Fluorescent chloride sensors
Fluorescent chloride sensors are used for chemical analysis. The discoveries of chloride (Cl−) participations in physiological processes stimulates the measurements of intracellular Cl− in live cells and the development of fluorescent tools referred below.
The quinolinium Cl− indicators are based on the capability of halides to quench the fluorescence of heterocyclic organic compounds with quaternary nitrogen. Fluorescence is quenched by a collision mechanism with a linear Stern–Volmer relationship:
F0/F=1+K[Cl] where: F0 is the fluorescence in the absence of halide F is the fluorescence in the presence of halide K is the Stern–Volmer quenching constant (in M-1)
Thus it is one wavelength dyes and radiometric measurement of halide concentration is not possible with quinolinium dyes. The kinetics of collision quenching is diffusion limited only. The indicators provide submillisecond time resolution. Quinolinium-based dyes are insensitive to physiological changes in pH, but they are prone to strong bleaching and need for ultraviolet excitation that could be damaging for the cell. The quinolinium-based halide indicators require cell loading. They are not retained perfectly in the cell and cannot be targeted easily to subcellular organelles.
The most used quinolinium-based Cl− indicators are 6-methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ), 6-methoxy-N-ethylquinolium Cl− (MEQ), and N-(6-methoxyquinolyl)-acetoethyl ester (MQAE).
YFP based Cl− sensors
Endogenously expressed fluorescent proteins such as Yellow fluorescent protein (YFP) based Cl− indicators. YFP based indicators are mutated forms of Green fluorescent protein (GFP) are alternatives to exogenous indicators. YFP contains four point mutations and has red-shifted excitation and emission spectra compared with GFP. YFP fluorescence is sensitive to various small anions with relative potencies iodine > nitrate > chloride > bromide > formate > acetate. YFP sensitivity to these small anions results from ground-state binding near the chromophore, which apparently alters the chromophore ionization constant and hence the fluorescence emission. The fluorescence of YFP is sensitive to [Cl− ] and pH. The effect is fully reversible.
YFP is exited at visible range and they are genetically encoded probes. YFP based Cl− sensors have rather low kinetics of Cl− association / dissociation. The half time association/dissociation constants for YFP mutant range from 50 ms (YFP-H148Q I152L) to 2 sec (YFP-H148Q V163S). They also does not allow ratiometric measurements.
FRET-based, genetically encoded Cl− indicators
Förster resonance energy transfer (FRET)-based Cl indicators consists of two fluorescent proteins, Cyan fluorescent protein (CFP) and YFP connected with polypeptide linker. This allows ratiometric Cl− measurements based on the Cl− sensitivity of YFP and Cl− insensivity of CFP. Clomeleon and Cl− Sensor are FRET-based Cl indicators that allow ratiometric non-invasive monitoring of chloride activity in living cells.
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