Study On High-throughput Suspension Array Technology For Simultaneous Detection Of Multiple Pesticide And Veterinary Drug Residues

All eyes have been focused on food safety globally especially in China are extremely and fiercely severe. Close and extensive concerns about pesticide and veterinary drug residues in environment and food are continuously arising. Some normal conventional technologies aren't capable to detect multiple residues rapidly, sensitively and accurately. To resolve the problem, a novel high-throughput suspension array technology and method has been established for multiple pesticide and veterinary drug residues. The aim of the establishment is to implement quantitative detection of the multiple residues rapidly, accurately, specifically and efficiently and for the foundation of the technical platform.Four kinds of veterinary drugs, including chloramphenicol, clenbuterol, 17-β-estradiol, tylosin and three kinds of pesticide including atrazine, imidacloprid and carbaryl which are commonly used are regarded as the targets. The experiment was based on the theoretic indirect competitive method. The conventional suspension array method that fixation on the beads by anatibody is improved. The goal that up to 7 kinds of residues can be detected at the same time has been achieved by competition of the specific biotinylated antibodies between the multiple antigens of pesticides and veterinary drugs and conjugates which labeled on the fluorescent beads in liquid phase. In addition, the shortage of conventional suspension array technology was abandoned by optimization and screening the experimental conditions. Conventional millipore filter plate and pump were non-necessary during the whole procedures by antigen-antibody complete reactions. Furthermore, ordinary 96-hole cell culture plates or ELISA plates can satisfy the requirement and without repeated pumping. Therefore, it's simple, rapid, saving lots of time, low-cost and reliable in the experiment. The followings have been concerned:1. Preparation and identification of the conjugates of pesticide and veterinary drugs2. Coupling, validation and optimization of the conjugate on fluorescent bead3. Determination of kinetic parameters of antigen-antibody reactions by SPR biosensor technology4. Characterization of the surface microstructure of the bead by scanning electron microscope5. Simultaneous detection for multiple pesticide and veterinary drug residues by high-throughput suspension array technology6. Methodological comparison of the detection for pesticide and veterinary drug residues Various chemical treatments such as active ester method, diazotization method, mixed anhydride and carbodiimide method, etc were carried out for the synthesis of the protein conjugates of the seven targets. The synthetic products were identified by ultraviolet spectrum and mass spectrum after purification of all the antibodies. The protein conjugates of the targets were identified by ultraviolet spectrum and mass spectrum. And the exact coupling ratios are obtained which provided criterion of conjugates amounts on the beads. And then, the seven protein conjugates were coupled and validated on the solid carrier-the polystyrene fluorescent beads by amino-coupling procedure.Afterwards, the important conditions such as adding amounts of the conjugates and the specific biotinylated antibodies, the activation buffers, antibody preservative and the dilution of streptavidin-phycoerythrin, etc are selected and optimized. By means of optimizing the condition and exploring a variety of the tested target concentration gradient, the standard curves of the single channel suspension array have been polted and the suspension array technique of single channel for the detection of pesticide and veterinary drug residues has been established. Logistic regression standard curves were able to be plotted respectively for the seven targets. All the coefficients of determination-R2 were high than 0.99, the minimum detection concentration and the detection arranges could be acquired respectively. The single channel detection method for pesticide and veterinary drug residue is simple, sensitive and rapid. At the same time, SPR biosensor technology was employed for the determination of antigen-antibody affinity constants of a variety of targets and their complementary antibodies.Specific recognition was carried on and it proved excellent recognition between various paired antigen-antibody on the beads. Meanwhile, specific competition occurred and no obvious cross-reactions with others in the high-throughput detection of suspension array. In the experiment of quenching and interfering by organic solvents, it indicated that when the volume ratio is not more than 40%, there is a degree of anti-quenching and anti-interference with the fluorescent beads.In the case of good response for the single channel of the suspension array, high-throughput detection and multi-analysis of suspension array for three kinds of veterinary drug residues, six kinds of pesticide and veterinary drug residues and seven pesticide and veterinary drug residues are respectively carried out. Standard curves of the multi-analysis were plotted and the coefficients of determination-R2 were investigated and performed well. All the detection range and the lowest detection limits of the targets were determined respectively. And in the aspect of specificity for multiplex analysis, it showed no significant cross-reaction and interference with other structurally similar and non-similar drugs. In the tests of different bland samples with multiple pesticide and veterinary drug residues, the relative standard deviations of the found and the real values are relatively small. Overall, suspension array can be employed for primary detection of real samples of pesticides and veterinary drug residues. It spends only 1 to 2 hours in the whole process. And the requirements for sensitive, specific, rapid and efficient detection for residues are basically satisfied.Scanning electron microscopy was employed for characterization on the micro-structure surface of the beads. It directly confirmed a series of events occurred, such as successful coupling of the conjugates on the beads, antigen-antibody reaction and the fluorescent reporter molecules-streptavidin-phycoerythrin in a combination on beads by biotinylated antibodies.In comparison with the conventional indirect ELISA, suspension array method takes relatively notable advantages in lowest detection limits, high-thought and spends less time. In comparison with LC-MS method, the lowest detection limits of the targets determined by suspension array are lower than national standard except Atrazine and Tylosin. When compared with LC-MS in practice, suspended array have a distinct advantage in the aspects of the lowest detection limit and detection range.The lowest detection limits of suspension array were lower than which of national standards by 2 order of magnitudes, and the detection ranges of suspension array exceeds 1~3 order of magnitudes which of the LC-MS method for imidacloprid and carbaryl detection. It can be concluded that suspension array technology takes the advantage in the detection of multiple pesticide and veterinary drug residues.Traditional indirect competitive ELISA method and LC-MS method have been employed for the detection of multiple pesticide and veterinary drug residues in comparison with suspension array. Compared with traditional indirect competitive ELISA method, suspension array has the advantage over the fomer method by broad detection ranges, high-throughput and sensitivities. Suspension array corresponds with LC-MS method in sensentivity and exceeds it in detection range. It hasn't been systemically reported at home and abroad yet, and to be considered as integrated innovation methodologically and theoretically.By virtue of establishment of multiple pesticide and veterinary drug residues by high-throughput suspension array technology, simultaneous high-throughput multi-analysis has been achieved and can be primarily employed on sensitive, specific, rapid and efficient determination of real samples and the elative standard deviations of detection values are relatively small by comparison with real values. It provides a novel method and foundation of the technical platform for multiple pesticide and veterinary drug residues with broad and promising application and development prospects.