CN107884573B - Preparation and detection method of high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement - Google Patents
Preparation and detection method of high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement Download PDFInfo
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Abstract
A high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement and a preparation and detection method thereof. The invention utilizes the reaction mechanism that the magnetic nanoparticles can quench the fluorescence signal under specific conditions, combines the advantages of visualization of the detection result of the superparamagnetic nanoparticle immunochromatographic test strip and high fluorescence detection sensitivity, and aims to construct a visualized fluorescence quantitative detection immunochromatographic test strip, by means of a reaction mechanism of quenching fluorescent signals, the interference of background fluorescence is eliminated, the rapid qualitative under the naked eye condition and the accurate quantitative wide-range bimodal detection under the dark field test are achieved through the signal intensity of fluorescence and the aggregation and color development of magnetic nanoparticles, a reverse fluorescence enhancement high-sensitivity visualization bimodal immunochromatographic test strip detection technology is built, the method is simple to operate, professional training is not needed, the method is convenient and rapid, the result is visual, the rapid and high-sensitivity detection can realize the accuracy of AMI early diagnosis and the universality of community screening.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to a preparation and detection method of a high-sensitivity visual bimodal immunochromatographic test strip based on reverse fluorescence enhancement.
Background
Acute Myocardial Infarction (AMI) is one of the most serious acute cardiovascular diseases, has acute morbidity and high mortality, and the mortality rate of the AMI increases with the age. National statistics show that after age 40, the mortality rate of acute myocardial infarction is increased rapidly, the prevalence rate of myocardial infarction of the old people is increased day by day, and the mortality rate and prevalence rate of myocardial infarction become common heart diseases of the old people and are mainly caused by the fact that: when the disease is developed for the first time, patients often have insufficient understanding on the disease and cannot see a doctor in time; the advanced symptoms of the old are more and less typical, the old is easy to misdiagnose into other system diseases, and a plurality of patients are painless, the proportion of the painless patients is increased along with the increase of the age, and the disease rate of the painless myocardial infarction is up to 60.69 percent for the old patients with the age of more than 80 years old; the complications and inclusion syndrome are more, and in the acute stage, the complications account for 90.3 percent, and the atypical and complicated symptoms are increased. It is statistically estimated that about 40% of patients die within 4 hours after onset of disease, and therefore, a more sensitive diagnostic method is important for early diagnosis, reduction of mortality of AMI patients, and improvement of prognosis, since an early correct diagnosis is made.
At present, the myocardial marker is an effective method for rapidly detecting acute myocardial infarction, and can be used as a necessary condition for diagnosing the acute myocardial infarction. The research of the method is helpful for finding the state of an illness as early as possible, and the ideal myocardial infarction marker is used for the evaluation of treatment efficacy and the prognosis evaluation, so that the method has high clinical application value. Troponin cTnI exists in myocardial cells, because the content of troponin cTnI in normal serum is very small, the troponin cTnI is obviously increased after 6-8 hours of onset of AMI and is released into blood circulation, the concentration in blood is rapidly increased and lasts for 10-14 days, the change of the content of troponin cTnI is closely related to the treatment effect, the troponin cTnI is the best index for diagnosing AMI and monitoring postoperative and observing the curative effect, the degree of myocardial injury can be judged according to the measured value of cTnI, risk stratification is carried out, and after healing, treatment measures are further taken. Therefore, the cTnI is used as a cardiac marker to screen high-risk people, and the work of early diagnosis, disease development, prognosis monitoring and the like of AMI is greatly facilitated. In summary, achieving high sensitivity detection of specific markers in living bodies is an important issue in the field of life science, and development of a new and highly sensitive detection method is also an objective of the present research efforts.
The immunochromatographic test strip is an immunological detection technology which is simple to operate, does not need professional training, is convenient and quick, has an intuitive result, and plays an important role in modern POCT. At present, the test strip technology is mature, is rapidly popularized in the field of diagnosis, is gradually developed into a new mode of testing medicine, and is successfully applied to detection of various biological micromolecules, heavy metals and tumor markers. For example, in the conventional colloidal gold test strip, gold nanoparticles show a certain color to realize immunoassay of specific detection, although the detection result is visible by naked eyes, the method cannot complete accurate quantitative detection; the fluorescence immunochromatographic test strip detection technology is a new technology which is started for realizing quantitative detection after the colloidal gold immunochromatographic test strip technology, but the existing fluorescence immunochromatographic detection technology is still difficult to meet the diagnosis requirement of the accurate quantification of trace markers in serum in the research process, and the reason is mainly that: 1) on signal reporting, the signal strength is still not high enough; 2) background noise interferes significantly with the signal-to-noise ratio. 3) Abandons the advantage of the rapid color development detection of the colloidal gold test strip. Therefore, how to establish a high-sensitivity visualized AMI marker immunochromatography technology becomes a key problem of the current AMI early diagnosis. The magnetic nanometer microsphere is a spherical nanometer particle coated with a certain chemical group or immune ligand through modifying the surface, the binding specificity of the magnetic nanometer microsphere and a target substance is good, and the size and the shape of the spherical magnetic nanometer particle have good uniformity, so the target substance can be quickly and effectively bound on the magnetic microsphere. In addition, the magnetic nanoparticles can be used as a fluorescence quencher and have stronger quenching performance on fluorescent dyes. Therefore, by combining the visual detection characteristic of the superparamagnetic nanoparticle test strip and the advantage of high fluorescence detection sensitivity, the reaction mechanism of quenching a fluorescence signal of the magnetic nanoparticles under a specific condition and the visual detection result of the magnetic nanoparticle immunochromatographic test strip are utilized to construct a visual fluorescent quantitative detection immunochromatographic test strip, the interference of background fluorescence is eliminated through the reaction mechanism of quenching the fluorescence signal, and the rapid qualitative under the naked eye condition and the accurate quantitative wide-range bimodal detection under the dark field test are achieved through the signal intensity of fluorescence and the aggregation and color development of the magnetic nanoparticles, so that the accuracy of AMI early diagnosis and the universality of community screening are improved.
Disclosure of Invention
The invention utilizes the reaction mechanism that the magnetic nanoparticles can quench the fluorescence signal under specific conditions, combines the advantages of visualization of the detection result of the superparamagnetic nanoparticle immunochromatographic test strip and high fluorescence detection sensitivity, aims to construct a visualized fluorescence quantitative detection immunochromatographic test strip, eliminates the interference of background fluorescence through the reaction mechanism of quenching the fluorescence signal, and achieves the rapid qualitative under naked eye conditions and the accurate quantitative wide-range bimodal detection under dark field test through the signal intensity of fluorescence and the aggregation and color development of the magnetic nanoparticles, thereby improving the accuracy of AMI early diagnosis and the universality of community screening.
The technical method of the invention is as follows:
a preparation method of a high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement comprises the following steps:
1) preparing a magnetic nanoparticle immune probe:
dissolving water-soluble magnetic nanoparticles obtained after modification of macromolecules (polymers of 2, 5-furandione and 1-octadecene), labeled antibodies cTnI and EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) of markers related to acute myocardial infarction into a reaction solution, carrying out suspension culture at room temperature, activating carboxyl on the surfaces of the magnetic nanoparticles, centrifuging, washing, and finally, blocking the magnetic nanoparticles coupled with the antibodies with BSA overnight;
2) assembling the test strip:
the test paper strip of the invention is composed of a sample pad, a binding pad coated with magnetic nanoparticles marked by an acute myocardial infarction specific monoclonal antibody on the surface, a cTnI coated antibody marked by fluorescein Cy5, namely a detection line, sprayed with an IgG antibody, namely a quality control line, a water absorption pad at the tail end and a plastic bottom plate in sequence.
The high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip detection method based on reverse fluorescence enhancement comprises the following steps:
and (3) qualitative detection: according to the principle of double-antibody sandwich immunoreaction, adding a mixture of the high-molecular modified water-soluble magnetic nanoparticles marked by the antibody and the object to be detected into a sample pool, carrying out immunoreaction for 2-3min, when a sample to be detected contains a target antigen, capturing the complex by a T line and a C line simultaneously, gathering and developing through the magnetic nanoparticles, and directly carrying out naked eye qualitative detection to obtain a positive result; on the contrary, when the detection sample does not contain the target antigen, the color is only gathered and developed at the C line position, and the detection result is negative; if neither the T line nor the C line is developed, the test result of the test strip is invalid.
And (3) quantitative detection: the detection steps are the same as above, target antigen is dripped into the sample pool, the sample pool is placed into an analyzer for fluorescent signal reading after continuous immunization for 15-20min, and the difference value of fluorescence quenching of a test strip detection line region without dripping the sample and a detection region after dripping the sample is taken as a read signal; a standard curve of the detection technology is established.
In the step 1), the mass fraction ratio of the oil-soluble magnetic nanoparticles to the polymer of the macromolecular 2, 5-furandione and 1-octadecene is 1: 4-8; and the mass fraction ratio of the dosage of the EDC to the dosage of the magnetic nano-particles is 1: 40-80.
In the step 1), the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 80-180: 1.
in the step 1), after the magnetic nanoparticle coupled antibody is subjected to suspension culture for 1.5-2 hours, centrifuging and washing.
In the step 1), the dosage of the BSA blocking solution is 1-3%.
In the step 2), the pretreatment solution of the sample pad is a mixed solution of sucrose, bovine serum albumin BSA, polyethylene glycol PEG and polyoxyethylene sorbitol monolaurate Tween-20.
In the step 5), different concentrations of cTnI standard curve antigen are selected as follows: 0.01-10 ng/ml.
The high-sensitivity visual bimodal immunochromatographic test strip detection technology based on the reverse fluorescence enhancement has the advantages that:
1. the detection sensitivity of the POCT fluorescence immunochromatography technology is obviously improved. The detection signal is mainly amplified, and the organic combination of the two aspects of background noise is reduced, namely the detection signal is obviously amplified by the efficient fluorescence quenching characteristic within the large space action range of the magnetic nanoparticles; background noise interference is overcome by a fluorescence signal quenching mode, so that the sensitivity of the fluorescence immunochromatography detection technology is obviously improved.
2. The rapid and sensitive detection of the serum marker cTnI is realized. Based on the immunochromatography technology, the rapid detection is realized; high sensitivity detection is realized by a reverse fluorescence enhancement technology.
3. Realize quick visual qualitative detection and high-sensitivity fluorescent quantitative detection. The rapid aggregation and color development of the magnetic nanoparticles on the T line are used for realizing rapid visual qualitative detection; and the high-sensitivity quantitative detection is realized by the fluorescence quenching intensity.
Drawings
FIG. 1 is a schematic diagram of a test strip assembly;
fig. 2 is a transmission electron microscope photograph of the magnetic nanoparticles after the macromolecular modification of the immunochromatographic test strip based on the magnetic nanoparticles.
FIG. 3 shows the specificity of the immunochromatographic test strip based on magnetic nanoparticles.
The experimental values of the sensitivity of the magnetic nanoparticle-based immunochromatographic test strip prepared in fig. 4 with respect to cTnI.
Detailed Description
The invention will be further illustrated in the following examples, without however being restricted thereto.
The test strip is assembled as shown in figure 1, a sample pad is sequentially arranged from left to right, a binding pad of a myocardial infarction specific monoclonal antibody marked by magnetic nanoparticles is coated on the surface of the sample pad, a cTnI coating antibody marked by fluorescein Cy5, namely a detection line, is sprayed on a specific area of a nitrocellulose membrane, an area sprayed with an IgG antibody, namely a quality control line, a water absorption pad at the tail end and a plastic bottom plate form the test strip; the novel test strip detection technology of the invention modifies oil-soluble magnetic nanoparticles into water-soluble magnetic nanoparticles by using macromolecules, the dispersion condition of the modified nanoparticles in aqueous solution is shown in figure 2, and the modified nanoparticles are uniform in size; the test strip assembled above is placed in a plastic base plate, different kinds of antigens are dripped into a sample pool of the plastic base plate, the test strip is placed in an analyzer for signal reading after reacting for a period of time, the specific experiment numerical value of the immunochromatographic test strip is shown in figure 3, and the change of the fluorescence signal intensity of an experiment group is far higher than that of a control group; and finally, dropwise adding cTnI antigens with different concentrations, and determining the detection sensitivity of the technology, wherein the sensitivity can reach 0.049ng/mL as shown in figure 4.
The implementation process of the invention comprises the following steps:
1) accurately weighing quantitative EDC, and using a pipette to take quantitative macromolecule modified water-soluble magnetic nanoparticle solution and a myocardial infarction related marker cTnI labeled antibody to dissolve in 260 mu L of reaction aqueous solution. The mixed solution was put in a 2mL EP tube, and the EP tube was put on a spinner culture apparatus, and the spinner culture apparatus was rotated at room temperature for about 1.5 to 2 hours at a rotation speed of about 50 r/min. The liquid was then transferred to a 1.5mL tipped EP tube, the precipitate was collected thoroughly, centrifuged, washed, and blocked with 65. mu.L of 1% -3% BSA solution in a refrigerator at 4 ℃ overnight.
2) Marking a myocardial infarction marker cTnI coated antibody by using fluorescein Cy5, spraying the fluorescently-marked antibody on a nitrocellulose membrane to serve as a detection line, spraying a 1mg/mL goat anti-mouse antibody solution above the detection line to serve as a quality control line, and drying the detection line and the quality control line in a 37 ℃ oven for 1.5-2h after the detection line and the quality control line are sprayed; the width of the cut glass cellulose membrane is about 2cm, the cut glass cellulose membrane is immersed in 0.01M Tris-HCl buffer solution with pH value of 7.4 containing 0.5% Tween-20 to be used as a treatment solution of a sample pad, and after the immersion for 10-15 min, the sample pad is placed in an oven at 37 ℃ to be dried for 1.5-2 h. The processed sample pad and the binding pad coated with the myocardial infarction specific monoclonal antibody marked by the magnetic nanoparticles are sequentially stuck on a test strip and assembled by a plastic bottom plate customized by a company.
3) And (3) dripping 50 mu L of antigen with different concentrations into a sample cell of an assembled plastic bottom plate by using a pipette, reacting for about 2 minutes, and carrying out naked eye qualitative detection according to whether black magnetic nanoparticles are aggregated in a detection line area.
4) And after continuing to react for 15-20min, placing the mixture into a gel analyzer for photographing, and counting and calculating the change of the fluorescence value.
The above reaction is a general step for preparing a high-sensitivity visualization bimodal immunochromatographic test strip detection technology based on reverse fluorescence enhancement, and is not detailed in the examples.
Example 1:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:4, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA confining liquid is 2%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is taken as a read signal, and the result of the fluorescence difference value is counted to be 36746; after reacting for 18min, the result of the fluorescence difference is 36927; after a further 20min reaction, the difference in fluorescence was 37001.
Example 2:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA blocking solution is 2%, and 50 μ L of cTnL standard antigen with the concentration of 5ng/mL is dripped into a sample pool for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is taken as a read signal, and the result of the fluorescence difference value is counted to be 39219; after reacting for 18min, the result of the difference of fluorescence is 39636; after a further 20min reaction, the difference in fluorescence was 39991.
Example 3:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1: 8, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nano-particles is 1:40, the dosage of BSA confining liquid is 2%, and 50 mu L of cTnI standard antigen with the concentration of 5ng/ml is dripped into a sample pool for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is taken as a read signal, and the result of the fluorescence difference value is counted to be 38020; after reacting for 18min, the result of the difference of fluorescence is 38461; after a further 20min reaction, the difference in fluorescence was 38997.
Example 4:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 80: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA confining liquid is 2%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is used as a read signal, the statistical fluorescence difference result is 38116, and after the reaction is carried out for 18min, the fluorescence difference result is 38476; after a further 20min reaction, the difference in fluorescence was 38849.
Example 5:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 180: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA confining liquid is 2%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is taken as a read signal, the statistical fluorescence difference value result is 38907, and after the reaction is carried out for 18min, the fluorescence difference value result is 39133; after a further 20min reaction, the difference in fluorescence was 39334.
Example 6:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:20, the dosage of BSA confining liquid is 2%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region without the dropwise added sample and the detection region after the dropwise added sample is taken as a read signal, the statistical fluorescence difference result is 38211, and after the reaction is carried out for 18min, the fluorescence difference result is 38577; after a further 20min reaction, the difference in fluorescence was 38914.
Example 7:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:60, the dosage of BSA confining liquid is 2%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region without the dropwise added sample and the detection region after the dropwise added sample is taken as a read signal, the statistical fluorescence difference result is 38804, and after the reaction is carried out for 18min, the fluorescence difference result is 39017; after the reaction was continued for 20min, the difference in fluorescence was 39243.
Example 8:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA blocking solution is 1%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region where the sample is not dripped and the detection region where the sample is dripped is taken as a read signal, the statistical fluorescence difference result is 39110, and after the reaction is carried out for 18min, the fluorescence difference result is 39472; after a further 20min reaction, the difference in fluorescence was 39899.
Example 9:
a preparation method based on a reverse fluorescence enhanced high-sensitivity visual bimodal immunochromatographic test strip detection technology; spraying a fluorescein Cy 5-labeled acute myocardial infarction related marker cTnI coated antibody at the detection line position of a nitrocellulose membrane, wherein the mass fraction ratio of magnetic nanoparticles to a polymer of macromolecule 2, 5-furandione and 1-octadecene is 1:6, the mass fraction ratio of the usage amount of the magnetic nanoparticles to the cTnI antibody is 120: 1, the mass fraction ratio of the dosage of EDC and the magnetic nanoparticles is 1:40, the dosage of BSA confining liquid is 3%, and 50 μ L of cTnI standard antigen with the concentration of 5ng/mL is dripped into a sample cell for three times. The qualitative detection method comprises the following steps: after the sample test strip is dripped for reaction for 2min, a reading frame is observed by naked eyes, black magnetic nanoparticles are gathered in the detection lines and the control lines to present black strips, the strip colors are close to 2min after 2.5min, and the strip colors are deepened a little bit after 3min when the strip colors in the detection lines and the control line are closer to 2 min. The quantitative detection method comprises the following steps: after the sample continuously reacts for 15min, the sample is put into an analyzer for signal reading, the fluorescence quenching difference value of the test strip detection line region without the dropwise added sample and the detection region after the dropwise added sample is taken as a read signal, the statistical fluorescence difference result is 36741, and after the reaction is carried out for 18min, the fluorescence difference result is 36902; after a further 20min reaction, the difference in fluorescence was 37111.
The invention relates to a preparation and detection method of a high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement; the method is simple to operate, does not need professional training, is convenient and rapid, has intuitive results, and can realize the accuracy of AMI early diagnosis and the universality of community screening by rapid and high-sensitivity detection.
Claims (8)
1. The preparation method of the high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on reverse fluorescence enhancement is characterized by comprising the following steps:
1) preparing a magnetic nanoparticle immune probe, dissolving water-soluble magnetic nanoparticles modified by 2, 5-furandione and 1-octadecene polymer, a labeled antibody cTnI of a related marker of Acute Myocardial Infarction (AMI), EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) in a reaction solution, suspending at room temperature, activating carboxyl on the surface of the magnetic nanoparticles, centrifuging, washing, and finally, blocking the magnetic nanoparticles coupled with the antibody by BSA overnight;
2) the assembled test strip of the test strip sequentially comprises a sample pad, a binding pad coated with magnetic nanoparticles marked by an acute myocardial infarction specific monoclonal antibody on the surface, a cTnI coated antibody marked by fluorescein Cy5, namely a detection line, sprayed on a specific area of a nitrocellulose membrane, a quality control line, a water absorption pad at the tail end and a plastic bottom plate.
2. The method as claimed in claim 1, wherein the mass fraction ratio of the magnetic nanoparticles, the high molecular 2, 5-furandione and the polymer of 1-octadecene is 1:4-8, and the mass fraction ratio of the dosage of EDC and the magnetic nano-particles is 1: 40-80.
3. The method of claim 1, wherein the amount of BSA blocking solution is 1% to 3%.
4. The method as set forth in claim 1, wherein the sample pad is immersed in Tris-HCl buffer containing Tween-20 (polyoxyethylene sorbitan monolaurate) and left to stand for 5-10min, and then put in a 37 ℃ constant temperature drying oven to be completely dried to obtain a pretreated sample pad, and the bag is sealed and stored at 4 ℃.
5. The method of claim 1, wherein the treatment solution of the conjugate pad is a mixed solution of sucrose, bovine serum albumin BSA, polyethylene glycol PEG, polyoxyethylene sorbitan monolaurate Tween-20.
6. The method for detecting the high-sensitivity visual bimodal acute myocardial infarction immunochromatographic test strip based on the reverse fluorescence enhancement of claim 1 is characterized in that the qualitative detection method comprises the following steps: according to the principle of double-antibody sandwich immunoreaction, adding a mixture of the high-molecular modified water-soluble magnetic nanoparticles marked by the antibody and the object to be detected into a sample pool, carrying out immunoreaction for 2-3min, when a sample to be detected contains a target antigen, capturing the complex by a T line and a C line simultaneously, gathering and developing through the magnetic nanoparticles, and directly carrying out naked eye qualitative detection to obtain a positive result; on the contrary, when the detection sample does not contain the target antigen, the color is only gathered and developed at the C line position, and the detection result is negative; if neither the T line nor the C line is developed, the test result of the test strip is invalid.
7. The method of claim 6, wherein the quantitative detection method is: and (3) dropwise adding a target antigen into the sample pool, continuously immunizing for 15-20min, then putting into an analyzer for reading a fluorescence signal, taking a fluorescence quenching difference value between a test strip detection line region without dropwise adding the sample and a detection region after dropwise adding the sample as a read signal, and establishing a standard curve of the detection technology.
8. The method of claim 1, wherein the various concentrations of the cTnI antigen are selected from the group consisting of: 0-10ng/mL, and establishing a standard curve.
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