U.S. patent application number 10/631573 was filed with the patent office on 2004-08-26 for method of detecting an analyte in a sample using semiconductor nanocrystals as a detectable label.
This patent application is currently assigned to Quantum Dot Corporation. Invention is credited to Bruchez, Marcel P., Daniels, R. Hugh, Empedocles, Stephen A., Phillips, Vince E., Wong, Edith Y., Zehnder, Donald A..
Application Number | 20040166505 10/631573 |
Document ID | / |
Family ID | 22456937 |
Filed Date | 2004-08-26 |
United States Patent
Application |
20040166505 |
Kind Code |
A1 |
Bruchez, Marcel P. ; et
al. |
August 26, 2004 |
Method of detecting an analyte in a sample using semiconductor
nanocrystals as a detectable label
Abstract
The use of semiconductor nanocrystals as detectable labels in
various chemical and biological applications is disclosed. The
methods find use for detecting a single analyte, as well as
multiple analytes by using more than one semiconductor nanocrystal
as a detectable label, each of which emits at a distinct
wavelength.
Inventors: |
Bruchez, Marcel P.; (Union
City, CA) ; Daniels, R. Hugh; (Palo Alto, CA)
; Empedocles, Stephen A.; (Mountain View, CA) ;
Phillips, Vince E.; (Sunnyvale, CA) ; Wong, Edith
Y.; (Danville, CA) ; Zehnder, Donald A.; (San
Carlos, CA) |
Correspondence
Address: |
ROBINS & PASTERNAK
1731 EMBARCADERO ROAD
SUITE 230
PALO ALTO
CA
94303
US
|
Assignee: |
Quantum Dot Corporation
|
Family ID: |
22456937 |
Appl. No.: |
10/631573 |
Filed: |
July 30, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10631573 |
Jul 30, 2003 |
|
|
|
09887914 |
Jun 21, 2001 |
|
|
|
6630307 |
|
|
|
|
09887914 |
Jun 21, 2001 |
|
|
|
09566014 |
May 5, 2000 |
|
|
|
6274323 |
|
|
|
|
60133084 |
May 7, 1999 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
G01N 33/54346 20130101;
G01N 2458/00 20130101; B82Y 15/00 20130101; G01N 33/588 20130101;
Y10S 977/924 20130101; G01N 33/533 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. A method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of: (a) providing the sample on a solid
support; (b) combining said sample with a semiconductor nanocrystal
conjugate, wherein said combining is performed under conditions
that allow formation of a complex comprising said conjugate and
said analyte, when present; (c) removing any unbound conjugate; (d)
detecting the presence of the complex, if present, by monitoring a
spectral emission mediated by the semiconductor nanocrystal in the
complex, wherein the emission indicates the presence of one or more
target analytes in the sample.
2. The method of claim 1, wherein there is a plurality of target
analytes and the method further comprises: (a) providing a
conjugate specific for each target analyte, wherein each
semiconductor nanocrystal conjugate has an emission spectrum
distinct from the other semiconductor nanocrystal conjugates; and
(b) detecting the presence of the target analytes by monitoring the
spectral emissions of the sample, wherein the emissions indicate
the presence of the target analytes in the sample.
3. The method of claim 1, wherein the one or more analytes is a
nucleic acid molecule.
4. The method of claim 3, wherein the nucleic acid molecule is
contained within a chromosome or chromosomal fragment.
5. The method of claim 2, wherein the one or more analytes is a
nucleic acid molecule.
6. The method of claim 5, wherein the nucleic acid molecule is
contained within a chromosome or chromosomal fragment.
7. The method of claim 6, wherein the target analytes are present
on more than one chromosome or chromosomal fragments.
8. The method of claim 7, wherein each of the target analytes is
different from the others.
9. The method of claim 6, wherein the target analytes are present
on a single chromosome.
10. The method of claim 9, wherein each of the target analytes is
different from the others.
11. The method of claim 3, wherein the nucleic acid molecule is a
DNA molecule.
12. The method of claim 3, wherein the nucleic acid molecule is a
RNA molecule.
13. The method of claim 3, wherein the conjugate comprises at least
one polymerase chain reaction primer.
14. The method of claim 1, wherein the one or more analytes is a
polypeptide.
15. The method of claim 14, wherein the semiconductor nanocrystal
conjugate comprises an antibody.
16. The method of claim 1, wherein the semiconductor nanocrystal
conjugate comprises an aptamer.
17. A method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of: (a) providing an unlabeled
specific-binding molecule on a solid support; (b) combining said
sample with said specific-binding molecule, wherein said combining
is performed under conditions that allow formation of a first
complex comprising said specific-binding molecule and said analyte,
when present; (c) removing any unbound sample; (d) combining said
first complex with a semiconductor nanocrystal conjugate, wherein
said combining is performed under conditions that allow formation
of a second complex comprising said conjugate and said analyte,
when present; (e) removing any unbound conjugate; (f) detecting the
presence of the second complex, if present, by monitoring a
spectral emission mediated by the semiconductor nanocrystal in the
second complex, wherein the emission indicates the presence of one
or more target analytes in the sample.
18. The method of claim 17, wherein there is a plurality of target
analytes and the method further comprises: (a) providing an
unlabeled specific-binding molecule on a solid support specific for
each target analyte and providing a semiconductor nanocrystal
conjugate specific for each target analyte, wherein each
semiconductor nanocrystal conjugate has an emission spectrum
distinct from the other semiconductor nanocrystal conjugates; and
(b) detecting the presence of the target analytes by monitoring the
spectral emissions of the sample, wherein the emissions indicate
the presence of the target analytes in the sample.
19. The method of claim 17, wherein the one or more analytes is a
nucleic acid molecule.
20. The method of claim 19, wherein the nucleic acid molecule is a
DNA molecule.
21. The method of claim 19, wherein the nucleic acid molecule is a
RNA molecule.
22. The method of claim 17, wherein the one or more analytes is a
polypeptide.
23. The method of claim 17, wherein the semiconductor nanocrystal
conjugate comprises an antibody.
24. The method of claim 17, wherein the semiconductor nanocrystal
conjugate comprises an aptamer.
25. A method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of: (a) providing the sample on a solid
support; (b) combining with said sample a specific-binding
molecule, wherein (i) said specific-binding molecule comprises a
first member of a binding pair, and (ii) said combining is
performed under conditions that allow formation of a first complex
comprising said specific-binding molecule and said analyte, when
present; (c) removing any unbound specific-binding molecule; (d)
combining said first complex with a second member of the binding
pair, wherein (i) said second member of the binding pair is linked
to a first semiconductor nanocrystal; and (ii) said combining is
performed under conditions that allow formation of a second complex
comprising the binding pair and said one or more analytes; and (e)
detecting the presence of the second complex, if present, by
monitoring a spectral emission mediated by the first semiconductor
nanocrystal in the second complex, wherein the emission indicates
the presence of one or more target analytes in the sample.
26. The method of claim 25, wherein the one or more analytes is a
nucleic acid molecule.
27. The method of claim 26, wherein the nucleic acid molecule is
contained within a chromosome or chromosomal fragment.
28. The method of claim 26, wherein the nucleic acid molecule is a
DNA molecule.
29. The method of claim 26, wherein the nucleic acid molecule is a
RNA molecule.
30. The method of claim 25, wherein the one or more analytes is a
polynucleotide.
31. The method of claim 30, wherein the specific-binding molecule
is a polymerase chain reaction amplification product and said first
member of the binding pair is incorporated in the amplification
product.
32. The method of claim 26, wherein the specific-binding molecule
is an aptamer.
33. The method of claim 25, wherein the one or more analytes is a
polypeptide.
34. The method of claim 33, wherein the first member of the binding
pair is a first antibody and the second member of the binding pair
is a second antibody reactive with the first antibody.
35. The method of claim 25, wherein the first member of the binding
pair is biotin and the second member of the binding pair is
streptavidin.
36. The method of claim 33, wherein the first member of the binding
pair is biotin and the second member of the binding pair is
streptavidin.
37. The method of claim 25, wherein the first member of the binding
pair is digoxygenin and the second member of the binding pair is an
antibody directed against digoxygenin.
38. The method of claim 25, wherein the first member of the binding
pair is flourescein and the second member of the binding pair is an
antibody directed against flourescein.
39. The method of claim 25, wherein there is more than one analyte,
wherein the method further comprises: combining with said sample a
second specific-binding molecule, wherein (i) said second
specific-binding molecule comprises a first member of a second
binding pair, and (ii) said combining is performed under conditions
that allow formation of a third complex comprising said second
specific-binding molecule and said analyte, when present; removing
any unbound second specific-binding molecule; combining said third
complex with a second member of the second binding pair, wherein
(i) said second member of the second binding pair is linked to a
second semiconductor nanocrystal that has an emission spectrum
distinct from the first semiconductor nanocrystal; and (ii) said
combining is performed under conditions that allow formation of a
fourth complex comprising the second binding pair and an analyte;
and detecting the presence of the fourth complex, if present, by
monitoring a second spectral emission mediated by the second
semiconductor nanocrystal in the fourth complex, wherein the second
emission indicates the presence of more than one target analyte in
the sample.
40. A method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of: (a) providing a first complex comprising
at least one specific-binding molecule to which is bound a
semiconductor nanocrystal conjugate, wherein said semiconductor
nanocrystal has a characteristic spectral emission and wherein said
conjugate specifically binds to said specific-binding molecule; (b)
combining said sample with said first complex, wherein said
combining is performed under conditions that allow formation of a
second complex comprising said specific-binding molecule and said
analyte, when present; (c) detecting the presence of the second
complex, if present, by monitoring the characteristic spectral
emission of the semiconductor nanocrystal, wherein a change in the
characteristic spectral emission indicates the presence of one or
more target analytes in the sample.
41. The method of claim 40, wherein a plurality of first complexes
are provided each comprising a different specific-binding molecule
each bound to a conjugate which specifically binds each
specific-binding molecule and where each specific-binding molecule
binds a different analyte, and wherein each conjugate bound to a
different specific-binding molecule comprises a semiconductor
nanocrystal that has a characteristic spectral emission distinct
from the other semiconductor nanocrystals; and wherein changes in
the spectral emission of any selected semiconductor nanocrystal
associated with a particular specific-binding molecule in a first
complex, indicates the presence of an analyte that binds to the
particular specific-binding molecule.
42. The method of claim 40, wherein said specific-binding molecule
is radiolabeled, wherein when said conjugate is bound to said
specific-binding molecule the semiconductor nanocrystal emits
light.
43. The method of claim 42, wherein a plurality of first complexes
are provided each comprising a different specific-binding molecule
each bound to a conjugate which specifically binds each
specific-binding molecule and where each specific-binding molecule
binds a different analyte, and wherein each conjugate bound to a
different specific-binding molecule comprises a semiconductor
nanocrystal that has a characteristic spectral emission distinct
from the other semiconductor nanocrystals; and wherein changes in
the spectral emission of any selected semiconductor nanocrystal
associated with a particular specific-binding molecule in a first
complex, indicates the presence of an analyte that binds to the
particular specific-binding molecule.
44. The method of claim 40, wherein said specific-binding molecules
are selected from the group consisting of a protein, an
oligonucleotide, a polysaccharide or a small molecule.
45. The method of claim 44, wherein said specific-binding molecules
are receptor molecules and a conjugate is provided that binds to
each receptor.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to provisional patent
application serial No. 60/133,084, filed May 7, 1999, from which
priority is claimed under 35 USC .sctn.119(e)(1) and which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the detection of
analytes in a sample. In particular, the invention relates to
assays that use semiconductor nanocrystals as a detectable label.
The invention further relates to assays in which multiple analytes
can be detected simultaneously by using more than one semiconductor
nanocrystal as a detectable label, each of which emits at a
distinct wavelength.
BACKGROUND OF THE INVENTION
[0003] A variety of chemical and biological assays exist to
identify an analyte of interest in a given sample. For example,
immunassays, such as enzyme-linked immunosorbent assays (ELISAs)
are used in numerous diagnostic, research and screening
applications. In its most common form, an ELISA detects the
presence and/or concentration of an analyte in a sample using an
antibody which specifically recognizes the analyte. An enzyme
label, capable of providing a detectable signal, is conjugated to
the antibody. The analyte is either immobilized directly onto a
solid support (direct-capture ELISA) or is bound to a different
specific antibody which itself is immobilized on a solid support.
The presence of the immobilized analyte is detected by binding to
it the detectably labeled antibody. A variety of different ELISA
formats have been described. See, e.g., U.S. Pat. No. 4,011,308 to
Giaever, U.S. Pat. No. 4,722,890 to Sanders et al., U.S. Pat. No.
Re. 032696 to Schuurs et al., U.S. Pat. No. 4,016,043 to Schuurs et
al., U.S. Pat. No. 3,876,504 to Koffler, U.S. Pat. No. 3,770,380 to
Smith, and U.S. Pat. No. 4,372,745 to Mandle et al.
[0004] Another technique for detecting biological compounds is
fluorescence in situ hybridization (FISH). Swiger et al. (1996)
Environ. Mol. Mutagen. 27:245-254; Raap (1998) Mut. Res.
400:287-298; Nath et al. (1997) Biotechnic. Histol. 73:6-22. FISH
allows detection of a predetermined target oligonucleotide, e.g.,
DNA or RNA, within a cellular or tissue preparation by, for
example, microscopic visualization. Thus, FISH is an important tool
in the fields of, for example, molecular cytogenetics, pathology
and immunology in both clinical and research laboratories.
[0005] This method involves the fluorescent tagging of an
oligonucleotide probe to detect a specific complementary DNA or RNA
sequence. Specifically, FISH involves incubating an oligonucleotide
probe comprising an oligonucleotide that is complementary to at
least a portion of the target oligonucleotide with a cellular or
tissue preparation containing or suspected of containing the target
oligonucleotide. A detectable label, e.g., a fluorescent dye
molecule, is bound to the oligonucleotide probe. A fluorescence
signal generated at the site of hybridization is typically
visualized using an epi fluorescence microscope. An alternative
approach is to use an oligonucleotide probe conjugated with an
antigen such as biotin or digoxygenin and a fluorescently tagged
antibody directed toward that antigen to visualize the
hybridization of the probe to its DNA target. A variety of FISH
formats are known in the art. See, e.g., Dewald et al. (1993) Bone
Marrow Transplantation 12:149-154; Ward et al. (1993) Am. J. Hum.
Genet. 52:854-865; Jalal et al. (1998) Mayo Clin. Proc. 73:132-137;
Zahed et al. (1992) Prenat. Diagn. 12:483-493; Kitadai et al.
(1995) Clin. Cancer Res. 1:1095-1102; Neuhaus et al. (1999) Human
Pathol. 30:81-86; Hack et al., eds., (1980) Association of
Cytogenetic Technologists Cytogenetics Laboratory Manual.
(Association of Cytogenetic Technologists, San Francisco, Calif.);
Buno et al. (1998) Blood 92:2315-2321; Patterson et al. (1993)
Science 260:976-979; Patterson et al. (1998) Cytometry 31:265-274;
Borzi et al. (1996) J. Immunol. Meth. 193:167-176; Wachtel et al.
(1998) Prenat. Diagn. 18:455-463; Bianchi (1998) J. Perinat. Med.
26:175-185; and Munne (1998) Mol. Hum. Reprod. 4:863-870.
[0006] FISH provides a powerful tool for the chromosomal
localization of genes whose sequences are partially or fully known.
Other applications of FISH include in situ localization of mRNA in
tissues sample and localization of nongenetic DNA sequences such as
telomeres.
[0007] Signal amplification is yet another method for sensitive
detection of nucleic acids and other receptor/ligand interactions.
Direct detection of a target nucleic acid is possible by
hybridization of a complementary nucleic acid probe to the target.
Detection of the complex can be achieved by numerous means, e.g., a
labeled probe or a reagent dye that specifically attaches to the
target/probe complex. Such "direct" detection systems are often not
sensitive enough to detect a target nucleic acid in a biological
sample. One method for overcoming this limitation is to employ
signal amplification. Signal amplification can be done, for
example, by indirectly binding multiple signal-generating molecules
to an analyte through a molecule which is (1) complementary to the
analyte and (2) contains multiple signal-generating molecule
binding sites and which signal-generating molecules (i) contain a
detectable label, (ii) bind to or otherwise activate a label or
(iii) contain sites for binding additional layers of molecules
which may in turn facilitate generation of a detectable signal.
Thus, rather than a single signal-generating label associated with
the target molecule, signal amplification results in the
association of multiple signal-generating labels associated with
the target molecule and, therefore, enhanced assay sensitivity.
[0008] Nucleic acid hybridization assays are described in, for
example, U.S. Pat. No. 5,681,697 to Urdea et al., U.S. Pat. No.
5,124,246 to Urdea et al., U.S. Pat. No. 4,868,105 to Urdea et al.,
and European Patent Publication No. 70.685, inventors Heller et
al.
[0009] There are many assays designed to obtain the sequence of a
DNA sample. Each of these methods shares some or all of a set of
common features. These features include: sequence specificity
derived from complementary oligonucleotide hybridization or
annealing; a solid support or solid phase which allows separation
of specifically bound assay reagents; and a label which is used for
detecting the presence or absence of the specific, intended assay
interaction. Examples of assays designed to detect the sequence of
a DNA sample can be found in U.S. Pat. No. 5,888,731 to Yager et
al., U.S. Pat. No. 5,830,711 to Barany et al., U.S. Pat. No.
5,800,994 to Martinelli et al., U.S. Pat. No. 5,792,607 to Backman
et al., U.S. Pat. No. 5,716,784 to Di Cesare, U.S. Pat. No.
5,578,458 to Caskey et al., U.S. Pat. No. 5,494,810 to Barany et
al., U.S. Pat. No. 4,925,785 to Wang et al., U.S. Pat. No.
4,9898,617 to Landegren et al.
[0010] Chemical compounds are typically evaluated for potential
therapeutic utility by assaying their ability to affect, for
example, enzyme activity, ligand-receptor interactions,
protein-protein interactions, or the like. Evaluating the effect of
each individual candidate compound on a variety of systems can be
tedious and time-consuming. Accordingly, protocols have been
developed to evaluate rapidly multiple candidate compounds in a
particular system and/or a candidate compound in a plurality of
systems. Such protocols for evaluating candidate compounds have
been referred to as high throughput screening (HTS).
[0011] In one typical protocol, HTS involves the dispersal of a
candidate compound into a well of a multiwell cluster plate, for
example, a 96-well or higher format plate, e.g., a 384-, 864-, or
1536-well plate. The effect of the compound is evaluated on the
system in which it is being tested. The "throughput" of this
technique, i.e., the combination of the number of candidate
compounds that can be screened and the number of systems against
which candidate compounds can be screened, is limited by a number
of factors, including, but not limited to: only one assay can be
performed per well; if conventional dye molecules are used to
monitor the effect of the candidate compound, multiple excitation
sources are required if multiple dye molecules are used; and as the
well size becomes small (e.g., the 1536-well plate can accept about
5 .mu.l of total assay volume), consistent dispensing of individual
components into a well is difficult and the amount of signal
generated by each assay is significantly decreased, scaling with
the volume of the assay.
[0012] A number of assay formats can be used for HTS assays. For
example, the inhibitory effect of the candidate compound on, e.g.,
enzyme activity, ligand-receptor binding, and the like, can be
measured by comparing the endpoint of the assay in the presence of
a known concentration of the candidate to a reference which is
performed in the absence of the candidate and/or in the presence of
a known inhibitor compound. Thus, for example, a candidate compound
can be identified which inhibits the binding of a ligand and its
receptor, or which inhibits enzyme activity, decreasing the
turnover of the enzymatic process. When this process (the inhibited
process) is of clinical significance, the candidates are identified
to be potential drugs for a particular condition.
[0013] A 1536-well plate is merely the physical segregation of
sixteen assays within a single 96 well plate format. It would be
advantageous to multiplex 16 assays into a single well of the 96
well plate. This would result in greater ease of dispensing
reagents into the wells and in high signal output per well. In
addition, performing multiple assays in a single well allows
simultaneous determination of the potential of a candidate compound
to affect a plurality of target systems. Using HTS strategies, a
single candidate compound can be screened for activity as, e.g., a
protease inhibitor, an inflammation inhibitor, an antiasthmatic,
and the like, in a single assay.
[0014] Each of the above-described assay formats utilizes
detectable labels to identify the analyte of interest. Radiolabeled
molecules and compounds are frequently used to detect biological
compounds both in vivo and in vitro. However, due to the inherent
problems associated with the use of radioactive isotopes,
nonradioactive methods of detecting biological and chemical
compounds are often preferable.
[0015] For example, fluorescent molecules are commonly used as tags
for detecting an analyte of interest. Fluorescence is the emission
of light resulting from the absorption of radiation at one
wavelength (excitation) followed by nearly immediate reradiation
usually at a different wavelength (emission). Organic fluorescent
dyes are typically used in this context. However, there are
chemical and physical limitations to the use of such dyes. One of
these limitations is the variation of excitation wavelengths of
different colored dyes. As a result, the simultaneous use of two or
more fluorescent tags with different excitation wavelengths
requires multiple excitation light sources.
[0016] Another drawback of organic dyes is the deterioration of
fluorescence intensity upon prolonged and/or repeated exposure to
excitation light. This fading, called photobleaching, is dependent
on the intensity of the excitation light and the duration of the
illumination. In addition, conversion of the dye into a
nonfluorescent species is irreversible. Furthermore, the
degradation products of dyes are organic compounds which may
interfere with the biological processes being examined.
[0017] Additionally, spectral overlap exists from one dye to
another. This is due, in part, to the relatively wide emission
spectra of organic dyes and the overlap of the spectra near the
tailing region. Few low molecular weight dyes have a combination of
a large Stokes shift, which is defined as the separation of the
absorption and emission maxima, and high fluorescence output. In
addition, low molecular weight dyes may be impractical for some
applications because they do not provide a bright enough
fluorescent signal.
[0018] Furthermore, the differences in the chemical properties of
standard organic fluorescent dyes make multiple, parallel assays
impractical as different chemical reactions may be involved for
each dye used in the variety of applications of fluorescent
labels.
[0019] Thus, there is a continuing need in the assay art for labels
with the following features: (i) high fluorescent intensity (for
detection in small quantities), (ii) adequate separation between
the absorption and emission frequencies, (iii) good solubility,
(iv) ability to be readily linked to other molecules, (v) stability
towards harsh conditions and high temperatures, (vi) a symmetric,
nearly gaussian emission lineshape for easy deconvolution of
multiple colors, and (vii) compatibility with automated analysis.
At present, none of the conventional fluorescent labels satisfies
all of these requirements.
SUMMARY OF THE INVENTION
[0020] The present invention is based on the discovery that
semiconductor nanocrystals can be used as reliable and sensitive
detectable labels in a variety of biological and chemical formats.
Semiconductor nanocrystals (also know as quantum dot and Qdot.TM.
nanocrystals) can be produced that have characteristic spectral
emissions. These spectral emissions can be tuned to a desired
energy by varying the particle size, size distribution and/or
composition of the particle. A targeting compound that has affinity
for one or more selected biological or chemical targets is
associated with the semiconductor nanocrystal. Thus, the
semiconductor nanocrystal will interact or associate with the
target due to the affinity of the targeting compound for the
target. The location and/or nature of the association can be
determined, for example, by irradiation of the sample with an
energy source, such as an excitation light source. The
semiconductor nanocrystal emits a characteristic emission spectrum
which can be observed and measured, for example,
spectroscopically.
[0021] Conveniently, 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
heterogeniety of the sample population, and lineshapes that are
symmetric, gaussian or nearly gaussian with an absence of a tailing
region. Accordingly, the above technology allows for detection of
one, or even several, different biological or chemical moieties in
a single reaction. The combination of tunability, narrow
linewidths, and symmetric emission spectra without a tailing region
provides for high resolution of multiply sized nanocrystals, e.g.,
populations of monodisperse semiconductor nanocrystals having
multiple distinct size distributions within a system, and
simultaneous detection of a variety of biological moieties.
[0022] 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 semiconductor nanocrystals.
Consequently, this 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 are also more robust than
conventional organic fluorescent dyes and are more resistant to
photobleaching than the organic dyes. The robustness of the
nanocrystal also alleviates the problem of contamination of
degradation products of the organic dyes in the system being
examined. Therefore, the present invention provides uniquely
valuable tags for detection of biological and chemical
molecules.
[0023] Accordingly, in one embodiment, the invention is directed to
a method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of:
[0024] (a) providing the sample on a solid support;
[0025] (b) combining the sample with a semiconductor nanocrystal
conjugate, wherein the combining is performed under conditions that
allow formation of a complex comprising the conjugate and the
analyte, when present;
[0026] (c) removing any unbound conjugate; and
[0027] (d) detecting the presence of the complex, if present, by
monitoring a spectral emission mediated by the semiconductor
nanocrystal in the complex, wherein the emission indicates the
presence of one or more target analytes in the sample.
[0028] In certain embodiments, the invention is directed to a
method where there is a plurality of target analytes and the method
further comprises:
[0029] (a) providing a conjugate specific for each target analyte,
wherein each semiconductor nanocrystal conjugate has an emission
spectrum distinct from the other semiconductor nanocrystal
conjugates; and
[0030] (b) detecting the presence of the target analytes by
monitoring the spectral emissions of the sample, wherein the
emissions indicate the presence of the target analytes in the
sample.
[0031] In yet another embodiment, the invention is directed to a
method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of:
[0032] (a) providing an unlabeled specific-binding molecule on a
solid support;
[0033] (b) combining the sample with the specific-binding molecule,
wherein the combining is performed under conditions that allow
formation of a first complex comprising the specific-binding
molecule and the analyte, when present;
[0034] (c) removing any unbound sample;
[0035] (d) combining the first complex with a semiconductor
nanocrystal conjugate, wherein the combining is performed under
conditions that allow formation of a second complex comprising the
conjugate and the analyte, when present;
[0036] (e) removing any unbound conjugate;
[0037] (f) detecting the presence of the second complex, if
present, by monitoring a spectral emission mediated by the
semiconductor nanocrystal in the second complex, wherein the
emission indicates the presence of one or more target analytes in
the sample.
[0038] In certain embodiments, the method is one where there is a
plurality of target analytes and the method further comprises:
[0039] (a) providing an unlabeled specific-binding molecule on a
solid support specific for each target analyte and providing a
semiconductor nanocrystal conjugate specific for each target
analyte, wherein each semiconductor nanocrystal conjugate has an
emission spectrum distinct from the other semiconductor nanocrystal
conjugates; and
[0040] (b) detecting the presence of the target analytes by
monitoring the spectral emissions of the sample, wherein the
emissions indicate the presence of the target analytes in the
sample.
[0041] In yet another embodiment, the invention is directed to a
method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes,
comprising the steps of:
[0042] (a) providing the sample on a solid support;
[0043] (b) combining with the sample a specific-binding molecule,
wherein (i) the specific-binding molecule comprises a first member
of a binding pair, and (ii) the combining is performed under
conditions that allow formation of a first complex comprising the
specific-binding molecule and the analyte, when present;
[0044] (c) removing any unbound specific-binding molecule;
[0045] (d) combining the first complex with a second member of the
binding pair, wherein (i) the second member of the binding pair is
linked to a first semiconductor nanocrystal; and (ii) the combining
is performed under conditions that allow formation of a second
complex comprising the binding pair and the one or more
analytes;
[0046] (e) detecting the presence of the second complex, if
present, by monitoring a spectral emission mediated by the first
semiconductor nanocrystal in the second complex, wherein the
emission indicates the presence of one or more target analytes in
the sample.
[0047] In certain embodiments, the first member of the binding pair
is a first antibody and the second member of the binding pair is a
second antibody reactive with the first antibody; or the first
member of the binding pair is biotin and the second member of the
binding pair is streptavidin; or the first member of the binding
pair is digoxygenin and the second member of the binding pair is an
antibody directed against digoxygenin; or the first member of the
binding pair is flourescein and the second member of the binding
pair is an antibody directed against flourescein.
[0048] In another embodiment, the above method is one where there
is more than one analyte, and the method further comprises:
[0049] combining with the sample a second specific-binding
molecule, wherein (i) the second specific-binding molecule
comprises a first member of a second binding pair, and (ii) the
combining is performed under conditions that allow formation of a
third complex comprising the second specific-binding molecule and
the analyte, when present;
[0050] removing any unbound second specific-binding molecule;
[0051] combining the third complex with a second member of the
second binding pair, wherein (i) the second member of the second
binding pair is linked to a second semiconductor nanocrystal that
has an emission spectrum distinct from the first semiconductor
nanocrystal; and (ii) the combining is performed under conditions
that allow formation of a fourth complex comprising the second
binding pair and an analyte; and
[0052] detecting the presence of the fourth complex, if present, by
monitoring a second spectral emission mediated by the second
semiconductor nanocrystal in the fourth complex, wherein the second
emission indicates the presence of more than one target analyte in
the sample.
[0053] In still a further embodiment, the invention is directed to
a method of detecting one or more target analytes in a sample
containing or suspected of containing the one or more analytes. The
method comprises the steps of:
[0054] (a) providing a first complex comprising at least one
specific-binding molecule to which is bound a semiconductor
nanocrystal conjugate, wherein the semiconductor nanocrystal has a
characteristic spectral emission and wherein the conjugate
specifically binds to the specific-binding molecule;
[0055] (b) combining the sample with the first complex, wherein the
combining is performed under conditions that allow formation of a
second complex comprising the specific-binding molecule and the
analyte, when present;
[0056] (c) detecting the presence of the second complex, if
present, by monitoring the characteristic spectral emission of the
semiconductor nanocrystal, wherein a change in the characteristic
spectral emission indicates the presence of one or more target
analytes in the sample.
[0057] In certain embodiments of the method above, the plurality of
first complexes are provided each comprising a different
specific-binding molecule each bound to a conjugate which
specifically binds each specific-binding molecule and where each
specific-binding molecule binds a different analyte, and wherein
each conjugate bound to a different specific-binding molecule
comprises a semiconductor nanocrystal that has a characteristic
spectral emission distinct from the other semiconductor
nanocrystals; and wherein changes in the spectral emission of any
selected semiconductor nanocrystal associated with a particular
specific-binding molecule in a first complex, indicates the
presence of an analyte that binds to the particular
specific-binding molecule.
[0058] In certain embodiments, the specific-binding molecule is
radiolabeled, and when the conjugate is bound to the
specific-binding molecule the semiconductor nanocrystal emits
light, as described in Example 13. Further, the method may be one
wherein a plurality of first complexes are provided each comprising
a different specific-binding molecule each bound to a conjugate
which specifically binds each specific-binding molecule and where
each specific-binding molecule binds a different analyte, and
wherein each conjugate bound to a different specific-binding
molecule comprises a semiconductor nanocrystal that has a
characteristic spectral emission distinct from the other
semiconductor nanocrystals; and wherein changes in the spectral
emission of any selected semiconductor nanocrystal associated with
a particular specific-binding molecule in a first complex,
indicates the presence of an analyte that binds to the particular
specific-binding molecule.
[0059] In the methods above, the one or more analytes may be one or
more polypeptides or nucleic acid sequences, either the same or
different; the nucleic acid sequences may be present on one or more
chromosomal fragments; the nucleic acid sequences may be DNA or
RNA; the semiconductor nanocrystal conjugate may comprise an
antibody, an aptamer, or at least one polymerase chain reaction
primer; the specific-binding molecule may comprise a nucleic acid
molecule, e.g., an oligonucleotide, DNA, RNA, an aptamer, a
protein, a receptor, an antibody, a polysaccharide or a small
molecule
[0060] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 is a pictorial representation of a Qdot.TM.
immunosorbent assay (QISA).
[0062] FIG. 1A is a pictorial representation of a sandwich QISA as
described in Example 1.
[0063] FIG. 1B is a pictorial representation of a direct capture
QISA as described in Example 2.
[0064] FIG. 1C is a pictorial representation of a fluid-phase QISA
as described in Example 3.
[0065] FIG. 2 shows the results of a QISA performed on a 96-well
plate and read in a standard fluorescent plate reader, as described
in Example 1A.
[0066] FIG. 3 shows the distribution of beads among three rows (A,
B and C) of eight wells with differing concentrations of
semiconductor nanocrystal conjugates, as described in Example
12.
[0067] FIG. 4 is a representation of results that can be obtained
when semiconductor nanocrystals are used in a homogeneous
scintillation inhibition assay, as described in Example 13. In the
Figure, lanes A, B and C contain different inhibitors.
[0068] FIG. 5 is a pictorial representation of a competitive
microsphere filter assay as described in Example 19.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry and
biochemistry within the skill of the art. Such techniques are
explained fully in the literature.
[0070] 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. Thus, for example,
reference to "a semiconductor nanocrystal" includes a mixture of
two or more such semiconductor nanocrystals, an "analyte" includes
more than one such analyte, and the like.
[0071] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0072] I. Definitions
[0073] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0074] The terms "semiconductor nanocrystal," "quantum dot" and
"Qdot.TM. 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,
preferably between about 2 nm and about 50 nm or any integer or
fraction of an integer therebetween, more preferably about 2 nm to
about 20 nm (such as about 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 will preferably have a bandgap energy
that is larger than the bandgap energy of the core material and may
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, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the
like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and
the like) and IV (Ge, Si, and the like) materials, and an alloy or
a mixture thereof.
[0075] A semiconductor nanocrystal is, optionally, surrounded by a
"coat" of an organic capping agent. The organic capping agent may
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 convey solubility, e.g.,
the ability to disperse a coated semiconductor nanocrystal
homogeneously into a chosen solvent, finctionality, 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.
[0076] Thus, the terms "semiconductor nanocrystal," "quantum dot"
and "Qdot.TM. nanocrystal" as used herein denote a coated
semiconductor nanocrystal core, as well as a core/shell
semiconductor nanocrystal.
[0077] 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 low-energy microwave
radiation to high-energy x-ray radiation. Typically, luminescence
refers to photons in the range from UV to IR radiation.
[0078] "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 preferably less
than 5% rms.
[0079] 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.
[0080] 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 nm, and preferably not
exceeding about 20 nm in width and symmetric about the center, in
contrast to the emission bandwidth of about 100 nm for a typical
dye molecule with a red tail which may 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 peak height (FWHM), and are appropriate in
the range of 200 nm to 2000 mn.
[0081] 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.
[0082] 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, Inc., Corvallis, Oreg. as Neugene) polymers, and other
synthetic sequence-specific 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 will be 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' P5' 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, labels which are known in the art,
methylation, "caps," substitution of one or more of the naturally
occurring nucleotides with an analog, intemucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoramidates, carbamates,
etc.), with negatively charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), and with positively charged linkages
(e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters),
those containing pendant moieties, such as, for example, proteins
(including nucleases, toxins, antibodies, signal peptides,
poly-L-lysine, etc.), those with intercalators (e.g., acridine,
psoralen, etc.), those containing chelators (e.g., metals,
radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide or oligonucleotide. In particular, DNA is
deoxyribonucleic acid.
[0083] 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.
[0084] 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.
[0085] As used herein, the term "nucleic acid probe" includes
reference to a structure comprised of a polynucleotide, as defined
above, that contains a 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.
[0086] 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" under assay conditions, generally where there is about
90% or greater homology.
[0087] The term "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. WO92/14843,
WO91/19813, and WO92/05285, the disclosures of which are
incorporated by reference herein.
[0088] "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.
[0089] As used herein, the term "binding pair" refers to first and
second molecules that specifically bind to each other. "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. Unless the context clearly indicates
otherwise, the terms "affinity molecule" and "target analyte" are
used herein to refer to first and second members of a binding pair,
respectively.
[0090] Exemplary binding pairs include 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) and nonimmunological binding pairs (e.g.,
biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine and
cortisol]-hormone binding protein, receptor-receptor agonist or
antagonist (e.g., acetylcholine receptor-acetylcholine or an analog
thereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme
cofactor, enzyme-enzyme-inhibitor, and complementary polynucleotide
pairs capable of forming nucleic acid duplexes) and the like.
[0091] 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,
aptamers, proteins, polysaccharides, sugars, and the like. See,.
e.g., Haugland, "Handbook of Fluorescent Probes and Research
Chemicals" (Sixth Edition), and any of the molecules capable of
forming a binding pair as described above.
[0092] A "semiconductor nanocrystal conjugate" is a semiconductor
nanocrystal which 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 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.
[0093] The term "antibody" as used herein includes antibodies
obtained from both polyclonal and monoclonal preparations, as well
as, the following: hybrid (chimeric) antibody molecules (see, for
example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No.
4,816,567); F(ab')2 and F(ab) fragments; Fv molecules (noncovalent
heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad
Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem
19:4091-4096); single-chain Fv molecules (sFv) (see, for example,
Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); dimeric
and trimeric antibody fragment constructs; minibodies (see, e.g.,
Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J
Immunology 149B:120-126); humanized antibody molecules (see, for
example, Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et
al. (1988) Science 239:1534-1536; and U.K. Patent Publication, No.
GB 2,276,169, published 21 Sep. 1994); and, any functional
fragments obtained from such molecules, wherein such fragments
retain specific-binding properties of the parent antibody
molecule.
[0094] 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
murine hybridomas, as well as human monoclonal antibodies obtained
using human rather than murine hybridomas. See, e.g., Cote, et al.
Monclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p.
77.
[0095] 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 may either be
directly linked to the specific-binding molecule or may be linked
via a linker moiety, such as via a chemical linker described below.
The terms indicate items that are 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,
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, nanocrystals can be associated with biotin
which can bind to the proteins, avidin and streptavidin. Also,
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 Wemmer (1995) Ann. 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, etc., 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. Photobiol. B
40:91-106; chemical and photochemical probes of DNA, see Nielsen
(1990) J. Mol. Recognit. 3:1-25.
[0096] As used herein, a "biological sample" refers to a sample of
isolated cells, tissue or fluid, including but not limited to, for
example, 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).
[0097] 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.
[0098] A "biomolecule" is a synthetic or naturally occurring
molecule, such as a protein, amino acid, nucleic acid, nucleotide,
carbohydrate, sugar, lipid and the like.
[0099] A "homogeneous assay" is one that is performed without
transfer, separation or washing steps. Thus, for example, a
homogeneous HTS assay involves the addition of reagents to a
vessel, e.g., a test tube or sample well, followed by the detection
of the results from that particular well. A homogeneous HTS assay
can be performed in the solution in the test tube or well, on the
surface of the test tube or well, on beads or cells which are
placed into the test tube or the well, or the like. The detection
system typically used is a fluorescence, chemiluminescence, or
scintillation detection system.
[0100] 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.
[0101] The term "barcode" as used herein refers to one or more
sizes, size distributions, compositions, or any combination
thereof, of semiconductor nanocrystals. Each size, size
distribution and/or composition of semiconductor nanocrystals has a
characteristic emission spectrum, e.g., wavelength, intensity,
FWHM, and/or fluorescent lifetime. In addition to the ability to
tune the emission energy by controlling the size of the particular
semiconductor nanocrystal, the intensities of that particular
emission observed at a specific wavelength are also capable of
being varied, thus increasing the potential information density
provided by the semiconductor nanocrystal barcode system. In
preferred embodiments, 2-15 different intensities may be achieved
for a particular emission at a desired wavelength, however, one of
ordinary skill in the art will realize that more than fifteen
different intensities may be achieved, depending upon the
particular application of interest. For the purposes of the present
invention, different intensities may be achieved by varying the
concentrations of the particular size semiconductor nanocrystal
attached to, embedded within or associated with an item, compound
or matter of interest. The "barcode" enables the determination of
the location or identity of a particular item, compound or matter
of interest. For example, semiconductor nanocrystals can be used to
barcode chromosomes, as well as portions of chromosomes, for
spectral karyotyping, as described further below.
[0102] "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 which 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.
[0103] II. Modes of Carrying Out the Invention
[0104] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific assay
formats, materials or reagents, as such may, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular embodiments of the invention only,
and is not intended to be limiting.
[0105] Although a number of compositions and methods similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0106] The present invention provides chemical and biological
assays which use semiconductor nanocrystals as detectable
luminescent labels to detect the presence or amount of one or more
molecules, as well as to detect biological interactions, biological
processes, alterations in biological processes, or alterations in
the structure of a chemical or biological compound.
[0107] 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 of 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.
Furthermore, semiconductor nanocrystals can be made highly
luminescent through the use of a shell material which efficiently
encapsulates the surface of the semiconductor nanocrystal core. A
"core/shell" semiconductor nanocrystal has a high quantum
efficiency and significantly improved photochemical stability. The
surface of the core/shell semiconductor nanocrystal can be modified
to produce semiconductor nanocrystals that can be coupled to a
variety of biological molecules or substrates by techniques
described in, for example, 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, 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, and described further below.
[0108] It is readily apparent that semiconductor nanocrystals can
be used to detect or track a single target. Additionally, a
population of semiconductor nanocrystals may be used for either
simultaneous detection of multiple targets or to detect particular
compounds and/or items of interest in, e.g., a library of
compounds.
[0109] For example, compositions of semiconductor nanocrystals
comprising one or more particle size distributions having
characteristic spectral emissions may be used as "barcodes" in
assays to either track the location or source of a particular item
of interest or to identify a particular item of interest. The
semiconductor nanocrystals used in such a "barcoding" scheme can be
tuned to a desired wavelength to produce a characteristic spectral
emission by changing the composition and size, or size
distribution, of the semiconductor nanocrystal. Additionally, the
intensity of the emission at a particular characteristic wavelength
can also be varied, thus enabling the use of binary or higher order
encoding schemes. The information encoded by the semiconductor
nanocrystals can be spectroscopically decoded, thus providing the
location and/or identity of the particular item or component of
interest.
[0110] The ability to use semiconductor nanocrystals in order to
detect multiple targets results from their unique characteristics.
Semiconductor nanocrystals have radii that are smaller than the
bulk exciton Bohr radius and constitute a class of materials
intermediate between molecular and bulk forms of matter. 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 nm
FWHM, 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 realize, the
linewidths are dependent on the size heterogeneity, i.e.,
monodispersity, of the semiconductor nanocrystals in each
preparation. Single semiconductor nanocrystal complexes have been
observed to have FWHM as narrow as 12 nm to 15 nm. In addition,
semiconductor nanocrystal distributions with larger linewidths in
the range of 35 nm to 60 nm can be readily made and have the same
physical characteristics as semiconductor nanocrystals with
narrower linewidths.
[0111] Semiconductor nanocrystals can be used to detect the
presence and/or amount of a biological moiety, e.g., a biological
target analyte; the structure, composition, and conformation of a
biological molecule; the localization of a biological moiety, e.g.,
a biological target analyte in an environment; interactions of
biological molecules; alterations in structures of biological
compounds; and/or alterations in biological processes.
[0112] Thus, it is readily apparent that semiconductor nanocrystals
find use in a variety of assays where other, less reliable,
labeling methods have typically been used, including, without
limitation, fluorescence microscopy, histology, cytology,
pathology, flow cytometry, FISH and other nucleic acid
hybridization assays, signal amplification assays, DNA and protein
sequencing, immunoassays such as competitive binding assays and
ELISAs, immunohistochemical analysis, protein and nucleic acid
separation, homogeneous assays, multiplexing, high throughput
screening, chromosome karyotyping, and the like.
[0113] Production of Semiconductor Nanocrystals
[0114] Semiconductor nanocrystals for use in the subject methods
are made using techniques known in the art. See, e.g., U.S. Pat.
Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of
which are incorporated herein in their entireties); 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, CaTe, SrS, SrSe, SrTe,
BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AIS, AlP,
AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures
thereof. The semiconductor nanocrystals are characterized by their
uniform nanometer size.
[0115] 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, InAs, 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.
[0116] 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 preferred embodiments, 5-20 discrete
emissions (five to twenty different size 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 could
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.
[0117] 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 preferred
embodiments for 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, which are hereby incorporated by reference
in their entireties. One of ordinary skill in the art will also
realize that 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.
[0118] However, for some applications high information density will
not be required and it may be more economically attractive to use
more polydisperse particles. Thus, for applications that do not
require high information density, the linewidth of the emission may
be in the range of 40-60 nm.
[0119] In a particularly preferred embodiment, the surface of the
semiconductor nanocrystal is also modified to enhance the
efficiency of the emissions, by adding an overcoating layer to the
semiconductor nanocrystal. The overcoating layer is particularly
preferred 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.
[0120] Suitable materials for the overcoating layer include
semiconductor materials having a higher bandgap energy than the
semiconductor nanocrystal core. In 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 may 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, may also be used. The preparation of
a coated semiconductor nanocrystal may be found in, e.g., Dabbousi
et al. (1997) J. Phys. Chem. B 101:9463) and Kuno et al. (1997) J.
Phys. Chem. 106:9869.
[0121] 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 is
readily soluble in organic solvents, such as toluene, chloroform
and hexane. As one of ordinary skill in the art will realize, these
functional moieties may 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 may be modified by a displacement
reaction, based upon the desired use therefor.
[0122] After selection of the composition of semiconductor
nanocrystal for the desired range of spectral emission and
selection of a desired surface functionalization 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 providing a sufficient number of semiconductor
nanocrystals that are bright (i.e., that emit light versus those
that are dark) and providing a sufficient number of semiconductor
nanocrystals to average out over the blinking effect observed in
single semiconductor nanocrystal emissions. Nirmal et al., (1996)
Nature 383:802.
[0123] 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. One of ordinary skill in the art
will realize, however, that fewer than eight semiconductor
nanocrystals of a particular composition and particle size
distribution may be utilized provided that a unique spectral
emission of sufficient intensity is observed, as determined by the
selection criteria set forth above.
[0124] 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.
[0125] 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 theory 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.
[0126] 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 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, M.sup.n 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.
[0127] 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.
[0128] 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. In
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.
[0129] 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 measurement
techniques, the bandgaps for nanocrystals of CdSe of sizes ranging
from 12.ANG. to 115.ANG. 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.
[0130] 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 UV 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. nm), 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.
[0131] 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.
[0132] 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
objective. The luminescence is put through a single monochromator
or a grating or prism to spectrally resolve the colors. The
detector is a 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.
[0133] Production of Semiconductor Nanocrystal Conjugates
[0134] The present invention uses a composition comprising
semiconductor nanocrystals associated with a specific-binding
molecule or affinity molecule, such that the composition can detect
the presence and/or amounts of biological and chemical compounds,
detect interactions in biological systems, detect biological
processes, detect alterations in biological processes, or detect
alterations in the structure of biological compounds. 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.
[0135] 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, may 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.
[0136] 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 carried out in aqueous media, a preferred embodiment of
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 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 may 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 (--CO.sup.2-), sulfonates
(SO.sup.3-), phosphates (--PO.sub.4.sup.2- and --PO.sub.3.sup.2-),
nitrates, ammonium salts (--NH.sup.4+), 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.,
International Publication No. WO 00/17655, published Mar. 30,
2000.
[0137] 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.
[0138] A displacement reaction may 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.
[0139] The surface layer may 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. A solid support, for the purposes of this invention,
is defined as an insoluble material to which compounds are attached
during a synthesis sequence, screening, immunoassays, etc. The use
of a solid support is particularly advantageous for the synthesis
of libraries because the isolation of support-bound reaction
products can be accomplished simply by washing away reagents from
the support-bound material and therefore the reaction can be driven
to completion by the use of excess reagents.
[0140] A solid support can be any material that is an insoluble
matrix and can have a rigid or semi-rigid surface. Exemplary solid
supports include but are not limited to pellets, disks,
capillaries, hollow fibers, needles, pins, solid fibers, cellulose
beads, pore-glass beads, silica gels, polystyrene beads optionally
cross-linked with divinylbenzene, grafted co-poly beads,
polyacrylamide beads, latex beads, dimethylacrylamide beads
optionally crosslinked with N-N'-bis-acryloylethylenediamine, and
glass particles coated with a hydrophobic polymer.
[0141] For example, the semiconductor nanocrystals of the present
invention can readily be finctionality to create styrene or
acrylate moieties, thus enabling the incorporation of the
semiconductor nanocrystals into 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.
[0142] For a detailed description of these linking reactions, see,
e.g., U.S. Pat. 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.
[0143] Semiconductor Nanocrystals as Detection Reagents in
Immunoassays
[0144] In one embodiment of the invention, immunoassays, such as
immunosorbent assays, are provided in which semiconductor
nanocrystal conjugates are used as the detection reagents. A
Qdot.TM. immunosorbent assay (QISA) has several advantages over
current immunosorbent assays including, but not limited to,
simultaneous multicolor detection and, hence, multiple analyte
detection, no requirement for enzyme development, increased
photostability over alternative fluorophores thereby allowing
increased detection sensitivity by virtue of the ability to monitor
the signal over a long period of time, increased sensitivity over
enzyme-based detection systems.
[0145] Semiconductor nanocrystals of varying core sizes (10-150
.ANG.), composition and/or size distribution are conjugated to
specific-binding molecules which bind specifically to an analyte of
interest. Any specific anti-analyte can be used, for example, an
antibody, an immunoreactive fragment of an antibody, and the like.
Preferably, the anti-analyte is an antibody. The semiconductor
nanocrystal conjugates are used in an immunosorbent assay to detect
any analyte for which a specific-binding agent exists.
[0146] More specifically, the specific-binding molecule may be
derived from polyclonal or monoclonal antibody preparations, may be
a human antibody, or may be a hybrid or chimeric antibody, such as
a humanized antibody, an altered antibody, F(ab').sub.2 fragments,
F(ab) fragments, Fv fragments, a single-domain antibody, a dimeric
or trimeric antibody fragment construct, a minibody, or functional
fragments thereof which bind to the analyte of interest. Antibodies
are produced using techniques well known to those of skill in the
art and disclosed in, for example, U.S. Pat. Nos. 4,011,308;
4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745,
[0147] For example, polyclonal antibodies are generated by
immunizing 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. Immunization is generally performed by mixing or
emulsifying the antigen in saline, preferably in an adjuvant such
as Freund's complete adjuvant, and injecting the 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 may also be
generated by in vitro immunization, using methods known in the art.
Polyclonal antiserum is then obtained from the immunized
animal.
[0148] Monoclonal antibodies are generally 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 may 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 unrelated antigens). The selected monoclonal antibody-secreting
hybridomas 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).
[0149] Human monoclonal antibodies are obtained by using human
rather than murine hybridomas. See, e.g., Cote, et al. Monclonal
Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77
[0150] Monoclonal antibodies or portions thereof may 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 may then be cloned and amplified to
obtain sequences that encode the antibody (or binding domain) of
the desired specificity.
[0151] As explained above, antibody fragments which retain the
ability to recognize the analyte of interest, will also find use in
the subject immunoassays. A number of antibody fragments are known
in the art which comprise antigen-binding sites capable of
exhibiting immunological binding properties of an intact antibody
molecule. For example, 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').sub.2 fragments. These fragments will contain two antigen
binding sites, but lack a portion of the constant region from each
of the heavy chains. Similarly, if desired, 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 F.sub.v. See, e.g., Inbar et al. (1972) Proc. Nat.
Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem
15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
[0152] A single-chain Fv ("sFv" or "scFv") polypeptide is a
covalently linked V.sub.H-V.sub.L heterodimer which is expressed
from a gene fusion including V.sub.H- and V.sub.L-encoding genes
linked by a peptide-encoding linker. Huston et al. (1988) Proc.
Nat. 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
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. Pat. 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) Proc. Nat. Acad. Sci. USA
85:5879-5883; U.S. Pat. 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. Pat. 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.
[0153] "Mini-antibodies" or "minibodies" will also find use with
the present invention. Minibodies are sFv polypeptide chains which
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
.alpha.-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) J Immunology 149B:120-126.
[0154] Once produced, the specific-binding molecules described
above may be used in immunoassays of varying formats. For example,
the antibodies may be linked to the semiconductor nanocrystal.
Alternatively, the semiconductor nanocrystals may be linked to
other affinity molecules, such as strepavidin, that will
specifically react with another molecule that is linked to the
analyte of interest.
[0155] The analyte of interest can be detected using standard
immunoassays such as competition, direct reaction, or sandwich type
assays. Such assays include, but are not limited to, ELISA-like
assays (termed QISA herein) and biotin/avidin type assays. The
reactions include the semiconductor nanocrystals in order to detect
the formation of a complex between the antigen and the antibody or
antibodies reacted therewith.
[0156] The aforementioned assays generally involve separation of
unbound antibody in a liquid phase from a solid phase support to
which labeled antigen-antibody complexes are bound. Solid supports
which can be used in the methods herein include substrates such as
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.
[0157] In one context, a solid support is first reacted with a
component that will bind to the solid support, i.e., the "solid
phase component," e.g., an antigen or an antibody, under suitable
binding conditions such that the component is sufficiently
immobilized to the support. Sometimes, immobilization to the
support can be enhanced by first coupling the antigen 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, amino acid copolymers, and the like.
Such molecules and methods of coupling these molecules to antigens,
are well known to those of ordinary skill in the art. See, e.g.,
Brinkley, M. A. (1992) Bioconjugate Chem. 3:2-13; Hashida et al.
(1984) J. Appl. Biochem. 6:56-63; and Anjaneyulu and Staros (1987)
International J. of Peptide and Protein Res. 30:117-124.
[0158] After reacting the solid support with the solid phase
component, any nonimmobilized solid-phase components are removed
from the support by washing, and the support-bound component is
then contacted with a biological sample suspected of containing
ligand moieties (e.g., antibodies toward the immobilized antigens,
or antigens toward immobilized antibodies) under suitable binding
conditions. After washing to remove any nonbound ligand, a
secondary binder moiety detectably labeled with the semiconductor
nanocrystals described herein is added under suitable binding
conditions, wherein the secondary binder is capable of associating
selectively with the bound ligand. The presence of the secondary
binder can then be detected using the techniques described above.
Alternatively, an indirect labeling technique can be used wherein
the presence of an unlabeled secondary binder moiety is detected by
the specific binding thereto of a detectably labeled tertiary
binder moiety.
[0159] More particularly, a QISA method can be used, wherein 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 plate(s) can be washed to remove unbound moieties and
a detectably labeled secondary binding molecule added. The
secondary binding molecule is allowed to react with any captured
sample antibodies, the plate washed and the presence of the
secondary binding molecule detected as described above.
[0160] Thus, in one particular embodiment, the presence of bound
antibody ligands from a biological sample can be readily detected
using a secondary binder comprising an antibody directed against
the antibody ligands, conjugated to semiconductor nanocrystals. A
number of immunoglobulin (Ig) molecules are known in the art which
can be readily conjugated to semiconductor nanocrystals as
described herein. In other related embodiments, competitive-type
QISA techniques can be practiced using methods known to those
skilled in the art.
[0161] Assays can also be conducted in solution, such that the
antigens and antibodies specific for those proteins form complexes
under precipitating conditions. In one particular embodiment,
antigens can be attached to a solid phase particle (e.g., an
agarose bead or the like) using coupling techniques known in the
art, such as by direct chemical or indirect coupling. The
antigen-coated particle is then contacted under suitable binding
conditions with a biological sample suspected of containing
antibodies for the antigen. Cross-linking between bound antibodies
causes the formation of particle-antigen-antibody complex
aggregates which can be precipitated and separated from the sample
using washing and/or centrifugation. The reaction mixture can be
analyzed to determine the presence or absence of antibody-antigen
complexes using any of a number of standard methods, such as those
immunodiagnostic methods described above.
[0162] In yet a further embodiment, 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 will thus only contain
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.
[0163] Accordingly, once the immunoglobulin molecules have been
immobilized to provide an immunoaffinity matrix, semiconductor
nanocrystal-labeled proteins are contacted with the bound
antibodies under suitable binding conditions. After any
nonspecifically bound antigen has been washed from the
immunoaffinity support, the presence of bound antigen can be
determined by assaying for label using methods described above.
[0164] 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.
[0165] In yet further embodiments, semiconductor
nanocrystal-antibody conjugates may be used to probe fixed tissue
samples or fixed cell populations for specific markers. In this
embodiment, prepared cells or tissue are incubated with an antibody
which is conjugated to a semiconductor nanocrystal. Semiconductor
nanocrystals allow stable, multicolor detection of markers in both
cell and tissue samples.
[0166] Semiconductor nanocrystal-conjugates (either a single
semiconductor nanocrystal conjugated to biomolecules or a plurality
of semiconductor nanocrystals) allow specific, sensitive,
photostable detection of antigens in staining procedures. This
offers a clear advantages over currently available stains.
Additionally the inherent properties of semiconductor nanocrystals,
i.e., single excitation source, narrow, gaussian spectra and
tunability of emission wavelength, mean that many more colors are
resolved than with conventional fluorescent dyes.
[0167] More particularly, multiple analysis staining can be
performed on a tissue sample, blood sample or any sample requiring
multiplexed analysis of cellular or extracellular markers. The
procedure may be carried out in a two-step reaction whereby a
primary antibody is followed by a semiconductor
nanocrystal-conjugated antibody or by using an antibody (or other
biomolecule) semiconductor nanocrystal conjugate to directly label
the sample. For 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 which specifically recognizes a
biomolecule of interest which may or may not be present in the
sample to be analyzed. Following standard staining 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 as described
above.
[0168] Since many semiconductor nanocrystals can be generated that
are spectrally distinct, it is possible to label different items
such as antibodies or cDNAs that can then be used to measure the
position and quantity of cellular compounds (as described above).
The number of compounds that can be followed is limited, however,
by the number of spectrally distinct colors that can be made. With
CdSe, semiconductor nanocrystals and the current synthetic
techniques, this is approximately 6-7 spectrally distinct colors.
If different compounds are not colocalized in the cell, however,
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
(see Example 18 below).
[0169] The semiconductor nanocrystals may also be used in
competitive microsphere filter assays, such as competitive latex
immunoassays. Such assays are described in, e.g., Stave J. W.
(1994) Immunoassay for priority pollutants. Analytica 94 Conference
Abstracts p. 339; and Bangs, L. B. (1996) Immunological
Applications of Microspheres. In The Latex Course 4196.
Traditionally, this assay uses antibody-conjugated latex particles
to detect industrial chemicals in soil or ground water samples at
parts per billion concentrations. In this application the antibody
is conjugated to, e.g., 3 .mu.m microspheres which are caught on 1
.mu.m pore filters. A sample is passed through the filter and the
immobilized antibodies catch any antigen present. An enzyme/antigen
conjugate is passed through the filter. If the sample contains no
antigen then the enzyme/antigen will bind to the free sites on the
antibody and added substrate will cause a color change on the
filter. Increases in antigen concentration will result in
corresponding decreases in the filter color change.
[0170] In the context of the present invention, the detection agent
is a semiconductor nanocrystal or a semiconductor
nanocrystal-encoded solid conjugate. The conjugate is a molecule
that specifically recognizes the analyte. The antigen conjugates
are direct semiconductor nanocrystal-antigen conjugates or
semiconductor nanocrystal-dyed microsphere-conjugates that are
small enough to pass through filter pores. This allows multiple
simultaneous detections using a light source for excitation of the
semiconductor nanocrystals and detection of emissions. The
detection takes place on the filter or in the filtrate and the
assay may be carried out in a high throughput multiwell
environment. The filters in this format are opaque to the
excitation light and allow detection of semiconductor nanocrystals
in the filtrate without the need for washes or sample removal.
Analyte concentration, in virtually any context where a specific
binder exists for that analyte, can be determined. Thus, almost any
analyte, chemical or biological, organic or inorganic may be
detected in this manner.
[0171] The detection is achieved by competition between analyte in
the sample and analyte conjugated to semiconductor nanocrystals or
conjugated to a semiconductor nanocrystal-encoded microsphere which
is small enough to pass through the filter pores. Separation is
achieved because the reaction is free to pass through a microporous
filter in which the pores are a smaller size than the diameter of
the microsphere. Thus the microsphere cannot pass through the
filter (see FIG. 5). Semiconductor nanocrystal-conjugates or
semiconductor nanocrystal encoded microspheres are of small enough
size to pass through the filter and will do so unless bound to the
microspheres. If a known amount of semiconductor nanocrystals are
applied to the upper level, with the analyte sample, allowed to
bind for a predetermined time and then passed through the filter,
the concentration of the analyte is determined by measuring the
level of fluorescence present in the lower level. Fluorescence in
the upper level is not detected either by removal of the upper
level or because the membrane is opaque to the excitation
source.
[0172] Detection is carried out by a spectral sensing device and
separation of different semiconductor nanocrystal spectra is
achieved by the use of band-pass filters or by measuring light
emission across the spectrum. Detection can be achieved by a static
detection device or by a scanning detector, as described above.
[0173] Semiconductor Nanocrystals as Detection Reagents in
Probe-based Assays
[0174] Semiconductor nanocrystals can also be used as sensitive
detection agents in probe-based assays for the detection of target
nucleic acid sequences in test samples. Probes for use in these
assays are designed from either conserved or nonconserved regions
of the target polynucleotide of interest, using techniques well
known in the art. Generally, probes are developed from nonconserved
or unique regions when maximum specificity is desired, and from
conserved regions when assaying for regions that are closely
related to, for example, different members of a multigene family or
in related species.
[0175] Polymerase chain reaction (PCR) is a technique for
amplifying a desired target nucleic acid sequence contained in a
nucleic acid molecule or mixture of molecules. In PCR, a pair of
primers is employed in excess to hybridize to the complementary
strands of the target nucleic acid. The primers are each extended
by a polymerase using the target nucleic acid as a template. The
extension products become target sequences themselves after
dissociation from the original target strand. New primers are then
hybridized and extended by a polymerase, and the cycle is repeated
to geometrically increase the number of target sequence molecules.
PCR is described in U.S. Pat. No. 4,683,195 and 4,683,202, which
are incorporated herein by reference in their entireties.
[0176] The Ligase Chain Reaction (LCR) is an alternate method for
nucleic acid amplification. In LCR, probe pairs are used which
include two primary (first and second) and two secondary (third and
fourth) probes, all of which are used in molar excess to target.
The first probe hybridizes to a first segment of the target strand,
and the second probe hybridizes to a second segment of the target
strand, the first and second segments being contiguous so that the
primary probes abut one another in 5' phosphate-3' hydroxyl
relationship. Thus, a ligase can covalently fuse or ligate the two
probes into a fused product. In addition, a third (secondary) probe
can hybridize to a portion of the first probe and a fourth
(secondary) probe can hybridize to a portion of the second probe in
a similar abutting fashion. If the target is initially
double-stranded, the secondary probes will also hybridize to the
target complement in the first instance. Once the ligated strand of
primary probes is separated from the target strand, it will
hybridize with the third and fourth probes which can be ligated to
form a complementary, secondary ligated product. By repeated cycles
of hybridization and ligation, amplification of the target sequence
is achieved. This technique is described in, e.g., European
Publication No. 320,308, published Jun. 16, 1989 and European
Publication No. 439,182, published Jul. 31, 1991
[0177] More particularly, in the above methods, once the primers or
probes have been sufficiently extended and/or ligated, they are
separated from the target sequence, for example, by heating the
reaction mixture to a "melt temperature" which dissociates the
complementary nucleic acid strands. Thus, a sequence complementary
to the target sequence is formed. A new amplification cycle can
then take place to further amplify the number of target sequences
by separating any double-stranded sequences, allowing primers or
probes to hybridize to their respective targets, extending and/or
ligating the hybridized primers or probes and reseparating. The
complementary sequences that are generated by amplification cycles
can serve as templates for primer extension or fill the gap of two
probes to further amplify the number of target sequences.
Typically, a reaction mixture is cycled between 20 and 100 times,
more typically between 25 and 50 times. In this manner, multiple
copies of the target sequence and its complementary sequence are
produced. Thus, primers initiate amplification of the target
sequence when it is present under amplification conditions.
[0178] mRNAs may be amplified by reverse transcribing the mRNA into
cDNA, and then performing PCR (RT-PCR), as described above.
Alternatively, a single enzyme may be used for both steps as
described in U.S. Pat. No. 5,322,770, which is incorporated herein
by reference. MRNA may also be reverse transcribed into cDNA,
followed by asymmetric gap ligase chain reaction (RT-AGLCR) as
described by Marshall et al. (1994) PCR Meth. App. 4:80-84.
[0179] Other known amplification methods which can be utilized in
probe-based assays include, but are not limited to, the "NASBA" or
"3SR" (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA
87:1874-1878 and Compton, J. Nature 350:91-92); Q-beta
amplification; strand displacement amplification (Walker et al.
Clin. Chem. 42:9-13 and European Patent Application No. 684,315);
and target mediated amplification (International Publication No. WO
93/22461).
[0180] Detection, both amplified and nonamplified, may be performed
using a variety of heterogeneous and homogeneous detection formats.
Examples of heterogeneous detection formats are disclosed in
Snitman et al., U.S. Pat. No. 5,273,882; Urdea et al., U.S. Pat.
No. 5,124,246; Ullman et al. U.S. Pat. No. 5,185,243; and Kourilsky
et al., U.S. Pat. No. 4,581,333, all of which are incorporated
herein by reference in their entireties. Examples of homogeneous
detection formats are described in Caskey et al., U.S. Pat. No.
5,582,989; and Gelfand et al., U.S. Pat. No. 5,210,015, which are
incorporated herein by reference in their entireties. Also
contemplated and within the scope of the present invention is the
use of multiple probes in hybridization assays, to improve
sensitivity and amplification of the target signal. See, for
example, Caskey et al., U.S. Pat. No. 5,582,989; and Gelfand et
al., U.S. Pat. No. 5,210,015; which are incorporated herein by
reference in their entireties.
[0181] Thus, in one embodiment, the present invention generally
comprises the steps of contacting a test sample suspected of
containing a target polynucleotide sequence with amplification
reaction reagents comprising an amplification primer, and a
detection probe that can hybridize with an internal region of the
amplicon sequences. Probes and primers employed according to the
method provided herein are labeled with capture and detection
labels, wherein probes are labeled with one type of label and
primers are labeled with another type of label. Additionally, the
primers and probes are selected such that the probe sequence has a
lower melt temperature than the primer sequences. The amplification
reagents, detection reagents and test sample are placed under
amplification conditions whereby, in the presence of target
sequence, copies of the target sequence (an amplicon) are produced.
In the usual case, the amplicon is double-stranded because primers
are provided to amplify a target sequence and its complementary
strand. The double-stranded amplicon then is thermally denatured to
produce single-stranded amplicon members. Upon formation of the
single-stranded amplicon members, the mixture is cooled to allow
the formation of complexes between the probes and single-stranded
amplicon members.
[0182] As the single-stranded amplicon sequences and probe
sequences are cooled, the probe sequences preferentially bind the
single-stranded amplicon members. After the probe/single-stranded
amplicon member hybrids are formed, they are detected. Standard
heterogeneous assay formats are suitable for detecting the hybrids
using the detection labels and capture labels present on the
primers and probes. The hybrids can be bound to a solid phase
reagent by virtue of the capture label and detected by virtue of
the detection label. Either the capture label or detection label
comprises semiconductor nanocrystals. In cases where the detection
label is directly detectable, the presence of the hybrids on the
solid phase can be detected using techniques described above. In
cases where the label is not directly detectable, the captured
hybrids can be contacted with a conjugate, which generally
comprises a binding member attached to a semiconductor nanocrystal
label. The conjugate becomes bound to the complexes and the
presence of the conjugate on the complexes can be detected with the
directly detectable semiconductor nanocrystal label. Thus, the
presence of the hybrids on the solid-phase reagent can be
determined. Wash steps are typically employed during the above
reactions to wash away unhybridized amplicon or probe as well as
unbound conjugate.
[0183] The heterogeneous assays can be conveniently performed using
a solid phase support that carries an array of nucleic acid
molecules. Such arrays are useful for high-throughput and/or
multiplexed assay formats, described in more detail below. Various
methods for forming such arrays from pre-formed nucleic acid
molecules, or methods for generating the array using in situ
synthesis techniques, are generally known in the art. (See, for
example, Dattagupta, et al., European Publication No. 234,726; U.S.
Pat. No. 5,700,637 to Souther; U.S. Pat. No. 5,143,854 to Pirrung,
et al.; International Publication No. WO 92/10092; and, Fodor, et
al. (1991) Science 251:767-777.
[0184] Although the target sequence above is described as
single-stranded, the target may also be double-stranded and
separated from its complement prior to hybridization with the
amplification primer sequences. Further, while the amplification
primers initiate amplification of the target sequence, the
detection (or hybridization) probe is not involved in
amplification. Detection probes are generally nucleic acid
sequences or uncharged nucleic acid analogs such as, for example,
peptide nucleic acids which are disclosed in International
Publication No. WO 92/20702; morpholino analogs which are described
in U.S. Pat. Nos. 5,185,444, 5,034,506 and 5,142,047; and the like.
Depending upon the type of label carried by the probe, the probe is
employed to capture or detect the amplicon generated by the
amplification reaction.
[0185] While the length of the primers and probes can vary, the
probe sequences are selected such that they have a lower melt
temperature than the primer sequences. Hence, the primer sequences
are generally longer than the probe sequences. Typically, the
primer sequences are in the range of between 20 and 50 nucleotides
long, more typically in the range of between 20 and 30 nucleotides
long. The typical probe is in the range of between 10 and 25
nucleotides long.
[0186] As explained above, 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. WO92/14843, WO91/19813, and WO92/05285. The SELEX
procedure, described in U.S. Pat. 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 the
SELEX procedure, an oligonucleotide is constructed wherein an
n-mer, preferably a randomized sequence of nucleotides thereby
forming a "randomer pool" of oligonucleotides, is flanked by two
polymerase chain reaction (PCR) primers. The construct is then
contacted with a target molecule under conditions which favor
binding of the oligonucleotides to the target molecule. Those
oligonucleotides which bind the target molecule are: (a) separated
from those oligonucleotides which do not bind the target molecule
using conventional methods such as filtration, centrifugation,
chromatography, or the like; (b) dissociated from the target
molecule; and (c) 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 achieved. The final aptamer sequence identified can then be
prepared chemically or by in vitro transcription. Semiconductor
nanocrystals are readily used in the detection of such
aptamers.
[0187] Specific embodiments of the above-described probe-based
assays are described in further detail below.
[0188] Semiconductor Nanocrystals for Detection Reagents in
Fluorescence in Situ Hybridization
[0189] In another embodiment of the invention, FISH assays using a
semiconductor nanocrystal as a detectable label are disclosed.
Techniques for performing various types of FISH assays are well
known in the art and described in, e.g., Raap, A. K. (1998)
Mutation Res. 400:287-298; Speel et al. (1998) Histochem. Cell.
Biol. 110:571-577; Nath and Johnson (1997) Biotech. Histochem.
73:6-22; Swiger and Tucker (1996) Environ. Molec. Mutagen.
27:245-254; Kitadai et al. (1995) Clin. Cancer Res. 1:1095-1102;
Heiskanen et al. (1995) Genomics 30:31-36; and Heiskanen et al.
(1994) BioTechniques 17:928-933. Semiconductor nanocrystals can be
substituted for the fluorescent labels normally used in each of
these techniques. The advantages of nucleic acid probes labeled
with semiconductor nanocrystals is that multiple probes directed at
distinct target oligonucleotides can be used simultaneously by
virtue of the fact that a plurality of populations of semiconductor
nanocrystals can be made with nonoverlapping emission spectra, each
of which can be excited with a single source and wavelength of
light. The ability to "multiplex" assays in this manner is
especially useful when the specimen to be analyzed contains a
limited source of cells or tissues, e.g., rare cells, fetal cells
in maternal blood, cancer cells in blood or urine samples,
blastomeres, or the like. By multiplexing, multiparametric
information at the single cell level may be collected. See, e.g.,
Patterson et al. (1998) Cytometry 31:265-274; Borzi et al. (1996)
J. Immunol. Meth. 193:167-176; Wachtel et al. (1998) Prenat. Diagn.
18:455-463; Bianchi (1998) J. Perinat. Med. 26:175-185; and Munne
(1998) Mol. Hum. Reprod. 4:863-870.
[0190] Semiconductor nanocrystals of many colors can be chemically
linked to nucleic acid (DNA or RNA) or indirectly linked to
streptavidin/biotin that binds to nucleic acid. Semiconductor
nanocrystals bind to DNA primers or incorporate into nucleic acid
by using semiconductor nanocrystal-linked nucleotide(s). PCR can be
used to generate nucleic acid fragments for FISH probes.
Semiconductor nanocrystals can also be chemically attached to a
nucleic acid containing the sequence of interest. Alternatively,
biotin molecules can be attached to oligonucleotide primers, or
incorporated into nucleic acid of interest by using biotinlyated
nucleotides in PCR. Semiconductor nanocrystals attached to
streptavidin will then be linked to biotin in the nucleic acid
probe. These semiconductor nanocrystal-FISH probes can be use for
in situ hybridization for DNA (see Example 5; see also Dewald et
al. (1993) Bone Marrow Transplantation 12:149-154; Ward et al.
(1993) Am. J. Hum. Genet. 52:854-865; Jalal et al. (1998) Mayo
Clin. Proc. 73:132-137; Zahed et al. (1992) Prenat. Diagn.
12:483-493; Neuhaus et al. (1999) Human Pathol. 30:81-86; Buno et
al. (1998) Blood 92:2315-2321; Munne (1998) Mol. Hum. Reprod.
4:863-870, and RNA (see Example 6; Kitadai et al. (1995) Clin.
Cancer Res. 1:1095-1102). The results can be analyzed, for example,
under an epi fluorescence microscope. Semiconductor
nanocrystal-FISH probe or probes for DNA and RNA together (see
Example 7; Wachtel et al. (1998) Prenat. Diagn. 18:455-463), or for
RNA and surface immunophenotyping together (see Example 8;
Patterson et al. (1998) Cytometry 31:265-274; Borzi et al. (1996)
J. Immunol. Meth. 193:167-176) can be used to identify, sort, and
analyze rare cells simultaneously. In the case where only short
oligonucleotide semiconductor nanocrystals-FISH probes (forward and
reverse primers) for RNA or DNA are available, sensitivity of
probes can be increased through PCR/FISH or RT-PCR/FISH. This can
be accomplished by incorporating a semiconductor nanocrystal-dNTP
into the in situ PCR or RT-PCR reaction. (see Example 9; Patterson
et al. (1993) Science 260:976-979; Patterson et al. (1998)
Cytometry 31:265-274). The detection system may be a microscope, or
flow cytometer, or detector capable of measuring the wavelength of
light emitted from the different semiconductor nanocrystals.
[0191] FISH technologies are widely used in research and clinical
molecular cytogenetics, pathology and immunology laboratories.
Semiconductor nanocrystal-DNA probes can be use to detect
amplification (e.g., HER2/neu, c myc genes amplification), addition
(e.g., trisomy 21, 13, 18), deletion (e.g., 45X, Turner's
Syndrome), translocation (e.g., BCR/ABL in CML) of DNA in the
nuclei.
[0192] Semiconductor nanocrystal-RNA probes can be used to localize
and to monitor expression of genes (mRNA) in the cell. This is
especially useful for detecting rare cells (e.g. fetal cells in
maternal blood, cancer cells for monitoring disease
recurrence).
[0193] In the case where only forward and reverse primers (with or
without semiconductor nanocrystals attached) are available,
PCR/FISH or RT-PCR/FISH can be used to amplify the target DNA or
RNA to increase sensitivity. This can be accomplished by
incorporating a semiconductor nanocrystal-dNTP into the in situ PCR
or RT-PCR reaction.
[0194] Semiconductor nanocrystals can be conjugated to antibodies,
to a protein of interest (antigen) to detect protein expression
and/or sort out cells of interest. Multiple semiconductor
nanocrystals-antibody(ies), semiconductor nanocrystals-nucleic acid
probes for RNA and or DNA can be used to hybridize with cells in
the same or sequential reaction. Cells of interest in the
population (rare cells) can then be identified and analyzed for DNA
(for genetic composition) or mRNA (for gene expression)
simultaneously.
[0195] Specimens for FISH Assays: Specimens can be cells (alive or
fixed) or nuclei in suspension or attached to microscope slides or
other solid supports or paraffin embedded tissue sections
containing one, or more than one specimen, or frozen tissue
sections or fine needle aspirate. FISH can be performed on
metaphase or interphase cells or directly onto DNA strands.
[0196] The specimen for the FISH assay is prepared using well known
methods depending on the specimen type, for example: peripheral
blood (Hack et al., eds., (1980), supra; Buno et al. (1998), supra;
Patterson et al. (1993), supra; Patterson et al. (1998), supra;
Borzi et al. (1996), supra); bone marrow (Dewald et al. (1993),
supra; Hack et al., eds., (1980), supra); amniocytes (Ward et al.
(1993), supra; Jalal et al. (1998), supra); CVS (Zahed et al.
(1992), supra); paraffin embedded tissue sections (Kitadai et al.
(1995); supra; Neuhaus et al. (1999), supra); fetal cells (Wachtel
et al. (1998), supra; Bianchi (1998), supra); and blastomeres
(Munne (1998), supra).
[0197] Optimization of Conditions for FISH Assays: The conditions
of the FISH assays exemplified below can be optimized depending on
the nature of different probes (e.g., whether the nucleic acid
sequence is highly repetitive or not) or mixtures of probes. The
assays are carried out at room temperature to 100.degree. C. but
are typically carried out in the range of 37.degree. C. to
80.degree. C.
[0198] Semiconductor Nanocrystals as Detection Reagents in Signal
Amplification Assays
[0199] In yet another embodiment of the invention, a method is
disclosed for using semiconductor nanocrystals as a
signal-generating label and semiconductor nanocrystal conjugates as
the detection reagent in signal amplification assay formats. This
type of signal amplification provides several advantages over
currently employed methods for detecting the signal in
signal-amplification assays. Among these advantages is the ability
to detect multiple analytes in the same sample simultaneously with
high sensitivity.
[0200] Semiconductor nanocrystals of one or more colors are
individually conjugated to distinct molecules (a "semiconductor
nanocrystal-conjugate") that specifically recognize an
amplification complex generated in response to the presence of an
analyte in a sample. A semiconductor nanocrystal-conjugate can be,
for example, the label in 1) a DNA hybridization assay, (see
Example 10), or 2) a biotin/avidin-layered amplification assay (see
Example 11). The detection system is a device capable of measuring
and distinguishing the wavelength of light emitted from
semiconductor nanocrystals of one or more colors.
[0201] Semiconductor Nanocrystals for use in Multiplexed, Single
Tube Assays
[0202] In still another embodiment of the invention, an HTS assay
using semiconductor nanocrystals as multiplexed detection reagents
is provided. Semiconductor nanocrystals of a particular color are
conjugated by one of the techniques described in Bruchez et al.
supra, Bruchez, supra, Chan et al., supra, or by any technique
known in the art for attaching or conjugating proteins, nucleic
acids, and the like. See, e.g., Hermanson (1996) Bioconjugate
Techniques (Academic Press).
[0203] The HTS assay is performed in the presence of various
concentrations of a candidate compound. The semiconductor
nanocrystal emission is monitored as an indication of the effect of
the candidate compound on the assay system. This technique is
amenable to any of the conventional techniques with the exception
of chemiluminscence. For example, fluorescence reading using a
semiconductor nanocrystal-conjugated ligand or receptor to monitor
binding thereof to a bead-bound receptor or ligand, respectively,
may be used as a flexible format to measure the semiconductor
nanocrystal emission associated with the beads. The measure of
semiconductor nanocrystal emission associated with the beads can be
a function of the concentration of candidate compound and, thus, of
the effect of the candidate compound on the system. In addition,
semiconductor nanocrystals can be used as a multicolor scintillant
to detect the binding of a radiolabeled ligand or receptor with a
semiconductor nanocrystal-conjugated receptor or ligand,
respectively. A decrease in scintillation would be one result of
inhibition by the candidate compound of the ligand-receptor pair
binding.
[0204] Semiconductor Nanocrystals for use in High-throughput
Sequence Analyses
[0205] Semiconductor nanocrystals conjugated to nucleic acids may
be used in high-throughput DNA sequencing and DNA fragment
analysis. To describe these sequencing reactions briefly, four
reactions are performed to determine the positions of the four
nucleotide bases within a DNA sequence. Using a DNA sample as a
template, a chain of DNA is synthesized from a pool of nucleotides
containing the four deoxynucleotides and one additional
dideoxynucleotide. For example, in the adenine sequencing reaction,
DNA is synthesized from a mixture that includes all four
deoxynucleotides (dATP, dGTP, dCTP, dTTP) plus dideoxyadenosine
triphosphate (ddATP). The enzyme DNA polymerase will synthesize the
new chain of DNA by linking dNTPs. Occasionally DNA polymerase will
incorporate a ddATP instead of a dATP. The ddATP in the nascent
chain will then terminate the synthesis of that chain of DNA due to
the lack of the 3' hydroxyl group as a connection to the next dNTP.
Thus the DNA products from the adenine sequencing reaction will be
a heterogeneous mixture of DNA that vary in length with each chain
terminated at a position corresponding to adenine.
[0206] The four DNA sequencing reactions are resolved by size by
polyacrylamide gel electrophoresis. With singly radiolabeled
(.sup.32p or .sup.35S) DNA, the four reactions are loaded into four
individual lanes. The resolved products of differing sizes result
in a pattern of bands that indicate the identity of a base at each
nucleotide position. This pattern across the four lanes can be read
like a simple code corresponding to the nucleotide base sequence of
the DNA template. With fluorescent dideoxynucleotides, samples
containing all four dideoxynucleotide chain-terminating reactions
can be loaded into a single lane. Resolution of the four
dideoxynucleotide reactions is possible because of the different
fluorescent labels for each sample. For example, ddATP can be
conjugated with a green fluorescent tag. The other three ddNTP
(dideoxynucleotide triphosphate) are tagged with three different
fluorescent colors. Thus, each chain-terminating ddNTP is coded
with a different color. When all four reactions are resolved in one
lane on a DNA sequencing gel, the result is one ladder of bands
having four different colors. Each fluorescent color corresponds to
the identity of the nucleotide base and can be easily analyzed by
automated systems.
[0207] However as previously discussed, multiple light sources are
needed for excitation of the four different fluorescent organic dye
markers. The use of semiconductor nanocrystals as the fluorescent
tags for each dideoxynucleotide chain-terminating reaction
simplifies the automation of high-throughput DNA sequencing since
only a single light source is needed to excite all four fluorescent
tags. In addition, multiplexing with semiconductor nanocrystals
permits multiple sequencing reactions to be conducted and analyzed
simultaneously, thereby further increasing the throughput of the
assay.
[0208] In PCR (polymerase chain reaction)-based DNA typing and
identification, short tandem repeat (STR) loci in the human genome
are amplified by PCR using primers that are labeled with
fluorescent tags. The size of these loci can differ or can coincide
from person to person, or from individual subject to individual
subject, and depends on genetic differences in the population.
Usually multiple loci are examined. Any locus that shows a size
difference with another sample conclusively indicates that the two
samples are derived from two different individuals. However,
demonstrating that two samples originate from the same individual
is less conclusive. Unlike fingerprint patterns, the size of STR
loci can coincide between two individuals. However, the statistical
probability of multiple loci coinciding in size between two
individuals decreases as the number of loci examined is increased.
Using conventional organic fluorescent dyes, a limitation to the
number of samples resolved in a single lane (and thus
high-throughput) is the number of the fluorescent tags available
and the resolution of the emission spectra. Increasing the
resolution of the fluorescent tags thus would increase the capacity
of the number of loci tested per lane on a gel.
[0209] Semiconductor Nanocrystals as Detection Reagents in
DNA-based Assays
[0210] In still further embodiments of the invention, semiconductor
nanocrystals can be used in other hybridization formats, sequence
specific extension and oligo ligation assays, for detecting the
presence/absence of specific DNA target sequences.
[0211] DNA Hybridization: The single strands within DNA helices are
held together by virtue of the hydrogen bonds between complementary
bases in each strand. When double-stranded DNA ("dsDNA") is
subjected to physical/chemical conditions which disrupt hydrogen
bonds (i.e., denature the DNA), the strands separate into
single-stranded ("ssDNA") molecules. When a mixture of different
dsDNA molecules is denatured and then returned to conditions in
which hydrogen bonds between complementary bases can reform, many
of the complementary ssDNA strands will "find" each other and
hybridize (reanneal) to form the original dsDNA molecules. The
process of nucleic acid hybridization serves as the basis for
countless diagnostic tests: e.g., Dot Blots, Southern Blots,
Northern Blots, and FISH Analysis.
[0212] Denaturation: DNA duplexes are held together by hydrogen
bonds between complementary base pairs as well as by hydrophobic
interactions between the stacked bases. These associations can be
disrupted (i.e., DNA can be denatured, or "melted") by raising the
temperature, decreasing the salt concentration, increasing the
alkalinity, or by adding various organic reagents. Since G-C base
pairs form three hydrogen bonds, whereas A-T base pairs form two
hydrogen bonds, A/T-rich DNA helices melt (denature) at lower
temperatures and/or higher salt concentrations than G/C-rich
sequences. The amount of dsDNA that has become denatured
(single-stranded, ssDNA) can be determined by a variety of physical
and chemical means. The temperature at which 50% of the total base
pairs within the dsDNA molecules in a solution have become
disrupted is called the melting temperature (Tm).
[0213] Renaturation ("reannealing," "hybridization"): The stability
of nucleic acid hybrids is dependent on environmental conditions;
two of the most important of which are temperature and salt
concentration. In general, DNA duplexes are more stable at lower
temperatures and higher salt concentrations. When denatured DNA is
cooled or subjected to higher salt concentrations, it will
spontaneously renature. Under ideal renaturation conditions and
given sufficient time, mixtures of single-stranded fragments of DNA
will reassociate (hybridize) with their complementary sequences.
This property of nucleic acids is the basis upon which the assays
disclosed herein are founded.
[0214] Stringency of Hybridization: When ssDNA molecules form
hybrids, the base sequence complementarity of the two strands does
not have to be perfect. Poorly matched hybrids (i.e. hybrids in
which only some of the nucleotides in each strand are aligned with
their complementary bases so as to be able to form hydrogen bonds)
can form at low temperatures, but as the temperature is raised (or
the salt concentration lowered) the complementary base-paired
regions within the poorer hybrids dissociate due to the fact that
there is not enough total hydrogen bond formation within the entire
duplex molecule to hold the two strands together under the new
environmental conditions. The temperature and/or salt
concentrations may be changed progressively so as to create
conditions where an increasing percentage of complementary base
pair matches is required in order for hybrid duplexes to remain
intact. Eventually, a set of conditions will be reached at which
only perfect hybrids can exist as duplexes. Above this stringency
level, even perfectly matched duplexes will dissociate. The
stringency conditions for each unique fragment of dsDNA in a
mixture of DNA depends on its unique base pair composition. The
degree to which hybridization conditions require perfect base pair
complementarity for hybrid duplexes to persist is referred to as
the "stringency of hybridization." Low stringency conditions are
those which permit the formation of duplex molecules having some
degree of mismatched bases. High stringency conditions are those
which permit only near-perfect base pair-matched duplex molecules
to persist. Manipulation of stringency conditions is key to the
optimization of sequence specific assays.
[0215] Nucleic acid probes: For each of the assay procedures
described it is necessary to have one or more nucleic acid
fragments that have been labeled with semiconductor nanocrystals,
so that when they bind to the target DNA sequence their presence
can be easily detected. Probes can consist of many nucleic acid
types, depending on subtle aspects of the detection method and
sample to be detected. They may be single-stranded or denatured
double-stranded DNA or RNA. Probes of dozens to several hundred
bases long can be artificially synthesized using oligonucleotide
synthesizing machines, or they may be derived from various types of
DNA cloning. The critical aspects are that the probe must contain a
nucleic acid strand that is at least partially complementary to the
target sequence to be detected, and the probe must be labeled with
the semiconductor nanocrystals of the invention so that its
presence can be visualized.
[0216] Semiconductor Nanocrystals for use in Spectral
Karyotyping
[0217] Spectral karyotyping is a FISH technique used on metaphase
preparations of chromosomes. See, e.g., Schrock et al. (1996)
Science 273:494-497; and Macville et al. (1997) Histochem. Cell.
Biol. 108:299-305. In spectral karyotyping and fluorescence in-situ
hybridization (FISH) measurements, chromosomes are stained using
combinations of dyes at different wavelengths and ratios. This acts
to "barcode" different chromosomes for detection. For example,
chromosome analysis is typically done by staining (e.g., Giesma
stain). In a similar way, semiconductor nanocrystals can be used to
barcode chromosomes using the techniques described above. In
particular, as explained above, an arbitrary number of codes can be
generated without the need to control the intensity of any spectral
peak within the code. In this case, DNA probes are labeled with
semiconductor nanocrystals that emit different colors to "paint"
different chromosomes. Since the chromosomes are spatially
separated during spectral karyotyping measurements, a barcoding
system can be used to label each chromosome. The chromosomes act as
the solid support for the coding semiconductor nanocrystal. By
using this type of coding system, it is possible to generate an
arbitrary number (more than 48) of unambiguous codes for spectral
karyotyping.
[0218] In addition to barcoding entire chromosomes, it is also
possible to barcode sections of a chromosome. In this way, this
technique can replace traditional chromosome staining.
[0219] Normally stains are used to label different spatially
distinct regions of a chromosome. Different colors are used to
stain different regions. With semiconductor nanocrystals, different
sections on each chromosome can be painted with different barcodes.
In this case, the spatial locations on the chromosome act as the
substrate for different codes. This allows a very large number of
regions to be labeled simultaneously.
[0220] Semiconductor nanocrystal-labeled probes for chromosome
painting can be generated using techniques well known in the art,
such as, but not limited to, micro-dissected PCR DNA or cloned DNAs
(eg. from YACS, BACS, PACs etc.).
[0221] III. Experimental
[0222] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0223] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
[0224] Reagents for use in the following examples may be purchased
from commercial sources, and used according to the manufacturers'
directions. In the cloning of DNA fragments, except where noted,
all DNA manipulations are done according to standard procedures.
See, e.g., Sambrook et al., supra. Restriction enzymes, T4 DNA
ligase, E. coli, DNA polymerase I, Klenow fragment, and other
biological reagents are purchased from commercial suppliers and
used according to the manufacturers' directions.
[0225] For the following examples, water-soluble core-shell
nanocrystals are synthesized as described in Bruchez et al. (1998)
Science 281:2013-2016. Affinity molecules are linked directly or
indirectly to a semiconductor nanocrysfal using chemical techniques
well known in the art. See, e.g., U.S. Pat. No. 5,990,479 to
Alivisatos et al; Bruchez et al. (1998) supra; and Chan et al.
(1998) Science 281:2016-2018; Haugland, supra; and Hermanson
"Bioconjugate Techniques" (Academic Press, NY).
EXAMPLE 1
Sandwich QISA
[0226] This example describes a sandwich QISA in which an
analyte-specific antibody, or a plurality of different
analyte-specific antibodies each of which is capable of
specifically recognizing and binding to a distinct analyte, are
immobilized on a solid support and analytes bound thereto are
detected using one or a plurality of semiconductor
nanocrystal/analyte-specific antibody conjugates.
[0227] A. A primary antibody (species 1 as illustrated in FIG. 1A)
specific for the analyte to be detected in the sample is
immobilized on a solid support, e.g., a well of a 96-well plate,
using well-established techniques. The concentration of
support-bound primary antibody is selected to be appropriate for
the assay (1 ng/ml to 10 mg/ml). Optionally, the support is washed
to remove unbound antibody.
[0228] Any portion of the solid support to which no antibody is
bound is treated with an agent to reduce nonspecific binding
thereto, e.g., 1% BSA+1% antibody species 2 antiserum (e.g., an
antibody that is distinct from the first capturing antibody
species) in phosphate-buffered saline (PBS). Optionally, the
support is washed to remove excess blocking agent.
[0229] A sample containing or suspected of containing the analyte
of interest is added to the treated solid support and incubated for
a time (30 seconds to 48 hours) sufficient to bind the analyte to
the primary antibody. Optionally, the support is again washed to
remove nonspecific and unbound analyte molecules in the sample.
[0230] A secondary antibody (species 3 distinct from the primary
antibody) conjugated to a single population of the same composition
of semiconductor nanocrystals is added at a known concentration (1
ng/ml to 10 mg/ml) and incubated for a time (30 seconds to 48
hours) sufficient to bind the secondary antibody to the
support-bound analyte. Optionally, the support is washed to remove
unbound semiconductor nanocrystal-conjugated secondary
antibody.
[0231] The results of assay are determined by measuring the
intensity of light emitted from the semiconductor nanocrystals
specifically bound to the solid support at wavelengths
corresponding to the semiconductor nanocrystals component of the
conjugate.
[0232] In particular, a QISA was performed by coating the wells of
a 96-well plate with 10 .mu.g/ml of rabbit IgG in PBS. After
coating, the plate was washed and unbound sites were blocked with
BSA. The plates were blocked for one hour at room temperature prior
to washing and concentrations of biotinylated goat anti-rabbit IgG
ranging from 0.1 nM to 100 nM (in PBS) were added. This was
incubated in the wells for one hour at room temperature and then
washed. 100 nM nanocrystal conjugates (streptavidin conjugates)
were added which had an emission peak at 580 nm. The nanocrystal
conjugates were incubated in the wells for one hour at room
temperature and then washed. The plate was read on a Molecular
Dynamics spectramax Gemini plate reader.
[0233] Results are shown in FIG. 2. As can be seen, fluorescent
intensity increased with the concentration of biotinylated antibody
used, demonstrating the usefulness of semiconductor nanocrystals in
this context.
[0234] B. A sample can be analyzed simultaneously for the presence
of more than one analyte in a single assay using a sandwich QISA as
disclosed herein. A species of primary antibody capable of
recognizing and specifically binding to one of each of the analytes
of interest is bound to the solid support, the support is,
optionally, washed, blocked and washed again, as described above.
The sample is then added to the support and incubated for a time
sufficient for each analyte to be recognized by and bind to its
corresponding primary antibody. The support is, optionally, washed
to remove unbound sample. A plurality of distinct secondary
antibodies, each of which is capable of recognizing and binding to
one of each of the analytes of interest and is conjugated to a
distinct species of a semiconductor nanocrystal. Each distinct
species of a semiconductor nanocrystal has distinctly detectable
emission properties
[0235] The support-bound semiconductor nanocrystal-antibody
conjugates are simultaneously irradiated with blue light and the
plurality of emission wavelengths corresponding to each species of
semiconductor nanocrystal is monitored using an appropriate
apparatus.
EXAMPLE 2
Direct Capture QISA
[0236] This example describes a direct capture QISA in which a
sample containing or suspected of containing an analyte of interest
or a plurality of different analytes of interest, is adsorbed onto
a solid support and adsorbed analytes are detected using one or a
plurality of semiconductor nanocrystal/analyte-specific conjugates,
respectively.
[0237] A sample is adsorbed onto a solid support using
well-established techniques for a time sufficient to effect
adsorption (30 seconds to 48 hours). Optionally, the support is
washed to remove unbound molecules. Nonspecific binding is
minimized by treating the solid support to which sample has been
adsorbed with an appropriate blocking agent, e.g., 1% BSA+1% serum
(same species as primary antibody) in PBS. The support is again,
optionally, washed to remove excess blocking agent,
[0238] For detecting a single analyte, analyte-specific antibody
conjugated to a single population of the same composition of
semiconductor nanocrystals is added at a known concentration (1
ng/ml to 10 mg/ml) and incubated with the sample-adsorbed solid
support for a time sufficient for the antibody to recognize and
bind to its corresponding antibody (30 seconds to 48 hours). The
support is, optionally, washed to remove unbound semiconductor
nanocrystal-conjugated antibody.
[0239] Results of the assay are determined by measuring the
intensity of light emitted at a wavelength that correspond to the
semiconductor nanocrystal in the conjugate.
[0240] In a manner similar to that described in Example 1B, the
direct capture QISA can be adapted to detect multiple analytes in a
single sample by using a plurality of primary antibody species,
each of which is conjugated to a semiconductor nanocrystal with
distinctly detectable emission properties. The support-bound
semiconductor nanocrystal-antibody conjugates are simultaneously
irradiated with blue light and the plurality of emission
wavelengths corresponding to each semiconductor nanocrystal is
detected using an appropriate apparatus.
EXAMPLE 3
Fluid-Phase QISA
[0241] This example describes a fluid-phase QISA in which a
fluid-phase sample containing or suspected of containing an analyte
of interest or a plurality of different analytes of interest, is
adsorbed onto a solid-phase substrate and adsorbed analytes are
detected using one or a plurality of semiconductor
nanocrystal/analyte-specific antibody conjugates.
[0242] A sample containing or suspected of containing analyte is
adsorbed onto, for example, latex microspheres (see, e.g., Bangs
Laboratories, Inc., TechNote#25) by incubating the microspheres
with the sample for an appropriate amount of time (30 seconds to 48
hours). Optionally, the microspheres are washed, e.g., by
centrifugation, to remove unbound sample. Microspheres to which
analyte have been adsorbed are incubated with semiconductor
nanocrystal-conjugated analyte-specific antibody (1 ng/ml to 10
mg/ml) for a period of time sufficient to allow binding of the
antibody to the adsorbed analyte (30 seconds 48 hours). Optionally,
the microspheres are washed to remove unbound semiconductor
nanocrystal-conjugated antibodies.
[0243] The amount of semiconductor nanocrystal fluorescence
associated with the microspheres can be detected using flow
cytometry or a static fluorimeter capable of distinguishing the
different spectrum of emissions from the semiconductor nanocrystal
conjugates.
[0244] In a manner similar to that described in Example 1B, the
fluid-phase QISA can be adapted to detect multiple analytes in a
single sample by using a plurality of primary antibody species,
each of which is conjugated to a semiconductor nanocrystal with
distinctly detectable emission properties. The support-bound
semiconductor nanocrystal-antibody conjugates are irradiated with
blue light and the plurality of emission wavelengths corresponding
to each semiconductor nanocrystal is detected using flow cytometry
or a static fluorimeter as described above.
EXAMPLE 4
Fluid-Phase OISA
[0245] This example describes a fluid-phase QISA in which a
fluid-phase sample containing or suspected of containing an analyte
of interest or a plurality of different analytes of interest, is
incubated with and bound to a primary antibody, or a plurality of
primary antibodies capable of specifically recognizing and binding
to an analyte. The primary antibody is immobilized on a
microsphere. Bound analyte or analytes are detected using one or a
plurality of semiconductor nanocrystal/analyte-specific antibody
conjugates.
[0246] A primary polyclonal or monoclonal antibody (species 1)
specific for the analyte to be detected in the sample is
immobilized onto microspheres using well-established techniques.
The concentration of primary antibody should be appropriate for the
assay (1 ng/ml to 10 mg/ml), as described in Example 1. Optionally,
the microspheres are washed to remove unbound primary antibody.
[0247] A sample containing or suspected of containing an analyte is
incubated with microspheres to which have been conjugated an
analyte-specific antibody for a period of time (30 seconds to 48
hours) sufficient to effect binding of an analyte to its respective
antibody. Optionally, the microspheres are washed to remove unbound
sample and/or analyte.
[0248] Primary antibody-microsphere conjugates which have been
incubated with the sample are then incubated with a semiconductor
nanocrystal/analyte-specific secondary antibody conjugates at a
concentration (1 ng/ml to 10 mg/ml) and for a period of time (30
seconds to 48 hours) sufficient to effect binding of the
semiconductor nanocrystal-conjugated to the microsphere-immobilized
analyte. Optionally, the microspheres are washed to remove unbound
semiconductor nanocrystal-conjugated antibodies.
[0249] The amount of semiconductor nanocrystal fluorescence
associated with the microspheres can be detected using flow
cytometry or a static fluorimeter capable of distinguishing the
different spectrum of emissions from the semiconductor nanocrystal
conjugates.
[0250] In a manner similar to that described in Example 3, the
fluid-phase QISA can be adapted to detect multiple analytes in a
single sample by using a plurality of primary antibody species,
each of which is specific for a preselected analyte, conjugated to
a microphere, and a plurality of semiconductor
nanocrystal/analyte-specific secondary antibody conjugates, in
which each conjugate contains a semiconductor nanocrystal having
distinctly detectable emission properties. The support-bound
semiconductor nanocrystal-antibody conjugates are irradiated with
blue light and the plurality of emission wavelengths corresponding
to each semiconductor nanocrystal is detected using flow cytometry
or a static fluorimeter as described above.
EXAMPLE 5
DNA-based FISH Assay
[0251] The specimen for FISH assay is prepared using various
published methods depending on the specimen type, as described
above.
[0252] A. Double-stranded specimen DNA and semiconductor
nanocrystal-FISH probe(s) can be denatured together or separately
to their single-stranded form. Denaturation of semiconductor
nanocrystal-FISH probes consisting of synthetic oligonucleotides
without secondary structure is not necessary. The specimen and
probes in single-stranded form are allowed to hybridize to their
complimentary sequence in hybridization buffer optionally
containing blocking DNA (5 minutes to overnight at 25.degree.
C.-70.degree. C.). The excess semiconductor nanocrystal-FISH probes
are removed using a series of stringent washes. (e.g.,
0.4.times.SSC to 2.times.SSC with or without formamide at
25.degree. C.-72.degree. C.). Optionally, the specimen is air dried
and counterstained to localize the nuclei.
[0253] More particularly, semiconductor nanocrystal-FISH probe(s)
is added to the hybridization mix containing 50% formamide, 10%
dextran sulphate, 1.times.SSC (saline citrate), 3 .mu.g Cot1 DNA.
The probe mixture is deposited on the slide and a coverslip is
placed on the probe. The slide is placed on a hot plate for 3
minutes at 72.degree. C. to denature the specimen and probe DNA.
The specimen and probes in single-stranded form are allowed to
hybridize to their complimentary sequence for 5 minutes to
overnight at 25.degree. C. to 45.degree. C. The excess
semiconductor nanocrystal-FISH probes are removed by washing the
slide in 2.times.SSC, 0.3% NP-40 for 2 minutes. The specimen is air
dried and counterstained to localize the nuclei.
[0254] The fluorescence signals of the semiconductor
nanocrystal-FISH probes is viewed under an epi-fluorescence
microscope. Alternatively, the signals are measured using a laser
detector.
[0255] B. Whole Chromosome Paints:
[0256] Human chromosomes 1, 2, and 3 were each microdissected, PCR
amplified and labeled with biotin-dUTP as described in Meltzer et
al. (1992) Mat. Genet. 1:24-28. Slides with metaphase spreads from
peripheral blood lymphocytes were prepared using conventional
cytogenetics techniques and denatured for 2 minutes at 72.degree.
C. in 70% formamide, 2.times.SSC (saline citrate). The slides were
then dehydrated 1 minute each with a series of 70%, 85% and 100%
ethanol and air-dried. Hybridization mixture (10 ng/.mu.l DNA of
each whole chromosome probe in 50% formamide, 10% dextran sulphate,
1.times.SSC, 3 .mu.g Cotl DNA) was denatured for 5 minutes at
72.degree. C. and placed on the slides. A cover slip was placed on
each slide and sealed with rubber cement. The slides were incubated
at 37.degree. C. overnight in a humidified chamber. After
hybridization, the excess probes were washed away three times with
2.times.SSC, 50% formamide at 42.degree. C. for 3 minutes each,
followed by a rinse with 2.times.SSC at ambient temperature for 3
minutes. The slides were then blocked with 4.times.SSC, 0.1% Tween
20, 1% BSA (Bovine Serum Albumin) for 30 minutes to prevent
nonspecific binding. Finally the slides were incubated with 40 nM
of 630 nm semiconductor nanocrystal-streptavidin in PBS (Phosphate
buffered saline), 1% BSA, 10 mM MgCl.sub.2 at ambient temperature
for 1 hour. The excess semiconductor nanocrystal-streptavidin and
salts were removed by rinsing the slides in PBS and in 10 mM
Phosphate buffer. The slides were then air dried and examined with
a fluorescence microscope.
[0257] Chromosome pairs 1, 2 and 3 were readily visualized using
the fluorescence microscope, demonstrating the utility of using
semiconductor nanocrystals in this context.
[0258] C. Fiber FISH
[0259] Semiconductor nanocrystals were used in a Fiber FISH format
essentially as described by Heiskanen et al. (1994) Biotechniques
17:928-933; and Heiskanen et al. (1995) Genomics 30:31-36. In
particular, a 10 kb DNA probe was labeled by nick-translation using
biotin-11-dUTP (Sigma Chemical). Fiber FISH slides were prepared as
described in Heiskanen et al., supra. The slides were denatured in
70% formamide, 2.times.SSC at 72.degree. C. for 4 minutes followed
by dehydration with a series of 70%, 85% and 100% ethanol. Biotin
labeled DNA probes 2.5-7.5 ng/.mu.l were added to hybridization mix
containing 50% formamide, 10% dextran sulfate, 2.times.SSC, 0.2
.mu.g/.mu.l herring sperm DNA, 0.25 .mu.g/.mu.l Cot-1 DNA. The
mixture was placed on slides, a coverslip was placed over each
slide and hybridized overnight at 37.degree. C. in a humidified
chamber. Excess probes were removed by washing the slides three
times with 50% formamide, 2.times.SSC for 5 minutes each, two times
with 2.times.SSC for 5 minutes each and a final wash in
0.5.times.SSC for 5 minute all at 45.degree. C. Slides were
incubated in 5% BSA, 4.times.SSC, 0.05% Tween 20 for 15 minute at
37.degree. C. to block nonspecific binding. Biotinylated probes
were detected by incubating in 40 nM semiconductor
nanocrystal-streptavidin in PBS, 1% BSA and 10 mM MgCl.sub.2,
followed by biotinylated anti avidin antibody and another layer of
semiconductor nanocrystal-streptavidin conjugate. The slides were
rinsed in PBS, stained with 5 ug/ml DAPI (Sigma Chemical) and
examined in a fluorescence microscope.
[0260] The DNA probes were readily visualized using a fluorescence
microscope, demonstrating the utility of semiconductor nanocrystals
for physical mapping positional cloning.
EXAMPLE 6
RNA-based FISH Assay
[0261] The specimen for FISH assay is prepared using various
published methods depending on the specimen type, as described
above.
[0262] Semiconductor nanocrystal-FISH probe(s) for RNA are
denatured to their single-stranded form. The specimen and probes in
single-stranded form are allowed to hybridized to their
complimentary sequence in hybridization buffer optionally
containing blocking DNA (e.g., 5 minutes to overnight at 25.degree.
C.-70.degree. C.).
[0263] The excess semiconductor nanocrystal-FISH probes are removed
using a series of stringent washes (e.g. 0.4.times.SSC to
2.times.SSC with or without formamide at 25.degree. C.-72.degree.
C.). Optionally, the specimen are air dried and counterstained to
localize the nuclei.
[0264] The fluorescence signals of the semiconductor
nanocrystal-FISH probes are viewed under an epi-fluorescence
microscope. Alternatively, the signals can be measured using a
laser detector.
[0265] In particular, synthetic oligonucleotide probes directed
against a certain mRNA are labeled with semiconductor nanocrystals
at the 3' or 5' end. The probes are added to the hybridization mix
containing 50% formamide, 10% dextran sulphate, 1% sarkosyl, 0.02 M
sodium phosphate, 4.times.SSC, 1.times.Denhardt's solution and 10
mg/ml ssDNA. The probe mixture is added to the specimen, a
coverslip is placed over the probe, and the probe and mRNA are
allowed to hybridized for 16-20 hours at 42.degree. C. in a
humidified chamber. After hybridization, the excess probes are
removed by washing several times in 1.times.SSC at 55.degree. C.
and air-dried. The slides are then examined under a fluorescence
microscope.
EXAMPLE 7
Multiplexed DNA- and RNA-based FISH Assay
[0266] The specimen for FISH assay is prepared using various
published methods depending on the specimen type, as described
above.
[0267] Double-stranded specimen DNA and semiconductor
nanocrystal-FISH probe(s) can be denatured together or separately
to their single-stranded form.
[0268] The specimen and semiconductor nanocrystal-FISH probes for
both RNA and DNA in single-stranded form are allowed to their
complimentary sequence in hybridization buffer optionally
containing blocking DNA (5 minutes to overnight at 25.degree.
C.-70.degree. C.).
[0269] The excess semiconductor nanocrystal-FISH probes for RNA and
DNA are removed using a series of stringent washes (e.g.,
0.4.times.SSC to 2.times.SSC with or without formamide at
37.degree. C.-72.degree. C.).
[0270] A flow cytometer or other similar detector can be used to
scan the specimens to detect the fluorescence from the
semiconductor nanocrystal RNA probe and the cell's DNA fluorescence
signals are detected simultaneously. Alternatively, the rare cells
are sorted aside for further DNA analysis using a microscope.
[0271] For example, semiconductor nanocrystal-FISH probes for DNA
and RNA are added to the hybridization mix containing 50%
formamide, 10% dextran sulphate, 1-4.times.SSC (saline citrate), 3
.mu.g Cot1 DNA, 10 mg/ml ssDNA. The probe mixture is deposited on
the slide and a coverslip is placed on the probe. The slide is
placed on a hot plate for 3 minutes at 72.degree. C. to denature
the specimen and probe DNA. The specimen and probes in
single-stranded form are allowed to hybridize to their
complimentary sequence overnight at 25.degree. C. to 45.degree. C.
After hybridization, the excess probes are removed by washing
several times in 1.times.SSC at 55.degree. C. and air-dried. The
specimen is counterstained to localize the nuclei.
[0272] The fluorescence signals of the semiconductor
nanocrystal-FISH probes are viewed under an epi-fluorescence
microscope. Alternatively, the signals are measured using a laser
detector.
EXAMPLE 8
Immunostaining and FISH Assays
[0273] Specimen cells are labeled with optimized concentration of
semiconductor nanocrystals-antibody(ies) or antibody with other dye
label specific to cell surface protein of interest (e.g. anti-CD4
or anti CD14).
[0274] The cells are fixed and permeabilized by addition of
reagents such as PERMEAFIX.RTM. (Ortho Diagnostics). After
incubation, the cells are pelleted and washed.
[0275] The cell pellet is resuspended for FISH assay with
hybridization buffer containing single-strand semiconductor
nanocrystal-RNA probes optionally containing blocking DNA. The
mixture is allowed to hybridized to their complimentary sequence (5
minutes to overnight at 25.degree. C.-70.degree. C.).
[0276] The excess semiconductor nanocrystal-FISH probes are removed
using a series of stringent washes (e.g., 0.4.times.SSC to
2.times.SSC with or without formamide at 37.degree. C.-72.degree.
C.).
[0277] A flow cytometer or other similar detector can be used to
scan the specimens to sort and/or to detect the surface
immunofluorescence and fluorescence from the semiconductor
nanocrystal RNA probe simultaneously.
[0278] In particular, specimen cells are fixed and permeabilized by
addition of reagents such as Penneafix (Ortho Diagnostics). After
incubation the cells are pelleted and washed. The cell pellet is
resuspended for FISH assay in hybridization buffer. The
hybridization mixture contains semiconductor nanocrystal-FISH
probes for DNA or RNA, 50% formamide, 10% dextran sulphate,
14.times.SSC, 3 .mu.g Cot1 DNA (for DNA probes) and or 10 mg/ml
ssDNA(for RNA probes). The mixture is allowed to denatured for 2
minutes at 72.degree. C. and hybridized overnight at 25.degree.
C.-45.degree. C. The excess FISH probes are removed by washing
several times in 1.times.SSC at 55.degree. C. Monoclonal antibody
from mouse to a protein of interest is used to bind the specimen
cells using standard immunohistochemistry procedures. The excess
antibody is removed, the specimen is incubated with semiconductor
nanocrystal-labeled anti-mouse IgG. The excess semiconductor
nanocrystal anti-mouse IgG is removed and the specimen cells can be
examined under a fluorescence microscope. Alternatively, a flow
cytometer or other similar detector can be used to scan the
specimens to sort and/or to detect the fluorescence from the
semiconductor nanocrystal-FISH probes and semiconductor
nanocrystal-labeled antibody.
EXAMPLE 9
FISH Assay of Amplified Specimen DNA
[0279] Specimen cells were fixed and permeabilized by addition of
reagents such as Permeafix (Ortho Diagnostics). After incubation
the cells are pelleted and washed.
[0280] The cell pellet is resuspended with PCR reaction mixture
along with optimized concentrations of dNTP and semiconductor
nanocrystal dUTP, semiconductor nanocrystal-forward primer and
semiconductor nanocrystal-reverse primer for DNA or RNA of
interest, Taq polymerase and gelatin.
[0281] The DNA in the above reaction mixture is amplified in a
thermocycler programmed for the specific conditions.
[0282] After in vitro amplification, cells are pelleted and
resuspended in hybridization mix along with semiconductor
nanocrystal-oligonucleotide probe directed against the specific
amplified product and, optionally, blocking DNA to reduce
background noise.
[0283] The product DNA is denatured and allowed to hybridized with
the semiconductor nanocrystal-oligonucleotide probe (5 minutes to
overnight at 25.degree. C.-70.degree. C.).
[0284] The excess semiconductor nanocrystal-oligonucleotide probes
are removed using a series of stringent washes (e.g., 0.4.times.SSC
to 2.times.SSC with or without formamide at 37.degree.
C.-72.degree. C.).
[0285] A flow cytometer, or other similar detector, or epi
fluorescence microscope is used to detect the fluorescence signals
from the semiconductor nanocrystal-oligonucleotide probes.
EXAMPLE 10
Signal Amplification in a DNA Hybridization Assay
[0286] A unique DNA sequence is chosen as a capture probe and is
designed to have a sequence complementary to a sequence in a
capture extender oligonucleotide. This sequence can range from 10
to 25 nucleotides in length. This DNA capture probe sequence is
chemically synthesized and purified.
[0287] The capture probe is conjugated to the surface of a well of
a microtiter plate, using any one of a variety of standard chemical
linkages and methods well known in the art. The analyte is
immobilized on the microtiter support through one or more
oligonucleotides (capture extenders) that have first and second
complementary sequences. The first sequence is complementary to a
sequence in the target analyte and the second sequence is
complementary to a sequence in the support-bound capture probe. The
capture extenders and the sample can be added to the assay
simultaneously. Alternatively, the capture extender can be added
and allowed to hybridize to the complementary sequence in the
support-bound capture probe prior to addition of the sample.
Hybridization is allowed to occur under conditions that favor
hybridization for a period of time sufficient to effect
hybridization of the capture extender to the capture probe and the
target analyte to the capture extender. The assay format can be
designed so that the target analyte will be immobilized to the
solid support only if two capture extenders, in which the first
complementary sequences thereof are complementary to distinct
segments in the target analyte, hybridize to the target. In
addition, the support-bound capture probe and the capture extenders
can be designed so that the second complementary sequence in the
capture extenders are complementary to distinct segments in a
capture probe. Thus, in order to immobilize the target to the solid
support, the second complementary sequences of two distinct capture
extenders hybridize to distinct complementary sequences in a
capture probe and the first complementary sequences of the capture
extenders hybridized to distinct sequences in the target
analyte.
[0288] After hybridization, an optional step is performed under
appropriate stringency conditions to separate material not retained
on the solid support.
[0289] One or more label extender oligonucleotide probes
complementary to the analyte sequence is chosen. Each of these
target probe oligonucleotides contains a second sequence segment
substantially complementary to a nucleic acid sequence within an
amplifier oligo. Hybridization is allowed to occur. After
hybridization, a wash step is performed under appropriate
stringency conditions.
[0290] The amplification oligo contains a sequence complementary to
a portion of each target probe oligo and also contains a
multiplicity of label probe sites. Hybridization is allowed to
occur. After hybridization, a wash step is performed under
appropriate stringency conditions.
[0291] Semiconductor nanocrystal-conjugated oligos which are
complementary to the amplifier oligos are allowed to hybridize to
the amplifier oligos. Hybridization is allowed to occur. After
hybridization, a wash step is performed under appropriate
stringency conditions.
[0292] Finally the well is scanned in a fluorimeter. The amount of
light emitted at the excitation wavelength of the semiconductor
nanocrystals is measured. Light emission at the detection
wavelength is proportional to the amount of target nucleic acid
present.
EXAMPLE 11
Signal Amplification in a Biotin/Avidin-Layered Amplification
Assay
[0293] The sample is immobilized on a solid support by established
techniques. Unbound sample is washed from the solid support.
[0294] A biotin-labeled antibody is reacted with an immobilized
antigen, followed by appropriate washes. Biotin-conjugated
semiconductor nanocrystals are mixed with avidin (or streptavidin)
in specific ratios to form complexes consisting of multiple
inter-linked avidin-semiconductor nanocrystal complexes with some
free biotin-binding sites still available. An aliquot of this
complex is added to the immobilized biotin-labeled antibody/antigen
complex. Unbound complex is washed using appropriate
conditions.
[0295] Result for assay is determined by measuring the intensity of
light emitted at wavelengths that correspond to the semiconductor
nanocrystals used.
EXAMPLE 12
Multiplexed Bead-Based Assay Using Multiple Semiconductor
Nanocrystal-Ligand Receptor Pairs
[0296] 5.times.10.sup.6 10 .mu.m polystyrene-divinylbenzene,
diaminododecane beads are rinsed with ethanol, then twice with
phosphate buffered saline (PBS) with 0.05% NaN.sub.3. They are then
resuspended in 1 ml of this solution, and incubated for one hour
sequentially with each of the following reagents, with two wash
steps between each incubation: 50 .mu.g/ml BSA-Biotin (Bovine serum
albumin-biotinylated) in PBS/ NaN.sub.3, 50 .mu.g/ml streptavidin
in PBS/NaN3, 50 .mu.g/ml of a biotinylated monoclonal antibody
which specifically binds the noninteracting region of the receptors
(e.g., MAb179), and 2 .mu.g/ml of the purified extracellular domain
of the receptor (e.g., IL-5).
[0297] Another 5.times.10.sup.6 10 .mu.m
polystyrene-divinylbenzene, diaminododecane beads are rinsed with
ethanol, then twice with PBS with 0.05% NaN.sub.3. They are then
resuspended in 1 ml of this solution, and incubated for one hour
sequentially with each of the following reagents, with two wash
steps between each incubation: 50 .mu.g/ml BSA-Biotin in
PBS/NaN.sub.3, 50 .mu.g/ml streptavidin in PBS/NaN.sub.3, 50
.mu.g/ml of a biotinylated monoclonal antibody which specifically
binds the noninteracting region of the receptors (e.g., MAb179),
and 2 .mu.g/ml of the purified extracellular domain of the receptor
(e.g., IL-1).
[0298] 2.times.10.sup.6 of each receptor bound bead are thoroughly
mixed into a single tube. This suspension was distributed into 8
wells containing 5000 beads each. 5000 beads of each of the
receptors (IL-1 and IL-5) are put into two further sets of 8 wells,
as shown in FIG. 3.
[0299] IL-1 receptor-antagonist and IL-5 receptor antagonist are
conjugated to red and green semiconductor nanocrystals using
techniques previously described. These are prepared in a stock
solution to 16 nM, then mixed in equal volumes with a dilution
series (0, 1 nM, 10 nM, 100 nM, 1 .mu.M, 10 .mu.M, 100 .mu.M, 1 mM)
of a competing molecule. 50 .mu.l of these solutions are added to
each of the wells, each well 1-8 containing a unique concentration
of the inhibitor (as above), each of the columns A-C (FIG. 3)
having identical conditions and only different beads. After
incubation, the beads from each well are transferred to tubes for
flow cytometric analysis, though other analysis techniques may be
preferable. The cytometer separates the beads into individual
events, allowing the detection of two wavelengths of light (e.g.
the fluorescence from the red and green semiconductor nanocrystals)
simultaneously upon excitation with a 488 nm laser. In this assay,
the ligand can inhibit one, both, or neither of the ligand-receptor
pairs. If the inhibitor is ineffective for both systems, the
results will be uniform across the concentration of inhibitor, and
should vary very little from the control wells. When the inhibitor
is effective in only one of these assays, for instance against IL-1
only, then the wells in column A have decreasing red fluorescence
associated with the beads at higher inhibitor concentrations.
[0300] A plot of the relative fluorescence with respect to the
standard as a function of concentration reveals the concentration
at which the normal receptor-ligand pair binding is inhibited by
50% (this is known as the IC50). In contrast, the wells in column C
(FIG. 3) are unaffected by the presence of the inhibitor at all
concentrations. In the center lane, the true multiplexed assay is
performed, and the results are seen as beads which fluoresce lower
intensities of red at the higher concentrations (due to inhibition
of binding), and beads which fluoresce with a homogeneous intensity
of green (due to the lack of inhibition of the IL-5 interactions).
Homogeneously stained green beads are present throughout each of
the inhibition series of the red emitting beads.
[0301] This format has advantages over standard formats using dye
molecules in that the number may be expanded significantly beyond
two using a variety of different colors (and thus different size or
composition) semiconductor nanocrystals, each conjugated to a
unique receptor or ligand, in order to maximize the throughput in a
single well. Furthermore, the fact that each and every
scintillation event is due to a ligand-receptor pair formation (in
the low concentration limit) makes this a very sensitive technique
for which there are no standard multiplexing methods.
EXAMPLE 13
Use of Semiconductor Nanocrystals in a Homogeneous Scintillation
Inhibition Assay
[0302] When the semiconductor nanocrystals are brought within 1
.mu.m of a radioactive nucleus, they can be made to emit light upon
the decay of that nucleus, which produces high energy radiation,
which is dissipated by the solvent, and can excite the
semiconductor nanocrystals, causing them to emit light. In order to
get the semiconductor nanocrystals to efficiently emit light, it is
necessary to have the nucleus remain in close proximity. This can
be accomplished through ligand-receptor interactions, where one is
radiolabeled, and the other is conjugated to a semiconductor
nanocrystal. This homogeneous assay format is advantageous in that
it allows free association in solution of all the receptor-ligand
pairs, each of which can be prepared to scintillate in a unique
region of the spectrum. Semiconductor nanocrystals which emit in
green, yellow, and red regions of the spectrum, and are well
resolved spectrally are conjugated to three different ligands
(receptor antagonists) to the IL-1, IL-5, and IL-12 receptors
respectively. A solution is prepared which is 1 nM in each of these
semiconductor nanocrystal-receptor antagonist conjugates. Aliquots
are mixed with a concentration series of the inhibitor of interest,
and brought to identical final volumes and concentrations of
RA-semiconductor nanocrystals (0.75 nM each). Radiolabeled
(.sup.3H) IL-1, IL-5, and IL-12 receptor extracellular domains are
prepared by proton exchange, as known in the art. A stock is
prepared which contains 1 nM of all three of these tritiated
proteins in phosphate buffered saline. This stock is added to
result in a final concentration of each receptor of 0.001 nM, a
large excess of ligand over receptor. Scintillation is seen only
from semiconductor nanocrystals which are bound to receptor
antagonist which is not inhibited by the candidate inhibitor. A
lower signal is seen in the characteristic color when the
inhibition is effective. These may be referenced to the control
well without inhibitor.
[0303] Signal may be detected in a dark box with a camera mounted
such that it can image the entire plate. All that is necessary is
for the camera to integrate the collected light for a long period,
as the scintillation is a low background technique. In addition,
multiple filters can be used to separate the signal arising from
each ligand-receptor pair. In FIG. 4, lanes A, B, and C contain
different inhibitors, lane A for IL-1 only, lane B for IL-5 and
IL-12, and lane C for all receptors.
[0304] The unique combination of colors which indicate inhibition
will determine which receptors the candidate may be an appropriate
drug for. One principal advantage of such a system is that it may
be performed with as many ligands as one can produce spectrally
resolved semiconductor nanocrystals. Since the range of 300 nm to
1.5 .mu.m is available for semiconductor nanocrystal manufacture,
well over 20 colors should be available for multiplexing in a
homogeneous reaction format. Furthermore, the fact that each and
every scintillation event is due to a ligand-receptor pair
formation (in the low concentration limit), this is a very
sensitive technique for which there are now no known techniques
that are capable of multiplexing.
EXAMPLE 14
Hybridization Assays
[0305] Hybridization assays are a convenient method for determining
the presence or absence of a specific DNA sequence in a sample.
Semiconductor nanocrystals are used to 5' end label a pair of
allele specific oligonucleotide primers. These primers are used in
PCR or other DNA amplification reaction, such that the target
sequence of interest is amplified, i.e., increased, in number. A
sequence-specific oligonucleotide probe is synthesized, and is
immobilized onto a surface to become a capture probe. The amplified
target is applied to the surface, and allowed to hybridize under
sufficiently stringent conditions of temperature and salt
concentration. After hybridization, unbound material is washed
away. The presence of semiconductor nanocrystals immobilized on the
surface is detected by exciting at an allowed absorbance
wavelength, and measuring at the semiconductor nanocrystal emission
wavelength. The presence of a semiconductor nanocrystal emission on
the surface is directly proportional to the amount of amplified
target DNA present.
[0306] A. A DNA sequence complementary to the target sequence is
chosen. This sequence can range from 15 to 25 nucleotides in
length. This DNA probe sequence is chemically synthesized and
purified. The probe is conjugated to the surface of a well of a
microtiter plate, using any one of a variety of standard chemical
linkages.
[0307] Two PCR primer sequences are selected, such that they
specifically amplify a portion of the target DNA sequence
containing the region to be probed. The 5' end of each of the two
primers is labeled with a semiconductor nanocrystal with peak
emission at 525 nm. These two primers are used to amplify a defined
portion of the target DNA in a standard PCR reaction. Since the
semiconductor nanocrystal is conjugated to the primer, and the
primer is extended to become the amplified target, the
semiconductor nanocrystal is connected to the 5' end of the
amplified target.
[0308] After the thermal cycling the target can be denatured or
made single-stranded by heating it for two minutes at 95.degree.
C.
[0309] The hybridization is performed by adding 20 .mu.l of the
amplified target and 480 .mu.l of 5.times.SSPE Buffer to the
microtiter well coated with DNA probe, and incubating for 60
minutes at 37.degree. C. After hybridization, a wash step under
stringent conditions is performed by adding 500 .mu.l of
3.times.SSPE, and incubating for 15 minutes at 37.degree. C. The
well is rinsed twice with water.
[0310] In places where the amplified DNA hybridized to the
immobilized probe, the semiconductor nanocrystal with 525 emission
is connected to the surface. The well is scanned in a fluorimeter.
UV light is shone upon the well, and the amount of phosphorescence
at 525 nm is measured. The presence of 525 nm emission indicates
that the immobilized probe-captured semiconductor
nanocrystal-labeled amplified target, and remained hybridized after
the stringent wash and water rinse steps.
[0311] B. Three DNA sequences complementary to the three different
target sequences are chosen. These sequences can range from 15 to
25 nucleotides in length. These three DNA probe sequences are
chemically synthesized and purified.
[0312] These three probe sequences are blended, and are conjugated
to the surface of the well of a microtiter plate, using any one of
a variety of standard chemical linkages.
[0313] Two PCR primer sequences are selected for each of three
targets. These primers are chosen such that each pair will
specifically amplify a portion of the target DNA sequence
containing the region complementary to the three DNA probes. The 5'
end of each of the first pair of primers is labeled with a
semiconductor nanocrystal with peak emission at 525 nm. The 5' end
of each of the second pair of primers is labeled with a
semiconductor nanocrystal with peak emission at 580 nm. The 5' end
of each of the third pair of primers is labeled with a
semiconductor nanocrystal with peak emission at 650 nm.
[0314] These three primer pairs are blended, and are used to
simultaneously amplify three defined portions of the target DNA in
a standard PCR reaction. Since each of the three semiconductor
nanocrystal types are conjugated to their respective primers, and
the primers are extended to become the amplified target, each of
the three amplified target sequences are labeled with the
semiconductor nanocrystal color with which their respective primers
were labeled.
[0315] After the thermal cycling the amplified target can be
denatured or made single-stranded by heating for two minutes at
95.degree. C.
[0316] The hybridization is performed by adding 20 .mu.l of the
amplified target and 480 .mu.l of 5.times.SSPE Buffer to the
microtiter well coated with three different sequences of DNA probe,
and incubating for 60 minutes at 37.degree. C. After hybridization,
a wash step under stringent conditions is performed by adding 500
.mu.l of 3.times.SSPE, and incubating for 15 minutes at 37.degree.
C. The well is rinsed twice with water.
[0317] The well is scanned in a fluorimeter. UV light is shone upon
the well, and the amount of phosphorescence at 525, 580 and 650 nm
wavelengths is measured. The presence of 525 nm emission indicates
that the immobilized probe captured the first semiconductor
nanocrystal labeled amplified target, and remained hybridized after
the stringent wash and water rinse steps. The presence of 580 nm
emission indicates that the immobilized probe captured the second
semiconductor nanocrystal-labeled amplified target. The presence of
650 nm emission indicates that the immobilized probe captured the
third semiconductor nanocrystal-labeled amplified target.
[0318] This multiplex assay allows simultaneous amplification,
labeling and detection of three different targets.
EXAMPLE 15
Sequence-specific Extension
[0319] Sequence-specific extension assays are a convenient method
for determining the presence or absence of a specific DNA sequence
in a sample. Two PCR primers are chosen, and the region of interest
such as the site of a Single Nucleotide Polymorphism (SNP) is
PCR-amplified. A sequence-specific extension primer is chosen, such
that the 3' end of the primer either ends immediately over the site
of the SNP, or is within 5 nucleotides of the SNP. This primer is
conjugated via its 5' end to a surface, such as a microtiter plate
well or bead. In the absence of complete sequence complementarity,
the base mismatch will disrupt the double-stranded complex formed
by the sequence-specific extension primer and the PCR amplified
target. The DNA polymerase has no site for attachment, so extension
will not occur. If the primer and amplified target sequences are
complementary, sequence-specific extension will occur. If the
extension reaction mixture includes a labeled dNTP(e.g.,
semiconductor nanocrystal-labeled dGTP), the presence or absence of
extension can be detected. This allows a determination of the
original target sequence.
[0320] Two different DNA sequence specific primer sequences are
chosen, such that the 3' end of the primers overlaps a SNP in the
target DNA sequence by 2 nucleotides. These sequence can range from
15 to 25 nucleotides in length. The first sequence will be
completely complementary to allele A, and will be called primer A.
The second sequence will be completely complementary to allele B,
and will be called primer B.
[0321] Primer A and Primer B sequences are chemically synthesized
and purified. The 5' end of primer A is conjugated to the surface
of the well of a microtiter plate, using any one of a variety of
standard chemical linkages.
[0322] The 5' end of this primer B is conjugated to the surface of
a second well of a microtiter plate, using any one of a variety of
standard chemical linkages. A semiconductor nanocrystal-dGTP
conjugate is prepared and purified. The semiconductor nanocrystal
is chosen with 525 nm emission. Two PCR primer sequences are
selected, such that they specifically amplify a portion of the
target DNA sequence containing the SNP to be evaluated. These two
primers are used to amplify a defined portion of the target DNA in
a standard PCR reaction(AmpliTaq Polymerase, dNTPs, Magnesium
Chloride, buffer and target DNA).
[0323] The sequence-specific extension is performed by combining
2.5 U AmpliTaq DNA polymerase, 2.5 mM magnesium chloride, buffer,
200 .mu.M dATP, 200 .mu.M dTTP, 200 .mu.M dCTP, 20 .mu.M dGTP, and
200 .mu.M semiconductor nanocrystal-dGTP conjugate. 95 .mu.l of
this solution is added to the microtiter well coated with
sequence-specific primer.
[0324] After the thermal cycling, the target is denatured or made
single-stranded by heating for two minutes at 95.degree. C. Add 5
.mu.l of denatured target to the 95 .mu.l of reaction solution
contained in the wells of the microtiter plate. The extension
reaction is allowed to occur for 5 minutes at 60.degree. C.
[0325] After extension, a wash step under denaturing conditions is
performed by adding 500 .mu.l of 3.times.SSPE, and incubating for 5
minutes at 95.degree. C. Rinse the well twice with water.
[0326] In places where the amplified DNA target annealed to the
immobilized primer and extended, the semiconductor nanocrystal with
525 emission is incorporated into the extension sequence, and is
thus connected to the surface of the plate via the
sequence-specific hybridization. The well is scanned in a
fluorimeter. UV light is shone upon the well, and the amount of
phosphorescence at 525 nm is measured.
[0327] The presence of 525 nm emission indicates that the
immobilized probe captured semiconductor nanocrystal-labeled
amplified target, and remained hybridized after the stringent wash
and water rinse steps.
EXAMPLE 16
Oligonucleotide Ligation Assay
[0328] Two oligonucleotide probes are hybridized to adjacent
sequences of PCR amplified target DNA with the join situated at the
position of a known mutation site. DNA ligase covalently joins the
two oligonucleotide probes only if they are perfectly hybridized,
which will occur if no mutation is present. Initial studies tagged
one probe with biotin and the other with a reporter molecule such
as digoxygenin. On transfer to streptavidin-coated microtiter
plates washing will remove the reporter labeled probe unless
ligation has occurred. Signal detection will therefore only occur
if the mutation is not present. This technology was improved by a
method of typing two alleles in a single microtiter well, by
marking each of the allele-specific primers with different enzyme
reporter molecules so that two different colors can be produced.
Advances to the ligation technique have replaced the biotin with
mobility modifying tails and digoxygenin with fluorescent tags
(e.g., FAM, HEX or TET) with the ligation products being
electrophoresed and analyzed on an automated sequencer. These
modifications have allowed the number of known point mutation sites
which can be screened at any one time to be increased.
[0329] An oligonucleotide sequence complementary to the wild type
of the target sequence is chosen. The 3' end of this oligo ends
with the last nucleotide over a Single Nucleotide Polymorphism
(SNP). This sequence can range from 15 to 25 nucleotides in
length.
[0330] A second oligonucleotide sequence is chosen, which is
complementary to the mutant sequence of the same SNP. The 3' end of
this oligo also ends with the last nucleotide over the SNP site.
This sequence can range from 15 to 25 nucleotides in length.
[0331] These two DNA probe sequences are chemically synthesized and
purified. These oligo probes are separately conjugated by their 5'
end to the surface of the wells of a microtiter plate, using any
one of a variety of standard chemical linkages.
[0332] A single ligation probe sequence is selected. The 5' end of
this probe will be directly adjacent to the 3' end of the two
selected oligonucleotide sequences. This sequence can range from 10
to 25 nucleotides in length.
[0333] The ligation probe is chemically synthesized, and the 5' end
is phosphorylated. The 3' end of the ligation probe is conjugated
to a semiconductor nanocrystal with emission at 525 nm.
[0334] A ligase reaction is performed in each of the two wells
containing the two sequences of oligonucleotides. This reaction
consists of the ligation probe, denatured or single-stranded target
DNA, T7 ligase, magnesium chloride and buffer in an aqueous
solution.
[0335] The plate can be thermally cycled 25 times from 60.degree.
C. to 95.degree. C., with a dwell time of 1 minute at each
temperature to achieve arithmetic amplification of the target.
[0336] In the presence of the complementary target sequence, the
target acts as a template for the ligase reaction. The
sequence-specific oligonucleotide and the ligation probe both
anneal to the single-stranded target DNA. The T7 enzyme ligates the
3' end of the sequence-specific oligo to the phosphorylated 5' end
of the ligation probe.
[0337] In the case where the template DNA is not complementary to
the sequence-specific oligonucleotide, the mismatch at the 3' end
of the annealed specific oligo is sufficient to prevent the
ligation reaction.
[0338] The wells of the microtiter plate are washed 3 times with
water.
[0339] In wells where the DNA ligation reaction occurred, the
semiconductor nanocrystal with 525 nm emission will be immobilized
to the bottom of the well. The well is scanned in a fluorimeter. UV
light is shone upon the well, and the amount of phosphorescence at
525 nm is measured.
[0340] The presence of 525 nm emission indicates that the ligation
probe/semiconductor nanocrystal-conjugate was immobilized to the
surface of the well via the ligation reaction, and remained after
the water rinse steps.
EXAMPLE 17
Tissue Microarrays for High-Throuphput Immunohistochemical Staining
of Tumor Specimens
[0341] Tissue microarrays were prepared as described in Kononen et
al. (1998) Nat. Med. 4:844-847. The tumor microarray block was cut
into 3-8 .mu.m sections, deparaffinized and prepared for
immunochemistry by using conventional techniques. Monoclonal
antibody from mouse to cytokeratin 8/18 was used to bind the
cytokeratin inside the cells. The excess antibody was removed, the
specimen was incubated with biotinylated anti-mouse IgG followed by
40 nM semiconductor nanocrystal-streptavidin. The excess
semiconductor nanocrystal-streptavidin was removed and the slides
examined under a fluorescence microscope.
[0342] The staining pattern of the cells was readily visualized
using a fluorescence microscope.
EXAMPLE 18
Use of Semiconductor Nanocrystals in Histochemical and Cytochemical
Analyses
[0343] As explained above, semiconductor nanocrystals may be used
to do multiple analysis staining on a tissue sample or blood sample
or any sample requiring multiplexed analysis of cellular or
extracellular markers. The procedure is carried out in a two-step
reaction whereby a primary antibody is followed by a semiconductor
nanocrystal-conjugated antibody or by using an antibody (or other
biomolecule) semi-conductor nanocrystal conjugate to directly label
the sample. For example five or more different populations of
semiconductor nanocrystals are synthesized with emission spectra
that spaced at 40 nm intervals from 490-650 nm. Each spectrally
distinct population of semiconductor nanocrystal is conjugated to a
different molecule which specifically recognizes the biomolecule of
interest which may or may not be present in the sample to be
analyzed. Following standard staining protocols, the sample is
labeled with the semiconductor nanocrystals and analyzed for the
location and quantity of the target molecule.
[0344] A. The sample is prepared for immunohistochemical analysis.
The incubation buffer is PBS+0.1% BSA+NaN.sub.3 (or similar
buffer). Active groups are blocked. The sample is washed and
incubated with semiconductor nanocrystal conjugates (the number
dependent on the number of parameters being assessed). The sample
is then washed, mounted and analyzed using conventional fluorescent
microscopy techniques or a spectral scanning device as described
above.
[0345] B. Cellular compounds that are known to exist in spatially
distinct regions of a cell are labeled with the same six colors. In
this case, six different compounds can be unambiguously monitored
in each distinct location within the cell.
[0346] C. Compounds in spatially distinct regions of the cell are
labeled with different sets of 6 colors, where each set has a small
wavelength shift relative to the others. For instance, proteins
located in the nucleus are labeled with semiconductor nanocrystals
that emit at 500, 530, 560, 590, 620 and 650 nm, while proteins
located in the cellular membrane are labeled with 505, 535, 565,
595, 625 and 655 nm emitting semiconductor nanocrystals. The
advantage to using nonidentical sets of colors is that if the
location of compounds is not known, this type of "barcoding" of the
different cellular compounds can be used to unambiguously determine
the location of each. A problem arises only when nonspectrally
distinct semiconductor nanocrystals (e.g. 550 and 555) colocalize.
If this happens, however, the information can be used in a second
experiment where the labels on the colocalized compounds are
rearranged to eliminate the spectral overlap. For instance, in the
example of colocalized 550 and 555 nm emitting semiconductor
nanocrystals, the 555 nm label is replaced in a second experiment
with 590 nm emitting semiconductor nanocrystals. By barcoding
different cellular compounds, it may be possible with CdSe
semiconductor nanocrystals to track the location of as many as 150
distinct compounds within a cell. Using other semi-conductor
nanocrystal materials such as CdTe, InP, InAs, CdS, etc. can
dramatically increase this number.
EXAMPLE 19
Competitive Microsphere Filter Assay
[0347] An explained above, semiconductor nanocrystals can be used
in competitive microsphere filter assays, such as competitive latex
immunoassays. In this application the detection agent is a
semiconductor nanocrystal or a semiconductor nanocrystal-encoded
solid conjugate. The conjugate is a molecule that specifically
recognizes the analyte. The antigen conjugates are direct
semiconductor nanocrystal-antigen conjugates or semiconductor
nanocrystal-dyed microsphere-conjugates that are small enough to
pass through filter pores.
[0348] This allows multiple simultaneous detections using a light
source for excitation of the semiconductor nanocrystals and
detection of emissions. The detection takes place on the filter or
in the filtrate and the assay may be carried out in a high
throughput multiwell environment. The filters in this format are
opaque to the excitation light and allow detection of semiconductor
nanocrystals in the filtrate without the need for washes or sample
removal.
[0349] In particular, antibodies to a specific analyte are
immobilized on microspheres with a diameter greater than that of
the filter pores using standard adsorption or conjugation
technologies. The antibody conjugates are placed in an appropriate
buffer (e.g. PBS) in the upper chamber or insert in a 96-well plate
separated from the bottom of the plate by a light impermeant porous
filter (e.g. BD fluoblock filters). The medium containing the
analyte(s) and a standard amount of analyte(s) labeled with
semiconductor nanocrystals that have a diameter less than that of
the filter pores are added to the upper chamber or insert. After an
appropriate incubation time (e.g., 30 seconds to 24 hours) the
amount of fluorescence in the lower chamber is quantitated by
spectral analysis systems described above.
[0350] This assay provides information on the concentration of
analyte(s) in any biological medium. It is especially useful for
measuring multiple analytes in a diagnostic type test and may also
be used for environmental, research or screening applications. The
greater the concentration of analyte in the sample the greater the
amount of fluorescence detected in the lower chamber. Thus, this
system provides a desirable increasing curve in terms of analyte
concentration.
[0351] FIG. 5 is a pictorial representation of a competitive
microsphere filter assay as described herein.
[0352] Thus, novel methods for using semiconductor nanocrystals are
disclosed. Although preferred embodiments of the subject invention
have been described in some detail, it is understood that obvious
variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
* * * * *