CN111638372B - Combined detection device for cardiac marker and preparation method thereof - Google Patents
Combined detection device for cardiac marker and preparation method thereof Download PDFInfo
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- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Abstract
The invention relates to a combined detection device of a cardiac marker and a preparation method thereof, belonging to the field of medical detection equipment. Is prepared by sticking solid phases of a nitrocellulose membrane with high specificity MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies and goat anti-mouse IgG polyclonal antibodies, a glass fiber membrane adsorbed with colloidal gold labeled MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies, a sample pad, absorbent paper and other auxiliary materials. The method comprises the steps of pretreating a nitrocellulose membrane by using polyethylene glycol glycerol treatment liquid, combining MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies with zinc sulfide nanoparticles modified by oleic acid, then adsorbing the zinc sulfide nanoparticles onto the nitrocellulose membrane, and preparing a proper gold spraying buffer solution and a proper sample pad treatment liquid.
Description
Technical Field
The invention relates to the field of medical detection equipment, in particular to a MPO, cTnI or cTnT, H-FABP and NT-proBNP combined detection device and a preparation method thereof, which utilize colloidal gold immunochromatography and a double-antibody sandwich method principle to quantitatively detect human Myeloperoxidase (MPO), cardiac troponin I or T (cTnI or cTnT), cardiac fatty acid binding protein (H-FABP) and N-terminal brain natriuretic peptide precursor (NT-proBNP) in whole blood, serum and plasma samples and a preparation method thereof, and can realize the sensitive, specific and rapid detection of cardiac markers.
Background
Acute Coronary Syndrome (ACS) refers to pathological phenomena of atherosclerotic plaque rupture, platelet aggregation, thrombosis leading to coronary stenosis, obstruction, myocardial ischemia and infarction, including Acute Myocardial Infarction (AMI) and Unstable Angina (UA). AMI is divided into acute ST elevation myocardial infarction (STEMI) and acute Non-ST elevation myocardial infarction (NSTEMI). In recent years, the incidence of ACS and the fatality rate have increased year by year, and have become serious diseases threatening human health and life. Therefore, establishing a more scientific and reasonable disease stratification system for early identification, early diagnosis and early treatment of ACS, thereby reducing the fatality rate is one of the core problems of clinical concern.
Acute Myocardial Infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary arteries, and seriously threatens human health. Early warning, rapid diagnosis and effective treatment evaluation of myocardial infarction are the keys for reducing the death rate of patients. For patients with myocardial infarction without typical chest pain and with insignificant changes of electrocardiogram, accurate diagnosis is difficult only by means of electrocardiogram, echocardiogram and cardiac nuclear magnetic resonance. Therefore, detection of serum cardiac markers is an essential basis for diagnosing AMI.
Coronary heart disease diagnosis and examination technology develops rapidly, except ECG, serum biochemical index, myocardial damage marker, in addition to heart color ultrasound, cardiovascular radiography, nuclear magnetic resonance imaging, computer tomography and so on. However, such inspection means are expensive and not suitable for dynamic continuous monitoring. Among the above various examination methods, the detection of ECG and myocardial damage markers is still the most inexpensive and widely used method in clinical practice, but the specificity of early diagnosis of acute myocardial infarction is only about 90%, the sensitivity is only about 45%, and no specific ST-T change is seen in the ECG results of a considerable part of the elderly patients with acute myocardial infarction. Recent clinical research data suggest that changes of markers such as myeloperoxidase are relevant to diagnosis and prognosis of patients with acute coronary syndrome, and the markers are new-generation early warning cardiac biomarkers. MPO as a biomarker reflecting atherosclerotic vulnerable plaques is relevant to the diagnosis of ACS and patient prognosis and can predict the risk of adverse cardiac events without myocardial necrosis. MPO is mainly present in azurophilic granules of neutrophils or monocytes, and during inflammation, the neutrophils are degranulated and release MPO, which can cause instability and even rupture of coronary atherosclerotic lesions, expose collagen tissues under vascular endothelium, and then cause platelet adhesion and thrombosis to cause coronary artery obstruction, generate ACS and cause severe irreversible ischemic injury of cardiac muscle. A large number of clinical research data show that the serum myeloperoxidase level of patients with acute coronary syndrome is remarkably increased, and the myeloperoxidase is a new prediction factor for predicting adverse cardiovascular events of patients with coronary heart disease.
Cardiac troponin (cTn) is a structural protein constituting striated myofilaments, has a function of regulating muscle cell contraction, and is composed of subunits of three different genes: cardiac troponin t (ctnt), cardiac troponin i (ctni) and troponin c (tnc) play an important role in controlling cardiac muscle contraction. When the integrity of a myocardial cell membrane is damaged due to severe myocardial ischemia, the cTnI or the cTnT is very easy to release into blood, the onset of chest pain is increased for 4-6 h, and the increase can be continued for 6-7 d. Currently, cardiac troponin is mainly used for clinical diagnosis, risk assessment and prognosis judgment of myocardial ischemia injury.
H-FABP is a novel small cytoplasmic protein abundant in the heart. Expressed primarily in cardiac tissue, it is highly heart specific. H-FABP can be found in blood 0.5-3H after myocardial ischemic injury, reaches a peak value 6-8H, and returns to normal within 24-30H. The significance of H-FABP in early diagnosis of myocardial injury is mainly based on the following characteristics that (1) the H-FABP has high concentration in the myocardium; (2) low molecular weight; (3) relative tissue specificity; (4) similar to the distribution of CK-MB in tissues other than the heart; (5) rapidly released into plasma after myocardial injury. Compared with myoglobin (Myo), the concentration of H-FABP in the heart is 2-10 times of that of skeletal muscle, and the concentration of Myo in heart cells is 1/2 of the skeletal muscle, so although the Myo level in plasma is increased and is a widely accepted early myocardial injury marker, the H-FABP is more specific to the heart. H-FABP is a cardiac marker reflecting myocardial cell injury, and can be used for early warning and diagnosis of AMI within 0.5-3H after AMI occurs; meanwhile, the H-FABP has the characteristics of high sensitivity, high negative prediction value and the like, so that heart damage can be found earlier and risks can be found earlier by combining the H-FABP with heart detection, the degree of myocardial damage can be evaluated according to the detection result, the detection sensitivity can be improved by combining the H-FABP with troponin, and the H-FABP has a diagnosis value on heart diseases; in addition, the H-FABP concentration can effectively identify high-risk patients with adverse events such as AMI, heart failure, unstable angina pectoris and the like in the long-term prognosis of ACS, and the detection of the high-risk patients is combined with the high-risk patients to realize great clinical diagnosis value.
The N-terminal pro-brain natriuretic peptide (NT-proBNP) is a cleavage product of the pro-brain natriuretic peptide (proBNP), and after the myocardial cells are stimulated, the proBNP is cleaved into NT-proBNP and BNP, which are closely related in secretion. The heart is the major source of circulating brain natriuretic peptides, BNP is primarily stored in ventricular myocytes. The physiological effects of the composition are natriuretic effect, vasodilatation effect, water retention effect and sodium retention effect of resisting epinephrine, renin-angiotensin and the like, and the composition can be used for evaluating the cardiac function. NT-proBNP has no similar physiological action, but is secreted into blood together with BNP, and is closely related. In recent years, it has been reported that NT-proBNP has higher plasma concentration, longer half-life (60-120min), higher stability and more sensitive detection marker than BNP.
At present, methods for detecting MPO, cTnI or cTnT, H-FABP and NT-proBNP mainly comprise an enzyme-linked immunosorbent assay, a chemiluminescence assay, an immunoturbidimetric immunoassay, a gold-labeled immunoassay and the like. The gold-labeled immunoassay method needs a small amount of specimens, is simple, convenient and quick, is suitable for quick detection of acute myocardial infarction, and is not limited by time and place. MPO may warn of the risk of 3 hours before myocardial infarction, but other inflammation may also lead to an increase in MPO markers. The elevation of cTnI or cTnT myocardial injury starting at 4-6 hours is a first-choice marker for evaluating myocardial necrosis and is a gold standard for detecting myocardial injury. The H-FABP can be rapidly released after myocardial injury, and the serum level can be rapidly increased, so that the method can be used for early diagnosis of acute myocardial infarction. NT-proBNP can be used for evaluating the cardiac function after myocardial infarction and has auxiliary effect on predicting disease prognosis. Products and literature reports exist in the general gold-labeled immunity method, and the products and the literature reports are all controlled aiming at a single index, only can detect or early warn a certain stage of myocardial infarction, but cannot comprehensively and specifically warn the whole generation and development process of the myocardial infarction; in addition, the index combination of the traditional combined detection project is unscientific and not compact, and the phenomena of missed diagnosis and misdiagnosis are easy to occur; therefore, the existing items aiming at heart detection have many traps, are not beneficial to the clinical diagnosis and are in urgent need of improvement.
The Gold Immunochromatography (GICA) is a solid-phase membrane immunoassay technique using a microporous membrane as a carrier, which combines a gold immunochromatography technique with a protein chromatography technique. The colloidal gold immunochromatographic assay is a commonly used immunochromatographic assay, is very suitable for field detection due to the characteristics of simple operation, time saving, low manufacturing cost, easy interpretation of results and the like, and is widely applied to the fields of biology, medicine, food and the like. Because the colloidal gold immunochromatographic assay is used for completing detection in one step, the interference factors in the detection process are more, the low sensitivity is a main factor for limiting the application range of the colloidal gold immunochromatographic assay, and the detection limit of the traditional colloidal gold immunochromatographic assay is higher than that of methods such as ELISA and the like.
In the colloidal gold immunochromatography assay, proteins are immobilized on a nitrocellulose membrane (NC membrane) as a capture reagent for a sample to be detected. Since the detection result completely depends on the good adsorption effect of the capture reagent on the membrane, the uniform and good adsorption of the protein on the membrane is very important for the detection result of the colloidal gold. If the amount of protein bound to the NC membrane is insufficient or the binding force of protein is not strong enough, a considerable problem occurs, and it is very obvious on the detection line of the detection result. If the amount of protein bound to the membrane is too low, the color development of the detection line is weak and the detection sensitivity is reduced in the result. If the protein is not firmly adsorbed to the NC membrane, the protein diffuses before adsorbing to the NC membrane, so that the detection line is wide, the color development is weak, the detection line is bright and clear, and the detection result is difficult to explain. Under extreme conditions, if the physical adsorption of the protein to the NC membrane is too weak, the protein assay and surfactant solution flowing through may wash the immobilized protein off the NC membrane, thereby revealing a wider or not clear detection line at all, making it difficult to interpret the detection results.
Disclosure of Invention
The invention provides a combined detection device for cardiac markers and a preparation method thereof, and aims to solve the problems of insufficient NC membrane protein adsorption amount and weak binding force in the prior art. The four-in-one combined detection device of the human Myeloperoxidase (MPO), the cardiac troponin I or T (cTnI or cTnT), the heart-type fatty acid binding protein (H-FABP) and the N-terminal brain natriuretic peptide precursor (NT-proBNP) can realize the sensitive, specific and rapid detection of cardiac markers, improve the reasonable comprehensive judgment of myocardial infarction risks of patients with acute myocardial infarction and quickly and accurately perform early warning of myocardial infarction and judgment of disease risks.
The technical scheme adopted by the invention is as follows: the sample pad 1, the immune colloidal gold glass fiber membrane 2, the nitrocellulose membrane 3 and the absorption pad 4 are respectively stuck on the plastic plate 5, two ends of the nitrocellulose membrane 3 are respectively lapped with the absorption pad 4 and the immune colloidal gold glass fiber membrane 2, and the other end of the immune colloidal gold glass fiber membrane 2 is lapped with the sample pad 1; a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4 and a quality control line C are arranged on the cellulose nitrate film 3; the solid phase on the first detection line T1 is provided with a high specificity MPO antibody; the solid phase on the second detection line T2 is provided with a high specificity cTnI or cTnT antibody; the solid phase on the third detection line T3 is provided with a high specificity H-FABP antibody; the solid phase on the fourth detection line T4 is provided with a high-specificity NT-proBNP antibody; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membrane 3 can be longitudinally arranged on the same nitrocellulose membrane 3 to form a combined detection device; the detection lines T1, T2, T3 and T4 arranged on the cellulose nitrate membranes 3 can also be combined in any two and respectively arranged on two cellulose nitrate membranes 3 and are arranged in parallel to form a combined detection device; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membranes 3 can also be respectively arranged on the four nitrocellulose membranes 3 and are arranged in parallel to form a combined detection device; and a goat anti-mouse IgG polyclonal antibody is spotted on the quality control line C.
A preparation method of a combined detection device of a myocardial marker comprises the following steps:
(a) preparing colloidal gold by a trisodium citrate reduction method;
(b) adopting the colloidal gold prepared in the step (a) to mark MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies to obtain immune colloidal gold;
(c) diluting the immune colloidal gold obtained in the step (b) by adopting a gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal gold glass fiber membrane;
(d) pretreating a nitrocellulose membrane by using a polyethylene glycol glycerol treatment solution, spraying MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies combined with zinc sulfide nano particles modified by oleic acid as a detection line, and spraying goat anti-mouse IgG antibodies as a quality control line to prepare an immune nitrocellulose membrane;
(e) and (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
The particle size of the colloidal gold particles prepared by the trisodium citrate reduction method in the step (a) is 20-60 nm.
The gold spraying buffer solution in the step (c) consists of Tris-HCl solution, sucrose, trehalose and bovine serum albumin BSA, and has a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02mol/L, the concentration of sucrose is 5-20%, the concentration of trehalose is 1-5%, and the concentration of bovine serum albumin BSA is 0.5-1%.
The pretreatment of the nitrocellulose membrane with the polyethylene glycol glycerol treatment solution in the step (d) of the invention is as follows: soaking the cellulose nitrate membrane in polyethylene glycol glycerol treatment solution for 1h, shaking at a low speed, taking out, washing with distilled water for 3 times, and finally drying in a vacuum drying oven.
The combination of the zinc sulfide nanoparticles in the step (d) of the invention with MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies respectively is as follows:
taking 1mL of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibody solution as a carrier by using ZnS modified by oleic acid, stirring for 1 hour, centrifuging at 12000rpm, 8500rpm and 7000rpm respectively for 10 minutes, and collecting, and washing 2 times by using deionized water respectively.
The sample pad treatment solution adopted by the pretreated sample pad in the step (e) of the invention consists of Tris-HCL solution, bovine serum albumin BSA, casein and surfactant (alkylphenol ethoxylates), wherein the concentration of the Tris-HCL solution is 0.1mol/L, the concentration of the bovine serum albumin BSA is 0.5-1%, the concentration of the casein is 0.1-0.2% and the concentration of the surfactant is 0.5-1%.
The polyethylene glycol glycerol treatment solution in the step (d) of the invention is formed by diluting polyethylene glycol glycerol to the concentration of 0.5%, and is filtered by a filter membrane of 0.22 mu m for later use.
The polyethylene glycol glycerol treatment solution in the step (d) of the invention is prepared by mixing polyethylene glycol glycerol and polylysine (SIGMA,150 KD-300 KD), wherein the concentration of the polyethylene glycol glycerol is 0.5 percent, and the concentration of the polylysine is 0.5 percent, and the polyethylene glycol glycerol treatment solution is filtered by a filter membrane of 0.22 mu m for standby.
The polyethylene glycol glycerol treatment liquid in the step (d) of the invention is formed by mixing polyethylene glycol glycerol, polylysine (SIGMA,150 KD-300 KD) and PEG20000, wherein the concentration of the polyethylene glycol glycerol is 0.5%, the concentration of the polylysine is 0.5%, the concentration of the PEG20000 is 0.1%, and the mixture is filtered by a 0.22 mu m filter membrane for later use.
The preparation method of the oleic acid modified zinc sulfide nano-particles in the step (d) comprises the following steps: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
The reaction of glycol and epichlorohydrin is catalyzed by alkali, the product is neutralized by dilute hydrochloric acid, extracted by carbon tetrachloride and distilled under reduced pressure, thus obtaining polyethylene glycol glycerol (PEGG) which is light yellow viscous substance. PEGG can be mixed with water in any proportion, can also be dissolved in common organic solvents such as ethanol, acetone, tetrahydrofuran and chloroform, and has certain surface activity. The polyethylene glycol glycerol has a structure containing a plurality of hydroxyl groups for coupling, the activation process is simple, and the protein can be conveniently fixed on the surface of the NC membrane. Under the conventional conditions, the number of the combined antibodies on the NC membrane per unit area is limited, and after the treatment by adopting the polyethylene glycol glycerol, the number of the combined antibodies on the NC membrane per unit area can be increased, so that higher detection sensitivity can be realized.
On the basis of improving the protein adsorption of the NC membrane, discussing the protein adsorption effect of the NC membrane is another way to improve the sensitivity of the colloidal gold. The ZnS modified by the oleic acid/lauryl sodium sulfate not only has a nano-scale particle size, but also has good water solubility and biocompatibility, and can be uniformly dispersed in an aqueous medium and combined with biological macromolecules by utilizing functional groups on the outer surface of the ZnS. The zinc sulfide nano-particles have the advantages of good stability, easy preparation, good biocompatibility, low immunogenicity and the like, and are widely researched in the field of biomedicine. However, the application of the method in the colloidal gold immunochromatography technology has not been reported. The research discusses the influence of zinc sulfide nanoparticles on NC membrane coated antibodies, firstly, the antibodies for membrane scribing are combined with the zinc sulfide nanoparticles, sealed, centrifugally purified, unbound antibodies are removed, then, the antibodies are redissolved to a certain proportion, and then, the membrane scribing is carried out, so that one zinc sulfide particle can be combined with a plurality of antibodies, the efficiency of coating the antibodies is increased, and the sensitivity is greatly improved.
In order to improve the sensitivity of the colloidal gold immunochromatography technology, the nitrocellulose membrane is pretreated by polyethylene glycol glycerol treatment fluid, and an antibody coated with NC is combined with zinc sulfide nanoparticles, so that the aim of improving the sensitivity of the test paper is fulfilled.
The invention has the beneficial effects that:
1. the detection device disclosed by the invention is simple in structure and novel in concept, MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies are coated on a nitrocellulose membrane, the specificity is strong, MPO, cTnI or cTnT, H-FABP and NT-proBNP in a sample can be detected simultaneously, and the complexity of production operation is not increased.
2. In the immune colloidal gold preparation step, the immune colloidal gold can be completely released by matching with a proper gold spraying buffer solution and a sample pad treatment solution, the reaction sensitivity is effectively improved, the using amount of the immune colloidal gold can be reduced under the same threshold value, and the cost is saved.
3. The invention pretreats the nitrocellulose membrane, modifies the antibody coated with the nitrocellulose membrane, and improves the sensitivity and specificity of the test paper.
4. The detection device does not need any special instrument and equipment, and has low detection cost.
5. The detection device is simple and convenient to operate, and does not need to be operated by professional staff. The practicability is strong.
Drawings
FIG. 1 is a schematic structural view of the present invention, in which detection lines T1, T2, T3 and T4 are longitudinally arranged on the same nitrocellulose membrane;
FIG. 2 is a cross-sectional view A-A of FIG. 1;
FIG. 3 is a schematic diagram of the structure of two combined detection lines T1, T2 and T3, T4 of the present invention, respectively disposed on two nitrocellulose membranes;
FIG. 4 is a cross-sectional view B-B of FIG. 3;
FIG. 5 is a cross-sectional view C-C of FIG. 3;
FIG. 6 is a schematic diagram of the structure of detection lines T1, T2, T3 and T4 of the present invention disposed on four nitrocellulose membranes, respectively;
FIG. 7 is a cross-sectional view D-D of FIG. 6;
FIG. 8 is a cross-sectional view E-E of FIG. 6;
FIG. 9 is a cross-sectional view F-F of FIG. 6;
fig. 10 is a sectional view G-G of fig. 6.
Detailed Description
As shown in the figure, the kit comprises a sample pad 1, an immune colloidal gold glass fiber membrane 2, a nitrocellulose membrane 3, an absorption pad 4 and a plastic plate 5, wherein the sample pad 1, the immune colloidal gold glass fiber membrane 2, the nitrocellulose membrane 3 and the absorption pad 4 are respectively adhered to the plastic plate 5, two ends of the nitrocellulose membrane 3 are respectively lapped with the absorption pad 4 and the immune colloidal gold glass fiber membrane 2, and the other end of the immune colloidal gold glass fiber membrane 2 is lapped with the sample pad 1; a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4 and a quality control line C are arranged on the cellulose nitrate film 3; the solid phase on the first detection line T1 is provided with a high specificity MPO antibody; the solid phase on the second detection line T2 is provided with a high specificity cTnI or cTnT antibody; the solid phase on the third detection line T3 is provided with a high specificity H-FABP antibody; the solid phase on the fourth detection line T4 is provided with a high-specificity NT-proBNP antibody; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membrane 3 can be arranged on the same nitrocellulose membrane 3 in a plurality of directions to form a combined detection device; the detection lines T1, T2, T3 and T4 arranged on the cellulose nitrate membranes 3 can also be combined in any two and respectively arranged on two cellulose nitrate membranes 3 and are arranged in parallel to form a combined detection device; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membranes 3 can also be respectively arranged on the four nitrocellulose membranes 3 and are arranged in parallel to form a combined detection device; and a goat anti-mouse IgG polyclonal antibody is spotted on the quality control line C.
A preparation method of a combined detection device of a cardiac marker comprises the steps of preparing a solid phase by adhering purified MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies (detection lines T1, T2, T3 and T4) with high specificity and a nitrocellulose membrane of goat anti-mouse IgG polyclonal antibody (control line), a glass fiber membrane adsorbed with colloidal gold labeled MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies, a sample pad, absorbent paper and other auxiliary materials. The method comprises the following specific steps:
(a) preparing colloidal gold by a trisodium citrate reduction method;
(b) adopting the colloidal gold prepared in the step (a) to mark MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies to obtain immune colloidal gold;
(c) diluting the immune colloidal gold obtained in the step (b) by adopting a gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal gold glass fiber membrane;
(d) pretreating a nitrocellulose membrane by using a polyethylene glycol glycerol treatment solution, spraying MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies combined with zinc sulfide nano particles modified by oleic acid as a detection line, and spraying goat anti-mouse IgG antibodies as a quality control line to prepare an immune nitrocellulose membrane;
(e) and (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
The particle size of the colloidal gold particles prepared by the trisodium citrate reduction method in the step (a) is 20-60 nm.
The gold spraying buffer solution in the step (c) consists of Tris-HCl solution, sucrose, trehalose and bovine serum albumin BSA, and has a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02mol/L, the concentration of sucrose is 5-20%, the concentration of trehalose is 1-5%, and the concentration of bovine serum albumin BSA is 0.5-1%.
The step (d) of pretreating the nitrocellulose membrane with a polyethylene glycol glycerol treatment solution comprises the following steps: soaking the cellulose nitrate membrane in polyethylene glycol glycerol treatment solution for 1h, shaking at a low speed, taking out, washing with distilled water for 3 times, and finally drying in a vacuum drying oven.
The zinc sulfide nanoparticles in the step (d) are respectively combined with MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies:
taking 1mL of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibody solution as a carrier by using ZnS modified by oleic acid, stirring for 1 hour, centrifuging at 12000rpm, 8500rpm and 7000rpm respectively for 10 minutes, and collecting, and washing 2 times by using deionized water respectively.
The sample pad treatment solution adopted by the pretreated sample pad in the step (e) consists of Tris-HCL solution, bovine serum albumin BSA, casein and surfactant (alkylphenol ethoxylates), wherein the concentration of the Tris-HCL solution is 0.1mol/L, the concentration of the bovine serum albumin BSA is 0.5-1%, the concentration of the casein is 0.1-0.2% and the concentration of the surfactant is 0.5-1%.
The polyethylene glycol glycerol treatment solution in the step (d) is formed by diluting polyethylene glycol glycerol to the concentration of 0.5%, and is filtered by a filter membrane of 0.22 mu m for later use.
The polyethylene glycol glycerol treatment liquid in the step (d) is formed by mixing polyethylene glycol glycerol and polylysine (SIGMA,150 KD-300 KD), wherein the concentration of the polyethylene glycol glycerol is 0.5 percent, and the concentration of the polylysine is 0.5 percent, and the polyethylene glycol glycerol treatment liquid is filtered by a filter membrane with the diameter of 0.22 mu m for standby.
The polyethylene glycol glycerol treatment liquid in the step (d) is prepared by mixing polyethylene glycol glycerol, polylysine (SIGMA,150 KD-300 KD) and PEG20000, wherein the concentration of the polyethylene glycol glycerol is 0.5%, the concentration of the polylysine is 0.5%, the concentration of the PEG20000 is 0.1%, and the mixture is filtered by a 0.22 mu m filter membrane for later use.
The preparation method of the oleic acid modified zinc sulfide nano-particles comprises the following steps: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
Example 1
(a) Colloidal gold prepared by trisodium citrate reduction method
Gold chloride was added rapidly to heated 100ml of purified water, and after the solution boiled again, trisodium citrate, gold chloride: trisodium citrate 1: 0.5, continuously boiling, observing that the color of the solution changes from yellow to black and then to purple, and finally changing to stable wine red, and continuously heating for 10 minutes at regular time, wherein the particle size of the colloidal gold is 20 nm;
(b) immune colloidal gold preparation
1) Respectively taking 100ml of 20nm colloidal gold solution, adding 140 mu l of pH regulator, and uniformly mixing; standing for 5 min;
2) adding MPO, cTnI, H-FABP and NT-proBNP into 20nm colloidal gold solution according to the proportion of 14 mu g of the colloidal gold solution per ml, and uniformly mixing the 1.4 mg; standing for 5 min;
3) respectively adding 0.4 ml of colloidal gold stabilizer according to the proportion of 0.4%, uniformly mixing, and standing for 5 minutes;
4) centrifuging at 10000, 12000 and 14000rpm for 10min, respectively collecting the precipitate, and combining the three collected precipitates;
(c) diluting immune colloidal gold by adopting an optimized gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal glass fiber membrane; the gold spraying buffer solution comprises: Tris-HCl solution with concentration of 20mM, sucrose concentration of 5%, trehalose concentration of 1%, BSA concentration of 1%, pH of 8.5;
(d) solid phase nitrocellulose membrane
1) Cellulose nitrate membrane pretreated by polyethylene glycol glycerol treatment liquid
Preparing a polyethylene glycol glycerol treatment solution: filtering with 0.22 μm filter membrane to obtain polyethylene glycol glycerol with concentration of 0.5%;
pretreating a nitrocellulose membrane by using polyethylene glycol glycerol treatment liquid: soaking the nitrocellulose membrane in a polyethylene glycol glycerol treatment solution for 1h, shaking at a low speed, taking out, washing with distilled water for 3 times, and finally drying in a vacuum drying oven;
2) zinc sulfide nanoparticle modified MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies
Preparing oleic acid modified zinc sulfide nanoparticles: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
Modifying MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies by zinc sulfide nanoparticles:
taking 1mL of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibody solution as a carrier by using ZnS modified by oleic acid, stirring for 1 hour, centrifuging at 12000rpm, 8500rpm and 7000rpm respectively for 10 minutes, and collecting, and washing 2 times by using deionized water respectively.
3) Coating of nitrocellulose membrane detection line and quality control line antibody
When the film spraying amount is 1.4 mul/cm, diluting a zinc sulfide nanoparticle-MPO antibody, a zinc sulfide nanoparticle-cTnI or cTnT antibody, a zinc sulfide nanoparticle-H-FABP antibody and a zinc sulfide nanoparticle-NT-proBNP antibody to 1.5mg/ml, diluting a quality control line goat anti-mouse IgG antibody to 1mg/ml, respectively coating a detection line and a quality control line of the cellulose nitrate film, drying at room temperature overnight, and storing for later use;
4) sample pad pretreatment
Soaking glass fiber in a sample pad treatment solution for 10min, wherein the sample pad treatment solution comprises: the concentration of Tris-HCL solution is 0.1M, the concentration of bovine serum albumin BSA is 0.5%, the concentration of casein is 0.1%, the concentration of surfactant is 0.5%, the drying is carried out for standby at 37 ℃, and the reaction sensitivity can be improved by the sample pad after the treatment;
e. assembly
Sequentially adhering the pretreated sample pad, the immune colloidal gold glass fiber membrane, the immune nitrocellulose membrane and the absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
f. And (3) quantitative detection: through the detection of a colloidal gold detector, the combined detection device detects that the minimum detection concentration of human MPO is 5ng/ml, the minimum detection concentration of cTnI or cTnT is 0.1ng/ml, the minimum detection concentration of H-FABP is 0.5ng/ml and the minimum detection concentration of NT-proBNP is 20 pg/ml.
Example 2:
preparing a polyethylene glycol glycerol treatment solution: is prepared by mixing polyethylene glycol glycerol with concentration of 0.5% and polylysine with concentration of 0.5% and filtering with 0.22 μm filter membrane.
The rest is the same as example 1.
Example 3:
preparing a polyethylene glycol glycerol treatment solution: is prepared by mixing polyethylene glycol glycerol (0.5% concentration), polylysine (0.5% concentration), and PEG20000 (0.1% concentration) with PEG2000, and filtering with 0.22 μm filter membrane.
The rest is the same as example 1.
The following experiment further illustrates the effects of the present invention.
Experimental example 1:
1. comparison of adsorption capacities of polyethylene glycol glycerol treatment solutions to nitrocellulose membrane protein
1.1 materials and methods
1.1 materials: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA
1.2 nitrocellulose Membrane treatment
1.2.1 preparing polyethylene glycol glycerol treating fluid
Preparing three polyethylene glycol glycerol treatment liquids: polyethylene glycol glycerol group, the concentration of polyethylene glycol glycerol is 0.5%; the polyethylene glycol glycerol treatment fluid polylysine group comprises polyethylene glycol glycerol with the concentration of 0.5 percent and polylysine with the concentration of 0.5 percent; polyethylene glycol glycerol, polylysine and PEG20000, wherein the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, the concentration of PEG20000 is 0.1%, and the three groups of treatment solutions are filtered with 0.22 μm filter membrane for use.
1.2.2 nitrocellulose Membrane treatment
And (3) putting the nitrocellulose membrane into the polyethylene glycol glycerol treatment solution, soaking for 1h, shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven.
1.3 Experimental methods
MPO, cTnI or cTnT, H-FABP and NT-proBNP combined test paper is prepared from the untreated and treated nitrocellulose membranes according to the process flows of the above examples, and the differences of the adsorption force and stability indexes of the untreated and treated nitrocellulose membranes are compared according to the specification of the test paper.
1.4 results
1.4.1 comparison of protein adsorption Capacity
And (3) taking the test paper of the treatment group and the test paper of the non-treatment group, respectively adding the test paper to be detected, and judging the protein adsorption capacity of the treated membrane by observing the color development condition, wherein the results are shown in table 1. The result shows that the treated nitrocellulose membrane is obviously better than the untreated membrane in the aspect of solution wettability, the color of the positive strip of the treated membrane is slightly dark, and particularly when the concentration is lower, the reaction sensitivity is improved, which shows that the protein adsorption capacity is obviously enhanced, and the reaction sensitivity is improved. The adsorption effect of the polyethylene glycol glycerol-polylysine-PEG 20000 treatment group is obviously better than that of the polyethylene glycol glycerol group and the polyethylene glycol glycerol-polylysine group.
TABLE 1 comparison of the adsorption Capacity of nitrocellulose membranes
1.4.2 comparison of stability of nitrocellulose membranes
The stability of the adsorbed protein on the nitrocellulose membrane after treatment was judged by observing the color development through an accelerated test at 37 ℃ using 3 groups of treated and untreated test papers, and the results are shown in table 2. The results in Table 2 and the results in Table 1 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 2 accelerated stability comparison of nitrocellulose membranes (10 days at 37 ℃ C.)
Experimental example 2:
2. oleic acid modified zinc sulfide nanoparticle modified MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies
2.1 materials and methods
2.1.1 materials: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA
2.1.2 preparation of oleic acid-modified Zinc sulfide nanoparticles
Adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
2.1.3 modification of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies with Zinc sulfide nanoparticles
Taking 1mL of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibody solution as a carrier by using ZnS modified by oleic acid, stirring for 1 hour, centrifuging at 12000rpm, 8500rpm and 7000rpm respectively for 10 minutes, and collecting, and washing 2 times by using deionized water respectively.
2.2 Experimental methods
MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies modified by zinc sulfide nanoparticles and unmodified MPO, cTnI or cTnT, H-FABP and NT-proBN antibodies are respectively subjected to the process flow of the above examples to prepare MPO, cTnI or cTnT, H-FABP and NT-proBNP combined test paper, and the test flow compares the differences of protein adsorption force and stability indexes of the treated zinc sulfide nanoparticles and the untreated zinc sulfide nanoparticles according to the specification of the test paper.
2.3 results
2.3.1 comparison of protein adsorption Capacity
The test paper of the zinc sulfide nanoparticle treatment group and the test paper of the non-treatment group are respectively added into a sample to be detected, and the protein adsorption capacity of the treated membrane is judged by observing the color development condition, and the result is shown in table 3. The result shows that the positive strip of the zinc sulfide nanoparticle modified membrane is slightly dark in color, and particularly when the concentration is low, the reaction sensitivity is improved, which shows that the protein adsorption capacity is obviously enhanced, and the reaction sensitivity is improved.
TABLE 3 comparison of protein adsorption Capacity for Zinc sulfide nanoparticle modification
2.3.2 stability comparison
Test paper of a zinc sulfide nanoparticle treatment group and test paper of an untreated group are taken, the stability of the protein adsorbed on the nitrocellulose membrane after zinc sulfide modification is judged by observing the color development condition through an accelerated experiment at 37 ℃, and the result is shown in table 4. The results in Table 4 and the results in Table 3 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 4 accelerated stability comparison (10 days at 37 ℃ C.)
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like of the present invention shall be included in the protection scope of the present invention.
Claims (8)
1. A preparation method of a combined detection device of a myocardial marker is characterized by comprising the following steps: the combined detection device for the myocardial markers comprises: the sample pad (1), the immune colloidal gold glass fiber membrane (2), the nitrocellulose membrane (3) and the absorption pad (4) are respectively adhered to the plastic plate (5), two ends of the nitrocellulose membrane (3) are respectively lapped with the absorption pad (4) and the immune colloidal gold glass fiber membrane (2), and the other end of the immune colloidal gold glass fiber membrane (2) is lapped with the sample pad (1); a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4 and a quality control line C are arranged on the nitrocellulose membrane (3); the solid phase on the first detection line T1 is provided with a high specificity MPO antibody; the solid phase on the second detection line T2 is provided with a high specificity cTnI or cTnT antibody; the solid phase on the third detection line T3 is provided with a high specificity H-FABP antibody; the solid phase on the fourth detection line T4 is provided with a high-specificity NT-proBNP antibody; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membrane 3 are longitudinally arranged on the same nitrocellulose membrane (3) to form a combined detection device; any two of detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membranes (3) are combined and respectively arranged on the two nitrocellulose membranes (3) and are arranged in parallel to form a combined detection device; the detection lines T1, T2, T3 and T4 arranged on the nitrocellulose membranes (3) are respectively arranged on the four nitrocellulose membranes (3) and are arranged in parallel to form a combined detection device; a goat anti-mouse IgG polyclonal antibody is spotted on the quality control line C; the preparation method comprises the following steps:
(a) preparing colloidal gold by a trisodium citrate reduction method;
(b) adopting the colloidal gold prepared in the step (a) to mark MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies to obtain immune colloidal gold;
(c) diluting the immune colloidal gold obtained in the step (b) by adopting a gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal gold glass fiber membrane;
(d) pretreating a nitrocellulose membrane by using a polyethylene glycol glycerol treatment solution, spraying MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies combined with zinc sulfide nano particles modified by oleic acid as a detection line, and spraying goat anti-mouse IgG antibodies as a quality control line to prepare an immune nitrocellulose membrane;
the preparation method of the oleic acid modified zinc sulfide nano-particles comprises the following steps: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, pouring the reaction solution into a 90ml hydrothermal kettle after uniform mixing, sealing the hydrothermal kettle, putting the hydrothermal kettle into a constant-temperature drying box, reacting at constant temperature for a certain time, cooling to 50 ℃ after the reaction is finished, taking out a product, washing by using acetone, deionized water, ethanol, performing centrifugal separation, performing vacuum drying at 50 ℃ for 2 hours to obtain powder ZnS, and storing for later use;
(e) and (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
2. The method for preparing a combined detection device for cardiac markers according to claim 1, wherein: the particle size of the colloidal gold particles prepared by the trisodium citrate reduction method in the step (a) is 20-60 nm.
3. The method for preparing a combined detection device for cardiac markers according to claim 1, wherein: the gold spraying buffer solution in the step (c) consists of Tris-HCl solution, sucrose, trehalose and bovine serum albumin BSA, and has a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02mol/L, the concentration of sucrose is 5-20%, the concentration of trehalose is 1-5%, and the concentration of bovine serum albumin BSA is 0.5-1%.
4. The method for preparing a combined detection device for cardiac markers according to claim 1, wherein: the step (d) of pretreating the nitrocellulose membrane with a polyethylene glycol glycerol treatment solution comprises the following steps: soaking the nitrocellulose membrane in polyethylene glycol glycerol treatment solution for 1h, shaking at a low speed, taking out, washing with distilled water for 3 times, and finally drying in a vacuum drying oven;
the oleic acid modified zinc sulfide nanoparticle is combined with MPO, cTnI or cTnT, H-FABP and NT-proBNP antibodies respectively and comprises the following components in percentage by weight: taking 1mL of MPO, cTnI or cTnT, H-FABP and NT-proBNP antibody solution as a carrier by using ZnS modified by oleic acid, stirring for 1 hour, centrifuging at 12000rpm, 8500rpm and 7000rpm respectively for 10 minutes, and collecting, and washing 2 times by using deionized water respectively.
5. The method for preparing a combined detection device for cardiac markers according to claim 1, wherein: the sample pad treatment liquid adopted by the pretreated sample pad in the step (e) consists of Tris-HCL liquid, bovine serum albumin BSA, casein and surfactant alkylphenol ethoxylates, wherein the concentration of the Tris-HCL liquid is 0.1mol/L, the concentration of the bovine serum albumin BSA is 0.5-1%, the concentration of the casein is 0.1-0.2%, and the concentration of the surfactant is 0.5-1%.
6. The method for preparing a combined detection device for cardiac markers according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment solution in the step (d) is formed by diluting polyethylene glycol glycerol to the concentration of 0.5%, and is filtered by a filter membrane of 0.22 mu m for later use.
7. The method for preparing a combined detection device for cardiac markers according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment liquid in the step (d) is formed by mixing polyethylene glycol glycerol and polylysine SIGMA with the concentration of 150 KD-300 KD, wherein the concentration of the polyethylene glycol glycerol is 0.5 percent, the concentration of the polylysine is 0.5 percent, and the polyethylene glycol glycerol treatment liquid is filtered by a filter membrane with the diameter of 0.22 mu m for standby.
8. The method for preparing a combined detection device for cardiac markers according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment liquid in the step (d) is prepared by mixing polyethylene glycol glycerol, polylysine SIGMA,150 KD-300 KD and PEG20000, wherein the concentration of the polyethylene glycol glycerol is 0.5%, the concentration of the polylysine is 0.5%, the concentration of the PEG20000 is 0.1%, and the mixture is filtered by a 0.22 mu m filter membrane for later use.
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