Multi-residue Rapid Detection Of Drugs In Meat Products Using Immunochip Assay Based On Smart Phone

“The people regard food as the heaven,food and security as the first”,and food safety is one of the major issues concerning people's livelihood and social harmony.In order to maximize the benefits,breeders often use banned drugs in violation of regulations.Excessive use of veterinary drugs is extremely serious.Therefore,veterinary drug residue problems and poisoning events occur from time to time.The state attaches greater importance to food safety.According to the“2017 Animal and Animal Products Veterinary Drug Residue Surveillance Plan”issued by the Ministry of Agriculture,there are as many as 30 veterinary drug residues to be tested for meat products,and the limit for certain veterinary drug residues is as low as 0.2?g/kg.The accuracy of the results of food safety residue detection and detection time have become the most concerned issues of the public at the moment.At present,the main methods for detecting veterinary drug residues in meat products include HPLC-MS and other instrumental validation methods and traditional immunoassay methods such as ELISA.However,these detection methods require the use of professional instruments and equipment,which are cumbersome to operate,and have low detection throughput and cannot meet the need for rapid on-site detection.Therefore,it is necessary to develop a rapid,simple and low-cost method for detecting multiple residues of veterinary drugs.Smart phones are closely related to people's lives and have a very wide range of popularity.It has high-speed analysis processing and high-definition image capture capabilities,combined with its open operating system.It is easy to develop,flexible to operate and easy to carry.Smartphones show great potential for rapid detection of food safety on the spot.In this paper,ractopamine,?₂-agonist,chloramphenicol,florfenicol and its metabolite florfenicol were used as the research object.Immune chip analysis methods were established separately.On this basis,we further developed an immunochip analysis method for simultaneous detection of four veterinary drug residues.We have also developed a smart phone detection APP,which can directly show the content of veterinary drug residues in the sample after analysis and analysis of the sample.This truly enables a simple,fast and low-cost quantitative test of veterinary drug residues.The main research contents and results are as follows:?1?Using nitrocellulose membrane as a solid-phase carrier and adopting the reaction principle of ic-ELISA,an immune chip analysis method was established for ractopamine,?2-agonist,chloramphenicol,florfenicol and its metabolite florfenicol amine.The concentration of ractopamine detection antigen was determined to be 20?g/mL,the antibody concentration was 0.22?g/mL,and the sensitivity was determined to be 2ng/mL;the concentration of?₂-agonist antigen was determined to be 25?g/mL,and the antibody concentration was determined.It was 0.15?g/mL,and its sensitivity was determined to be 2 ng/mL;the dot concentration of the chloramphenicol detection antigen was 3.2?g/mL,the antibody concentration was 0.022?g/mL,and the sensitivity was determined to be 0.5 ng/mL;Florfenicol and its metabolite florfenicol amine detection antigen lattice concentration of 10?g/mL,antibody concentration of 0.025?g/mL,and determine the sensitivity of 50ng/mL.?2?Based on the establishment of four methods for the detection of target analyte immunochips,we established an analytical method for the simultaneous detection of these four veterinary drugs residual immunochips.Optimize the series of factors that affect the analysis of the immune chip.The results showed that 0.22?m nitrocellulose membrane was used;CBS was used as spotting solution for ractopamine and chloramphenicol;PBS was used as?₂-agonist and florfenicol.the optimal coating method is a 37°C water bath;the optimal reaction time is coating 1 h,blocking 30 min,the primary antibody was incubated for 15 min and the secondary antibody was incubated for15 min.The optimal antibody dilution was T solution.The best times of washes was 4.For the four antibodies,the specificity of the coating was evaluated and no cross-reactions were apparent,as expected.At the same time,using Photoshop software to read the gray value of the color results,a digital colorimetric detection method was established for the four target analytes.The linear ranges of the four spectrophotometry were 0.25-2 ng/mL,0.25-2 ng/mL,0.0625-0.5 ng/mL and 6.25-50 ng/mL respectively.The linear equations were y=-3.12x+3.18,y=-2.92x+3.335,y=-6.88x+2.265 and y=-0.0352x+1.63 respectively.The linear equations are y=-3.12x+3.18,y=-2.92x+3.335,y=-6.88x+2.265,and y=-0.0352x+1.63,respectively.The intra-and inter-batch recovery rates of pig urine samples were 70.4%¹38.67%and 70.02%¹37.60%,respectively,and the coefficient of variation was 3.81%³3.4%and 4.54%³1.2%,respectively.The variation coefficient of recovery of the four target analytes was less than 35%,so the method has good accuracy.?3?The rapid and quantitative detection of four veterinary drug residues was achieved using the laboratory's smart phone APP software and ancillary picture collection devices.The recovery rate in the pig urine sample was 76.56%¹39.88%,and the coefficient of variation was less than 30%.Compared with the colloidal gold test card results,the compliance rate was 98.75%.Compared with the ELISA kit test results,the linear coefficients were y=0.9295x+0.035,y=0.8792x+0.0518,y=1.1115x-0.012 and y=0.784x+2.2432,respectively.The correlation coefficients were R²=0.9476,R²=0.9194,R²=0.9495,and R²=0.935 in turn.It shows that the method has good consistency and reliability.