Impact/Purpose:

Develop rapid, practical and sensitive biosensor, immunoassay, chemiluminescence and HPLC methods to detect caffeine, urobilin, and coprostanol, using them as non-microbial indicators of human fecal contamination in water.

Description:

Limitations exist in applying the conventional microbial methods to the detection of human fecal contamination in water. Recently, there has been an increased interest in developing supplemental and/or alternate indicators of human contamination to better define water quality and predict the risk of human disease outbreaks. Non-microbial indicators such as caffeine, urobilin, and coprostanol have been proposed and have shown great potential as alternate markers of human contamination. The employment of these organic compounds has been hampered by the absence of a rapid, sensitive, and inexpensive method to detect their presence in water. This may be rectified by the application of molecular imprinting and biosensor technologies. Molecularly-imprinted polymers are used in molecular recognition, separation and sensor technology to detect specific molecules. Electrochemical, piezoelectric, and spectroscopic methods using this method can potentially improve sensitivity, cost and time of analysis. Conventional methods such as immunoassays, chemiluminescence, fluorescence, and high performance liquid chormatography (HPLC) were developed. Developed methods were tested in the field and compared with the standard microbial procedures.

The developed methods will allow immediate assessment of water treatment deficiencies so remedial measures can be put in place. Moreover, the biosensor methods developed can potentially be made portable and "real time," thereby allowing greater flexibility in assessing fecal contamination.

Record Details:

Record Type:PROJECT

Start Date:01/01/1999

Completion Date:09/30/2002

Record ID: 15800

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Project Information:

Progress :Completed. A pilot study, in collaboration with the US Geological Survey, was initiated to determine the suitability of the HPLC with photodiode array (PDA) detection method to simultaneously detect caffeine and urobilin. Sixty-four water samples were collected in Ohio in June-August 2000. Solid-phased extraction (SPE) was performed to preconcentrate caffeine and urobilin using 100-200 ml of each water samples followed by high-performance liquid chromatography (HPLC) with photodiode array and fluorescence detection methods to simultaneously detect caffeine and urobilin. Enterococci, total coliform (TC), and Escherichia coli (E. coli) were measured in each sample using standard procedures. Caffeine was detected in 33% (21/64) of samples and urobilin in 5% (3/64) of samples. Caffeine level and the presence of E. coli showed strong correlation (r=0.98), whereas enterococci and total coliform levels were poorly correlated with caffeine (r=0.22 and -0.01, respectively).

Follow-up water samples (105 samples) were collected on May-August 2001 to verify caffeine and E. coli correlation. Three sites were selected based on earlier findings. Water samples were collected at three different points on each site weekly. About 94% of the samples were positive for caffeine with levels ranging from 6.32 to 101.52 ng/100 ml water sample.

Three highly sensitive, selective, and reagent-free transduction methods to detect caffeine in source and drinking water samples have been developed. In method one, caffeine was detected by fluorescence competitive inhibition. Anti-caffeine antibodies in physical contact with caffeine labeled with coumarin cause a dramatic drop in coumarin fluorescence intensity, apparently due to a self-quenching effect. Unlabelled caffeine in water samples competitively inhibit caffeine-coumarin conjugate, thereby influencing fluorescence intensity. Method two uses a high molecular weight polyvinyl chloride with a chloroparaffin plasticizer as a thin polymeric phase on the attenuated total reflectance-Fourier transformation infrared (ATR-FTIR) silicon plate surface to capture caffeine in solution. Trace amounts of caffeine (10-6 M) on the plate surface was detected by their infrared vibrational modes. Method three involves a continuous liquid-phased piezoelectric biosensor using AT-cut crystals with a fundamental resonant frequency of 10 MHz. Protein A-anti-caffeine antibody immobilized on a gold surface was used to capture caffeine in solution. The resonant frequency was followed continuously using a piezoelectric detector and a decrease in resonant frequency was observed when caffeine binds. At least 10-7 M of caffeine was detected depending on surface functionalization.

Relevance :Doubts on the suitability of the traditional total and fecal coliform indicators of human fecal pollution has stimulated research into alternative markers of human contamination. Three low molecular weight organic compounds, coprostanol, urobilin, and caffeine, have been proposed as potential markers of human contamination. The development of methods to detect these novel indicators may provide more rapid, specific and cost-effective monitoring techniques to better assess water quality and predict the risk of transmission of human waterborne diseases. These methods would be of great value in assessing the sanitary quality of recreational waters and in aquacultural practices. These methods could potentially be made portable. Chemical-based water qualtiy monitoring might represent a less expensive, more robust methodology that could be automated and useful for field use or real-time, in-line monitoring of water, providing fast, presumptive results. Continued validation of this monitoring technology with the standard bacteriological methods should result in the broad adoption of this methodology. Such a robust methodology would be used by State and local public health authorities in continual monitoring programs assessing water quality.

These tasks meet the need of the Agency's strategic goal of clean and safe water, ORD's High-Priority Research Areas - "Improve Health Risk Assessment," ORD's strategic goal of "Discovery and innovation revolutionize environmental decision-making," and NERL's long term goal to provide improved exposure assessment methods and models and information needed for guidance on exposure evaluation.

M/DBP Research Plan Relationship: Topic area Ex.M.13a.

Clients :Office of Ground Water and Drinking Water (Dr. Paul Berger)

Research Component :M/DBP (MICROBIAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :EX.M.13a

Project type :ORD-DW Plan

ID Code :5793

Project type :OMIS