PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 12867149

Highly sensitive fluorescence detection technology currently available for HTS.

Christian Eggeling·Leif Brand·Dirk Ullmann·Stefan Jäger
View original source ↗Export metadata ↓

Abstract

Homogeneous fluorescence methods are providing an important tool for HTS technologies. A wide range of different techniques have been established on the market, with read-outs ranging from total fluorescence intensity to statistical analysis of fluorescence fluctuations for biochemical assays or fluorescence imaging techniques for cellular systems. Each method has its own advantages and limitations, which have to be accounted for when designing a specific assay. Here, recently developed fluorescence techniques and some of their applications, with a particular focus on sensitivity, are summarized and their principles are presented.

Explore related subjects

Fluorescence PolarizationResearchTechnology, PharmaceuticalJournal ArticleReview

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Christian Eggeling, Leif Brand, Dirk Ullmann, Stefan Jäger. 2003-07-15. Highly sensitive fluorescence detection technology currently available for HTS.. https://doi.org/10.1016/s1359-6446(03)02752-1

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Characterization of chiral interactions using fluorescence anisotropy.

This work details a study whereby the characterization of chiral selectors and identification of optimal separation conditions is evaluated by steady-state fluorescence anisotropy measurements. Earlier studies in our laboratory have shown fluorescence anisotropy to be an effective tool in evaluating chiral recognition, and in this study, the feasibility of characterizing chiral separation systems by the technique is evaluated. Four chiral selectors were examined under various conditions to explore correlation between chiral separation ability and differences in the steady-state fluorescence anisotropy of the enantiomers measured under similar conditions. A good correlation between the fluorescence anisotropy data and separation data was observed with R2 values ranging from 0.9279 to 0.9959. The fluorescence anisotropy measurements were examined under conditions that mimicked chiral separation conditions and the feasibility of a priori optimization of chiral separations is discussed.

Fluorescence Polarization↗
PubMed2006

Single-molecule detection and identification of multiple species by multiparameter fluorescence detection.

Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.

Fluorescence Polarization↗
PubMed2006

A fluorescence polarization assay for screening inhibitors against the ribonuclease H activity of HIV-1 reverse transcriptase.

A fluorescence polarization (FP) microplate assay suitable for screening compounds against the ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase has been developed. This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labeled with 6-carboxytetramethyl rhodamine at the 3' end that is annealed to a complementary unlabeled DNA strand. The labeled RNA/DNA duplex demonstrated Michaelis-Menten kinetics with a Km value of 9.6+/-2.8 nM. Substrate cleavage by RNase H to produce small RNA fragments (1-4 mer) resulted in a large change in the measured FP value. This FP assay was amenable to kinetics protocols as well as stopped endpoint measurements. When using the latter for conducting robotics runs, Z' values greater than 0.8 typically were observed. The stopped endpoint FP assay was used successfully in a high-throughput screening campaign to screen 1.8 million compounds for RNase H inhibition.

Fluorescence Polarization↗