Fluorescence-based assays

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Flavonoids, physiological and nutritional aspects, 14 (1977) 285 Fluorescence-based assays, 43 (2005) 19 Fluoroquinolone antibacterial agents,... 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

The process of combining cyanine and squaraine dyes by encapsulation, or covalent or noncovalent attachment with macrocyclic hosts, macromolecules, and micro- or nano-particles is a promising way to design novel probes and labels with substantially improved properties and for the development of advanced fluorescence-based assays. Nevertheless, the physicochemical properties of these dye-compositions are strongly dependent on the dye structure as well as the nature of the host macrocycle, macromolecule, or particle. Finally, development of new methods to synthesize these tracers can also be considered a challenging task. 

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

It is apparent that signal amplification provides increased sensitivity over direct labeling. This is especially true for fluorescent-based assays. One of the most sensitive signal detection technologies is the immunoRCA (Schweitzer et al., 2000). Rolling circle amplificahon (RCA) is combined with antibody detection. RCA involves the amplification of circularized oligonuceotide probes under isothermal conditions by DNA polymerase (Lizardi et al., 1998). With immunoRCA, the 5 primer is attached to the reporter antibody. Initiation of the amplification starts when circular DNA template binds to the attached primer. 

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

Fig. 8. Scheme for fluorescence-based assay used in the determination of enantioselectivity of ester hydrolysis (80). 

Robertson, B. and Knox, R.J. (2004) A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells. Journal of Biomolecular Screening, 9, 671-677. 

DeLange RJ, Glazer AN. Phycoerythrin fluorescence-based assay for peroxy radicals a screen for biologically relevant protective agents. Analytical Biochemistry. 1989 177(2) 300-306. 

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e],... 

Endo, T., A. Okuyama, Y. Matsubara, et al. 2005. Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls. Anal. Chim. Acta 531 7-13. 

G10. Glazer, A. N., Phycoerythrin fluorescence-based assay for reactive oxygen species. Meth. Enzymol. 186, 161-168 (1990). 

BP Holskin, M Bukhtiyarova, BM Dunn, P Baur, J de Chastonay, MW Pennington. A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate. Anal Biochem 226 148-155, 1995. 

Cihlar T, Ho ES. Fluorescence-based assay for the interaction of small molecules with the human renal organic anion transporter 1. Anal Biochem 2000 283 49-55. 

The sensitivity of fluorescence-based assays can hence be greatly improved by the use of dye-doped silica nanoparticles and this approach has been pioneered and subsequently deeply investigated by Tan and coworkers.15 Their luminophore of choice was the water-soluble, positively charged tris(2,2 -bipyridyl)dichlororuthenium(II) [Ru(bpy)3]2+ hydrochloride that can be easily incorporated into silica nanoparticles prepared using the reverse microemulsion method. The charge complementarity between the dye and the silica matrix prevents leaching from the particles.15... 

Several approaches have been reported for the screening of polymerase activity, for example radioisotope assays such as scintillation proximity assays [56, 57] or fluorescence-based assays [58-61], Most of these assays, however, suffer from use of tedious procedures, the use of radioisotopes, or use of expensive reagents for fluorescence signal generation. A convenient means of online monitoring of DNA polymerase activity has recently been presented by Andreas Marx and Daniel Summerer [62]. Their technique involves a DNA template that forms a stable hairpin structure labeled at two positions ... 

Fluorescent-Based Assay Formats to Identify CFTR Potentiators... 

Labeled chemokines have been generated by a variety of procedures including fusion with alkaline phosphatase (1), derivatization with fluorescent probes (2), and radiolabeling (3,4). While homogeneous fluorescent based assays (fluorescence polarization, fluorescence energy transfer, etc.) will become... 

High throughput flash luminescence readers such as the FDS6000 and 7000, as well as the Lumax Flash HT, enable functional GPCR and calcium channel testing. A sensitive photon-counting CCD camera enables aequorin and luciferase activity to be measured by flash luminescence. Thus, these advances allow fluorescence-based assays to be replaced by luminescence-based assays,... 

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