Biomonitoring of organophosphorus agent exposure by reactivation of cholinesterase enzyme based on carbon nanotube-enhanced flow-injection amperometric detection

Abstract

A portable, rapid, and sensitive assessment of subclinical organophosphorus (OP) agent exposure based on reactivation of cholinesterase (ChE) from OP-inhibited ChE using rat saliva (in vitro) was developed using an electrochemical sensor coupled with a microflow-injection system. The sensor was based on a carbon nanotube (CNT)-modified screen printed carbon electrode (SPE), which was integrated into a flow cell. Because of the extent of interindividual ChE activity variability, ChE biomonitoring often requires an initial baseline determination (noninhibited) of enzyme activity which is then directly compared with activity after OP exposure. This manuscript describes an alternative strategy where reactivation of the phosphorylated enzyme was exploited to enable measurement of both inhibited and baseline ChE activity (after reactivation by an oxime, i.e., pralidoxime iodide) in the same sample. The use of CNT makes the electrochemical detection of the products from enzymatic reactions more feasible with extremely high sensitivity (5% ChE inhibition) and selectivity. Paraoxon was selected as a model OP compound for in vitro inhibition studies. Some experimental parameters, e.g., inhibition and reactivation time, have been optimized such that 92-95% of ChE reactivation can be achieved over a broad range of ChE inhibition (5-94%) with paraoxon. The extent of enzyme inhibition using this electrochemical sensor correlates well with conventional enzyme activity measurements. On the basis of the double determinations of enzyme activity, this flow-injection device has been successfully used to detect paraoxon inhibition efficiency in saliva samples (95% of ChE activity is due to butyrylcholinesterase), which demonstrated its promise as a sensitive monitor of OP exposure in biological fluids. Since it excludes inter- or intraindividual variation in the normal levels of ChE, this new CNT-based electrochemical sensor thus provides a sensitive and quantitative tool for point-of-care assessment and noninvasive biomonitoring of the exposure to OP pesticides and chemical nerve agents.

Figure 1

(A) The entire analyzing system. (B) The part of sensing area.

Figure 2

(A) Cyclic voltammograms of ATCh at (a) bare and (b) CNT modified electrodes in PBS buffer and at (c) bare and (d) CNT modified electrodes in PBS containing 3 nM AChE. (B) Cyclic voltammograms of ATCh at CNT modified electrodes (a) in PBS containing 3 nM AChE and (b) in PBS containing 5 mM 2-PAM for reaction of 15 min after dispersing 25 nM paraoxon in 3 nM AChE for 30 min and (c) in PBS dispersing 25 nM paraoxon in 3 nM AChE for 30 min without adding 5 mM 2-PAM.

Figure 3

Effect of inhibition time on inhibition efficiency by adding (a) 50 nM, (b) 10 nM and (c) 1 nM paraoxon to 3 nM AChE.

Figure 4

Amperometric responses 3 nM AChE in PBS containing 5 mM ATCh (a) before paraoxon exposure and after 25 nM paraoxon inhibition of 30 min followed by treating with 5 mM 2-PAM for (b) 0 min, (c) 2 min, (d) 5 min, (e) 8 min, (f) 10 min, (g) 12 min, (h) 15 min and (i) 18 min. Inset: plot of reactivation efficiency vs different incubation time.

Figure 5

Reactivation efficiency of AChE treated with 5 mM 2-PAM for 15 min after paraoxon inhibition of (a) 5.11%, (b) 12.84%, (c) 25.99%, (d) 47.96%, (e) 65.05%, (f) 80.53% and (g) 93.15%.

Figure 6

(A) Amperometric responses of 3 nM AChE in PBS containing 455 5 mM ATCh (a) before paraoxon exposure and after exposure to (b) 50 nM, (d) 25 nM, (f) 10 nM, (h) 5 nM, (j) 1 nM, (l) 0.5 nM and (n) 0.1 nM paraoxon for 30 min and (c, e, g, i, k, m, o) following reactivation by 5 mM 2-PAM incubation for 15 min. (B) Relationship between inhibition efficiencies calculated from reactivated AChE (I_r %) and those from control intact AChE (I %).

Figure 7

Amperometric responses of 3-fold diluted saliva in PBS containing 5 mM ATCh (a) before paraoxon exposure and after exposure to (b) 25 nM, (d) 10 nM, (f) 5 nM and (h) 0.5 nM paraoxon for 30 min and (c, e, g, i) following reactivation by 5 mM 2-PAM incubation for 15 min.

Figure 8

Reactivation efficiency of AChE by 2-PAM incubation for 15 min after exposing (a) 25 nM and (b) 1 nM paraoxon to AChE for different periods. Inset: enlarged plot of reactivation efficiency before 10 hours exposure of paraoxon.

Scheme 1

Schematic illustration of OP-AChE formation and AChE regeneration process by reactivator.