Fluorescent methods and materials for directed biomarker signal amplification
Inventors
Gaylord, Brent S. • Hong, Janice W. • Fu, Tsu-Ju • SUN, Chengjun • Baldocchi, Russell
Assignees
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Publication Number
US-9383353-B2
Publication Date
2016-07-05
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 an assay method using a multichromophore complex for fluorescent biomarker detection. The multichromophore comprises a water-soluble, light harvesting π conjugated polymer with substituted or unsubstituted multiple units in a π conjugated state, and the polymer has a linker with a bioconjugation site that enables covalent attachment to a sensor biomolecule capable of recognizing a target biomolecule.
An optional signaling chromophore is covalently bound either to the multichromophore or to the sensor biomolecule, and it receives energy from the multichromophore upon excitation of the multichromophore. The assay includes contacting a sample suspected of containing a target biomolecule, applying a light source to excite the multichromophore, and detecting whether light is emitted from the multichromophore complex.
The multichromophore complex is configured to amplify fluorescent detection by using energy transfer from the multichromophore to the signaling chromophore, with reduced background compared to electrostatic or indirect approaches. Multiplexing is supported by using multiple signaling chromophores emitting different wavelengths from a single excitation wavelength, and bioconjugate formats include donor-dye coupling and donor/acceptor arrangements, including dual bioconjugate bridging formats using first and second bioconjugates.
Claims Coverage
The independent claim covers an assay method with four inventive features: a water-soluble π conjugated polymer with a linker bioconjugation site, covalent linkage of a target-recognizing sensor biomolecule, an optional signaling chromophore capable of receiving energy upon excitation, and sample contact with light excitation and emitted-light detection.
Multichromophore complex with water-soluble π conjugated polymer and bioconjugation linker
A multichromophore comprising a water-soluble, light harvesting π conjugated polymer comprising substituted or unsubstituted multiple units in a π conjugated state, each unit having a structure with a linker having a bioconjugation site, where A is independently an aromatic, heteroaromatic, optionally substituted conjugated polymer segment or oligomeric structure with at least one A being a phenyl linked to the linker, and where R1 is a water solubilizing group elected from ethylene glycol oligomers, ethylene glycol polymers, ω-ammonium alkyl/alkoxy/oligoether salts, ω-sulfonate alkyl/alkoxy/oligoether salts, and combinations thereof.
Covalent linkage of sensor biomolecule to multichromophore via bioconjugation site
A sensor biomolecule covalently linked to the multichromophore by the bioconjugation site on the conjugated polymer, where the sensor biomolecule is capable of recognizing the target biomolecule.
Optional signaling chromophore energy recipient for excitation of multichromophore
An optional signaling chromophore covalently bound to the multichromophore or covalently bound to the sensor biomolecule, where the optional signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore.
Assay method: contact sample, excite multichromophore, and detect emitted light
Contacting the sample with the multichromophore complex under conditions in which the sensor biomolecule can bind to the target biomolecule or a target-associated biomolecule if present, applying a light source to the sample that can excite the multichromophore, and detecting whether light is emitted from the multichromophore complex.
The claims center on a multichromophore assay format in which a water-soluble light harvesting π conjugated polymer covalently links to a target-recognizing sensor biomolecule, with an optional signaling chromophore configured to receive energy upon multichromophore excitation, followed by excitation and detection of emitted light.
Stated Advantages
Amplified emitted-light detection using energy transfer from the multichromophore to the signaling chromophore.
Reduced background compared with electrostatic or indirect approaches.
Allows target recognition by a sensor biomolecule covalently linked to the multichromophore through a bioconjugation site.
Supports multiplexing by using multiple signaling chromophores emitting different wavelengths from a single excitation wavelength.
Documented Applications
Sandwich immunoassay.
Protein array.
Microarray configurations.
Immunohistochemistry.
FISH.
Flow cytometry.
ELISA.
Protein arrays.
DNA microarrays.
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