U.S. patent application number 11/206403 was filed with the patent office on 2006-08-03 for synthesis of highly luminescent colloidal particles.
Invention is credited to Imad Naasani.
Application Number | 20060172133 11/206403 |
Document ID | / |
Family ID | 35511132 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172133 |
Kind Code |
A1 |
Naasani; Imad |
August 3, 2006 |
Synthesis of highly luminescent colloidal particles
Abstract
The present invention includes compositions and methods for
their used wherein the compositions include clusters of coated
fluorescent nanocrystals having a select size formed by controlled
aggregation of individual coated nanocrystals.
Inventors: |
Naasani; Imad; (Eugene,
OR) |
Correspondence
Address: |
PEPPER HAMILTON; LLP; CHRISTOPHER J. BUNTEL;;AND KOREN ANDERSON
ONE MELLON CENTER, 50TH FLOOR GRANT STREET
PITTSBURGH
PA
15219
US
|
Family ID: |
35511132 |
Appl. No.: |
11/206403 |
Filed: |
August 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60602271 |
Aug 17, 2004 |
|
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|
Current U.S.
Class: |
428/403 ;
427/212 |
Current CPC
Class: |
C09K 11/883 20130101;
B82Y 5/00 20130101; C30B 7/005 20130101; C30B 29/605 20130101; B82Y
15/00 20130101; B82Y 30/00 20130101; C09K 11/565 20130101; G01N
33/588 20130101; C09K 11/02 20130101; Y10T 428/2991 20150115; Y10S
977/896 20130101; Y10S 977/882 20130101 |
Class at
Publication: |
428/403 ;
427/212 |
International
Class: |
B32B 5/16 20060101
B32B005/16; B05D 7/00 20060101 B05D007/00 |
Claims
1. A composition comprising an aggregate of nanocrystals; wherein:
the nanocrystals comprise a coating layer; the coating layer
comprises one or more imidazole groups; and the nanocrystals
interact through the coating layer to form the aggregate.
2. The composition of claim 1, wherein the aggregate is cross
linked.
3. The composition of claim 2, wherein a cross linking agent is
used to cross link the aggregate.
4. The composition of claim 3, wherein the cross linking agent is
tris(hydroxy methyl) phosphine.
5. The composition of claim 3, wherein the cross linking agent is
beta-[tris (hydroxymethyl)phosphino]propionic acid.
6. The composition of claim 1, wherein the coating layer is bound
to the nanocrystal by the one or more imidazole groups.
7. The composition of claim 1, wherein the aggregated nanocrystals
are crosslinked by one or more organophosphine compounds.
8. The composition of claim 1, wherein the nanocrystals are
luminescent.
9. The composition of claim 1, wherein the nanocrystals are
fluorescent.
10. The composition of claim 1, wherein the aggregate is dispersed
in an aqueous based solution.
11. The composition of claim 1, wherein the aggregate further
comprises at least one functional group on the surface of the
aggregate.
12. The composition of claim 1, wherein the aggregate further
comprises a functional group selected from the group consisting of
a hydroxyl, thiol, amino, acetylenic, carboxyl, ester, amide,
dicarboxylic, carboxamide selenol, hydrazide, aldehyde and a
combination thereof on the surface of the coated nanocrystal
aggregate.
13. The composition of claim 1, wherein the nanocrystals are
semiconductor core nanocrystals.
14. The composition of claim 1, wherein the nanocrystals are
semiconductor core/shell nanocrystals.
15. The composition of claim 1, wherein the coating layer comprises
histidine, carnosine, polyhistidine, polyimidazole, or glycyl
histidine.
16. A composition comprising a nanocrystal aggregate and at least
one affinity molecule; wherein: the aggregate comprises two or more
coated nanocrystals; the nanocrystals comprise a coating layer
comprising one or more imidazole groups; the nanocrystals interact
through the coating layer to form the aggregate; the aggregate
comprises at least one functional group on its surface; and the at
least one affinity molecule is linked to the functional group.
17. The composition of claim 16, wherein the affinity molecule is
selected from the group consisting of a polyclonal antibody, a
monoclonal antibody, a peptide, an aptamer, a nucleic acid, a
lectin, a lipid, a small organic molecule, a polysaccharide,
avidin, neutravidin, streptavidin, an avidin derivative, biotin, a
biotin derivative, and combinations thereof.
18. The composition of claim 16, wherein the affinity molecule is
covalently linked to the functional group.
19. The composition of claim 16, wherein the functional group is
selected from the group consisting of a hydroxyl, thiol, amino,
acetylenic, carboxyl, ester, amide, dicarboxylic, carboxamide
selenol, hydrazide, aldehyde and a combination thereof.
20. A composition comprising two to about twenty aggregated
fluorescent semiconductor nanocrystals; wherein: the fluorescent
nanocrystals comprise a coating layer; the coating layer comprises
one or more imidazole groups; and the nanocrystals interact through
their coating layers to form an aggregate, the aggregate being
composed of from about two to about twenty fluorescent
semiconductor nanocystals.
21. The composition of claim 20, wherein the nanocrystals are
crosslinked by an organophosphine compound in the aggregate.
22. A method of preparing a nanocrystal aggregate, the method
comprising: contacting two or more nanocrystals in a solvent, the
nanocrystals comprising a coating layer comprising at least one
imidazole group; and contacting the nanocrystals to prepare an
aggregate of coated nanocystals.
23. The method of claim 22, further comprising controlling the
aggregate size by modifying the solvent.
24. The method of claim 22, wherein the nanocrystals interact
through their coating layers.
25. The method of claim 22, wherein the contacting step further
comprises placing the two or more nanocrystals in a solvent
mixture.
26. The method of claim 22, wherein the contacting step further
comprises placing the two or more nanocrystals in an aqueous
solvent mixture.
27. A method of detecting a target molecule in a sample, the method
comprising: providing a sample suspected of containing a target
molecule; providing one or more coated nanocrystal aggregates, said
coated nanocrystal aggregates comprising a nanocrystal, an
imidazole containing coating, and an affinity ligand having binding
specificity for the target molecule; contacting the sample and the
aggregates to form a treated sample, wherein the aggregates form a
complex with the target molecule; exciting the complex with a
wavelength of energy to form an excited complex; and detecting the
excited complex.
28. The method of claim 27, where the detecting step comprises
detecting light emitted by the excited complex.
29. The method of claim 27, where the detecting step comprises
using a Scintillation Proximity assay.
30. The method of claim 27, where the detecting step further
comprises quantifying the amount of target molecule in the
sample.
31. The method of claim 27, wherein the wavelength is less than
about 500 nm.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Application Ser. No. 60/602,271 filed Aug. 17, 2004 the
contents of which are incorporated herein by reference in their
entirety.
BACKGROUND
[0002] Fluorescence-based analyses and nonisotopic detection
systems have become a powerful tool for scientific research and
clinical diagnostics for the detection of biomolecules using
various assays including, but not limited to, flow cytometry,
nucleic acid hybridization, DNA sequencing, nucleic acid
amplification, immunoassays, histochemistry, and functional assays
involving living cells. Fluorescent semiconductor nanocrystals have
found widespread use due to their high fluorescent intensity and
the ability of different nanocrystals to be excited by a single
light source. It would be desirable to increase the signal from
these non-isotopic materials to increase the sensitivity of a
variety of assays and analyses utilizing them. It would be
advantageous to controllably link numbers of nanocrystals and other
small particles into structures for analytical applications that
can be used to label a target molecule to be detected.
[0003] Mirkin et al, in WO 98/04740 discloses nanoparticles having
oligonucleotide attached to them. Methods are disclosed that
comprise contacting a nucleic acid with one or more types of
nanoparticles having oligonucleotides attached to them. The
oligonucleotides are attached to nanoparticles and have sequences
complementary to portions of the sequence of the nucleic acid. A
detectable change, a color change, is brought about as a result of
the hybridization of the oligonucleotides on the nanoparticles to
the nucleic acid. The compositions disclosed do not include
core/shell semiconductor nanocrystals and use oligonucleotides,
specifically complementary oligonucleotides, to form conjugates of
the nanoparticles.
[0004] Mirkin et al. in U.S. Pat. No. 6,361,944 disclose
nanoparticles having oligonucleotides attached to them and uses for
the compositions. Again, the disclosure provides oligonucleotides
attached to nanoparticles that include core/shell semiconductor
nanocrystals and where the oligonucleotide sequences are
complementary to portions of the sequence of a nucleic acid to be
detected. A detectable change is brought about as a result of the
hybridization of the oligonucleotides on the nanoparticles to the
nucleic acid. The disclosure purports to illustrate the formation
of nanoparticle aggregates, nanomaterials, and nanostructure by
combining nanoparticles having complementary oligonucleotides
attached to them, the nanoparticles being held together in the
aggregates as a result of the hybridization of the complementary
oligonucleotides.
[0005] Hansen et al. in WO 98/33070 disclose a homogeneous binding
assay. The disclosure describes a homogeneous method of measuring
chemical binding that relies on resonant, or "amplified", optical
extinction (light scattering plus absorption) from a defined,
specific class of colloidal particles where the real term n of the
complex refractive index n-ik approaches zero while the imaginary
term k approaches 2.sup.(1/2). Chemical binding partners are coated
onto the particles, which either aggregate or disperse during the
binding reaction, causing an optical extinction change at one
wavelength that is quantitatively related to the number of single
colloidal particles and another at a second wavelength that is
quantitatively related to the number of doublet colloidal
particles. The disclosure describes the uses of optical extinction
to measure the formation of particle dimers (through the appearance
of increased extinction at the split resonant wavelength) and the
concomitant disappearance of the singlet particles (through the
decrease of extinction at the original resonant wavelength).
[0006] Bawendi et al. in EP0990903 disclose biological applications
of semiconductor nanocrystals. The disclosure describes
compositions comprising fluorescent semiconductor nanocrystals
associated to a compound, where the nanocrystals have a
characteristic spectral emission that is tunable to a desired
wavelength by controlling the size of the nanocrystal, and where
the emission provides information about a biological state or
event.
[0007] Barbera-Guillem et al in U.S. Pat. No. 6,261,779 discloses
nanocrystals having polynucleotide strands and their use to form
dendrimers in a signal amplification system. The disclosure
provides compositions and assay kits comprising functionalized
nanocrystals having a plurality of polynucleotide strands of known
sequence extending from them. The disclosure describes primary dots
that are used to operably link to a molecule, and secondary dots
comprise a plurality of polynucleotide strands which are
complemetary to the plurality of polynucleotide strands of the
primary dots. The disclosure provides a method for detecting the
presence or absence of target molecules in a sample comprising
operably linking primary dots to molecules, contacting the complex
formed with the sample, contacting the sample with successive
additions of secondary dots and primary dots. If a target molecule
is present in the sample, the primary dots and secondary dots will
form dendrimers that can be detected by fluorescence emission.
[0008] Peng et al. U.S. Pat. No. 6,872,249 disclose the synthesis
of colloidal nanocrystals. A method of synthesizing colloidal
nanocrystals is disclosed using metal oxides or metal salts as a
precursor. The metal oxides or metal salts are combined with a
ligand and then heated in combination with a coordinating
solvent.
[0009] Peng et al. U.S. Pat. No. 6,869,545 discloses colloidal
nanocrystals with high photoluminescence quantum yields and methods
of preparing the same. The disclosure provides compositions
containing colloidal nanocrystals with high photoluminescence
quantum yields, synthetic methods for the preparation of highly
luminescent colloidal nanocrystals, as well as methods to control
the photoluminescent properties of colloidal nanocrystals.
[0010] Bawendi et al. in U.S. Pat. No. 6,306,610 disclose quantum
dot white and colored light emitting diodes. The disclosure
describes an electronic device comprising a population of quantum
dots embedded in a host matrix and a primary light source which
causes the dots to emit secondary light of a selected color, and a
method of making such a device. The size distribution of the
quantum dots is chosen to allow light of a particular color to be
emitted from the structure. The dots can be composed of an undoped
semiconductor such as CdSe, and may optionally be overcoated to
increase photoluminescence. The host matrix for the device includes
isolated dots within the matrix and not defined aggregates of
nanocrystals.
[0011] U.S. Pub. No. 20040110220 to Mirkin et al. discloses
nanoparticles having oligonucleotides attached to them and uses for
such coated nanoparticles. The disclosure provides methods of
detecting a nucleic acid that comprise contacting the nucleic acid
with one or more types of nanoparticles having oligonucleotides
attached to them. The disclosure describes a method where
oligonucleotides are attached to nanoparticles and have sequences
complementary to portions of the sequence of the nucleic acid. A
detectable change is brought about as a result of the hybridization
of the oligonucleotides on the nanoparticles to the nucleic acid.
The disclosure describes methods of synthesizing
nanoparticle-oligonucleotide conjugates and methods of using the
conjugates. The disclosure describes nanomaterials and
nanostructures comprising nanoparticles and methods of
nanofabrication utilizing nanoparticles. The disclosure describes a
method of separating a selected nucleic acid from other nucleic
acids.
SUMMARY
[0012] There is a need to form colloidal aggregates or cluster of
particles in a controlled manner using inexpensive coating
materials. Such clusters could be used in a variety of nonisotopic
detection systems to increase the signal comprising fluorescence
emission of high quantum yield. Such clusters may be used to
provide tailored signal amplification that is not limited as to the
chemical nature of the target molecule to be detected. It would be
desirable that such non-isotopic probes could be used to bind
target molecules of various and that they can be excited with a
single excitation light source and with resultant fluorescence
emissions with discrete fluorescence peaks.
[0013] Embodiments of the invention include nanocrystal aggregates.
These compositions can include two or more aggregated nanocrystals;
where the nanocrystals includes a coating layer, and the coating
layer can include one or more imidazole groups. The coated
nanocrystals interact or associate through their coating layers to
form an aggregate.
[0014] The composition of aggregated nanocrystals may further
include a cross linking agent. The aggregated nanocrystals can be
crosslinked by one or more organophosphine compounds. The cross
linking agent can include tris(hydroxy methyl) phosphine,
beta-[tris(hydroxymethyl)phosphino]propionic acid, any combination
of these, or other suitable organophosphine compounds.
[0015] The coating layer on the nanocrystals can be bound or
operably linked to the nanocrystal by the one or more imidazole
groups. The coating layer on the nanocrystals can include
histidine, carnosine, polyhistidine, polyimidazole, glycyl
histidine or other similar imidizole containing compounds. In some
embodiments one or more imidazole groups of the coating layer bond
or otherwise operably link the imidazole coating compound to the
nanocrystal.
[0016] The aggregated nanocrystals can be luminescent, fluorescent,
magnetic, or may include one or more these properties by
aggregating two or more different nanocrystals that include any of
these properties. In some embodiments, the aggregates can include
nanocrystals that are semiconductor core nanocrystals or
semiconductor core/shell nanocrystals.
[0017] The aggregate may further comprise at least one functional
group on the surface of the aggregate. The aggregate may further
comprise functional groups on the surface of the coated nanocrystal
aggregate such as but not limited to hydroxyl, thiol, amino,
acetylenic, carboxyl, ester, amide, dicarboxylic, carboxamide
selenol, hydrazide, aldehyde, or combinations of any of these. The
aggregates can be dispersed in a variety of organic solvents,
mixtures of organic solvent and water, or in aqueous based
solutions.
[0018] Embodiments of the invention can include aggregates that
have been functionalized with reactive groups. A functionalized
aggregate composition can include a nanocrystal aggregate and at
least one affinity molecule. The nanocrystal aggregate comprises
two or more coated nanocrystals; the nanocrystals comprise a
coating layer comprising one or more imidazole groups where the
nanocrystals interact through their coating layers to form an
aggregate. In some embodiments, the aggregate may include at least
one functional group on its surface with at least one affinity
molecule is linked to the functional group.
[0019] In embodiments of the invention the affinity molecule can be
but is not limited to a polyclonal antibody, a monoclonal antibody,
a peptide, an aptamer, a nucleic acid, a polynucleotide, a lectin,
a lipid, a small organic molecule, a polysaccharide, avidin,
neutravidin, streptavidin, an avidin derivative, biotin, a biotin
derivative, or any combination of these affinity molecules. The
affinity molecule can be covalently linked to the functional group.
The functional group can include but is not limited to hydroxyl,
thiol, amino, carboxyl, ester, amide, dicarboxylic, carboxamide,
selenol, hydrazide, aldehyde, or any combination of these.
[0020] Some embodiments of the invention can include an aggregate
having a defined number of coated nanocrystals, coated
nanoparticles, or any combination of these in the aggregate. The
aggregate composition can include from about two or more to about
twenty aggregated coated fluorescent semiconductor nanocrystals.
The coated nanocrystals and or coated nanoparticles in the
aggregate comprise a coating layer that includes one or more
imidazole groups and the coated nanocrystals interact through their
coating to form the aggregate. In some embodiments one or more
imidazole groups of the coating layer bond or otherwise operably
link the imidazole coating compound to the nanocrystals or
nanoparticles. The number of coated nanocrystals, coated
nanoparticles, or combination of these in the aggregate or cluster
can separately form a cluster of a defined size, preferably between
2 and 20 coated nanocrystals of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 coated nanometer sized particles.
The cluster or aggregate is a colloidal particle and the
nanocrystals and/or nanoparticles in the aggregate can be
crosslinked by an organophosphine compound.
[0021] One embodiment of the invention is a method of preparing
aggregates comprised of coated nanocrystals. A method of preparing
these aggregates can include the acts or steps of providing two or
more nanocrystals, the nanocrystals include a coating layer
comprising at least one imidazole group, and then contacting or
combining the nanocrystals to prepare an aggregate coated
nanocrystals. The nanocrystals interact through their coating
layers to form the aggregate. The method can further include the
act of placing the two or more nanocrystals in a solvent mixture
and contacting or combining them to form an aggregate or an
aggregate of a predetermined size. The solvent mixture can be an
aqueous solvent mixture. Similar acts or steps can be used to
prepare clusters or aggregates that include coated nanoparticles,
or any combination of coated nanoparticles and coated nanocrystals
where the coating layer comprises at least one imidazole group.
[0022] One embodiment of the invention is a method of using
nanocrystal aggregates. The method can include detecting a target
molecule in a sample with these nanocrystal aggregates. The method
can include the acts of providing a sample suspected of containing
a target molecule and providing one or more aggregates of coated
nanocrystals to the sample. Preferably the nanocrystals are coated
with a material with one or more imidazole groups and where the
coating further includes an affinity ligand or other reactive
functionality having binding specificity for the target molecule.
Contacting the sample and the aggregates forms a treated sample. A
treated sample can be excited with energy (electromagnetic
radiation, an electric field and or magnetic field, high energy
particles) and an excited complex formed that is used to detect the
presence of a target molecule complexed with the aggregate in the
treated sample. Similar acts or steps can be taken to use clusters
or aggregates to detect target molecules where the aggregates
include coated nanoparticles, or any combination of coated
nanoparticles and coated nanocrystals where the coating layer
comprises at least one imidazole group.
[0023] The detecting step or act can include detecting light
emitted by the excited aggregate or cluster complex. The detecting
step can include detecting light emitted by a nanocrystal aggregate
complex with a radio labeled target molecule in a Scintillation
Proximity assay. The detecting step or act may further include
quantifying the amount of target molecule in the sample. In some
embodiments, the energy used to excite the nanocrystals can have
wavelength that is less than about 500 nm.
[0024] Embodiments of the present invention include fluorescent
nanocrystals which have high fluorescence intensity and can for
example be dispersed or solubilized in water or water containing
solutions. The present invention provides for the synthesis of
colloidal particles using functionalized fluorescent nanocrystal
compositions. Embodiments of the invention provide methods for
making and using these compositions in biological detection
applications, material separations, and in the production of
biosensors. The compositions are colloidal particles produced by
clustering two or more nanocrystals and preferably two or more
fluorescent nanocrystals together. The compositions and method for
making them provides for colloidal fluorescent nanocrystal
compositions which are water dispersible, chemically stable, and
emit light with a high quantum yield and/or luminescence efficiency
when excited with light or other sources of energy. The colloidal
material, which comprises clusters or aggregates of coated
nanocrystals, may also have chemical functional groups, compounds
or ligands with moieties for bonding to target molecules and
cells.
[0025] A highly luminescent, chemically functionalized, and water
dispersible colloidal particle of clustered or aggregated coated
nanocrystals in embodiments of the present invention may include a
cluster of two or more nanocrystals, preferably those nanocrystals
that have size dependent properties including fluorescent semi
conductor nanocrystals, and more preferably fluorescent
semiconductor nanocrystals coated with an organic coating. The
coating on the fluorescent semiconductor nanocrystal is one that
permits controlled aggregation of individual coated nanocrystals
and the coating may be formed by complexation between the
nanocrystal inorganic compound and organic material. Examples of
such organic coatings include but are not limited to imidazole
containing compounds like carnosine and histidine, polymers coating
the nanocrystal including but not limited to imidazole (or an
imidazole-mimicking compound) and phosphine or amine cross linking
compounds.
[0026] The highly luminescent, chemically functionalized and water
dispersible colloidal particle comprised of nanocrystals having a
coating containing one or more imidazole groups in embodiments of
the present invention can include those where the cluster is
comprised of two or more quantum dots such as but not limited to a
Group II-VI semiconductor material (of which ZnS, and CdSe are
non-limiting illustrative examples), a Group m-V semiconductor
material (of which GaAs is a non-limiting illustrative example), a
Group IV semiconductor nanocrystal, colloidal gold, silver,
ferromagnetic, ferrimagnetic nanoparticles, or any combination of
these particles.
[0027] In the highly luminescent, chemically functionalized and
water dispersible colloidal particles, aggregates of coated
nanocrystals or clusters of coated nanocrystals, the cluster can
include two or more core, core/shell, or a combination including
these. The quantum dots may be chosen from an inorganic material
such as but not limited to a Group II-VI semiconductor material (of
which ZnS, and CdSe are illustrative examples), a Group III-V
semiconductor material (of which GaAs is an illustrative example),
a Group IV semiconductor material, colloidal gold, silver,
ferromagnetic, ferrimagnetic nanoparticles, or a combination these.
Preferably an organic material complexes with the inorganic
material of the quantum dot by the formation of chemical adducts
and bonds.
[0028] The adduct forming organic coating over the nanocrystals is
preferably formed by an imidazole-containing compound (of which
histidine, carnosine, polyhistidine, polyimidazole are illustrative
examples), or an imidazole-mimicking compound compound (of which
thiazole, oxazole, pyrrole, thiophene, furan, pyridine, pyrimidine,
pyrazine, triazine, triazole, thiophene, phthalocyanine, porphyrin,
and their derivatives are illustrative examples). Preferably the
imidazole containing compound, or the imidazole mimicking compound
in the coating is bonded to the nanocrystal through the imidazole
or imidazole mimicking group. Optionally the organic coating may
include and an alkyl phosphine-containing compound (of which
tris(hydroxy methyl) phosphine and
beta-[Tris(hydroxymethyl)phosphino]propioninc acid are illustrative
examples). The highly luminescent, chemically functionalized and
water dispersible colloidal cluster particles, aggregates or
clusters of coated nanocrystals, can have the imidazole-containing
compound (or an imidazole-mimicking compound) and the alkyl
phosphine containing compound crosslinked.
[0029] The highly luminescent, chemically functionalized, and water
dispersible colloidal cluster particles preferably have a coating
on the individual nanocrystals or the cluster that is
functionalized so that the functionalized colloidal particle is
capable of linking to a target molecule, affinity molecule, a
sensor molecule or sensor substrate; and capable of, in response to
excitation by a first energy, providing a second energy used for
detection.
[0030] The target or affinity molecules linked to the colloidal
aggregate of coated nanocrystals or cluster of coated nanocrystals
may include but are not limited to those such as of polyclonal
antibodies, monoclonal antibodies, a peptide, an aptamer, a nucleic
acid, a lectin, a lipid, a small organic molecule, a
polysaccharide, avidin, neutravidin, streptavidin, an avidin
derivative, biotin, a biotin derivative. The target may also be a
sensor surface diode, a nanodevice, an optical fiber, or any one of
these whose surface has been functionalized to interact with
reactive groups on the surface of the coating on the cluster.
[0031] The highly luminescent, chemically functionalized, and water
dispersible colloidal particle, aggregate of coated nanocrystals or
cluster of coated nanocrystals, may be used for detecting target
molecules or surfaces including the acts of contacting one or more
of the functionalized colloidal cluster particles with a sample
(for example a molecule, cell, tissue, or substrate) being
analyzed. The presence or absence of a target molecule or target
substrate in the sample being determined by the affinity ligand on
the cluster that has binding specificity for the molecule or
substrate. If the target molecule or substrate is present in the
sample a complex is formed comprising the functionalized colloidal
particles bound to the substrate. Exposing the contacted sample in
a detection system to a wavelength of light or a source of energy
suitable for exciting the complexed functionalized colloidal
cluster particles bound to the sample to emit a highly luminescent
peak will signal the presence or absence of the target. The
luminescence peak emitted by the complexes, if present, can be made
by a detection system for detecting the luminescence peak; wherein
the detection of luminescence peak is indicative of the presence of
the target in the sample. For example, one detection method using
the highly luminescent, chemically functionalized and water
dispersible colloidal cluster particles for detection may include a
detection system such as a luminescence counter used in a
Scintillation Proximity assay.
[0032] Advantages of the present invention include enhanced
brightness due to the effect of imidazole/THP (as in the case of
standard functionalized nanocrystals) and multiple nanocrystals.
Unlike embedded beads, colloidal fluorescent nanocrystals are
coated with a thin transparent layer without fillers. This results
in a minimum light shielding effect (inward and outward) and a
brighter emission. Nanocrystals of different emissions can be
clustered in one cluster particle with a unique signal
(fingerprint) from the combination of the spectra of the clustered
nanocrystals. The clusters of the present invention may be
derivatized with carboxyl groups for covalent or electrostatic
conjugation to a target protein or bio-agent. Because the clusters
include more than one nanocrystal, and unlike polymer embedded
beads, they have high density and advantageously collection and
separation by centrifugation is facilitated. Dual detection
potential by visual (like colloidal gold) and/or fluorescent
approaches can be used.
[0033] The above and other objects, features, and advantages of the
present invention will be apparent in the following detailed
description of the invention when read in conjugation with
accompanying drawings in which reference numerals denote the same
or similar parts throughout the several illustrated views and
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The file of this patent contains at least one photograph or
drawing executed in color. Copies of this patent with color
drawing(s) or photograph(s) will be provided by the Patent and
Trademark Office upon request and payment of necessary fee.
[0035] FIG. 1 Illustrates schematically the production of colloidal
nanocrystals that are an aggregate of coated nanocrystals or a
cluster of coated nanocrystals;
[0036] FIG. 2 Shows test results where colloidal nanocrystals,
comprised of an aggregate of coated nanocrystals or a cluster of
coated nanocrystals, were conjugated to avidin using EDC chemistry
and were tested by dot blot assay to detect biotinylated antibody
blotted on nitrocellulose membrane; the top row is the biotinylated
antibodies and the bottom row is the non biotinylated antibodies
(control).
DETAILED DESCRIPTION OF THE INVENTION
[0037] Before the present compositions and methods are described,
it is to be understood that this invention is not limited to the
particular molecules, compositions, methodologies or protocols
described, as these may vary. It is also to be understood that the
terminology used in the description is for the purpose of
describing the particular versions or embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
[0038] It must also be noted that as used herein and in the
appended claims, the singular forms "a", "an", and "the" include
plural references unless the context clearly dictates otherwise.
Thus, for example, reference to a "cell" is a reference to one or
more cells and equivalents thereof known to those skilled in the
art, and so forth. Unless defined otherwise, all technical and
scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of embodiments of the
present invention, the preferred methods, devices, and materials
are now described. All publications mentioned herein are
incorporated by reference. Nothing herein is to be construed as an
admission that the invention is not entitled to antedate such
disclosure by virtue of prior invention.
[0039] "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 occurs or material
is present and instances where the event does not occur or where
the material is not present.
[0040] Throughout the specification of the application, various
terms are used such as "primary", "secondary", "first", "second",
and the like. These terms are words of convenience in order to
distinguish between different elements, and such terms are not
intended to be limiting as to how the different elements may be
utilized.
[0041] Embodiments of the present invention are directed to
nanocrystals with organic linkers or polymer coatings that are able
to make the nanocrystals aggregate. While in preferred embodiments,
the organic coating is an imidazole containing compound, the
present invention is not limited to any particular coating
disclosed, and any coating on the nanocrystals capable of forming
aggregates of controlled size or a narrow distribution of sizes can
be used.
[0042] Compositions of the invention can include colloidal
particles produced by clustering two or more nanocrystals and
preferably two or more fluorescent nanocrystals together. Colloidal
aggregates or clusters in other embodiments of the present
invention can be comprised of coated nanocrystals, coated
nanoparticles, or any combination of these. A dispersion of coated
nanocrystals, coated nanparticles, or any combination of these can
associate to form larger sized aggregates or clusters that include
two or more coated nanocrystals, coated nanoparticles, or any
combination of these.
[0043] The preparation of clusters of aggregated coated
nanocrystals includes fluorescent nanocrystals or quantum dots
coated with an organic shell or capping layer comprised of ligands
formed by self assembly, where preferably the ligand bonds to the
nanocrystal via a functional group of the ligand. Next, the
induction of controlled aggregation of the coated nanocrystals is
initiated. Clusters of aggregated coated nanocrystals (van der
Waals type interaction) may be facilitated for example by using a
solvent mixture that favors aggregation of the individually coated
nanocrystals. Cross-linking and capping of the cluster can be used
to link or bond together the individual nanocrystals in the
cluster. The coated nanocrystals may also have their aggregation
controlled or modified by functional groups on the coating
compound. Similar materials and processes may be used to prepare
clusters or aggregates of coated nanoparticles or aggregates that
include any combination of coated nanoparticles and coated
nanocrystals.
[0044] The preparation of nanocrystals of the present invention is
illustrated with reference to nanocrystals coated by an imidazole
ligand or imidazole mimicking ligand by self assembly, where
preferably the ligand bonds to the nanocrystal via the imidazole or
imidazole mimicking group. The coating of the nanocrystals and
extraction from a solvent can be performed using the methods
disclosed in U.S. Publication No. 2004-0009341 A1 filed Sep. 17,
2002 Titled HIGHLY LUMINESCENT FUNCTIONALIZED SEMICONDUCTOR
NANOCRYSTALS FOR BIOLOGICAL AND PHYSICAL APPLICATIONS the contents
of which are incorporated into the present application by reference
in their entirety. The induction of controlled aggregation of the
coated nanocrystals is initiated to form clusters of coated
nanocrystals (van der Waals type interaction) using for example a
solvent mixture like 50% ethanol/50% water (the ratio of ethanol to
water can be used to determine the final size of the colloidal
particle). The number of nanocrystals per particle (i.e., the size
of the formed particle) can be manipulated by changing the ratio of
ethanol. Larger particles can be formed by adding higher
concentrations of ethanol. For approximately 80 nm size (5-10
nanocrystals per particle), 50% of ethanol (final concentration)
can be used. Other solvents or mixtures of solvents may be used to
initiate the controlled aggregation of the coated nanocrystals to
form the clusters. Finally, surface oriented cross-linking and
capping of the cluster can be used to link or bond together the
individual coated nanocrystals. The imidazole coated nanocrystals
or nanocrystals coated by imidazole mimicking compound may also
have their aggregation controlled or modified by functional groups
on the coating compound. Advantageously, the coating compounds of
the present invention do not require that the nanoparticles be
further functionalized with individual recognition groups that are
complementary to each other, have recognition groups that are not
complementary but bridged through a bispecific linker, or require a
bivalent linker that recognizes the surface of one or more
nanoparticles and is used for aggregation--this simplifies the
process for making clusters. Preferably the imidazole coated
nanocrystals or nanocrystals coated by imidazole mimicking compound
are those that can be linked together to form the cluster by a
compound such as but not limited to tris(hydroxy methyl) phosphine
or other cross linking compounds disclosed in U.S. Ser. No.
10/410,108, filed Apr. 9, 2003, titled HIGHLY LUMINESCENT
FUNCTIONALIZED SEMICONDUCTOR NANOCRYSTALS FOR BIOLOGICAL AND
PHYSICAL APPLICATIONS, the contents of which are incorporated by
reference in their entirety into the present application.
[0045] FIG. 1 illustrates an embodiment of a composition of the
present invention and a method for making it. Nanocrystals and/or
nanoparticles 104, 108, and 112, which can be the same or
different, include a ligand coating that comprises one or more
coating molecules 116 and or 118 operably linked with each
nanocrystal or nanoparticle. The molecules 116 and or 118 can
optionally have reactive functionalities to link with target
molecules, affinity molecules or cross-linking agents. The coated
nanocrystals can aggregate 120 by adjusting the concentration of
solvents that suspend the coated nanocrystals or nanoparticles. The
suspended particles aggregate to form a colloidal cluster 124 of
the coated nanocrystals and or coated nanoparticles interacting or
associated through their coating. The colloidal cluster or
aggregate 124 can be optionally crosslinked 126 to form a
crosslinked colloidal cluster or aggregate 136. For example, one or
more crosslinking molecules 128 and or 132 can be used to operably
link the coated nanocrystals in the aggregate 124 together. The
cross linking molecules 128 and or 132 can be the same or different
and can have reactive functionalities to link the aggregate 136 to
one or more linking groups, affinity groups, target molecules, or a
substrate.
[0046] One embodiment of the invention is a composition comprising
one or more colloidal particles. The colloidal particle can include
of two or more fluorescent semiconductor nanocrystals. Each
fluorescent semiconductor nanocrystal is coated with an organic
shell that can be formed by complexation between the fluorescent
semiconductor nanocrystal and an imidazole containing molecule and
an optional organophosphine compound. In the coating, one or more
imidazole groups from the imidazole containing molecule are bonded
to the fluorescent semiconductor nanocrystal. The coated
fluorescent semiconductor nanocrystals aggregate or form a
colloidal cluster that can optionally be bonded to each other in
the colloid by an organophosphine containing compound. The size of
the colloid can be modified by controlled aggregation of two or
more imidazole containing compound coated fluorescent semiconductor
nanocrystals. In some embodiments, the colloid is a cluster or
aggregate of coated nanocrystals that is comprised of two or more
quantum dots or semiconductor nanocrystals. In some embodiments,
the colloidal particle or aggregate of coated nanocrystals is
comprised of two or more core or core/shell quantum dots or
core/shell semiconductor nanocrystals. The colloidal particle or
aggregate comprised of coated nanocrystals can be luminescent. In
some embodiments of the composition, the colloid cluster or
nanocrystal aggregate includes more than 3, 4, 5, 6, 7, 8, 9 or
more than 10 quantum dots or semiconductor nanocrystals.
[0047] The clusters may include two or more coated nanocrystals,
coated nanoparticles, or any combination of these linked together.
Dimers, trimers, and large n-mers may be formed where n is an
integer 2 or larger, preferably from 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and most preferably from
about 2, 3, 4, 5, 6, 7, 8, 9 or 10. Where a distribution of sizes
for cluster occurs, the size of the cluster can be further selected
for example by size selective precipitation or sieving
filtration.
[0048] The term nanocrystal refers to an inorganic crystallite
having a largest dimension of from between about 1 nm and about
1000 nm, more typically between about 2 nm and about 20 nm
including but not limited to doped metal oxide, semiconductor, and
doped semiconductor nanocrystals. A semiconductor nanocrystal or
quantum dot 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.
Preferably the coated nanocrystals are fluorescent nanocrystals. A
semiconductor nanocrystal core surrounded by a semiconductor shell
is referred to as a "core/shell" semiconductor nanocrystal. The
surrounding "shell material typically has a bandgap energy that is
larger than the bandgap energy of the core material and can be
chosen to have an atomic spacing close to that of the core
substrate. The core and/or shell can be a semiconductor material
including, but not limited to, those of the Groups 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), and alloys or mixtures thereof.
[0049] Nanoparticle refers to particles less than 1000 nm in size
which may include regions that are semicrystalline amorphous, any
combination of these regions, or any combination of these regions
that further includes crystalline regions. The association between
the coated nanocrystals, coated nanoparticles, or any combination
of these in an aggregate or cluster can be through a variety of
chemical, physical, or a combination of these bonding interactions
including but not limited to one or more of: covalent, ionic,
hydrogen bonding, van der Waals, chemisorption, physisorption, and
the like.
[0050] Colloidal aggregates or colloidal clusters in embodiments of
the present invention can be comprised of coated nanocrystals,
coated nanoparticles, or any combination of these. The coated
nanocrystals and or nanoparticles may be semiconductors, metallic,
magnetic, or ceramic materials. The clusters of aggregated coated
nanocrystals, coated nanoparticles, or any combination of these may
include those having fluorescent, luminescent, ferromagnetic,
antiferromagnetic, ferrimagnetic, antiferrimagnetic or
superparamagnetic properties.
[0051] In embodiments of the aggregate or cluster compositions the
coating on the nanocrystals or nanoparticles includes an imidazole
containing molecule or an imidazole containing compound. In some
embodiments of the composition, the coating material on the
nanocrystals or nanoparticles that is used to aggregate or
flocculate them is comprised of an imidazole-containing compound
(of which histidine, carnosine, polyhistidine, polyimidazole are
illustrative examples), or an imidazole-mimicking compound compound
(of which thiazole, oxazole, pyrrole, thiophene, furan, pyridine,
pyrimidine, pyrazine, triazine, triazole, thiophene,
phthalocyanine, porphyrin, and their derivatives are illustrative
examples). Coatings in embodiments of the present invention may
include imidazole and imidazole mimicking compounds with more than
one imidazole group. For example, a di-imidazole called
1,1'-carbonyldiimidazole (or Wang imidazolide carbamate resin) that
can be used to induce aggregation. Another example includes the
imidazole compound called 4-imidazoleacrylic acid (or urocanic
acid) that can be polymerized by heat or other polymerization
initiators to induce nanocrystal aggregation.
[0052] In some embodiments of the composition, the aggregated
coated nanocrystals, aggregated coated nanoparticles, or aggregated
coated nanocrystals and aggregated coated nanoparticles are bonded
or crosslinked one to the other in the colloid by an
organophosphine containing compound. In some embodiments the
imidazole-containing compound (or an imidazole-mimicking compound)
and an alkyl phosphine containing compound are bonded by
crosslinking. In some embodiments the aggregated coated quantum
dots can be cross linked with an alkyl phosphine-containing
compound such as tris(hydroxy methyl) phosphine, or
beta-[Tris(hydroxymethyl) phosphino]propioninc acid. Various
trivalent and/or bivalent linkers may be used in coating the
nanocrystals or nanoparticles. For example, controlled aggregation
of nanometer sized particles may be achieved by using trivalent
crosslinkers like TSAT (Tris-succinimidyl aminotriacetate) and THP
(Tris(hydroxymethyl)phosphine).
[0053] Embodiments of the aggregated coated nanocrystals,
aggregated coated nanoparticles, or aggregated coated nanocrystals
and aggregated coated nanoparticles are capable of, in response to
excitation by a first energy, providing a second energy used for
detection. These colloidal aggregates can be optionally cross
linked, and can have functional groups on a surface of the colloid
aggregate capable of linking to target molecules, affinity
molecules, a sensor surface, a well plate, surface of a diode, a
MEMS sensor, an optical fiber, or to other similar molecules and
substrates. The target or affinity molecules can separately include
but are not limited to polyclonal antibodies, monoclonal
antibodies, peptides and polypeptides, an aptamer, a nucleic acid
or polynucleotide, a lectin, a lipid, a small organic molecule, a
polysaccharide, avidin, neutravidin, streptavidin, an avidin
derivative, biotin, a biotin derivative, or other affinity and
target groups.
[0054] A method of using chemically functionalized and water
dispersible, colloidal clusters of coated nanocrystals and or
coated nanoparticles in a detection system can include the acts of
contacting the functionalized colloidal cluster particles with a
sample being analyzed for the presence or absence of a substrate or
target molecule for which an affinity ligand on the colloidal
cluster has binding specificity. If the substrate or target
molecule is present in the sample, a complex is formed comprising
the functionalized colloidal cluster particles bound to the
substrate. The method can include exposing the complex, if formed,
in the detection system to a wavelength of light or a source of
energy that causes the functionalized colloidal particles of the
complexes to emit a second energy. In some embodiments, the second
energy can be a highly luminescent peak emitted by colloidal
clusters comprising coated fluorescent core/shell semiconductor
nanocrystals. The method can further include detecting the second
energy which indicates the presence of the target or substrate. For
example, the luminescence peak emitted by the complexes of
colloidal clusters comprising coated fluorescent core/shell
semiconductor nanocrystals bound to a target, can be used to
indicate the presence of the target molecule. In some embodiments,
the aggregate or cluster may be excited with a single excitation
light source and with resultant fluorescence emissions with
discrete fluorescence peaks detected.
[0055] Excitation sources suitable for characterizing aggregates or
clusters with one or more coated nanocrystals, coated
nanoparticles, or a combination of these optionally linked to one
or more target molecules or substrates in various embodiments of
this invention can include but are not limited to polychromatic
ultraviolet and visible lamps, substantially monochromatic sources
of light, polarized light, beta emitters including but not limited
to .sup.33P, .sup.125I, and .sup.3H. Sources of light may include
low, medium, and high pressure lamps as well as lasers. In some
embodiments the energy used to excite the nanocrystals or
nanoparticles can have wavelength that is less than about 500 nm.
Electric current and electron bombardment of the nanocrystals or
nanoparticles may also be used for excitation. One energy source
can be an alternating magnetic field generator for producing an
alternating magnetic field that may be guided to a specific
location (well plates, a tissue sample, or location within a
patient) by a magnetic circuit. Suitable detectors may include but
are not limited to visual detection, photodiodes, photomultipliers,
heat detectors and charge coupled device detectors (CCDs);
detectors may also include the use of polarizing filters. The
emission of light and its intensity from excited functionalized
fluorescent nanocrystals in colloidal aggregates and clusters may
be measured in any direction with respect to the excitation source;
preferably the emission intensity is measured parallel,
perpendicular, or in both directions with respect to the excitation
source. The location and concentration of nanocrystal and or
nanoparticle aggregates that include one or more coated
ferromagnetic, antiferromagnetic, ferrimagnetic, antiferrimagnetic
or superparamagnetic nanoparticles or nanocrystals may each be
determined using an existing technique, such as magnetic resonance
imaging, or another diagnostic technique can be established and
performed using a suitable magnetometer, such as a Superconducting
Quantum Interference Device (SQUID).
[0056] The detecting step or act can include detecting light or
energy emitted by the excited complex. The detecting step or act
may include detecting the magnetic flux and or energy such as light
emitted from an aggregate or cluster with one or more coated
fluorescent nanocrystals, one or more coated nanocrystals, one or
more coated nanoparticles, or any combination of these.
[0057] In one embodiment, the detection step or act can include
using an aggregate or cluster with one or more coated fluorescent
nanocrystals in a Scintillation Proximity assay. For example, a
target molecule may include a site that can react with a
radiolabel. The aggregated nanocrystal with an affinity group for
the target molecule can act as a scintillant and emit light when
the radiolabeled target molecule bonds or associates with the
aggregate or cluster with one or more coated fluorescent
nanocrystals. When the radiolabled target molecules forms a complex
with the aggregate or cluster with one or more coated fluorescent
nanocrystals, then light characteristic of the fluorescent
nanocrystal is emitted from the aggregate. For example, a
biotinylated target pepetide with a phosphorylation site may be
radiolabled using an enzyme with a [.gamma.-.sup.33P] source and
combined with a streptavidin affinity group linked to an aggregate
or cluster with one or more coated core shell fluorescent
nanocrystals. The .sup.33P labeled peptide can excite the aggregate
or cluster with one or more coated core shell fluorescent
nanocrystals to emit light which can be detected. The detecting
step or act may further include quantifying the amount of target
molecule in the sample.
[0058] Kits with one or more compositions that include an aggregate
or colloidal cluster with one or more coated fluorescent
nanocrystals, one or more coated nanocrystals, one or more coated
nanoparticles, or any combination of these are provided. Kits with
compositions that include colloidal sized cluster or aggregates may
be used to detect target molecules (for example by hybridization)
or they can be used for various diagnostic purposes or microarray
analysis. Such colloidal sized aggregate or cluster with one or
more reactive functionalities or affinity molecules may be bound
directly or indirectly (for example by hybridization) to one or
more solid supports or one or more arrays. These bound colloidal
sized aggregates or bound colloidal clusters may be included with
the kit. The kits containing these colloidal aggregates or clusters
may include one or more blank solid supports or one or more arrays
that can be functionalized or impregnated with colloidal aggregates
or target substrates.
[0059] The kits can be used with embodiments of the methods of the
invention. The kits can be used for detecting target molecules or
other substrates. The kits may include one or a number (for example
two, three, four or more) of different types of colloidal aggregate
or colloidal cluster with one or more coated fluorescent
nanocrystals. Each type of aggregate or cluster may include
different coated fluorescent nanocrystals and or functional groups
and or affinity molecules. These may be stored (in one or more
separate containers). The kits of the invention may also comprise,
in the same or different containers, at least one component
selected from one or more DNA polymerases (preferably thermostable
DNA polymerases), one or more primers, one or more templates, a
suitable buffer, enzymes (for example but not limited to a kinase),
a combination of these or other reagents.
[0060] The following examples will serve to illustrate various
embodiments of the present invention, but should not be construed
as a limitation in the scope thereof. One skilled in the art will
appreciate that although specific reagents and conditions are
outlined in the following examples, modifications can be made which
are meant to be encompassed by the spirit and scope of the
invention.
EXAMPLE 1
[0061] This example illustrates the preparation of colloidal
nanocrystals (cluster of aggregated coated nanocrystals).
[0062] 2 mg of CdSe/ZnS core/shell nanocrystals were suspended in
an organic solvent (e.g., pyridine) were extracted by 1.6 ml of 500
mM imidazole containing compound (e.g., Gly-His). After adding
chloroform (5 volumes of pyridine), the preparation was slowly
mixed by a rotary mixer for 30 minutes. The upper aqueous layer was
transferred to a different tube and diluted with 6 ml of distilled
water. Then, the preparation was dialyzed against distilled water
through a 10 kD for 70 minutes. Following dialysis, 10% v/w of
glycerol is added. To the resulting solution equal volume of
absolute ethanol was added. The number of nanocrystals per particle
(i.e., the size of the formed particle) could be manipulated by
changing the ratio of ethanol. Larger particles could be formed by
adding higher concentrations of ethanol. For about 80 nm size (5-10
nanocrystals per particle), 50% of ethanol (final concentration)
was used. After 10 minutes, 5 mM of tris-(hydroxymethylphosphine)
(THP) was added to the preparation and then the solution was mixed
for 15 hours at room temperature using a rotary mixer. The THP
treatment was repeated one time using the same conditions. A
semi-transparent solution was formed. The colloidal nanocrystals
were finally washed using centrifugation filters (MWCO 10 kD) and
re-suspended in the desired buffer.
EXAMPLE 2
[0063] This example illustrates the preparation of colloidal
cluster of aggregated coated nanocrystals conjugated to avidin.
[0064] A probe molecule having a free carboxyl-reactive group may
be operably linked to a molecule of an alkyl phosphine- or
imidazole-containing compound including the coating of the
colloidal particle (cluster of aggregated coated nanocrystals)
using methods known in the art (e.g., treatment with EDC
(1-ethyl-3-[3-dimethyl-aminopropyl]carbdiimide), followed by
treatment with sulfo-NHS (sulfo-N-hydroxysuccinimide)).
[0065] Alternatively a probe molecule having a free amine-reactive
group may be operably linked to molecule of an alkyl phosphine- or
imidazole-containing compounds comprising the colloidal particle
(cluster of aggregated coated nanocrystals) using methods known in
the art (e.g., treatment with EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbdiimide), followed by
treatment with sulfo-NHS (sulfo-N-hydroxysuccinimide).
[0066] The avidin was operably linked using these reactions to the
colloidal nanocrystals (cluster of aggregated coated nanocrystals)
previously prepared (Example 1) to form a complex. Procedures
similar to these were also used to operably bond functionalized
fluorescent nanocrystals in the cluster of aggregated coated
nanocrystals from Example 1 to the following: ConA, lectin, IgG,
and nucleic acids. The colloidal nanocrystals (cluster of
aggregated coated nanocrystals) comprising a 4-8 nanocrystals per
particle (produced by the methods described in Example 1 herein),
were operably bound to avidin. The amino groups of avidin were
operably bound to the carboxyl groups of the colloidal nanocrystals
(cluster of aggregated coated nanocrystals).
[0067] 1 mg of colloidal nanocrystals (cluster of aggregated coated
nanocrystals) suspended in 2 ml conjugation buffer (MES 50 mM, NaCl
250 mM was treated by 2 mM of EDC and 5 mM sulfo NHS. The resulting
solution was mixed at room temperature for 15 minutes, and then was
dialyzed against the conjugation buffer for 90 minutes using
dialysis membrane with a molecular weight cut off (MWCO) of 10,000
daltons. To the resulting solution was added 100-200 microgram
avidin (dissolved in 500 ul conjugation buffer), and the entire
solution was mixed at room temperature for 30 minutes. The reaction
was terminated by adding 25 mM glycine and mixing for another 30
minutes. The solution was then purified from excess avidin and
reagents using ultra-filtration centrifugal membranes with a MWCO
of 10 KD.
EXAMPLE 3
[0068] This example illustrates the covalent linking of colloidal
biocrystals (cluster of aggregated coated nanocrystals) to
avidin.
[0069] Activation of carboxyl groups on colloidal nanocrystals
(cluster of aggregated coated nanocrystals) as prepared in Example
1. In a 1.5 ml tube 1 ml of colloidal nanocrystals (1 ml 100 ug/ml
of colloidal nanocrystals (cluster of aggregated coated
nanocrystals) were briefly vortexed in a linking buffer (MES (50
mM), NaCl (200 mM), pH 6.7) with 0.05% Tween-20) for about 5
seconds to ensure uniform suspension.
[0070] Added to the colloidal nanocrystals (cluster of aggregated
coated nanocrystals) was 100 .mu.l EDC
(1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide Hydrochloride,
Pierce) 20 mM in water, freshly prepared before use, and 100 ul
Sulfo-NHS (freshly prepared Sulfo-NHS, (Pierce)) 50 mM in water to
obtain a final concentration of 2 mM and 5 mM, respectively for EDC
and Sulfo-NHS. Then, this was incubated with mild mixing at room
temperature for 10 minutes manually (a rotary mixer could also be
used).
[0071] Linking reaction: The excess of of EDC and Sulfo-NHS was
removed by 10 kD MWCO dialysis (Slide-A-Lyzer (MWCO 10,000 Daltons;
Pierce) against the linking buffer (MES (50 mM), NaCl (200 mM), pH
6.7) with 0.05% Tween-20 for 70 to about 90 minutes (final volume
of colloidal nanocrystal (cluster of aggregated coated
nanocrystals) solution is 1.2 ml).
[0072] Then the suspension was transferred from the dialysis
cassette into a 15 ml tube and agitated by adding 200 .mu.l avidin
solution (20-40 .mu.g Avidin in 200 .mu.l of linking buffer with
0.05% Tween-20 (Sigma)). This was incubated for about 1 hour at
room temperature with mild mixing. An incubation time was
determined for each ligand; to determine activity and preferred
conditions for linking different ligands to the cluster of
aggregated coated nanocrystals.
[0073] The reaction with glycine was quenched at a final
concentration of 10 mM (for 1.4 ml add 14 .mu.l of a 1 M glycine
solution) and final pH was adjusted to 7.5 by sodium carbonate
(about 10 .mu.l of 1M Na.sub.2CO.sub.3 for 1.4 ml) and mixing
continued for an additional 30 minutes.
[0074] The mixture was transferred into a 4 ml Millipore
centrifugation filter (MWCO 10k) and TBS was added with 0.05%
Tween-20 up to 4 ml, and was mixed and spun at 2000 rpm/5 min. The
supernatant was then carefully removed to a different collection
tube. The fluffy sediment was resuspended in TBS with 0.05%
Tween-20 and spun at 2000 rpm for about 5 min. The supernatant was
carefully removed and it was pooled with the first supernatant.
Finally, the fluffy sediment was resuspended with a suitable volume
(about 1 ml) of TBS with 0.05% Tween-20 and stored at 4.degree. C.
until its use.
[0075] The concentration of protein can be determined by
determining the 280 nm absorbance of the pooled supernatants and
calculating the conjugated fraction.
[0076] For other targets or proteins, the suitable amounts for
conjugation can be determined empirically. The MW of avidin is
about 65 kD. For antibodies (MW about 150 kD), replacing the same
number of molecules can be used. For example, instead of 20-40
.mu.g it might be suitable to start with 50-100 .mu.g of antibodies
to match with 100 .mu.g colloidal nanocrystals (cluster of
aggregated coated nanocrystals).
EXAMPLE 4
[0077] This example shows the detection of biotin target antigens
with avidin-conjugated nanocrystal colloids (avidin conjugated
cluster of aggregated coated nanocrystals).
[0078] Colloidal cluster biotin-conjugated (target) and
unconjugated (negative control) antibodies on nitrocellulose in a
range of concentrations from 1 .mu.g to 30 ng were placed on sheets
in 6 well plates.
[0079] The sheets were rinsed with TBS with 0.1% Tween. The TBS was
blocked in with 0.1% Tween with 1% Perfect Block (MoBiTec) for 1
hour at room temperature. The avidin-conjugated cluster colloids
were diluted to 5 .mu.g/ml in a blocking solution. As a positive
control, one can dilute avidin-conjugated FITC (Sigma) in the same
manner.
[0080] 2 ml of the fluorescent conjugate was added to each well and
incubated for 1 hour at room temperature without agitation. This
was then rinsed 3 times using TBS with 0.1% Tween while avoiding
the application of the rinse buffer directly onto the
nitrocellulose. The membranes were then illuminated with UV light
from above. The colloids appeared orange without UV illumination.
FIG. 2 shows the test results where colloidal nanocrystals were
conjugated to avidin using EDC chemistry and were tested by dot
blot assay to detect biotinylated antibody blotted on a
nitrocellulose membrane; the top row was the biotinylated
antibodies and the bottom row was the non biotinylated antibodies
(control). Binding to the top row of dots were specific
avidin-biotin interaction; binding to the lower row of dots
indicated the presence of unconjugated colloidal clusters. The same
buffers and reagent concentrations can be used in a lateral flow
assay system, or to detect biotinylated antigens in a Western
Blot.
EXAMPLE 5
[0081] This example illustrates the functionalization or coating of
core-shell nanocrystals that was used to form colloids (cluster of
aggregated coated nanocrystals).
[0082] 500 mM Gly-His was dissolved TTin 1M Na.sub.2CO.sub.3.
Sonnicat The core-shell nanocrystals were sonicated and then 2 mg
core-shell nanocrystals were added to Gly-His solution in an inert
atmosphere. Then 6 ml chloroform about 3 ml/mg NCs was added and
extracted for 30 min. on a rotator platform at room temperature. A
centrifuge was then used for 2 minutes at 500 rpm. The top layer
was then removed to a 50 ml tube. Then the interface was removed to
eppendorf tubes and a centrifuge was used for 1 minute at 6000 rpm.
The top layer was collected and added to previously collected
material. (Total recovery about 1 ml). 5 ml ddH.sub.2O was added
and dialyzed in a 10 kD MWCO Slide-Lyzer against 2 L ddH.sub.2O for
70 minutes at room temperature. The dialyzed material was then
moved to a 50 ml tube and 12 ml 95% EtOH/5% Isopropanol was added
quickly while vorxeting and mixed gently 10 minutes. 4 mg
H.sub.2N-PEG-COOH (Nektar, mw 3,400) was added and then mixed
gently 3 minutes. Glycerol was then added to a final concentration
of 10% and the pH was adjusted to 9.0+/-0.5 with 1M Citric Acid.
The tube was then filled with argon and sonnicated for 5 minutes. 5
mM THP (freshly made) was added and incubated on rotator platform
overnight (while protecting from light). 5 mM THP (freshly made)
was then added and incubated on rotator platform overnight (while
protecting from light). A Centrifuge was used for 2 minutes at 500
rpm in an Amicon filter (10 kD MWCO). Do not allow a true pellet to
prevent aggregation of the colloids. The supernatant (check to be
sure it is not fluorescent) was removed. Resuspend pellet in
ddH.sub.2O and use a centrifuge 2 minutes at 500 rpm. The colloids
were resuspended in a desired buffer.
* * * * *