A biosensor platform for rapid detection of E. coli in water

The need for rapid, sensitive and specific assays that provide a near real-time detection of bacterial indicators are of primary importance for monitoring microbiological activities and water quality. Biochemical reactions allow rapid detection, identification and characterization of bacteria using the proposed strategy/scheme. E. coli is a preferred indicator of water quality. The objective of this study was to develop a bio-sensing platform for the detection and characterization of E. coli in drinking water. The strategy developed is based on a series of fluorescence assays using several specific fluorogenic substrates and the biochemical properties of live bacteria. Numerous comparisons have shown that the proposed assays may serve as a suitable alternative to the conventional techniques for rapid detection of bacteria. The enzymatic machinery and the biochemical properties of bacterial cells are used as basis for the newly developed the bio-sensing process. Many fluorogenic substrates exist and can be used for the specific detection of such biochemical activities in bacteria. In this study, rapid assays for the detection of E. coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by the specific E. coli glucuronidase enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells in water samples. The system is based on monitoring the response of bacterial enzymatic machinery to the added specific fluorogenic substrates. The generated fluorescence signals are detected using a custom designed fluorescence based biosensor. The detection time required for the biosensor response versus the culture methods ranges from 30 to 120 minutes and 18 to 24 hours, respectively. The specificity of the MUG based biosensor platform assay was examined by pure cultures of Klebsiella, Salmonella, Enterobacter and Bacillus, and as expected, no enzymatic activities were observed. In addition, the experiment performed to identify patterns of enzymatic activities of E. coli in pure cultures resulted in no enzymatic interference when 6-Chloro-4-methylumbelliferyl β-D-glucuronide, 4-methylumbelliferyl-β- D-galactopyranoside, or L-Leucine β-naphthylamide were used. The sensitivity of the fluorescence assay in a biosensor platform was repeatedly determined to be less than 30 E. coli cells in a reaction cuvette. This system can be used independently or in conjunction with other methods as part of an array of biochemical assays in order to reliably detect E. coli in water. The data show that the fluorescence signals generated in samples using specific substrate molecules can be utilized as a platform to design a fluorescence biosensor for the detection and characterization of E. coli in environmental water samples.