Methods and compositions for the detection of biological molecules using a two particle complex

• US 20060078912A1
• Filed: 06/23/2005
• Published: 04/13/2006
• 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
• Granted Patent: US 8,188,243 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

The invention provides methods of detecting analytes of interest in a sample using electrogenerated chemiluminescence. The invention also provides compositions comprising at least one solid support that entraps or contains an electrogenerated chemiluminescent moiety.

Claims

1-47. -47. (canceled)

48. A composition for detecting analyte of interest in a sample comprising:

(A)k, (B)u, (C), (D)_x

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

B is at least one first carrier containing A, wherein A can be released from B and B is either linked to

(1) an analog of the analyte of interest;

or

(2) 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 at least one second carrier and is either linked to

(3) the analog of the analyte of interest;

or

(4) 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,

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

provided that if B is linked to the analog of the analvte of interest, then D is linked to the second specific binding partner and if D is linked to the analog of the analyte of interest, then B is linked to the first specific binding partner.

49. The composition of claim 48, wherein the analog of the analyte of interest is linked to B and wherein the second specific binding Partner is linked to D.

50. The composition of claim 48, wherein the analog of the analyte of interest is linked to D and wherein the first specific binding partner is linked to B.

52. The composition of claim 48, wherein at least one of B and D is a solid support.

53. The composition of claim 52, wherein B is a first solid support and D is a second solid support.

56. The composition of claim 48, wherein the first specific binding partner is linked to B and the second specific binding partner is linked to D.

61. The composition of claim 48, wherein the analyte of interest is a protein.

62. The composition of claim 48, wherein the ECL moiety is soluble in an organic solvent.

63. The composition of claim 48, wherein the ECL moiety is insoluble in an aqueous solvent.

64. The composition of claim 48, wherein the ECL moiety comprises rubrene and/or 9,10-diphenylanthracene.

65. The composition of claim 48, wherein B and D are independently a bead, a gel, a membrane, a liposome, or a micelle.

66. The composition of claim 53, wherein the first and second solid supports are independently comprised of a compound chosen from polystyrene, polypropylene, agarose, acrylamide, nitrocellulose, nylon, PVDF, Sepharose®

, Sephadex®

, hyaluronic acid, chitin, acyl gellan, dextran, carboxymethylcellulose, carboxymethyl starch, carboxymethyl chitin, silica, and poly(lactide-co-ethylene glycol).

67. The composition of claim 53, wherein at least one of the first and second solid supports is a bead.

68. The composition of claim 53, wherein the second solid support is a magnetizable bead.

69. The composition of claim 53, wherein the first solid support is a polystyrene bead.

71. The composition of claim 48, wherein the ECL moiety comprises a metal ion selected from osmium and ruthenium.

73. The composition of claim 64, wherein the ECL moiety comprises Ru(bpy)₃[B(C₆F₅)₄]₂.

77. The composition of claim 56, wherein said first and said second specific binding partners are oligonucleotides.

85. The composition of claim 56, wherein the first specific binding partner of the protein of interest and the second specific binding partner of the protein of interest are independently selected from antibodies and binding proteins.

205. The composition of claim 48 where k is an integer between 1×

10² and 1×

10¹⁵.

206. The kit of claim 205, comprising 1×

10²-1×

10¹⁵ molecules of the at least one ECL moiety.

207. The composition of claim 48, wherein B is soluble in an organic solvent.

208. The composition of claim 207, wherein the organic solvent is acetonitrile, ether, chloroform, tetrahydrofuran (THF), benzene, or a mixture thereof.

209. The composition of claim 52 or 53, wherein the solid support is insoluble in an aqueous environment.

210. The composition of claim 65, wherein the gel is low melting temperature agarose.

211. The composition of claim 48, wherein at least one of B and D comprises a liquid.

212. The composition of claim 211, wherein the liquid comprises at least one amphiphilic molecule.

213. The composition of claim 48, wherein at least one of B and D does not comprise an antibody or an oligonucleotide.

214. The composition of claim 48, further comprising a coreactant selected from tripropylamine (TPrA), persulfate, piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), and N,N,N′

,N′

-Tetrapropyl-1,3-diaminopropane.

215. The composition of claim 48, wherein B is permeable to a coreactant and impermeable to a binding partner of the analyte of interest.

219. The composition of claim 48, wherein k is between 1×

10² and 1×

10¹⁰.

220. The composition of claim 48, wherein a partition coefficient for the ECL moiety between the first carrier and an aqueous solution is greater than 10.

222. The composition of claim 48, wherein the analog of the analyte of interest is a known amount of the analyte of interest itself.

51. (canceled)

54-55. -55. (canceled)

57-60. -60. (canceled)

70. (canceled)

72. (canceled)

76. (canceled)

78-82. -82. (canceled)

86-177. -177. (canceled)

179-185. -185. (canceled)

186. A kit for detecting an analyte of interest in a sample, comprising

at least one ECL moiety which can be induced to repeatedly emit electromagnetic radiation by direct exposure to an electrochemical energy source;

at least one first carrier containing the ECL moiety, wherein the ECL moiety can be released from the first carrier and the first carrier is either linked to

(1) an analog of the analyte of interest;

or

(2) a first specific binding partner of the analyte of interest;

at least one second carrier which is either linked to

(3) the analog of the analvte of interest;

or

(4) a second specific binding partner of the analyte of interest;

provided that the first carrier and the second carrier are not both linked to the analog of the analyte of interest, and

provided that if the first carrier is linked to the analog of the analvte of interest, then the second carrier is linked to the second specific binding partner and if the second carrier is linked to the analog of the analyte of interest, then the first carrier is linked to the first specific binding partner.

187. The kit of claim 186, wherein the ECL moiety comprises Ru(bpy)₃[B(C₆F₅)₄]₂, rubrene, and/or 9,10-diphenylanthracene.

188. The kit of claim 186, wherein the first and second carriers are solid supports.

189. The kit of claim 186, wherein the first carrier is a polystyrene bead.

190. The kit of claim 186, wherein the second carrier is a magnetizable bead.

191. The kit of claim 186, wherein the first carrier is a polystyrene bead and the second carrier is a magnetizable bead.

193. The kit of claim 186, wherein the first specific binding partners is an oligonucleotides.

194. The kit of claim 186, wherein the analog of the analyte of interest is an oligonucleotide.

196. The kit of claim 186, wherein the first specific binding partners is an antibody or a binding proteins.

216. The kit of claim 187, wherein the ECL moiety comprises a metal ion chosen from osmium and ruthenium.

217. The kit of claim 186, wherein the second specific binding partner is an oligonucleotide.

218. The kit of claim 186, wherein the second specific binding partner is an antibody or a binding protein.

221. The kit of claim 186, comprising 1×

10²-1×

10¹⁰ molecules of the at least one ECL moiety.

223. The kit of claim 186, wherein the analog of the analyte of interest is a known amount of the analyte of interest itself.

192. (canceled)

195. (canceled)

197. A composition comprising a polystyrene bead containing an electrochemiluminescent compound comprising an aromatic hydrocarbon.

198. The composition of claim 197, wherein the aromatic hydrocarbon is chosen from diphenylanthracene and rubrene.

199. The composition of claim 197, wherein the polystyrene bead contains 1×

10²-1×

10¹⁵ molecules of the electrochemiluminescent compound.

200. The composition of claim 197, wherein the polystyrene bead contains 1×

10²-1×

10¹⁰ molecules of the electrochemiluminescent compound.

201-204. -204. (canceled)