US 8,946,389 B2Granted

Compositions and methods for multiplex biomarker profiling

AssigneeUniversity of Washington

InventorsXiaohu Gao, Pavel Zrazhevskiy

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Abstract

Provided herein are compositions and methods for identifying or quantitating one or more analytes in sample. The composition can comprise an affinity molecule reversibly conjugated to a label moiety via a double-stranded nucleic acid linker or via an adaptor molecule. The affinity molecule and the label moiety can be linked to different strands of the double-stranded nucleic acid linker. Compositions can be used in any biological assays for detection, identification and/or quantification of target molecules or analytes, including multiplex staining for molecular profiling of individual cells or cellular populations. For example, the compositions can be adapted for use in immunofluorescence, fluorescence in situ hybridization, immunohistochemistry, western blot, and the like.

Claims26

Dependent claims

A composition comprising an affinity molecule reversibly conjugated to a label moiety, in which the affinity molecule is linked to the label moiety via a linker comprising first and second strands of nucleic acid that specifically hybridize to each other,

wherein the first nucleic acid strand is linked to the affinity molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH2 groups triazole (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof,
and wherein the second nucleic acid strand is linked to the label via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof,
such that the affinity molecule is conjugated to the label moiety under conditions that permit hybridization between the first and second nucleic acid strands, but is not conjugated to the label under conditions that do not permit such hybridization.

A method of analyzing a sample for a plurality of analytes, the method comprising, in order:

(a) contacting the sample with a first plurality of different affinity molecules under conditions that permit specific analyte binding by the different affinity molecules, wherein each different affinity molecule specifically binds a different member of the plurality of analytes and members of the plurality of different affinity molecules become bound to members of the plurality of analytes present in the sample, wherein each different affinity molecule is reversibly conjugated to a different luminescent nanoparticle, in which the nanoparticle is covalently linked to an adaptor molecule and the adaptor molecule is non-covalently linked to the affinity molecule, the affinity molecule and the adaptor molecule are present in a 1:1 ratio, and wherein the luminescent nanoparticle is a colloidal water-soluble nanoparticle comprising a stable non-fouling coating wherein non-specific binding of the nanoparticle to a cell or a tissue sample is 5% or less relative to a nanoparticle lacking a non-fouling coating, and detectable properties of the luminescent nanoparticles are distinguishable from one another;
and
(b) detecting signal from luminescent nanoparticles associated with the first plurality of affinity molecules bound to the sample, thereby detecting the presence or amount of the plurality of analytes.

Dependencies are inferred from the claim text.

Facts

Application: 13/455,674Filed 25 Apr 2012 · us provisional application
Priority: 25 Apr 2011
Published: US 2015/0004598 A11 Jan 2015
Granted: 3 Feb 2015
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12Q 1/6841
Hybridisation assays: in situ hybridisation
C12Q 1/6804
Involving nucleic acids: nucleic acid analysis using immunogens
G01N 33/588
Involving labelled substances: with semiconductor nanocrystal label, e.g. quantum dots
G01N 33/587
With a particulate label, e.g. coloured latex: nanoparticles