Assessment of activities of mitochondrial
electron transport enzymes
is important for understanding mechanisms of metabolic diseases, but
structural organization of mitochondria and low sample availability
pose distinctive challenges for in situ functional studies. We report
the development of a tandem microfluidic respirometer that simultaneously
tracks both the reduction of mediators on the electrode and the ensuing
reduction of O2 by complex IV in the inner mitochondrial
membrane. The response time of O2 consumption to multiple
alternating potential steps is of approximately 10 s for a 150 μm-thick
sample. Steady O2 depletion shows good quantitative correlation
with the supplied electric charge, Pearson’s r = 0.994. Reduction of mediators on biocompatible gold electrodes
modified with carbon ink or fumed silica can compete with the oxidation
of mediators by mitochondria, yielding an overall respiratory activity
comparable to that upon chemical reduction by ascorbate. The dependence
of O2 consumption on mediator and mitochondrial suspension
concentrations shows that mass transport between the electrode and
mitochondria does not limit biological activity of the latter. The
mediated electrochemical approach is validated by the radiometric
measurements of simulated changes in the intrinsic mitochondrial activity
upon partial inhibition of complex IV by NaN3. This approach
enables the development of O2-independent, biomimetic electrochemical
assays narrowly targeting components of the electron transport chains
in their native environments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.