The overall goal of this Phase II SBIR project is to develop a platform biosensor technology for rapid, multianalyte detection of biological and chemical analytes such as proteins, peptides, nucleic acids, toxins, bacteria, viruses, drugs, narcotics, explosive materials, and other molecules of interest in the biodefense and biomedical areas. We propose to build a hand-held, robust device similar to a palm computer, equipped with cartridges designed for simultaneous detection of up to 100 analytes in a microliter-size sample. The sensitivity of the envisioned sensor is at the level of single molecule detection, with a typical response time from several seconds to a few minutes. An inexpensive, disposable sensor cartridge will have an array of receptor molecules and set of reagents for a one-step detection process. In contrast to existing technologies that utilize antibodies and require multiple stages of reagent injection and washing, the envisioned biosensor system employs a novel type of receptors - molecular and aptamer beacons constructed of nucleic acids. The proposed assay is based on a single stage process that allows detection of molecular signatures in a matter of seconds. This project builds upon the experience of the investigators at BioElectroSpec in developing total internal reflection fluorescence (TIRF) biosensor systems. We have built and currently are testing a prototype of a bench-top, analytical grade, versatile TIRF biosensor instrument, which has sensitivity at the level of single molecule detection. In Phase II we will use our previous experience and the results obtained in Phase I to develop rapid multianalyte biosensors capable of detecting single target molecules. In Phase I we demonstrated the feasibility of the envisioned TIRF biosensor and developed a 'single-pixel' model of the multianalyte TIRF biosensor. Detection of single target molecules with a response time of several seconds has been demonstrated in Phase I. The TIRF biosensor platform in conjunction with molecular beacons and aptamer beacons demonstrated superior sensitivity and excellent selectivity. A new conceptual design for the multianalyte biosensor has been developed and prototyped in Phase I. Based on successful results of Phase I we propose accelerated development of TIRF biosensors devices. In Year 1 of Phase II we will develop and test a portable TIRF sensor, an array printing station, an automated fluidics, respective methods and standard operation protocols (SOP) for simultaneous detection of up to 100 bioanalytes. In parallel with BioElectroSpec's efforts, our collaborator, the Ellington lab, will adjust existing molecular and aptamer beacons to the TIRF platform and develop new aptamers for emerging biothreats. Year 2 of Phase II will complete prototyping and testing of a hand-held TIRF biosensor. Manufacturing and marketing of the portable and hand-held sensors can start in Year 2 of Phase II and Year 1 of Phase III, respectively. The envisioned TIRF biosensor technology has the potential to significantly enhance the biodefense potential of the Department of Homeland Security. However, the prospective market for the TIRF biosensor is not limited to biodefense applications. The envisioned portable and hand-held TIRF biosensors can become indispensable tools for rapid detection of a large variety of bioanalytes, such as pathogens, disease markers, toxins, drugs, and food additives. A broad customer base including DHS, DOD, NIH, FDA, EPA, USDA, clinical and research laboratories, state and local governments, and many commercial as well as non-profit organizations will benefit from the envisioned biosensor devices
