CN114264817A - Manganese dioxide immune rapid detection kit for pesticide carbendazim in fruits and vegetables and application thereof - Google Patents
Manganese dioxide immune rapid detection kit for pesticide carbendazim in fruits and vegetables and application thereof Download PDFInfo
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- CN114264817A CN114264817A CN202111602148.9A CN202111602148A CN114264817A CN 114264817 A CN114264817 A CN 114264817A CN 202111602148 A CN202111602148 A CN 202111602148A CN 114264817 A CN114264817 A CN 114264817A
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Abstract
The invention discloses a manganese dioxide immune rapid detection kit for pesticide carbendazim in fruits and vegetables and application thereof. The test strip comprises a water absorption pad (1), a nitrocellulose membrane (2), a PVC (polyvinyl chloride) back plate (3), a combination pad (4) and a sample pad (5). The sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially stuck on the PVC backboard. The nitrocellulose membrane is respectively coated with (6) a quality control line formed by goat anti-mouse antibodies and (7) a detection line formed by carbendazim antigens. Mixing the manganese dioxide-carbendazim antibody probe and a sample to be detected, then dropwise adding the mixture into a sample pad, carrying out chromatography on a water absorption pad through capillary action, allowing an antigen of the object to be detected and the carbendazim to compete and combine with the manganese dioxide-carbendazim antibody probe, allowing manganese dioxide nanosheets to be intercepted on a detection line to generate colors, and realizing qualitative and semi-quantitative analysis on the carbendazim through visual observation. The invention has the advantages of high specificity, good sensitivity and simple operation.
Description
Technical Field
The invention relates to a manganese dioxide immune rapid detection kit for carbendazim pesticide in fruit and vegetable products and application thereof, belonging to the technical field of analytical chemistry.
Background
Carbendazim (bcm), a low-toxicity, spectrally antimicrobial agent, is commonly used in the control of fungi-caused toxicities. Carbendazim is mainly applied by means of foliar spraying, seed treatment or soil treatment, etc., so that carbendazim can penetrate into the interior of plants. The residues of carbendazim can cause liver diseases and chromosome aberration, and are toxic to mammals. If the vegetables with pesticide residues exceeding the standard are eaten for a long time, adverse effects on human health can be caused. Therefore, establishing a rapid detection means of the healthy carbendazim has very important practical significance for ensuring food safety.
Although there are many methods for detecting carbendazim, they are limited by their expensive equipment, cumbersome operation and time-consuming. In recent years, the immunochromatography method has the characteristics that the operation is simple, auxiliary instruments and reagents are not needed, the result is obtained in 3-5 minutes, and the judgment can be carried out by naked eyes. The manganese dioxide nanosheet has the advantages of good water solubility, simplicity in preparation and strong adsorption capacity, and can be coupled with an antibody to prepare an immunosensing probe.
Disclosure of Invention
In view of the above, the invention provides a manganese dioxide immune rapid detection kit for qualitative and semi-quantitative detection of carbendazim in fruit and vegetable products and a detection method thereof, and in order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a manganese dioxide immunity rapid detection kit for pesticide carbendazim in fruits and vegetables comprises a box body, wherein a test strip, a manganese dioxide-carbendazim antibody probe, an extracting solution and a centrifuge tube are arranged in the box body. The test strip comprises a water absorption pad, a nitrocellulose membrane, a PVC (polyvinyl chloride) back plate and a combination pad, and is characterized in that the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the PVC back plate; the nitrocellulose membrane is respectively coated with a quality control line formed by goat anti-mouse antibodies and a detection line formed by carbendazim antigens, and the interval distance between the two lines is 4-6 mm; the lengths of the overlapped areas of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are respectively 0.8-1.3mm, and the width of the test paper strip is 3.0-4.0 mm; the lengths of the overlapped areas of the sample pad, the combination pad, the nitrocellulose membrane and the absorbent pad are respectively 1 mm. Mixing the manganese dioxide-carbendazim antibody probe and a sample to be detected, then dropwise adding the mixture into a sample pad, carrying out chromatography on a water absorption pad through capillary action, allowing an antigen of the object to be detected and the carbendazim to compete and combine with the manganese dioxide-carbendazim antibody probe, allowing manganese dioxide nanosheets to be intercepted on a detection line to generate colors, and realizing qualitative and semi-quantitative analysis on the carbendazim through visual observation.
Preferably, the manganese dioxide-carbendazim antibody probe is composed of a manganese dioxide nanosheet and a carbendazim monoclonal antibody. The preparation method comprises the following specific steps:
(1) 500. mu.L of a 50. mu.g/mL manganese dioxide nanosheet solution was taken, and 2. mu.L of 0.2M K was added thereto2CO3The solution and 20. mu.L of 0.65mg/mL analyte antibody were added to the above solution, mixed well, and allowed to stand at room temperature for 1 h.
(2) To this was added 10. mu.L of 20% BSA and 8. mu.L of 10% PEG20000And standing at room temperature for 30min after mixing.
(3) The ampoule was trimmed, centrifuged at 8000rpm for 40min at 4 deg.C, the supernatant discarded, and the solution redissolved to 200. mu.L of 0.5% BSA, 0.5% PVP, 1% Tween 20, and 0.5% PEG200And (5) mixing the solution for later use.
Preferably, the goat anti-rabbit or goat anti-mouse antibody diluted by the PBS solution is coated on a nitrocellulose membrane as a quality control line, the coating amount is 0.7 muL/cm respectively, the carbendazim antigen diluted by the PBS solution is coated on the nitrocellulose membrane as a detection line, the coating amount is 0.7 muL/cm respectively, the test strip is dried at 37 ℃, cut into test strips with the width of 3.7mm, and packaged for later use.
Preferably, the use method of the test strip of the invention is as follows: 10 μ L of the prepared manganese dioxide-carbendazim antibody probe was added to 100 μ L of the sample solution, mixed and dropped on the sample pad, and visual results were obtained within 10 minutes with the naked eye. The detection limit of the invention is defined as the concentration of carbendazim when the color on the detection line disappears.
Furthermore, the detection limit of the kit on the carbendazim is 5 ng/mL.
Compared with the existing carbendazim detection technology at home and abroad, the invention has the following outstanding advantages:
1. the invention can be used for high sensitivity and quantitative detection of carbendazim;
2. the method has the advantages of good accuracy and high sensitivity, is suitable for rapid screening of a large number of samples, and can be used as an effective screening means for rapid detection of micromolecule substances to be detected in food.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the invention without limitation. In the drawings:
FIG. 1 is a schematic diagram of the preparation of a manganese dioxide-antibody probe.
FIG. 2 is an assembly diagram of the test strip of the present invention.
FIG. 3 is a graph showing the results of the detection of a carbendazim standard solution according to the present invention (carbendazim addition concentrations: 0, 5, 10, 20, 50ng/mL from left to right).
FIG. 4 is a graph showing the results of detecting carbendazim in Chinese cabbage according to the present invention (from left to right, carbendazim addition concentrations: 0, 25, 50, 100, 250 ng/g).
Detailed Description
The invention will be described in more detail hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown, but the examples are not intended to limit the invention.
Example l (preparation example)
Preparation of manganese dioxide-antibody Probe
(1) As shown in FIG. 1, 500. mu.L of a 50. mu.g/mL manganese dioxide nanosheet solution was taken, and 2. mu.L of 0.2M K was added thereto2CO3The solution and 20. mu.L of 0.65mg/mL analyte antibody were added to the above solution, mixed well, and allowed to stand at room temperature for 1 h.
(2) To this was added 10. mu.L of 20% BSA and 8. mu.L of 10% PEG20000And standing at room temperature for 30min after mixing.
(3) The ampoule was trimmed, centrifuged at 8000rpm for 40min at 4 deg.C, the supernatant discarded, and the solution redissolved to 200. mu.L of 0.5% BSA, 0.5% PVP, 1% Tween 20, and 0.5% PEG200And (5) mixing the solution for later use.
Example 2 (preparation example)
Preparation of carbendazim manganese dioxide immune rapid detection test strip
1. Coating of nitrocellulose membranes
Diluting carbendazim antigen to 0.1mg/mL by using a two-dimensional plane film-drawing instrument, and coating the carbendazim antigen on a nitrocellulose membrane 2 as a detection line 7, wherein the coating amount is 0.7 muL/cm; coating goat anti-mouse antibody diluted to 0.81mg/mL by PBS solution on nitrocellulose membrane 2 as quality control line 6, wherein the coating amount is 0.7 muL/cm, drying at 37 ℃, chopping into test strips with the width of 3.7mm, and packaging for later use.
2. Assembly of test strips
As shown in fig. 2, a sample pad 5, a combination pad 4, a nitrocellulose membrane 2 and a water absorption pad 1 are sequentially adhered to a PVC back plate 3 in the order shown in fig. 1, and the lengths of the overlapped regions of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are respectively 1 mm. Cutting into small strips with the width of 3.7mm, and vacuum packaging.
Example 3 (application example)
Application technology of rapid detection kit for carbendazim
1. Pretreatment of fruit and vegetable samples
Placing 1.0-5.0g of fruit and vegetable sample in 5-25mL of extracting solution, and extracting for 1-5min with shaking to obtain the solution as sample extracting solution.
2. Detection step
And adding 10 mu L of prepared manganese dioxide-carbendazim antibody probe into 100 mu L of sample solution, mixing, dripping on a sample pad, and observing the color change on the detection line by naked eyes within 10 minutes to obtain a visual result.
3. Determination of results
The detection principle of the carbendazim manganese dioxide immune rapid detection kit provided by the invention is as follows: carbendazim in the sample is combined with specific antibodies marked by manganese dioxide in the flowing process, and the combination of the antibodies and carbendazim coating antigens on the nitrocellulose membrane detection line is inhibited, so that the color of the detection line is changed. And when the sample does not contain carbendazim or the concentration is lower than the detection limit, the color development of the detection line is consistent with that of the quality control line. When the concentration of the carbendazim in the sample is equal to or higher than the detection limit, the color development of the detection line is obviously weaker than that of the quality control line or the detection line does not develop color. And no matter whether the sample contains the carbendazim or not, the quality control line can be developed to show that the detection is effective. As shown in figure 3, the detection limit of the kit on the carbendazim standard solution is 5ng/mL, and as shown in figure 4, the detection limit of the kit on the carbendazim in the fruit and vegetable samples is 25 ng/g.
Claims (7)
1. A manganese dioxide immunity rapid detection kit for pesticide carbendazim in fruits and vegetables comprises a box body, wherein a test strip, a manganese dioxide-carbendazim antibody probe, an extracting solution and a centrifuge tube are arranged in the box body, the test strip comprises a water absorption pad, a nitrocellulose membrane, a PVC (polyvinyl chloride) back plate and a combination pad, and the manganese dioxide immunity rapid detection kit is characterized in that a sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the PVC back plate; the nitrocellulose membrane is respectively coated with a quality control line formed by goat anti-mouse antibodies and a detection line formed by carbendazim antigens, and the interval distance between the two lines is 4-6 mm; the lengths of the overlapped areas of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are respectively 0.8-1.3mm, and the width of the test paper strip is 3.0-4.0 mm; the lengths of the overlapped areas of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are respectively 1 mm; mixing the manganese dioxide-carbendazim antibody probe and a sample to be detected, then dropwise adding the mixture into a sample pad, carrying out chromatography on a water absorption pad through capillary action, allowing an antigen of the object to be detected and the carbendazim to compete and combine with the manganese dioxide-carbendazim antibody probe, allowing manganese dioxide nanosheets to be intercepted on a detection line to generate colors, and realizing qualitative and semi-quantitative analysis on the carbendazim through visual observation.
2. The preparation method of the manganese dioxide immune rapid detection kit for fruit and vegetable pesticide carbendazim, which is disclosed by claim 1, comprises the following steps: the method comprises the following steps:
(1) adsorbing a carbendazim antibody on the surface of a manganese dioxide nanosheet by utilizing the adsorption performance of the manganese dioxide nanosheet to prepare a manganese dioxide nanosheet-carbendazim antibody probe;
(2) sequentially adhering the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad to the PVC back plate; the nitrocellulose membrane is respectively coated with a detection line formed by carbendazim antigen and a quality control line formed by goat anti-mouse antibody;
(3) and mixing the probe and the extracting solution, then dropwise adding the mixture into a sample pad, carrying out chromatography on the sample pad through capillary action, competitively combining the carbendazim antigen and the carbendazim with the probe, and realizing qualitative and semi-quantitative analysis on the carbendazim through naked-eye observation.
3. The preparation method of claim 2, wherein the preparation of the manganese dioxide nanosheet-carbendazim antibody photothermal probe comprises the following steps:
(1) 500. mu.L of a 50. mu.g/mL manganese dioxide nanosheet solution was taken, and 2. mu.L of 0.2M K was added thereto2CO3Adding the solution and 20 mu L of 0.65mg/mL antibody of the substance to be detected into the solution, uniformly mixing, and standing for 1h at room temperature;
(2) to this was added 10. mu.L of 20% BSA and 8. mu.L of 10% PEG20000Standing at room temperature for 30 min;
(3) the ampoule was trimmed, centrifuged at 8000rpm for 40min at 4 deg.C, the supernatant discarded, and the solution redissolved to 200. mu.L of 0.5% BSA, 0.5% PVP, 1% Tween 20, and 0.5% PEG200And (5) mixing the solution for later use.
4. The manganese dioxide immunoassay kit for fruit and vegetable pesticide carbendazim according to claim 1, wherein goat anti-mouse antibodies diluted by PBS solution are coated on a nitrocellulose membrane as a quality control line, the coating amount is 0.7 μ L/cm, the test strips are dried at 37 ℃, cut into test strips with the width of 3.7mm, and packaged for later use.
5. The application of the manganese dioxide immune rapid detection kit for the pesticide carbendazim in fruits and vegetables, which is disclosed by claim 1, is characterized in that the manganese dioxide immune rapid detection kit is used for detecting the pesticide carbendazim, and the specific method is as follows: mixing the manganese dioxide-carbendazim antibody with 200 mu L of carbendazim extracting solution, dripping the mixture to a sample pad, carrying out chromatography for 5-10min, and observing color change at a detection limit to realize visual colorimetric detection of carbendazim.
6. The use of the kit according to claim 5, wherein the carbendazim extract is obtained by the following steps: placing 1.0-5.0g of fruit and vegetable sample in 5-25mL of extracting solution, and extracting for 1-5min with shaking to obtain the solution as sample extracting solution.
7. The application of the manganese dioxide immune rapid detection kit for the pesticide carbendazim in fruits and vegetables according to claim 5 is characterized in that the detection limit of the kit on the carbendazim standard solution is 5ng/mL, and the detection limit of the kit on the carbendazim in fruit and vegetable samples is 25 ng/g.
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| CN202111602148.9A CN114264817A (en) | 2021-12-24 | 2021-12-24 | Manganese dioxide immune rapid detection kit for pesticide carbendazim in fruits and vegetables and application thereof |
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| CN202111602148.9A CN114264817A (en) | 2021-12-24 | 2021-12-24 | Manganese dioxide immune rapid detection kit for pesticide carbendazim in fruits and vegetables and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116449026A (en) * | 2023-06-19 | 2023-07-18 | 成都信息工程大学 | Method for detecting cardiac troponin I based on manganese dioxide nano-sheet |
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- 2021-12-24 CN CN202111602148.9A patent/CN114264817A/en active Pending
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| CN109900890A (en) * | 2019-03-28 | 2019-06-18 | 天津科技大学 | A kind of black phosphorus-gold nanoparticle compound photo-thermal quantitative immunochromatographic test strips and preparation method thereof detecting small-molecule substance |
| CN113804887A (en) * | 2021-08-25 | 2021-12-17 | 中国农业科学院油料作物研究所 | Immunoassay kit with multiple detection scales and application thereof |
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| CN116449026A (en) * | 2023-06-19 | 2023-07-18 | 成都信息工程大学 | Method for detecting cardiac troponin I based on manganese dioxide nano-sheet |
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