A library of Rhodamine6G-based pH-sensitive fluorescent probes with versatile in vivo and in vitro applications

W. Benton Swanson, Margaret Durdan, Miranda Eberle, Seth Woodbury, Ava Mauser, Jason Gregory, Boya Zhang, David Niemann, Jacob Herremans, Peter X. Ma, Joerg Lahann, Megan Weivoda, Yuji Mishina, Colin F. Greineder

Research output: Contribution to journalArticlepeer-review


Acidic pH is critical to the function of the gastrointestinal system, bone-resorbing osteoclasts, and the endolysosomal compartment of nearly every cell in the body. Non-invasive, real-time fluorescence imaging of acidic microenvironments represents a powerful tool for understanding normal cellular biology, defining mechanisms of disease, and monitoring for therapeutic response. While commercially available pH-sensitive fluorescent probes exist, several limitations hinder their widespread use and potential for biologic application. To address this need, we developed a novel library of pH-sensitive probes based on the highly photostable and water-soluble fluorescent molecule, Rhodamine 6G. We demonstrate versatility in terms of both pH sensitivity (i.e., pKa) and chemical functionality, allowing conjugation to small molecules, proteins, nanoparticles, and regenerative biomaterial scaffold matrices. Furthermore, we show preserved pH-sensitive fluorescence following a variety of forms of covalent functionalization and demonstrate three potential applications, both in vitro and in vivo, for intracellular and extracellular pH sensing. Finally, we develop a computation approach for predicting the pH sensitivity of R6G derivatives, which could be used to expand our library and generate probes with novel properties.

Original languageEnglish (US)
Pages (from-to)748-764
Number of pages17
JournalRSC Chemical Biology
Issue number6
StatePublished - Apr 29 2022

ASJC Scopus subject areas

  • Chemistry (miscellaneous)
  • Biochemistry
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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