An Acetylcholinesterase/Choline Oxidase-Based Amperometric Biosensor as a Liquid Chromatography Detector for Acetylcholine and Choline Determination in Brain Tissue Homogenates

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Abstract
A liquid chromatography (LC) detector based on a fast response and sensitive bienzyme amperometric biosensor for acetylcholine (ACh) and choline (Ch) is described. The detector fabrication consisted of glutaraldehyde co-cross-linking of acetylcholinesterase and choline oxidase with bovine serum albumin on the Pt working electrode of a conventional thin-layer electrochemical flow cell. The influence of some experimental parameters (e.g., enzyme loading, thickness of the bienzyme layer, flow rate) on the detector characteristics has been studied in order to optimize the analyte response while minimizing band-broadening and distortion. A mobile phase consisting of a phosphate buffer (I, 0.1 M; pH, 6.5) containing 5 mM sodium hexane sulfonate and 10 mM tetramethylammonium phosphate was found to give very satisfactory resolution and peak shape in ion-pair, reversed-phase LC. Linear responses were observed over at least four decades and absolute detection limits (at a signal-to-noise ratio of 3) were 12 and 27 fmol injected for Ch and ACh, respectively. After one month of intensive use in the LC system, the detector retained about 70% of its initial sensitivity. The potential of the described approach is demonstrated by the simultaneous determination of Ch and ACh in rat brain tissue homogenates.

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