Ratiometric measurements

Square-650-pH having a pKa in the physiological pH range (pKa = 7.1 for free dye and the pKa 6.1 when labeled to an antibody) was recently introduced by SETA BioMedicals [119]. This dye is commercially available as a free carboxylic acid and a mono-NHS ester. Square-650-pH has spectral properties similar to those of the CypHer dyes but is fluorescent in both the protonated and deproto-nated forms. This dye displays reasonable molar absorptivities (135,000 and 48,000 M-1cm-1) and quantum yields (16% and 9%) for the protonated and deprotonated forms, an extremely large Stokes shift of more than 100 nm for the deprotonated form, and enables excitation and emission ratiometric measurement... 

A different approach to design a self-calibrating dye was proposed [70], in which a viscosity-sensitive molecular rotor (2-cyano-3-(4-dimethylaminophenyl) prop-2-enoic acid) was covalently linked to a reference dye, 7-methoxycoumarin-3-carboxylic acid, which exhibited no viscosity sensitivity (40, Fig. 13). A ratiometric measurement, that is, rotor emission relative to reference emission, was shown to be widely independent of dye concentration [70]. However, the design of such a ratiometric dye poses some challenges because of resonance energy transfer from... 

Klymchenko AS, Stoeckel H, Takeda K et al (2006) Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential. J Phys Chem B 110 13624-13632... 

The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

A reference channel (quantum counter or photodiode) has two advantages (i) it compensates for the time fluctuations of the lamp via a ratiometric measurement (ratio of the output signals of the photomultiplier detecting the fluorescence of the sample to the output signal of the reference detector) (ii) it permits correction of excitation spectra (see below). 

It is recommended, whenever possible, to carry out ratiometric measurements, i.e. to determine the ratio of the fluorescence intensities measured at two wavelengths according to the following methods ... 

Fig. 10.3. Principles of ratiometric measurements. A doubleexcitation measurements. B double-emission measurements.

Tab. 10.1. Examples of fluorescent pH indicators allowing ratiometric measurements. It should be recalled that the values of pKa (given here at room temperature for dilute solutions) can be more or less affected by changes in ionic strength and temperature...

The ratiometric measurements are preferable because the ratio of the fluorescence intensities at two wavelengths is in fact independent of the total concentration of the dye, photobleaching, fluctuations of the source intensity, sensitivity of the instrument, etc. The characteristics of some fluorescent pH indicators allowing ratiometric measurements are given in Table 10.1. 

For ratiometric measurements, the following equation may be used to fit the calibration curve (see Appendix A) ... 

The pyranine derivative DHPDS (l,3-dihydroxypyrene-6,8-disulfonic acid) retains the high pH sensitivity of pyranine but its acido-basic properties are much less sensitive to ionic strength. This indicator has two pKa values 7.33 + 0.04 and 8.55 + 0.2. Ratiometric measurements in both excitation and emission are possible. 

Introduction of an additional phenyl ring in the SNAFL structure leads to carboxynaphthofluorescein (CNF), whose absorption and fluoresence spectra are further red-shifted. CNF has a pKa of 7.5 and can be used in either excitation or emission ratiometric measurements. 

Attention should be paid to the possible existence of several complexes having different stoichiometries. A necessary preliminary experiment consists of recording the fluorescence and/or excitation spectra under experimental conditions (nature of the solvent, composition of the medium, ionic strength, pH (if it has an effect on the stability constant), etc.) as close as possible to the medium in which a cation must be detected. The variations in the fluorescence intensity for an appropriate couple of excitation and emission wavelengths (or for several emission or excitation wavelengths) as a function of cation concentration must be analyzed in order to determine the stoichiometry and the stability constant of the complexes (Appendix B). As in the case of pH determination (see Section 10.2.1), ratiometric measurements are recommended. 

An interesting feature of PET-5 is its ability to form exaplexes characterized by an additional band at higher wavelengths, thus allowing ratiometric measurements at two different observation wavelengths. 

The Na+ sensor M-9 has a structure analogous to that of compound E-4, but instead of two identical pyrene fluorophores, it contains two different fluorophores with a pyrene group and an anthroyloxy group. Resonance energy transfer (see Chapter 9) from the former to the latter is then possible because of the spectral overlap between the fluorescence spectrum of the pyrene moiety and the absorption spectrum of the anthroyloxy moiety. Upon addition of Na+ to a solution of M-9 in a mixture of MeOH and THF (15 1 v/v), the fluorescence of the anthroyloxy group increases significantly compared with that of the pyrene group, which permits a ratiometric measurement. 

Another feature is that the absence of spectral change precludes ratiometric measurements. However, dual-wavelength Cl sensors have been constructed. For instance, in compound A-6 (Figure 10.30), 6-methoxyquinolinium (MQ) as the Cl -sensitive fluorophore (blue fluorescence) is linked to 6-aminoquinolinium (AQ) as the Cl--insensitive fluorophore (green fluorescence), the spacer being either rigid or flexible. 

A ratiometric measurement consists of monitoring the ratio Y(X )/Y(A2) given by... 

In the ratiometric method, the fluorescence intensity of the liposomes containing pyranine (F) and in the presence of the quencher DPX was determined at 520 nm upon excitation at two wavelengths 460 nm (of the charged unprotonated pyranine) and 415 nm (of the pH-independent isosbestic wavelength that describe the total pyranine concentration). The ratio of is described as F. The ratiometric measurement is used to determine the intraliposome aqueous phase pH (18,22). Then nigericin (or nonactine) at final concentration of 5pM was added to disrupt the pH and/or ammonium ion gradient that induce complete gradient collapse and the measurement at the above two excitations was repeated, and indeed it demonstrated a shift of the intraliposome aqueous pH to be identical to the extraliposome medium pH (10). 

Ruthenium complexes with mixed bipyridyl ligands, immobilized inside a Nation film, may also be used as pH-sensitive sensor layers [90]. A completely different approach for a ratiometric imaging of pH sensor foils was developed for diagenetic studies of marine sediments, using the dual fluorescence excitation ratio of the pH-sensitive fluorophore 8-hydroxypyrene-l,3,6-trisulfonic acid (HPTS) [91]. Commonly used dual fluorophors with different absorption and emission maxima in the protonated and basic form for ratiometric measurements are the naphthofluorescein and seminaphthofluorescein derivates (SNARF and SNAFL) [92], It should be noted that ammonia or carbon dioxide can also be detected by some of these pH-sensitive materials [55,93]. 

The method may also be used the other way round with GOx as the enzyme label, glucose as substrate, and [Eu(Tc)] as the fluorescent indicator. In that type of assay, the presence of enzyme-labeled antibodies is indicated by an increase in the observed fluorescence intensity. It should be noted that commercially available fluorescence microplate readers, preferable equipped with time resolution, are also suited for the screening of all the microwell plate-based assays presented in this chapter. Nevertheless, the imaging process is much faster, accomplished in the order of one second, and enables ratiometric measurements. 

Ge XD, Tolosa L, Rao G. Dual-labeled glucose binding protein for ratiometric measurements of glucose. Analytical Chemistry 2004, 76, 1403-1410. 

An advantage of this readout is the opportunity to carry out ratiometric measurements, because two true fluorophores with different excitation and emission maxima are used. This strongly increases the robustness of the assay. In general, TR-FRET-based assays can easily be miniaturized to the 1536-well format. The disadvantage of APC as a fluorescence acceptor is the size of the... 

It is our experience that the resnlts obtained by using fluorescent techniques that do not use ratiometric measurement and/or time resolution should be carried out and validated with great care in order to avoid false positive or negative results. 

All nicotinic receptors are somewhat calcium permeable the most permeable are neuronal homomeric receptors (a7, a9) and the least permeable, embryonic muscle receptors. It must be noted that the measurement of relative calcium permeability by the simplest technique (reversal potential shift induced by changes in extracellular calcium concentrations) is error-prone for neuronal nicotinic receptors because their extreme inward rectification makes it difficult to measure reversal potentials accurately. A further technical difficulty (for recombinant receptors) arises from the presence in Xenopus oocytes of a calcium-dependent chloride conductance that has to be suppressed or minimized by either intracellular calcium chelation or chloride depletion. Some of these problems can be overcome by expressing the receptors in mammalian cell lines and using ratiometric measurements of intracellular calcium and coupled with wholecell recording, to obtain a measure of what proportion of the nicotinic current is carried by calcium (a measure that also has the advantage of being physiologically more relevant). 

The ratiometric measurement principle is independent of the configuration of the input stage, which therefore can be adapted to the specific sensor application. The impedance range is restricted by stray capacitance in general and by the input capacitance of the measurement channels in particular. 

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