Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline.

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). 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 microM 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 microM) suitable for the measurement of choline and acetylcholine in vivo.

Luciferin incorporation in the structure of acrylic microspheres with subsequent confinement in a polymeric film: A new method to develop a controlled release-based biosensor for ATP, ADP and AMP.

The controlled release of coreactants at the sensing tip of a biosensor is a possible approach to develop self-contained devices. For luciferin which is a firefly luciferase cosubstrate, a new method of retention is evaluated. The two-step procedure consists of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a PVA SbQ film. When associated with a compartmentalised trienzymatic sequence (adenylate kinase, creatine kinase and luciferase), such a complex matrix ensures the internal delivery of the cosubstrate in the enzymatic microenvironment at a controlled rate. For the three adenylic nucleotides (ATP, ADP and AMP), the self-containment working time is 3 h of continuous and reproducible assays. The sensitivity of the fibre optic biosensor represents, for ATP, 30% of that obtained when luciferin is supplied in solution whereas for ADP and AMP, the values are about 80% of the reference ones.