An electrochemical enzyme immunoassay for aflatoxin B1 based on bio-electrocatalytic reaction with room-temperature ionic liquid and nanoparticle-modified electrodes

Abstract

A new electrochemical immunosensor for the determination of aflatoxin B1 (AFB1) based on bio-electrocatalytic reaction was proposed. An imidazolium cation room-temperature ionic liquid (RTIL), 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIm][BF4]), was initially immobilized on the surface of a glassy carbon electrode (GCE) through titania sol and Nafion film, then nanogold particles were adsorbed onto the titania surface, and then horseradish peroxidase (HRP)-labeled anti-AFB1 antibodies (HRP-anti-AFB1) were attached on the nanogold surface. With a non-competitive immunoassay format, the formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-AFB1 and AFB1 in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface, thus local current variations could be detected by the HRP bio-electrocatalytic reaction in 0.1 M PBS (pH 6.8) containing 0.28 M H2O2. Under optimal conditions, the electrochemical immunosensor exhibited a good current response relative to AFB1 concentration in a linear range from 0.1 to 12 ng/mL with a relatively low detection limit of 0.05 ng/mL at 3δ. The inter-assay coefficients of variation are 7.1% and 5.4% for 1.0 ng/mL and 8.0 AFB1, respectively. Naturally contaminated samples were screened with the developed immunosensor, and results were compared with those obtained by validated ELISA method. The assay was demonstrated to be accurate and reliable giving no false compliant and only a low percentage of false non-compliant results. The described method offers a simple, rapid and cost-effective screening tool, thus contributing to a better consumers’ health protection.