Aggregation sensor and solutions and kits comprising the same

• US 8,110,673 B2
• Filed: 02/23/2010
• Issued: 02/07/2012
• Est. Priority Date: 01/31/2005
• 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): Horlick, Kenneth R.
• Application Number:
  US12/711,233
• Publication Number:
  US 20100255477A1
• Time in Patent Office: 714 Days
• Field of Search: 536/24.3
• US Class Current: 536/24.3
• 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 61/123 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 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to an aggregation sensor useful for the detection and analysis of aggregants in a sample, and methods, articles and compositions relating to such a sensor. The sensor comprises first and second optically active units, where energy may be transferred from an excited state of the first optically active unit to the second optically active unit. The second optically active unit is present in a lesser amount, but its relative concentration is increased upon aggregation, increasing its absorption of energy from the first optically active units. This increase in energy transfer can be detected in variety of formats to produce an aggregation sensing system for various aggregants, including for quantitation. Other variations of the inventions are described further herein.

Claims

1. An aggregation sensor soluble in a polar medium comprising:

(a) a conjugated polymer comprising

a plurality of first optically active units forming a conjugated system, having a first absorption wavelength at which the first optically active units absorbs light to form an excited state, and

a plurality of solubilizing functionalities; and

(b) one or more second optically active units that can receive energy from the excited state of the first optically active unit;

said aggregation sensor comprising at least three first optically active units per second optically active unit;

wherein the second optically active unit is grafted to the conjugated polymer.

2. The aggregation sensor of claim 1, wherein the conjugated polymer comprises a sufficient amount of repeat units to provide a two-fold, three-fold, four fold, five fold, or greater increase in emission form an optically active species to which it can transfer energy.

3. The aggregation sensor of claim 1, wherein the second optically active unit forms part of the conjugated system.

4. The aggregation sensor of claim 1, wherein the one or more second optically active units disperse energy non-radiatively.

5. The aggregation sensor of claim 1, wherein the one or more second optically active units emit light of a characteristic wavelength.

6. The aggregation sensor of claim 1, wherein the one or more second optically active units are used to transfer energy to a subsequent fluorophore, series of fluorophores, or quencher.

7. The aggregation sensor of claim 1, wherein the aggregation sensor further comprises a component having an affinity for an aggregant.

8. The aggregation sensor of claim 7, wherein the component comprises an ionic region.

9. The aggregation sensor of claim 7, wherein the component comprises one member of a binding pair.

10. The aggregation sensor of claim 9, wherein the member of a binding pair is selected from a member of an immunological binding pair.

11. The aggregation sensor of claim 10, wherein the member of an immunological binding pair is an antibody or binding portion thereof.

12. The aggregation sensor of claim 9, wherein the member of a binding pair is selected from biotin, avidin, streptavidin, a hormone, a hormone binding protein, a receptor, a receptor agonist, a receptor antagonist, IgG, protein A, a lectin, a carbohydrate, an enzyme, an enzyme cofactor, an enzyme inhibitor, ad a polynucleotide.

13. The aggregation sensor of claim 7, wherein the component is a sensor.

14. The aggregation sensor of claim 7, wherein the aggregant is a biomolecule.

15. A solution comprising the aggregation sensor of claim 1 and a solvent.

16. The solution of claim 15, wherein the solvent is water.

17. The solution of claim 15, wherein the aggregation sensor is present at a concentration at which the first emission wavelength is detectable when the solution is contacted with light comprising the first absorption wavelength.

18. The solution of claim 15, wherein effectively all of the first optically active units in the solution are within energy-transferring distance of at least one second optically active unit.

19. The solution of claim 15, wherein the solution further comprises one or more additional components selected from a solvent, a buffer, a biomolecule, a polynucleotide, and a fluorophore.

20. The solution of claim 15, wherein the solution further comprises a second solvent that can reduce hydrophobic interactions between components of the solution.

21. The solution of claim 15, further comprising a probe that can bind specifically to a particular species of a class of aggregants that comprises the conjugated polymer aggregation sensor, said probe attached to a fluorophore or quencher that can receive energy from the second optically active unit.

22. The solution of claim 15, further comprising a sample suspected of comprising an aggregant.

23. The solution of claim 22, wherein the sample comprises cells.

24. An aggregate comprising the aggregation sensor of claim 1 and an aggregant.

25. The aggregate of claim 24, wherein the aggregation sensor comprises a first member of a binding pair and the aggregant comprises a second member of a binding pair.

26. A kit for assaying a sample for an aggregant comprising:

the aggregation sensor of claim 1; and

a probe that can bind specifically to a particular species of a class of aggregants that can bind to the aggregation sensor.

27. The kit of claim 26, wherein the kit further comprises a substrate.

28. The kit of claim 26, further comprising a plurality of different probes each specific for a different species of aggregant, each of said different probes comprising a different label.