Moscone_2011_Methods.Mol.Biol_739_217

A novel method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE, from electric eel) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). To select and optimize the analytical procedures, the investigation on type of AChE inhibition by AFB(1) was carried out. The AChE degree of inhibition by AFB(1) was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB(1). In fact, only a 3-min analysis is required. For the development of AFB(1) assay, the pH, the reaction time, the temperature, and the substrate concentration were evaluated and optimized. The linear range of 10-60 ng/mL was assessed. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins, such as AFB(2) (aflatoxin B(2)), AFG(1) (aflatoxin G(1)), AFG(2) (aflatoxin G(2)), and AFM(1) (aflatoxin M(1)), was investigated. The suitability of the assay for AFB(1) quantification in barley was also evaluated. This study shows a new approach to detect aflatoxins based on enzyme inhibition with several advantages, such as the easiness of use, the rapidity, and the cost-effectiveness, demonstrating a possible use as screening method for this type of mycotoxins.