A Strategy to Establish a Quality Assurance/Quality Control Plan for the Application of Biosensors for the Detection of E. coli in Water.

Rapid bacterial detection using biosensors is a novel approach for microbiological testing applications. Validation of such methods is an obstacle in the adoption of new bio-sensing technologies for water testing. Therefore, establishing a quality assurance and quality control (QA/QC) plan is essential to demonstrate accuracy and reliability of the biosensor method for the detection of E. coli in drinking water samples. In this study, different reagents and assay conditions including temperatures, holding time, E. coli strains and concentrations, dissolving agents, salinity and pH effects, quality of substrates of various suppliers of 4-methylumbelliferyl glucuronide (MUG), and environmental water samples were included in the QA/QC plan and used in the assay optimization and documentation. Furthermore, the procedural QA/QC for the monitoring of drinking water samples was established to validate the performance of the biosensor platform for the detection of E. coli using a culture-based standard technique. Implementing the developed QA/QC plan, the same level of precision and accuracy was achieved using both the standard and the biosensor methods. The established procedural QA/QC for the biosensor will provide a reliable tool for a near real-time monitoring of E. coli in drinking water samples to both industry and regulatory authorities.

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

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-ß-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli ß-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-ß-d-galactopyranoside (MUGal) and l-leucine ß-naphthylamide aminopeptidase (LLß-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.

Biochemical signature assay for use in a biosensor platform to detect bacteria in drinking water biofilms.

The objective of the study was to identify the enzymatic-biochemical (enz-bio) signatures of Escherichia coli and Salmonella for rapid detection of these bacteria in drinking water biofilms. The relative potency of lipophilic, glucosidic, and proteolytic activities in biofilms containing single bacterial species and mixture of different bacterial was used to identify the enz-bio signatures of Escherichia coli and Salmonella. The enz-bio signatures identified were: Lipophilic < Glucosidic < Proteolytic (for Escherichia coli); and Glucosidic = Lipophilic < Proteolytic (for Salmonella). The enz-bio assays were performed sequentially for detecting Escherichia coli and Salmonella in pure and mixed biofilm cultures formed on the coupons incubated in a batch reactor. The results obtained were substantiated by culture-based assays indicating comparable data. The enz-bio sensing method described here is a proof of principle and the results of this study provide a platform for the fabrication of a biosensor for bacterial detection in biofilms. The detection time required for the biosensor platform versus culture methods ranged from 10 to 120 min and 24 to 48 h, respectively.