Research - Category III: Water Quality

DNA Aptamers-Based Detection of Atrazine in Water
(Funded 2002-2003)

Principal Investigators:
Ashok Mulchandani
Department of Chemical & Environmental Engineering
UC Riverside
(909) 787-6419
adani@engr.ucr.edu

Wilfred Chen
Department of Chemical & Environmental Engineering
UC Riverside
(909) 787-2473
wilfred@engr.ucr.edu

Executive Summary:
Agricultural producers across the country rely on s-triazines such as atrazine, as the foundation of their week control programs. Many crops, commodities and services in the U.S. could not be supplied in an economic fashion without the use of these herbicides. However, the economic benefits from herbicide usage are not achieved without potential risks to human health and the environment due to the toxicity and potency of these herbicides. Extensive pollution of the environment by atrazine is of major concern as an increasing number of studies reveal contamination of rivers and groundwaters by these herbicides (Squillance and Thurman, 1992; Domagalski and Dubrovsky, 1992). Because of their extreme toxicity, EPA has set the maximum contaminant level (MCL) for these herbicides at 3 parts per billion (ppb). A survey by the Cal/EPA's Department of Pesticide Regulation (DPR) on water samples taken from 3,564 wells in 48 of California's 58 counties revealed the detection of atrazine above regulatory limits in many areas, where the soil conditions favor movement of pesticides to ground water (Cal EPA, 1996).

Analytical tools for rapid and on-site monitoring of atrazine is necessary to ensure the safe drinking water supply and to monitor the progress of any treatment process being implemented to decontaminate sources of drinking water supply. Currently used analytical techniques, HPLC, immunoassays and tandem mass spectrometry, are inadequatte since they are time consuming, require expensive instrumentation and trained personnel and are unsuitable for rapid and on-site monitoring.

The objective of the proposed research is to develop a novel bioassay based on DNA aptamers and fluorescence polarization that will allow sensitive, selective, rapid, precise, accurate and cost-effective quantitative detection of atrazine in the environment and particularly in drinking water supply sources such as, surface- and ground-water. Such a field-portable, sensitive, selective, rapid and cost-effective analytical technique will be useful to monitor the quality of drinking water supply sources and to follow the progress of any decontamination process.

A 40-mer random DNA library will be screened for its binding affinity to the atrazine modified magnetic beads followed by recovery and amplification of the recovered ssDNA will be subjected to the ame binding and selecxtion procedure with more stringent conditions for four to five rounds. The selected ssDNA aptamer will be efvaluated for the binding affinity, selectivity and rate of binding and then applied for ht edevelopment of the bioassay. The bioassay will be based on competitive FP in which fluorescently-labeled atrazine will be compared to atrazine analyte for binding to the aptamer. The polarization of the fluorescence resulting from the binding of the labeled atrazine to the receptor will be correlated to the concentration of the atrazine analyte. Investigations will be performed to evaluate the bioassay lower detection limit, sensitivity, selectivity against other s-atrazine and pesticides in the environment, precision (deviation in replicate analyses) and accuracy (synthetic and environmental smaples will be evaluated and results compared to EPA recommended analytical protocol.)