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Chloride Probes

Ursa BioScience offers a range of high quantum yield fluorophores with differing water solubility’s for a range of customer chloride sensing applications.

Our chloride probes are readily fluorescence quenched (intensity and lifetime) by the presence of aqueous, complexed or pseudo chloride and are ideally suited for physiological or environmental chloride determination.

Saline Blue™

Saline Blue™, our highly water soluble, highly fluorescent chloride sensor is readily excited at 300-380 nm, with emission in the blue, ≈ 440 nm, with a dynamic Stern-Volmer chloride quenching constant of 118 mol L-1.

absorption spectra and contour emission graph for Saline Blue

Figure 1. (A) Absorption spectra recorded for Saline Blue™ dissolved in water. (B) Contour emission graph recorded for Saline Blue™ dissolved in water.

 

Saline Blue 2™

Saline Blue 2™ is a highly fluorescent water soluble probe which is sensitive to aqueous, complexed or pseudo chloride, and is ideally suited for researchers wishing to analytically determine chloride in aqueous media. Saline Blue 2™ can readily be excited from 300-380 nm, with an emission centered ≈ 440 nm and a Stern-Volmer chloride quenching constant of ≈ 101 M-1. Saline Blue 2 contains a -(CH2)7COOH terminal chain, allowing insertion into membranes, lipid Bilayers etc.

absorption spectra and contour emission graph for Saline Blue 2

Figure 2. (A) Absorption spectra recorded for Saline Blue 2™ dissolved in water. (B) Contour emission graph recorded for Saline Blue 2™ dissolved in water.

 

Saline Blue 3™

Saline Blue 3™ is a highly fluorescent probe, with fair water solubility, which is sensitive to aqueous, complexed or pseudo chloride, and is ideally suited for researchers wishing to analytically determine chloride in a variety of media. Saline Blue 3™ can readily be excited from 300-380 nm, with an emission centered ≈ 440 nm and a Stern-Volmer chloride quenching constant (aq) of ≈ 85 M-1. Saline Blue 3™ is highly soluble in both MeOH and EtOH and readily soluble in plastic sensor supports, such as PolyVinyl Alcohol (PVA). Saline Blue 3™ contains a -(CH2)10COOH terminal chain, allowing insertion into membranes, lipid Bilayers etc.

absorption spectra and contour emission graph for Saline Blue 3

Figure 3. (A) Absorption spectra recorded for Saline Blue 3™ dissolved in water. (B) Contour emission graph recorded for Saline Blue 3™ dissolved in water.

 

Saline Blue 4™

Saline Blue 4™ is a highly fluorescent probe, partly soluble in water and sensitive to complexed or pseudo chloride, and is ideally suited for researchers wishing to analytically determine chloride in a variety of media and cellular/lipid applications. Saline Blue 4™ can readily be excited from 300-380 nm, with an emission centered ≈ 440 nm. Saline Blue 4™ is highly soluble in both MeOH and EtOH and readily soluble in plastic sensor supports, such as PolyVinyl Alcohol (PVA). Saline Blue 4’s insolubility in water makes it ideal for plastic sensor design, the probe not leaching and favoring the sensor support. Saline Blue 4™ contains a -(CH2)15COOH terminal chain, allowing insertion into membranes, lipid Bilayers etc.

Saline Blue 2-4 are progressively less water soluble, respectively, with Saline Blue 4™ ideal for cellular surface chloride determination, the probes hydrophobic tail anchoring the probe within the cellular membrane.

absorption spectra and contour emission graph for Saline Blue

Figure 4. (A) Absorption spectra recorded for Saline Blue 4™ dissolved in water. (B) Contour emission graph recorded for Saline Blue 4™ dissolved in water.

 

Stern-Volmer Fluorescence Quenching

The fluorescence quenching of a fluorophore by halide was first described by George Stokes [1] as early as 1869 when he observed that the fluorescence of quinine in dilute sulphuric acid was reduced after the addition of hydrochloric acid, i.e. chloride ions. The process that he observed is now commonly referred to as 'dynamic fluorescence quenching', where both the lifetime and intensity of fluorescence are reduced in the presence of a quencher, Q. This process is known to follow Stern–Volmer kinetics [2-4]:

Equation 1

(1)

Equation 2

(2)

which can be used to obtain values of kqτ0 (the Stern–Volmer onstant, KSV , units mol L-1) by plotting I0/I as a function of [Q]. I0 and I are the fluorescence intensities in the absence and presence of Q respectively, kq is a specific constant describing bimolecular collisional deactivation of electronic energy and τ0 is the fluorescence lifetime in the absence of a quencher.

If quenching occurs only by a dynamic mechanism and τ0 is a monoexponential decay time, then the ratio τ0/τ will also be equal to 1 +KSV[Q], where τ is the lifetime in the presence of quencher, hence,

Equation 2

(3)

This equation illustrates an important characteristic of collisional quenching, which is the equivalent reduction in fluorescence intensity and lifetime.

A detailed description of fluorescence quenching can be found elsewhere (4).

Understanding the Biomolecular Quenching Constant

The dynamic or bimolecular quenching rate constant, kq, is a useful parameter which can reflect the efficiency of quenching or the accessibility of the fluorophores to the quencher, and can provide useful information on halide diffusion through membranes, or polymer supports etc.

Diffusion controlled quenching usually results in kq values ≈ 1010 M−1 s−1. Smaller values are usually indicative of shielding of the fluorophore whilst larger apparent values of kq usually suggest some form of binding interaction (5).

The bimolecular quenching rate constant is the product of the quenching efficiency, γ, multiplied by the diffusion limited bimolecular rate constant for collision, k:

Equation 4

(4)

References

[1] Stokes G G 1869 On a certain reaction of quinine J. Chem. Soc. 22, 174–85.

[2] Stern O and Volmer M 1919 Uber die abklingungszeit der fluoreszenz Phys. Z. 20 183–8 (in German).

[3] Birks J B (ed) 1975 Organic Molecular Photophysics (New York: Wiley) pp 409–613.

[4] Geddes, C.D. (2001) Optical halide sensing using fluorescence quenching: Theory, simulations and applications – A review, Measurement Science and Technology, 12(9), R53-R88.

[5] Lakowicz, JR, (ed), 1999 Principles of Fluorescence Spectroscopy 2nd edn, (New York: Kluwer–Plenum).

 

 

 

Saline Blue without and with sodium chloride

Saline Blue™ dissolved in water without and with sodium chloride present.

Illustration of the Saline Blue probes of various chain lengths

Cartoon illustration of the Saline Blue™ probes.