Final Report: Biosensor for Field Monitoring of Pesticides in Water

EPA Contract Number: EPD06077
Title: Biosensor for Field Monitoring of Pesticides in Water
Investigators: Carter, Michael T.
Small Business: Eltron Research & Development Inc.
EPA Contact: Manager, SBIR Program
Phase: II
Project Period: May 1, 2006 through April 30, 2008
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2006) Recipients Lists
Research Category: Hazardous Waste/Remediation , SBIR - Waste , Small Business Innovation Research (SBIR)


This SBIR Phase II proposal addressed development of a field-portable, amperometric biosensor for monitoring organophosphorus (OP) and carbamate insecticides in water. The miniaturized biosensor used a modified, screen-printed microelectrode. The enzyme used to generate the amperometrically detectable species is selectively inhibited by the pesticides, but not by other common classes of agricultural chemicals, such triazine herbicides. The device is a single-use, disposable monitoring chip, because some of the active pesticides (e.g., the Ops) irreversibly inhibit enzyme activity, although carbamate inhibition is more reversible.

The purpose of this SBIR Phase II program was to develop a carbamate and organophosphorus insecticide biosensor for monitoring surface water. This device was intended to be incorporated into a hand-held analysis kit. The biosensor was based on a sol-gel modified, enzyme-immobilized microelectrode array designed to enhance sensitivity and limit of detection. The monitoring kit would consist of disposable biosensor probes, which interface to a hand-held electrochemical instrument containing provision for introduction of water sample to the biosensor surface. The proposed sensor will provide rapid, on-site field monitoring of pesticides arising from nonpoint source pollution in the form of runoff from agricultural areas. This type of device could be especially useful for cost reduction via screening, by eliminating samples that contain permissible level of pesticide prior to more expensive laboratory confirmation.

Summary/Accomplishments (Outputs/Outcomes):

Work carried out in the Phase II focused on the production of a reliable biosensor element based on the enzyme inhibition approach. The sensor consisted of a sol-gel film and conductive carbon matrix into which the enzyme (acetylcholinesterase, AChE) and redox mediator (TCNQ) were incorporated. Work performed during Phase II involved detailed, systematic studies of the biosensor components and fabrication strategies for the sensor in an effort to achieve a reproducible inhibition response to low concentrations of representative pesticides.

Reproducible sensor response proved elusive, not only for inhibition of AChE on acetylthiocholine at realistic inhibitor concentrations, but also in the simple, uninhibited chronoamperometric response of the fully functioning enzyme impregnated layer. A sufficiently reliable sensing device to warrant prototype analysis system development was not achieved during Phase II. For example, with appropriate manipulation of the sensor film formulation (i.e., adjustment of the sol-gel composition, nature and loading of conductive carbon material, loading of electron transfer mediator and enzyme), we were able to detect 25–100ppb Aldicarb (carbamate pesticide) with reasonable linearity on high-performing electrodes. However, in most cases, all electrodes from the same batch did not perform the same to within a reasonable variability (e.g., 5–10% relative standard deviation). Batches that contained some high performers also contained unresponsive or marginally or anomalously responsive sensors. Exhaustive systematic tests of the biosensor film formulation failed to uncover the source of this variability.


The fabrication of inhibition biosensor chips with incorporated conductive materials, sol-gel framework, electron transfer mediator, enzyme and substrate for the detection of low levels of pesticides was shown to be feasible, with respect to qualitative aspects, at 25–100ppb levels for Aldicarb. However, on a quantitative basis, we do not believe that it is feasible to achieve the level of repeatability required to fabricate a reliable sensor. Variability in the degree of electrochemical signal obtained in the uninhibited chip was often greater than the change in signal obtained as a result of enzyme inhibition by pesticide. This made it impossible to reliably measure the presence of the pesticide at sub-ppm concentrations or to reliably quantify pesticide concentration by its proportionality to extent of enzyme inhibition. Even when this problem was solved, the variability of sensor performance was still unfavorable for fabrication of a reliable system. While the studies undertaken in this Phase II program were systematic and fairly exhaustive, we were unable to sort out exactly what factors in the formulation of the biosensor components (sol-gel, conductive carbon, enzyme, mediator and substrate) were responsible for the variation in results that were observed. It is possible that some improvement could be obtained in the case of organophosphate pesticides, which bind more irreversibly to the active site of AChE; however, the poor results obtained with Aldicarb reduced enthusiasm for expectations of this possibility.

Supplemental Keywords:

small business, SBIR, EPA, pesticide, biosensor, field, monitoring, organophosphate, amperometric biosensor, organophosphorus insecticide, carbamate insecticide, sensor electrode, environmental monitoring, contaminated water sites, chemical air emissions, ecosystems, toxics, environmental monitoring, field monitoring, herbicides, insecticides, neurotoxic, agriculture,, RFA, Scientific Discipline, Air, Toxics, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, PESTICIDES, Environmental Chemistry, Monitoring/Modeling, Environmental Monitoring, Agroecosystems, Engineering, Chemistry, & Physics, Environmental Engineering, Pesticide Types, neurotoxic, amperometric, field monitoring, biosensing system, insecticides, agriculture, organophosphorus hydrolase, herbicides, biosensors, biosensor, organophosphate pesticides

SBIR Phase I:

Biosensor for Field Monitoring of Pesticides in Water