We are proposing to develop devices for the quantitative detection of botulinum neurotoxins (BoNT) at attomolar sensitivity with fully automated bench-top instrumentation. We will adopt the existing bead-based BoNT ALISSA technology, developed by the Kalkum lab at City of Hope, into a monolithic microcolumn format to be utilized by common liquid handling robots. We will also develop an automated, highly sensitive, and inexpensive wave-guide-based optical read-out system for integration in the robots. The resulting functional prototype system is expected to automatically process and analyze multiple samples simultaneously and repeatedly (up to 96 per hour). Automated calibration routines and controls for botulinum toxin and non-botulinum toxin proteases will be included. Our assay system will be tested and validated with complex biological matrices such as serum, solid organ and food extracts as well as clinical samples.