Confocal detection in ultra-high throughput screening

Today, ultra-high throughput screening (uHTS) methods enable screening of 100,000 samples/day or more to meet the demands for speed, with assay volumes miniaturised to 10 μl or below to reduce reagent consumption. Various methods have been employed and are now implemented on a common instrumental platform (EVOscreen™ by Evotec Technologies, Hamburg), which allows efficient selection of the best read-out mode for particular assay types, or even multiplexing of methods. Fluorescence correlation spectroscopy (PCS) is sensitive to changes in diffusion as seen when a fluorescent ligand binds to a receptor protein, for example. Fluorescence intensity distribution analysis (FIDA) allows detection of changes in specific brightness which could be caused for example by quenching, fluorescence resonance energy transfer (FRET) or accumulation of fluorophores on particles with multiple binding sites. By extending this method to 2-channel detection (2D-FIDA) using a polarising beamsplitter, anisotropy changes can be monitored, which is often the method of choice for small-ligand binding assays. Finally, using pulsed laser sources and single-photon counting electronics, fluorescence lifetimes can be measured providing a robust read-out parameter in various assay formats. We will present examples of assays and screens based on these detection modes which include the most important target classes and cover a variety of assay types. Several examples for these assay types and their validation for uHTS will be discussed.