CN113721024A - Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food - Google Patents
Fluorescence immunoassay rapid detection kit and detection method for enrofloxacin carbon quantum dots in animal derived food Download PDFInfo
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Abstract
The invention discloses a rapid fluorescence immunoassay kit and a rapid fluorescence immunoassay method for enrofloxacin carbon quantum dots in animal derived food, and belongs to the field of fluorescence immunoassay. The kit consists of a reaction cup and a reagent; wherein the reaction cup is coated with a coating antigen, and the reagent is marked with an antibody working solution by horseradish peroxidase; enrofloxacin series standard solutions; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B. The carbon quantum dot is used as a fluorescent substrate, and the enrofloxacin fluorescence quenching immunoassay method is established. The detection time of the kit is 30min, the detection limit is 0.3ng/mL, the intra-batch variation coefficient is less than 8%, the inter-batch variation coefficient is less than 15%, and the recovery rate of the aquatic product sample is 90% +/-30%. The fluorescence immunoassay rapid detection kit has the advantages of high specificity, good sensitivity, simple and convenient operation and capability of realizing quantitative detection.
Description
Technical Field
The invention relates to a rapid fluorescence immunoassay kit and a rapid fluorescence immunoassay method for enrofloxacin carbon quantum dots in a biological food, and belongs to the technical field of immunoassay.
Background
Enrofloxacin (Enrofloxacin, ENR) is a broad-spectrum bactericidal drug specially used for treating animal tissue infection, and with the wide use in recent years, side effects caused by drug use are increasingly shown, and after being ingested by human bodies, the residue of Enrofloxacin in animal food is more harmful to human health, so that the effective and rapid detection of Enrofloxacin is necessary.
Immunoassay based on antigen-antibody specific binding reaction has been widely focused and studied due to its unique recognition form and its excellent characteristics such as specificity, reversibility and reaction stage, and has gradually become an important detection means for clinical disease diagnosis. Immunodiagnosis technology also constitutes three major fields of in vitro diagnosis, along with biochemical and molecular diagnostic techniques. Under the recommendation of the world Food and Agriculture Organization (FAO), the immunoassay method is also gradually applied to the field of food safety detection and analysis, and becomes an important component of food safety detection. Various immunoassays including ELISA, chemiluminescence enzyme immunoassay, fluorescence immunoassay, immunochromatographic sensing assay (ICS) have been widely used for on-site rapid detection of food contaminant residues in agricultural and animal products.
Immunoassay using fluorescent signals instead of colorimetric signals has the characteristics of high sensitivity, strong resistance to mass interference and the like, and is widely applied. At present, the main labeling materials for fluorescence analysis are mainly Fluorescent Microspheres (FM), Quantum Dots (QD), upconversion nanoparticles (UCNP) and the like, but the problems of high heavy metal toxicity of QDs, few fluorescence types of UCNPs and the like become the key limiting factors of the QDs and the UCNPs. Carbon quantum dots (CDs), as a class of zero-dimensional (OD) nanomaterials with excellent photoluminescence properties, are fluorescent nanomaterials that have been widely studied since the successful synthesis of semiconductor quantum dots. Based on the method, the carbon quantum dots are prepared in the aqueous solution environment by a simple method, and the rapid and high-sensitivity fluorescent immune rapid detection kit for the enrofloxacin carbon quantum dots in the biogenic food is established. At present, a carbon quantum dot fluorescence immunoassay rapid detection kit is not reported.
Disclosure of Invention
In view of the above, the present invention provides a carbon quantum dot fluorescence immunoassay rapid detection kit for detecting enrofloxacin in animal derived food and a detection method thereof, and in order to achieve the above object, the technical scheme of the present invention is as follows:
a rapid detection kit for enrofloxacin carbon quantum dot fluorescence immunoassay in animal derived food comprises a kit body, a reaction cup arranged in the kit body and a reagent arranged in the kit body; wherein the reaction cup is coated with a coating antigen, and the reagent comprises: horse radish peroxidase labeled antibody working solution; enrofloxacin series standard solutions; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; a carbon quantum dot fluorescence reaction solution A and a carbon quantum dot fluorescence reaction solution B.
Preferably, the carbon quantum dot fluorescence reaction solution a contains hydrogen peroxide and carbon quantum dots, and the carbon quantum dot fluorescence reaction solution B contains 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
The preparation of the carbon quantum dot comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
The reaction cup is a single-hole or 8-linked reaction cup with the volume of 300 muL or 500 muL or other volumes; the enrofloxacin series standard solutions are respectively obtained by diluting enrofloxacin pure products, and the enrofloxacin concentrations in the enrofloxacin standard products are respectively as follows: 0.3ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, and 24.3 ng/mL.
The method for detecting the substance to be detected by using the kit is based on antigen-antibody reaction. Adding a standard substance to be detected or a processed sample extracting solution and a horseradish peroxidase labeled antibody working solution into the reaction cup in sequence, adding a carbon quantum dot fluorescence reaction solution A and a carbon quantum dot fluorescence reaction solution B after incubation and washing, detecting a fluorescence value, solving an inhibition rate by using the fluorescence value of the added enrofloxacin standard substance, drawing a standard curve, and calculating the content of the substance to be detected in the detected sample from the standard curve by using the fluorescence value of the added detected sample.
The detection method of the kit comprises the steps of adding a standard solution of an object to be detected or a processed sample to be detected into a reaction cup, adding an antibody working solution, incubating for 20min at 37 ℃, washing with a phosphate washing solution, beating the phosphate washing solution on filter paper to dry, adding 50 mu L of each of a fluorescence reaction solution A and a fluorescence reaction solution B into the reaction cup, incubating for 10min at 37 ℃, and detecting a fluorescence value.
The method for calculating the inhibition rate of the kit comprises the following steps of (F)x-F0)/(F0-Fb) X 100% where F0To determine the fluorescence value of the cuvette in the standard solution without enrofloxacin, FxIn order to detect the fluorescence value of the reaction cup when detecting the standard solution or the sample solution to be detected containing enrofloxacin, FbThe method comprises the steps of calculating inhibition rates by using fluorescence values of enrofloxacin standard substances with different concentrations, taking the inhibition rates as vertical coordinates and logarithm of the enrofloxacin standard substance concentration as horizontal coordinates, and taking a standard curve as detection limits and sensitivity, namely the enrofloxacin concentration (IC50) corresponding to the inhibition rate of 50% from the standard curve, wherein the detection fluorescence values of a reaction cup are detected when a standard solution of a substance to be detected or a sample to be detected is not added; the concentration of each sample can be calculated by measuring the fluorescence value of the sample to calculate the inhibition rate, and the inhibition rate is read from the standard curve.
Further, the detection limit of the kit on enrofloxacin is 0.3ng/mL, and the sensitivity is 1.02 ng/mL.
Further, the fluorescence value is measured at 560nm for each reaction cup with a fluorescence detector at an excitation wavelength of 365 nm.
Compared with the existing domestic and foreign micromolecular substance immunoadsorption detection technology, the invention has the following outstanding advantages:
1. the invention utilizes the carbon quantum dot fluorescent signal to replace the colorimetric signal of enzyme-linked immunoassay, thus realizing the high-sensitivity fluorescence immunoassay analysis of enrofloxacin;
2. the kit of the invention can be read without acid solution termination.
3. The quantity of the reaction cups in the reagent kit can be selected according to the detection requirement, the rapid detection and analysis of 100-500 samples can be completed within 1h, the reagent kit is suitable for rapid screening of a large number of samples, and the reagent kit can be used as an effective screening means for rapid detection of micromolecular substances to be detected in food.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the invention without limitation. In the drawings:
FIG. 1 is a fluorescence spectrum of the carbon quantum dot of the present invention.
FIG. 2 is a result chart of enzyme-linked immunoassay and carbon quantum dot fluorescence immunoassay rapid detection kit for detecting enrofloxacin (the concentrations of the substances to be detected are 0.3, 0.9, 2.7, 8.1 and 24.3ng/mL from left to right).
Detailed Description
The invention will be described in more detail hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown, but the examples are not intended to limit the invention.
Example l (preparation example)
Carbon quantum dot preparation
(1) And (3) synthesis of carbon quantum dots: 3g of citric acid and 5g of urea were added to 10mL of ultrapure water to form a clear solution. The solution was then heated in a 800W microwave oven for 5 minutes during which time the solution changed from a colorless liquid to brown and finally to a dark brown clustered solid. After cooling to room temperature, 20mL of water was added to dissolve the product.
(2) And (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence spectrum shown in FIG. 1, and fluorescence is observed at 400-600 nm under excitation of 365nm excitation light.
Example 2 (preparation example)
Preparation method of rapid detection kit for enrofloxacin carbon quantum dot fluorescence immunity
(1) Coating antigen: the enrofloxacin-coated antigen was diluted to 0.1. mu.g/mL with pH9.6 sodium carbonate buffer, and 300. mu.L of the diluted enrofloxacin-coated antigen was added to the reaction cup and incubated overnight at 4 ℃.
(2) Washing the reaction cup: the reaction solution is discarded the next day, then 200-.
(3) And (3) sealing: add 200. mu.L of casein blocking solution to the reaction cup, incubate at 37 ℃ for 1.5h, discard the casein blocking solution, vacuum seal and store at 4 ℃.
Example 3 (application example)
Application of rapid detection kit for enrofloxacin carbon quantum dot fluorescence immunoassay
1. Detection step
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard dilution containing enrofloxacin with certain concentration into each reaction cup, adding 100 mu L of enzyme-labeled antibody mixture into each reaction cup, wherein each concentration is two in parallel, incubating for 20min at 37 ℃, removing the reaction solution, adding 200-500 mu L of phosphate washing solution, shaking for 2min, removing the phosphate washing solution, repeating the process for 3 times, and finally, drying the plate on filter paper.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
2. Determination of results
The carbon quantum dot fluorescence immunoassay rapid detection kit inhibits substrate oxidation by using enrofloxacin-combined enzyme labeled antibody so as to cause fluorescence recovery, and the fluorescence intensity value of 560nm in a reaction cup to be detected is along with the fluorescence intensity value of a sample to be detected under the excitation of 365nmThe enrofloxacin content is increased. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb)×100％，F0To determine the fluorescence value detected in the cuvette in a standard solution without enrofloxacin, FxTo examine the fluorescence value of the cuvette in the case of a standard solution containing enrofloxacin, FbThe fluorescence value detected by the reaction cup when the standard solution of the substance to be detected is not added is represented by the standard curve with the inhibition rate as the ordinate and the logarithm of the concentration of the substance to be detected as the abscissa, and the sensitivity, namely the enrofloxacin concentration (IC) corresponding to the inhibition rate of 50 percent50) Can be read from the standard curve. As shown in FIG. 2, IC of the method of the present invention50It was 1.02 ng/mL.
Example 4 (application example)
Application technology of enrofloxacin enzyme-linked immunoassay method
1. Detection step
(1) Adding a standard solution and a horseradish peroxidase labeled antibody working solution: adding 100 mu L of standard dilution containing enrofloxacin with certain concentration into each reaction cup, adding 100 mu L of enzyme-labeled antibody mixture into each reaction cup, wherein each concentration is two in parallel, incubating for 20min at 37 ℃, removing the reaction solution, adding 200-500 mu L of phosphate washing solution, shaking for 2min, removing the phosphate washing solution, repeating the process for 3 times, and finally, drying the plate on filter paper.
(2) Color development: to each reaction cup, 50. mu.L each of a urea peroxide solution and a 3,3',5,5' -tetramethylbenzidine solution was added, and color development was performed at 37 ℃ for 10 min.
(3) And (4) terminating: the color reaction was stopped with 50. mu.L of sulfuric acid stop solution and read within 10 min.
(4) Reading: the absorbance value at 450nm of each reaction cup was measured with a microplate reader.
2. Determination of results
The enzyme-linked immunosorbent assay rapid detection method provided by the invention uses enrofloxacin and enzyme-labeled antibody to inhibit substrate oxidation, so that the absorbance value is reduced, and the absorbance value of the solution in the reaction cup to be detected at 450nm is reduced along with the increase of enrofloxacin content in the sample to be detected. Criteria for judging results: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) ═ a0-AX)/(A0-Ab)×100％，A0To determine the absorbance value of the cuvette in a standard solution without enrofloxacin, AxTo determine the absorbance value of the cuvette in a standard solution containing enrofloxacin, AbThe absorbance value of the reaction cup when the standard solution of the substance to be detected is not added is determined, the inhibition rate is used as the ordinate, the logarithm of the concentration of the substance to be detected is used as the abscissa, the standard curve is made, and the sensitivity is the enrofloxacin concentration (IC) corresponding to the inhibition rate of 50 percent50) Can be read from the standard curve. As shown in FIG. 2, IC of the ELISA assay of the present invention50It was 2.15 ng/mL.
Example 5 (application example)
Use technology of enrofloxacin carbon quantum dot fluorescence immunoassay rapid detection kit
1. Pretreatment of aquatic products
Placing 2g of the fat-removed meat homogenate in a 50mL centrifuge tube, adding 2mL of saturated sodium chloride solution and 4mL of acetonitrile-0.1M NaOH (v/v ═ 84: 16), mixing and shaking for 5min, centrifuging at 4000rpm for 5min, transferring 1mL of supernatant to the 5mL centrifuge tube, evaporating and drying at 60 ℃ by using a gentle air flow, dissolving the residue in 1mL of n-hexane, and adding 1mL of sample reconstituted solution. Vortex for 30s and centrifuge at 4000rpm for 3 min. Pipette 100. mu.L of the lower layer solution for detection. The final assay result should be multiplied by the dilution factor 3.
2. Pretreatment of livestock and poultry products
Placing 2g of the fat-removed meat homogenate in a 50mL centrifuge tube, adding 4mL acetonitrile-0.1M NaOH (v/v is 84: 16), mixing and shaking for 5min, centrifuging at 4000rpm for 5min, transferring 1mL of supernatant into a 5mL centrifuge tube, evaporating and drying at 60 ℃ by using a gentle air flow, dissolving the residue in 1mL n-hexane, and adding 1mL of sample reconstituted solution. Vortex for 30s and centrifuge at 4000rpm for 3 min. Pipette 100. mu.L of the lower layer solution for detection. The final assay result should be multiplied by the dilution factor 2.
3. Detection step
(1) Adding enrofloxacin standard solution or sample to be detected and horseradish peroxidase labeled antibody working solution: adding 100 mu L of enrofloxacin standard solution or sample solution to be detected into each reaction cup, adding 100 mu L of enzyme-labeled antibody mixture into each reaction cup, wherein the concentration is two in parallel, incubating for 20min at 37 ℃, removing the reaction solution, adding 200-mu L of phosphate washing solution, shaking for 2min, throwing away the phosphate washing solution, repeating the process for 3 times, and finally, beating the reaction cups on filter paper to be dried.
(2) Color development: 50 μ L of each of the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B was added to each reaction cup, and color development was carried out at 37 ℃ for 10 min.
(3) Fluorescence reading: the fluorescence of each reaction cup at 560nm was measured with a fluorescence detector at an excitation wavelength of 365 nm.
4. Determination of results
Under the excitation of 365nm, the fluorescence intensity value of the solution in the reaction cup to be detected at 560nm is increased along with the increase of the enrofloxacin content in the sample to be detected. And (4) judging a result standard: the formula for calculating the inhibition rate is as follows: inhibition ratio (%) - (F)x-F0)/(F0-Fb)×100％，F0To determine the fluorescence value of the cuvette in the standard solution without enrofloxacin, FxIn order to detect the fluorescence value of the reaction cup when detecting the standard solution or the sample solution to be detected containing enrofloxacin, FbThe method comprises the steps of calculating inhibition rates by using fluorescence values of enrofloxacin standard substances with different concentrations, taking the inhibition rates as vertical coordinates and taking logarithm of the enrofloxacin standard substance concentration as horizontal coordinates, calculating the inhibition rates by using the fluorescence values of the samples according to the concentration of each sample, and reading out the inhibition rates from a standard curve according to the inhibition rates. The detection limit of the kit for aquatic product samples is 0.9ng/g, and the detection limit of the livestock and poultry product samples is 0.6 ng/g. As shown in Table 1, the detection value and the addition value of the enrofloxacin in the aquatic product sample detected by the kit are consistent.
TABLE 1 carbon quantum dot fluorescence immunoassay rapid detection kit for detecting recovery rate of enrofloxacin in aquatic products
Claims (9)
1. A fluorescence immunoassay rapid detection kit for enrofloxacin carbon quantum dots in animal derived food comprises a kit body, wherein a reaction cup and a reagent are arranged in the kit body; the kit is characterized in that the reaction cup is coated with an antigen, and the reagent is marked with an antibody working solution by horseradish peroxidase; enrofloxacin series standard solutions; 20 times of concentrated phosphate washing solution; concentrating the phosphate complex solution by 10 times; the carbon quantum dot fluorescence reaction solution A and the carbon quantum dot fluorescence reaction solution B.
2. The rapid fluorescence immunoassay kit for detecting enrofloxacin carbon quantum dots in animal derived food as claimed in claim 1, wherein the reaction cup is a single-hole or 8-linked reaction cup made of polystyrene, and the volume is 300 μ L or 500 μ L or other volumes.
3. The rapid fluorescence immunoassay kit for detecting enrofloxacin carbon quantum dots in animal derived food as claimed in claim 1, wherein the carbon quantum dot fluorescence reaction solution A comprises hydrogen peroxide and carbon quantum dots, and the carbon quantum dot fluorescence reaction solution B comprises 3,3',5,5' -tetramethylbenzidine and carbon quantum dots.
4. The rapid fluorescence immunoassay kit for detecting enrofloxacin carbon quantum dots in animal derived food as claimed in claim 1, wherein the preparation of the carbon quantum dots comprises the following steps:
(1) and (3) synthesis of carbon quantum dots: adding 3g of citric acid and 5g of urea to 10mL of ultrapure water to form a transparent solution, then heating the solution in a 800W microwave oven for 5 minutes during which the solution changes from a colorless liquid to brown and finally to a dark brown cluster solid, after cooling to room temperature, adding 20mL of water to dissolve the product;
(2) and (3) purifying the carbon quantum dots: centrifuging the dissolved product at 5000rpm for 10min, placing into 3500Da dialysis bag, dialyzing with water at room temperature overnight to obtain brown solution with fluorescence at 400-600 nm under excitation of 365nm excitation light.
5. The rapid fluorescence immunoassay kit for detecting enrofloxacin carbon quantum dots in animal derived food as claimed in claim 1, wherein the step of coating antigen in the reaction cup comprises: diluting the enrofloxacin coating antigen to 0.1 mu g/mL by using a pH9.6 sodium carbonate buffer solution, adding 300 mu L of 100 plus materials into each reaction cup, placing the reaction cups in a refrigerator at 4 ℃ for coating overnight, adding 250 mu L of phosphate washing solution into the reaction cups coated with the antigen the next day, shaking for 2min, then throwing off the phosphate washing solution, repeating the steps for 3 times, finally, after drying the reaction cups on filter paper, adding 250 mu L of 100 plus materials casein sealing buffer solution into each reaction cup, sealing at 37 ℃ for 1-2h, then discarding the casein sealing solution in the reaction cups, and preserving at 4 ℃ after vacuum sealing.
6. The method for detecting enrofloxacin by using the kit of claim 1, wherein a standard solution of a test substance or a treated test sample is added to a reaction cup coated with an antigen, adding horseradish peroxidase labeled antibody working solution, incubating at 37 deg.C for 20min, adding 500 μ L phosphate washing solution into the reaction cup coated with antigen, shaking for 2min, removing the phosphate washing solution, repeating the above steps for 3 times, finally, patting the reaction cup on filter paper to dry, adding 50 μ L of each of the fluorescence reaction solution A and the fluorescence reaction solution B, incubating at 37 ℃ for 10min, detecting the fluorescence value, the inhibition rate is calculated by adding the fluorescence values of enrofloxacin standard substances with different concentrations, the inhibition rate is used as an ordinate, the logarithm of the concentration of the enrofloxacin standard substances is used as an abscissa to make a standard curve, and the corresponding enrofloxacin concentration IC50 can be read from the standard curve when the detection limit and the sensitivity, namely the inhibition rate, are 50%.
7. The method for detecting enrofloxacin as claimed in claim 6, wherein the inhibition rate is:
inhibition ratio (%) - (F)x-F0)/(F0-Fb) X 100% where F0To determine the fluorescence value of the cuvette in the case of a solution without enrofloxacin, FxTo measure the fluorescence value of the cuvette in the case of measuring the solution containing enrofloxacin, FbThe method is characterized in that the detection fluorescence value of a reaction cup when no standard solution of an object to be detected or a sample to be detected is added is taken, the inhibition rate of the enrofloxacin standard solution is taken as an ordinate, the logarithm of the concentration of the enrofloxacin standard is taken as an abscissa, a standard curve is taken, the concentration of each sample can be calculated through the fluorescence value measured by the sample, and the inhibition rate is read from the standard curve according to the inhibition rate.
8. The method for detecting enrofloxacin by using the kit as claimed in claim 7, wherein the detection limit of enrofloxacin by the kit is 0.3ng/mL, and the sensitivity is 1.02 ng/mL.
9. The method for detecting enrofloxacin by using the kit of claim 7, wherein the fluorescence value is measured at 560nm in each reaction cup by a fluorescence detector at an excitation wavelength of 365 nm.
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