Development Of A Biomimetic Enzyme-Linked Immunosorbent Assay For The Determination Of Citrinin

In this research, a citrinin molecularly imprinted membrane was synthesized on the well surface of polystyrene 96-well plate with bulk polymerization method using 3,5-dihydroxy-2-naphthoic acid as template, methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker, in which has introduced the molecular imprinting technique into immunoassay and developed a biomimetic enzyme-linked immunosorbent assay for citrinin detection in food samples. The synthesis parameters and conditions of the membrane were optimized in details and the SEM were employed for the structure characterization. The adsorption capability of the molecularly imprinted membrane was evaluated in the isotherm and kinetic adsorption experiments, which has confirmed that the synthesized membrane was with good selective adsorption ability and fast mass-transfer process. Good adsorption capacity (1.07 μg well-1) was obtained higher than that of non-imprinted film (0.213 μg well-1). At the citrinin concentration of 50 mg L-1, 54.9% of the saturated binding capacity was obtained within 10 min,97.3% within 30 min and achieved adsorption equilibrium within 40 min. A direct competitive biomimetic enzyme-linked immunosorbent assay (BELISA) for citrinin detection was developed using molecularly imprinted film as artificial antibody.Under the optimum conditions, the sensitivity (IC50) and the limit of detection (IC15) achieved to 109.2 μg L-1 and 0.50 μg L-1 respectively. The cross-reactivity (CR) with five other biotoxins was less than 4.7%. This BELISA was successfully applied to citrinin determination in steamed rice, corn and rice vinegar, the recoveries of which ranged from 73.2% to 89.9% in steamed rice,64.3 to 86.2% in corn and 82.3 to 92.8% in rice vinegar under three spiked conditions. Compared with the results obtained from the traditional HPLC method, good correlation coefficient (R2= 0.9942) was achieved, demonstrating that the developed BELISA method has offered one stable, feasible and cheap method for citrinin determination in food samples.