CN110794137A - Alpha fetoprotein immunochromatographic test strip and preparation method and application thereof - Google Patents
Alpha fetoprotein immunochromatographic test strip and preparation method and application thereof Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention provides an alpha fetoprotein immunochromatographic test strip and a preparation method and application thereof, the alpha fetoprotein immunochromatographic test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a backing plate, wherein a composite fluorescent microsphere probe is combined on the combination pad, the composite fluorescent microsphere probe is a microsphere probe formed by an alpha fetoprotein monoclonal antibody, a carbon nano tube and water-soluble quantum dots, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line contains an alpha fetoprotein antibody, and the quality control line contains goat anti-mouse IgG. The alpha fetoprotein immunochromatographic test strip is simple in preparation method and easy to operate, the fluorescence intensity of the alpha fetoprotein detection is obviously enhanced, the detection is accurate, the sensitivity is obviously enhanced, and the minimum detection limit is 0.02 ng/mL.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and relates to an alpha fetoprotein immunochromatography test strip, and a preparation method and application thereof.
Background
The primary liver cancer is one of the most common clinical malignant tumors, the incidence rate of the primary liver cancer is on the rise in the world, and the method has important significance for the accurate diagnosis of the primary liver cancer and the treatment of the liver cancer.
Alpha fetoprotein is a specific clinical index for diagnosing primary liver cancer, currently, immunoassay is mainly adopted for detecting the alpha fetoprotein, and CN103558396A discloses a quantitative detection method of the alpha fetoprotein, which comprises the following steps: (1) mixing the alpha fetoprotein antibody with a hydroformylation glucoamylase solution to prepare an enzyme-labeled antibody; loading the alpha fetoprotein antibody on the nano gold magnetic particles; (2) adding the alpha-fetoprotein antigen sample and a series of alpha-fetoprotein antigen standard samples with different concentrations into an immunoreaction interface of the antibody-loaded nano gold magnetic particles for incubation, measuring the glucose concentration by a glucometer to obtain a linear equation of the corresponding relation between the alpha-fetoprotein concentration and the glucose concentration, and substituting the glucose concentration value of the alpha-fetoprotein antigen sample into the linear equation to calculate the alpha-fetoprotein concentration of the alpha-fetoprotein antigen sample.
CN108709914A discloses a method for rapidly detecting alpha fetoprotein, which comprises the steps of coating a single-arm carbon nanotube alpha fetoprotein antibody dispersion liquid on a paper base material, determining the resistance of a paper-based carbon nanotube sensor according to the resistance change of the paper-based sensor before and after the antibody is combined with an antigen by utilizing the characteristic of specific combination of the alpha fetoprotein and the antibody thereof, and detecting the content of a target antigen by recording the resistance change of the sensor within a certain time to realize the detection of the alpha fetoprotein.
Although these detection methods can detect alpha-fetoprotein, they are complicated to operate and time-consuming, and therefore, it is desired in the art to develop a method for detecting alpha-fetoprotein simply and rapidly.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an alpha fetoprotein immunochromatographic test strip, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
on one hand, the invention provides an alpha fetoprotein immunochromatographic test strip which comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a backing plate, wherein a composite fluorescent microsphere probe is combined on the combination pad, the composite fluorescent microsphere probe is a microsphere probe formed by an alpha fetoprotein monoclonal antibody, a carbon nano tube and water-soluble quantum dots, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line contains an alpha fetoprotein antibody, and the quality control line contains goat anti-mouse IgG.
In the invention, the alpha fetoprotein immunochromatographic test strip developed by a microspherical probe formed by the alpha fetoprotein monoclonal antibody, the carbon nano tube and the water-soluble quantum dot takes the carbon nano tube as a core, the water-soluble quantum dot and the alpha fetoprotein monoclonal antibody are coupled, and a large number of water-soluble quantum dots are combined on the surface of the carbon nano tube, so that the fluorescence intensity is obviously enhanced, and the sensitivity of the test strip for detecting the alpha fetoprotein is obviously enhanced.
Preferably, the carbon nanotubes are multi-walled carbon nanotubes or single-walled carbon nanotubes.
Preferably, the water-soluble quantum dots are carboxyl-coated CdTe quantum dots or carboxyl-coated CdTe/ZnSe core-shell quantum dots.
In the invention, the carboxyl-coated CdTe quantum dot or the carboxyl-coated CdTe/ZnSe core-shell quantum dot can be prepared by the prior art.
Preferably, the preparation method of the composite fluorescent microsphere probe comprises the following steps: incubating aminated carbon nanotubes and alpha fetoprotein monoclonal antibodies at 37 ℃ under the action of a condensing agent, centrifuging, redissolving precipitates after centrifugation, adding the condensing agent and carboxylated water-soluble quantum dots, incubating at 37 ℃, and centrifuging to obtain the composite fluorescent microsphere probe.
Preferably, when the aminated carbon nanotube and the alpha fetoprotein monoclonal antibody are incubated at 37 ℃ under the action of a condensing agent, the condensing agent used is any one of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and N-hydroxythiosuccinimide or the combination of at least two of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide and the N-hydroxythiosuccinimide.
Preferably, the time for incubating the aminated carbon nanotube and the alpha fetoprotein monoclonal antibody at 37 ℃ under the action of the condensing agent is 30min-2h, such as 30min, 35min, 45min, 1h, 1.2h, 1.5h, 1.8h or 2 h.
Preferably, when the condensing agent and the carboxylated water-soluble quantum dot are added, the condensing agent is any one or a combination of at least two of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and N-hydroxythiosuccinimide.
Preferably, the condensing agent and the carboxylated water-soluble quantum dot are added, and the incubation time at 37 ℃ is 30min-2h, such as 30min, 35min, 45min, 1h, 1.2h, 1.5h, 1.8h or 2 h.
In another aspect, the invention provides a preparation method of the above-mentioned alpha fetoprotein immunochromatographic test strip, which comprises the following steps:
and (2) spraying the composite fluorescent microsphere probe on the bonding pad to obtain the bonding pad sprayed with the composite fluorescent microsphere probe, then respectively scribing the alpha fetoprotein antibody and goat anti-mouse IgG on a nitrocellulose membrane to obtain a detection line and a quality control line, and assembling the sample pad, the bonding pad sprayed with the composite fluorescent microsphere probe, the nitrocellulose membrane with the detection line and the quality control line and a water absorption pad on a back plate to obtain the alpha fetoprotein immunochromatographic test strip.
Preferably, the scribing is performed with a scribe.
In the invention, the preparation method of the alpha fetoprotein immunochromatographic test strip is simple, efficient and easy to operate.
In another aspect, the invention provides an application of the above-mentioned alpha-fetoprotein immunochromatographic test strip in the preparation of an alpha-fetoprotein detection device.
Compared with the prior art, the invention has the following beneficial effects:
the alpha fetoprotein immunochromatographic test strip developed by a microsphere probe formed by an alpha fetoprotein monoclonal antibody, a carbon nano tube and water-soluble quantum dots takes the carbon nano tube as a core, is coupled with the water-soluble quantum dots and the alpha fetoprotein monoclonal antibody, and a large number of water-soluble quantum dots are combined on the surface of the carbon nano tube, so that the fluorescence intensity is obviously enhanced, the detection is accurate, the sensitivity of the detection of the alpha fetoprotein is obviously enhanced, and the minimum detection limit is 0.02 ng/mL.
Drawings
FIG. 1 is a schematic structural diagram of the alpha fetoprotein immunochromatographic test strip of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this embodiment, an alpha fetoprotein immunochromatographic test strip is provided, the alpha fetoprotein immunochromatographic test strip includes a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a backing plate, the combination pad is combined with a composite fluorescent microsphere probe, the composite fluorescent microsphere probe is a microsphere probe formed by an alpha fetoprotein monoclonal antibody, a multiwalled carbon nanotube and a water-soluble quantum dot carboxyl-coated CdTe quantum dot, the nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line contains an alpha fetoprotein antibody, and the quality control line contains goat anti-mouse IgG. The structure of the device is schematically shown in figure 1.
The preparation method of the carboxyl-coated CdTe quantum dot comprises the following steps: 244mL concentration at nitrogen saturation was 2X 10-2mol/LCdCl2·2.5H2To the O solution (114.2mg) was added 104.4. mu.L of mercaptopropionic acid, and the pH of the solution was adjusted to about 9.2 with 1mol/L of LNaOH. Freshly prepared NaHTe was injected and N was used with vigorous stirring2Deoxidizing for 20min, then quickly adding the newly prepared sodium hydrogen telluride solution, injecting into a reaction system, and refluxing for 2h under the protection of nitrogen at 100 ℃ to obtain the carboxyl-coated CdTe quantum dot.
The preparation method of the composite fluorescent microsphere probe comprises the following steps: dispersing 0.02g of carbon nanotube with PBS (pH 7.4), activating with 0.1mL of EDC and 0.15mL of NHS for 10min, adding 100mL of alpha fetoprotein monoclonal antibody, incubating at 37 ℃ for 1h, centrifuging to remove unbound antibody and other impurities, redissolving the centrifuged precipitate with PBS (pH 7.4), activating with 0.1mL of EDC and 0.15mL of NHS for 10min, adding 5mL of carboxyl-coated CdTe quantum dots, incubating at 37 ℃ for 1h, centrifuging to remove free quantum dots and impurities, redissolving the precipitate with PBS (pH 8.0) containing BSA, sucrose, PEG20000, Tween-80, sodium azide and the like, and storing in a refrigerator at 4 ℃ for later use.
And respectively scribing the alpha fetoprotein antibody and goat anti-mouse IgG onto a nitrocellulose membrane by using a scribing instrument to obtain a detection line and a quality control line, and assembling the sample pad, the binding pad sprayed with the composite fluorescent microsphere probe, the nitrocellulose membrane with the detection line and the quality control line and a water absorption pad on a back plate to obtain the alpha fetoprotein immunochromatography test strip.
Example 2
In this embodiment, an alpha fetoprotein immunochromatographic test strip is provided, and the alpha fetoprotein immunochromatographic test strip includes a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a backing plate, wherein the combination pad is combined with a composite fluorescent microsphere probe, the composite fluorescent microsphere probe is a microsphere probe formed by an alpha fetoprotein monoclonal antibody, a multiwall carbon nanotube and a water-soluble quantum dot carboxyl-coated CdTe/ZnSe core-shell quantum dot, and the nitrocellulose membrane is provided with a detection line and a quality control line, wherein the detection line contains an alpha fetoprotein antibody, and the quality control line contains goat anti-mouse IgG. The structure of the device is schematically shown in figure 1.
The preparation method of the carboxyl-coated CdTe/ZnSe core-shell quantum dot comprises the following steps: synthesizing oil-soluble CdTe/CdSe core-shell quantum dots in a high-temperature organic phase, then performing ligand exchange by using MPA, and transferring the oil-soluble quantum dots to a water phase. To obtain the water-soluble CdTe/CdSe quantum dots coated by carboxyl.
The preparation method of the composite fluorescent microsphere probe comprises the following steps: dispersing 0.02g of carbon nanotube with PBS (pH 7.4), activating with 0.1mL of EDC and 0.2mL of NHS for 10min, adding 100mL of alpha fetoprotein monoclonal antibody, incubating at 37 ℃ for 1h, centrifuging to remove unbound antibody and other impurities, redissolving the centrifuged precipitate with PBS (pH 7.4), activating with 0.1mL of EDC and 0.2mL of NHS for 10min, adding 5mL of carboxyl-coated CdTe/ZnSe core-shell quantum dots, incubating at 37 ℃ for 1h, centrifuging to remove free quantum dots and impurities, redissolving the precipitate with PBS (pH 8.0) containing BSA, sucrose, PEG20000, Tween-80, sodium azide and the like, and storing at 4 ℃ in a refrigerator for later use.
And respectively scribing the alpha fetoprotein antibody and goat anti-mouse IgG onto a nitrocellulose membrane by using a scribing instrument to obtain a detection line and a quality control line, and assembling the sample pad, the binding pad sprayed with the composite fluorescent microsphere probe, the nitrocellulose membrane with the detection line and the quality control line and a water absorption pad on a back plate to obtain the alpha fetoprotein immunochromatography test strip.
Example 3
In this example, the alpha-fetoprotein immunochromatographic test strips of example 1 and example 2 were used for semiquantitative and quantitative detection.
Semi-quantitative detection: and (3) dropwise adding a sample to be detected onto the sample pad, carrying out chromatographic expansion on the sample to be detected along the directions of the combination pad, the nitrocellulose membrane and the water absorption pad under the action of the water absorption pad, wherein a red line appearing on the detection line is positive, otherwise, the detection line is negative, and if the quality control line does not show red, the detection is invalid.
Preparing a series of alpha-fetoprotein standard solutions: 0ng/mL, 0.001ng/mL, 0.005ng/mL, 0.01ng/mL, 0.02ng/mL, 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 0.8ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, and then dropped on the sample pad of the fetoprotein immunochromatographic test strip of example 1 and example 2, respectively, to perform detection, and the test was performed on average 5 times at each concentration, and color development was observed, the color development results of which are shown in Table 1 below.
TABLE 1
According to the initial judgment of the results in the table 1, the lowest detection limit of the alpha fetoprotein immunochromatographic test strip is 0.02 ng/mL.
The embodiment can realize the quantitative detection of the alpha fetoprotein, and the detection method comprises the following steps:
and (3) quantitative detection: preparing a series of alpha-fetoprotein standard solutions: 0ng/mL, 0.005ng/mL, 0.01ng/mL, 0.02ng/mL, 0.03ng/mL, 0.04ng/mL, 0.08ng/mL, 0.5ng/mL, 0.8ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, then respectively dripping the test paper on the sample pad of the alpha fetoprotein immunochromatography test paper strip of the example 1 and the example 2, photographing with an olympus camera in a reflective ultraviolet dark box after 15 minutes, and then, the photo is led into a computer, the photo shop software is used for processing the photo shop photo to be gray, then the Scion Image software is used for converting the strip into a peak value signal, the corresponding concentration of each peak area is respectively used for making a standard curve to obtain a peak area and concentration corresponding formula, the detection sample is processed in the same way to obtain a peak area, and the content of the alpha fetoprotein in the sample can be obtained according to the formula.
According to the detection method, an alpha-fetoprotein sample solution with known concentration is prepared, wherein the concentration of an alpha-fetoprotein sample in a sample to be detected No. 1 is 0.02ng/mL, the concentration of an alpha-fetoprotein sample in a sample to be detected No. 2 is 0.05ng/mL, the concentration of an alpha-fetoprotein sample in a sample to be detected No. 3 is 0.5ng/mL, the concentration of an alpha-fetoprotein sample in a sample to be detected No. 4 is 12ng/mL, the concentration of an alpha-fetoprotein sample in a sample to be detected No. 5 is 50ng/mL, the samples to be detected are respectively dripped on sample pads of the alpha-fetoprotein immunochromatography test strips of example 1 and example 2, after 15 minutes, an orinbis used for photographing in a reflective ultraviolet camera, then the photos are led into a computer, are processed into gray scale by photoshop software, then the strips are converted into peak signals by ScionImage software, a signal value standard curve, the alpha-fetoprotein content was obtained and the results are shown in table 2.
TABLE 2
As can be seen from the table 2, the alpha fetoprotein immunochromatographic test strip can well perform quantitative detection, and has high detection accuracy.
The applicant states that the invention is illustrated by the above examples to the alpha fetoprotein immunochromatographic test strip of the invention, the preparation method and the application thereof, but the invention is not limited to the above examples, that is, the invention is not limited to the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. The utility model provides an alpha fetoprotein immunochromatographic test strip, which is characterized in that, alpha fetoprotein immunochromatographic test strip includes sample pad, combination pad, cellulose nitrate membrane and water absorption pad and backing plate, it has compound fluorescence microsphere probe to combine on the combination pad, compound fluorescence microsphere probe is the microsphere probe that alpha fetoprotein monoclonal antibody and carbon nanotube and water-soluble quantum dot formed, last detection line and the quality control line of cellulose nitrate membrane, wherein contain the alpha fetoprotein antibody on the detection line, contain sheep anti mouse IgG on the quality control line.
2. The alpha fetoprotein immunochromatographic test strip of claim 1, wherein the carbon nanotubes are multi-walled carbon nanotubes or single-walled carbon nanotubes.
3. The alpha fetoprotein immunochromatographic test strip according to claim 1 or 2, wherein the water-soluble quantum dots are carboxyl-coated CdTe quantum dots or carboxyl-coated CdTe/ZnSe core-shell quantum dots.
4. The alpha fetoprotein immunochromatographic test strip according to any one of claims 1 to 3, wherein the preparation method of the composite fluorescent microsphere probe is as follows: incubating aminated carbon nanotubes and alpha fetoprotein monoclonal antibodies at 37 ℃ under the action of a condensing agent, centrifuging, redissolving precipitates after centrifugation, adding the condensing agent and carboxylated water-soluble quantum dots, incubating at 37 ℃, and centrifuging to obtain the composite fluorescent microsphere probe.
5. The alpha fetoprotein immunochromatographic test strip according to claim 4, wherein when the aminated carbon nanotube and the alpha fetoprotein monoclonal antibody are incubated at 37 ℃ under the action of a condensing agent, the condensing agent used is any one of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and N-hydroxythiosuccinimide or a combination of at least two of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide and the N-hydroxythiosuccinimide.
6. The alpha fetoprotein immunochromatographic test strip according to claim 4 or 5, wherein the incubation time of the aminated carbon nanotube and the alpha fetoprotein monoclonal antibody at 37 ℃ under the action of the condensing agent is 30min-2 h.
7. The immunochromatographic strip for alpha fetoprotein according to any one of claims 4 to 6, wherein when a condensing agent and a carboxylated water-soluble quantum dot are added, the condensing agent is any one of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and N-hydroxythiosuccinimide or a combination of at least two of the above.
8. The alpha fetoprotein immunochromatographic test strip according to any one of claims 4 to 7, wherein the condensing agent and the carboxylated water-soluble quantum dot are added, and the incubation time at 37 ℃ is 30min-2 h.
9. The method for preparing the alpha fetoprotein immunochromatographic test strip according to any one of claims 1 to 8, comprising the steps of:
spraying the composite fluorescent microsphere probe on a bonding pad to obtain the bonding pad sprayed with the composite fluorescent microsphere probe, then respectively scribing the alpha fetoprotein antibody and goat anti-mouse IgG on a nitrocellulose membrane to obtain a detection line and a quality control line, and assembling a sample pad, the bonding pad sprayed with the composite fluorescent microsphere probe, the nitrocellulose membrane with the detection line and the quality control line and a water absorption pad on a back plate to obtain the alpha fetoprotein immunochromatography test strip;
preferably, the scribing is performed with a scribe.
10. Use of the alpha-fetoprotein immunochromatographic test strip according to any one of claims 1 to 8 in the preparation of an alpha-fetoprotein detection device.
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