Optimized Biosensors Based on Purified Enzymes and Engineered Yeasts: Detection of Inhibitors of Cholinesterases on Grapes

Abstract

Purified acetyl cholinesterase (AChE) from electric eel or engineered yeasts (Kluyveromyces lactis) expressing cholinesterase activity from rat, were immobilised on a nylon membrane for the measurement of residual cholinesterase activity after inhibition by the organophosphorous insecticide Paraoxon (diethyl p‐nitrophenyl phosphate). Measurement was separately operated after the incubation (inhibition) step. The measurement of the residual enzyme activity was therefore performed in a standardised solution with a choline electrochemical biosensor, without any interference, avoiding the use of protecting selective membranes. Both commercial amperometric sensor and screen‐printed electrodes, produced and optimized in the laboratory, were used. The time consuming incubation step was simultaneously performed on several samples, thus lowering the analysis time per sample. Good limit of detection (LOD) (0.1 µg/L) and reproducibility were obtained for the analysis of paraoxon. A simple procedure for the detection of cholinesterase inhibitors on grapes was then developed. The procedure itself could be further extended to several fruits and vegetables, giving a simple but effective tool to verify the absence of residues of anticholinesterasic insecticides.

Keywords:

• Cholinesterase inhibitors
• Acetyl cholinesterase
• Gas chromatography
• Limit of detection
• Enzyme inhibition‐based biosensors

Acknowledgments

This work was partly supported by COSMIC Project (ENEA “Target Project on Biosensors and Bioelectronics”) and RoseProMilk (the 5th FW UE‐QLK1‐CT2001‐01617). We would like to acknowledge Prof. C. Palleschi of the University of Rome “La Sapienza” for his valuable suggestions and for providing us with engineered K. lactis yeast.