Fluorescent Methods and Materials for Directed Biomarker Signal Amplification

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a sensor protein conjugated to a signaling chromophore and capable of interacting with the target biomolecule or a target-associated biomolecule;
• providing a conjugated polymer selected from the group consisting of
• wherein R and R1 are solubilizing groups selected from the group consisting of ω-ammonium alkyl salts, ω-ammonium alkoxy salts, ω-ammonium oligoether salts, ω-sulfonate alkyl salts, ω-sulfonate alkoxy salts, and ω-sulfonate oligoether salts, and wherein the polymer electrostatically interacts with the sensor protein and upon excitation is capable of transferring energy to the signaling chromophore;
• contacting the sample with the sensor protein and the conjugated polymer in a solution under conditions in which the sensor protein can bind to the target biomolecule or a target-associated biomolecule if present;
• applying a light source to the sample that can excite the conjugated polymer;
• and
• detecting whether light is emitted from the signaling chromophore.

A multichromophore complex for identifying a target biomolecule comprising:

• a multichromophore, wherein the multichromophore comprises the structure
• wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugated polymers or conjugated polymer segments or oligomeric structures, that are the same or different from one another,
• a sensor biomolecule covalently linked to the multichromophore,
• a signaling chromophore comprising the multichromophore or optionally comprising a signaling chromophore covalently bound to the multichromophore or optionally comprising a signaling chromophore covalently bound to the sensor biomolecule, wherein the signaling chromophore, except where comprising the multichromophore, 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.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a multichromophore complex comprising a multichromophore, covalently bound to a biomolecule, and a signaling chromophore covalently linked to the multichromophore complex, wherein the multichromophore comprises the structure
• wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugated polymers or conjugated polymer segments or oligomeric structures, that are the same or different from one another, and 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;
• 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 signaling chromophore.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a first bioconjugate conjugated to a signaling chromophore;
• providing a second bioconjugate conjugated to a multichromophore, wherein the multichromophore comprises the structure
• wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugated polymers or conjugated polymer segments or oligomeric structures, that are the same or different from one another, wherein the second bioconjugate can bind to the first bioconjugate and wherein upon such binding excitation of the multichromophore is capable of transferring energy to the signaling chromophore, and wherein either the first bioconjugate or the second bioconjugate, but not both, is a target-binding bioconjugate configured to interact with the target biomolecule or a target-associated biomolecule;
• contacting the sample with the target-binding bioconjugate under conditions which support binding of the target-biding bioconjugate to the target biomolecule or target-associated biomolecule if present;
• contacting the sample with either the first bioconjugate or second bioconjugate depending on which is not a target-binding bioconjugate;
• applying a light source to the sample that can excite the multichromophore;
• and
• detecting whether light is emitted from the signaling chromophore.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a multichromophore complex comprising a multichromophore and a first bioconjugate covalently linked thereto and optionally further comprising a signaling chromophore covalently linked to the multichromophore complex, wherein the multichromophore comprises the structure
• wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugated polymers or conjugated polymer segments or oligomeric structures, that are the same or different from one another;
• providing a sensor biomolecule complex comprising a sensor biomolecule capable of interacting with the target biomolecule or a target-associated biomolecule, a covalently linked second bioconjugate capable of binding with the first bioconjugate, and optionally further comprising a signaling chromophore,
• wherein either the multichromophore complex or the sensor biomolecule complex, but not both, comprises a signaling chromophore;
• contacting the sample with the sensor biomolecule complex under conditions in which the sensor biomolecule can bind to the target biomolecule or target-associated biomolecule if present;
• contacting the sample with the multichromophore complex under conditions in which the first bioconjugate can bind with the second bioconjugate on the sensor biomolecule complex if present;
• applying a light source to the sample that can excite the multichromophore, wherein the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore;
• and
• detecting whether light is emitted from the signaling chromophore.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a multichromophore complex comprising a multichromophore and a sensor biomolecule covalently linked thereto, wherein the multichromophore comprises the structure
• wherein CP1, CP2, CP3, and CP4 are optionally substituted conjugated polymers or conjugated polymer segments or oligomeric structures, that are the same or different from one another,
• wherein the sensor biomolecule is capable of interacting with the target biomolecule;
• 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.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a first bioconjugate labeled with a biotin which is capable of binding the target biomolecule or a target-associated biomolecule;
• providing a second bioconjugate wherein the second bioconjugate is comprised of a multichromophore covalently bound to an avidin, or avidin derivative, selected from the group consisting of streptavidin, neutravidin, avidin DN, and avidinD;
• providing a signaling chromophore comprising the multichromophore or optionally comprising a signaling chromophore covalently bound to the multichromophore or optionally comprising a signaling chromophore bound to the avidin or avidin derivative, selected from the group consisting of streptavidin, neutravidin, avidin DN, and avidinD;
• and
• contacting the second bioconjugate with the first bioconjugate indicates the presence of the target biomolecule.

An assay method comprising:

• providing a sample that is suspected of containing a target biomolecule;
• providing a first bioconjugate labeled with a digoxigenin which is capable of binding the target biomolecule or a target-associated biomolecule;
• providing a second bioconjugate, wherein the second bioconjugate is comprised of a multichromophore covalently bound to an anti-digoxigenin antibody;
• providing a signaling chromophore comprising the multichromophore or optionally comprising a signaling chromophore covalently bound to the multichromophore or optionally comprising a signaling chromophore bound to the anti-digoxigenin antibody;
• and
• contacting the second bioconjugate with the first bioconjugate indicates the presence of the target biomolecule.