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

Abstract

The unique optical properties of semiconductor quantum dots (QDs) are highly advantageous for biological imaging and analysis, particularly when combined with Forster resonance energy transfer (FRET). A recent innovation in this area has been concentric FRET (cFRET), wherein QDs are assembled with multiple copies of two different types of fluorescent label. Although multifunctional biological probes have been developed utilizing cFRET, a detailed photophysical analysis of cFRET has not been undertaken, and energy transfer in these probes has been understood only qualitatively. Here, we characterize a prototypical QD-(A555) M -(A647) N cFRET configuration through photoluminescence (PL) intensity, decay, and photobleaching measurements. This cFRET configuration combines a central, green-emitting QD with Alexa Fluor 555 (A555) and Alexa Fluor 647 (A647) dyes that are assembled to QDs through peptide linkers, where M and N are the numbers of A555 and A647 per QD. Following initial photoexcitation of the QD, the energy transfer pathways are QD-to-A555 and QD-to-A647, which compete with one another, and A555-to-A647, which occurs subsequent to QD-to-A555 energy transfer. A rate analysis, calibrated to the conventional QD-(A555) M and QD-(A647) N FRET systems, accurately predicts quenching efficiencies and permits a first approximation of dye/ QD PL intensity ratios in the cFRET configurations. CdSe/CdS/ZnS QDs and CdSeS/ZnS QDs of different sizes but similar emission characteristics are used for these experiments, and they demonstrate the general applicability of the analysis. The interplay between the three FRET pathways and nonideal behavior within this system is discussed with directions for future research. Overall, this study provides a framework and predictive power for the rational design and optimization of novel cFRET probes and biosensors for biological applications. ■ INTRODUCTIONColloidal semiconductor nanocrystals, or "quantum dots" (QDs), are of great interest for biological imaging and analysis. 1−3 These materials are well-known for their bright, spectrally narrow photoluminescence (PL), which is also resistant to photobleaching. 4,5 As recent reviews attest, QDs have been widely utilized as labels for multicolor fluorescence measurements and imaging, single-particle tracking, and superresolution imaging, with applications spanning in vitro assays, cellular imaging, and in vivo imaging. 1,6−8 The chemistry associated with these materials is also well developed: methods for the synthesis of CdSe/ZnS and related core/shell nanocrystals are established, 9,10 several QD materials are available commercially, a variety of ligand and polymer coatings can be used to transfer QDs into aqueous media, 11−13 and numerous methods for bioconjugation have been reported. 11,14,15 The cumulative optical properties of QDs, which additionally include larger one-photon and two-photon absorption coefficients, spectrally broad absorption profiles, good quantum yields, and precise wavelength-tuning of PL through size and comp...

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

“…The A555 PL is directly quenched by increasing A555-to-A647 FRET, and indirectly quenched through a reduction in the efficiency of QD-to-A555 FRET with increasingly competitive and efficient QD-to-A647 FRET. 537 Resolution of A555-to-A647 FRET requires either a correction for this competitive effect, or direct excitation of the A555 without excitation of the QD. 537 Experiments have shown that the A555-to-A647 E FRET depends strongly on N A647 , but its dependence on N A555 appears to be much smaller and is still unclear in its details.…”

Portable Spectroscopy

“…184,185 Case 4 has largely been explored in university and other research institutions to construct portable clinical analyzers. [513][514][515][516][517][518][519][520][521][522][523][524][525][526][527][528][529] The fluorescence and colorimetric test reagents for clinical assays and analyses are well-known and widely available. In principle, therefore, interfacing microfluidics and a wavelength-selective device to a smartphone camera, with appropriate software, produces a mobile clinical analyzer with built-in communications abilities.…”

Sensing with photoluminescent semiconductor quantum dots

“…This concentric relay design (cFRET) was used for multiplexed protease sensing [34]. While the attachment schemes for these systems are relatively simple, analysis can become difficult because the dyes can interact with each other as well as the quantum dot [28,193]. In 2017, a three-acceptor cFRET system was demonstrated by attaching three different enzyme cleavable peptides labeled with three discrete dyes to a QD donor [28].…”