Study On SPR Biosensor For Detection Of Ractopamine In Animal Food

Ractopamine is a kind of phenethanolamineβ-adrenergic agonist with N-alkylphenyl substituents. As other P-adrenergic agonists, when the dosage is 4-9 times higher than its clinical dosage, it can also regulate growth rate and redistribute nutrition. When the residues come to food though the food chain, it's dangerous for people's health. At present, usage ofβ-adrenergic agonists as growth accelerant is strictly prohibited in China. But some poisoning events which caused by illegal usage of P-adrenergic are still happening. Because of spending more attention on forbiding the usage of Clenbuterol, more and more Ractopamine is used to attend the same utility. In China, the official regulatory determinative method of ractopamine analysis is SN/T 1924-2007, . The rapid screening method is enzyme-linked immunosorbent assay (ELISA). The HPLC-MS/MS method needs high costs, complicated pre-treatment technique and time-consuming operation. It is impossible to achieve a large samples analysis. ELISA format is considered as cost-effective complement techniques to chromatographic methods. However, long incubation periods and repeated washing steps make it time-consuming.Otherwise, it needs to lable either antigen or antibody, and some reagents are harmful to the operator. So to develop a rapid, sensitive, effective and specific analytic method for ractopamine detection is very significant.With the development of biotechnology, bio-sensor technology was born. A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component. It consists of 3 parts:the sensitive biological element (biological material (eg. enzymes, antibodies, microorganisms, cell receptors, organelles, tissue, nucleic acids, etc), a biologically derived material or biomimic).The sensitive elements can be created by biological engineering; the transducer or the detector element (works in a physicochemical way; optical, piezoelectric, electrochemical, etc.) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal (i.e., transducers) that can be more easily measured and quantified; associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. SPR sensing is an optical biosensor that uses antibodies as the recognition element SPR sensing has several advantages over conventional ELISA. First, the analytical procedures are direct and simple without multiple labeling separation steps; second, a real-time, automatic detection mode can be achieved in combination with a auto-injection system, allowing high-throughput analysis; third, SPR is (in most cases) less time-consuming than ELISA. Thus, SPR biosensor analysis combines the advantages of conventional immunochemical procedures with a high degree of auto-mation and the generation of results in real time. In most cases, sample preparations for these biosensor assays are relatively simple and straightforward. On the other hand, the higher consumption of antiserum or antibodies (about 10-100 times) is generally a disadvantage of SPR assays compared with ELISA tests. SPR based biosensor immunoassays is highly sensitive and stability, so it has been used in the rapid detection of low molecular-weight analytes and the interaction of biomolecules.One of the key steps for immunoassay is the synthesis of immunogen and the gain of the highly effective and specific antibodies.In our study, immunogens were synthesized by mixed anhydride. RAC-BSA was used as the immunogen, and the final compounds were identified by UV spectrophotometer, infrared spectrometer and SDS-PAGE. The immunogens was synthesized successfully.The rabbits were immunized by RAC-BSA. Sunthesized the coating antigen in the same method as the synthesis of immunogens. The subcutaneous five points injection method was used. Anti-sera with high titers 1:200000 were obtained after seven times immunization. ELISAs showed that the cross-reactivity with Gentamicin sulfate,Salbutamol and Clenbuterol are all bellow 0.01%. The LOD for detection of ractopamine was 0.400 ng/mL.The kinetics of the binding between the Ractopamine antibody and the antigen were analyzed by SPR. The kinetic constants were calculated, affinity constant was 4.48275E-7M. This antiserum had high affinity to Ractopamine.A fabricate the sensing surface of the SPR immunosensor simply by covalent amide binding of RAC-OVA on the Au-thiolate self-assembly of simply and commercially available 11-mercaptopropionic acid (MUA). The optimization immobilization condition is:Using 10 mmol/L pH4.5 NaAC as coupling buffer; EDC/NHS as activating solution for 7 min; The concentration of ligand is 10μg/ml, and immobilization time is 20 min. The immobilization is reproducibility.An indirect competitive immunoassay method that assures the detection of low molecular-weight analytes was accomplished for the detection of Ractopamine. The antibody was mixed with Ractopamine of different concentrations prepared with HBS to construct a standard curve. The calibration curve gave an IC50 of 1.17 ng/mL, and the LOD is 0.12 ng/mL. Then compared with the results of biosensor and the results obtained from ELISA and HPLC-MS-MS. The method of SPR has good veracity and precision, and the recovery rate is acceptable. The biosensor assay is an excellent analytical tool to screen ractopamine residues in animal food, and the results should be extendible to other species with suitable validation.