A New Microfludic System for the Determination of Cryptosporidium Oocysts in Water

Cryptosporidium, originating from contamination of public drinking water supplies, ponds, rivers, or swimming pools, have cause large-scale and deadly outbreaks, which became a major cause of waterborne infectious diseases. Since 1991, the percent of outbreaks attributable to Cryptosporidium has doubled, and in the 1993-1994 period, 17 percent of all outbreaks were caused by Cryptosporidium. In 1996, 42 states reported 2,426 cases to the Centers for Disease Control. Such outbreaks prompted EPA to establish new regulations for monitoring raw and finished water systems. For instance, EPA's Information Collection Rule and Enhanced Surface Water Treatment Rule require that all large public water systems routinely conduct microbiological monitoring of raw and finished waters. Smaller utilities are likely to face compliance deadlines before 2004. Even though EPA's current recommended detection limit is 10 oocysts/L, the Agency likely will impose stricter requirements in the future. The currently available method for detection of Cryptosporidium oocysts, Method 1622, is very laborious, yields poor oocysts recovery, cannot be performed in the field, and needs highly trained professionals to accurately perform the analysis. This fluorescent microscopy-based method is strongly affected by nonspecific fluorescence by other organisms and chlorine compounds present in the sample. The proposed new method is based on two innovations, one which utilizes (di)electrophoretic separation, concentration, and focusing of Cryptosporidium oocysts in a microfluidic sensor, and the other using on-chip polymerase chain reaction (PCR) and electrochemical detection of DNA amplicons. The Phase I results clearly demonstrated the feasibility of electrical separation of Cryptosporidium oocysts as well as an enhanced and highly sensitive method for electrochemical detection of Cryptosporidium DNA amplicons. The results indicated that the full system development would provide competitive detection limits for in-field testing of oocysts and cysts. The Phase II project will further optimize the design parameters for separation and detection systems. A prototype microfluidic analyzer with on-chip PCR detection will be designed, built, and thoroughly tested. It is planned to perform an interlaboratory comparison of results obtained using the proposed method and the current EPA Method 1622/1623. Because of the simplicity of the design of new PCR primers and selective recognition of characteristic DNA templates, the proposed microfluidic analyzer will find a large number of applications in detection of other microorganisms in the field or on production lines, e.g., in food, chemical, environmental, or biotechnological industries. The same principles could be used for detection of cells in biological fluids; thus, the analyzer offers numerous biomedical and pharmaceutical applications.