Limitations of available indicators [such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl− are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl− and/or I− sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(β-d-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400–470 and 490–560 nm, molar extinction 11,100 M−1 ⋅ cm−1(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I− by a collisional mechanism (Stern-Volmer constant 60 M−1) and is not affected by other halides, nitrate, cations, or pH changes (pH 5–8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I− (50% quenching at 38 mM I−). LZQ was used to measure forskolin-stimulated I−/Cl−and I−/[Formula: see text]exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.
Visible-Light-Excited Room Temperature Phosphorescent Carbon Dots