CN114675015A - Parathion pesticide colloidal gold marker based on nano antibody and application thereof - Google Patents
Parathion pesticide colloidal gold marker based on nano antibody and application thereof Download PDFInfo
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Abstract
The invention provides a parathion pesticide colloidal gold marker based on a nano antibody and application thereof. The invention is developed for the first time by combining the parathion nano antibody and the colloidal gold to prepare the parathion nano antibody-colloidal gold marker for detecting parathion pesticides. The parathion nano antibody-colloidal gold marker is adopted for detection, so that the stability is good, and the sensitivity is high; the nano antibody colloidal gold test strip for preparing the parathion pesticide has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, easy storage and long quality guarantee period. The sensitivity of the test strip for detecting the parathion pesticide residue provided by the invention reaches 0.0013-0.085 mg/kg, the detection limit is 0.00069mg/kg, the test strip can be used for quickly detecting the parathion pesticide in agricultural products such as vegetables and fruits, and the method is simple, convenient, quick, intuitive, accurate, wide in application range, low in cost and easy to popularize and use.
Description
Technical Field
The invention belongs to the technical field of pesticide residue immunoassay detection. More particularly, relates to a parathion pesticide colloidal gold marker based on a nano antibody and application thereof.
Background
Parathion is a broad-spectrum high-toxicity organic phosphate insecticide and acaricide, has strong contact poisoning and stomach poisoning effects, and has a certain fumigation effect. Because of the pest control effect of the parathion pesticide on crops such as fruits, vegetables and grains, the parathion pesticide is widely used in agricultural production, and the ecological safety risk and the potential safety hazard to human beings and other non-target organisms are concerned at present. Parathion can inhibit the activity of acetylcholinesterase, so that a great amount of acetylcholine of neurotransmitter in vivo is accumulated, a series of nerve reactions are caused, toxic symptoms such as nausea, headache, weakness, chest distress and the like are caused, and death can be caused seriously. If the parathion is accumulated in the environment and food, direct or potential serious harm is certainly caused to the health of human beings, and the parathion belongs to a high-toxicity pesticide variety and is forbidden to be used on vegetables and fruits at present, so that the research and development of a rapid detection method and technology for the parathion pesticide residue have important practical significance.
At present, the detection method of parathion pesticide residue is mainly instrument methods such as High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS) and the like, but although the detection result of the instrument detection method is accurate and sensitive, the defects of complex operation process, expensive instrument, high detection cost and the like cause the instrument detection to be not suitable for on-site instant detection. The colloidal gold immunochromatography technology is an instant detection technology combining the immunity technology and the chromatography technology, which is applied from the 80 th of the last century. The principle is that a sample containing an analyte flows through the whole test strip through capillary action and performs high-specificity and high-affinity immunoreaction with a coating substance (antigen or antibody) on a reaction membrane of the test strip, and an immune complex is trapped on the test strip for color development, so that a detection result can be obtained. The antibody used by the existing colloidal gold immunochromatography technology is almost all monoclonal antibody or polyclonal antibody, for example, the prior art CN 106970217B discloses an immunochromatography method for quantitatively detecting organophosphorus pesticides, which is an immunochromatography method for preparing the quantitatively detecting organophosphorus pesticides by using a monoclonal antibody as a fixed detection line and using acetylcholinesterase to modify an indicator, and the method is directed at the multi-residue detection of the organophosphorus pesticides; although the detection method using the antigen-antibody can only realize the detection of a single target object, the maximum residual limit of the national standard for the relatively severe residual limit of parathion pesticides is 0.01mg/kg, so that the current immunochromatography method for detecting parathion by adopting a monoclonal antibody or a polyclonal antibody can not meet the national standard. Compared with the traditional antibody, the nano antibody (Nbs) has the advantages of small relative molecular mass, simple humanization, high affinity, high stability, microbial expression, low immunogenicity, strong penetration, good solubility and the like, and is concerned in basic medical research, disease diagnosis and treatment. At present, no report of the detection and analysis of the parathion pesticide by the nano antibody exists, so that more new products and methods which are simpler, more convenient and lower in cost and can be used for detecting the parathion pesticide residue need to be developed, and the parathion detection method meets the national standard requirements.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing problems and provides a parathion pesticide colloidal gold marker based on a nano antibody and application thereof.
The invention aims to provide a parathion pesticide colloidal gold marker based on a nano antibody.
The second purpose of the invention is to provide the application of the parathion nano antibody colloidal gold marker.
The third purpose of the invention is to provide a parathion pesticide detection nano antibody colloidal gold test strip.
The fourth purpose of the invention is to provide the application of the test strip.
The fifth purpose of the invention is to provide a method for detecting parathion pesticides in crops.
The above purpose of the invention is realized by the following technical scheme:
a parathion pesticide colloidal gold marker based on a nano antibody is prepared by the following steps: adding 1-10 mu g/mL parathion nano antibody into a colloidal gold solution with the pH value of 8-10, uniformly mixing and standing, adding 20-40 mu L10% BSA, standing and centrifuging, discarding supernatant, and carrying out heavy suspension precipitation by using a colloidal gold redissolution buffer solution to obtain a parathion nano antibody colloidal gold marker; the volume ratio of the colloidal gold solution is 1: 6 of 1% chloroauric acid and 1% trisodium citrate; the formula of the colloidal gold redissolution buffer solution is as follows: 0.01-0.5M Tris-HCl, 0.1-2% BSA, 0.1-2% Tween-20, 0.1-2% polyvinylpyrrolidone and 1-15% sucrose.
The parathion nano antibody is combined with the parathion colloidal gold for the first time, and the prepared parathion nano antibody colloidal gold marker can be used for detecting parathion pesticides; the phosphorus nano antibody colloidal gold marker with good stability and high sensitivity is finally prepared by optimizing and screening various conditions in the preparation process of the phosphorus nano antibody colloidal gold marker.
Preferably, the pH value of the colloidal gold solution is 8.5-9.5, the adding amount of the parathion nano antibody is 3-7 mu g/mL, and the adding amount of 10% BSA is 30-40 mu L.
Preferably, the particle size of the colloidal gold solution is 10-20 nm.
Preferably, the centrifugation conditions are 9000rpm, 4 ℃ and 15 min.
Preferably, the resuspended pellet is resuspended with a volume of colloidal gold 1/5 of colloidal gold reconstitution buffer.
Preferably, the formula of the colloidal gold redissolution buffer solution is as follows: 0.05M Tris-HCl, 0.2-1% BSA, 2-10% sucrose, 200.1-1% Tween-and 0.3-1% polyvinylpyrrolidone.
The invention provides an application of a parathion nano antibody colloidal gold marker in the detection of parathion pesticides by an immunochromatography technology.
The invention provides application of a parathion nano antibody colloidal gold marker in preparation of parathion pesticide products.
The invention provides a nano antibody colloidal gold test strip for detecting parathion pesticide, which contains the parathion nano antibody colloidal gold marker on a combined release pad.
Preferably, the kit further comprises a sample pad, a reaction membrane, a water absorption pad and a bottom plate.
Preferably, the reaction membrane is provided with a detection line coated with a parathion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel antibody.
The nano antibody colloidal gold test strip for detecting parathion pesticide provided by the invention combines a parathion nano antibody colloidal gold marker with a substance to be tested, and detects the parathion pesticide by carrying out competitive inhibition reaction with a parathion pesticide hapten-carrier protein and rabbit anti-camel anti-antibody. Adding a sample solution to be detected onto the test strip from the sample pad, enabling the sample solution to flow through the whole test strip through capillary force, and firstly combining a substance to be detected in a sample with a parathion nano antibody colloidal gold marker on the combined release pad to form a drug-antibody-colloidal gold marker. And then the mixed solution flows through a reaction membrane, the drug and the parathion pesticide hapten-carrier protein on the detection line compete to combine with the parathion nano antibody colloidal gold marker, and whether the parathion pesticide residue exists in the sample solution to be detected is judged according to whether the strip of the detection line is developed or the development depth.
The test strip is prepared by the following method:
s1, preparing a combined release pad sprayed with a parathion nano antibody colloidal gold marker;
s2, preparing a reaction membrane with a detection line coated with a parathion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel anti-antibody;
s3, assembling the combined release pad and the reaction membrane prepared in the steps S1 and S2, the sample pad, the water absorption pad and the bottom plate into the test strip.
Preferably, the formula of the colloidal gold redissolution buffer solution is as follows: 0.05M Tris-HCl, 0.2-1% BSA, 2-10% sucrose, 200.1-1% Tween-and 0.3-1% polyvinylpyrrolidone.
Preferably, the parathion pesticide hapten solution is prepared by a method described in the literature (Xu et al. The hapten-carrier protein of the parathion pesticide is obtained by chemically coupling the hapten of the parathion pesticide and ovalbumin by using chemical reagents DMF and NHS.
Preferably, the rabbit anti-camel antibody solution is an antibody solution obtained by injecting parathion nano-antibodies serving as immunogens into rabbits and finally separating the rabbit sera.
Preferably, the parathion pesticide hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the rabbit anti-camel antibody is coated on the reaction membrane to form a quality control line.
When the test strip prepared by the invention is used for detection, a sample solution is added into the test strip from the sample pad and passes through the whole test strip through the capillary action force. When the concentration of the parathion pesticide in the sample treatment liquid is lower than the detection limit or zero, the parathion nano antibody colloidal gold marker is combined with the parathion pesticide hapten-carrier protein and the rabbit anti-camel anti-antibody in the chromatography process to respectively develop color on a detection line and a quality control line; when the concentration of the parathion pesticide in the sample treatment liquid is equal to or higher than the detection limit, the pesticide in the sample can be completely combined with the parathion nano antibody colloidal gold marker, so that the parathion hapten-carrier protein on the detection line can not be combined with the parathion nano antibody colloidal gold marker due to the competitive reaction and the color development is not generated.
The invention provides application of the test strip in detection of parathion pesticide residues in fruits and vegetables.
The invention provides a method for detecting parathion pesticide in crops, which adopts the test strip to detect a sample and judges the residue condition of the parathion pesticide according to the color development result of the test strip.
The invention has the following beneficial effects:
the parathion nano antibody and the colloidal gold are combined for preparing the parathion nano antibody colloidal gold marker for the first time, the parathion pesticide detection stability is good by adopting the parathion nano antibody colloidal gold marker, the detection sensitivity is high, and the parathion pesticide detection colloidal gold test strip based on the nano antibody has the advantages of high sensitivity, strong specificity, low cost, short detection time, suitability for rapid detection in the market, easiness in storage and long quality guarantee period and is simple to operate. The sensitivity of the test strip provided by the invention for detecting parathion pesticide residues reaches 0.0013-0.085 mg/kg, the detection limit is 0.00069mg/kg, the national standard detection requirement is met, the reaction time is 5-8 min, and the test strip is suitable for quickly detecting parathion pesticides in agricultural products such as vegetables and fruits.
Drawings
FIG. 1 shows five colloidal gold solutions prepared in different proportions;
FIG. 2 shows the results of combining five kinds of colloidal gold solutions with parathion nano-antibodies in different proportions;
FIG. 3 shows the results of parathion drug solution detection by five different colloidal gold solutions to prepare colloidal gold-parathion nano-antibody probes;
FIG. 4 is pH optimization of parathion nano antibody colloidal gold marker preparation;
FIG. 5 is the optimized addition of nanometer antibody for the preparation of parathion nanometer antibody colloidal gold marker;
FIG. 6 shows the BSA content optimization for the preparation of parathion nano antibody colloidal gold markers;
FIG. 7 is the preparation of gold colloidal re-solution buffer optimization for parathion nano antibody gold colloidal marker;
FIG. 8 is a schematic structural diagram of the test strip of the present invention (wherein, 1-sample pad, 2-conjugate release pad, 3-reaction membrane, 4-absorbent pad, 5-detection line of parathion pesticide hapten and carrier protein conjugate, 6-quality control line coated with rabbit anti-camel anti-antibody, 7-bottom plate);
FIG. 9 is a schematic diagram showing the detection result of the nano antibody colloidal gold test strip for parathion pesticide of the present invention;
FIG. 10 shows the test results of test strips for different parathion standard pesticide concentrations;
FIG. 11 is a standard curve for different concentration gradient measurements;
FIG. 12 is a stability test of a nano-antibody colloidal gold test strip for parathion pesticide;
FIG. 13 shows the detection of a test strip of colloidal gold with a nano antibody for parathion pesticide.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 preparation and optimization of parathion Nanobody colloidal gold marker
1. Preparation of colloidal gold
Putting ultrapure water into a conical flask, adding 1% chloroauric acid, heating to boil with a constant-temperature electromagnetic stirrer, adding 1% trisodium citrate under continuous stirring at a high temperature, continuously stirring at a constant speed to heat the solution, wherein the solution is firstly changed from yellow to colorless, then changed into black and then slowly changed into wine red, continuing to react for 10min after the solution is not changed any more in color, cooling to room temperature, recovering the original volume with the ultrapure water, and storing at 4 ℃. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating material.
The particle size of the colloidal gold solution finally obtained in the preparation process of the colloidal gold solution varies with the content of the added trisodium citrate. The volume ratio of 1% chloroauric acid to 1% trisodium citrate used in the preparation of colloidal gold solutions for mabs in the prior art is generally 1: 1. 1: 1.25 and 1: 1.5. in order to prepare the colloidal gold solution suitable for the nano-antibody, the colloidal gold solution is prepared by adopting 1% of chloroauric acid and 1% of trisodium citrate in different volume ratios, and the volume ratio of the 1% of chloroauric acid to the 1% of trisodium citrate is set as 1: 1. 1: 1.25, 1: 1.5 and 1: 2 and 1: and 6, preparing five kinds of colloidal gold solutions with different particle sizes, respectively combining the colloidal gold solutions with parathion nano antibody marks, and judging the combination condition of the colloidal gold solutions and the nano antibodies according to results.
The five prepared colloidal gold solutions with different volume ratios are shown in figure 1, the combination result of the five colloidal gold solutions with different volume ratios and the parathion nano-antibody is shown in figure 2, the colloidal gold solution can be aggregated under an unsuitable condition, and the color of the colloidal gold solution is changed from wine red to black and has precipitate; the volume ratio of 1: 1. 1: 1.25, 1: 1.5 adding parathion nano antibody solution into common colloidal gold solution of the monoclonal antibody, the color of the solution is changed from red to black, which shows that the volume ratio is 1: 1. 1: 1.25, 1: 1.5 the colloidal gold solution commonly used for the monoclonal antibody prepared by the method is not suitable for the combination of parathion nano-antibody. The volume ratio is 1: 2, after the colloidal gold solution is combined with the parathion nano antibody, the solution is changed from red to purple, and the volume ratio is 1: 2, the prepared colloidal gold solution is combined with the parathion nano antibody and then has unstable condition; and when the volume ratio of 1: 6 after the parathion nano antibody is added into the prepared colloidal gold solution, the color of the solution is not obviously changed, which shows that the mixed solution can be kept stable.
And then adopting the five prepared colloidal gold solutions with different volume ratios to prepare a colloidal gold-parathion nano antibody probe for parathion drug solution detection, wherein the results are shown in figure 3, and the volume ratio is 1: the colloidal gold solution of 6 and the parathion nano antibody have the best combination stability and detection effect.
2. Preparation of parathion nano antibody colloidal gold marker
The parathion nano antibody solution is prepared by combining the description in the reference literature and the conventional method (Zhangyuqi et al, analytical chemistry, 2019,47(9): 1419-. And (2) adjusting the pH value of the colloidal gold solution by using 0.2mol/L potassium carbonate solution, adding 1-10 mu g of parathion nano antibody into each milliliter of the colloidal gold solution, oscillating, uniformly mixing, standing for 30min, adding 10% BSA (bovine serum albumin) to enable the final concentration of the BSA in the colloidal gold solution to be 0.5% (volume fraction), and standing for 30 min. After that, centrifugation was carried out, the supernatant was discarded, and the precipitate was resuspended in a volume of 1/5 colloidal gold reconstitution buffer and kept at 4 ℃ for further use.
3. Optimization of parathion nano antibody colloidal gold marker
And optimizing the pH, the nano antibody adding amount, the BSA adding amount, the centrifugation condition and the final colloidal gold complex solution system in the whole labeling process of the parathion nano antibody colloidal gold marker, wherein the optimization result is determined according to the sensitivity and the stability of the finally obtained nano antibody colloidal gold marker.
(1) Optimizing the pH: the pH of the colloidal gold solution was adjusted using 0.2M potassium carbonate solution by adding 20, 25, 30, 35, 40 μ L potassium carbonate solution, pH: 8.12, 8.45, 8.78, 9.12 and 9.67, and obtaining colloidal gold solutions with different pH values. And then obtaining different colloidal gold compounds according to the reaction process in the step 2, and determining proper pH according to the sensitivity and stability of the finally obtained nano antibody colloidal gold marker.
The results of the detection of parathion drug solutions by using the five kinds of colloidal gold-parathion nano antibody probes with different pH values are shown in FIG. 4, the colloidal gold-nano antibody compound obtained when the addition amount of potassium carbonate solution is 30 muL has the best effect, and the binding stability and detection effect of the colloidal gold with the pH value of 8.5-9.5 and the parathion nano antibody are the best.
(2) Optimizing the addition amount of the nano antibody: and respectively adding 3, 5, 7 and 10 mu L of 1mg/mL parathion nano antibody solution according to the optimized pH value of 9.12 to obtain different colloidal gold compounds, and determining the appropriate parathion nano antibody addition amount according to the sensitivity and stability of the finally obtained nano antibody colloidal gold marker.
The results of the detection of the parathion drug solution by using the 4 colloidal gold-parathion nano antibody probes of the parathion nano antibody solutions with different contents are shown in figure 5, and the binding stability and the detection effect of the colloidal gold and the parathion nano antibody with the parathion nano antibody solution content of 3-7 mug/mL are the best.
(3) And (3) optimizing the BSA content: and (3) blocking by using 25, 30, 35 and 40 mu L of 10% BSA respectively in the blocking process by using the optimized conditions, and determining the appropriate BSA (bovine serum albumin) adding amount according to the sensitivity and stability of the finally obtained nano antibody colloidal gold marker.
The 4 colloidal gold-parathion nano antibody probes with different BSA contents are used for parathion drug solution detection, and the results are shown in figure 6, wherein the combination stability and the detection effect of the colloidal gold with the BSA content of 30-40 mu L and the parathion nano antibody are the best.
(4) Optimizing colloidal gold redissolution buffer solution: optimizing the colloidal gold redissolution buffer solution, which comprises a colloidal gold redissolution system (adopting different buffer solutions): PBS, Tris-HCl, BB, PB; different Tris-HCl concentrations 0.5, 0.2, 0.1, 0.05, 0.02M; different BSA contents 0.1, 0.2, 0.5, 1, 2%; sucrose content 1, 2, 5, 10, 15%; tween-20 content 0.1, 0.2, 0.5, 1, 2%; and PVP contents 0.1, 0.2, 0.5, 1, 2%. The sensitivity and stability of the nanobody-gold colloid marker were also used to determine the appropriate re-solution formulation.
After the formula of the colloidal gold redissolution buffer solution is optimized, the colloidal gold-parathion nano antibody probe prepared from the colloidal gold redissolution buffer solution is respectively adopted to detect parathion drug solution, the result is shown in figure 7, and the formula of the colloidal gold redissolution buffer solution is as follows: the combination stability and detection effect of the colloidal gold and the parathion nano antibody are best when 0.05M Tris-HCl, 0.2-1 percent of BSA, 2-10 percent of sucrose, 200.1-1 percent of Tween-and 0.3-1 percent of polyvinylpyrrolidone are used.
(5) And (3) centrifugal condition optimization: and optimizing the centrifugal conditions on the basis of the optimized conditions, wherein the centrifugal conditions comprise centrifugal temperature, centrifugal rotating speed and centrifugal time. Respectively setting: the temperature includes 0, 4, 16, 25 and 37 ℃; the rotating speeds comprise 8000, 9000, 10000 and 12000 rpm; the centrifugation time includes 10, 15 and 20 min. And determining proper centrifugation conditions according to the sensitivity and stability of the finally obtained nano antibody-colloidal gold marker.
The parathion drug solution detection is carried out by utilizing colloidal gold-parathion nano antibody probes under different centrifugation conditions, and the parathion nano antibody combination stability and detection effect are the best when the centrifugation conditions are 9000rpm, 4 ℃ and 15 min.
In conclusion, after the preparation of the parathion nano antibody colloidal gold marker is optimized, the final optimization result is as follows: the pH value is 8.5-9.5, the addition amount of the parathion nano antibody is 3-7 mu g/mL, the content of 10% BSA is 30-40 mu L, and the centrifugation condition is 9000rpm, 4 ℃ and 15 min. The formulation of the colloidal gold redissolution buffer solution is 0.05M of Tris-HCl, 0.2-1% of BSA, 2-10% of sucrose, 200.1-1% of Tween-and 0.3-1% of PVP; the prepared parathion nano antibody colloidal gold marker has good stability and high sensitivity.
Example 2 preparation of a Nanobody colloidal gold test strip for detecting parathion pesticides
1. Preparation of conjugate Release pad
Firstly, soaking the bonding release pad in a colloidal gold re-solution for 30min, then drying in a 45 ℃ oven, then spraying the parathion nano antibody colloidal gold marker solution prepared in the embodiment 1 on the treated bonding release pad by using an AUTOKUN membrane scribing gold spraying machine in an amount of 5 mu L/cm per hole, then placing the sprayed bonding release pad in a 37 ℃ oven, placing for 12h, sealing, adding a drying agent, and drying for storage.
2. Preparation of the reaction film
The parathion pesticide hapten solution is prepared by a method described in a literature (Xu et al. analytical Chimica Acta,2009,647(1): 90-96); the hapten-carrier protein of the parathion pesticide is obtained by chemically coupling the hapten of the parathion pesticide and ovalbumin by using chemical reagents DMF and NHS.
The rabbit anti-camel antibody solution adopted by the invention is an antibody solution which is obtained by injecting parathion nano-antibodies into a rabbit body as immunogens and finally separating rabbit serum.
The parathion pesticide hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the rabbit anti-camel antibody is coated on the reaction membrane to form a quality control line.
Coating process: diluting the parathion pesticide hapten-ovalbumin conjugate to 0.2mg/mL by using a phosphate buffer solution, and coating the parathion pesticide hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an AUTOKUN membrane-scribing gold-spraying label machine, wherein the coating amount is 0.8 mu L/cm; the rabbit anti-camel anti-antibody was diluted to 1mg/mL with phosphate buffer, and the membrane-sprayed gold label was scratched with AUTOKUN to coat the control line (line C) on the nitrocellulose membrane in an amount of 0.8. mu.L/cm. And (3) drying the coated reaction membrane at 37 ℃ for 4 hours for later use.
3. Preparation of sample pad
The sample pad was soaked in phosphate buffer containing 1% bovine serum albumin (volume fraction), 1% sucrose (volume fraction), 0.3% polyvinylpyrrolidone (volume fraction), 0.5% triton (volume fraction), pH7.4, 0.2mol/L for 1h, and baked at 37 ℃ for 4h for use.
4. Assembly of test strips
Sequentially adhering the reaction membrane, the combined release pad, the sample pad and the water absorption pad on the PVC bottom plate; the tail end of the sample pad is connected with the initial end of the combined release pad, the tail end of the combined release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the combined release pad, the water absorption pad and the reaction membrane are overlapped by 2mm, and the sample pad and the combined release pad are overlapped by 2 mm. The initial end of the sample pad is aligned with the initial 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 and a quality control line, 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 positioned at one side close to the tail end of the sample pad; the quality control line is positioned on one side close to the head end of the water absorption pad; the assembled test strip is shown in fig. 8, and is cut into small strips with the width of 4mm by a machine, and the small strips are arranged in a special sealing bag and can be stored for 12 months at the temperature of 4-30 ℃.
Example 3 detection of Phosphothion pesticide residue with Nanobody colloidal gold test strip for detecting Phothion pesticide
1. Limit of detection test
Parathion standard pesticides were added to PBS buffer to final concentrations of 0.2mg/kg, 0.1mg/kg, 0.05mg/kg, 0.025mg/kg, 0.0125mg/kg, 0.00625mg/kg, 0.003125mg/kg, 0.00156mg/kg and 0.00078mg/kg, respectively, and the test strips prepared in example 2 were further tested and each sample was repeated three times.
And (3) vertically dropwise adding the sample solution onto the sample pad, starting timing when the liquid flows, reacting for 5-8 min, judging the result, wherein a schematic diagram of the test strip detection result is shown in fig. 9. And accurate data can be obtained through the reading of the colloidal gold analyzer. A standard curve can be obtained by Origin software according to the data.
The detection result of the test strip is shown in figure 10, and the color of the detection line of the detection result of the parathion standard pesticide on the test strip is gradually deepened along with the reduction of the concentration of the parathion standard pesticide; the standard curve obtained by detecting different concentration gradients is shown in fig. 11, and the corresponding result of the curve shows that the detection range of the method for detecting parathion pesticide residues is 0.0013-0.085 mg/kg, and the detection limit is 0.00069mg/kg, so that the national standard detection requirement is met.
2. Specificity test
The nano antibody colloidal gold test strip of the parathion pesticide prepared by the invention is used for respectively detecting 5mg/kg of quinalphos, methyl parathion, isocarbophos, fenitrothion, profenofos, carbofuran and the like. The pesticides used above were diluted to a concentration of 5mg/kg using PBS buffer.
And (3) vertically dropwise adding the sample solution onto the sample pad, timing when the liquid flows, reacting for 5-8 min, and judging the result.
Reading the detection result by a colloidal gold analyzer:
negative (-): indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
and (4) invalidation: indicating that retesting is required.
The result shows that the quality control line and the detection line of the test strip are both colored and negative. The test strip has no cross reaction to 5mg/kg quinalphos, triazophos, methyl parathion, isocarbophos, fenitrothion, profenofos, carbofuran and other medicaments.
3. Stability test
The nano antibody colloidal gold test strip of the parathion pesticide prepared in the embodiment 2 is filled into a sealed bag, a drying agent is arranged in the sealed bag, the test strip is stored in a baking oven at the temperature of 45 ℃, the test strip is taken out in 3 days, 7 days, 14 days, 21 days, 28 days and 30 days respectively, the parathion standard substance is diluted to 0.01mg/kg of solution by PBS, and then the stability of the test strip is detected.
As shown in FIG. 12, the results were found to be unchanged when the gold-labeled antibody was stored at 45 ℃ for 3 days, 7 days, 14 days, 21 days, and 28 days, but the redissolution effect of the gold-labeled antibody on the gold-labeled pad was reduced, the color of the T-line and C-line became lighter, and the sensitivity was slightly reduced when the gold-labeled antibody was stored at 45 ℃ for 30 days. The storage at 45 ℃ for 30 days is equivalent to the storage at normal temperature for 12 months.
Example 4 detection analysis of parathion pesticide residues in fruit and vegetable crops
After samples of commercially available cabbage, cucumber and orange samples are cleaned and dried, 10g (+ -0.1 g) of homogeneous samples are weighed into a 50mL plastic centrifuge tube, each sample is divided into two groups, namely a negative sample and a positive sample, the negative sample is not treated, and the positive sample is added with a parathion standard substance until the drug concentration reaches 0.01 mg/kg. Then adding 10mL of acetonitrile into the negative sample and the positive sample, and uniformly mixing by vortex; adding extraction package, continuing vortex and shaking for 2min, centrifuging at 5000rpm/min for 3min, and standing at room temperature. Adding the supernatant solution into a purification tube, performing vortex oscillation for 2min, centrifuging at 5000rpm/min for 3min, taking the supernatant, blowing nitrogen at 37 ℃, and then performing primary redissolution by using an EDTA solution.
And then taking the test strip prepared in the embodiment 2 for detection, vertically dropping the treated sample solution on the sample pad, starting timing when the liquid flows, reacting for 5-8 min, and judging the result.
Reading the detection result by a colloidal gold analyzer:
negative (-): indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
and (4) invalidation: indicating that retesting is required.
The result schematic diagram of the detection by adopting the test strip prepared by the invention is shown in fig. 13, when detecting cabbage, cucumber and orange samples, we can see that the T line and the C line in the negative sample are developed, and the C line development of the positive sample is obviously stronger than the development result of the T line. The parathion detection method of the invention can meet the requirement for the detection result of the actual sample.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A parathion pesticide colloidal gold marker based on a nano antibody is characterized in that the preparation method comprises the following steps: adding 1-10 mu g/mL parathion nano antibody into a colloidal gold solution with the pH value of 8-10, uniformly mixing and standing, adding 20-40 mu L10% BSA, standing and centrifuging, discarding supernatant, and carrying out heavy suspension precipitation by using a colloidal gold redissolution buffer solution to obtain a parathion nano antibody colloidal gold marker;
the volume ratio of the colloidal gold solution is 1: 6, 1% of chloroauric acid and 1% of trisodium citrate;
the formula of the colloidal gold redissolution buffer solution is as follows: 0.02-0.5M Tris-HCl, 0.1-2% BSA, 0.1-2% Tween-20, 0.1-2% polyvinylpyrrolidone and 1-15% sucrose.
2. The parathion nano-antibody colloidal gold marker as claimed in claim 1, wherein the colloidal gold solution has a pH of 8.5-9.5, the parathion nano-antibody is added in an amount of 3-7 μ g/mL, and 10% BSA is added in an amount of 30-40 μ L.
3. The parathion nano antibody colloidal gold marker according to claim 1, wherein the formulation of the colloidal gold redissolution buffer is: 0.05M Tris-HCl, 0.2-1% BSA, 2-10% sucrose, 200.1-1% Tween-and 0.3-1% PVP.
4. The application of the parathion nano antibody colloidal gold marker in any one of claims 1 to 3 in immunochromatography or detection of parathion pesticides.
5. The application of the parathion nano antibody colloidal gold marker in the preparation of parathion pesticide products according to any one of claims 1 to 3.
6. A nano antibody colloidal gold test strip for detecting parathion pesticide, which is characterized in that a binding release pad contains the parathion nano antibody colloidal gold marker of claim 1.
7. The test strip of claim 6, further comprising a sample pad, a reaction membrane, a wicking pad, and a bottom plate.
8. The test strip of claim 7, wherein the reaction membrane has a detection line coated with a parathion pesticide hapten-carrier protein conjugate and a quality control line coated with a rabbit anti-camel antibody.
9. The use of the test strip of any one of claims 6 to 8 in the detection of parathion pesticide residues in fruit and vegetable crops.
10. A method for detecting parathion pesticides in crops is characterized in that a sample is detected by adopting the test strip of any one of claims 6 to 8, and the residue condition of the parathion pesticides is judged according to the color development result of the test strip.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116643049A (en) * | 2023-07-27 | 2023-08-25 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
| CN116769043A (en) * | 2023-06-09 | 2023-09-19 | 华南农业大学 | Antigen triggering type fluorescent probe based on quetiapine nano antibody and preparation method and application thereof |
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2022
- 2022-04-06 CN CN202210357010.5A patent/CN114675015A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116769043A (en) * | 2023-06-09 | 2023-09-19 | 华南农业大学 | Antigen triggering type fluorescent probe based on quetiapine nano antibody and preparation method and application thereof |
| CN116643049A (en) * | 2023-07-27 | 2023-08-25 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
| CN116643049B (en) * | 2023-07-27 | 2023-09-29 | 云南省农业科学院质量标准与检测技术研究所 | Profenofos pesticide colloidal gold marker based on modified nano gold material and application thereof |
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