A Hybrid Pathogen Detection System

Cryptosoridium is a resilient waterborne protozoan pathogen that caused gastrointestinal disease and is one of five major causes for 5 million deaths each year worldwide. Recently, a Cryptosoridium outbreak has sickened more than 1,700 people, mostly children and teenagers in New York. Cryptosoridium oocysts are of particular interest in the water industry because the infectious dose is low (1to 132 oocysts) and it does not respond to common drug treatments. Self-contained microelectrochemical assays are desirable because precise detection can be performed down to a single oocyst on colored and turbid samples. In addition, electrochemical detection, that depend only on loss and gain of electrons, immediately adjacent to surface-bound assay components provide rapid signals even for very low analyte concentrations based on short transport distances for reporter molecules. In Phase I, we successfully demonstrated our chip-based self-contained microelectrochemical hybrid assay (SMEHA) that involved immunoassay followed by DNA-hybridization assay to confirm viability of the oocysts. Both assays which took ~90 min to complete were demonstrated in an 8 x 3 array of 50-µm diameter cavities. The ability to detect live C. parvum oocysts down to 5 oocysts/10 L of pelleted surface water samples in less than 90 min were proven in Phase I of this project. This Small Business Innovations Research Phase II project proposes to develop disposable SMEHA cartridges for the quantification and viability assessment of water borne pathogens to meet the escalating need for fast warming of pathogen outbreaks and possible dispersal of bioterrorism agents. Although the disposable SMEHA cartridges can be specified for nearly any pathogen of interest, this Phase II project will focus on the detection of Cryptosoridium oocysts and Giardia cysts. Both protozoans seriously threaten the nation¿s water supply because they resist ordinary water treatment processes and do not respond to common antibiotics. Because of the speed and specificity of our proposed device, investigators will be able to act quickly to suspected Cryptosoridium or Giardia contamination. Both the source and the path of contamination flow can be efficiently traced. The development of the disposable SMHA cartridge and reagent kits will advance the state-of-the-art over existing EPA methods 1622 and 1623 by providing a previously unavailable combination of speed, sensitivity, affordability, ease-of-use, and test of viability of waterborne pathogens.