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

Abstract

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.