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Cytochrome C Oxidase Deficiency (COXD) is a disorder caused by the mutation in genes encoding for the cytochrome c oxidase enzyme (COX), 1 a mitochondrial multimeric enzyme which acts as the terminal electron acceptor in the electron transport chain. 2 COXD causes serious health issues in affected individuals, including severe muscle weakness, brain damage and disorders in organs such as heart, liver and kidneys. 1 Diagnosis is mostly performed by muscle biopsy, 3,4 which is invasive and time-consuming. Finding new effective and early diagnostic strategies is therefore crucial to minimize symptoms and long-term disabilities. This research proposes the use of scanning electrochemical microscopy (SECM) as a promising method to quantify COX activity in normal and COXD fibroblasts as cell models. We explore the interaction of the redox probe N,N,N’,N’-tetramethyl- p -phenylene diamine (TMPD) with COX as an indicator of enzymatic activity. 5,6 A platinum ultramicroelectrode was used to monitor electrochemical currents at different scan speeds, which were later used to determine an apparent heterogeneous rate constant ( k 0 ) for TMPD regeneration by each cell line using numerical modelling. A significant difference in k 0 values (p < 0.1) was observed when comparing normal and COXD fibroblasts. The further treatment of normal fibroblasts with a COX inhibitor, NaN 3 , 7 induced significant decreases in k 0 (p < 0.01). The COX activity results determined using SECM were in good agreement with a standard spectrophotometric assay. Taken together, our findings confirm SECM as an effective and rapid method to detect COXD in living tissues and provides a foundation for the development of new assays to diagnose COXD in infants. References M. Brischigliaro and M. Zeviani, Biochimica et Biophysica Acta (BBA) - Bioenergetics , 1862 , 148335 (2021). A. Timón-Gómez et al., Semin Cell Dev Biol , 76 , 163–178 (2018). M. L. Simard, A. Mourier, L. C. Greaves, R. W. Taylor, and J. B. Stewart, Journal of Pathology , 245 , 311–323 (2018). S. B. Wortmann, J. A. Mayr, J. M. Nuoffer, H. Prokisch, and W. Sperl, Neuropediatrics , 48 , 309–314 (2017). S. Kuss, E. E. L. Tanner, M. Ordovas-Montanes, and R. G. Compton, Chem Sci , 8 , 7682–7688 (2017). S. Thind et al., Proc Natl Acad Sci U S A (2023). In press. M. J. Fei et al., Acta Crystallogr D Biol Crystallogr , 56 , 529–535 (2000).
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