Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor in Live Animals

Regulation of physiological pH is integral for proper whole body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, composed of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8 and 8.4, making it a viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished using both common luminescence spectroscopy and advanced optical imaging methods. Using an IVIS Spectrum, pH can be measured through tissue with Ratio-pHCL-1, which is shown in vitro and calibrated in sacrificed mouse models. Intraperitoneal injections of Ratio-pHCL-1 into live mice show high photon outputs and consistent increases in the flux ratio when measured at pH 6, 7, and 8.