Since the intentional distribution of Bacillus anthracis spores through the US postal system in 2001, the prospect of bioterrorism in food, water, and agriculture has identified critical needs in prevention, protection, and mitigation for homeland security. Towards that end, we recently demonstrated that our DNA-based biosensors, which utilize gold nanoparticles for signal amplification and magnetic nanoparticles for easy and clean separation from samples, can detect the non-PCR amplified genomic DNA of the bacterial foodborne pathogen Salmonella Enteritidis within basic and acidic liquid food matrices (2% milk and 100% orange juice)-something that had not been demonstrated before. These findings strongly suggest that our DNA-biosensor detection system is one step closer toward providing a new, simple, fast, reliable, economical, and field-ready technology for the detection of foodborne pathogens. Therefore, the main goal of this project is to improve upon the sample preparation and DNA-biosensor sensitivity and fully validate its detection capabilities to achieve a system that can establish high sensitive detection within hours. In particular, the specific objectives of this proposal are to: 1) improve and reduce the amount of time it takes to isolate the pathogenic genomic DNA from food matrix samples; 2) improve upon the hybridization steps involved in the DNA-biosensor scheme to reduce the overall time to detection; 3) test the ability of the DNA-biosensor to detect nonPCR amplified pathogenic genomic DNA target concentrations in the range of 7-50 ng/mL, similar to those achieved with a PCR method; 4) establish the statistical variability of the DNA-biosensor detection sensibility; and 5) validate the ability of the DNA-biosensor to detect non-PCR amplified pathogenic genomic DNA from solid food matrices, such as egg yolks, yogurt, spinach, and ground beef. As our previous work using this DNA-biosensor has been very promising, we believe that the work proposed here will be instrumental in helping us move towards transitioning this detection method towards prototype and commercialization. The work proposed in this study will serve to fully validate our DNAbiosensor for the detection of foodborne pathogens, using a true field-based and resource-limited setting detection system.