Fluorescent methods and materials for directed biomarker signal amplification
Inventors
Gaylord, Brent S. • Hong, Janice W. • Fu, Tsu-Ju • Sun, Cheng-Jun • Baldocchi, Russell
Assignees
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Publication Number
US-8158444-B2
Publication Date
2012-04-17
Expiration Date
Abstract
Methods and compositions are provided that include a multichromophore and/or multichromophore complex for identifying a target biomolecule. A sensor biomolecule, for example, an antibody can be covalently linked to the multichromophore. Additionally, a signaling chromophore can be covalently linked to the multichromophore. The arrangement is such that the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore. Since the sensor biomolecule is capable of interacting with the target biomolecule, the multichromophore and/or multichromophore complex can provide enhanced detection signals for a target biomolecule.
Core Innovation
The invention relates to multichromophore complexes in which a multichromophore is covalently bound by a unique bioconjugation site pendant to at least one biomolecule selected from a sensor biomolecule, a bioconjugate, and a target biomolecule. The multichromophore is used as a reporter and can further comprise a signaling chromophore covalently bound to the multichromophore or the biomolecule.
The multichromophore comprises a π-conjugated backbone with π-conjugated units and ethylene glycol oligomer or ethylene glycol polymer R1 groups, and the π-conjugated units are linked by a linker in the π-conjugated backbone, with optional angled π-conjugated linkers. In one described composition, the π-conjugated units include fluorene and benzyl groups substituted with the bioconjugation site.
The disclosed complexes support identifying a target biomolecule, where the sensor biomolecule covalently linked to the multichromophore through the unique bioconjugation sites interacts with the target biomolecule or a target-associated biomolecule. The signaling chromophore is capable of receiving energy from the multichromophore upon excitation, and the document describes multiplexing with multiple signaling dyes and dye signal amplification under indirect excitation.
Claims Coverage
The independent claims consistently require a multichromophore covalently attached to biomolecules via a unique bioconjugation-site pendant, with a π-conjugated multichromophore architecture including ethylene glycol oligomer/polymer substituents or specified fluorene and benzyl substitutions. Across the independent claims, there are 4 main inventive features: unique bioconjugation-site pendant attachment, optional signaling chromophore energy reception, π-conjugated multichromophore structure with ethylene glycol R1 groups, and sensor biomolecule interaction with a target biomolecule or target-associated biomolecule.
Unique bioconjugation-site pendant multichromophore reporter complex
A multichromophore covalently bound by a unique bioconjugation site pendant to at least one biomolecule selected from a sensor biomolecule, a bioconjugate, and a target biomolecule, wherein the multichromophore is used as a reporter or further comprises a signaling chromophore covalently bound to the multichromophore or the biomolecule.
π-conjugated multichromophore with ethylene glycol oligomer or polymer R1 groups
The multichromophore comprises m, n, and p each at least 1, each R1 independently an ethylene glycol oligomer or ethylene glycol polymer, and π-conjugated units independently selected from benzene, naphthalene, anthracene, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene, and oxadiazole, each optionally substituted, linked by a linker and optionally angled π-conjugated linkers in the π-conjugated backbone.
Energy-transfer signaling chromophore for identifying a target biomolecule
A multichromophore complex for identifying a target biomolecule comprising a multichromophore used as a reporter or further comprising a signaling chromophore covalently bound to the multichromophore or to the sensor biomolecule, wherein the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore and the sensor biomolecule is capable of interacting with the target biomolecule or a target-associated biomolecule.
Sensor biomolecule covalently linked via unique bioconjugation sites
A multichromophore complex for identifying a target biomolecule comprising a sensor biomolecule covalently linked to the multichromophore through said unique bioconjugation sites.
Specific multichromophore composition with fluorene and benzyl bioconjugation-site pendant
A multichromophore complex comprising a multichromophore covalently bound by a bioconjugation site pendant to at least one biomolecule, with the multichromophore used as a reporter or further comprising a signaling chromophore covalently bound to the multichromophore or the biomolecule, wherein each R1 is (CH2CH2O)3CH2CH2OCH3, wherein one π-conjugated unit is a fluorene substituted with at least one (CH2CH2O)3CH2CH2OCH3, and wherein another π-conjugated unit comprises a π-conjugated benzyl group substituted with said bioconjugation site selected from maleimide, thiol, succinimidyl ester (NHS ester), amine, azide chemistry, carboxylate, carboxy/EDC, Sulfo-SMCC, amine/BMPH, and Sulfo-SBED.
Across the independent claims, the coverage centers on covalent multichromophore reporter complexes that use unique bioconjugation-site pendants to attach to sensor, bioconjugate, or target biomolecules, with optional signaling chromophores that covalently couple to the multichromophore system and, in the identification claim, are capable of receiving energy upon excitation. The claims also require multichromophore structural definitions using π-conjugated units and ethylene glycol oligomer or polymer substituents, with one independent claim further specifying a fluorene and π-conjugated benzyl bioconjugation-site composition and an explicit set of bioconjugation-site chemistries.
Stated Advantages
Reduced background signal compared to electrostatic approaches is discussed.
Fixed donor-acceptor distances are discussed as contributing to improved detection.
Dye signal amplification under indirect excitation is reported, with example performance trends including 5-fold and 10-fold dye signal amplification.
Documented Applications
Identifying a target biomolecule using a multichromophore complex comprising a sensor biomolecule covalently linked through unique bioconjugation sites, with optional signaling chromophore energy transfer upon excitation.
Biorecognition and assay formats using biotin-streptavidin systems.
Immunoassay formats including sandwich immunoassays.
Multiplexing contexts using a single excitation source.
DNA microarrays.
FISH assays.
PCR assays.
Logic device and block diagram contexts described in the document.
Kit contexts described in the document.
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