Detection of paraoxon by immobilized organophosphorus hydrolase in a Langmuir–Blodgett film

Abstract

Langmuir–Blodgett (LB) film deposition technique was employed for the immobilization of organophosphorus hydrolase (OPH). OPH enzyme was covalently bonded to a fluorescent probe, fluorescein isothiocyanate (FITC), and used as a biological recognition element. Under optimal experimental conditions, OPH monolayers were deposited onto the surface of silanized quartz slides as LB film and utilized as a bioassay for the detection of paraoxon. Two different methods were employed for detection of paraoxon: the fluorescence quenching of the fluorescence probe (FITC) covalently bonded to OPH and the UV–vis absorption spectrum of the paraoxon hydrolysis product. The UV–vis absorption measurement demonstrated a linear relationship between the absorbance at 400 nm and the concentration of paraoxon solutions over the range of 1.0 × 10 −7–1.0 × 10 −5 M (0.27–27 ppm). By observing the FITC fluorescence quenching, the concentration of paraoxon can be detected as low as 10 −9 M (S/N = 3). The research described herein showed that the LB film bioassay had high sensitivity, rapid response time and good reproducibility.