Method for the detection of biological molecules using a two particle complex

• US 9,442,117 B2
• Filed: 08/21/2014
• Issued: 09/13/2016
• Est. Priority Date: 06/23/2004
• Status: Active Grant

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Bard, Allen J., Miao, Wujian
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US14/465,326
• Publication Number:
  US 20150044669A1
• Time in Patent Office: 754 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods, compositions and kits for detecting analytes of interest in a sample using electrogenerated chemiluminescence are provided. Compositions comprising at least one solid support that entraps or contains an electrogenerated chemiluminescent moiety also provided.

Claims

1. A method of detecting an analyte of interest in a sample, comprising

(a) forming a composition comprising:

(A)_k (B)_u,(C),(D)_x

wherein A is an electrogenerated chemiluminescent (ECL) moiety which can be induced to repeatedly emit electromagnetic radiation by direct exposure to an electrochemical energy source; and

the ECL moiety is soluble in organic solvent and insoluble in aqueous solvent;

B is a synthetic organic polymer bead comprising a plurality of the ECL moieties entrapped within its interior; and

B is either linked to the analyte of interest or linked to a first specific binding partner of the analyte of interest;

C is the sample which may contain the analyte of interest; and

D is a magnetizable bead which is either linked to the analyte of interest or linked to a second specific binding partner of the analyte of interest;

wherein k, u, and x are each an integer equal to or greater than 1;

(b) separating a complex comprising A, B, D and the analyte of interest from other components of the composition;

(c) inducing the ECL moiety in the complex to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy; and

(d) detecting the emitted electromagnetic radiation and thereby detecting the presence of the analyte of interest,

provided that B and D are not both linked to the analyte of interest.

2. The method of claim 1, wherein B is linked to the first specific binding partner;

and D is linked to the second specific binding partner.

3. The method of claim 1, wherein the analyte of interest is a protein.

4. The method of claim 3, wherein the first specific binding partner is an antibody, a part of an antibody, or a binding protein;

and the second specific binding partner is an antibody, a part of an antibody, or a binding protein.

5. The method of claim 1, wherein the analyte of interest is a nucleic acid.

6. The method of claim 5, wherein the first specific binding partner is a nucleic acid;

and the second specific binding partner is a nucleic acid.

7. The method of claim 5, wherein the first specific binding partner is an oligonucleotide;

the second specific binding partner is an oligonucleotide.

8. The method of claim 1, wherein the synthetic organic polymer bead comprises a compound chosen from polystyrene, polyacrylic, nylon, polymethacrylate, polyacrylamide, polyacrylonitrile, PVDF, poly(lactide-co-ethylene glycol), and polyolefins.

9. The method of claim 1, wherein the ECL moiety comprises a metal ion selected from osmium or ruthenium and at least one polydendate ligand.

10. The method of claim 1, wherein the ECL moiety comprises bis(2,2′

-bipyridyl)-ruthenium(II) or tris(2,2′

-bipyridyl)ruthenium(II).

11. The method of claim 1, wherein the composition further comprises an amine ECL coreactant.

12. The method of claim 11, wherein the amine ECL coreactant comprises tripropylamine (TPrA), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N,N,N′

,N′

-tetrapropyl-1,3-diaminopropane or a mixture thereof.

13. The method of claim 11, wherein the synthetic organic polymer bead is a polystyrene bead;

the amine ECL coreactant comprises tripropylamine; and

the ECL moiety comprises bis(2,2′

-bipyridyl)-ruthenium(II) or tris(2,2′

-bipyridyl) ruthenium(II).

14. The method of claim 11, wherein each ECL moiety comprises Ru(bpy)₃[B(C₆F₅)₄]₂;

and the amine ECL coreactant comprises tripropylamine.

15. The method of claim 1, wherein the analyte of interest is a protein;

the first specific binding partner is an antibody, a part of an antibody, or a binding protein; and

the second specific binding partner is an antibody, a part of an antibody, or a binding protein; and

the ECL moiety comprises a ruthenium ion and three bidentate ligands.

16. The method of claim 15, wherein the ECL moieties comprise Ru(bpy)₃[B(C₆F₅)₄]₂.

17. The method of claim 1, wherein the analyte of interest is a nucleic acid;

the first specific binding partner is an oligonucleotide; and

the second specific binding partner is an oligonucleotide; and

the ECL moiety comprises a ruthenium ion and three bidentate ligands.

21. The method of claim 1, wherein the ECL moieties comprise a ruthenium ion and at least one polydendate ligand;

the synthetic organic polymer bead is a polystyrene bead;

the magnetizable bead is a magnetic polymeric bead; and

the composition further comprises an amine ECL coreactant.

18. A method of detecting an analyte of interest in a sample, comprising

(a) forming a composition comprising:

(A)_k (B)_u,(C),(D)_x

wherein A is an ECL moiety which can be induced to repeatedly emit electromagnetic radiation by direct exposure to an electrochemical energy source; and

the ECL moiety is soluble in organic solvent and insoluble in aqueous solvent;

B is a polystyrene bead, which is either linked to the analyte of interest or linked to a first specific binding partner of the analyte of interest;

wherein a plurality of the ECL moieties are entrapped within the polystyrene bead;

C is the sample which may contain the analyte of interest; and

D is a magnetic polymeric bead, which is either linked to the analyte of interest or linked to a second specific binding partner of the analyte of interest;

wherein u and x are each an integer equal to or greater than 1;

k is an integer greater than or equal to 2; and

provided that B and D are not both linked to the analyte of interest;

(b) separating a complex comprising A, B, D and the analyte of interest from other components of the composition;

(c) dissolving B from the separated complex in an organic solvent;

(d) inducing the ECL moieties from the dissolved B to repeatedly emit electromagnetic radiation by exposing the ECL moieties to electrochemical energy in the presence of an ECL coreactant, which comprises tertiary amine; and

(e) detecting the emitted electromagnetic radiation.

19. The method of claim 18, wherein the ECL moieties comprise bis(2,2′

-bipyridyl)ruthenium(II) or tris(2,2′

-bipyridyl) ruthenium(II);

the ECL coreactant comprises tripropylamine; and

the analyte of interest is a protein or a nucleic acid molecule.

20. The method of claim 18, wherein the first specific binding partner is an antibody, nucleic acid, receptor or enzyme.