A new quantitative detection methodology for nucleic acid targets based on hybridization of single-stranded target with immobilized single-stranded probe will provide a visual signal for very low concentrations of multiple targets in seconds to minutes. Sensitive detection of hybridization will be achieved by relying on fluorescence from hybrids. Using a new laser developed in our labs, a novel approach based on Total Internal Reflection Fluorescence Microscopy will be examined to provide a capability that approaches single molecule detection. An electrokinetically controlled microfluidic chip will dispense samples of nanoliter volumes to a detection element consisting of a linear strip of immobilized probe nucleic acid on one wall of a microfluidics channel. Electroosmotic flow control in combination with surface charge on the flow channel and the geometry of the flow channel provides for: a simultaneous adjustment of shearing effects at the immobilized probes; electrophoretic effects that move the target; and Joule heating that controls the hybridization reaction. The simultaneous adjustment of these three parameters provides for outstanding ability to dynamically control selectivity, and to accelerate hybridization kinetics by moving from diffusion control to convective control of target delivery to the immobilized strip of probe molecules. Quantitative analysis can then be based on a new approach that obviates the reliance on fluorescence intensity. Instead, measurement is based on determination of the length of occupancy of the linear strip of probe molecules. This would substantially simplify instrumental and calibration requirements while maintaining the advantages of speed for higher sample throughput in an on-line configuration.
Expected Impact of Project Outcomes on Food Safety in Ontario:
Two key challenges exist that must be overcome prior to the implementation of practical biosensor and biochip technologies for testing of food samples for nucleic acids that are indicative of pathogenic contaminants. These are: (1) the provision of stable, reproducible and self-contained detection technologies to achieve the desired device performance, in a form that is commensurate with large scale manufacture of such devices, and, (2) to rapidly process on-line statistically representative samples (e.g. liters of fluid or grams of tissue) and deliver isolated target molecules to the sensing device in a small volume aliquot. In an earlier project funded by OMAFRA, a methodology was developed for rapidly concentrating target nucleic acids from bacteria such as E. coli from food and water samples. This new project will address the key challenge of creation of new technology for near real-time automated and high throughput analysis of pathogens in foodstuffs for the Ontario food industry. This has generated significant interest by diagnostic test and service providers in Ontario. The new technology developed in this research will be transferred for commercialization. The research is jointly supported by Safeguard Biosystems, an Ontario company that has diagnostics as its primary market niche and is developing a variety of tests for animal pathogens. Safeguard brings international partners to Ontario, including the Veterinary Laboratory Agency of the United Kingdom and the Argonne National Laboratories in the United States.
<P> For more information, please visit the <a href="http://www.omafra.gov.on.ca/english/research/foodsafety/index.html" target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.