CN110927133A - Fluorescence immunochromatography test paper for rapidly detecting heavy metal lead in food - Google Patents
Fluorescence immunochromatography test paper for rapidly detecting heavy metal lead in food Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food, which comprises a bottom plate, wherein a sample pad, a combination pad, a coating film and absorbent paper are sequentially and mutually stuck on the bottom plate in a staggered manner, the upper end face of one end of the coating film is stuck with one end of the combination pad, the upper end face of the other end of the coating film is stuck with one end of the absorbent paper, the coating film is coated with a detection line and a quality control line, the upper end face of the other end of the combination pad is stuck with one end of the sample pad, the combination pad is coated with a lead specific antibody marked by fluorescent microspheres, the detection line is composed of a lead ion complete antigen, and the quality control line is composed of goat anti-mouse IgG. According to the invention, the fluorescent microspheres are used as the markers, the fluorescence is used as the detection signal, and the detection result is observed under an ultraviolet lamp, so that the influence of the detection matrix is reduced, the anti-interference performance is improved, the detection can be rapidly carried out, the sensitivity is high, and the defects of the existing detection mode are overcome.
Description
Technical Field
The invention relates to the technical field of rapid detection of heavy metal in food safety, in particular to a fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food.
Background
With the development of urbanization and industrialization, the problem of heavy metal pollution is increasingly serious in the atmosphere, soil, water and food, and the serious influence is caused on food safety, human health and economic benefit. Lead is one of the most potentially harmful heavy metal ions known. Heavy metal lead exists in food in the form of compounds, enters a human body through the digestive tract, is not easy to metabolize and accumulates in the body, is harmful to the human body in multiple systems, multiple organs and irreversible, and seriously harms human health.
The traditional lead detection methods comprise atomic absorption spectrometry, fluorescence spectrometry, inductively coupled plasma mass spectrometry and the like, and the detection methods need complex sample pretreatment, are time-consuming, high in cost and difficult to popularize, and are difficult to realize field detection and portability. At present, some emerging rapid detection means such as a colorimetric method, an electrochemical method, an enzyme-linked immunosorbent assay, a colloidal gold immunochromatography test paper and the like have low sensitivity, are easily influenced by the complexity of a matrix and have low anti-interference performance although the operation is simple. Therefore, it is necessary to establish a method for detecting the heavy metal lead in the food, which is rapid, reliable, simple and convenient to operate and low in cost.
Chinese patent CN204882566U discloses an immune colloidal gold reagent plate for rapidly detecting heavy metal lead, and specifically discloses the following technical features; including two piece upper and lower plastic formwork, the PVC end liner, the sample pad, gold mark antibody conjugate pad, nitrocellulose membrane and absorbent paper, the PVC end liner bonds the sample pad from left to right, gold mark antibody conjugate pad, nitrocellulose membrane and absorbent paper, and form the overlap joint structure, spout parallel detection line and control line on the nitrocellulose membrane, both are 5mm apart from, the detection line is the sign of a mark of artifical peridium antigen, the control line is goat anti mouse IgG's sign of a mark, spout the specific antibody that has the colloidal gold mark on the gold mark antibody conjugate pad, sample pad and gold mark antibody conjugate pad all have glass fiber to make. According to the technical scheme, the detection result is judged according to the color depth degree on a control line observed by naked eyes by the condition that a gold-labeled antibody reacts with the specificity of an artificial antigen, so that the advantages of good specificity, strong anti-interference performance and the like are mainly achieved, but the detection mode of the detection reagent plate mainly has the following defects:
(1) the sensitivity is low, and the sensitivity of the detection reagent plate is as follows: the minimum detection limit of the detection reagent plate for heavy metal lead in water is 100 mug/L, namely 100 mug/kg, although the national limit requirement is met, the detection reagent plate can not be applied to the detection environment with lead content lower than 100 mug/kg, so that the sensitivity is low, and the detection reagent plate can not be used for detecting the lead content in the food field with lead content lower than 100 mug/kg;
(2) the detection reagent plate is easily influenced by the complexity of the matrix, when the result is judged, the result is judged according to the color depth of the detection line observed by naked eyes, and the detection reagent plate is obviously easily subjected to the coloring interference of the detection matrix and easily generates the judgment error, so the detection reagent plate is easily influenced by the complexity of the matrix;
(3) the cost is high, the colloidal gold used by the gold-labeled antibody is made of colloidal gold particles, the gold is made of expensive noble metal, and the expensive noble metal is used for labeling and detecting, so the manufacturing and using costs are obviously greatly increased, and the cost is high.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the fluorescence immunochromatographic test paper for rapidly detecting the heavy metal lead in the food is provided, the fluorescent microspheres are used as the markers, the fluorescence is used as the detection signals, the detection result is observed under an ultraviolet lamp, the influence of the matrix of the sample extracting solution is further reduced, the anti-interference performance of the sample extracting solution is improved, and meanwhile, the detection mode is adopted, so that the rapid detection can be carried out, the sensitivity of the sample extracting solution is greatly improved, and the defects of the existing detection mode are overcome.
The technical scheme adopted by the invention is as follows: the utility model provides a fluorescence immunochromatographic test paper of heavy metal lead in short-term test food, includes the bottom plate, paste sample pad, combination pad, envelope membrane and absorbent paper in proper order and crisscross each other on the bottom plate, the up end of envelope membrane one end is laminated with the one end of combination pad, and the up end of the other end of envelope membrane is laminated with the one end of absorbent paper, and envelope membrane upper cladding has detection line and quality control line, and the up end of the combination pad other end is laminated with the one end of sample pad, and detection line and quality control line are not crisscross each other, the combination is filled up and is wrapped up the plumbum specificity antibody that has the fluorescence microballon mark, the detection line comprises the complete antigen of plumbum ion, the quality control line comprises goat anti mouse IgG.
Further, the sample pad is glass fiber cotton, the combination pad is a polyester fiber film, and the coating film is a nitrocellulose film.
Further, the detection line and the quality control line are parallel to each other with a gap, the detection line is close to the combination pad, and the quality control line is close to the absorbent paper.
Further, the lead specific antibody is a mouse-derived monoclonal antibody, the fluorescent microsphere is a microsphere doped with 100-300 nm-diameter rare earth europium ion chelate, and the surface of the microsphere is provided with active group carboxyl.
Further, the lead ion complete antigen is a coupling compound formed by lead ions, a chelating agent and carrier protein.
Further, the chelating agent is ethylenediamine tetraacetic acid, and the carrier protein is bovine serum albumin.
Further, the method for forming the fluorescent microsphere marked lead-specific antibody spray coating on the bonding pad comprises the following steps:
step 1, adding a designed amount of europium ion chelate fluorescent microspheres into a prepared boric acid buffer solution in advance, and then carrying out ultrasonic dispersion;
step 2, after the step 1 is finished, adding carbodiimide and N-hydroxysuccinimide in designed amount into the solution, carrying out centrifugal washing after oscillation reaction, and then dispersing the solution into boric acid buffer solution;
and 5, mixing the storage liquid according to the volume ratio of 1: (90-100) diluting in boric acid buffer solution containing bovine serum albumin, Tween-20, polyvinylpyrrolidone, trehalose and sodium azide, spraying on the bonding pad by using a quantitative film spraying instrument, drying by blowing at 30-40 ℃, and drying in dark at 3-6 ℃ to obtain the product.
Further, in the step 5, the boric acid buffer solution contains bovine serum albumin with the mass concentration of 0.4-0.6%, Tween-20 with the volume fraction of 2-3%, polyvinylpyrrolidone with the mass concentration of 0.4-0.6%, trehalose with the mass concentration of 1-3% and sodium azide with the mass concentration of 0.01-0.03%, and the pH value of the boric acid buffer solution is 8.0-8.5.
The detection principle of the fluorescence immunochromatographic test paper provided by the invention is as follows: the invention applies the principle of competitive inhibition immunochromatography, takes fluorescence as a detection signal, dropwise adds the sample solution to be detected on a sample pad, the sample to be detected contains Pb (II) -EDTA (i.e. a coupling compound of lead ions (Pb (II)) and chelating agent Ethylene Diamine Tetraacetic Acid (EDTA)) by electrophoresis on the test paper through chromatography, then Pb (II) -EDTA is combined with the lead specific antibody marked by the fluorescent microspheres in the combination pad in an immune manner, further blocking the antigen binding site of Pb (II) -EDTA on the fluorescent microsphere-antibody, inhibiting the antibody from binding with the antigen (Pb (II) -EDTA-BSA conjugate) on the detection line, reducing the amount of the antibody bound on the detection line with the increase of the content of Pb (II) -EDTA in the sample, and correspondingly reducing the fluorescence intensity on the detection line, and the goat anti-mouse IgG on the quality control line is not influenced by the concentration of Pb (II) -EDTA to bind the fluorescent microsphere labeled antibody.
Because the europium ion chelate fluorescent microspheres emit bright red light under an ultraviolet lamp, the liquid to be detected is dripped for 5 minutes and then is observed under the ultraviolet lamp of 365nm, the negative result presents two red fluorescent lines, namely a detection line and a quality control line, when Pb (II) -EDTA is higher than or equal to 10 mug/kg, the detection line disappears, only one quality control line appears, and at the moment, the result is a positive result, the detection process is rapid, simple and convenient, and the result is visual.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. the fluorescence immunochromatographic test paper has high sensitivity, can be used for qualitative detection of heavy metal lead, has the sensitivity as low as 10 mu g/kg, is far higher than the sensitivity of a detection mode using a gold marker in the background technology, and can meet the requirement of detection of heavy metal lead in the food field;
2. the influence of the complexity of the matrix is small, and the invention adopts fluorescent microspheres as the marker, fluorescence as the detection signal and then observes the detection result under an ultraviolet lamp, thereby completely avoiding the problem of judging the coloring influence of the detected matrix and greatly weakening the influence of the detected matrix;
3. the fluorescent immunochromatographic test paper has simple and quick detection and strong anti-interference performance, the processes of detection, treatment, judgment and the like are simple, the detection result can be obtained within 10min, the detection is simple and quick, and meanwhile, because the competitive inhibition immunochromatographic technology is adopted, the detection of the heavy metal lead in the food is realized through specific immunoreaction, the specificity is good, and the anti-interference performance is strong;
4. the invention has the advantages of low cost of the used materials such as the marker, the antibody, the antigen and the like, uncomplicated structure, simple production process and mature flow, and is worthy of application and popularization.
Drawings
FIG. 1 is a schematic structural diagram of a fluorescence immunochromatographic test strip of the present invention.
The labels in the figure are: 1 is a bottom plate, 2 is a sample pad, 3 is a combination pad, 4 is a coating film, 5 is a detection line, 6 is a quality control line, and 7 is absorbent paper.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1, the fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food comprises a base plate 1, wherein a sample pad 2, a combination pad 3, a coating film 4 and absorbent paper 7 are sequentially and mutually staggered adhered on the base plate 1, the upper end face of one end of the coating film 4 is adhered to one end of the combination pad 3, the upper end face of the other end of the coating film 4 is adhered to one end of the absorbent paper 7, the coating film 4 is coated with a detection line 5 and a quality control line 6, the upper end face of the other end of the combination pad 3 is adhered to one end of the sample pad 2, the detection line 5 and the quality control line 6 are not crossed with each other, the combination pad 3 is coated with a lead specific antibody marked by fluorescent microspheres, the detection line 5 is composed of a lead ion complete antigen, and the quality control line 6 is composed of goat anti-mouse IgG.
In the above, the sample pad 2 may be made of glass fiber cotton, the conjugate pad 3 may be made of a polyester fiber film, and the envelope 4 may be made of a nitrocellulose film, but these specific materials do not have to be considered to be the only limiting function, and other materials having the same performance as the above materials may be used.
Further, for the convenience of detection and comparative observation, the detection line 5 and the quality control line 6 are parallel to each other and spaced at a distance of 4-8 mm, the detection line 5 is close to the bonding pad 3, and the quality control line 6 is close to the absorbent paper 7.
As a preferable embodiment, the lead-specific antibody is preferably a murine monoclonal antibody, the fluorescent microsphere is a microsphere doped with a rare earth europium ion chelate with a diameter of 100-300 nm, and the surface of the microsphere is provided with active group carboxyl.
Further, the lead ion complete antigen is a coupling compound formed by lead ions, a chelating agent and a carrier protein. Preferably, the chelating agent is ethylenediamine tetraacetic acid, and the carrier protein is bovine serum albumin.
Further, the method for forming the sprayed layer of the fluorescent microsphere-labeled lead-specific antibody on the conjugate pad 3 includes the following steps:
step 1, adding a designed amount of europium ion chelate fluorescent microspheres into a prepared boric acid buffer solution in advance, and then carrying out ultrasonic dispersion;
step 2, after the step 1 is finished, adding carbodiimide and N-hydroxysuccinimide in designed amount into the solution, carrying out centrifugal washing after oscillation reaction, and then dispersing the solution into boric acid buffer solution;
and 5, mixing the storage liquid according to the volume ratio of 1: (90-100) diluting in boric acid buffer solution containing bovine serum albumin, Tween-20, polyvinylpyrrolidone, trehalose and sodium azide, spraying on the bonding pad by using a quantitative film spraying instrument, drying by blowing at 30-40 ℃, and drying in dark at 3-6 ℃ to obtain the product.
In the above, the boric acid buffer solution in step 5 contains bovine serum albumin with a mass concentration of 0.4-0.6%, Tween-20 with a volume fraction of 2-3%, polyvinylpyrrolidone with a mass concentration of 0.4-0.6%, trehalose with a mass concentration of 1-3%, and sodium azide with a mass concentration of 0.01-0.03%, and the pH value of the boric acid buffer solution is 8.0-8.5.
In order to better practice the invention, the following examples are given.
Example 1
A fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food is prepared by the following steps:
(1) preparation of lead ion antibody compound marked by fluorescent microsphere
Adding 200 mu L of 1% (w/v) europium ion chelate fluorescent microspheres into 800 mu L of boric acid buffer solution with the concentration of 0.2mol/L, pH being 8.2, adding 20mg of carbodiimide (EDC) and 10mg of N-hydroxysuccinimide (NHS) after ultrasonic dispersion, carrying out oscillation reaction for 2 hours, carrying out centrifugal washing, dispersing in 1mL of boric acid buffer solution, adding 160 mu L of 1.0mg/mL lead ion antibody solution, and carrying out oscillation reaction overnight (more than 8 hours) to obtain the europium ion chelate fluorescent microspheres;
(2) preservation of complexes of fluorescent microsphere-labeled lead ion antibodies
Centrifugally separating the solution obtained in the step (1), blocking the solution for 2 hours by using a boric acid buffer solution containing 0.5% (w/v) BSA, centrifugally washing the solution, and storing the solution in 1mL of boric acid buffer solution at 4 ℃ to obtain a storage solution;
(3) bonding pad 3 spray coating
The stock solution was mixed as follows: 100 percent of the total weight of the solution is diluted in boric acid buffer solution containing 0.5 percent (w/v) BSA, 2.5 percent (v/v) Tween-20, 0.5 percent (w/v) polyvinylpyrrolidone, 2 percent (w/v) trehalose, 0.02 percent (w/v) sodium azide and pH8.2, the solution is sprayed on a bonding pad 3 by using a quantitative film spraying instrument, and after being dried by air blowing at 37 ℃ for 2 hours, the solution is dried and stored at 4 ℃ in a dark place for standby;
(4) form a detection line 5 and a quality control line 6
Lead ion complete antigen (Pb (II) -EDTA-BSA conjugate) and goat anti-mouse IgG are respectively coated on different positions of the coating film 4 to form a detection line 5 and a quality control line 6;
the preparation method of the coating film 4 comprises the following steps: spraying Pb (II) -EDTA-BSA conjugate 0.5mg/mL and goat anti-mouse IgG 0.5mg/mL on the coating film 4 (i.e. nitrocellulose membrane) at an interval of 0.50cm to form a detection line 5 and a quality control line 6, drying at 37 ℃ for 2 hours, and placing into a dryer for storage;
(5) test paper assembly
Sequentially and alternately sticking a sample pad 2, a bonding pad 3, a coating film 4 and absorbent paper 7 on a bottom plate 1, cutting into 0.4cm size, and drying and storing at 4 deg.C in dark.
Detecting by using test paper:
the fluorescence immunochromatographic test paper is used for detecting heavy metal lead in food, and comprises sample pretreatment, use of fluorescence test paper and result judgment, wherein in the detection process, the lead content detection in a commercially available seasoning, namely cumin powder is taken as an example:
pre-treating cumin powder: weighing 1g of cumin powder, adding 2mL of 2% (v/v) nitric acid, carrying out ultrasonic extraction for 3 minutes, standing, taking supernatant, adjusting the supernatant to be neutral by using a sodium hydroxide solution, then taking 100 mu L of the neutral solution, putting the neutral solution into a 9-time volume phosphoric acid buffer solution (0.02mol/L, pH7.4) containing 10mg/L of Ethylene Diamine Tetraacetic Acid (EDTA), and uniformly mixing to obtain a sample solution to be detected.
During detection, 70 μ L of sample solution to be detected is dropped on the sample pad 2, and the sample solution is electrophoresed on the test paper through chromatography, if the sample solution to be detected contains Pb (II) -EDTA, the Pb (II) -EDTA is immunologically combined with the lead specific antibody marked by the fluorescent microsphere in the combination pad, so that the antigen binding site of the Pb (II) -EDTA on the fluorescent microsphere-antibody is closed, the antibody is inhibited from being combined with the antigen (Pb (II) -EDTA-BSA conjugate) on the detection line 5, the amount of the antibody combined on the detection line 5 is reduced along with the increase of the content of the Pb (II) -EDTA in the sample, the fluorescence intensity on the corresponding detection line is reduced, and the goat anti-mouse IgG on the quality control line 6 is not influenced by the concentration of the Pb (II) -EDTA to combine with the antibody marked by the fluorescent microsphere.
Because the europium ion chelate fluorescent microspheres emit bright red light under an ultraviolet lamp, after the liquid to be detected is dripped for 5 minutes, the liquid to be detected is placed under the 365nm ultraviolet lamp for observation, a negative result presents two red fluorescent lines, namely a detection line 5 and a quality control line 6, when Pb (II) -EDTA is higher than or equal to 10 mu g/kg, the detection line 5 disappears, only one quality control line 6 appears, and at the moment, the result is a positive result, the detection process is rapid, simple and convenient, and the result is visual.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food comprises a bottom plate (1), it is characterized in that the bottom plate (1) is sequentially and mutually stuck with a sample pad (2), a combination pad (3), a coating film (4) and absorbent paper (7) in a staggered way, the upper end face of one end of the coating film (4) is attached to one end of the combination pad (3), the upper end face of the other end of the coating film (4) is attached to one end of the absorbent paper (7), the coating film (4) is coated with a detection line (5) and a quality control line (6), the upper end face of the other end of the combination pad (3) is attached to one end of the sample pad (2), the detection line (5) and the quality control line (6) are not crossed with each other, the combination pad (3) is coated with a lead specific antibody marked by fluorescent microspheres, the detection line (5) is composed of a lead ion complete antigen, and the quality control line (6) is composed of goat anti-mouse IgG.
2. The fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food according to claim 1, wherein the sample pad (2) is glass fiber cotton, the conjugate pad (3) is a polyester fiber membrane, and the coating membrane (4) is a nitrocellulose membrane.
3. The fluorescence immunochromatographic test strip for rapid detection of heavy metal lead in foods according to claim 2, wherein the detection line (5) and the quality control line (6) are parallel to each other with a space left therebetween, the detection line (5) is adjacent to the binding pad (3), and the quality control line (6) is adjacent to the absorbent paper (7).
4. The fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food according to any one of claims 1 to 3, wherein the lead-specific antibody is a murine monoclonal antibody, the fluorescent microspheres are microspheres doped with rare earth europium ion chelate with a diameter of 100-300 nm, and the surfaces of the microspheres are provided with active group carboxyl.
5. The fluorescence immunochromatographic test strip for rapidly detecting heavy metal lead in food according to claim 4, wherein the lead ion complete antigen is a conjugate formed by lead ions, a chelating agent and a carrier protein.
6. The fluorescence immunochromatographic test paper for rapidly detecting heavy metal lead in food according to claim 5, wherein the chelating agent is ethylenediaminetetraacetic acid and the carrier protein is bovine serum albumin.
7. The fluorescence immunochromatographic test strip for rapid detection of heavy metal lead in foods according to claim 6, wherein the method for forming the sprayed layer of lead-specific antibody labeled with fluorescent microspheres on the conjugate pad (3) comprises the steps of:
step 1, adding a designed amount of europium ion chelate fluorescent microspheres into a prepared boric acid buffer solution in advance, and then carrying out ultrasonic dispersion;
step 2, after the step 1 is finished, adding carbodiimide and N-hydroxysuccinimide in designed amount into the solution, carrying out centrifugal washing after oscillation reaction, and then dispersing the solution into boric acid buffer solution;
step 3, after the step 2 is completed, adding a designed amount of lead ion antibody solution into the solution, and carrying out oscillation reaction for more than 8 hours;
step 4, performing centrifugal separation on the solution obtained in the step 3, sealing and storing the solution for 1-3 hours by using a boric acid buffer solution containing bovine serum albumin, then performing centrifugal washing, and storing the solution in the boric acid buffer solution at the temperature of 3-6 ℃ to obtain a storage solution;
and 5, mixing the storage liquid according to the volume ratio of 1: (90-100) is diluted in boric acid buffer solution containing bovine serum albumin, Tween-20, polyvinylpyrrolidone, trehalose and sodium azide, sprayed on the bonding pad (3) by using a quantitative film spraying instrument, dried by blowing at 30-40 ℃, and dried in the dark at 3-6 ℃ to obtain the product.
8. The fluorescence immunochromatographic test strip for rapid detection of heavy metal lead in food according to claim 7, wherein in step 5, the boric acid buffer solution contains bovine serum albumin with a mass concentration of 0.4-0.6%, Tween-20 with a volume fraction of 2-3%, polyvinylpyrrolidone with a mass concentration of 0.4-0.6%, trehalose with a mass concentration of 1-3% and sodium azide with a mass concentration of 0.01-0.03%, and has a pH value of 8.0-8.5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112505323A (en) * | 2021-02-03 | 2021-03-16 | 南京市产品质量监督检验院 | Raman immunochromatographic test strip for detecting lead ions and preparation method and application thereof |
| CN112526122A (en) * | 2020-11-13 | 2021-03-19 | 山东美正生物科技有限公司 | Fluorescent quantitative test strip for rapidly detecting heavy metal lead |
| CN113607951A (en) * | 2021-07-29 | 2021-11-05 | 上海师范大学 | Immunomacrosphere chromatography test strip for rapidly detecting heavy metal cadmium and preparation method thereof |
| WO2023179696A1 (en) * | 2022-03-24 | 2023-09-28 | 利多(香港)有限公司 | Test reagent plate and method |
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| WO2023179696A1 (en) * | 2022-03-24 | 2023-09-28 | 利多(香港)有限公司 | Test reagent plate and method |
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