An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube-cross-linked chitosan composite

A simple method has been devised for immobilization of acetylcholinesterase (AChE)-covalent bonding to a multiwall carbon nanotube (MWNT)-cross-linked chitosan composite (CMC)-and a sensitive amperometric sensor for rapid detection of acetylthiocholine (ATCl) has been based on this. Fourier-transform infrared spectroscopy proved that the native structure of the immobilized enzyme was preserved on this chemically clean and homogeneous composite film, because of the excellent biocompatibility and non-toxicity of chitosan. Glutaraldehyde was used as cross-linker to covalently bond the AChE, and efficiently prevented leakage of the enzyme from the film. Because of the inherent conductive properties of the MWNT, the immobilized AChE had greater affinity for ATCl and excellent catalytic effect in the hydrolysis of ATCl, with a K-m(app) value of 132 mu mol L-1, forming thiocholine, which was then oxidized to produce a detectable and rapid response. Under optimum conditions the amperometric current increased linearly with the increasing concentration of ATCl in the range 2.0-400 mu mol L-1, with a detection limit of 0.10 mu mol L-1. Fabrication reproducibility of the sensor was good and the stability was acceptable. The sensor is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis.