Methods and compositions for assaying a sample for an aggregant

• US 8,835,113 B2
• Filed: 09/03/2013
• Issued: 09/16/2014
• 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:
  US14/017,127
• Publication Number:
  US 20140087480A1
• Time in Patent Office: 378 Days
• Field of Search: 435/6.1, 435/7.1
• US Class Current: 435/6.1
• 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 61/123 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. A method of assaying a sample for an aggregant, the method comprising:

(a) combining the sample with an aggregation sensor comprising

(i) a 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 absorb light to form an excited state that can emit light of a first emission wavelength, and a plurality of solubilizing functionalities; and

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

wherein said aggregation sensor comprises at least three first optically active units per second optically active unit and the second optically active unit is grafted to the conjugated system;

(b) contacting the sample with light of the first absorption wavelength; and

(c) detecting the optical properties of the aggregation sensor to assay the sample for the aggregant.

2. The method of claim 1, wherein detecting the optical properties of the aggregation sensor comprises detecting if the aggregation sensor emits light at the first emission wavelength.

3. The method of claim 1, wherein the polymer comprises a sufficient amount of repeat units to provide a two-fold or greater increase in emission from an optically active species to which it can transfer energy.

4. The method of claim 3, wherein the polymer comprises a sufficient amount of repeat units to provide a three-fold or greater increase in emission from an optically active species to which it can transfer energy.

5. The method of claim 4, wherein the polymer comprises a sufficient amount of repeat units to provide a four-fold or greater increase in emission from an optically active species to which it can transfer energy.

6. The method of claim 5, wherein the polymer comprises a sufficient amount of repeat units to provide a five-fold or greater increase in emission from an optically active species to which it can transfer energy.

7. The method of claim 1, wherein the one or more second optically active units transfer energy to a subsequent fluorophore, a series of fluorophores, or a quencher.

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

9. The method of claim 1, wherein detecting the optical properties of the aggregation sensor comprises detecting if the aggregation sensor emits decreased light at the first emission wavelength.

10. The method of claim 1, wherein:

the second optically active unit forms an excited state upon receiving energy from the first optically active unit and emits light of a second emission wavelength; and

detecting the optical properties of the aggregation sensor comprises detecting light emission at the second emission wavelength.

11. The method of claim 1, further comprising combining a probe with the aggregation sensor and the sample, wherein the probe specifically binds to the aggregant.

12. The method of claim 11, wherein the probe comprises a binding pair member.

13. The method of claim 12, wherein the binding pair member is selected from the group consisting of a polypeptide, a polynucleotide and a peptide nucleic acid (PNA).

14. The method of claim 12, wherein the binding pair member is selected from a member of an immunological binding pair.

15. The method of claim 14, wherein the member of an immunological binding pair is an antibody or binding portion thereof.

16. The method of claim 11, wherein said probe is attached to a fluorophore that receives energy from the second optically active unit when in proximity thereto and then emits light of a third emission wavelength.

17. The method of claim 16, wherein detecting the optical properties of the aggregation sensor comprises detecting if the solution emits light of the third emission wavelength.

18. The method of claim 17, wherein the amount of light emitted from the fluorophore is greater than can be achieved by direct excitation of the fluorophore with light of an appropriate wavelength.

19. The method of claim 11, wherein said probe is attached to a quencher that receives energy from the aggregation sensor when in proximity thereto and then emits decreased light.

20. The method of claim 1, wherein the conjugated polymer comprises at least one optionally substituted repeat unit selected from the group consisting of 2,1,3- benzothiadiazole, benzoselenadiazole, naphthoselenadiazole, 4,7 -di(thien-2-yl)-2, 1 ,3- benzothiadiazole, an olefin, a cyano-substituted olefin, 2,7-carbazolene-vinylene, 2,7-fluorene and 4,4′

-biphenyl.

21. The method of claim 1, wherein the aggregation sensor comprises a conjugated polymer comprising optionally substituted poly(fluorene-co-phenylene) repeat units with 2,1,3-benzothiadiazole as the second optically active unit.

22. The method of claim 1, wherein the aggregation sensor comprises a ratio of first optically active units per second optically active units selected from the group consisting of at least four first optically active units per second optically active unit, at least six first optically active units per second optically active unit, at least nine first optically active units per second optically active unit, and at least nineteen first optically active units per second optically active unit.

23. The method of claim 1, wherein the aggregation sensor contains one second optically active unit.

24. The method of claim 1, wherein the first absorption wavelength is about 488 nm.

25. The method of claim 1, wherein the aggregant is selected from the group consisting of a polypeptide and a polynucleotide.

26. The method of claim 25, wherein the aggregant is a polypeptide.

27. The method of claim 1, further comprising quantitating the aggregant in the sample.

28. The method of claim 1, further comprising using a result of the detecting to diagnose a disease state of a patient.

29. The method of claim 28, wherein using a result comprises reviewing or analyzing data relating to the presence of an aggregant in a sample and providing a conclusion to a patient, a health care provider or a health care manager, the conclusion being based on the review or analysis of data regarding a disease diagnosis.