Amperometric acetylthiocholine sensor based on acetylcholinesterase immobilized on nanostructured polymer membrane containing gold nanoparticles

Abstract

Poly(acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were chemically modified and loaded with gold nanoparticles. Acetylcholinesterase was immobilized on the prepared membranes in accordance with two distinctive procedures, the first of which involved immobilization of the enzyme by convection, and the other by diffusion. The prepared enzyme carriers were used for the construction of amperometric biosensors for detection of acetylthiocholine. Two sets of experiments were carried out. The first set was designed so that to evaluate the effects of the gold nanoparticle deployment and the immobilization procedures over the biosensor effectiveness. The other set of experiments was conducted in order to determine the influence of the individual components of the enzyme mixture, containing gold nanoparticles, acetylcholinesterase, bovine serum albumin and glutaraldehyde, over the current output of the constructed acetylthiocholine biosensors. The optimum composition of the mixture was determined to be as follows: enzyme, 0.1 U ml −1; gold nanoparticles, 0.50 ml (per 1 ml enzyme mixture); albumin, 0.5% and glutaraldehyde, 0.7%. On the basis of the experimental results, the most efficient enzyme membrane was selected and used for the preparation of an acetylthiocholine biosensor. Its basic amperometric characteristics were investigated. A calibration plot was obtained for ATCh concentration ranging from 10 to 400 μM. A linear interval was detected along the calibration curve from 10 to 170 μM. The sensitivity of the constructed biosensor was calculated to be 0.066 μA μM −1 cm −2. The correlation coefficient for this concentration range was 0.996. The detection limit with regard to ATCh was calculated to be 1.80 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit for Paraoxon was determined, 7.39 × 10 −11 g l −1, as well as the concentration interval (10 −10 to 10 −7 g l −1) within which the biosensor response was linearly dependant on Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 50 days. After storage for 20 days the sensor retained 75% of its initial current response and after 30 days −25%.