An FRET-based ratiometric chemosensor for in vitro cellular fluorescence analyses of pH

Abstract

Ratiometric fluorescence sensing is an important technique for precise and quantitative analysis of biological events occurring under complex conditions by simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios. Herein, we design a ratiometric chemosensor for pH that is based on photo-induced electron transfer (PET) and binding-induced modulation of fluorescence resonance energy transfer (FRET) mechanisms. This ratiometric chemosensor was constructed by introduction of a pH-insensitive coumarin fluorophore as a FRET donor into a pH-sensitive amino-naphthalimide derivative as the FRET acceptor. The sensor exhibited clear dual-mission signal changes in blue and green spectral windows upon pH changes. The pH sensor was applied for not only measuring cellular pH, but also for visualizing stimulus-responsive changes of intracellular pH values.

Poly(ε-caprolactone)-containing graft copolymers for ratiometric extracellular oxygen sensing

“…Herein, we extended the AIE fluorophores as internal references for oxygen sensors to achieve ratiometric sensing. The ratiometric method is capable to alleviate the environmental influences, such as fluctuations in excitation source intensity, variance in probe concentration, and uncontrollable variations in background fluorescence, and as a result, increases the measurement accuracy [43][44][45][46][47].…”

An indole-carbazole-based ratiometric emission pH fluorescent probe for imaging extreme acidity

“…In contrast, a ratiometric measurement through simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios, could endow a self-calibration effect for most of the aforementioned interferences, and thus allow for a more precise measurement of intracellular pH in quantitative manner. Indeed, there are many reports on ratiometric pH probes for imaging weak acidic or near neutral intracellular pH values [31][32][33][34][35][36][37][38][39]. However, up to now still very limited ratiometric fluorescent pH probes have been developed for extreme acidity in living cells [40].…”

“…So far, a number of excellent pH probes working for weak acidic organelles (such as lysosomes, pH 4.5-5.0) [7][8][9][10][11][12][13][14][15][16][17] or near neutral cytosol (pH 6. 8-7.4) [7,[18][19][20][21][22][23] have been exploited, and some are commercially available [7].…”

“…Cell cytotoxicity assayMTT assay is used to test the cytotoxicity of CzBI on HeLa cells[32]. The cells with a density of 1×10 5 cells mL -1 are cultured in a 96-well microplate to a total volume of 200 μL well -1 and 50 μM in fresh medium are incubated with HeLa cells for 3 h, respectively.After washing the cells with cold phosphate buffered saline (PBS, pH 7.4) three times,10 μL of MTT solution (10 mg/ml, PBS) is added into each well of 96-well microplate for another 4 h. Then the remaining MTT solution is removed from the wells, and 150 μL of DMSO is added into each well to dissolve the intracellular blue-violet formazan crystals. The absorbance of the solution is measured at 490 nm wavelength with a microplate reader.…”

The study of a curcumin-resembling molecular probe for the pH-responsive fluorometric assay and application in cell imaging

“…In their study, the ratiometric fluorescence analysis was applied as an optimized method for the quantitative determination of biological events occurring under complex conditions by simultaneously recording fluorescence intensities at two wavelengths and calculating their ratios. Zhou et al designed a ratiometric chemosensor for pH that was constructed by introducing a pH-insensitive coumarin fluorophore as a fluorescence resonance energy transfer (FRET) donor into a pHsensitive amino-naphthalimide derivative as the FRET acceptor [10]. Tang et al prepared a complex probe in which the fluorescence lifetime of CdTeSe/ZnS quantum dots was modulated by NIR organic chromophores for generating reproducible pH-sensing nanomaterials.…”