Fluorescence kinetic-based

Fluorescence Kinetic-Based Measurements. Our studies of the reaction rate determination of thiamine (vitamin Bl) will be used to demonstrate the unique capabilities of rapid acquisition of spectra in kinetic measurements. The kinetic method is based on the oxidation of thiamine by Hg + in basic solutions to highly fluorescent thiochrome (16) The initial rate, taken as the change in fluorescence signal at 444 nm that occurs in a fixed time after mixing the sample and reagents, is directly proportional to the thiamine concentration. 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

Kinetics of Energy and Electron Transfer. A semi-quantitative estimate for the rate constants of the various photophysical processes can be obtained from fluorescence quenching. Based on the quenching ratios of the OPV fluorescence and the OPVn singlet excited state lifetimes, the rate constants for energy transfer reactions in toluene solutions were estimated to lie between 1.1 x 1012 and 2.1 x 1012 s-1 for OPV3 Cgo and OPV4 Cgo (Table... 

Leissring, M.A. et al. 2003. Kinetics of amyloid /3-protein degradation determined by novel fluorescence polarization-based assays. J. Biol. Chem. 278, 37314—37320. 

A more detailed picture of the energy-transfer sequence was revealed by additional kinetic analysis. Mimuro et al. obtained rise and decay curves for the individual chromophores by deconvolution ofthe time-resolved fluorescence spectra based on the relative intensities obtained by photon counting. The time-resolved fluorescence spectra could be computer-fitted to the reported emission spectra of nine major chromophores four belonging to the rod PC and the remaining five to the APC-core complex. 

For measurement of steady-state fluorescence, the rapid acquisition of spectra is a convenience but not essential. However, the rapid acquisition of spectral information is essential for measurement of transient species or where the luminescence signal continually changes with time. This will be demonstrated first for kinetic-based luminescence measurements and then chemiluminescence measurements. 

In order to describe the fluorescence kinetics at F (PS II centers closed), four exponential components were required. The corresponding DAS are shown in Fig. 1. The fastest component (t =100 ps) has been attributed to PS I previously (7,8). The second component (x =380 ps) has been resolved for the first time and together with the 1.34 ns and 2.08 ns component we attribute it, based on its spectrum and its dependence on the state of the PS II center, to PS II (8). No lifetime >2.1 ns could be resolved. Extending the fitting window from 6 ns to 18 ns yields virtually identical results, indicating the absence of any longer-lived fluorescence component within the limits of resolution. 

Many experimental methods such as fluorescence, reflection Fourier transform infrared, NMR, quartz resonators, and acoustic wave admittance analysis, are used to study diffusion of solvents. " Special models have been developed to study process kinetics based on experimental data. 

Fig. 7.3 Response kinetics of optical (fluorescence) sensors based on ruthenium complex incorporated in IL-modified sol-gel matrix (Reprinted with permission from Oter et al. (2009). Copyright 2009 Elsevier)...

According to reference [13], at low temperatures the R.>ta6>tam fluorescence kinetics change with the observation wavelength in case of both open and closed (special pair oxidized) RCs. With closed RCs most fluorescence kinetics at 77 K could be well approximated by the sum of two exponents with 15-5 ps and 170-20 ps decay constants. Based on our model, the former of these constants allow us to estimate the average time for excitation stay in the lattice site, which is 5-8 ps, and the approximate value of the lattice constant, which is 32 8. 

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Based on analogies we have cited, the kinetic scheme proposed for heavy-atom fluorescence quenching is reasonable and would predict the following relationship for fluorescence quenching ... 

A method has been proposed for evaluation of LIP concentration in living cells based on the fluorescent probe calcein (Breuer etai, 1995 Epsztejn etai, 1997). The authors assumed that calcein bound Fe2+. However, a more recent thermodynamic and kinetic study indicates that the preferred form bound is Fe3+, and that calcein is a good chemosensor of Fe3+ (Thomas, etai., 1999). The iron... 

Kinetic treatments are usually based on the assumption that reaction does not occur across the micelle-water interface. In other words a bimolecular reaction occurs between reactants in the Stern layer, or in the bulk aqueous medium. Thus the properties of the Stem layer are of key importance to the kineticist, and various probes have been devised for their study. Unfortunately, many of the probes are themselves kinetic, so it is hard to avoid circular arguments. However, the charge transfer and fluorescence spectra of micellar-bound indicators suggest that the micellar surface is less polar than water (Cordes and Gitler, 1973 Fernandez and Fromherz, 1977 Ramachan-dran et al., 1982). 

The micellar surface appears to be less polar than water, based largely on shifts in fluorescence or charge-transfer spectra (Section 1). Although it may not be reasonable to apply bulk solvent parameters such as Z or dielectric constant to submicroscopic species such as micelles, the spectral and kinetic evidence are self-consistent. An additional point is that these reactions have... 

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