Fluorescent readout

Expression systems with direct or indirect fluorescence readout... 

Pope, A.J., Haupts, U.M., Moore, K.J. Homogenous fluorescence readouts for miniaturized high-throughput screening theory and practice. Drug Discov. Today 1999, 4, 350-362. 

Fig. 28 a Single-photon fluorescence readout of data recorded by single-photon writing scale bar 100 ixm) b intensity profile (the direction is shown hy the arrows) of (a) c two-photon fluorescence readout of data recorded by single-photon writing (scale bar 100 xm) d intensity profile (the direction is shown by the arrows) of (c) e quadratic dependence of up-converted fluorescence of fluorene 17 on the input intensity. The smallest readout pattern achieved in this system was 3.5 xm... 

Fluorescence-based readouts build by far the most important basis of protease assays for HTS and compound profiling in the drug discovery industry today. These assay formats became increasingly popular in the 1990s (Burbaum and Sigal, 1997 Silverman et al., 1998). The basics of fluorescence are very well described in detail by Lakowicz (2006). This discussion will be restricted to biochemical protease assays in homogeneous formats based on fluorescence readouts that are suitable for HTS and automated compound profiling. 

With more than 500 members, the proteases constitute one of the largest families of enzymes in the human genome and are an important class of targets for drug discovery. Biochemical assays based on fluorescence readouts are the most frequently used technologies to elaborate the enzymatic mechanisms of proteases and to test the inhibitory potencies of drug-like chemical compounds. These assay formats exist for all classes of proteases and substrate specificities. 

Moger, J. et al. 2006. The application of fluorescence readouts in high-throughput screening. J. Biomol. Screen. 11, 765-772. 

For reading data, a similar two-photon process was employed. When isomer B molecules were excited by absorbing two 1064 nm photons, the excitation will cause only the written molecules to emit fluorescence. Although the fluorescence readout method is sensitive, the memory is erased during the reading process because the reverse reaction from the photoexcited isomer B to isomer A takes place, to some extent. Such a destructive readout... 

In the first example we have multiplexed two primary bioassays that utilize different reporter systems, i.e., visible and/or fluorescence readout. In... 

Chromogenic assays are colorimetric assays which use a substrate containing a chromophore. The substrate itself is designed such that it is colorless, and during the enzymatic reaction the chromophore is released and the color change is measured by a plate reader. This technique is applied for certain enzyme reactions for which a conversion of assays to fluorescent readout for HTS is Hkely to be problematic or even impossible, e.g., when it would require labehng of a small substrate with a large fluorophore. 

An integrated pTAS system for the detection of bacteria including lysis, DNA purification, PCR and fluorescence readout has also been published recently [113]. A microfluidic plastic chip with integrated porous pol5mier monoliths and silica particles for lysis and nucleic acid isolation was used for detection (Fig. 8). A custom-made base device provided liquid actuation and off-chip valving by stopping liquid flow from the exits of the chip, utilizing the incompressibility of liquids. Detection of 1.25 x 10 cells of B. subtilis was demonstrated with all assay steps performed on-chip. 

Many other detection techniques rely on fluorescence readouts. However, other types of measurements can be used too. Receptors have a long history of being amenable to... 

Gribbon, P., Sewing, A. Fluorescence readouts in HTS No gain without pain Drug Discov. Today 2003, 8,... 

There are four basic types of classical assays recep-tor/ligand assays, enzyme/substrate assays, anti-body/antigen assays, and cell-based assays, which use live cells and measure a cellular response. Assays which measure the association of two molecules are the simplest types of assays to develop, because the binding reaction can be followed by radioactivity or colorimetric or fluorescent readouts. There are several technical developments which reduce or eliminate the need for radioactivity. 

The most sophisticated mode of fluorescence readout in reasonably common use. Intensity = measurement of fluorescence intensity at fixed wavelengths Exc. ratio = measurement of fluorescence at two separate excitation wavelengths and a common emission band and ratioing of the two excitation amplitudes Emission ratio = measurement of fluorescence at a common excitation wavelength and two separate emission bands and ratioing of the two emission amplitudes. Some intensity measurements may be replaced in the future by excited-state lifetime measurements. 

High-content screen (HCS) is a phenotypic screening that monitors multiple cellular parameters simultaneously. HCS employs fluorescence-based reagents (antibodies, dyes that bind or localize to a given cell component, sensors) to generate a multicolor fluorescence readout that is usually recorded using automated optical image acquisition devices. 

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