Summary Reactive oxygen species (ROS) have a ubiquitous impact on human health. ROS are directly involved in maintenance of homoeostasis and implicated in disease when misregulated. Currently there is a gap in technologies capable of characterizing and quantifying these important analytes in physiologically relevant conditions (e.g., whole blood). This Phase II SBIR continues development of a quantum sensing technology to perform accurate and accessible kinetic measurements of ROS in minimally processed biofluids. Once developed, the new technology will improve the understanding the roles these species play in disease pathogenesis. It is also expected to enable new modalities for drug and biomarker discovery which could potentially aid in clinical diagnostics. The translational capability of quantum sensors lies in equipping biomedical researchers and clinicians with new measurement tools offering improved sensitivity, resolution, and speed compared to classical sensors. The proposed new family of robust quantum sensors is based on nitrogen- vacancy (NV) centers fabricated in nanodiamonds (NDs). NDNV sensors possess unique magneto-optical quantum properties, where the optical output of the NV depends on its spin state, while the spin state, in turn, is highly sensitive to paramagnetic species/free radicals present nearby. The value of the proposed technology is that the measurements may be reproducibly performed in a media with a high background fluorescence, like whole blood. To achieve this goal, this Phase II SBIR aims at developing a prototype instrument which will be further commercialized in Phase III of this project. We will also continue development of the ND-NV sensor and validation of its performance with measurements in translationally relevant whole blood samples. Our team has complementary expertise in ND processing and commercialization and instrumentation prototyping (Adámas), development of cutting-edge detection/validation schemes in electron paramagnetic resonance (EPR) and chemical synthesis (NCSU), and clinical research (Duke Medical Center). The outcome of this project will be a sensor with the capability to perform measurements of ROS in whole blood with minimal processing. We anticipate that the greatly reduced assay time and preserved sample integrity will increase the reliability of ROS measurement in basic biomedical research and clinical diagnostics. We aim to provide the full technology solution to end users including an instrument and consumable reagents for conducting Quantum Sensing ROS assays.