Fluorescence polarity parameter

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

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. 

This important parameter can provide information on the excited states. For instance, when the dipole moment of a fluorescent molecule is higher in the excited state than in the ground state, the Stokes shift increases with solvent polarity. The consequences of this in the estimation of polarity using fluorescent polarity probes is discussed in Chapter 7. 

Fluorescence polarization measurements can thus provide useful information on molecular mobility, size, shape and flexibility of molecules, fluidity of a medium, and order parameters (e.g. in a lipid bi layer)2 . 

Fluorescence polarization 1) steady state 2) time-resolved emission anisotropy rotational diffusion of the whole probe simple technique but Perrin s Law often not valid sophisticated technique but very powerful also provides order parameters... 

Fluorescence polarization is the subject of Chapter 5. Factors affecting the polarization of fluorescence are described and it is shown how the measurement of emission anisotropy can provide information on fluidity and order parameters. 

W. van der Meer, R. B. van Hoeven, and W. J. van Blitterswijk, Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores, Biochim. Biophys. Acta 854, 38-44 (1986). 

M. Shinitsky and Y. Barenholtz, Fluidity parameters of lipid regions determined by fluorescence polarization, Biochim. Biophys. Acta 515, 367-394(1976). ... 

Keywords Fluorescence probing Hemimicelle Micellar fluidity Micelle Organized assemblies in solution and interfaces Polarity parameter Pyrene... 

Table 1. Binding constants (K) for host-guest complexes of artificial receptors with 11, and microenvironmental polarity parameters ( ) and steady-state fluorescence polarization values (P) for the guest bound to hosts in aqueous solution at 30.0 °C ...

Many of the fully aromatic 6-6-5 tricyclics show good fluorescence properties, and in some instances this has been utilized in biochemical applications. The reader is directed to the literature cited in Section 7.22.12.1 (benzo-separated purines) for examples of how the sensitivity of the fluorescence properties of tricyclic fluorophores to environmental conditions can be utilized to analyze the binding parameters associated with biomacromolecules. Of particular interest in this regard is a method known as fluorescence-polarization titration (83B2347). 

Differential solvent interactions with ground- and excited-state molecules not only lead to shifts in the fluorescence maxima but also to perturbation of the relative intensities of the vibrational fine structure of emission bands. For instance, symmetry-forbidden vibronic bands in weak electronic transitions can exhibit marked intensity enhaneements with increasing solute/solvent interaction [320, 359]. A particularly well-studied ease is the solvent-influenced fluorescence spectrum of pyrene, first reported by Nakajima [356] and later used by Winnik et al. [357] for the introduction of an empirical solvent polarity parameter, the so-called Py scale cf. Section 7.4. 

M. Shnitzki and G. Barenholz, Fluidity parameters of lipid regions determinated by fluorescence polarization, Biochim.Biophys.Acta. 515 (1978) 367-394. 

FIGURE 9. Correlation of the fluorescence spectra of HPTA in pure solvents with the Kamlet-Taft solvent-polarity parameters ... 

FIGURE 20. The poor correlation found between the fluorescence maximum of IN and solvent polarity using Kamlet-Taft solvent-polarity parameters of 15 common solvents (from Reference 91)... 

The polarization of the fluorescence emitted at 90° is determined by measuring the intensities of the vertically polarized fluorescence (J ) and of the horizontally polarized fluorescence (li), i.e., the intensities after the fluorescence has passed through a polarizer with the polarizing axis set at the vertical and horizontal orientations, respectively. The resultant parameter is ihe fluorescence polarization p. 

Enzyme activities may also be measured in urine, cerebrospinal fluid, bone marrow cells or fluid, amniotic cells or fluid, red blood cells, leukocytes, and tissue cells. Cytochemical localization is possible in leukocytes and biopsy specimens (e.g., from liver and muscle). Under ideal conditions, both the concentration of the enzyme and its activity would be measured. Radioimmunoassay (RIA) and its alternative modes such as fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), and chemiluminescence immunoassay (CLIA) (discussed later), can be used to measure enzyme concentration as well as other clinically important parameters. 

Plots of the energy E ) of the excited singlet state and of the fluorescence quantum yield ( 0 t ) as function of the solvent polarity parameter (is t (30)) for three N-arylaminonaphthalene sulfonate probes in dioxane/water mixtures... 

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