Biosensors and Bioelectronics 21 (2005) 87–94 Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline O.N. Schuvailo a , S.V. Dzyadevych a , A.V. El’skaya a , S. Gautier-Sauvign´ e b , E. Cs ¨ oregi c , R. Cespuglio b , A.P. Soldatkin a,∗ a Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of Ukrainian NAS, 150 Zabolotnogo Street, Kyiv 03143, Ukraine b Laboratory of Neurobiology of the vigilance states, EA 3734, C. Bernard University, 8 av. Rockefeller, Lyon 69373, Cedex 08, France c Department of Biotechnology, Lund University, Getingev¨ agen 60, S-221 00 Lund, Sweden Received 5 July 2004; received in revised form 22 September 2004; accepted 22 September 2004 Abstract This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H 2 O 2 (detection limit at 0.5 M). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800–1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 M for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3–100 M) suitable for the measurement of choline and acetylcholine in vivo. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbon fibre electrode; Amperometric microbiosensor; Mediated electron transfer; Acetylcholine; Choline; Sensitivity; Selectivity 1. Introduction Direct monitoring of neurotransmitters in living organ- isms is of great interest and still remains a challenge, despite intensive research efforts over the last two decades (Gonon et al., 1980; Cespuglio et al., 1981; Burlet et al., 1999). The plu- rality and low concentrations at which neurotransmitters are released into the extracellular space within the brain neuronal network make their detection technically difficult. Acetyl- choline is a well known neurotransmitter in brain signalling that is synthesised in various neuronal sets extending from ∗ Corresponding author. Tel.: +380 44 2660749; fax: +380 44 2660759. E-mail address: a soldatkin@yahoo.com (A.P. Soldatkin). the pons to rostral brain structures (Tucek, 1984; Roisin et al., 1991; McNally and Wong, 2001), and its release char- acteristics vary with the behavioural situation of the animal (e.g. sleep–wake alternance, attention and stress) (Kodama et al., 1992). A meant interest in the follow-up in vivo of this neurotransmitter appears also with clarity when considering the pathologies (Alzheimer and Parkinson diseases, epilepsy, etc.), in which reductions in the availability of acetylcholine are recognised (Pomora, 1986; Varadarajan et al., 2000). Many methods have been developed for the detection of neurotransmitters, such as biochemical analysis using ra- dioactive labels (Guyenet et al., 1975; Haubrich et al., 1981), HPLC analysis (Bullock et al., 1991; Murai et al., 1995; Izaki et al., 1998) and microdialysis with electrochemical 0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.09.017