Study On Microfluidic Immunoassay Of Multiple Veterinary Drug Residues In Animal Products

Veterinary drugs play an important role in the prevention and treatment of animal diseases,promoting the growth of animal,improving the quality of edible animal meat and so on.However,the food safety problems caused by veterinary drug residues are seriously harmful to human health,which may cause toxic reaction,allergy,carcinogenic,teratogenic and mutagenic effects.With the improvement of people’s attention to food safety,the detection of veterinary drug residues has been paid more attention.Since the the residues of Ractopamine（RAC）,clenbuterol（CLB）,chloramphenicol（CAP）in animals are mainly excreted in the urine,detection of veterinary drug residues in animal urine can be used to monitor the rveterinary drug residues in animals.As reported at home and abroad at present,the methods for the detection of RAC、CLB、CAP are mainly instrumental confirmation methods such as GC/MS,HPLC-MS and rapid detection methods such as ELISA and GICA.However,instrument validation methods usually require professionals and large-scale instrument,while rapid detection methods such as ELISA and GICA cannot achieve the purpose of rapid and accurate detection of multiple veterinary drug residues.In this study,ractopamine,clenbuterol and chloramphenicol were selected as research objects.Based on microfluidic and immunocompetitive analysis technology,a microfluidic immunoassay method was established t o simultaneously detect three veterinary drug residues by self-driving microfluidic chip.The main research contents and results are as follows:（1）Measuring the contact angle and flow rate to identify the performance of self-driving microfluidic chip.The contact angles of the non-microchannel surface,the microchannel surface and the viscous surface of the cover plate are respectively greater than 90°,less than 25°and less than 10°,which indicats that each surface is hydrophobic,hydrophilic and near superhydrophilic.This result shows that the microfluidic chip meets the basic design requirements of preparing self-driving microfluidic chip based on different hydrophilicity and capillary force of microchannel.The results of flow velocity measurement show that the coefficient of variation（CV）of time of microfluidics flowing through different areas of the chip is less than 1.2%,which shows no significant difference in the flow velocity of microfluidics between different chips,and microfluidics can be stably driven.Therefore,this chip can be applied to more accurate detection and analysis.（2）Selecting red time-resolved fluorescent microsphere with suitable diameter,and preparing time-resolved fluorescent microsphere-antibody conjugates（TRFNs-Ab）based the method of EDC/NHS.After optimizing the reaction conditions,identifying the prepared TRFNs-Ab,a method of detecting single veterinary drug was established.The most suitable diameter of microspheres is 200 nm,and the optimal dosage of EDC is 0.5μg EDC per microliter microsphere.Furthermore,the maximum antibody labeling quantities of RAC、CLB、CAP were 2.4μg、4μg、2.8μg per micro-liter microspheres respectively,and the labeling time was 30-40 min.Besides,the identification test shows that the all prepared TRFNs-Ab could specifically bind to the corresponding antigen respectively and could be used for immunoassay.（3）Verifying the interference of simultaneous detection of three veterinary drug residues,and preliminarily establishing a method for simultaneous detection of three veterinary drug residues.The validation results shows no significant interference between each detection when detect three veterinary drug residues simultaneously on the same chip.The optimum sample-dispensing diluent for the chip was CBS solution,and the optimum coating conditions were 37℃for 1 h.In addition,the optimum formulation of TRFNs-Ab solution was PB（0.02M,PH=7.4）containing 0.5%PVP,5%trehalose,1%BSA,0.5%Tween-20 and 0.5%Pro Clin 300.Furthermore,the optimum antigen spot concentration of RAC、CLB、CAP were 25,10 and 15μg/mL respectively,while the the optimal antibody labeling amount of TRFNs-Ab was 1.2,1 and 2μg per microliter microspheres respectively,and the optimum secondary antibody spot concentration was 0.096 mg/mL.（4）Establishing the standard curve of each object.According to the fitting standard curve,the IC50 of RAC、CLB、CAP were 0.57 ng/mL,0.46 ng/mL and 0.49 ng/mL respectively,and the linear range(IC₂₀～IC₈₀)was 0.14～2.25 ng/mL,0.15～1.40 ng/mL,0.13～1.43 ng/mL respectively.（5）Evaluating the established microfluidic analysis method for simultaneous detection of three veterinary drugs residues.The results show that the detection limits of RAC、CLB、CAP were 0.08 ng/mL,0.05 ng/mL and 0.03 ng/mL respectively.The precision detection shows that the detection coefficient of variation（CV）of different chips was less than 7%.The accuracy evaluation showed that the average recovery rate was 80%～120%,and the coefficient of variation（CV）was less than 25%.Compared with GICA,the coincidence rate of two test results is 100%,and the correlation coefficient between the the microfluidic immunoassay and ELISA was more than 0.98.These results show that the microfluidic immunoassay method established in this study has good sensitivity,precision and accuracy,and can be used to screen a variety of veterinary drug residues in the field.