Project Grant R44ES034301

DEVELOPMENT OF A PORTABLE, RAPID, EASY-TO-USE, QUANTITATIVE DIHYDROANATOXIN-A DETECTION SYSTEM - ABSTRACT ALGAE BLOOMS THROUGHOUT THE WORLD CAN PRODUCE TOXINS (HABS) THAT IMPACT ANIMAL AND HUMAN HEALTH. MONITORING PROGRAMS FOR THE TOXIN'S PRESENCE CAN HELP MITIGATE BOTH SHORT- AND LONG-TERM EXPOSURE WHICH MAY HAPPEN THROUGH DIRECT CONTACT OR INGESTING CONTAMINATED FOOD/WATER. THE PRESENCE OF DIHYDROANATOXIN-A (DHATX) WHICH BIND TO THE NICOTINIC ACETYLCHOLINE RECEPTOR IS IMMENSELY IMPORTANT BECAUSE OF ITS INCREASING PREVALENCE IN WATER BODIES, ITS EXTREME TOXICITY TO HUMANS AND ANIMALS AND NO READILY AVAILABLE METHOD FOR ITS DETECTION. FOR EXAMPLE, THE ONLY METHOD TO TEST THE PRESENCE OF DHATX DERIVED FROM TOXIN-PRODUCING CYANOBACTERIA IS INTENSIVE, TIME CONSUMING, ASTRONOMICALLY EXPENSIVE, COMPLICATED, AND REQUIRES HIGHLY EXPENSIVE EQUIPMENT THAT IS LOW THROUGHPUT MAKING ROUTINE SCREENING IMPOSSIBLE. THIS SMALL BUSINESS INNOVATIVE RESEARCH GRANT WILL ENABLE ATTOGENE TO DEVELOP A UNIQUE, ROBUST, STABLE, AND SCALABLE COMPETITIVE LATERAL FLOW SYSTEM THAT DETECTS THE ACETYLCHOLINE RECEPTOR LIGAND TOXIN, DHATX. DURING PHASE I, ATTOGENE DEVELOPED METHODS TO STABLY ASSEMBLE A FUNCTIONAL RECOMBINANTLY EXPRESSED ACETYLCHOLINE RECEPTOR (ACHR) SURROGATE TO THE HUMAN 42 AND 7 NICOTINIC ACETYLCHOLINE RECEPTOR ONTO COLLOIDAL GOLD. THE COMPLEX OF ACHR COLLOIDAL GOLD WAS EMBEDDED INTO A RELEASE MATRIX PAD AND DRIED. THESE ACHR BOUND GOLD RELEASE MATRIX PADS WERE APPLIED TO BACKING CARDS CONTAINING NITROCELLULOSE SPRAYED WITH A SPECIALLY DESIGNED NICOTINE CONJUGATE (TEST LINE) AND AN INDEPENDENT CONTROL LINE. A WICK AND SAMPLE PAD WERE APPLIED TO THE BACKED CARDS AND THEN CUT INTO STRIPS. USING THESE STRIPS, WE DEMONSTRATED THAT IN THE PRESENCE OF DHATX THE ACHR BOUND GOLD SPECIFICALLY REDUCES ITS ABILITY TO ASSOCIATE WITH THE TEST LINE, RESULTING IN A DIMINISHED SIGNAL. WE HAVE EVALUATED THIS RESPONSE TO BE BOTH CONCENTRATION DEPENDENT AND ISOMER SPECIFIC. ATTOGENE HAS ALSO DEMONSTRATED THIS TEST IS ABLE TO SPECIFICALLY DETECT ACHR LIGAND TOXINS PRESENT IN ALGAE CULTURES. WE ARE NOW PERFECTLY POSITIONED TO MOVE THIS PROJECT INTO A BIOMANUFACTURING AND PRODUCT DEVELOPMENT PROGRAM. DURING PHASE II WE WILL DEVELOP THE BIOMANUFACTURING CAPABILITY TO MAKE THIS UNIQUE SCREENING TEST SYSTEM BASED ON OUR LATERAL FLOW ARCHITECTURE IN A RELIABLE AND HIGHLY REPRODUCIBLE FASHION IN THE USA AND INTEGRATE THIS ASSAY SYSTEM INTO AN EXTREMELY WELL OPTIMIZED WORKFLOWS FOR SPECIFIC SAMPLE TYPES. THE TESTING SYSTEM WILL ENABLE COST EFFECTIVE, QUANTITATIVE, ROUTINE MONITORING FOR DHATX, NEONICOTINOID PESTICIDE RESIDUES, AND CYCLIC IMINES IN BOTH A LAB AND FIELD SETTING WHILE AT THE SAME TIME HAVING SIGNIFICANT APPLICATIONS IN THE DRUG AND BIODEFENSE INDUSTRIES. IN CONCLUSION, THE WORK OUTLINED IN THIS GRANT WILL ENABLE ATTOGENE TO MANUFACTURE A COMMERCIALLY READY, EASY TO USE, ROBUST, STABLE, TECHNOLOGICALLY INNOVATIVE SYSTEM AVAILABLE FOR MULTIPLE LARGE AND IMPORTANT ADDRESSABLE MARKETS.