Conjugated polymers for use in homogeneous and solid state assays

• US 8,338,532 B2
• Filed: 03/01/2011
• Issued: 12/25/2012
• 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
• Primary Examiner(s): Truong, Duc
• Application Number:
  US13/038,378
• Publication Number:
  US 20120035066A1
• Time in Patent Office: 665 Days
• Field of Search: 525/54.1, 525/54.2, 525/535, 524/800, 435/6, 428/690
• US Class Current: 525/54.1
• CPC Class Codes: C08G 61/02 Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
• CPC Class Codes: C08G 61/122 derived from five- or six-membered heterocyclic compounds, other than imides
• CPC Class Codes: C08G 61/123 derived from five-membered heterocyclic compounds
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Abstract

The invention further relates to multichromophores, which may be 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. A water-soluble conjugated polymer comprising a plurality of solubilizing functionalities, said polymer soluble in a polar medium,

wherein said polymer comprises a plurality of low bandgap repeat units sufficient for the polymer to form an excited state when the polymer is contacted with incident light in the region of about 450 nm to about 1000 nm in the absence of a target biomolecule, and wherein the polymer can transfer energy from its excited state to a fluorophore to provide a greater than 50% increase in fluorescence emission from the fluorophore than can be achieved by direct excitation.

2. The polymer of claim 1, wherein the polymer is water soluble.

3. The polymer of claim 1, wherein the peak absorbance of the polymer shifts no more than about 0.10 eV upon binding to the target.

4. The polymer of claim 1, wherein the polymer comprises a solid phase.

5. The polymer of claim 4, wherein the solid phase is a film.

6. The polymer of claim 1, wherein the polymer is purified.

7. A solution comprising the polymer of claim 1 and a solvent.

11. A detection complex comprising the polymer of claim 1 and a target biomolecule.

8. A kit for assaying a sample for a target biomolecule comprising:

a water-soluble conjugated polymer comprising a plurality of solubilizing functionalities, wherein said polymer absorbs light of greater than about 450 nm to about 1000 nm in the absence of target biomolecule and forms an excited state; and

a sensor biomolecule that can bind to the target biomolecule.

9. The kit of claim 8, wherein the sensor biomolecule is conjugated to a substrate.

10. The kit of claim 8, wherein the sensor biomolecule is provided in a solution.

12. A polymer prepared by Suzuki copolymerization of 2,7-bis [9,9′

-bis(6″

-bromohexyl)-fluorenyl]-4,4,5,5-tetramethy-[1.3.2]di-oxaborolane and 4,7- dibromo-2,1,3-benzothiadiazole.

13. A method for the detection of a target biomolecule, the method comprising contacting a water-soluble conjugated polymer with the target biomolecule,

wherein the polymer comprises a plurality of low bandgap repeat units sufficient for the polymer to form an excited state when the polymer is contacted with incident light in the region of about 450 nm to about 1000 nm in the absence of a target biomolecule, and

wherein the polymer can transfer energy from its excited state to a fluorophore to provide a greater than 50% increase in fluorescence emission from the fluorophore than can be achieved by direct excitation.

14. The method of claim 13, wherein the peak absorbance of the polymer shifts no more than about 0.10 eV upon binding to the target.

15. The method of claim 13, further comprising contacting the target biomolecule with a sensor biomolecule that can bind to the target biomolecule.

16. The method of claim 15, wherein the sensor biomolecule is conjugated to a substrate.

17. The method of claim 13, wherein the conjugated polymer is prepared by Suzuki copolymerization of 2,7-bis[9,9′

-bis(6″

-bromohexyl)-fluorenyl]-4,4,5,5-tetramethyl-[1.3.2]di-oxaborolane and 4,7-dibromo-2,1,3-benzothiadiazole.