Spectro-temporal optical encoding of information using a time-gated fluorescence resonance energy transfer (FRET)
Inventors
Algar, W. Russ • Hildebrandt, Niko • Huston, Alan L. • Medintz, Igor L.
Assignees
Publication Number
US-9752986-B2
Publication Date
2017-09-05
Expiration Date
2032-05-18
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Abstract
Described herein is a time-gated, two-step FRET relay effective to provide temporal transference of a prompt FRET pathway, or provide spectro-temporal encoding analytical signals and other information. A FRET relay assembly includes a long lifetime FRET donor (for example, a lanthanide complex), a semiconductor quantum dot (QD) configured as an intermediate acceptor/donor in FRET, and a fluorescent dye configured as a terminal FRET acceptor, wherein the long lifetime FRET donor has an excited state lifetime of at least one microsecond and the QD and fluorescent dye each have excited state lifetimes of less than 100 nanoseconds.
Core Innovation
The invention is a time-gated, two-step FRET relay assembly that provides temporal transference of a prompt FRET pathway and enables spectro-temporal encoding of analytical signals and other information. The assembly includes a long lifetime FRET donor, a semiconductor quantum dot as an intermediate acceptor/donor, and a fluorescent dye as a terminal FRET acceptor, with the long lifetime FRET donor having an excited state lifetime of at least one microsecond, and the quantum dot and fluorescent dye each having excited state lifetimes of less than 100 nanoseconds.
The problem solved addresses limitations in conventional FRET assays, which predominantly use single donor-acceptor pairs and are limited to prompt (nanosecond scale) fluorescence observation. Multi-step FRET relays had been described previously but only for extending net FRET range or serving as photonic wires, without temporal gating or spectro-temporal encoding capabilities. The invention provides a novel temporal dimension by leveraging a long lifetime FRET donor to enable time-gated observation and thereby resolve distinct FRET processes that were previously indistinguishable in time.
This technique employs a central quantum dot acting simultaneously as an intermediate FRET acceptor from a long lifetime donor (e.g., a lanthanide complex such as Tb3+) and as a donor to a terminal fluorescent dye (e.g., Alexa Fluor 647). The time gating allows the quantum dot to relax to ground state before acting as a FRET acceptor, enabling detection of energy transfer over microsecond to millisecond timescales. The two FRET steps are approximately independent and resolvable using spectral measurements in distinct observation windows, enabling multiplexed biosensing and reducing background fluorescence. The invention also includes bioconjugation strategies using peptides and oligonucleotides labeled with these luminophores and assembled with the quantum dots via His6 metal-affinity motifs for controlled valence and spacing.
Claims Coverage
The patent contains one independent method claim covering the use of a FRET relay assembly comprising three key components for two distinct FRET processes with specific lifetime characteristics.
FRET relay assembly structure and composition
A FRET relay assembly comprising a long lifetime FRET donor, a semiconductor quantum dot (QD) acting as an intermediate acceptor/donor assembled in proximity to the donor, and a fluorescent dye as a terminal FRET acceptor assembled in proximity to the QD. The long lifetime FRET donor has an excited state lifetime of at least one microsecond, while the QD and the fluorescent dye have excited state lifetimes of less than 100 nanoseconds.
Method of spectral excitation and emission detection in spectro-temporal windows
Spectrally exciting the FRET relay assembly and receiving spectral emissions from the assembly, including measurement of emissions in at least two distinct observation windows, thereby enabling observation of both FRET processes.
Bioconjugation and assembly specifics
The long lifetime FRET donor may comprise metal ions such as Tb3+, Eu3+, Sm3+, Tm3+, or complexes such as Ru2+. The donor and the fluorescent dye can be bound to the QD using peptides and/or oligonucleotides, with at least one His6 motif adapted to bind them to the QD. The QD can be functionalized with groups such as carboxylate, amine, or poly(ethylene glycol).
The independent claim covers a method employing a specifically assembled FRET relay comprising a long lifetime donor, an intermediary quantum dot acceptor/donor, and a terminal fluorescent dye acceptor, with spectral excitation and emission detection that enables spectro-temporal encoding through two distinct FRET processes occurring within defined lifetime parameters.
Stated Advantages
The use of a long-lifetime FRET donor enables time-gated observation that allows independent measurement of a second, approximately independent FRET process that normally is only observable with prompt detection.
Time-gated measurements permit reduction of background fluorescence and scattered source light, which is particularly beneficial for complex biological samples and in vivo tissue imaging.
The design allows spectral observation of both FRET steps at characteristic wavelengths in both prompt and time-gated windows, facilitating multiplexed biosensing without requiring multiple quantum dot colors.
Independent tuning of the FRET efficiencies is possible through control of donor and acceptor numbers and proximity, providing flexibility and precise modulation of energy transfer.
The quantum dot acts as both an energy relay and a nanoscaffold enabling assembly and precise spatial organization of biomolecules, improving reproducibility and simplifying bioconjugation.
Ratiometric detection in the FRET relay allows kinetic measurements with improved sensitivity and reduced instrumental drift effects.
Documented Applications
Labeling, assays, or chemo/biosensing on the surface of or within cells, tissues (in vitro or in vivo), environmental samples, and other complex sample matrices prone to optical scattering and autofluorescence.
Multiplexed detection in biological environments via spectro-temporal resolution of FRET signals, including monitoring of enzymatic activities such as protease activity and nucleic acid hybridization.
Optical barcoding for commercial or shipping use, anti-counterfeit measures, and forgery deterrents through unique spectro-temporal luminescent signatures.
Measurement of biomolecular binding and dissociation, enzymatic function, protein folding, and cellular processes including endocytosis and protein synthesis.
Use in diagnostic probes, theranostics, therapeutics, and biomedical technologies involving nanoparticles like quantum dots.
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