Robert D. Stedtfeld
Ph.D. Candidate
2005-contd.
CV
stedtfel@msu.edu |
PCR-chip for point-of-sampling detection of multiple microbial threat agents
Diagnostic devices for point-of-use applications are in increasing demand for monitoring safety of water, air, and food. This project aims to develop a microfluidic PCR-biochip and hand-held device to simultaneously quantitatively detect and identify pathogenic microorganisms in the field. The technology has a sensitivity of 1-10 copies and high sample and assay throughput. Specificity, accuracy, and robustness of the system are obtained by targeting multiple virulence/toxin and marker genes (VMGs) simultaneously. Figure 1 depicts a small chip that could serve as the central piece in a hand-held gene analyzer.
Figure 1. Photo of the first generation microPCR chip for hand-held gene analyzer
There are several factors that are being studied to develop this device including the following:
- optimization of biological and microfluidic compatibility of substrate material, eliminating evaporation, minimizing cross talk, and uniform sample distribution. Therefore, a number of microfluidic designs are currently being tested for effortless dispensing of multiple samples into multiple assay wells;
- Development of a hand-held device for field deployment of PCR-biochip. The PCR-biochip must be integrated with a hand-held device that allows point-of-use, cost-effective, automated, rapid, simple, and rugged analysis. The current device has a low cost source of light, CCD camera, and temperature cycling, which is performed by applying voltage directly to the biochip. It will be controlled via serial ports with a PDA or with a cellular phone and Bluetooth connection.
- Design and validation of assays targeting virulence and marker genes. Primers are designed to target all sequenced alleles of a given gene. Validation includes testing each set of primers for specificity, accuracy, limits of detection, range, and quantification. Specificity and accuracy is assessed using an extensive matrices of organisms spiked into various environmental water samples.
- Treatment of reagents for long term field deployment. We are developing procedures for freeze drying PCR reagents to increase the shelf life. Users of the biochip can simply add sample directly to the biochip, and previously dried PCR reagents diffuse into solution for amplification.
- The finished device is expected to significantly improve strategies for monitoring biological quality of source waters, food, and air. It can also be used for analysis of microbial community applications such bioremediation and bioreactors.
- For further details and publications, please go to my webpage at:
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