Enzymatic spectrophotometric method for aflatoxin B detection based on acetylcholinesterase inhibition

A new method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). Cholinesterases (ChEs) from various sources were tested using AFB(1) as reference aflatoxin. AChE from electric eel has shown the highest sensitivity to AFB(1), and it was chosen for the rest of the work. To select and optimize the analytical procedures, an investigation on the 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), requiring only 3 min. For the development of the AFB(1) assay, the pH, the time of reaction, temperature, and substrate concentration were evaluated and optimized. The linear range of 10-60 ng mL(-1) was determined. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins such as aflatoxin B-2, aflatoxin G(1), aflatoxin G(2), and aflatoxin M-1 was investigated. Finally, the suitability of the assay for AFB(1) quantification in barley was evaluated. This study shows a new approach to detect aflatoxins based on enzyme inhibition and has advantages such as the ease of use, rapidity, and cost effectiveness. Thus, it could find a possible use as a screening method for this type of mycotoxins.