期刊
ACS NANO
卷 14, 期 5, 页码 5956-5967出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsnano.0c01410
关键词
FRET; lanthanides; molecular diagnostics; molecular ruler; protein structure; binding assay
类别
资金
- French National Research Agency (ANR project Neutrinos)
- French National Research Agency (ANR project AlphaSense)
- French National Research Agency (ANR project AMPLIFY)
- French National Research Agency (ANR project PhenX)
- VBFF program through the OSD
- Institut Universitaire de France (IUF)
Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of Forster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (alpha Rep bidomains) labeled with a Tb complex (Tb) donor on the C- terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the alpha Reps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET switches were used in a separation-free solution-phase assay to quantify different protein targets at sub-nanomolar concentrations and to measure the conformational changes with sub-nanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the alpha Rep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.
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