Concentric forster resonance energy transfer relay for the parallel detection of two bio/physicochemical process
Inventors
Medintz, Igor L. • Algar, W. Russ
Assignees
Publication Number
US-9120967-B2
Publication Date
2015-09-01
Expiration Date
2034-03-07
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Abstract
Described herein is a Förster (or fluorescence) resonance energy transfer (FRET) configuration with three energy transfer pathways between three luminescent components, where two of the energy transfer steps occur in sequence as a relay, and the first step of the relay is in competition with a third energy transfer process (energy transfer from the donor to the intermediary is in competition with energy transfer from the donor directly to the terminal acceptor).
Core Innovation
Described herein is a Förster resonance energy transfer (FRET) configuration with three energy transfer pathways between three luminescent components, where two of the energy transfer steps occur in sequence as a relay, and the first step of the relay is in competition with a third energy transfer process. The configuration involves a semiconductor quantum dot (QD) as the FRET donor, a first fluorescent dye as a FRET acceptor to the QD in a first FRET process, and a second fluorescent dye serving as a FRET acceptor in both a second FRET process from the first dye and a relatively inefficient third FRET process directly from the QD. The two fluorescent dyes are assembled to the QD at approximately the same distance, allowing all three FRET processes to occur.
Conventional FRET configurations typically use two fluorescent dyes in discrete donor-acceptor pairs, with energy transfer observed in a single step. More complex FRET arrangements involve multi-step sequential transfers along a linear or pseudo-linear pathway extending the range of energy transfer. In contrast, the described invention presents a concentric FRET relay that differs by not moving excitation energy further from the donor than the first step, and by competing energy transfer pathways where the donor simultaneously interacts with two acceptors at similar distances. This unique design allows multiplexed detection of two bio/physicochemical processes with one probe, unlike current methods relying on separate probe entities.
The concentric FRET relay enables determination of the number of each fluorescent dye assembled per QD through characteristic photoluminescence (PL) ratios. The assembly can be tuned by varying the average dyes per QD, which alters the efficiencies of the competing FRET pathways. A reduction to practice demonstrated the use of CdSe/ZnS QDs with Alexa Fluor 555 and Alexa Fluor 647 dyes to detect activities of two proteolytic enzymes independently and in parallel. This system allows simultaneous monitoring of two proteolytic enzymes and their interactions, showcasing its ability to parallel detect multiple biological processes using a single FRET relay assembly.
Claims Coverage
The claims cover one independent FRET relay assembly, one independent method of preparing such an assembly, and one independent method of using the assembly. Each claims the unique three-pathway FRET process involving a semiconductor quantum dot donor and two fluorescent dyes assembled at similar distances enabling the FRET relay.
FRET relay assembly with three overlapping energy transfer pathways
An assembly comprising a semiconductor quantum dot as a FRET donor, a first fluorescent dye as a FRET acceptor in a first FRET process, and a second fluorescent dye as a FRET acceptor in both a second FRET process from the first dye and a relatively inefficient third FRET process directly from the quantum dot. The first and second fluorescent dyes are assembled to the quantum dot at approximately the same distance allowing for the three FRET processes.
Method of preparing the FRET relay assembly
A method comprising binding semiconductor quantum dots to a first fluorescent dye and to a second fluorescent dye to form the described FRET relay assembly where the dyes and quantum dot are assembled to enable the three FRET processes at comparable distances.
Method of using FRET relay assemblies with separable linkages
Providing a population of FRET relay assemblies with the quantum dot and two fluorescent dyes assembled via first and second separable linkages, respectively, at similar distances allowing the three FRET processes, then subjecting these linkages to separation, such as proteolysis, to monitor changes.
The independent claims establish a FRET relay assembly and its methods of preparation and use based on the unique tri-pathway energy transfer configuration with dyes assembled at similar distances around a quantum dot donor, enabling parallel detection methods.
Stated Advantages
Provides two analytic optical signals (photoluminescence ratios) from a single probe entity rather than separate probes.
Allows excitation with a single wavelength, simplifying instrumentation requirements.
Avoids issues arising from using two different probes, such as differing biological activities or distribution.
Offers tunability of competing FRET pathways through controlling dye numbers, spectral overlaps, and molecular linkages.
Compatible with multiple detection environments including solution, interfaces, and cellular or tissue contexts.
Enables detection based on simple photoluminescence intensity measurements compatible with standard fluorescence instruments.
Directly amenable to multiphoton excitation to reduce background interference and enhance in vivo application potential.
Documented Applications
Multiplexed detection in biological applications requiring luminescent probes or reporters.
Imaging and assays involving chemo/biosensing in vitro or within cells and tissues.
Parallel detection and quantitative measurement of activities of two proteolytic enzymes.
Monitoring enzyme interactions such as activation of chymotrypsinogen by trypsin.
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