Detection of Protein鈥揝mall Molecule Binding Using a Self-Referencing External Cavity Laser Biosensor

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• 作者：Meng Zhang ; Jessie Peh ; Paul J. Hergenrother ; Brian T. Cunningham
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：April 23, 2014
• 年：2014
• 卷：136
• 期：16
• 页码：5840-5843
• 全文大小：320K
• 年卷期：v.136,no.16(April 23, 2014)
• ISSN：1520-5126

文摘

High-throughput screening has enabled the identification of small molecule modulators of important drug targets via well-established colorimetric or fluorimetric activity assays. However, existing methods to identify small molecule binders of nonenzymatic protein targets lack either the simplicity (e.g., require labeling one of the binding partners with a reporter) or throughput inherent in enzymatic assays widely used for HTS. Thus, there is intense interest in the development of high-throughput technologies for label-free detection of protein鈥搒mall molecule interactions. Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar. Finally, a 鈥渘eedle-in-the-haystack鈥?screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.