Fluorescent chloride sensors
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Fluorescent chloride sensors are used for
chemical analysis Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separati ...
. The discoveries of
chloride The chloride ion is the anion (negatively charged ion) Cl−. It is formed when the element chlorine (a halogen) gains an electron or when a compound such as hydrogen chloride is dissolved in water or other polar solvents. Chloride sa ...
(Cl) participations in physiological processes stimulates the measurements of intracellular Cl in live cells and the development of fluorescent tools referred below.


Quinoline-based dyes

quinolinium - based Cl indicators are based on the capability of halides to quench the
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
of heterocyclic organic compounds with quaternary
nitrogen Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
. Fluorescence is quenched by a collision mechanism with a linear Stern–Volmer relationship: \frac = 1 + K( l^- where:
F_0 is the fluorescence in the absence of halide
F is the fluorescence in the presence of halide
K is the Stern–Volmer quenching constant, which depends on the chloride concentration, l^-/math>. in a linear manner. Thus, quinoline-based indicators are one-wavelength dyes - the signal results from monitoring the fluorescence at a single wavelength. Ratiometric measurement of halide concentration is not possible with quinolinium dyes. The kinetics of collision quenching are diffusion-limited only, and these indicators provide submillisecond time resolution. Quinolinium-based dyes are insensitive to physiological changes in pH, but they are prone to strong bleaching and demand
ultraviolet Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiation ...
excitation, which is harmful for living organisms. Because quinolinium is not occurring in the cells naturally, cell loading is necessary. However, quinolinium-based dyes aren't retained perfectly in the cell and can't be targeted easily to subcellular organelles. Also, they cannot be designed specific to a certain type of cell. 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

Clindicators can be designed on the basis of endogenously expressed fluorescent proteins such as
Yellow fluorescent protein Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish '' Aequorea victoria''. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP ...
(YFP). An advantage of endogenously expressed probes over dye-based probes is their ability to achieve cell-type-specificity by the choice of
Promoter (genetics) In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of ...
promotor. YFP based indicators are mutated forms of Green fluorescent protein (GFP). YFP contains four point mutations and has a red-shifted excitation and emission spectrum compared with GFP. YFP fluorescence is sensitive to various small
anion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conve ...
s with relative potencies iodine > nitrate >
chloride The chloride ion is the anion (negatively charged ion) Cl−. It is formed when the element chlorine (a halogen) gains an electron or when a compound such as hydrogen chloride is dissolved in water or other polar solvents. Chloride sa ...
>
bromide A bromide ion is the negatively charged form (Br−) of the element bromine, a member of the halogens group on the periodic table. Most bromides are colorless. Bromides have many practical roles, being found in anticonvulsants, flame-retardant ...
> formate > acetate. YFP sensitivity to these small
anion An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conve ...
s results from ground-state binding near the chromophore, which apparently alters the chromophore
ionization Ionization, or Ionisation is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons, often in conjunction with other chemical changes. The resulting electrically charged atom or molecul ...
constant and hence the fluorescence emission. The fluorescence of YFP is sensitive to l and pH. The effect is fully reversible. YFP is excited at visible range and is a genetically encoded probe. 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). If a fluorescent indicators is based on one fluorescent protein only, it doesn't allow for ratiometric measurements. Hence, a rationale for ratiometric fluorescent indicators results.


FRET-based, genetically encoded Cl indicators

Förster resonance energy transfer Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). ...
(FRET)-based Cl indicators consist of two fluorescent proteins,
Cyan fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequore ...
(CFP) and YFP connected via a 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.


Notes


References

* * * * * {{cite journal , last1=Markova , first1=O , last2=Mukhtarov , first2=M , last3=Real , first3=E , last4=Jacob , first4=Y , last5=Bregestovski , first5=P , year=2008 , title=Genetically encoded chloride indicator with improved sensitivity , journal= Journal of Neuroscience Methods , volume=170 , issue=1 , pages=67–76 , doi=10.1016/j.jneumeth.2007.12.016 , pmid=18279971 , s2cid=15427384 Analytical chemistry Chlorides Fluorescent dyes