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Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing

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    Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing
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    † †Center for Bio/Molecular Science and Engineering, Code 6900, ‡Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington DC 20375, United States
    § College of Science, George Mason University, Fairfax, Virginia 22030, United States
    ∥ Institut d’Electronique Fondamentale, Université Paris-Sud, 91405 Orsay Cedex, France
    ⊥ Departments of Chemistry and Cell Biology, The Scripps Research Institute La Jolla, California 92037, United States
    # Sotera Defense Solutions, Crofton, Maryland 21114, United States
    E-mail: [email protected]
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    Cite this: J. Am. Chem. Soc. 2012, 134, 3, 1876–1891
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    https://doi.org/10.1021/ja210162f
    Published January 5, 2012
    Copyright © 2012 American Chemical Society
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    Abstract

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    The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD–A647 donor–acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.

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    Supporting Information

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    Detailed descriptions of reagents, labeling chemistry, bioconjugate and assay preparation, instrumentation, data analysis, equations for calculation of FRET efficiencies, FRET ratios, FRET rates, additional PL intensity and PL decay data, further discussion of peptide design, characterization of QD-oligonucleotide conjugates, further description of the two-plex hybridization assay, and alternate analysis methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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    Cite this: J. Am. Chem. Soc. 2012, 134, 3, 1876–1891
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