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

Abstract

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.

Colloidal nanoparticles as advanced biological sensors

“…They have been used on both QDs and AuNPs (54). A short sequence of histidine amino acids can be included in a recombinant protein (54), in a peptide sequence (55), or as part of an oligonucleotide construct (56), and its high affinity for metal nanoparticle surfaces allows attachment with excellent control over number and orientation of attached species. The many emerging highly site-or sequence-selective conjugation techniques, such as click reactions (57,58), strain-promoted cycloadditions (59), and enzyme-mediated ligations (60,61), are excellent prospects for nanoparticle bioconjugation, as they offer rapid, efficient, and strong binding.…”

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

“…The time-gated FRET relay was applied in a model two-plex DNA hybridization assay (Fig. 5B), 105 and in two-plex assays for protease activity. 136 The latter assay included tracking the activation of an inactive pro-protease by another protease, where the activity of both the upstream activating protease and downstream activated protease were quantitatively measured.…”

Photophysical evaluation of a new functional terbium complex in FRET-based time-resolved homogenous fluoroassays

“…The environmental factors such as the presence of particular proteins, ions or pH change can also influence the overall fluoroassay performance. Recently, a very interesting example of a (FRET relay)-based immunoassay has been presented, 31,32 in which FRET first proceeds from a lanthanide complex to QD and then from QD to a dye. The here presented complex can be applied in such systems to improve FRET efficiency and, therefore, sensitivity and robustness for such assay.…”