Project Grant R43ES036049

MULTIPLEXED NUCLEAR RECEPTOR BIOASSAY PLATFORM TO INEXPENSIVELY AND BROADLY SURVEY THE NATION'S WATER SUPPLY FOR CONTAMINATION BY ENDOCRINE DISRUPTING CHEMICALS - THIS SBIR PROJECT WILL TEST AND EXPAND A NOVEL MULTIPLEX BIOASSAY PROTOCOL TO SAFEGUARD THE NATION'S WATER SUPPLY AGAINST CHEMICAL CONTAMINANTS AT CONCENTRATIONS THAT ILLICIT A BIOLOGIC EFFECT. THE US SAFE DRINKING WATER ACT ESTABLISHES THE METHODS AND FREQUENCY OF WATER MONITORING FOR CHEMICAL CONTAMINANTS IN US DRINKING WATER. 100,000 CHEMICALS CURRENTLY ARE CONSIDERED AS POTENTIAL "CONTAMINANTS OF EMERGING CONCERN" (CECS). PRACTICALLY, THE SCALE AND COST OF MONITORING 100,000 UNIQUE CECS BY EXISTING METHODS NECESSITATES EVIDENCE-BASED PRIORITIZATION. CURRENTLY, THE EPA MANDATES PUBLIC WATER AGENCIES TO REGULARLY MONITOR THE AMOUNTS OF >60 SPECIFIC INORGANIC AND ORGANIC CHEMICALS, DISINFECTANTS AND BYPRODUCTS THROUGH THE WATER PROCUREMENT PROCESS FROM WATER SOURCING, STORAGE, TREATMENT TO TAP. ANOTHER 66 CHEMICALS CURRENTLY ARE LISTED IN THE EPA'S "CONTAMINANT CANDIDATE LIST-5 (CCL-5)" FOR LESS FREQUENT, EXPLORATORY EVALUATION. METHODS THAT ENABLE AFFORDABLE MONITORING OF ALL 100,000 SUSPECT CHEMICALS ARE DESIRED. HIGH THROUGHPUT MOLECULAR BIOASSAYS, WHICH REPORT ON THE ACTIVITY OF A PROTEIN, PROVIDE ONE SOLUTION IN WHICH THE PRESENCE OF ANY ACTIVITY-ALTERING CONTAMINANT(S) CAN BE DETECTED IN A WATER SAMPLE. NATIONAL AND INTERNATIONAL WATER MONITORING AUTHORITIES HAVE SHOWN INTEREST IN BIOASSAYS AS BROAD MONITORS OF THE WATER SUPPLY. THE MAJOR IMPEDIMENT IS THAT CURRENT COMMERCIAL BIOASSAYS COST AROUND $150 TO REPORT ON JUST ONE BIOASSAY-SPECIFIC SIGNALING PATHWAY AND MANY OF THOSE COSTLY BIOASSAYS ARE NEEDED TO SCREEN ACROSS MANY DIFFERENT CHEMICAL CLASSES. XCELLASSAY'S MAJOR INNOVATION HAS BEEN TO DEVELOP HIGHLY MULTIPLEXED PROTOCOLS TO ACCURATELY MEASURE MANY DIFFERENT 'BAR-CODED' BIOASSAYS AFFORDABLY AND SIMULTANEOUSLY ON THE SAME WATER SAMPLE ADDED TO A SINGLE WELL OF A 384-WELL DISH. IN PRACTICE, 15 OR MORE DIFFERENT BIOASSAYS COULD BE READILY SURVEYED AT A TOTAL COMMERCIAL PRICE OF JUST $30 TO MEET WATER MONITORING NEEDS. FOR THIS SBIR APPLICATION, WE WILL DEVELOP A COST-EFFECTIVE, COMPREHENSIVE BROAD BIOASSAY SURVEY OF WATER QUALITY. OUR INITIAL MOLECULAR TARGETS WILL BE PROTEINS OF THE NUCLEAR RECEPTOR (NR) CLASS BECAUSE OF THE PERVASIVE CONCERNS ABOUT THE PREVALENCE OF ENDOCRINE DISRUPTING CHEMICALS (EDCS) IN OUR SURROUNDINGS. IN PHASE 1, WE PROPOSE TO TROUBLESHOOT THE DEVELOPMENT OF THE TOOLS FOR WATER MONITORING BY: AIM 1: DEVELOPING WATER MEASUREMENT PROTOCOLS WITH OUR EXISTING PANEL OF FIVE BAR-CODED NR BIOASSAYS AIM 2: IMPLEMENTING INTO OUR INSTRUMENT DATABASE THE BAR-CODE UNMIXING PROCESSES FOR RAPIDLY AND ACCURATELY SEPARATING AN EXPANDABLE NUMBER OF SPECTRALLY DISTINCT 'GREEN' AND 'RED' FLUORESCENT PROTEIN TAGGED BIOASSAYS IF SPECIFICITY, SENSITIVITY AND REPRODUCIBILITY MILESTONES ARE MET FOR THE PILOT PHASE I STUDY, PHASE II WOULD DEVELOP A SET OF NUCLEAR AND XENOBIOTIC RECEPTOR BIOASSAYS FOR COMPREHENSIVE EDC MONITORING OF THE WATER SUPPLY. ONCE THE EDC PLATFORM IS FULLY DEVELOPED AND VALIDATED (PHASE II), THE PLUG-AND-PLAY NATURE OF THE PLATFORM WILL ALLOW EXPANSION TO BIOASSAY PANELS FOR NON-EDC CONTAMINANTS IN THE WATER SUPPLY.