Immobilized acetylcholinesterase in magnetic nanoparticles for in-line inhibition studies using a capillary electrophoresis system

Enzyme assays can be performed with the capillary electrophoresis technique (CE) in many approaches, such as the immobilized enzyme micro-reactor. Acetylcholinesterase is a promising enzyme to be used when pursuing such a method, as it has already been explored in the proposal of similar methods of miniaturizing enzyme assays. The present work proposes a novel enzyme micro-reactor, based on the anchorage of the enzyme on magnetic nanoparticles of MnFe2O4, with chitosan and glutaraldehyde as the cross-linker in the capillary by means of an arrange of neodymium magnets. The calculated Km of the enzyme evaluated by this method was 1.12 mmol L-1, comparable to other studies in the literature that utilizes immobilized enzymes. Also, IC50 for neostigmine was assessed in 3 different micro-reactors, with an average of 29.42 & PLUSMN; 3.88 & mu;mol L-1. In terms of the micro-reactor stability, it was possible to perform at least 25 experiments with assembled micro-reactor. The method was applied to hydroalcoholic extracts of 7 plant species. Plinia cauliflora had the best result, with 42.31 & PLUSMN; 6.81% of enzyme inhibition in a concentration of 100 mg L-1.

Automated summary

This study proposes a novel enzyme micro-reactor using acetylcholinesterase anchored on magnetic nanoparticles and immobilized in capillaries using chitosan and glutaraldehyde. The enzyme showed a Km value of 1.12 mmol L-1, comparable to other immobilized enzyme studies. The method was also used to evaluate IC50 for neostigmine and showed good stability with at least 25 experiments possible. The method was applied to hydroalcoholic extracts of 7 plant species, with Plinia cauliflora showing the best enzyme inhibition of 42.31±6.81% at a concentration of 100 mg L-1.