Rapid and Sensitive Electrochemical-Based Method for Improved Detection of Cryptosporidium parvum in Water

Cryptosporidium parvum, a waterborne pathogen, is a serious threat to the Nation¿s water supply. It does not respond to common antibiotics and resists water purification treatments. It is of interest to not only monitor C. parvum oocysts in drinking water, but also to study their fate in the environment from various sources (e.g., cattle) in lakes and streams. This is especially important today in assessing vulnerability to biological terrorism. However, current U.S. Environmental Protection Agency (EPA) methods (ICR, 1622, and 1623) for the detection of C. parvum in water are inadequate for such studies. Several aspects inhibit extensive use of the methods: expense ($400¿$750 per sample), time (a few days to several weeks), the need for highly skilled personnel, and poor sensitivity and repeatability (approximately 100 oocysts/L detection limit). Vegrandis, Inc.¿s approach promises to provide significant improvements over current EPA methods. During Phase I, a new concept will be evaluated for capturing oocysts and tested on a macroscale, first on laboratory-prepared samples, and second on resuspended pellets from the filtration and concentration portions of EPA Method 1623. Extrapolation of macroscale results to the microscale will allow for the estimation of detection limits and response time, and thus, determine feasibility for Phase II. Parameters based on macroscale studies also will be considered for optimizing a microelectrochemical method that may substitute for the current immunofluorescence assay and microscope detection of the EPA method. If Phase I is successful, Phase II will involve downsizing to the microscale, developing direct sampling from environmental water sources, and automating the process using miniaturization strategies that allow filtration to be minimized or eliminated and make onsite analysis possible. The commercial application includes developing water monitoring methodologies and devices. Several innovations that contrast greatly from EPA methods are involved. First, detection of C. parvum oocysts will utilize electrochemical detection instead of fluorescence methods that suffer from interfering background fluorescence (e.g., algae). Second, the capturing antibody (IgG) is based on recognition of oocyst coat proteins instead of carbohydrates (IgM), thereby further minimizing interferences from other organisms. Third, the capture approach holds promise for high-volume throughput capability, which may lead to the elimination of filter use. Fourth, with microscale electrochemical detection, immunoassembly sites will be located within a few microns of the detector, which will greatly improve speed and sensitivity of detection. These are patent-pending approaches originally developed by the Principal Investigator and her research team, to which Vegrandis, Inc., has an exclusive license.