Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer.
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 Förster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (αRep 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 αReps 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 αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.
['Léger|Corentin|C|', 'Yahia-Ammar|Akram|A|', 'Susumu|Kimihiro|K|0000-0003-4389-2574', 'Medintz|Igor L|IL|0000-0002-8902-4687', 'Urvoas|Agathe|A|', 'Valerio-Lepiniec|Marie|M|', 'Minard|Philippe|P|', 'Hildebrandt|Niko|N|0000-0001-8767-9623']
D015374:Biosensing Techniques D031541:Fluorescence Resonance Energy Transfer D045663:Quantum Dots D012666:Semiconductors D013725:Terbium
FRET binding assay lanthanides molecular diagnostics molecular ruler protein structure