This Small Business Innovation Research (SBIR) Phase II project proposes to develop a prototype botulinum toxin detector based on a novel ultra-sensitive Surface Plasmon Resonance (SPR) technology. The botulinum toxin will be detected by means of the specific cleavage of a peptide substrate attached to the sensor surface. The system will provide results in a fraction of the time and at a much lower cost compared to currently available methods. The feasibility of this technology was successfully demonstrated in Phase I. The research in Phase II will include assay optimization for detection of botulinum types A and B, development of a toxin extraction protocol from complex solutions, and construction of an instrument for multiplexed detection of botulinum toxins. The developed instrument will have the capability for ultrasensitive detection of Botulinum A and B (comparable to the sensitivity of the mouse LD50 assay) on a single chip. The principal commercial application of this project will be in the detection of biothreat agents. The proposed work, though initially aimed at rapid detection of botulism in individuals and in foods, will be extendable to other biothreat agents such as anthrax and mycotoxins. Additional applications are expected in drug discovery and biomedical research, and for potency testing of botulinum products in medical and cosmetic applications.