HTS fluorescence

Bard etaL 5S6>5571 and Visco etaL 558) have quantitatively analyzed the intensity of pulsed ECL of 9,10-diphenylanthracene, tetraphenylpyrene and rubrene. By computer simulation of the electrode process and the subsequent chemical reactions the rates for chemical decay of the radical ions could be determined. Weaker ECL with fluorescence emission 559 or electrophosphorescence S60) occurs if the radical anion R - reacts with a dissimilar radical cation R,+ of insufficient high oxidation potential to gain enough energy for fluorescence emission, that is, if ht fluorescence) >23.06 (Ej >+. -Ej -.), e.g., in the annihilation of the anthracene radical anion with Wurster s blue. For these process the following schemes are assumed (Eq. (242) ) ... 

The following sections give an overview and some examples of the fluorescence detection methods currently used in HTS fluorescence intensity (FI) fluorescence polarization (FP) or fluorescence anisotropy (FA) fluorescence resonance energy transfer (FRET) Hfetime-based measurements (TRF and FLT) fluorescence corre-... 

HT FLUORESCENCE-BASED INVESTIGATION OF THE IMPACT OF LATEX COMPONENTS ON THE EFFICACY OF BIOCIDES... 

HT Fluorescence Nonselective - fluorogenic Single rCYP Fluorescence High... 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

The truth most likely lies somewhere in between. Bender [67] published the most quantitative study to date on the success of HTS at Novartis. Several conclusions could be drawn. Particular target types and assay technologies have a great impact on screening success, and this was not always correlated to the number of identifying hits in the HTS runs. For assay formats used a minimum of five times, LC/MS readouts succeed 83% of the time, followed by FP assays, which succeed in 72% of the cases. TR-FRET showed a success rate of 70%, with FLIPR assays (61%), fluorescence intensity readouts (59%), and AlphaScreen (60%) performing... 

Turconi S, Bingham RP, Haupts U, Pope AJ (2001) Developments in fluorescence for lifetime-based analysis for ultra-HTS. Drug Discov Today 6 27-39... 

Huang CC, Chang HT (2006) Selective gold-nanoparticle-based tum-on fluorescent sensors for detection of mercury(II) in aqueous solution. Anal Chem 78 8332-8338... 

Clapp AR, Medintz IL, Uyeda HT, Fisher BR, Goldman ER, Bawendi MG, Mattoussi H (2005) Quantum dot-based multiplexed fluorescence resonance energy transfer. J Am Chem Soc 127 18212-18221... 

HTS screening is often based on miniaturized cell assays that enable chemical libraries to be screened for molecules that present different biological activities and sometimes-different biological targets using fluorescence, scintillation proximity assays (SPA) and luminescence as detection techniques. 

One very important device is the plate reader, which can be rate limiting in HTS. Most laboratories use multimodal readers that can detect various forms of fluorescence as well as luminescence and absorbance. The traditional readers are photomultiplier-based devices that usually read from one well to the next. This process can take considerable time for 384-well and higher-density plates. A more desirable HTS reader type images the entire plate with a charge-coupled device (CCD) camera. The latter device is usually a faster reader for 384-well and higher-density plates. Imagers can capture significant cross talk from one well to another, but with proper set up, they can produce data of equal quality. 

Many scientists would argue that the flexibility and reliability of SPA more than offset these potential disadvantages, and SPA is still in wide use for HTS today. Nevertheless, methods that generate fluorescent and luminescent signals without radioisotopes have become favorites in some HTS laboratories. These fluorescence- and luminescence-based methods,... 

Eggeling, C., Brand, L., Ullmann, D., and Jager, S., Highly sensitive fluorescence detection technology currently available for HTS, Drug Discov. Today, 8, 632, 2003. 

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

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