CN113156127A - Test strip and method for detecting chlorpyrifos - Google Patents

Abstract

The invention discloses a test strip and a method for detecting chlorpyrifos. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and a chlorpyrifos monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad. The invention also provides a method for detecting chlorpyrifos in plant-derived food by applying the test strip. The test strip provided by the invention has the advantages of simple operation, high sensitivity, high detection speed, low cost, suitability for screening large-batch samples and the like, and can meet the requirements of food supervision departments in China on-site monitoring and detection.

Description

Test strip and method for detecting chlorpyrifos

Technical Field

The invention relates to a test strip and a method for detecting chlorpyrifos, in particular to a colloidal gold test strip for detecting chlorpyrifos, which is particularly suitable for detecting the chlorpyrifos in plant-derived foods such as vegetables, fruits and the like.

Background

The chlorpyrifos is a low-toxicity organophosphorus insecticide with contact poisoning, stomach poisoning and fumigation effects, and is widely applied as a substitute due to low toxicity as 5 kinds of high-toxicity organophosphorus pesticides (methamidophos, parathion, methyl parathion, monocrotophos and ammonium phosphate) are prohibited from being produced and sold in China in recent years, but pesticide residue verification test results show that the application of the chlorpyrifos not only easily causes the pesticide residue of vegetables and fruits to exceed the standard, but also has influence on the environment, the living things and the human health. Therefore, in order to better guarantee the public life health and reduce the risk to the maximum extent, the national standard GB 2763 'maximum pesticide residue limit in food safety national standard food' stipulates the maximum residue limit of chlorpyrifos in different foods.

At present, the chlorpyrifos detection mainly adopts analysis methods such as a high performance liquid chromatography, a liquid chromatography-mass spectrometry combined method, a gas chromatography-mass spectrometry combined method and the like, the methods are required to be operated under laboratory conditions, the sample pretreatment is complicated and time-consuming, expensive instruments and equipment are required, the detection cost is high, the time consumption is long, the operation is complex, the method has great limitation in the practical application process, and the requirement of rapid detection of a large number of samples and field samples is difficult to meet. Therefore, the colloidal gold test strip which is simple and quick and is suitable for detecting the chlorpyrifos in the food is developed, the field screening and monitoring of a large number of samples can be met, and the detection work of food supervision departments and the like in China can be better met.

Disclosure of Invention

The invention aims to provide a colloidal gold test strip capable of detecting chlorpyrifos in plant-derived food, and provides a detection method which is efficient, accurate, simple and convenient, and is suitable for field monitoring and large-scale sample screening.

The test strip for detecting chlorpyrifos provided by the invention comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate; the reaction membrane is provided with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody; the conjugate release pad is sprayed with a chlorpyrifos monoclonal antibody-colloidal gold marker.

The chlorpyrifos monoclonal antibody is prepared by taking a chlorpyrifos hapten-carrier protein conjugate as an immunogen.

The chlorpyrifos hapten-carrier protein conjugate is obtained by coupling chlorpyrifos hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, the chlorpyrifos hapten is obtained by taking trichlorosulfur as a starting raw material and carrying out condensation reaction with (2,3, 5-trichloro-6-hydroxy-pyridine-4-yl) -acetic acid and ethanol in sequence, and the molecular structural formula is as follows:

the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate, and the conjugate release pads 1/3-1/2 are covered under the sample absorption pad.

The bottom plate is a PVC bottom plate or other hard non-absorbent materials; the sample absorption pad is made of polyester fiber or glass fiber; the conjugate release pad is a polyester cellulose membrane or a glass cellulose membrane; the water absorption pad is water absorption filter paper; the reaction membrane is a nitrocellulose membrane or a cellulose acetate membrane.

Another object of the present invention is to provide a method for preparing the test strip, which comprises the steps of:

1) preparing a conjugate release pad sprayed with a chlorpyrifos monoclonal antibody-colloidal gold marker;

2) preparing a reaction membrane with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

Specifically, the steps include:

1) taking trichloro-sulfur as a starting material, and carrying out condensation reaction on the trichloro-sulfur as well as (2,3, 5-trichloro-6-hydroxy-pyridin-4-yl) -acetic acid and ethanol in sequence to prepare chlorpyrifos hapten;

2) coupling the chlorpyrifos hapten with carrier protein to prepare a chlorpyrifos hapten-carrier protein conjugate;

3) immunizing a mouse by using a chlorpyrifos hapten-carrier protein conjugate, and fusing and screening splenocytes of the mouse and myeloma cells of the mouse to obtain a hybridoma cell strain secreting a chlorpyrifos monoclonal antibody;

4) extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibodies;

5) coating the chlorpyrifos hapten-carrier protein conjugate and the goat anti-mouse anti-antibody on a detection line (T) and a quality control line (C) of a reaction membrane respectively;

6) preparing colloidal gold by reacting trisodium citrate with chloroauric acid;

7) adding the prepared chlorpyrifos monoclonal antibody into the prepared colloidal gold to obtain a chlorpyrifos monoclonal antibody-colloidal gold marker;

8) spraying the chlorpyrifos monoclonal antibody-colloidal gold marker on a conjugate release pad, drying at 37 ℃ for 2h, taking out, and storing in a dry environment for later use;

9) soaking a sample absorption pad in 0.02mol/L phosphate buffer solution containing 1% bovine serum albumin and having pH of 7.2 for 2h, and drying at 37 ℃ for 2h for later use;

10) a sample absorbing pad, a conjugate releasing pad, a reaction membrane and a water absorbing pad are sequentially adhered on the bottom plate, and the conjugate releasing pad is covered by the sample absorbing pad from the 1/3 area at the starting end. Finally cutting into small strips with the width of 3.95mm, putting the small strips into a special plastic card shell, sealing the card shell by an aluminum foil bag, and storing the card shell for 12 months at the temperature of 4-30 ℃.

The invention also aims to provide a method for detecting chlorpyrifos in plant-derived food by using the test strip, which comprises the following steps:

(1) pretreating a sample;

(2) detecting by using a test strip;

(3) and analyzing the detection result.

The test strip for quickly detecting the chlorpyrifos adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology, a chlorpyrifos monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and the chlorpyrifos in a sample is combined with the chlorpyrifos monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form the chlorpyrifos-antibody-colloidal gold marker. The chlorpyrifos in the sample and the chlorpyrifos hapten-carrier protein conjugate on the reaction film detection line compete to be combined with the chlorpyrifos monoclonal antibody-colloidal gold marker, and whether the chlorpyrifos is contained in the sample liquid to be detected or not is judged according to the depth of a red strip of the detection line.

During detection, a sample is treated and then dropped into a test strip card hole, when the concentration of chlorpyrifos in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with a chlorpyrifos hapten-carrier protein conjugate fixed on a reaction membrane in the chromatography process, a red strip is respectively formed on a detection line (T) and a quality control line (C), and the color development of the T line is deeper than that of the C line or is consistent with that of the C line; if the concentration of chlorpyrifos in the sample is equal to or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the chlorpyrifos, so that no red band or less coloration will appear at line T due to the competition reaction with the chlorpyrifos hapten-carrier protein conjugate. As shown in fig. 3.

Negative: when the quality control line (C) shows a red strip, the detection line (T) also shows a red strip, and the color of the line (T) is close to or deeper than that of the line (C), the line (C) is judged to be negative.

Positive: and when the quality control line (C) shows a red strip, the detection line (T) does not show color or the color of the line (T) is lighter than that of the line (C), the test line is judged to be positive.

And (4) invalidation: when the quality control line (C) does not show a red strip, the test strip is judged to be invalid whether the detection line (T) shows a red strip or not.

The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long quality guarantee period. The method for detecting chlorpyrifos by using the test strip is simple, convenient, rapid, visual, accurate, wide in application range, low in cost and easy to popularize and use.

Drawings

FIG. 1 is a diagram of synthesis of chlorpyrifos hapten.

FIG. 2 is a schematic cross-sectional view of a test strip.

FIG. 3 is a diagram showing the test result of the test strip.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 preparation of test strip for detecting Chlorpyrifos

The preparation method of the test strip mainly comprises the following steps:

1) preparing a conjugate release pad sprayed with a chlorpyrifos monoclonal antibody-colloidal gold marker;

2) preparing a reaction membrane with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

The following steps are detailed:

1. synthesis of Chlorpyrifos hapten (the synthetic route is shown in figure 1)

Taking 2.56g of (2,3, 5-trichloro-6-hydroxy-pyridin-4-yl) -acetic acid, adding 80mL of acetone for dissolving, adding 1.71g of trichloro sulfur phosphorus, fully stirring at room temperature, adding 10mL of pyridine, continuing stirring for 2h, stopping the reaction, performing rotary evaporation, removing organic solvents such as acetone and the like to obtain a red oily substance, namely a crude intermediate compound b, directly dissolving the red oily substance in 60mL of absolute ethyl alcohol, fully stirring, adding 3mL of triethylamine, stirring, heating at 55 ℃ for reaction for 4h, stopping the reaction, performing rotary evaporation, removing the ethyl alcohol and the triethylamine to obtain an oily substance, loading on a silica gel column, and performing elution and separation by using a mixed solution of dichloromethane and methanol with the volume ratio of 10:1 to obtain 1.16g of chlorpyrifos hapten.

2. Preparation of immunogens

Taking 15mg of chlorpyrifos hapten, adding 1mL of dimethyl sulfoxide (DMSO) for dissolving, adding 11.7mg of N-hydroxysuccinimide (NHS) and 13.2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and reacting at room temperature for 3h to obtain a hapten solution A; dissolving 50mg of Human Serum Albumin (HSA) in 0.05mol/L PB buffer solution to obtain solution B; dripping the solution A into the solution B, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution three times every day to obtain the chlorpyrifos hapten-HSA conjugate which is the immunogen, and storing at-20 ℃ for later use.

3. Preparation of coating antigen

Taking 8.6mg of chlorpyrifos hapten, adding 1mL of N, N-Dimethylformamide (DMF) for dissolving, adding 6.1mg of NHS and 8.8mg of EDC, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving Ovalbumin (OVA)50mg in PB buffer solution 0.05mol/L to obtain solution B; dripping the solution A into the solution B, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution three times every day to obtain the chlorpyrifos hapten-OVA conjugate which is the coating antigen, and storing at-20 ℃ for later use.

4. Preparation of chlorpyrifos monoclonal antibody

(1) Animal immunization

Injecting the immunogen obtained in the step 2 into Balb/c mice at an immune dose of 150 mug/mouse to generate antiserum.

(2) Cell fusion and cloning

Taking immune Balb/c mouse spleen cells, fusing the immune Balb/c mouse spleen cells with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative ratio), measuring cell supernatant by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining the hybridoma cell strain which stably secretes the monoclonal antibody.

(3) Cell cryopreservation and recovery

Making hybridoma cell into 1 × 10 with frozen stock solution⁶Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.

(4) Preparation and purification of monoclonal antibodies

An incremental culture method: placing the hybridoma cell in cell culture medium, culturing at 37 deg.C, purifying the obtained culture solution by octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibody, and storing at-20 deg.C.

The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium to make the final concentration of calf serum in the cell culture medium 20% (mass fraction) and the final concentration of sodium bicarbonate in the cell culture medium 0.2% (mass fraction); the pH of the cell culture medium was 7.4.

5. Preparation of goat anti-mouse anti-antibody

The sheep is taken as an immune animal, and the pathogen-free sheep is immunized by taking the murine antibody as an immunogen to obtain the goat anti-mouse antibody.

6. Preparation of chlorpyrifos monoclonal antibody-colloidal gold marker

(1) Preparation of colloidal gold

Diluting 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, placing 100mL into a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 1.5mL of 1% trisodium citrate under continuous stirring at high temperature, stirring and heating at constant speed until the solution is bright red, cooling to room temperature, recovering the volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, is transparent and bright, has no sediment or floating objects, and is wine red when observed in sunlight.

(2) Preparation of chlorpyrifos monoclonal antibody-colloidal gold marker

Under magnetic stirring, adjusting the pH value of the colloidal gold to 7.2 by using 0.2mol/L potassium carbonate solution, adding the chlorpyrifos monoclonal antibody into the colloidal gold solution according to the standard that 5-50 mu g of antibody is added into each milliliter of the colloidal gold solution, and continuously stirring and uniformly mixing for 30 min; after standing for 10min, 10% BSA was added to make the final concentration of the solution in colloidal gold 1%, and the solution was allowed to stand for 10 min. Centrifuging at 12000r/min at 4 deg.C for 40min, discarding supernatant, washing the precipitate with redissolving buffer twice, resuspending the precipitate with redissolving buffer whose volume is 1/10 of the original volume of colloidal gold, and standing at 4 deg.C for use.

Redissolving buffer solution: 0.1 to 0.5 percent of BSA, 2 to 4 percent of sucrose and 0.02mol/L of pH 7.2.

7. Preparation of conjugate Release pad

The conjugate release pad was soaked in 0.02mol/L phosphate buffer containing 0.5% BSA, 5% sucrose, pH 7.4, soaked for 2h, and dried at 37 deg.C for use. And (3) uniformly spraying the prepared chlorpyrifos monoclonal antibody-colloidal gold marker on a conjugate release pad by using a Bio dot film scratching instrument, spraying 0.01mL of the chlorpyrifos monoclonal antibody-colloidal gold marker on every 1cm of the conjugate release pad, placing the mixture in an environment (the humidity is less than 20%) at 37 ℃ for 2h, taking out the mixture, and placing the mixture in a dry environment (the humidity is less than 20%) for storage for later use.

8. Preparation of sample absorbent pad

The sample absorbing pad is soaked in 0.02mol/L phosphate buffer solution containing 1% BSA and having pH of 7.2 for 2h, and dried at 37 ℃ for 2h for later use.

9. Preparation of the reaction film

And coating the chlorpyrifos hapten-OVA conjugate on a reaction membrane to form a detection line, and coating the goat anti-mouse anti-antibody on the reaction membrane to form a quality control line.

Coating process: diluting the chlorpyrifos hapten-OVA conjugate to 1mg/mL by using 0.01mol/L, pH 7.2.2 of phosphate buffer, and coating the chlorpyrifos hapten-OVA conjugate on a detection line (T line) on a nitrocellulose membrane by using a Bio dot film cutting instrument, wherein the coating amount is 1.0 mu L/cm; the goat anti-mouse anti-antibody was diluted to 200. mu.g/mL with 0.01mol/L, pH 7.2.2 phosphate buffer and coated on a control line on nitrocellulose membrane (line C) with a Bio dot striping machine in an amount of 1.0. mu.L/cm. And (3) drying the coated reaction membrane at 37 ℃ for 16h for later use.

10. Assembly of test strips

According to the section structure of the test strip shown in the attached figure 2, a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3) and a water absorption pad (4) are sequentially adhered to a PVC bottom plate (7); the binder release pad is covered by the sample absorption pad from the 1/3 area at the starting end, the tail end of the binder release pad is connected with the starting end of the reaction film, the tail end of the reaction film is connected with 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 base plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC base plate; the reaction membrane is provided with a detection line (5) and a quality control line (6), and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are vertical to the long phase of the test strip; the detection line is located on the side near the end of the conjugate release pad; the quality control line is positioned on the side away from the end of the conjugate release pad; cutting the test strip into small strips with the width of 3.95mm by a machine, placing the small strips in a specially-made plastic card shell, sealing the card shell by an aluminum foil bag, and storing the card shell in an environment at the temperature of 4-30 ℃ for 12 months.

Example 2 detection of Chlorpyrifos in plant-derived foods

1. Sample pretreatment

Taking a fresh sample, wiping off soil, and cutting the fresh sample into fragments with the square length of less than 1 cm; weighing (1.00 +/-0.05) g of sample into a 15mL polystyrene centrifuge tube, adding 5mL of 0.02mol/L, pH 7.4.4 PBS buffer solution, covering the cover, manually oscillating up and down for 30s, and standing for 1min to obtain the upper layer liquid, namely the sample liquid to be detected.

2. Detection with test strips

Sucking 100 mu L of sample liquid to be detected by a micropipettor and vertically dripping the sample liquid into the sample adding hole; the liquid flow was started, the reaction was carried out for 10min, and the results were judged.

3. Analyzing the results of the detection

Negative (-): the color development of the T line is darker than that of the C line or consistent with that of the C line, which indicates that the concentration of chlorpyrifos in the sample is lower than the detection limit, as shown in FIGS. 3a and 3 b.

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 chlorpyrifos in the sample is equal to or higher than the detection limit, as shown in FIGS. 3C and 3 d.

And (4) invalidation: the absence of line C indicates an incorrect procedure or the test strip has deteriorated and failed, as shown in FIGS. 3e and 3 f.

Example 3 sample testing example

1. Limit of detection test

Taking samples of blank leeks, spinach, common cabbages, leaf lettuce, Chinese cabbages, cucumbers, apples, pears and oranges, respectively adding chlorpyrifos to the samples until the final concentration is 0.05mg/kg, 0.1mg/kg and 0.2mg/kg, taking test strips for detection, and repeatedly measuring each sample for three times.

When the test paper is used for detecting samples of Chinese chives, spinach, common cabbages, leaf lettuce, Chinese cabbages, cucumbers, apples, pears and oranges, when no chlorpyrifos is added and the addition concentration of the chlorpyrifos is 0.05mg/kg, the test paper shows that the color development of a T line is darker than that of a C line or is consistent with that of the C line, and the test paper is negative; when the adding concentration of the chlorpyrifos is 0.1mg/kg and 0.2mg/kg, the test strip shows that the color development of the T line is lighter than that of the C line or the T line is not developed and is positive, which indicates that the detection limit of the test strip on the chlorpyrifos in the plant-derived food is 0.1 mg/kg.

2. Test for false positive and false negative rates

Taking samples of blank leeks, spinach, common cabbages, leaf lettuce, Chinese cabbages, cucumbers, apples, pears and oranges, adding 20 parts of positive leeks, spinach, common cabbages, leaf lettuce, Chinese cabbages, cucumbers, apples and oranges which are respectively added with chlorpyrifos to the final concentration of 0.1mg/kg, respectively detecting by using 3 test strips produced in batches, and calculating the negative and positive rates of the samples.

The results show that: when 3 batches of test strips are used for detecting positive samples, the results are all positive, the positive coincidence rate is 100 percent, and the false negative rate is 0; when negative samples are detected, the results are all negative, and the negative coincidence rate is 100 percent and the false positive rate is 0. The test strip for detecting chlorpyrifos can be used for quickly detecting the chlorpyrifos in plant-derived food.

3. Specificity test

When the test strip is used for detecting other organophosphorus pesticides such as 10mg/kg parathion, triazophos, isocarbophos, malathion, fenitrothion, dimethoate, trichlorfon and the like, the test strip shows that the color development of a T line is darker than or consistent with that of a C line and is negative, so that the test strip has no cross reaction to the drugs.

Claims (5)

1. A test strip for detecting chlorpyrifos comprises a sample absorption pad, a conjugate release pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the reaction membrane is provided with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody, and a chlorpyrifos monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad; the chlorpyrifos monoclonal antibody is prepared by taking a chlorpyrifos hapten-carrier protein conjugate as an immunogen; the chlorpyrifos hapten-carrier protein conjugate is obtained by coupling chlorpyrifos hapten and carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroid protein or human serum albumin, and is characterized in that the chlorpyrifos hapten is obtained by taking trichlorothion as a starting raw material and carrying out condensation reaction on the trichlorothion, the (2,3, 5-trichloro-6-hydroxy-pyridin-4-yl) -acetic acid and ethanol in sequence, and the molecular structural formula is as follows:

2. the strip of claim 1, wherein the sample absorbing pad, the conjugate releasing pad, the reaction membrane, and the absorbent pad are sequentially attached to the base plate.

3. The strip of any one of claims 1-2, wherein the conjugate release pad 1/3-1/2 is covered under a sample absorbing pad.

4. A method of making the test strip of any one of claims 1-3, comprising the steps of:

1) preparing a conjugate release pad sprayed with a chlorpyrifos monoclonal antibody-colloidal gold marker;

2) preparing a reaction membrane with a detection line coated with a chlorpyrifos hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;

3) assembling the conjugate release pad and the reaction membrane prepared in the steps 1) and 2) with a sample absorption pad, a water absorption pad and a base plate to form the test strip.

5. A method for detecting chlorpyrifos in plant-derived food comprises the following steps:

1) pretreating a sample;

2) performing a test using the test strip of any one of claims 1-3;

3) and analyzing the detection result.

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