Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay

Abstract

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL^-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

Keywords: 4-methylumbelliferyl butyrate; Alicyclobacillus acidocaldarius; EST2; Organophosphate pesticides; biomonitoring; biosensor; esterase-2; fluorescence; human urine; paraoxon.

Scheme 1

Mechanism of (A) the spontaneous hydrolysis of 4-methylumbelliferyl butyrate (4-MUBu), in 2-[4-(2 Hydroxyethyl)-1-piperazino]-ethansulfonic acid (HEPES) buffer and (B) by enzymatic hydrolysis after the addition of esterase-2 (EST2). The release of 4-methylumbelliferone (4-MU) produces the increase in fluorescence.

Figure 1

EST2 assay using 4-MUBu. (A) Fluorescence development of 4-MUBu by spontaneous hydrolysis in HEPES buffer (curve 1) and enzymatic hydrolysis after the addition of EST2 (curve 2). (B) Calibration curve of fluorescence intensity at increasing concentration of 4-MU in HEPES buffer for the calculation of the coefficient value necessary for the determination of the amount of reaction products from the enzymatic hydrolysis (R square value for the regression line = 0.996). The error bars represent the standard deviation (SD) from three independent experiments.

Figure 2

Docking analysis with binding free energies (shown as kcal mol^-1) of complexes between EST2 and substrates. Trace representation of EST2 backbone (black), residues in the catalytic site are represented as stick in gray, the substrates MUBu (dark gray) and pNitrophenyl-butyrate (gray) are depicted as a van der Waals surface (WDV) representation. (A) Accommodation of 4-MUBu in the alcohol pocket of EST2, with respect the pNitrophenyl-butyrate bonding to the acyl one; and (B) arrangement of 4-MUBu in the catalytic site with less affinity with respect to the alcohol pocket.

Figure 3

Inhibition of EST2 by diethyl (4-nitrophenyl) phosphate (paraoxon). Description of the kinetic (A) and molecular (B) mechanism of the inhibition of EST2 by paraoxon. The inhibited enzyme is frozen in a stable covalent intermediate with the organophosphate group of pesticide. (C) Plot of EST2 residual activity in presence of an increasing concentration of paraoxon. In the insert, the percentage of the inhibition of EST2 in the presence of this increasing concentration of paraoxon. The error bars represent the standard deviation (SD) from three independent experiments.

Figure 4

Determination of paraoxon in human urine. (A) Plot of EST2 residual activity in presence of increased amount of urine. (B) Plot of EST2 residual activity in 4% urine, in the presence of the increasing concentration of paraoxon. In the insert, the percentage of inhibition of EST2 in the presence of this increasing concentration of paraoxon. The error bars represent the SD from three independent experiments.