Development of Nano-Structured Biosensors for Rapid Detection of Disease-Causing Agents in Food and Water

NON-TECHNICAL SUMMARY: Conventional detection techniques are time-consuming and laborious. Real-time field-based detection of pathogenic contaminants provides immediate interactive information about the sample being tested, enabling food and water facility administrators to take corrective measures (recall, disinfection of production environment, alternative supplies, etc.) before the product is released for consumption, thus protecting the public from disease-causing organisms with serious chronic impacts on the population. This umbrella project proposes to develop novel field-based nano-structured biosensors for the purpose of preventing, protecting, and minimizing risks from any intentional or inadvertent contamination of disease-causing agents in food sources, food products, and water supply. The project is at the heart of the mission of the Michigan Agricultural Experiment Station, which is to generate knowledge through strategic research to enhance agriculture, natural resources, families, and communities in Michigan. Rapid detection of pathogens has potential for minimizing these deadly organisms from being passed on up the food chain and preventing their transfer from the source to the table. Beneficiaries of the technology are the consumers, food industries, farm animal industries, and tourism (for water-based tourist attractions). Direct benefits to Michigan and the U.S. include a safer food supply, cleaner water system, a healthier population, and more energetic work force. Such benefits will translate to a better society, economy, and environment.
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APPROACH: Techniques will be developed to prepare (synthesize and fabricate) polyaniline, polypyrrole, nanoporous silicon, and other emerging nanomaterials (NM) for use as transducers. The synthesis will follow techniques that have been developed in the Biosensors Lab and other appropriate methods from the literature. The procedures will be modified to prepare polyaniline nanocomposites incorporating carbon nanotubes (single-walled and multi-walled), magnetic nanoparticles, and other emerging materials. To confirm successful synthesis, scanning electron microscopy and transmission electron microscopy imaging will be used to visualize the NM structures. Various sensing elements will be evaluated, such as antibodies, DNA probes, aptamers, and whole cells, to determine the appropriate sensing technique with respect to the target and transducer. Monoclonal and polyclonal antibodies will be purchased from appropriate vendors and evaluated for their specificity. DNA probe of various base-pair lengths will be synthesized at the MSU Genomics Facility and evaluated for target hybridization. Aptamers will be synthesized using the SELEX method. Whole cells will be tested as appropriate in the detection architecture based on previous works. The NM will be conjugated with the antibodies, DNA probes, or aptamers according to the protocols developed in the Biosensors Lab as well as other methods to be identified in the literatures. For the direct-charge-transfer biosensor design using lateral flow, application, capture, and absorption membrane pads will be put together according to the existing design. For the nanoporous silicon (PS) structure, electrochemical anodization will be used under varying etchant composition, elapsed time, and forcing function. Probe DNA (pDNA) will be immobilized on the PS substrate. After electrochemically immobilizing pDNA, target DNA (tDNA) will be extracted from the target bacteria and hybridized with the pDNA at different hybridizations times such as Escherichia coli O157:H7, Salmonella ssp., Bacillus cereus, and Helicobacter pylori. Characterized strains of these bacteria will be grown in appropriate media dn serially diluted to obtain populations of 100-108 CFU/ml. One hundred microliters of these cultures will be placed on the biosensor. A dose-response curve showing cell concentration and signal output will be plotted. Fresh produce recently implicated in foodborne disease outbreaks will be purchased from various food outlets. Water samples will be collected from various drinking water sites. These food products and water samples will be used for testing the biosensor performance in food and water matrices. Sample preparation will follow published standard procedures. The number of false positive and false negative results from the biosensors will be monitored to generate a statistics on these performance criteria.