CN113484513A - Immunochromatography test strip for rapidly detecting glyphosate and preparation method thereof - Google Patents
Immunochromatography test strip for rapidly detecting glyphosate and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of biological immune antibodies and detection, in particular to an immunochromatographic test strip for detecting glyphosate and a preparation method thereof. The PVC waterproof board comprises a PVC base plate, and a sample pad, a colloidal gold combination pad, an NC membrane and a water absorption pad which are sequentially spliced on the PVC base plate; the NC membrane is coated with a detection line and a quality control line which are parallel to the end face of the PVC bottom plate, and the detection line is arranged below the quality control line. The invention also discloses a preparation method of the immunochromatographic test strip for detecting glyphosate. The method is simple and convenient to operate, short in time consumption, simple and easy to operate by using equipment, only needs 15 minutes in the whole detection process, and is suitable for rapid detection of glyphosate residues in individual growers, food safety quality inspection departments, customs and other plant-derived foods.
Description
Technical Field
The invention relates to the field of biological immune antibodies and detection, in particular to an immunochromatographic test strip for detecting glyphosate and a preparation method thereof.
Background
Glyphosate (also called Nongda and Nicotinine) belongs to a systemic conduction type broad-spectrum biocidal herbicide, is a non-selective herbicide, and is widely applied to various agricultural and non-agricultural fields in the world. Glyphosate can be combined with metal ions such as aluminum, iron and the like in soil quickly to lose chemical activity, and is enriched in environment and organisms continuously, researches show that glyphosate can destroy the normal functions of human bodies and cause diseases, and the harmful effects are gradually obvious along with the time. The national standard GB 2763 and 2019, maximum limit of pesticide residue in food safety national standard food, limit the glyphosate.
At present, methods for detecting glyphosate include gas chromatography, ion chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ion chromatography-mass spectrometry and the like. Most of the methods require derivatization reaction, the pretreatment operation procedure is complicated, the derivatization reaction time is long, and the detection efficiency is low. Although the ion chromatography does not require derivatization, the sensitivity is low, and the ion chromatography mass spectrometry has high sensitivity but is inconvenient to popularize. Therefore, the establishment of a high-sensitivity rapid detection method for glyphosate residues in food is of great significance.
The immunochromatography is a new technology for rapid inspection and diagnosis in recent years, and the principle is that a specific antibody is loaded on a fixed area belt of a nitrocellulose membrane, after a detection end is immersed in a sample solution, a sample moves forwards along the nitrocellulose membrane, when the sample moves to an antibody area, a corresponding antigen in the sample solution is specifically combined with the antibody, and at the moment, colloidal gold can enable the area belt to display a certain color, so that the judgment and identification of specificity can be carried out.
At present, the research of combining the glyphosate monoclonal antibody with immunochromatography for rapid detection is not available. Therefore, it is necessary to invent an immunochromatographic test strip for detecting glyphosate and a preparation method thereof to solve the above problems.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an immunochromatographic test strip for detecting glyphosate and a preparation method thereof, so as to solve the problems in the background art.
In order to solve the technical problems, the technical scheme of the invention is as follows: an immunochromatography test strip for detecting glyphosate comprises a PVC (polyvinyl chloride) base plate, and a sample pad, a colloidal gold combination pad, an NC (numerical control) membrane and a water absorption pad which are sequentially spliced on the PVC base plate; the NC membrane is coated with a detection line and a quality control line which are parallel to the end face of the PVC bottom plate, and the detection line is arranged below the quality control line.
Preferably, the coincidence of the sample pad and the colloidal gold combined pad in the length direction is 0.2cm, the coincidence of the colloidal gold combined pad and the NC membrane in the length direction is 0.2cm, and the coincidence of the NC membrane and the water absorption pad in the length direction is 0.3 cm.
A preparation method of an immunochromatographic test strip for detecting glyphosate comprises the following steps:
1) preparing a colloidal gold solution: uniformly mixing 100mL of a solution containing 0.05% chloroauric acid, heating to boiling, rapidly adding 0.07mL of 1% sodium citrate into the solution, stirring uniformly, continuously heating until the solution turns into wine red, switching a heater to a lower power, continuously heating for 5min to stabilize the particle size of the colloidal gold in the solution, cooling to room temperature, and storing at 2-8 ℃ for later use;
2) preparation of monoclonal antibody-colloidal gold marker: taking 0.5mg of glyphosate monoclonal antibody, adding 1mL of 0.01mol/L PB solution, shaking up and dilutingReleasing; taking 8mL of colloidal gold solution, and using 0.2mol/L K2CO3Adjusting the pH value of the solution to be the optimal value, fully stirring, then dropwise adding the diluted monoclonal antibody solution, uniformly stirring, and standing for 20 min; dropwise adding a proper amount of BSA solution containing 1% into the colloidal gold, stirring and uniformly mixing, standing for 20min, and centrifuging; discarding the supernatant, and adding 0.8mL of the heavy suspension for redissolving;
3) preparing a colloidal gold bonding pad: putting the colloidal gold conjugate pad into PBS buffer solution containing 0.5% BSA, pH7.2, and 0.02mol/L, soaking for 1h, drying at 37 deg.C for 3h, spraying glyphosate monoclonal antibody-colloidal gold marker onto the colloidal gold conjugate pad with a film spraying instrument according to 5.5 μ L/cm amount, and drying at 37 deg.C for 1 h;
4) preparing an NC film: taking a glyphosate hapten-ovalbumin conjugate as a detection line on a nitrocellulose membrane, taking a goat anti-rabbit anti-antibody as a quality control line, diluting the glyphosate hapten-ovalbumin conjugate by 20 times by using PBS buffer solution with pH7.4 and 0.01mol/L, spraying the glyphosate hapten-ovalbumin conjugate on an NC membrane by using a membrane spraying instrument with the spraying amount of 1 mu L/cm, spraying the goat anti-rabbit anti-antibody (0.07mg/mL) with the spraying amount of 1 mu L/cm, and placing the coated NC membrane at 37 ℃ for drying for 2 hours for later use;
5) assembling the immunochromatography test strip: taking a PVC base plate, and sequentially splicing a sample pad, a colloidal gold combination pad, an NC membrane and a water absorption pad on the PVC base plate; coating a detection line and a quality control line on the NC film, wherein the detection line and the quality control line are parallel to the end face of the PVC bottom plate during coating, and the detection line is arranged below the quality control line.
In the step 2, the preparation method of the glyphosate monoclonal antibody comprises the following steps:
preparing immune antigen: dissolving 15mg of hapten in 1mL of N, N-dimethylformamide; adding 8mg of carbodiimide, and stirring at room temperature for 24 hours to obtain a reaction solution A; weighing 30mg of bovine serum albumin, fully dissolving the bovine serum albumin in 4mL of 0.1mol/L phosphate buffer solution, dropwise and slowly adding the reaction solution A into the protein solution, stirring for 24 hours at room temperature, dialyzing for 3 days at 4 ℃ by using 0.01mol/L phosphate buffer solution, and changing the dialyzate for 3 times every day to remove unreacted micromolecular substances to obtain an immunogen B;
preparing a glyphosate monoclonal antibody: taking spleen of a mouse after immunization and fusing myeloma cells, establishing a hybridoma cell strain, preparing ascites of a monoclonal antibody, purifying and identifying to obtain the monoclonal antibody.
Compared with the prior art, the invention has the beneficial effects that:
1. the monoclonal antibody for detecting glyphosate can identify glyphosate antigen, and further reflects whether a detection sample contains glyphosate substances or not through a colloidal gold immunochromatography detection test strip, so that the detection time of glyphosate is shortened, the detection sensitivity is high, the applicability is strong, and the prevention and supervision effects of a crop production source can be popularized.
2. The monoclonal antibody of the invention is only directed against glyphosate antigens, and is combined with other organophosphates such as: methyl parathion, phoxim, triazophos and the like do not react, and the kit has high specificity, high titer and high sensitivity, thereby enhancing the specificity of detection and the accuracy of detection results and ensuring the reliability of detection test paper strip results in application.
3. The method is simple and convenient to operate, short in time consumption, simple and easy to operate by using equipment, only needs 15 minutes in the whole detection process, and is suitable for rapid detection of glyphosate residues in individual growers, food safety quality inspection departments, customs and other plant-derived foods.
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FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a graph showing the detection sensitivity and detection limit of the present invention for detecting glyphosate drug;
FIG. 3 is a state diagram of the immunochromatographic test strip in the detection of the present invention.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Referring to fig. 1, the immunochromatographic test strip for detecting glyphosate of the present invention comprises a PVC base plate 1, and a sample pad 2, a colloidal gold conjugate pad 3, an NC membrane 4 and a water absorption pad 5 which are sequentially spliced on the PVC base plate 1; the NC membrane 4 is coated with a detection line 6 and a quality control line 7 which are parallel to the end face of the PVC bottom plate 1, and the detection line 6 is arranged below the quality control line 7.
In the invention, the sample pad 2 and the colloidal gold bonding pad 3 are overlapped by 0.2cm in the length direction, the colloidal gold bonding pad 3 and the NC membrane 4 are overlapped by 0.2cm in the length direction, and the NC membrane 4 and the water absorption pad 2 are overlapped by 0.3cm in the length direction.
The monoclonal antibody for detecting glyphosate is prepared according to the following steps:
1. preparing immune antigen: dissolving 15mg of hapten in 1mL of N, N-Dimethylformamide (DMF); adding 8mg of carbodiimide (EDC), and stirring at room temperature for 24h to obtain a reaction solution A; weighing 30mg of Bovine Serum Albumin (BSA), fully dissolving the BSA in 4mL of 0.1mol/L phosphate buffer solution (PB, pH 7.0), dropwise and slowly adding the reaction solution A into the protein solution, stirring at room temperature for 24h, dialyzing with 0.01mol/L Phosphate Buffer Solution (PBS) at 4 ℃ for 3d, and changing the dialysate 3 times a day to remove unreacted micromolecular substances to obtain an immunogen B;
2. preparing a glyphosate monoclonal antibody;
(1) mouse immunization: mixing the glyphosate immunogen prepared in the step 1 with Freund's adjuvant in the same volume to prepare water-in-oil emulsion, injecting 0.5mL of the mixed solution (containing 100 mu g of protein fragment) into the abdominal cavity of each mouse, and injecting 6 BALB/c mice in total; two weeks later, the same dose of antigen is added with Freund incomplete adjuvant to strengthen the immunity, 7 days later, the titer of mouse serum head protein polyclonal antibody is detected by indirect ELISA, the head vein with high titer is impacted and immunized for 1 time, each 20 mu g, and cell fusion is carried out after 3 days;
(2) establishment of hybridoma cell strain: fusing spleen cell suspension of an immune mouse and SP2/0 myeloma cells in a ratio of 5: 1 under the action of polyethylene glycol according to a conventional method, and screening and culturing by using HAT complete 1640 culture medium; taking glyphosate (1 microgram/mL) as an antigen to coat an enzyme-labeled 96-well plate, detecting a hybridoma cell culture supernatant by an indirect ELISA method, performing subcloning on a clone with the detected OD490 (absorption at 490 nm) being 10 times higher than that of a negative control, and performing amplification and cryopreservation; after 3 times of limiting dilution cloning, a large amount of hybridoma cell lines secreting specific antibodies are amplified and frozen, and after long-term subculture, cloning identification is carried out again by the same method;
(3) preparing the monoclonal antibody ascites: injecting 0.5mL incomplete Freund's adjuvant into the abdominal cavity of BALB/c mouse, injecting 0.5mL incomplete Freund's adjuvant containing 2 × 10 into the abdominal cavity of each mouse after 5-7 days6Observing the abdominal expansion degree of the mouse after 7-10 days by using the hybridoma cell solution, and collecting ascites;
(4) purification of monoclonal antibodies: the ascites of the monoclonal antibody of glyphosate is purified by 50-60% (mass volume ratio) saturated ammonium sulfate salting-out and Protein G affinity chromatography, and the purity of the obtained monoclonal antibody is determined by Protein electrophoresis.
After the monoclonal antibody for detecting glyphosate is prepared, an immunochromatographic test strip for detecting glyphosate is prepared, and the preparation method specifically comprises the following steps:
1. preparing a colloidal gold solution: uniformly mixing 100mL of a solution containing 0.05% chloroauric acid, heating to boiling, rapidly adding 0.07mL of 1% sodium citrate into the solution, stirring uniformly, continuously heating until the solution turns into wine red, switching a heater to a lower power, continuously heating for 5min to stabilize the particle size of the colloidal gold in the solution, cooling to room temperature, and storing at 2-8 ℃ for later use;
2. preparation of monoclonal antibody-colloidal gold marker (gold-labeled antibody): taking 0.5mg of glyphosate monoclonal antibody, adding 1mL of 0.01mol/L PB solution, shaking up and diluting; taking 8mL of colloidal gold solution, and adding 0.2mol/LK2CO3Adjusting the pH value of the solution to the optimum value, fully stirring, then dropwise adding the diluted monoclonal antibody solution, uniformly stirring, and standing for 20 min. Adding appropriate amount of 1% BSA (blocking solution) solution dropwise into colloidal gold, stirring, mixing, and standing for 20 min. Centrifuging (12000 r/min; 8 min); discarding the supernatant, and adding 0.8mL of the heavy suspension for redissolving;
3. preparation of the colloidal gold conjugate pad 3: putting the colloidal gold combined pad 3 into PBS buffer solution containing 0.5% BSA, pH7.2, 0.02mol/L, soaking for 1h, drying for 3h at 37 ℃, then using a film spraying instrument to spray the glyphosate monoclonal antibody-colloidal gold marker on the colloidal gold combined pad 3 according to the amount of 5.5 muL/cm, and drying for 1h at 37 ℃ for later use;
5) assembling the immunochromatography test strip: taking a PVC base plate 1, and sequentially splicing a sample pad 2, a colloidal gold combination pad 3, an NC membrane 4 and a water absorption pad 5 on the PVC base plate 1; coating a detection line 6 and a quality control line 7 on the NC film 4, wherein the detection line 6 and the quality control line 7 are parallel to the end face of the PVC bottom plate 1 during coating, and the detection line 6 is arranged below the quality control line 7.
The prepared immunochromatography test strip for detecting glyphosate is evaluated for sensitivity and detection limit: the detection sensitivity of the glyphosate medicament reaches 1ng/ml, and the lowest detection line in tea and vegetable and fruit samples reaches 0.5 mg/kg; as shown in table 1, fig. 2.
TABLE 1 test results for sensitivity of test strip for rapid examination of glyphosate
| Sensitivity of the probe | T/C value | Color development degree of CT line |
| 0(ddH2O) | 1.20 | - |
| 1μg/ml | 0.01 | ++++ |
| 100ng/ml | 0.23 | +++ |
| 10ng/ml | 0.41 | ++ |
| 1ng/ml | 0.56 | + |
Note: ("-" indicates that the T line is slightly darker than the C line, and "+" indicates that the T line is lighter than the C line, the more "+" the T line is lighter)
And (3) evaluating the specificity of the test strip: the glyphosate belongs to herbicide pesticides, and the experiment tests the similar drugs and has no specificity to other herbicide pesticides except 3-hydroxy glyphosate; as in table 2.
Table 2 test results on specificity of immunochromatographic test strip for rapidly detecting glyphosate
| Name of drug | Results |
| Acetochlor | - |
| Herbicidal ethers | - |
| Alachlor | - |
| Fluorine ammonia | - |
| 3-hydroxy glyphosate | + |
| Dichloromethane | - |
The immunochromatographic test strip for detecting glyphosate by using the method comprises the following steps:
1. before detection, the sample and the test strip are required to be recovered to the room temperature of 20-25 ℃;
2. preparing a glyphosate derivatization reagent solution: a derivatization reagent of FMOC (fluorenylmethoxycarbonylcarbonyl chloride) with a concentration of 1mg/ml was prepared with acetone, and the mixture was shaken to dissolve it sufficiently for use.
3. The sample processing method comprises the following steps:
(1) before detection, cutting fresh tea leaves, vegetables and fruits samples into fragments smaller than 1cm or finely divided small pieces, and directly weighing dry tea leaves;
(2) weighing 1 plus or minus 0.05g of sample into a 10ml centrifuge tube, adding 5ml of acetonitrile, covering the centrifuge tube, manually oscillating for 30s, and standing for 1min to obtain a sample mixed solution for later use;
(3) and adding another 10 mu L of the currently prepared 1mg/mL glyphosate derivatization solution into a tube filled with 1mL of 0.02mol/L PBS buffer solution, uniformly mixing, adding 200 mu L of sample mixed solution, and standing for 1min to obtain the sample to-be-detected solution.
4. The detection method comprises the following steps:
(1) taking the test strip out of the original packaging bag, and using the test strip as soon as possible within one hour after the test strip is opened;
(2) 70 mu L of sample solution to be detected (or 3 drops of sample solution are taken by a dropper) is vertically dripped into the sample adding hole;
(3) timing when the liquid flows, reacting for 10min, and judging the result, wherein the result is more than 10min and can be used as a reference.
As shown in fig. 3, 5. result judgment method: by means of visual inspection, the method of measurement,
negative (-): the color development of the T line is darker than that of the C line or is consistent with that of the C line, and both indicate that the concentration of the substance to be detected in the sample is lower than the detection limit.
Positive (+): the color development of the T line is lighter than that of the C line or the T line is not developed, which indicates that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit.
And (4) invalidation: the absence of a C-line indicates an incorrect procedure or the test strip has failed. In this case, the test is retested using a new test strip.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (4)
1. An immunochromatographic test strip for detecting glyphosate, which is characterized in that: comprises a PVC base plate (1), and a sample pad (2), a colloidal gold combination pad (3), an NC membrane (4) and a water absorption pad (5) which are spliced on the PVC base plate (1) in sequence; the NC film (4) is coated with a detection line (6) and a quality control line (7) which are parallel to the end face of the PVC bottom plate (1), and the detection line (6) is arranged below the quality control line (7).
2. The immunochromatographic test strip for detecting glyphosate according to claim 1, characterized in that: the sample pad (2) and the colloidal gold combined pad (3) are overlapped by 0.2cm in the length direction, the colloidal gold combined pad (3) and the NC membrane (4) are overlapped by 0.2cm in the length direction, and the NC membrane (4) and the water absorption pad (2) are overlapped by 0.3cm in the length direction.
3. A preparation method of an immunochromatographic test strip for detecting glyphosate is characterized by comprising the following steps:
1) preparing a colloidal gold solution: uniformly mixing 100mL of a solution containing 0.05% chloroauric acid, heating to boiling, rapidly adding 0.07mL of 1% sodium citrate into the solution, stirring uniformly, continuously heating until the solution turns into wine red, switching a heater to a lower power, continuously heating for 5min to stabilize the particle size of the colloidal gold in the solution, cooling to room temperature, and storing at 2-8 ℃ for later use;
2) preparation of monoclonal antibody-colloidal gold marker: taking 0.5mg of glyphosate monoclonal antibody, adding 1mL of 0.01mol/L PB solution, shaking up and diluting; taking 8mL of colloidal gold solution, and using 0.2mol/L K2CO3Adjusting the pH value of the solution to be the optimal value, fully stirring, then dropwise adding the diluted monoclonal antibody solution, uniformly stirring, and standing for 20 min; dropwise adding a proper amount of BSA solution containing 1% into the colloidal gold, stirring and uniformly mixing, standing for 20min, and centrifuging; discarding the supernatant, and adding 0.8mL of the heavy suspension for redissolving;
3) preparing a colloidal gold bonding pad (3): putting the colloidal gold combined pad (3) into PBS buffer solution containing 0.5% BSA, pH7.2 and 0.02mol/L for uniform soaking for 1h, drying for 3h at 37 ℃, then uniformly spraying the glyphosate monoclonal antibody-colloidal gold marker on the colloidal gold combined pad (3) according to the amount of 5.5 muL/cm by using a film spraying instrument, and drying for 1h at 37 ℃ for later use;
4) NC film preparation (4): taking a glyphosate hapten-ovalbumin conjugate as a detection line (6) on a nitrocellulose membrane, taking a goat anti-rabbit anti-antibody as a quality control line (7), diluting the glyphosate hapten-ovalbumin conjugate by using PBS buffer solution with pH7.4 and 0.01mol/L by 20 times, spraying the glyphosate hapten-ovalbumin conjugate on an NC membrane by using a membrane spraying instrument with the spraying amount of 1 mu L/cm, spraying the goat anti-rabbit anti-antibody (0.07mg/mL) with the spraying amount of 1 mu L/cm, and drying the coated NC membrane (4) at 37 ℃ for 2 hours for later use;
5) assembling the immunochromatography test strip: taking a PVC base plate (1), and sequentially splicing a sample pad (2), a colloidal gold bonding pad (3), an NC membrane (4) and a water absorption pad (5) on the PVC base plate (1); coating a detection line (6) and a quality control line (7) on the NC film (4), wherein the detection line (6) and the quality control line (7) are parallel to the end face of the PVC bottom plate (1) during coating, and the detection line (6) is arranged below the quality control line (7).
4. The method for preparing the immunochromatographic test strip for detecting glyphosate according to claim 3, wherein in the step 2, the preparation method of the glyphosate monoclonal antibody is as follows:
(1) preparing immune antigen: dissolving 15mg of hapten in 1mL of N, N-dimethylformamide; adding 8mg of carbodiimide, and stirring at room temperature for 24 hours to obtain a reaction solution A; weighing 30mg of bovine serum albumin, fully dissolving the bovine serum albumin in 4mL of 0.1mol/L phosphate buffer solution, dropwise and slowly adding the reaction solution A into the protein solution, stirring for 24 hours at room temperature, dialyzing for 3 days at 4 ℃ by using 0.01mol/L phosphate buffer solution, and changing the dialyzate for 3 times every day to remove unreacted micromolecular substances to obtain an immunogen B;
(2) preparing a glyphosate monoclonal antibody: taking spleen of a mouse after immunization and fusing myeloma cells, establishing a hybridoma cell strain, preparing ascites of a monoclonal antibody, purifying and identifying to obtain the monoclonal antibody.
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