Homeostasis inside Single Activated Phagolysosomes: Quantitative and Selective Measurements of Submillisecond Dynamics of Reactive Oxygen and Nitrogen Species Production with a Nanoelectrochemical Sensor

Abstract

Reactive oxygen and nitrogen species (ROS/RNS) are generated by macrophages inside their phagolysosomes. This production is essential for phagocytosis of damaged cells and pathogens, i.e., protecting our organism and maintaining immune homeostasis. The ability to quantitatively and individually monitor the four primary ROS/RNS (ONOO -, H2O2, NO, and NO2 -) with sub-millisecond resolution is clearly warranted to elucidate the still unclear mechanisms of their rapid generation and to track their concentrations variations over time inside phagolysosomes, in particular, to document the origin of ROS/RNS homeostasis during phagocytosis. A novel nanowire electrode (NWE) has been specifically developed for this purpose. It consisted of wrapping a SiC NW with a mat of 3 nm platinum nanoparticles (Pt NPs) whose high electrocatalytic performances allow the characterization and individual measurements of each of the four primary ROS/RNS. This allowed for the first time a quantitative, selective and statistically robust determination of the individual amounts of ROS/RNS present in single dormant phagolysosomes. Additionally, the sub-millisecond resolution of the nanosensor allowed confirmation and measurement of the rapid ability of phagolysosomes to differentially mobilize their enzyme pools of NADPH oxidases (NOX) and inducible nitric oxide synthases (iNOS) to finely regulate their homeostasis. This reveals an essential key to immune responses and immunotherapies and rationalizes its biomolecular origin. ASSOCIATED CONTENT Supporting InformationThe Supporting Information is available free of charge at http://pubs.acs.org.Experimental details and supplement data including characterization analysis, electrochemical measurements and cell experiments (PDF)

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