Abstract
Monitoring intracellular pH has drawn much attention due to its undeniably important function in cells. The widespread development of fluorescent imaging techniques makes pH sensitive fluorescent dyes valuable tools, especially red-emitting dyes which help to avoid the overcrowded green end of the spectral band. Herein, we present H-Rubies, a family of pH sensors based on a phenol moiety and a X-rhodamine fluorophore that display a bright red fluorescence upon acidification with pKa values spanning from 4 to 9. Slight structural modifications led to dramatic changes in their physicochemical properties and a relationship between their structures, their ability to form H-aggregates, and their apparent pKa was established. While molecular form H-Rubies can be used to monitor mitochondrial acidification of glioma cells, their functionalised forms were linked via click chemistry to dextrans or microbeads containing a near infrared Cy5 (Alexa-647) in order to provide ratiometric systems that were used to measure respectively the phagosomal and endosomal pH in macrophages (RAW 264.7 cells) using flow cytometry.
Highlights
E-mail: mayeul.collot@ unistra.fr bEcole Normale Superieure, Institut de Biologie de l'ENS (IBENS), INSERM U1024, CNRS UMR 8197, Paris F-75005, France cInserm U836, LabEx Ion Channels, Science and Therapeutics, Grenoble Institute of Neuroscience, chemin fortune ferrini, batiment Edmond Safra, 38042 Grenoble Cedex 09, France dUniversite Joseph Fourier, Grenoble, France eSmartox Biotechnology, Saint Martin d’Heres, France fINSERM U932, Institute Curie, 75248, Paris, Cedex 05, France † Electronic supplementary information (ESI) available: Details of the organic synthesis, characterisations (HPLC, NMR, mass spectra) as well as measurements of their photophysical properties can be found in the supplementary information
Boens' group successfully converted an ortho-chlorophenol to obtain a pH-sensitive BODIPY with a pKa of 7.6.31 Only few examples of phenolic rhodamines are given in the literature[32] and none of those were exploited as pH probes, leading us to investigate the potential efficiency of these entities as uorescent pH sensors
The red-emitting uorophore X-rhodamine was chosen for its highly desirable spectral properties including a high molar absorption, high quantum yield and good photo-stability as well as its reduced hydrophobicity compared to BODIPY
Summary
Intracellular pH plays many different and important roles in cellular activity and is especially involved in ion transport,[1] apoptosis,[2,3] multidrug resistance[4] and muscle contraction.[5,6] abnormal intracellular pH values are associated with diseases such as Alzheimer's7 and cancer.[8,9] Monitoring variations of intracellular pH in living cells is of the utmost importance and is an essential parameter for studying phagocytosis,[10] endocytosis[11] and cellular aLaboratory of Biomolecules (LBM), UPMC Universite Paris 06, Ecole Normale Superieure (ENS), CNRS, UMR 7203, Paris F-75005, France. Many uorescent indicators have already been developed (for an extensive review see: Han and Burgess),[12] several factors have to be taken into account in order to achieve efficient use within cells, including solubility in biological media, hydrophobicity, photostability and brightness. Whilst recent progresses have been reported in the development of red emitting genetically encoded pH sensors,[16] there is still a demand for efficient molecular red uorescent indicators. Redemitting BODIPYs have recently attracted special attention due to their photostability and high brightness.[17,18] Having said this they tend to suffer from high lipophilicity making them inefficient for cellular experiments as their apparent pKa shi s in lipophilic environment.[19]
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