METHODS AND ARTICLES FOR STRAND-SPECIFIC POLYNUCLEOTIDE DETECTION WITH CONJUGATED POLYMERS

• US 20110027898A1
• Filed: 10/12/2010
• Published: 02/03/2011
• Est. Priority Date: 01/10/2005
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Bazan, Guillermo C., Liu, Bin
• Granted Patent: US 8,669,055 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2563/119 the label being proteinic
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention further relates to conjugated polymers, and methods, articles and compositions employing them as described herein. In some aspects, the invention relates to methods, articles and compositions for the detection and analysis of biomolecules in a sample. Provided assays include those determining the presence of a target biomolecule in a sample or its relative amount, or the assays may be quantitative or semi-quantitative. The methods can be performed on a substrate. The methods can be performed in an array format on a substrate, which can be a sensor. In some embodiments, detection assays are provided employing sensor biomolecules that do not comprise a fluorophore that can exchange energy with the cationic multichromophore. In some aspects biological assays are provided in which energy is transferred between one or more of the multichromophore, a label on the target biomolecule, a label on the sensor biomolecule, and/or a fluorescent dye specific for a polynucleotide, in all permutations. The multichromophore may interact at least in part electrostatically with the sensor and/or the target, and an increase in energy transfer with the polymer may occur upon binding of the sensor and the target. Other variations of the inventions are described further herein.

Claims

1. An assay method comprising:

providing a sample that is suspected of containing a target polynucleotide;

providing a polycationic multichromophore that comprises a conjugated polymer or dendrimer, wherein said multichromophore has a detectable excitation emission;

providing a sensor that is a polynucleotide that is complementary to the target polynucleotide, wherein said sensor is conjugated to a substrate and is not conjugated to a fluorescently detectable label that can receive energy from an excited state of the multichromophore;

contacting the substrate with the sample;

contacting the substrate with the multichromophore under conditions wherein the multichromophore can bind to the sensor and/or target polynucleotide, if present;

detecting whether target has bound to the sensor.

2. The method of claim 1, wherein detecting whether target has bound to the sensor comprises applying a light source that can excite the multichromophore and detecting whether light is emitted in the vicinity of the substrate.

3. The method of claim 1, wherein detecting whether target has bound to the sensor comprises applying a light source that can excite the multichromophore and detecting whether energy has been transferred from the multichromophore.

4. The method of claim 3, wherein the method comprises detecting whether energy has been transferred from the multichromophore to a fluorophore.

5. The method of claim 1, wherein the polycationic multichromophore has a detectable excitation emission when excited with light comprising a wavelength in the region of about 450 nm to about 1000 nm.

6. The method of claim 2, the method comprising detecting whether the multichromophore emits light in the vicinity of the substrate.

7. The method of claim 1, wherein the multichromophore comprises a conjugated polymer.

9. The method of claim 1, wherein light emitted from the multichromophore above a threshold level indicates that the target polynucleotide is present in the sample.

10. The method of claim 1, wherein the amount of light emitted from the multichromophore is quantitated and used to determine the amount of the target polynucleotide in the sample.

11. A kit for assaying a sample for a target polynucleotide comprising:

a conjugated polymer and that can interact with a target polynucleotide;

a substrate comprising a sensor polynucleotide that is complementary to the target polynucleotide, wherein said sensor polynucleotide is not conjugated to a fluorescently detectable label that can receive energy from an excited state of the multichromophore.

12. The kit of claim 11, further comprising a plurality of different sensor polynucleotides complementary to a corresponding plurality of different target polynucleotides conjugated to the substrate.

13. A detection method comprising:

providing a sample that is suspected of containing a target biomolecule;

providing a chromophore that comprises a conjugated polymer, wherein said chromophore has a detectable excitation emission;

providing a sensor biomolecule capable of recognition pairing with the target biomolecule, wherein the sensor is conjugated to a substrate and is not conjugated to a fluorescently detectable label that can receive energy from an excited state of the chromophore;

contacting the substrate with the sample under conditions in which the sensor can pair with the target, if present;

contacting the substrate with the multichromophore under conditions wherein the multichromophore can bind to the sensor and/or target polynucleotide, if present;

detecting whether target has bound to the sensor.

14. The method of claim 13, wherein the substrate is further contacted with an intercalating dye in a solution under conditions in which the dye can intercalate with the sensor and the target when paired and wherein the target is conjugated to a fluorescently detectable label that can receive energy from an excited state of the chromophore.

15. The method of claim 13, wherein the sensor is selected from the group consisting of a peptide and a polynucleotide.

16. The method of claim 13, wherein the chromophore has a detectable excitation emission when excited with light having a wavelength in the region of about 450 nm to about 1000 nm.