19F Activity-Based Barcoding Enables Simultaneous Measurement of pH and Analyte Reactivity

Activity-based sensing (ABS) probes are designed to report on analyte activity, but their readouts are often influenced by local pH. Because both probe response and analyte reactivity can vary across physiological pH ranges, signal changes can reflect altered chemistry rather than differences in analyte abundance. Here, we present a 19F magnetic resonance imaging (MRI) platform that converts analyte activation into a pH-dependent chemical shift barcode, enabling simultaneous assignment of both properties within a single measurement. Using nuclear magnetic resonance spectroscopy and MRI, we achieve accurate pH mapping across a broad physiological range and show that F-pHOx, an H2O2-responsive probe, enables quantitative inference of hydrogen peroxide activity across varying pH conditions. We further demonstrate its performance in an ex vivo mouse stomach model, which is a complex environment exhibiting substantial pH heterogeneity. Importantly, the modular fluorophenol reporter is compatible with diverse ABS triggers. This strategy reveals how environmental conditions can influence apparent chemical activity, which is often overlooked.