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Fluo-Green™ Wavelength Ratiometric Fluoride Probe

Ursa BioScience offers a unique wavelength Ratiomeric Fluoride Probe, namely Fluo-Green™. Fluo-Green™ readily chelates dissolved fluoride ions and as such gives a unique fluorescence wavelength ratiometric response with a dissociation constant of 42 mM. While the other halides, Cl-, Br- and I- are readily known to quench fluorescence [1-10], fluoride seldom quenches fluorescence and hence its detection and quantitation is usually limited to other non-fluorescence techniques such as ion-selective electrodes [11].

Absorption spectrum of Ursa BioSciences' probe Fluo-Green™

Figure 1. Absorption spectrum of Fluo-Green™ with increasing fluoride concentration.

Ratiometric plot for Fluo-Green™

Figure 2. Wavelength ratiometric plot based on the A342/A388 absorption bands of Fluo-Green™.

 

Fluo-Green™ responds in both an absorption wavelength ratiometric manner, 325-450 nm, or a fluorescence emission wavelength ratiometric manner, ~400-700 nm, and is ideal for researches determining environmental, waste water or physiological fluoride. In addition, Fluo-Green™ is selective to fluoride in the presence of high concentrations of dissolved chloride and other blood analytes, such as glucose. The Stern-Volmer fluorescence quenching constants for I-, Br- and Cl- are 34, 1.4 and 1.0 M-1 respectively. The mean and amplitude weighted lifetimes for aqueous (deionized water) Fluo-Green™ are 2.52 and 2.39 ns respectively.

Emission spectra Fluo-Green™

Figure 3. Fluorescence emission spectra of Fluo-Green™ with increasing fluoride concentrations.

Fluo-Green C™ - Control Fluorophore

Ursa BioScience also offers a Control Fluorescence Compound for Fluo-Green™. Our Fluo-Green C™ has the same chromophore fluorescent backbone, but its fluorescence is not influenced by the presence of dissolved fluoride ions, making the Control Probe ideal for researchers wishing to study any background interferences on the fluorescence of the parent chromophore, Fluo-Green™. Subsequently, Fluo-Green C™ does not show any ratiometric response to fluoride. Fluo-Green C™ can readily be excited at 400 nm, with an emission centered at ~560 nm.

Physiological Fluoride

Fluoride is present in biological fluids and tissues and especially in bone and tooth. Typical fluoride levels in blood have been reported to be in the range 20–60 µg l-1 using a fluoride ion-selective electrode [11]. Fluoride is easily absorbed but it is excreted slowly from the body, which can result in chronic poisoning [12]. Fluoride has a strong effect on most enzymes and acute poisoning by fluoride is almost always caused by blockage of the enzyme functions. Hence monitoring fluoride in the human body is important. Some published methods for the determination of fluoride in biological fluids include ion chromatography [12]; fluoride ion-selective electrodes [11,13,14]; spectrophotometry [15] and gas chromatography [16].

Advantages of the Fluo-Green™ Fluorophore

Steady-state Stern-Volmer plot for Fluo-Green™

Figure 4. Steady-state Stern-Volmer plot for Fluo-Green™ with increasing concentrations of sodium halide.

It is not just the physiological significance of chloride that drives workers to mostly report the chloride sensitivity of some fluorescent probes, but because the quenching of fluorescence is not a selective process, and any fluorophore quenched by chloride is also quenched by bromide to a greater extent and also by iodide to an even greater extent. Therefore, for dynamic quenching, the sensitivity of fluorophores to halide is well known to be I- > Br- > Cl- [5]. The explanation of this effect lies in the fact that the efficiency of intersystem crossing to the excited triplet state, promoted by spin-orbit coupling of the excited singlet fluorophore and halide upon contact, depends on the mass of the quencher atom, hence the expression "heavy-atom effect" is sometimes used [1,2,5]. It is for this reason that fluoride does not typically quench fluorescence. As such, traditional halide sensitive probes are not very sensitive to fluoride and are therefore not suited for detecting fluoride < 50 mM [5]. Only a few fluorescent probes can be found in the literature which are sensitive to fluoride [1,5], all based on either the benzene or naphthalene backbone and therefore showing absorption in the deep UV (~270 nm), which is not practical for many sensing applications [1,5,17]. In contrast, Ursa BioScience's unique Fluo-Green™ Fluoride Sensor detects dissolved fluoride in the low mM concentration range, responds in both an absorption and emission wavelength ratiometric manner and can be excited 325-450 nm, with practical and user friendly emission wavelengths ~400-700 nm.

Advantages of the Fluo-Green™ Fluorophore Ratiometric Response

It is widely recognized that ratiometric or lifetime-based methods offer intrinsic advantages for both chemical and biomedical sensing [1,18]. Fluorescence intensity measurements are typically unreliable away from the laboratory and can require frequent calibration/s due to a variety of chemical, optical or other instrumental related factors [1,18]. Unfortunately, while fluorescent probes are known to be very useful for many applications [1,18], such as in fluorescence microscopy, DNA technology and fluorescence sensing, most sensing fluorophores only display changes in intensity in response to analytes, and relatively few wavelength ratiometric and lifetime based probes are available [1,18]. Some useful wavelength ratiometric probes are available for pH, Ca2+, and Mg2+ [19,20], but the probes for Na+ and K+ generally display small spectral shifts and negligible lifetime changes and are subsequently inadequate for quantitative sensing measurements. Dynamic quenchers such as O2 and the halides usually occur with a change in intensity and lifetime but without an emission spectral shift [5]. In addition, due to its low mass, the fluoride ion is not an effective collisional quencher and only a few probes are known to be quenched by fluoride in the low mM concentration range [5].

 

 

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References

[1]  Valeur, B and Berberan-Santos, MN, Eds, “Molecular Fluorescence”, second edition, Wiley-VCH, Weinheim, Germany 2012.
[2] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd  Edition, Springer, New York, 2006.
[3] Optical thin film polymeric sensors for the determination of aqueous chloride, bromide and iodide ions at high pH, based on the quenching of fluorescence of two acridinium dyes, Geddes, C.D., Dyes and Pigments, (2000), 45(3), 243-251.
[4] Naim J O, Lanzafame R J, Blackman J R and Hinshaw R J, The in vitro quenching effects of iron and iodine on fluorescein fluorescence J. Surg. Res. (1986), 40 225–8.
[5] Optical halide sensing using fluorescence quenching: Theory, simulations and applications – A review, Geddes, C.D., Meas. Sci. Technol., (2001), 12(9), R53-R88.
[6] Najbar J. and Mac M. 1991, Mechanisms of fluorescence quenching of aromatic molecules by potassium iodide and potassium bromide in methanol–ethanol solutions J. Chem. Soc. Faraday Trans, (1991), 87 1523–9.
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[8] Moriya T., Excited-state reactions of coumarins in aqueous solutions III. The fluorescence quenching of 7-ethoxycoumarins by the chloride ion in acidic solutions Bull. Chem. Soc. Japan, (1986), 59, 961–8.
[9] Moriya T., Excited-state reactions of coumarins in aqueous solutions VI. The fluorescence quenching of 7-hydroxycoumarins by chloride ions in acidic solutions Bull. Chem. Soc. Japan (1988), 61, 753–9.
[10] Carrigan S., Doucette S., Jones C., Marzzacco C. J. and Halpern A. M., The fluorescence quenching of 5,6-benzoquinoline and its conjugate acid by Cl-, Br-, SCN- and I- ions J. Photochem. Photobiol. A (1996), 99 29–35.
[11] Kissa E., Determination of inorganic fluoride in blood with a fluoride ion-selective electrode Clin. Chem. (1987), 33 253–5.
[12] Michigami Y, Kuroda Y, Ueda K. and Yamamoto Y., Determination of urinary fluoride by ion chromatography Anal. Chim. Acta (1993), 274, 299–302.
[13] Tyler J. E. and Poole D. F. G., The rapid measurement of fluoride concentrations in stored human saliva by means of a differential electrode cell Arch. Oral Biol., (1989), 34 995–8.
[14] Tyler J. E., Poole D. F. G. and Kong K. L., Determination of fluoride in blood plasma J. Dent. Res., (1988), 67, 677.
[15] Culik B., Microdiffusion and spectrophotometric determination of fluoride in biological samples Anal. Chim. Acta., (1986), 189, 329–37.
[16] Ikenishi R., and Kitagawa T., Gas chromatographic method for the determination of fluoride ion in biological samples II. Stability of fluoride-containing drugs and compounds in human plasma Chem. Pharm. Bull., (1988), 36, 810–14.
[17] C. R. Cooper, N. Spencer, T. D. James, Chem. Commun., (1998), 13, 1365.
[18] Z. Gryczynski, I. Gryczynski, J. R. Lakowicz, Methods in Enzymology, (2002), 360, 44.
[19] R. Y Tsien, T. J. Rink, M. Poenie, Cell Calcium, (1985), 6, 145.
[20] J. P. Y. Kao, Methods Cell Biol., (1994), 40, 155.