On-the-flow pathogen detection in water

Abstract

Water-quality monitoring is an essential priority for global health. It is estimated that worldwide more than 5000 people die daily from drinking contaminated water. With microorganisms a primary cause for the occurrence of infectious diseases, the concentrations of harmful bacterial cells should be routinely monitored to maintain microbiological quality control of drinking water. Because of the difficulty and cost of directly measuring all microbial pathogens in water samples, organisms like E.coli, Giardia and Cryptosporiduim that indicate the presence of sewage and fecal contamination have been targeted for measurement. Bacterial quantitation is currently performed by labs that primarily use plate-culture assay techniques which can take up to 24 hours to produce test result. In order to achieve more timely assessment of water quality, PARC is developing a compact and robust platform for rapid pathogen identification and quantitation in water. The anticipated microfluidic system should provide sample concentration, on-chip sample preparation, and uses a micro-fluidic based flow cytometer for detection. The suggested approach is suitable for point-of-need testing and in-line water monitoring. The technique enabling the on-the-flow detection of the pathogens is termed spatially modulated fluorescence detection and delivers high signal-to-noise discrimination without precision optics. Relative movement between analyte and a predefined patterned environment generates a time-dependent signal, and correlating the detected signal with the known pattern achieves high discrimination of the particle signal from background noise. The detection technique has been evaluated with measurements of absolute CD4+ and percentage CD4 counts in human blood. Our results are in excellent agreement with cell counts of the same samples performed with a commercial instrument (BD FACSCount). We have assembled and tested a working prototype of a micro-fluidic based flow cytometer. The detection subsystem includes a basic pin photodiode rather than a PMT or APD. The prototype was assembled with off-the-shelf components (total cost of all active parts <$350). Measurements of the sensitivity and dynamic range were conducted with calibration particles and yielded a detection limit of ~200 MEPE, which meets the needs for a wide range of bio-particle-detection applications. By using an avalanche rather than a pin photodiode the sensitivity has been further improved to ~50-100 MEPE which is promising for detection of very dim objects, e.g., specifically tagged E-coli. First measurements with water-borne pathogens clearly show that this instrument can be used to reliably identify and count specifically-tagged pathogens in water. Acknowledgment: This project is partly funded by ARO.