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Addendum: Hochreiter, B.; Pardo-Garcia, A.; Schmid, J.A. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences. Sensors 2015, 15, 26281–26314
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Sensors 2015, 15(12), 30457-30468; doi:10.3390/s151229810

Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly

1
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, 4555 Overlook Ave, S.W., Washington, DC 20375, USA
2
Sotera Defense Solutions, Inc., 7230 Lee DeForest Drive, Columbia, MD 21046, USA
3
Optical Sciences Division, Code 5600 U.S. Naval Research Laboratory, Washington, DC 20375, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Alexander Star
Received: 8 October 2015 / Revised: 23 November 2015 / Accepted: 26 November 2015 / Published: 4 December 2015
(This article belongs to the Special Issue FRET Biosensors)
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Abstract

Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing. View Full-Text
Keywords: fluorescence; sensor; FRET; quantum dot; protein; membrane; assembly; noncovalent; ligand fluorescence; sensor; FRET; quantum dot; protein; membrane; assembly; noncovalent; ligand
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Field, L.D.; Walper, S.A.; Susumu, K.; Oh, E.; Medintz, I.L.; Delehanty, J.B. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly. Sensors 2015, 15, 30457-30468.

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