Intensity quenching

Figure 4.9. Stem-Volmer intensity quenching of (a) Ru(bpy)32+, (b) Ru(phen)32 and (c) Ru(Ph2phen)32+ on hydrophilic Cab-O-Sil silica disks. The solid lines are the best fits using a two-parameter Freundlich adsorption model. (Reprinted from Ref. 33 with permission. Copyright 1991, American Chemical Society.)...

We turn now to intensity quenching measurements (/o// versus [Q]). As we will show, these measurements are even less sensitive for detecting complex models than are lifetime measurements. On the plus side, however, they show a remarkable ability... 

Even though the two-site model has shortcomings, it is excellent for fitting intensity quenching curves. Thus, it has excellent predictive and calibration properties, has a chemically sound basis, and (at least for inorganic complex sensors) is preferable to the less accurate power law calibration equation. 

Our results demonstrated clearly that the lifetime data are more sensitive to subtleties of the micromechanistic photophysics. In this case we were able to establish inadequacies of the two-component model that were not detected by intensity quenching measurements alone. It is also clear that resolution of the detailed mechanism in these complex polymer systems will require even better lifetime data than we are able to obtain with a conventional flash lamp-based time-correlated photon counting system. 

We have examined numerous models including discrete and continuous z distributions. Remarkably, the best fit to all the intensity quenching data involved quenching by surface-bound O2 where the adsorption was described by a Freundlich isotherm ... 

A single Gaussian distribution of conformations (unless pathologically wide (R > 1)) shows little detectable nonlinearity in Stern-Volmer intensity quenching curves. 

An advantage of the inability to detect single Gaussian distributions by intensity data is that intensity quenching data (even complex distribution functions of two sites) can be reliably modeled using a discrete two-site model. This has obvious practical implications in sensor design and calibration. 

The Ksv value shows the importance of fluorophore accessibility to the quencher, while the value of A q gives an idea of the importance of the diffusion of the quencher within the medium. Figure 10.1 shows a Stern-Volmer plot of fluorescence intensity quenching with iodide of flavin free in solution and of flavin bound to flavocytochrome ba- The Ksv values found are 39 and 14.6 M-1 for free and bound flavins, respectively, i.e., values of kq equal to 8.3 x 109 and 3.33 x 109 M-1 s-1, respectively. Accessibility of flavin to KI is more important when it is free in solution, and the presence of protein matrix prevents frequent collisions between iodide and FMN thereby decreasing the fluorophore accessibility to the quencher. Also, as revealed by the Aq values, diffusion of iodide in solution is much more important than in flavocytochrome b2. The protein matrix inhibits iodide diffusion, thereby decreasing the A q value. 

Figure 10.2 shows a Stern-Volmer plot for the fluorescence intensity quenching by oxygen of zinc protoporphyrin IX embedded in the heme pocket of apomyoglobin (Mbdes Fe). The slope of the plot yields Ksv and A q values of 15.96 M-1 s-1 and 7.6 x 109 M-1 s-1,... 

Figure 10.1 Stern-Volmer plots of fluorescence intensity quenching with iodide of FMN free in solution (plot b) and of FMN bound to flavocytochrome b2 (plot a). Reproduced from Albani, J.R., Sillen, A., Engel borghs, Y. and Gervais, M. (1999). Photochemistry and Photobiology, 69, 22-26, with the permission of the American Society for Photobiology.

Figure 10.4 Fluorescence intensity quenching of L-Trp by Kl. Stock solution = 4 M Kl + 10 3 Na2SC>4.

Figure 10.9 Stern-Volmer plots of the fluorescence intensity quenching of by oxygen. The Arg...

If students have access to a fluorescence lifetime instrument, it would be useful to see how one can measure fluorescence lifetime. In this case, it will be useful if students can perform the experiments described by following fluorescence lifetime quenching with KI and compare their results with intensity quenching experiments. 

Figure 13.6 Fluorescence intensity quenching of fluorescein bound to LVA, as a result of STF-LCA (a) and of LCF-LCA (b) interactions. [LCA] = 0.7 /xM. Source Albani, J. R., Debray, H., Vincent, M. and Gallay, J. (1 997). Journal of Fluorescence, 7, 293-298. Albani, J.R., Debray, H., Vincent, M. and Gallay, J. (1997). Journal of Fluorescence, 7, 293-298. Figure No. 1. With kind permission of Springer Science and Business Media (1, 2, and 3).

Figure 14.2 shows fluorescence intensity quenching of 4,6-diamidino-2-phenylindole (DAPI) complexed to DNA in the presence of two concentrations of Acridine Orange. In fact, one can see that while the fluorescence intensity of DAPI decreases, that of Acridine... 

Figure 15.9 Stern-Volmer plot of fluorescence-intensity quenching of free Trp in solution with TNS.

Figure 2 Fluorescence intensity quenching [(/q//)- 1] of AF probes in SC as a function of iodide (KI) concentration. Data represents the average S.D. and are obtained from three different pieces of skin. Symbols solid triangles (2-AF) open triangles (6-AF) solid circles (9-AF) open circles (12-AF) solid squares (16-AF).

The amount of fluorescence intensity quenching can be quantitatively related to the partial pressure of oxygen in a sample from the simplified Stern-Volmer equation ... 

If the fluorescent emission spectrum of the bound labeled ligand is sufficiently displaced, enhanced or decreased in intensity (quenched) relative to that of the free labeled ligand, the resulting spectroscopic measurements can be used for quantitation without a separation step. Additionally, the techniques previously described in enzyme immunoassays, such as reactant-labeled immimoassay, can form the bases of fluorescent... 

The curves in Fig. 1 demonstrate the decrease of PL intensity (quenching) and the red shift of PL maximum with the voltage increased. At the values of electrical field strength E up to 10 V/cm the PL of nanorods is quenched more than PL of QDs. However, the wavelength shift of PL maximum with applied electric field for nanorods increases very weak. Evidently, due to the elongated shape of nanorods, the external electric field effect may differ for S- and P-polarized PL. This property is important for application of this material in optoelectronic nanodevices. To understand reasons of the electric field effect difference between QDs and nanorods, the mechanism of nanorods PL quenching has to be studied. The quantum-confined Stark effect is probably not the single factor in force. 

Titration curves of HS fluorescence quenching versus concentration of added metal quencher have been used to obtain the CC values of HS ligands and the stability constants of HS-metal complexes (Saar and Weber, 1980, 1982 Underdown et al., 1981 Ryan et al., 1983 Weber, 1983 Dobbs et al., 1989 Grimm et al., 1991 Hernandez et al., 2006 Plaza et al., 2005, 2006). Two fluorescence techniques, lanthanide ion probe spectroscopy (LIPS) and fluorescence quenching of HSs by Cu-+, have been used in conjunction with a continuous distribution model to study metal-HS complexation (Susetyo et al., 1991). In the LIPS technique, the HS samples are titrated by Eu-+ ions, and the titration plot of the ratio of the intensities of two emission lines of Eu + is used to estimate the amount of bound and free species of the probe ion. In the other technique, titration curves of fluorescence intensity quenched by Cu versus the logarithm of total added Cu2+ are used. 

Fluorescence intensity quenching with iodide can be used to find out whether ethidium bromide added to DNA is intercalated into the double helical DNA or present at the surface of the DNA. In fact, since DNA is negatively charged, addition of iodide ion to a DNA-EB complex will not decrease the fluorescence intensity of the ethidium bromide if the fluorophore is intercalated into the DNA. However, if the fluorophore is bound to the DNA surface, its fluorescence will be quenched by iodide ion. 

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