A layer-by-layer tyrosinase biosensor for assay of carboxylesterase and neuropathy target esterase activities in blood

Abstract

A tyrosinase-based phenol biosensor was formed via layer-by-layer (LBL) deposition of poly(dimethyldiallylammonium chloride) (PDDA) and tyrosinase onto polished graphite, followed by glutaraldehyde (GA) crosslinking. The PDDA/tyrosinase/GA graphite-substrate biosensor exhibited good reproducibility (6% relative SD for 1 × 10−5 M phenol, n = 10) and high sensitivity (6 nM detection limit, S/N = 3). The low detection limit enabled phenol measurements in highly diluted blood samples, which also minimized interference from extraneous (non-analyte) substances in blood (e.g., adrenaline, ascorbate, glucose, L-tyrosine, and urate). Applicability of the biosensor to analysis of carboxylesterase (CaE) and neuropathy target esterase (NTE) activities in mouse and human blood was demonstrated, and parallel biosensor and spectrophotometric CaE analyses were carried out 1 h after intraperitoneal injection of mice with the model dialkylphosphate, (C2H5O)2P(O)OCH(CF3)2 (DEHFPP). Dose-related inhibition of CaE activity was observed, yielding ED50 values [mean (95% CI) (n)] of 25.5 (23.2, 28.0) mg kg−1 (6) and 21.1 mg kg−1 (18.9, 23.5) (4) for spectrophotometric and electrochemical assays, respectively. Although the ED50 values were significantly different from each other (p < 0.007), excellent correlation between biosensor and spectrophotometric measurements was found (r = 0.99), indicating that the differences between the two methods are systematic and providing validation of the biosensor assay.