Nanodiamond Quantum Sensors for Free Radical Detection

Summary Reactive oxygen species (ROS) are key mediators in human health but when misregulated can contribute to the progression of many diseases (e.g., cardiovascular disease, Parkinsonandapos;s disease, Alzheimerandapos;s disease, cancer, Downandapos;s syndrome, cataract, several neurological disorders, etc.). While biological effects of ROS are thought to be determined by their both spatial (subcellular localization) and temporal (duration of exposure) levels, detailed understanding of site-specific ROS intracellular concentrations and their relationship to the disease pathogenesis is currently missing. The main reason for this is the commercial unavailability of experimental tools to detect and characterize ROS at specific cellular locations with sufficient sensitivity and spatial and temporal resolution. Electron paramagnetic resonance (EPR) is considered to be the gold standard for unambiguous chemical identification of ROS by spin-trapping methods. However, the technical barriers for implementation are high, and efforts toward developing EPR-based imaging of ROS within a biological environment have proven difficult. Methods based on changes in fluorescence emission upon reactions of a dye with ROS are more accessible than EPR. While such optical methods can be readily combined with cellular imaging, the current implementations are riddled with difficulties including lack of specificity in ROS detection, toxicity concerns, artefactual ROS production by the probes themselves, signal variability due to high levels of background fluorescence and, importantly, photobleaching. This phase I proposal advances the field of ROS detection by developing a new family of nanodiamond (ND) based bright fluorescent ROS sensors that will combine the specificity and information content of EPR spin trapping with the advanced imaging capabilities enabled by optical probes without the problems of phototoxicity and photobleaching. Our pathway to commercialization assembles a team with expertise in ND processing and commercialization, development of cutting-edge ROS detection schemes in EPR and chemical synthesis, and expertise in free radical biology and oxidative stress. Phase I is aimed at demonstrating a proof-of-principle prototype ROS sensor which consists of spin-reactive molecules crafted on ND surface and correlating fluorescence and EPR data. The ROS sensor will then be tested in vitro to detect superoxide radical produced by a xanthine oxidase system and then detection and imaging ROS in RAW264.7 macrophages. Benchmarking of ND over conventional ROS optical probes will be aimed to demonstrate advantages of ND ROS sensors in extending the observation period and reducing resultsandapos; variability. Commercialization of these new ROS detection tools will enable longitudinal studies of site-specific ROS production in cells and tissue to advance the understanding of the roles of ROS and oxidative stress in the pathogenesis and progression of diseases not otherwise achievable. Moreover, the adaption ND-NV-based spin probes to ex vivo clinical diagnostics has high a commercial potential.Narrative This project demonstrates the feasibility of a new class of reactive oxygen species (ROS) spin sensors that significantly improve the reliability and resolution of measurement. The probes are based on non-toxic photostable fluorescent nanodiamond particles which enable optical detection and imaging of ROS in biological specimens with the capability for cross-examination by electron paramagnetic resonance (EPR) spectroscopy to significantly enhance the fidelity of ROS determination. These probes will contribute to understanding the mechanisms controlling ROS production at the cellular level, which can more readily elucidate the role of these reactive species in the pathogenesis and the progression of diseases such as cardiovascular diseases, diabetes, Alzheimerandapos;s disease, and cancer among others.