Isofenphos-methyl test strip and application thereof in detection of isofenphos-methyl residue

Abstract

The invention provides an isofenphos-methyl test strip and application of the isofenphos-methyl test strip in detection of isofenphos-methyl residues, and belongs to the technical field of isofenphos-methyl residue detection. The test strip for detecting isofenphos-methyl comprises a sample absorption pad, a conjugate release pad, a reaction film, a water absorption pad and a bottom plate, and a detection line of the reaction film is coated with an isofenphos-methyl hapten-carrier protein conjugate; a quality control line of the reaction film is coated with a goat anti-mouse antibody; an isofenphos-methyl monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; and assembling the conjugate release pad, the reaction film, the sample absorption pad, the water absorption pad and the bottom plate into the test strip. The test strip for detecting the isofenphos-methyl residue is provided based on the isofenphos-methyl hapten, and the provided test strip has the characteristics of simplicity in operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.

Description

Technical Field

[0001] The invention relates to the technical field of isofenphos-methyl residue detection, and in particular to an isofenphos-methyl test strip and application thereof in detecting isofenphos-methyl residue. Background Art

[0002] Isofenphos-methyl, chemical name: N-isopropyl-O-methyl-O-[(2-isopropoxycarbonyl)phenyl]thiophosphoramidate. Pure isofenphos-methyl is a light yellow oily liquid. It is relatively stable at room temperature, and is easily decomposed in strong acid or alkaline medium. Light and heat can accelerate the decomposition. It is difficult to dissolve in water, but easily soluble in various organic solvents such as toluene, xylene and ether. Isofenphos-methyl is a new type of soil insecticide, which belongs to the highly toxic thiophosphate organophosphorus pesticide.

[0003] Methods for detecting isofenphos-methyl include gas chromatography, gas chromatography-mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, high performance liquid chromatography, etc. Compared with the above methods, immunochemical detection has the advantages of rapid, specific, sensitive, accurate, batch monitoring, simple sample processing, and automated operation for drug detection. The premise of immunochemical detection of isofenphos-methyl is that antibodies against isofenphos-methyl are required. So far, no artificial antigen for isofenphos-methyl detection has been found.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The invention aims to provide a test strip for detecting isofosfos-methyl residues with high sensitivity, simple operation, low cost and short detection time.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a test strip for detecting isofenphos-methyl, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the detection line of the reaction membrane is coated with a isofenphos-methyl hapten-carrier protein conjugate; the quality control line of the reaction membrane is coated with a goat anti-mouse anti-antibody; and the conjugate release pad is sprayed with a isofenphos-methyl monoclonal antibody-colloidal gold marker;

[0008] The conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate are assembled into a test strip.

[0009] Preferably, the isofosfosfomethyl hapten-carrier protein conjugate is obtained by coupling the isofosfosfosfomethyl hapten with a carrier protein.

[0010] Preferably, the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin.

[0011] Preferably, the structural formula of the methyl isofosfate hapten is as follows:

[0012]

[0013] Preferably, the isofosfosfo-methyl monoclonal antibody is prepared using isofosfosfo-methyl hapten-carrier protein conjugate as an immunogen.

[0014] The present invention also provides an application of the test strip in detecting isofenphos-methyl residues.

[0015] The methyl isofenphos rapid detection test strip provided by the present invention adopts highly specific antibody-antigen reaction and competitive inhibition immunochromatographic analysis technology, fixes methyl isofenphos monoclonal antibody-colloidal gold marker on the conjugate release pad, and the methyl isofenphos in the sample combines with the methyl isofenphos monoclonal antibody-colloidal gold marker on the conjugate release pad during the flow process to form a drug-antibody-colloidal gold marker. The drug in the sample competes with the methyl isofenphos hapten-carrier protein conjugate on the reaction membrane detection line to bind to the methyl isofenphos monoclonal antibody-colloidal gold marker, and whether the sample liquid to be tested contains methyl isofenphos residue is determined according to the presence or absence of a red strip on the detection line or the depth of the color.

[0016] During the test, the sample is treated and then dropped into the well of the test strip. When the concentration of methyl isofenphos in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker will bind to the methyl isofenphos hapten-carrier protein conjugate fixed on the reaction membrane during the chromatography process, and a red strip will appear in the test line (T) and the quality control line (C). If the concentration of methyl isofenphos in the sample is equal to or higher than the detection limit, the monoclonal antibody-colloidal gold marker will completely bind to the methyl isofenphos, so that it will not bind to the methyl isofenphos hapten-carrier protein conjugate at the T line due to the competitive reaction, and no red strip will appear.

[0017] The test strip of the present invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitable for use by various units, simple storage and long shelf life. The method of using the test strip of the present invention to detect the residue of isofosfos-methyl is simple, rapid, intuitive, accurate, widely applicable, low cost and easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a synthetic diagram of the methyl isoflavone hapten.

[0019] Figure 2 This is the H NMR spectrum of methyl isofosfosinate hapten.

[0020] Figure 3It is a schematic diagram of the cross-sectional structure of the test strip, 1 is the sample absorption pad, 2 is the conjugate release pad, 3 is the reaction membrane, 4 is the water absorption pad, 5 is the detection line (T), 6 is the quality control line (C), and 7 is the bottom plate. DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0022] Example 1

[0023] Preparation of methyl isofosfate hapten, hapten synthesis diagram as shown in Figure 1 shown.

[0024] Take 2.25 g of isopropyl 2-hydroxy-5-nitrobenzoate (CAS: 508211-75-4), add 200 mL of chloroform to dissolve, add 2.05 g of N-isopropylphosphoramidochlorosulfate O-methyl ester (CAS: 35236-17-0) and 1.3 mL of triethylamine, heat at 55 ° C for 2 h, stop the reaction, evaporate to dryness, and apply to a silica gel column. Elution and separation with methanol and dichloromethane in a volume ratio of 10:1 to obtain 2.16 g of intermediate 1 with a yield of 57.44%.

[0025] Take 2.16g of intermediate product 1, add 150mL of ethanol to dissolve, add 0.65g of zinc powder, add 1mL of 1mol / L hydrochloric acid and stir well, heat to 80℃ for reaction for 2h, stop the reaction, add 1mol / L sodium hydroxide to neutralize to neutrality, rotary evaporate to remove ethanol, add 200mL of chloroform, add 100mL of water, shake, let stand, separate the water phase, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness to obtain an oily substance, i.e. intermediate product 2.

[0026] Take all the intermediate product 2 obtained in the previous step, dissolve it in 50 mL of pyridine, add 1.14 g of succinic anhydride, heat at 90°C for 4 hours, stop the reaction, remove pyridine by rotary evaporation, and apply it to a silica gel column. Elution and separation with a volume ratio of dichloromethane to methanol of 6:1 are obtained to obtain 0.92 g of methyl isoflavone hapten with a yield of 35.9%.

[0027] The hapten product was analyzed by H NMR ( Figure 2 ) determination 1HNMR (500MHz, Chloroform-d) δ9.29 (s, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 8.4, 2.2 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 5.05 (dq, J = 11.5, 5.9 Hz, 1H), 5.00 (d, J = 7.7 Hz, 1H), 3.87 (d, J = 11.0 Hz, 2H), 3.64 (dp, J = 7.7, 6.6 Hz, 1H), 2.63–2.50 (m, 4H), 1.29 (dd, J = 12.5, 6.1 Hz, 12H). The existence of chemical shift δ absorption peaks of 2.63–2.50 (m, 4H) and 9.29 (s, 1H) proves that the spacer arm is successfully coupled.

[0028] The structural formula of the methyl isofosfate hapten is as follows:

[0029]

[0030] Example 2

[0031] Preparation of isofosfosinate-methyl hapten-carrier protein conjugate

[0032] Take 33.3 mg of the methyl isoflavone hapten prepared in Example 1, add 2 ml of DMF to dissolve and clarify, add NHS18 mg, EDC31 mg, fully dissolve and mix, react at room temperature for 2 hours, and obtain hapten activation solution A; take 100 mg of bovine serum albumin BSA, add 4 ml of 0.1M PB 8.0 buffer to dissolve, obtain solution B, add solution A dropwise into solution B, react at room temperature (20-25° C.) for 6 hours, stop the reaction, dialyze and purify with 0.02 M PBS for 3 days (the molecular weight cutoff of the dialysis bag is 8000-10000 Da), change the solution 3 times a day, centrifuge and pack, and obtain methyl isoflavone-BSA conjugate, which is the immunogen.

[0033] In the same manner, 19.84 mg of the methyl isoflavone hapten prepared in Example 1 was coupled with 100 mg of ovalbumin OVA in PB8.0 buffer, and the methyl isoflavone-OVA conjugate was obtained after purification by dialysis and used as the coating source.

[0034] Example 3

[0035] Preparation of test strips for isofosfos-methyl

[0036] 1. Preparation of monoclonal antibody against isofosfosinate-methyl

[0037] (1) Animal immunization

[0038] The immunogen of Example 2 was injected into Balb / c mice at an immunization dose of 150 μg / mouse to produce antiserum.

[0039] (2) Cell fusion and cloning

[0040] Take spleen cells of immune Balb / c mice, fuse them with SP2 / 0 myeloma cells at a ratio of 8:1 (quantity ratio), use indirect competitive ELISA to measure the cell supernatant, and screen the positive wells. Use limiting dilution method to clone the positive wells until a hybridoma cell line that stably secretes monoclonal antibodies is obtained. The present invention has nothing special about the screening of hybridoma cells. With the maturity of hybridoma cell technology, the present invention can obtain a hybridoma cell line that produces monoclonal antibodies corresponding to the immunogen according to conventional hybridoma construction and screening methods.

[0041] (3) Cell cryopreservation and recovery

[0042] Hybridoma cells were cryopreserved in 1×10 6 Cell suspension of 100 cells / ml is stored in liquid nitrogen for a long time. When thawing, take out the cryopreservation tube and immediately put it into a 37℃ water bath to thaw quickly. After centrifugation to remove the cryopreservation solution, transfer it into a culture bottle for culture.

[0043] (4) Preparation and purification of monoclonal antibodies

[0044] Incremental culture method: hybridoma cells are placed in a cell culture medium and cultured at 37°C. The culture fluid is purified using the caprylic acid-saturated ammonium sulfate method to obtain methyl isoflavone monoclonal antibody, which is stored at -20°C.

[0045] The cell culture medium is prepared by adding calf serum and sodium bicarbonate to RPMI1640 culture medium, so that the final concentration of calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH value of the cell culture medium is 7.4.

[0046] 2. Preparation of goat anti-mouse antibodies

[0047] Sheep are used as immune animals, and mouse antibodies are used as immunogens to immunize pathogen-free sheep to obtain sheep anti-mouse antibodies.

[0048] 3. Preparation of colloidal gold labelled monoclonal antibody against isofosfosinate-methyl

[0049] (1) Preparation of colloidal gold

[0050] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, take 100 ml and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, add 2.5 ml 1% trisodium citrate under continuous high temperature and continuous stirring, continue to stir and heat at a uniform speed until the solution turns translucent red, stop, cool to room temperature, restore to the original volume with deionized water, and store at 4°C. The prepared colloidal gold has a pure, translucent appearance, without precipitation and floating matter.

[0051] (2) Preparation of methyl isofosfosinate monoclonal antibody-colloidal gold label

[0052] Under magnetic stirring, adjust the pH value of colloidal gold to 7.0 with 0.2 mol / L potassium carbonate solution, add the prepared methyl isofosfosinate monoclonal antibody to the colloidal gold solution according to the standard of adding 25 μg per ml of colloidal gold solution, continue stirring and mixing for 30 minutes, add 10% BSA to make the final concentration in the colloidal gold solution 1% (volume fraction), and stand for 10 minutes. Centrifuge at 12000r / min and 4°C for 40 minutes, discard the supernatant, wash the precipitate twice with resolubilization buffer, resuspend the precipitate with resolubilization buffer of 1 / 10 of the initial colloidal gold volume, and set at 4°C for use.

[0053] Reconstitution buffer: containing 0.05% (mass fraction) casein, 0.1% (mass fraction) Tween-80, 0.02 mol / L phosphate buffer at pH 7.2.

[0054] 4. Preparation of conjugate release pad

[0055] The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer was 0.5%), pH 7.2, 0.5 mol / L, and evenly soaked for 1 hour, and then dried at 37°C for 3 hours for use. The prepared isofosfomethyl monoclonal antibody-colloidal gold marker was evenly sprayed on the conjugate release pad using an Isoflow film sprayer, and 0.01 ml of isofosfomethyl monoclonal antibody-colloidal gold marker was sprayed per 1 cm of the conjugate release pad, and then placed in a 37°C environment (humidity <20%) for 60 minutes, taken out, and placed in a dry environment (humidity <20%) for storage.

[0056] 5. Preparation of reaction membrane

[0057] The methyl isoflavone hapten-ovalbumin conjugate is coated on the reaction membrane to form a detection line, and the goat anti-mouse antibody is coated on the reaction membrane to form a quality control line.

[0058] Coating process: dilute the methyl isofosfate hapten-ovalbumin conjugate to 10 mg / ml with phosphate buffer, and coat it on the test line (T line) on the nitrocellulose membrane with an Isoflow film dispenser, with a coating amount of 0.8 μl / cm; dilute the goat anti-mouse anti-antibody to 200 μg / ml with 0.01 mol / L, pH 7.4 phosphate buffer, and coat it on the quality control line (C line) on the nitrocellulose membrane with an Isoflow film dispenser, with a coating amount of 1.0 μl / cm. Dry the coated reaction membrane at 37°C for 2 hours for use.

[0059] 6. Preparation of sample absorption pad

[0060] The sample absorption pad was immersed in a phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH 7.2, and 0.1 mol / L for 2 h, and then dried at 37° C. for 2 h for later use.

[0061] 7. Assembly of test strips

[0062] like Figure 3 As shown, the sample absorption pad, the conjugate release pad, the reaction membrane, and the water absorption pad are sequentially pasted on the PVC bottom plate; 1 / 3 of the conjugate release pad is covered by the sample absorption pad from the starting end, the end of the conjugate release pad is connected to the starting end of the reaction membrane, the end of the reaction membrane is connected to the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC bottom plate, and the end of the water absorption pad is aligned with the end of the PVC bottom plate; the reaction membrane is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are both strips perpendicular to the length of the test strip; the detection line is located on the side close to the end of the conjugate release pad; the quality control line is located on the side away from the end of the conjugate release pad; the test strip is cut into 3 mm wide strips by a machine, and is placed in a special plastic card, which can be stored for 12 months at 4-30°C.

[0063] Example 4

[0064] Detection Method of Isothioate-Methyl Residue in Samples

[0065] 1. Sample pretreatment method

[0066] (1) Before testing, the samples should be returned to room temperature and the Chinese medicine samples should be fully crushed;

[0067] (2) Weigh 1.00 g of the crushed sample into a 10 mL centrifuge tube, add 4 mL of extract (pure acetonitrile), vortex for 1 min, and centrifuge at 3000 r / min for 5 min at room temperature;

[0068] (3) Take 2 mL of the supernatant and transfer it to a 10 mL centrifuge tube and blow dry it in a 50°C water bath under a nitrogen flow;

[0069] (4) Add 500 μL of reconstitution solution (0.1 mol / L phosphate buffer, pH 7.2) and vortex for 30 s to mix thoroughly to obtain the test solution.

[0070] 2. Test with test strips

[0071] Use a pipette to draw up the test liquid and vertically drip 3 drops into the sample hole of the test strip. Start timing when the liquid flows, and let the reaction take 5 to 10 minutes to determine the result.

[0072] 3. Analyze the test results

[0073] Negative (-): The color of the T line is higher than or equal to that of the C line, indicating that the concentration of the analyte in the sample is lower than the detection limit;

[0074] Positive (+): The color of the T line is lower than that of the C line, or the T line does not show color, which means that the concentration of the analyte in the sample is equal to or higher than the detection limit;

[0075] Invalid: No C line appears, indicating that the operation process is incorrect or the test strip has expired. In this case, you should read the instructions carefully again and retest with a new test strip.

[0076] Example 5

[0077] Sample testing example

[0078] 1. Detection limit test

[0079] Take blank honeysuckle, wolfberry, and tangerine peel samples, add methyl isothioate to the final concentrations of 10, 20, and 40 μg / L, respectively, take test strips for detection, and repeat the measurement three times for each sample.

[0080] When using test strips to detect traditional Chinese medicine samples, when the added concentration of methyl isothioate is 10 μg / L, the color of the T line is higher than or equal to the color of the C line, and the result is negative; when the added concentration of methyl isothioate is 20 or 40 μg / L, the color of the T line is lower than the color of the C line or the T line does not show color, and the result is positive.

[0081] 2. False positive rate and false negative rate test

[0082] Take 50 positive honeysuckle, wolfberry, and tangerine peel samples with a known methyl isofenphos content of 20 μg / L and 50 negative honeysuckle, wolfberry, and tangerine peel samples with a content of less than 10 μg / L, and use three batches of test strips to test and calculate their positive and negative rates. The results are shown in Table 1.

[0083] Table 1 Results of Chinese medicine test samples

[0084]

[0085] The results show that when the test strips produced in three batches are used to detect Chinese medicine samples, the false negative rate of the Chinese medicine samples is 0%, and the false positive rate is 0%-4%. A small number of false positive samples appear in the above samples, but the false positive rate is low, indicating that the test strip for detecting methyl isofenphos can be used to quickly detect methyl isofenphos residues in Chinese medicine.

[0086] 3. Specificity test

[0087] The specificity with phorate, phoxim, carbendazim, chlorpyrifos, procymidone, chlorfenapyr, alachlor, dicofol and other substances was determined, and the results are shown in Table 2. The test results show that the test strip has good specificity for isofenphos-methyl.

[0088] Table 2 Specificity test

[0089] Drug Name Test strip results Isothioate-methyl Positive Phorate Negative Phoxim Negative Carbendazim Negative Chlorpyrifos Negative Procymidone Negative Chlorfenapyr Negative Alachlor Negative Dicofol Negative

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A test strip for detecting isofosfos-methyl, comprising a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, characterized in that: The detection line of the reaction membrane is coated with a methyl isoflavone hapten-carrier protein conjugate; the quality control line of the reaction membrane is coated with a goat anti-mouse anti-antibody; and the conjugate release pad is sprayed with a methyl isoflavone monoclonal antibody-colloidal gold marker; The conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate are assembled into a test strip.

2. A test strip for detecting isofosfos-methyl as claimed in claim 1, characterized in that: The isofosfomethyl hapten-carrier protein conjugate is obtained by coupling the isofosfomethyl hapten with a carrier protein.

3. A test strip for detecting isofosfos-methyl as claimed in claim 2, characterized in that: The carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin.

4. A test strip for detecting isofosfos-methyl as claimed in claim 3, characterized in that: The structural formula of the isofosfate-methyl hapten is as follows:

5. The test strip according to claim 1, characterized in that: The isofosfosinate-methyl monoclonal antibody is prepared by using isofosfosinate-methyl hapten-carrier protein conjugate as an immunogen.

6. Use of the test strip according to claim 1 in detecting isofosfos-methyl residues.

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