Methods for assaying a sample for an aggregant

• US 7,666,594 B2
• Filed: 01/31/2006
• Issued: 02/23/2010
• 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:
  US11/344,942
• Publication Number:
  US 20060204984A1
• Time in Patent Office: 1,484 Days
• Field of Search: 435/6, 435/7.1
• US Class Current: 435/6.11
• 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:

providing an aggregation sensor soluble in a polar medium, the aggregation sensor comprising

(a) a polyionic 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 first charges; and

(b) one or more second optically active units that can receive energy from the excited state of the first optically active unit, wherein the second optically active unit is grafted to the conjugated system or the second optically active unit forms part of the conjugated system;

said aggregation sensor comprising at least three first optically active units per second optically active unit, wherein a solution of the aggregation sensor emits detectably less light at the first emission wavelength when the concentration of first optically active units within energy-transferring distance of at least one second optically active unit is increased;

combining the aggregation sensor and the sample in solution, wherein the aggregation sensor is present at a concentration at which there is detectable emission at the first emission wavelength in the absence of sample;

contacting the aggregation sensor with light of the first absorption wavelength; and

detecting whether the optical properties of the aggregation sensor are altered in the presence of the sample.

2. The method of claim 1, where detecting whether the optical properties of the aggregation sensor are altered in the presence of the sample comprises detecting if the aggregation sensor emits less light of the first emission wavelength.

3. The method of claim 1, where 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 where detecting whether the optical properties of the aggregation sensor are altered in the presence of the sample comprises detecting if the aggregation sensor emits light of the second emission wavelength.

4. The method of claim 3, further comprising combining a probe with the aggregation sensor and the sample in solution, wherein the probe binds specifically to a particular species of a class of aggregants that binds to the aggregation sensor, said probe 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, where detecting whether the optical properties of the aggregation sensor are altered in the presence of the sample comprises detecting if the solution emits light of the third emission wavelength.

5. The method of claim 4, 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.

6. The method of claim 4, wherein the probe comprises a component selected from the group consisting of a polypeptide, a polynucleotide, and a peptide nucleic acid (PNA).

7. The method of claim 1, further comprising combining an intercalating dye with the aggregation sensor and the sample in solution under conditions in which the dye intercalates with a sequence-specific duplex aggregate comprising a probe comprising a polynucleotide bound to a polynucleotide aggregant, wherein the intercalated dye exchanges energy with at least one other optically active species present in the aggregate when formed.

8. 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.

9. 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.

10. 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.

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

12. The method of claim 1, wherein the first charges are selected from the group consisting of cations and anions.

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

14. The method of claim 1, wherein the class of aggregants is selected from the group consisting of polypeptides and polynucleotides.

15. The method of claim 14, wherein the aggregant detected is a polynucleotide produced via an amplification reaction.

16. The method of claim 1, wherein the second optically active unit is grafted to the conjugated system.

17. The method of claim 1, wherein the second optically active unit forms part of the conjugated system.

18. The method of claim 1, wherein a single nucleotide polymorphism (SNP) is detected in the aggregant.

19. The method of claim 1, wherein expression of a gene is detected upon detection of the aggregant.

20. The method of claim 1, wherein a result is used to diagnose a disease state of a patient.

21. The method of claim 20, wherein the use of the result to diagnose a disease state 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.

22. The method of claim 21, wherein providing a conclusion comprises transmission of the data over a network.

23. The method of claim 1, where an aggregant is present in the sample and an aggregate is formed comprising the aggregant and the aggregation sensor.

24. The method of claim 1, wherein the method is selected from the group consisting of an assay of relative amount, a quantitative assay and a semi-quantitative assay.

25. The method of claim 1, wherein the aggregant is a polynucleotide,

wherein the second optically active unit forms part of the conjugated system,

where 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 where detecting whether the optical properties of the aggregation sensor are altered in the presence of the sample comprises detecting if the aggregation sensor emits light of the second emission wavelength.

26. The method of claim 25, wherein the first absorption wavelength is about 488 nm.

27. The method of claim 25, wherein the method is quantitative.

28. The method of claim 1, wherein the aggregant comprises a plurality of second charges opposite in charge to the first charges.