A dendrimer-based platform for simultaneous dual fluorescence imaging of hydrogen peroxide and pH gradients produced in living cells

We present a modular dendrimer-based platform for simultaneous dual fluorescence imaging of hydrogen peroxide (H2O2) and pH gradients produced in living cells. Acetyl-capped G5 PAMAM dendrimers functionalized with boronate-caged Peroxyfluor-1 (PF1) fluorophores for H2O2 detection and semi-naphthorhodafluor (SNARF2) dyes for pH sensing provide a single probe system that is capable of sensing multiple analytes at a time by multicolor fluorescence imaging. Spectroscopic measurements of the doubly-labeled dendrimer conjugates establish their ability to simultaneously monitor changes in both H2O2 and pH using different excitation/emission profiles. Moreover, this dual-probe platform allows for selective discrimination between H2O2 and pH changes in live RAW 264.7 macrophage cells when stimulated by an immune insult. Further imaging experiments show that pharmacological inhibition of NADPH oxidase (Nox) proteins triggers a decrease in both oxidative burst and in pH regulation within phagocytic compartments and leads to disruptions of endocytic activity, suggesting that Nox-derived H2O2 signaling is critical to the maintenance of multiple components of the immune response. This work establishes a general molecular platform for simultaneous, real-time imaging of multiple analytes associated with redox biology in living systems and should be applicable to a wide range of chemosensor constructs.