CN111965366A - Method and kit for rapidly detecting exosome troponin by saliva - Google Patents
Method and kit for rapidly detecting exosome troponin by saliva Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
Abstract
The invention provides immunochromatographic test paper for rapidly detecting exosome troponin by saliva, which comprises a sample pad area, an antibody area, a microfluidic paper chip area, a gold-labeled pad area, a cellulose nitrate membrane area and a water absorption pad area, wherein the sample pad area, the antibody area, the microfluidic paper chip area, the gold-labeled pad area, the cellulose nitrate membrane area and the water absorption pad area are sequentially connected. The immunochromatographic test paper can quickly detect troponin in saliva secretion, and can judge whether acute myocardial infarction exists through the test paper without depending on instruments and equipment. The test paper can distinguish the troponin contents of 15mg/mL, 50mg/mL and 100mg/mL in people at the same time of diagnosis, and can accurately judge high risk people. The invention not only shortens the detection time, but also improves the detection precision, and realizes mobile detection and home self-service detection.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method and a kit for rapidly detecting exosome troponin by saliva.
Background
Cardiovascular disease (CVD) is the "first killer" of global health, leading to 30% of deaths each year (1730 million people in 2008), and is expected to increase further by 2030 to 2330 million people. Cardiovascular disease is 80% of the coronary atherosclerotic heart disease (coronary heart disease), and acute myocardial infarction is one of the most severe of coronary heart diseases. Acute Myocardial Infarction (AMI) is a life-threatening acute disease caused by acute obstruction of coronary artery and continuous ischemia and hypoxia of heart, which leads to the damage of heart function, and belongs to the category of acute coronary syndrome. The Chinese cardiovascular disease report 2018 indicates that the prevalence and mortality of the cardiovascular diseases are still in the rising trend and are younger, at present, the number of patients suffering from the cardiovascular diseases in China is 2.9 hundred million, the mortality is the first place, the death rate accounts for more than 40% of the death rate of the cardiovascular diseases, and one person dies from acute myocardial infarction every 37 seconds. The coronary heart disease death rate of residents in cities and rural areas in China keeps on rising trend since 2012, and the coronary heart disease death rate in rural areas has obvious rising trend. From 2009, the mortality rate of cardiovascular diseases in rural areas has continued to be higher than at the urban level. In 2016, the mortality rate of cardiovascular diseases in rural areas is 309.33/10 ten thousand, the mortality rate of cardiovascular diseases in urban areas is 265.11/10 ten thousand, and the mortality rates of cardiovascular diseases in rural areas and urban areas account for 45.5 percent and 43.16 percent of all death causes respectively.
According to the World Health Organization (WHO) standard: two indexes of three indexes of typical chest pain, electrocardiogram change and cardiac troponin abnormity are met, and AMI can be diagnosed. The clinical manifestations of AMI include angina pectoris and painless, the asymptomatic condition of AMI patients in China accounts for about 25% of the population suffering from general diseases, the cases without typical angina pectoris accounts for about 30%, the clinical diagnosis is generally carried out by adopting an electrocardiogram, and the accuracy of AMI diagnosis by the electrocardiogram is about 60%. Pathological Q waves with definite diagnosis significance often appear after 6-8 hours of disease attack, but the conventional electrocardiogram lacks specificity for diagnosing non-Q-wave AMI, unstable angina and small-range AMI are difficult to identify, and atypical early AMI cannot be used for definite diagnosis by relying on the electrocardiogram; 1-6 hours after the AMI is taken as the gold time for thrombolytic therapy and interventional operation therapy, so that the rapid and accurate diagnosis of the AMI in the early stage within 6 hours of the onset of the AMI is a key link for determining the therapy. The detection of biochemical markers of myocardial injury plays an important role in diagnosis, monitoring, risk assessment, prognosis, treatment guidance and the like of acute myocardial infarction. The existing common methods for detecting troponin (cTnI or CTNI) comprise a chemiluminescence method, a rapid detection method (POCT) and the like, wherein blood samples are adopted, and traumatic examination is carried out. Therefore, at present, a diagnosis means which is convenient for collecting samples and has high specificity and good sensitivity is urgently needed, and has important clinical significance for the early diagnosis of AMI.
New studies by foreign Miller scholars show that: the heart disease can be detected by saliva, the accuracy of AMI detection by combining the saliva test and electrocardiogram detection is up to 100%, and the AMI accuracy of saliva determination is up to 95% (C.S. Miller, et al.JDR clinical Research Supplement,2014,93(7): 72-79). Although the electrocardiogram is one of the best methods for diagnosing AMI at present, the accuracy of AMI determination from the electrocardiogram is only 60%, and accurate detection and diagnosis of AMI is an important aspect to avoid its harm. The Mohd Aizart et al study reported that saliva measures the clinical value of AMI, when a person suffers from ischemic heart disease, myocardial muscle tissue becomes hypoxic and produces cardiac enzymes, including troponins, which are first transported to salivary glands by circulating in the body (Mohd Aizart Abdul Rahim, et al, int.J.Med.Sci.2015,12(4): 329-. Therefore, rapid saliva detection of troponin can provide AMI patients with valuable time savings and gold time to save lives.
Exosomes (exosomes) can be used as markers of cardiovascular diseases, have the diameter of about 30-150nm and the density of 1.13-1.21g/m1, are membrane vesicles actively secreted by cells, and are formed through a series of regulation processes such as endocytosis, fusion and efflux. In recent years, a large number of domestic and foreign researches report that exosomes represent the physiological and pathological states of source cells and can be used as biomarkers for early diagnosis of diseases. Exosomes are naturally present in body fluids, including blood, saliva, urine, ascites, and breast milk. The exosomes carry unique or key functional molecules of the source cells, resulting in exosomes of different sources with different biological functions. Therefore, it is possible to measure changes in certain proteins and nucleic acids in certain cells based on the detection of substances contained in exosomes. At present, many diseases including cardiovascular diseases, tumors and the like can be detected from exosomes in serum or plasma of a patient, protein content in a sample is researched through a certain experiment and data analysis method, early diagnosis is realized by comparing sample data of a normal individual, and important basis is provided for curative effect judgment and prognosis.
Currently, differential centrifugation is considered the gold standard for exosome separation, but has some drawbacks. The method is time-consuming, labor-consuming, highly dependent on manpower, low in recovery rate, prone to damage to vesicles of exosomes due to repeated centrifugation operation, low in quality, prone to cause pollution due to agglomeration and unfavorable for downstream analysis. Current methods of exosome extraction and enrichment using immunomagnetic beads, ultrafiltration, precipitation or kits have limitations because each cell secretes at least 10 different types of nanovesicles, which are difficult or impossible to distinguish based on similar size, density and surface markers. Therefore, at present, scholars at home and abroad actively explore an experimental method capable of quickly and efficiently separating exosomes for early diagnosis of acute myocardial infarction.
201811060763, which is disclosed by X, is an in-vitro instant detection platform based on exosomes and a detection method thereof, and the platform is used for semi-quantitatively detecting specific markers of tumors by separating and extracting exosomes through an air filter membrane.
At present, peripheral blood samples are collected by using chemiluminescence technology, enzyme-linked immunosorbent assay, flight mass spectrometry and colloidal gold immunochromatography for detecting troponin markers applied to hospitals of all levels (major, middle, minor and community hospitals), and relevant reports of AMI specific markers for quickly separating, extracting and detecting the salivary exosome troponin are not reported in China.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides the immunity layer system test paper and the kit for rapidly detecting the troponin of the saliva exosomes based on the test paper of the galactose agglutinin-microfluidic chip (paper chip) for rapidly separating, purifying, extracting the exosomes and quantitatively detecting the troponin.
In order to achieve the purpose, the invention adopts the following technical scheme:
the utility model provides an immunochromatography test paper of saliva short-term test exosome troponin, includes that sample fills up district, antibody district, micro-fluidic paper chip district, gold mark fills up district, nitrocellulose membrane district, absorbs water and fills up the district, wherein:
sample pad area: for dripping saliva or soaking saliva. Preferably, the saliva sample is subjected to saliva sample pretreatment liquid to remove abundant proteins and mucins in the saliva.
Antibody region: the antibody region is coated with a galectin 3 antibody for the first enrichment and isolation of saliva exosomes, and the coated galectin 3 antibody region is connected with the sample pad region. The galectin 3 antibody coated on the antibody region is enriched with a glycoprotein (galectin 3 antigen) on saliva exosomes and saliva exosomes are isolated.
A chip area: the chip area is provided with a micro-fluidic paper chip for further separation and purification and obtaining the saliva exosome with the size less than 200-250nm, and the micro-fluidic paper chip area is connected with the coated galectin 3 antibody area.
Gold mark pad area: the gold-labeled pad area is provided with a troponin antibody marked by colloidal gold particles, the troponin antibody marked by the colloidal gold particles is combined with a troponin antigen on exosomes in saliva, and the gold-labeled pad area is connected with the microfluidic paper chip area.
Cellulose nitrate membrane zone: a detection T line and a quality control C line are arranged on the nitrocellulose membrane area, and the multiple groups of T lines are used for detecting the troponin content on exosomes in saliva; and the line C is used as a control line, and the nitrocellulose membrane pasting area is connected with the gold-labeled pad area.
Preferably, the microfluidic paper chip (referred to as paper chip) uses a high molecular polymer material: including Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), polystyrene, filter paper.
Preferably, the microfluidic paper chip is provided with a hydrophilic/hydrophobic micro-channel network and is connected with the chromatographic paper.
Preferably, the microfluidic paper chip is provided with a hydrophilic/hydrophobic micro-channel network and is connected with the chromatographic paper. A round hole with the diameter of 200-250nm is designed in the middle of the microfluidic paper chip, an exosome is extracted and purified, and protein and impurities with the diameter of more than 200-250nm are separated. Meanwhile, a nitrocellulose filter membrane is selected as a substrate of the paper chip.
Preferably, the particles in the antibody labeled with particles in the gold-labeled pad region are any one of colloidal gold bodies, nanogold or fluorescent materials.
Preferably, the streaked antibody of the T-line is a double anti-sandwich antibody; the streak antibody of the C line is marked goat anti-rabbit antibody.
Preferably, three groups of T lines (71, 72, 73) are arranged on the T lines, and the content of troponin on exosomes in saliva is detected to be 15-100 mg/mL.
Preferably, the troponin content on exosomes in saliva measured on the T-line (71) is 15 mg/mL; the content of troponin on exosomes in saliva measured on the T line (72) is 50 mg/mL; the troponin content on exosomes in saliva measured on the T-line (73) was 100 mg/mL.
The invention has the beneficial effects that: the immunochromatographic test paper can quickly detect troponin in saliva secretion, and can judge whether acute myocardial infarction exists through the test paper without depending on instruments and equipment. The test paper can distinguish the troponin contents of 15mg/mL, 50mg/mL and 100mg/mL in people at the same time of diagnosis, and can accurately judge high risk people. The invention not only shortens the detection time, but also improves the detection precision, and realizes mobile detection and home self-service detection.
Drawings
Fig. 1 is a front view of a test strip for detecting troponin of saliva exosomes according to an embodiment of the present invention.
Fig. 2 is a top view of a test strip for detecting troponin of saliva exosomes according to an embodiment of the present invention. Wherein: 1. sample pad area, 2. coating galactose agglutinin 3 area, 3. microfluidic paper chip area, 4. gold mark pad area, 5. nitrocellulose membrane area, 6. water absorption pad area, 7.T line area, first T line: 7.1, second T line: 7.2, third T line: line 7.3, 8.C region.
FIG. 3 shows specificity and sensitivity of troponin of saliva exosomes detected by a test paper provided in the embodiment of the present invention.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the technical solutions of the present invention is provided with reference to specific embodiments and accompanying drawings.
Example 1
Preparing a saliva sample pretreatment liquid, which comprises the following specific contents: 0.2422g of 0.01-0.2 mol/L Tris is dissolved in 100ml of distilled water; then 0.149g of 0.4% EDTA and 0.16g of SDS are added, 10-100 mg of cross-linked polyvinylpyrrolidone (USNF), 1% -5% BSA and HCl are added to adjust the pH value to 7.0-8.0. Namely the saliva sample pretreatment liquid.
Saliva is rich in various proteins such as alpha-amylase, mucin, histatin, immunoglobulin, cystatin, cells, etc., because saliva contains 99% of water, and therefore, it is necessary to treat saliva with a saliva sample pretreatment liquid. The method removes high-abundance proteins and mucins of saliva, reduces the aggregation of related proteins and the adhesion of saliva exosomes, and solves the problem that saliva samples are viscous and contain various interference substances and other hindering factors, thereby greatly improving the extraction efficiency of exosomes.
Collecting 1-2 mL of saliva and placing the saliva into a 2mL sealed plastic tube, wherein the tube contains saliva sample pretreatment liquid, after fully shaking up, dropwise adding (2 drops or 3 drops) onto a test strip by using a straw or soaking the test strip into the saliva of the plastic tube, and after 5-10, observing that a red detection result appears on the test strip.
Example 2
The embodiment provides a troponin test paper for detecting saliva exosomes and a preparation method thereof, and the test paper comprises the following components:
1. the sample pad (1) is glass fiber with the thickness of 2.5cm multiplied by 0.4cm and comprises a galactose agglutinin 3 area (2) and a microfluidic paper chip (3) area;
2. the gold label pad (4) is a 0.4cm multiplied by 0.4cm polyester film containing the troponin monoclonal antibody marked by the colloidal gold;
3. the size of the cellulose nitrate membrane (5) is 2.5cm multiplied by 0.4cm, troponin monoclonal antibody and goat anti-mouse IgG are sequentially coated, the distance between the two is 4mm, and a detection line T1(7.1), a detection line T2(7.2), a detection line T3(7.3) and a quality control line C (8) are respectively formed;
4. the water absorption pad (6) is water absorption filter paper with the thickness of 2.7cm multiplied by 0.4 cm;
the following steps are organized in sequence from 1 to 7: the sample pad (1,2,3) is a sample adding area, the gold label pad (4) is a troponin monoclonal antibody area, and the nitrocellulose membrane is a reaction area (5,7,8), so that a troponin detection test paper strip and a colloidal gold immunochromatography method are formed.
The preparation method comprises the following steps:
(1) sample pad preparation and coating with galectin 3 antibody:
the preparation method of the sample pad and the coated galectin 3 antibody zone comprises the following steps: spraying 100 μ l of the galectin 3 antibody on glass fiber of 1.5cm × 0.4cm, and drying at 37 deg.C for 8-24 hr to obtain a sample pad and a galectin 3 antibody coating region. Sealing the aluminum foil bag, and storing at room temperature for later use.
The first step is to enrich and isolate saliva exosomes after the saliva sample is bound to the coated galectin 3 antibody.
(2) Making microfluidic paper chip area
Micro-fluidic paper chip structure: the micro-fluidic chip is a square paper chip 5cm multiplied by 5cm and is tightly attached to the sample pad, the middle hole is 200-250nm, and the micro-fluidic paper chip is made of high molecular polymer materials: including Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), polystyrene, filter paper, high polymer, etc.
Because the flow channel pattern area is still the filter paper, the main component cellulose of the paper enables the solution to flow through the capillary action, and the flow channel pattern area is a hydrophilic area and does not need an external driving pump, the detected solution can flow in the hydrophilic area of the filter paper; the areas outside the flow channel patterns are coated with glue and then form a cross-linked network through exposure, the cross-linked network is fixed on filter paper to form a hydrophobic area, and the solution cannot infiltrate and flow, so that the solution flows only along the designed flow channel areas.
The microfluidic paper chip used in the invention is donated by hong Kong City university. Commercial microfluidic paper chips are also sold in the market. Microfluidic paper chips can be purchased or customized from the tertiary microfluidic technologies, inc.
The microfluidic paper chip is used for further separation and purification and obtaining saliva exosomes with the size less than 200 nm. The micro flow channel of the paper chip is 200 nm-250 nm, the micro flow paper chip adopts a nitrocellulose filter membrane as a substrate to replace materials such as silicon, glass, high polymer and the like, and through various processing technologies, the paper chip is a hydrophilic/hydrophobic micro channel with a certain structure processed on paper, can separate, extract and purify exosome, and is one of micro flow control analysis systems. The paper chip is adhered to the glass fiber to manufacture the microfluidic paper chip. Compared with the common microfluidic chip, the microfluidic chip has the advantages of low cost, simple preparation, no need of complex peripheral equipment, capability of performing disposable, low-price and portable analysis, and is the development trend of field real-time diagnosis (POCT) at present.
(3) Making gold label pad
In this example, the troponin monoclonal antibody is a mouse anti-human troponin monoclonal antibody, and the specific operation preparation method is as follows:
10mL of the prepared colloidal gold solution was added with 1.0% K2CO3After the pH value is adjusted to 8.2, 120 mu L of troponin monoclonal antibody of 1mg/mL is absorbed, slowly added into the colloidal gold solution, shaken up and then kept stand for 30 min. Adding 10 μ L of 1% PEG20000, shaking, adding 10 μ L of 10% BSA, shaking, standing, centrifuging, collecting precipitate, and re-dissolving with re-dissolving solution at 10% of the volume of the colloidal gold solution used in the original labeling. Spraying 1ml of redissolved colloidal gold marker on a polyester film of a gold marker pad with the thickness of 0.4cm multiplied by 0.4cm, drying for 8-24 hours at 37 ℃, sealing an aluminum foil bag, and storing at room temperature for later use.
Experiments prove that the optimal binding pH value of the troponin monoclonal antibody colloid marker is 8.2, and the ratio of the colloidal gold to the antibody is 12 mu g/ml colloidal gold.
The complex solution is prepared from Tris with the final concentration of 0.01-0.2 mol/L, 1-5% BSA or 0.01-0.2% complexing protein, 1-3% trehalose, 1-5% sucrose, 0.1-2% triton X-100, 0.1-0.5% Tween-20, 0.1-1% PEG20000, 0.1-2% sodium chloride or 0.1-2% sodium hydroxide and 0.05-0.1% sodium azide, the pH value is adjusted to be 7.0-8.0 by HCl, and the complex solution is stored in a dark place at the temperature of 4 ℃ for later use.
(4) Preparation of the reaction film
The troponin monoclonal antibody is diluted by antibody diluent to the concentration of 0.3-1.5 mg/mL, 0.5-3 mg/mL and 1.0-5.0 mg/mL respectively, goat anti-mouse IgG is diluted to the concentration of 0.2-1.2 mg/mL and sprayed on a nitrocellulose membrane to form detection T lines 71,72 and 73 and a quality control C line (8) respectively. The T line is 4mm apart from the C line. Then respectively adding the mixture into a T cup and a C cup of a film-scribing metal spraying instrument; and (5) debugging a film-scribing metal spraying instrument, and setting the liquid spraying amount to be 1.0 mu L/cm. After debugging is finished, film cutting and coating are carried out to form a detection line T: 71,72 and 73, respectively corresponding to troponin contents of 15mg/mL, 50mg/mL and 100mg/mL and a quality control line C, drying for 8-24 hours at 37 ℃; and sealing the aluminum foil bags to be tested to be qualified, and storing at room temperature for later use.
(5) The immunochromatographic test strip of the invention is prepared
Example 3
The invention adopts immunochromatographic test paper to detect the content of troponin of saliva exosomes, and the technical principle applies a specific immunoreaction double-antibody sandwich method. The detection method comprises the following steps: (1) after test saliva is soaked in or dripped into the sample pad of the test strip, the galactose agglutinin 3 antibody zone is coated through the capillary action of the absorbent fibers, the test saliva quickly wets the gold label pad, and the colloidal gold on the gold label pad is dissolved in combination with the troponin antibody and moves forwards along the chromatographic material along with the test liquid. (2) If the antigen to be tested exists in the test saliva, the test saliva and the colloidal gold-conjugated troponin antibody have specific immunoreaction to form an immune complex of the colloidal gold-conjugated troponin antibody and the antigen to be tested. The microfluidic paper chip area was then flowed through a T-line capture reagent immobilized on a nitrocellulose membrane with the test saliva. (3) The capture reagent of the T line captures the colloidal gold-conjugated troponin antibody-antigen immune complex to be detected, namely, the capture reagent and the immune complex generate specific immune reaction to form the colloidal gold troponin antibody-antigen to be detected-capture reagent immune complex, so that the colloidal gold troponin antibody-antigen to be detected immune complex is retained at the T line. (4) The test saliva moves forward continuously, when the test saliva flows through a quality control C line on which a goat anti-mouse IgG antibody is fixed on a nitrocellulose membrane, the colloidal gold troponin antibody which does not react with the antigen to be tested and the goat anti-mouse antibody on the C line have immunoreaction, so that free colloidal gold troponin antibody is retained at the C line. At the moment, colloidal gold troponin antibodies are retained at the T line and the C line, namely, red can be observed at the T line and the C line, and the detection result is a positive result, namely, the test saliva contains the antigen to be tested; if only the color is observed at the C line and no color is observed at the T line, the detection result is a negative result, namely the test solution does not contain the antigen to be detected; (5) the content of troponin in the saliva to be detected is different, and the content of troponin corresponding to lines 71,72,73 and 71,72 or 73 respectively appears red respectively is 15mg/mL, 50mg/mL and 100 mg/mL. (6) After the test saliva is soaked or dripped into the sample pad of the test strip for 5-10 minutes, the C, T lines can be directly observed by naked eyes, and then the quantitative detection result can be judged.
The sensitivity of the test paper can reach 10mIU/ml, and the quantitative range is 10-10000 mIU/ml. The detection test paper is adopted to detect 50 clinical AMI samples, and Roche chemiluminescence quantitative detection troponin is taken as a control, and the result shows that the sensitivity of the invention is 10mIU/ml, the specificity is 100%, and compared with the Roche chemiluminescence quantitative detection troponin result: among 50 AMI patients, 8 chemiluminescence test results showed normal, and the coincidence rate with clinical diagnosis AMI was 84%; in 50 cases of AMI patients tested in the patent, the salivary exosome troponin shows abnormal results, and the coincidence rate with clinical diagnosis AMI is 100% (see figure 3).
The results show that the detection test strip provided by the invention takes human saliva as a detection sample, effectively monitors troponin of exosomes in the saliva, and the minimum detection amount reaches 10 mg/L. The troponin detection test strip can rapidly diagnose and distinguish the content of troponin in people of less than 15mg/mL, 50mg/mL and 100mg/mL, and can accurately judge high risk people. The test strip has the advantages of simple operation, intuition, rapidness, high detection efficiency, simple method, no pollution, low cost, high sensitivity, strong specificity and the like, does not need special instruments and equipment, does not need professional training, has clear and easily identified results, is easy to popularize, and is widely applied to clinical on-site detection (POCT), field on-site and family self-detection.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. The utility model provides an immunochromatography test paper of saliva short-term test exosome troponin which characterized in that, includes that sample fills up district, antibody district, micro-fluidic paper chip district, gold mark fills up the district, cellulose nitrate membrane district, absorbs water and fills up the district, wherein:
sample pad area: used for dripping saliva or soaking saliva;
antibody region: the antibody region is coated with a galectin 3 antibody for the first step of enriching and separating saliva exosomes, and the coated galectin 3 antibody region is connected with the sample pad region;
a chip area: the chip area is provided with a micro-fluidic paper chip for further separation and purification and obtaining saliva exosomes with the size less than 200-250nm, and the micro-fluidic paper chip area is connected with the coated galectin 3 antibody area;
gold mark pad area: the gold-labeled pad area is provided with a troponin antibody marked by colloidal gold particles, the troponin antibody marked by the colloidal gold particles is combined with a troponin antigen on exosomes in saliva, and the gold-labeled pad area is connected with the microfluidic paper chip area;
cellulose nitrate membrane zone: a detection T line and a quality control C line are arranged on the nitrocellulose membrane area, a plurality of groups of T lines are used for detecting the troponin content on exosomes in saliva, and the C line is used as a control line; the nitrocellulose membrane pasting area is connected with the gold-labeled pad area.
2. The immunochromatographic test strip of claim 1, wherein the microfluidic paper chip uses a high molecular polymer material: comprises polydimethylsiloxane, polymethyl methacrylate, polystyrene and filter paper.
3. The immunochromatographic test strip of claim 2, wherein the microfluidic paper chip is provided with a hydrophilic/hydrophobic micro-channel network and is connected with a chromatographic paper.
4. The immunochromatographic test strip of claim 3, wherein a circular hole with a diameter of 200-250nm is designed in the middle of the microfluidic paper chip for extracting and purifying exosomes and separating proteins and impurities with a diameter of more than 200-250nm, and the nitrocellulose filter membrane is used as a substrate of the paper chip.
5. The immunochromatographic test strip of claim 1, wherein the particles in the antibody labeled with particles in the gold-labeled pad region are any one of colloidal gold or nanogold or a fluorescent material.
6. The immunochromatographic test strip of claim 1, wherein the streaked antibody of the T-line is a double-antibody sandwich antibody; the streak antibody of the C line is marked goat anti-rabbit antibody.
7. The immunochromatographic test strip of claim 1, wherein the T-lines have three sets of T-lines (71, 72, 73) for detecting the troponin content on exosomes in saliva at 15-100 mg/mL.
8. The immunochromatographic test strip according to claim 5, wherein the content of troponin on exosomes in saliva measured on the T-line (71) is 15 mg/mL; the content of troponin on exosomes in saliva measured on the T line (72) is 50 mg/mL; the troponin content on exosomes in saliva measured on the T-line (73) was 100 mg/mL.
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