Ratiometric probes

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

Grieshaber, S., Swanson, J. A. and Hackstadt, T. (2002). Determination of the physical environment within the Chlamydia trachomatis inclusion using ion-selective ratiometric probes. Cell. Microbiol. 4, 273-283. 

In principle, the problems of intensity-based sensing can be avoided using wavelength-ratiometric probes, i.e., fluorophores that display spectral changes in the absorption or emission spectrum on binding or interaction with the analytes (Figure 1.1). In this case, the analyte concentration can be determined independently of the probe concentration by the ratio of intensities at two excitation or two emission wavelengths. 

The difficulties of intensity-based flow cytometry are illustrated by the present difficulties of cell-by-cell measurements of intracellular calcium. This can be accomplished using the calcium probe Indo-l,(34 38) but requires an ultraviolet (UV) laser source which is not routinely available in flow cytometry (Indo-1 is an emission wavelength ratiometric probe). Flow cytometers routinely have argon ion laser sources with outputs of 488 or 514 nm. Measurement of intracellular ions other than Ca2+ is nearly impossible. (The SNAFL and SNARF probes should allow pH measurement from the wavelength-ratiometric data.(15))... 

E. U. Akkaya and J. R. Lakowicz, Styryl-based wavelength ratiometric probes A new class of fluorescent calcium probes with long wavelength emission and a large stokes shift, Anal. Biochem. 213, 285-289 (1993). 

Calibration method. The measured fluorescence parameter should be independent of indicator concentration, geometry of sample, and sensitivity of detection system. Thus, an intensity-based method requires wavelength-ratiometric probes. Lifetime and anisotropy methods do not require wavelength-ratiometric probes, but the lifetime or anisotropy must be sensitive to analyte. 

Bretonniere, Y, Cann, M.J., Barker, D., and Slater, R. (2002) Ratiometric probes for hydrogencarbonate analysis in intracellular or extracellular environments using europium luminescence. Chemical Communications, 1930-1931. 

Q.29.10 What arc the advantages of a ratiometic fluorescent dye over a non-ratiometric probe ... 

Ueberfeld J, Walt DR (2004) Reversible ratiometric probe for quantitative DNA measurements. Anal Chem 76 947-952... 

A more recent class of pH probes are the seminaphtofluoresceins (SNAFL) and the seminaphtorhodafluors (SNARF). These probes were reported for use as wavelength-ratiometric probes (86), Their absorption and emission spectra change significantly in response to pH. 

Fluorescein tetramethylrhodamine dextran (FRD) was used as a ratiometric probe, which provides a pH-independent signal (tetramethylrhodamine), to compensate for different organelle volumes, and a pH-dependent signal (fluorescein). The ratio of the pH-independent and pH-dependent fluorescence was expected to provide a quantitative measure of the pH. Acidic organelles (lysosomes and endosomes) were specifically labeled by FRD, because the cells endocytose small amounts of the extracellular medium, which is accumulated in endosomes and eventually lysosomes. CE was performed in a poly((V-acryloyl aminopropanol, AAP) modified capillary to reduce both adsorption at the capillary surface and the electroosmotic flow. LIF detection was performed off-column using a... 

A disadvantage of the use of fluorescein in sensors is that fluorescein is difficult to enqrloy as a wavelength-ratiomet-ric probe. This is because the absotption and emission intensity are tow for 4S0-mn excitation (Figure 19.32). The pH probe S-hydroxypyrene-l,3,6,8-trisulfonate (HPTS) displays more favorable properties as a wave-tongth-ratiometric probe. In HPTS (Figure 19.34), the sulfonate groups provide solubility in water, and the hydroxyl group provides sensitivity to pH. 

For an emission wavelength-ratiometric probe, one can use Eq. 19.11 with the values of Sx(X2) and SsfAj) these values ace tdated to the relative intensities of die fine and bound forms of the probe ... 

Such ratiometric probes are already in widespread use for measurement of Ca and pH (Chapter 19), but these are not MLC probes and they dlqilay nanosecond deciQr times. [Ru(bpy)2(deabpy)] is the first MLC probe suitable for use as a ratiometric probe. 

Figure S.7 Intrinsic fluorescence of CQ-susceptibie (a) and CQ-resistant (b) strains of P. falciparum in both the absence and presence of ratiometric probe.

Recently, a ratiometric probe based on coumarin-cyanine hybrid system was selected for the study of intracellular redox processes in the absence or presence of CQ or FQ [80]. This probe was selected for its selectivity for HO radicals compared with other species of interest [81], This probe for HO radicals ratio-metric imaging displays an emission band centered at 495 nm (green) (Figure 5.7). The presence of HO elicited a marked decrease of emission intensities at 495 nm, but a significant enhancement of emission intensities at 651 nm (red). Previously, Ribaut et al. [53] have shown that erythrocytes infected by P. falciparum produce reactive oxygen species, whereas healthy erythrocytes do not However, these studies do not specify the nature of the ROS. It was suggested that some of these ROS could be HO radicals [80]. 

The aforementioned probe show fluorescent turn-on signals upon interaction with benzenethiols. Moreover, probes 28 and 29 bear sulfoxide function at 3-position of BODIPY core. They are ratiometric probes for benzenethiols based on thiol-sulfoxide transduction. The two probes feature a distinct absorption and emission redshift upon reduction with benzenethiols to give... 

One of the most widely used photo-chemicals for pH measmement is fluorescein [6], which displays complex optical properties dependent upon its ionic form (See Chap. 4, compound 4.3). In particular only the two anionic forms present at high pH are fluorescent. Fluorescein can be used a wavelength ratiometric probe... 

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