U.S. patent number RE47,983 [Application Number 16/022,679] was granted by the patent office on 2020-05-12 for compositions and methods for multiplex biomarker profiling.
This patent grant is currently assigned to University of Washington through its Center for Commercialization. The grantee listed for this patent is University of Washington. Invention is credited to Xiaohu Gao, Pavel Zrazhevskiy.
View All Diagrams
United States Patent |
RE47,983 |
Gao , et al. |
May 12, 2020 |
Compositions and methods for multiplex biomarker profiling
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.
Inventors: |
Gao; Xiaohu (Shoreline, WA),
Zrazhevskiy; Pavel (Redmond, WA) |
Applicant: |
Name |
City |
State |
Country |
Type |
University of Washington |
Seattle |
WA |
US |
|
|
Assignee: |
University of Washington through
its Center for Commercialization (Seattle, WA)
|
Family
ID: |
52115941 |
Appl.
No.: |
16/022,679 |
Filed: |
June 28, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
13455674 |
Feb 3, 2015 |
8946389 |
|
|
|
61478618 |
Apr 25, 2011 |
|
|
|
|
61478626 |
Apr 25, 2011 |
|
|
|
Reissue of: |
14577507 |
Dec 19, 2014 |
9376717 |
Jun 28, 2016 |
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6804 (20130101); G01N 33/588 (20130101); C12Q
1/6804 (20130101); C12Q 1/6841 (20130101); G01N
33/588 (20130101); C12Q 1/6841 (20130101); C12Q
1/6804 (20130101); C12Q 1/6804 (20130101); C12Q
2563/155 (20130101); C12Q 2563/155 (20130101); G01N
33/587 (20130101); G01N 33/587 (20130101); G01N
2458/10 (20130101); G01N 2458/10 (20130101) |
Current International
Class: |
G01N
33/53 (20060101); C12Q 1/68 (20180101); C12Q
1/6841 (20180101); G01N 33/58 (20060101); C12Q
1/6804 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2010/115089 |
|
Oct 2010 |
|
WO |
|
WO 2010/115089 |
|
Oct 2010 |
|
WO |
|
Other References
Alivisatos, A.P., "Perspectives on the Physical Chemistry of
Semiconductor Nanocrystals," Journal of Physical Chemistry
100(31)13226-13239, 1996. cited by applicant .
Alivisatos, A.P., "Semiconductor Clusters, Nanocrystals, and
Quantum Dots," Science 271(5251):933-937, 1996. cited by applicant
.
Bailey, R.C., et al., "DNA-Encoded Antibody Libraries: A Unified
Platform for Multiplexed Cell Sorting and Detection of Genes and
Proteins," Journal of the American Chemical Society
129(7):1959-1967, 2007. cited by applicant .
Bruchez, M., et al., "Semiconductor Nanocrystals as Fluorescent
Biological Labels," Science 281(5385):2013-2016, 1998. cited by
applicant .
Cady, N.C., et al., "Optimized Linkage and Quenching Strategies for
Quantum Dot Molecular Beacons," Molecular and Cellular Probes
21(2):116-124, 2007. cited by applicant .
Chan, P., et al., "Method for Multiplex Cellular Detection of mRNAs
Using Quantum Dot Fluorescent in Situ Hybridization," Nucleic Acids
Research 33(18):e161, 2005. cited by applicant .
Chan, W.C., et al., "Luminescent Quantum Dots for Multiplexed
Biological Detection and Imaging," Current Opinions in
Biotechnology 13(1):40-46, 2002. cited by applicant .
Chen, C., et al., "Quantum-Dot-Based Immunofluorescent Imaging of
HER2 and ER Provides New Insights Into Breast Cancer
Heterogeneity," Nanotechnology 21(9):095101, 2010. cited by
applicant .
Chen, Y., et al., "Selection and Analysis of an Optimized Anti-VEGF
Antibody: Crystal Structure of an Affinity-Matured Fab in Complex
With Antigen," Journal of Molecular Biology 293:865-881, Nov. 1999.
cited by applicant .
Christensen, N.K., and L. Winther, "Multi-Staining
Immunohistochemistry," Chapter 15 in Kumar and Rudbeck (eds.),
"Immunohistochemical (IHC) Staining Methods," .COPYRGT.2009 Dako
North America, 5th Edition, pp. 103-108. cited by applicant .
Duose, D.Y., et al., "Configuring Robust DNA Strand Displacement
Reactions for in situ Molecular Analyses," Nucleic Acids Research
40(7):3289-3298, 2012. cited by applicant .
Duose, D.Y., et al., "Multiplexed and Reiterative Fluorescence
Labeling via DNA Circuitry," Bioconjugate Chemistry 21:2327-2331,
2010. cited by applicant .
Englert, C.R., et al., "Layered Expression Scanning: Rapid
Molecular Profiling of Tumor Samples," Cancer Research
60(6):1526-1530, 2000. cited by applicant .
Fountaine, T.J., et al., "Multispectral Imaging of Clinically
Relevant Cellular Targets in Tonsil and Lymphoid Tissue Using
Semiconductor Quantum Dots," Modern Pathology 19(9):1181-1191,
2006. cited by applicant .
Furuya, T. et al., "A Novel Technology Allowing Immunohistochemical
Staining of a Tissue Section With 60 Different Antibodies in a
Single Experiment," Journal of Histochemistry and Cytochemistry
52(2):205-210, 2004. cited by applicant .
Gao, X. and S. Nie, "Molecular Profiling of Single Cells and Tissue
Specimens With Quantum Dots," Trends in Biotechnology
21(9):371-373, 2003. cited by applicant .
Ghazani, A.A., et al., "High Throughput Quantification of Protein
Expression of Cancer Antigens in Tissue Microarray Using Quantum
Dot Nanocrystals," Nano Letters 6(12):2881-2886, 2006. cited by
applicant .
Glass, G. et al. "SIMPLE: A Sequential Immunoperoxidase Labeling
and Erasing Method," Journal of Histochemistry and Cytochemistry
57(10):899-905, 2009. cited by applicant .
Goldman, E.R., et al., "Avidin: A Natural Bridge for Quantum
Dot-Antibody Conjugates," Journal of the American Chemical Society
124:6378-6382, 2002. cited by applicant .
Goldman, E.R., et al., "Conjugation of Luminescent Quantum Dots
With Antibodies Using an Engineered Adaptor Protein to Provide New
Reagents for Fluoroimmunoassays," Analytical Chemistry 74:841-847,
2002. cited by applicant .
Gueroui, Z., and A. Libchaber, "Single-Molecule Measurements of
Gold-Quenched Quantum Dots," Physical Review Letters 93(16):166108,
2004. cited by applicant .
Guo, J., et al., "Multispectral Labeling of Antibodies With
Polyfluorophores on a DNA Backbone and Application in Cellular
Imaging," Proceedings of the National Academy of Sciences of the
USA (PNAS) 108(9):3493-3498, 2011. cited by applicant .
Han, K.-C. et al., "An Approach to Multiplexing an Immunosorbent
Assay With Antibody--Oligonucleotide Conjugates" Bioconjugate
Chemistry 21(12):2190-2196, 2010. cited by applicant .
Hendrickson, E.R., et al., "High Sensitivity Multianalyte
Immunoassay Using Covalent DNA--Labeled Antibodies and Polymerase
Chain Reaction," Nucleic Acids Research 23(3):522-529, 1995. cited
by applicant .
Huang, D.H., et al., "Comparison and Optimization of Multiplexed
Quantum Dot-Based Immunohistofluorescence," Nano Research
3(1):61-68, 2010. cited by applicant .
Jaiswal, J.K., et al., "Long-Term Multiple Color Imaging of Live
Cells Using Quantum Dot Bioconjugates," Nature Biotechnology
21:47-51, 2003. cited by applicant .
Jin, T., et al., "Anlibody-ProteinA Conjugated Quantum Dots for
Multiplexed Imaging of Surface Receptors in Living Cells,"
Molecular Biosystems 6:2325-2331, 2010. cited by applicant .
Kang, W.J., et al., "Multiplex Imaging of Single Tumor Cells Using
Quantum-Dot-Conjugated Aptamers," Small 5:2519-2522, 2009. cited by
applicant .
Lim, S.H., et al. "Simultaneous Intracellular Delivery of Targeting
Antibodies and Functional Nanoparticles With Engineered Protein G
System," Biomaterials 30:1197-1204, 2009. cited by applicant .
Lim, S.H., et al. "Specific Nucleic Acid Detection Using
Photophysical Properties of Quantum Dot Probes," Analytical
Chemistry 82(3):886-891, 2010. cited by applicant .
Lind, K., et al "Development and Evaluation of Three Real-Time
Immune-PCR Assemblages for Quantification of PSA," Journal of
Immunological Methods 304(1-2):107-116, 2005. cited by applicant
.
Liu, A.Y., et al. "Heterogeneity in Primary and Metastatic Prostate
Cancer as Defined by Cell Surface CD Profile," American Journal of
Pathology 165(5):1543-1556, 2004. cited by applicant .
Liu, J., et al., "Molecular Mapping of Tumor Heterogeneity on
Clinical Tissue Specimens With Multiplexed Quantum Dots," ACS Nano
4(5):2755-2765, 2010. cited by applicant .
Liu, J., et al., "Multiplexed Detection and Characterization of
Rare Tumor Cells in Hodgkin's Lymphoma With Multicolor Quantum
Dots," Analytical Chemistry 82(14):6237-6243, 2010. cited by
applicant .
Matsuno, A., et al., "Three-Dimensional Imaging of the
Intracellular Localization of Growth Hormone and Prolactin and
Their Mma Using Nanocrystal (Quantum Dot) and Confocal Laser
Scanning Microscopy Techniques," Journal of Histochemistry and
Cytochemistry 53(7):833-838, 2005. cited by applicant .
Nash, D.R., et al., "Sequential Immunofluorescent Staining: A
Simple and Useful Technique," Immunology 16:785-790, 1969. cited by
applicant .
Niemeyer, C.M., "Semisynthetic DNA-Protein Conjugates for
Biosensing and Nanofabrication," Angewandte Chemie International
Edition 49:1200-1216, 2010. cited by applicant .
Pirici, D., et al., "Antibody Elution Method for Multiple
Immunohistochemistry on Primary Antibodies Raised in the Same
Species and of the Same Subtype," Journal of Histochemistry and
Cytochemistry 57(6):567-575, 2009. cited by applicant .
Schubert, W.M., et al., "Analyzing Proteome Topology and Function
by Automated Multidimensional Fluorescence Microscopy," Nature
Biotechnology 24(10):1270-1278, Oct. 2006. cited by applicant .
Schwamborn, K., and R.M. Caprioli, "Molecular Imaging by Mass
Spectrometry--Looking Beyond Classical Histology," Nature Reviews:
Cancer 10(9):639-646, 2010. cited by applicant .
Schweller, R.M., et al., "Multiplexed in situ Immunofluorescence
via Dynamic DNAComplexes," Angew Chem Int Ed Engl.
51(37):9292-9296, Sep. 2012 (Author manuscript, 8 pages). cited by
applicant .
Shi, C., et al., "Quantum Dots-Based Multiplexed
Immunohistochemistry of Protein Expression in Human Prostate Cancer
Cells," European Journal of Histochemistry 52(2):127-134, 2008.
cited by applicant .
Smith, A.M., et al. "Multicolor Quantum Dots for Molecular
Diagnostics of Cancer," Expert Review of Molecular Diagnostics
6(2):231-244, 2006. cited by applicant .
Sweeney, E., et al., "Quantitative Multiplexed Quantum Dot
Immunohistochemistry," Biochemical and Biophysical Research
Communications 374(2):181-186, 2008. cited by applicant .
Tholouli, E., et al., "Imaging of Multiple mRNA Targets Using
Quantum Dot Based in Situ Hybridization and Spectra Deconvolution
in Clinical Biopsies," Biochemical and Biophysical Research
Communications 348(2):628-636, 2006. cited by applicant .
Toth, Z.E., and E. Mezey, "Simultaneous Visualization of Multiple
Antigens With Tyramide Signal Amplification Using Antibodies From
the Same Species," Journal of Histochemistry and Cytochemistry
55(6):545-554, 2007. cited by applicant .
True, L.D., and X. Gao, "Quantum Dots for Molecular Pathology:
Their Time Has Arrived," Journal of Molecular Diagnostics
9(1):7-11, Feb. 2007. cited by applicant .
Wahlby, C., et al., "Sequential Immunofluorescence Staining and
Image Analysis for Detection of Large Numbers of Antigens in
Individual Cell Nuclei," Cytometry 47(1):32-41, 2002. cited by
applicant .
Wollscheid, B., et al., "Mass-Spectrometric Identification and
Relative Quantification of N-Linked Cell Surface Glycoproteins,"
Nature Biotechnology 27(4):378-386, 2009. cited by applicant .
Wu, X., et al., "Immunofluorescent Labeling of Cancer Marker Her2
and Other Cellular Targets With Semiconductor Quantum Dots," Nature
Biotechnology 21(1):41-46, 2003. cited by applicant .
Xing, Y., and J. Rao, "Quantum Dot Bioconjugates for in Vitro
Diagnostics & in Vivo Imaging," Cancer Biomarkers 4:307-319,
2008. cited by applicant .
Xing, Y., et al., "Bioconjugated Quantum Dots for Multiplexed and
Quantitative Immunochemistry," Nature Protocols 2:1152-1165, 2007.
cited by applicant .
Yezhelyev, M.V., et al., "In Situ Molecular Profiling of Breast
Cancer Biomarkers With Multicolor Quantum Dots," Advanced Materials
19(20):3146-3151, 2007. cited by applicant .
Zhang, Q., and L.-H. Guao, "Multiple Labeling of Antibodies With
Dye/DNA Conjugate for Sensitivity Improvement in Fluorescence
Immunoassay," Bioconjugate Chemistry 18:1668-1672, 2007. cited by
applicant .
Zimak, J., et al., "Programming in Situ Immunofluorescence
Intensities through Interchangeable Reactions of Dynamic DNA
Complexes," ChemBioChem 13:2722-2728, 2012. cited by applicant
.
Zrazhevskiy, P., and X. Gao, "Multifunctional Quantum Dots for
Personalized Medicine," Nano Today 4(5):414-428, 2009. cited by
applicant .
Zrazhevskiy, P., and X. Gao, "Quantum Dots for Cancer Molecular
Imaging," Minerva Biotecnologica 21(1):37-52, 2009. cited by
applicant .
Zrazhevskiy, P., et al., "Designing Multifunctional Quantum Dots
for Bioimaging, Detection, and Drug Delivery," Chemical Society
Reviews 39:4326-4354, 2010. cited by applicant.
|
Primary Examiner: Campell; Bruce R
Attorney, Agent or Firm: Christenseon O'Connor Johnson
Kindness PLLC
Government Interests
GOVERNMENT SUPPORT
This invention was made with government support under grant no.
W81XWH-07-1-0117 awarded by the Department of Defense, no.
5R01CA131797 and no. 1R01CA140295 awarded by the National
Institutes of Health, and no. 0645080 awarded by the National
Science Foundation. The government has certain rights in the
invention.
Parent Case Text
.Iadd.This application is an reissue of U.S. patent application
Ser. No. 14/577,507, filed on Dec. 19, 2014, now U.S. Pat. No.
9,376,717, which is a divisional application of U.S. application
Ser. No. 13/455,674, filed on Apr. 25, 2012, now U.S. Pat. No.
8,946,389, which claims benefit under 35 U.S.C. .sctn. 119(e) of
the U.S. Provisional Application No. 61/478,618, filed Apr. 25,
2011 and U.S. Provisional Application No. 61/478,626, filed Apr.
25, 2011, all of which are incorporated herein by reference in
their entirety. .Iaddend.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application
Ser. No. 13/455,674, filed on Apr. 25, 2012, which claims benefit
under 35 U.S.C. .sctn.119(e) of the U.S. Provisional Application
No. 61/478,618, filed Apr. 25, 2011 and U.S. Provisional
Application No. 61/478,626, filed Apr. 25, 2011, the content of
both of which is incorporated herein by reference in its entirety
Claims
What is claimed is:
1. A composition comprising a plurality of affinity molecules,
wherein: (i) each member of the plurality binds a target, wherein
each affinity molecule is conjugated via hybridized first and
second nucleic acid strands to a label moiety, wherein detectable
properties of the label moieties are distinguishable from one
another, wherein the targets are distinguishable from one another,
wherein the hybridized first nucleic acid strands are
distinguishable from one another, wherein members of the plurality
of affinity molecules are distinguishable from one another, wherein
the affinity molecule is linked to the first nucleic acid strand
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 --NH.sub.2 groups triazole bond from "click"
reaction), a phosphodiester linkage, a phosphorothioate linkage, or
a combination thereof, and wherein the label moiety is linked to
the second nucleic acid strand 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-hydrazinonicotinamide
(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 --NH.sub.2 groups triazole bond from "click"
reaction a phosphodiester linkage a phosphorothioate linkage, or a
combination thereof; or (ii) each member of the plurality binding a
target, wherein each affinity molecule is conjugated to a first
single strand nucleic acid molecule that is specifically hybridized
to a second single strand nucleic acid molecule, wherein the second
single strand nucleic acid molecule is conjugated to a label
moiety, such that each affinity molecule is conjugated via
hybridized first and second nucleic acid strands to a label moiety,
wherein detectable properties of the label moieties are
distinguishable from one another, wherein the targets are
distinguishable from one another, wherein the first single strand
nucleic acid molecules are distinguishable from one another,
wherein members of the plurality of affinity molecules are
distinguishable from one another, and wherein the affinity molecule
is linked to the first single strand nucleic acid 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 --NH.sub.2 groups, triazole bond (from "click" reaction), a
phosphodiester linkage, a phosphorothioate linkage, or a
combination thereof, and wherein the label is linked to the second
single strand nucleic acid 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 --NH.sub.2 groups triazole bond from "click"
reaction a phosphodiester linkage, a phosphorothioate linkage, or a
combination thereof.
2. A composition comprising a solid support comprising a sample for
analysis for the presence of analytes, and a plurality of affinity
molecules, each member of the plurality of affinity molecules
conjugated to a first single-strand nucleic acid, wherein each
member of the plurality of affinity molecules is bound to a
different analyte in the sample, and wherein each first
single-strand nucleic acid has a different nucleotide sequence,
wherein members of the plurality of affinity molecules are
distinguishable from one another, and wherein the affinity molecule
is linked to the first single-strand nucleic acid 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 --NH.sub.2 groups, triazole bond (from
"click" reaction), a phosphodiester linkage, a phosphorothioate
linkage, or a combination thereof.
3. The composition of claim 2, wherein the composition further
comprises a plurality of second single strand nucleic acid
molecules, each conjugated to a label moiety, wherein each second
single strand nucleic acid molecule specifically hybridizes to a
first single strand nucleic acid molecule conjugated to a member of
the plurality of affinity molecules, such that at least a subset of
the plurality of affinity molecules bound to the analytes in the
sample is specifically associated with a plurality of label
moieties, wherein detectable properties of the label moieties are
distinguishable from one another, wherein members of the plurality
of second single stand nucleic acid molecules are distinguishable
from one another, and wherein the label moiety is linked to the
second single strand nucleic acid 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 --NH.sub.2 groups, triazole bond (from
"click" reaction), a phosphodiester linkage, a phosphorothioate
linkage, or a combination thereof.
4. A method of analyzing a sample for a plurality of analytes, the
method comprising, in order: (a) contacting the sample with a
plurality of affinity molecules under conditions that permit
specific analyte binding by the affinity molecules, wherein each
affinity molecule specifically binds a different member of the
plurality of analytes, and wherein each affinity molecule is
conjugated to a first single strand nucleic acid, such that members
of the plurality of affinity molecules become bound to members of
the plurality of analytes present in the sample, wherein the first
single strand nucleic acids are distinguishable from each other,
wherein members of the plurality of affinity molecules are
distinguishable from one another, and wherein the affinity molecule
is linked to the first single strand nucleic acid via an adaptor
molecule or via a linker
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 --NH.sub.2 groups triazole bond from "click"
reaction), a phosphodiester linkage, a phosphorothioate linkage, or
a combination thereof; (b) contacting the sample, under conditions
that permit specific nucleic acid hybridization, with a first set
of second single strand nucleic acid molecules, each conjugated to
a label moiety, wherein each second single strand nucleic acid
molecule specifically hybridizes to a first single strand nucleic
acid molecule conjugated to a member of the plurality of affinity
molecules, such that at least a subset of the plurality of affinity
molecules bound to members of the plurality of analytes in the
sample becomes specifically associated with a plurality of label
moieties, wherein detectable properties of the label moieties are
distinguishable from one another, wherein members of the first set
of second single strand nucleic acid molecules are distinguishable
from one another, and wherein the label moiety is linked to the
second single strand nucleic acid 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 --NH, groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phosphorothioate linkage, or
a combination thereof; and (c) detecting signal from label moieties
associated with affinity molecules bound to the sample, thereby
detecting the presence or amount of at least a subset of the
plurality of analytes.
5. The method of claim 4, further comprising the steps of: (d)
erasing the signal from the label moieties conjugated to the first
set of second single strand nucleic acid molecules; (e) contacting
the sample, under conditions that permit specific nucleic acid
hybridization, with a second set of second single strand nucleic
acid molecules, each conjugated to a label, wherein each second
single strand nucleic acid molecule specifically hybridizes to a
first single strand nucleic acid molecule conjugated to a member of
the plurality of affinity molecules, such that a subset of the
plurality of affinity molecules bound to members of the plurality
of analytes in the sample becomes specifically associated with a
plurality of label moieties, wherein members of the second set of
second single strand nucleic acid molecules are distinguishable
from one another and from members of the first subset of single
strand nucleic acid molecules of step (b), and wherein the label
moiety is linked to the second single strand nucleic acid 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 --NH.sub.2 groups, triazole bond (from
"click" reaction), a phosphodiester linkage, a phosphorothioate
linkage, or a combination thereof; (f) detecting signal from label
moieties associated with affinity molecules bound to the sample,
thereby detecting the presence or amount of the subset of the
plurality of analytes; and (g) optionally repeating steps (d)-(f)
with a further set of second single strand nucleic acid
molecules.
6. 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 affinity molecules under conditions that permit
specific analyte binding by the affinity molecules, wherein each
affinity molecule specifically binds a different member of the
plurality of analytes, wherein each affinity molecule is conjugated
to a first single strand nucleic acid molecule that is specifically
hybridized to a second single strand nucleic acid molecule, wherein
the second single strand nucleic acid molecule is conjugated to a
label moiety, such that each affinity molecule is conjugated via
hybridized first and second nucleic acid strands to a label moiety
and members of the plurality of different affinity molecules become
bound to members of the plurality of analytes present in the
sample, wherein detectable properties of the label moieties are
distinguishable from one another, wherein the first single strand
nucleic acids are distinguishable from each other, and wherein the
affinity molecule is linked to the first single strand nucleic acid
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-hydrazinonicotinamide
(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 --NH.sub.2 groups triazole bond from "click"
reaction), a phosphodiester linkage, a phosphorothioate linkage, or
a combination thereof, and wherein the label is linked to the
second single strand nucleic acid 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-hydrazinonicotinamide
(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 --NH.sub.2 groups, triazole bond (from
"click" reaction), a phosphodiester linkage, a phosphorothioate
linkage, or a combination thereof; and (b) detecting signal from
label moieties associated with first plurality of affinity
molecules bound to the sample, thereby detecting the presence or
amount of the plurality of analytes.
7. The method of claim 6, further comprising the steps of: (c)
erasing the signal from the label molecules conjugated to the first
set of second single strand nucleic acid molecules; (d) contacting
the sample with a second plurality of affinity molecules under
conditions that permit specific analyte binding by the affinity
molecules wherein members of the second plurality of affinity
molecules are distinguishable from one another and from members of
the first plurality of affinity molecules of step (a); (e)
detecting signal from label moieties associated with second
plurality of affinity molecules bound to the sample, thereby
detecting the presence or amount of at least a subset of the
plurality of analytes; and (f) optionally repeating steps (c)-(e)
with a further second set of the affinity molecules.
.Iadd.8. A method comprising: (1) contacting a sample being tested
for the presence of one or more analytes with one or more affinity
molecules, wherein each of the affinity molecules is linked to an
encoding molecule, wherein the encoding molecule 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 bond (from "click"
reaction), a phosphodiester linkage, a phosphorothioate linkage,
and a combination thereof, and wherein affinity molecules of
different specificity are linked to different encoding molecules to
produce one or more analytes bound to one or more affinity
molecules; (2) optionally removing unbound affinity molecules; (3)
contacting the sample with labeled nucleic acid strands conjugated
to a label moiety that bind to encoding molecules to produce label
moieties bound to encoding molecules; wherein each of the labeled
nucleic acid strands are linked to the label moiety 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,
and a combination thereof, (4) optionally removing unbound labeled
nucleic acid strands; (5) imaging the sample to detect the label
moieties bound to encoding molecules; (6) removing the label
moieties from the encoding molecules, wherein the labeled nucleic
acid strands are removed from the encoding molecules by chemical,
physical, or enzymatic means; and (7) repeating at least some of
steps (3)-(6) at least once with a labeled nucleic acid strand
having a unique composition relative to at least one other labeled
nucleic acid strand of step (3). .Iaddend.
.Iadd.9. The method of claim 8, wherein the label moieties are
removed by enzymatic means. .Iaddend.
.Iadd.10. The method of claim 9, wherein the encoding molecules are
first nucleic acid strands and the labeled nucleic acid strands are
second nucleic acid strands, and wherein the first nucleic acid
strands and the second nucleic acid strands are complementary
nucleic acids relative to each other. .Iaddend.
.Iadd.11. The method of claim 10, wherein the labeled nucleic acid
strands are cleaved by a single-stranded nuclease. .Iaddend.
.Iadd.12. The method of claim 10, wherein the label moieties are
removed by at least one restriction endonuclease. .Iaddend.
.Iadd.13. The method of claim 8, wherein the label moieties are
removed by cleavage at one or more ribonucleotide by RNase.
.Iaddend.
.Iadd.14. The method of claim 8, wherein the label moieties are
capable of being quenched by photo-bleaching or cleavage.
.Iaddend.
.Iadd.15. The method of claim 8, wherein removing the label
moieties comprises: a. cleavage and photo-bleaching; or b. cleavage
and chemical modification. .Iaddend.
.Iadd.16. The method of claim 10, wherein the sample is contacted
with more than one affinity molecule in (1). .Iaddend.
.Iadd.17. The method of claim 10, wherein the one or more affinity
molecules comprise an antibody or portion thereof. .Iaddend.
.Iadd.18. The method of claim 10, wherein the one or more affinity
molecules is a ligand, an aptamer, a peptide, or an
oligonucleotide. .Iaddend.
.Iadd.19. The method of claim 10, wherein the labeled nucleic acid
strands are labeled identically. .Iaddend.
.Iadd.20. The method of claim 10, wherein the labeled nucleic acid
strands comprise a distinguishable label moiety. .Iaddend.
.Iadd.21. The method of claim 8, wherein the label moieties are
fluorescent molecules. .Iaddend.
.Iadd.22. The method of claim 8, wherein the one or more analytes
are proteins and/or the sample is a cell or tissue sample.
.Iaddend.
.Iadd.23. The method of claim 10, wherein the sample is imaged in
step (5) using fluorescence microscopy. .Iaddend.
.Iadd.24. The method of claim 8, wherein the label moieties are
removed by strand displacement. .Iaddend.
.Iadd.25. The method of claim 24, wherein the strand displacement
is performed using a displacement nucleic acid complementary to the
labeled nucleic acid strands or the encoding molecules.
.Iaddend.
.Iadd.26. The method of claim 25, wherein the sample is contacted
with more than one affinity molecule in (1). .Iaddend.
.Iadd.27. The method of claim 25, wherein the one or more affinity
molecules comprise an antibody or portion thereof. .Iaddend.
.Iadd.28. The method of claim 25, wherein the one or more affinity
molecules comprise a ligand, an aptamer, a peptide, or an
oligonucleotide. .Iaddend.
.Iadd.29. The method of claim 25, wherein the labeled nucleic acid
strands are labeled identically relative to each other.
.Iaddend.
.Iadd.30. The method of claim 25, wherein each of the labeled
nucleic acid strands comprises a distinguishable label moiety.
.Iaddend.
.Iadd.31. The method of claim 25, wherein the label moieties are
fluorescent molecules. .Iaddend.
.Iadd.32. The method of claim 25, wherein the one or more analytes
are proteins and/or the sample is a cell or tissue sample.
.Iaddend.
.Iadd.33. The method of claim 25, wherein the sample is imaged in
step (5) using fluorescence microscopy. .Iaddend.
Description
TECHNICAL FIELD
The present disclosure relates generally to compositions and
methods for visualization and staining of analytes in a sample.
BACKGROUND
Comprehensive and accurate molecular profiling on a single-cell
level is highly sought after within the fields of cell biology,
pathology, and clinical diagnostics, especially for untangling the
complex interactions underlying cancer, neurological disorders, and
immune system disorders. A key challenge is presented by the
complexity and heterogeneity of these diseases (Liu, A. Y., M. P.
Roudier, and L. D. True, Heterogeneity in primary and metastatic
prostate cancer as defined by cell surface CD profile. American
Journal of Pathology, 2004. 165(5): p. 1543-1556 and True, L. D.
and X. Gao, Quantum dots for molecular pathology: their time has
arrived. J Mol Diagn, 2007. 9(1): p. 7-11), which is hard to assess
using conventional biomedical techniques that suffer from a
limitation in the number of biomarkers that can be analyzed
simultaneously, provide limited single-cell information, and often
utilize qualitative rather than quantitative analysis. See for
example, Englert, C. R., G. V. Baibakov, and M. R. Emmert-Buck,
Layered expression scanning: rapid molecular profiling of tumor
samples. Cancer Research, 2000. 60(6): p. 1526-30; Furuya, T., et
al., A novel technology allowing immunohistochemical staining of a
tissue section with 50 different antibodies in a single experiment.
Journal of Histochemistry and Cytochemistry, 2004. 52(2): p.
205-10; Glass, G., J. A. Papin, and J. W. Mandell, SIMPLE: a
sequential immunoperoxidase labeling and erasing method. Journal of
Histochemistry and Cytochemistry, 2009. 57(10): p. 899-905; Pirici,
D., et al., Antibody elution method for multiple
immunohistochemistry on primary antibodies raised in the same
species and of the same subtype. Journal of Histochemistry and
Cytochemistry, 2009. 57(6): p. 567-75; Schwamborn, K. and R. M.
Caprioli, Molecular imaging by mass spectrometry--looking beyond
classical histology. Nature Reviews. Cancer, 2010. 10(9): p.
639-46; Toth, Z. E. and E. Mezey, Simultaneous visualization of
multiple antigens with tyramide signal amplification using
antibodies from the same species. Journal of Histochemistry and
Cytochemistry, 2007. 55(6): p. 545-54; Wahlby, C., et al.,
Sequential immunofluorescence staining and image analysis for
detection of large numbers of antigens in individual cell nuclei.
Cytometry, 2002. 47(1): p. 32-41; and Wollscheid, B., et al.,
Mass-spectrometric identification and relative quantification of
N-linked cell surface glycoproteins. Nature Biotechnology, 2009.
27(4): p. 378-86. Consequently, fundamental understanding of
pathological processes as well as clinical diagnostics are limited
by the lack of knowledge about the predictive biomarkers that would
unambiguously discriminate between disease and normal function,
distinguish different disease types, and provide information about
possible progression of the pathological process.
Advances in nanotechnology have enabled the design of
nanoparticle-based tools for improved molecular characterization of
physiological and pathological processes. In particular,
semiconductor fluorescent nanoparticles (quantum dots, or QDs) have
emerged as a new platform for high-throughput quantitative
characterization of multiple biomarkers in cells and clinical
tissue specimens (Zrazhevskiy, P. and X. Gao, Multifunctional
Quantum Dots for Personalized Medicine. Nano Today, 2009. 4(5): p.
414-428 and Zrazhevskiy, P. and X. Gao, Quantum dots for cancer
molecular imaging. Minerva Biotecnologica, 2009. 21(1): p. 37-52).
Having size of only 2 to 10 nm in diameter, QDs possess unique
photo-physical properties drastically different from those of
single atoms or bulk materials. Size-tunable and spectrally narrow
light emission, simultaneous excitation of multiple colors,
improved brightness, resistance to photobleaching, and large Stokes
shift enable simultaneous parallel detection and reliable
quantification of up to 10 spectrally distinct QD probes
Alivisatos, A. P., Perspectives on the physical chemistry of
semiconductor nanocrystals. Journal of Physical Chemistry, 1996.
100(31): p. 13226-13239; Alivisatos, A. P., Semiconductor clusters,
nanocrystals, and quantum dots. Science, 1996. 271(5251): p.
933-937; Bruchez, M., Jr., et al., Semiconductor nanocrystals as
fluorescent biological labels. Science, 1998. 281(5385): p. 2013-6;
Chan, W. C., et al., Luminescent quantum dots for multiplexed
biological detection and imaging. Curr Opin Biotechnol, 2002.
13(1): p. 40-6; and Gao, X. and S. Nie, Molecular profiling of
single cells and tissue specimens with quantum dots. Trends
Biotechnol, 2003. 21(9): p. 371-3). However, utilization of such
multiplexing capability has been hampered by the inability to
uniquely match each QD probe with the corresponding biomarker, thus
yielding simultaneous detection of only a modest number of
biomarkers. See for example, Chen, C., et al., Quantum-dot-based
immunofluorescent imaging of HER2 and ER provides new insights into
breast cancer heterogeneity. Nanotechnology, 2010. 21(9): p.
095101; Fountaine, T. J., et al., Multispectral imaging of
clinically relevant cellular targets in tonsil and lymphoid tissue
using semiconductor quantum dots. Modern Pathology, 2006. 19(9): p.
1181-91; Ghazani, A. A., et al., High throughput quantification of
protein expression of cancer antigens in tissue microarray using
quantum dot nanocrystals. Nano Letters, 2006. 6(12): p. 2881-6;
Huang, D. H., et al., Comparison and Optimization of Multiplexed
Quantum Dot-Based Immunohistofluorescence. Nano Research, 2010.
3(1): p. 61-68; Liu, J., et al., Multiplexed detection and
characterization of rare tumor cells in Hodgkin's lymphoma with
multicolor quantum dots. Analytical Chemistry, 2010. 82(14): p.
6237-43; Liu, J., et al., Molecular mapping of tumor heterogeneity
on clinical tissue specimens with multiplexed quantum dots. ACS
Nano, 2010. 4(5): p. 2755-65; Shi, C., et al., Quantum dots-based
multiplexed immunohistochemistry of protein expression in human
prostate cancer cells. European Journal of Histochemistry, 2008.
52(2): p. 127-34; Sweeney, E., et al., Quantitative multiplexed
quantum dot immunohistochemistry. Biochemical and Biophysical
Research Communications, 2008. 374(2): p. 181-6; Wu, X., et al.,
Immunofluorescent labeling of cancer marker Her2 and other cellular
targets with semiconductor quantum dots. Nat Biotechnol, 2003.
21(1): p. 41-6; and Yezhelyev, M. V., et al., In situ molecular
profiling of breast cancer biomarkers with multicolorquantum dots.
Advanced Materials, 2007. 19(20): p. 3146-3151.
Accordingly, there is need in the art for compositions and methods
for multiplex detection, identification or quantitation of analytes
in sample. This disclosure provides such compositions and
methods.
SUMMARY
Multiplexed staining requires unique assignment of biomarkers to
corresponding detectable label probes (e.g., QD probes or other
fluorescent propbes). This can be achieved either via direct
QD-affinity molecule (e.g., antibody (Ab)) conjugation (covalent or
non-covalent) or encoding of each Ab with a different unique
encoding molecule (such as DNA or other nucleic acid) that can be
identified by complementary QD-nucleic acid probe. Each strategy,
suited for either 1-step or 2-step staining modalities, features
its own important benefits. For example, 1-step staining with QD-Ab
conjugates provides quick direct labeling of biomarkers in a
stoichiometric manner.
In contrast, while multiplexed staining can become available
through further optimization of direct biomarker labeling with
QD-antibody probes, a two-step staining modality, where biomarker
recognition with primary antibody (1'Ab) and labeling with
fluorescent probes (FL probes) are done in separate steps, provides
several important benefits: (i) staining conditions can be
optimized for Ab and FL probe separately; (ii) FL probe size can be
kept significantly smaller, as FL-Ab complex does not need to be
pre-assembled prior to staining; (iii) signal amplification
capability can be incorporated within the second step; and (iv)
de-staining/re-staining can be performed by cycling the FL labeling
step, without the need for biomarker recognition on each cycle.
Accordingly, provided herein are compositions and methods for
multiplex biomarker profiling. In one aspect, provided herein is a
composition comprising an affinity molecule reversibly conjugated
to a label moiety. The affinity molecule can be linked to the label
moiety via a linker comprising first and second strands of nucleic
acid that specifically hybridize to each other. The first nucleic
acid strand can be linked to the affinity molecule and the second
nucleic acid strand can be linked to the nanoparticle. This allows
the affinity molecule to be conjugated to the label moiety under
conditions that permit hybridization between the first and second
nucleic acid strands, but is not conjugated to the nanoparticle
under conditions that do not permit such hybridization.
In another aspect provided herein is a composition comprising an
affinity molecule reversibly conjugated to a 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 can be present in a 1:1 ratio. In some embodiments of this
aspect, the luminescent nanoparticle is a colloidal water-soluble
nanoparticle comprising a stable non-fouling coating such that
non-specific binding of the nanoparticle to a cell or a tissue
sample is reduced relative to a nanoparticle lacking a non-fouling
coating.
In still another aspect, provided herein is a composition
comprising a plurality of different affinity molecules, wherein:
(i) each member of the plurality binds a different target, wherein
each different affinity molecule is conjugated via different
hybridized first and second nucleic acid strands to a different
label moiety, wherein detectable properties of the different label
moieties are distinguishable; or (ii) each member of the plurality
binding a different target, wherein each different affinity
molecule is conjugated to a different first single strand nucleic
acid molecule that is specifically hybridized to a second single
strand nucleic acid molecule, wherein the second single strand
nucleic acid molecule is conjugated to a label moiety, such that
each different affinity molecule is conjugated via different
hybridized first and second nucleic acid strands to a different
label moiety, wherein detectable properties of the different label
moieties are distinguishable.
In yet another aspect, provided herein is a composition comprising
a plurality of different affinity molecules, each member of the
plurality binding a different target. Each different affinity
molecule can be reversibly conjugated to a 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 can be present in a 1:1 ratio. The luminescent
nanoparticle can be a colloidal water-soluble nanoparticle
comprising a stable non-fouling coating such that non-specific
binding of the nanoparticle to a cell or a tissue sample is reduced
relative to a nanoparticle lacking a non-fouling coating. Further,
emission spectra of the different luminescent nanoparticles can be
distinguishable.
In yet still another aspect, described herein are solid supports
bearing a plurality of different affinity molecules. Depending upon
the particular embodiment, the solid support can comprise a
plurality of different affinity molecules bearing, for example,
nucleic acid molecules, adaptor molecules and/or label moieties in
any of the various combinations or arrangements as described
herein. Such solid supports can comprise samples, e.g., biological
samples to be tested for the presence and/or amounts of any of a
plurality of analytes. A non-limiting example of a sample on a
solid support is a tissue or cell sample, e.g., a tissue section,
on a slide or coverslip. Additional non-limiting examples of solid
supports include, for example, a plate, dish, well, membrane,
grating, bead or particle (including, but not limited to an agarose
or latex bead or particle, a magnetic particle, etc.).
To fully utilize extensive multiplexing potential of fluorescent
tags for molecular profiling of cells and tissue specimens and
exploit the benefits of 2-step staining modality, the inventors
have also developed a novel hybrid IF/FISH 2-step staining
procedure featuring unique assignment of each biomarker to a
corresponding fluorescent probe. This method is exemplified herein
using antibody affinity molecule and quantum dot fluorescent
nanoparticles, but can be generalized to other affinity molecule
labels as known in the and described herein. Immunofluorescence
(IF) has been widely utilized for labeling of protein biomarkers in
cells and tissue specimens; however, utilization of primary
antibodies (1'Ab) for biomarker recognition and fluorophore-labeled
secondary antibodies (2'Ab-FL) for subsequent staining hampered
multiplexing capability of this technique due to limited
availability of unique 1'Ab/2'Ab pairs. At the same time,
fluorescence in situ hybridization (FISH) has been optimized for
multiplexed detection of DNA and mRNA, but it cannot be used for
analysis of cell phenotype. In the novel hybrid IF/FISH method
described herein, during the first step, biomarkers are detected by
specific 1'Ab carrying unique DNA tags, thus converting
antigenicity information into DNA sequence code. During the second
step, multiplexed FISH with QD-DNA or DNA-FL probes is performed,
thus overcoming limitations imposed by the choice of 1'Ab/2'Ab
pairs. Furthermore, multiplexing capability can be significantly
expanded by using the sequential staining/imaging method described
herein. Notably, hybrid IF/FISH method is equally amenable for
QD-based probes as well as conventional FISH DNA-probes labeled
with organic fluorophores, making this technique widely applicable
to a large number of studies with different aims and
parameters.
Accordingly, provided herein is also a method of analyzing a sample
for a plurality of analytes. In some embodiments, the method
comprises: (a) contacting the sample with a 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 wherein each different affinity molecule
is conjugated to a different first single strand nucleic acid, such
that members of the plurality of different affinity molecules
become bound to members of the plurality of analytes present in the
sample; (b) contacting the sample, under conditions that permit
specific nucleic acid hybridization, with a first set of different
second single strand nucleic acid molecules, each conjugated to a
different label moiety, wherein each different second single strand
nucleic acid molecule specifically hybridizes to a different first
single strand nucleic acid molecule conjugated to a member of the
plurality of different affinity molecules, such that at least a
subset of the plurality of different affinity molecules bound to
members of the plurality of analytes in the sample becomes
specifically associated with a plurality of different label
moieties, wherein detectable properties of the different label
moieties are distinguishable; and (c) detecting signal from label
moieties associated with affinity molecules bound to the sample,
thereby detecting the presence or amount of at least a subset of
the plurality of analytes. In some further embodiments of this, the
method further comprises the steps of: (d) quenching the signal
from the label moieties conjugated to the first set of second
single strand nucleic acid molecules; (e) contacting the sample,
under conditions that permit specific nucleic acid hybridization,
with a second set of different second single strand nucleic acid
molecules, each conjugated to a different label, wherein each
different second single strand nucleic acid molecule specifically
hybridizes to a different first single strand nucleic acid molecule
conjugated to a member of the plurality of different affinity
molecules, such that a different subset of the plurality of
different affinity molecules bound to members of the plurality of
analytes in the sample becomes specifically associated with a
different plurality of different label moieties relative to those
detected in step (c); (f) detecting signal from label moieties
associated with affinity molecules bound to the sample, thereby
detecting the presence or amount of the different subset of the
plurality of analytes; and (g) optionally repeating steps (d)-(f)
with a further set of second single strand nucleic acid
molecules.
In some other embodiments, the method comprises: (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,
wherein each different affinity molecule is conjugated to a
different first single strand nucleic acid molecule that is
specifically hybridized to a second single strand nucleic acid
molecule, wherein the second single strand nucleic acid molecule is
conjugated to a label moiety, such that each different affinity
molecule is conjugated via different hybridized first and second
nucleic acid strands to a different label moiety and members of the
plurality of different affinity molecules become bound to members
of the plurality of analytes present in the sample, wherein
detectable properties of the label moieties are distinguishable;
and (b) detecting signal from label moieties associated with first
plurality of affinity molecules bound to the sample, thereby
detecting the presence or amount of the plurality of analytes. In
some further embodiments of this, the method further comprises: (c)
quenching the signal from the label molecules conjugated to the
first set of second single strand nucleic acid molecules; (d)
contacting the sample with a second plurality of different affinity
molecules under conditions that permit specific analyte binding by
the different affinity molecules; detecting signal from label
moieties associated with second plurality of affinity molecules
bound to the sample, thereby detecting the presence or amount of at
least a subset of the plurality of analytes; and (f) optionally
repeating steps (c)-(e) with a further second set of the affinity
molecules.
In yet some other embodiments, the method comprises: (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 such that non-specific binding of the
nanoparticle to a cell or a tissue sample is reduced relative to a
nanoparticle lacking a non-fouling coating, and detectable
properties of the luminescent nanoparticles are distinguishable;
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. In some further embodiments of this, the method further
comprises: c) quenching the signal from the luminescent
nanoparticles conjugated to the first set of different second
single strand nucleic acid molecules; (d) contacting the sample
with a second set of the plurality of different affinity molecules
under conditions that permit specific analyte binding by the
different affinity molecules; (e) detecting signal from luminescent
molecules associated with the second set of affinity molecules
bound to the sample, thereby detecting the presence or amount of at
least a subset of the plurality of analytes; and (f) optionally
repeating steps (c)-(e) with a further second set of the affinity
molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of an embodiment of the hybrid
IF/FISH procedure for multiplexed 2-step cell staining. In the
first step, biomarkers of interest are simultaneously encoded by
unique DNA tags via labeling with 1'Ab-DNA conjugates. On the
second step a subset of biomarkers is labeled with complementary
QD-DNA probes. Subsequently, spectral imaging is used to unmix and
quantify staining produced by individual QD probes. Finally,
incubation with a longer oligonucleotide leads to DNA bond
displacement and QD-DNA probe dissociation, thus achieving specimen
de-staining for the next staining cycle. Notably, de-staining does
not remove the first layer of Ab-DNA tags, thus eliminating the
need for biomarker re-detection on each staining cycle.
FIG. 2 shows exemplary strategies for conjugation of Ab with
quantum dots via DNA mediated linkage including covalent
conjugation with reduced IgG, amine cross-linking using whole
antibodies, and non-covalent self-assembly with protein A (SpA).
Each of these approaches can be used to conjugate nucleic acid,
e.g., DNA oligonucleotides to antibody. It is important to
recognize the limitations inherent in each approach. For example,
covalent conjugation with reduced IgG can be used, but might lead
to loss of Ab affinity. Amine cross-linking can be used using whole
antibodies, but there may be little control over the final
bioconjugate structure. Non-covalent self-assembly with SpA-DNA can
provide a route for labeling of intact antibodies, but the
bioconjugate may possess only short-term stability.
FIG. 3 shows lack of cross-talk between free dye-labeled SpA. Only
free SpA pre-assembled with anti-androgen receptor (anti-AR)
antibody produced characteristic nuclear staining, while competing
SpA failed to stain the target. SpA labeled with Alexa Fluor 488
was imaged with FITC filter, while SpA labeled with Alexa Fluor 568
was imaged with a Rhodamine filter.
FIGS. 4A-4D show covalent conjugation of amine-modified
oligonucleotides to PMAL-coated QDs. QD-oligo conjugates prepared
in .about.100% DMF showed distinct DNA absorption peak after
purification (FIG. 4A) and increase in hydrodynamic size
corresponding to deposition of oligonucleotide-PEG on the QD
surface (FIG. 4C). Reaction performed under identical conditions in
aqueous Bicarbonate buffer failed to produce any QD-oligo
conjugates showing no changes in light absorption (FIG. 4B) and
particle size (FIG. 4D) in purified samples vs. control.
FIG. 5 shows preparation of QD-DNA probes on QD-Streptavidin
platform. QD-Str efficiently capture biotinylated ssDNA even at
slight DNA excess, as indicated by formation of QD-DNA bands on
agarose gel. QD-Str show minimal gel motility (Ox line); however,
upon DNA binding, particles become more negatively charged, thus
moving faster.
FIG. 6 shows 1-color staining with covalently linked Ab-DNA and
self-assembled Ab/SpA-DNA probes. Androgen receptor (AR) and
monoamine oxidase A (MAOA) staining patterns obtained with both
types of probes were consistent with conventional 2-step staining
with QD-2'Ab.
FIG. 7 shows 1-color staining with QD-DNA probes on PEG-coated
(left) and zwitterionic (right) particle platform. Clear AR and
MAOA staining patterns are produced, while only minimal
non-specific binding is observed in controls.
FIG. 8 shows off-target nuclear biding of SpA-DNA probes due to
high DNA load. Increasing activation of SpA with succinimidyl
4-hydrazinonicotinate acetone hydrazine (SANH) leads to increased
loading of ssDNA per protein, which, in turn, results in enhanced
nuclear binding (top row). Partial blocking of ssDNA tag with short
oligonucleotide completely eliminates this effect (bottom row).
FIG. 9 shows 3-color staining with covalently conjugated Ab-DNA
probes. Spectral imaging clearly separated signals from individual
QD probes (bottom row), demonstrating consistent staining patterns
for MAOA, AR, and pAkt.
FIG. 10 shows 4-color staining with self-assembled Ab/SpADNA
probes. Featuring good Ab/SpA bond stability, these probes
exhibited no cross-talk. As a result, multiplexed 2-step staining
(middle row) produced biomarker expression patterns consistent with
those obtained with single-biomarker staining (bottom row).
FIG. 11 shows specimen de-staining according to an embodiment of
the method. MB-Str (top left) were decorated with ssDNA, which was
labeled with complementary QD probes (top right). Incubation with
matching displacement probe yielded complete de-staining (bottom
left), while mismatch probe had no effect (bottom right).
FIGS. 12A and 12B show QD-based staining and de-staining on fixed
cells. FIG. 12A, bright nuclear AR staining is achieved with
Ab/SpA-DNA and QD-DNA probes. FIG. 12B, signal drop of up to 60-80%
could be achieved. Further improvement of de-staining procedure can
be achieved by quenching or removing residual non-specifically
bound QD probes
FIG. 13 shows AR staining and de-staining with dye-labeled ssDNA
probes. Only complementary probe produced staining, whereas
matching displacement probe yielded complete de-staining. Notably,
re-staining of AR could be achieved during the second cycle,
indicating that Ab/SpA-DNA probes remained intact throughout
staining/de-staining procedure.
FIGS. 14A-14F shows a schematic representation of an embodiment of
the QD-based cyclic multiplexed staining method for comprehensive
single-cell molecular profiling. FIG. 14A, universal QD/SpA
platform that can be used for 1-step purification-free assembly of
functional QD-Ab probes via capture of free antibodies from
solution by SpA. Once bound, Ab cannot be exchanged with other
QD/SpA probes, thus enabling mixing of multicolor probes within a
single cocktail (FIG. 14B) for a 1-step parallel multiplexed
staining (FIG. 14C). In this procedure, each QD-Ab probe
specifically labels corresponding biomarker without showing
interference or cross-talk with other probes. FIG. 14D, spectral
imaging can be used for unmixing the individual QD peaks,
quantitative analysis of biomarker expression, and depiction of
relative biomarker distribution within the specimen. FIG. 14E,
brief washing with low-pH buffer yields complete specimen
de-staining, thus enabling another cycle of parallel multiplexed
staining. FIG. 14F, sequential repetition of N-biomarker parallel
staining for M cycles enables labeling of N.times.M biomarkers.
With utilization of QD probes featuring narrow emission profiles,
molecular profiling for over 100 biomarkers can be achieved.
FIG. 15 shows multiplexed staining of 4 cancer biomarkers (HSP90,
MAOA, AR, and pAkt) and housekeeping biomarker B-tubulin. Both
parallel (top panel) and sequential (bottom panel) staining yielded
consistent molecular profiles with sub-cellular resolution.
Sequential images were taken on the same cell sub-population with
identical imaging parameters, thus allowing direct comparison of
biomarker relative expression levels and intracellular
distribution. However, intensity of individual channels (for
parallel staining) or frames (for sequential staining) was adjusted
to achieve clear biomarker representation in false-color images.
Scale bar, 50 .mu.m.
FIG. 16 shows comparison of QD/SpA-1'Ab staining with conventional
IF. For all 5 biomarkers, individual staining patterns and relative
expression levels detected with QD/SpA-1'Ab probes in a 1-step
staining procedure (top row) were consistent with those obtained
with either QD-2'Ab probes (middle row) or Alexa Fluor568-labeled
2'Ab (bottom row) in a 2-step staining, confirming robustness and
specificity of QD/SpA-Ab probes. Scale bar, 50 .mu.m.
FIG. 17 shows AR staining with QD/SpA-Ab probes. Androgen receptor
was stained with 5 different QD/SpA probes in either 1-step (top
row) or 2-step (middle row) staining procedure. Both procedures
revealed characteristic nuclear localization of this biomarker and
yielded similar staining intensity, confirming QD/SpA-Ab binding
exclusively via SpA-Ab bond. At the same time, no significant
non-specific staining with non-functionalized QD/SpA probes was
observed (bottom row). Scale bar, 50 .mu.m.
FIGS. 18A-18C shows SPR analysis of SpA-Ab bond stability. Either
rabbit anti-mouse antibody or SpA was immobilized on the surface of
C5 chip. Then binding and dissociation of free SpA to immobilized
Ab (FIG. 8A) or free Ab to immobilized SpA (FIGS. 18B and 18C) were
monitored. FIGS. 18A and 18B, at high analyte concentration (100
nM) fast saturation of surface binding sites was observed, thus
leading to analyte capture via weak binding in addition to strong
interactions. As a result, quick initial dissociation of analyte
accounting to nearly 5% loss was detected (likely due to breaking
of weak interactions) followed by very slow dissociation kinetics.
FIG. 18C, at low analyte concentration (10 nM) only slow
dissociation was observed. Overall retention of 97% bound analyte
after 1 hour of washing confirmed sufficient stability of SpA-Ab
bond within the time-frame of staining experiment.
FIG. 19 shows lack of cross-talk between QD/SpA-Ab probes in
parallel staining. To determine the possibility of the Ab exchange
between probes and examine the extent of cross-talk staining, the
inventors pre-assembled anti-AR IgG with either QD525/SpA (top row)
or QD565/SpA (middle row). Then the competing QD/SpA probe of
opposite color was added, and QD mixtures were incubated with
cells. In both cases only pre-assembled probe showed specific
nuclear staining, while the competing probe failed to capture Ab
and produce any detectable staining. At the same time, when IgG,
QD525/SpA, and QD565/SpA were mixed simultaneously and immediately
applied to cells, both probes successfully captured Ab and produced
AR staining with nearly 50% contribution each (bottom row).
Spectral imaging and unmixing was used to extract individual QD
signals and remove background. Intensity of QD525 channel was
increased 4 times relative to QD565 channel to compensate for
differential brightness of QD probes. Scale bar, 50 .mu.m.
FIGS. 20A and 20B show consistent size (FIG. 20A) and biomarker
staining kinetics (FIG. 20B) with 5 different QD-SpA probes.
FIG. 20C shows consistency of staining kinetics of HSP90 biomarker
(HeLa cells) with small (QD525) and large (QD605) QD/SpA/Ab.
FIGS. 21A-21D show efficiency of QD/SpA-Ab elution for specimen
de-staining. FIG. 21A, characteristic nuclear staining was obtained
with QD545/SpA probes pre-assembled with anit-AR antibody. FIG.
21B, brief incubation with pH2 Glycine-HCl/0.1% casein buffer
achieved complete specimen de-staining, leaving no detectable QD
fluorescence signal. FIG. 21C, re-staining of cells with anti-AR
QD545/SpA-Ab probe during the second cycle yielded nearly complete
restoration of fluorescence signal, indicating that all biomarkers
were vacated during de-staining procedure. FIG. 21D, another
re-staining with non-functionalized QD545/SpA probes produced only
background staining, confirming that no vacant 1' antibodies were
left on the specimen and, thus, ensuring no cross-talk between
staining cycles. Same sub-population of cells was imaged after each
step using consistent imaging parameters to aid in direct
comparison of staining intensity and distribution. Scale bar, 50
.mu.m.
FIG. 22 shows long-term stability of staining with QD/SpA-Ab
probes. Androgen receptor was stained with QD/SpA probes
pre-assembled with anti-AR Ab using a 1-step procedure. Then
true-color images of the same sub-population of cells were taken
immediately after staining (at t.sup.-0), and after 4, 24, and 48
hours of incubation in TBS buffer at 4.degree. C. Nearly complete
retention of QD probes even after 48 hours showed high durability
of staining. Scale bar, 50 .mu.m.
FIGS. 23A-23D show QD quenching vs. elution during low-pH-mediated
de-staining. FIG. 23A, AR staining with Alexa Fluor568-labeled
QD/SpA-Ab probes showed characteristic nuclear staining pattern
with both Wide UV (QD channel, left) and Rhodamine LP (Alexa
Fluor568 channel, right) filter cubes. FIG. 23B, after de-staining
with pH2 Glycine-HCl/0.1% casein buffer QD signal was completely
eliminated due to probe elution and quenching, while Alexa Fluor568
staining highlighted diffuse distribution of residual
non-specifically bound QD/SpA probes. FIG. 23C, cross-linking of
QD/SpA-Ab probes to biomarkers did not affect staining pattern or
intensity. However, following de-staining (FIG. 23D) cross-linked
probes could not be eluted, as indicated by retained Alexa Fluor568
signal, assigning removal of QD signal solely to low-pH-mediated
quenching. Scale bar, 50 .mu.m.
FIG. 24 shows biomarker re-staining and target exchange with cyclic
parallel staining During the first cycle (top row) parallel
staining of AR with QD525 and MAOA with QD565 was achieved.
Spectral imaging and unmixing revealed distinct AR staining pattern
in QD525 channel (middle panel) and MAOA pattern in QD565 channel
(right panel). Following de-staining, during the second cycle
(middle row) same biomarkers were stained with counterpart QD
probes. In this case, clear MAOA pattern was detected in QD525
channel (middle panel) and AR in QD565 channel (right panel).
Therefore, complete target re-staining with a different probe was
achieved (bottom row), while no cross-talk between probes within
the same cycle or between cycles was observed. Intensity of QD525
channel was increased 4 times relative to QD565 channel to
compensate for differential brightness of QD probes. Scale bar, 50
.mu.m.
FIGS. 25A-25D show biomarker stability during cyclic exposure to
low-pH degradation conditions. FIG. 25A, high-magnification
microscopy of AR staining after 1 and 10 degradation cycles
revealed only a minor loss of signal intensity with preserved
nuclear biomarker localization. To aid in signal intensity
comparison, spectral imaging and unmixing (right panels) was used
to extract QD signal and remove background autofluorescence present
in true-color images (left panels). Scale bar, 50 u.m. FIG. 25B,
quantitative analysis of AR staining intensity showed no more than
10% signal loss due to cyclic low-pH treatment. Some variability in
AR staining between different cells was observed, which could be
assigned to natural variability in biomarker expression. FIGS. 25C
and 25D, qualitative comparison of unmixed low-magnification images
of cells exposed to different number of degradation cycles
correlates well with quantitative analysis in (FIG. 25B) and shows
good preservation of biomarker antigenicity for at least 10
staining cycles. Scale bar, 250 .mu.m.
FIGS. 26A and 26B show effect of pre-staining cell processing on
biomarker preservation during sequential staining. Cells were fixed
with 4% formaldehyde/TBS for 10 minutes (FIG. 26A) or 20 minutes
(FIG. 26B) at room temperature, followed by permeabilization with
2% DTAC and 0.5% TritonX-100. Insufficient cell fixation in (FIG.
26A) resulted in significant loss of biomarker antigenicity after
only a few low-pH degradation cycles, while properly processed
cells showed high biomarker stability throughout at least 10
cycles. Low-magnification microscopy with spectral imaging was done
after each degradation cycle. Signal unmixing, background removal,
and average AR staining intensity measurements were done to
quantitatively examine biomarker stability. Scale bar, 500
.mu.m.
FIG. 27 shows sequential staining of 5 biomarkers performed in
different order. To evaluate dependence of sequential staining
performance on the order of biomarkers stained, procedure was
performed in the order from low-abundance to high-abundance
biomarker (top row) and from high-abundance to low-abundance
biomarker (bottom row). Independent of order, all biomarkers were
reliably stained showing correct staining pattern and relative
staining intensity. Also, no carry-over fluorescence, build-up of
background fluorescence, or cross-talk between cycles was observed.
Scale bar, 50 .mu.m.
FIG. 28A shows hyperspectral imaging (HSI) can be used to
accurately identify a composition comprising a mix of QDs.
FIG. 28B shows quantitative analysis of biomarker expression
(LaminA and HSP90) with QD probes is consistent with differential
QD brightness (measured by HSI from bulk QD solution).
DESCRIPTION OF EXEMPLARY EMBODIMENTS
In-depth understanding of the nature of cell physiology and ability
to diagnose and control the progression of pathological processes
heavily rely on untangling the complexity of intracellular
molecular mechanisms and pathways. Therefore, comprehensive
molecular profiling of individual cells within the context of their
natural tissue or cell culture microenvironment is required. In
principle, this goal can be achieved with immunofluorescence
staining and imaging by tagging each biomarker with a unique
fluorescent probe and detecting its localization with high
sensitivity at sub-cellular resolution. Yet, neither widely used
conventional techniques nor more advanced nanoparticle-based
methods have been able to address this task up to date. High
multiplexing potential of fluorescent probes is significantly
restrained by the inability to uniquely match probes with
corresponding biomarkers. This issue is especially relevant for
quantum dot probes--while simultaneous spectral imaging of up to 10
different probes is possible, only a few can be used concurrently
for staining with existing methods. To fully utilize multiplexing
potential of label moieties or fluorescent labels, exemplified
herein by quantum dots fluorescent nanoparticles (QDs), it is
necessary to design new staining methods featuring unique
assignment of each biomarker to a corresponding QD probe. The
Hybrid IF/FISH procedure described here achieves this objective by
encoding each biomarker with a unique nucleic acid (e.g. DNA) tag,
thus converting IF into a highly multiplexable FISH-like staining
method. Furthermore, multiplexing capability of this method can be
expanded by performing several staining cycles in a sequential
manner with no interference between different cycles. The method is
directly applicable for a wide range of molecular profiling
studies. Utilization of the compositions and methods described
herein can benefit both biomedical research and clinical
diagnostics by providing a tool for addressing phenotypic
heterogeneity within large cell populations, opening access to
studying low-abundance events often masked or completely erased by
batch processing, and elucidating biomarker signatures of diseases
critical for accurate diagnostics and targeted therapy.
Accordingly, described herein are compositions and methods for
detecting and/or quantitating biomarkers in a biological sample,
e.g., a cellular biological sample, whether comprising isolated
cells or cells in a tissue sample. The compositions and methods
described herein are well suited to assay the presence or level of
a plurality of biomarkers simultaneously. In one aspect,
compositions and methods are described in which affinity moieties
specific for a plurality of target biomarkers are coupled, either
directly or indirectly, to label moieties such that each biomarker
is paired with a corresponding label moiety via a corresponding
affinity molecule. The biomarker/label moiety pairing for this
aspect and others described herein is reversible as that term is
defined herein.
As noted, the reversible coupling between affinity molecule and
label moiety can be indirect or direct. Both the indirect and
direct approaches are described in more detail herein below, and
each has properties beneficial in certain applications described
herein.
One approach to indirect coupling takes advantage of the
information-carrying capacity of nucleic acid sequences to uniquely
encode the coupling of affinity molecule to label moiety. In this
approach, the affinity molecule and label moiety are each coupled
to a separate complementary strand of nucleic acid, such that
hybridization of the separate, complementary strands reversibly
couples, links or conjugates the affinity molecule to the label
moiety. This approach has the advantage that the exacting
specificity of nucleic acid hybridization permits a large number of
different affinity molecules to be coded with different nucleic
acids, each specific for only one corresponding complementary
sequence joined to a respective different label moiety. When
applied to a sample simultaneously, the affinity molecules
specifically bind their respective biomarker targets present on the
sample, and the hybridization of the complementary nucleic acid
sequences links the different label moieties to the different
affinity molecules in a way that permits multiplex detection. The
coupling is reversible by any approach that separates hybridized
strands of nucleic acid, e.g., displacement by competing
complementary sequence, heating, changes in salt concentration,
etc. as described elsewhere herein.
One approach to direct coupling of affinity molecule and label
moiety takes advantage of the strong, specific, non-covalent
binding of the constant domains of antibody affinity molecules by
certain polypeptides. As but one example (others are provided
herein below), the S. aureus Protein A (SpA) polypeptide tightly
binds immunoglobulin molecules via determinants on their constant
domains. In this aspect, an antibody can be reversibly coupled to a
label moiety by coupling the label moiety to SpA, and then mixing
the label-SpA complexes with the antibody to effect linkage of the
antibody and label moiety through the SpA.
Various aspects and embodiments employing either the indirect or
direct coupling of affinity molecule and label moiety are described
in further detail herein below that each permit multiplex detection
and/or quantitation of biomarker molecules in a cellular sample.
The various components and considerations necessary for these
biomarker detection compositions and methods are described in the
sections that follow.
Unless stated otherwise, or implicit from context, the following
terms and phrases include the meanings provided below. Unless
explicitly stated otherwise, or apparent from context, the terms
and phrases below do not exclude the meaning that the term or
phrase has acquired in the art to which it pertains. The
definitions are provided to aid in describing particular
embodiments of the aspects described herein, and are not intended
to limit the claimed invention, because the scope of the invention
is limited only by the claims. Further, unless otherwise required
by context, singular terms shall include pluralities and plural
terms shall include the singular.
As used herein the terms "comprising" or "comprises" means
"including" or "includes" and are used in reference to
compositions, methods, and respective component(s) thereof, that
are useful to the invention, yet open to the inclusion of
unspecified elements, whether useful or not.
As used herein the term "consisting essentially of" refers to those
elements required for a given embodiment. The term permits the
presence of additional elements that do not materially affect the
basic and novel or functional characteristic(s) of that embodiment
of the invention.
The term "consisting of" refers to compositions, methods, and
respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction
conditions used herein should be understood as modified in all
instances by the term "about." The term "about" when used in
connection with percentages may mean.+-.5% of the value being
referred to. For example, about 100 means from 95 to 105.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Thus for example, references to "the method"
includes one or more methods, and/or steps of the type described
herein and/or which will become apparent to those persons skilled
in the art upon reading this disclosure and so forth.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of this
disclosure, suitable methods and materials are described below. The
abbreviation, "e.g." is derived from the Latin exempli gratia, and
is used herein to indicate a non-limiting example. Thus, the
abbreviation "e.g." is synonymous with the term "for example."
As used herein, the term "linker" means an organic moiety that
connects two parts of a compound. Linkers typically comprise a
direct bond or an atom such as oxygen or sulfur, a unit such as
NR.sup.1, C(O), C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of
atoms, such as substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,
heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,
alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl, alkynylheteroarylalkyl,
alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or
more methylenes can be interrupted or terminated by O, S, S(O),
SO.sub.2, N(R.sup.1).sub.2, C(O), cleavable linking group,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocyclic; where
R.sup.1 is hydrogen, acyl, aliphatic or substituted aliphatic.
As used herein, the term "nucleic acid linker" refers to a nucleic
acid that connects two parts of a compound, e.g., an affinity
molecule to a label moiety. A nucleic acid linker can be
single-stranded, fully double-stranded, or partially
double-stranded. A nucleic acid linker can be any length. For
example, a nucleic acid linker can be from 1 nucleotide to about
100 nucleotides in length. When the nucleic acid linker is
double-stranded, the linker can comprise a double stranded region
of about 6 to about 100 consecutive base pairs. However, the duplex
region can be interrupted by one or more single-stranded regions in
one or both of the strands of the duplex. Further, a
double-stranded nucleic acid linker can comprise a single-stranded
overhang on one or both ends of the double-stranded region.
Moreover, a nucleic acid linker can comprise one or more nucleic
acid modifications described herein. A nucleic acid linker can be
attached to a compound by a non-nucleic acid linker.
As used herein, the term "non-nucleic acid linker" refers to any
linker that is not a nucleic acid linker.
Compositions
In one aspect, provided herein is a composition comprising an
affinity molecule reversibly conjugated to a label moiety. As used
herein, the term "reversibly conjugated" means that the two parts
are conjugated by non-covalent bonding only. Generally such
non-covalent bonding comprises one or more of hydrogen bonding, Van
der Waals forces, electrostatic forces, hydrophobic forces, and the
like. As such, reversible conjugation does not require breaking of
a covalent bond to remove or separate the affinity molecule from
the label moiety.
The affinity molecule and the label moiety can be present in any
ratio in the composition. For example, the affinity molecule and
the label moiety can be in a ratio from about 1:1 to about 1:100,
from about 1:1 to about 1:50, from about 1:1 to about 1:25, from
about 1:1 to about 1:20, or from about 1:1 to about 1:15 (affinity
molecule:label moiety). In some embodiments, the affinity molecule
and the label moiety can be in a ratio of about 1:1, 1:2, 1:3, 1:4,
1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. Generally, when the composition
is to be used for quantitative labeling or detection of a target
molecule in a sample, the affinity molecule and the label moiety
are in a 1:1 ratio. On the other hand, when the composition is to
be used for qualitative labeling or detection of a target molecule
in a sample, a higher number of label moieties per affinity
molecule can be used to provide signal amplification.
The affinity molecule can be conjugated with more than one first
nucleic acid strands. For example, the affinity molecule can be
conjugated with one, two, three, four, five, six, seven, eight,
nine, ten or more first nucleic acid strands. When the affinity
molecule is conjugated with two or more first nucleic acid strands,
the first nucleic acid stands can all be the same, all different,
or some same and some different. In some embodiments, the affinity
molecule is conjugated with one first nucleic acid strand, i.e.,
the affinity molecule and the conjugated first nucleic acid strand
have a 1:1 ratio. Generally, when the composition is to be used for
quantitative labeling or detection of a target molecule in a
sample, the affinity molecule and the first nucleic acid strand are
in a substantially 1:1 ratio. On the other hand, when the
composition is to be used for qualitative labeling or detection of
a target molecule in a sample, affinity molecule can be conjugated
with two or more first nucleic acid strands to provide signal
amplification.
The label moiety can also be conjugated with more than one second
nucleic acid strand. For example, the label moiety can be
conjugated with one, two, three, four, five, six, seven, eight,
nine, ten or more second nucleic acid strands. When the label
moiety is conjugated with two or more second nucleic acid strands,
the second nucleic acid stands can all be the same, all different,
or some same and some different. In some embodiments, the label
moiety is conjugated with one second nucleic acid strand, i.e., the
label moiety and the conjugated second nucleic acid strand have a
substantially 1:1 ratio.
In some embodiments, the affinity molecule is conjugated to the
label moiety via a nucleic acid linker. In some embodiments, the
nucleic acid linker comprises first and second strands of nucleic
acid (i.e., a nucleic acid linker) that specifically hybridize to
each other. The affinity molecule and the label moiety can be
attached to different nucleic acid strands. For example, the
affinity molecule can be bound to a first strand of the nucleic
acid linker and the label moiety can be attached to the second
strand of the nucleic acid linker. Thus, the affinity molecule can
be conjugated to a label moiety molecule under conditions that
permit hybridization between the first and second nucleic acid
strands. However, the affinity molecule is not conjugated to the
lab molecule under conditions that do not permit such
hybridization. As described in more detail below, the affinity
molecule or the label moiety can be linked to the respective
nucleic acid strand covalently or non-covalently.
As used herein, the term "specifically hybridize" refers to the
ability of a nucleic acid molecule to hybridize, under moderately
or highly stringent conditions, to a desired nucleic acid molecule,
without substantial hybridization under the same conditions with
nucleic acid molecules that are not the desired nucleic acid
molecule. Those skilled in the art can readily determine whether
the first nucleic acid strand hybridizes to the second nucleic acid
strand under stringent conditions by performing a hybridization
assay in the presence of other nucleic acid molecules, such as
total cellular nucleic acid molecules, and detecting the presence
or absence of hybridization to the other nucleic acid molecules.
The terms "hybridize" and "hybridization" refer to the formation of
complexes between nucleotide sequences which are sufficiently
complementary to form complexes via Watson-Crick base pairing.
As used herein the term "stringent conditions" refers to conditions
under which a nucleic acid strand will hybridize preferentially to,
or specifically bind to, its complementary binding partner, and to
a lesser extent to, or not at all to, other sequences. Put another
way, the term "stringent hybridization conditions" as used herein
refers to conditions that are compatible to produce duplexes
between complementary nucleic acid strands, e.g., between DNA
probes and complementary targets in a sample or between a PCR
primer and a nucleic acid molecule to be amplified with a
substantial lack of duplexes formed between non-complementary
nucleic acid strands.
Suitable stringent hybridization buffers and conditions are well
known to those of skill in the art and are described, for example,
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York (1992) and in Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md. (1998).
In some embodiments, non-stringent conditions can be used. Without
wishing to be bound by a theory, due to DNA linker design (each
pair is significantly different than others with no more than 4 bp
hetero-dimer), non-stringent conditions can also be used for
specifically binding a nucleic acid strands with its complementary
binding partner. Non-stringent conditions can include standard
biological buffers, such as PBS or TBS, with physiological ionic
strength and pH. The term "physiological ionic strength" is well
known to those skilled in the art. It is generally equivalent to
about 137 mM NaCl, 3 mM KCl and about 10 mM phosphate.
Physiological pH is about 7.4.
In some embodiments, the affinity molecule is conjugated to the
label moiety via an adaptor molecule, in which the label moiety is
covalently linked to the adaptor molecule via a non-nucleic acid
linker and the adaptor molecule is non-covalently linked to the
affinity molecule. The non-covalent binding between the affinity
molecule and the adaptor molecule can be disrupted and the label
moiety disassociated from the affinity molecule. Thus, the affinity
molecule can be conjugated to the label moiety molecule under
conditions that permit non-covalent binding between the affinity
molecule and the adaptor molecule, but is not conjugated to the
label moiety under conditions that do not permit such non-covalent
binding.
When the conjugation is via an adaptor molecule, the adaptor
molecule and the affinity molecule can be present in any ratio in
the composition. For example, the adaptor molecule and the affinity
molecule can be in a ratio from about 1:1 to about 1:100, from
about 1:1 to about 1:50, from about 1:1 to about 1:25, from about
1:1 to about 1:20, or from about 1:1 to about 1:15 (affinity
molecule:adaptor molecule). In some embodiments, the adaptor
molecule and the affinity molecule can be in a ratio about 1:1,
1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some
embodiments, it can be useful to limit the adaptor molecule to
affinity molecule ratio in order to increase the stability of the
complex. High stability of the complex can be advantageous to avoid
cross-talk and/or rearrangement between different affinity
molecules bound to adaptor molecules. For example, Staphylococcus
protein A (SpA) has four immunoglobulin binding sites, but the
stability of immunoglobulin to SpA binding is greater when there
are two, or even one immunoglobulin per SpA molecule, as opposed to
four immunoglobulin molecules per SpA. Thus, in some embodiments,
an adaptor molecule to affinity molecule ratio of 1:1 or not more
than 1:2 can be useful. Put slightly differently, it can be useful
to have an adaptor molecule to affinity molecule ratio of 1:2 or
less. It is noted that when preparing pre-assembled affinity
molecule:adaptor molecule conjugates, one can actually include a
ratio of less than one affinity molecule per adaptor molecule in
order to limit the number of affinity molecules conjugated per
adaptor molecule.
The inventors have discovered that binding of no more than 1
affinity molecule to an adaptor molecule forms a stable bond that
shows slow dissociation kinetics. In some embodiments, the stable
bond shows very little or no dissociation within 2-4 hours. For
example, less than 5%, less than 10%, less than 15%, less than 20%,
less than 25%, less than 30%, less than 35%, less than 40%, less
than 45%, or less than 50% of the affinity molecule--adaptor
molecule complex is dissociated in 12 hours, 10 hours, 9 hours, 8
hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1
hour. In other words, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90% or atleast 95% of the affinity molecule--adaptor
molecule is still in the form of a complex after 12 hours, 10
hours, 9 hours, 8 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3
hours, 2 hours or 1 hour.
Nucleic Acid
As used herein, the term "nucleic acid" refers to a polymer or
oligomer of nucleotide or nucleoside monomers consisting of
naturally occurring bases, sugars and inter-sugar linkages. The
term "nucleic acid" also includes polymers or oligomers comprising
non-naturally occurring monomers, or portions thereof, which
function similarly. At a minimum, a nucleic acid useful in the
compositions and methods described herein for coupling an affinity
molecule and label moiety is capable of sequence specific
hydrogen-bonded hybridization between complementary nucleic acid
strands. A nucleic acid can be DNA, RNA or chimeric, i.e.,
comprising both deoxy- and ribo-nucleotides.
The affinity molecule can be conjugated to the first strand of the
nucleic acid linker at any position in the first nucleic acid
strand. For example, the affinity molecule can be conjugated to the
3' or 5' end of the first nucleic acid strand. Similarly, the label
moiety can be conjugated at any position in the second nucleic acid
strand. For example, the label moiety can be conjugated to the 3'
or 5' end of the second nucleic acid strand. Generally, the
affinity molecule and the label moiety are conjugated to the
nucleic acid strand at positions such that hybridizing the two
strands does not interfere with functioning of the affinity
molecule or the label moiety. Accordingly, in some embodiments, the
affinity molecule is conjugated to the 3' terminus of the first
nucleic acid strand and the label moiety is conjugated to the 3'
terminus of the second nucleic acid strand. In other embodiments,
the affinity molecule is conjugated to the 5' terminus of the first
nucleic acid strand and the label moiety is conjugated to the 5'
terminus of the second nucleic acid strand. One or both strands of
the double-stranded nucleic acid can comprise a modification. When
both strands comprise a modification, such a modification can be
the same or different.
As indicated above, the affinity molecule or the label moiety can
be linked to the respective nucleic acid strand covalently or
non-covalently. Accordingly, in some embodiments, the affinity
molecule and the first strand of the nucleic acid linker are
covalently linked together using a non-nucleic acid linker. For
example, the affinity molecule and the first stand of the nucleic
acid linker can be covalently linked together via a linker selected
from the group consisting of a bond,
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 (from S-HyNic/S-4FB reaction), zero-length
peptide bond (between --COOH and --NH2 directly on affinity
molecule and nucleic acid), two peptide bonds on a spacer (from
cross-linking of two --NH2 groups), triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, and
any combination thereof.
Alternatively, the affinity molecule and the first stand of the
nucleic acid linker can be non-covalently linked together via an
adaptor molecule, in which the adaptor molecule binds
non-covalently with the affinity molecule and the first strand of
the nucleic acid is conjugated (covalently or non-covalently) with
the adaptor molecule. For example, the first strand of the nucleic
acid can be covalently linked to the adaptor molecule by a linker
selected from the group consisting of a bond,
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 (from S-HyNic/S-4FB reaction), zero-length
peptide bond (between --COOH and --NH2 directly on affinity
molecule and nucleic acid), two peptide bonds on a spacer (from
cross-linking of two --NH2 groups), triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, and
any combination thereof.
The label moiety and the second strand of the nucleic acid linker
can be covalently linked together via a linker selected from the
group consisting of a bond,
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 (from S-HyNic/S-4FB reaction), zero-length
peptide bond (between --COOH and --NH2 directly on affinity
molecule and nucleic acid), two peptide bonds on a spacer (from
cross-linking of two --NH2 groups), triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, and
any combination thereof.
Alternatively, the label moiety and the second strand of the
nucleic acid linker can be non-covalently linked together via a
coupling pair in which the label moiety can be linked to one member
of the coupling pair and the second stand of the nucleic acid can
be linked to other member of the coupling pair. For the label
moiety can be linked to either streptavidin or biotin and the
affinity molecule can be bound to the other, e.g., if the label
moiety is bound to streptavidin then the affinity molecule is
linked to biotin and vice versa.
The nucleic acid strand conjugated with the label moiety can
comprise additional molecules conjugated to it. In some
embodiments, the nucleic acid strand conjugated with the label
moiety does not comprise any additional molecule which can quench a
detectable signal from the label moiety. For example, the nucleic
acid strand conjugated with the label moiety does not comprise any
additional molecule that can act as a fluorophore acceptor.
Each nucleic acid strand can be independently from about 6 to about
50 nucleotides in length or more. What should be considered is the
length necessary to provide specific pairing with a complementary
nucleic acid strand that is stable under the conditions employed
for label binding and detection. Generally, the more different
affinity molecules included, the higher temperature or lower the
salt concentration, the longer the sequence will need to be to
provide specificity and stability sufficient for a given assay. For
example, each strand can be independently about 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49 or 50 nucleotides in length. In some embodiments, at
least one nucleic acid strand is about 11 nucleotides in length. In
some embodiments, at least one nucleic acid strand is about 21
nucleotides in length.
The first and second strands of the nucleic acid linker can
hybridize over at least part of their length to form a
double-stranded region that is stable under moderately or highly
stringent conditions. For example, the first and second strand of
the nucleic acid can hybridize to form a double-stranded region
that is stable at low temperatures but is unstable at high
temperatures, i.e., the double-stranded region has a high melting
temperature (Tm). For example, the first and second strand of the
nucleic acid can hybridize to form a double-stranded region having
a Tm of 30.degree. C. or higher (e.g., 30.degree. C., 31.degree.
C., 32.degree. C., 33.degree. C., 34.degree. C., 35.degree. C.,
36.degree. C., 37.degree. C., 38.degree. C., 39.degree. C.,
40.degree. C., 41.degree. C., 42.degree. C., 43.degree. C.,
44.degree. C., 45.degree. C., 46.degree. C., 47.degree. C.,
48.degree. C., 49.degree. C., 50.degree. C. or higher). One of
skill in the art is well aware of computational and experimental
methods for determining the melting temperature of double stranded
nucleic acids.
The first and second strand of the nucleic acid can hybridize to
form a double-stranded region of about 6 to about 30 consecutive
base-pairs. However, the double-stranded region can be interrupted
by one or more (e.g., one two, three or more) single-stranded
nucleotides in one or both of the strands. This can be useful in
dissociating the label moiety from the affinity molecule, if
needed. When both strands comprise single-stranded nucleotide(s) in
the double-stranded region, they can be opposite to each other
(i.e., forms a mismatch) or not next to each other (i.e., forms a
bulge, loop, or hairpin).
In some embodiments, the first and second strand of the nucleic
acid can hybridize to form a double-stranded region of about 10-18
base-pairs. In some embodiments, the first and second strand of the
nucleic acid can hybridize to form a double-stranded region of
about 12-16 base-pairs. In some embodiments, the first and second
strand of the nucleic acid can hybridize to form a double-stranded
region of about 11 base-pairs. The double-stranded nucleic acid
linker can also comprise a single stranded overhang of about 1 to
about 25 nucleotides, (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25). As used
herein, the term "overhang" refers to a double-stranded structure
where at least one end of one strand is longer than the
corresponding end of the other strand forming the double-stranded
structure. The single-stranded overhang can be located at the 3' or
the 5' end of either strand. The double-stranded nucleic acid can
also have a blunt end, i.e., no single-stranded over hang. A
single-stranded overhang can be used to facilitate or dissociating
the label moiety from the affinity molecule via strand-displacement
with a longer complementary sequence that that hybridized to join
the affinity molecule and label moiety. Strand-displacement is
discussed in more detail below.
As stated above, the term "nucleic acid" also includes polymers or
oligomers comprising non-naturally occurring monomers, or portions
thereof, which function similarly. Accordingly, in some
embodiments, the nucleic acid comprises at least one modification.
Typical nucleic acid modifications can include one or more of: (i)
alteration, e.g., replacement, of one or both of the non-linking
phosphate oxygens and/or of one or more of the linking phosphate
oxygens in the phosphodiester intersugar linkage; (ii) alteration,
e.g., replacement, of a constituent of the sugar, e.g., at the 2'
position of the sugar; (iii) wholesale replacement of the phosphate
moiety with "dephospho" linkers; (iv) modification or replacement
of a naturally occurring base with a non-natural base; (v)
replacement or modification of the ribose-phosphate backbone, e.g.
peptide nucleic acid (PNA); (vi) modification of the 3' end or 5'
end of the nucleic acid, e.g., removal, modification or replacement
of a terminal phosphate group or conjugation of a moiety, e.g.,
conjugation of a ligand, to either the 3' or 5' end of nucleic
acid; and (vii) modification of the sugar, e.g., six membered
rings.
In some embodiments, the nucleic acid is not modified relative to
naturally-occurring nucleic acid molecules.
The terms replacement, modification, alteration, and the like, as
used in this context, do not imply any process limitation, e.g.,
modification does not mean that one must start with a reference or
naturally occurring nucleic acid and modify it to produce a
modified nucleic acid but rather "modified" simply indicates a
difference from a naturally occurring molecule.
In some embodiments, the modification is selected from the group
consisting of nucleobase modifications, sugar modification,
inter-sugar (or inter-nucleoside) linkage modifications, backbone
modifications (or sugar-phosphodiester replacement), and any
combinations thereof. Exemplary sugar modifications at the sugar
moiety include but are not limited to, modifying the 2' position of
the sugar, such as 2'-O-Me (2'-O-methyl), 2'-O-MOE
(2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl]
(2'-O-NMA), 2'-S-methyl, 2'-O--CH.sub.2-(4'-C) (LNA),
2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE),
2'--NH.sub.2, and 2-SH; arabinose sugar; threose sugar; and acyclic
sugar (e.g., glycol nucleic acids).
Exemplary inter-sugar linkage and backbone modifications include,
but are not limited to, replacing one or both of the non-bridging
phosphate oxygen atoms in the intersugar linkage can be replaced by
the following: S, Se, BR.sub.3 (R is hydrogen, alkyl, aryl), C
(i.e. an alkyl group, an aryl group, etc. . . .), H, NR.sub.2;
replacing one or both of bridging oxygen, (i.e. oxygen that links
the phosphate to the nucleoside), with nitrogen (bridged
phosphoroamidates), sulfur (bridged phosphorothioates) or carbon
(bridged methylenephosphonates); replacing the phosphate group with
amides (for example amide-3 (3'-CH.sub.2--C(.dbd.O)--N(H)-5') and
amide-4 (3'-CH.sub.2--N(H)--C(.dbd.O)-5')), hydroxylamino, siloxane
(dialkylsiloxxane), carboxamide, carbonate, carboxymethyl,
carbamate, carboxylate ester, thioether, ethylene oxide linker,
sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal
(3'-S--CH.sub.2--O-5'), formacetal (3'-O--CH.sub.2--O-5'), oxime,
methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI,
3'-CH.sub.2--N(CH.sub.3)--O-5'), methylenehydrazo,
methylenedimethylhydrazo, methyleneoxymethylimino, ethers
(C3'-O--C5'), thioethers (C3'-S--C5'), thioacetamido
(C3'-N(H)--C(.dbd.O)--CH.sub.2--S--C5'), C3'-O--P(O)--O--SS--C5',
C3'-CH.sub.2--NH--NH--C5', 3'-NHP(O)(OCH.sub.3)-O-5' and
3'-NHP(O)(OCH.sub.3)--O-5'; and replacing the phosphate linker and
sugar by nuclease resistant nucleoside or nucleotide surrogates,
such as morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid
(PNA), amino-ethylglycyl PNA (aegPNA), and backnone-extended
pyrrolidine PNA (bepPNA) nucleoside surrogates.
While one or both strands of the nucleic acid linker can comprise
ribonucleotides, in some embodiments it is preferred that a strand
is not comprised of all ribonucleotides. For example, a strand can
comprise one, two, three, four, five, six, seven, eight, nine, ten,
or more ribonucleotides as long as there is at least one (e.g.,
one, two, three, four, five, six, seven, eight, nine, ten, or more)
2'-deoxynucleotides in the strand. If more than one ribonucleotide
is present, they can be present consecutively, i.e., next to each
other, or non-consecutively.
Label Moiety
As used herein, the term "label moiety" refers to any molecule that
has a detectable property or is capable of producing a detectable
signal. The term "detectable property" means a physical or chemical
property of a molecule that is capable of independent detection or
monitoring by an analytical technique after being conjugated with
an affinity molecule, i.e., the property is capable of being
detected in the presence of a sample under analysis. The property
can be light emission after excitation, quenching of a known
emission sites, electron spin, radio activity (electron emission,
positron emission, alpha particle emission, etc.), nuclear spin,
color, absorbance, near IR absorbance, UV absorbance, far UV
absorbance, etc. Suitable label moieties include fluorescent
molecules, luminescent molecules and nanoparticles, radio-isotopes,
chromophores, nucleotide chromophores, enzymes, substrates,
chemiluminescent moieties, magnetic microbeads, magnetic
nanoparticles, plasmonic nanoparticles, upconverting nanoparticles,
bioluminescent moieties, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and the like.
Means of detecting such labels are well known to those of skill in
the art. For example, radiolabels can be detected using
photographic film or scintillation counters, fluorescent markers
can be detected using a photo-detector to detect emitted light.
Enzymatic labels are typically detected by providing the enzyme
with an enzyme substrate and detecting the reaction product
produced by the action of the enzyme on the enzyme substrate, and
calorimetric labels can be detected by visualizing the colored
label. As such, a label moiety is any moiety detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Any method known in the art
for detecting the particular label moiety can be used for
detection.
The term "analytical technique" means an analytical chemical or
physical approach or instrument for detecting and/or monitoring the
property. Such instruments can be based on spectroscopic analytical
methods such as UV and visible light spectrometry, far IR, IR and
near IR spectrometry, X-ray spectrometry, electron spin resonance
spectrometry, nuclear magnetic resonance (NMR) spectrometry,
etc.
In some embodiments, the label moiety is a luminescent
nanoparticle. As used herein, the term "luminescent nanoparticle"
refers to luminescent materials that generate light upon the
combination of holes and electrons. Luminescent nanoparticles are
generally nanocrystals such as quantum dots, nanorods, nanobipods,
nanotripods, nanomultipods or nanowires.
Luminescent nanoparticles can be made from compound semiconductors
which include Group II, Group III, group IV, Group V, or Group VI
materials. For example, Luminescent nanoparticles can be made from
compound semiconductors which include Group II-VI, II-IV and III-V
materials. Exemplary luminescent nanoparticles include, but are not
limited to cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium
telluride (CdTe), cadmium zinc sulfide (CdZnS), cadmium telluride
silicone (CdTeSi), cadmium mercury telluride (CdHgTe), zinc
selenide (ZnSe), zinc sulfide (ZnS), zinc oxide (ZnO), lead sulfide
(PbS), lead selenide (PbSe), gallium arsenide (GaAs), indium
phosphide (InP), indium arsenide (InAs), silicon (Si), Ge, SiGe,
and the like.
The luminescent nanoparticles can be core type or core-shell type.
In a core-shell nanoparticle, the core and shell are made from
different materials. Both core and shell can be made from compound
semiconductors. In some embodiments, the luminescent nanoparticle
comprises a core; and a shell forms a colloidal particle. Without
wishing to be bound by a theory, colloidal properties can come from
surface coating. Inorganic shell only confines electrons/holes to
the core for improved fluorescent properties. In some embodiments,
the luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS.
The shape of the luminescent nanoparticle is not limited and can be
in the shape of a sphere, a rod, a wire, a pyramid, a cube, or
other geometric or non-geometric shapes. Additionally, the particle
size of the luminescent nanoparticle is typically from about 1 nm
to about 1000 nm. For example, the luminescent nanoparticle can be
from about 1 nm to about 100 nm, or from about 1 nm to about 50 nm
in size. In some embodiments, the luminescent nanoparticle is from
about 2 nm to about 25 nm in size. In some embodiments, the
luminescent nanoparticle can be about 10 nm in size.
In some embodiments, hydrodynamic size of the luminescent
nanoparticle in aqueous buffer can range from about 5 nm to about
30 nm. As used herein, "hydrodynamic size" refers to the apparent
size of a molecule based on the diffusion of the molecule through
an aqueous solution. The diffusion or motion of the molecule
through solution can be processed to derive an apparent size of the
molecule, where the size is given by the "Stokes radius" or
"hydrodynamic radius" of the molecule. The "hydrodynamic size" of a
molecule depends on both mass and shape (conformation), such that
two molecules having the same mass may have differing hydrodynamic
sizes based on the overall conformation. Hydrodynamic size can be
measured, for example, by size exclusion chromatography.
The color of the light emitted by the luminescent nanoparticle
depends on a number of factors that include the size and shape of
the luminescent nanoparticle. As is in known in the art,
luminescent nanoparticles having the same composition but having
different diameters absorb and emit radiation at different wave
lengths. For example, a luminescent nanoparticle with a larger
particle size emits light with a lower energy as compared to a
luminescent nanoparticle made of the same material but with a
smaller particle size. Thus, the small luminescent nanoparticles
absorb and emit in the blue portion of the spectrum, whereas the
medium and large quantum dots absorb and emit in the green and red
portions of the visible spectrum, respectively. It is to be noted
that unlike organic fluorophores, QDs can absorb all light with
energy higher than the band-gap (i.e. wavelengths lower than
emission wavelength): larger QDs absorb blue light very
efficiently.
Alternatively, or in addition, the luminescent nanoparticles can be
essentially the same size but made from different materials. For
example, UV-absorbing luminescent nanoparticles can be made from
zinc selenide, whereas visible and IR luminescent nanoparticles can
be made from cadmium selenide and lead selenide, respectively.
Nanoparticles having different size and/or composition can be used
in each of the nanoparticle layers.
Emission spectra of different luminescent nanoparticles are
distinguishable. For example, emission spectra of a first
luminescent nanoparticle can have a peak emission wavelength that
differs by least 5 nm, at least 10 nm, at least 15 nm, at least 20
nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm,
at least 45 nm, or at least 50 nm from the peak emission wavelength
of a second nanoparticle.
The luminescent nanoparticles are typically hydrophobic in nature.
Accordingly, luminescent nanoparticles can be coated with a polymer
to provide desirable properties to the luminescent nanoparticles.
For example, coating the luminescent nanoparticle can aid in
allowing the luminescent nanoparticle to exist in an aqueous medium
while retaining its optical properties. A coating can also provide
functional groups for conjugating the nanoparticle with a molecule,
such as a nucleic acid or an adaptor molecule. The luminescent
nanoparticles can be coated with an amphiphilic polymer or lipids.
Surface can be made positively charged, negatively charged,
neutral, or zwitterionic.
In some embodiments, the luminescent nanoparticle comprises a
polyethylene glycol (PEG) coating layer. As used herein, the term
"polyethylene glycol" refers to a polymer containing ethylene
glycol monomer units of formula --O--CH.sub.2--CH.sub.2--.
Polyethylene glycols can be polymers of any chain length or
molecular weight, and can include branching. In some embodiments,
the average molecular weight of the polyethylene glycol is from
about 200 to about 9000. In some embodiments, the average molecular
weight of the polyethylene glycol is from about 200 to about 5000.
In some embodiments, the average molecular weight of the
polyethylene glycol is from about 200 to about 900. In some
embodiments, the average molecular weight of the polyethylene
glycol is about 400. Suitable polyethylene glycols include, but are
not limited to polyethylene glycol-200, polyethylene glycol-300,
polyethylene glycol-400, polyethylene glycol-600, and polyethylene
glycol-900. The number following the dash in the name refers to the
average molecular weight of the polymer. In some embodiments, the
polyethylene glycol is polyethylene glycol-400.
Polyethylene glycols can have a free hydroxyl group at each end of
the polymer molecule, or can have one or more hydroxyl groups
etherified with a lower alkyl, e.g., a methyl group. Also suitable
are derivatives of polyethylene glycols having esterifiable
carboxyl groups.
In some embodiments, the polyethylene glycol is functionalized with
amine groups. Such amine groups can be used for conjugating the
luminescent nanoparticle with a molecule, such as a nucleic acid or
an adaptor molecule.
Suitable polyethylene glycols include, but are not limited to the
CARBOWAX.TM. and CARBOWAX.TM. Sentry series (available from Dow),
the LIPDXOL.TM. series (available from Brenntag), the LUTROL.TM.
series (available from BASF), and the PLURIOL.TM. series (available
from BASF).
Further, as used herein, the term "polyethylene glycol" is
synonymous with the terms "polyethylene oxide" (PEO). These terms
are used interchangeably, regardless of molecular weight, chain
length, viscosity, branching structures if any, and the like. The
terms "oligo ethylene glycol" and "oligo ethylene oxide" are also
used for shorter versions of polyethylene glycol.
In some embodiments, the luminescent nanoparticle has a
substantially neutral charge. The inventors have discovered that
luminescent nanoparticles having a substantially neutral charge
have reduced non-specific binding to a cell or tissue sample.
In some embodiments, the luminescent nanoparticle has a
substantially non-fouling surface. The inventors have discovered
that luminescent nanoparticles having a substantially non-fouling
surface have reduced non-specific binding to a sample. As used
herein, the term "non-fouling surface" is defined as a surface that
does not substantially adsorb to or attract sample or substrate
molecules non-specifically under the conditions for a given assay.
Generally, a non-fouling surface will attract or absorb
non-specifically less than 5% as much as a corresponding surface
not treated to be non-fouling, e.g., less than 4%, less than 3%,
less than 2%, less than 1% or even less relative to a non-treated
corresponding surface. Non-fouling properties can be achieved, for
example, by shielding the surface with a layer of non-fouling
polymer (such as polyethylene glycol) or by utilizing zwitterionic
surface coatings (i.e. coatings with alternating positive and
negative charges, which yield highly hydrated, but neutrally
charged surfaces).
In some embodiments, the luminescent nanoparticle is a colloidal
water-soluble nanoparticle with a stable non-fouling coating such
that non-specific binding of the luminescent nanoparticle to a
sample (e.g., cell or tissue sample) is reduced or eliminated)
relative to a nanoparticle lacking such coating. A nanoparticle
lacking a stable-non-fouling coating usually has a highly charged
surface, a surface with exposed hydrophobic areas, and/or a poorly
stable coating.
In some embodiments, the label moiety can be a magnetic
nanoparticle, a plasmonic nanoparticle, or an upconverting
nanoparticle. As used herein, the term "plasmonic nanoparticle"
refers to a nanoparticle that has very strong absorption (and
scattering) spectrum that is tunable by changing the shape, the
composition or the medium around their surfaces. It will be
appreciated that the term includes all plasmonic nanoparticles of
various shapes and surface surrounding which gives them surface
plasmon absorption and scattering spectrum in the visible-near
infra-red region of the spectrum. As used herein, an "upconverting
nanoparticle" means a nanoparticle which is a combination of an
absorber which is excited by infrared (IR) light and an emitter ion
in a crystal lattice, which converts IR light into visible
radiation.
In some embodiments, the label moiety is a fluorophore or
fluorescent molecule or dye. A wide variety of fluorescent
molecules are known in the art. Typically, the fluorophore is an
aromatic or heteroaromatic compound and can be a pyrene,
anthracene, naphthalene, acridine, stilbene, indole, benzindole,
oxazole, thiazole, benzothiazole, cyanine, carbocyanine,
salicylate, anthranilate, coumarin, fluorescein, rhodamine or other
like compound. Exemplary fluorophores include, but are not limited
to, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;
5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);
5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine
(5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine
(HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA
(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G;
6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine;
ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);
Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;
Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor
350.TM.; Alexa Fluor 430.TM.; Alexa Fluor 488.TM.; Alexa Fluor
532.TM.; Alexa Fluor 546.TM.; Alexa Fluor 568.TM.; Alexa Fluor
594.TM.; Alexa Fluor 633.TM.; Alexa Fluor 647.TM.; Alexa Fluor
660.TM.; Alexa Fluor 680.TM.; Alizarin Complexon; Alizarin Red;
Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin);
AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl
stearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange
R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG.TM.
CBQCA; ATTO-TAG.TM. FQ; Auramine; Aurophosphine G; Aurophosphine;
BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);
Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H);
BG-647; Bimane; Bisbenzamide; Blancophor FFG; Blancophor SV;
BOBO.TM.-1; BOBO.TM.-3; Bodipy 492/515; Bodipy 493/503; Bodipy
500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy
558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy
630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy F1; Bodipy FL
ATP; Bodipy F1-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X
conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X
SE; BO-PRO.TM.-1; BO-PRO.TM.-3; Brilliant Sulphoflavin FF; Calcein;
Calcein Blue; Calcium Crimson.TM.; Calcium Green; Calcium Green-1
Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium
Green-C18 Ca2+; Calcium Orange; Calcofluor White;
Carboxy-X-rhodamine (5-ROX); Cascade Blue.TM.; Cascade Yellow;
Catecholamine; CFDA; CFP-Cyan Fluorescent Protein; Chlorophyll;
Chromomycin A; Chromomycin A; CMFDA; Coelenterazine; Coelenterazine
cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h;
Coelenterazine hcp; Coelenterazine ip; Coelenterazine O; Coumarin
Phalloidin; CPM Methylcoumarin; CTC; Cy2.TM.; Cy3.1 8; Cy3.5.TM.;
Cy3.TM.; Cy5.1 8; Cy5.5.TM.; Cy5.TM.; Cy7.TM.; Cyan GFP; cyclic AMP
Fluorosensor (FiCRhR); d2; Dabcyl; Dansyl; Dansyl Amine; Dansyl
Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;
Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH
(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine
123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di16-ASP); DIDS;
Dihydorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7));
Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP;
ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1
(EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Fast
Blue; FDA; Feulgen (Pararosaniline); FITC; FL-645; Flazo Orange;
Fluo-3; Fluo-4; Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold
(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43.TM.; FM 4-46;
Fura Red.TM. (high pH); Fura-2, high calcium; Fura-2, low calcium;
Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl
Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shifted (rsGFP);
GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV
excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue;
Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580;
HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);
Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine
(DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan;
LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine
Rhodamine; Lissamine Rhodamine B; LOLO-1; LO-PRO-1; Lucifer Yellow;
Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium
Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10
GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin;
Mitotracker Green FM; Mitotracker Orange; Mitotracker Red;
Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);
Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD
Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast
Red; Nuclear Yellow; Nylosan Brilliant lavin EBG; Oregon Green.TM.;
Oregon Green 488-X; Oregon Green.TM. 488; Oregon Green.TM. 500;
Oregon Green.TM. 514; Pacific Blue; Pararosaniline (Feulgen);
PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin
B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite
RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin
R [PE]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3;
PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodid
(PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin
7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2;
Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine
6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra; Rhodamine
BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;
Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;
R-phycoerythrin (PE); red shifted GFP (rsGFP, S65T); S65A; S65C;
S65L; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B;
Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange;
Sevron Yellow L; sgBFP.TM.; sgBFP.TM. (super glow BFP); sgGFP.TM.;
sgGFP.TM. (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene
Isothiosulphonic Acid); SPQ
(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine
B can C; Sulphorhodamine G Extra; Tetracycline;
Tetramethylrhodamine; Texas Red.TM.; Texas Red-X.TM. conjugate;
Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;
Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole
Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3;
TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC
(TetramethylRodamineIso ThioCyanate); True Blue; TruRed; Ultralite;
Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-Rhodamine; XRITC;
Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1;
YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these
fluorescent molecules are available and can be used.
In some embodiments, the label moiety is a nanoparticle comprising
a fluorophore or fluorescent molecule.
Other exemplary label moieties include radiolabels (e.g., .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g.,
galactosidases, glucorinidases, phosphatases (e.g., alkaline
phosphatase), peroxidases (e.g., horseradish peroxidase), and
cholinesterases), and calorimetric labels such as colloidal gold or
colored glass or plastic (e.g., polystyrene, polypropylene, and
latex) beads. Patents teaching the use of such label moieties
include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345,
4,277,437, 4,275,149, and 4,366,241, content of each of which is
incorporated herein by reference in its entirety.
In some embodiments, the label moiety is not a gold
nanoparticle.
Affinity Molecule
As used herein, the term "affinity molecule" refers to any molecule
that is capable of specifically interacting or binding with another
molecule, i.e. a target molecule. Generally, the nature of
interaction or binding between the affinity molecule and the target
molecule is non-covalent, such as one or more of hydrogen bonding,
Van der Waals forces, electrostatic forces, hydrophobic forces, and
the like. However, interaction or binding can also be covalent.
As used herein, the term "specifically binding" and "specific
binding" means that an affinity molecule binds to the target
molecule with greater affinity than it binds to other molecules
under the same conditions. Specific binding is generally indicated
by a dissociation constant of 1 .mu.M or lower, e.g., 500 nM or
lower, 400 nM or lower, 300 nM or lower, 250 nM or lower, 200 nM or
lower, 150 nM or lower, 100 nM or lower, 50 nM or lower, 40 nM or
lower, 30 nM or lower, 20 nM or lower, 10 nM or lower, or 1 nM or
lower.
An affinity molecule can be a naturally-occurring, recombinant or
synthetic molecule. However, an affinity molecule need not comprise
an entire naturally occurring molecule but can consist of only a
portion, fragment or subunit of a naturally or non-naturally
occurring molecule. Exemplary affinity molecules include, but are
not limited to, ligand receptors, ligands for a receptor, one
member of a coupling pair, nucleic acids (e.g., aptamers),
peptides, proteins, peptidomimetics, antibodies, portion of an
antibody, antibody-like molecules, antigens, and the like.
In some embodiments, the affinity molecule is an antibody or a
portion thereof. In some embodiments, the affinity molecule is an
antigen binding fragment of an antibody. As used herein, the term
"antibody" or "antibodies" refers to an intact immunoglobulin or to
a monoclonal or polyclonal antigen-binding portion with the Fc
(crystallizable fragment) region or FcRn binding fragment of the Fc
region. The term "antibodies" also includes "antibody-like
molecules", such as portions of the antibodies, e.g.,
antigen-binding portions. Antigen-binding portions can be produced
by recombinant DNA techniques or by enzymatic or chemical cleavage
of intact antibodies. "Antigen-binding portions" include, inter
alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining
region (CDR) fragments, single-chain antibodies (scFv), single
domain antibodies, chimeric antibodies, diabodies, and polypeptides
that contain at least a portion of an immunoglobulin that is
sufficient to confer specific antigen binding to the polypeptide.
Linear antibodies are also included for the purposes described
herein. The terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with
standard immunological meanings (Klein, Immunology (John Wiley, New
York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations
of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt,
I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific
Publications, Oxford)). Antibodies or antigen-binding portions
specific for various antigens are available commercially from
vendors such as R&D Systems, BD Biosciences, e-Biosciences and
Miltenyi, or can be raised against these cell-surface markers by
methods known to those skilled in the art.
In some embodiments, the affinity molecule is not protein A,
streptavidin, avidin, or biotin.
An affinity molecule can be generated by any method known in the
art. For example, antibodies can be found in an antiserum, prepared
from a hybridoma tissue culture supernatant or ascites fluid, or
can be derived from a recombinant expression system, as is well
known in the art. Fragments, portions or subunits of e.g., an
antibody, receptor or other species, can be generated by chemical,
enzymatic or other means, yielding for example, well-known (e.g.,
Fab, Fab') or novel molecules. The present invention also
contemplates that affinity molecules can include recombinant,
chimeric and hybrid molecules, such as humanized and primatized
antibodies, and other non-naturally occurring antibody forms. Those
skilled in the art will recognize that the non-limiting examples
given above describing various forms of antibodies can also be
extended to other affinity molecules such that recombinant,
chimeric, hybrid, truncated etc., forms of non-antibody molecules
can be used in the compositions and methods of the present
invention.
In some embodiments, the affinity molecule and the label moiety can
be reversibly conjugated to each other by an at least partially
double-stranded nucleic acid comprising a first strand and a second
strand. The first and second strand of the nucleic acid can
specifically hybridize to each other. Without limitations, the
affinity molecule and/or the label moiety can be linked to the
nucleic acids strands covalently or non-covalently.
In some embodiments, the affinity molecule can be covalently linked
to a strand of the double-stranded nucleic acid via a linker. When
the affinity molecule is covalently linked to the nucleic acid via
a linker, the linker can be a bond or bifunctional molecule. In
some embodiments, the affinity molecule is covalently linked to a
strand of the double-stranded oligonucleotide via a linker selected
from the group consisting of a bond,
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, also
referred to as SFB herein) linker, and any combination thereof.
Methods of linking a nucleic acid covalently or non-covalently with
another molecule are well known in the art and available to an
ordinarily skilled artisan. For example, DNA conjugated antibodies
prepared with a variety of covalent and non-covalent procedures
have been described for bio-analytical methods (Bailey, R. C., et
al., DNA-encoded antibody libraries: a unified platform for
multiplexed cell sorting and detection of genes and proteins.
Journal of the American Chemical Society, 2007. 129(7): p. 1959-67
and Lind, K. and M. Kubista, Development and evaluation of three
real-time immuno-PCR assemblages for quantification of PSA. Journal
of Immunological Methods, 2005. 304(1-2): p. 107-16).
In a non-limiting example, an amine-functionalized nucleic acid can
be covalently linked to an affinity molecule comprising a free
sulfhydryl group. As shown in FIG. 2A, one such covalent
conjugation approach involves reduction of IgG followed by
maleimide mediated reaction between pre-activated oligonucleotide
and a sulfhydryl group in the Fc region of an antibody. One benefit
of this approach is the controlled stoichiometry and structure of
antibody-nucleic acid bioconjugate. As reaction is limited to 1-2
sites on the Fc region, the preparation of antibodies with intact
Fab antigen recognition region and a single nucleic acid is readily
achievable. This can be important for minimizing potential
off-target binding of antibody-nucleic acid bioconjugates.
Alternatively, amine cross-linking can be used for covalently
linking a nucleic acid to an affinity molecule comprising an amine
group. FIG. 2B illustrate covalent linking of a nucleic acid with
an antibody using amine cross-linking.
While, covalent linking can produce stable and functional affinity
molecule-nucleic acid conjugates, complexity, high cost, and low
yield can hamper preparation of different affinity molecule-nucleic
acid conjugates. Accordingly, in some embodiments, the affinity
molecule can be non-covalently linked to the nucleic acid via an
adaptor molecule to which the nucleic acid is linked, covalently or
non-covalently. An adaptor molecule can be covalently linked to the
nucleic acid using a linker.
Adaptor Molecule
As used herein, the term "adaptor molecule" means any molecule that
is capable of specifically binding with an affinity molecule.
Generally, an appropriate adaptor molecule does not inhibit or
reduce binding of the affinity molecule to its target, i.e.,
binding of the affinity molecule to its target is reduced very
little, if at all, when said affinity molecule is bound by an
adaptor molecule. In one sense, an adaptor molecule can be an
affinity molecule as that term is used herein. Exemplary adaptor
molecules include, but are not limited to protein A, protein G,
antibody, portion of an antibody, antigen, receptor ligand,
receptor, ligand binding fragment of a receptor, one member of a
coupling pair, an aptamer, and the like. In some embodiments, when
the affinity molecule is an antibody or antibody derivative, the
adaptor molecule is protein A, streptavidin, avidin, biotin, an
antibody, or a portion of an antibody.
In some embodiments, the adaptor molecule is not protein A,
streptavidin, avidin, biotin, an antibody, or a portion of an
antibody.
Compositions Comprising a Plurality of Labeled Molecules
Provided herein is also a composition comprising an affinity
molecule covalently conjugated with a single-stranded nucleic acid.
Further, a composition comprising a plurality of different such
affinity molecules is also provided herein. Each member of the
plurality is conjugated with a single-stranded nucleic acid which
is different for each member of the plurality.
Further provided herein is a composition comprising an affinity
molecule non-covalently conjugated with an adaptor molecule,
wherein the adaptor molecule is conjugated (covalently or
non-covalently) with a single-stranded nucleic acid. A composition
comprising a plurality of different such affinity molecules is also
provided herein. Each member of the plurality is conjugated with a
single-stranded nucleic acid which is different for each member of
the plurality.
A composition comprising a label moiety covalently conjugated with
a single-stranded nucleic acid is also provided herein. Further, a
composition comprising a plurality of different such label moieties
is also provided herein. Each member of the plurality is conjugated
with a single-stranded nucleic acid which is different for each
member of the plurality. In some embodiments, the composition
comprising plurality of nucleic acid conjugated affinity molecules
is a dried composition. Further, at least one detectable property
of the label moiety on each member of the plurality is different or
distinguishable from at least one detectable property of a label
moiety on the other members of the plurality. For example, the
label moiety on one member can have a peak emission wavelength that
differs by least 5 nm, at least 10 nm, at least 15 nm, at least 20
nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm,
at least 45 nm, or at least 50 nm from the peak emission wavelength
of the label moiety on another member.
Also provided herein is composition comprising a label moiety
non-covalently conjugated with an adaptor molecule via a
double-stranded nucleic acid. A composition comprising a plurality
of different such label moieties is also provided. Each member of
the plurality is conjugated with an adaptor molecule via a
double-stranded nucleic acid that is different for each member of
the plurality such that a nucleic acid strand of the
double-stranded nucleic acid of a first member of the plurality
does not specifically bind or hybridize with either nucleic strand
of a second member of the plurality.
Further provided herein is composition comprising an adaptor
molecule conjugated with a single-stranded nucleic acid. A
composition comprising a plurality of different such adaptor
molecules is also provided herein. Each member of the plurality is
conjugated with a single-stranded nucleic acid which is different
for each member of the plurality
Provided herein is also a composition comprising a plurality of
different affinity molecules, wherein each member of the plurality
is capable of binding a different target. Each member of the
plurality comprising an affinity molecule reversibly conjugated to
a label moiety. At least one detectable property of the label
moiety on each member is different or distinguishable from at least
one detectable property of a label moiety on the other members. For
example, the label moiety on one member can have a peak emission
wavelength that differs by least 5 nm, at least 10 nm, at least 15
nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm,
at least 40 nm, at least 45 nm, or at least 50 nm from the peak
emission wavelength of the label moiety on another member. Further,
the inventors have discovered that no detectable re-arrangement
takes place between different members of the composition. In other
words, there is no detectable re-arrangement of a label moiety from
one affinity molecule to a different affinity molecule in the
composition.
In some embodiments, the affinity molecule on a member of the
plurality is conjugated to the label moiety via hybridized first
and second nucleic acid strands. The first and second nucleic acid
strands of each member specifically hybridize with each other,
i.e., the first and second strands of one member do not
specifically hybridize with a first or a second strand of a
different member under moderately or highly stringent conditions.
The affinity molecule is conjugated, covalently or non-covalently,
with the first nucleic acid strand and the label moiety is
conjugated, covalently or non-covalently, with the second nucleic
acid stand.
In some embodiments, the affinity molecule in a composition
comprising a plurality of affinity molecules can be conjugated via
an adaptor molecule to the label molecule, wherein the label
molecule is conjugated (covalently or non-covalently) with the
adaptor molecule and the adaptor molecule is non-covalently bound
with the affinity molecule.
As used herein, the term "plurality" means "two or more", unless
expressly specified otherwise. For example, a plurality can
comprise 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25 or more different members.
Further, a composition comprising a plurality of different
molecules can be in any form, e.g., liquid or solid. Accordingly,
in some embodiments, the composition comprising a plurality of
different molecules is a dried composition, e.g., a powder.
Labeled Cell or Tissue Sample
Provided herein is also a composition comprising a cell or a tissue
sample and a plurality of different affinity molecules, each member
of the plurality comprising an affinity molecule conjugated to a
first single-strand nucleic acid and wherein each member of the
plurality is bound to a different analyte or biomarker in the cell
or tissue sample. The first single-strand nucleic acid is different
from the first single-strand nucleic acid conjugated to each other
member of the plurality. As used herein, the term "different
nucleic acid strand" means that the nucleotide sequence of one
strand is different from another strand, i.e., two different
strands have less than 80% (e.g., less than 70%, less than 60%,
less than 50%, less than 40%, less than 30%, less than 20%, or less
than 10%) sequence homology or complementarity. Further, the first
nucleic acid strands from different members of the plurality can be
of different lengths. Moreover, different first nucleic acid
strands will specifically hybridize to different second
strands.
In some embodiments, the composition further comprises a plurality
of second single strand nucleic acid molecules, wherein each member
of the plurality of second single-strand nucleic acids is
conjugated to a different label moiety, and wherein each different
second single strand nucleic acid molecule specifically hybridizes
to a different first single strand nucleic acid molecule conjugated
to a member of the plurality of different affinity molecules. In
this composition, at least a subset of the plurality of different
affinity molecules bound to the analytes in the cell or tissue
sample is specifically associated with a plurality of different
label moieties, wherein detectable properties of the different
label moieties are distinguishable. In this manner, each different
biomarker recognized by a different affinity molecule can be
specifically and reversibly conjugated to a distinguishable label
moiety.
Method
The compositions described herein can be used in 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,
immunohistochemistry, western blot, and the like. Accordingly,
disclosed herein is also a method for staining one or more analytes
in a sample for identification or quantitation.
Generally, the methods comprise contacting a plurality of analytes
in a sample with a first plurality of affinity molecules, wherein
each affinity molecule of the plurality binds to a different
analyte if present. The affinity molecules can be pre-conjugated
with label moieties before contacting or binding to the analytes or
the affinity molecules can be conjugated to label moieties after
affinity molecules are bound with the analytes. Conjugated label
moieties provide detectable signal for identification or
quantitation of bound analyte. At least one detectable signal from
each label moiety is different from at least one detectable signal
of other label moieties. In other words, in the subject method of
this embodiment, each different analyte is bound to a different
label moiety such that each different analyte has detectable signal
that is different from at least one detectable signal associated
with other analytes.
Signal from the conjugated label moieties can be detected using any
method known in the art for detecting the particular label moiety
to provide identification or quantitation of at least a subset of
the plurality of analytes in the sample. The number of analytes
identified or quantitated can range from 1 to 100's. For example,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25 or more different analytes can be identified
or quantitated at the same time.
After detecting the signal from the conjugated label moieties, the
signal can be erased and a different plurality of analytes can be
detected using a second plurality of affinity molecules, wherein
each affinity molecule of the plurality binds to a different
analyte. Again an affinity molecule in the second plurality can be
pre-conjugated with a label moiety before binding to the analyte or
the affinity molecule can be conjugated to a label molecule after
binding of the analyte. Signal from the second set of conjugated
label moieties can be detected to provide identification or
quantitation of a second plurality of analytes in the sample.
The erasing and relabeling steps can be cycled or repeated as many
times as needed to detect the desired number of different analytes
in the sample. For example, erasing and relabeling steps can
repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more times. Additionally, number
of analytes identified or quantitated in each cycle can range from
1 to 100's. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more different
analytes can be identified or quantitated in each cycle. Thus, the
method provides multiplexing capabilities to identified or
quantitate from 1 to 100's of analytes in a sample.
If the affinity molecule is not pre-conjugated with the label
moiety, nucleic acid conjugated affinity molecules for binding all
the different desired analytes can be added to the sample in one
contacting step. Then, for detection, a subset of the analyte-bound
affinity molecules can be specifically conjugated with different
label moieties via complementary nucleic acid conjugated label
moieties, and signal from the conjugated label moieties can be
detected to provide identification or quantitation of a subset of
the analytes. The signal can be quenched or erased and a different
subset of the bound affinity molecules detected by specific binding
of a new set of distinguishable label moieties bearing different
nucleic acid strands that hybridize to nucleic acids on a different
subset of affinity molecule bound to the sample, to identify or
quantitate a different subset of analytes. This process can be
repeated as many times as needed.
In some embodiments, when the affinity molecule is not
pre-conjugated to the label moiety before contacting with the
analyte, the method comprises, in order: (a) contacting the sample
with a 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 wherein
each different affinity molecule is conjugated to a different first
single strand nucleic acid, such that members of the plurality of
different affinity molecules become bound to members of the
plurality of analytes present in the sample; (b) contacting the
sample, under conditions that permit specific nucleic acid
hybridization, with a first set of different second single strand
nucleic acid molecules, each conjugated to a different label
moiety, wherein each different second single strand nucleic acid
molecule specifically hybridizes to a different first single strand
nucleic acid molecule conjugated to a member of the plurality of
different affinity molecules, such that at least a subset of the
plurality of different affinity molecules bound to members of the
plurality of analytes in the sample becomes specifically associated
with a plurality of different label moieties, wherein detectable
properties of the different label moieties are distinguishable; and
(c) detecting signal from label moieties associated with affinity
molecules bound to the sample, thereby detecting the presence or
amount of at least a subset of the plurality of analytes.
In some embodiments, the method further comprises the steps of: (d)
erasing the signal from the label moieties conjugated to the first
set of second single strand nucleic acid molecules; (e) contacting
the sample, under conditions that permit specific nucleic acid
hybridization, with a second set of different second single strand
nucleic acid molecules, each conjugated to a different label,
wherein each different second single strand nucleic acid molecule
specifically hybridizes to a different first single strand nucleic
acid molecule conjugated to a member of the plurality of different
affinity molecules, such that a different subset of the plurality
of different affinity molecules bound to members of the plurality
of analytes in the sample becomes specifically associated with a
different plurality of different label moieties relative to those
detected in step (c); (f) detecting signal from label moieties
associated with affinity molecules bound to the sample, thereby
detecting the presence or amount of the different subset of the
plurality of analytes; and (g) optionally repeating steps (d)-(f)
with a further set of second single strand nucleic acid
molecules.
In some embodiments, when the affinity molecule is reversibly
conjugated with the label moiety before contacting the sample, the
method comprises, 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, wherein each
different affinity molecule is conjugated to a different first
single strand nucleic acid molecule that is specifically hybridized
to a second single strand nucleic acid molecule, wherein the second
single strand nucleic acid molecule is conjugated to a label
moiety, such that each different affinity molecule is conjugated
via different hybridized first and second nucleic acid strands to a
different label moiety and members of the plurality of different
affinity molecules become bound to members of the plurality of
analytes present in the sample, wherein detectable properties of
the label moieties are distinguishable; and (b) detecting signal
from label moieties associated with the first plurality of affinity
molecules bound to the sample, thereby detecting the presence or
amount of the plurality of analytes.
In some embodiments, the method further comprises the steps of: (c)
erasing the signal from the label molecules conjugated to the first
set of second single strand nucleic acid molecules; (d) contacting
the sample with a second plurality of different affinity molecules
under conditions that permit specific analyte binding by the
different affinity molecules; (e) detecting signal from label
moieties associated with a second plurality of affinity molecules
bound to the sample, thereby detecting the presence or amount of at
least a subset of the plurality of analytes; and (f) optionally
repeating steps (c)-(e) with a further second set of the affinity
molecules.
Conditions that permit specific analyte binding by affinity
molecules are well known in the art. A, but one example, conditions
that permit specific antibody binding are well known in the art.
Exemplary conditions are also described in the examples provided
herein below.
Similarly, conditions that permit specific nucleic acid
hybridization are also well known in the art. Exemplary conditions
are also described in the examples provided herein below.
As used herein, the term "analyte" refers to a molecule, substance
or chemical constituent of interest in a sample or a biological
cell. Thus, the term "analyte" includes any substance which is
desired to be detected in a given sample. The analyte can be
attached to or present on a solid support surface. For example, an
analyte can be attached to or present on surface of a plate, dish,
well, membrane, grating, bead or particle (including, but not
limited to an agarose or latex bead or particle, a magnetic
particle, etc.). In some embodiments, the solid support can be an
ELISA plate or a western blot membrane. In some embodiments, an
analyte can be present on surface of a cell or a biological sample.
The cell or biological sample can be unfixed or fixed.
In some embodiments, the analyte is biomarker. As used herein, the
term "biomarker" refers to any biological feature from tissue
sample or a cell to be identified or quantitated. A biomarker can
be useful or potentially useful for measuring the initiation,
progression, severity, pathology, aggressiveness, grade, activity,
disability, mortality, morbidity, disease sub-classification or
other underlying feature of one or more biological processes,
pathogenic processes, diseases, or responses to a therapeutic
intervention. A biomarker is virtually any biological compound,
such as a protein and a fragment thereof, a peptide, a polypeptide,
a proteoglycan, a glycoprotein, a lipoprotein, a carbohydrate, a
lipid, a nucleic acid, an organic on inorganic chemical, a natural
polymer, and a small molecule, that is present in the sample to be
analyzed and that can be isolated from, or measured in, the
sample.
As used therein, the term "contacting" refers to any suitable means
for delivering, or exposing, to a sample, the specified composition
or molecule. In some embodiments, the term "contacting" refers to
adding specific composition or molecules (e.g., suspended in a
solution) directly to the sample. In some embodiments, the term
"contacting" can further comprise mixing the sample with the
specific composition or molecules by any means known in the art
(e.g., vortexing, pipetting, and/or agitating). In some
embodiments, the term "contacting" can further comprise incubating
the sample together with the specific composition or molecules for
a sufficient amount of time, e.g., to allow binding of the affinity
molecules to the target analytes. The contact time can be of any
suitable length, depending on the binding affinities and/or
concentrations of the affinity molecules or the analytes,
concentrations of the affinity molecules, or incubation condition
(e.g., temperature).
In some embodiments, the contact time between the sample and the
affinity molecules can be at least about 30 seconds, at least about
1 minute, at least about 5 minutes, at least about 10 minutes, at
least about 15 minutes, at least about 30 minutes, at least about 1
hour, at least about 2 hours, at least about 3 hours, at least
about 4 hours, at least about 6 hours, at least about 8 hours, at
least about 10 hours, at least about 12 hours, at least about 24
hours, at least about 48 hours or longer. One of skill in the art
can adjust the contact time and conditions accordingly.
Similarly, contact time between the sample and the second
single-strand nucleic acid can be at least can be at least about 30
seconds, at least about 1 minute, at least about 5 minutes, at
least about 10 minutes, at least about 15 minutes, at least about
30 minutes, at least about 1 hour, at least about 2 hours, at least
about 3 hours, at least about 4 hours, at least about 6 hours, at
least about 8 hours, at least about 10 hours, at least about 12
hours, at least about 24 hours, at least about 48 hours or longer.
Again, one of skill in the art can adjust the contact time and
conditions accordingly.
As used herein, the term "detecting" refers to observing a signal
from a label moiety to indicate the presence of an analyte in the
sample. Any method known in the art for detecting a particular
label moiety can be used for detection. Exemplary detection methods
include, but are not limited to, spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical
methods.
In some embodiments, detecting the signal comprises imaging
spectral emission from the label moiety. Systems and methods for
imaging spectral emission from a label moiety are well known in the
art and available to an ordinarily skilled artisan.
Systems and methods for multispectral imaging are described, for
example, in U.S. application Ser. No. 10/965,209; Ser. No.
12/092,670; Ser. No. 12/456,022; Ser. No. 12/525,059; and Ser. No.
12/985,161, and U.S. Pat. No. 6,208,749; no. 6,480,273; no.
6,639,665; no. 6,825,930; no. 7,019,777; no. 7,145,124; no.
7,473,334; no. 7,786,440; no. 7,589,772; and no. 8,027041, content
of all of which is incorporated herein by reference.
In some embodiments, imaging spectral emission comprises
hyperspectral imaging. Hyperspectral imaging is an extension of
multispectral imaging and is also referred to as imaging
spectrometry. Whereas multispectral imaging consists of
measurements from two to about ten discrete wavelengths for a given
image, hyperspectral imaging measures more than 10 contiguous
wavelengths, often many more. Like multispectral imaging,
hyperspectral imaging is an imaging technique that combines aspects
of conventional imaging with spectrometry and radiometry. The
result is a technique that is capable of providing an absolute
radiometric measurement over a contiguous spectral range for each
and every pixel of an image. Thus, data from a hyperspectral image
contains two-dimensional spatial information plus spectral
information over the spectral image. These data can be considered
as a three dimensional hypercube which can provide physical and
geometric observations of size dimension, orientation, shape,
color, and texture, as well as chemical/molecular information. As
shown in FIG. 27, hyperspectral imaging can accurately identify
different components of a composition comprising a mix of QDs.
Systems and methods for hyperspectral imaging are described, for
example, in U.S. application Ser. No. 11/774,704; Ser. No.
11/912,361; Ser. No. 11/933,253; Ser. No. 11/987,574; Ser. No.
12/162,486; Ser. No. 12/284,462; and Ser. No. 12/370,557, and U.S.
Pat. No. 6,008,492; no. 6,552,788; no. 6,831,688; no. 6,998,614;
no. 7,013,172; no. 7,149,366; no. 7,420,679; and no. 7,835,002,
content of all of which is incorporated herein by reference.
In some embodiments, the detecting can further comprise spectral
unmixing. Spectral unmixing corresponds to a linear decomposition
of an image or other data set into a series of contributions from
different spectral contributors. For example, image of a sample can
include multiple different contributions from label moieties
conjugated with affinity molecules which are bound with the
analytes in the sample. Each of these contributions can be unmixed
or decomposed into a separate spectral channel, forming an image of
the sample that corresponds almost entirely to signal contributions
from single spectral sources. When the contributions are unmixed
into separate channels or images, signal strengths can be
accurately quantified and analyzed. Methods and apparatus for
spectral unmixing are known in the art. See for example, U.S.
patent application Ser. No. 11/844,920; Ser. No. 12/757,831; Ser.
No. 11/649,292; Ser. No. 12/564,857; and Ser. No. 11/999,914, and
U.S. Pat. No. 6,665,438; no. 6,930,773; no. 7,072,770; no.
7,471,386; and no. 7,555,155, content of all of which is
incorporated herein by reference.
As used herein, the term "erasing" a signal means removing the
signal or reducing strength of the signal so as not to interfere
with signal from another label moiety. A signal can be erased by
quenching the signal. Alternatively, or in addition, the signal can
be erased by removing the label moiety generating the signal or the
affinity molecule conjugated with the label moiety generating the
signal from the sample. A signal can be erased by any method known
to the artisan including, but not limited to, chemical, physical,
or enzymatic means. For example, a signal can be erased by elution,
denaturation, washing, displacement, cleavage, photo-bleaching,
heating, quenching with a fluorophore acceptor, or a combination
thereof. Generally, a suitable method employed for erasing the
signal does not affect the sample or its biomarkers.
In some embodiments, the label moiety or the affinity molecule
conjugated with the label moiety can be physically removed from the
sample. This can be accomplished by inhibiting or reducing or
overcoming the binding between the affinity molecule and the
analyte or between the affinity molecule and the label moiety.
Methods of unbinding an affinity molecule from its target are well
known in the art. For example, washing with a low pH buffer can
reduce the binding between an affinity molecule and its target. In
other examples, changing the salt concentrations can reduce the
binding strength between an affinity molecule and its target.
Heating can also dissociate an affinity molecule from its
target.
In some of the compositions described herein, the affinity molecule
is conjugated with the label moiety via a nucleic acid linker,
wherein the nucleic acid comprises first and second strands of
nucleic acid which are specifically hybridized to each other. When
such compositions are used, the signal can be erased by denaturing
the double-stranded nucleic acid linker to release or unconjugate
the label moiety from the affinity molecule.
As an ordinarily skilled artisan is well aware, a double-stranded
nucleic acid can be denatured into single strands by heating.
Accordingly, in some embodiments, erasing is by heating the sample
to "melt" the double-stranded nucleic acid linker. Thus,
unconjugating or removing the label moiety from the affinity
molecule which can still be bound to the analyte in the sample. As
an example, washing or rinsing with a suitable wash buffer at a
temperature greater than the melting temperature of the hybridized
nucleic acid strands can permit label removal.
In some embodiments, strand displacement can be used for
dissociating the two strands of the nucleic acid linker. As used
herein, the term "strand displacement" refers to replacing one of
the nucleic acid strands in a double-stranded nucleic acid by third
strand. For example, if strands A and B are in the original
double-stranded nucleic acid, then strand displacement comprises
replacing either strand A or B in the duplex with strand C to
obtain either a A:C or B:C duplex.
Accordingly, in some embodiments, erasing the signal comprises
adding a nucleic acid to the sample, e.g., a single-stranded
nucleic acid. The nucleic acid to be added ("displacement nucleic
acid") has a nucleotide sequence which is substantially
complementary to one of the strands of the nucleic acid linker.
Further, the displacement strand can specifically hybridize with
the one of the strands of the nucleic acid linker more strongly
relative to the hybridization of the two strands of the linker to
each other.
Generally, strand displacement works best when the displacing
strand is complementary to a longer stretch of one of the nucleic
acid strands than the strand being displaced. Thus, the most
readily displaced strand is that which generates a partially
double-stranded configuration between the first and second nucleic
acid strands linking affinity molecule and label moiety. This can
be accomplished by having the nucleic acid linker comprise a
single-strand overhang on one of the stands and displacing strand
being complementary to said strand over a length that is longer
than the other strand of the linker.
In addition, or alternatively, the displacing stand can comprise on
or more modifications that promote duplex stability. Exemplary such
modifications include, but are not limited to, 2-amino-A; 2-thio U;
5-Me-thio-U; G-clamp (an analog of C having 4 hydrogen bonds);
psuedo uridine; 2' modifications, e.g., 2'F; "locked" nucleic acids
(LNA) in which the oxygen at the 2' position is connected by
(CH.sub.2).sub.n, wherein n=1-4, to the 4' carbon of the same
ribose sugar, preferably n is 1 (LNA) or 2 (ENA); inter-sugar
modifications, such as phosphorothioates.
After addition of the displacing strand, the sample can be
incubated for a sufficient period of time to allow the displacing
strand to hybridize with its complementary strand of the nucleic
acid linker. For example, the incubation time can be at least about
30 seconds, at least about 1 minute, at least about 5 minutes, at
least about 10 minutes, at least about 15 minutes, at least about
30 minutes, at least about 1 hour, at least about 2 hours, at least
about 3 hours, at least about 4 hours, at least about 6 hours, at
least about 8 hours, at least about 10 hours, at least about 12
hours, at least about 24 hours, at least about 48 hours or longer.
One of skill in the art can adjust the contact time
accordingly.
In other embodiments, label removal or signal erasure can include
treatment with a molecule that cleaves nucleic acids. For example,
label removal can include treatment with a nuclease that cleaves
double-stranded sequences. Such nucleases can recognize a specific
sequence, e.g., as effected by a restriction endonuclease, or,
alternatively, can recognize double-stranded sequences in sequence
independent manner, e.g., as effected by RNaseH.
When the nucleic acid linker comprises a single-stranded region in
the duplex region, a single-stranded nuclease can be used to cleave
the single-stranded region. Cleavage at an internal position of one
strand can reduce the binding affinity of the two strands of the
linker for each other thus "melting" the linker and the affinity
molecule and the label molecules can dissociate from each
other.
As is well known in the art, methods for staining of biomarkers or
analytes in sample can comprise one or more washing or blocking
steps. For example, a sample can be subjected to one or more
blocking steps before contacting the sample with the affinity
molecule to reduce or inhibit non-specific binding of the affinity
molecule to the sample. One or more blocking steps can also be used
before contacting the sample with the DNA-conjugated label moiety
to reduce or inhibit non-specific binding of the label moiety to
undesired affinity molecule or the sample. One or more washing
steps can be performed after a contacting step to wash away any
leftover reagents from the contacting step.
Embodiments of the aspects disclosed herein can be also be
described by any of the following numbered paragraphs: 1. 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 and
the second nucleic acid strand is linked to the nanoparticle, 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 nanoparticle
under conditions that do not permit such hybridization. 2. The
composition of paragraph 1, wherein the first and second nucleic
acid strands hybridize to form a double-stranded region of about 6
base-pairs to about 30 basepairs. 3. The composition of paragraph
2, wherein the first and second nucleic acid strands hybridize to
form a double-stranded region of about 12 to about 16 base-pairs.
4. The composition of any of paragraphs 1-3, wherein the first and
second nucleic acid strands hybridize to form a double-stranded
region having a melting temperature about 40.degree. C. or above.
5. The composition of any of paragraphs 1-4, wherein the first and
second nucleic acid strands hybridize to form a double-stranded
region having a 3' or 5' single-stranded overhang of about 6 to
about 20 nucleotides. 6. The composition of any of paragraphs 1-5,
wherein the affinity molecule and the label moiety are in a 1:1
(affinity molecule: label moiety) ratio. 7. The composition of any
of paragraphs 1-6, wherein (i) the affinity molecule is conjugated
to 3' terminus of the first nucleic acid strand and the label
moiety is conjugated to 3' terminus of the second nucleic acid
strand; or (ii) the affinity molecule is conjugated to 5' terminus
of the first nucleic acid strand and the label moiety is conjugated
to 5' terminus of the second nucleic acid strand. 8. The
composition of any of paragraphs 1-7, wherein at least one of the
first or the second strands of nucleic acid comprises a
modification selected from the group consisting of nucleobase
modifications, sugar modifications, inter-sugar linkage
modifications, backbone modifications, and any combinations
thereof. 9. The composition of any of paragraphs 1-8, wherein the
affinity molecule is an antibody or antigen-binding portion
thereof. 10. The composition of any of paragraphs 1-9, wherein the
affinity molecule is covalently linked to the first nucleic acid
strand through a linker or the affinity molecule is non-covalently
linked to the first nucleic acid strand through an adaptor
molecule. 11. The composition of paragraph 10, wherein the linker
is a bond,
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,
bisaryl hydrazone bond, an amide bond, tow amide bonds on a spacer
for cross-linking two --NH2 groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, or
any combination thereof. 12. The composition of paragraph 10,
wherein the adaptor molecule is selected from the group consisting
of protein A, protein G, antibody, antibody fragment, antigen,
receptor ligand, receptor, ligand binding fragment of a receptor,
member of a coupling pair, aptamer, biotin-streptavidin pair, and
combinations thereof. 13. The composition of any of paragraphs
1-12, wherein the label moiety is covalently linked to the second
nucleic acid strand through a linker or the label moiety is
non-covalently linked to the second nucleic acid via a coupling
pair. 14. The composition of any of paragraphs 1-13, wherein the
label moiety is selected from the group consisting of a luminescent
nanoparticle, fluorescent molecule, chemiluminescent moiety,
bioluminescent moiety, luminescent molecule, radioisotope,
chromophore, magnetic nanoparticles, plasmonic nanoparticles,
upconverting nanoparticles, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and any
combination thereof. 15. The composition paragraph 14, wherein the
luminescent nanoparticle is an inorganic semiconductor nanoparticle
chosen from group consisting of a Group II, Group III, Group IV,
Group V, and Group VI semiconductor nanoparticles. 16. The
composition of paragraph 14 or 15, wherein the luminescent
nanoparticle is selected from the group consisting of cadmium
selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe),
cadmium zinc sulfide (CdZnS), cadmium telluride silicone (CdTeSi),
cadmium mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc
sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide
(PbSe), gallium arsenide (GaAs), indium phosphide (InP), indium
arsenide (InAs), silicon (Si), Ge, SiGe, and a combination thereof.
17. The composition of any of paragraphs 14-16, wherein the
luminescent nanoparticle is core type or core-shell type. 18. The
composition any of paragraphs 14-17, wherein the luminescent
nanoparticle comprises a core and a shell forms a colloidal
particle. 19. The composition any of paragraphs 14-17, wherein the
luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS. 20. The composition any of paragraphs 14-19, wherein
the luminescent nanoparticle is about 1 nm to about 100 nm in size.
21. The composition any of paragraphs 14-21, wherein the
luminescent nanoparticle comprises a polymer coating layer. 22. The
composition of paragraph 21, wherein the polymer is polyethylene
glycol. 23. The composition any of paragraphs 14-22, wherein the
luminescent nanoparticle is a colloidal water-soluble nanoparticle
comprising a stable non-fouling coating such that non-specific
binding of the nanoparticle to a cell or a tissue sample is reduced
relative to a nanoparticle lacking a non-fouling coating. 24. A
composition comprising an affinity molecule reversibly conjugated
to a 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, wherein 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
such that non-specific binding of the nanoparticle to a cell or a
tissue sample is reduced relative to a nanoparticle lacking a
non-fouling coating. 25. The composition of paragraph 24, wherein
the affinity molecule is an antibody or antigen-binding portion
thereof. 26. The composition any of paragraphs 24-25, wherein the
adaptor molecule is selected from the group consisting of protein
A, protein G, antibody, antibody fragment, antigen, receptor
ligand, receptor, ligand binding fragment of a receptor, member of
a coupling pair, aptamer, biotin-streptavidin pair, and
combinations thereof. 27. The composition any of paragraphs 24-26,
wherein the luminescent nanoparticle is an inorganic semiconductor
nanoparticle chosen from group consisting of a Group II, Group III,
Group IV, Group V, and Group VI semiconductor nanoparticles. 28.
The composition any of paragraphs 24-27, wherein the luminescent
nanoparticle is selected from the group consisting of cadmium
selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe),
cadmium zinc sulfide (CdZnS), cadmium telluride silicone (CdTeSi),
cadmium mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc
sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide
(PbSe), gallium arsenide (GaAs), indium phosphide (InP), indium
arsenide (InAs), silicon (Si), Ge, SiGe, and a combination thereof.
29. The composition any of paragraphs 24-28, wherein the
luminescent nanoparticle is core type or core-shell type. 30. The
composition any of paragraphs 24-29, wherein the luminescent
nanoparticle comprises a core and a shell forms a colloidal
particle. 31. The composition any of paragraphs 24-30, wherein the
luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS. 32. The composition any of paragraphs 24-31, wherein
the luminescent nanoparticle is about 1 nm to about 100 nm in size.
33. The composition any of paragraphs 24-33, wherein the
luminescent nanoparticle comprises a polymer coating layer. 34. The
composition any of paragraphs 24-33, wherein the polymer is
polyethylene glycol. 35. A composition comprising a plurality of
different affinity molecules, wherein: (i) each member of the
plurality binds a different target, wherein each different affinity
molecule is conjugated via different hybridized first and second
nucleic acid strands to a different label moiety, wherein
detectable properties of the different label moieties are
distinguishable; or (ii) each member of the plurality binding a
different target, wherein each different affinity molecule is
conjugated to a different first single strand nucleic acid molecule
that is specifically hybridized to a second single strand nucleic
acid molecule, wherein the second single strand nucleic acid
molecule is conjugated to a label moiety, such that each different
affinity molecule is conjugated via different hybridized first and
second nucleic acid strands to a different label moiety, wherein
detectable properties of the different label moieties are
distinguishable. 36. The composition of paragraph 35, wherein the
first and second nucleic acid strands hybridize to form a
double-stranded region of about 6 base-pairs to about 30
base-pairs. 37. The composition of any of paragraphs 35-36, wherein
the first and second nucleic acid strands hybridize to form a
double-stranded region of about 12 to 16 base-pairs. 38. The
composition of any of paragraphs 35-37, wherein the first and
second nucleic acid strands hybridize to form a double-stranded
region having a melting temperature about 40.degree. C. or above.
39. The composition of any of paragraphs 35-38, wherein the first
and second nucleic acid strands hybridize to form a double-stranded
region having a 3' or 5' single-stranded overhang of about 6 to
about 20 nucleotides. 40. The composition of any of paragraphs
35-39, wherein the affinity molecule and the label moiety are in a
1:1 (affinity molecule: label moiety) ratio. 41. The composition of
any of paragraphs 35-40, wherein (i) the affinity molecule is
conjugated to 3' terminus of the first nucleic acid strand and the
label moiety is conjugated to 3' terminus of the second nucleic
acid strand; or (ii) the affinity molecule is conjugated to 5'
terminus of the first nucleic acid strand and the label moiety is
conjugated to 5' terminus of the second nucleic acid strand. 42.
The composition of any of paragraphs 35-41, wherein at least one of
the first or the second strands of nucleic acid comprises a
modification selected from the group consisting of nucleobase
modifications, sugar modifications, inter-sugar linkage
modifications, backbone modifications, and any combinations
thereof. 43. The composition of any of paragraphs 35-42, wherein
the affinity molecule is an antibody or antigen-binding portion
thereof. 44. The composition of any of paragraphs 35-43, wherein
the affinity molecule is covalently linked to the first nucleic
acid strand through a linker or the affinity molecule is
non-covalently linked to the first nucleic acid strand through an
adaptor molecule. 45. The composition of paragraph 44, wherein the
linker is a bond,
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, tow amide bonds on a spacer
for cross-linking two --NH2 groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, or
any combination thereof. 46. The composition of paragraph 44,
wherein the adaptor molecule is selected from the group consisting
of protein A, protein G, antibody, antibody fragment, antigen,
receptor ligand, receptor, ligand binding fragment of a receptor,
member of a coupling pair, aptamer, biotin-streptavidin pair, and
combinations thereof. 47. The composition of any of paragraphs
35-46, wherein the label moiety is covalently linked to the second
nucleic acid strand through a linker or wherein the label moiety is
non-covalently linked to the second nucleic acid via a coupling
pair. 48. The composition of any of paragraphs 35-47, wherein the
label moiety is selected from the group consisting of a luminescent
nanoparticle, fluorescent molecule, chemiluminescent moiety,
bioluminescent moiety, luminescent molecule, radioisotope,
chromophore, magnetic nanoparticles, plasmonic nanoparticles,
upconverting nanoparticles, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and any
combination thereof. 49. The composition of paragraph 48, wherein
the luminescent nanoparticle is an inorganic semiconductor
nanoparticle chosen from group consisting of a Group II, Group III,
Group IV, Group V, and Group VI semiconductor nanoparticles. 50.
The composition of paragraph 48 or 49, wherein the luminescent
nanoparticle is selected from the group consisting of cadmium
selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe),
cadmium zinc sulfide (CdZnS), cadmium telluride silicone (CdTeSi),
cadmium mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc
sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide
(PbSe), gallium arsenide (GaAs), indium phosphide (InP), indium
arsenide (InAs), silicon (Si), Ge, SiGe, and a combination thereof.
51. The composition of any of paragraphs 48-50, wherein the
luminescent nanoparticle is core type or core-shell type. 52. The
composition of any of paragraphs 48-51, wherein the luminescent
nanoparticle comprises a core and a shell forms a colloidal
particle. 53. The composition of any of paragraphs 48-52, wherein
the luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS. 54. The composition of any of paragraphs 48-53,
wherein the luminescent nanoparticle is about 1 nm to about 100 nm
in size. 55. The composition of any of paragraphs 48-54, wherein
the luminescent nanoparticle comprises a polymer coating layer. 56.
The composition of paragraph 55, wherein the polymer is
polyethylene glycol. 57. The composition of any of paragraphs
48-56, wherein emission spectra of the different luminescent
nanoparticles are distinguishable. 58. The composition of any of
paragraphs 48-57, wherein the luminescent nanoparticle is a
colloidal water-soluble nanoparticle comprising a stable
non-fouling coating such that non-specific binding of the
nanoparticle to a cell or a tissue sample is reduced relative to a
nanoparticle lacking a non-fouling coating. 59. A composition
comprising a plurality of different affinity molecules, each member
of the plurality binding a different target, wherein each different
affinity molecule is reversibly conjugated to a 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, wherein 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 such that non-specific
binding of the nanoparticle to a cell or a tissue sample is reduced
relative to a nanoparticle lacking a non-fouling coating, and
wherein emission spectra of the different luminescent nanoparticles
are distinguishable. 60. The composition of paragraph 59, wherein
the affinity molecule is an antibody or antigen-binding portion
thereof. 61. The composition of any of paragraphs 59 or 60, the
adaptor molecule is selected from the group consisting of protein
A, protein G, antibody, antibody fragment, antigen, receptor
ligand, receptor, ligand binding fragment of a receptor, member of
a coupling pair, aptamer, biotin-streptavidin pair, and
combinations thereof. 62. The composition of any of paragraphs
59-61, wherein the luminescent nanoparticle is an inorganic
semiconductor nanoparticle chosen from group consisting of a Group
II, Group III, Group IV, Group V, and Group VI semiconductor
nanoparticles. 63. The composition of paragraph 59, wherein the
luminescent nanoparticle is selected from the group consisting of
cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride
(CdTe), cadmium zinc sulfide (CdZnS), cadmium telluride silicone
(CdTeSi), cadmium mercury telluride (CdHgTe), zinc selenide (ZnSe),
zinc sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead
selenide (PbSe), gallium arsenide (GaAs), indium phosphide (InP),
indium arsenide (InAs), silicon (Si), Ge, SiGe, and a combination
thereof. 64. The composition any of paragraphs 59-63, wherein the
luminescent nanoparticle is core type or core-shell type. 65. The
composition any of paragraphs 59-64, wherein the luminescent
nanoparticle comprises a core and a shell forms a colloidal
particle. 66. The composition any of paragraphs 59-65, wherein the
luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS. 67. The composition any of paragraphs 59-66, wherein
the luminescent nanoparticle is about 1 nm to about 100 nm in size.
68. The composition any of paragraphs 59-67, wherein the
luminescent nanoparticle comprises a
polymer coating layer. 69. The composition of paragraph 68, wherein
the polymer is polyethylene glycol. 70. A composition comprising a
solid support comprising a sample for analysis for the presence of
analytes, and a plurality of different affinity molecules, each
member of the plurality of different affinity molecules conjugated
to a first single-strand nucleic acid, wherein each member of the
plurality of different affinity molecules is bound to a different
analyte in the sample, and wherein each first single-strand nucleic
acid has a different nucleotide sequence. 71. The composition of
paragraph 70, wherein the composition further comprises a plurality
of different second single strand nucleic acid molecules, each
conjugated to a different label moiety, wherein each different
second single strand nucleic acid molecule specifically hybridizes
to a different first single strand nucleic acid molecule conjugated
to a member of the plurality of different affinity molecules, such
that at least a subset of the plurality of different affinity
molecules bound to the analytes in the cell or tissue sample is
specifically associated with a plurality of different label moiety,
wherein detectable properties of the different label moieties are
distinguishable. 72. The composition of paragraph 71, wherein the
first and second nucleic acid strands hybridize to form a
double-stranded region of about 6 base-pairs to about 30
base-pairs. 73. The composition of paragraph 72, wherein the first
and second nucleic acid strands hybridize to form a double-stranded
region of about 12 to about 16 base-pairs. 74. The composition of
any of paragraphs 71-73, wherein the first and second nucleic acid
strands hybridize to form a double-stranded region having a melting
temperature about 40.degree. C. or above. 75. The composition of
any of paragraphs 71-74, wherein the first and second nucleic acid
strands hybridize to form a double-stranded region having a 3' or
5' single-stranded overhang of about 6 to about 20 nucleotides. 76.
The composition of any of paragraphs 71-75, wherein the affinity
molecule and the label moiety are in a 1:1 (affinity molecule:
label moiety) ratio. 77. The composition of any of paragraphs
71-76, wherein (i) the affinity molecule is conjugated to 3'
terminus of the first nucleic acid strand and the label moiety is
conjugated to 3' terminus of the second nucleic acid strand; or
(ii) the affinity molecule is conjugated to 5' terminus of the
first nucleic acid strand and the label moiety is conjugated to 5'
terminus of the second nucleic acid strand. 78. The composition of
any of paragraphs 71-77, wherein at least one of the first or the
second strands of nucleic acid comprises a modification selected
from the group consisting of nucleobase modifications, sugar
modifications, inter-sugar linkage modifications, backbone
modifications, and any combinations thereof. 79. The composition of
any of paragraphs 70-78, wherein the affinity molecule is an
antibody or antigen-binding portion thereof. 80. The composition of
any of paragraphs 70-79, wherein the affinity molecule is
covalently linked to the first nucleic acid strand through a linker
or the affinity molecule is non-covalently linked to the first
nucleic acid strand through an adaptor molecule. 81. The
composition of paragraph 80, wherein the linker is a bond,
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, tow amide bonds on a spacer
for cross-linking two --NH2 groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, or
any combination thereof. 82. The composition of any of paragraphs
70-81, wherein the adaptor molecule is selected from the group
consisting of protein A, protein G, antibody, antibody fragment,
antigen, receptor ligand, receptor, ligand binding fragment of a
receptor, member of a coupling pair, aptamer, biotin-streptavidin
pair, and combinations thereof. 83. The composition of any of
paragraphs 71-82, wherein the label moiety is covalently linked to
the second nucleic acid strand through a linker or the the label
moiety is non-covalently linked to the second nucleic acid via a
coupling pair. 84. The composition of any of paragraphs 71-83,
wherein the label moiety is selected from the group consisting of a
luminescent nanoparticle, fluorescent molecule, chemiluminescent
moiety, bioluminescent moiety, luminescent molecule, radioisotope,
chromophore, magnetic nanoparticles, plasmonic nanoparticles,
upconverting nanoparticles, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and any
combination thereof. 85. The composition of paragraph 84, wherein
the luminescent nanoparticle is an inorganic semiconductor
nanoparticle chosen from group consisting of a Group II, Group III,
Group IV, Group V, and Group VI semiconductor nanoparticles. 86.
The composition of paragraph 84 or 85, wherein the luminescent
nanoparticle is selected from the group consisting of cadmium
selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe),
cadmium zinc sulfide (CdZnS), cadmium telluride silicone (CdTeSi),
cadmium mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc
sulfide (ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide
(PbSe), gallium arsenide (GaAs), indium phosphide (InP), indium
arsenide (InAs), silicon (Si), Ge, SiGe, and a combination thereof.
87. The composition of any of paragraphs 84-86, wherein the
luminescent nanoparticle is core type or core-shell type. 88. The
composition of any of paragraphs 84-87, wherein the luminescent
nanoparticle comprises a core and a shell forms a colloidal
particle. 89. The composition of any of paragraphs 84-88, wherein
the luminescent nanoparticle core comprises CdSe and the shell
comprises ZnS. 90. The composition of any of paragraphs 84-89,
wherein the luminescent nanoparticle is about 1 nm to about 100 nm
in size. 91. The composition of any of paragraphs 84-90, wherein
the luminescent nanoparticle comprises a polymer coating layer. 92.
The composition of paragraph 91, wherein the polymer is
polyethylene glycol. 93. The composition of any of paragraphs
84-92, wherein the luminescent nanoparticle is a colloidal
water-soluble nano-particle comprising a stable non-fouling coating
such that non-specific binding of the nanoparticle to a cell or a
tissue sample is reduced relative to a nanoparticle lacking a
non-fouling coating. 94. A kit comprising a composition of
paragraph 1, 24, or 35. 95. A method of analyzing a sample for a
plurality of analytes, the method comprising, in order: (b)
contacting the sample with a 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 wherein each different affinity molecule is
conjugated to a different first single strand nucleic acid, such
that members of the plurality of different affinity molecules
become bound to members of the plurality of analytes present in the
sample; (i) contacting the sample, under conditions that permit
specific nucleic acid hybridization, with a first set of different
second single strand nucleic acid molecules, each conjugated to a
different label moiety, wherein each different second single strand
nucleic acid molecule specifically hybridizes to a different first
single strand nucleic acid molecule conjugated to a member of the
plurality of different affinity molecules, such that at least a
subset of the plurality of different affinity molecules bound to
members of the plurality of analytes in the sample becomes
specifically associated with a plurality of different label
moieties, wherein detectable properties of the different label
moieties are distinguishable; and (j) detecting signal from label
moieties associated with affinity molecules bound to the sample,
thereby detecting the presence or amount of at least a subset of
the plurality of analytes. 96. The method of paragraph 95, further
comprising the steps of: (k) quenching the signal from the label
moieties conjugated to the first set of second single strand
nucleic acid molecules; (l) contacting the sample, under conditions
that permit specific nucleic acid hybridization, with a second set
of different second single strand nucleic acid molecules, each
conjugated to a different label, wherein each different second
single strand nucleic acid molecule specifically hybridizes to a
different first single strand nucleic acid molecule conjugated to a
member of the plurality of different affinity molecules, such that
a different subset of the plurality of different affinity molecules
bound to members of the plurality of analytes in the sample becomes
specifically associated with a different plurality of different
label moieties relative to those detected in step (c); (m)
detecting signal from label moieties associated with affinity
molecules bound to the sample, thereby detecting the presence or
amount of the different subset of the plurality of analytes; and
(n) optionally repeating steps (d)-(f) with a further set of second
single strand nucleic acid molecules. 97. The method of paragraph
95 or 96, wherein said detecting comprises imaging spectral
emissions. 98. The method of paragraph 97, wherein said imaging is
hyperspectral imaging or multispectral imaging. 99. The method of
any of paragraphs 95-98, wherein said detecting comprises spectral
unmixing. 100. The method of any of paragraphs 96-99, wherein said
quenching comprises removing the label moieties from the sample or
quenching fluorescent signal from the label moieties. 101. The
method of any of paragraphs 96-100, wherein said quenching is
chemical or physical. 102. The method of any of paragraphs 96-101,
wherein said quenching is by elution, denaturation, washing,
displacement, cleavage, photo-bleaching, heating, quenching with a
fluorophore acceptor, or a combination thereof. 103. The method of
any of paragraphs 96-102, wherein said quenching is by washing with
a low pH buffer. 104. The method of any of paragraphs 96-103,
wherein said quenching is by nucleic acid strand displacement or
nucleic acid cleavage. 105. The method of paragraph 104, wherein
said strand displacement is by adding an oligonucleotide comprising
a nucleic acid sequence complementary to the first or second
strands of nucleic acid. 106. The method of any of paragraphs
95-105, further comprising one or more wash steps. 107. The method
of any of paragraphs 95-107, further comprising one or more
blocking steps. 108. The method of any of paragraphs 95-107,
wherein the first and second nucleic acid strands hybridize to form
a double-stranded region of about 6 base-pairs to about 30
base-pairs. 109. The method of any of paragraphs 95-108, wherein
the first and second nucleic acid strands hybridize to form a
double-stranded region of about 12 to about 16 base-pairs. 110. The
method of any of paragraphs 95-109, wherein the first and second
nucleic acid strands hybridize to form a double-stranded region
having a melting temperature about 40.degree. C. or above. 111. The
method of any of paragraphs 95-110, wherein the first and second
nucleic acid strands hybridize to form a double-stranded region
having a 3' or 5' single-stranded overhang of about 6 to about 20
nucleotides. 112. The method of any of paragraphs 95-111, wherein
the affinity molecule and the label moiety are in a 1:1 (affinity
molecule: label moiety) ratio. 113. The method of any of paragraphs
95-112, wherein (i) the affinity molecule is conjugated to 3'
terminus of the first nucleic acid strand and the label moiety is
conjugated to 3' terminus of the second nucleic acid strand; or
(ii) the affinity molecule is conjugated to 5' terminus of the
first nucleic acid strand and the label moiety is conjugated to 5'
terminus of the second nucleic acid strand. 114. The method of any
of paragraphs 95-113, wherein at least one of the first or the
second strands of nucleic acid comprises a modification selected
from the group consisting of nucleobase modifications, sugar
modifications, inter-sugar linkage modifications, backbone
modifications, and any combinations thereof. 115. The method of any
of paragraphs 95-114, wherein the affinity molecule is an antibody
or antigen-binding portion thereof. 116. The method of any of
paragraphs 95-115, wherein the affinity molecule is covalently
linked to the first nucleic acid strand through a linker or the
affinity molecule is non-covalently linked to the first nucleic
acid strand through an adaptor molecule. 117. The method of
paragraph 116, wherein the linker is a bond,
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, tow amide bonds on a spacer
for cross-linking two --NH2 groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, or
any combination thereof. 118. The method of any of paragraphs
95-117, wherein the adaptor molecule is selected from the group
consisting of protein A, protein G, antibody, antibody fragment,
antigen, receptor ligand, receptor, ligand binding fragment of a
receptor, member of a coupling pair, aptamer, biotin-streptavidin
pair, and combinations thereof. 119. The method of any of
paragraphs 95-118, wherein the label moiety is covalently linked to
the second nucleic acid strand through a linker or the label moiety
is non-covalently linked to the second nucleic acid via a coupling
pair. 120. The method of any of paragraphs 95-119, wherein the
label moiety is selected from the group consisting of a luminescent
nanoparticle, fluorescent molecule, chemiluminescent moiety,
bioluminescent moiety, luminescent molecule, radioisotope,
chromophore, magnetic nanoparticles, plasmonic nanoparticles,
upconverting nanoparticles, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and any
combination thereof. 121. The method of paragraph 120, wherein the
luminescent nanoparticle is an inorganic semiconductor nanoparticle
chosen from group consisting of a Group II, Group III, Group IV,
Group V, and Group VI semiconductor nanoparticles. 122. The method
of paragraph 120 or 121, wherein the luminescent nanoparticle is
selected from the group consisting of cadmium selenide (CdSe),
cadmium sulfide (CdS), cadmium telluride (CdTe), cadmium zinc
sulfide (CdZnS), cadmium telluride silicone (CdTeSi), cadmium
mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc sulfide
(ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide (PbSe),
gallium arsenide (GaAs), indium phosphide (InP), indium arsenide
(InAs), silicon (Si), Ge, SiGe, and a combination thereof. 123. The
method of any of paragraphs 120-122, wherein the luminescent
nanoparticle is core type or core-shell type. 124. The method of
any of paragraphs 120-123, wherein the luminescent nanoparticle
comprises a core and a shell forms a colloidal particle. 125. The
method of any of paragraphs 120-124, wherein the luminescent
nanoparticle core comprises CdSe and the shell comprises ZnS. 126.
The method of any of paragraphs 120-125, wherein the luminescent
nanoparticle is about 1 nm to about 100 nm in size. 127. The method
of any of paragraphs 120-126, wherein the luminescent nanoparticle
comprises a polymer coating layer. 128. The method of 120-127,
wherein the polymer is polyethylene glycol. 129. The method of any
of paragraphs 120-128, wherein the luminescent nanoparticle is a
colloidal water-soluble nanoparticle comprising a stable
non-fouling coating such that non-specific binding of the
nanoparticle to a cell or a tissue sample is reduced relative to a
nanoparticle lacking a non-fouling coating. 130. The method of any
of paragraphs 120-129, wherein emission spectra of the different
luminescent nanoparticles re distinguishable. 131. A method of
analyzing a sample for a plurality of analytes, the method
comprising, in order: (g) 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, wherein each
different affinity molecule is conjugated to a different first
single strand nucleic acid molecule that is specifically hybridized
to a second single strand nucleic acid molecule, wherein the second
single strand nucleic acid molecule is conjugated to a label
moiety, such that each different affinity molecule is conjugated
via different hybridized first and second nucleic acid strands to a
different label moiety and members of the plurality of different
affinity molecules become bound to members of the plurality of
analytes present in the sample, wherein detectable properties of
the label moieties are distinguishable; and (h) detecting signal
from label moieties associated with first plurality of affinity
molecules bound to the sample, thereby detecting the presence or
amount of the plurality of analytes. 132. The method of paragraph
131, further comprising the steps of: (i) quenching the signal from
the label molecules conjugated to the first set of second single
strand nucleic acid molecules; (j)
contacting the sample with a second plurality of different affinity
molecules under conditions that permit specific analyte binding by
the different affinity molecules; (k) detecting signal from label
moieties associated with second plurality of affinity molecules
bound to the sample, thereby detecting the presence or amount of at
least a subset of the plurality of analytes; and (l) optionally
repeating steps (c)-(e) with a further second set of the affinity
molecules. 133. The method of paragraph 131 or 132, wherein said
detecting comprises imaging spectral emissions. 134. The method of
paragraph 133, wherein said imaging is hyperspectral imaging or
multispectral imaging. 135. The method of any of paragraphs
131-134, wherein said detecting comprises spectral unmixing. 136.
The method of any of paragraphs 132-135, wherein said quenching
comprises removing the label moieties from the sample or quenching
fluorescent signal from the label moieties. 137. The method of any
of paragraphs 132-136, wherein said quenching is chemical or
physical. 138. The method of any of paragraphs 132-137, wherein
said quenching is by elution, denaturation, washing, displacement,
cleavage, photo-bleaching, heating, quenching with a fluorophore
acceptor, or a combination thereof. 139. The method of any of
paragraphs 132-138, wherein said quenching is by washing with a low
pH buffer. 140. The method of any of paragraphs 132-138, wherein
said quenching is by nucleic acid strand displacement or nucleic
acid cleavage. 141. The method of paragraph 140, wherein said
strand displacement is by adding an oligonucleotide comprising a
nucleic acid sequence complementary to the first or second strands
of nucleic acid. 142. The method of any of paragraphs 131-141,
further comprising one or more wash steps. 143. The method of any
of paragraphs 131-142, further comprising one or more blocking
steps. 144. The method of any of paragraphs 131-143, wherein the
first and second nucleic acid strands hybridize to form a
double-stranded region of about 6 base-pairs to about 30
base-pairs. 145. The method any of paragraphs 131-144, wherein the
first and second nucleic acid strands hybridize to form a
double-stranded region of about 12 to about 16 base-pairs. 146. The
method any of paragraphs 131-145, wherein the first and second
nucleic acid strands hybridize to form a double-stranded region
having a melting temperature about 40.degree. C. or above. 147. The
method any of paragraphs 131-146, wherein the first and second
nucleic acid strands hybridize to form a double-stranded region
having a 3' or 5' single-stranded overhang of about 6 to about 20
nucleotides. 148. The method any of paragraphs 131-147, wherein the
affinity molecule and the label moiety are in a 1:1 (affinity
molecule: label moiety) ratio. 149. The method any of paragraphs
131-148, wherein (i) the affinity molecule is conjugated to 3'
terminus of the first nucleic acid strand and the label moiety is
conjugated to 3' terminus of the second nucleic acid strand; or
(ii) the affinity molecule is conjugated to 5' terminus of the
first nucleic acid strand and the label moiety is conjugated to 5'
terminus of the second nucleic acid strand. 150. The method any of
paragraphs 131-149, wherein at least one of the first or the second
strands of nucleic acid comprises a modification selected from the
group consisting of nucleobase modifications, sugar modifications,
inter-sugar linkage modifications, backbone modifications, and any
combinations thereof. 151. The method any of paragraphs 131-150,
wherein the affinity molecule is an antibody or antigen-binding
portion thereof. 152. The method of any of paragraphs 131-151,
wherein the affinity molecule is covalently linked to the first
nucleic acid strand through a linker or the affinity molecule is
non-covalently linked to the first nucleic acid strand through an
adaptor molecule. 153. The method of paragraph 152, wherein the
linker is a bond,
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, tow amide bonds on a spacer
for cross-linking two --NH2 groups, triazole bond (from "click"
reaction), a phosphodiester linkage, a phsophothioate linkage, or
any combination thereof. 154. The method of any of paragraphs
131-153, wherein the adaptor molecule is selected from the group
consisting of protein A, protein G, antibody, antibody fragment,
antigen, receptor ligand, receptor, ligand binding fragment of a
receptor, member of a coupling pair, aptamer, biotin-streptavidin
pair, and combinations thereof. 155. The method of any of
paragraphs 131-154, wherein the label moiety is covalently linked
to the second nucleic acid strand through a linker or the label
moiety is non-covalently linked to the second nucleic acid via a
coupling pair. 156. The method of any of paragraphs 131-155,
wherein the label moiety is selected from the group consisting of a
luminescent nanoparticle, fluorescent molecule, chemiluminescent
moiety, bioluminescent moiety, luminescent molecule, radioisotope,
chromophore, magnetic nanoparticles, plasmonic nanoparticles,
upconverting nanoparticles, nanoparticles comprising fluorescent
molecules, nanoparticles comprising fluorophores, and any
combination thereof. 157. The method of paragraph 156, wherein the
luminescent nanoparticle is an inorganic semiconductor nanoparticle
chosen from group consisting of a Group II, Group III, Group IV,
Group V, and Group VI semiconductor nanoparticles. 158. The method
of any of paragraphs 156-157, wherein the luminescent nanoparticle
is selected from the group consisting of cadmium selenide (CdSe),
cadmium sulfide (CdS), cadmium telluride (CdTe), cadmium zinc
sulfide (CdZnS), cadmium telluride silicone (CdTeSi), cadmium
mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc sulfide
(ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide (PbSe),
gallium arsenide (GaAs), indium phosphide (InP), indium arsenide
(InAs), silicon (Si), Ge, SiGe, and a combination thereof. 159. The
method of any of paragraphs 156-158, wherein the luminescent
nanoparticle is core type or core-shell type. 160. The method of
any of paragraphs 156-159, wherein the luminescent nanoparticle
comprises a core and a shell forms a colloidal particle. 161. The
method of any of paragraphs 156-160, wherein the luminescent
nanoparticle core comprises CdSe and the shell comprises ZnS. 162.
The method of any of paragraphs 156-161, wherein the luminescent
nanoparticle is about 1 nm to about 100 nm in size. 163. The method
of any of paragraphs 156-162, wherein the luminescent nanoparticle
comprises a polymer coating layer. 164. The method of paragraph
163, wherein the polymer is polyethylene glycol. 165. The method of
any of paragraphs 156-164, wherein the luminescent nanoparticle is
a colloidal water-soluble nanoparticle comprising a stable
non-fouling coating such that non-specific binding of the
nanoparticle to a cell or a tissue sample is reduced relative to a
nanoparticle lacking a non-fouling coating. 166. The method of any
of paragraphs 156-165, wherein emission spectra of the different
luminescent nanoparticles are distinguishable. 167. 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 such that
non-specific binding of the nanoparticle to a cell or a tissue
sample is reduced relative to a nanoparticle lacking a non-fouling
coating, and detectable properties of the luminescent nanoparticles
are distinguishable; 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. 168. The method of paragraph
167, further comprising the steps of: (c) quenching the signal from
the luminescent nanoparticles conjugated to the first set of
different second single strand nucleic acid molecules; (d)
contacting the sample with a second set of the plurality of
different affinity molecules under conditions that permit specific
analyte binding by the different affinity molecules; (e) detecting
signal from luminescent molecules associated with second set of
affinity molecules bound to the sample, thereby detecting the
presence or amount of at least a subset of the plurality of
analytes; and (f) optionally repeating steps (c)-(e) with a further
second set of the affinity molecules. 169. The method of paragraph
167 or 168, wherein said detecting comprises imaging spectral
emissions. 170. The method of paragraph 169, wherein said imaging
is hyperspectral imaging or multispectral imaging. 171. The method
of any of paragraphs 167-170, wherein said detecting comprises
spectral unmixing. 172. The method of any of paragraphs 167-171,
wherein said quenching comprises removing the luminescent
nanoparticles from the sample or quenching fluorescent signal from
the label moieties. 173. The method of any of paragraphs 167-172,
wherein said quenching is chemical or physical. 174. The method of
any of paragraphs 167-173, wherein said quenching is by elution,
denaturation, washing, displacement, cleavage, photo-bleaching,
heating, quenching with a fluorophore acceptor, or a combination
thereof. 175. The method of any of paragraphs 167-174, wherein said
quenching is by washing with a low pH buffer. 176. The method of
any of paragraphs 167-175, further comprising one or more wash
steps. 177. The method of any of paragraphs 167-176, further
comprising one or more blocking steps. 178. The method of any of
paragraphs 167-177, wherein the affinity molecule is an antibody or
antigen-binding portion thereof. 179. The method of any of
paragraphs 167-178, wherein the adaptor molecule is selected from
the group consisting of protein A, protein G, antibody, antibody
fragment, antigen, receptor ligand, receptor, ligand binding
fragment of a receptor, member of a coupling pair, aptamer,
biotin-streptavidin pair, and combinations thereof. 180. The method
of any of paragraphs 167-179, wherein the luminescent nanoparticle
is an inorganic semiconductor nanoparticle chosen from group
consisting of a Group II, Group III, Group IV, Group V, and Group
VI semiconductor nanoparticles. 181. The method of any of
paragraphs 167-180, wherein the luminescent nanoparticle is
selected from the group consisting of cadmium selenide (CdSe),
cadmium sulfide (CdS), cadmium telluride (CdTe), cadmium zinc
sulfide (CdZnS), cadmium telluride silicone (CdTeSi), cadmium
mercury telluride (CdHgTe), zinc selenide (ZnSe), zinc sulfide
(ZnS), zinc oxide (ZnO), lead sulfide (PbS), lead selenide (PbSe),
gallium arsenide (GaAs), indium phosphide (InP), indium arsenide
(InAs), silicon (Si), Ge, SiGe, and a combination thereof. 182. The
method of any of paragraphs 167-181, wherein the luminescent
nanoparticle is core type or core-shell type. 183. The method of
any of paragraphs 167-182, wherein the luminescent nanoparticle
comprises a core and a shell forms a colloidal particle. 184. The
method of paragraph 183, wherein the luminescent nanoparticle core
comprises CdSe and the shell comprises ZnS. 185. The method of any
of paragraphs 167-184, wherein the luminescent nanoparticle is
about 1 nm to about 100 nm in size. 186. The method of any of
paragraphs 167-185, wherein the luminescent nanoparticle comprises
a polymer coating layer. 187. The method of paragraph 186, wherein
the polymer is polyethylene glycol. 188. The method of any of
paragraphs 70-93, wherein the solid support an ELISA plate, a
magnetic bead, an agarose bead, a western blot membrane, or a
combination thereof. 189. The method of any of paragraphs 70-93 or
188, wherein the sample is a cell or a tissue sample.
To the extent not already indicated, it will be understood by those
of ordinary skill in the art that any one of the various
embodiments herein described and illustrated can be further
modified to incorporate features shown in any of the other
embodiments disclosed herein.
The following examples illustrate some embodiments and aspects of
the invention. It will be apparent to those skilled in the relevant
art that various modifications, additions, substitutions, and the
like can be performed without altering the spirit or scope of the
invention, and such modifications and variations are encompassed
within the scope of the invention as defined in the claims which
follow. The following examples do not in any way limit the
invention.
EXAMPLES
Example 1
Hybrid IF/FISH Procedure for Molecular Profiling of Cells and
Tissue Specimens
Conventional 2-step staining procedures utilize links with no (e.g.
streptavidin/biotin) or limited (e.g. primary/secondary antibodies)
selectivity, thus prohibiting highly multiplexed biomarker
detection. Therefore, the inventors designed a hybrid IF/FISH
2-step staining procedure featuring highly selective links for
unique assignment of FL probes to corresponding biomarkers. This
functionality can be achieved by encoding each biomarker with a
unique DNA tag via specific recognition by oligonucleotide-labeled
1'Ab. Conversion of biomarker antigenicity information into a DNA
sequence code enables performance of a highly multiplexed FISH-like
staining procedure with complementary FL-oligonucleotide probes
(FIG. 1). With this method design, simultaneous encoding of a large
number of biomarkers is possible, several of which can be labeled
with FL probes in parallel and analyzed by spectral imaging. The
inventor have optimized this method for QD-DNA probes to gain
higher multiplexing potential, while demonstrating applicability of
conventional FISH probes based on organic fluorophores for staining
with lower parallel multiplexing capability. Furthermore, the
utility of cyclic staining is demonstrated herein for expanding the
number of biomarkers analyzed on the same specimen. After staining
and imaging, specimen is de-stained at gentle conditions via DNA
link displacement with longer oligonucleotides (FIG. 1), which
achieves complete removal of FL probes, while exhibiting no
interference with antigen-antibody binding, thus leaving the first
layer of Ab-DNA tags intact. Therefore, de-staining is achieved at
physiological conditions, preserving biomarker antigenicity and
eliminating the need for antigen re-detection on each cycle. After
de-staining, another sub-set of biomarkers can be stained via
detection of corresponding DNA tags. The hybrid IF/FISH staining
system described in this example generally involves: (i) method for
preparation of oligonucleotide-labeled primary antibodies (Ab-DNA);
(ii) preparation of oligonucleotide-functionalized QD probes
(QD-DNA); and (iii) optimization of multiplexed 2-step staining
procedure on formalin-fixed and permeabilized cells. The method
provides substantial flexibility in probe design depending on
particular application needs. For example, the length and structure
of DNA link can be optimized to offer selectivity of bond
formation, sufficient stability, and capacity for quick
displacement with longer oligonucleotide for de-staining purposes.
Labeling of primary antibodies with ssDNA can be achieved using,
among others, the following strategies: (i) covalent conjugation
with reduced IgG via reaction with sulfhydryl group in Fc region;
(ii) covalent conjugation with whole IgG via cross-linking with
primary amines; and (iii) non-covalent self-assembly of intact
whole IgG with DNA-carrying adaptor protein (on the basis of
protein A from Staphylococcus aureus, SpA, or Fc-specific Fab
antibody fragments). Preparation of QD-DNA probes, in turn, can be
done based on 3 different QD platforms: (i) compact zwitterionic
QDs can be used for EDC-mediated conjugation; (ii) larger
PEG-coated QDs can be used for BS3-mediated cross-linking with
amine groups; and (iii) streptavidin-functionalized QDs can be used
for non-covalent capture of biotinylated oligonucleotides. Each
approach has benefits and drawbacks in terms of complexity and
yield of conjugation reaction, control over the final structure of
bioconjugates, and stability of probes; yet every combination of
probes satisfies basic criteria imposed by the hybrid IF/FISH
method, while enabling flexibility in system design for addressing
specific needs of a wide range of applications.
DNA Link Design:
DNA-encoded antibodies prepared with a variety of covalent and
non-covalent procedures have been used for a number of
bio-analytical methods (Bailey, R. C., et al., DNA-encoded antibody
libraries: a unified platform for multiplexed cell sorting and
detection of genes and proteins. Journal of the American Chemical
Society, 2007. 129(7): p. 1959-67 and Lind, K. and M. Kubista,
Development and evaluation of three real-time immuno-PCR
assemblages for quantification of PSA. Journal of Immunological
Methods, 2005. 304(1-2): p. 107-16). At the same time,
QD-oligonucleotide probes have been developed for DNA detection and
FISH applications (Chan, P., et al., Method for multiplex cellular
detection of mRNAs using quantum dot fluorescent in situ
hybridization. Nucleic Acids Res, 2005. 33(18): p. e161; Matsuno,
A., et al., Three-dimensional imaging of the intracellular
localization of growth hormone and prolactin and their mRNA using
nanocrystal (Quantum dot) and confocal laser scanning microscopy
techniques. J Histochem Cytochem, 2005. 53(7): p. 833-8; Tholouli,
E., et al., Imaging of multiple mRNA targets using quantum dot
based in situ hybridization and spectral deconvolution in clinical
biopsies. Biochemical and Biophysical Research Communications,
2006. 348(2): p. 628-36; Cady, N. C., A. D. Strickland, and C. A.
Batt, Optimized linkage and quenching strategies for quantum dot
molecular beacons. Molecular and Cellular Probes, 2007. 21(2): p.
116-2; Gueroui, Z. and A. Libchaber, Single-molecule measurements
of gold-quenched quantum dots. Physical Review Letters, 2004.
93(16): p. 166108; and Lim, S. H., et al., Specific nucleic acid
detection using photophysical properties of quantum dot probes.
Analytical Chemistry, 2010. 82(3): p. 886-91). Among these studies,
the length and structure of DNA link varies significantly, as
different criteria have to be satisfied. However, 12-16 base-pair
overlap appears to be optimal for providing sufficient bond
strength, while minimizing formation of unfavorable secondary
structures or significant cross-hybridization between non-matching
probes. Therefore, to provide a foundation for further
application-specific optimization the inventors designed a panel of
10 unique 16 bp oligonucleotide pairs. Each oligonucleotide was
functionalized with a primary amine group on a short PEG spacer to
enable covalent conjugation to antibodies and QDs. Each probe was
designed to have balanced base content, melting temperature above
45.degree. C., no secondary structures at room temperature, and no
more than 4 bp homodimers or hetero-dimers with non-matching
probes. Those parameters ensured the unique match between
DNA-encoded biomarker and complementary QD without probe cross-talk
even at non-stringent hybridization conditions. For cyclic staining
applications, the link length was shortened to 11 bp and additional
10 bp handle for displacement oligonucleotide was incorporated to
enable specimen de-staining at physiological conditions via DNA
displacement. The 21 bp displacement probe can quickly and
efficiently break the 11 bp bridge between Ab and QD, thus
releasing QDs into solution. HPLC-purified oligonucleotides were
purchased from IDT DNA. All DNA analysis was performed with an IDT
DNA Oligo Analyzer.
Labeling of Primary Antibodies with DNA Tags:
Encoding of biomarkers was achieved via specific recognition and
binding by primary antibodies labeled with DNA tags. Preparation of
a library of DNA-labeled antibodies was done either via covalent
conjugation between Ab and amine-functionalized oligonucleotides or
non-covalent self-assembly with SpA-DNA. One covalent conjugation
approach involved reduction of IgG followed by maleimidemediated
reaction between pre-activated oligonucleotide and sulfhydryl group
in Fc region of antibody (FIG. 2A). In particular, antibody was
incubated with either dithiothreitol (DTT) or 2-mercaptoethylamine
(2-MEA) for 1 hour at ambient conditions and desalted twice with
Pierce protein desalting spin columns. Concurrently,
amine-functionalized oligonucleotides were activated with
sulfo-SMCC for 1 hour and desalted. At least 2 consecutive rounds
of desalting were required to completely eliminate excess
cross-linker. Reduced Ab and activated DNA at 20 times molar excess
were reacted for 5 hours, quenched with mercaptoethanol, and
buffer-exchanged into 1.times. PBS. One benefit of this approach is
the controlled stoichiometry and structure of Ab-DNA bioconjugate.
As reaction is limited to 1-2 sites on Fc region, preparation of
antibodies with intact Fab antigen recognition region and single
oligonucleotide attached is readily achievable. This can be
important for minimizing potential off-target nuclear binding of
Ab-DNA probes. The inventors discovered that a large number of
oligonucleotides deposited on biomolecule (e.g. IgG or SpA) led to
enhanced nuclear binding in fixed cells, which could not be
eliminated by DNA blocking or use of stringent hybridization
conditions. To test the off-target binding of Ab-DNA probes
prepared via amine-sulfhydryl cross-linking, the inventors
functionalized secondary rabbit anti-mouse IgG, incubated with
fixed and permeabilized cells after DNA blocking, and detected
location of those antibodies with anti-rabbit QD-2'Ab
conjugates.
Only minimal nuclear staining was observed, indicating good probe
specificity. However, this conjugation strategy showed varied
efficiency with different antibodies, exhibiting differences in
reaction and purification yield and potentially reducing Ab
affinity.
To preserve affinity and specificity of antibodies and improve the
yield of conjugation reaction, the inventors utilized an amine
cross-linking strategy. While direct reaction with
homo-bifunctional cross-linker Bis(Sulfosuccinimidyl) suberate
(BS3, which involved initial activation of oligonucleotides
followed by incubation with Ab, proceeded with low efficiency
(likely due to hydrolysis of active NHS groups during activation
and purification steps), an alternative conjugation system
utilizing reaction between hydrazide residues and aldehydes
successfully produced Ab-DNA conjugates with varying degrees of
modification (FIG. 2B). In this procedure Ab was activated by SANH
cross-linker, which converted primary amines on antibodies into
hydrazide residues reactive towards aldehydes. Excess cross-linker
was removed by protein desalting spin column with exchange of
buffer to pH5 MES. Amine-modified oligonucleotides were, in turn,
reacted with SFB, which converted primary amines into aldehydes,
and buffer-exchanged into pH5 MES. Activated antibodies were mixed
with modified oligonucleotides at 10-20 DNA molar excess and
incubated at room temperature for 5 hours. High stability of
reactive intermediates and prolonged reaction time ensured
efficient conjugation. The degree of DNA modification could be
controlled by the amount of SANH moieties introduced to the
antibody; however, the location of oligonucleotide attachment was
random. It should be noted that over-modification with DNA led to
significant off-target nuclear binding in cells. Therefore,
conjugation procedure was optimized to minimize the number of DNA
tags on each Ab. Generally, the optimal number of DNA tags
provides: unaffected Ab specificity/affinity; very little or no
non-specific binding: maximum number of binding sites for labels.
Accordingly, in some embodiments, the affinity molecule can
comprise from 1 to about 7, e.g., one, two, three, four, five, six
or seven, DNA tags. In one embodiment, the affinity molecule can
comprise from 2 to 5 DNA tags.
Both covalent conjugation procedures tried produced stable and
functional Ab-DNA probes. However, the complexity, high cost, and
low yield of such custom probe synthesis hampers preparation of a
large library of DNA-encoded antibodies. To resolve this issue the
inventors have also utilized a universal adaptor protein (SpA) as
well as species-matched Fc-specific Fab antibody fragments modified
with different DNA strands for on-demand preparation of
non-covalent Ab/DNA probes (FIG. 2C). Covalent modification of SpA
with oligonucleotides using SANH/SFB procedure is required.
However, since SpA is significantly less expensive and more stable
compared to primary antibodies, a library of SpA-DNA adaptors can
be prepared in large quantities and stored for a long time.
Essential to the success of this approach is the lack of SpA-Ab
cross-talk and Ab exchange. To demonstrate that SpA-Ab complex
exhibit sufficient stability and does not allow Ab exchange, the
inventors labeled SpA with organic dyes (either Alexa Fluor 488 or
Alexa Fluor 568) and performed cross-talk studies. In particular,
the inventors pre-assembled SpA-dye universal probes with 1'Ab
raised against androgen receptor (AR, a biomarker with
characteristic nuclear localization), then mixed in free SpA
labeled with a different dye as a competitor probe and performed
staining on fixed cancer cells. Only SpA pre-assembled with anti-AR
antibody produced characteristic nuclear staining, while competing
SpA failed to capture Ab and showed only minor non-specific
staining (FIG. 3). Similarly, the inventors have replicated these
studies with oligonucleotide-labeled SpA, demonstrating absolutely
no crosstalk between Ab/SpA-DNA probes (data not shown). Therefore,
SpA-DNA adaptors can be used for preparation of Ab-DNA libraries
for multiplexed 2-step staining.
QD-Oligonucleotide Conjugation:
A complementary library of QD-oligonucleotide probes was prepared
using covalent conjugation between amine-modified ssDNA and either
carboxy-functionalized or amine-functionalized QDs. At the same
time use of streptavidin-coated QDs was validated for flexible
on-demand preparation of QD-oligonucleotide probes. Conjugation to
carboxylic acid groups of compact zwitterionic QDs was achieved via
EDC-mediated coupling. In one experiment, negatively-charged
QD-PMAT particles featuring readily-accessible carboxylic acid
groups on the surface were incubated with a large excess of EDC and
10-20 molar excess of oligonucleotides overnight. Following the
conjugation reaction, the QD surface was back-filled with tertiary
amines to yield a zwitterionic coating. In another experiment, QDs
pre-coated with zwitterionic polymer PMAL were used. However,
unlike PMAT-coated QDs, access to carboxylic acid groups on PMAL is
sterically hindered by an abundance of bulky tertiary amines spaced
away from the QD surface by C3 linkers. Therefore, while providing
a very good barrier to non-specific interaction with biomolecules,
chemical modification of such surface can be challenging.
Furthermore, an oligonucleotide amine group placed at the end of a
PEG spacer on a hydrophobic C6 linker exhibited poor accessibility
in aqueous buffers. For example, colorimetric TNBS testing for
primary amines detected a significantly higher number of accessible
amines on oligonucleotides dissolved in DMSO compared to
bicarbonate buffer. Therefore, to resolve accessibility issues,
EDC-mediated conjugation between PMAL-coated QDs and amine-modified
oligonucleotides was performed in 100% DMSO or DMF solution. Unlike
PMAT-coated QDs that showed severe aggregation in DMSO/DMF, QD-PMAL
remained single even after addition of small amount of EDC. Some
aggregation could happen upon addition of larger amounts of EDC;
however, upon resuspension in Borate buffer aggregates broke back
into single QDs. QD-oligonucleotide probes were extensively
purified with ultrafiltration. Successful conjugation was confirmed
by detection of DNA absorption peak at 260 nm in QD-oligo solution
and increase in hydrodynamice size from 10 to 11-13 nm (depending
on number of DNA tags conjugated) (FIGS. 4A&C). Interestingly,
a conjugation reaction with identical conditions performed in
Bicarbonate buffer did not produce any QD-oligo conjugates (FIGS.
4B&D). Based on the oligonucleotide and QD absorption it was
calculated that 3-4 oligonucleotides were conjugated to each
QD.
PEG-coated QDs can represent a more flexible and stable platform
for preparation of QD probes, despite larger size and lower
accessibility of surface functional groups. Prolonged reaction time
necessary for efficiently reaching primary amine groups on the PEG
shell can necessitate the use of stable reactive intermediates or
repeated re-activation throughout the conjugation procedure. To
implement the first approach, the inventors utilized the SANH/SFB
system used previously for Ab-DNA conjugation. Similarly, QDs were
activated with SANH, while oligonucleotides were modified with SFB.
Reaction under mild acidic conditions yielded QD-DNA conjugates.
However, modification of PEG shell with SANH moieties caused some
increase in non-specific QD cell staining, while a lower degree of
activation yielded poor conjugation efficiency. Therefore, the
inventors used a second strategy to avoid significant alterations
of the QD coating. The second approach consisted of oligonucleotide
activation with BS3 followed by quick purification and reaction
with PEG-coated QDs. To reverse the effect of hydrolysis of
reactive NHS groups, EDC was repeatedly added to reaction mixture
in 1-hour intervals, resulting in efficient conjugation. However,
modification yield was not as significant as with zwitterionic
particles, resulting in no changes in absorption properties and
particle size. Surprisingly, besides promoting the conjugation
reaction EDC also interacted with carboxylic acid groups buried
underneath PEG shell, causing irreversible reduction of QD negative
charge and producing nearly neutral particles. Such a side reaction
was found to be quite favorable, as the increase in QD negative
charge associated with DNA conjugation could induce undesirable
electrostatic interactions with fixed cells.
Both covalent conjugation strategies permitted efficient production
of stable QD-oligonucleotide probes. Use of those approaches can be
suitable for preparation of large quantities of QD-DNA for longterm
storage and use with established staining protocols.
Streptavidin-coated QDs, on the other hand, offer more flexibility
in preparing on-demand probes immediately prior to staining, thus
enabling quick testing of new DNA sequences and staining
modalities. Straightforward probe self-assembly with relatively
controlled stoichiometry can be achieved by simply mixing
biotinylated oligonucleotides in slight excess to QD-streptavidin
particles and incubating for 30-60 minutes. The inventors
discovered that even a small excess of ssDNA (e.g. 2DNA:1QD)
resulted in efficient formation of QD-DNA conjugates (FIG. 5).
Depending on the application, larger numbers of DNA can be
deposited on QDs. However, purification away from unbound
oligonucleotides might be difficult (primarily due to small scale
of on-demand probe preparation). Therefore, the inventors also
designed a small-scale purification method with streptavidin-coated
magnetic beads (MB-Str). Upon mixing with QD-DNA, MB-Str depleted
all free ssDNA, thus leaving pure QD-DNA probes. Furthermore, to
prevent probe binding to endogenous biotin, the inventors blocked
pre-formed and purified QD-DNA probes with excess biotin for 10
minutes prior to staining.
Multiplexed 2-Step Staining of Fixed Cells:
Embodiments of the Hybrid IF/FISH methods described herein involve
recognition and DNA encoding of biomarkers in a first step by
appropriate Ab-DNA probes and labeling of DNA tags with
complementary QD-DNA probes (or FL-DNA probes) in a second step.
Since the link between first and second step is established by DNA
hybridization, sequence-specific recognition of multiple targets
without cross-talk between probes is possible, thus limiting the
maximum number of biomarkers detected in parallel only by the
current capabilities of spectral imaging and unmixing of different
fluorescent probes.
In most generic staining of fixed cells any Ab-DNA and QD-DNA probe
can be successfully used. To demonstrate this, the inventors
prepared formalin-fixed and permeabilized prostate cancer LNCaP
cells. Prior to staining, cells were blocked with 6% BSA/TBS buffer
with 0.1 mg/mL of sheared salmon sperm DNA to prevent off-target
binding of oligonucleotide-functionalized probes. Then cells were
contacted with Ab-DNA probes for 1 hour at room temperature,
washed, and stained with QD-DNA probes for another hour.
Single-color patterns obtained with both types of probes were
consistent. Staining of AR and MAOA achieved with either directly
conjugated Ab-DNA or self-assembled Ab/SpA-DNA probes showed
biomarker staining patterns consistent with those obtained in a
2-step staining procedure with QD-2'Ab conjugates (FIG. 6).
Similarly, QD-DNA probes based on both zwitterionic and PEG-coated
platforms produced clear staining with minimal nonspecific
background (FIG. 7). It should be noted that SpA-DNA probes
exhibited slightly enhanced off-target nuclear binding when
carrying a large number of oligonucleotides per SpA (FIG. 8, top
row). The inventors have developed two solutions to this problem.
First, as discussed above, DNA loading of SpA should be minimized,
as only a few QDs can sterically fit on SpA regardless of the DNA
density. Second, in cases when high DNA loading is required (e.g.
when detection is done by organic dyes instead of QDs), SpA-DNA can
be hybridized to a short blocking ssDNA sequence, thus forming
partial dsDNA tag. Such a construct efficiently eliminated nuclear
binding (FIG. 8, bottom row), while being easily removable by a
longer complementary ssDNA on QD probe. In multiplexed staining
experiments, the hybrid IF/FISH method described herein proved to
be robust and specific, exhibiting sufficient DNA link stability
and no cross-talk between probes. For example, the inventors have
achieved 3-color staining of AR, MAOA, and pAkt with covalently
conjugated Ab-DNA probes and QD-PEG-DNA labels (FIG. 9). Spectral
imaging clearly extracted signals of green QDs spectrally separated
by only 20 nm, yielding consistent biomarker staining patterns in
respective QD channels. Similarly, the inventors utilized
Ab/SpA-DNA probes for 4-color staining (FIG. 10). Ab/SpA-DNA probes
demonstrated good stability and specificity of staining, exhibiting
absolutely no cross-talk.
Specimen De-Staining Via DNA Link Displacement:
Hybrid IF/FISH methods described herein are amenable for highly
multiplexed parallel biomarker staining. However, to overcome the
limitation imposed by the maximum number of probes that can be
unambiguously distinguished and further enhance the multiplexing
capability of QD-based or FL-based molecular profiling, multiple QD
or fluorophore staining cycles can be implemented. Specimen
de-staining by low-pH treatment, heat degradation, and washing with
alcohol and detergents utilized in previous studies often causes
specimen degradation. Therefore, DNA link displacement via
hybridization with a longer oligonucleotide can provide faster
de-staining under non-degrading conditions, as it can efficiently
proceed at room temperature in standard neutral buffers. Moreover,
as antibody-antigen bond is not broken with this approach (in
contrast to other methods), time-consuming re-detection of
biomarkers on each cycle is unnecessary.
To test whether QD-DNA probe can be displaced with a longer
oligonucleotide, the inventors constructed a model system
consisting of streptavidin-coated magnetic beads (MB-Str), to which
biotinylated ssDNA was bound. MB-DNA particles were then labeled
with complementary QD-DNA probes. While magnetic beads did not show
any endogenous fluorescence, QD labeling turned MBs bright red
(FIG. 11, top row). Brief incubation with matching displacement
probe eliminated QD signal nearly completely, whereas mismatch
probe had no effect on MB-QD complex (FIG. 11, bottom row). This
shows that de-staining with DNA link displacement works well in the
model system and can be used on fixed cells.
De-staining of fixed cells proved to be more challenging, as a
number of additional interactions could influence dissociation of
QD-DNA probe and access to the DNA link for displacement probe
could be sterically hindered. Still, the inventors were able to
achieve up to 80% reduction in staining intensity during
de-staining (FIG. 12). This is in contrast to the quick, specific,
and complete de-staining achieved with ssDNA probes labeled with
Alexa Fluor dye (FIG. 13). Notably, complete re-staining was also
achieved, indicating that Ab/SpA-DNA tag remained intact throughout
2 staining cycles. The data demonstrates the feasibility of using
cyclic staining with QD-DNA and dye-labeled DNA probes via DNA link
displacement.
Improvements in such method can be achieved by further optimization
of QD surface coating and staining conditions to minimize potential
non-specific interactions between cells and QDs. While zwitterionic
and PEG-coated QDs do not show significant non-specific staining,
prolonged residence near cell surface due to formation of DNA link
might promote binding to cell components. Further, steric access to
the DNA linker for displacement can also be improved. For example,
DNA tags can be placed on longer PEG spacers and the length of
displacement handle can be increased to promote hybridization of
displacement oligonucleotide. Finally, residual non-specifically
bound QDs can also be quenched. To avoid dissociation of Ab-DNA
from biomarkers, DNA-tagged specimen can be cross-linked with BS3,
thus converting cells into stable spatially-encoded DNA arrays.
The highly flexible hybrid IF/FISH 2-step staining platform
described herein permits optimization for application-specific
criteria due to availability of a number of strategies for
preparation of Ab-DNA and QD-DNA probes. Among these, covalent
modifications are suitable for preparation of large quantities of
probe stocks that can be routinely used for established molecular
profiling studies. At the same time, more flexible non-covalent
probe self-assembly offers on-demand synthesis of custom probes for
validation tests and small studies. Regardless of the probe
preparation method, multiplexed parallel staining is readily
achievable, thus providing a powerful and yet simple tool for
molecular profiling of fixed cells and tissue specimens.
Utilization of unique DNA sequences for linking 1'Ab with
fluorescent probe permits further sequence-specific signal
amplification via successive deposition of another layer of FL
probes. In most one of the most straightforward implementations
with QD-based probes, second staining with QD-DNA probes
complementary to first layer of QD-DNA can be performed. Since each
QD carries multiple oligonucleotides, original Ab-DNA sequences
linked with QD-DNA will be converted into a yet larger number of
DNA tags present on QDs. Detection of those tags with same-color
QDs can provide a signal amplification mechanism. As second layer
of staining is also driven by specific DNA hybridization, it is
completely compatible with multiplexed staining modalities. In
another implementation with organic dye-labeled ssDNA probes,
additional amplifier molecule carrying a high number of scarcely
distributed DNA tags is added during the second step, thus
converting single Ab-DNA into a large DNA array, labeling of which
during the 3.sup.rd step should yield much enhanced signal. We have
implemented this approach, for example, on the basis of SpA for
labeling of each biomarker with multiple dye molecules, thus
achieving good signal-to-noise ratio of staining.
Additionally, ability to significantly expand multiplexing
capability of hybrid IF/FISH method with incorporation of cyclic
staining approach has been demonstrated. Complete or nearly
complete specimen de-staining can be achieved solely by
introduction of displacement DNA probes under physiological
conditions, thus preserving specimen antigenicity and permitting
reliable cyclic staining. This technique can be especially powerful
with utilization of flow-chambers for semi-automated cyclic
staining and real-time monitoring of staining and de-staining. We
have already tested the performance of glass-bottom channel slides
and flow-chambers for cell staining with QDs and cyclic staining
with dye-labeled ssDNA probes and demonstrated feasibility of
real-time staining monitoring.
Comprehensive molecular diagnostics and targeted therapy are
essential for making progress towards combating such complex
diseases as cancer, immune system disorders, and neurological
disorders. Incorporation of novel QD-based tools will undoubtedly
play a major role in this process. Unique photo-physical properties
and versatile bio-functionalization capabilities make QDs well
suited for sensitive quantitative molecular profiling of cells and
tissue specimens. Unfortunately, current limitations imposed by
either multistep staining modalities or complex QD-antibody
bioconjugation procedures hamper wide adaptation of QD technology
for fundamental research and clinical diagnostics. The highly
multiplexed staining method described here overcomes such
limitations by providing straightforward routes for unique matching
of biomarkers and QD probes, thus opening access to single-cell
molecular profiling within the context of preserved tissue or cell
culture morphology.
Example 2
Quantum-dot Based Cyclic Multiplexed Staining for Comprehensive
Molecular Profiling of Individual Cells and Cellular
Populations
In-depth understanding of the nature of cell physiology and ability
to diagnose and control the progression of pathological processes
heavily rely on untangling the complexity of intracellular
molecular mechanisms and pathways. To achieve this goal,
comprehensive molecular profiling of individual cells within the
context of their natural tissue or cell culture microenvironment is
required. Neither widely used conventional techniques nor more
advanced nanoparticle-based methods have been able to address this
task up to date. We have developed a highly multiplexed imaging
method, potentially capable of creating single-cell molecular
profiles consisting of over 100 biomarkers, and engineered
complementary universal quantum dot-based platforms for quick and
easy preparation of an extensive library of biomarker-specific
fluorescent probes. As our method consists of simple steps
requiring no advanced technical skills, it can be directly applied
for a wide range of molecular profiling studies, enabling direct
analysis of low-abundance events, heterogeneity within cell
populations, and interplay between different molecular pathways on
a single-cell level.
Conventional biomedical techniques suffer from a limitation in the
number of biomarkers that can be analyzed simultaneously (e.g.
immunohistochemistry, or IHC), provide limited single-cell
information resulting from the need to analyze signals averaged
over many cells (e.g. gene chips, protein chips, biomolecular mass
spectrometry, etc.), and often utilize qualitative rather than
quantitative analysis. Consequently, fundamental understanding of
pathological processes as well as clinical diagnostics are limited
by the lack of knowledge about the predictive biomarkers that would
unambiguously discriminate between disease and normal function,
distinguish different disease types, and provide information about
possible progression of the pathological process.
Realizing the importance of examining biomarker expression patterns
within the context of preserved specimen morphology, a variety of
multiplexed imaging techniques have been proposed. Building upon
conventional IHC (a mostly single-biomarker imaging method),
parallel and sequential staining techniques have been utilized to
determine not only expression levels of multiple biomarkers, but
also their distribution within individual cells or tissues. See,
for example, Englert, C. R., Baibakov, G. V. & Emmert-Buck, M.
R. Layered expression scanning: rapid molecular profiling of tumor
samples. Cancer Res 60, 1526-1530 (2000); Furuya, T. et al. A novel
technology allowing immunohistochemical staining of a tissue
section with 50 different antibodies in a single experiment. J.
Histochem Cytochem 52, 205-210 (2004); Pirici, D. et al. Antibody
elution method for multiple immunohistochemistry on primary
antibodies raised in the same species and of the same subtype. J.
Histochem Cytochem 57, 567575 (2009); Toth, Z. E. & Mezey, E.
Simultaneous visualization of multiple antigens with tyramide
signal amplification using antibodies from the same species. J.
Histochem Cytochem 55, 545-554 (2007); Glass, G., Papin, J. A.
& Mandell, J. W. SIMPLE: a sequential immunoperoxidase labeling
and erasing method. J. Histochem Cytochem 57, 899-905 (2009);
Wahlby, C., Erlandsson, F., Bengtsson, E. & Zetterberg, A.
Sequential immunofluorescence staining and image analysis for
detection of large numbers of antigens in individual cell nuclei.
Cytometry 47, 32-41 (2002); Micheva K D, Busse B. Weiler N C,
O'Rourke N, Smith S J: Single-synapse analysis of a diverse synapse
population: proteomic imaging methods and markers. Neuron 2010,
68:639-653; and Schubert W, Bonnekoh B, Pommer A J, Philipsen L,
Bockelmann R, Malykh Y, Gollnick H, Friedenberger M, Bode M, Dress
AWM: Analyzing proteome topology and function by automated
multidimensional fluorescence microscopy. Nat Biotechnol 2006,
24:1270-1278. However, while being highly laborious and
time-consuming, these methods still hold a limited multiplexing
capability. Recently developed imaging with Raman probes offers
more flexibility in obtaining multiplexed data (Liu, Z. et al.
Multiplexed Five-Color Molecular Imaging of Cancer Cells and Tumor
Tissues with Carbon Nanotube Raman Tags in the Near-Infrared. Nano
Research 3, 222-233 (2010) and Zavaleta, C. L. et al. Multiplexed
imaging of surface enhanced Raman scattering nanotags in living
mice using noninvasive Raman spectroscopy. Proc Natl Acad Sci USA
106, 13511-13516 (2009)); yet, the large size often significantly
hampers probe diffusion within the cross-linked cells and tissues,
compromising detection of biomarkers located deep within the
specimen. Imaging mass spectrometry offers the greatest
multiplexing capability and has a potential for quantitative
analysis (Schwamborn, K. & Caprioli, R. M. Molecular imaging by
mass spectrometry--looking beyond classical histology. Nat Rev
Cancer 10, 639-646 (2010) and Wollscheid, B. et al.
Mass-spectrometric identification and relative quantification of
N-linked cell surface glycoproteins. Nat Biotechnol 27, 378-386
(2009)), but it comes at a price of low lateral resolution, high
equipment costs, and requirement of correlating obtained
biomolecule composition data with optical imaging.
Fluorescent probes based on semiconductor nanoparticles (quantum
dots, or QDs) offer advantages for multiplexing over chemical
fluorophore probes. Having size of only 2 to 10 nm in diameter, QDs
possess unique photo-physical properties, such as size-tunable and
spectrally narrow light emission, simultaneous excitation of
multiple colors, improved brightness, resistance to photobleaching,
and large Stokes shift. Due to these properties, simultaneous
parallel detection of up to 10 spectrally distinct QD probes is
possible. However, utilization of such multiplexing capability has
been hampered by the inability to uniquely match each QD probe with
the corresponding biomarker.
To fully utilize extensive multiplexing potential of QD fluorescent
tags and exploit wide selection of validated primary antibodies for
molecular profiling with IF, the inventors have developed a
universal QD/adaptor protein platform for flexible and fast
preparation of a library of functional QD-Ab probes. These probes
have been used in a multi-cycle 1-step staining procedure described
herein below. In a method described herein, QD-Ab probes are
prepared prior to staining in a straightforward 1-step procedure
via self-assembly between universal QD/adaptor protein platform and
virtually any Ab of choice, thus requiring no chemical modification
of antibodies and eliminating the need for costly and
time-consuming QD-Ab probe purification. Direct 1-step parallel
multiplexed staining performed with such probes can utilize a full
range of spectrally distinguishable probes without Ab species or
buffer composition limitations. Furthermore, multiplexing
capability is dramatically expanded by performing
staining/imaging/de-staining procedure on the same specimen in
several cycles. Capability of the QD-Ab probe to disassemble under
low-pH conditions enables complete removal of the QD signal after
imaging, thus restoring the specimen to its original state and
keeping it unperturbed for the next round of staining. The
inventors have used this new multiplexed imaging technique for
molecular profiling of fixed cancer cells, showing that at least 50
biomarkers can be reliably imaged at sub-cellular resolution, with
potential of expanding this method to imaging of over 100
biomarkers.
Materials and Methods
Synthesis of QD/SpA Probes:
Amine-functionalized PEG-coated QDs (Qdot ITK amino (PEG) quantum
dots, Invitrogen) with emission peaks centered at 525, 545, 565,
585, and 605 nm were used for preparation of universal fluorescent
probes. First, QDs were activated with bifunctional cross-linker
BS3(Bis [sulfosuccinimidyl] suberate, Thermo Scientific), followed
by covalent conjugation with SpA (Protein A from Staphylococcus
aureus, Sigma-Aldrich). 100 uL 1 uM QD solution in PBS was mixed
with 500-1000 molar excess of BS3 and incubated for 30 minutes at
RT. Free cross-linker was removed by passing QD/BS3 mixture through
a NAP-5 column (GE Healthcare) pre-equilibrated with PBS. Handheld
UV lamp was used to aid in collection of QD-containing elution
fraction. Eluted QDs were concentrated down to 70 uL with a 100 KDa
MWCO concentrator (GE Healthcare), and 30 uL of 100 uM SpA solution
in PBS was added. Reaction was incubated overnight at room
temperature, quenched for 30 minutes with ethanolamine
(Sigma-Aldrich), and purified by ultrafiltration for at least 7
times with a 100 KDa MWCO concentrator. Purified QD-SpA probes were
stored in PBS solution at 4'C.
Cell Culture and Processing:
Human prostate cancer cell line LNCaP (ATCC) was used as a model
for optimization of a multiplexed IF protocol. Cells were grown in
glass-bottom 24-well plates (Greiner Bio-One) for 2-3 days to a
density of .about.60%. Humidified atmosphere at 37.degree. C. with
5% CO.sub.2 was maintained. RPMI-1640 culture medium with
L-Glutamine and 25 mM HEPES (Lonza) supplemented with 10% Fetal
Bovine Serum (PAA Laboratories) and antibiotics (60 lig/mL
streptomycin and 60 U/mL penicillin) was used. For IF staining
procedure, cells were fixed with formaldehyde and permeabilized
with detergents. First, cells were washed twice with pre-warmed TBS
(1 mL per well). Solution was added carefully to the wall of each
well to avoid cell detachment from the glass coverslip. Next, 400
uL of 4% formaldehyde in TBS (prepared from methanol-free 16%
stock, Thermo Scientific) was added to each well, incubated for 20
min at room temperature, and rinsed with TBS. Finally, cells were
permeabilized with 2% DTAC/TBS (Dodecyltrimethylammonium chloride,
Sigma-Aldrich) for 20 min and 0.25% TritonX-100/TBS (prepared from
10% stock, Thermo Scientific) for 5 min and washed 5 times with
TBS. Fixed cells were stored in TBS at 4.degree. C.
Imaging:
An IX-71 inverted fluorescence microscope (Olympus) equipped with
true-color camera (QColorS, Olympus) and spectral imaging camera
(Nuance, CRI) was used for cell imaging. Low-magnification images
were obtained with 10.times. and 20.times. dry objectives (NA 0.40
and 0.75 respectively, Olympus) and high-magnification with
100.times. oil-immersion objective (NA 1.40, Olympus). Wide UV
filter cube (330-385 nm band-pass excitation, 420 nm long-pass
emission, Olympus) was used for imaging of all QD probes, while
Rhodamine LP cube (530-560 nm band-pass excitation, 572 nm
long-pass emission, Chroma) was used for Alexa Fluor 568 detection.
All images were acquired with cells attached to the coverslip
bottom of the well and immersed in TBS. No anti-fading reagents
were used. For parallel multiplexed staining, images were obtained
with the spectral imaging camera scanning through the full 420-720
nm spectral range. NUANCE.TM. image analysis software was used to
unmix obtained images based on the reference spectra of each QD
component along with an extra channel for background fluorescence.
In a false-color composite image, brightness and contrast of each
channel was adjusted for best visual representation and clear
depiction of relative biomarker distribution. For sequential
staining a permanent reference point was marked on the bottom of
the well to aid in finding the same cell subset for each imaging
cycle. Minor misalignment between different frames was adjusted
manually, and frames were merged into a false-color composite image
in Photoshop (Adobe Systems). Each frame was imaged using the same
parameters for direct comparison of signal intensity and biomarker
expression levels. However, for false-color composite image,
brightness and contrast of each frame was again adjusted to achieve
the best clarity in relative biomarker distribution.
2-Step Single-Color Immunofluorescence:
Polyclonal rabbit antibodies raised against AR, MAOA, pAkt, and
P-tubulin were purchased from Santa Cruz Biotechnology. Monoclonal
mouse anti-HSP90 Ab was from Thermo Scientific. All buffers were
prepared with deionized water (>18 M0-cm). Blocking buffer
composition was 2% BSA (from Bovine Serum Albumin powder,
Sigma-Aldrich), 0.1% casein (from 5% solution, Novagen), and 1
.times. TBS (from 10.times. solution, Fisher Scientific). Staining
buffer composition was 6% BSA in 1.times. TBS. Staining was
performed with either dye-labeled secondary antibodies
(rabbit-anti-mouse and goat-anti-rabbit IgG, Sigma-Aldrich, labeled
with Alexa Fluor 568 carboxylic acid succinimidyl ester,
Invitrogen), QDs functionalized with secondary Ab fragments (Qdot
goat F(ab')2 anti-mouse or anti-rabbit IgG conjugates (H+L),
Invitrogen), or QD/SpA probes prepared as described above. All
staining steps were performed directly inside the wells of
glass-bottom 24-well plates at ambient conditions.
For a 2-step IF, cells were blocked with blocking buffer for 30
minutes and incubated with 300 .mu.l 1 .mu.g/mL primary antibodies
(diluted 1:200 from 0.2 mg/mL stock in staining buffer) for 1 hour.
Then, cells were washed 3 times with TBS and incubated with either
300 .mu.l 4 .mu.g/mL dye-labeled 2'Ab, 4 nM QD-2'Ab, or 10 nM
QD/SpA in staining buffer for 1 hour in the dark. Extra fluorescent
labels were removed by rinsing cells with 1% BSA/0.1% casein/TBS
twice and washing with TBS 3 times. Fluorescence imaging was done
immediately following staining, unless stated otherwise.
Multiplexed 1-Step Immunofluorescence:
Multiplexed IF studies were performed in parallel, sequential, or
combined parallel/sequential manner. Regardless of the staining
type, cells were blocked with blocking buffer for 30 minutes as
described for 2-step IF. Concurrently, QD-Ab probes were prepared
by incubating 5 .mu.l 500 nM QD/SpA with 1.5 .mu.1 0.2 mg/mL
primary Ab for 1 hour at room temperature. Each QD _Ab probe was
prepared in a separate microcentrifuge tube. For parallel
multiplexed staining all probes were combined in a single tube,
diluted to 300 .mu.l with staining buffer, and immediately applied
to pre-blocked cells. After 1-2 hour staining, cells were rinsed
with 1% BSA/0.1% casein/TBS twice and washed with TBS 3 times.
Imaging was done immediately following staining. For single-color
sequential staining, QD-Ab probes were prepared using the same
QD/SpA incubated with different antibodies in separate
microcentrifuge tubes. Then each probe was used for a 1-step
single-color staining and imaging. Each staining cycle consisted of
(i) pre-blocking, (ii) staining, (iii) imaging, and (iv)
de-staining. The first three steps were identical to those for
parallel staining. De-staining was performed by incubating stained
cells in 400 .mu.l pH2 Glycine-HCI buffer with 0.1% casein for 15
minutes. Following de-staining the pH was gradually brought back to
neutral by rinsing with pH3, 4, and 5 Glycine-HCI buffers and
washing 5 times with TBS. Combined parallel/sequential staining
utilized multiple QD-Ab probes and spectral imaging on each
cycle.
QD-Ab probe cross-talk and stability studies: Probe cross-talk
studies were performed in a same manner as parallel 2-color
staining described above, except that one QD/SpA conjugate was
incubated with primary Ab, while the other one was not. Following
QD-Ab probe assembly, QD.sub.1-Ab and QD.sub.2/SpA were combined in
the same microcentrifuge tube, diluted to 300 .mu.L in staining
buffer, and added to pre-blocked cells. After 1-hour staining,
cells were washed and imaged. Spectral imaging was used to unmix
and quantitatively compare individual QD signals. QD525 and QD565
were used for this study. Intensity of the dimmer QD525 channel was
scaled up 4 times compared to brighter QD565 channel to correct for
differential brightness of QD probes.
For examination of long-term staining stability with QD-Ab probes,
single-color 1-step staining was performed as described above. AR
was selected as a target biomarker due to its clearly defined
nuclear localization. Permanent reference point was marked on the
bottom of the well, and cell subset in the vicinity of reference
point was imaged. Stained cells were then left in TBS at 4'C.
Images of the same subset of cells were taken at 0, 4, 24, and 48
hours post-staining, keeping camera exposure time and other imaging
parameters the same for all time points.
Biomarker degradation studies: To study biomarker degradation (i.e.
loss of antigenicity) due to cyclic washing/blocking/de-staining
treatment and prolonged exposure to ambient conditions, staining of
AR was performed with QD-Ab probes immediately after treating cells
with 1 to 10 degradation cycles. Each treatment condition was
tested on cells in different wells of the same 24-well plate.
Degradation cycle consisted of the following steps: washing cells
with TBS for 10 minutes, de-staining at pH2 for 15 minutes, rinsing
with pH3,4,5 buffers, washing with TBS, and blocking for 30 minutes
with 2% BSA/0.1% casein/TBS. To avoid inconsistency in staining and
imaging conditions, all cells were stained and imaged at the same
time (as a result, exposure to 1 degradation cycle implied
incubation in TBS for 9 cycles, then treatment with degradation
conditions, and then staining and imaging). All cells were imaged
at low and high magnification using true-color and spectral imaging
cameras. Exposure time and other imaging conditions were kept the
same for all images taken, thus enabling direct quantitative
comparison of signal intensity. NUANCE.TM. image analysis software
was used to identify regions of interest (ROIs) that included
nuclear AR staining and excluded background fluorescence. Average
signal from multiple ROIs in low-magnification images was
recorded.
SPR Analysis of SpA/IgG Bond Stability:
SPR measurements of SpA/IgG bond stability were done on a Biacore
T100 instrument (GE Healthcare). SpA or rabbit-anti-mouse IgG were
immobilized on CMS sensor chip via covalent conjugation between
primary amines on SpA or IgG and carboxylic acid groups on the
dextran coating of the chip. All studies were done in 150 mM NaCI,
10 mM HEPES pH7.4 buffer under continuous flow at 104/min. Surface
regeneration was performed with 10 mM Glycine-HCI pH2 buffer. For
binding/dissociation studies SpA was injected over IgG-modified
surface and control unmodified surface, whereas IgG was injected
over SpA-modified and control surfaces. Interaction between ligand
and analyte at high (100 nM) and low (10 nM) analyte concentrations
were monitored. For high analyte concentration, binding was
monitored for 10 minutes and dissociation for 30 minutes, while for
low analyte concentration, times were 30 and 60 minutes
respectively. During the dissociation step, the surface was
continuously washed with running buffer. The data analysis was
carried out using the BlAevaluation 2.0 software.
Results
Molecular profiling of individual cells involves detection and
quantification of multiple relevant biomarkers, which in principle
can be achieved with IF imaging by tagging each biomarker with
unique fluorescent probe and detecting its localization with high
sensitivity at sub-cellular resolution. Utilization of QD probes
featuring narrow symmetrical emission profiles permits simultaneous
detection of up to 10 different probes within the visible spectral
range. However, multiplexing capacity of QD-based IF can be further
expanded to over 100 biomarkers by performing multiple
staining/imaging cycles on the same specimen. In order to achieve
this: (i) each QD probe should be uniquely matched to corresponding
biomarker, exhibiting no cross-talk between different probes in a
staining cocktail; (ii) QD signal should be completely removed
after imaging, providing no interference with the next staining
cycle; (iii) biomarker antigenicity and specimen integrity should
be retained throughout multiple staining/imaging/de-staining
cycles. Practical considerations associated with these conditions
should also be addressed. In particular, molecular profiling
requires preparation of a large library of unique QD-Ab probes.
Therefore, preparation of such probes should be easy, flexible, and
relatively inexpensive.
In a new cyclic multiplexed imaging approach described herein, the
inventors addressed these conditions by engineering a universal
QD/adaptor protein platform for 1-step, purification-free
preparation of QD-Ab probes via self-assembly, utilizing QD-Ab
probes for direct biomarker labeling in parallel multiplexed
staining, and performing multiple cycles of staining for obtaining
a comprehensive molecular profiles of individual cancer cells at
sub-cellular resolution. The method can be described as follows.
First, universal QD/adaptor protein platform is used to capture
intact Ab from solution during a pre-staining step to form a
functional QD-Ab fluorescent probe (FIG. 14A). Once formed, all
different probes are mixed in a single cocktail (FIG. 14B) and
incubated with cells for parallel multiplexed staining (FIG. 14C).
Second, following staining, fluorescence microscopy with spectral
imaging is utilized to acquire and unmix signal from each QD probe,
generate quantitative biomarker expression profiles, and depict
relative biomarker distribution (FIG. 14D). Third, complete
de-staining of the specimen is done by brief washing with low-pH
buffer, permitting the next full cycle of IF staining for a
different subset of biomarkers (FIG. 14E). With each staining cycle
N biomarkers can be analyzed, where N depends on the number of
spectrally distinct fluorescent probes that can be detected
simultaneously. Performing IF staining for M sequential cycles
generates M subsets of data for the same specimen, thus yielding an
overall molecular profile consisting of N.times.M biomarkers (FIG.
14F). Therefore, the method permits extensive molecular profiling
of specimens with preserved morphology at multiplexing levels far
exceeding even the most advanced imaging techniques.
Multiplexed Biomarker Imaging:
To demonstrate the utility of the method for molecular profiling,
the inventors imaged 4 cancer biomarkers (heat shock protein 90,
HSP-90; androgen receptor, AR; monoamine oxidase A, MAOA; and
phosphorAKT, pAkt) and a housekeeping biomarker .beta.-tubulinin
formalin-fixed prostate cancer cells (LNCaP). Parallel staining of
these biomarkers with 5 different QD-Ab probes (FIG. 15, top panel)
as well as sequential staining with same-color QD-Ab probes (FIG.
15, bottom panel) yielded consistent molecular profiles with
sub-cellular resolution.
For parallel multiplexing, the inventors utilized QDs emitting in a
visible spectral range (at 525, 545, 565, 585, and 605 nm). 20 nm
separation between emission peaks ensured reliable spectral
unmixing of individual QDs, while permitting imaging of multiple
probes within a narrow spectral window. QD-Ab probe assembly was
done in separate tubes for 1 Hr immediately prior to staining. At
the same time, cells were blocked with BSA (bovine serum
albumin)/casein blocking buffer to eliminate QD-Ab probe
non-specific binding. Parallel multiplexing involved spectral
imaging and unmixing of QD signals, which produced most accurate
results when all signals were of similar intensity. Therefore,
brighter red QD probes were used for less abundant (or more
diffusely distributed) biomarkers, while dimmer green QDs were
reserved for more abundant (or more densely packed) biomarkers. In
a case when differences in biomarker expression could not be
compensated by brighter probes, less abundant biomarkers were
imaged in a separate cycle. For example, staining intensity of
.beta.-tubulin was significantly dimmer compared to cancer
biomarkers. Therefore, for parallel imaging this biomarker was
labeled with brightest QD605 probes (FIG. 15, top panel). Yet, its
contribution to an unmixed image was less reliable, while
expression and distribution of B-tubulin was clearly visualized
when imaged on a separate cycle (FIG. 15, bottom panel).
Nonetheless, spectral imaging provides sufficient flexibility for
simultaneous imaging and unmixing of high- and low-abundance
biomarkers, and re-staining on a separate cycle can be performed
when the quality of low-intensity signal is significantly
compromised.
Sequential multiplexing involved imaging of the same area of the
specimen and matching biomarker distribution patterns obtained at
different imaging cycles. This was particularly challenging to
achieve when high-magnification imaging was performed. To aid in
finding the same subset of cell for each imaging cycle, the
inventors placed a reference mark on the bottom of each well and
stepped a set distance from the mark for imaging. Minor mismatch in
frame alignment was manually corrected with image-processing
software. After alignment each frame was false-colored, and all
frames were overlaid to yield a composite 5-color image for the
study of relative biomarker distribution. It should be noted, that
sequential imaging provided direct quantitative comparison of
biomarker expression levels, as identical QD probes and imaging
conditions were used for all biomarkers. In contrast, quantitative
analysis of spectrally unmixed parallel staining was not as
straightforward as each QD probe exhibited different relative
brightness (due to differences in absorption cross-section, quantum
yield, and camera sensitivity to different wavelengths of light).
Therefore, signal intensity of each QD channel in composite image
should be scaled appropriately when quantitative analysis is
necessary.
QD-Ab Probe Preparation:
Multiplexed IF necessarily requires preparation of a large library
of QD-labeled antibodies. Common 2-step staining procedure utilizes
QD-labeled secondary antibodies for detection of intact primary
antibodies. However, multiplexing capability of this approach is
limited by the number of suitable primary/secondary antibody
combinations. To overcome this limitation, several direct covalent
and non-covalent QD-Ab conjugation methods have been developed, yet
all methods suffer from serious limitations that hamper wide use of
QD-based multiplexed imaging techniques for biomedical research and
clinical diagnostics. For example, covalent QD-Ab conjugation
yields stable and unique fluorescent probes, though chemical
modification of antibodies is not only highly complex and
prohibitively expensive, but also often results in reduction in
antibody affinity and/or specificity. Widely used
streptavidin-biotin pairing creates a strong link in a
straightforward procedure, but it requires the preparation of
biotinilated antibodies and often leads to cross-linking of
QD/Streptavidin due to binding to multiple biotins on a single Ab.
Functionalization of QDs with intact antibodies can be achieved via
self-assembly with adaptor protein. Yet, preparation of QD/adaptor
protein nanoparticles was done either via strong electrostatic
interactions (Goldman, E. R. et al. Conjugation of luminescent
quantum dots with antibodies using an engineered adaptor protein to
provide new reagents for fluoroimmunoassays. Anal Chem 74, 841-847
(2002); Goldman, E. R. et al. Avidin: a natural bridge for quantum
dot-antibody conjugates. J Am Chem Soc 124, 6378-6382 (2002); and
Jaiswal, J. K., Mattoussi, H., Mauro, J. M. & Simon, S. M.
Long-term multiple color imaging of live cells using quantum dot
bioconjugates. Nat Biotechnol 21, 47-51 (2003)), HIS-tag-mediated
binding (Lim, Y. T., Cho, M. Y., Lee, J. M., Chung, S. J. &
Chung, B. H. Simultaneous intracellular delivery of targeting
antibodies and functional nanoparticles with engineered protein G
system. Biomaterials 30, 1197-1204 (2009)), or covalent conjugation
to carboxylic acid groups on the QD surface (Jin, T. et al.
Antibody-protein A conjugated quantum dots for multiplexed imaging
of surface receptors in living cells. Mol Biosyst 6, 2325-2331
(2010)), all of which utilized negatively-charged QDs that often
exhibit extremely high non-specific binding to fixed cells and
tissue specimens. Therefore, such probes were successfully used for
immunoassays and live-cell imaging, but not for multiplexed IF.
Moreover, potential for Ab exchange between QDs and probe
cross-talk due to relatively low stability of non-covalent
interactions have not been carefully evaluated. Despite these
concerns, we regarded adaptor protein as the most flexible and
promising linker for engineering of universal QD-based imaging
probes for multiplexed IF applications.
Shielding of the QD core with PEG shell and neutralization of the
QD charge are critical for eliminating non-specific staining during
IF. Therefore, as a platform for our method we used highly stable,
bright, and water-soluble PEG-coated QDs. With proper specimen
blocking, those particles produced no detectable non-specific
staining. It should be noted that strong non-specific QD binding
represents a serious and persistent problem for QD-based staining
of fixed cells and tissue specimens, often hampering utilization of
QD probes for IF applications. In our experience careful shielding
of the QD core by poly(ethylene glycol) (PEG) shell or complete
neutralization of the QD surface charge by functionalization with
zwitterionic groups efficiently eliminated non-specific
interactions (Zrazhevskiy, P., Sena, M. & Gao, X. H. Designing
multifunctional quantum dots for bioimaging, detection, and drug
delivery. Chemical Society Reviews 39, 4326-4354 (2010)). As it is
hard to achieve precise control over the QD surface properties,
variations in composition of different PEG-coated QD lots led to
variations in non-specific staining observed with those probes. To
compensate for this effect, in addition to commonly used BSA the
inventors included 0.1% casein in a blocking step. While not
wishing to be bound by a theory, being more negatively charged and
hydrophobic than BSA, casein serves as a more stable blocking
reagent for QDs, which also carry a net negative charge. However,
since use of higher casein content during the blocking step or
incorporation of casein in staining buffer often resulted in
decreased staining intensity, utilization of this blocking reagent
was kept to a minimum.
At the same time, the PEG shell featured primary amine groups for
covalent conjugation with adaptor protein. A wide range of native
and engineered adaptor proteins capable of binding intact
antibodies is available. The inventors chose the most widely used
and well-characterized adaptor protein--Protein A from
Staphylococcus aureus (SpA)--for demonstration of multiplexed
imaging technology, while other adaptor proteins (e.g. Protein G,
Protein A/G, etc.) can also be used in a similar fashion. In some
embodiments, the number of sterically accessible binding points
between adaptor protein and Ab can be limited to one, as to prevent
cross-linking of different QD/SpA probes via single Ab. In this
regard, SpA possesses five IgG binding sites (Moks, T. et al.
Staphylococcal protein A consists of five IgG-binding domains. Eur
1 Biochem 156, 637-643 (1986)), two of which are accessible
simultaneously, while IgG has two SpA binding sites on its Fc
region. In solution, formation of polymeric SpA/IgG complexes is
possible (Mota, G., Ghetie, V. & Sjoquist, J. Characterization
of the soluble complex formed by reacting rabbit IgG with protein A
of S. aureus. Immunochemistry 15, 639-642 (1978)). However, when
SpA/IgG is located on the QD surface, binding of additional SpA
molecules is likely to be sterically hindered, thus eliminating the
possibility of QD cross-linking.
Sufficiently high excess of cross-linker was added to prevent
cross-linking of primary amines on different QDs or on the surface
of the same QD. Extra cross-linker was removed with a NAP-5
desalting column. Since purification with the desalting column led
to significant dilution of the QD sample, activated QDs were
concentrated to 1 .mu.M and allowed to react with SpA at ambient
conditions overnight. While the reaction could be performed at
lower QD concentrations (e.g. 100-200 nM), the conjugation yield
was noticeably higher when at least 1 .mu.M QD solution was used
(especially for lower-quality QD lots). Vortexing or other
mechanical agitation was avoided on all steps, as it led to QD
aggregation. Finally, QD/SpA conjugates were purified from excess
SpA by repeated ultrafiltration in 100 kDa MWCO concentrators.
Six-seven rounds of ultrafiltration (with 10.times. sample dilution
each) completely eliminated SpA (as tested with Alexa Fluor
647-labeled SpA, data not shown).
Performance of QD/SpA-Ab probes was assessed by staining 5
different biomarkers individually (FIG. 16, top row) and comparing
the relative staining intensity and biomarker distribution patterns
with those obtained with conventional 2-step IF using QD-2'Ab (FIG.
6, middle row) or Alexa Fluor 568-labeled 2'Ab (FIG. 16, bottom
row). As expected, overall signal intensity obtained with QD/SpA-Ab
probes was lower than that of QD-2'Ab due to the absence of
amplification mechanism in 1-step staining. Therefore, 1-step
staining procedure was most suitable for characterization of
high-abundance biomarkers, while staining of low-abundance
biomarkers (e.g. .beta.-tubulin) was less reliable. Nonetheless,
staining patterns and relative biomarker expression were consistent
throughout all three procedures, indicating preserved specificity
and affinity of antibodies in a QD/SpA-Ab complex. No aggregation
of QD probes was observed throughout prestaining and staining
steps, confirming that binding of more than one QD/SpA per Ab was
sterically hindered.
According to the method design, all QD-Ab probes were formed via
non-covalent binding between SpA and Ab. However, direct QD-Ab
self-assembly via other routes (e.g. electrostatic or hydrophobic
interactions) could be possible. To confirm that assembly happened
indeed due to SpA-Ab binding, we performed several control staining
experiments. First, all 5 different QD/SpA probes were used in
1-step staining of androgen receptor, showing consistent
predominantly nuclear localization of this biomarker (FIG. 17, top
row) and indicating that QD-Ab complex successfully formed. Second,
2-step AR staining was done. On the first step cells were incubated
with 1'Ab, then excess Ab was washed away, and cells were stained
with QD/SpA probes. Consistent nuclear staining with similar signal
intensity was obtained in this case as well (FIG. 17, middle row),
confirming that QD/SpA probes successfully recognized target-bound
antibodies inside cells. Finally, control staining with QD/SpA
probes alone showed only minimal diffuse non-specific binding (FIG.
17, bottom row). Same 1-step and 2-step staining experiments
performed with non-modified PEG-coated QDs produced only minimal
non-specific staining and failed to bind to Ab in either procedure
(data not shown). Therefore, we confirmed with certainty that QD-Ab
complex formation was mediated exclusively by SpA-Ab binding.
Moreover, QD/SpA exhibited high specificity in Ab binding, showing
no enhanced off-target binding beyond that observed for unmodified
PEG-coated QDs.
QD-Ab Probe Stability and Cross-Talk:
Assembly of the QD/SpA-Ab probe was done in a microcentrifuge tube
by simple mixing of QD/SpA and 1'Ab raised against a biomarker of
interest. SpA binds a wide range of antibodies with a reasonably
high affinity (Kd on the order of 10.sup.-8 M). Therefore, by
adding QD/SpA in a slight excess to antibodies and performing
incubation in concentrated solution prior to staining, complete
capture of antibodies by QD/SpA can be achieved, thus, uniquely
matching each QD-biomarker pair and preventing binding of free IgG
to vacant SpA sites on different QD/SpA probes. However,
considering that all QD-Ab probes were formed via the same
non-selective and non-covalent SpA-Ab bond, we were concerned that
spontaneous QD-Ab dissociation, Ab exchange, and cross-talk between
different probes could occur. In fact, probability of such
cross-talk could be high if free antibodies were present in
solution, as we observed that QD/SpA could bind Ab and produce
nearly identical staining even when mixed in dilute concentrations
and immediately applied to cells. To address this concern the
inventors monitored SpA-Ab dissociation kinetics using SPR (surface
plasmon resonance) measurements on a Biacore T100 instrument and
assessed QD-Ab probe stability and potential for cross-talk
experimentally with cell staining.
For SPR studies, either SpA or rabbit anti-mouse 2'Ab were
immobilized on the dextran-coated surface of C5 chip. Binding of
excess 2'Ab to immobilized SpA and excess SpA to immobilized 2'Ab
in reference to control unmodified surface was recorded. The
invetors observed overall lower binding affinity when analyte was
injected at high concentration (250 nM-4 .mu.M) with Kd 5-10 times
higher than that for low-concentration analyte (10 nM-250 nM).
Measurements of binding/dissociation kinetics showed nonlinear
behavior which was especially pronounced for high-concentration
analyte. This observation was consistent with previous reports
suggesting that slow binding/dissociation by strong binding sites
was accompanied with fast binding/dissociation via weak sites,
especially when strong sites were saturated (Myhre, E. B. &
Kronvall, G. Immunochemical aspects of Fc-medicated binding of
human IgG subclasses to group A, C and G streptococci. Mol Immunol
17, 1563-1573 (1980)). As a result, dissociation curves (recorded
for 30 minutes) for high-concentration analyte (either SpA or 2'Ab
at 100 nM) showed an initial fast drop in signal accounting for up
to 5% loss of bound analyte, followed by a very slow dissociation
(FIGS. 18A and 18B). Same measurements performed with
low-concentration analyte (2'Ab at 10 nM, which is consistent with
final Ab concentration in staining buffer) showed nearly no SpA-Ab
dissociation for 60 minutes, retaining over 97% of bound Ab (FIG.
18C). These results indicated that SpA-Ab dissociation kinetics is
sufficiently slow at the concentration range and time-frame of the
cell staining procedure to prevent QD-Ab complex disassembly and
release of free antibodies in solution.
To test QD/SpA-Ab probe stability and examine cross-talk
experimentally with cell staining, the inventors prepared QD525-Ab
and QD565-Ab probes in a pre-staining step. The inventors chose
androgen receptor as staining target for its high expression in
LNCaP cells and distinct nuclear localization. Immediately before
staining, inventors mixed QD-Ab probes with counterpart
non-complexed QD/SpA (i.e. QD525-Ab were mixed with QD565/SpA,
while QD565-Ab were mixed with QD525/SpA) in staining buffer and
incubated with cells. With this setup, a large excess of vacant
counterpart QD/SpA probes can bind any free Ab released from QD-Ab
probes and compete for biomarker binding, producing cross-talk.
However, the inventors did not observe any cross-talk staining with
either QD525-Ab or QD565-Ab (FIG. 19, top and middle rows). At the
same time, QD525/SpA and QD565/SpA probes mixed together with Ab
and incubated with cells efficiently captured free Ab from solution
and produced mixed-color AR staining with nearly 50% contribution
by each (FIG. 19, bottom row). Therefore, SPR data analysis and
staining results show that pre-formed QD/SpA-Ab complex, featuring
very slow dissociation kinetics and sterically blocking access to
bound Ab, could not release free Ab into solution or exchange bound
Ab with other vacant QD/SpA probes within the concentration range
and time-frame of a cell staining experiment, thus producing no
detectable cross-talk between different probes and permitting
reliable and specific parallel multiplexed staining. Further, as
seen in FIGS. 20A-20C, QD-SpA probes provided consistent size and
biomarker staining kinetics on HeLa cells.
It should be noted that the success of 1-step purification-free
preparation of QD/SpA-Ab probes depended on the optimal combination
of QD/SpA and Ab parts. Having QD/SpA in slight excess over Ab
ensured complete capture of antibodies, which was essential for
eliminating probe cross-talk. Even though QD/SpA (likely featuring
multiple SpA per QD) could accommodate a range of Ab concentrations
in preparation of functional QD-Ab probes, large excess of
antibodies saturated all SpA binding sites, thus leaving free Ab in
solution. In cases when Ab concentration or QD/SpA Ab binding
capacity were not known, each Ab/QD combination was optimized and
checked using cross-talk staining experiments as described above.
However, such an approach might not be practical for highly
multiplexed staining experiments. Therefore, in an alternative
approach, QD/SpA and antibodies were mixed in roughly 1:1 ratio
during the pre-staining procedure. Then, SpA-functionalized
magnetic beads (MB/SpA) were added and briefly incubated QD-Ab
mixture to capture free antibodies. As QD-bound antibodies were not
accessible to MB/SpA, no QD-Ab probes were consumed by this step,
while all unbound antibodies were efficiently removed from staining
solution, completely eliminating probe cross-talk (data not
shown).
Sequential Cyclic Staining with QD/SpA-Ab Probes:
Cyclic staining involves multiple rounds of complete IF staining on
the same specimen. Therefore, complete de-staining after each IF
cycle is can provide for accurate biomarker detection. Generally,
the de-staining step should remove the fluorescence signal. It can
also be useful to remove all the probe components, including 1'Ab,
to ensure no carry-over fluorescence signal and preclude binding of
vacant QD/SpA probes to left-over 1'Ab. At the same time,
de-staining procedure should be gentle enough to preserve specimen
morphology and biomarker antigenicity. Microwave treatment.sup.s,
strong acidic conditions.sup.4, 6, 7, and specimen
dehydration.sup.6 have been used with some success for sequential
staining procedures based on conventional IF and IHC. See, for
example, Pirici, D. et al. Antibody elution method for multiple
immunohistochemistry on primary antibodies raised in the same
species and of the same subtype. J Histochem Cytochem 57, 567575
(2009); Toth, Z. E. & Mezey, E. Simultaneous visualization of
multiple antigens with tyramide signal amplification using
antibodies from the same species. J Histochem Cytochem 55, 545-554
(2007); Glass, G., Papin, J. A. & Mandell, J. W. SIMPLE: a
sequential immunoperoxidase labeling and erasing method. J
Histochem Cytochem 57, 899-905 (2009); and Wahlby, C., Erlandsson,
F., Bengtsson, E. & Zetterberg, A. Sequential
immunofluorescence staining and image analysis for detection of
large numbers of antigens in individual cell nuclei. Cytometry 47,
32-41 (2002). However, elimination of large staining complexes
(often consisting of cross-linked primary and secondary antibodies
conjugated with an enzyme or streptavidin and surrounded by
precipitated dye) can be challenging and can require extensive
chemical or thermal treatment, which can led to biomarker
degradation.
With QD/SpA-Ab probes, quick and efficient de-staining can be
achieved by brief exposure to low-pH buffer since the staining
procedure does not involve formation of large precipitates. The
SpA-Ab bond can be easily broken by exposure to low pH and
biomarker-bound antibodies are free to dissociate from the
specimen. Thus, any residual QD fluorescence could be completely
quenched. The inventors stained AR with QD545/SpA (FIG. 21A) and
de-stained by incubation with pH2 Glycine-HCl/0.1% casein buffer
for 15 minutes. Imaging of the same sub-population of cells
revealed no residual staining (FIG. 21B). To test whether the
original biomarker was de-occupied, the inventors re-stained AR
with the same QD545/SpA-Ab probes during the second cycle,
achieving nearly complete restoration of fluorescence signal (FIG.
21C), whereas re-staining with QD545/SpA alone during the third
cycle produced only minor background staining (FIG. 21D). At the
same time, without de-staining procedure QD staining persisted with
nearly no signal loss for at least 48 hours when cells were
incubated in TBS at 4.degree. C. (FIG. 22). These results show that
low-pH de-staining can be achieved by dissociation of QD/SpA-Ab
probe, either as separate components or as a whole complex, from
the biomarker, thus leaving behind unoccupied biomarkers.
To test whether QD-SpA probe was washed away from the specimen
following dissociation from the biomarker, the inventors labeled
QD/SpA with pH-stable Alexa Fluor 568 dye (as QD fluorescence could
not be used for imaging of QD/SpA probes after de-staining due to
QD quenching). QD525/SpA probes were used for this study to achieve
clear spectral separation between the QD and dye fluorescence. AR
staining with dye-labeled QD/SpA-Ab probes produced characteristic
nuclear staining pattern detectable in both QD and dye channels
(FIG. 23A). Following de-staining QD signal was completely
eliminated, as expected, while barely detectable nuclear signal in
dye channel suggested that minor amount of QD/SpA was left in the
specimen (FIG. 23B). At the same time, QD/SpA-Ab probes
cross-linked to the specimen with BS3 showed similar dye signal
before (FIG. 23C) and after (FIG. 23D) de-staining, confirming that
localization of QD/SpA probe can be reliably detected with organic
dye. Therefore, complete de-staining was achieved primarily by
dissociation of QD/SpA-Ab probe from the biomarker and washing away
from the specimen, while low-pH mediated QD quenching eliminated
minor fluorescence of residual QD probes retained within the
specimen due to non-specific interactions.
The results demonstrate robustness of multiplexed cyclic staining
procedure by performing 2-color AR/MAOA staining on the first cycle
and achieving complete target exchange on the second cycle. In
particular, during the first cycle AR was stained with QD525/SpA-Ab
probes and MAOA was stained with QD565/SpA-Ab. Spectral imaging and
signal unmixing produced characteristic AR and MAOA staining
patterns in QD525 and QD565 channels respectively (FIG. 24, top
row). Then the specimen was de-stained, and biomarker/QD pairs were
switched (i.e. AR was stained with QD565, while MAOA--with QD525).
Imaging of the same sub-population of cells revealed re-staining of
each biomarker with a counterpart probe (FIG. 24, middle row), thus
achieving complete target exchange with no probe cross-talk within
one cycle or between different cycles (FIG. 24, bottom row).
Finally, the inventors assessed the effect of cyclic treatment on
specimen morphology and biomarker antigenicity. The inventors
performed this experiment on cells located in 10 separate wells of
the same 24-well glass-bottom plate, thus ensuring that all cells
were prepared in identical conditions. Then cells were exposed to M
degradation cycles (where M ranged from 1 to 10). Each cycle
consisted of washing, de-staining, and blocking, thus imitating
full IF staining cycle, but skipping the staining step. Treatment
was performed in 5-cycle portions over 2 days, with overnight
incubation in TBS at 4.degree. C. Cells treated for less than 10
cycles were kept in TBS for (10-M) cycles and then were exposed to
degradation conditions. Following cyclic treatment, AR staining was
performed simultaneously on all cells using identical QD545/SpA-Ab
probes. Low- and high-magnification microscopy and image
acquisition was done with the same camera exposure time and other
imaging parameters to enable consistent signal quantification and
comparison of staining intensity between cycles. High-magnification
true-color and unmixed spectral images revealed just minor loss of
staining intensity after 10 degradation cycles (FIG. 25A). To
quantify biomarker degradation, the inventors measured average
nuclear staining intensity throughout a large number of cells
imaged at low magnification. Spectral imaging and deconvolution
were used to remove cell autofluorescence and measure only QD
fluorescence signal. Quantitative analysis revealed no more than
10% signal loss during 10 degradation cycles (FIG. 25B), which was
consistent with qualitative evaluation of low-magnification unmixed
images (FIGS. 25C and 25D). Some variability in nuclear staining
observed here could be assigned to natural variability in AR
expression. It should be noted that successful preservation of
biomarker antigenicity relied on appropriate pre-staining cell
preparation. In particular, the inventors found that optimized cell
fixation and permeabilization were critical. Incomplete cell
fixation with formaldehyde/TBS yielded over 40% signal drop after
10 degradation cycles (FIG. 26), while fixation with
formaldehyde/PBS and Triton X-100 permeabilization failed to
preserve biomarker antigenicity even after one de-staining (data
not shown). At the same time, over-fixation often led to hampered
QD intracellular access, reduced staining efficiency, and enhanced
non-specific nuclear binding.
In order to confirm that complete staining/imaging/de-staining
cycles did not lead to alterations in biomarker antigenicity and
staining efficiency, the inventors repeated the same 1-color
5-cycle sequential staining described above in an opposite
biomarker order, i.e. instead of staining from low-abundance to
high abundance biomarker (FIG. 27, top row), the inventors
performed staining from high-abundance to low-abundance biomarker
(FIG. 27, bottom row). Not only absence of carry-over or crosstalk
signal was observed throughout the 5 cycles, but also consistent
biomarker distribution and staining intensity was demonstrated
independent of the staining sequence. This confirmed the robustness
and reliability of the multiplexed cyclic staining method described
here.
The highly multiplexed cyclic staining methods described herein
provides access to single-cell molecular profiling within the
context of preserved tissue or cell culture morphology. Even within
the same cell culture each cell may exhibit its own unique
features, unique molecular portrait (at least due to variations in
cell state and cycle). Batch analysis of large cell populations
erases such variations, providing simplistic and often deceptive
averaged information. This issue is more pronounced when analysis
of tissue specimens, consisting of multiple cell types, is
attempted, or when the discovery of scarce cells with distinct
molecular profiles is desired (e.g. identification of cancer
progenitor cells). Throughout the studies on LNCaP cell cultures
described herein, the inventors routinely observed variations in
biomarker expression levels between different cells.
Accordingly, disclosed herein is a method for single-cell molecular
profiling. In one aspect, single-cell molecular profiling can be
achieved by (i) parallel multiplexed staining method capable of
utilizing full range of spectrally distinguishable QD probes
simultaneously in a simple 1-step procedure; (ii) sequential
multiplexed staining method permitting performance of multiple
parallel staining procedures on the same specimen, thus
dramatically increasing the number of biomarkers that could be
analyzed; and (iii) a universal QD/SpA platform featuring quick and
simple preparation of an extensive library of functional QD-Ab
probes while exhibiting no cross-talk between different probes
either within a staining cocktail or between different staining
cycles. The utility of the method was demonstrated by performing
5-biomarker staining either in parallel or sequential format,
confirming potential for obtaining at least 25-biomarker profiles.
The number of biomarkers imaged simultaneously was limited by the
current availability of suitable QD probes. While QDs in 500-600 nm
range featured narrow fluorescence emission profiles, thus
permitting reliable signal unmixing, the quality of QDs in 600-700
nm range was lower. With synthesis of higher-quality QDs covering
full visible spectral range and advances in spectral imaging,
simultaneous imaging of over 10 biomarkers can be achieved. At the
same time, the inventors also demonstrated that up to 10 staining
cycles could be performed without significant loss of biomarker
antigenicity and cell morphology. Therefore, comprehensive
single-cell molecular profiles consisting of over 100 biomarkers
can be performed using the methods described herein.
The universal QD/SpA platform, as described herein, provides
flexibility to the method. Preparation of unique biomarker-specific
fluorescent probes represents a significant hurdle for highly
multiplexed detection methods, as direct QD-Ab assembly (either via
non-covalent or covalent bonds) is technically complex,
time-consuming, and prohibitively expensive, while providing
limited flexibility in matching QD-biomarker pairs and requiring
long-term storage of QD-Ab probes. In contrast, QD/SpA featured
on-demand 1-step purification-free QD-Ab assembly along with high
probe stability and specificity. Notably, the SpA-Ab bond proved to
be sufficiently stable to avoid QD-Ab probe cross-talk, while
easily breakable for de-staining purposes. Overall, PEG-coated QDs
combined with SpA exhibited several features useful for an IF
imaging probe: (i) QD-Ab preparation was fast, inexpensive, and
suitable for a wide range of users without specialized technical
skills; (ii) direct biomarker labeling ensured 1:1 correlation
between biomarker expression and fluorescence signal, while high
brightness of QD probes made imaging without a signal amplification
step possible; (iii) the SpA-Ab bond could be easily broken at
low-pH conditions, thus providing a suitable route for QD-Ab probe
disassembly during de-staining step; and (iv) QD fluorescence could
be efficiently quenched by low-pH conditions, eliminating even
minor non-specific staining after each cycle and preventing
build-up of background signal. With such design flexibility, newly
developed QDs and biomarker-specific antibodies can be easily
incorporated within the methods described herein. A wide range of
laboratories can use the cyclic multiplexed staining method
described herein for addressing their specific research goals.
Utilization of a reliable comprehensive single-cell molecular
profiling technique based on cyclic multiplexed staining with QD-Ab
probes can provide a great benefit for both biomedical research and
clinical diagnostics by providing a tool for addressing phenotypic
heterogeneity within large cell populations, opening access to
studying low-abundance events often masked or completely erased by
batch processing, and elucidating biomarker signatures of diseases
critical for accurate diagnosis and targeted therapy.
To the extent not already indicated, it will be understood by those
of ordinary skill in the art that any one of the various
embodiments herein described and illustrated can be further
modified to incorporate features shown in any of the other
embodiments disclosed herein.
All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
* * * * *