Spectro-temporal optical encoding of information using a time-gated fluorescence resonance transfer (FRET)
Inventors
Algar, W. Russ • Hildebrandt, Niko • Huston, Alan L • Medintz, Igor L.
Assignees
Publication Number
US-8476083-B1
Publication Date
2013-07-02
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 described is a time-gated, two-step fluorescence resonance energy transfer (FRET) relay assembly that enables temporal transference of a prompt FRET pathway and provides spectro-temporal encoding of analytical signals and other information. The assembly includes a long lifetime FRET donor, such as a lanthanide complex, a semiconductor quantum dot (QD) functioning as an intermediate acceptor/donor, and a fluorescent dye as a terminal acceptor. The long lifetime FRET donor exhibits an excited state lifetime of at least one microsecond, while the QD and fluorescent dye have excited state lifetimes of less than 100 nanoseconds.
The problem addressed arises from limitations in traditional FRET configurations which typically use single donor-acceptor pairs and lack the capability for multiplexed information encoding or extended observation windows. Multi-step FRET relays previously described only utilized prompt fluorescence on nanosecond scales mainly to extend FRET range or serve as photonic wires. However, they do not provide spectro-temporal encoding or time-gated capabilities that enable separation of energy transfer processes in time and spectrum.
The present invention overcomes these limitations by leveraging a long lifetime donor to enable time-gated observation. This approach facilitates the spectral measurement of two approximately independent FRET steps in distinct temporal windows, enhancing the ability to distinguish and quantify complex interactions such as biomolecular binding events. The QD acts as a nanoscaffold enabling co-assembly of donor and acceptor components, and the time-gated FRET relay allows multiplexed sensing applications by utilizing temporal and spectral resolution of energy transfer steps.
Claims Coverage
The patent contains one independent claim defining a FRET relay assembly and one independent claim defining a population of quantum dots comprising such assemblies. Three main inventive features are identified.
FRET relay assembly with long lifetime donor and intermediate QD acceptor/donor
A FRET relay assembly comprising a long lifetime FRET donor having an excited state lifetime of at least one microsecond, a semiconductor quantum dot (QD) configured as an intermediate acceptor/donor assembled in sufficient proximity to the donor to allow a first FRET process, and a fluorescent dye as a terminal acceptor assembled in sufficient proximity to the QD to allow a second FRET process. The QD and fluorescent dye each have excited state lifetimes of less than 100 nanoseconds.
Long lifetime FRET donor composition
The long lifetime FRET donor comprises lanthanide ions such as Tb3+, Eu3+, Sm3+, Tm3+, ruthenium complexes (Ru2+), or combinations thereof.
Bioconjugation and QD functionalization
The long lifetime FRET donor and/or fluorescent dye are bound to the QD using peptides and/or oligonucleotides, potentially employing a His6 motif for binding. The QD is functionalized with carboxylate, amine, or poly(ethylene glycol) coatings and may comprise semiconductor materials including ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InN, InP, InAs, or InSb.
The claims cover a time-gated FRET relay assembly comprising a long lifetime donor, an intermediate semiconductor QD acceptor/donor, and a terminal fluorescent dye acceptor arranged for two-step FRET processes, including variations in donor composition, assembly methods using biomolecules such as peptides and oligonucleotides with His6 motifs, and functional coatings on QDs. The claims also cover populations of QDs including such assemblies.
Stated Advantages
The QD intermediate acceptor/donor has broad resonant absorption with multiple emission lines of the long lifetime donor, enabling large spectral overlap and Förster distances.
The QD acts as a nanoscaffold permitting approximately centrosymmetric distribution of donor and acceptor molecules, facilitating precise assembly and analysis.
Use of His6 self-assembly enables simple, efficient, and reproducible binding with control over bioconjugate valence, allowing tuning of FRET efficiencies.
Time-gated observation eliminates background fluorescence and autofluorescence to improve signal-to-noise in complex biological samples.
The relay allows multiplexed biosensing exploiting independent tuning of two FRET mechanisms in a single QD-bioconjugate without requiring multiple QD colors.
Ratiometric detection is provided, which is insensitive to variations in excitation intensity or instrument differences and useful for kinetic assays.
The time-gated FRET relay reduces spectral bandwidth and channel requirements to encode multiplexed analytical information.
Spectro-temporal encoding of information is more secure and difficult to replicate compared to spectral encoding alone, useful for authentication/anti-counterfeit.
Documented Applications
Labeling, assays, and chemo/biosensing in biological samples including within cells and tissues, benefiting from extended FRET observation windows via time-gating.
Detection of protease activity by monitoring proteolysis in a kinetic and time-gated manner.
Nucleic acid hybridization assays, including two-plex multiplexed sensing using spectro-temporal resolution of FRET steps.
Orthogonal multiplexed biosensing with single color QD-based probes measuring two different biomolecular targets simultaneously.
Applications where luminescence is used for unique identification, tracking, validation, authentication, optical barcoding, and anti-counterfeit measures.
Measurements of binding, dissociation, enzymatic activity, protein folding, and cellular processes such as endocytosis and protein synthesis.
Potential use in non-biological complex samples with high optical scattering or autofluorescence that can be ameliorated by time-gating.
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