U.S. patent application number 14/173138 was filed with the patent office on 2014-08-07 for mass dots: nanoparticle isotope tags.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University Avenue. Invention is credited to Sean C. Bendall, Bernd Bodenmiller, Garry P. Nolan, Erin F. Simonds.
Application Number | 20140221241 14/173138 |
Document ID | / |
Family ID | 46455569 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140221241 |
Kind Code |
A1 |
Nolan; Garry P. ; et
al. |
August 7, 2014 |
Mass Dots: Nanoparticle Isotope Tags
Abstract
Compositions and methods are provided for the use of
nanoparticles, which may be referred to herein as mass dots, as
mass tags for probes such as antibodies, aptamers, nucleic acids,
etc. in multiplexed bioassays with ICP-MS detection.
Inventors: |
Nolan; Garry P.; (San
Francisco, CA) ; Simonds; Erin F.; (Stanford, CA)
; Bendall; Sean C.; (San Mateo, CA) ; Bodenmiller;
Bernd; (Zurich, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior University
Avenue |
Palo Alto |
CA |
US |
|
|
Family ID: |
46455569 |
Appl. No.: |
14/173138 |
Filed: |
February 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13348512 |
Jan 11, 2012 |
8679858 |
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14173138 |
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61461017 |
Jan 11, 2011 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/7.2; 435/7.91; 530/350; 530/367; 530/391.3; 536/23.1 |
Current CPC
Class: |
G01N 2458/20 20130101;
G01N 33/587 20130101; B82Y 5/00 20130101; G01N 33/566 20130101 |
Class at
Publication: |
506/9 ; 530/350;
530/391.3; 536/23.1; 530/367; 435/7.2; 435/6.11; 435/7.91 |
International
Class: |
G01N 33/566 20060101
G01N033/566 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0001] This invention was made with Government support under
contract HV028183 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A probe comprising a specific binding member for an analyte,
conjugated to a mass dot, wherein the mass dot is a high-atomic
mass, non-biological element of a size and uniformity sufficient to
enable highly sensitive detection of very small quantities of
analyte.
2. The probe of claim 1, wherein said high-atomic mass,
non-biological element is a metal with isotopic purity between
about 90 and 100%.
3. The probe of claim 2, wherein said mass dot is at least 100
metal atoms.
4. The probe of claim 2 wherein said mass dot is at least about
10.sup.3 metal atoms.
5. The probe of claim 2, wherein said mass dot is at least about
10.sup.4 metal atoms.
6. The probe of claim 2, wherein said mass dot is free of high
atomic mass dopants.
7. The probe of claim 2, wherein said mass dot is comprised of
non-fluorescent nanocrystals with two or more atomic mass
metals.
8. The probe of claim 2 where said mass dot is comprised of
multiple high atomic mass metals at a known ratio and absolute
quantity.
9. The probe of claim 2, wherein said mass dot comprises a
nanocrystal.
10. The probe of claim 2, wherein said mass dot comprises a
nanoparticle core of said metal, and a low molecular weight
counter-anion.
11. The probe of claim 2, wherein said mass dot comprises a coating
selected from an amphipathic polymer, PEG, silane, siloxane,
silica, or lipids.
12. The probe of claim 2, wherein a single mass dot is conjugated
to said specific binding member.
13. The probe of claim 12, wherein not more than 10 mass dots are
conjugated to said specific binding member.
14. The probe of claim 12, wherein said specific binding member is
a polypeptide.
15. The probe of claim 14, wherein said specific binding member is
an antibody.
16. The probe of claim 12, wherein said specific binding member is
a polynucleotide.
17. The probe of claim 12, wherein said specific binding member is
avidin or streptavidin.
18. A method for the sensitive detection of an analyte, the method
comprising: binding said analyte through specific binding to a
probe as set forth in claim 1; and detecting the presence of said
mass dot.
19. The method of claim 18, wherein said detecting is performed by
ICP-MS.
20. A probe comprising a specific binding member for an analyte,
conjugated to a daughter assayable detector species (DADS), wherein
the DADS is (1) associated with the analyte particle or (2) can be
separated from the analyte particle, measured and registered to the
particle they were derived from.
21. The probe of claim 20, wherein the DADS is a metal encoded nano
object with definable mass over charge ratio.
22. The probe of claim 20, wherein the DADS is a structural unit
encoded nano-object with definable mass over charge ratio.
23. A method for the sensitive detection of an analyte, the method
comprising: binding said analyte through specific binding to a
probe as set forth in claim 20; and detecting the presence of said
probe.
Description
BACKGROUND OF THE INVENTION
[0002] Conventional detection reagents for biological assays
frequently consist of a binding moiety having specificity for the
molecule of interest, conjugated to a moiety with enzymatic or
optical properties. To date, these determinations are generally
facilitated through the use of radiological, fluorescent or
enzymatic tags.
[0003] Among methods of interest for analysis, flow cytometry
provides the means for simultaneous multiparametric analysis of the
physical and/or chemical characteristics of up to thousands of
particles per second, and is routinely used for research and
clinical diagnostic applications, including both particle analysis
and particle sorting. The analysis of cells is of particular
interest. Modern instruments usually have multiple lasers and
fluorescence detectors. Increasing the number of lasers and
detectors allows for simultaneous analysis of multiple labeled
antibodies, and can more precisely identify a target population by
their phenotypic markers.
[0004] In traditional flow cytometry, fluorescently labeled
particles such as live cells, fixed cells, beads, etc. are
individually distinguished and separated based on their
fluorescence and light scatter characteristics. The phenotype of
the particles can be further investigated after they are isolated.
Such traditional flow cytometry methods are limited by the number
of simultaneous parameters that can be measured on a single
particle, and there are problems with overlap of fluorescence
emissions during simultaneous measurement; and background
fluorescence or enzymatic activity. As the number of simultaneous
parameters increases, this spectral overlap severely convolutes
analysis impinging on both the accuracy as well as sensitivity of
the assay.
[0005] In alternative methods of detection, atomic mass
spectrometry measurements have been used in conjunction with stable
isotope tags of rare elements. Existing elemental tagging capture
reagents for use in ICP-MS are based on chelators, such as
ethylenediamine tetraacetic acid (EDTA),
tetraazacyclododecane-tetraacetic acid (DOTA) or
diethylenetriaminepentaacetic acid (DTPA), for example a
maleimide-functionalized polymer of DTPA, with an average length of
between 10 and 30 monomers. Such protocols allow conjugation to a
typical antibody of 6 or 7 polymers, thereby conjugating an average
of 200 tagging isotope atoms per antibody. The sensitivity of this
method is directly related to the number of elemental isotope tags
per detection reagent molecule. The number of polymers that can be
attached is limited to the number of disulfide bonds that can be
broken on the immunoglobulin without disrupting its function. The
number of metal chelating units that can be conjugated to a
detection reagent is also limited because increased numbers can
interfere with the detection reagent or induce nonspecific
interactions and thus interfering or inducing high background in an
assay.
[0006] Other existing nanocrystal labeling reagents include
luminescent nanocrystals, also known as upconversion nanocrystals,
quantum dots, luminescent nanocrystals, and Raman composite
organic-inorganic nanoparticles (COINs), all of which were designed
for optical or electromagnetic labeling. To achieve useful optical
properties, the nanocrystals used in these products contain
high-atomic mass dopants, such as cadmium, tellurium, selenium,
europium, terbium, or neodymium, and contain undefined mixtures of
these rare metals. The high-atomic mass dopants occupy otherwise
useful channels of instrument detection. The mixed nature of the
elements and their isotopes in these reagents makes them less
desirable for atomic mass spectrometry measurement as it would
dilute the signal, thus reducing sensitivity, as well as occupy
multiple measurement channels per reagent that could otherwise be
used as individual reporters.
[0007] There is interest in methods of analysis that provide for
highly sensitive detection of molecules in biological assays, where
multiple parameters can be simultaneously analyzed without signal
overlap. The present invention addresses this issue.
Publications
[0008] U.S. Pat. No. 7,135,296 Baranov: Elemental analysis of
tagged biologically active materials. Winnik et al., J. Anal. At.
Spectrom. 2008; 23(4): 463-469, Development of analytical methods
for multiplex bio-assay with inductively coupled plasma mass
spectrometry. Winnik et al., Angew. Chem. Int. Ed. 2007; 46 (32):
6111-6114, Polymer-Based Elemental Tags for Sensitive Bioassays.
Winnik et al., J. Am. Chem. Soc. 2007; 129(44): 13653-13660,
Lanthanide-containing polymer nanoparticles for biological tagging
applications: Nonspecific endocytosis and cell adhesion; Thickett
et al. (2010) Bio-functional, lanthanide labeled polymer particles
by seeded emulsion polymerization and their characterization by
novel ICP-MS detection. Journal of Analytical Atomic Spectrometry
25 (3):269-281; Abdelrahman et al. (2010) Lanthanide-Containing
Polymer Microspheres by Multiple-Stage Dispersion Polymerization
for Highly Multiplexed Bioassays (vol 131, pg 15276, 2009). Journal
of the American Chemical Society 132 (7):2465-2465, 2010; Berger et
al. (2010) Hybrid nanogels by encapsulation of lanthanide-doped
LaF3 nanoparticles as elemental tags for detection by atomic mass
spectrometry. Journal of Materials Chemistry 20 (24):5141-5150;
Ornatsky et al. (2010) Highly multiparametric analysis by mass
cytometry. J Immunol Methods 361 (1-2):1-20; Bandura et al. (2009)
Mass Cytometry: Technique for Real Time Single Cell Multitarget
Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass
Spectrometry. Analytical Chemistry 81:6813-6822
SUMMARY OF THE INVENTION
[0009] Elemental mass spectrometry-based detection analyzes cells
with binding reagents that are "mass tagged", i.e., tagged with an
element or isotope having a defined mass, e.g. a high-atomic mass,
non-biological element. In the methods of the invention, labeled
particles are introduced into a detector, e.g. a mass cytometer,
atomic mass spectrometer (ICP-MS), etc., where they are atomized
and ionized. The particles or solution are then subjected to
elemental analysis, which identifies and measures the abundance of
the mass tags used. The identities and the amounts of the isotopic
elements associated with each particle or solution sample are then
stored and analyzed.
[0010] The present invention allows for extremely sensitive highly
multiplexed applications, where a large number of elemental tags
are used in simultaneous or sequential detection and measurement of
biologically active material. Multiplex applications may analyze
more than about 10, more than about 15, more than about 20, more
than about 25, more than about 30, more than about 40, more than
about 50 different probes. Analysis of cells is of particular
interest, where the probes may bind to the cell surface, or to
cytoplasmic and nuclear components of permeabilized cells.
Alternatively bead and solution based assays are performed. In some
embodiments the components of signaling pathways are analyzed, e.g.
for changes in post-translational modification of proteins, such as
phosphorylation and the like. Other embodiments include imaging
applications where the detector is a LA-ICPMS (LA-laser ablation),
where the laser scans a sample stained with elemental isotope
reporters
[0011] Compositions and methods are provided for the use of
nanoparticles, which may be referred to herein as mass dots, as
mass tags for probes such as antibodies, aptamers, nucleic acids,
etc. in multiplexed bioassays with ICP-MS detection. A probe may be
conjugated to one or more mass dots; usually not more than about 10
mass dots are conjugated to a single probe. In a preferred
embodiment, the mass dots are comprised of substantially pure
isotope preparations, e.g. at least about 90%, at least about 95%,
at least about 99%, at least about 99.9%, at least about 99.99%, at
least about 99.999%, at least about 99.9999% purity or more.
Preferably the mass dot is free of high atomic mass dopants, as
such dopants can occupy otherwise useful channels of instrument
detection that could be used to detect other species of tagging
isotope. Alternatively, when contamination by additional high
atomic mass elements is unavoidable, the sum of atoms of all of the
tagging isotopes in a composite isotope tag may be calculated
during elemental analysis, and correlated with the abundance of the
mass dot. In a third scenario, two or more isotopes or elements may
be combined in a single reagent at a known stoichiometric ratio and
total abundance for the purpose of multiplexing beyond the limit of
discrete analysis channels on the instrument, with the identity and
abundance of each tag being determined by subsequent deconvolution
of the composite signal by Fourier transformation.
[0012] Mass dots are generally of a size large enough to enable
highly sensitive detection of very small quantities of analyte,
e.g. comprising at least about 500 metal atoms, at least about
10.sup.3 metal atoms, at least about 2.5.times.10.sup.3 metal
atoms, at least about 5.times.10.sup.3 metal atoms, at least about
7.5.times.10.sup.3 metal atoms, at least about 10.sup.4 metal
atoms, and not more than about 10.sup.5 metal atoms. The size may
vary with the specific isotope and chemical formulation, including
surface coatings. The use of a dot of this size increases the
sensitivity of ICP-MS-based assays over conventional chelation
methods by 10- or 100-fold. Solid metal nanoparticles can be
attached to biomolecules at a single linkage site, thereby
minimizing the detrimental effects of protein denaturation which is
required to attach multiple chelators per molecule of detection
reagent. The mass dots in the present invention may be optimized to
contain the highest concentration per unit volume of the desired
high-mass metal atom of a particular isotopic mass. Counter-anions
with small atomic radii and low masses may be used preferentially,
for example fluorine, in the formulation of nanocrystal cores.
Alternatively, counter-anions may be selected that provide useful
functions, such as modification of solubility, hydrophobicity,
hydrophilicity, electrical conductance, magnetic, paramagnetic or
supermagnetic qualities, electrical conducting or superconducting
qualities.
[0013] In some embodiments the mass dots are doped with organic or
amphipathic molecules (e.g. chitosan, oleic acid) to increase
hydrophilicity, or to decorate the surface with moieties that are
amenable to further chemistry (e.g. carboxylic acids, sulfhydryls,
amines, aldehydes, esters, aromatic hydrazides, aromatic aldehydes,
hydroxides, etc.). These types of derivatization moieties may be
incorporated into mass dots by many methods, including
co-crystallization, surface chemistry, or polymer coating. Once
incorporated, these moieties can be functionalized using reagents
such as hetero-bifunctional cross linkers to facilitate covalent
conjugation to detection reagents such as antibodies.
[0014] In other embodiments the mass dot is coated with a silica or
siloxane coating, optionally functionalized with thiol, amine,
carboxyl, aromatic hydrazide, or aromatic aldehyde groups. Silica
is a convenient material for coating metal nanoparticles due to the
ability to form a stable shell around the particle and the relative
non-reactivity of silica in biological environments.
[0015] In other embodiments the mass dot is coated with an
amphipathic polymer coating. The hydrophobic portion of the
amphipathic polymer facilitates binding of the coating to the
hydrophobic metal core, while the hydrophilic portion limits
non-specific binding with biological substrates.
[0016] In some embodiments of the invention, a composition of probe
conjugated to a mass dot as described herein is provided, where the
probe may be dry, or provided in a suitable excipient. In some
embodiments a plurality of such probes are provided, e.g. as a kit
for detection of a cellular phenotype of interest, e.g. a panel of
cancer-associated antigens; a panel of epitopes; a panel of
reagents selective for a signaling pathway of interest, a panel of
pathogen antigens or antibodies specific for pathogen antigens, and
the like. Alternatively a kit may comprise a plurality of mass dots
suitable for conjugation, with such reagents as are require for
conjugating to probes of interest. Kits may also comprise buffers,
controls, instructions for use, and the like.
[0017] In other embodiments of the invention, a DADS: Daughter
Assayable Detector Species is utilized for labeling, where the DADS
may be a metal encoded nano-object (MENOS), as described above, or
may be a structural unit encoded nano-object (SUENOS), which is a
unique molecular weight organic molecule that is detected in mass
spectrometry as a distinct species. Various linkages are useful in
attached a DADS to the particle for analysis, including without
limitation covalent linkage, affinity linkage such as a DNA or
protein ligand, etc.
[0018] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the invention will become apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Equimolar solutions of quantum dots of increasing
size (indicated by increasing emission wavelength--bottom) were
measured by ICP-MS (FIG. 1). As expected, increasing particle size
lead to a proportional increase in Cd signal (Intensity counts per
second) as reported by the Cd.sup.114 measurement, Cd's most
abundant naturally occurring isotope--summarized in the bar graph.
Units are counts per minute.
[0020] FIG. 2. Peripheral blood mononuclear cells (PBMCs) were
stained with the indicated Invitrogen Qdot reagents, or the same
antibody clones conjugated with traditional MaxPAR reagents and the
indicated reported elemental isotope. The results of the MaxPAR and
quantum dot staining as measured by mass cytometry are shown in the
left and center columns, respectively. The same quantum dot labeled
samples was then measured on the LSR-II collecting Qdot655
fluorescence, right column. Positive cell populations are indicated
with the blue box and population frequency as a percentage of the
whole samples is listed.
[0021] FIG. 3. Closer comparison of the CD19 and CD3 quantum dot
results from PBMC staining as measured by mass cytometry (top) and
fluorescence flow cytometry (bottom). The left contour plots show
the difference in background signal on the two platforms as
exemplified by analysis of unstained PBMC samples. It is this lack
of background signal in the mass cytometry analysis with
contributes to the enhanced measurement resolution. The center and
right contour plots show the results of CD3 and CD19 staining on
the same PBMC samples. The median intensity of the positive cell
population (blue box) as measured by atomic mass spectrometry or
fluorescence is shown. CD3 is 15 fold stronger than CD19 by mass
cytometry where CD3 is only 8.5 fold brighter of LSR-II using the
same Qdot 655. Overall, this indicates at least a 76% improvement
of sample resolution between different cell populations by mass
cytometry using the same reagent.
[0022] FIG. 4. Scanning electron micrograph of LaF.sub.3
nanoparticles synthesized in a high-temperature ICP reactor through
evaporation and subsequent condensation.
[0023] FIG. 5: Left: general view of reactor; Right: closer view at
target temperature.
[0024] FIG. 6: Plot of vapor pressure vs temperature for
PrF.sub.3(s)=PrF.sub.3(g).
[0025] FIG. 7: SEM micrographs of PrF3 particles as collected from
the walls.
[0026] FIG. 8: EDX spectrum.
[0027] FIG. 9A: Daughter Assayable Detector Species (DADS), which
can either be Metal Encoded Nano-Objects (MENOs) or Structural Unit
Encoded Nano-Objects-(SUENOs) with definable mass over charge
ratios (m/z), are coupled to binding reagents (BR) via attachment
sites (AS). The BR specifically recognize and bind epitopes on the
mother particle (MP) B. SUENOs with unique mass over charge rations
can be small molecule compounds that for example differ in their
isotopic composition, small peptide tags (here shown with the amino
acid sequence MASSTAG) or polymers consisting of small molecule
compounds/peptides linked with specifically cleavable linkers
(-L-). C. As a result each SUENO or subunits thereof can be
uniquely identified and quantified via its mass to charge
ratio.
[0028] FIG. 10. DADS amplification (in this example the DNA strands
are bound to an antibody) A. The DNA tag is amplified by using
primers which are coupled to DADS via standard PCR approaches.
After n-rounds of amplification the amplified DNA is captured and
separated from the free DADS primer. Subsequently the DADS can be
cleaved and analyzed using mass spectrometry. B. In an alternative
approach, DNA-DADS probes complementary to the amplified DNA strand
are added during the PCR reaction. In every PCR cycle, the probe is
degraded and the DADS is released. After PCR either the remaining
DNA-DADS probes can be captured and subsequently the free DADS can
be measured using mass spectrometry.
DEFINITIONS
[0029] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are described.
[0030] Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
[0031] The headings provided herein are not limitations of the
various aspects or embodiments of the invention. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0032] Mass Dots.
[0033] Mass dots are nanoparticles of a high-atomic mass,
non-biological element. Mass dots are of a size large enough to
enable highly sensitive detection of very small quantities of
analyte by ICP-MS, e.g. comprising at least about 500 metal atoms,
at least about 10.sup.3 metal atoms, at least about
2.5.times.10.sup.3 metal atoms, at least about 5.times.10.sup.3
metal atoms, at least about 7.5.times.10.sup.3 metal atoms, at
least about 10.sup.4 metal atoms, and not more than about 10.sup.5
metal atoms. Mass dots may alternatively be 2+ elements in defined
stochoimetric ratios for multiplexing, or due to impurities.
Preferred counter-anions are of a molecular weight outside the
sensitive detection range of the ICP-MS instrument, and have a
small atomic radius, which provides for a higher number of metal
atoms per unit of space than counter-anions with a larger atomic
radius. Elements of interest include, without limitation, the
lanthanide series of the periodic table, which comprises 15
elements, 14 of which have stable isotopes (La, Ce, Pr, Nd, Sm, Eu,
Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu), and are useful as tagging isotopes
due to their rarity in the biosphere. In other embodiments, tagging
isotopes comprise non-lanthanide elements that can form stable
nanoparticles or nanocrystals for the applications described
herein. These may include the high molecular weight members of the
transition metal (e.g. Rh, Ir, Cd, Au, Ag, Pd, Hf, In),
post-transition metals (e.g. In, Sb, Sn, Pb), metalloids (e.g. Te,
Bi), alkaline metals, halogens, or actinides. In some embodiments,
tagging isotopes may comprise radioactive elements that can form
nanoparticles for the applications described herein (e.g.
.sup.131I). In other embodiments, tagging isotopes comprise any
metal isotope not present in the biological analyte under study,
ideally in the mass range of 100-240 A.M.U.
[0034] Mass dots are generally comprised of substantially pure
isotope preparations, e.g. at least about 90%, at least about 95%,
at least about 99%, at least about 99.9%, at least about 99.99%, at
least about 99.999%, at least about 99.9999% purity or more.
Preferably the mass dot is free of high atomic mass dopants.
[0035] In alternative embodiments, mass dots contain a defined
mixture of a plurality of high-mass elements or isotopes. For
example, mass dots may contain a 1:2 molar ratio of .sup.169Tm and
.sup.159Tb. Objectives of using a defined mixture of high-mass
elements or isotopes may include increasing the molar ratio of
high-mass elements to low-mass elements, conferring stability to
nanoparticles, achieving desired crystal geometries, or avoiding
the expense of eliminating natural impurities. In some embodiments,
the composite nanoparticles are synthesized such that the total
abundance of each element is restricted to a narrow range, allowing
multiplexing of reagents beyond the number of discrete analysis
channels on the instrument. In some embodiments, mass dots may
contain a plurality of high mass elements in the nanoparticle core
(e.g. EuTe nanocrystals), serving as a composite isotope tag.
[0036] Multiple metals at known ratios and abundances can be used
for multiplexing. This is a desirable feature of nanoparticles
because they can be constructed and characterized within very tight
tolerances. By contrast, solution-based methods are subject to
stochasticity and mass action, so each reagent preparation will
have a range of labeling intensities per molecule.
[0037] Although not required, in some embodiments, the
nanoparticles are nanocrystals. Nanocrystals may be formed by de
novo crystallization of an ionic solution, or by micronization of a
larger crystalline solid. It is recognized that ionic interactions
with other elements are required to form a crystal lattice. It is
desirable that the nanocrystal lattices be compact, containing a
high density of tag atoms, e.g. at least about 20, at least about
25, at least about or more 30 tag atoms/nm.sup.3, and containing
only elements that will neither interfere with the signal from
other isotope tags, nor with the detection method itself. For
example, lanthanide elements form stable nanocrystals with a high
density of tag atoms per unit volume, e.g. lanthanum trifluoride
(LaF.sub.3) forms compact, water insoluble nanocrystals with a high
density of lanthanum atoms. Exemplary synthetic methods are
provided in the Examples.
[0038] Crystal structures contain counter-anions that may also be
detected by an elemental analysis platform such as ICP-MS. In some
embodiments of the present invention, nanocrystals may be
synthesized using a single high-mass element as the tagging
isotope, and one or more low-mass elements as counter-anions. An
objective of using low-mass elements as counter-anions is to limit
the amount of signal on detection channels that would be useful for
multiplexed analysis of high-mass isotope tags. Counter-anions with
small atomic radii and low masses may be used preferentially in the
formulation of nanocrystal cores. Alternatively, counter-anions are
selected that provide useful functions, such as modifying
solubility hydrophobicity, hydrophilicity, electrical conductance,
magnetic or supermagnetic qualities, electrical conducting or
superconducting qualities.
[0039] In some embodiments the mass dots are doped with organic or
amphipathic molecules (e.g. chitosan, oleic acid) to increase
hydrophilicity, or to decorate the surface with moieties that are
amenable to further chemistry (e.g. carboxylic acids, sulfhydryls,
amines, aldehydes, esters, hydroxides, aromatic hydrazides,
aromatic aldehydes, etc.). These types of derivatization moieties
may be incorporated into mass dots by many methods, including
co-crystallization, surface chemistry, or polymer coating. The
number of derivatization moieties per mass dot may be tightly
controlled to limit the number of detection reagent molecules that
may be conjugated per mass dot in a later step. Once incorporated,
these moieties can be functionalized using reagents such as
hetero-bifunctional cross linkers to facilitate covalent
conjugation to detection reagents such as antibodies.
[0040] In other embodiments the mass dot is coated with a silica or
siloxane coating, optionally functionalized with thiol, amine,
carboxyl, aromatic hydrazide, or aromatic aldehyde groups. The
number of functional groups per mass dot may be tightly controlled
to limit the number of detection reagent molecules that may be
conjugated per mass dot in a later step. Silica is a convenient
material for coating metal nanoparticles due to the ability to form
a stable shell around the particle and the relative non-reactivity
of silica in biological environments.
[0041] In other embodiments the mass dot is coated with an
amphipathic polymer coating. The hydrophobic portion of the
amphipathic polymer facilitates binding of the coating to the
hydrophobic metal core, while the hydrophilic portion limits
non-specific binding with biological substrates.
[0042] The nanoparticle core of the mass dot may have a defined
size range, which may be substantially homogeneous, where the
variability may be not more than 100% of the diameter, not more
50%, not more than 10%, etc.
[0043] Probes.
[0044] As used here, the term "probe" refers to a specific binding
partner for an analyte of interest, where the probe is generally
conjugated to one or more mass dots as described above. In some
embodiments a single mass dot is conjugated to a probe. In other
embodiments 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mass dots are
conjugated, usually not more than 10, and more usually not more
than 5 mass dots are conjugated.
[0045] MENOs: Metal Encoded Nano-Objects. Nanoscale size particles
with defined ratios of 1 or more isotopes. Each nanoparticle's
signature ratio is distinct such that it would act as a unique DADS
when bound to an BR. To use MENOs, one would create 100s, or 1000s
of MENOs with unique signatures. These would be attached to BRs.
The entire set of MENOs from a given cell are assayed individually
in a manner that allows the cumulative number of given MENOs with a
given ratio of elements to be determined on a per cell basis.
[0046] SUENOs: Structural Unit Encoded Nano-Objects (FIGS. 1B &
C). A A SUENO can be: a) a small molecule of a defined molecular
weight unique from other SUENOs. b) A polymeric substance of a
defined molecular weight unique from other SUENOs. c) A small
molecule of a defined molecular weight, or whose derived ions
generated during mass spectrometry are uniquely generated or have
unique masses and/or charges unique from other SUENOs. d) A polymer
of distinct subunits that together have a defined molecular weight,
or whose derived (i.e. by collision induced dissociation--CID) ions
generated during mass spectrometry are uniquely generated or have
unique masses and/or charges unique from other SUENOs.
[0047] Linkage between the mass dot, MENOS or SUENOS may be to a
covalent, ionic, and/or amphipathic coating, preferably
functionalized with a low molecular weight dopant, using any
suitable linker. Illustrative entities include: Trioctylphosphine
oxide, mercaptopropyltris(methyloxy)silane,
aminopropyltris(methyloxy)silane, tetramethylammonium hydroxide,
tetramethylammonium hydroxide pentahydrate, (trihydroxysilyl)propyl
methylphosphonate, chlorotrimethylsilane, mercaptopropionic acid,
4-(dimethylamino)pridine, 5,5'-dithiobis(2-nitrobenzoic acid),
azidobenzoyl hydrazide,
N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide),
bis-sulfosuccinimidyl suberate, dimethyladipimidate,
disuccinimidyltartrate, N-.gamma.-maleimidobutyryloxysuccinimide
ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl
[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl
[4-iodoacetyl]aminobenzoate, glutaraldehyde, NHS-PEG-MAL;
succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate;
3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP)
or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid
N-hydroxysuccinimide ester (SMCC). Chemical groups that find use as
couplings of Q to X include amide (amine plus carboxylic acid),
ester (alcohol plus carboxylic acid), thioether (haloalkane plus
sulfhydryl; maleimide plus sulfhydryl), Schiff's base (amine plus
aldehyde), urea (amine plus isocyanate), thiourea (amine plus
isothiocyanate), sulfonamide (amine plus sulfonyl chloride),
hydrazide (aromatic hydrazide plus aromatic aldehyde) and the like,
as known in the art.
[0048] The term "specific binding member" as used herein refers to
a member of a specific binding pair, i.e. two molecules, usually
two different molecules, where one of the molecules through
chemical or physical means specifically binds to the other
molecule. The complementary members of a specific binding pair are
sometimes referred to as a ligand and receptor; or receptor and
counter-receptor. Specific binding indicates that the agent can
distinguish a target antigen, or epitope within it, from other
non-target antigens. It is specific in the sense that it can be
used to detect a target antigen above background noise
("non-specific binding"). For example, a specific binding partner
can detect a specific sequence or a topological conformation. A
specific sequence can be a defined order of amino acids or a
defined chemical moiety (e.g., where an antibody recognizes a
phosphotyrosine or a particular carbohydrate configuration, etc.)
which occurs in the target antigen. The term "antigen" is issued
broadly, to indicate any agent which elicits an immune response in
the body. An antigen can have one or more epitopes.
[0049] Binding pairs of interest include antigen and antibody
specific binding pairs, complementary nucleic acids,
peptide-MHC-antigen complexes and T cell receptor pairs, biotin and
avidin or streptavidin; carbohydrates and lectins; complementary
nucleotide sequences; peptide ligands and receptor; effector and
receptor molecules; hormones and hormone binding protein; enzyme
cofactors and enzymes; enzyme inhibitors and enzymes; and the like.
The specific binding pairs may include analogs, derivatives and
fragments of the original specific binding member. For example, an
antibody directed to a protein antigen may also recognize peptide
fragments, chemically synthesized peptidomimetics, labeled protein,
derivatized protein, etc. so long as an epitope is present.
[0050] Immunological specific binding pairs include antigens and
antigen specific antibodies; and T cell antigen receptors, and
their cognate MHC-peptide conjugates. Suitable antigens may be
haptens, proteins, peptides, carbohydrates, etc. Recombinant DNA
methods or peptide synthesis may be used to produce chimeric,
truncated, or single chain analogs of either member of the binding
pair, where chimeric proteins may provide mixture(s) or fragment(s)
thereof, or a mixture of an antibody and other specific binding
members. Antibodies and T cell receptors may be monoclonal or
polyclonal, and may be produced by transgenic animals, immunized
animals, immortalized human or animal B-cells, cells transfected
with DNA vectors encoding the antibody or T cell receptor, etc. The
details of the preparation of antibodies and their suitability for
use as specific binding members are well-known to those skilled in
the art.
[0051] A nucleic acid based binding partner such as an
oligonucleotide can be used to recognize and bind DNA or RNA based
analytes. The term "polynucleotide" as used herein may refer to
peptide nucleic acids, locked nucleic acids, modified nucleic
acids, and the like as known in the art. The polynucleotide can be
DNA, RNA, LNA or PNA, although it is not so limited. It can also be
a combination of one or more of these elements and/or can comprise
other nucleic acid mimics.
[0052] Binding partners can be primary or secondary. Primary
binding partners are those bound to the analyte of interest.
Secondary binding partners are those that bind to the primary
binding partner.
[0053] Analytes.
[0054] As used herein, analytes refers to quantifiable components
of cells or biological material, particularly components that can
be accurately measured. An analyte can be any cell component or
cell product including cell surface determinant, receptor, protein
or conformational or posttranslational modification thereof, lipid,
carbohydrate, organic or inorganic molecule, nucleic acid, e.g.
mRNA, DNA, etc. or a portion derived from such a cell component or
combinations thereof. Some variability may be expected and a range
of values may be obtained using standard statistical methods with a
common statistical method used to provide single values.
[0055] Analytes of interest include cytoplasmic, cell surface or
secreted biomolecules, frequently biopolymers, e.g. polypeptides,
polysaccharides, polynucleotides, lipids, etc. In some embodiments,
analytes include specific epitopes. Epitopes are frequently
identified using specific monoclonal antibodies or receptor probes.
In some cases the molecular entities comprising the epitope are
from two or more substances and comprise a defined structure;
examples include combinatorially determined epitopes associated
with heterodimeric proteins. An analyte may be detection of a
specifically modified protein or oligosaccharide, e.g. a
phosphorylated protein, such as a STAT transcriptional protein; or
sulfated oligosaccharide, or such as the carbohydrate structure
Sialyl Lewis x, a selectin ligand. The presence of the active
conformation of a receptor may comprise one analyte while an
inactive conformation of a receptor may comprise another, e.g. the
active and inactive forms of heterodimeric integrin.
[0056] Analytes of interest include biological molecules in a
variety of spatial configurations, on a variety of substrates, and
in a variety of degraded states. An analyte may be a naturally
occurring protein in its native conformation or chemically altered,
denatured state. An analyte may be affixed to the cell in its
native orientation, or it may be adhered to a variety of
substrates, including synthetic substrates e.g. glass, plastic, or
metal. An analyte may be affixed to planar substrates or bead-like
substrates in a suspension. An analyte may be affixed to a
substrate in a particular orientation by a second binding reagent,
such as an antibody, as in the case of a sandwich ELISA.
[0057] Analytes of interest include polypeptides, and the epitope
that is being quantitated by be a primary amino acid epitope, an
epitope formed by protein secondary or tertiary structure, an
epitope formed by two or more interacting polypeptides, or an
epitope that results from posttranslational modification of a
polypeptide.
[0058] Among the post-translational modifications that can be
probed, are protein specific glycoslyation. Membrane associated
carbohydrate is exclusively in the form of oligosaccharides
covalently attached to proteins forming glycoproteins, and to a
lesser extent covalently attached to lipid forming the glycolipids.
Many proteins are modified at their N-termini following synthesis;
in most cases the initiator methionine is hydrolyzed and an acetyl
group is added to the new N-terminal amino acid. Post-translational
methylation occurs at lysine residues in some proteins.
Post-translational phosphorylation is one of the most common
protein modifications that occurs in animal cells, often as a
transient mechanism to regulate the biological activity of a
protein. In animal cells serine, threonine and tyrosine are the
amino acids subject to phosphorylation. Sulfate modification of
proteins occurs at tyrosine residues such as in fibrinogen and in
some secreted proteins. Prenylation refers to the addition of the
15 carbon farnesyl group or the 20 carbon geranylgeranyl group to
acceptor proteins, both of which are isoprenoid compounds derived
from the cholesterol biosynthetic pathway. Modifications of
proteins that depend upon vitamin C as a cofactor include proline
and lysine hydroxylations and carboxy terminal amidation. Vitamin K
is a cofactor in the carboxylation of glutamic acid residues that
results in the formation of a .gamma.-carboxyglutamate
(gamma-carboxyglutamate), referred to as a gla residue.
[0059] Cells.
[0060] Cells for use in the assays of the invention can be an
organism, a single cell type derived from an organism, or can be a
mixture of cell types. Included are naturally occurring cells and
cell populations, genetically engineered cell lines, cells derived
from transgenic animals, etc. Virtually any cell type and size can
be accommodated. Suitable cells include bacterial, fungal, plant
and animal cells. In one embodiment of the invention, the cells are
mammalian cells, e.g. complex cell populations such as naturally
occurring tissues, for example blood, liver, pancreas, neural
tissue, bone marrow, skin, and the like. Some tissues may be
disrupted into a monodisperse suspension. Alternatively, the cells
may be a cultured population, e.g. a culture derived from a complex
population, a culture derived from a single cell type where the
cells have differentiated into multiple lineages, or where the
cells are responding differentially to stimulus, and the like.
[0061] Cell types that can find use in the subject invention
include stem and progenitor cells, e.g. embryonic stem cells,
hematopoietic stem cells, mesenchymal stem cells, neural crest
cells, etc., endothelial cells, muscle cells, myocardial, smooth
and skeletal muscle cells, mesenchymal cells, epithelial cells;
hematopoietic cells, such as lymphocytes, including T-cells, such
as Th1 T cells, Th2 T cells, Th0 T cells, cytotoxic T cells; B
cells, pre-B cells, etc.; monocytes; dendritic cells; neutrophils;
and macrophages; natural killer cells; mast cells; etc.;
adipocytes, cells involved with particular organs, such as thymus,
endocrine glands, pancreas, brain, such as neurons, glia,
astrocytes, dendrocytes, etc. and genetically modified cells
thereof. Hematopoietic cells may be associated with inflammatory
processes, autoimmune diseases, etc., endothelial cells, smooth
muscle cells, myocardial cells, etc. may be associated with
cardiovascular diseases; almost any type of cell may be associated
with neoplasias, such as sarcomas, carcinomas and lymphomas; liver
diseases with hepatic cells; kidney diseases with kidney cells;
etc.
[0062] The cells may also be transformed or neoplastic cells of
different types, e.g. carcinomas of different cell origins,
lymphomas of different cell types, etc. The American Type Culture
Collection (Manassas, Va.) has collected and makes available over
4,000 cell lines from over 150 different species, over 950 cancer
cell lines including 700 human cancer cell lines. The National
Cancer Institute has compiled clinical, biochemical and molecular
data from a large panel of human tumor cell lines, these are
available from ATCC or the NCI (Phelps et al. (1996) Journal of
Cellular Biochemistry Supplement 24:32-91). Included are different
cell lines derived spontaneously, or selected for desired growth or
response characteristics from an individual cell line; and may
include multiple cell lines derived from a similar tumor type but
from distinct patients or sites.
[0063] Cells may be non-adherent, e.g. blood cells including
monocytes, T cells, B-cells; tumor cells, etc., or adherent cells,
e.g. epithelial cells, endothelial cells, neural cells, etc. In
order to profile adherent cells, they must be dissociated from the
substrate that they are adhered to, and from other cells, in a
manner that maintains their ability to recognize and bind to probe
molecules.
[0064] Such cells can be acquired from an individual using, e.g., a
draw, a lavage, a wash, surgical dissection etc., from a variety of
tissues, e.g., blood, marrow, a solid tissue (e.g., a solid tumor),
ascites, by a variety of techniques that are known in the art.
Cells may be obtained from fixed or unfixed, fresh or frozen, whole
or disaggregated samples. Disaggregation of tissue may occur either
mechanically or enzymatically using known techniques.
[0065] Various methods and devices exist for pre-separating
component parts of the sample. These methods include filters,
centrifuges, chromatographs, and other well-known fluid separation
methods; gross separation using columns, centrifuges, filters,
separation by killing of unwanted cells, separation with
fluorescence activated cell sorters, separation by directly or
indirectly binding cells to a ligand immobilized on a physical
support, such as panning techniques, separation by column
immunoadsorption, and separation using magnetic immunobeads.
[0066] As used herein, the term "elemental analysis" refers to a
method by which the presence and/or abundance of elements of a
sample are evaluated. "Capacitively coupled plasma" (CCP) means a
source of ionization in which a plasma is established by capacitive
coupling of radiofrequency energy at atmospheric pressure or at a
reduced pressure (typically between 1 and 500 Torr) in a graphite
or quartz tube. The term "inductively coupled plasma" (ICP) means a
source of atomization and ionization in which a plasma is
established in an inert gas (usually argon) by the inductive
coupling of radiofrequency energy. The frequency of excitation
force is in the MHz range. The term "plasma source" means a source
of atoms or atomic ions comprising a hot gas (usually argon) in
which there are approximately equal numbers of electrons and ions,
and in which the Debye length is small relative to the dimensions
of the source. The term "flow cell" refers to a conduit in which
particles flow, in a medium, one by one in single file. The term "a
diverter" refers to a branch of a flow cell in which particles can
be separated from other components passing through the flow cell.
"Laser ablation" means a source of combusted material liberated
from an otherwise intact surface by exposure to laser radiation,
optionally used in conjunction with microscopy to preserve spatial
information. "Mass spectrometer" means an instrument for producing
ions in a gas and analyzing them according to their mass/charge
ratio. "Microwave induced plasma" (MIP) means a source of
atomization and ionization in which a plasma is established in an
inert gas (typically nitrogen, argon or helium) by the coupling of
microwave energy. The frequency of excitation force is in the GHz
range. "Glow discharge" (GD) means a source of ionization in which
a discharge is established in a low pressure gas (typically between
0.01 and 10 Torr), typically argon, nitrogen or air, by a direct
current (or less commonly radiofrequency) potential between
electrodes. "Graphite furnace" means a spectrometer system that
includes a vaporization and atomization source comprised of a
heated graphite tube. Spectroscopic detection of elements within
the furnace may be performed by optical absorption or emission, or
the sample may be transported from the furnace to a plasma source
(e.g. inductively coupled plasma) for excitation and determination
by optical or mass spectrometry.
[0067] Preferred methods for analysis of mass dots utilize ICP-MS.
In some embodiments the ICP-MS is performed with solution analysis,
for example ELAN DRC II, Perkin-Elmer. In other embodiments the
analysis is performed with a mass cytometer (e.g. CyTOF, DVS
Sciences), which uses a nebulizer to administer a suspension of
cells, beads, or other particles in a single-particle stream to an
ICP-MS chamber, thereby yielding single particle/cell data similar
to a flow cytometer. Alternatively the analysis is performed by an
elemental analysis-driven imaging system (e.g. laser ablation
ICP-MS). Devices for such analytic methods are known in the
art.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0068] Compositions and methods are provided for the sensitive
detection of an analyte through specific binding to a probe labeled
with a mass dot, i.e. a nanoparticle of a high-atomic mass,
non-biological element of a size and uniformity sufficient to
enable highly sensitive detection of very small quantities of
analyte, and usually substantially pure isotope of the high-atomic
mass element.
[0069] Analytes of interest include any molecule in which a
specific binding partner can be devised and labeled with a mass
dot, e.g. arrays of polynucleotides or proteins, histochemistry
slides, ELISA plates, and the like. The methods of the invention
find particular use in highly multiplexed applications, where
multiple different probes are applied to a sample.
[0070] In one embodiment, the analyte is present on cells or beads
in a single particle suspension. Examples of cells of interest are
described above. If beads are employed, the beads can range in size
from 20 nM to 200 .mu.M or larger, and may be made of polystyrene,
but other materials such as polymethylmethacrylate (PMMA),
polyvinyltoluene (PVT), styrene/butadiene (S/B) copolymer,
styrene/vinyltoluene (S/VT) can also used. Alternatively analytes
may be bound to a solid substrate, e.g. glass, plastic, etc.
[0071] The analyte is contacted with the mass dot labeled probe,
and incubated for a period of time sufficient to bind the available
analyte. The incubation will usually be at least about 2 minutes
and usually less than about 24 hours. It is desirable to have a
sufficient concentration of probe in the reaction mixture so that
the efficiency of detection is not limited by lack of probe. The
appropriate concentration is determined by titration. Where the
labeling is direct, the probe is labeled with a mass dot. Where the
labeling is indirect, a second stage probe or label can be used, by
washing and resuspending in medium prior to incubation with the
second stage probes.
[0072] Where the analyte is present in or on cells, the cells may
be labeled on the surface, or may be located in the cytoplasm or
nucleus of the cell. For such intracellular labeling it is
generally desirable to fix and permeabilize the cells. For example,
where transient signaling pathways are being analyzed, it is
desirable to fix the cells at the desired timepoint, then
permeabilize to allow the probes access to the intracellular
environment. Various fixatives are known in the art, including
formaldehyde, paraformaldehyde, formaldehyde/acetone,
methanol/acetone, etc. Paraformaldehyde used at a final
concentration of about 1 to 2% has been found to be a good
cross-linking fixative. Permeabilizing agents are known in the art,
and include mild detergents, such as Triton X-100, NP-40, saponin,
etc.; methanol, and the like. It may also be desirable to label
cells with a positive heavy metal control, e.g. a DNA intercalator
labeled with a heavy metal, e.g. iridium, etc. Cells may also be
stained with a viability dye prior to fixation, e.g. ethidium
bromide, RhCl.sub.3, etc., as known in the art.
[0073] The analyte is washed of unbound probe using any suitable
method known in the art. For example cells or beads may be pelleted
and washed in PBS or normal saline; polynucleotide arrays, beads,
blots and the like are washed with a buffer of suitable stringency;
and the like as known in the art.
[0074] The labeled analyte is then analyzed by, for example,
inductively coupled plasma mass spectrometry (ICP-MS) identity to
determine the abundance of the mass tag for the particle or other
element, methods for performance of which are readily adapted from
known methods. In particular embodiments the mass dots are
vaporized, atomized and ionized by plasma (e.g., inductively
coupled plasma) to produce ions that are subsequently analyzed by a
mass spectrometer or emision spectroscopy to provide the identity
and/or determine the abundance of the mass dots. The data produced
by the elemental analysis of the mass dots.
[0075] Where particles or cells are being analyzed, they may be
analyzed on a mass cytometer, in which cells are introduced into a
fluidic system and introduced into the mass cytometer one cell at a
time. In one embodiment, cells are carried in a liquid suspension
and sprayed into a plasma source by means of a nebulizer. In
another embodiment, the cells may be hydrodynamically focused one
cell at a time through a flow cell using a sheath fluid. In
particular embodiments, the particle may be compartmentalized in
the flow cell by introduction of an immiscible barrier, e.g., using
a gas (e.g., air or nitrogen) or oil, such that the particle is
physically separated from other particles that are passing through
the flow cell. The particles may be compartmentalized prior to or
during introduction of the particle into the flow cell by
introducing an immiscible material (e.g., air or oil) into the flow
path.
[0076] The general principles of mass cytometry, including methods
by which single cell suspensions can be made, methods by which
cells can be labeled using, e.g., mass-tagged antibodies, methods
for atomizing particles and methods for performing elemental
analysis on particles, as well as hardware that can be employed in
mass cytometry, including flow cells, ionization chambers,
reagents, mass spectrometers and computer control systems are known
and are reviewed in a variety of publications including, but not
limited to Bandura et al Analytical Chemistry 2009 81 6813-6822),
Tanner et al (Pure Appl. Chem 2008 80: 2627-2641), U.S. Pat. No.
7,479,630 (Method and apparatus for flow cytometry linked with
elemental analysis) and U.S. Pat. No. 7,135,296 (Elemental analysis
of tagged biologically active materials); and published U.S. patent
application 20080046194, for example, which publications are
incorporated by reference herein for disclosure of those methods
and hardware.
[0077] The results of such analysis may be compared to results
obtained from reference compounds, concentration curves, controls,
etc. The comparison of results is accomplished by the use of
suitable deduction protocols, Al systems, statistical comparisons,
etc.
[0078] In particular embodiments, the method described above may be
employed in a multiplex assay in which a heterogeneous population
of cells is labeled with a plurality of distinguishably mass dot
labeled binding agents (e.g., a number of different antibodies). As
there are more than 80 naturally occurring elements having more
than 250 stable isotopes, the population of cells may be labeled
using at least 5, at least 10, at least 20, at least 30, at least
50, or at least 100, up to 150 or more different binding agents
(that bind to, for example different cell surface markers) that are
each tagged with a different isotopically pure mass dot. After the
population of cells is labeled, the cells are introduced into the
flow cell, and individually analyzed using the method described
above.
[0079] A database of analytic information can be compiled. These
databases may include results from known cell types, references
from the analysis of cells treated under particular conditions, and
the like. A data matrix may be generated, where each point of the
data matrix corresponds to a readout from a cell, where data for
each cell may comprise readouts from multiple mass dot labels. The
readout may be a mean, median or the variance or other
statistically or mathematically derived value associated with the
measurement. The output readout information may be further refined
by direct comparison with the corresponding reference readout. The
absolute values obtained for each output under identical conditions
will display a variability that is inherent in live biological
systems and also reflects individual cellular variability as well
as the variability inherent between individuals.
Kits
[0080] Also provided by the present disclosure are kits for
practicing the method as described above. The subject kit contains
reagents for performing the method described above and in certain
embodiments may contain a plurality of labeled specific binding
reagents, wherein each of the labeled specific binding reagent
specifically binds a different target and each of the mass dot tags
are distinguishable from one another by elemental analysis. The kit
may also contain a reference sample to which results obtained from
a test sample may be compared.
[0081] In addition to above-mentioned components, the subject kit
may further include instructions for using the components of the
kit to practice the methods described herein. The instructions for
practicing the subject method are generally recorded on a suitable
recording medium. For example, the instructions may be printed on a
substrate, such as paper or plastic, etc. As such, the instructions
may be present in the kits as a package insert, in the labeling of
the container of the kit or components thereof (i.e., associated
with the packaging or subpackaging) etc. In other embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, e.g. CD-ROM,
diskette, etc. In yet other embodiments, the actual instructions
are not present in the kit, but means for obtaining the
instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate. In addition to above-mentioned components, the subject
kit may include software to perform comparison of data.
Utility
[0082] Exemplary analytic methods employing the above-described
method include, for example, antigen identification, disease
diagnostics, and the like, particularly methods in which high
levels of sensitivity and multiplexing are required.
[0083] It is to be understood that this invention is not limited to
the particular methodology, protocols, cell lines, animal species
or genera, and reagents described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0084] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. All technical and scientific terms used herein have the
same meaning as commonly understood to one of ordinary skill in the
art to which this invention belongs unless clearly indicated
otherwise.
[0085] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
EXPERIMENTAL
Example 1
Quantitative Comparison of CyTOF and LSR2 Sensitivity to
Qdot-Stained Cells
[0086] In order to assess the feasibility of applying nanoparticles
to bioassays with atomic mass spectrometry detection we used
commercially available quantum dots (QDots.TM.) as a test material
because they fall within the general physical requirements for mass
dot applications. Non-isotopically-enriched Qdots were were
measured by summing the abundance of multiple cadmium isotopes,
which are detected as distinct masses by the instrument. Quantum
dots are nanocrystals primarily composed of the element cadmium
doped with small amounts of the element tellurium. By controlling
the amount of dopant and the size of the quantum dot the
fluorescent properties can be modulated--where larger quantum dots
generally have longer emission wavelengths. Cadmium contains 8
stable mass isotopes between the atomic masses of 106 and 116.
While not ideal, the signal for these particles was measured by
monitoring the 6 most abundant isotopes. Also of note, if
isotopically pure Qdots were available, they could potentially add
8 additional mass channels for simultaneous quantification by
atomic mass spectrometry in biological assays.
[0087] First, to assess efficiency of the ability to measure
reporter elements from nanocrystals/nanoparticles, equimolar
solutions of quantum dots of increasing size (hence increasing
emission wavelength) were measured by ICP-MS (FIG. 1). As expected,
increasing particle size lead to a proportional increase in Cd
signal as measured by Cd.sup.114, Cd's most abundant naturally
occurring isotope. These results indicate that increasing number of
atoms present in a solid nanoparticle, as predicted, will result in
a proportion increase in measured reporter signal.
[0088] To assess the utility of these particles in bioassays,
single cell analysis was performed on samples stained with
nanoparticle reagents (Quantum Dots, Invitrogen) in comparison to
conventional elemental reporter labeling reagents (MaxPAR, DVS
sciences) as measured by a single cell CyTOF.TM. mass cytometer
(FIG. 2). The same samples stained with the quantum dot reagents
were then measured by traditional fluorescence flow cytometry to
confirm the accuracy of measurement and compare the fluorescence
and atomic mass spectrometry detection platforms (FIG. 2-3). Here,
peripheral blood mononuclear cells (PBMCs) were stained with the
indicated QDot reagents, or the same antibody clones conjugated
with traditional MaxPAR reagents and the indicated reported
elemental isotope. For the quantum dot reagents the same tube was
first run on CyTOF (collecting cadmium ion abundance across the 6
most abundant isotopes), then on the LSR-II (collecting Qdot655
fluorescence) (FIG. 2-3).
[0089] Quantum dot measurements by mass cytometry revealed similar
cell frequencies and a higher overall signal compared to
conventional mass cytometry reagents (FIG. 2 left and center). The
fact that the quantum dot reagents already yield a higher
single-cell signal as compared to conventional mass cytometry
(MaxPAR) reagents is promising as Cd falls into the range of lower
sensitivity, as compared to the Lanthanide metals used in the
MaxPAR reagents. Additionally, the Cd signal here is split between
8 analysis channels, requiring 8, as opposed to 1, atomic mass
spectrometry limits of detection to be overcome. Again, as high MW
elements of a purely monoisotopic nature are utilized we expect the
benefits of the mass dot technology to become evermore apparent.
Further to this, the accuracy of mass dot analysis by mass
cytometry was confirmed by the similar frequencies observed through
fluorescence analysis of the same samples by LSR-II fluorescence
flow cytometry (FIG. 2-left).
[0090] Finally, quantitative comparison of the mass cytometry and
fluorescence values for the same samples (FIG. 3) revealed that,
when the mass cytometry measurements are on-scale, mass dot
analysis as measure on the CyTOF has 76% better resolution between
positive and negative cell populations compared to the same
analysis by fluorescence. (CD3 is 15 fold stronger than CD 19 on
the CyTOF using the Cd channels to measure Qdot 655; CD3 is only
8.5 fold brighter of LSR-II using Qdot 655).
[0091] Collectively, these examples demonstrate Mass Dot
feasibility, utility, as well as their potential improvement over
existing technologies as a reporter in single cell analysis and
other bioassays.
Example 2
Preparation of 20 Nm-Diameter, Chitosan-Impregnated EuF.sub.3
Nanocrystals
[0092] Combine:
TABLE-US-00001 Volume Concentration Compound Source 10 ml 0.2M
.sup.151EuCl.sub.3 98% enriched isotope Sigma 25 ml 1% w/v (in
0.05M HCl) Chitosan, highly Fluka viscous 10 ml 0.12 mols (0.2222 g
in NH.sub.4F Sigma 10 mL water)
Titrate pH to 6.5 with dilute ammonia. React 2 hrs at 75*C.
Nanocrystals will form spontaneously. Centrifuge to purify
nanocrystals. Wash multiple times with deionized water with 0.5%
v/v acetic acid. Nanocrystals can be stored in deionized water at
this point. In 1 ml of phosphate buffered saline (PBS) react 1 mg
of antibody with 0.4 mg of
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDAC)
and 0.6 mg N-hydroxysuccinimide (NHS) to conjugate. After 15 min
add 4 mg of nanocrystals and react for 2 hrs at ambient
temperature. Quench reaction by adding 0.2 mL of 1M glycine pH 8.
Wash several times in PBS.
Example 3
Preparation of 8 nm-Diameter, Oleic Acid-Impregnated LaF.sub.3
Nanocrystals
[0093] Combine:
TABLE-US-00002 Volume Concentration Compound Source 1 ml 0.5M
.sup.139La(NO.sub.3).sub.3 Sigma 2 ml 1.0M NaF Sigma 20 ml 100%
Ethanol Sigma 1.2 ml 100% Deionized water Fisher 10 ml 100% Oleic
Acid Sigma
Stir the solution thoroughly until it becomes milky colloidal.
Transfer it to a 50 mL Teflon lined autoclave and heat at 190*C for
6 h. Allow cooling to room temperature and collect the final
product by means of centrifugation. Wash multiple times with
ethanol to remove any possible remnants, and then dispersed in
cyclohexane. At this point nano crystals could be functionalized
with a photoreactive cross-linker. Alternatively, the carboxylic
acid in the nanocrystal embedded oleic acid is reduced to an
alcohol for further functionalized through treatment with a
compound such as LiAlH under anhydrous conditions.
Example 4
Alternative Antibody Cross-Linking for Chitosan-Impregnated
Nanocrystals
[0094] Synthesize nanocrystals according to Example 2 protocol,
steps 1 through 5. Resuspend 2 mg of chitosan-impregnated
nanocrystals (as in Example I) in 0.3 mL of 0.1M phosphate, pH 7.0.
Add 16 mg (approximately 100-fold molar excess) of
4-(N-Maleimidomethyl)cyclohexanecarboxylic acid
N-hydroxysuccinimide ester (Sigma) dissolved in 20 ul of
N,Ndimethylformamide (DMF). Incubate for 1 hr stirring continuously
at 30 degrees Celsius. Remove any precipitated by low speed
centrifugation. Remove any free succinimide ester linker by gel
filtration using a PD-10 column (Pharmacia) with a 0.1M phosphate
buffer, pH 6.0. Concentrate the nanocrystal-containing fraction via
centrifugal filtration. Lyophilize for long-term storage. In 0.1M
sodium phosphate buffer, pH 6.5 with 5 mM (EDTA), combine a
pre-reduced antibody preparation with maleimide-containing
nanocrystals in a 1:1 ratio by weight. Incubate for 1 hr at 37
degrees Celsius. Purify the antibody/nanocrystal conjugate using
size exclusion chromatography or differential
ultra-centrifugation.
Example 5
Nanoparticle Synthesis by Evaporative Condensation
[0095] To synthesize nanoparticles of LaF.sub.3 and other RE
fluorides of about 50 nm diameter.
[0096] We selected two approaches to produce nanoparticles of this
material. One is based on evaporation condensation, and the other
in the synthesis in the gas phase using a variation of the
atmospheric pressure CVD using organometallic precursors. This
report describes the work using evaporation condensation
process.
[0097] Powders of LaF3 of high purity were loaded in a high purity
graphite crucible with polished walls. The loaded crucible was
thermally insulated using a layer of graphite felt and placed on a
ceramic pedestal in a gas tight water cooled quartz jacket. Argon
gas can be flown pass the crucible and injected through the top of
the quartz jacket. The system was heated by direct induction
provided by external coils powered by a Westinghouse 450 KHz Radio
Frequency power supply. The temperature was measured by an Ircon
dual wave length pyrometer.
[0098] The system was heated to temperatures above 1500.degree. C.
to melt the LaF.sub.3 and obtain vapor pressures in the tens of
millitorr range. The hot molecules of LaF.sub.3 in the gas phase
emerge from the crucible to find a much cooler region. Assuming
that the vapor pressure inside the crucible was near the
equilibrium value, as the gas molecules cool down the equivalent
equilibrium pressure will drop by several orders of magnitude and a
degree of supersaturation will occur and, the molecules will have a
tendency to condense to form droplets (above 1440.degree. C.) and
solids below that temperature. Using the Kelvin equation we can get
an approximate idea of the diameter of the droplets/powders
formed:
Dp=4*Surf Tension 8.molec volume)/kB T Ln(Saturation Ratio)
[0099] The diameter of the first to form particles, and the rate of
nucleation are heavily dependent on the saturation ratio and on the
surface energy of the species. Note that at the initial stages of
condensation, the supersaturation has to be significantly high to
compensate for the tendency of small (Angstrom radius) droplets to
evaporate. As the cooling progresses, already formed particles will
collide with each other and form agglomerates. By the time that the
supersaturation is over 3 times, the condensation will expedite. It
is expected that significant formation can happen while the
LaF.sub.3 is liquid and very fast nucleation will happen after it
solidifies.
[0100] The incoming Ar gas is significantly cooler that the
effluent vapors and the wall of the reactor are kept close to room
temperature. Once formed, the nuclei grow by agglomeration (the
rate of agglomeration is proportional to the concentration of
nuclei to the square as in bimolecular collision theory) and then
the agglomerates are propelled to the cool walls by thermophoretic
forces, where they form a porous coating of independent
particles.
[0101] The particles were collected by washing the walls of the
reactor with alcohol and kept in dispersion in a glass vial.
Samples were taken by drying a droplet on a graphite substrate and
analyzing the resulting particles by SEM. Examples of these
particles are shown in FIG. 4.
Example 6
PrF.sub.3 Nanoparticle Synthesis by Chemical Vapor Generation in a
Fluidized Bed Reactor (CVG-FBR)
[0102] Powders of PrF.sub.3 of high purity were loaded in a high
purity graphite crucible with polished walls. The loaded crucible
(18 mm ID, 30 mm OD, h=70 mm) was thermally insulated using a layer
of graphite felt (total OD=40 mm) and placed on a porous graphite
pedestal in a gas tight water cooled quartz jacket (72 mm ID) as
shown in FIG. 5. Argon gas can be flown pass the crucible and
injected through the top of the quartz jacket. The system was
heated by direct induction provided by external coils powered by a
Westinghouse 450 KHz radio frequency power supply. The temperature
was measured at the crucible wall (close to the base, through an
orifice in the felt insulator) by means of a dual wave length
pyrometer.
[0103] Ar gas was fed through different ports in the reactor: 1000
sccm were supplied from the top directly into the main reactor
space; 1100 sccm were fed through the graphite center tube, which
had the open end at the same height as the top part of the
crucible; finally, 350 sccm were fed through the tube holding the
pedestal, which had lateral openings at its wide section
(funnel-like zone). The system was heated at 1580.degree. C. for
one hour. This temperature is above the melting point of PrF.sub.3
and the expected vapor pressure is of the order of several
Torr.
[0104] As explained in Example 5 for the synthesis of LaF.sub.3
nanoparticles, the hot molecules of PrF.sub.3 in the gas phase
emerge from the crucible to find a much cooler region, where
supersaturation occurs and the molecules have a tendency to form
droplets or, below the melting point, solids (FIG. 6). In our
reactor, the incoming Ar gas is significantly cooler that the
effluent vapors and the wall of the reactor are kept close to room
temperature. The nuclei grow by agglomeration and then are
propelled to the cool walls by thermophoretic forces, where they
form a porous coating of independent particles. The particles were
scraped from the walls and dispersed in isopropyl alcohol.
[0105] FIG. 7 shows SEM micrographs of particles as collected from
the walls, and also particles dispersed in alcohol after the
suspension have been let to settle for one day. Most of the
particles have sizes of the order of tens of nanometers and, as we
observed in the case of LaF.sub.3, several of the particles have
sizes in the range 100-200 nm. From the practical point of view,
the larger particles can be allowed to settle or centrifuged and
only that naturally dispersed particles used for this application.
FIG. 8 depicts the EDX spectrum corresponding to an area like the
one shown in FIG. 7. As expected, Pr and F peaks are observed, as
well as an O peak corresponding to the background (conductive
tape).
Example 7
Deca, Centi, Through Mega Parameter Detection on Single Particles
Using Mass Spectrometry
[0106] Traditional flow cytometry allows for fluorescently labeled
live cells, fixed cells, beads, or objects (referred herein as
mother particles (MP)) to be individually distinguished and
separated using cytometric sorting technology based on their
fluorescent and light scatter characteristics. This approach is
particularly advantageous because it allows for further functional
or analytical characterization of individually purified MPs on a
phenotypic basis. At the same time, the number of simultaneous
parameters that can be measured on a single MP limits this
phenotype. In the case of fluorescence, as high as 17 have been
reported, however, due to spectral overlap considerations, and a
consequent need for a form of correction called "compensation",
10-12 parameters is often thought of as a practical limit.
[0107] Elemental mass spectrometry-based flow cytometry (mass
cytometry), implemented and established using an instrument
(commercial name CyTOF) at the University of Toronto, offers a new
approach to analyze MPs via the replacement of fluorochrome-labeled
binding reagents (BR--i.e. antibodies, aptamers, chemical linkers,
or other affinity reagents) with elemental metal isotope-labeled
binding reagents (EmisoL-BR). The MPs to which these EmisoL-BRs
bind are then injected into the CyTOF device wherein they are
completely atomized, ionized and the resulting elemental metal ions
of the (former) EmisoL-BR bound to the MP are mass measured and the
individual elemental isotopes are quantified. The relative or
absolute number of each isotopic elements associated with each
individual MP are then enumerated and stored.sub.3. Due to the
achieved resolution (full width at half-maximum) of mass
measurement combined with the number of non-biological rare-earth
elemental isotopes available for creating different IsoL-BRs, it is
theoretically only possible to measure less than 100 parameters
simultaneously on a MP-by-MP basis. The CyTOF device uses an
inductively coupled plasma time-of-flight mass spectrometer
(ICPTOF-MS), which differs from other MS devices in use for
elemental analysis by first, its capability to record mass spectra
with 76,400 Hz, allowing to resolve single cell transients, second,
its improved ion transmission capabilities and thereby at least
.about.10-fold increased sensitivity compared to prior ICP-TOF-MS
instruments and third and finally, the ability to process and
record the generated data stream in real time.
[0108] The value of this mass cytometry-based technique, including
the routine measurement of 33 parameters, has recently been
documented.sub.3-9. The limitation, however, is that still the
number of measurable parameters is limited by the number of
available non-biological rare-earth elemental isotopes
(approximately 100). For a thorough phenotypic characterization of
a single cell hundreds or even thousands of parameters should be
ideally measured and quantified. Consequently, all current flow
cytometry approaches, including mass cytometry, fall short in
yielding a more complete phenotypic signature of single cells. As
such, there would be great value in allowing for an approach that
overcomes the limitations of measurable parameters set by the
availability of non-biological rare-earth elemental isotopes.
Terms (FIG. 9A):
[0109] MP: Mother Particle. Cell or unit object that hosts or
houses multiple epitopes or determinants to be measured.
[0110] BR: Binding Reagent. Reagents with specificity for epitopes
on the MP, and which have a moiety allowing attachment of a DADS.
BRs can be aptamers, antibodies, diabodies, constrained binding
loops, or affinity reagents of any kind. Depending on the method
used, can either a) harbor a conjugatable moiety for attaching a
DADS directly by covalent bond or chelation; b) harbor an attached
double strand DNA sequence "bar code" that uniquely identifies the
BR distinctly from other BRs; or c) harbor an attached single
strand "sense" DNA sequence "bar code" that uniquely identifies the
BR distinctly from other BRs.
[0111] DADS: Daughter Assayable Detector Species. A unique
assayable (i.e. by mass or mass to charge ratio--m/z) species
detectable by mass spectrometry. Can be attached directly to a BR
via a covalent linkage. Can be attached to a BR via a linker to the
bar coded DNA. Can be attached to a BR via a complementary
"anti-sense" strand to the "sense" DNA strand (after denaturation
to free up the available DNA "sense" strand). Can be attached to a
BR via a complementary "anti-sense" strand to the "sense" DNA
strand. DADS are either in the current form either MENOs or
SUENOs.
[0112] MENOs: Metal Encoded Nano-Objects. Nanoscale size particles
with defined ratios of 1 or more isotopes. Each nanoparticle's
signature ratio is distinct such that it would act as a unique DADS
when bound to an BR. To use MENOs, one would create 100s, or 1000s
of MENOs with unique signatures. These would be attached to BRs.
The entire set of MENOs from a given cell are assayed individually
in a manner that allows the cumulative number of given MENOs with a
given ratio of elements to be determined on a per cell basis.
[0113] SUENOs: Structural Unit Encoded Nano-Objects (FIGS. 10B
& C). A unique molecular weight organic molecule that is
detected in mass spectrometry as a distinct species. A SUENO can
be: a) a small molecule of a defined molecular weight unique from
other SUENOs. b) A polymeric substance of a defined molecular
weight unique from other SUENOs. c) A small molecule of a defined
molecular weight, or whose derived ions generated during mass
spectrometry are uniquely generated or have unique masses and/or
charges unique from other SUENOs. d) A polymer of distinct subunits
that together have a defined molecular weight, or whose derived
(i.e. by collision induced dissociation--CID) ions generated during
mass spectrometry are uniquely generated or have unique masses
and/or charges unique from other SUENOs.
[0114] The approaches of the invention can greatly extend the
number of simultaneous measured parameters. All these approaches
have in common that multiple individual Daughter Assayable Detector
Species (DADS) are bound via a BR to given epitopes on a MP. The
DADS can be quantified using MS techniques as they are (1)
associated with the MP or (2) can be separated from the MP,
measured and registered to the MP they were derived from. In
addition, the DADS in (1) or (2) can be amplified before MS
analysis to achieve greater sensitivity.
[0115] Such assayable objects as DADS can either be Metal Encoded
Nano-Objects (MENOs) or Structural Unit Encoded
Nano-Objects-(SUENOs) with definable mass over charge ratios (m/z).
Both MENOs and SUENOs have in common that 100s, 1000s to millions
or any other number of unique distinguishable and detectable
species can be created. As a result, the limitations given by the
lack of availability of rare-earth metal isotopes is overcome and
an unlimited number of phenotypic features can be analyzed per MP
(in a preferred case, the MP is a cell) via the MENOs/SUENOs. In
addition, by separating the assayable DADS from the MP, we realize
an important signal to noise increase, since most cellular
constituents that could interfere with the ionization, ion
transmission/storage and ion detection (e.g. overlapping the
spectral region defined by the DADS) are "left behind" with the MP
during the process. The separation further bears the advantage that
DADS can be, if e.g. nucleic acids are used as linkers before the
final binding event, amplified or otherwise processed prior to the
mass measurement, thereby greatly expanding the usable DADS and
overcoming detection limits.
Technical Specifications:
[0116] Measurement and quantification of DADS by mass spectrometry.
Analysis of MENOs.
[0117] Currently, mass cytometry instruments can detect under
optimal conditions 1 out of 10,000 ions of a given metal isotope at
a concentration of approximately 0.1 part per trillion,
corresponding to a detection limit of .about.100 zeptomol.
Therefore, if a MENO contains per used metal isotope equal or more
than 10,000 atoms, single molecules can be detected on a MP. The
MENOs derived from a single MP can be analyzed as described in
Bandura et al. Two modes for the analysis of the MENOs exist. (1)
The MENOs are separated from the MP as described under J.2.d/e and
quantified using mass cytometry or (2) The MENOs are still attached
to the MP. In the first variant the MENOs can be separated and
analyzed one by one. Alternatively, for both approaches, all MENOs
derived from a single MP can be measured concomitantly and the
presence of the MPs is mathematically inferred based on the metal
isotope composition and corresponding ion counts present in the
recorded composite mass spectrum.
[0118] Different approaches can be used to separate and align the
MPs for a sequential analysis. These include separation by the
following: Generating Micro-bubbles; Placement in Micro-cavities;
Dilution; Flow system, e.g. Capillary; Laser guidance; Sonic
alignment; Magnetic alignment; Compartmentalization with oil or
air; Others well known in the art. The same approaches can also be
used to separate the DADS/SUENOs (see below) derived from one MP to
ensure their sequential analyses if needed.
[0119] Analysis of SUENOs. Current non-ICP-MS set ups can typically
detect 1 out of 1,000,000 ions corresponding to a low attomole
sensitivity. Therefore, in order to detect a molecule which is
present in a cell with 100 copies, a BR has to carry 10,000 SUENOs.
To reduce the number of SUENOs which are bound to a MP via the BR
and to overcome any detection limits imposed by the sensitivity of
a given MS instrument an alternative approach can be taken: Here a
barcode on the DADS are amplified prior to their analyses. As there
are no upper limits in the amplification of e.g. DNA based DADS the
presented approach allows for single molecule detection per MP,
irrespective of the sensitivity of the used MS instrument. A wide
variety of methods can be used to analyze and quantify the SUENOs
using MS. First, the SUENOs have to be ionized. This can be
achieved in the gas phase, solid state or in solution, for example
by: Electron and chemical ionization; Spray ionization--e.g.
electrospray ionization; Desorption ionization--e.g.
matrix-assisted laser desorption ionization; Gas discharge
ionization; Ambient ionization; Any other used methods to ionize
analytes for MS.
[0120] Appropriate MS instruments are either able to directly
measure and quantify the SUENOs or can be combined with devices
which allow to breakdown the DADS into the SUENOs (see J.2.d/e) and
allowing for the sensitivity and scanning speed to detect the
SUENOs of single MPs. The following mass analyzers can be used to
determine the m/z of the SUENOs: Time of flight (TOF); Quadrupole;
Ion trap; Fourier transform ion cyclotron resonance; Orbitrap;
Sector; Any other mass analyzer; And combinations thereof. One MS
instrument set-up which is particularly suitable to measure and
quantify the SUENOs is a quadrupole-time of flight MS. It achieves
low attomole sensitivity, allows detecting transients of single MPs
due to the high scanning speed and can either be directly used to
break down the DADS into SUENOs via collision induced dissociation
(CID) or can be easily interfaced with additional instruments
needed to e.g. break down the DADS into the SUENOs. Another
instrument set-up exploits laser ionizable SUENOs derived from a
MP. The SUENOS from a given MP are either already located (see H.1)
or can be deposited on a chip and MALDI is used to ionize them. If
the DADS also consist of SUENOs linked via UV or acid sensitive
linkers to the MP, the DADS are concomitantly broken down into
their SUENOs. The ions are preferably detected using a TOF
instrument.
[0121] Amplification of DADS and subsequent analysis using mass
spectrometry. The DNA signature of the DADS can either be amplified
in the gas, solid or liquid phase. The amplification process can be
performed in a mixture of MPs if no cross-talk between the
MP-BR-DADS is possible. Otherwise, the MPs are in localizable
reaction chambers separated from each other to perform manipulation
of the DADS. These reaction chambers can be in the solid, gas or
liquid phase, for example on/in: Microarray; Microfluidics device;
Emulsions (e.g. water-oil); Droplets in gas phase; Any other
technique which allows the separation needed to amplify the
DADS.
[0122] Alternatively, the DADS can be first separated from the MPs
as described above before amplification. By using standard
molecular biology approaches (such as frequently employed in
emulsion polymerase chain reactions (PCR) the DNA-DADS present in
the reaction chamber are amplified using primers which carry a
unique non-DNADADS. After amplification, DNA sequences
complementary to the DNA-DADS (but different from the primer
sequence) are used to capture and retain the reaction product in
the reaction chamber while all left over primers are removed. This
can e.g. be achieved with magnetic beads or with the microarray
wells spotted with the complementary DNA strands. In the last step,
the DADS are released from the DNA strands. Alternatively, an
approach analogous to the commonly used real time PCR method using
a "Tag-man probe" can be employed: besides the primers needed to
amplify the DNA-DADS, a short DNA probe complementary to the
DNA-DADS can be added during amplification. This probe is coupled
to an non-DNA-DADS. If the target sequence is present, the DNA
sequences anneal and by using polymerases with 5'-3' exonuclease
activity, the probe is degraded during amplification in every round
of PCR, thereby releasing the non-DNA-DADS. As a result in both
cases free DADS are generated which can be directly analyzed,
separated into the SUENOs or separated from the MP and measured as
described above.
Generation and Manipulation of DADS.
[0123] The MENOs attached to the MPs are nanoparticles or particles
of any other size with a defined combination and number of metal
isotopes. As such n100 different particles can be generated (100 is
the number of available metal isotopes measurable by CyTOF and "n"
describes the number of distinct molar quantities (relative to an
internal standard per particle). Even if the nanoparticles are
generated by using each metal in a digital manner (present or not
present) 2100 unique particles can be generated.
[0124] MENOs can be generated in a variety of manners: (1) Cooled
vapor deposition (CVD) of heated ratiometrically determined
mixtures of isotopes to crystalline or other solid-packed forms.
(2) Liquid phase condensation of dissolved ratiometrically
determined mixtures of isotopes with appropriate salts or
conditions to create a crystal structure of appropriate final
ratios. (3) Layering of pure or ratiometrically determined mixtures
of isotopes to attain a final ratiometrically determined mixture of
isotopes in a given nanoparticle. 3. In a final step MENOs are
treated, or made, with a tagging group that directly, or
indirectly, allows for their attachment to a BR.
[0125] SUENOs with a defined mass to charge ratio. 1. An
alternative DADS consists of SUENOs to detect and quantify epitopes
present on the MPs. Each SUENO has a defined and unique m/z and can
be specifically released from the MP and is subsequently injected
into a more "standard" MS instrument, where it is detected and
measured relative or absolutely quantified. To increase the
sensitivity of the detected targets, the DADS can be provided as a
polymer of detectable SUENOs that, in the monomeric form are 1%,
10%, 100% or any other percentage ionized. Alternatively, the DADS
and/or SUENOs can be amplifiable. In either event, the SUENO is the
detected unit in the MS.
[0126] Generation of DADS-BR: a) First, each BR is labeled with a
DADS/SUENO, which can be a: (1) Small molecule compound; (2) Amino
acid; (3) Peptide; (4) Nucleotide; (5) DNA; (6) RNA; (7)
Metabolite; (8) Mono-saccharide; (9) Poly-saccharide; (10) Any
other sort of enzyme cleavable substrate; (11) Metalo-organic
compound; (12) Any other chemically producible compound; (13) Any
combination of the above mentioned. Alternatively, in order to
increase the number of ions bound to a BR, the BR can be labeled
with DADS composed of SUENOs, such as a polymer (or variants
thereof such as dendrimers), consisting of the compounds mentioned
above.
[0127] Different forms of linkers can be used to connect the SUENOs
into the DADS. These include: (1) Covalent linkers as (used in
protein chemistry), including Peptide bonds; Nucleotide-nucleotide
bonds; Small molecules; Acid/base labile bonds; Electromagnetic
labile bonds; Electron-radical labile bond; DNA; Peptide;
Saccharides; Polyethylene glycol (PEG); Any other sort of enzyme
cleavable substrate; Or any other linker. Alternatively affinity
based linkers may be used, e.g. Biotin-streptavidin; Protein A or G
or L; Calmodulin; Glutathione S-transferase; Poly-Histidine;
Peptide tags (e.g. FLAG-tag); Metal affinity based linkers; Or any
other affinity based linker.
[0128] The linkers between the SUENOs are cleavable by any rapid
inducible separation technique, including; (1) Chemical; (2)
Mechanical; (3) Enzymatic; (4) Sonic; (5) Electromagnetic methods;
(6) Any other method; e) If the linkers between the SUENOs are
broken down within the mass spectrometer, the separation methods
can more specifically include (1) Collision induced dissociation;
(2) Infrared multiphoton dissociation; (3) Blackbody infrared
radiative dissociation; (4) Electron-capture dissociation; (5)
(Negative) electron-transfer dissociation; (6) Electron-detachment
dissociation; (7) Surface induced dissociation.
[0129] The cleavage either adds or subtracts a defined number of
charges to/from each SUENO or does not alter the charge state at
all. The cleavage can happen in the gas, liquid or solid phase.
[0130] The DADS can be broken down into the SUENOs in gas, liquid
or solid phase, while (1) in the reaction chamber; (2) still
attached to the MP and transported to the MS; (3) injected into the
MS instrument; (4) in the MS instrument.
[0131] Each SUENO has a defined and distinct m/z and can be
singly-, doubly-, triply- or n-times charged, depending on the
design of the experimental detection protocol. The SUENO can be any
of the compounds as described for the DADS. To increase the number
of producible DADS and therefore measurable parameters the SUENOs
can be synthesized with defined isotopes of any existing element
yielding SUENOs with 1 or n Dalton mass differences that allow to
uniquely identify and quantify them in a MS measurement, therefore
their mass difference will be defined by the achievable resolution
of the MS instrument. Preferred elements with their isotopes are
(1) hydrogen; (2) carbon; (3) nitrogen; (4) oxygen; (5) chlorine;
(6) fluorine; (7) bromide; (8) any other available element and its
isotopes that can be synthesized into an SUENO. Similarly, only a
unique DNA sequence is attached to the BR and used to stain the
MPs, allowing for further amplification of the DADS.
[0132] Staining/labeling of MPs with DADS/SUENO-BRs: The staining
of MPs with DADS-BRs or SUENO-BRs follows standard staining
protocols for antibodies (as BRs) to cells. The following
references contain exemplary staining protocols: 1. Fluorescent
cell barcoding for multiplex flow cytometry. Krutzik P O, Clutter M
R, Trejo A, Nolan G P. Curr Protoc Cytom. 2011 January; Chapter
6:Unit 6.31. Phospho flow cytometry methods for the analysis of
kinase signaling in cell lines and primary human blood samples.
Krutzik P O, Trejo A, Schulz K R, Nolan G P. Methods Mol Biol.
2011; 699:179-202. Duration of antigen receptor signaling
determines T-cell tolerance or activation. Katzman S D, O'Gorman W
E, Villarino A V, Gallo E, Friedman R S, Krummel M F, Nolan G P,
Abbas A K. Proc Natl Acad Sci USA. 2010 Oct. 19; 107(42):18085-90.
Epub 2010 Oct. 4. Tyramide signal amplification for analysis of
kinase activity by intracellular flow cytometry. Clutter M R,
Heffner G C, Krutzik P O, Sachen K L, Nolan G P. Cytometry A. 2010
November; 77(11):1020-31. B-cell signaling networks reveal a
negative prognostic human lymphoma cell subset that emerges during
tumor progression. Irish J M, Myklebust J H, Alizadeh A A, Houot R,
Sharman J P, Czerwinski D K, Nolan G P, Levy R. Proc Natl Acad Sci
USA. 2010 Jul. 20; 107(29):12747-54. Epub 2010 Jun. 11. New
technologies for autoimmune disease monitoring. Maecker H T, Nolan
G P, Fathman C G. Curr Opin Endocrinol Diabetes Obes. 2010 August;
17(4):322-8. Review. Characterization of patient specific signaling
via augmentation of Bayesian networks with disease and patient
state nodes. Sachs K, Gentles A J, Youland R, Rani S, Irish J,
Nolan G P, Plevritis S K. Conf Proc IEEE Eng Med Biol Soc. 2009;
2009:6624-7.
[0133] Methods to couple and separate DADS from the MPs 1. The DADS
are coupled to the MPs such that they allow for the direct analysis
using MS or alternatively, are separated from the MP for further
manipulation (such as amplification) needed for the subsequent MS
quantification. 2. The DADS can be linked to the BR. 3. In order to
release the DADS from the BR, the methods as described above can be
employed. 4. The DADS can be released from the BR in the gas phase,
solid state or in solution. 5. Depending on the methods used to
release and separate the DADS from the MP, distinct methods can be
employed to ensure that a given MS spectrum can be assigned to a
MP. a) In case the DADS was separated from the BR in the solid
phase, the coordinates of the MP position can be used to assign a
MS spectrum to the MP.
[0134] In case the DADS is decoupled and separated from the MP in a
liquid microsphere (e.g. emulsion, microfluidics device, droplets
in gas phase), then the exact location of the release transfer must
be known as well as a) the accurate time to the MS analysis of the
DADS and b) the accurate time at which the MP is further process
(e.g. sorted). Importantly, time in a)>b) to allow for the data
processing of the MS spectrum and controlling the destiny of the
MP. a) To ensure an accurate timing, e.g. bifunctional
fluorescent-DADS calibration beads can be added to the
microcavities. Thereby a) and b) can be calibrated in real
time.
[0135] In an example set up, the BR could be coupled to a short DNA
tether by standard chemical approaches, and then coupled to the
DADS. Similarly, the above described DNA tethers are only used to
tie/decouple the binding reagent to the DADS. This "tri" reagent is
then used to stain the MPs. To `decouple` the BR from the DADS and
to generate the quantifiable SUENOs, one would use DNAse which
specifically cleaves DNA and would release the BR from the DADS and
also generates the SUENOs.
[0136] Preferred set-ups a) Separation and amplification b) MENOs
or SUENOs Direct measurement of DADS In a preferred configuration,
the DADS are not naturally occurring DNA sequences and are attached
to BRs. These DADSBRs are used to stain the cells and the unbound
DADS-BR reagents are removed. Subsequently, the MPs are separated
from each other into distinct reaction chambers using a
hydrocarbon/water emulsion and the bound DADS are amplified. In the
final step the DADS are analyzed by mass spectrometry.
[0137] References: Herzenberg, L. A. et al. The history and future
of the fluorescence activated cell sorter and flow cytometry: a
view from Stanford. Clin. Chem 48, 1819-1827 (2002); Perfetto, S.
P., Chattopadhyay, P. K. & Roederer, M. Seventeen-colour flow
cytometry: unravelling the immune system. Nature Rev. Immunol. 4,
648-655 (2004); Bandura, D. R. et al. Mass Cytometry Technique for
Real Time Single Cell Multitarget Immunoassay Based on Inductively
Coupled Plasma Time-of-Flight Mass Spectrometry. Anal. Chem
(2009).doi:10.1021/ac901049w; Razumienko, E. et al. Element-tagged
immunoassay with ICP-MS detection: evaluation and comparison to
conventional immunoassays. J. Immunol. Methods 336, 56-63 (2008);
Ornatsky, O. I. et al. Study of cell antigens and intracellular DNA
by identification of element-containing labels and
metallointercalators using inductively coupled plasma mass
spectrometry. Anal. Chem 80, 2539-2547 (2008). 6. Ornatsky, O. I.
et al. Development of analytical methods for multiplex bio-assay
with inductively coupled plasma mass spectrometry. J Anal At
Spectrom 23, 463-469 (2008); Ornatsky, O., Baranov, V. I., Bandura,
D. R., Tanner, S. D. & Dick, J. Multiple cellular antigen
detection by ICP-MS. J. Immunol. Methods 308, 68-76 (2006);
Baranov, V. I., Quinn, Z., Bandura, D. R. & Tanner, S. D. A
sensitive and quantitative element-tagged immunoassay with ICPMS
detection. Anal. Chem 74, 1629-1636 (2002); Bendall S C, Simonds,
Qiu Amir E D, Krutzik P O, Finck R, Bruggner R V, Melamed R, Trejo
A, Ornatsky O I, Balderas R S, Plevritis S K, Sachs K, Pe'er D,
Tanner S D & Nolan G P. Single-cell Mass Cytometry of
Differential Immune and Drug Responses Across a Human Hematopoietic
Continuum. Science In Press; Williams, R., Peisajovich, .S. G,
Miller, O. J., Magdassi, S., Tawfik, D. S. & Griffiths A. D.
Amplification of complex gene libraries by emulsion PCR. Nat.
Methods 7, 545-50 (2006); Holland, P. M., Abramson, R. D., Watson,
R., & Gelfand, D. H. Detection of specific polymerase chain
reaction product by utilizing the 5'-3' exonuclease activity of
Thermus aquaticus DNA polymerase. PNAS 88, 7276-80 (1991).
[0138] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in chemistry,
medicine, and molecular biology or related fields are intended to
be within the scope of the following claims.
* * * * *