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Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

submitted on 02.05.2020, 19:47 and posted on 05.05.2020, 11:57 by Lucas S. Ryan, Jeni Gerberich, Uroob Haris, ralph mason, Alexander Lippert

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, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.


CAREER: Triggered Energy Transfer Chemiluminescence for In Vivo Imaging

Directorate for Mathematical & Physical Sciences

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NIH 1S10RR024757

NIH P30 CA142543


Email Address of Submitting Author


Southern Methodist University


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

A.R.L. discloses a financial stake in BioLum Science, LLC.