Cationic conjugated polymers suitable for strand-specific polynucleotide detection in homogeneous and solid state assays

• US 20060183140A1
• Filed: 01/10/2006
• Published: 08/17/2006
• 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 7,897,684 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 polycationic 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 polycationic conjugated polymer that can electrostatically interact with a biomolecule comprising a plurality of anionic groups, 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 the 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 polymer comprises a plurality of optionally substituted 2,1,3-benzothiadiazole repeat units.

4. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted benzoselenadiazole repeat units.

5. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted naphthoselenadiazole repeat units.

6. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole repeat units.

7. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted olefinic repeat units.

8. The polymer of claim 1, wherein the polymer comprises a plurality of cyano-substituted olefinic repeat units.

9. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted 2,7-carbazolene-vinylene repeat units.

10. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted 2,7-fluorene repeat units.

11. The polymer of claim 1, wherein the polymer comprises a plurality of optionally substituted 4,4′

-biphenyl repeat units.

12. The polymer of claim 1, wherein the polymer comprises greater than about 15 mol % of the low bandgap repeat unit.

13. The polymer of claim 1, wherein the polymer comprises greater than about 20 mol % of the low bandgap repeat unit.

14. The polymer of claim 1, wherein the polymer comprises greater than about 25 mol % of the low bandgap repeat unit

15. The polymer of claim 1, wherein the polymer comprises greater than about 35 mol % of the low bandgap repeat unit.

16. The polymer of claim 1, wherein the polymer comprises at least about 50 mol % of the low bandgap repeat unit.

17. The polymer of claim 1, wherein the polymer forms an excited state upon contact with incident light having a wavelength selected from the group consisting of about 488 nm, about 532 nm, about 594 nm and about 633 nm.

18. 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.

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

20. The polymer of claim 19, wherein the solid phase is a film.

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

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

23. The solution of claim 22, wherein the solution is predominantly aqueous.

24. The solution of claim 22, wherein the solution further comprises a second solvent that can reduce nonionic interactions with the polymer.

25. The solution of claim 24, wherein the second solvent is an alcohol.

26. The solution of claim 24, wherein the second solvent is 1-methyl-2-pyrrolidinone.

35. The polymer of claim 1, wherein the polymer provides at least a two-fold, three-fold, four-fold, or five-fold increase in fluorescence emission from the fluorophore.

36. A detection complex comprising the polymer of claim 1 and a target biomolecule, wherein said target biomolecule is bound to a sensor biomolecule.

37. The detection complex of claim 36, wherein the sensor biomolecule is conjugated to a substrate.

38. The detection complex of claim 37, where the sensor biomolecule is not labeled with a fluorophore that can receive energy from an excited state of the multichromophore.

58. Use of the polymer of claim 1 for the detection of a target biomolecule.

59. The use according to claim 58, wherein the target biomolecule is a polynucleotide.

60. The use according to claim 59, wherein the polynucleotide is amplified.

61. The use according to claim 60, wherein the polymer is used for the detection of a target biomolecule in a real-time amplification reaction.

62. The use according to claim 58, wherein the polymer is used for the quantitation of a target biomolecule.

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

a polycationic conjugated polymer, wherein said polymer can electrostatically interact with a polyanionic target biomolecule, 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 polynucleotide that can bind to the target biomolecule.

28. The kit of claim 27, wherein the sensor polynucleotide is conjugated to a substrate.

29. The kit of claim 27, wherein the sensor polynucleotide is provided in a solution.

30. The kit of claim 27, wherein the sensor polynucleotide comprises PNA.

31. The kit of claim 27, wherein the sensor polynucleotide comprises DNA.

32. The kit of claim 27, wherein the sensor polynucleotide is conjugated to a fluorescent label that can receive energy from an excited state of the multichromophore.

33. The kit of claim 27, wherein the sensor polynucleotide is not conjugated to a fluorescent label that can receive energy from an excited state of the multichromophore.

34. The kit of claim 27, further comprising a plurality of different sensor polynucleotides.

39. An aqueous solution comprising a polycationic conjugated polymer that can electrostatically interact with a biomolecule comprising a plurality of anionic groups,

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 the target biomolecule.

40. A polymer that can interact with a target biomolecule, the polymer being soluble in a polar medium and wherein the polymer and the target interact electrostatically,

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 the target biomolecule, and wherein the polymer can transfer energy from its excited state to a fluorophore, the fluorophore generating more fluorescence with the polymer present than if the polymer is not present.

41. The polymer of claim 40, wherein the polymer forms an excited state upon contact with incident light having a wavelength selected from the group consisting of 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 700 nm, and 700 nm to 1000 nm.

42. The polymer of claim 40, wherein the polymer forms an excited state upon contact with incident light having a wavelength selected from the group consisting of about 488 nm, about 532 nm, about 594 nm and about 633 nm.

43. The polymer of claim 42, wherein the wavelength is about 488 nm.

44. The polymer of claim 40, wherein the plurality of low bandgap repeat units comprise repeat units selected from the group consisting of optionally substituted 2,1,3-benzothiadiazole, optionally substituted benzoselenadiazole, optionally substituted naphthoselenadiazole, optionally substituted 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, optionally substituted olefinic, cyano-substituted olefinic, and optionally substituted 2,7-carbazolene-vinylene.

45. The polymer of claim 40, wherein the increased fluorescence emission from the fluorophore is greater than 50% over the emission that can be achieved from direct excitation of the fluorophore.

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

a polycationic conjugated polymer, wherein said polymer can electrostatically interact with a polyanionic target comprising a first biomolecule, wherein said polymer absorbs light of about 450 nm to about 1000 nm in the absence of target and forms an excited state; and

a sensor biomolecule capable of recognition pairing with the target.

47. The kit of claim 46, wherein the sensor is conjugated to a substrate.

48. The kit of claim 46, wherein the polymer forms an excited state upon contact with incident light having a wavelength selected from the group consisting of 450 nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, 600 nm to 700 nm, and 700 nm to 1000 nm.

49. The kit of claim 46, wherein the polymer forms an excited state upon contact with incident light having a wavelength selected from the group consisting of about 488 nm, about 532 nm, about 594 nm and about 633 nm.

50. The kit of claim 49, wherein the wavelength is about 488 nm.

55. The kit of claim 46, wherein the sensor biomolecule comprises a protein or polypeptide.

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

a polycationic conjugated polymer, wherein said polymer can electrostatically interact with a polyanionic target polynucleotide, wherein said polymer absorbs light at a wavelength of about 488 nm in the absence of target polynucleotide and forms an excited state; and

a sensor polynucleotide capable of recognition pairing with the target.

52. The kit of claim 51, wherein the target polynucleotide comprises single-stranded DNA.

53. The kit of claim 51, wherein the sensor polynucleotide comprises DNA.

54. The kit of claim 51, wherein the sensor polynucleotide comprises PNA.

56. A polymer having the structure:

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

-bis(6″

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

63. Use of the polymer of claim 57 for the detection of a target biomolecule.