Polarisation fluorescence

We have also used a non-radiometric-binding approach based on fluorescence polarisation [29], where a fluorescent label is used in place of a radiolabel. As the fluorescently tagged oxytocin binds to the receptor, its rotational velocity is reduced and the polarisation of the fluorophore increases. The displacement of the ligand may be measured by a decrease in polarisation. 

The availability of MIP microparticles through this synthetic method has also stimulated the development of analytical techniques that make use of them as sensing elements. Apart from competitive radioassays [30] and immunoassays [32], which were already performed with ground bulk polymers, the small, regular size of the beads prepared by dispersion/precipitation polymerisation enables their use in CEC [45, 46], scintillation proximity assays [35], fluorescent polarisation assays [47], and chemiluminescence imaging [48]. 

Several configurations for the sensor are possible. An especially viable alternative would seem to be the competitive displacement of fluorescent label. Since this is an equilibrium, fouling or contamination of the surface should not alter the absolute result. Krull et al (75) have reported the reproducible immobilisation of a stable phospholipid membrane containing fluorophore in this context. Concurrent fluorescence polarisation measurements can offer the possibility of multidimensional analysis (76) and are in any case experiencing a rejuvenation of interest as a highly selective technique, when the effective molecular weight of the antibody is increased relative to the antigen, by immobilisation on a latex or metal particle (77)... 

The results discussed in this chapter demonstrate that 2H NMR is a powerful technique for investigating microscopic properties in rubber networks. Most of the experiments described here are easy to handle on standard NMR equipment. Due to the absence of interactions between 2H nuclei, spectra and line shapes are easy to interpret and give quite direct information, at least in the first step of analysis, which is that generally required to correlate microscopic to macroscopic properties in these systems. Additionally, in contrast to optical techniques (as birefringence, infrared dichroism, fluorescence polarisation) the information which is obtained is very specific, because spatial and temporal averaging processes are clearly distinguishable in NMR. 

X-band cavity changes the fluorescence polarisation, which can be detected. The main features of the apparatus are illustrated in figure 11.12. One of the advantages of excitation with an electron beam, rather than with conventional monochromatic or white light sources, is that transitions between electronic states of different spin multiplicity are allowed consequently both singlet and triplet excited states can be populated. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

In hair analysis, three methodically different lA with different kinds of labeling are used radioimmunoassay (RIA), which is the most common lA in hair analysis, enzymeimmunoassay (EIA), and fluorescence polarisation immunoassay (FPIA). In hair analysis, lA have the same advantages and disadvantages as compared to their use in urinalysis. They are fast, easy to handle, and can be automated. Their use can save time and expenses when a great number of negative samples has to be expected. 

Maurer, H. H., Fritz, C. R, Toxicological Detection of Pholcodine and Its Metabolites in Urine and Hair Using Radio Immunoassay, Fluorescence Polarisation Immunoassay, Enzyme Immunoassay and Gas Chromatography-Mass Spectrometry, Int. J. Leg. Med., 104, 43,1990. 

H.H. Maurer and C.E Fritz. Toxicological detection of pholcodine and its metabolites in urine and hair using radio immunoassay, fluorescence polarisation immunoassay, enzyme immunoassay and gas chromatography-mass spectrometry. Int. ]. Leg. Med. 104, 43 6, 1990. 

Fluorescence polarisation immunoassay, 154 Fluorescence spectrophotometers, 233 standardisation, 234... 

Fluorescence Polarisation coupled with confocal spectroscopy... 

FPIA = Fluorescence Polarisation Immunoassay (Abbott, TD,). El A = Enzyme Immunoassay. 

Fig. 2 Details of the crystal structures of the active site of Nt-Hsp90 (see legend to Fig. lc) and selected solvent molecules for virtual screening hit compounds 3,4,5 and 6 binding to Nt-Hsp90. The affinities quoted are IC50 values for binding in a fluorescence polarisation assay [32]...

IC50 values measured using a fluorescence polarisation assay... 

Fig. 6 Fragment growth to identify the clinical candidate AUY922. IC50 was measured in a fluorescence polarisation assay GI50 values are for growth inhibition of HCT116 cells. See text for details...

Fluorescence polarisation studies on acenaphthylene labelled polyacrylic acid has demonstrated complexation occurs with polyethylene oxide to give a tightly packed and sterically restricted conformation SS. Neutralisation of the acidic groups restored the polarisation of the fluorescence and together with time resolved fluorescence analysis indicated that the motion of the probe is anisotropic. Solvent partitioning has been observed between glycol methacrylate copolymers from the fluorescence anisotropy changes of... 

A similar effect occurs in the detection light path. A part of the Z component of the fluorescence is detected both in the Is and in the Ip channel. As a result, a substantial amount of fluorescence polarised in the Z direction is recorded, i.e. additional Is. The total intensity is therefore not / = 7, 4- 2 /j as in the case of narrow beam angles but... 

Two-color primer extension assay with real time monitoring of fluorescent polarisation Mass spectrosopy - (MS)... 

A falsely elevated imipramine level was recorded when HPLC was used to determine serum imipramine levels in a patient taking imipramine, quetiapine, fluvoxamine, lithium and docusate. The abnormal readings were found to have been caused by a metabolite of quetiapine, and normal readings were obtained by altering the wavelength for detection of imipramine. Nortriptyline levels have been found to be falsely elevated in a patient also taking quetiapine when blood was analysed using fluorescence polarisation immunoassay, but were normal when an HPLC... 

Asian or Chinese ginseng (Panax ginseng) and Siberian ginseng (Eleu-therococcus senticosus) have both been found to interfere with some digoxin assays including fluorescence polarisation immunoassay (FPIA) and microparticle enzyme immunoassay (MEIA). ... 

Thus, the time-resolved measurement of such membrane probes contains information on the dynamics of the hindered probe rotation, often interpreted as the micro-viscosity, and about the hindrance of this rotation, usually interpreted as the static packing arrangement of the lipids or the so-called membrane order [136, 137]. Fluorescence polarisation studies in membranes, however, exhibit some major limitations the experimentally determined steady-state and time-re-solved anisotropies characterize the motional restrictions of the reporter molecule itself and give therefore only indirect information about the dye environment, with the consequence that, if the probe is bound covalently to the lipid (TMA-DPH), this attachment may dominate the recorded depolarisation behaviour. The membrane order parameters obtained from freely mobile probes like (DPH) result from a broad distribution of localisation within the hydrophobic interior, the detailed characterisation of which reveals inherent ambiguities [138]. 

Fluorescence polarisation spectroscopy is still very much used to probe the rotational dynamics of single molecules, either on surfaces or in solution [152]. In bioa-nalytical assays the fluorescence emission intensity is measured as a function of rotational speed. When a solution of fluorophores is excited with polarised light, the fluorophores selectively absorb those photons that are parallel to the transition moment of the fluorophore, resulting in photoselective excitation. The fluorophore molecules rotate to varying extents during the fluorophore lifetime. If the fluores-... 

There have been several new developments in fluorescence polarization immunoassay (FPIA). Lackowicz and Terpetschnig reviewed the use of long-lifetime metal-ligand complexes in fluorescence polarization assays [154, 155]. New complexes with Re(l) and Ru(II) were described for the highly sensitive detection of high-molecular weight analytes by FPIA. Laser-induced fluorescence polarisation detection has been used by Yatscoff and coworkers in capillary electrophoresis detection (CE-LIFP) [156]. For the analyte cyclosporin picomolar detection limits were attained. 

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