Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh). A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C-E: 70 U mL(-1): C-conA: 1.5 mg mL(-1) and C-MWCN: 11 mg mL(-1), with an amperometric current 0.418 mu A. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 mu M. A linear interval was detected along the calibration curve from 5 to 200 mu M. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 mu A mu M-1 cm(-2). The detection limit with regard to ATCh was calculated to be 0.34 mu M. 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 was determined to be 1.39 x 10(-12) g L-1 of Paraoxon, as well as the interval (10(-11) to 10(-8) g L-1) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days-68% and after 120 days-61%. (C) 2010 Elsevier B.V. All rights reserved.