Fluorescent methods and materials for directed biomarker signal amplification
Inventors
Gaylord, Brent S. • Hong, Janice W. • Fu, Tsu-Ju • SUN, Chengjun • Baldocchi, Russell A.
Assignees
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Publication Number
US-10859578-B2
Publication Date
2020-12-08
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 a fluorescent assay approach that uses a water soluble multichromophore that is a conjugated polymer having a backbone of π-conjugated repeat units. The multichromophore comprises CP1, CP2, and CP3, where CP1 provides water-soluble groups selected from ethylene glycol oligomer, ethylene glycol polymer, ω-ammonium alkoxy salt, and ω-sulfonate alkoxy salt, CP2 is an aromatic repeat unit different from CP1, and CP3 is an aromatic repeat unit comprising a bioconjugation functional group capable of covalently linking to a biomolecule or dye and does not comprise a biotin group.
The multichromophore enables light harvesting and energy transfer between chromophores, yielding amplified emission. In the described conjugates, a sensor biomolecule is covalently linked to the light harvesting multichromophore via a bioconjugation site, and a signaling chromophore is positioned in energy-receiving proximity to the light harvesting multichromophore through the bioconjugation functional group on CP3.
The polymer architecture is defined to control properties through the selection of repeat unit types and substituents. The multichromophore includes parameters m and n for repeat unit counts, with m+n>1, and parameters b, a, and c for structural variables subject to a+b+c>1, with covalent attachment to the unsaturated backbone marked by an asterisk as a site for covalent attachment.
Claims Coverage
The independent claims identified are two in number. Across these claims, the invention is directed to a defined water soluble multichromophore as a π-conjugated conjugated polymer with specified repeat units and covalent bioconjugation functionality, and a multichromophore-biomolecule conjugate in which a sensor biomolecule is covalently linked to the multichromophore and a signaling chromophore is arranged for energy-receiving proximity.
Water soluble multichromophore as a π-conjugated conjugated polymer with specified repeat units and bioconjugation site
A water soluble multichromophore that is a conjugated polymer having a backbone of π-conjugated repeat units, comprising CP1, CP2, and CP3 where each R1 is independently a water-soluble group selected from ethylene glycol oligomer, ethylene glycol polymer, ω-ammonium alkoxy salt and ω-sulfonate alkoxy salt; CP2 is an aromatic repeat unit different from CP1; CP3 is present and is an aromatic repeat unit comprising a bioconjugation functional group capable of covalently linking to a biomolecule or dye and does not comprise a biotin group; m and n are independently 0 to 10,000 with m+n>1; and b is 0 to 250 and a and c are independently 1 to 250 with a+b+c>1.
Multichromophore-biomolecule conjugate with light harvesting multichromophore and energy-receiving signaling chromophore
A multichromophore-biomolecule conjugate comprising a sensor biomolecule covalently linked to a light harvesting multichromophore via a bioconjugation site, where the light harvesting multichromophore is a conjugated polymer having a backbone of π-conjugated repeat units comprising CP1, CP2, and CP3 defined so that each R1 is independently selected from ethylene glycol oligomer, ethylene glycol polymer, ω-ammonium alkoxy salt and ω-sulfonate alkoxy salt; CP2 is an aromatic repeat unit different from CP1; CP3 is present and is an aromatic repeat unit comprising the bioconjugation site having a bioconjugation functional group capable of covalently linking to a biomolecule or dye and does not comprise a biotin group; and m+n>1, with b, a, and c subject to the stated ranges.
The claim set is centered on a water soluble π-conjugated conjugated polymer multichromophore with specific repeat units and a covalent bioconjugation functional group lacking biotin, and on a conjugate form where a sensor biomolecule is covalently linked to that light harvesting multichromophore, with a signaling chromophore positioned for energy-receiving proximity.
Stated Advantages
Large fluorescence amplification is described in the document, including reported fluorescence amplification (e.g., 5–10× for electrostatic cases and up to ~19× for biotinylated polymer versus direct dye excitation).
Background reduction is described by using covalent bioconjugation versus electrostatic binding.
Enhanced signal via amplified emission.
Improved control of donor–acceptor distance via covalent attachment [procedural detail omitted for safety].
Documented Applications
Biomolecule assays using multichromophore-enabled signal amplification with π-conjugated polymer light-harvesting donors and signaling chromophores associated with biomolecular recognition elements.
Sandwich assays using antibodies (primary/secondary) as sensor biomolecule recognition elements.
Biotin/streptavidin systems described for biomolecule recognition (including biotin–avidin/streptavidin binding).
Nucleic acid sensing contexts referenced (including nucleic acids such as FISH/PCR/microarrays mentioned).
Multiplexing using multiple acceptor dyes under single donor excitation is described.
Directed biomarker detection using fluorescent assay methods with multichromophore materials configured for sensor biomolecule binding and signaling chromophore emission amplification.
Multiplexing with multiple signaling chromophores emitting different wavelengths for assay formats.
Multichromophore-based assay formats using covalent sensor–multichromophore–signaling-chromophore complexes and bioconjugation pairing strategies.
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