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NOTE: This document is provided for historical purposes only.
The authors ask you:
Biomonitoring is a critical tool to assess chemical exposures for a variety of potential toxicants, including pesticides, and there is a critical need to develop new approaches. This project is developing and validating non-invasive biomonitoring capability to evaluate real-time human exposures to insecticides utilizing sensitive, micro-analytical instruments. In order to evaluate occupational exposures to the insecticide chlorpyrifos (CPF), electrochemical assays are being developed to measure a key metabolite of CPF, trichloropyridinol (TCP), and cholinesterase (ChE) inhibition in saliva. Two techniques are being developed capable of measuring TCP: a bioelectrochemical magnetic immunoassay (detection limits (DL): low ppt range) and a quantum-dot (QD)-based electrochemical immunoassay (DL: low ppb range). To biomonitor ChE inhibition, an electrochemical sensor coupled with a micro-flow injection system was developed to characterize ChE enzyme activity (DL: low pM range). To interpret sensor output, efforts are also underway to quantitatively evaluate salivary clearance of OP insecticides with in vivo pharmacokinetic studies using the rat as an animal model. Data are being incorporated into an existing physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for CPF, which will be coupled with sensor platforms to facilitate quantifying dosimetry based on a saliva analysis. It is envisioned that this approach will be used to both monitor and assess risk to agricultural workers that are occupationally exposed to pesticides.
This research project is developing a non-invasive biomonitoring capability to evaluate exposure to organophosphorus (OP) insecticides utilizing a sensitive, non-invasive, micro-analytical instrument for real-time analysis of biomarkers of exposure and response in saliva. This project will create a miniaturized nanobioelectronic biosensor that is highly selective and sensitive for the target analyte(s). In addition, a physiologically based pharmacokinetic and pharmacodynamic model (PBPK/PD) for the OP insecticide chlorpyrifos will be modified to incorporate a salivary gland compartment that will be used to quantitatively predict blood chlorpyrifos concentration and saliva cholinesterase (ChE) inhibition to estimate systemic dose based on a saliva specimen. The utilization of saliva as a biomonitoring matrix, coupled to real-time quantitation and PBPK/PD modeling represents a novel approach with broad application for evaluating both occupational and environmental exposures to insecticides.
Sensors: Several types of electrochemical immunoassays capable of measuring key metabolites of the OP insecticide chlorpyrifos and cholinesterase (ChE) inhibition in saliva at relevant occupational exposure concentrations are being developed. This includes a bioelectrochemical magnetic immunosensing method which measure the metabolite trichloropyridinol (TCP) by capturing and accumulating the TCP along with horseradish peroxidase (HRP) labeled TCP as immunocomplexes associated with magnetic beads. The activity of HRP is monitored by square-wave voltammetry (SWV) by scanning the electroactive enzyme product (H2O2).
A second approach utilizes a quantum-dot (QD)-based electrochemical immunoassay to detect TCP. QD conjugated with TCP derivative, triclopyr, was used as a label in the competitive immuno-recognition event. TCP and the QD-labeled triclopyr competitively bind with the limited TCP antibodies on the magnetic beads (TCP-Ab-MB). The captured QD labels were quantified by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable-screen-printed electrode.
To biomonitor ChE inhibition, an electrochemical sensor coupled with a micro-flow injection system was developed to characterize ChE enzyme activity. The sensor is based on a carbon nanotube (CNT)-modified screen-printed carbon electrode (SPE) which is integrated into a flow cell.
Pharmacokinetic Analysis: To validate this approach there is a need to more fully understand the pharmacokinetics of chlorpyrifos excretion and ChE inhibition in saliva under various physiological conditions and dose levels to ensure that the quantitation of chlorpyrifos metabolites in saliva and ChE inhibition are an accurate predictor of internal dose. To accomplish this goal, in vivo studies are being conducted to evaluate the disposition of chlorpyrifos and ChE inhibition kinetics in blood, plasma, parotid gland, and saliva following chlorpyrifos exposure. The in vivo salivary clearance rate for chlorpyrifos metabolites in rats are underway to evaluate the impact of alterations in salivation rate based on the stimulation of cholinergic receptor agonist as well as through the modulation of ChE activity following acute chlorpyrifos exposure. It is anticipated that these data will provide some understanding of the mechanism for saliva chlorpyrifos clearance and ChE inhibition in the rat, which can be extrapolated to humans, and will be used to facilitate further development and refinement of the PBPK/PD model and the biosensor.
I.
Bioelectrochemical magnetic immunosensing protocol. (A) Liquid phase competitive
immunoreaction among TCP antibody-coated magnetic beads, TCP analyte and HRP
labeled TCP. (B) Magnetic accumulation of immunoreaction complex to magnet/glassy
carbon electrode surface, enzyme catalytic reaction, and accumulation of catalytic
product. (C) Square-wave voltammetric detection of enzyme catalytic product.
II.
(A) Typical SVW responses of increasing TCP concentration in incubation solution.
Bottom to top concentration of TCP is: 0, 0.01, 0.1, 1, 3, and 5 mg/L, respectively.
(B) Calibration curve of TCP. The concentration of TCP ranged from 5 ng/L to
40 mg/L.
III. A.
Scheme of Quantum dot (QD)-based electrochemical immunoassay of TCP. (A) Competitive
immunoreaction among TCP, trichlopry-QD and TCP antibody coated magnetic bead.
(B) Magneticseparation and collection of the TCP and triclopyr-QD associated
magnetic beads. (C) Dissolution of the captured QD with 1M HCL. (D) Electrochemical
stripping analysis of the released cadmium (Cd) ions. Detection limits: 0.02
ng/mL.
Model simulation of blood and saliva concentrations and blood CPF concentration
following a repeated (3-day) dietary exposure (12hr/day) to 0.003 mg/kg/day
(RfD). The detection limit (*) for the ELISA assay is based on the value reported
by manufacturer of the TCP RaPID AssayÒ kit (0.25 mg/L or 1E-3 mmol/L).
Time-course of TCP in blood (A) and saliva (B) following oral administration.
Data points mean ± s.d. of TCP for 4 animals/treatment/time-point. The
line is the PK model fit to the experimental data (note: saliva 12 hr sample
from 10 mg/kg group not included due to analytical error).
The major chlorpyrifos metabolite, TCP, is detectable in saliva and the kinetics parallel the TCP blood kinetics over the range evaluated. Electrochemical immunoassays have been developed to monitor for TCP, and theoretical model simulations suggest that the detection limits may be in the range to detect TCP in blood and saliva at relevant exposure levels. Results are encouraging and suggest that once fully developed and validated a field deployable immunosensor platform may enable real-time biomonitoring of organphosphorus insecticides following occupational or environmental exposure.
Supported by CDC/NIOSH Grant R01 OH008173.
The findings and conclusions in this poster are those of the author(s) and do not necessarily represent the views of the National Institute for Occupational Safety and Health. Citations to Web sites external to NIOSH do not constitute NIOSH endorsement of the sponsoring organizations or their programs or products. Furthermore, NIOSH is not responsible for the content of these Web sites.
