High-throughput biosensing of neurotoxic insecticides using polystyrene microplate-immobilized acetylcholinesterase

Acetylcholinesterase (AChE) inhibition-based biosensors can be used as a screening test for neurotoxic insecticides. A high-throughput AChE inhibition assay was developed via AChE immobilized on a microplate using enzyme-linked immunosorbent assay (ELISA) reader. The presence of insecticide was confirmed by reactivation of the inhibited AChE immobilized on the microplate. The polystyrene (PS) surface of the microplates was first modified with amine groups. The aminated 96-well microplate was modified further with chitosan to generate more amine groups. Thus, AChE could be covalently immobilized onto the aminated microplate via cross-linking with glutaraldehyde. The activity of the immobilized AChE upon surface modification with chitosan was 12-fold higher than without spacer molecules. The activity of the immobilized AChE was measured before and after incubation with test samples to calculate the inhibition rate. The calibration curves showed a linear response ranging between 1.0 and 20.0 mu g L-1 for paraoxon with detection limits (3 sigma) of 0.5 mu g L-1 in buffer or organic milk. This assay format was applied directly to determine the insecticide in real milk samples with recovery rate 89-108%. The developed microplate assay format could be used as a convenient tool for high-throughput prescreening of insecticides in samples to increase the number of tested samples and ensure consumer safety.