ORIGINAL PAPER An amperometric acetylthiocholine sensor based on immobilization of acetylcholinesterase on a multiwall carbon nanotube–cross-linked chitosan composite Dan Du & Xi Huang & Jie Cai & Aidong Zhang & Jiawang Ding & Shizhen Chen Received: 21 July 2006 / Revised: 21 October 2006 / Accepted: 30 October 2006 / Published online: 22 December 2006 # Springer-Verlag 2006 Abstract A simple method has been devised for immobi- lization 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 app m value of 132 μmol 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 μmol L -1 , with a detection limit of 0.10 μmol 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. Keywords Acetylthiocholine sensor . Acetylcholinesterase . Chitosan . Multiwall carbon nanotube . Composite film Introduction One of the products of hydrolysis of acetylthiocholine chloride (ATCl) with acetylcholinesterase (AChE) is thio- choline. Detection of thiocholine can be used to assess the activity of AChE, a biomarker of the effect of pesticides (organophosphates (OPs) and carbamates) which inhibit cholinesterases [1]. Analysis of ATCl is, therefore, of great importance, particularly in the development of sensors for detection of environmental pollutants such as OPs and carbamates [2]. Traditional methods available for determination of ATCl include colorimetry and spectrophotometry [3–7]. Because electrochemistry is a powerful tool for real-time detection compared with fluorescence and spectrophotometry, amper- ometric sensors have been regarded as most suitable for biochemical analysis, because of their good selectivity, rapid response, miniature size, and reproducible results. Combina- tion of enzymatic reactions with electrochemical methods has enabled the development of a variety of enzyme-based electrochemical biosensors for sensitive and rapid determi- nation of environmental pollutants [8, 9]. During fabrication of biosensors, immobilization of the enzyme on the solid electrode surface is a crucial step. According to the literature, AChE can be immobilized on electrode surfaces by use of a variety of matrices, for example cross-linked polymers [10], cross-linked bovine serum albumin [11, 12], chitosan [13], cellulose [14] and different support matrices, for example Nylon [15–17], controlled-pore glass [18], magnetic particles Anal Bioanal Chem (2007) 387:1059–1065 DOI 10.1007/s00216-006-0972-6 D. Du (*) : X. Huang : A. Zhang : J. Ding : S. Chen Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, Central China Normal University, Wuhan 430079, China e-mail: dudan@mail.ccnu.edu.cn J. Cai The Technology Center of Wuhan Iron & Steel Company, Wuhan 430080, China