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  • Title: Fluorescence-quenching-based homogeneous caspase-3 activity assay using photon upconversion.
    Author: Vuojola J, Riuttamäki T, Kulta E, Arppe R, Soukka T.
    Journal: Anal Chim Acta; 2012 May 06; 725():67-73. PubMed ID: 22502613.
    Abstract:
    Caspase proteases are key mediators in apoptosis and thus of great interest in pharmaceutical industry. Enzyme-activity assays are commonly employed in the screening of protease inhibitors that are potential drug candidates. Conventional homogeneous fluorescence-based assays are susceptible to autofluorescence originating from biological material. This background autofluorescence can be eliminated by using upconverting phosphors (UCPs) that emit visible light upon excitation at near-infrared. In the assay energy was transferred from a UCP-donor to a conventional fluorophore acceptor that resided at one end of a caspase-3-specific substrate peptide. Attached to the other end was a quencher molecule that was used to attenuate the acceptor emission through intramolecular energy transfer in an intact peptide. In non-inhibitory conditions the enzyme reaction separated the fluorophore from the quencher and the emission of the fluorophore was recovered. The method was applied for the detection and characterization of a known caspase-3 inhibitor Z-DEVD-FMK, and the assay gave IC(50) values of approximately 13 nM for this inhibitor. We have demonstrated the applicability of UCPs on a fluorescence-quenching-based homogeneous enzyme-activity assay for the detection of caspase-3 inhibitors. The use of near-infrared excitable UCPs enables inexpensive instrumentation and total elimination of autofluorescence, while the use of an internally quenched substrate molecule diminishes the background resulting from radiatively excited acceptor molecules. The reduction of autofluorescence and radiative background result in high signal-to-background ratios (ratios of approximately 100 were obtained). By further utilizing assay miniaturization and signal enhancement in a white microtitration plate, a significant reduction in the reagent consumption can be achieved rendering the assay applicable for high-throughput screening.
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