Final Report: A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water

EPA Contract Number: 68D00249
Title: A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water
Investigators: Hodko, Dalibor
Small Business: Lynntech Inc.
EPA Contact:
Phase: I
Project Period: September 1, 2000 through March 1, 2001
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2000) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

The alarming increase in the recent number of outbreaks arising from exposure to waterborne pathogens gives rise to a pressing demand for the development of reliably efficient testing protocols for water monitoring. With the recent implementation of stringent environmental regulations, the currently available methods of testing have come under scrutiny due to their inefficient filtration procedures and unreliable detection strategies. Lynntech has proposed to develop a microfluidic device capable of filtering and concentrating infectious microorganisms such as Cryptosporidium oocysts from water samples, performing amplification of target DNA material, and quantitatively determining contamination levels using conventional electrochemical methodologies. The use of electrical filtration via simultaneous AC and DC polarization of waterborne particles will eliminate the need for cartridge filters that prevent complete recovery of microorganisms. The use of PCR to amplify target DNA will allow for the analysis of more detectable material, thus improving upon on the sensitivity level of other methods. Furthermore, electrochemical detection will allow for the quantitative determination of Cryptosporidium levels in water sources without the need for delicate fluorescent labels and expensive equipment (e.g., fluorescence microscopy).

Summary/Accomplishments (Outputs/Outcomes):

The Phase I effort was able to illustrate the potential of employing electrical filtration for waterborne particle manipulation. Using flow devices machined from polymer composites, the effectiveness of AC and DC electric fields to influence particle migration was explored. In the presence of DC electric fields, charged particles were easily influenced to traverse in specific directions. As expected, the application of homogeneous DC fields had no effect on uncharged particles. However, when non-uniform AC electric fields were applied both charged and uncharged particles were forced to migrate in the direction of the highly divergent field, effectively resulting in particle accumulation and concentration.

Effective lysis of Cryptosporidium oocysts and subsequent PCR of the corresponding DNA was shown to be effective in both controlled (i.e., commercial PCR vials) and uncontrolled (i.e., molded polymer wells) environments. Beginning with minute quantities of DNA, adequate amplification was achievable, allowing for the qualitative detection of DNA using gel electrophoresis.

Electrochemical detection of amplified Cryptosporidium DNA was performed to quantitatively determine the contamination level of a corresponding water sample. In the absence of DNA, the electrochemical signal generated by ruthenium oxidation is diffusion limited and concentration dependent. However, the faradaic electrochemical current is substantially enhanced when trace amounts of DNA are present. In addition, impedance transients were shown to significantly change when DNA was present in solution, and in accord with quantitative detection, it was shown that the impedance signal is dependent upon DNA concentration.

Conclusions:

Based on the evidence of electrical filtration, it is evident that superimposed AC and DC electric fields can be applied to effectively concentrate Cryptosporidium oocysts from water samples. Proper control over the magnitude and frequency of the electrical signals allows for precise control of separation efficiency. As a result, it will be possible to incorporate electrical filtration into a detection protocol, hereby replacing cartridges and immunomagnetic labels. PCR amplification of genetic material from the microorganisms will allow for enhanced sensitivity and potentially lead to detection limits on the single organism level. Furthermore, electrochemical detection has been shown to be repeatedly sensitive to trace levels of DNA, thus offering a more reliable method of quantitative contamination level determination in comparison to immunofluorescence. In essence, the Phase I effort suggests that the all of the aforementioned approaches can be incorporated into a single microfluidic assembly capable of efficient filtration and concentration of Cryptosporidium oocysts from a water sample as well as perform extremely sensitive (and quantitative) detection of infection levels.

Supplemental Keywords:

Water monitoring, Wastewater analysis, Contamination levels, Waterborne microorganisms, Cryptosporidium detection, Electrophoretic/Dielectrophoretic separation, PCR amplification, Electrochemical detection, Microfluidic device., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, exploratory research environmental biology, Ecosystem/Assessment/Indicators, Ecosystem Protection, Chemistry, Microbiology, Monitoring/Modeling, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Ecological Effects - Human Health, Environmental Monitoring, Biology, Drinking Water, Ecological Indicators, monitoring, microbiological organisms, Giardia cysts, exposure and effects, detect, exposure, microorganisms, cryptosporidium , treatment, microbial risk management, microorganism, Giardia, water treatment, environmental monitoring data, cryptosporidium

SBIR Phase II:

A New Microfluidic System for the Determination of Cryptosporidium Oocysts in Water  | 2000 Progress Report  | Final Report