinical Biochemistry,2013, 46, 26-30).

The growth stimulatory gene 2 protein (growth stimulation expression gene, ST 2) is a member of the interleukin-1 receptor family of cytokines, with a soluble form (soluble growth STimulation expressed gene, sST 2) and a transmembrane form (ST 2L) subtype, and IL-33 is a functional ligand for sST2 and ST 2L. IL-33 is released from cells rapidly when they become damaged or necrotic, and binds to ST2L receptors, forming an IL-33/ST2L signaling pathway. Normally, ST2 is not expressed in cardiac myocytes; however, expression of sST2 and ST2L can be induced when an inflammatory response occurs and the myocardium is damaged. IL-33/ST2L signaling pathway controls cardiomyocyte hypertrophy and fibrosis to protect the heart, while sST2 acts as a decoy receptor, can competitively bind to IL-33, preventing expression of IL-33/ST2L signaling pathway (N Martin et al, nature Immunology,2016, 17 (2), 122-131). The sST2 level is not affected by weight, sex, renal function and the like, and can be used as a novel biomarker index for detecting AAD. Compared to acute myocardial infarction patients, acute phase AAD patients with higher sST2 levels within 24 hours of onset of symptoms (129.2 ng/mL at AAD, 14.7 ng/mL at AMI, P < 0.001); compared to several other cardiovascular diseases, sST2 was most significantly changed in AAD patients (P < 0.001). 34.6 ng/mL can be used as a sST2 threshold diagnostic AAD with a sensitivity of 99.1% and a specificity of 84.9% (F Suzuki et al AMERICAN HEART Association Journal et al, 2018, 137 (4), 270-272). cTnI and sST2 can be used as biomarkers for patient risk stratification, and have important value in rapidly distinguishing AMI from AAD patients.

The high mobility group protein (high-mobility group box, hmgb1) is a highly conserved nucleoprotein, playing a different role in different cellular compartments. HMGB1, a danger-related molecular pattern (DAMP) molecule, can enhance immune responses during tissue injury, also has pro-angiogenic activity, is involved in platelet activation and various cellular functions, including binding to phosphatidylserine and phosphatidylethanolamine, mediating signaling of neuronal growth by RAGE, and possibly helping to extend the accumulation of polyglutamine (polyQ) proteins, which are closely related to the occurrence of body inflammation (B Wang et al, pharmacology & Therapeutics,2019, 196, 160-182). HMGB1 is a key inflammatory factor of myocardial remodeling, has extremely strong pro-inflammatory effect, and the HMGB1 gene is up-regulated in the acute aortic dissection, so that the HMGB1 level in the acute aortic dissection is rapidly increased. The HMGB1 level of the AAD patient is obviously increased (P < 0.05), the HMGB1 level is obviously reduced (P < 0.05) after treatment, the HMGB1 level can reflect the disease condition of the AAD, and the AAD patient has good warning effect clinically (Z Zeng et al REVISTA DA Associa ç ã o M e dica Brasileira,2021, 67 (1), 1251-1255).

Biomarkers can provide information about the health development and disease progression of an individual, playing an important role in early diagnosis, risk assessment and therapeutic effect detection of cardiovascular disease. Compared with the tedious and inconvenient imaging examination and blood component analysis, the lateral flow immunochromatographic test strip detection method is widely applied to the clinical rapid diagnosis field due to simple operation, rapidness and effectiveness.

Disclosure of Invention

Therefore, the invention aims to provide a chest pain detection kit capable of rapidly distinguishing AMI and AAD, and a preparation method and application thereof. The chest pain detection kit takes fluorescent composite microspheres as markers, is respectively coupled with four cTnI, h-FABP, HMGB1 and sST2 monoclonal antibodies, and three multichannel immunochromatography test strips are prepared, so that the rapid diagnosis of AMI and AAD is realized.

The invention provides a chest pain detection kit capable of rapidly distinguishing AMI and ADD, which comprises three multi-channel immunochromatographic test strips (namely a primary screening strip for detecting cTnI and sST2, an AMI strip for detecting cTnI and h-FABP and an AAD strip for detecting HMGB1 and sST 2) which are sequentially adhered to a PVP bottom plate, a nitrocellulose membrane and a water absorption pad; the nitrocellulose membrane is provided with 1 quality control line C and 2 detection T lines, wherein the 2 detection T lines are respectively coated cTnI and coated sST2 (primary screening bars), coated cTnI and coated h-FABP (AMI bars), coated HMGB1 and coated sST2 antibodies (AAD bars); and the quality control line C is coated with goat anti-mouse IgG protein.

The preparation method of the ZrO ₂ @PEI-QDs hollow quantum dot composite microsphere mainly comprises the following steps: firstly, dispersing self-made hollow ZrO ₂ nano microspheres in deionized water; then, a positively charged branched Polyethylenimine (PEI) molecular chain is adsorbed on the surface of the hollow ZrO ₂ by an electrostatic adsorption method; and then adding negatively charged carboxylated CdSe/ZnS quantum dots to form the fluorescent nano composite microsphere with high fluorescence and high stability.

Four immune probes based on ZrO ₂ @PEI-QDs nano-microspheres are prepared by the following steps: taking ZrO ₂ @PEI-QDs composite microsphere of 0.8-0.12 mg, centrifuging with 2- (N-morpholine) -ethanesulfonic acid buffer (MES), cleaning, and dispersing in MES buffer of 1 mL. Adding 5-50 mu L of EDC solution and 10-100 mu L N-hydroxy thiosuccinimide (NHS) into the dispersion liquid respectively, and carrying out ultrasonic reaction at room temperature for 15 min; centrifuging and washing the activated ZrO ₂ @PEI-QDs composite microsphere for 2 times by using an MES buffer solution, finally dispersing in a PBS buffer solution of 1 mL, adding 50-800 mug chest pain marker antibody Ab1, and reacting 0.2-4 h under the oscillation condition to form an amide bond between a carboxyl group activated on the microsphere surface and an amino group on the antibody surface, wherein the amide bond is shown in figure 1; then adding 1.2-3 mL of antibody protein blocking solution, and oscillating to block 0.2-3.0 h; after centrifugation and washing of the obtained immune probe with PBS for 2 times, the immune probe is redispersed in 200 mu L of preservation solution containing 10% of sucrose and 1% of BSA, and the immune probe is preserved at a low temperature until being used.

The immunochromatographic test strip consists of a sample pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate. Two different coating antibodies Ab1 and Ab2 (cTnI and sST2, cTnI and h-FABP, HMGB1 and sST 2) were streaked on nitrocellulose membrane at 0.1-5.0 μl/cm, 0.5-2 mg/mL as T1 line and T2 line using a streaking machine, while 0.5-5.0 mg/mL of goat anti-mouse IgG was streaked on one end of nitrocellulose membrane at 0.5-2.5 μl/cm rate as quality control line C, and streaked NC membrane was dried at 37 ℃ for 24 h. After drying, the sample pad, nitrocellulose membrane and absorbent pad were assembled sequentially with a PVC base plate, and the overlapping length of each part was 1 to 4 mm as shown in fig. 2. The obtained test strip is divided into test strips with the width of 3-5 mm by a strip cutter, and the test strips are put into a plastic packaging card shell, sealed, dried and stored for use.

The invention provides application of the kit in rapid diagnosis and identification of acute myocardial infarction and acute aortic dissection. The invention provides three fluorescence immunochromatography reagent strips for simultaneously detecting cTnI and sST2, or cTnI and h-FABP, or HMGB1 and sST2, wherein 1 quality control line C and 2 detection T lines are arranged on a nitrocellulose membrane; the 2 detection T lines are respectively coated with a cTnI antibody and a sST2 antibody, or a cTnI antibody and an h-FABP antibody, or an HMGB1 antibody and a sST2 antibody; the fluorescent probe takes ZrO ₂ @PEI-QDs NPs as a marking material, respectively marks four antibodies of cTnI, h-FABP, HMGB1 and sST2, has good fluorescence intensity and fluorescence stability, and can greatly improve the detection sensitivity; the target antigen is detected by using a double-antibody sandwich method, so that the qualitative and quantitative detection of two different target antigens can be realized, and the accuracy of diagnosing chest pain diseases can be greatly improved; the test paper box has fewer immune probes, reduces the detection cost, and can greatly improve the detection sensitivity, thereby realizing the rapid and accurate detection and resolution of AMI and AAD. Firstly, the primary screening test strip capable of detecting cTnI and sST2 is used for primarily screening the conditions of chest pain patients (namely, primarily judging myocardial infarction or aortic dissection), then the AMI chest pain patients are further determined by the test strip capable of detecting cTnI and h-FABP through the AMI strip, or the AAD chest pain patients are determined by the test strip capable of detecting sST2 and HMGB1 through the AAD strip, so that the accuracy of acute chest pain blood sample detection is improved.

The chest pain detection kit prepared by the method is used for detecting multiple clinical human serum. In the course of the assay, 20 ‒. Mu.L of patient serum, 5 ‒. Mu.L of immuno-probe dispersion, and 20 ‒ 90. Mu.L of immunochromatographic fluid (PBS solution of 0.1 ‒% Tween 20 to promote chromatographic flow of the serum in the strip) were added to the sample wells of the strips, and each strip was assayed three times. After reaction 10 ‒ 15, 15min, fluorescence photographs of the multichannel immunochromatographic test strips and fluorescence intensity data of the detection lines were recorded. According to the fitted linear relation curve, the signal to noise ratio of the multichannel immunochromatographic test strip is brought, so that the trace concentration of cTnI, h-FABP, HMGB1 or sST2 specific proteins in patient serum is obtained, and further, the accurate and rapid diagnosis and resolution of AMI patients, AAD patients and healthy people can be realized. The kit provided by the invention can be used for non-disease diagnosis and treatment purposes of AMI and AAD, such as research of corresponding medicaments, further research of diagnosis and treatment methods, and the like.

Drawings

FIG. 1 is a schematic diagram of probe preparation.

FIG. 2 is a schematic diagram of a multi-channel immunochromatographic test strip.

FIG. 3 is a diagram of the morphology of ZrO ₂ @PEI microspheres: a) Hollow zirconium dioxide microspheres; b) ZrO ₂ @ PEI composite microsphere.

FIG. 4 is a graph of the fluorescent composite microsphere shape of ZrO ₂ @ PEI-QDs: a) Microsphere low power diagram; b) High resolution transmission electron microscopy.

FIG. 5 is a fluorescence emission spectrum of ZrO ₂ @PEI-QDs composite microspheres.

FIG. 6 shows the specificity of cTnI/sST2 immunochromatographic test strip: a) Fluorescent photographs; b) Fluorescence data.

FIG. 7 shows the specificity of cTnI/h-FABP immunochromatographic test strip: a) Fluorescent photographs; b) Fluorescence data.

FIG. 8 shows the specificity of sST2/HMGB1 immunochromatographic test strip: a) Fluorescent photographs; b) Fluorescence data.

FIG. 9 is a graph of three multi-channel immunochromatographic test strip reproducibility: a) cTnI/sST2 test strip; b) cTnI/h-FABP test strip; c) sST2/HMGB1 test strip.

FIG. 10 is a graph showing the visual detection limit and quantitative analysis of three multi-channel immunochromatographic test strips: a. b) a cTnI/sST2 test strip; c. d) sST2/HMGB1 test strip; e. f) cTnI/h-FABP test strip.

FIG. 11 is a fluorescence photograph and fluorescence data of three different sera detected by three multi-channel immunochromatographic test strips: a) AAD patient; b) AMI patients; c) Healthy people.

FIG. 12 is a schematic diagram of three multi-channel immunochromatographic test strips.

FIG. 13 shows the results of three two-channel immunochromatographic test strips for detecting five different sera.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the following description will be made in detail with reference to the technical solutions of the embodiments of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. It will be apparent that the described embodiments 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.

The features and properties of the present invention are described in further detail with reference to the examples, and the following list of multi-step experimental procedures of the present invention.

The preparation method of the ZrO ₂ @PEI-QDs composite microsphere comprises the following specific steps: 1) The self-made hollow ZrO ₂ nano microsphere is shown in figure 3 a; 2) Dispersing 10 mg hollow ZrO ₂ microspheres in 150 mL deionized water containing 2.5 mL branched PEI, ultrasonically coating 20-60 min at room temperature, washing with deionized water, centrifuging for multiple times to remove excessive PEI, and obtaining ZrO ₂ @PEI, as shown in figure 3 b; 3) Dispersing the obtained ZrO ₂ @PEI in CdSe/ZnS Quantum Dot (QDs) dispersion liquid containing excessive red carboxylation, ultrasonically coating 20-60 min at room temperature, and washing with deionized water for multiple times to obtain ZrO ₂ @PEI-QDs composite microspheres, as shown in figure 4; after electrostatic adsorption of QDs, the fluorescence intensity of ZrO ₂ @ PEI-QDs is greatly improved, as shown in FIG. 5.

Four ZrO₂@PEI-QDs-cTnI、ZrO₂@PEI-QDs-sST2、ZrO₂@PEI-QDs-h-FABP、ZrO₂@PEI-QDs-HMGB1 immunological probes were prepared, specifically as follows: dispersing 1 mg mu L of ZrO ₂ @PEI-QDs composite microsphere in 1 mL MES (10 mM, pH=5.0) buffer solution, sequentially adding 5 mu L of EDC (10 mg/mL) and 10 mu L of NHS (10 mg/mL) solution, and carrying out ultrasonic treatment at room temperature for 15 min; centrifugation was performed 2 times with MES (10 mm, ph=5.5) buffer at 6000 rpm, unreacted EDC and NHS were rapidly removed, and finally dispersed in 1 mL PBS (0.1 m, ph=7.2) buffer; then adding 50 mu L of a monoclonal labeled antibody into the activated fluorescent composite microsphere, and carrying out oscillation reaction at room temperature for 2 h; to block the excess binding sites, a further 1.5 mL% mixture of PBS (0.1 m, ph=7.2) containing 5% BSA and 0.5% PEG was added, after blocking reaction 1h, the collected immunoprobes were centrifuged and washed 2 times with PBS buffer, and the collected immunoprobes were stored in PBS buffer containing 10% sucrose, 1% BSA and 0.1% PVP, and stored at low temperature of 4 ℃ until use.

The immunochromatographic test strip consists of a sample pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate. Two different coating antibodies Ab1 and Ab2 (cTnI and sST2, cTnI and h-FABP, HMGB1 and sST 2) were streaked onto the nitrocellulose membrane as T1 line and T2 line at a streaking rate of 1 μl/cm, 1 mg/mL, and a sheep anti-mouse IgG was streaked onto the other end of the nitrocellulose membrane as quality control line C at the same streaking rate and streaking concentration, followed by drying of the streaked NC membrane at 37 ℃ for 24 h. After the drying is finished, the sample pad, the nitrocellulose membrane and the absorption pad are sequentially assembled by using a PVC base plate, and the overlapping length of the tail ends of each part is 2 mm. The obtained test strip is divided into test strips with the width of 3.5 mm by a strip cutting machine, and the test strips are put into a plastic packaging card shell, sealed, dried and stored for use.

Three multi-channel immunochromatography test strips are constructed by taking ZrO ₂ @PEI-QDs fluorescent composite microspheres as fluorescent marking materials. The specific operation is as follows: uniformly mixing a sample to be detected containing a marker antigen, a 5 mu L immune probe and 85 mu L immune chromatography liquid, wherein the immune probe can be rapidly combined with a target antigen specifically to form a binary immune complex; then dripping the mixture into a sample hole of a test strip, and forming a ternary immune complex by combining the binary immune complex with the coated antibody on the detection line in the process that the mixed liquid flows from the sample pad to the absorption pad under the action of capillary force when the binary immune complex passes through the corresponding detection T line. Under 365 nm ultraviolet light, a bright red strip appears on the corresponding detection T line; along with the increase of the concentration of the target antigen, the fluorescence signal of the corresponding T line is gradually enhanced; the goat anti-mouse IgG of the quality control C line can directly perform immune reaction with the labeled antibody, and no matter whether the target antigen exists in the sample to be detected, the C line can have bright red stripes, so that the effectiveness of the immunochromatography test strip can be judged. Finally, the fluorescent intensity of the T line and the C line can be measured by a dry fluorescent immunoassay analyzer to obtain the fluorescent intensity ratio of the T line and the C line, and the linear correlation relation between the antigen concentration and the fluorescent intensity ratio is realized according to the fluorescent intensity ratio, so that quantitative analysis is performed.

To evaluate the specificity of three cTnI/sST, cTnI/h-FABP, sST2/HMGB1 multichannel immunochromatographic test strips, we used different cardiac markers to detect them, including cTnI, h-FABP, sST2, HMGB1 and CRP antigens. The concentration of the target antigen is 100 ng/mL, and the concentration of the interference antigen is 1 mug/mL. In the process of evaluating the specificity of three immunochromatographic test strips, for a sample liquid containing target antigens, three other irrelevant antigens are sequentially added, and the influence of other interference antigens on the immunochromatographic test strip is deeply explored. As shown in FIGS. 6-8, the fluorescence photographs and fluorescence intensity data statistics of three multi-channel immunochromatographic test strips under ultraviolet irradiation can show that 15 immunochromatographic test strips all have bright red strips on the C line, and the effectiveness of the immunochromatographic test strips is proved. For three multi-channel immunochromatographic test strips respectively provided with cTnI, sST, cTnI, h-FABP, sST2 and HMGB1 antibodies, when two target antigens exist simultaneously, bright red strips appear on two T lines, and the fluorescence intensity is high; when only one of the target antigens exists, the corresponding T line also has bright red stripes, and the other T line has no obvious change, which indicates that a plurality of proteins have no obvious cross reaction and have better specificity; when the tested liquid does not contain the target antigen, no bright red strip is generated on two T lines of the multichannel immunochromatographic test strip. These results indicate that the three dual-channel immunochromatographic test strips established based on ZrO ₂ @PEI-QDs NPs have good specificity.

To evaluate the specificity of three multichannel immunochromatographic test strips containing cTnI and sST, cTnI and h-FABP, sST2 and HMGB1 respectively, we prepared a mixed solution containing 100 ng/mL of cTnI, sST2, h-FABP and HMGB1 antigens as the test solution, and the test strip was repeated for 5 times. The detection results are shown in fig. 9, and bright red bands appear on the C line and the two T lines of the three double-channel immunochromatographic test strips. The fluorescence intensity analysis is carried out by the dry type fluorescence immunochromatography test strip, and the fluorescence intensities of the C line, the T1 line and the T2 line of the 5 multichannel immunochromatography test strips are basically similar, and the variation coefficient in batches is within 15%. These results show that three multi-channel immunochromatographic test strips established based on ZrO ₂ @PEI-QDs fluorescent composite microspheres have good repeatability.

Through detecting a series of cTnI and sST2, cTnI and h-FABP with different concentration gradients, the concentration of the cTnI and sST2 is respectively 100/100, 75/75, 50/50, 25/25, 10/10, 1/1, 0.1/0.1, 0.075/0.075 and 0.05/0.05 ng/mL, and the control group is a negative sample; the concentration of cTnI and h-FABP is 100/100, 75/75, 50/50, 25/25, 10/10, 1/1, 0.1/0.1, 0.05/0.05 ng/mL, and the control group is a negative sample; the concentrations of sST2 and HMGB1 are 100/100, 75/75, 50/50, 25/25, 10/10, 1/1, 0.1/0.1, 0.05/0.05 ng/mL, respectively, and the control group is a negative sample, and the detection results are shown in FIG. 10. As can be seen from fig. 10, the quality control C line of all immunochromatographic test strips had clear red fluorescence bands, indicating that the detection of these test strips was effective; meanwhile, the fluorescent colors of the two T detection lines and the corresponding fluorescent signal intensities are gradually weakened along with the reduction of the concentration of the target antigen. The results of FIG. 10a show that for the cTnI and sST2 dual-channel immunochromatographic test strip, when the cTnI antigen concentration was as low as 0.1 ng/mL and the sST2 antigen concentration was as low as 0.075 ng/mL, it was almost difficult to visually observe a fluorescent band on the T line. Therefore, we determined that the visual detection limits of cTnI and sST2 in the multichannel immunochromatographic primary screening test strips for cTnI and sST2 were 0.1 ng/mL and 0.075 ng/mL, respectively. For the cTnI and h-FABP multichannel immunochromatography ANI test strip, when the concentration of the cTnI antigen is as low as 0.1 ng/mL and the concentration of the h-FABP antigen is as low as 1.1 ng/mL, fluorescent bands are hardly observed on a T line with naked eyes; therefore, the visual detection limits of the cTnI and the h-FABP in the cTnI and h-FABP multichannel immunochromatographic test strip are respectively 0.1 ng/mL and 1 ng/mL. For the sST2 and HMGB1 dual-channel immunochromatography AAD test strip, when the sST2 antigen concentration is as low as 0.05 ng/mL and the HMGB1 antigen concentration is as low as 1 ng/mL, the fluorescent band is hardly observed on the T line with naked eyes. Therefore, we determine that the visual detection limits of sST2 and HMGB1 in the sST2/HMGB1 double-channel immunochromatographic test strip are 0.05 ng/mL and 1 ng/mL respectively.

The clinical reliability of three multichannel immunochromatographic test strips was investigated by testing five different serum samples, including 1 AAD patient, 1 AMI patient and three healthy human serum. The detection results are shown in table 1, and compared with the serum of a healthy person, the concentration of cTnI and the concentration of h-FABP of an AMI patient are obviously increased, and the concentrations of the cTnI in the cTnI/sST2 and the concentration of the cTnI/h-FABP in the two multi-channel immunochromatographic test strips are approximately the same; for AAD patients, it can be seen from Table 1 that the concentrations of sST2 and HMGB1 increased significantly, and that the sST2 concentration exceeded the critical value of 34.6 ng/mL for the judged AAD patients, where the concentration of sST2 was essentially the same in the cTnI/sST2 and sST2/HMGB1 multi-channel immunochromatographic test strips. For three healthy human serum, the concentrations of cTnI, h-FABP, sST2 and HMGB1 were all within the normal range. Meanwhile, the test results of the test strips of the AMI patient, the AAD patient and a healthy person are selected for photographing and data statistics, and the test results correspond to the test results shown in the table 1 as shown in fig. 11. The results show that the chest pain detection kit prepared by the method can realize the accurate and rapid diagnosis and resolution of AMI patients, AAD patients and healthy people, and has good clinical application prospect.

The kit provided by the invention can be used for non-disease diagnosis and treatment purposes of AMI and AAD, such as research of corresponding medicaments, further research of diagnosis and treatment methods, and the like.

Claims (10)

1. An immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection is characterized in that the kit takes ZrO ₂ @PEI-QDs fluorescent composite nano-microspheres as markers, four cTnI, h-FABP, HMGB1 and sST2 monoclonal antibodies are respectively coupled, and the rapid detection of AMI and AAD is realized through three multi-channel immunochromatography test strips; preferably, the kit is formed by carrying out surface modification on the ZrO ₂ @PEI-QDs fluorescent composite nano-microsphere; further preferably, the kit consists of three multi-channel immunochromatographic test strips, which are respectively: primary screening strips useful for detecting cardiac troponin (cTnI) and soluble growth stimulation expressed gene 2 protein (sST 2), AMI strips useful for detecting cTnI and cardiac fatty acid binding protein (h-FABP), AAD strips useful for detecting sST2 and high mobility group protein (HMGB 1); further preferably, the primary screening strip, the AMI strip and the AAD strip are sequentially adhered to a sample pad, a nitrocellulose membrane and a water absorption pad on the PVP bottom plate; the nitrocellulose membrane is provided with 1 quality control line C and 2 detection T lines, wherein the 2 detection T lines are respectively coated cTnI and coated sST2 (primary screening bars), coated cTnI and coated h-FABP (AMI bars), coated HMGB1 and coated sST2 antibodies (AAD bars); and the quality control line C is coated with goat anti-mouse IgG protein.

2. The method for preparing the immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 1, comprising the following steps:

(1) Preparing the fluorescent composite nano microsphere;

(2) Preparing the four fluorescent immune probes;

(3) Three multi-channel immunochromatography test strips are prepared; preferably, the immunochromatographic test strip consists of a sample pad, a nitrocellulose membrane, a water absorption pad and a PVC bottom plate; more preferably the specific operation is: two different coating antibodies Ab1 and Ab2 (cTnI and sST2, cTnI and h-FABP, HMGB1 and sST 2) are marked on a nitrocellulose membrane with the concentration of 0.1-5.0 [ mu ] L/cm and the concentration of 0.5-2 mg/mL to be used as a T1 line and a T2 line by using a marking machine, sheep anti-mouse IgG is marked on one end of the nitrocellulose membrane with the concentration of 0.5-2.5 [ mu ] L/cm and the concentration of 0.5-5.0 mg/mL to be used as a quality control line C, and the marked NC membrane is dried at 37 ℃ for 24 h; after the drying is finished, the sample pad, the nitrocellulose membrane and the absorption pad are sequentially assembled by using a PVC base plate, the overlapping length of the tail end of each part is 1-4 mm, the obtained test strip is divided into test strips with the width of 3-5 mm by using a strip cutting machine, and the test strips are put into a plastic packaging card shell, sealed, dried and stored for use.

3. The preparation method of the immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 2 is characterized by comprising the following steps of:

1) Adding branched Polyethylenimine (PEI) into deionized water dispersion liquid of hollow nano ZrO ₂ microsphere; then, under the condition of ultrasonic oscillation or an oscillation table, the branched PEI molecular chains are coated on the inner and outer surfaces of the hollow nano ZrO ₂ microsphere electrostatically; then, centrifuging, separating, washing the coated product with deionized water, and preparing into ZrO ₂ @PEI composite nano microsphere dispersion liquid with the concentration of 1-10 mg/mL and the surface having positive charges;

2) Dispersing ZrO ₂ @PEI composite nano-microspheres with positive charges on the surfaces in deionized water containing CdSe/ZnS or CdTe/ZnS Quantum Dots (QDs) with carboxylated surfaces, and carrying out electrostatic coating to obtain QDs-coated fluorescent composite nano-microspheres (ZrO ₂ @PEI-QDs); and washing with deionized water for 3-5 times, dispersing in a small amount of deionized water, and preserving at low temperature of 2-8deg.C for use.

4. The method for preparing an immunochromatographic chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 3, wherein in the step 1) of preparing the ZrO ₂ @PEI-QDs fluorescent composite nano-microsphere, the relative weight average molecular weight of branched PEI is 0.4-200 kDa.

5. The method for preparing the immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction from acute aortic dissection according to claim 2, wherein the four fluorescent immuno-probes are prepared by the following steps:

1) Adding N-hydroxy thiosuccinimide (or N-hydroxy succinimide) and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide (EDC) into 4-morpholinoethanesulfonic acid (MES) dispersion liquid of ZrO ₂ @PEI-QDs fluorescent composite nano-spheres to perform an activation reaction; then centrifugally separating out a reaction product, washing the reaction product for 3 times by using 2- (N-morpholine) -ethane sulfonic acid buffer solution (MES), and dispersing the reaction product in Phosphate Buffer Solution (PBS) to obtain EDC activated ZrO ₂ @PEI-QD fluorescent composite nano microsphere dispersion liquid, wherein the concentration of the dispersion liquid is 0.1-10 mg/mL;

2) Four different chest pain marker antibodies (namely cTnI, h-FABP, HMGB1 and sST2 monoclonal antibodies) are respectively added into PBS dispersion liquid of EDC activated ZrO ₂ @PEI-QD fluorescent composite nano-microspheres for coupling reaction, so that four fluorescent immune probe microspheres are obtained;

3) Adding a blocking solution to terminate the coupling reaction, wherein the blocking reaction time is 10-180 min;

4) After washing for 3-5 times by PBS buffer solution, the four prepared fluorescent immune probes are respectively redispersed by antibody protection solution, and then are preserved at a low temperature of 2-8 ℃ for standby.

6. The method for preparing an immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 5, wherein in the step 1) of the preparation method of four fluorescence immunological probes, the concentration of the fluorescent quantum dot hollow nanoparticle dispersion liquid is 0.1-10 mg/mL, the concentration of added N-hydroxysulfosuccinimide (or N-hydroxysuccinimide) is 0.1-50 mg/mL, the concentration of added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 0.1-50 mg/mL, the volume of added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 2-500 mL, the volume ratio of the two added reactant solutions is 1:1-5, and the activation reaction time is 1-60 min.

7. The method for preparing an immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 6, wherein in the preparation step 2) of four fluorescent immune probes, EDC activated ZrO ₂ @PEI-QD fluorescent composite nano microsphere dispersion liquid is diluted by 2-10 times, and chest pain marker antibody with the concentration of 1-200 mg/mL is added for connection reaction, wherein the reaction time is 5-60 min.

8. The method for preparing an immunochromatography chest pain detection kit capable of simultaneously and rapidly detecting acute myocardial infarction and acute aortic dissection according to claim 7, wherein in the step 3) of the preparation method of four fluorescent immuno probes, the blocking solution can be calf serum with the concentration of 0.1-50 mg/mL, fetal bovine serum albumin with the concentration of 0.1-50 mg/mL, polyvinylpyrrolidone with the concentration of 0.1-10 mg/mL, PBS buffer with the concentration of 0.1-1 mg/mL polyethylene glycol with the molecular weight of 20000 and PBS buffer with the concentration of 0.1-50 mg/mL bovine serum albumin with the added blocking solution with the volume of 0.5-5 mL.

9. The immunochromatography chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection obtained by the preparation method of claim 1 or any one of claims 2 to 8, which can be used for the diagnosis and treatment of non-diseases and the application of the kit for detecting acute myocardial infarction and acute aortic dissection, and is characterized in that the detection method is as follows:

1) The prepared four fluorescent immune probes are respectively applied to three multi-channel immunochromatographic test strips of a primary screening strip (for detecting cTnI and sST), an AMI strip (for detecting cTnI and h-FABP) and an AAD strip (for detecting sST2 and HMGB 1), and different antigen concentrations are detected within the range of 100-0.05 ng/mL, so that six linear fitting curves of the fluorescent intensities corresponding to the three detection strips and the corresponding protein concentrations are obtained;

2) Detecting patient serum, including serum of AAD patient, AMI patient and healthy person, recording fluorescence photo and fluorescence intensity data thereof, and obtaining the concentrations of four chest pain marker antigens detected in three multichannel immunochromatography test strips according to the linear fitting curves of the six fluorescence intensities and the corresponding chest pain marker protein concentrations, wherein each test strip is detected three times, thereby determining chest pain symptoms of a serum provider.

10. The immunochromatographic chest pain detection kit capable of simultaneously and rapidly distinguishing acute myocardial infarction and acute aortic dissection according to claim 9, which can be used for the detection of acute myocardial infarction and acute aortic dissection for non-disease diagnosis and treatment purposes, wherein in step 2) of the detection method, 20 ‒ mu L of patient serum, 5 ‒ 40 mu L of immune probe dispersion liquid and 20 ‒ mu L of immune chromatographic liquid (PBS solution of 0.1 ‒ 10 wt% Tween 20) are added to promote chromatographic flow of blood serum in a test strip; and meanwhile, the trace concentrations of cTnI, h-FABP, HMGB1 and sST2 specific proteins in serum are detected according to six linear fitting curves of the fluorescence intensity of the test strip and the corresponding protein concentrations, so that the accurate and rapid symptom determination and resolution of AMI patients, AAD patients and healthy people can be realized.