Reactive oxygen species (ROS) are key mediators in human health but when misregulated can contribute to theprogression of many diseases (e.g., cardiovascular disease, Parkinson's disease, Alzheimer's disease, cancer,Down's syndrome, cataract, several neurological disorders, etc.). While biological effects of ROS are thought tobe determined by their both spatial (subcellular localization) and temporal (duration of exposure) levels, detailedunderstanding of site-specific ROS intracellular concentrations and their relationship to the disease pathogenesisis currently missing. The main reason for this is the commercial unavailability of experimental tools to detect andcharacterize 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 chemicalidentification of ROS by spin-trapping methods. However, the technical barriers for implementation are high, andefforts 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 accessiblethan EPR. While such optical methods can be readily combined with cellular imaging, the currentimplementations 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 backgroundfluorescence and, importantly, photobleaching. This phase I proposal advances the field of ROS detection bydeveloping a new family of nanodiamond (ND) based bright fluorescent ROS sensors that will combine thespecificity and information content of EPR spin trapping with the advanced imaging capabilities enabled byoptical probes without the problems of phototoxicity and photobleaching. Our pathway to commercializationassembles a team with expertise in ND processing and commercialization, development of cutting-edge ROSdetection 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-reactivemolecules crafted on ND surface and correlating fluorescence and EPR data. The ROS sensor will then betested in vitro to detect superoxide radical produced by a xanthine oxidase system and then detection andimaging ROS in RAW264.7 macrophages. Benchmarking of ND over conventional ROS optical probes will beaimed to demonstrate advantages of ND ROS sensors in extending the observation period and reducing results'variability. Commercialization of these new ROS detection tools will enable longitudinal studies of site-specificROS production in cells and tissue to advance the understanding of the roles of ROS and oxidative stress in thepathogenesis and progression of diseases not otherwise achievable. Moreover, the adaption ND-NV-based spinprobes to ex vivo clinical diagnostics has high a commercial potential.
Public Health Relevance Statement: 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,
Alzheimer's disease, and cancer among others.
Project Terms: Affect ; Alzheimer's Disease ; AD dementia ; Alzheimer ; Alzheimer Type Dementia ; Alzheimer disease ; Alzheimer sclerosis ; Alzheimer syndrome ; Alzheimer's ; Alzheimer's disease dementia ; Alzheimers Dementia ; Alzheimers disease ; Primary Senile Degenerative Dementia ; dementia of the Alzheimer type ; primary degenerative dementia ; senile dementia of the Alzheimer type ; Biology ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cardiovascular Diseases ; cardiovascular disorder ; Cells ; Cell Body ; chemical synthesis ; Chemistry ; Diabetes Mellitus ; diabetes ; Disease ; Disorder ; DNA ; Deoxyribonucleic Acid ; Down Syndrome ; Down's Syndrome ; Downs Syndrome ; Langdon Down syndrome ; Mongolism ; Trisomy 21 ; chromosome 21 trisomy syndrome ; congenital acromicria syndrome ; morbus Down ; pseudohypertrophic progressive muscular dystrophy ; trisomy 21 syndrome ; Dyes ; Coloring Agents ; Electron Spin Resonance Spectroscopy ; EPR spectroscopy ; ESR Spectroscopy ; Electron Paramagnetic Resonance ; Electron Spin Resonance ; Paramagnetic Resonance ; electron paramagnetic resonance spectroscopy ; Environment ; Exhibits ; Family ; Fluorescence ; Free Radicals ; Gold ; Health ; Human ; Modern Man ; Hydroxylamine ; In Vitro ; Kinetics ; Ligands ; Lipids ; Longitudinal Studies ; long-term study ; longitudinal outcome studies ; longterm study ; macrophage ; MÏ ; Methods ; Mitochondria ; mitochondrial ; Molecular Probes ; nervous system disorder ; Nervous System Diseases ; Neurologic Disorders ; Neurological Disorders ; neurological disease ; Nitrogen ; Optics ; optical ; Parkinson Disease ; Paralysis Agitans ; Parkinson ; Parkinson's disease ; Parkinsons disease ; Primary Parkinsonism ; Production ; Proteins ; Publications ; Scientific Publication ; Research ; Research Personnel ; Investigators ; Researchers ; RNA ; Non-Polyadenylated RNA ; RNA Gene Products ; Ribonucleic Acid ; Role ; social role ; Salvelinus ; Chars ; Signal Pathway ; Signal Transduction ; Cell Communication and Signaling ; Cell Signaling ; Intracellular Communication and Signaling ; Signal Transduction Systems ; Signaling ; biological signal transduction ; Specificity ; Superoxides ; Superoxide Anion ; Superoxide Radical ; Tissues ; Body Tissues ; Xanthine Oxidase ; Hypoxanthine Dehydrogenase ; Hypoxanthine Oxidase ; Hypoxanthine-Xanthine Oxidase ; Purine-Xanthine Oxidase ; Diamond ; Measures ; Cataract ; cataractogenesis ; cataractous lenses ; Reactive Oxygen Species ; Active Oxygen ; Oxygen Radicals ; Pro-Oxidants ; base ; density ; Label ; sensor ; improved ; Site ; Surface ; Phase ; Biological ; Chemicals ; Measurement ; Oxidative Stress ; Disease Progression ; Spin Trapping ; tool ; Research Specimen ; Specimen ; Protocol ; Protocols documentation ; Reaction ; Source ; System ; Location ; Best Practice Analysis ; Benchmarking ; magnetic ; Magnetism ; adduct ; biocompatibility ; biomaterial compatibility ; chemical stability ; particle ; Toxicities ; Toxic effect ; novel ; novel technologies ; new technology ; Pathogenesis ; Reporting ; Modeling ; Property ; Photobleaching ; Mediator ; Mediator of Activation ; Mediator of activation protein ; Data ; Detection ; Reproducibility ; Resolution ; Optical Methods ; Scheme ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Development ; developmental ; Phototoxicity ; cellular imaging ; cell imaging ; Image ; imaging ; Pathway interactions ; pathway ; design ; designing ; quantum ; Outcome ; Coupled ; prototype ; commercialization ; oxidative damage ; oxidative injury ; in vitro testing ; temporal measurement ; temporal resolution ; time measurement ; clinical diagnostics ; imaging capabilities ; nanodiamond ; implementation barriers ; implementation challenges ;
