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

Lucas S. Ryan, Jeni Gerberich, Uroob Haris, Daphne Nguyen, Ralph P. Mason, Alexander R. Lippert

Research output: Contribution to journalArticle


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.

Original languageEnglish (US)
Pages (from-to)2925-2932
Number of pages8
JournalACS Sensors
Issue number9
StatePublished - Sep 25 2020


  • 1,2 dioxetanes
  • chemiluminescence
  • CRET
  • in vivo imaging
  • pH imaging
  • ratiometric

ASJC Scopus subject areas

  • Bioengineering
  • Instrumentation
  • Process Chemistry and Technology
  • Fluid Flow and Transfer Processes

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