Compact flow cytometer for rapid pathogen characterization in water based on spatial modulation technique

Abstract

Water quality monitoring is an essential priority for global health. With microorganisms a primary cause for the occurrence of infectious diseases, the concentration of harmful pathogens should be routinely monitored to maintain microbiological quality control of drinking water. Currently, testing is conducted in central labs by using 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 characterization in water. The presented approach is suitable for point-of-need testing and is able to provide test results in less than 20min. The enabling technique is termed spatially modulated emission and generates a time-dependent signal as a continuously fluorescing bio-particle traverses a predefined pattern for optical transmission. Correlating the detected signal with the known pattern achieves high discrimination of the particle signal from background noise. In conventional flow cytometry, the size of the excitation area is restricted approximately to the size of the particle. Our method allows a large excitation area to increase the total flux of fluorescence light that originates from a particle. Despite the large excitation area, the mask pattern enables a high spatial resolution which permits independent detection and characterization of near-coincident particles, with a separation (in the flow direction) that can approach the dimension of individual particles. In addition, the concept is intrinsically tolerant to background fluorescence originating from fluorescent components in solution or contaminants on the chip.

We have demonstrated pre-concentration of Giardia and Cryptosporidium which substantially reduces the analyte volume (~1000 times) while retaining most of the pathogens (>90%). For pathogen detection we have assembled and tested a working prototype of a micro-fluidic-based flow cytometer which analyzes water samples with a throughput of 50ul/min. Measurements of the sensitivity and dynamic range were conducted with calibration particles and yielded a detection limit of ~500 MEPE, which clearly meets the requirements for a wide range of bio-particle-detection applications. Tests with water-borne pathogens clearly show that this instrument can be used to reliably identify and count specifically-tagged pathogens at meaningfully low concentrations. We will show results for Giardia, Cryptosporidium, and E. coli. Incubation studies with anti-body based reagents show that for Giardia incubation times as short as 2min and analyte-to reagent-ratios as low as 1:100 are sufficient for reliable detection. We will also show that the antibody-based reagents are highly stable, with little degradation over a period of months at 37 degrees C. * Acknowledgment: the research is funded by the U.S. Army Research Office, Contract # W911NF-10-1-0479. (PM Wallace G Buchholz.).