Project Grant R43OD034180

DYE-LOADED NANOPARTICLE PLATFORM FOR RAPID AND SENSITIVE VIVARIUM PATHOGEN DETECTION - PROJECT SUMMARY THIS PROPOSAL DEVELOPS KEY ASPECTS OF AN ULTRASENSITIVE PATHOGEN DETECTION SYSTEM FOR RODENT ANIMAL HEALTH SURVEILLANCE AND IS IN RESPONSE TO THE NIH FOA PAR-21-225 TO DEVELOP NOVEL TOOLS AND DEVICES FOR ANIMAL RESEARCH FACILITIES AND TO SUPPORT THE CARE OF ANIMAL MODELS. THE GOAL OF THE PROJECT IS TO DEVELOP A MICROFLUIDIC DEVICE FOR RAPID AND INEXPENSIVE PATHOGEN DETECTION DIRECTLY FROM AN ANIMAL OR FROM SOILED BEDDING. THE DEVICE WILL BE USED CAGE-SIDE AND WILL COMPLEMENT CURRENT RODENT HEALTH MONITORING PROGRAMS WHICH ARE DESIGNED TO MONITOR ROOMS OR CAGE RACKS QUARTERLY. USE OF THE DEVICE WILL ENCOURAGE RAPID OUTBREAK MITIGATION AND RESOLUTION AS WELL AS CONFIRM HEALTH STATUS FOR ANIMAL IMPORTATION. THE ADVANTAGES OF THE PLATFORM WILL BE SPECIFICITY, SIMPLICITY, SPEED, AND SENSITIVITY IN A "3RS" CONSISTENT APPROACH. THE SPECIFICITY OF THE DETECTION SYSTEM RELIES ON DNA HYBRIDIZATION OF A SYNTHETIC OLIGONUCLEOTIDE DESIGNED SPECIFICALLY FOR THE PATHOGEN OF INTEREST WITH DNA CAPTURED FROM INFECTIOUS AGENTS DIRECTLY FROM THE RODENT OR FROM BEDDING SAMPLES. THE DETECTION STEP WILL BE RAPID SINCE THE DNA HYBRIDIZATION WILL NOT RELY ON CYCLIC AMPLIFICATION OF THE DNA BUT INSTEAD WILL SHIFT THE SENSITIVITY REQUIREMENTS TO RELEASE OF DYE FROM MESOPOROUS SILICA NANOPARTICLES USING PH-DEPENDENT DYE RELEASE. IN THIS PHASE I PROPOSAL, THE KEY ASPECTS OF THE PLATFORM WILL BE OPTIMIZED AND TESTED FOR FEASIBILITY IN 4 AIMS. FIRST, THE PATHOGEN DNA PREPARATION PROTOCOL WILL BE ESTABLISHED USING A BACTERIAL AND VIRAL DNA SOURCE, AND THE LIMIT OF PATHOGEN DETECTION QUANTIFIED FOR DNA COLLECTION. SECOND, THE SYNTHETIC OLIGONUCLEOTIDE THAT SERVES IN PATHOGEN DETECTION WILL BE OPTIMIZED AND BOUND TO MAGNETIC NANOPARTICLES. THIRD, INTERNAL PORES AND EXTERNAL SURFACES OF MESOPOROUS SILICA NANOPARTICLES (MSNPS) WILL BE DIFFERENTIALLY LABELLED SO THAT THE INTERNAL SURFACES ARE SATURATED WITH REVERSIBLY BOUND DYE MOLECULES WHILE THE EXTERNAL SURFACES ARE COATED WITH DNA THAT WILL HYBRIDIZE WITH THE PATHOGEN SPECIFIC DETECTION OLIGONUCLEOTIDE. LASTLY, PROOF OF FEASIBILITY OF THE PLATFORM WILL BE TESTED BY COMBINING THE PATHOGEN DETECTION AND ULTRASENSITIVE PH-RESPONSIVE DYE RELEASE ASPECTS. THE INNOVATION OF THIS PLATFORM IS IN THE USE OF DNA SECONDARY STRUCTURE TO PROVIDE PATHOGEN SPECIFICITY, THE USE OF MSNPS DESIGNED SPECIFICALLY FOR SATURATED DYE LOADING WITH PH-SENSITIVE DYE RELEASE, AND THE USE OF DNA HYBRIDIZATION TO PHYSICALLY BIND THE SPECIFICITY AND SENSITIVITY FEATURES TOGETHER, ALLOWING ENRICHMENT OF THE DETECTION COMPLEXES WITH THE USE OF MAGNETIC NANOPARTICLES.