Concentric Förster Resonance Energy Transfer Imaging

Abstract

Concentric Förster resonance energy transfer (cFRET) configurations based on semiconductor quantum dots (QDs) are promising probes for biological sensing because they offer multiplexing capability in a single vector with robust ratiometric detection by exploiting a network of FRET pathways. To expand the scope and utility of cFRET probes, it is necessary to develop and validate cFRET imaging methodology. In this technical note, we present such a methodology using a protease-sensitive cFRET configuration that comprises a green-emitting QD, Alexa Fluor 555 (A555), and Alexa Fluor 647 (A647). Photoluminescence (PL) images were acquired with three filter-based emission channels to permit measurement of A555/QD and A647/QD PL ratios. With reference to calibration samples, these PL ratios were used to calculate quantitative progress curves for proteolytic activity in regions of interest in the acquired images. Importantly, the imaging methodology reproduces quantitative results obtained with a monochromator-based fluorescence plate reader. Spatiotemporal resolution is demonstrated by tracking the activity of two prototypical proteases, trypsin and chymotrypsin, as they diffuse down the length of a capillary. This methodology is expected to enable the future use of cFRET probes for cellular sensing and other imaging assays.

Integration of isothermal amplification with quantum dot-based fluorescence resonance energy transfer for simultaneous detection of multiple microRNAs

“…Some typical examples include the combination of two QD donors with two dye acceptors, the integration of three QD donors with three dye acceptors, 46 – 48 the interaction of multiple QD donors with one dye acceptor, 49 multi-FRET distribution systems made of a central QD surrounded by pendant dyes in a sequential order, 50 – 53 and concentric FRET configurations with multiple dye acceptors arranged symmetrically around a central QD. 54 – 56 These multiplex FRET configurations give rise to the possibilities of multiple energy transfer between the QDs and the dyes along with dye-to-dye steps. 39 The linear and concentric configurations enable energy transfer from a QD donor to the first dye and then to the second dye.…”

Energy Transfer Pathways in a Quantum Dot-Based Concentric FRET Configuration

“…26 A cFRET imaging method was also recently developed. 28 In each case, energy transfer has been understood only qualitatively and analytical applications have relied on empirical calibration.…”

Nanoparticles as energy donors and acceptors in bionanohybrid systems

“…The individual or simultaneous activities of trypsin and chymotrypsin, which recognize the proteolytic cleavage sites on the different peptides, could be assessed by comparison of 555/520 and 647/520 fluorescence intensity ratios. The same conjugate was used for sensing and imaging the diffusion of proteases in the glass capillary (Wu & Algar, 2015). This co-called concentric FRET scheme was additionally extended by employing three molecular fluorophores, linked to the QD either by peptides with different protease recognition sites or oligonucleotides with different restriction sites (Massey et al, 2017;Hu et al, 2019).…”