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An integrated food safety biosensor system is an important tool for preserving public health and confidence in Idaho's food and agricultural products. The primary goal of this project is to enhance the detection of food safety targets through the development and integration of emerging nanotechnologies into comprehensive electronic biosensor systems. Nanotechnology and the unique properties of nanomaterials make them especially promising for the development/ improvement of biological sensing and diagnostic technologies. As a result, four nanotechnologies will be investigated in this work to improve the detection of food-related bio-threats within the framework of previously established biosensor detection systems. The four technologies are: immunomagnetic nanoparticles, streptavidin/ streptavidin polystyrene particles, nanospring-based biosensors, and locked nucleic acid (LNA). Each technology is designed to function as one of the three components of the detection systems (sample preparation/ target capture, signal production or signal detection). <P>The proposed work consists of four objectives, one for each nanotechnology to be evaluated. The four objectives are: <OL> <LI> Evaluation of magnetic nanoparticles for sample preparation/ target capture<LI> Optimization of streptavidin nanoscaffold for enhanced signal production<LI> Development of nanospring-based electrochemical biosensors for nucleic acid signal detection<LI> Integration of affinity-enhancing LNA probes as nucleic acid signal detection components
NON-TECHNICAL SUMMARY: Idaho has built an international reputation for providing high quality food and agricultural products. However, maintaining the expected quality and safety is often challenged due to potential chemical and biological contamination. The purpose of this study is to improve the detection of foodborne pathogens and toxins by enhancing biosensor detection systems through the use of nanotechnology. <P>APPROACH: The impact of four nanotechnologies on the detection of pathogens and toxins important to food safety will be evaluated in the context of two biosensor systems. The two biosensor detection systems integrate immunomagnetic separation with electronic biosensor detection for low-cost, rapid and sensitive food pathogen detection. The first system, enzymatic bio-nanotransduction, provides a multiplexed readout of multiple targets from a single sample based on the production of nucleic acid signal sequences from specific biological recognition events. The second system allows rapid and sensitive detection of single targets on a screen printed electrode. Evaluation of magnetic nanoparticles for sample preparation/ target capture will compare immunomagnetic nanoparticles with microparticles on the basis of overall target capture and the speed of the capture event. The nanoparticles will also be investigated for target specific detection using enzyme labeled and DNA template labeled antibodies. Micro and nano magnetic particles will be used to test the ability of the detection system to discriminate live from dead pathogenic cells (Escherichia coli O157) using two time point detection separated by culturing. Streptavidin will be explored as a nanoscaffold for the production of novel enzyme or DNA template/ antibody conjugates for signal production. Magnetic streptavidin particles will be used determine the sensitivity difference between using a signaling element that produces RNA nano-signals (detected through enzyme linked fluorescence) versus a signaling element that directly produces fluorescence. This evaluation will be used to optimize an alternative detection approach for Staphylococcal enterotoxin B (SEB) that is based on the application of biotinylated antibodies and streptavidin. Dual functional complexes with recognition and signaling elements will be produced with individual streptavidin molecules or multiple streptavidin functionalized microparticles and used for detection of SEB. The novel properties of nanosprings for biosensor application will be explored and used to develop nanospring-based electrochemical biosensors for nucleic acid signal detection. Immobilization of DNA probe sequences and surface hybridization on nanosprings will be determined using fluorescence spectroscopy. This information will be used to establish an approach for enzyme linked electrochemical detection of nucleic acid signals on nanospring electrodes. In parallel work, an approach to pathogen detection based on immunomagnetic capture and asymmetric PCR will be developed to provide an alternative method for food pathogens in conjunction with nanospring-based nucleic acid detection. LNA probes will be designed and utilized as nucleic acid signal detection components. Improvements to nucleic acid detection using surface bound and functionalized LNA probes will be determined and optimized to provide the most sensitive detection of DNA targets and transcription produced RNA nano-signals. The implementation of LNA probes for electrochemical detection of nucleic acids will also be investigated.