High fluorescence polarization

L-pyrenyldiazomethane to form stable, highly fluorescent L-pyrenyhnethyl monoesters (87). These esters have been analy2ed in human blood by ce combined with lif detection. To mimini e solute adsorption to the capillary wall, they were coated with polyacrjiamide, and hydroxypropyl methylceUulose and dimethylfoTTnamide were used as buffer additives to achieve reflable separations. Separation was performed in tris-citrate buffer, pH 6.4, under reversed polarity conditions. The assay was linear for semm MMA concentrations in the range of 0.1—200 p.mol/L. 

Figure 12 Resynthesized library hits identified from the high throughput fluorescence-polarization assay along with their Src SH2 binding data.

Jameson DM, Croney JC (2003) Fluorescence polarization past, present and future. Comb Chem High Throughput Screen 6 167-173... 

Guo XQ, Castellano FN, Li L, Lakowicz JR (1998) Use of a long lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular weight analytes. Anal Chem 70 632-637... 

Most of the reported methods of analysis of valproic acid and its sodium salt in biological fluids rely on the use of chromatography, especially gas chromatography, although high performance liquid chromatography (HPLC) is also reported. Other methods, such as flow injection analysis, enzyme-immunoassay, fluorescence-polarization capillary electrophoresis, and potentiometry are sometimes used. The reported methods can be classified as follows. 

Nitrobenzoxadiazole. NBD derivatives are environmentally sensitive. In apolar solvents, they are highly fluorescent. When dissolved in polar solvents, the fluorescence intensity decreases significantly. Semi-empirical calculations can explain these changes of quantum yields in terms of decreased energy gaps between the... 

Fisz, J. J. (2007). Fluorescence polarization spectroscopy at combined high-aperture excitation and detection Application to one-photon-excitation fluorescence microscopy. J. Phys. Chem. A 111, 8606-21. 

Rizzo, M. A. and Piston, D. W. (2005). High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy. Biophys. J. 88, L14—6. 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Both vacuoles and cytoplasm can be visualized by ester-loading imper-meant fluorochromes. The fluorochrome 6CF can be introduced by ester-loading with 6CF-diacetate, which is not fluorescent or polar and readily permeates cells. Once inside the cell, it is cleaved into the highly fluorescent and charged anion 6CF, which is ion-trapped. Lipophilic FITC derivatives are compartmented in patterns that depend on the subcellular location of esterases, and in different cells may be compartmented by cytoplasm or vacuole (51). 

One of the most well known polarity probes is ANS (l-anilino-8-naphthalene sulfonate), discovered by Weber and Lawrence in 1954. It exhibits the interesting feature of being non-fluorescent in aqueous solutions and highly fluorescent in solvents of low polarity. This feature allows us to visualize only hydrophobic regions of biological systems without interference from non-fluorescent ANS molecules remaining in the surrounding aqueous environment. 

Nasir, M.S. and Jolley, M.E., Fluorescence polarization an analytical tool for immunoassay and drug discovery, Comb. Chem. High Throughput Screen., 2,177,1999. 

Owicki, J.C., Fluorescence polarization and anisotropy in high throughput screening perspectives and primer, /. Biomol. Screen., 5, 297, 2000. 

Burke, T.J., Loniello, K.R., Beebe, J.A. and Ervin, K.M. (2003) Development and application of fluorescence polarization assays in drug discovery. Combinatorial Chemistry High Throughput Screening, 6, 183-194. 

Figure 2.3 Relationship between the concentration of teicoplanin in the serum of patients as determined using high-performance liquid chromatography (HPLC) versus fluorescence polarization immunoassay (FPIA) (r = 0.974). (From McCann et al., 2002.)...

McCann S.J., O. White, and B. Keevil (2002). Assay of teicoplanin in serum Comparison of high-performance liquid chromatography and fluorescence polarization immunoassay. Journal of Antimicrobial Chemotherapy 50 107-110. 

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