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Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening.

Buehler, Christof and Dreessen, Joerg and Mueller, Kurt and So, Peter T C and Schilb, Alain and Hassiepen, Ulrich and Stoeckli, Kurt and Auer, Manfred (2005) Multi-photon excitation of intrinsic protein fluorescence and its application to pharmaceutical drug screening. Assay and Drug Development Technologies, 3 (2). pp. 155-167. ISSN 1540-658X

Abstract

The majority of proteins contain intrinsic fluorophores as natural sensors of molecular structures, dynamics, and interactions. The intrinsic protein fluorescence signal allows for the label-free and, hence, undisturbed and rapid study of protein-ligand interactions. Ultraviolet-based drug screening is hampered by the background, photobleaching, light scattering, inner filter effects, and interfering assay compounds. Such problems can be overcome by means of molecular three-photon excitation (3PE) with infrared femtosecond light pulses since longer excitation wavelengths result in less Raleigh scattering, and the subfemtoliter (confocal-like) 3PE volume minimizes out-of-focus photobleaching, background generation, and inner filter effects. We demonstrate the general feasibility of 3PE for protein spectroscopy and illustrate the technique's excellent potential for high-throughput screening. By using the intrinsic fluorescence intensity of a protein-substrate, we were able to discriminate between ligands of different affinities in binding assays.

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Date Deposited: 14 Dec 2009 13:56
Last Modified: 31 Jan 2013 01:11
URI: https://oak.novartis.com/id/eprint/641

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