Arthritis is a heterogeneous disease of the 'whole joint', whereby different combinations of proteolytic enzymes are active in different patient subpopulations leading to progressive joint degeneration. Upfront identification and stratification is critical for appropriate intervention with specific inhibitors, several of which are being evaluated in clinical trials. Nesher Technologies, Inc. (NTI) proposes a next-generation multiplex test for protease activity profiling in minute synovial fluid samples from arthritic joints, using alternating laser excitation (ALEX) single molecule fluorescence spectroscopy. This test should prove highly useful for facilitating treatment (as well as clinical trials) of arthritis with suitale protease inhibitors, or combinations thereof, personalized according to an individual patient's profile. NTI has exclusively licensed the intellectual property for ALEX, a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing and amplification steps). Target recognition molecules are tagged with different color fluorescent dyes (and quenchers). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct, amplification-free quantification via molecule counting. NTI recently achieved expansion from 2- color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for detection of proteins and nucleic acids. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization termed single molecule optofluidics, and ii) enhanced throughput using a multifoci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated diagnostic tests requiring minimal sample sizes. For this SBIR Phase I grant application we propose multiplexed detection and quantification of protease activities for MMP- 13 and ADAMTS-4 as proof-of-principle, to be expanded in Phase II to a panel of up to 10 enzymes. Our Specific Aims are: 1. MMP-13 and ADAMTS-4 substrate peptide synthesis and fluorophore/quencher conjugation; 2. Separate as well as multiplexed protease activity detection and quantification using spiked samples; 3. ALEX-based analysis of 42 archived clinical synovial fluid samples (PRoBE study design implementation). Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. Sub- sequent technology commercialization will target pharmaceuticals, diagnostics, and basic research markets.