A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission.
A set of three pH-responsive ratiometric Eu(III)complexes has been synthesised incorporating a coordinated azathioxanthone sensitiser and a pH dependent alkylsulfonamide moiety. Emission properties, anion binding affinities, pH response curves and protein binding constants were studied in detail in aqueous media, and solutions containing various concentrations of interfering anions and protein were also examined. The complex, [EuL3] exhibited some interference from protein and endogenous anions, e.g. lactate and hydrogen carbonate, but possessed a protonation constant of 7.2 in human serum solution. A suitable calibration curve was obtained and was used to determine the local pH using a 680/589 nm intensity ratio vs. pH plot. Confocal fluorescence microscopy images revealed fast uptake of the complex and a well distributed localisation within the cell; fast egress also occurred. Ribosomal localisation, with a high concentration within the protein-dense nucleoli was observed, in a similar manner to structurally related complexes bearing the same coordinated sensitising moiety. An IC(50) value of 67 (+/-20) microM was estimated using an MTT assay. Selected emission band ratio versus pH plots allow pH measurement in the range 6 to 8, enabling intracellular pH to be measured by microscopy. A value of 7.4 was estimated for NIH 3T3 cells in the protein rich regions of the nucleolus and ribosomes.
Pal
Robert
R
Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK.
Parker
David
D
D001667:Binding, Competitive; D002138:Calibration; D005063:Europium / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism; D049408:Luminescent Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D049408:Luminescent Agents; D009942:Organometallic Compounds; D005063:Europium