Kinetic approach of aflatoxin B1-acetylcholinesterase interaction: a tool for developing surface plasmon resonance...

This work presents a kinetic approach of the interaction between acetylcholinesterase (AChE) from electric eel and aflatoxin B1 (AFB1) or its protein conjugate (e.g., AFB1-HRP [horseradish peroxidase]) in order to develop a simple and sensitive detection method of these compounds. The dissociation constant K(d) of the AChE/AFB1-HRP interaction (0.4 μM) obtained with the surface plasmon resonance (SPR) technique is very close to the inhibition constant reported in amperometric assay (K(i)=0.35 μM), proving that the conjugation of AFB1 to a carrier protein does not significantly influence the affinity of AFB1 for AChE. Thus, the AChE/AFB1-HRP couple can be used as mimic system for the binding of AChE to other AFB1-protein adducts and further used for developing biosensors for AFB1 bound to plasma proteins. The immobilization protocol was designed to minimize the nonspecific adsorption on the self-assembled monolayer (SAM) functionalized surface of the SPR chip without an additional hydrophilic linker, whereas the interaction protocol was designed to mark out the possible occurrence of mass transport limitation (MTL) effects. The detection limits (LODs) were 0.008 μM for AFB1-HRP (2.5 ng ml⁻¹ AFB1) and 0.94 ng ml⁻¹ for AFB1 itself, which is lower than recently reported values in spectrophotometric and amperometric assays.