Note: Descriptions are shown in the official language in which they were submitted.
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Microarray Methods Utilizing Semiconductor Nanocrystals
CROSS-REFERENCES TO RELATED APPLICATTONS
This application claims priority to U.S. Patent Application entitled
"Microarray Methods Utilizing Semiconductor Nanocrystals," filed September 29,
2000,
and having attorney docket number 019916-001200US, and claims the benefit of
U.S.
Provisional Patent Application No. 60/182,845, filed February 16, 2000, both
of which
are incorporated by reference in their entirety for all purposes. This
application is related
to U.S. Patent Application No. 09/566,014, filed May 5, 2000, which claims the
benefit of
U.S. Provisional Application No. 60/133,084, filed May 7, 1999, both of which
are also
incorporated by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
Bioassays are used to probe the quantity of a target analyte present in a
biological sample. Surface-based assays, in which the amount of target is
quantified by
capturing it on a solid support and then labeling it with a detectable label,
are especially
important since they allow for the facile separation of bound and unbound
labels.
Recently a number of surface-based assays have been developed that utilize
different
types of arrays.
Array-based assays are of importance because they permit a very large
number of interrogations to be performed simultaneously by placing different
"assays" on
spatially distinct locations of an array. Addressable arrays can be fabricated
to study
many different analytes including proteins, DNA and RNA. W general, the sample
to be
tested is spread over the entire array so that target biomolecules in the
sample can form
complexes with their binding partner on the array. The target is typically
labeled with
some type of detectable tag (e.g., a fluorescent or radioactive label) so that
the amount of
each target analyte in the sample can be quantified by detecting the labeled
complexes on
the array.
One example of a surface based assay is a DNA microarray. The use of
DNA microarrays has become widely adopted in the study of gene expression and
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genotyping due to the ability to monitor large numbers of genes simultaneously
(see, e.g.,
Schena et al. (1995) Science 270:467-470; and Pollack et al. (1999) Nat.
Genet. 23:41-
46). More than 100,000 different probe sequences can be bound to distinct
spatial
locations across the microarray surface, each spot corresponding to a single
gene (Schena
et al. (1998) Tibtech 16:301-306). When a fluorescently labeled DNA target
sample is
placed over the surface of the array, individual DNA strands hybridize to
complementary
strands within each array spot. The level of fluorescence detected quantifies
the number
of copies bound to the array surface and therefore the relative presence of
each gene,
while the location of each spot determines the gene identity. Using arrays, it
is
theoretically possible to simultaneously monitor the expression of all genes
in an
organism's genome. The use of DNA microarrays is an extremely powerful
techiuque,
with applications spanning all areas of genetics (see, e.g., the Chipping
Forecast
supplement to Nature Genetics 21 (1999)). Arrays can also be fabricated using
other
binding moieties such as antibodies, proteins, haptens or aptamers, in order
to facilitate a
wide variety of bioassays in array format.
Other surface based assays include microtiter plate-based ELISAs
(enzyme-linked immunosorbent assays) in which the bottom of each well is
coated with a
different antibody. A protein sample is then added to each well along with a
fluorescently
labeled secondary antibody for each protein. Target proteins are captured on
the surface
of each well and secondarily labeled with a fluorophore. Fluorescence at the
bottom of
each well quantifies the amount of each target molecule in the sample.
Similarly,
antibodies or DNA can be bound to a microsphere such as a polymer bead and
assayed as
described above.
Often detection of binding complexes in array-based assays involves the
detection of a fluorescently labeled species that is part of the binding
complex. Currently,
two fluorescent dyes predominate the field of addressable array assay
techniques: cy3,
having an emission peak at 565 nrn, and cy5, having an emission peak at 670
nm. There
are many properties of these dyes that are typical of most organic dyes and
that can limit
their use for providing quantitative results using addressable arrays. Fast
photobleaching
of the dyes is one problem. Photobleaching refers to the deterioration of
fluorescence
intensity upon prolonged and/or repeated exposure to excitation light.
Photobleaching is
dependent on the intensity of the excitation light and the duration of the
illumination.
Conversion of the dye into a nonfluorescent species is irreversible.
Furthermore,
photobleaching limits the amount of signal that can be collected from a given
region of
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the microarray, thereby limiting discrimination of very low level signals. The
chemical
instability of cy5 makes this dye very unpredictable, and consequently makes
it difficult
to quantify accurately assay results obtained using this dye.
Low quantum yield is another shortcoming of organic dyes which limits
the amount of light that can be collected. The broad emission spectra
associated with
organic dyes create overlap between different colored dyes. Such overlap
requires
complex deconvolution of the signal to quantify assay results, thereby
limiting the
dynamic range of the assay. While emission spectra are quite broad, excitation
spectra of
organic dyes tend to be quite narrow. Consequently, different wavelengths of
light are
required for excitation of each dye. Additionally, organic dyes have a small
Stokes shift
(the separation of the absorption and emission maxima) that can result in high
autofluorescence and create problems with scattered excitation light, thereby
increasing
the signal background in these measurements.
Furthermore, with cy3 and cy5, a maximum of two colors can be used in
detection. Consequently, it is difficult to perform analyses in a multiplex
format in which
multiple species are examined simultaneously in a single assay. For array-
based assays,
this limitation means that if multiple samples are to be tested for the same
analytes
multiple identical arrays must be used. For instance, if one seeks to
determine the level of
gene expression under 20 different conditions, it is necessary to run 20
different assays
using 20 arrays, each with a comparison between a test condition and a
reference
condition. This technique requires more arrays and materials and is therefore
very costly.
It also introduces additional noise with each measurement since comparisons
between
different conditions are not made directly on the same array.
In addition to these problems associated with organic fluorescent dyes,
limitations with regard to sensitivity and dynamic range is another
problematic area in
array-based assays. Dynamic range refers to the ability to simultaneously
measure
analyte over a wide range of concentrations. Using current detection
technology, it is
usually necessary to sacrifice linearity in the high concentration regime for
detection
sensitivity in the low concentration regime. This limits the dynamic range of
a single
experiment.
The performance of an assay is typically measured by its ability to
specifically and quantitatively measure vanishingly small quantities of the
target species
under investigation. This is especially true for genetic analysis such as gene
expression
or genotyping, where the available quantity of genetic material is limited.
For instance,
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using current detection technology with organic dye labels, gene expression
analysis on
DNA microarrays requires between 50 and 200 p,g of total RNA for single array
hybridization. This requires as many as 105 cells (Duggan et al. (1999) Nature
Genetics
21(n1 s):10-14). In many instances, such as samples extracted through
microdissection
(Sgroi et al. (1999) Cancer Res. 59:5656-5661), these large quantities of
material are
simply not available. This greatly complicates the detection of such samples
labeled with
standard organic fluorophores.
Thus, there is a need to address the limitations associated with existing
organic fluorescent dyes and to increase sensitivity and dynamic range in
order to
improve the results that can be obtained using surface-based arrays such as
those
conducted with addressable arrays.
SUMMARY OF THE INVENTION
Methods for conducting a variety of array assays utilizing semiconductor
nanocrystals as labels are provided herein. Various features of the
semiconductor
nanocrystals enhance signal detection relative to conventional organic dyes.
For
example, the semiconductor nanocrystals emit an intense signal that aids
detection. In
some instances, signals are sufficiently intense that a single semiconductor
nanocrystal
can be detected. By controlling the size and composition of the semiconductor
nanocrystals, one can obtain semiconductor nanocrystals that emit at
particular
wavelengths. Further, while the semiconductor nanocrystals have large
absorption cross
sections, they have narrow, symmetric emission spectra. This means that a
number of
different semiconductor nanocrystals can be excited at a single wavelength but
emit at a
variety of distinct wavelengths. This feature is useful for assays conducted
in multiplex
formats. Because the semiconductor nanocrystals can be readily attached to a
variety of
different biomolecules, the semiconductor nanocrystals can be utilized in a
variety of
different microarray analyses. For example, the semiconductor nanocrystals can
be
utilized to label target molecules that are probed using nucleic acid arrays,
protein arrays,
tissue arrays or other arrays that utilize labeled targets and optical
detection.
Accordingly, certain methods for detecting a ligand of interest in a sample
involve initially providing a first plurality of antiligands immobilized on a
solid support at
positionally distinct locations thereon to provide a first array, wherein the
plurality of
antiligands comprises a first antiligand capable of binding specifically to a
first ligand of
interest. This array is then contacted with a sample containing or suspected
of containing
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the first ligand, wherein the first ligand is linked through a linker to a
first semiconductor
nanocrystal before, during or after the contacting, under conditions in which
the first
ligand binds specifically to the first antiligand to form a first complex.
Unbound ligand is
optionally removed from the array. The location of the first complex is then
identified by
detecting, and optionally quantifying, the presence in the first complex of
the first
semiconductor nanocrystal.
Certain methods are used in analyzing variations in nucleic acids such as
single nucleotide polymorphisms. Some of these methods involve providing a
first
plurality of nucleic acid primers having a 3' end and a 5' end and which
primers are
immobilized on a solid support at positionally distinct locations thereon to
provide a first
array, wherein the plurality of primers comprise a first primer complementary
to a first
target nucleic acid having an allelic site. The first array is then contacted
with a sample
containing or suspected of containing the first target nucleic acid, in the
presence of a first
terminating nucleotide linked to a first semiconductor nanocrystal through a
linker, under
conditions such that the first target nucleic acid hybridizes to the first
primer to form a
first target-primer complex and such that if the first terminating nucleotide
is
complementary to the nucleotide at the allelic site the first primer is
extended to
incorporate the first terminating nucleotide to provide an extended primer.
The location
or locations that includes extended primer is identified by detecting the
presence therein
of the first semiconductor nanocrystal.
Other methods are secondary interrogation or sandwich type assays.
These methods typically involve providing a first plurality of antiligands
immobilized on
a solid support at positionally distinct locations thereon to provide a first
array, wherein
the first plurality of antiligands comprises a first antiligand that is a
binding partner of a
first ligand. The array is then contacted with a sample containing or
suspected of
containing the first ligand, whereby the first antiligand and the first ligand
interact to form
a first binary complex. The binary complex in turn is contacted with a second
antiligand
wherein the second antiligand is (i) a binding partner of the first ligand and
(ii) linked to a
first semiconductor nanocrystal through a linker, whereby the second
antiligand binds to
the first ligand in the first binary complex to form a first ternary complex.
The location of
the array that includes the first ternary complex is identified by detecting
the presence
therein of the first semiconductor nanocrystal.
Still other methods involve labeling a ligand after it has become bound to
an array. Certain of these methods involve providing a first plurality of
antiligands
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immobilized on a solid support at positionally distinct locations thereon to
provide a first
array, wherein the plurality comprises a first antiligand that is a binding
partner of a first
ligand. The first array is then contacted with a sample containing or
suspected of the first
Iigand, whereby the first Iigand and the first antiligand interact to form a
first complex.
The first ligand in the first complex is subsequently labeled with a first
semiconductor
nanocrystal. The location of the array that includes the first complex is
identified by
detecting the presence therein of the first semiconductor nanocrystal.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graphical representation that depicts the results of a particular
immunological assay involving a secondary interrogation of a complex between a
capture
antibody and a protein labeled with a semiconductor nanocrystal and a
secondary
antibody labeled with another semiconductor nanocrystal.
FIG. 1B is a graphical representation of a tertiary complex (capture
1 S antibody, protein labeled with semiconductor nanocrystal, secondary
antibody labeled
with another semiconductor nanocrystal) formed in a secondary interrogation
according
to one method of the invention.
FIGS. 2A-2B illustrate the optical properties associated with
semiconductor nanocrystals as a consequence of the phenomenon of quantum
confinement. FIGS. 2A and 2B show the absorption and emission spectra from
different
semiconductor nanocrystal samples, illustrating how the emission wavelength
varies as a
function of size. Absorption spectra have been normalized to the height of the
first
absorption peak and have been vertically offset for clarity. Inset numbers
correspond to
the average diameter of the quantum dots within each ensemble sample.
FIG. 2C illustrates how the material from which a semiconductor
nanocrystal is constructed affects the wavelength at which it emits. Emission
spectrum
from semiconductor nanocrystals of three different materials are shown: CdSe
(visible),
InP (visible-near infrared) and InAs (infraxed).
FIG. 3 provides a graph that illustrates photodegradation of semiconductor
nanocrystals vs fluorescein under identical excitation conditions. Sample
concentrations
were matched (~10-5 mol/1) and each was excited with ~1 W/cm2 of 488 nm light
from an
Ar+ laser. Note that while fluorescein photobleaches within the first few
seconds,
quantum dots actually increase slightly in intensity over the first minute.
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FIGS. 4A and 4B illustrate single semiconductor nanocrystal detection.
FIG. 4A is a photograph of single semiconductor nanocrystals using a laser
epifluorescence microscope. Each individual spot corresponds to the
fluorescence from a
single semiconductor nanocrystal. FTG. 4B depicts spectra from single
semiconductor
nanocrystals. Wavelength is dispersed on the x-axis and position on the y-
axis. Each
horizontal line corresponds to the fluorescence spectrum from a single
semiconductor
nanocrystal. Note that different size semiconductor nanocrystals are easily
identified by
small changes in emission wavelength.
FIGS. 5A and SB show a comparison between the absorption and emission
spectra of fluorescein (FIG. 5A) and a comparable color semiconductor
nanocrystal (FIG.
5B). Note that while the emission spectrum of the semiconductor nanocrystal is
significantly narrower than that for fluorescein, the absorption spectrum
extends far to the
blue, allowing efficient excitation with all wavelengths shorter than the
emission
wavelength.
FIGS. 6A-6C illustrate the extension of dynamic range that can be
achieved through single hybridization counting. FIG. 6A is a graphic
representation of
the transition from the ensemble concentration regime to the single copy
hybridization
regime. FIG. 6B is a graph showing simulated data demonstrating the improved
sensitivity achieved through single hybridization detection. FIG. 6C is a plot
of the
theoretical number of discrete points detected within a 100 ~,m diameter array
spot as the
total number of bound labels increases. The calculation assumes that
individual labels
cannot be distinguished if they reside within the same 0.5 ~,m diameter region
and a
random distribution of label locations with an average density that is uniform
across the
array spot. Saturation becomes significant above 6000 as the probability of
finding 2 or
more labels within the same diffraction limited spot increases.
FIG. 7 presents a schematic drawing of single quantum dot microscope.
FIGS. 8A-8E illustrate and summarize the steps in certain automated array
scanning methods of the invention. Initially, sequential images are taken at
periodic
positions across the array (FIG. 8A). The array is then reconstructed (FIG.
8B). Pattern
recognition is utilized to identify the location of the array spots relative
to alignment spots
(FIG. 8C). Within each spot the average intensity is measured as well as the
total number
of discrete points (FIG. 8D). Values for the average intensity and the total
number of
discrete points are exported (FIG. 8E).
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DETAILED DESCRIPTION
I. Definitions
As used in this specification and the appended claims, the singular forms
"a," "an" and "the" include plural references unless the content clearly
dictates otherwise.
S Thus, for example, reference to "a semiconductor nanocrystal" includes a
mixture of two
or more such semiconductor nanocrystals, and an "analyte" includes more than
one such
analyte.
Unless defined otherwise, all technical and scientific terms used herein
have the meaning commonly understood by a person skilled in the art to which
this
invention belongs. The following references provide one of skill with a
general definition
of many of the terms used in this invention: Singleton et al., DICTIONARY OF
MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE
DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE
GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag
(1991);
and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).
As used herein, the following terms have the meanings ascribed to them unless
specified
otherwise.
The terms "semiconductor nanocrystal," "quantum dot," "QdotTM
nanocrystal" or simply "nanocrystal" are used interchangeably herein and refer
to an
inorganic crystallite between about 1 nm and about 1000 nm in diameter or any
integer or
fraction of an integer therebetween, generally between about 2 nm and about 50
nm or
any integer or fraction of an integer therebetween, more typically about 2 nm
to about 20
nm (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 nm). A
semiconductor nanocrystal is capable of emitting electromagnetic radiation
upon
excitation (i.e., the semiconductor nanocrystal is luminescent) and includes a
"core" of
one or more first semiconductor materials, and may be surrounded by a "shell"
of a
second semiconductor material. A semiconductor nanocrystal core surrounded by
a
semiconductor shell is referred to as a "core/shell" semiconductor
nanocrystal. The
surrounding "shell" material typically has a bandgap energy that is larger
than the
bandgap energy of the core material and can be chosen to have an atomic
spacing close to
that of the "core" substrate. The core and/or the shell can be a semiconductor
material
including, but not limited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS,
CdSe,
CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, Case, Care, SrS, SrSe, SrTe, BaS,
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Base, Bare, and the like) and III-V (GaN, GaP, GaAs, GaSb, W N, InP, InAs,
InSb, and
the like) and IV (Ge, Si, and the like) materials, and an alloy or a mixture
thereof.
A semiconductor nanocrystal is, optionally, surrounded by a "coat" of an
organic capping agent. The organic capping agent can be any number of
materials, but
has an affinity for the semiconductor nanocrystal surface. In general, the
capping agent
can be an isolated organic molecule, a polymer (or a monomer for a
polymerization
reaction), an inorganic complex, and an extended crystalline structure. The
coat is used
to confer solubility, e.g., the ability to disperse a coated semiconductor
nanocrystal
homogeneously into a chosen solvent, functionality, binding properties, or the
like. In
addition, the coat can be used to tailor the optical properties of the
semiconductor
nanocrystal. Methods for producing capped semiconductor nanocrystals are
discussed
further below.
Thus, the terms "semiconductor nanocrystal," "quantum dot" and "QdotTM
nanocrystal" as used herein denote a coated semiconductor nanocrystal core, as
well as a
core/shell semiconductor nanocrystal.
By "luminescence" is meant the process of emitting electromagnetic
radiation (light) from an object. Luminescence results from a system which is
"relaxing"
from an excited state to a lower state with a corresponding release of energy
in the form
of a photon. These states can be electronic, vibronic, rotational, or any
combination of
the three. The transition responsible for luminescence can be stimulated
through the
release of energy stored in the system chemically or added to the system from
an external
source. The external source of energy can be of a variety of types including
chemical,
thermal, electrical, magnetic, electromagnetic, physical or any other type
capable of
causing a system to be excited into a state higher than the ground state. For
example, a
system can be excited by absorbing a photon of light, by being placed in an
electrical
field, or through a chemical oxidation-reduction reaction. The energy of the
photons
emitted during luminescence can be in a range from Iow-energy microwave
radiation to
high-energy x-ray radiation. Typically, luminescence refers to photons in the
range from
W to IR radiation.
"Monodisperse particles" include a population of particles wherein at least
about 60% of the particles in the population, more preferably 75% to 90% of
the particles
in the population, or any integer in between this range, fall within a
specified particle size
range. A population of monodispersed particles deviate less than 10% rms (root-
mean-
square) in diameter and typically less than 5% rms.
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The phrase "one or more sizes of semiconductor nanocrystals" is used
synonymously with the phrase "one or more particle size distributions of
semiconductor
nanocrystals." One of ordinary skill in the art will realize that particular
sizes of
semiconductor nanocrystals are actually obtained as particle size
distributions.
By use of the term "a narrow wavelength band" or "narrow spectral
linewidth" with regard to the electromagnetic radiation emission of the
semiconductor
nanocrystal is meant a wavelength band of emissions not exceeding about 40
nrn, and
typically not exceeding about 20 nm in width and symmetric about the center,
in contrast
to the emission bandwidth of about 100 mn for a typical dye molecule with a
red tail that
can extend the bandwidth out as much as another 100 nm. It should be noted
that the
bandwidths referred to are determined from measurement of the full width of
the
emissions at half maximum peak height (FWHM), and are appropriate in the range
of 200
nm to 2000 nm.
By use of the term "a broad wavelength band," with regard to the
excitation of the semiconductor nanocrystal is meant absorption of radiation
having a
wavelength equal to, or shorter than, the wavelength of the onset radiation
(the onset
radiation is understood to be the longest wavelength (lowest energy) radiation
capable of
being absorbed by the semiconductor nanocrystal). This onset occurs near to,
but at
slightly higher energy than the "narrow wavelength band" of the emission. This
is in
contrast to the "narrow absorption band" of dye molecules which occurs near
the
emission peak on the high energy side, but drops off rapidly away from that
wavelength
and is often negligible at wavelengths further than 100 nm from the emission.
The terms "polynucleotide," "oligonucleotide," "nucleic acid" and
"nucleic acid molecule" are used herein to include a polymeric form of
nucleotides of any
length, either ribonucleotides or deoxyribonucleotides. This term refers only
to the
primary structure of the molecule. Thus, the term includes triple-, double-
and single
stranded DNA, as well as triple-, double- and single-stranded RNA. It also
includes
modifications, such as by methylation and/or by capping, and unmodified forms
of the
polynucleotide. More particularly, the terms "polynucleotide,"
"oligonucleotide,"
"nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides
(containing
2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type
of
polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base,
and other
polymers containing nonnucleotidic backbones, for example, polyamide (e.g.,
peptide
nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-
Virals,
CA 02400379 2002-08-14
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Inc., Corvallis, Oregon, as Neugene) polymers, and other synthetic sequence-
specif c
nucleic acid polymers providing that the polymers contain nucleobases in a
configuration
which allows for base pairing and base stacking, such as is found in DNA and
RNA.
There is no intended distinction in length between the terms "polynucleotide,"
"oligonucleotide," "nucleic acid" and "nucleic acid molecule," and these terms
are used
interchangeably. These terms refer only to the primary structure of the
molecule. Thus,
these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide
N3' PS'
phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA,
as
well as double- and single-stranded RNA, DNA:RNA hybrids, and hybrids between
PNAs and DNA or RNA, and also include known types of modifications, for
example,
Iabels that are known in the art, methylation, "caps," substitution of one or
more of the
naturally occurring nucleotides with an analog, internucleotide modifications
such as, for
example, those with uncharged linkages (e.g., methyl phosphonates,
phosphotriesters,
phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g.,
phosphorotluoates, phosphorodithioates), and with positively charged linkages
(e.g.,
aminoallclyphosphoramidates, aminoall~ylphosphotriesters), those containing
pendant
moieties, such as, for example, proteins (including nucleases, toxins,
antibodies, signal
peptides, poly-L-lysine), those with intercalators (e.g., acridine, psoralen),
those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals), those
containing alkylators, those with modified linkages (e.g., alpha anomeric
nucleic acids),
as well as unmodified forms of the polynucleotide or oligonucleotide. In
particular, DNA
is deoxyribonucleic acid.
The terms "polynucleotide analyte" and "nucleic acid analyte" are used
interchangeably and include a single- or double-stranded nucleic acid molecule
that
contains a target nucleotide sequence. The analyte nucleic acids may be from a
variety of
sources, e.g., biological fluids or solids, chromosomes, food stuffs,
environmental
materials, etc., and may be prepared for the hybridization analysis by a
variety of means,
e.g., proteinase K/SDS, chaotropic salts, or the like.
As used herein, the term "target nucleic acid region" or "target nucleotide
sequence" includes a probe-hybridizing region contained within the target
molecule. The
term "target nucleic acid sequence" includes a sequence with which a probe
will form a
stable hybrid under desired conditions.
As used herein, the term "nucleic acid probe" or simply "probe" includes
reference to a structure comprised of a polynucleotide, as defined above, that
contains a
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nucleic acid sequence complementary to a nucleic acid sequence present in the
target
nucleic acid analyte. The polynucleotide regions of probes may be composed of
DNA,
and/or RNA, and/or synthetic nucleotide analogs.
It will be appreciated that the hybridizing sequences need not have perfect
complementarity to provide stable hybrids. In many situations, stable hybrids
will form
where fewer than about 10% of the bases are mismatches, ignoring loops of four
or more
nucleotides. Accordingly, as used herein the term "complementary" refers to an
oligonucleotide that forms a stable duplex with its "complement" tinder assay
conditions,
generally where there is about 90% or greater homology.
An "array" broadly refers to an arrangement of antiligands in positionally
distinct locations on a substrate. Typically the location of the antiligands
on the array are
spatially encoded so that the identity of an antiligand of an array can be
deduced from its
location on the array. A "microarray" generally refers to an array in which
detection
requires the use of microscopic detection to detect complexes formed between
antiligands
and ligands. A "location" on an array refers to a localized area on the array
surface that
includes antiligands, each defined so that it can be distinguished from
adjacent locations
(e.g., being positioned on the overall array or having some detectable
characteristic that
allows the location to be distinguished from other locations). Typically, each
location
includes a single type of antiligand. The location can have any convenient
shape (e.g.,
circular, rectangular, elliptical or wedge-shaped). The size of a.n area can
vary
significantly. In some instances, the area of a location is greater than 1
cm2, such as 2-20
cm2, including any area within tlus range. More typically, the area of the
location is less
than 1 cma, in other instances less than 1 mrn2, in still other instances less
than 0.5 mm2,
in yet still other instances less than 10,000 ~.m2 , or less than 100 gm~'.
A "solid support" includes planar or nonplanar substrates such as glass,
nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride
(e.g., sheets
or microtiter wells); polystyrene latex (e.g., beads or microtiter plates);
polyvinylidine
fluoride; diazotized paper; nylon membranes; activated beads, magnetically
responsive
beads, and the Like.
The teen "aptamer" (or nucleic acid antibody) is used herein to refer to a
single- or double-stranded DNA or a single-stranded RNA molecule that
recognizes and
binds to a desired target molecule by virtue of its shape. See, e.g., PCT
Publication Nos.
W092/14843, W091/19813, and W092/05285.
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The term "aptazyme" includes allosteric ribozymes that are activated in the
presence of an effector molecule (either chemical or biological). Aptazyrnes
are capable
of transducing a noncovalent molecular recognition event into a catalytic
event, for
example, the production of a new covalent bond via ligation.
"Polypeptide" and "protein" are used interchangeably herein and include a
molecular chain of amino acids linked through peptide bonds. The terms do not
refer to a
specific length of the product. Thus, "peptides," "oligopeptides," and
"proteins" are
included within the definition of polypeptide. The terms include post-
translational
modifications of the polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. In addition, protein fragments, analogs,
mutated or variant
proteins, fusion proteins and the like are included within the meaning of
polypeptide.
A "ligand" generally refers to any molecule that binds to an antiligand to
form a ligand/antiligand pair. Thus, a ligand is any molecule for which there
exists
another molecule (i.e., the antiligand) that specifically binds to the ligand,
owing to
recognition of some portion or feature of the ligand.
An "antiligand" is a molecule that specifically or nonspecifically interacts
with another molecule (i.e., the ligand).
A "target molecule" or "analyte" refers to the species whose presence,
absence and/or concentration is being detected or assayed. In the array-based
assays,
described herein, the target molecule or analyte is also referred to as the
ligand.
As used herein, the term "binding pair" or "binding partners" refers to first
and second molecules that specifically bind to each other such as a ligand and
an
antiligand. The term binding pair or binding partners can refer to the
antiligand and
ligand that form a complex on an array. The terms can also refer to a first
molecule
attached to a ligand and a second molecule attached to a semiconductor
nanocrystal that
interact such that the ligand becomes attached to the semiconductor
nanocrystal via the
interacting binding pair members. "Specific binding" of the first member of
the binding
pair to the second member of the binding pair in a sample is evidenced by the
binding of
the first member to the second member, or vice versa, with greater affinity
and specificity
than to other components in the sample. The binding between the members of the
binding pair is typically noncovalent. Binding partners need not necessarily
be limited to
pairs of single molecules. For example, a single ligand can be bound by the
coordinated
action of two or more antiligands. The result of binding between bind pairs or
binding
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partners is a binding complex, sometimes referred to as a ligand/antiligand
complex or
simply as ligand/antiligand.
Exemplary binding pairs include: (a) any haptenic or antigenic compound
in combination with a corresponding antibody or binding portion or fragment
thereof
(e.g., digoxigenin and anti-digoxigenin; fluorescein and anti-fluorescein;
dinitrophenol
and anti-dinitrophenol; bromodeoxyuridine and anti-bromodeoxyuridine; mouse
immunoglobulin and goat anti-mouse immunoglobulin), (b) nonimmunological
binding
pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-Neutravidin); (c)
hormone [e.g.,
thyroxine and cortisol]-hormone binding protein; (d) receptor-receptor agonist
or
antagonist (e.g., acetylcholine receptor-acetylcholine or a~1 analog thereof);
(e) IgG-
protein A (f) lectin-carbohydrate; (g) enzyme-enzyme cofactor; (h) enzyme-
enzyme-
inhibitor; (i) and complementary polynucleotide pairs capable of forming
nucleic acid
duplexes and the like.
The terms "specific-binding molecule" and "affinity molecule" are used
interchangeably herein and refer to a molecule that will selectively bind,
through
chemical or physical means to a detectable substance present in a sample. By
"selectively
bind" is meant that the molecule binds preferentially to the target of
interest or binds with
greater affinity to the target than to other molecules. For example, an
antibody will
selectively bind to the antigen against which it was raised; A DNA molecule
will bind to
a substantially complementary sequence and not to unrelated sequences. The
affinity
molecule can comprise any molecule, or portion of any molecule, that is
capable of being
linked to a semiconductor nanocrystal and that, when so linked, is capable of
recognizing
specifically a detectable substance. Such affinity molecules include, by way
of example,
such classes of substances as antibodies, as defined below, monomeric or
polymeric
nucleic acids, aptarriers, proteins, polysaccharides, sugars, and the like.
See,. e.g.,
Haugland, "Handbook of Fluorescent Probes and Research Cheynicals" (Sixth
Edition),
and any of the molecules capable of forming a binding pair as described above.
A "semiconductor nanocrystal conjugate" is a semiconductor nanocrystal
that is linked to or associated with a specific-binding molecule, as defined
above. A
"semiconductor nanocrystal conjugate" includes, for example, a semiconductor
nanocrystal linked or otherwise associated, through the coat, to a member of a
"binding
pair" or a "specific-binding molecule" that will selectively bind to a
detectable substance
present in a sample, e.g., a biological sample as defined herein: The first
member of the
binding pair linked to the semiconductor nanocrystal can comprise any
molecule, or
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portion of any molecule, that is capable of being linked to a semiconductor
nanocrystal
and that, when so linked, is capable of recognizing specifically the second
member of the
binding pair.
The term "antibody" as used herein includes antibodies obtained from both
polyclonal and monoclonal preparations, as well as, the following: (i) hybrid
(chimeric)
antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299;
and U.S.
Patent No. 4,816,567); (ii) F(ab')2 and Flab) fragments; (iii) Fv molecules
(noncovalent
heterodimers, see, for example, mbar et al. (1972) Pz~oc Natl Acad Sci USA
69:2659-
2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); (iv) single-chain Fv
molecules
(sFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-
5883); (v)
dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack
et al.
(1992) Biochezn 31:1579-1584; Cumber et al. (1992) J. Immunology 149B:120-
126); (vi)
humanized antibody molecules (see, for example, Riechmann et al. (1988) Nature
332:323-327; Verhoeyan et al. (1988) Scienee 239:1534-1536; and U.K. Patent
Publication No. GB 2,276,169, published 21 September 1994); and, (vii) any
functional
fragments obtained from such molecules, wherein such fragments retain specific-
binding
properties of the parent antibody molecule.
Functional antibody fragments can be produced by cleaving a constant
region, not responsible for antigen binding, from the antibody molecule, using
e.g.,
pepsin, to produce F(ab')2 fragments. These fragments contain two antigen
binding sites,
but lack a portion of the constant region from each of the heavy chains.
Similarly, Fab
fragments, comprising a single antigen binding site, can be produced, e.g., by
digestion of
polyclonal or monoclonal antibodies with papain. Functional fragments,
including only
the variable regions of the heavy and light chains, can also be produced,
using standard
techniques such as recombinant production or preferential proteolytic cleavage
of
immunoglobulin molecules. These fragments are known as Fv. See, e.g., mbar et
al.
(1972) Pz~oc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochezn
15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
A single-chain Fv ("sFv" or "scFv") polypeptide is a covalently linked
VH-VL heterodimer which is expressed from a gene fusion including VH- and VL-
encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc.
Natl.
Acad. Sci. USA 85:5879-5883. A number of methods have been described to
discern and
develop chemical structures (linkers) for converting the naturally aggregated,
but
chemically separated, light and heavy polypeptide chains from an antibody V
region into
CA 02400379 2002-08-14
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an sFv molecule which will fold into a three dimensional structure
substantially similar to
the structure of an antigen-binding site. See, e.g., U.S. Patent Nos.
5,091,513, 5,132,405
and 4,946,778. The sFv molecules may be produced using methods described in
the art.
See, e.g., Huston et al. (1988) P~oc. Nat. Acad. Sci. USA 85:5879-5883; U.S.
Patent Nos.
5,091,513, 5,132,405 and 4,946,778. Design criteria include determining the
appropriate
length to span the distance between the C-terminus of one chain and the N-
terminus of
the other, wherein the linker is generally formed from small hydrophilic amino
acid
residues that do not tend to coil or form secondary structures. Such methods
have been
described in the art. See, e.g., U.S. Patent Nos. 5,091,513, 5,132,405 and
4,946,778.
Suitable linkers generally comprise polypeptide chains of alternating sets of
glycine and
serine residues, and may include glutamic acid and lysine residues inserted to
enhance
solubility.
"Mini-antibodies" or "minibodies" are sFv polypeptide chains that include
oligomerization domains at their C-termini, separated from the sFv by a hinge
region.
Pack et al. (1992) Biochem 31:1579-1584. The oligomerization domain comprises
self
associating a-helices, e.g., leucine zippers, that can be further stabilized
by additional
disulfide bonds. The oligomerization domain is designed to be compatible with
vectorial
folding across a membrane, a process thought to facilitate in vivo folding of
the
polypeptide into a functional binding protein. Generally, minibodies are
produced using
recombinant methods well known in the art. See, e.g., Pack et al. (1992)
Biochem
31:1579-1584; Cumber et al. (1992) Jlmsnunology 149B:120-126.
As used herein, the term "monoclonal antibody" refers to an antibody
composition having a homogeneous antibody population. The term is not limited
regarding the species or source of the antibody, nor is it intended to be
limited by the
manner in which it is made. Thus, the term encompasses antibodies obtained
from
marine hybridomas, as well as human monoclonal antibodies obtained using human
rather than marine hybridomas. See, e.g., Cote, et al. Mofaclonal Antibodies
ayad Cahcef°
Tlaef~apy, Alan R. Liss, 1985, p. 77.
A semiconductor nanocrystal is "linked" or "conjugated" to, or
"associated" with, a specific-binding molecule or member of a binding pair
when the
semiconductor nanocrystal is chemically coupled to, or associated with the
specific-
binding molecule. Thus, these terms intend that the semiconductor nanocrystal
can either
be directly linked to the specific-binding molecule or can be linked via a
linker moiety,
such as via a chemical linker described below. The terms indicate species that
are
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physically linked by, for example, covalent chemical bonds, physical forces
such van der
Waals or hydrophobic interactions, encapsulation, embedding, or the like. As
an example
without limiting the scope of the invention, semiconductor nanocrystals can be
conjugated to molecules that can interact physically with biological compounds
such as
cells, proteins, nucleic acids, subcellular organelles and other subcellular
components.
For example, semiconductor nanocrystals can be associated with biotin which
can bind to
the proteins, avidin and streptavidin. Also, semiconductor nanocrystals can be
associated
with molecules that bind nonspecifically or sequence-specifically to nucleic
acids (DNA,
RNA). As examples without limiting the scope of the invention, such molecules
include
small molecules that bind to the minor groove of DNA (for reviews, see
Geierstanger and
Wemrner (1995) Aran. Rev. Biophys. Biomol. Struct. 24:463-493; and Baguley
(1982)
Mol. Cell. Biochem 43:167-181), small molecules that form adducts with DNA and
RNA
(e.g. CC-1065, see Henderson and Hurley (1996) J. Mol. Recognit. 9:75-87;
aflatoxin, see
Garner (1998) Mutat. Res. 402:67-75; cisplatin, see Leng and Brabec (1994)
IARC Sci.
Publ. 125:339-348), molecules that intercalate between the base pairs of DNA
(e.g.
methidium, propidium, ethidium, porphyrins; for a review see Bailly et al. J.
Mol.
Recognit. 5:155-171), radiomimetic DNA damaging agents such as bleomycin,
neocarzinostatin and other enediynes (for a review, see Povirk (1996) Mutat.
Res. 355:71-
89), and metal complexes that bind and/or damage nucleic acids through
oxidation (e.g.
Cu-phenanthroline, see Perrin et al. (1996) Prog. Nucleic Acid Res. Mol. Biol.
52:123-
151; Ru(II) and Os(II) complexes, see Moucheron et al. (1997) J. Photochem.
P7zotobiol.
B 40:91-106; chemical and photochemical probes of DNA, see Nielsen (1990) J.
Mol.
Recognit. 3:1-25.
As used herein, a "biological sample" refers to a sample of isolated cells,
tissue or fluid, including but not limited to, plasma, serum, spinal fluid,
semen, lymph
fluid, the external sections of the skin, respiratory, intestinal, and
genitourinary tracts,
tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro
cell culture
constituents (including, but not limited to, conditioned medium resulting from
the growth
of cells in cell culture medium, putatively virally infected cells,
recombinant cells, and
cell components).
A "small molecule" is defined as including an organic or inorganic
compound either synthesized in the laboratory or found in nature. Typically, a
small
molecule is characterized in that it contains several carbon-carbon bonds, and
has a
molecular weight of less than 1500 grams/Mol.
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A "biomolecule" is a synthetic or naturally occurring molecule, such as a
protein, amino acid, nucleic acid, nucleotide, carbohydrate, sugar, lipid and
the like.
The term "multiplexing" is used herein to include conducting an assay or
other analytical method in which multiple analytes or biological states can be
detected
simultaneously by using more than one detectable label, each of which emits at
a distinct
wavelength, with a distinct intensity, with a distinct FWHM, with a distinct
fluorescence
lifetime, or any combination thereof. Preferably, each detectable label is
linked to one of
a plurality of first members of binding pairs each of which first members is
capable of
binding to a distinct corresponding second member of the binding pair. A
multiplexed
method using semiconductor nanocrystals having distinct emission spectra can
be used to
detect simultaneously in the range of 2 to 1,000,000, preferably in the range
of 2 to
10,000, more preferably in the range of 2 to 100, or any integer between these
ranges, and
even more preferably in the range of up to 10 to 20, e.g., 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20, of analytes, biological compounds or
biological states.
Multiplexing also includes assays or methods in which the combination of more
than one
semiconductor nanocrystal having distinct emission spectra can be used to
detect a single
analyte.
"Optional" or "optionally" means that the subsequently described event or
circumstance may or may not occur, and that the description includes instances
where the
event or circumstance occurs and instances in wluch it does not. For example,
the phrase
"optionally overcoated with a shell material" means that the overcoating
referred to may
or may not be present in order to fall within the scope of the invention, and
that the
description includes both the presence and absence of such overcoating.
A "site of variation," "variant site" or "allelic site" when used with
reference to a nucleic acid broadly refers to a site wherein the identity of
nucleotide at the
+e..,....~"t.~+._.......,.......~..:......:a....~...~._ai_________,_____
_____.,_________._,-._._ ,"
CA 02400379 2002-08-14
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"Polymorphic marker" or "site" refers to a genetic locus at which
divergence occurs. Preferred markers have at least two polymorphic forms, each
occurring at frequency of greater than 1%, and more preferably greater than
10% or 20%
of a selected population. A genetic locus can be as small as one base pair, if
the
polymorphism is a nucleotide substitution or deletion, or many base pairs if
the
polymorphism is, e.g., deletion, inversion or duplication of part of a
chromosome.
Polymorphic markers include, e.g., restriction fragment length polymorphisms,
variable
number of tandem repeats (VNTR's), hypervariable regions, minisatellites,
dinucleotide
repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence
repeats, and
insertion elements such as Alu. One identified allelic form is arbitrarily
designated as a
the reference allele and other allelic forms are designated as alternative or
variant alleles.
The allelic form occurring most frequently in a selected population is
sometimes referred
to as the wild-type form. Diploid organisms may be homozygous or heterozygous
for
allelic forms. A di-allelic polymorphism has two forms. A tri-allelic
polymorphism has
three forms.
A single nucleotide polymorphism (SNP) occurs at a polymorphic site
occupied by a single nucleotide, which is the site of variation between
allelic sequences.
The site is usually preceded by and followed by highly conserved sequences of
the allele
(e.g., sequences that vary in less than 1/100 or 1/1000 members of the
populations). A
single nucleotide polymorphism usually arises due to substitution of one
nucleotide for
another at the polymorphic site. A transition is the replacement of one purine
by another
purine or one pyrimidine by another pyrimidine. A transversion is the
replacement of a
purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also
arise
from a deletion of a nucleotide or an insertion of a nucleotide relative to a
reference
allele.
A "primer" is a single-stranded polynucleotide capable of acting as a point
of initiation of template-directed DNA synthesis under appropriate conditions
(i.e., in the
presence of four different nucleoside triphosphates and an agent for
polymerization, such
as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer
and at a
suitable temperature. The appropriate length of a primer depends on the
intended use of
the primer but typically is at least 7 nucleotides long and, more typically
range from 10 to
30 nucleotides in length. Short primer molecules generally require cooler
temperatures to
form sufficiently stable hybrid complexes with the template. A primer need not
reflect
the exact sequence of the template but must be sufficiently complementary to
hybridize
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with a template. The term "primer site" or "primer binding site" refers to the
segment of
the target DNA to which a primer hybridizes. The term "primer pair" means a
set of
primers including a 5' "upstream primer" that hybridizes with the complement
of the 5'
end of the DNA sequence to be amplified and a 3' "downstream primer" that
hybridizes
with the 3' end of the sequence to be amplified.
A primer that is "perfectly complementary" has a sequence fully
complementary across the entire length of the primer and has no mismatches.
The primer
is typically perfectly complementary to a portion (subsequence) of a target
sequence. A
"mismatcli" refers to a site at which the nucleotide in the primer and the
nucleotide in the
target nucleic acid with which it is aligned are not complementary. The term
"substantially complementary" when used in reference to a primer means that a
primer is
not perfectly complementary to its target sequence; instead, the primer is
only sufficiently
complementary to hybridize selectively to its respective strand at the desired
primer-
binding site.
A "specimen" is a small part, or sample, of any substance or material
obtained for analysis.
A "tissue" is an aggregation of similar cells united in the performance of a
particular function. The four basic tissues are epithelium, connective tissues
(including
blood, bone and cartilage), muscle tissue and nerve tissue.
A "cellular specimen" is one that contains whole cells, and includes
tissues. Examples include, but are not limited to, cells from the skin,
breast, prostrate,
blood, testis, ovary and endometrium.
A "cellular suspension" is a liquid in which cells are dispersed, and can
include a uniform or non-uniform suspension. Examples of cellular suspensions
are those
obtained by fine-needle aspiration from tumor sites, cytology specimens ,
washes, urine
that contains cells, ascitic fluid, or other bodily fluids.
A "cytological preparation" is a pathological specimen in which a cellular
suspension can be converted into a smear or other form for pathological
examination or
analysis."
A "tumor" is a neoplasm that may be either malignant or non-malignant.
"Tumors of the same tissue type" refers to primary tumors originating in a
particular
organ (such as breast, prostrate, bladder or lung).
The term "naturally occurring" as applied to an object means that the
object can be found in nature.
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The term "subject" and "individual" are used interchangeably herein to
refer to any type of organism, including, but not limited to, plants, animals
and
microorganisms.
II. Overview
The present invention provides a variety of methods for conducting assays
with different types of addressable arrays using semiconductor nanocrystals
(also referred
to herein simply as a quantum dot or a QdotTM) as a label to enhance detection
of various
complexes formed on the array. Semiconductor nanocrystals can be used to label
various
ligands or target molecules for use in nucleic acid arrays, protein arrays,
tissue arrays or
essentially any other type of array that utilizes optical detection methods.
The
semiconductor nanocrystal labels can be directly incorporated into, or
directly attached to,
the ligands of interest through covalent or non-covalent attachment,or
indirectly attached
via a linker. By labeling ligands in different samples with different
semiconductor
nanocrystals, the methods can be used in multiplex formats to simultaneously
evaluate a
plurality of samples with a single array. Some methods also utilize ligands
that each bear
a single semiconductor nanocrystal.
By controlling various parameters, semiconductor nanocrystals utilized to
label ligands or antiligands for use in array-based assays can be tailored to
have a number
of desired properties. For example, semiconductor nanocrystals can be produced
that
have characteristic spectral emissions. These spectral emissions can be tuned
to a desired
wavelength by varying the particle size, size distribution and/or composition
of the
particle. This means that multiple emission colors can be achieved, a feature
that can be
utilized in separately detecting ligands from different samples. The emission
spectra of a
population of semiconductor nanocrystals can be manipulated to have linewidths
as
narrow as 25-30 nm, depending on the size distribution heterogeneity of the
sample
population, and lineshapes that are symmetric, gaussian or nearly gaussian
with an
absence of a tailing region. The combination of tunability, narrow linewidths,
and
symmetric emission spectra enables high resolution of multiply sized
semiconductor
nanocrystals (e.g., populations of monodisperse semiconductor nanocrystals
having
multiple distinct size distributions within a system) and simultaneous
detection of a
variety of species.
In addition, the range of excitation wavelengths of such nanocrystals is
broad and can be higher in energy than the emission wavelengths of all
available
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semiconductor nanocrystals. This feature allows the use of a single energy
source, such
as light, usually in the ultraviolet or blue region of the spectrum, to effect
simultaneous
excitation of all populations of semiconductor nanocrystals in a system having
distinct
emission spectra. Semiconductor nanocrystals can also be more robust than
conventional
organic fluorescent dyes by having a high quantum yield, and typically are
more resistant
to photobleaching than the organic dyes conventionally utilized in array-based
assays.
The robustness of the nanocrystal also alleviates the problem of contamination
of
degradation products of the organic dyes in the system being examined.
Moreover,
semiconductor nanocrystals have a relatively large Stokes shift, thereby
significantly
reducing problems with autofluorescence and scattered excitation light.
Therefore, array-
based technology used in combination with semiconductor nanocrystals can be
used as a
sensitive way to conduct a variety of assays and in certain instances the
methods can be
designed to allow for quantification of complexes formed on an array.
These various aspects of semiconductor nanocrystals also permit flexibility
in methods for detecting and quantifying ligands as assayed using arrays. For
example,
the ability to detect single nanocrystals means that in some instances single
ligands bound
to the array can be individually counted. This capability means that one can
quantitate
the amount of ligand bound to the array, as well as quantifying the amount of
ligand in
the original sample containing the ligand by calibration against samples of
known
concentration.
The use of semiconductor nanocrystals also enables the dynamic range of
detection to be extended relative to assays conducted with other types of
labels.
Depending upon the density of labeled ligand bound to the array, detection can
involve
counting of single ligands (lower densities) or determining the total emission
intensity
from each location of the array (higher ligand density). The ability to chose
between
these detection regimes results in significant expansion of the dynamic range
of detection,
thus allowing a greater range in the concentration of ligands that can
accurately be
quantified either as attached to the array or in the original sample.
III. Arran-Based Methods Utilizing Semiconductor Nanocr s
A. General Methods
The present invention in general provides a variety of methods for
assaying for ligands or target molecules using various array formats.
Semiconductor
nanocrystals are used as a labeling agent to enhance detection in several
respects. The
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methods utilize arrays that include a substrate or support upon which a
plurality of
antiligands are placed or attached. If attached, the antiligands can be
directly attached to
the support, or attached via a linker. The array includes a variety of
distinct locations to
which the antiligands are placed or attached, hence the identity of the
antiligands on the
array is spatially encoded. Each location has at least one antiligand, but
often there are a
plurality of antiligands at each location. The antiligands at the various
locations can be
the same or different.
The array is contacted with a sample that contains, or potentially contains,
one or more ligands. As the ligands in the sample are brought into contact
with the
antiligands of the array, ligands and antiligands that are members of a
binding pair
interact to form complexes. The ligands can be labeled with semiconductor
nanocrystals
either before or after the sample containing the ligands is contacted with the
array.
The array is then typically rinsed to remove uncomplexed ligand and other
assay components. Complexes formed on the array axe identified by detecting a
signal
mediated by the semiconductor nanocrystals contained within the complexes. The
identity of antiligands that have bound to a ligand can be determined based
upon the
location of the antiligand on the array.
Various modifications upon this general scheme can be made. For
example, a sample containing one or more unlabeled ligands can be contacted
with an
array including multiple antiligands. As described above, ligands and
antiligands that are
binding partners form binary complexes. Since the ligands are unlabeled,
complexes can
be detected by contacting the binary complexes with a sample that contains
secondary
antiligands labeled with semiconductor nanocrystals. The secondary antiligands
can bind
to ligands in the binary complexes that are binding partners to form a
tertiary complex.
Those locations of the array in which an antiligand is complexed with a ligand
can then
be detected by a signal from the semiconductor nanocrystal in the tertiary
complexes.
This approach is a sandwich type assay in which the antiligand serves to
capture a ligand
which is its binding partner. The ligand is then bound to the labeled
secondary antiligand
such that the ligand is sandwiched between the two antiligands.
As described in greater detail infra, the ability to tune different
semiconductors to emit at a distinctive wavelength by adjusting their size
enables a
variety of different multiplex analyses to be conducted. For example,
different ligands
from different samples can be separately labeled and then mixed together. The
mixture
can be applied to the array and complexes containing ligands from different
samples
23
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identified on the basis of the color of the semiconductor nanocrystal within
the complex.
Alternatively, different ligands within a single sample can be differentially
labeled by
selectively attaching a first member of different binding pairs to the
different ligands.
The second member of the various binding pairs can than be selectively
attached to
different semiconductor nanocrystals. The resulting ligands and semiconductor
nanocrystals can then be mixed. Different ligands within the sample become
differentially labeled because each ligand only joins to a label that bears a
complementary
binding pair member.
B. Semiconductor Nanocr ss
Semiconductor nanocrystals are typically nanometer sized semiconductor
crystals that have optical properties that are strongly dependent on both the
size and the
material of the crystal (see, e.g., Alivisatos (1996) Science 271:933-937).
One feature of
semiconductor nanocrystals is that the absorption and emission spectra from
' semiconductor nanocrystals can be tuned across a broad range of the
electromagnetic
spectrum by changing their size. For example, semiconductor nanocrystals
manufactured
from CdSe can emit light in a narrow wavelength band at any chosen wavelength
between
490 nm and 640 nm.
The principle behind the size dependent optical properties of
semiconductor nanocrystals is an effect called "quantum confinement" (see,
e.g., Efros, et
al. (192) Sov. Phyr. Semicoyad. 16:772-775). Light emission from bulk
semiconductors
is generated through the creation and annihilation of an electron and anti-
electron (hole)
within the semiconductor lattice. In bulk semiconductors, the energy of this
"electron-
hole pair" is govenled entirely by the composition of the semiconductor
material. If,
however, the physical size of the semiconductor is reduced so that it is
smaller than the
intrinsic size of the electron-hole pair, additional energy is required to
confine this
excitation within the semiconductor structure. In the size~range of
semiconductor
nanocrystals, the confinement energy can be extremely large, and becomes one
of the
dominant factors affecting the absorption and emission energies of the
material.
Therefore, by changing the size of the quantum dots, the absorption and
emission can be
modified due to changes in the confinement energy. Figures 2A - 2B demonstrate
this
effect by showing a series of absorption and emission spectra from different
size
semiconductor nanocrystals of the same material (CdSe). Changing the material
of the
semiconductor nanocrystal can also affect the emission energy. By using a few
different
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materials, it is possible to generate semiconductor nanocrystals with emission
spectra that
are tunable from the ultraviolet into the infrared (see FIG. 2C).
Semiconductor nanocrystals demonstrate quantum confinement effects in
their luminescent properties. When semiconductor nanocrystals are illuminated
with a
primary energy source, a secondary emission of energy occurs at a frequency
that
corresponds to the bandgap of the semiconductor material used in the
semiconductor
nanocrystal. In quantum confined particles, the bandgap energy is a function
of the size
and/or composition of the nanocrystal. A mixed population of semiconductor
nanocrystals of various sizes and/or compositions can be excited
simultaneously using a
single wavelength of light and the detectable luminescence can be engineered
to occur at
a plurality of wavelengths. The luminescent emission is related to the size
and/or the
composition of the constituent semiconductor nanocrystals of the population.
More specifically, quantum confinement of both the electron and hole in
all three dimensions leads to an increase in the effective band gap of the
material with
decreasing crystallite size. Consequently, both the optical absorption and
emission of
semiconductor nanocrystals shift to the blue (higher energies). Upon exposure
to a
primary light source, each semiconductor nanocrystal distribution is capable
of emitting
energy in narrow spectral linewidths, as narrow as 12 nm to 60 mn full width
of
emissions at half peak height (FWI~VI), and with a symmetric, nearly Gaussian
line
shape, thus providing an easy way to identify a particular semiconductor
nanocrystal. As
one of ordinary skill in the art will recognize, the linewidths are dependent
on, among
other things, the size heterogeneity, i.e., monodispersity, of the
semiconductor
nanocrystals in each preparation. Certain single semiconductor nanocrystal
complexes
have been observed to have F~~VHM as narrow as 12 nm to 15 nm. Semiconductor
nanocrystal distributions with larger linewidths in the range of 35 nm to 60
mn can be
readily made and have the same physical characteristics as semiconductor
nanocrystals
with narrower linewidths.
Because the emission characteristics of semiconductor nanocrystals are
dependent upon size and composition one can detect and/or distinguish between
different
semiconductor nanocrystals in a number of ways, including for example,
emission
intensity, emission wavelength, full width at half maximum peak height,
absorption,
scattering, fluorescence lifetime, or any combination of the foregoing.
A core/shell semiconductor nanocrystal is one made from one material
such as CdSe that has been coated with a shell of a second, higher bandgap
material such
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as ZnS (see, e.g., Hines et al. (1996) J. Phys. Chem. 100:468-471; Peng, et
al. (1997) J.
Am. Chem. Soc. 119:7019-7029; and Dabbousi, et al. (1997) J. Phys. Chem. B
101:9463-
9475). The higher bandgap shell material protects the fluorescent electron-
hole pair from
interacting with the surface and surrounding environment (such interactions
can produce
fluorescence quenching in semiconductor nanocrystals). This results in
significantly
enhanced fluorescence quantum yields, typically from 50% to 80% . These
core/shell
structures have a surface that is intrinsically functionalized with organic
ligands.
As described further below, modification of these ligands allows one to
make water soluble semiconductor nanocrystals that can be directly conjugated
to
biologically relevant molecules such as biotin, streptavidin and antibodies.
Techniques
for coupling semiconductor nanocrystals and a variety of biological molecules
or
substrates are described, for example, by Bruchez et. al. (1998) Scieyace
281:2013-2016,
Chan et. al. (1998) Sciefzce 281:2016-2018, Bruchez "Luminescent Semiconductor
Nanocrystals: Intermittent Behavior and use as Fluorescent Biological Probes"
(1998)
Doctoral dissertation, University of California, Berkeley, Mikulec
"Semiconductor
Nanocrystal Colloids: Manganese Doped Cadmium Selenide, (Core)Shell Composites
for Biological Labeling, and Highly Fluorescent Cadmium Telluride" (1999)
Doctoral
dissertation, Massachusetts W stitute of Technology.
Several optical properties make semiconductor nanocrystals useful for
detecting complexes in array-based methods. These properties include:
1) Large absorption cross sections. Semiconductor nanocrystals have very
large absorption cross-sections relative to comparable organic dyes. For
instance, Cy5
has a maximum cross section at approximately 630 nm of ~250,OOOM-1 cni 1 while
a red
CdSe semiconductor nanocrystal (emission at 640 nm) has a cross section at 630
nm of
800,000 M-lcrri 1 and greater than 2x106 M-lcrri 1 at 488 nm. This means that
with
comparable excitation intensities at 488 nm and 632 nm for semiconductor
nanocrystals
and Cy5 respectively, semiconductor nanocrystals can absorb more than 8 times
the
amount of incident light.
2) High quantum yield. As described above, semiconductor nanocrystals
can have quantum yields as high as 80%, hence a significant number of the
absorbed
photons are re-emitted as fluorescent signal.
3) High photostability. Unlike many organic dyes, semiconductor
nanocrystals exhibit high photostability. Figure 3 shows a comparison of
photobleaching
between fluorescein and water soluble semiconductor nanocrystals under
identical
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excitation conditions. Enhanced photostability means that fluorescence from
semiconductor nanocrystals can be integrated for long periods of time, thereby
significantly enhancing detection sensitivity.
High absorption cross section, high quantum yield and high photostability
combine to make an extremely bright fluorophore. With certain semiconductor
nanocrystals, one can detect the fluorescence from single semiconductor
nanocrystals
(FIGS. 4A and 4B). In fact, the fluorescence from certain single semiconductor
nanocrystals is sufficiently bright, that it can be seen by eye. This high
fluorescence
intensity can be exploited to allow the detection of single bound target
molecules labeled
with semiconductor nanocrystals as described in greater detail infra.
Additional useful characteristics of semiconductor nanocrystals include:
4) Narrow, symmetric emission spectra. The emission spectra from
semiconductor nanocrystals are significantly narrower than most organic dyes,
and do not
have asymmetric tails extending to longer wavelengths. Figures SA and SB show
a
comparison between the absorption and emission spectra of fluorescein (FIG.
5A) and a
comparable color semiconductor nanocrystal analogue (FIG. 5B). Narrow,
symmetric
emission spectra significantly reduce the overlap of adjacent colors in
multiplexed assays,
thereby increasing detection sensitivity.
5) Large "Stokes-shifts." Contrary to the performance observed with
organic dyes, shows that semiconductor nanocrystals actually absorb more light
the
farther away from the emission that they are excited (see FIGS. 5A and SB).
Hence, one
can excite very far to the blue, minimizing any interference at the emission
wavelength
from scattered excitation light or autofluorescence, which generally results
in emissions at
wavelengths close to the excitation wavelength.
6) Tunable emission. Semiconductor nanocrystals can be synthesized to
control the wavelength at which they emit. Emission wavelengths can therefore
be
selected to avoid overlap with autofluorescence. In addition, since
semiconductor
nanocrystals can also be excited at wavelengths shorter than the emission
wavelength,
excitation can also be chosen to avoid exciting autofluorescence.
Appropriately chosen
excitation and emission wavelengths can significantly reduce autofluorescence,
thereby
increasing detection sensitivity.
7) Multiplexible emission. Broad excitation spectra allow a large number
of semiconductor nanocrystal colors to be simultaneously excited with a single
excitation
wavelength. For instance, all semiconductor nanocrystal samples in FIGS. 2A
and 2B
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can be efficiently excited with 457 nm light. This characteristic is unique to
semiconductor nanocrystals, and simplifies the development of optical systems
for
detection of multiplexed bioassays.
C. Labeling
A variety of methods are available for labeling biomolecules with
semiconductor nanocrystals. In certain methods, the semiconductor nanocrystal
and the
biomolecule have appropriate functional groups that allow the two molecules to
be
coupled. Certain biomolecules can be labeled by labeling a component of the
biomolecule (e.g., a monomer of a polymer) which becomes incorporated into the
final
biomolecule during synthesis (e.g., incorporation of a nucleotide labeled with
a
semiconductor nanocrystal into a nucleic acid). The semiconductor nanocrystal
and
biomolecule can also be linked via a linker. The linkers typically are
bifunctional, having
a functional group at each end. One end of the linker becomes attached to the
semiconductor nanocrystal and the other end to the biomolecule.
Alternatively, the semiconductor nanocrystal can bear one member of a
binding pair and the biomolecule the other member of the binding pair. The
biomolecule
and nanocrystal can thus be joined via the binding pair members.
Additional detail regarding labeling is set forth for the various types of
array-based assays and in the conjugation section iraf~a.
D. Arravs
As indicated above, an array broadly refers to an arrangement of
biomolecules in positionally distinct locations on a substrate such that the
identity of the
various biomolecules in the array can be determined based upon their location
in the
array. The biomolecules of the array are attached to a support that maintains
the relative
position of the biomolecules of the array either directly or via a linker.
The support can be any material which can support a plurality of
biomolecules and maintain the biomolecules such that they remain positionally
distinct.
Hence, the support can be manufactured from a wide variety of materials. For
example,
the support can be made of organic, inorganic, biological, or nonbiological
materials or
combinations of these materials. Specific examples of suitable supports
include, but are
not limited to, various plastics, polymers, Pyrex~, quartz, resins, silicon,
silica or silica
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based materials, carbon, metals, inorganic glasses, inorganic crystals,
cellulose, nylon and
the like.
The form of the support can also vary. The support can have essentially
any configuration. It may include a substantially planar surface or lack a
planar surface.
The substrate can have raised or depressed regions at which a reaction can
occur or at
which a solution or suspension can be placed. A specific example of such a
support is a
microtiter plate as is known in the art. Other suitable shapes for the support
include, but
are not limited to, beads, particles, strands, gels, sheets, membranes,
tubing, capillaries,
pads, films, plates and slides, for example. The array can also be in the form
of a bundle
of optical fibers, each fiber in the bundle having an end that is
substantially planar or that
includes a cavity etched into the end (see, e.g., U.S. Pat. No. 5,837,196 and
PCT
Publication WO 98/50782).
IV. Nucleic Acid Arrays
A. General Methods with Nucleic Acid Arrays
1. Contacting hybridization
A sample containing or potentially containing target nucleic acids from
one or more sources is contacted with an array of nucleic acid probes attached
at different
locations on the array. The target nucleic acids are typically labeled with
one or more
semiconductor nanocrystals prior to contacting the array with a sample, or
include a
modified nucleotide that permits the facile labeling of the target nucleic
acids after they
have become hybridized to complementary probes attached to the array. For
example, a
modified nucleotide can be a nucleotide (e.g., dATP, dTTP, dGTP and dCTP) that
has
been functionalized with a group that reacts with a complementary functional
group borne
by the semiconductor nanocrystal. Alternatively, the modified nucleotide is
attached to
one member of a binding pair that specifically binds to the other member of
the binding
pair that is attached to a semiconductor nanocrystal.
After the target nucleic acids have been allowed to hybridize with
complementary probes attached to the array, the array is optionally washed
with a
stringency buffer to remove unbound or non-specifically bound target nucleic
acids.
Hybridization complexes formed on the array are detected by detecting a signal
associated or mediated by semiconductor nanocrystals attached to target
nucleic acids that
axe within the hybridization complexes.
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These series of steps can be automated utilizing various automated
systems. These systems can include temperature controllers and mixers to
regulate the
reaction conditions as appropriate to the particular analysis being conducted.
The
systems can be programmable to program the temperature and mixing conditions,
as well
as to automatically dispense reagents, wash the array and perform detection
assays.
Information regarding such systems and components is described, for example,
in PCT
publication WO 95/3386.
As just noted, the array typically is stringency washed following
application of sample to the array to remove unbound target nucleic acids and
to at least
partially remove target nucleic acids that are not perfectly complementary to
the probe
nucleic acid to which they are bound. The stringency of selected hybridization
conditions
depends on various factors known in the art, including, e.g., temperature,
ionic strength
and pH. An extensive guide to the hybridization of nucleic acids is found in
Tijssen,
Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic
Probe,
"Overview of principles of hybridization and the strategy of nucleic acid
assays" (1993).
Generally, "stringent conditions" are selected to be about 5-10 °C
lower
than the thermal melting point (Tm) for the specific sequence at a defined
ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic
acid
concentration) at which 50% of the probes complementary to the target
hybridize to the
target sequence at equilibrium (as the target sequences are present in excess,
at Tm, 50%
of the probes are occupied at equilibrium). Stringent conditions are those in
which the
salt concentration is less than about 1.0 M sodium ion, typically about 0.01
to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature
is at least
about 30 °C for short probes (e.g.; 10 to 50 nucleotides) and at least
about 60 °C for long
probes (e.g., greater than 50 nucleotides). Stringent conditions can also be
achieved with
the addition of de-stabilizing agents such as formamide. For selective or
specific
hybridization, a positive signal is at least two times background, preferably
10 times
background hybridization.
"Moderately stringent hybridization conditions" include hybridization in a
buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 °C, and a wash in 1X
SSC at 45 °C.
A positive hybridization is at least twice background. Those of ordinary skill
will readily
recognize that alternative hybridization and wash conditions can be utilized
to provide
conditions of similar stringency.
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2. Labeling of Nucleic Acids
A variety of methods can be utilized to incorporate semiconductor
nanocrystals into the target nucleic acids to be analyzed by use of an array.
One approach
involves enzymatically incorporating semiconductor nanocrystal-dNTPs into
reverse-
transcribed cDNA by reverse transcribing a template nucleic acid in the
presence of
dNTPs labeled with semiconductor nanocrystals. In a second approach, the
desired
cDNA is amplified using polymerase chain reaction (PCR) primers labeled with
one or
more semiconductor nanocrystals.
Because the intensity of the emission of certain semiconductor
nanocrystals is sufficiently high such that a single semiconductor nanocrystal
can be
detected, in some methods it is advantageous to incorporate just a single
semiconductor
into the nucleic acid. Incorporation of a single semiconductor nanocrystal
into a nucleic
acid can be achieved using primers that are labeled with a single
semiconductor
nanocrystal during amplification of target. As described in greater detail
below, labeling
with a single semiconductor nanocrystal allows one to quantify the amount of
ligand (e.g.,
cDNA) that has hybridized to the array using the total fluorescence intensity.
With
existing organic dyes, low sensitivity requires that multiple fluorophores be
attached to
each cDNA for detection. This prevents a quantitative measure of cDNA
hybridization,
since the number of fluorophores/cDNA is not known.
A third nucleic acid labeling approach involves synthesizing cDNA from
active group-functionalized dNTP using reverse transcriptase. The resulting
unlabeled
form is then labeled by directly conjugating the active groups to the surface
of
semiconductor nanocrystals. As used herein, an "active group" or "functional
group" has
means an atom or group of atoms that define the structure of a particular
molecule or
family of molecules and, at the same time, determines their properties.
Exemplary
functional groups include hydroxyl, sulfhydryl, carbonyl, carboxyl, amino and
double or
triple bonds. In one specific example of such an approach, an amine-
functionalized
dNTP is covalently bound to succinamidyl ester-functionalized semiconductor
nanocrystals. By first generating the cDNA using unlabeled dNTPs and
subsequently
labeling the synthesized product, one can avoid the possibility of steric
hindrance
preventing transcription of template into cDNA.
Another option is to postpone labeling until after a target has become
hybridized to a complementary probe nucleic acid borne by the array. For
example, one
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can incorporate different active groups into different cDNA chains by reverse
transcribing
a template nucleic acid in the presence of different functionalized dNTPs. The
synthesized cDNA is then hybridized to probes on the array, followed by
conjugation of
the semiconductor nanocrystals to the functional groups borne by the dNTPs
that were
incorporated into the synthesized cDNA. For example, four semiconductor
nanocrystals
each of a different color and each with a different surface functionalization
can be washed
over a hybridized array to label four different sets of cDNA. This approach
has the
advantage that the semiconductor nanocrystals are not present during the
hybridization
step, thereby minimizing potential interference by the nanocrystals with
hybridization.
This rri'ethod also minimizes the possibility of cross-linking different cDNA
strands
during labeling if individual semiconductor nanocrystals have more than one
functional
group on the surface. This type of labeling can also be done for single color
detection by
using a single active group and conjugating after hybridization.
Another labeling approach is to fragment transcribed DNA and then end-
label the fragments with a semiconductor nanocrystal-dTTP conjugate using
terminal
transferase.
In all of the above procedures, except those in which labeling occurs after
hybridization, multiplexed assays can be performed by preparing and labeling
different
cDNA samples separately, and then blending the samples together prior to
hybridization
on the array.
Certain labeling option involve joining the target nucleic acid to a
semiconductor nanocrystal via some type of linker. The linker can be any of a
number of
different homo- and hetero-bifunctional moieties that include a functional
group at either
end of a chain of molecules; the functional groups at each end can be the same
or
different. Examples of suitable linkers include, but are not limited to,
straight or
branched-chain carbon linkers, heterocyclic linkers and peptide linkers.
Suitable linkers
are available from Pierce Chemical Company in Rockford, Illinois and are
described in
EPA 188,256; U.S. Pat. Nos. 4,671,958; 4,659,839; 4,414,148; 4,669,784;
4,680,338,
4,569, 789 and 4,589,071, and by Eggenweiler, H.M, (1998) Df~ugDiscovery
Today, 3:
552.
A number of labeling options involve the use of binding pair members, in
which one member of the pair is attached to the semiconductor nanocrystal and
the other
member is attached to a nucleic acid or nucleotide incorporated therein. The
binding pair
members can be any set of molecules that specifically bind to one another.
Suitable
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binding pair members include, but are not limited to, antigen/antibodies,
biotin/streptavidin (or avidin or neutravidin) and oligosaccharide/lectin.
Specific examples of such labeling methods include the following. One
option is to bind streptavidin-coated semiconductor nanocrystals to
biotinylated cDNA.
The reverse approach can also be taken in which biotin-coated semiconductor
nanocrystals are bound to biotinylated cDNA through a streptavidin bridge.
Another
approach is to bind antibody-labeled semiconductor nanocrystals to antigen-
labeled
cDNA. For instance, digoxigenin-labeled cDNA can be bound to semiconductor
nanocrystal-labeled anti-digoxigenin antibody. The member of the binding pair
can be
incorporated at internal locations within a cDNA by conducting the reverse
transcription
of a nucleic acid template in the presence of dNTPs labeled with the binding
pair
member(s). Alternatively, the binding pair member can be incorporated near the
terminus
of the target by using primers labeled with the binding pair member to amplify
the target
nucleic acid.
Target nucleic acids bearing multiple labels can be prepared by conducting
transcription in the presence of dNTPs bearing a binding pair member or by
amplifying
the target with primers bearing multiple binding pairs. Target nucleic acids
bearing a
single semiconductor nanocrystal can be prepared by using primers bearing a
single
binding pair member to conduct amplification of the target nucleic acid.
Furthermore,
attachment of the semiconductor nanocrystals bearing a binding pair member to
target
nucleic acids bearing the other binding pair member can be done either before
or after
hybridization of target nucleic acids to probes on the arrays.
One can conduct multiplexing analyses using various mufti-color
approaches to distinguish between different samples. For example, one can
synthesize
different cDNAs in different reaction vessels by using dNTPs bearing different
binding
pair members for different samples. These cDNAs can then be labeled with
different
semiconductor nanocrystals in a single reaction vessel (or after hybridization
to arrays) by
adding semiconductors bearing different binding pair members that are
complementary to
the different binding pair members borne by the different target nucleic
acids.
Alternatively, target nucleic acids from different samples can be separately
reacted with
different semiconductors (conjugated to a binding pair member) in different
reaction
vessels. Even if the target nucleic acids bear the same binding pair member,
target
nucleic acids can be differentially labeled by adding different semiconductor
nanocrystals
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into the different reaction vessels. The resulting labeled target nucleic
acids can then be
mixed together prior to applying the labeled target nucleic acids to the
array.
For a detailed description of other methods for conjugating ligands such as
nucleic acids to semiconductor nanocrystals, see, e.g., U.S. Patent No.
5,990,479;
Bruchez et. al. (1998) Science 281:2013-2016., Chan et. al. (1998) Science
281:2016-
2018, Bruchez "Luminescent Semiconductor Nanocrystals: Intermittent Behavior
and
use as Fluorescent Biological Probes" (1998) Doctoral dissertation, University
of
California, Berkeley, and Mikulec "Semiconductor Nanocrystal Colloids:
Manganese
Doped Cadmium Selenide, (Core)Shell Composites for Biological Labeling, and
Highly
Fluorescent Cadmium Telluride" (1999) Doctoral dissertation, Massachusetts
Institute of
Technology.
3. Array S, thesis
The arrays typically utilized when assaying target nucleic acids according
to the methods of the present invention typically include some type of solid
support to
which nucleic acid probes are attached. The nucleic acid probes attached to
the solid
support are generally nucleic acids or uncharged nucleic acid analogs such as,
for
example, peptide nucleic acids that are disclosed in International Publication
No. WO
92/20702; morpholino analogs that are described in U.S. Patents Nos.
5,185,444,
5,034,506 and 5,142,047.
Nucleic acid arrays can be prepared in two general ways. One approach
involves binding DNA from genomic or cDNA libraries to some type of solid
support,
such as glass for example. [See for example, Meier-Ewart, et al., Nature
361:375-376
(1993); Nguyen, C. et al., Genomics 29:207-216 (1995); Zhao, N. et al., Gene,
158:207-
213 (1995); Takahashi, N., et al., Gene 164:219-227 (1995); Schena, et al.,
Science
270:467-470 (1995); Southern et al., Nature GetZetics Supplentent 21:5-9
(1999); and
Cheung, et al., Nature Genetics Supplement 21:15-19 (1999)].
The second general approach involves the synthesis of nucleic acid probes.
One method involves synthesis of the probes according to standard automated
techniques
and then post-synthetic attachment of the probes to a support. See for
example,
Beaucage, Tetrahedron Lett., 22:1859-1862 (1981) and Needham-VanDevanter, et
al.,
Nucleic Acids Res., 12:6159-6168 (1984). A second category is the so-called
"spatially
directed" oligonucleotide synthesis approach. Methods falling within this
category
further include, by way of illustration and not limitation, light-directed
oligonucleotide
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synthesis, microlithography, application by ink jet, microchannel deposition
to specific
locations and sequestration by physical barriers.
Light-directed combinatorial methods for preparing nucleic acid probes are
described in U.S. Pat. Nos. 5,143,854 and 5,424,186 and 5,744,305; PCT patent
publication Nos. WO 90/15070 and 92/10092; Fodor et al., Science 251:767-777
(1991);
and Lipshutz, et al., Natu>"e Genetics Supplement 21:20-24 (1999). These
methods
combine solid-phase chemical synthesis and semiconductor-based lithography.
Various
masking strategies are utilized to reduce the number of synthesis cycles such
as described
in U.S. Pat. Nos. 5,571,639 and 5,593,839 to Hubbel et al., and by Fodor et
al., Science
251:767-777 (1991).
Other combinatorial methods which can be used to prepare arrays include
spotting reagents on the support using ink jet printers [see, e.g., Pease et
al., EP 728, 520,
and Blanchard, et al. Biosenso~s and Bioelect~onics II: 687-690 (1996)].
Arrays can also
be synthesized utilizing combinatorial chemistry by utilizing mechanically
constrained
flowpaths or microchannels to deliver monomers to cells of a support (see,
e.g., Winkler
et al., EP 624,059; WO 93/09668; and U.S. Pat. No. 5,885,837).
4. Target Nucleic Acid Amplification
In some instances, the samples contain such a low level of target nucleic
acids that it is useful to conduct a pre-amplification reaction to increase
the concentration
of the target nucleic acids. As described supra, amplification using primers
or
nucleotides labeled with semiconductor nanocrystals also provides a facile way
to label
the target nucleic acids of interest.
If samples are to be amplified, amplification is typically conducted using
the polymerase chain reaction (PCR) according to known procedures. See
generally,
PCR Technology: Principles and Applications fo>~ DNA Anzplification (H.A.
Erlich, Ed.)
Freeman Press, NY, NY (1992); PCR Protocols: A Guide to Methods and
Applications
(Innis, et al., Eds.) Academic Press, San Diego, CA (1990); Mattila et al.,
Nucleic Acids
Res. 19: 4967 (1991); Eckert et al., PCR Methods and Applications 1: 17
(1991); PCR
(McPherson et al. Ed.), IRL Press, Oxford; and U.S. Patent Nos. 4,683,202 and
4,683,195. Other suitable amplification methods include the ligase chain
reaction (LCR)
(see, e.g., Wu and Wallace, Genoznics 4:560 (1989) and Landegren et al.,
Science
241:1077 (1988); transcription amplification [see, e.g., Kwoh et al., P~oc.
Natl. Acad. Sci.
LISA 86:1173 (1989)); self sustained sequence replication (see, e.g., Guatelli
et al., P~oc.
CA 02400379 2002-08-14
WO 01/61040 PCT/USO1/04871
Natl. Acad. Sci. USA, 87:1874 (1990)); and nucleic acid based sequence
amplification
(NABSA) (see, e.g., Sooknanan, R. and Malek, L., Bio Techyaology 13: 563-65
(1995)].
Further guidance regarding nucleic sample preparation is described in
Sambrook, et al., Moleculay~ Clotzirag: A LaboYato~y Mayaual, 2"d Ed., Cold
Spring Harbor
Laboratory Press, (1989).
B. Expression Arrays
1. General
Certain methods of the present invention involve the analysis of gene
expression levels. In general terms, expression analysis involves the
detection and
quantification of mRNA levels (or cDNA derived therefrom) in one or more
samples. In
some instances, differently colored labels are used simultaneously to
quantitate changes
in gene expression levels, or to estimate changes in expression of one gene
relative to
another, in the cell under different conditions. For instance, cDNA prepared
from a first
specimen under one set of environmental conditions can be labeled with a first
color tag
and the cDNA derived from a second specimen, or the same first specimen under
a
different set of environmental conditions, can be labeled with a second color
tag. Both
samples are co-hybridized to the array and the differentially labeled first
and second
cDNA molecules compete to bind at each spot. The ratio of the two colors at
each
location gives a quantitative measure of the relative change in expression for
the gene.
Expression analysis provides key insight into a variety of biological
phenomenon. Cellular development and differentiation is one area in which
expression
analysis fords particular utility. In any given cell, only a fraction of all
the encoded genes
are expressed. The levels and timing of expression control cellular
development,
differentiation, function and physiology. Thus, monitoring gene expression can
be used
to analyze these processes. As an example, expression analysis can be utilized
to assess
differences in expression between different types of tissue. Expression
studies can also
provide important information into the genetic basis for aging and phenotypic
differences.
Expression analysis can also be of value in studies of various diseases. For
instance, expression analysis can be used to analyze the development and
progression of
cancer, since these events are accompanied by complex changes in the pattern
of gene
expression. Such studies can involve, for example, a comparison of gene
expression in
diseased tissue and normal tissue or infected tissue and normal tissue.
Expression
analysis can also be used in other clinical applications, including evaluating
the effects of
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various drug treatments on expression. For instance, differences in gene
expression for
normal tissue treated with drugs or a drug candidate and normal tissue can be
compared.
Similar drug studies could be performed with diseased tissue and normal
tissue. By
determining which genes are expressed in various diseases, one can identify
genes or their
protein products that have potential utility as drugs or as drug targets. In
other clinical
applications, expression analysis can be used in toxicological evaluations by
comparing
expression levels between tissue treated with potential poisons or toxins and
normal
tissue. Of course, expression analysis can be used in a variety of other
comparative
studies to assess the impact of variations in gene expression.
2. Sample Preparation
a. RNA Isolation
Samples can be obtained from essentially any source from which nucleic
acids can be obtained. Cells in the sample can be disrupted in a variety of
ways to release
the RNA therein (see for example, Watson, et al., Recombinant DNA, 2nd
Edition,
Scientific American Books, NY 1992). For example, nucleic acids may be
released by
mechanical disruption (such as repeated freeze/thaw cycles, abrasion and
sonication),
physical/chemical disruption, such°as treatment with detergents (e.g.,
Triton, Tween, or
sodium dodecylsulfate), osmotic shock, heat, or enzymatic lysis (e.g.,
lysosyme,
proteinase K, and pepsin).
Once nucleic acids have been obtained, they typically are reversed
transcribed into cDNA, although mRNA can be used directly. Following formation
of the
cDNA, generally sequences of interest are amplified according to any of the
various
amplification techniques that are known in the art such as those described
infra. Labeled
RNA can be prepared from a cDNA template using RNA polymerase, or methods
known
in the art.
3. Methods
Differential gene expression analysis in which gene expressiomnder
differing sets of conditions is monitored can be accomplished in two general
ways. One
approach, and the approach traditionally utilized, is to use multiple arrays.
In this
approach, a different array is utilized for each of the different samples,
each sample
corresponding to a different set of conditions. For example, as described
above, one
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sample might contain nucleic acids obtained from a healthy cell, while a
second sample
contains nucleic acids from a diseased cell. In such an investigation, one
array would be
used to determine expression in the healthy cell and the other array used to
determine
expression in the diseased cell. The problem with this approach is that each
measurement
made for the different arrays has some error associated with it. By making
comparisons
between different arrays, the error in each measurement becomes cumulative,
thereby
increasing the total error. Semiconductor nanocrystals can be used to reduce
error
generated in this approach. For example, the nucleic acids from each sample
can be
labeled with one or more semiconductor nanocrystals according to any of the
direct or
indirect methods just described, either before or after hybridization of the
nucleic acids to
the array. Furthermore, such error reduction can be accomplished using samples
individually labeled with detectably distinct semiconductor nanocrystals and
by making
simultaneous measurements with the same array.
However, because semiconductor nanocrystals can readily be tuned to emit
at different wavelengths, one can simultaneously analyze numerous different
samples on
a single array by differentially labeling the target nucleic acids from
different samples
using the labeling methods set forth above. This abilitywith the use of
semiconductors
afford results in significant improvements in the noise level when comparing
hybridization results from different expression conditions because the
cumulative error
associated with measurements for multiple arrays is avoided.
Details regarding methods for using arrays of nucleic acid probes for
monitoring expression of mRNA molecules is set forth in PCT/US96/143839 and WO
97/17317. With these methods the polynucleotides selected to hybridize with
target
polynucleotides are selected to be complementary to the mRNA targets of
interest or
amplification products therefrom. Additional discussion regarding the use of
microarrays
in expression analysis sufficient to guide one skilled in the art to conduct
such analyses is
set forth for example in Duggan, et al., Natuz~e Genetics Supplemezzt 21:10-14
(1999);
Bowtell, Nature Genetics Supplement 21:25-32 (1999); Brown and Botstein,
Nature
Gezietics Supplement 21:33-37 (1999); Cole et al., Natuz°e Genetics
Supplement 21:38-41
(1999); Debouck and Goodfellow, NatuYe Genetics Supplement 21:48-50 (1.999);
Bassett,
Jr., et al., Nature Genetics Supplement 21:51-55 (1999); and Chakravarti,
Nature
Genetics Supplenzent 21:56-60 (1999).
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C. Genotypin~/SNP Anal~s
1. General
It has been found that relatively minor changes in the genome of an
organism, including changes as small as a single nucleotide, can result in
substantially
different phenotypes. For example, these changes or mutations can be
responsible for a
variety of different diseases, influence the efficacy of different therapeutic
treatments and
alter the pathogencity of a microorganism or change the resistance of a
microorganism to
therapeutics directed towards it. Often such effects are the result of
alteration of a single
nucleotide. Such alterations are generally referred to as single nucleotide
polymorphisms,
or simply SNPs. The site at which an SNP occurs is referred to as a
polymorphic site or
an allelic site. A number of SNPs have been correlated with various human
diseases (see,
e.g., Publication WO 93/02216 wluch provides an extensive list of such SNPs).
Because
SNPs appear regularly throughout the genome, they also serve as useful genetic
markers.
The ability to detect specific nucleotide alterations or mutations in DNA
sequences has a number of medical and non-medical utilities. For example,
methods
capable of identifying nucleotide alterations provide a means for screening
and
diagnosing many common diseases that are associated with SNPs. Such methods
are also
valuable in identifying individuals susceptible to disease, those who could
benefit from
prophylactic measures, and thus obtaining information useful in patient
counseling and
education. Methods for detecting alterations and mutations have further value
in the
detection of microorganisms, and making correlations between the DNA in a
particular
sample and individuals having related DNA. This latter capability can be
useful in
resolving paternity disputes and in forensic analysis.
2. Methodology
General Considerations: The invention provides a number of different
methods for detecting one or more target nucleic acids having a particular
sequence. In
general, these methods involve providing an array that bears a plurality of
nucleic acid
probes having different sequences. Normally, probes of different sequence are
positioned
at different locations so that the identity of the probe is spatially encoded.
A sample
containing target nucleic acids labeled with semiconductor nanocrystals is
contacted with
the probe. Hybridization complexes between complementary probes and target
nucleic
acids is detected by detecting a signal associated with the semiconductor
nanocrystal. As
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described further below, through the use of appropriate probes, one can detect
the
presence or absence of a particular target nucleic acid of interest.
Allele Specific Hybridization: One method for detecting a target nucleic
acid that has a particular polymorphic form is to utilize allele specific
probes that each
specifically hybridize with a particular polymorphic form of a target nucleic
acid. By
detecting which of the probes on the array form hybridization complexes, one
can
determine the presence or absence of particular target nucleic acids. Samples
from
different individuals can be probed on a single array by differentially
labeling nucleic
acids from the different individuals with different semiconductor
nanocrystals. The
labeled probes necessary for conducting the reaction can be prepared according
to the
methods set forth above, by synthesizing the probes with functionalized
nucleotides that
permit the post-synthetic attachment of the semiconductor nanocrystals or
using one or
more labeled nucleotides in the synthesis of the probes by standard methods.
The group of probes attached to the array support can include all the allelic
probes that specifically hybridize to each of the different polymorphic forms
of a target
nucleic acid. Since most polymorphisms are biallelic, this means that the
array includes
two probes for each polymorphic form. If, however, the target nucleic acid is
triallelic,
then three probes each complementary to one of the three polymorphic forms can
be
utilized. Similarly, if the target nucleic acid is tetra-allelic, then the
four probes
complementary to the four different polymorphic forms can be included in the
array. By
labeling the target nucleic acids with semiconductor nanocrystals, detection
of the
hybridization complexes is enhanced; the ability to use different colored
semiconductor
nanocrystals means that samples from a number of individuals can be analyzed
simultaneously.
Further guidance regarding allele-specific hybridization is set forth, for
example, by Erlich, et al. (1991) Eur. J. Immuraogenet. 18:33-55; Zhang, et
al. (1991)
Nucleic Acids Res. 19:3929-3933; Impraim, et al. (1987) Biochem. Bioplays.
Res.
Commun. 142:710-716; Saiki, et al. (1986) Nature 324:163-166; Wu, et al.
(1989) DNA
8:135-142; Thein, et al. (1988) B~. J. Haematol. 70:225-231; and Connor, et
al. (1983)
Pr~oc. Natl. Acad. Sci. LISA 80:278-282, and in PCT Publication 95/11995.
Allele-Specific Li~ation: Other methods of the invention utilize
semiconductor labeled probes to conduct allele-specific ligation reactions to
detect the
CA 02400379 2002-08-14
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presence or absence of a particular target nucleic acid and distinguish
between different
polymorphic forms. In general, these methods involve contacting a target
nucleic acid
(which can be amplified prior to analysis) with a first probe that is
complementary to a
sequence adjacent the polymorphic site under hybridization conditions. This
first probe
hybridizes to a sequence that is possessed by all the target nucleic acids
regardless of the
nucleotide at the polymorphic site (i.e., regardless of the polymorphic form
of the target).
The target nucleic acid is also contacted with a second probe that is an
allele-specific
probe, i. e., a probe that only hybridizes to a particular polymorphic form of
the target
nucleic acid. The first and second probes are selected such that when the
allele-specific
probe is complementary with the nucleotide at the polymorphic site the two
probes
hybridize directly adjacent one another. In particular, the 3' terminus of one
probe is
immediately adjacent the S' terminus of the other probe. So long as the allele-
specific
probe is complementary to the nucleotide at the polymorphic site, added ligase
can join
the two probes. By labeling either or both of the probes with semiconductor
nanocrystals,
detection of ligated product can be enhanced. The probes necessary for
conducting these
types of analyses can be prepared as described above in the section on allele-
specific
hybridization.
In some instances, one of the probes is attached to a solid support. In other
methods, the ligation reaction is conducted in solution and the ligated
products are
detected after being captured by capture reagents attached to an array. In
this latter
instance, the capture reagents specifically recognize a tag attached to one or
both of the
probes. Different targets can be distinguished by different tags and/or by
labeling
different target nucleic acids with different semiconductor nanocrystals.
These analyses
can be conducted in multiplex format by differentially labeling the probes
with different
semiconductor nanocrystals. For example, the presence or absence of specific
alleles can
rapidly be determined by differentially labeling the allele-specific probes
used to conduct
the ligation reaction. ,
The general methods set forth above can be modified by conducting a
series of ligase chain reactions or by coupling the ligase chain reaction with
one or more
PCR amplifications of the target nucleic acid as well. Further guidance on
such methods
is set forth, for example, in U.S. Pat. Nos. 5,830,711; 6,027,889; and
5,869,252.
Primer Extension/Mini-Sequencing: Certain methods of the invention
involve conducting mini-sequencing reactions or primer extension reactions to
identify
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the nucleotide present at a polymorphic site in a target nucleic acid. In
general, in these
methods a primer complementary to a segment of a target nucleic acid is
extended if the
reaction is conducted in the presence of a nucleotide that is complementary to
the
nucleotide at the polymorphic site. More specifically, the primer extension
assays or
mini-sequencing assays of the invention typically involve hybridizing a primer
to a
complementary target nucleic acid such that the 3' end of the primer is
immediately
adjacent the polymorphic site or is a few bases upstream of the polymorphic
site. The
extension reaction is conducted in the presence of one or more nucleotides
labeled with a
semiconductor nanocrystal and a polymerise. Often the nucleotide is a
dideoxynucleotide that prevents further extension by the polymerise once it is
incorporated onto the 3' end of the primer. If one of the added non-extendible
nucleotides is complementary to the nucleotide at the polymorphic site, then a
labeled
nucleotide is incorporated onto the 3' end of the primer to generate a labeled
extension
product. Because the incorporated nucleotide is complementary to the
nucleotide at the
polymorphic site, extended primers provide an indication of which nucleotide
is present
at the polymorphic site of target nucleic acids. Methods utilizing this
general approach
are discussed, for example, in U.S. Patent Nos. 5,981,176; 5,846,710;
6,004,744;
5,888,819; 5,856,092; 5,710,028; and 6,013,431; and in PCT publication WO
92/16657.
In the array format of the present invention, the primers are typically
attached to a support. These primers can be of random sequence or selected to
be
complementary to the target nucleic acids of interest. A sample containing
target nucleic
acids is contacted with the array of primers under conditions in which target
nucleic acids
become hybridized to complementary primers. Primers of the appropriate
sequence
hybridize to the target nucleic acid so that the 3' end of the primer is
adjacent to the
polymorphic site of the target. Preferably, the 3' end of the primer is
immediately
adjacent (but does not span) the polymorphic site (i.e., the 3' end hybridizes
to the
nucleotide just upstream of the polymorphic site). If not already present, one
or more
nucleotides labeled with semiconductor nanocrystals are added. As indicated
above, if
the labeled nucleotides added include a nucleotide complementary to the
nucleotide at the
polymorphic site, the primer is extended by incorporation of a nucleotide
bearing a
semiconductor nanocrystal. By using four different semiconductor nanocrystals,
each
attached to a different nucleotide (e.g., ddATP, ddTTP, ddCTP and ddGTP), all
possible
alleles can be tested on a single array simultaneously.
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In some instances methods can be performed with primers that simply
hybridize adjacent to, but do not span, the polymorphic site. This is possible
so long as
none of the nucleotides on the target nucleic acid located between the 3' end
of the primer
and the polymorphic site are the same as the nucleotide at the variant site.
The extension
reaction mixture in such instances must also include nucleotides complementary
to those
nucleotides positioned between the 3' primer end and the polymorphic site.
In related methods, the primers include two general regions: a 5' end
region that includes a tag, and a 3' region that is complementary to a target
nucleic acid of
interest. The array includes one or more capture reagents that can
specifically bind with a
tag borne by the primers. Typically, different capture reagents specific fox
different
extension products are positioned at different locations on the array so that
the identity of
the capture reagents is spatially encoded. In general, the tag and capture
reagent can be
selected from any type of binding pairs in which the members of the pair
specifically bind
to one another. For example, the capture reagent can be a nucleic acid that is
complementary to a nucleic acid segment of a primer (i. e., the primer tag).
In methods of
the invention utilizing this general approach, the extension reactions
described above can
be conducted in solution rather than on the array.
As set forth above, the reactions are conducted in the presence of one or
more nucleotides (typically ddNTPs) labeled with a semiconductor nanocrystal.
Following extension, the extension products in the extension reaction are
contacted with
the array. The capture reagents on the array capture primers bearing tags that
are
specifically recognized by the capture reagent. Those primers that have been
extended
can be detected by the semiconductor nanocrystal incorporated into the primer.
The
identity of the nucleotide at the polymorphic site of the target nucleic acid
can be
determined from the location on the array at which the extended product binds
(see, e.g.,
U.S. Pat. No. 5,981,176).
In other methods for analyzing SNPs, an array of nucleic acid probes that
are complementary to subsequences of a target sequence can be utilized to
determine the
identity of a target sequence, measure its amount, and detect differences
between the
target and a reference sequence using a procedure referred to as "tiling." In
brief, tiling
strategies utilize a tiled array in which multiple nucleic acids that are
identical except for
one location are utilized. For example, in a 4L tiled array that typically is
used for SNP
analyses, there is a set of four probes of relatively short length (for
example, 15-mers)
which vary at the SNP position but which otherwise are identical and are
perfect
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complements to a segment of the target nucleic acid being screened. A
perfectly
complementary probe binds more tightly to a target nucleic acid than those
probes that
have a single mismatch. Hence, the labeled probe generating the most intense
signal
corresponds to the probe having a nucleotide complementary to the nucleotide
at the
polymorphic site. The target nucleic acids can be labeled with semiconductor
nanocrystals either before or after target nucleic acids have hybridized to
the nucleic acid
probes of the array.
The tiling approach to SNP analysis can be extended to examine long
nucleic acid targets and detect numerous polymorphisms/mutations relative to a
characterized consensus sequence. Additional guidance regarding such methods
is
generally available [see, e.g., WO 95/11995; U.S. Pat. No. 5,858,659; Chee, et
al. Scierace
274:610-614 (1996); U.S. Pat. No. 5,837,832; and Lipshutz, et al.,
BioTechfZiques 19:442-
447 (1995)].
The labeled nucleotides utilized in the primer extension reactions can be
prepared by directly attaching a semiconductor nanocrystal to the nucleotides
via
functional groups present on the naturally occurnng nucleotides, or through
different
functional groups introduced onto the nucleotides. Alternatively, different
nucleotides
can bear different binding pair members (e.g., biotin or antibodies); the
other
complementary binding pair members are attached to the semiconductor
nanocrystals. If
functionalized nucleotides or nucleotides bearing binding pair members are
used to
conduct the extension reaction, then extension products can be labeled either
before or
after extension has occurred.
3. Target Nucleic Acid Preparation
The target nucleic acids utilized in SNP analyses can be extracted and
isolated according to the methods generally described supra for expression
analysis. If
necessary, target nucleic acids can be amplified according to the various
amplification
methods also described above.
D. Sequencin~g~.Anal,
1. General
Traditional sequencing technologies involve complicated and time
consuming procedures requiring electrophoretic size separation of labeled DNA
fragments (see, e.g., Alphey, DNA Sequencing: From Experimental Methods to
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Biolnformatics, Springer-Verlag, New York, 1997). An alternative approach
involves
using nucleic acid arrays to conduct hybridization studies with fragments of a
target
nucleic acid. From the hybridization results obtained, one can reconstruct the
sequence of
a target nucleic acid. In general, SBH uses a set of short nucleic acid probes
of defined
sequence to probe for complementary sequences on a longer target nucleic acid
strand.
The defined sequences that hybridize to the target can then be aligned using
computer
algorithms to construct the sequence of the target nucleic acid.
2. Methodology
The strategy of SBH can be illustrated by the following example. A 12-
mer target DNA sequence, AGCCTAGCTGAA, is mixed with a complete set of
octanucleotide probes. If only perfect complementarity is considered, five of
the 65,536
octamer probes -TCGGATCG, CGGATCGA, GGATCGAC, GATCGACT, and ,
ATCGACTT will hybridize to the target. Alignment of the overlapping sequences
from
the hybridizing probes reconstructs the complement of the original 12-mer
target:
TCGGATCG
CGGATCGA
GGATCGAC
GATCGACT
ATCGACTT
TCGGATCGACTT
SBH can be performed in two formats. Hybridization methodology can be
carned out by attaching target DNA to a surface. The target is then
interrogated with a
set of oligonucleotide probes, one at a time [see Strezoska et al., Proc.
Natl. Acad. Sci.
USA ~5:100~9-10093 (1991); and Drmanac et al., Science 260:1649-1652, (1993)].
Although this approach can be implemented with well established methods of
immobilization and hybridization detection, it involves a large number of
manipulations.
For example, to probe a sequence utilizing a full set of octanucleotides, tens
of thousands
of hybridization reactions must be performed. In the second format, SBH is
carned out
by attaching probes to a surface in an array format where the identity of the
probes at
each site is known. Target nucleic acid, typically fragmented, is then added
to the array
CA 02400379 2002-08-14
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of probes. The hybridization pattern determined in a single experiment can
directly
reveal the identity of all complementary probes.
The methods of the invention utilize target nucleic acids labeled with
semiconductor nanocrystals using either of these two formats. Most typically,
however,
the latter approach is utilized. Hence, in certain methods, sequencing begins
with the
fragmenting of the target nucleic acid into fragments using various techniques
known in
the art (e.g., the use of restriction enzymes, or heating in the presence of
high salt
concentrations). The resulting fragments are labeled with semiconductor
nanocrystals,
diluted in buffer and then applied to an array bearing nucleic acid probes.
The fragments
are allowed to hybridize to the probes, typically using an automated apparatus
to control
temperature and sample mixing. The array is then optionally rinsed with a
stringency
buffer to remove unbound fragments and hybridization complexes detected by
detecting a
signal from the semiconductor nanocrystal used to label the fragments. The
target nucleic
acid can be labeled with semiconductor nanocrystals using any of the various
methods
described supra in the section on expression analysis.
Variations of the SBH procedure have been developed, primarily to
address a problem with SBH, namely the problem of mismatches creating errors
in the
sequence determination. One such method termed "positional SBH" (PSBH)
utilizes
duplex probes having 3' single-stranded overhangs to capture the target, and
is followed
by enzymatic ligation of the target to the duplex probe. This approach is
designed to
reduce mismatches (see for example, Broude, et al., P~oc. Natl. Acad. Sci. USA
91:3072-
3076 (1994); and U.S. Patent No. 5,631,134 to Cantor). PSBH itself has been
further
modified so that following the ligation reaction, DNA polymerase is added to
extend the
immobilized probe as a way of further reducing mismatches during capture of
the target
[see, e.g., Kuppuswamy, et al., Proc. Natl. Acad. Sci. USA 86:1143-1147
(1991)]. The
target nucleic acid fragments, probes or nucleotides used in such approaches
can be
labeled with semiconductor nanocrystals to enhance detection.
Additional guidance regarding sequencing by hybridization is provided,
for example ,by Lysov et al., Dokl. Akad. Nauk SSSR 303:1508-1511 (1988);
Bains et al.,
J. Theor. Biol. 135:303-307 (1988); Drmanac et al., Geho~2ics 4:114-128
(1989);
Barinaga, Science 253:1489 (1991); Stresoska et al., Pr~oc. Natl. Acad. Sci.
USA 88:10089
(1992); Bains, BioTeclzraology 10:757-758 (1992); and U.S. Pat. No. 5,202,231.
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V. Aptamers
Aptamers are single- or double-stranded DNA or single-stranded RNA
molecules that recognize and bind to a desired target molecule by virtue of
their shapes.
See, e.g., PCT Publication Nos. W092/14843, W091/19813, and W092/05285. The
SELEX procedure, described in U.S. Patent No. 5,270,163 to Gold et al., Tuerk
et al.
(1990) Science 249:505-510, Szostak et al. (1990) Nature 346:818-822 and Joyce
(1989)
Gene 82:83-87, can be used to select for RNA or DNA aptamers that are target-
specific.
In certain methods, an oligonucleotide pool is constructed wherein an n-
mer, typically a randomized sequence of nucleotides thereby forming a
"randomer pool"
of oligonucleotides, is flanked by two polymerase chain reaction (PCR)
primers. The
oligonucleotides in the pool are labeled with semiconductor nanocrystals
according the
methods described above. The array utilized in the screening of aptamers,
typically bears
the target molecules (e.g., proteins or small molecules) to be screened. The
array is then
contacted with the oligonucleotide pool under conditions that favor binding of
the
oligonucleotides to the target molecules on the array. Those oligonucleotides
that bind
the target molecule are separated from non-binding oligonucleotide using
stringency
washes. Those oligonucleotides that bind to the target molecules on the array
are
dissociated from the array using known techniques and then amplified using
conventional
PCR technology to form a ligand-enriched pool of oligonucleotides. Further
rounds of
binding, separation, dissociation and amplification are performed until an
aptamer with
the desired binding affinity, specificity or both is aclueved. The final
aptamer sequence
identified can then be prepared chemically or by in vitro transcription.
VI. Protein Arrays
A. General
Protein arrays can be used to investigate interactions between proteins and
a wide variety of different types of molecules such as nucleic acids, various
small
molecules and other proteins, for example. Protein arrays can be designed
according to
the aims of the particular investigation. For instance, an array can contain
all the
combinatorial variants of a bioactive protein or specific variants of a single
protein
species (e.g., splice variants, domains, or mutants), a family of protein
orthologs from
different species, a protein pathway, or even the entire protein complement of
an
organism. The arrays can also include antibodies, recombinant proteins,
purified proteins
and receptors, for example.
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Protein arrays can be assigned to two general types. One type is referred
to as a nonliving or chemical array. These protein arrays are composed of
synthetic
proteins. Arrays of this type are useful to investigate specific interactions
between
relatively small proteins with other proteins, particular nucleic acids or
metal ions, for
example. The second type is the biological or living protein array. These
arrays include
living entities that express proteins, including, but not limited to, viruses
or cells. The
arrays can include pools of proteins, cell fractions or intact cells. This
type of array can
be used to investigate more complex biological activities, including, but not
limited to, '
activities involving multicomponent complexes or multistep enzymatic or
signaling
pathways. Regardless of type, the proteins typically are placed at
positionally distinct
locations on the array so that the different proteins are spatially encoded.
Certain features of semiconductor nanocrystals make them useful in
protein arrays. For example, tunability permits multicolor simultaneous
detection and,
hence, multiple sample detection. There is also no need for enzyme development
as with
certain traditional ELISA methods. The semiconductor nanocrystals have
increased
photostability relative to organic fluorophores, thereby increasing detection
sensitivity by
virtue of the ability to motitor the signal over a long period of time.
B. Arra~paration
A variety of options are available for synthesizing proteins for use in
nonliving arrays. One approach is to utilize various solid state synthesis
approaches to
synthesize the proteins on a desired support. For example, a method for
synthesizing an
array of peptides in microtiter wells has been described (see, e.g., Geysen,
H.M. et al.
(1984) Proc. Natl. Acad. Sci. USA 81:3998-4002. Other solid state methods are
discussed, for example, by Merrifield, J. Am. Chem. Soc. (1963) 85:2149-2154;
Atherton,
Solid Phase Peptide Synthesis, IRL Press, Oxford (1989); Erickson, B.W. and
Mernfield,
R.B. (1976) in The Proteiyas, (Neurath, H. and Hill, R.L., eds.) Academic
Press, New
York, vol. 2, pp. 255-527; and Meienhofer, J. (1973) in Hormonal Proteins acrd
Peptides,
(Li, C.H., ed.) Academic Press, New York, vol. 2, pp. 45-267.
A technique sometimes referred to as "spot synthesis" has also been
developed using chemistry similar to the solid state methods. This particular
approach
takes advantage of the abundant hydroxyl groups present on cellulose filters
to derivatize
Fmoc-[3-alanine groups. Peptide arrays can then be synthesized via the
cellulose-bound
alanine following deprotection (see, e.g., Gausepohl, H., et al., Pept. Res.
5:315-320
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(1992); and Kramer, A. and Schneider-Mergener, J., Methods in Molecular
Biology
87:25-39 (1998)). Other methods can be used to synthesize proteins on
polymeric rods
using solid state chemistry (see, e.g., Geysen, et al., Proc. Natl. Acad. Sci.
USA 81:3998
4002 (1984)).
Methods allowing for the preparation and purification in parallel of large
numbers of recombinant proteins can also be utilized to generate proteins for
arrays.
Such methods are discussed, for example, by Martzen, M. R., et al., Science
286:1153-
1155 (1999).
Proteins can be synthesized directly on a solid support using various
photolithographic methods. These techniques allow the number of proteins
synthesized
per unit area to be greatly increased. Examples of such methods are discussed,
for
example, in U.S. Pat. Nos. 5,143,854; 5,489,678; and 5,744,305; PCT
Publications WO
90/15070 and WO 92/10092; and by Fodor, S.P. et al., Science 251:767-773
(1991).
Instead of synthesizing the proteins on an array, preformed proteins can be
directly deposited on a support to form the arrays. Thus, for example,
recombinant
proteins, purified proteins and the like can be blotted onto a support.
The proteins synthesized for use in non-living protein arrays need not be
limited to proteins composed of the L-amino acids. Other building blocks, such
as D-
amino acids and modified amino acids can also be used.
Living arrays can also be prepared utilizing different methods. As
indicated above, the arrays can be formed from pools of proteins, cellular
extracts or
intact cells. The pools, extracts or intact cells typically are placed in some
type of support
having a series of depressions (e.g., a microtiter plate) to contain the
proteins. Methods
for producing genome-wide protein arrays have also been described. Certain of
these
methods involves transformation events in which one of the open-reading frames
(ORF)
from an organism (e.g., yeast) is inserted into plasmid encoding for
glutathione-S-
transferase (GST). This plasmid is subsequently used to transform the organism
which
then expresses different GST-ORF fusion proteins. The resulting transformed
cell
cultures or colonies can be used as an element of an array (i.e., different
colonies or
groups of colonies are placed at different locations of the array; see, e.g.,
Martzen, M.R. ,
et al., (1999) Science 286:1153-1155).
C. General Assays
1. Background
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Protein arrays can be used to assay or screen for a variety of different types
of activity or to conduct other types of analyses. For example, protein arrays
can be used
to: (1) screen for various molecules that interact with a protein of interest
(e.g., agonists
or antagonists). Molecules to be screened can include, but are not limited to,
other
proteins, nucleic acids, drugs, macromolecules or small molecules (see, e.g.,
Framer, A.,
et al., (1993) Pept. Res. 6:314-319); (2) to map antibody epitopes (see, e.g.,
Framer, A.,
et al., (1994) Methods 6:388-395; Reineke, U., et al., Mol. Diversity 1:141-
148;
Schneider-Mergener, J., et al., (1996) "Peptide Libraries Bound to Continuous
Cellulose
Membranes: Tools to Study Molecular Recognition" in Combihato~ial Libraries,
(Cortese, R., ed.), W. de Gruyter, Berlin, pp. 53-68; and Kramer, A., et al.,
(1995) Mol.
Immunol. 32:459-465); and (3) determine the cellulax location of proteins of
interest.
Each of these applications is discussed in additional detail below.
2. Immunoassays
One major utility of the semiconductor nanocrystals is to utilize them to
label either the antigen or an antibody in various types of immunological
assays. As
noted generally above, the use of semiconductor nanocrystals can permit
multicolor
detection thereby allowing multiple assays to be conducted simultaneously.
Unlike
standard ELISA methods, there is no need to wait for an enzyme to generate a
detectable
signal. Further, the photostability of semiconductor nanocrystals provides for
increased
detection sensitivity relative to organic fluorophores because the resistance
to
photobleaching allows for longer signal acquisition.
The immunoassays can be conducted in a vaxiety of different formats. W
some methods, the ligands (potential antigens) to be screened are attached to
an array and
then contacted with antibodies that are labeled with semiconductor
nanocrystals.
Alternatively, antibodies can be positioned on a support and then contacted
with samples
containing ligands that are labeled with semiconductor nanocrystals. In a
third format,
the ligand being screened is an antibody and it is attached to a support and
then screened
with a known antigen of interest. Regardless of the particular approach,
ligands or
antibodies that do not form a binding complex on the array are typically
washed from the
array. Complexes on the array are then detected by detecting a signal from a
semiconductor nanocrystal within a complex.
The assays can also be performed in a "sandwich" type format in which
antibodies positioned on an array are contacted with a sample containing
ligands. The
CA 02400379 2002-08-14
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ligands in the sample can be labeled or unlabeled. Ligands that specifically
bind to an
antibody form a binary antibody-ligand complex. Thus, the antibodies on the
array act to
capture a ligand to which it specifically binds; hence, such antibodies are
sometimes
called "capture antibodies." The binary complex formed between a capture
antibody and
a ligand can optionally be detected if the ligands are labeled. The array is
also contacted
with a secondary antibody that is labeled with a semiconductor nanocrystal. In
those
instances in which a secondary antibody specifically binds to a binary
complex, a tertiary
complex is formed. Tertiary complexes can be detected by detecting ari
emission
mediated by the semiconductor nanocrystal in the tertiary complex.
As a further example, with certain immunoassays the wells of a microtiter
plate are coated with a selected antigen. A biological sample containing or
suspected of
containing antibodies to the antigen is then added to the coated wells. After
a period of
incubation sufficient to allow antibody binding to the immobilized antigen,
the plates)
can be washed to remove unbound antibodies and other sample components and a
detection moiety labeled with a semiconductor nanocrystal is added. The
detection
moiety is allowed to react with any captured sample antibodies, the plate
washed and the
presence of the secondary binding molecule detected as described above.
Thus, in one particular embodiment, the presence of antibodies bound to
antigens immobilized on a solid support can be readily detected using a
detection moiety
that comprises an antibody labeled with a semiconductor nanocrystal that
specifically
binds to the antigen/antibody complex.
In still other methods, an immunoaffinity matrix can be provided, wherein
a polyclonal population of antibodies from a biological sample suspected of
containing a
particular antigen is immobilized to a substrate. In this regard, an initial
affinity
purification of the sample can be carried out using immobilized antigens. The
resultant
sample preparation thus only contains specific antibodies, avoiding potential
nonspecific
binding properties in the affinity support. A number of methods of
immobilizing
immunoglobulins (either intact or in specific fragments) at high yield and
good retention
of antigen binding activity are known in the art. Not being limited by any
particular
method, immobilized protein A or protein G can be used to immobilize
immunoglobulins.
Accordingly, once the immunoglobulin molecules have been immobilized
to provide an immunoaffinity matrix, semiconductor nanocrystal-labeled
proteins (i. e.,
potential antigens) are contacted with the bound antibodies under suitable
binding
conditions. After any nonspecifically bound antigen has been washed from the
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immunoaffinity support, the presence of bound antigen can be determined by
assaying for
a signal mediated by a semiconductor nanocrystal.
Additionally, antibodies raised to particular antigens, rather than the
antigens themselves, can be used in the above-described assays in order to
detect the
presence of a protein of interest in a given sample. These assays are
performed
essentially as described above and are well known to those of skill in the
art.
The ligands or antibodies used to prepare the array can be placed on a
variety of different supports. Suitable supports for use in the methods of the
invention
include, but are not limited to, nitrocellulose (e.g., in membrane or
microtiter well form);
polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g.,
beads or
microtiter plates); polyvinylidine fluoride; diazotized paper; nylon
membranes; activated
beads; and magnetically responsive beads.
Sometimes, immobilization to a support can be enhanced by first coupling
the ligand or antibody to a protein with better solid phase-binding
properties. Suitable
coupling proteins include, but are not limited to, macromolecules such as
serum albumins
including bovine serum albumin (BSA), keyhole limpet hemocyanin,
immunoglobulin
molecules, thyroglobulin, ovalbumin, and other proteins well known to those
skilled in
the art. Other reagents that can be used to bind molecules to the support
include
polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids
and amino
acid copolymers. Additional details regarding molecules and coupling methods
are
provided by, for example, Brinkley, M.A. (1992) Bioconjugate Chen2. 3:2-13;
Hashida et
al. (1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987)
International J.
ofPeptide and P~oteifa Res. 30:117-124.
3. Antibody Generation for Immunoassay
Antibodies that are used in the methods of the invention are produced
using established techniques and disclosed in, for example, U.S. Patent Nos.
4,011,308;
4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. For example,
polyclonal
antibodies axe generated by irmnunizing a suitable animal, such as a mouse,
rat, rabbit,
sheep or goat, with an antigen of interest. In order to enhance
immunogenicity, the
antigen can be linked to a carrier prior to immunization. Such carriers are
well known to
those of ordinary skill in the art.
Tmmunization is generally performed by mixing or emulsifying the antigen
in saline, preferably in an adjuvant such as Freund's complete adjuvant, and
injecting the
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mixture or emulsion parenterally (generally subcutaneously or
intramuscularly). The
animal is generally boosted 2-6 weeks later with one or more injections of the
antigen in
saline, preferably using Freund's incomplete adjuvant. Antibodies can also be
generated
by in vitYO immunization, using methods known in the art. Polyclonal antiserum
is then
obtained from the immunized animal.
Monoclonal antibodies can be prepared using the method of Kohler and
Milstein (1975) Nature 256:495-497, or a modification thereof. Typically, a
mouse or rat
is immunized as described above. However, rather than bleeding the animal to
extract
serum, the spleen (and optionally several large lymph nodes) is removed and
dissociated
into single cells. If desired, the spleen cells can be screened (after removal
of
nonspecifically adherent cells) by applying a cell suspension to a plate or
well coated with
the antigen. B-cells, expressing membrane-bound immunoglobulin specific for
the
antigen, will bind to the plate, and are not rinsed away with the rest of the
suspension.
Resulting B-cells, or all dissociated spleen cells, are then induced to fuse
with myeloma
cells to form hybridomas, and are cultured in a selective medium (e.g.,
hypoxanthine,
aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by
limiting dilution, and are assayed for the production of antibodies which bind
specifically
to the immunizing antigen (and which do not bind to mirelated antigens). The
selected
monoclonal antibody-secreting hybridoinas are then cultured either in vitro
(e.g., in tissue
culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in
mice).
Human monoclonal antibodies are obtained by using human rather than
marine hybridomas. See, e.g., Cote, et al. (1985) Monoclonal Antibodies and
CanceY
Therapy, Alan R. Liss, p. 77
Monoclonal antibodies or portions thereof can be identified by first
screening a B-cell cDNA library for DNA molecules that encode antibodies that
specifically bind to p185, according to the method generally set forth by Huse
et al.
(1989) Science 246:1275-1281. The DNA molecule can then be cloned and
amplified to
obtain sequences that encode the antibody (or binding domain) of the desired
specificity.
As indicated supra, a variety of other types of antibodies can also be
utilized. For example, the antibodies can be recombinant antibodies from phage
libraries.
A variety of antibody fragments can also be used. Such fragments include Fab
fragments,
scFv fragments and mini-antibodies, for example.
Once formed, the antibodies can be labeled with semiconductor
nanocrystals using the conjugation methods set forth infra.
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D. Exemplar~Assays
1. Whole Genome Screening
Certain techniques can be utilized to prepare large numbers of fusion
proteins that subsequently can be screened for their ability to interact with
macromolecules such as other proteins, nucleic acids, small molecules,
oligosaccharides,
drugs and other biological molecules. As noted supra, an array of cells
expressing
different fusion proteins comprising different ORFs from various organisms
(e.g., yeast)
can be constructed (see, e.g., Martzen, M. R., et al., (1999) Sciehce 286:1153-
1155). The
proteins expressed by the cells can then be screened with ligands of interest
to identify
those capable of interacting with the expressed proteins. Such methods allow
one to
identify unknown ligands to known proteins, as well permitting one to identify
unknown
proteins (i.e., rio activity has yet been assigned to the ORF) capable of
binding known or
unknown ligands.
2. Cellular Localization of Proteins
One can also conduct experiments to identify the location of various
proteins throughout a cell using semiconductor nanocrystal labeled reagents.
For
example, recently developed transposon tagging schemes can be utilized. In
this
approach, transposon constructs that include a transposon flanked by
recombination sites
are prepared. The construct also includes an epitope tag segment adjacent one
of the
recombination sites. By the process of homologous recombination, a transposon
construct can become integrated into the genome of the organism (e.g., yeast)
being
transformed. When the construct is inserted in frame with a coding region, a
full-length
epitope tagged protein is generated.
An array of such cells can then be assayed to determine the subcellular
localization of various proteins. Since in frame insertions of the construct
generates
fusion proteins that include the epitope, the fusion proteins can be localized
by contacting
cells with antibodies that specifically bind to the epitope. In this way, one
can localize
proteins to various regions of the cell, such as the nucleus, mitochondria and
plasma
membrane, for example. See, e.g., Ross-Macdonald, P., et al. (1999) Nature
402:413-
418. Detection of complexes formed between the antibodies and the localized
proteins is
enhanced by using antibodies that are labeled with semiconductor nanocrystals.
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3. Epitope Determination
Semiconductor labeled antibodies can also be utilized to determine the
antigenic epitope of antigens of interest. An antigenic epitope is defined as
a region of a
protein to which an antibody can bind. An immunogenic epitope refers to those
parts of a
protein that elicit the antibody response when the whole protein is the
immunogen.
Certain of these methods involve the synthesis of overlapping proteins that
cover the
entire amino acid sequence of protein known to elicit an antigenic response.
These
proteins can be synthesized in an array format using solid state protein
synthesis methods
such as described supra. The immobilized proteins are then tested for
antigenicity using
established ELISA techniques and various different anti-sera in which the
antibodies are
labeled with semiconductor nanocrystals. Once a peptide is identified that
forms a stable
complex with an antibody generated against the natural antigen, a replacement
set of
proteins is generated. The replacement set includes all the proteins
corresponding to the
identified protein except that a single amino acid replacement is introduced.
Each
replacement set also can be synthesized as an array. The replacement set is
then
rescreened using antibodies labeled with semiconductor nanocrystals to
determine if
antigenicity is retained. From the collective results, one can determine the
location and
identity of amino acids that play a critical role in antigenicity. See, e.g.,
Geysen, H. M.,
et al. (1984) P~oc. Natl. Acad. Sci. USA 81:3998-4002.
VII. Tissue Arrays
A. Back_r
Histopathological examination in which tissue specimens are subjected to
microscopic examination has enabled the biological mechanisms of many diseases
to be
clarified and consequently aided in the development of effective medical
treatment for a
variety of illnesses. In traditional pathological analysis, a diagnosis is
made on the basis
of cell morphology and staining characteristics. Such analyses can sometimes
be limited
because of the sensitivity of standard staining procedures.
Developments in the field of molecular medicine have generated new
methods for investigating the cellular mechanisms of disease and to determine
the most
appropriate treatment course. For example, often certain diseases are
associated with an
alteration (i.e., decrease or elevation) in the expression level of certain
proteins. As a
specific example, it has been found that certain hormone dependent breast
tumor cells
have increased expression of estrogen receptors. Other diseases have been
correlated
CA 02400379 2002-08-14
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with the production of cell surface antigens or the generation of other
cellular proteins.
Various detection techniques have been developed to identify and detect such
markers,
including, for example, immunophenotyping with monoclonal antibodies and in
situ
hybridization with probes specific for the nucleic acid sequences encoding
such markers.
To improve the throughput at which samples can be analyzed, tissue arrays can
be
utilized to permit the rapid screening of a large number of tissue samples.
Semiconductor nanocrystals can be used in all of the foregoing methods to
enhance detection, sensitivity and, in some instances, to provide quantitative
information.
The semiconductor. nanocrystals can be conjugated to a number of different
species to
stain particular cellular components. For example, as described above,
semiconductor
nanocrystals can be conjugated to nucleic acids and antibodies and then used
to probe the
presence or absence of particular proteins or nucleic acids within a tissue
specimen.
B. Tissue Arra,~paration
Tissue arrays can be prepared in a number of different ways. A clinician
can obtain tissue samples from various sources using standard anatomical
procedures and
individually place the tissue samples at different locations on some type of a
support (e.g.,
a glass slide or microtiter wells). The process can be automated in certain
respects using
devices designed to obtain tissue samples and then place the samples within an
array. An
example of suitable devices for preparing arrays in this manner are discussed
in PCT
publications WO 00/24940, 99/44062 and 99/44063. In general these devices
utilize a
punch apparatus to bore into a tissue sample and then dislodge the sample into
a
receptacle in an array support.
The array can be prepared in a variety of different formats. For example,
the array can include tissue samples from a single individual. Different
labeled
biomolecules bearing a semiconductor nanocrystal can be added to each location
to detect
the presence of a different cellular species. Other arrays can include tissue
samples from
a number of different individuals. For instance, the array can include tissue
samples
obtained from the same type of tissue from different individuals to screen for
particular
disease marlcers. A specific example of such an array is an array of breast
tissue samples
taken from women suspected of having breast cancer. Sets of tissue arrays can
also be
prepared, each set of arrays including the same tissue samples. Each array can
be
subjected to a different type of analysis to detect different markers. In this
way, results
for a number of different markers can rapidly be compiled for a population of
different
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individuals. As discussed further below, however, the multicolor capabilities
of
semiconductor nanocrystals permits a number of different types of
interrogations to be
conducted with a single tissue sample. Thus, utilization of the multiplexing
capabilities
of semiconductor nanocrystals coupled with an array format in which sets of
arrays
having the same tissue samples on each array allows for very lugh throughput
analysis of
a number of different markers.
Various types of tissue samples can be utilized. Suitable tissue samples
include, but are not limited to, tissue sections excised using known surgical
procedures,
one or more whole cells, cellular suspensions, cytological preparations (e.g.,
smears
obtained from cellular suspensions), as well as cellular extracts or
homogenates. Cell
suspensions can be utilized, for example, by pelleting the suspension and then
fixing it to
a support. The samples can also be obtained from specific tissues (e.g.,
slcin, breast,
prostrate, testes and ovaries) or from various bodily fluids (e.g., blood,
plasma, urine that
contains cells, semen, vaginal fluids, bronchial washings and ascitic fluids).
In the case
or cell suspensions or extracts, the cells can be directly obtained from an
organism or can
be obtained from a cell culture.
C. Assays
Tissue arrays can be used to rapidly profile hundreds or thousands of tissue
samples at the DNA, RNA and protein levels. However, the assays are not
limited to
detecting these three major classes of biomolecules. The presence and
concentration of
oligosaccharides, glycoproteins, particular fatty acids and other targets can
all be detected
using an appropriate molecule that specifically binds to the target of
interest Because
semiconductor nanocrystals can be tuned to a variety of different wavelengths,
it is
possible to use semiconductor nanocrystal labeled nucleic acids, proteins
(e.g.,
antibodies) and other biomolecules to probe multiple different DNAs, RNAs,
proteins
and/or other biomolecules within a single tissue sample. The results from such
investigations can be compiled in a database.
The use of semiconductor nanocrystal-conjugates (either a single
semiconductor nanocrystal conjugated to biomolecules or a plurality of
semiconductor
nanocrystals) allow specific, sensitive, photostable detection of target
molecules, factors
that can be problematic using currently available stains. Additionally the
inherent
properties of semiconductor nanocrystals, i. e., single excitation source,
narrow, gaussian
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spectra and tenability of emission wavelength, mean that many more colors are
resolved
than with conventional fluorescent dyes.
Various formats can be used to conduct analyses utilizing tissue arrays.
As indicated above, samples can be interrogated to detect the levels of target
nucleic acids
using either semiconductor labeled nucleic acids whose sequence is
complementary to the
target nucleic acid being interrogated or a labeled protein that specially
binds to the target
nucleic acid. When the level of a protein is to be monitored, typically an
antibody that
specifically binds to the target protein and bears a semiconductor nanocrystal
is used to
detect the presence of the target protein. However, if the target protein is a
DNA binding
protein, then the nucleic acid that binds to the protein can be labeled with a
semiconductor nanocrystal and used to probe for the target protein.
When labeled antibodies are utilized, analyses can be carned out in a two-
step reaction in which a primary antibody is initially contacted with the
tissue sample,
followed by addition of a semiconductor nanocrystal-conjugated antibody.
Alternatively,
an antibody (or other biomohecule) semiconductor nanocrystal conjugate can be
used to
directly label proteins of interest within the sample. As a specific example,
five (or more
with increased spectral use or reduced spectral separation) different
populations of
semiconductor nanocrystals can be synthesized with emission spectra that are
spaced at
40 nm intervals from, e.g., 490-650 nm. Each spectrally distinct population of
semiconductor nanocrystals is conjugated to a different molecule that
specifically
recognizes a biomolecule of interest that may or may not be present in the
sample to be
analyzed. Following standard protocols, the sample is labeled with the
semiconductor
nanocrystals and analyzed for the location and quantity of the target
molecule. This
analysis may be carried out by conventional fluorescent microscopy techniques
or by use
of a spectral scanning device.
Since many semiconductor nanocrystahs can be generated that are
spectrally distinct, it is possible to label different biomolecules such as
multiple
antibodies and/or multiple nucleic acid probes that can then be used to
measure the
position and quantity of cellular compounds. When different compounds are not
colocalized in the cell, many more semiconductor nanocrystal colors can be
used by
taking advantage of the known spatial separation of the targets to be
analyzed. For
example, no overlap would occur between nuclear localized targets and membrane
localized targets. Hence organelle- specific groups of semiconductor
nanocrystals can be
employed to increase dramatically the number of discernable targets.
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More specifically, the multi-color capability of semiconductor
nanocrystals can be used, for example, to conduct multi-color in-situ
hybridization to
measure the levels of tRNA, mRNA, DNA, protein or any other cellular compound
that
can be stained in a tissue array. By using multiple colors on one array rather
than using
multiple arrays with single color detection, greater accuracy is achieved,
since the tissue
sections are not homogeneous and can therefore vary slightly from array to
array. This
variance can easily effect assay results. Utilization of multi-color detection
with
semiconductor nanocrystals reduces this problem. In addition, as described in
greater
detail below, by utilizing semiconductor nanocrystal conjugates that include a
single
semiconductor nanocrystal, one can make quantitative measurements of the
target
biomolecule(s) under investigation. Multiplexing also allows rapid analysis of
many
molecular markers in the same set of specimens with greater accuracy.
The use of semiconductor nanocrystal labeled biomolecules in assays of
tissue arrays can be utilized in a variety of applications. One application is
to use tissue
arrays in various types of correlation studies. For instance, by obtaining
samples from
individuals known to have a particular disease, analyses with various
semiconductor
nanocrystal conjugates can be used to identify potential markers associated
with the
disease or illness. In general, such methods involve the use of semiconductor
nanocrystal
conjugates to determine the levels of selected DNA, RNA and/or protein levels
in tissues
from diseased individuals. Detection of elevated or reduced levels of various
genes or
gene products can be used to make initial correlations between such genes or
proteins
with the particular disease under investigation
In a related fashion, tissue array assays utilizing semiconductor
nanocrystal conjugates can be used to establish correlations between certain
markers and
patient prognosis. The levels of various DNA, RNA and/or protein levels can be
monitored over time in tissue samples obtained from patients known to have a
disease as
the patients receive different treatments. By monitoring the health history of
the patients
and the various levels of markers over time, one can establish correlations
between
certain markers and disease outcomes. Similarly, correlations between levels
of markers
° and the efficacy of different therapeutic treatments can established.
In this instance, the
level of different markers is tracked for individuals receiving different
treatments. The
photostability of semiconductor nanocrystals allows tissue arrays t be read
repeatedly and
archived for the purpose of comparison at a later date.
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Once a correlation has been established between a disease and one or more
markers, semiconductor nanocrystal conjugates that specially recognize such
markers can
be used to screen and identify individuals that have a disease or are
susceptible to
acquiring the disease. In like manner, once correlations between marker
profiles and
treatment efficacy have been established, assays utilizing semiconductor
nanocrystal
conjugates can be utilized to determine the marker profile for an individual
and thus
identify the most appropriate treatment option.
Tissue array analysis can also be used in combination with other analyses.
For example, the initial correlation studies just described can be conducted
using the
differential gene expression methods described supra. Hence, nucleic acid
arrays can be
utilized to identify a nucleic acid whose expression is altered in diseased
individuals.
Additional validation studies can then be conducted using tissue axrays. If
the nucleic
acid is shown to be a boyaa fide marker for a disease, then semiconductor
nanocrystal
conjugates that specifically bind to the marlcer can be used to screen
individuals for the
presence of, or susceptibility to, the disease.
VIII. Secondary Interco ations
Semiconductor nanocrystals can be utilized to label various target
biomolecules for use in various types of secondary interrogations. Such
investigations
generally involve conducting an additional analysis once a binding a binding
complex
between two or more biomolecules have already been formed. The array in such
investigations typically bears a biomolecule that captures a target molecule
in preparation
for a secondary interrogation. Suitable targets in this type of study include,
but are not
limited to, nucleic acids (e.g., DNA, RNA), proteins, or antibodies.
A specific example of a secondary interrogation is the use of an array of
antibodies to probe for multiple epitopes of a protein. In this instance, each
spot on the
array contains a different antibody. A complex protein target is labeled with
a single
color semiconductor nanocrystal and allowed to bind to the array bearing the
different
antibodies. In the case of FIG. 1A, the three shaded regions correspond to the
positive
signals from the array. Once the target molecules have been bound to the
array, a
secondary antibody (labeled with a semiconductor nanocrystal having a second
color) is
brought into contact with the array and given the opportunity to bind to a
second epitope
of the bound proteins (FIG. 1B). In the specific example shown in FIG. 1A,
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colors are used to label each of the three active antibodies identified in the
first round of
screening.
This procedure can be used, for example, to find complementary antibody
pairs. In the first step, several antibodies are identified that bind
efficiently to the target
molecule. Since each of these antibodies may bind to a different epitope of
the target
molecule, the second step can interrogate the bound proteins by contacting the
array with
the same antibodies identified in the first step. Those locations on the array
that emit
signals from both semiconductor nanocrystals are those locations in which
there are two
antibodies that recognize different epitopes of the target molecule.
This technique of capture and secondary interrogation can also be used to
do simultaneous genotyping of multiple SNPs. If 2 SNPs are located far apart
on the
target nucleic acid under analysis, both polymorphic sites can be interrogated
simultaneously by using a nucleic acid probe on an array that is complimentary
to the first
SNP to capture the target nucleic acid labeled with a semiconductor
nanocrystal. Once
the labeled target nucleic acid is captured, it can be probed with a second
set of labeled
probes that is complimentary to the various polymorphic forms of the second
SNP.
Probes for different polymorphic forms can each be labeled with a different
color. By
contacting the array with the second set of probes, each SNP can be identified
by color
and position, and the haplotype can be determined.
IX. Expansion of Dynamic Range and Sin lg-a Copy Counting
A. General
As noted above, two key shortcomings associated with existing array
methods concern limitations on sensitivity at low concentrations and
limitations on
dynamic range, namely the ability to accurately and simultaneously measure
target
concentration over a wide range of concentrations. With the fluorescent labels
currently
utilized to conduct array analyses, one often is forced to sacrifice linearity
at high
concentrations for detection sensitivity at low concentrations.
The use of semiconductor nanocrystals can provide significant
improvement in both sensitivity and dynamic range. The fluorescence from
semiconductor nanocrystals is extremely bright and stable, and permits routine
detection
of single semiconductor nanocrystals (see, e.g., Empedocles et al. (1999a),
"Three-
dimensional orientation measurements of symmetric single chromophores using
polarization microscopy," Nature 399:126-130; Empedocles et al. (1999b),
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"Spectroscopy of Single CdSe Nanocrystallites," Acc. Chem. Res. 32:389-396;
Empedocles et al. (1997), "Quantum-confined Stark effect in single CdSe
nanocrystallite
quantum dots," Science 278:2114-2117; Empedocles et al. (1996),
"Photoluminescence
spectroscopy of Single CdSe nanocrystallite quantum dots," Phys. Rev. Lett.
77:3873-
3876). The ability to detect single labels dramatically increases detection
sensitivity.
This feature of semiconductor nanocrystals means detection can be extended
into the
single copy counting regime. The increase in sensitivity afforded by
semiconductor
nanocrystals enables the detection of minute quantities of target molecule.
This is
important in a variety of assays in which the target is present at only very
low
concentrations, such as detecting subtle changes in gene expression (changes
caused, for
example, by disease.or environmental changes).
Even when dealing with target molecule concentrations that can be
detected using current fluorophores, there are still problems associated with
the
quantitative analysis of array results using organic dyes. Inconsistent
performance of
dyes such as Cy5 can result in unreliable array results. Semiconductor
nanocrystals can
act as a more stable and reliable substitute for existing fluorophores such as
Cy3 and CyS.
In addition, typically semiconductor nanocrystals do not self quench to the
extent that
organic dyes do, remaining capable of producing strong luminescence even when
they are
packed into solid semiconductor nanocrystal films. This means that in some
instances
semiconductor nanocrystals can also expand the high-end limit of concentration
detection
over what can be achieved using standard fluorophores. Hence, the
characteristics of
semiconductor nanocrystals allows detection to occur at lower and higher
concentration
levels, thus expanding the dynamic range of detection.
The single target counting assays and methods thereof described herein do
not necessarily have to be performed using semiconductor nanocrystal labels.
Any
fluorescent label capable of being detected on the single molecule level can
be utilized for
the type of measurement described herein. Hence, suitable labels include, but
is not
limited to, organic dye molecules, metal colloid scattering particles, and
surface-enhanced
Raman spectroscopy (SERS) particles. However, the many unique features of
semiconductor nanocrystals described supra, make them particularly useful as
labels in
single target counting assays. In addition, certain methods described herein
do not
require the ability to detect a single label, but rather a single target
molecule. Therefore,
the methods described herein can be used to detect single target molecules
that are labeled
with a single detectable label, or with multiple detectable labels.
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B. Basis for Sin 1~ a Copy Countin~ynamic Range Expansion
The high stability, detection sensitivity and ease of multiplexing make
semiconductor nanocrystals useful as multi-color fluorophores for use in ultra-
sensitive
surface based assays. The ability to easily detect single semiconductor
nanocrystals
means that semiconductor nanocrystals are particularly useful as fluorophores
in
bioassays in which single target molecules bound to the assay surface are
counted one at a
time.
"Single target counting" does not mean the counting of all of the target
molecules within a sample, but rather the counting of the target molecules
that are bound
to the surface of the array substrate. While this number is not necessarily
the same as the
total number of target molecules in the sample, the actual target level can be
determined
through calibration against a sample of known concentration. By enabling the
detection
and counting of single bound target molecules, one can extend the sensitivity
of surface-
based assays beyond what is possible using current detection techniques. For
instance,
current microarray technology allows the detection of target at a density of
as low as 0.1
labels/~.mz ( ~8 labels per 10~,m diameter confocal spot). With single target
counting, the
theoretical limit of detection is 1 label per array spot, extending the
detection sensitivity
by as much as 3 orders of magnitude for a 100~.m diameter array element.
In order to properly understand how the detection of single bound target
molecules improves the sensitivity and dynamic range of a surface-based assay,
it is
important to understand what is actually measured at the high and low end of
the
concentration range on an assay surface. For the purposes of illustration, a
microarray
will be considered; however, the ideas presented for the microarray also hold
true for any
surface-based assay. Figure 6A shows a graphic representation of a series of
microarray
spots with decreasing concentrations of bound target. The left side
corresponds to the
high concentration regime (ensemble regime), in which the entire array spot is
covered
with target and the average emission intensity is dependent on the average
density of label
across the surface of the array. In tlus regime, sample concentration is
proportional to
average emission intensity (ensemble intensity). The right side corresponds to
the single
copy counting regime, where individual bound target molecules are separated
from each
other by distances that are greater than the diffraction limit of light and
can be detected
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one at a time. In this regime, sample concentration is proportional to the
number of
individual targets counted on the surface of the array.
Figure 6B shows data simulating the relative signal vs concentration
detected using ensemble intensity and single copy counting over the entire
concentration
range. Ensemble measurements yield a linear concentration dependence at lugh
concentrations, but saturate at low concentrations. This saturation occurs
when the total
signal from bound target in the detection region is lower than the noise
generated from
the integrated background across that entire region. Detecting single
molecules bound to
the array with high-resolution microscopy, however, can dramatically reduce
the
integrated background noise by comparing the signal from a single fluorophore
to the
background from an extremely small (diffraction limited) area of the array
spot.
As an example, if the background signal increases linearly with total
detection area, then the background generated over a standard 10~,m diameter
ensemble
probe spot is 400 times higher than the background generated from a high
resolution
image of a single fluorophore (~O.S~,m diameter). This results in a decrease
in noise (and
therefore an increase in sensitivity) of a factor of 20. This effect is
further enhanced if the
ensemble signal is integrated over the entire array spot. For a 100~m diameter
spot, the
background signal is 40000 times higher than for a diffraction limited spot
resulting in
approximately 200 times higher sensitivity. The background over the bottom of
an entire
well of a 96 well plate is ~10$ times higher yielding an enhancement of 104.
To achieve
these enhancements, however, one needs to be able to detect the fluorescence
from a
single bound target molecule with high spatial resolution.
In contrast to ensemble intensity measurements, the single target counting
signal saturates at high concentrations. This occurs when the concentration
increases to
the point where individual target molecules are so close together that they
can not be
distinguished. This means that some individual spots actually contain more
than one
bound target molecule and therefore results in an undercounting of the total
number of
target molecules. The result is an underestimate of the total sample
concentration (see
FIG. 6C).
Between the ensemble and single target counting regime, there is a regime
in which the concentration is low enough to count individual targets, but high
enough to
be detectable in an ensemble measurement. This is referred to as the
transition regime.
Preliminary measurements of single semiconductor nanocrystals nonspecifically
adsorbed
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to a surface over a wide range of concentrations suggests that it should be
possible to
calibrate the transition regime using either ensemble or single target
counting, allowing
the user to calibrate concentrations across all regimes.
By combining single copy counting and ensemble intensity measurements,
detection sensitivity can be increased, as well as the dynamic range of
microarray assays.
In standard measurements, detection sensitivity at the low end is achieved at
the expense
of dynamic range at the high end due to detector saturation. However, by
combining
single target counting with ensemble intensity measurements, one can cover the
entire
dynamic range in a single experiment. The reason is that in the single copy
counting
regime, as the concentration increases, the peak intensity does not -- only
the number of
detected spots increases. As such the entire dynamic range of the detector can
be used to
cover the ensemble concentration regime, where peak intensity varies linearly
with
concentration.
As an example, consider a detection system capable of detecting the
fluorescence from single semiconductor nanocrystals over the entire area of a
100p,m
microarray spot, with a spatial resolution of less than 0.5~,m. This system
uses a 2-
dimensional CCD camera with a dynamic range of 65,536 counts per pixel and a
read
noise of ~2 counts/pixel. If excitation intensity and integration time are
selected to yield
30 counts/pixel/semiconductor nanocrystal, then in the single copy counting
regime,
individual semiconductor nanocrystals are detected with a signal to noise
ratio of ~15.
Assuming an even distribution of bound molecules and a spatial resolution of
~0.5~,m, at
best one can detect 40,000 individual spots within each 100p,m array spot. In
an ideal
system, this would result in a dynamic range within the single target counting
regime of
more than 104. As the concentration increases into the ensemble regime, the
average
intensity increases linearly with concentration. The detector then provides an
additional
dynamic range of 103 before saturating. As a result, a total dynamic range of
107 is
theoretically possible in a single experiment. Of course, multiple integration
times can be
used to extend the dynamic range to higher concentrations if necessary.
C. Detection
Single molecule fluorescence detection can be achieved using either laser
scanning confocal microscopy or wide-field imaging with a 2D CCD camera. One
distinct advantage of wide-field imaging over scanning confocal microscopy for
these
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applications is that fluorescence can be collected from all points within one
or more array
spots simultaneously. This means that the signal can be integrated for
relatively long
periods of time without increasing the read-time for the array. This is
particularly
beneficial when detecting semiconductor nanocrystals, since they do not
photobleach. It
is therefore possible to integrate the signal from each array spot for a
relatively long time
compared to organic dyes. This can provide significantly higher fluorescence
signal and
is one of the things that allows one to easily detect the fluorescence from
single
semiconductor nanocrystals. At the same time, however, a relatively high
spatial
resolution (<O.S~.m) is needed in order to be able to spatially separate the
fluorescence
from individual target molecules and maximize the signal to background ratio.
The
combination of high spatial resolution with long integration times can be
prohibitive
when using a confocal scanning optical system. For instance, using a confocal
scanning
system with a O.S~,m resolution and an integration time of 100 ms would take
more than
minutes to scan a single 100~.m diameter microarray spot. This is a
prohibitively long
15 time when considering arrays with 10000 spots 0100 days).
Using wide-field detection and a high numerical aperture microscope
objective, the same image of a single spot can be obtained in a single 100 ms
exposure.
Once taken, the array can be translated to an adjacent region and the next
image acquired.
By precisely controlling the scanning stage and stitching the images together,
the entire
array image can be produced. This procedure dramatically decreases the total
read-time,
allowing an entire array to be read in Iess than 20 minutes. In addition, in
some instances
multiple array spots can be imaged simultaneously, further reducing the total
collection
time.
While the methods of the present invention focus on array based assays in
which an assay occurs on an array surface, the methods also apply to other
bioassays
performed on a surface support such as the bottom of a microtitre plate or a
polymer
bead. The considerations just described apply generally to any of the assays
set forth
above. Assays typically are conducted with semiconductor nanocrystals, but as
noted
above, can also be performed with other fluorophores such as an organic dye or
metal
colloid. Semiconductor nanocrystals can be incorporated into the ligands or
antiligands
of the assay via a plurality of techniques described herein. Each bound target
molecule is
labeled with 1 or more semiconductor nanocrystals.
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Once the assay is complete and one or more complexes containing
semiconductor nanocrystals have been formed, the fluorescence from the sample
is
detected. If the density of bound target molecules is greater than ~1
target/~,m2, then the
assay signal is measured and calibrated using the total emission intensity
from the entire
assay region (e.g. the total signal from a single microarray spot or from an
entire
microtitre well). If the target density is less than ~1 target/~,m2, so that
individual target
molecules can be spatially resolved using standard far-field optics, then the
assay signal is
measured and calibrated by counting the total number of bound target
molecules. The
assay signal can be measured from all assays using both ensemble and single
target
counting methods. A calibration curve can then be used to identify which
assays fall in
the ensemble, single and transition regimes.
Typically, complexes including semiconductor nanocrystals are detected
with an optical detection system capable of detecting the fluorescence from
single
semiconductor nanocrystals (or other labels) with a spatial resolution of lpm
or less. In
some instances, this optical system is comprised of a wide-field imaging
system with a
2D CCD camera and a high numerical apertuxe microscope objective. A laser
based
microscope system capable of detecting and spectrally resolving the
fluorescence from
single semiconductor nanocrystals can also be utilized (see, e.g., Empedocles
et al.
(1999x), Nature 399:126-130; Empedocles et al. (1999b), Acc. Chem. Res. 32:389-
396;
Empedocles et al. (1997), Science 278:2114-2117; and Empedocles et al. (1996),
Phys.
Rev. Lett. 77:3873-3876).
The optical design of the laser based microscope system is based on a.
wide-field epifluorescence microscope. Figure 7 is a schematic drawing of the
significant
optical components of such a laser microscope system 100. Excitation light 102
from a
laser source (488 nm Ar+) 104 is transmitted through a dispersing prism 106
and a 500
nm short pass dichroic mirror 108 at an angle of 45°. The excitation
light is then focused
by a high numerical aperture microscope objective 110 onto the sample surface
112. An
additional lens in the excitation path (i.e., the dispersing prism 106) causes
the laser 104
to illuminate a wide area of the sample surface 112. The fluorescent image is
collected
by the same objective lens 110. The image is reflected by the dichroic mirror
108, passes
through a wavelength specific filter 114 to remove any excitation light, and
is focused by
a final lens 116 onto the detection system 118. The detection system 118
consists of a 2D
CCD camera 120 and a tunable bandpass filter 122. Spectral images are obtained
by
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acquiring multiple images at a different wavelength. With this system, it is
possible to
simultaneously obtain spectra at every point within the image with a spectral
resolution of
2 nm and a spatial resolution of less than ~O.S~,m. Uniform excitation
intensity in this
system can be generated either through the use of a lamp light source or a
laser excitation
source that has been transformed from a Gaussian intensity profile to a "top-
hat" profile
through the use of a series of 2 Powel lenses each oriented at 90 degrees
relative to each
other. Alternatively, the optical system can be comprised of a scanning
confocal
microscope system with a spatial resolution of less than ~O.S~,m.
Another detection option utilizes an optical system that comprises a
microscope with an immersion microscope objective in which the sample is
viewed from
the backside of the sample substrate (e.g. from the underside of a microarray
substrate or
from the bottom of a microtitre well). In certain instances, the sample can be
located on
the surface of a glass or quartz substrate and is detected with a high
numerical aperture
oil-immersion microscope and index matching immersion oil (n=1.51). This can
yield an
increase in collection efficiency of as much as 800%. Alternatively, detection
can be with
a water- or other fluid-immersion lens, also detecting from the baclc-side of
the sample
substrate.
For ultrasensitive detection of single target molecules, it is not only
necessary to have a bright fluorophore, but also to minimize the collection of
background
fluorescence from the substrate surface and assay materials. Autofluorescence
from the
array substrate and assay materials can be minimized by (a) using low
fluorescence array
substrates such as quartz or low fluorescence glass, (b) choosing a
fluorescent label that
does not overlap significantly with the autofluorescence from the substrate
and assay
materials, and (c) choosing an excitation wavelength that does not
significantly excite
autofluorescence. Since semiconductor nanocrystals can be synthesized to
absorb and
emit at any wavelengths, they are an ideal fluorophore for minimizing
interference from
autofluorescence.
An important issue in detecting assays on the single target counting level is
how to locate assay regions with very low signal. For instance, if a
microarray is labeled
at a density in the single target counting regime, in some instance it can be
difficult to
locate the array spots for quantitative detection. In certain methods,
kinematic alignment
of the array slide combined with the use of "alignment spots" is used to
automatically
locate the edges of the array and register the first image so that the array
spots are each
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located within the center of each image. Alignment spots are array spots that
are not
complementary to any sequence of interest. To each hybridization sample, one
can add a
labeled target that is specific for these alignment spots at a known
concentration. These
spots therefore have a high signal and can be detected and used for alignment
purposes.
A pattern of alignment spots can be placed across each array that
unambiguously identify
the absolute position of the array.
Software can then be used to locate and analyze each spot within the array.
Using pattern recognition algorithms, the alignment spots can be identified
and all other
spot locations will be determined from the lcnown periodicity of the array.
Once the array
pattern is determined, each spot on the array can be located according to its
position
within a periodic lattice. The radius of all spots is the same and can be
predetermined or
extracted from the radius of the alignment spots. Two separate algorithms,can
then be
used to analyze the signal from within each spot area. First, the total
integrated signal
from within each spot can be measured and compared to either an equivalent
area outside
of the array spot or to a calibration spot of known intensity. Second, an
algorithm can be
used to count individual fluorescent point within each array spot. Using
pattern
recognition, the algorithm can identify and count fluorescent points that fit
a set of
predetermined characteristics of shape, size and threshold intensity that are
specific for
the fluorescence from single semiconductor nanocrystals. A data file can be
exported
containing the ensemble intensity and the "count number" (i.e. the number of
discrete
fluorescent points) for each spot. Figures 8A-8E describe the complete array
scanning
procedure.
For some surface based assays such as microtitre plate assays,
macroscopic alignment of the optical system can be used (i.e. scanning the
entire bottom
of each microtitre well). For bead-based assays, it is possible to use a
second
semiconductor nanocrystal color that does not spectrally overlap with the
detection label.
This second color can be added to each bead, either internally, or bound to
the surface at a
known concentration. This color can then be used to locate individual beads.
Once
found, a bandpass filter can be used to block the fluorescence from the
alignment color
and allow single target detection of only the label semiconductor
nanocrystals. This 2-
color technique can also be used for microarrays.
One additional requirement for a successful assay system capable of
detecting single bound target molecules is the minimization of nonspecific
binding of the
detection label. Too many nonspecifically bound semiconductor nanocrystals can
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interfere with the quantitative measure of target concentrations on the level
of single
target counting. Consequently, labeling of these assays typically is with a
fluorophore
with extremely low nonspecific binding. Because the surface of semiconductor
nanocrystals can be modified to have virtually any functionality, one can
optimize the
surface characteristics of the semiconductor nanocrystals to minimize
nonspecific
binding.
In other methods, each target molecule is labeled with two different
semiconductor nanocrystal colors via two different binding interactions.
Specifically
bound labels can then be identified through the detection of both colors
colocalized
within the same fluorescent spot.
D. Nonspecific Binding Identified by Sin~Ie Color Fluorescence
The primary shortcoming of surface based assays such as nucleic acid
microarrays is the lack of appropriate sensitivity needed to detect extremely
low levels of
target concentration. For instance, as much as 40% of the known genes of
interest studied
using gene expression microarrays are expressed at a level of between 1 and 10
copies per
cell, just at or below the limit of detection using current detection schemes.
In addition to
low expression levels, the costs incurred in extracting material for genetic
testing creates
pressure to minimize sample size requirements for genetic analysis. The
ability to
measure vanishingly small quantities of expressed DNA significantly improves
one's
ability to identify and treat diseases at an early stage. Ultra-sensitive
detection in
microarray assays can also assist in identifying new genes of interest in all
areas of
disease. A system for labeling and high sensitivity detection of fluorescence
from DNA
microarrays can significantly reduce the costs associated with expression
analysis while
simultaneously increasing the available information content.
Currently, the preferred method for detection of surface based assays such
as microarrays is by labeling target molecules with organic dyes. For DNA
microarrays
using organic dyes, the current state-of the-art detection can only detect a
minimum of
approximately 10 molecules in a 10~.m x 10~,m region of a microarray spot
(Duggan et
al. (1999) Nature Geyaetics 21(nls):10-14). This means that the minimum number
of
bound DNA molecules required in order to detect signal from a standard 100p,m
diameter
microarray spot is approximately 1000. In order to generate this signal, more
than 10
million cells may be required. In many instances, it is not possible to
extract this much
CA 02400379 2002-08-14
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cellular material. Using single target counting with semiconductor
nanocrystals, the
theoretical limit of detection is 1 molecule per array spot, reducing the
amount of cellular
material required by three orders of magnitude and significantly improving our
ability to
identify and monitor important expression profiles. Similarly, the ability to
detect single
bound target molecules in all types of bioassays will dramatically improve the
sensitivity
and dynamic range of these measurements, enhancing the information content and
minimizing costs.
X. Preparation of Semiconductor Nanocrystals
Semiconductor nanocrystals for use in the subject methods are made using
techniques known in the art. See, e.g., U.S. Patent Nos. 6,048,616; 5,990,479;
5,690,807;
5,505,928; 5,262,357; as well as PCT Publication No. 99/26299 (published May
27,
1999). In particular, exemplary materials for use as semiconductor
nanocrystals in the
biological and chemical assays of the present invention include, but are not
limited to
those described above, including group II-VI, III-V and group IV
semiconductors such as
ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, Case, Care, SrS, SrSe,
SrTe, BaS, Base, Bare, GaN, GaP, GaAs, GaSb, InP, In.As, InSb, A1S, A1P, AISb,
PbS,
PbSe, Ge and Si and ternary and quaternary mixtures thereof. The semiconductor
nanocrystals are characterized by their uniform nanometer size.
As discussed above, the selection of the composition of the semiconductor
nanocrystal, as well as the size of the semiconductor nanocrystal, affects the
characteristic
spectral emission wavelength of the semiconductor nanocrystal. Thus, as one of
ordinary
skill in the art will realize, a particular composition of a semiconductor
nanocrystal as
listed above will be selected based upon the spectral region being monitored.
For
example, semiconductor nanocrystals that emit energy in the visible range
include, but are
not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs.
Semiconductor nanocrystals that emit energy in the near IR range include,
but are not limited to, InP, In.As, InSb, PbS, and PbSe. Finally,
semiconductor
nanocrystals that emit energy in the blue to near-ultraviolet include, but are
not limited to,
ZnS and GaN.
For any particular composition selected for the semiconductor nanocrystals
to be used in the inventive methods, it is possible to tune the emission to a
desired
wavelength by controlling the size of the particular composition of the
semiconductor
nanocrystal. In some instances, 5-20 discrete emissions (five to twenty
different size
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populations or distributions distinguishable from one another), more
preferably 10-15
discrete emissions, are obtained for any particular composition, although one
of ordinary
skill in the art will realize that fewer than five emissions and more than
twenty emissions
can be used depending on the monodispersity of the semiconductor nanocrystal
particles.
If high information density is required, and thus a greater number of distinct
emissions,
the nanocrystals are preferably substantially monodisperse within the size
range given
above.
As explained above, "monodisperse," as that term is used herein, means a
colloidal system in which the suspended particles have substantially identical
size and
shape. In certain high information density applications, monodisperse
particles deviate
less than 10% rms in diameter, and preferably less than 5%. Monodisperse
semiconductor nanocrystals have been described in detail in Murray et al.
(1993) J. Am.
Chem. Soc. 115:8706, and in Murray, "Synthesis and Characterization of II-VI
Quantum
Dots and Their Assembly into 3-D Quantum Dot Superlattices," (1995) Doctoral
dissertation, Massachusetts Institute of Technology. The number of discrete
emissions
that can be distinctly observed for a given composition depends not only upon
the
monodispersity of the particles, but also on the deconvolution techniques
employed.
Semiconductor nanocrystals, unlike dye molecules, can be easily modeled as
Gaussians
and therefore are more easily and more accurately deconvoluted.
However, for some applications, high information density is not required
and it is more economically attractive to use more polydisperse particles.
Thus, for
applications that do not require high information density, the linewidth of
the emission
can be in the range of 40-60 nm.
In certain methods, the surface of the semiconductor nanocrystal is
modified to enhance the efficiency of the emissions, by adding an overcoating
layer to the
semiconductor nanocrystal. The overcoating layer is typically utilized because
at the
surface of the semiconductor nanocrystal, surface defects can result in traps
for electrons
or holes that degrade the electrical and optical properties of the
semiconductor
nanocrystal. An insulating layer at the surface of the semiconductor
nanocrystal provides
an atomically abrupt jump in the chemical potential at the interface that
eliminates energy
states that can serve as traps for the electrons and holes. This results in
higher efficiency
in the luminescent process. '
Suitable materials for the overcoating layer include semiconductor
materials having a higher bandgap energy than the semiconductor nanocrystal
core. In
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addition to having a bandgap energy greater than the semiconductor nanocrystal
core,
suitable materials for the overcoating layer should have good conduction and
valence
band offset with respect to the core semiconductor nanocrystal. Thus, the
conduction
band is desirably higher and the valence band is desirably lower than those of
the core
semiconductor nanocrystal. For semiconductor nanocrystal cores that emit
energy in the
visible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g., InP,
InAs,
InSb, PbS, PbSe), a material that has a bandgap energy in the ultraviolet
regions can be
used. Exemplary materials include ZnS, GaN, and magnesium chalcogenides, e.g.,
MgS,
MgSe, and MgTe. For a semiconductor nanocrystal core that emits in the near
IR,
materials having a bandgap energy in the visible, such as CdS or CdSe, can
also be used.
The preparation of a coated semiconductor nanocrystal can be found in, e.g.,
Dabbousi et
al. (1997) J. Phys. Chem. B 101:9463) and Kuno et al. (1997) J. Phys. Chem.
106:969.
Most semiconductor nanocrystals are prepared in coordinating solvent,
such as trioctylphosphine oxide (TOPO) and trioctyl phosphine (TOP) resulting
in the
formation of a passivating organic layer on the nanocrystal surface comprised
of the
organic solvent. This layer is present on semiconductor nanocrystals
containing an
overcoating and those that do not contain an overcoating. Thus, either of
these classes of
passivated semiconductor nanocrystals are readily soluble in organic solvents,
such as
toluene, chloroform and hexane. These functional moieties can be readily
displaced or
modified to provide an outer coating that renders the semiconductor
nanocrystals suitable
for use as the detectable labels of the present invention, as described
further below.
Furthermore, based upon the desired application, a portion of the
semiconductor
nanocrystal functionality, or the entire surface of the semiconductor
nanocrystal
functionality can be modified by a displacement reaction, based upon the
desired use
therefor.
After selection of the composition of semiconductor nanocrystal for the
desired range of spectral emission and selection of a desired surface
fiznctionalization
compatible with the system of interest, it may also be desirable to select the
minimum
number of semiconductor nanocrystals needed to observe a distinct and unique
spectral
emission of sufficient intensity for spectral identification. Selection
criteria important in
determining the minimum number of semiconductor nanocrystals needed to observe
a
distinct and unique spectral emission of sufficient intensity include: (1)
providing a
sufficient number of semiconductor nanocrystals that are bright (i.e., that
emit light
versus those that are dark) and, (2) providing a sufficient number of
semiconductor
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nanocrystals to average out over the blinking effect observed in single
semiconductor
nanocrystal emissions. Nirmal et al., (1996) Nature 383:802.
For example, eight or more semiconductor nanocrystals of a particular
composition and particle size distribution can be provided. If, for example,
the desired
method of use utilizes three different particle size distributions of a
particular
composition, eight of each of the three different particle size distributions
of a
semiconductor nanocrystal is used, in order to observe sufficiently intense
spectral
emissions from each to provide reliable information regarding the location or
identity of a
particular analyte of interest. Fewer than eight semiconductor nanocrystals of
a particular
composition and particle size distribution can be utilized provided that a
unique spectral
emission of sufficient intensity is observed, as determined by the selection
criteria set
forth above.
The above method can be used to prepare separate populations of
semiconductor nanocrystals, wherein each population exhibits a different
characteristic
photoluminescence spectrum. Each of a plurality of populations of
semiconductor
nanocrystals can be conjugated to distinct first members of binding pairs for
use in a
multiplexed assay or analytical method in which each of a plurality of
corresponding
second members of the binding pairs can be detected simultaneously.
The narrow spectral linewidths and nearly gaussian symmetrical
lineshapes lacking a tailing region observed for the emission spectra of
nanocrystals
combined with the tunability of the emission wavelengths of nanocrystals
allows high
spectral resolution in a system with multiple nanocrystals. In .general up to
10-20 or more
different-sized nanocrystals or different size distributions of monodisperse
populations of
nanocrystals from different preparations of nanocrystals, with each sample
having a
different emission spectrum, can be used simultaneously in one system, i.e.,
multiplexing,
with the overlapping spectra easily resolved using techniques well known in
the art, e.g.,
optically with or without the use of deconvolution software.
As discussed previously, the ability of the semiconductor nanocrystals to
produce discrete optical transitions, along with the ability to vary the
intensity of these
optical transitions, enables the development of a versatile and dense encoding
scheme.
The characteristic emissions produced by one or more sizes of semiconductor
nanocrystals attached to, associated with, or embedded within a particular
support,
compound or matter enables the identification of the analyte of interest
and/or its location.
For example, by providing N sizes of semiconductor nanocrystals (each having a
discrete
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optical transition), each having M distinguishable states resulting from the
absence of the
semiconductor nanocrystal, or from different intensities resulting from a
particular
discrete optical transition, Mn different states can be uniquely defined. In
the case
wherein M is 2, in which the two states could be the presence or absence of
the
semiconductor nanocrystal, the encoding scheme would thus be defined by a base
2 or
binary code. In the case wherein M is 3, in which the three states could be
the presence
of a semiconductor nanocrystal at two distinguishable intensities and its
absence, the
encoding scheme would be defined by a base 3 code. Herein, such base M codes
wherein
M is greater than 2 are termed higher order codes. The advantage of higher
order codes
over a binary order code is that fewer identifiers are required to encode the
same quantity
of information.
As one of ordinary skill in the art will realize, the ability to develop a
higher order encoding system is dependent upon the number of different
intensities
capable of detection by both the hardware and the software utilized in the
decoding
system. In particularly preferred embodiments, each discrete emission or
color, is
capable of being detectable at two to twenty different intensities. In a
particularly
preferred embodiment wherein ten different intensities are available, it is
possible to
employ a base 11 code comprising the absence of the semiconductor nanocrystal,
or the
detection of the semiconductor nanocrystal at 10 different intensities.
Clearly, the advantages of the semiconductor nanocrystals, namely the
ability to observe discrete optical transitions at a plurality of intensities,
provides a
powerful and dense encoding scheme that can be employed in a variety of
disciplines. W
general, one or more semiconductor nanocrystals may act as a barcode, wherein
each of
the one or more semiconductor nanocrystals produces a distinct emissions
spectrum.
These characteristic emissions can be observed as colors, if in the visible
region of the
spectrum, or may also be decoded to provide information about the particular
wavelength
at which the discrete transition is observed. Likewise, for semiconductor
nanocrystals
producing emissions in the infrared or ultraviolet regions, the characteristic
wavelengths
that the discrete optical transitions occur at provide information about the
identity of the
particular semiconductor nanocrystal, and hence about the identity of or
location of the
analyte of interest.
The color of light produced by a particular size, size distribution and/or
composition of a semiconductor nanocrystal can be readily calculated or
measured by
methods which will be apparent to those skilled in the art. As an example of
these
CA 02400379 2002-08-14
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measurement techniques, the bandgaps for nanocrystals of CdSe of sizes ranging
from
12~ to 1151 are given in Murray et al. (1993) J. Am. Chem. Soc. 115:8706.
These
techniques allow ready calculation of an appropriate size, size distribution
and/or
composition of semiconductor nanocrystals and choice of excitation light
source to
produce a nanocrystal capable of emitting light device of any desired
wavelength.
An example of a specific system for automated detection for use with the
present methods includes, but is not limited to, an imaging scheme comprising
an
excitation source, a monochromator (or any device capable of spectrally
resolving the
image, or a set of narrow band filters) and a detector array. In one
embodiment, the
apparatus consists of a blue or UV source of light, of a wavelength shorter
than that of the
luminescence detected. This may be a broadband IJV light source, such as a
deuterium
lamp with a filter in front; the output of a white light source such as a
xenon lamp or a
deuterium lamp after passing through a monochromator to extract out the
desired
wavelengths; or any of a number of continuous wave (cw) gas lasers, including
but not
limited to any of the Argon Ion laser lines (457, 488, 514, etc. mn), a HeCd
laser; solid
state diode lasers in the blue such as GaN and GaAs (doubled) based lasers or
the doubled
or tripled output of YAG or YLF based lasers; or any of the pulsed lasers with
output in
the blue, to name a few.
The luminescence from the dots may be passed through an imaging
subtracting double monochromator (or two single monochromators with the second
one
reversed from the first), for example, consisting of two gratings or prisms
and a slit
between the two gratings or prisms. The monochromators or gratings or prisms
can also
be replaced with a computer controlled color filter wheel where each filter is
a narrow
band filter centered at the wavelength of emission of one of the dots. The
monochromator assembly has more flexibility because any color can be chosen as
the
center wavelength. Furthermore, a CCD camera or some other two dimensional
detector
records the images, and software color codes that image to the wavelength
chosen above.
The system then moves the gratings to a new color and repeats the process. As
a result of
this process, a set of images of the same spatial region is obtained and each
is color-coded
to a particular wavelength that is needed to analyze the data rapidly.
In another embodiment, the apparatus is a scanning system as opposed to
the above imaging scheme. In a scanning scheme, the sample to be analyzed is
scanned
with respect to a microscope obj ective. The luminescence is put through a
single
monochromator or a grating or prism to spectrally resolve the colors. The
detector is a
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diode array that then records the colors that are emitted at a particular
spatial position.
The software then ultimately recreates the scanned image and decodes it.
XI. Production of Semiconductor Nanocrystal Conjugates
The present invention utilizes various conjugates that generally comprise a
biological molecule and one or more semiconductor nanocrystals, such that the
conjugate
can detect the presence, absence and/or amounts of various complexes formed on
addressable arrays. Without limitation, semiconductor nanocrystal conjugates
comprise
any molecule or molecular complex, linked to a semiconductor nanocrystal, that
can
interact with a biological target, to detect biological processes, or
reactions, as well as
alter biological molecules or processes. Preferably, the molecules or
molecular
complexes or conjugates physically interact with a biological compound.
Preferably, the
interactions are specific. The interactions can be, but are not limited to,
covalent,
noncovalent, hydrophobic, hydrophilic, electrostatic, van der Waals, or
magnetic.
Preferably, these molecules are small molecules, proteins, or nucleic acids or
combinations thereof.
Semiconductor nanocrystal conjugates can be made using techniques
known in the art. For example, moieties such as TOPO and TOP, generally used
in the
production of semiconductor nanocrystals, as well as other moieties, can be
readily
displaced and replaced with other functional moieties, including, but not
limited to
carboxylic acids, amines, aldehydes, and styrene to name a few. One of
ordinary skill in
the art will realize that factors relevant to the success of a particular
displacement reaction
include the concentration of the replacement moiety, temperature and
reactivity. Thus,
for the purposes of the present invention, any functional moiety may be
utilized that is
capable of displacing an existing functional moiety to provide a semiconductor
nanocrystal with a modified functionality for a specific use.
The ability to utilize a general displacement reaction to modify selectively
the surface functionality of the semiconductor nanocrystals enables
functionalization for
specific uses. For example, because detection of biological compounds is most
preferably
carned out in aqueous media, typically the present invention utilizes
semiconductor
nanocrystals that are solubilized in water. In the case of water-soluble
semiconductor
nanocrystals, the outer layer includes a compound having at least one linking
moiety that
attaches to the surface of the particle and that terminates in at least one
hydrophilic
moiety. The linking and hydrophilic moieties are spanned by a hydrophobic
region
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sufficient to prevent charge transfer across the region. The hydrophobic
region also
provides a "pseudo-hydrophobic" environment for the nanocrystal and thereby
shields it
from aqueous surroundings. The hydrophilic moiety can be a polar or charged
(positive
or negative) group. The polarity or charge of the group provides the necessary
hydrophilic interactions with water to provide stable solutions or suspensions
of the
semiconductor nanocrystal. Exemplary hydrophilic groups include polar groups
such as
hydroxides (-OH), amines, polyethers, such as polyethylene glycol and the
like, as well as
charged groups, such as carboxylates (-COZ ), sulfonates (S03'), phosphates (-
PO4z- and -
P032-), nitrates, ammonium salts (-NHø~), and the like. A water-solubilizing
layer is
found at the outer surface of the overcoating layer. Methods for rendering
semiconductor
nanocrystals water-soluble are known in the art and described in, e.g., PCT
Publication
No. WO 00/17655, published March 30, 2000.
The affinity for the nanocrystal surface promotes coordination of the
linking moiety to the semiconductor nanocrystal outer surface and the moiety
with
affinity for the aqueous medium stabilizes the semiconductor nanocrystal
suspension.
A displacement reaction can be employed to modify the semiconductor
nanocrystal to improve the solubility in a particular organic solvent. For
example, if it is
desired to associate the semiconductor nanocrystals with a particular solvent
or liquid,
such as pyridine, the surface can be specifically modified with pyridine or
pyridine-like
moieties to ensure solvation.
The surface layer can also be modified by displacement to render the
semiconductor nanocrystal reactive for a particular coupling reaction. For
example,
displacement of TOPO moieties with a group containing a carboxylic acid moiety
enables
the reaction of the modified semiconductor nanocrystals with amine containing
moieties
(commonly found on solid support units) to provide an amide linkage.
Additional
modifications can also be made such that the semiconductor nanocrystal can be
associated
with almost any solid support such as those describe supra.
For example, the semiconductor nanocrystals of the present invention can
readily be functionalized to create styrene or acrylate moieties, thus
enabling the
incorporation of the semiconductor nanocrystals onto polystyrene, polyacrylate
or other
polymers such as polyimide, polyacrylamide, polyethylene, polyvinyl,
polydiacetylene,
polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole,
polyimidazole, polythiophene, polyether, epoxies, silica glass, silica gel,
siloxane,
polyphosphate, hydrogel, agarose, cellulose, and the like.
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For a detailed description of these linking reactions, see, e.g., U.S. Patent
No. 5,990,479; Bruchez et. al. (1998) Science 281:2013-2016., Chan et. al.
(1998)
Science 281:2016-2018, Bruchez "Luminescent Semiconductor Nanocrystals:
Intermittent Behavior and use as Fluorescent Biological Probes" (1998)
Doctoral
dissertation, University of California, Berkeley, and Mikulec "Semiconductor
Nanocrystal Colloids: Manganese Doped Cadmium Selenide, (Core)Shell Composites
for Biological Labeling, and Highly Fluorescent Cadmium Telluride" (1999)
Doctoral
dissertation, Massachusetts Institute of Technology.
The following examples are provided to illustrate certain aspects of the
invention and are not to be interpreted so as to limit the scope of the
invention.
EXAMPLE 1
Preparation of Detectable Probes
For cDNA Array Labeling
cDNA microarray slides were prepared as described in the Fabrication
section of www.nh r~h.~ov/DIR/microarrax. Further guidance on fabrication,
sample
labeling and conditions for hybridization using microarrays is provided, for
example, by
Bittner M., et al. (2000) Nature 406:536-540; Khan J., et al. (1999)
Electrophoresis
20:223-9; Duggan, D.J. (1999) Science 283:83-87; and DeRisi, J. et al. (1996)
14:457-60.
A. cDNA labeled with biotin.
The preparation of RNA, sample labeling by reverse transcription and
hybridization were performed by using method as described in Khan et al.
(1999)
Biochim. Biophys. Acta 1423:17-28 or from www.nhgri.nih.gov/DIR/microarray or
other
published methods. The cDNA was labeled with biotin. For labeling with
semiconductor
nanocrystals (SCNC)-streptavidin conjugates, Cy~3 dUTP was replaced with
biotin-16-
dUTP (Roche Molecular Biochemicals, Indianapolis, III.
After hybridization, the slide was incubated in 4X SSC, 0.1% Tween~ 20,
1 % bovine serum albumin (BSA) at room temperature for a minimal of 30 minutes
and
rinsed in 1X phosphate-buffered saline (PBS), 1% BSA, 1 mM MgCl2. The slide
was
incubated in 25 nM 630 nm SCNC-streptavidin in 1X PBS, 1% BSA, 10 mM MgCl2 for
1
hour at room temperature. The slide was rinsed in 1X PBS, 1% BSA, 1 mM MgCl2
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followed by IO mM Phosphate buffer pH 7.4 and spin-dried in centrifuge at 500
rpm for 5
minutes.
The spots on the microarray were viewed under a fluorescence microscope
and scanner optimized for SCNCs.
B. cDNA labeled with biotin and Cy~3 or C.~.
The preparation of RNA, sample labeling by reverse transcription and
hybridization were performed using methods described in Khan et al. (1999)
Biochifn.
Biophys. Acta 1423:17-28 or from www.nhgri.nih.gov/DIR/microarray. One cDNA
was
labeled with biotin and the other with Cy3 or CyS. For labeling with
semiconductor
nanocrystal (SCNC)-streptavidin, Cy5- or Cy3-dUTP was replaced with biotin-16-
dUTP
(Roche Molecular Biochemicals, Indianapolis, IN'.
After the hybridization step, the microarray slide was incubated in 4X
SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes at room temperature, rinsed in
0.06X
1 S SSC, and spun dry at 500 rpm for 5 minutes in a centrifuge with a
horizontal rotor for
microplates. SCNC-streptavidin were added to 6X SSPE, 1% BSA, 10 mM MgCl2 to a
final concentration of 25 nM. Depending on the size of the array, 40-80 ~L of
the SCNC-
streptavidin was applied on the array area, a coverslip was added and the
covered
microarray was incubated in a humidified container for 1 hour at room
temperature. The
slide was rinsed in 1X SSPE followed by 0.06X SSPE and spin-dried in
centrifuge at 500
rpm for 5 minutes.
The microarray was viewed on a fluorescence microscope. SCNC-labeled
cDNA hybridized to the microarray was easily detected under fluorescence
microscopy.
C. The preparation of RNA, sample labeling by reverse transcription and
hybridization are performed by using method as described in Khan et al. (1999)
Biochim.
Bioplays. Acta 1423:17-28 or from web site www.nhgri.nih.gov/DIR/rnicroarray,
or by
any suitable method known to those skilled in the art. One cDNA is labeled
with biotin
and the other with a hapten (e.g., fluorescein, digoxygenin, or estradiol).
For labeling
with SCNC-streptavidin, and SCNC-anti-hapten, Cy5- and Cy3-dUTP is replaced
with
biotin-16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) and dUTP-
hapten
(e.g., fluorescein-12-dUTP, DIG 11-dUTP, estradiol-15-dUTP).
After hybridization, the slide is incubated in blocking solution 4X SSC,
0.1% Tween 20,1% BSA for 30-60 minutes at room temperature, rinsed in 0.06X
SSC,
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and dried by spinning at 500 rpm for 5 minutes in a centrifuge with a
horizontal rotor for
microplates. SCNC-streptavidin and SCNC-anti-hapten are added to 6X SSPE, 1%
BSA,
mM MgCl2 to a final concentration of 25 nM each. Depending on the size of the
array, 40-80 ~L of the mixture is applied on the array area, a coverslip is
added over the
array area and the covered array is incubated in a humidified container for 1
hour at room
temperature. The slide is rinsed in 1X SSPE followed by 0.06X SSPE and dried
by
spinning in centrifuge at 500 rpm for 5 minutes. The microarray is read on a
scanner
optimized for SCNC emission.
10 D. cDNA labeled with SCNC1-dUTP and SCNC2-dUTP
The preparation of RNA, sample labeling by reverse transcription and
hybridization are performed by using method as described in Khan et al. (1999)
BiocIZim.
Biophys. Acta 1423:17-28 or from web site www.nhgri.nih.gov/DIR/microarray or
by
other methods well known in the art. One cDNA is labeled with a first SCNC
(SCNC 1)
have a first emission spectrum and the other with a second SCNC (SCNC2) having
an
emission spectrum distinct from SCNC1. For labeling with SCNC1 and SCNC2, Cy5-
or
Cy3-dUTP is replaced with SCNCl-dUTP and SCNC2-dUTP, respectively. After
hybridization and washes, the dried slide is read on a scamler optimized for
SCNCs.
It is anticipated that each distinctly labeled cDNA will be easily
distinguished.
EXAMPLE 2
cDNA for oligonucleotide microarray
Oligonucleotide microarray chips can purchased from Operon
Technologies, Inc. or from other sources.
A. cDNA labeled with biotin and Cy~3 or C
The preparation of RNA and sample labeling by reverse transcription are
performed using method described in Khan et al. (1999) Biochim. Biophys. Acta
1423:17-
28, from web site www.nh ri.~rn'h.~ov/DIR/microarraX, or using methods known
to those
of skill in the art. Alternatively, preparation of cDNA and hybridization can
be
performed using protocols described in web site
www.pangloss.com/seidel/Protocols.
One cDNA is labeled with biotin and the other with Cy3 or CyS. For labeling
with
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SCNC-streptavidin, Cy5- or Cy3-dUTP is replaced with biotin-16-dUTP (Roche
Molecular Biochemicals, Indianapolis, IN).
The hybridization buffer contains the labeled cDNAs in 4X SSC and 1
mg/ml poly(dA) (Pharmacia) and 0.2 mg /ml yeast tRNA (Sigma). The probe
mixture is
denatured at 98 °C for 2 minutes, cooled to 45 °C and a small
volume of 10% SDS
solution is added to a final concentration of 0.2% SDS. The volume of 15-30
~,L
depending on the size of the array is applied onto microarray area and the
microarray area
is covered with a glass cover-slip. The covered microarray is placed in a
humidified
chamber and incubated overnight at 65 °C. After hybridization, the
slide is sequentially
rinsed in 1X SSC with 0.03 % SDS, 0.2X SSC and O.OSX SSC. The slide is dried
by
spinning in a centrifuge with horizontal rotor at 500 rpm for 5 minutes. The
slide is
incubated in blocking solution 4X SSC, 0.1% Tween 20, 1% BSA for 30-60 minutes
at
room temperature, rinsed in O.OSX SSC, and dried by spinning in centrifuge.
SCNC-
streptavidin is added to 6X SSPE, 1% BSA, 10 mM MgCl2 to a final concentration
of 25
nM. Depending on the size of the array, 40-80 ~L of the SCNC-streptavidin is
applied on
the array area, a coverslip is applied over the array area and the cover array
is incubated
in a humidified container for 1 hour at room temperature. The slide is rinsed
in 1X SSPE
followed by 0.06X SSPE and dried by spiiming in centrifuge at 500 rpm for 5
minutes.
The microarray is read in a scanner optimized for SCNCs and Cy dyes.
B. cDNA labeled with biotin and hapten
The preparation of RNA and sample labeling by reverse transcription are
performed using method described in Khan et al. (1999) Biochi~z. Bioplays.
Acta 1423:17-
28 or from web site www.nh ri.nih.~ov/DIR/microarray. Alternatively,
hybridization can
be performed using protocols described in web site
www.pan~loss.comlseidel/Protocols.
One cDNA is labeled with biotin and the other with a hapten such as
digoxygeiun,
fluorescein, estradiol. For labeling with SCNC-streptavidin and SCNC-anti-
hapten, Cy5-
or Cy3-dUTP is replaced with biotin-16-dUTP (Roche Molecular Biochemicals,
Indianapolis, Il~ and hapten-dUTP (e.g., fluorescein-12-dUTP, DIG 11-dUTP,
estradiol-
15-dUTP).
The hybridization buffer contains the labeled cDNAs in 4X SSC and 1
mg/ml poly(dA) (Pharmacia), 0.2 mg /ml yeast tRNA (Sigma), the probe mixture
is
denatured at 98 °C for 2 minutes, cool to 45 °C and a small
volume of 10% SDS solution
is added to a final concentration of 0.2% SDS. The volume 15-30 ~,L depending
on the
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size of the array is applied on microarray, a cover slip is placed over the
array area, and
the covered array is placed in a humidified chamber and incubated overnight at
65 °C.
After hybridization, the slide is sequentially rinsed in 1X SSC with 0.03 %
SDS, 0.2X
SSC and O.OSX SSC. The slide is dried by spinning in centrifuge with
horizontal rotor for
microplates at 500 rpm for 5 minutes.
The slide is incubated in blocking solution 4X SSC, 0.1% Tween 20, 1%
BSA for 30-60 minutes at room temperature, rinsed in O.OSX SSC, and dried by
spinning
in centrifuge. SCNC-streptavidin and SCNC-anti-hapten is added to 6X SSPE, 1%
BSA,
mM MgCl2 to a final concentration of 25 nM each. Depending on the size of the
10 array, 40-80 p.L of the mixture is applied on the array area, a cover slip
is applied over the
array area and the covered array is incubated in a humidified container for 1
hour at room
temperature. The slide is rinsed in 1X SSPE followed by 0.06X SSPE and dried
by
spinning in centrifuge at 500 rpm for 5 minutes.
The microarray is read on a scanner optimized for SCNCs.
C. cDNA labeled with SCNCl-dUTP and SCNC2-dUTP
The preparation of RNA and sample labeling by reverse transcription are
performed by using method as described in Khan et al. (1999) Biochim. Biophys.
Acta
1423:17-28 or from web site www.nh rig~nih.~ov/DIR/microarray. Alternatively,
hybridization can be performed using protocols described in web site
www.pan~loss.com/seidel/Protocols. One cDNA is labeled with SCNC1 and the
SCNC2.
For labeling cDNA with SCNC1 and SCNC2, Cy5- or Cy3-dUTP is replaced with
SCNC1-dUTP and SCNC2-dUTP.
The hybridization buffer contains the labeled cDNAs in 4X SSC and 1
mg/m1 poly(dA) (Pharmacia), yeast tRNA 0.2 mg /ml, the probe mixture is
denatured at
98 °C for 2 minutes, cooled to 45 °C and a small volume of 10%
SDS solution is added to
a final concentration of 0.2% SDS. The volume of hybridization mixes various
from 15-
~,L depending on the size of the array. The mixture is applied on microarray,
a cover
slip is applied over the array area, the covered microarray is placed in a
humidified
30 chamber and incubated overnight at 65 °C. After hybridization, the
slide is sequentially
rinsed in 1X SSC with 0.03% SDS, 0.2X SSC and O.OSX SSC. The slide is dried by
spinning in centrifuge with horizontal rotor for microplates at 500 rpm for 5
minutes.
The microarray is scanned on a scanner optimized for SCNCs.
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D. Oligonucleotide microarrays were purchased from Operon Technologies;
Inc. These test arrays contained forty spots each, ten spots from each of the
four 70 mers
selected from Caspase 9-Genbank U56390, Laminin gamma 3 chain precusor, LAMC3-
Genbank AF041835, Alpha-tubulin-Genbank K00558 and Ribosomal protein S9-
Genbank U14971. 50-mer complementary oligonucleotides biotinylated at the 3'
end
were made from each of the four 70 mers.
The hybridization buffer contained the biotin-labeled 50 mer
complementary sequences, 4 mg/ml herring sperm DNA as carrier in 4X SSC and 1
mg/ml poly(dA) (Phannacia), 0.2 mg /ml yeast tRNA (Sigma). The probe mixture
was
denatured at 98 °C for 2 minutes, cooled to 45 °C and a small
volume of 10% SDS
solution is added to a final concentration of 0.2% SDS. The volume of 15-30 ~L
depending on the size of the array was applied on the microarray area and a
cover slip
was applied over the microarray area. The covered microarray slide was placed
in a
humidified chamber and incubated overnight at 65 °C. After
hybridization, the slide was
sequentially rinsed in 1X SSC with 0.03% SDS, 0.2X SSC and O.OSX SSC. The
slide
was dried by spinning in centrifuge with horizontal rotor at 500 rpm for 5
minutes. The
slide was incubated in blocking solution 4X SSC, 0.1% Tween 20, 1% BSA for 30-
60
minutes at room temperature, rinsed in O.OSX SSC, and dried by spinning in
centrifuge.
SCNC-streptavidin is added to 6X SSPE, 1% BSA, 10 mM MgCl2 to a final
concentration
of 25 nM. Depending on the size of the array, 40-80 ~L of the SCNC-
streptavidin was
applied on the array area and the array area was covered with a glass
coverslip. The
cover array slide was incubated in a humidified container for 1 hour at room
temperature.
The slide was rinsed in 1X SSPE followed by 0.06X SSPE and dried by spnnung in
centrifuge at 500 rpm for 5 minutes.
The microarray was scanned in a scanner optimized for SCNCs.
EXAMPLE 3
Protein Array: Detection Using Semiconductor Nanocrystals
A protein array was prepared to interrogate protein species on a spatially
addressed array. Protein generated from any source (e.g., a recombinant
expression
system, a differentially treated cell supernatants, or the like) can be
immobilized in a
small, spatially addressed spot on a substrate. The spot size can vary from
micrometer to
millimeter diameter dependent on the assay substrate.
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In this Example, 1 ~,L of rabbit IgG or mouse IgG was spotted onto
nitrocellulose and allowed to dry; 50 spots of each different IgG dilution
were addressed
in a 5 x 10 array. The nitrocellulose was then blocked by incubation in
phosphate-
buffered saline (PBS)/1% bovine serum albumin (BSA) for 30 minutes at room
temperature. A 1 ~g/ml solution of biotinylated anti-rabbit IgG (Vector) was
then applied
for 30 minutes and the membrane subsequently washed in excess PBS. The array
was
then exposed to 5 mls of a 25 nM solution of streptavidin-conjugated 630 nm
emitting
semiconductor nanocrystals in PBS/1% BSA for 30 minutes at room temperature.
The
membrane was then washed in excess PBS and the luminescence from the
semiconductor
nanocrystals was detected using an ultraviolet transilluminator (Stratagene
Eagle Eye~)
and a microarray scanner set to excite at 488 nm with an argon ion laser.
Less than 1 ng of antibody was specifically detected using each detection
device.
EXAMPLE 4
Tissue Array: Detection Using Semiconductor Nanocrystals
Multiple intracellular markers can be simultaneously analyzed with
semiconductor nanocrystal-labeled ligands. This, coupled with a spatial
arraying of tissue
samples in a defined area, allows a further increase in the throughput of
analyzing cellular
markers. Small sections of tissue can be immobilized on a microscope slides or
some
other support as is well known in the art. The tissue source can be derived
from a living
organism, from a population of cultured cells treated in various ways, or the
like.
In this Example, a specific intracellular antigen has been detected on a
tissue section attached to a microscope slide. The tissue section was mouse
stomach and
kidney and was purchased from InovaDX (Sam Diego, CA). The goal of this
Example
was to detect the presence or absence of auto-immune markers, antinuclear
antibodies
(ANA), that recognize nuclear antigens. The anti-nuclear antibodies can be
specifically
detected using a biotinylated anti-human antibody followed by a binding
thereto of a
streptavidin-conjugated semiconductor nanocrystal.
The tissue section was incubated for one hour with a positive control
containing ANA (InovaDX) or with a human serum sample diluted in phosphate-
buffered
saline (PBS)/1% bovine serum albumin (BSA). A negative control sample was also
provided by InovaDX and this is also incubated with a section to provide
background or
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non-specific binding information. The section was then washed by repeated
immersion in
PBS. The section was then incubated with 3 ~g/ml biotinylated anti-human
antibody
(Vector) for a further 30 minutes at room temperature. The slide was washed in
PBS.
Finally, the section was incubated with streptavidin-conjugated semiconductor
nanocrystals (both 525 nm and 630 nm emitting nanocrystals have been used) for
30
minutes at room temperature and finally washed in PBS. A solution of 50%
glycerol in
PBS was used to mount a coverslip and the section is examined under an epi-
fluorescent
microscope.
The nuclei were clearly observed as brightly stained whereas the cytosol
and surrounding tissue were not stained. No nuclei were observed in the
negative control
section.
EXAMPLE 5
Single Target Counting
The goal of these studies was to demonstrate that single analyte targets can
be detected and quantified.
10 ~,glml of rabbit IgG diluted in PBS was passively adsorbed to the
surface of standard glass coverslips. Excess antibody was removed and the
surfaces were
blocked with bovine serum albumin (3% BSA in PBS overnight at 4 °C or 2
hours at
room temperature). Each coverslip was immersed in different concentrations of
biotinylated anti-rabbit IgG (10 nM to 100 fM in PBS/1% BSA). After incubating
for 15
minutes at room temperature, the cover slips were washed in excess PBS and
incubated
with 10 nM streptavidin functionalized semiconductor nanocrystals (580 rim
emission) at
room temperature for 10 minutes. After 30 minutes of washing in PBS/1%BSA/0.1%
Igepal~ at room temperature, samples were imaged with a fluorescence
microscope.
Signals from single bound analyte molecules and the density of molecules
decreased as a
function of analyte concentration.
The results were quantified by courting analyte molecules in a defined
area of the assay surface (a circular region of about 60 ~m in diameter
defined by the
illumination pattern of our single molecule microscope). The results were
linear with
concentration of biotinylated rabbit IgG and the sensitivity extended to
densities of about
0.001 molecules/~,mz.
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It is understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or changes in
light thereof
will be suggested to persons skilled in the art and are to be included within
the spirit and
purview of this application and scope of the appended claims. All
publications, patents
and patent applications cited herein are hereby incorporated by reference in
their entirety
for all purposes to the same extent as if each individual publication, patent
or patent
application were specifically and individually indicated to be so incorporated
by
reference.
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