Improving the Safety and Shelf Life of Agricultural Commodities (FY2009)

NON-TECHNICAL SUMMARY: The primary goal of this project is to develop electronic biosensors that can quickly detect the presence of microbial pathogens and toxins in agricultural, food and environmental systems. The early, fast and accurate detection of biological pathogens is an important element for ensuring the safety and shelf life of agricultural commodities resulting from both accidental and intentional food poisoning. The work will play a major role in preventing bioterrorism in our food supply. In two earlier USDA projects, we have developed a biosensor system that can detect multiple organisms in a multiplex system allowing simultaneous screening for several organisms and toxins at one time. We propose to further the development of these electronic biosensor systems through the development and application of new nanotechnology and microelectronics toward several agricultural, food, environmental and health systems. The proposed project will support our goal through the completion of four objectives: development of a real-time food-sampling sensor characterization of pathogen detection assays utilizing silica nanosprings and locked nucleic acids (LNAs); biofunctionalization of nanomaterials for pathogen detection in food products; integrated nanosensors for ultra-sensitive, multiplex detection of bacterial toxins (shiga-toxin) in meat products; and improving shelf-life of meat through pre-harvest regulation of post-harvest fatty acid oxidation. The four projects are unique, yet complementary, and cover a broad range of food pathogen detection methodologies. The research teams bring a breath of experience in electronic detection, nanomaterials development and characterization, and biological sciences. The integrated teams will establish new protocols for pathogen detection that dovetail with advanced food safety nanotechnology in sensing.

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APPROACH: <BR> Project 1: Real Time Food Sampling Sensors <BR> Objective 1: Measure and model the electrical characteristics of a nanospring biosensor<UL> <LI> Fabrication and modification of the multi-electrode/terminal devices <LI>Electrochemical modeling of device electrical properties </ul> Objective 2: Bio-functionalization and characterization of a nanospring-based biosensor<UL> <LI> Immobilization of avidin on nanospring mat -Characterize the bio-functionalized surface <LI>Measure Staphylococcal enterotoxin B detection by enzyme linked immunoassay on the nanosprings <LI>Measure Staphylococcal enterotoxin B detection with nanospring-based biosensor </ul>Project 2: Enhancement of Biosensor Systems through Biofunctionalization of Nanomaterials <BR> Objective 1: Development of fluorescent nanoparticles for optical biosensor platforms <ul><LI> Optimize synthesis of fluorescent silica nanoparticles<LI>Characterize properties of silica nanoparticles -Characterize the conjugation of biological molecules to the nanoparticles <LI>Use the nanoparticles for Staphlococcal enterotoxin B (SEB) detection<LI>Integrate nanoparticles in detection using a Lateral Flow device </ul> <BR> Objective 2: Development and application of Locked Nucleic Acid (LNA) building blocks for nucleic acid specific detection of food pathogens<UL> <LI>Synthesize LNA probe strands and signaling strands <LI>Determine thermal denaturation temperatures (Tm) and fluorescence properties of nucleic acid complexes<LI>Evaluate surface hybridization properties using LNA probes<LI>Develop protocol for detection of PCR amplicons from food pathogens using LNA <LI>Use anthraquinone labeled LNA for electrochemical detection of gene sequences </ul> Project 3: Nanomaterials for Gene Knock-down in Muscle: Improving Shelf-life of Meat through Pre-harvest Regulation of Post-harvest Fatty Acid Oxidation <BR> Objective 1: Synthesis and physicochemical characterization of functionalized gold nanoparticles for targeted delivery of gene knockdown agents <UL> <LI> Gold nanoparticles of specific sizes (5-100 nm) will be conjugated to antisense oligonucleotides with selected backbone chemistries and to muscle-binding peptides. </ul> Objective 2: Evaluate specificity and gene knockdown in cell models <UL> <LI> Multifunctionalized gold nanoplatforms with antisense oligonucleotides and muscle-binding peptide (obj. 1) will be targeted to muscle cells and effect knockdown of targeted genes (Nox, Nos, TNF-á and Hsp-90). </ul>Project 4: Nanoelectronic Biosensors for Shiga Toxin Detection <BR> Objective 1: Fabrication and modification of nanowire devices <UL> <LI> Fabrication and characterization of nanowire devices <LI> Modification of sensing surface </UL> Objective 2: Molecular signal transduction and electronic detection of Shiga toxins<UL> <LI> Electro-spin fabrication and nano-fiber functionalization<LI> Molecular signal transduction;</ul> Task 3: Bio-conjugation of Fab fragment and streptavidin <UL> <LI> Electronic detection of Shiga toxins </ul>Objective 3: Integration of micro- and nano-electronics <ul><LI> Design and fabrication of microelectronics <LI>Micro- and nano-electronic integration and packaging </ul>Objective 4: Evaluation of electronic detection method<UL> <LI> Compare detection sensitivity with existing methods<LI> Compare detection specificity with existing methods