Development Of A Biomimetic Enzyme-Linked Immunosorbent Assay Method Of Molecularly Imprinted Film For Ractopamine

In this thesis, a highly selective imprinted membrane was synthesized by an organic-inorganic hybrid technology using ractopamine (RAC) as template, methacrylic acid as functional monomer, γ-(methacryloyloxyethyl) trimethoxysilane as cross-linker. The polymerization conditions were discussed in details. The imprinted membrane was evaluated by FT-IR and static and kinetic adsorption experiments. A sensitive direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) was developed with the molecularly imprinted film as artificial antibody. And the imprinted film exhibited an antibody-like binding ability, rapid adsorption speed, high stability and hydrophilic nature, which was particularly advantageous and suitable for the BELISA development.The results showed that the molecularly imprinted film exhibit higher adsorption capacity and selective ability than that of non-imprinted film. The imprinted film had fast uptake kinetics,0.46μg well-1of binding capacity was obtained within a short shaking period of10min at RAC concentration of20mg L-1, which was67.5%of the saturated binding capacity, and the adsorption equilibrium was almost reached within60min. The established ELISA method gave a good sensitivity (IC50:15.77μg L-1) and a low detection limit (IC15:0.01μg L-1) for RAC under the optimum condition and it was applied to the determination of RAC in urine and pork samples with recoveries ranging from77.7%to108.9%(urine) and from93.5%to101.1%(pork). The obtained results, which correlated well with those gained from the traditional HPLC method (R2=0.9974(urine),0.9922(pork)), demonstrated that the developed ELISA method was reliable for RAC determination in real samples.