Issue 12, 2016

A long-wavelength quantum dot-concentric FRET configuration: characterization and application in a multiplexed hybridization assay

Abstract

Quantum dot-based concentric Förster resonance energy transfer (cFRET) is a promising modality for the development of multifunctional fluorescent probes for bioanalysis and bioimaging. To date, the scope of cFRET has been largely limited to a prototypical configuration with a particular combination of quantum dot (QD) and fluorescent dyes linked through peptides. Expansion of the scope of cFRET is critical for its further development. Here, we expand the scope of cFRET in two capacities. First, we design and characterize a new long-wavelength cFRET configuration that combines red- and deep-red fluorescent dyes, Alexa Fluor 633 and Alexa Fluor 680, with an orange-emitting QD. Sequential and competitive energy transfer pathways are characterized through a rate analysis, where the balance of these rates more strongly favours competitive energy transfer in the new long-wavelength configuration versus sequential energy transfer in the previous prototypical configuration. Although the new cFRET configuration is more susceptible to photobleaching, its superior brightness and longer-wavelength excitation and emission provide an order of magnitude higher signal-to-background ratios in biological matrices (e.g., serum, blood) than the previous prototypical configuration. Second, we demonstrate that an oligonucleotide-linked, long-wavelength cFRET configuration has energy transfer similar to an analogous peptide-linked configuration, where the oligonucleotide-linked cFRET configuration can be combined with toehold-mediated strand displacement for the multiplexed detection of unlabeled nucleic acid targets as a single vector. Overall, this work establishes the general applicability of cFRET and introduces new strategies for its bioanalytical application.

Graphical abstract: A long-wavelength quantum dot-concentric FRET configuration: characterization and application in a multiplexed hybridization assay

Supplementary files

Article information

Article type
Paper
Submitted
29 Nah 2016
Accepted
30 Cig 2016
First published
06 Agd 2016

Analyst, 2016,141, 3636-3647

A long-wavelength quantum dot-concentric FRET configuration: characterization and application in a multiplexed hybridization assay

J. J. Li and W. R. Algar, Analyst, 2016, 141, 3636 DOI: 10.1039/C6AN00492J

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements