U.S. patent number 8,637,082 [Application Number 13/119,170] was granted by the patent office on 2014-01-28 for methods for preparation of znte nanocrystals.
This patent grant is currently assigned to Life Technologies Corporation. The grantee listed for this patent is Joseph Bartel, Joseph Treadway, Eric Tulsky. Invention is credited to Joseph Bartel, Joseph Treadway, Eric Tulsky.
United States Patent |
8,637,082 |
Tulsky , et al. |
January 28, 2014 |
**Please see images for:
( Certificate of Correction ) ** |
Methods for preparation of ZnTe nanocrystals
Abstract
Nanocrystals having a ZnTe core and methods for making and using
them to construct core-shell nanocrystals are described. These
core-shell nanocrystals are highly stable and provide quantum
yields and stability suitable for applications such as flow
cytometry, cellular imaging, and protein blotting, medical imaging,
and other applications where cadmium toxicity is an issue.
Inventors: |
Tulsky; Eric (Berkeley, CA),
Bartel; Joseph (Eugene, OR), Treadway; Joseph (Eugene,
OR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Tulsky; Eric
Bartel; Joseph
Treadway; Joseph |
Berkeley
Eugene
Eugene |
CA
OR
OR |
US
US
US |
|
|
Assignee: |
Life Technologies Corporation
(Carlsbad, CA)
|
Family
ID: |
42074228 |
Appl.
No.: |
13/119,170 |
Filed: |
October 2, 2009 |
PCT
Filed: |
October 02, 2009 |
PCT No.: |
PCT/US2009/059346 |
371(c)(1),(2),(4) Date: |
August 29, 2011 |
PCT
Pub. No.: |
WO2010/040032 |
PCT
Pub. Date: |
April 08, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20110300076 A1 |
Dec 8, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61102599 |
Oct 3, 2008 |
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Current U.S.
Class: |
424/489 |
Current CPC
Class: |
B82Y
30/00 (20130101); C09K 11/883 (20130101); C01B
19/007 (20130101); C01P 2004/04 (20130101); C01P
2002/84 (20130101); C01P 2004/80 (20130101); C01P
2004/64 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
A61K
9/14 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Navarro, Investigating uptake of water-dispersible CdSe/ZnS quantum
dot nanoparticles by Arabidopsis thaliana plants , Journal of
Hazardous Materials, 2012, 211-221, 427-435. cited by examiner
.
Dabbousi et al., J. Phys. Chem. B 101(46):9463-9475 (1997). cited
by applicant .
Guan, J, Submitted for the Degree of Master of Science in
Chemistry, Massachusetts Institute of Technology, pp. 16, 23-27,
31, 38 and 54 (2008). cited by applicant .
Hines et al., J. Phys. Chem. 100(2):468-471 (1996). cited by
applicant .
Kuno et al., J. Phys. Chem. 106(23):9869-9882 (1997). cited by
applicant .
Peng et al., J. Am. Chem. Soc. 119(30):7019-7029 (1997). cited by
applicant .
Stankova et al., J. Peptide Sci. 5:392-398 (1999). cited by
applicant .
Molecular Probes, Inc., "Qdot.RTM. ITK.TM. Carboxyl Quantum Dots
Product Sheet", 2007, pp. 1-11. cited by applicant.
|
Primary Examiner: Dickinson; Paul
Attorney, Agent or Firm: Life Technologies Corporation
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH FUNDING
This invention was made, in part, with government support under
cooperative agreement No. 70NANB4H3053 with the National Institute
of Standards and Technology and the U.S. Department of Commerce.
The government may have certain rights in this invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATION
This application is a national stage filing of International
Application No. PCT/2009/059346, filed Oct. 2, 2009, which claims
priority to U.S. Provisional Application No. 61/102,599, filed Oct.
3, 2008; which disclosures are incorporated herein by reference in
their entirety.
Claims
We claim:
1. A semiconductor nanocrystal, comprising: a semiconductor core
comprising zinc and tellurium; a semiconductor shell surrounding
the core comprising zinc and selenium; and a hydrophilic surface
coating on the shell that renders the nanocrystal water
dispersible.
2. The semiconductor nanocrystal of claim 1, wherein the
nanocrystal has a quantum yield of at least 20%.
3. The semiconductor nanocrystal of claim 1, further comprising an
additional shell, which is a shell of ZnS applied over the ZnSe
shell.
4. The semiconductor nanocrystal of claim 1, wherein the
semiconductor core consists essentially of zinc and tellurium.
5. The semiconductor nanocrystal of claim 1, wherein the shell
consists essentially of zinc and selenium.
6. A water-stable semiconductor nanocrystal composition, comprising
the semiconductor nanocrystal of claim 1, wherein the surface
coating is a water-stabilizing layer.
7. A population of semiconductor nanocrystals, the population
comprising a plurality of semiconductor nanocrystals of according
to claim 1, and having a quantum yield of at least 20%.
8. A composition, comprising the population of semiconductor
nanocrystals of claim 7, wherein the composition is photochemically
and chemically stable and non-toxic to cells or tissue.
9. The composition of claim 8, further comprising an organic
solvent, water, polymer, or glass.
10. The composition of claim 8 formulated for use in an in vitro
biological assay, an in vivo assay, or for ophthalmic or topical
administration.
11. A kit, comprising: a) the composition of claim 8; and b)
instructions for use.
12. A population of ZnTe semiconductor nanocrystals produced by a
method comprising contacting a Zn.sup.2+ salt with a Te.sup.0
precursor in a solvent at a temperature that is sufficiently high
to induce nanocrystal formation, wherein each nanocrystal further
comprises a hydrophilic surface coating on the shell that renders
the nanocrystal water dispersible.
13. The population of claim 12, wherein the method further
comprises adding an amount of a strong reducing agent to the
solvent to initiate nanocrystal formation.
14. The population of claim 12, wherein the method further
comprises contacting the semiconductor ZnTe core in a solvent with
a Zn.sup.2+ salt and a Se.sup.0 precursor, at a temperature
sufficiently high to induce formation of a ZnSe shell on the ZnTe
nanocrystal core.
15. A method to form the semiconductor nanocrystal of claim 1,
comprising contacting a semiconductor ZnTe core in a solvent with a
Zn.sup.2+ salt and a Se.sup.0 precursor , at a temperature
sufficiently high to induce formation of a ZnSe shell on the ZnTe
nanocrystal core.
16. The method of claim 15, wherein the Se.sup.0precursor is added
after the Zn.sup.2+ salt.
17. The method of claim 15, wherein the Zn.sup.2+ salt is
ZnCl.sub.2, ZnCl(O.sub.2CR) or Zn(O.sub.2CR).sub.2, wherein R is an
alkyl group, or comprises an alkyl carboxylate anion or an
oleate.
18. The method of claim 17, wherein the alkyl carboxylate anion
comprises 4-24 carbon atoms or at least one unsaturated group.
19. The method of claim 15, wherein the alkyl group comprises at
least one unsaturated group.
20. The method of claim 15, further comprising applying an outer
shell of ZnS over the ZnTe/ZnSe core/shell nanocrystal.
21. A method of detecting a target in a biological sample, the
method comprising: contacting a biological sample with the
semiconductor nanocrystal of claim 1; and detecting the spectral
emission of the semiconductor nanocrystal.
22. A population of ZnTe/ZnSe semiconductor nanocrystals produced
by the method of claim 15.
Description
FIELD OF THE INVENTION
Provided herein are semiconductor nanocrystal compositions
containing zinc telluride and methods of making and using such
compositions.
BACKGROUND ART
Semiconductor nanocrystals have a wide variety of applications. Of
the many unique properties of these materials, the photophysical
characteristics may be the most useful. Specifically, these
materials can absorb light and then emit an intense luminescent
emission that is particle size-dependent and particle
composition-dependent. This fluorescent emission can have an
extremely narrow luminescence bandwidth, can be environmentally
sensitive or insensitive depending on the nanocrystal's structure,
and can be resistant to photobleaching under intensive light
sources. Emissions can be efficiently excited with electromagnetic
radiation having a shorter wavelength than the highest energy
emitter in the material, and by varying the size and composition of
the nanocrystal, a user can use many different types of
nanoparticles mixed together and can still distinguish each type.
These properties allow semiconductor nanocrystals to be used as
markers or as ultra-sensitive luminescent reporters of biological
states and processes in highly multiplexed systems.
Nanocrystals are typically spherical or nearly so (though methods
of making nanocrystals of other shapes are known), and can have
multiple layers, such as a central core, a surrounding shell, and
optional capping groups, linkers, and other surface-conjugated
materials. Typically, core/shell nanocrystals are described
according to the composition of the core and of a semiconductor
shell applied outside the core; the shell usually stabilizes the
nanocrystal and protects its photophysical properties. It may also
provide an attachment surface for linking the nanocrystal to a
molecule, cell, subcellular organelle, and the like that is to be
tracked or observed.
The nanocrystal core largely determines its critical light
absorption and emission characteristics. Nanocrystal cores have
been broadly studied and improvements in synthesis have led to the
optimization of key physiochemical properties resulting in
nanocrystal cores with uniform size distributions and intense,
narrow emission bands following photo-excitation. However,
nanocrystal cores alone lack sufficiently intense or stable
emission intensities for most applications, and nanocrystal cores
are particularly sensitive to their environment; for example, the
aqueous environment required for many biological applications can
lead to the complete destruction of the luminescence of nanocrystal
cores. Thus, methods to photostabilize nanocrystal cores (e.g.,
protect their luminescent properties) and make them stable and
useful in aqueous media are of great interest for biological
applications. Commonly, this is achieved by applying a shell over
the core, to form a so-called core/shell nanocrystal.
The choice of shell material must be made to match the core
material. For example, the shell material may have a wider band gap
than the core, which enables it to protect the activated state that
the core occupies when it has been photoactivated, forming a
separated electron and hole. The shell may ideally be chosen to
have an atomic spacing and lattice structure that closely match
those of the core material to best preserve the photophysical
attributes of the core, since irregularities in the interface
between core and shell may be responsible for non-radiative energy
dissipation mechanisms that reduce luminescent efficiency.
Core/shell nanocrystals having a CdX core wherein X is S, Se, or Te
coated with a YZ shell where Y is Cd or Zn, and Z is S, Se, or Te
are commonly discussed and used, and have been shown to have good
emission characteristics and stability. This may largely be due to
the YZ coating material's band-gap energy which spans that of the
core relatively symmetrically. `Symmetry` as used in this sense
means that the wider bandgap of the shell material fully
encompasses the narrower bandgap of the core material and extends
both above the high end of the core material's bandgap and below
the low end of the core material's bandgap.
One limitation of CdSe-based core/shell nanocrystals is that the
blue emitting particles have lower extinction coefficients than red
emitting particles. This is due to the fact that emission
wavelength are tuned by changing the CdSe core particle size:
smaller particles have a blue-shifted emission, but also typically
absorb light less efficiently than larger, red-shifted particles.
Several researchers have shown that by utilizing alloy cores (e.g.,
CdSSe or ZnCdSe) one can tune the wavelength by adjusting the
elemental composition rather than size, and can thus decouple
emission color from extinction coefficient. One can also utilize a
semiconductor material with a larger bulk band gap such that the
largest nanocrystals emit in the blue/green portion of the visible
spectrum (e.g., ZnSe).
A more serious limitation of CdSe nanocrystals for certain
applications such as in vivo imaging or diagnostic tests is
toxicity. Cadium is a toxic metal. The toxicity of cadmium, and to
a lesser extent selenium, raises concerns about using a nanocrystal
containing cadmium and selenium for in vivo applications in live
organisms or in living cells. Therefore, bright and stable
nanocrystals that do not contain cadmium are of special value for
such uses, and for any uses involving large scale production or use
of nanocrystals, in order to minimize environmental impact and
associated health concerns.
Accordingly, for certain applications it is advantageous to use
different core materials that do not have attendant toxicity
concerns. Additionally, for some applications, very small or very
large nanocrystals (relatively speaking) may be advantageous; for
example, if used to label a biomolecule like DNA or a protein, it
may be preferable to have a very small nanocrystal, less than about
10 nm in overall size, including the core/shell nanocrystal and a
coating used on the shell to adapt the particle for use in a
suitable medium. For biomolecules, the most relevant medium is
frequently water; thus the nanocrystals must often be specially
treated and/or coated so they are readily suspended or dissolved in
water. For other applications, such as tracking a large cell such
as a bacterium, flow cytometry, cellular imaging, protein blotting,
and other protein detection methods, it may be advantageous to use
a single, very bright nanoparticle, which may sometimes be a larger
particle.
BRIEF SUMMARY OF THE INVENTION
Provided herein are nanoparticles that are particularly useful in
certain in vivo applications where toxicity concerns are paramount,
and in applications where visualizing a labeled molecule is
important. More particularly, provided herein are nanocrystals that
are small, bright, stable and versatile, as well as convenient
methods for making such nanocrystals. The nanoparticles can be
larger than typical CdSe core nanocrystals having similar emission
wavelengths, which is achieved by using a different core
material.
The methods provided herein are particularly applicable to
preparation of ZnTe core nanocrystals having a ZnSe shell, and
optionally certain additional features. The compositions and
related methods solve numerous unexpected difficulties caused by
the properties of the ZnTe core.
In one aspect, provided herein are nanocrystals having a core that
comprises, or consists essentially of, a semiconductor material
containing zinc and tellurium (ZnTe). In some embodiments, the core
is made from zinc and tellurium precursors (zinc salts, for
example, and Te salts or tellurium dissolved in a trialkylphosphine
to form a phosphine telluride) under conditions selected to
minimize or prevent incorporation of other elements into the core.
Use of ZnTe as the material for the core can provide nanocrystals
that are larger than CdSe cores emitting at the same wavelength and
are free of concerns about cadmium toxicity. Moreover, the
nanocrystals provided herein are bright and are photostable enough
for many applications where quantum dots have been used.
The ZnTe nanocrystals provided herein can be particularly useful in
certain applications, such as flow cytometry, cellular imaging, and
protein blotting. In particular, these nanocrystals are
particularly useful in certain in vivo applications where toxicity
concerns are paramount, e.g., in ophthalmology and live cell
imaging and in certain applications where visualizing a labeled
molecule is important. In addition, the nanocrystals provided
herein are useful in application where environmental disposal is
particularly problematic, such as lighting and display technology,
and in other high volume consumer electronics.
ZnTe has been suggested as a suitable material for some
nanocrystals; however, few reports of ZnTe nanocrystal cores have
achieved the high quantum yield (>20%) needed for most practical
applications, and few have provided a core/shell nanocrystal having
a ZnTe core with a protective semiconductor shell. In particular,
there are few reports of stable, bright (high quantum yield plus
good light absorption) ZnTe core nanocrystals that are stable and
usable in an aqueous environment. The nanocrystal cores as well as
methods for stabilizing these cores with a passivating shell to
form a core/shell nanocrystal are provided herein. In some
embodiments, the core/shell nanocrystal has a core of ZnTe and a
shell of ZnSe, and needs no interface layer between the core and
shell to achieve the desired properties. The ZnSe shell is thus
applied directly onto and in contact with the ZnTe core.
Advantageously, the nanocrystals described herein have a quantum
yield of at least about 10%, sometimes at least 20%, sometimes at
least 30%, sometimes at least 40%, and sometimes at least 50% or
greater. The quantum yield can change over time, and in some
embodiments it does not decrease by more than 50% after three weeks
in organic solution. Preferably, it does not decrease by more than
about 35% over three weeks, and in some embodiments the quantum
yield decreases by less than 25% over a period of three weeks in
solution.
Certain compositions provided herein include fluorescent
semiconductor nanocrystals having a ZnTe core and a ZnSe shell.
Nanocrystals prepared by the methods disclosed herein have been
shown to be chemically and electrically stable in organic medium
and provide cadmium-free core/shell nanocrystals with fluorescence
emissions in the green to orange-red part of the visible spectrum,
from about 420 nm to about 670 nm, sometimes from about 525 nm to
about 560 nm. In certain embodiments, nanocrystals with ZnTe core
are provided exhibiting fluorescence emission in the green region
of the spectrum (e.g., about 510-550 nm).
In one example, ZnTe/ZnSe nanocrystals disclosed herein provided a
quantum yield of at least about 20%, along with good photochemical
and chemical stability. Nanocrystals provided herein can have
fluorescence maxima in the visible wavelength range, typically
between about 420 nm and about 670 nm, sometimes between about 525
nm and about 560 nm. Sometimes, the fluorescence maxima is greater
than or equal to 500 nm, greater than or equal to 525 nm, greater
than or equal to 550 nm, greater than or equal to 575 nm, or
greater than or equal to 600 nm.
In one aspect, a semiconductor nanocrystal is provided, comprising
a semiconductor core comprising zinc and tellurium (ZnTe) and a
shell comprising zinc and selenium (ZnSe). In certain embodiments,
the semiconductor nanocrystal can have a quantum yield of at least
20%. The semiconductor nanocrystal can be a member of a
substantially monodisperse population. Any of the core/shell
nanocrystals provided herein can be less than about 10 nm in
diameter. In some cases, the core is less than 6 nm in diameter.
The semiconductor nanocrystal can have a fluorescence emission
wavelength in the range from about 420 nm to about 670 nm In some
cases, the nanocrystal has a fluorescence emission wavelength in
the range from about 525 nm to about 560 nm. The semiconductor
nanocrystal can further comprise a coating of organic ligands.
Alternatively, the nanocrystal is provided with a coating that
makes the nanocrystal water dispersible. In certain embodiments,
the semiconductor nanocrystal further comprises an additional shell
(e.g., a shell of ZnS applied over the ZnSe shell). In certain
embodiments, the semiconductor core that consists essentially of
zinc and tellurium. In certain embodiments, the shell consists
essentially of zinc and selenium.
In yet another aspect, a semiconductor nanocrystal is provided
comprising a ZnTe core, and a shell comprising MgX or BeX, wherein
X represents O, S or Se and can further include an additional shell
of MgX or BeX, wherein X represents O, S or Se.
In yet another aspect, a core/shell nanocrystal is provided
comprising a core of ZnTe and a shell of ZnSe or ZnSe/ZnS, further
comprising a coating of phosphonic acid ligands.
In another aspect, a composition is provided, comprising a
ZnTe/ZnSe core/shell semiconductor nanocrystal, wherein the
composition is non-toxic to cells or tissue. The composition is
photochemically and chemically stable. Such compositions can
include a plurality of semiconductor nanocrystals. In certain
embodiments, the composition includes a substantially monodisperse
particle population of ZnTe/ZnSe nanocrystals. The nanocrystals in
the composition can be water-dispersible and can have a quantum
yield of 20% or greater. In some case, the quantum yield is 40% or
greater. In certain embodiments, the composition further comprises
an organic solvent, water, polymer, or glass. For example,
compositions are provided in which the semiconductor nanocrystals
are embedded in or applied to the surface of a solid or semi-solid
matrix (e.g., polymer matrix, bead, or resin). Alternatively, the
composition can be in the form of a liquid, gel, paste, cream,
patch, film, or a powder (e.g., lyophilized powder). In certain
embodiments, compositions are provided that include one or more
semiconductor nanocrystals that emit light in a wavelength range
that is substantially non-absorbent to animal fluid, cells, or
tissue. In certain embodiments, the composition is adapted for
inserting into a mammalian body, while in other cases the
composition is adapted or formulated for use in an in vitro
biological assay or an in vivo assay.
In yet another aspect, a pharmaceutical composition is provided,
comprising a semiconductor nanocrystal as described herein (e.g.,
ZnTe/ZnSe). The composition can be formulated for administration to
a patient. For example, the composition can be for ophthalmic
administration (e.g., as an ophthalmic solution) or for topical
administration (e.g., as an ophthalmic solution, skin cream, or
surgical paste). The pharmaceutical composition can further include
a pharmaceutically acceptable carrier for the nanocrystals (e.g.,
water, a saline solution, or a buffer).
In yet another aspect, kits containing ZnTe nanocrystals are
provided. The kits can be for pharmaceutical uses or for use in a
biological assay. An exemplary kit for pharmaceutical use includes
a) one or more pharmaceutically acceptable containers; b) a
pharmaceutical composition as provided herein; and c) instructions
for use. Kits for biological assay can include, in addition to the
nanocrystals, other reagents, such as solvents, standards, buffers,
dyes, and the like.
In yet another aspect, a water-stable semiconductor nanocrystal
composition is provided, comprising: at least one semiconductor
ZnTe/ZnSe nanocrystal as described herein; wherein the at least one
nanocrystal further comprises a water-stabilizing layer. The
water-stabilizing layer can include a hydrophobic portion for
interacting with the surface of the semiconductor nanocrystal and a
hydrophilic portion for interacting with an aqueous medium.
In yet another aspect, provided herein are methods to make ZnTe
nanocrystal cores, and methods to add a shell such as ZnSe to these
cores. It has been found that ZnTe cores are unexpectedly sensitive
to certain types of reaction conditions often used for making and
shell-coating fluorescent nanocrystals. Consequently, good quality
nanocrystals having ZnTe cores are not readily made by simply
modifying the typical reaction conditions used to make other types
of nanocrystals, e.g., CdSe cores. Similarly, their chemical
reactivity leads to poor results when typical shell-forming
reaction conditions are used. Provided herein are methods to make
ZnTe crystalline and colloidally stable cores and to add a
protective shell coating to produce useful fluorescent nanocrystals
with high quantum yields.
In yet another aspect, a method to make a ZnTe semiconductor
nanocrystal is provided, comprising contacting a Zn.sup.2+ salt
with a Te.sup.0 precursor at a temperature that is sufficiently
high to induce nanocrystal formation. The method can further
include contacting the Zn.sup.2+ and Te.sup.0 precursor in a
solvent (e.g., an amine, a phosphine, or an alkyl carboxylic acid).
The amine can be a secondary or tertiary amine. In some
embodiments, the amine is an alkyl amine comprising 4-24 carbon
atoms. In some embodiments, the alkyl amine is a dialkylamine or a
trialkylamine. In some embodiments, the amine comprises 10 or more
carbon atoms. The Zn.sup.2+ salt can include an alkyl carboxylate
anion. For example, the alkyl group can include 4-24 carbon atoms.
The Zn.sup.2+ salt can include at least one unsaturated group
(e.g., oleate). The Te.sup.0 precursor can be Te.sup.0 or
R.sub.3PTe. The temperature is typically above 200.degree. C. The
method can further include adding an amount of a strong reducing
agent (e.g., lithium triethylborohydride) to the solvent to
initiate nanocrystal formation. The amount of the strong reducing
agent can be less than 1 equivalent of the Zn.sup.2+ or less than 2
equivalents of the Zn.sup.2+. In some embodiments, the method
involves further adding a weak reductant to promote nanocrystal
growth. The amount of weak reducing agent can be in excess of the
number of equivalents of the Zn.sup.2+. The method can further
involve adding an amount of a Te.sup.2- precursor to the solvent to
initiate nanocrystal formation, wherein the amount of the Te.sup.2-
precursor is less than 1 equivalents of the Zn.sup.2+. In certain
embodiments, the method further comprises doping the ZnTe core with
Se. For example, the core of the nanocrystal comprises an alloy of
Zn, Te, and Se.
In yet another aspect, a method to form a ZnSe shell on a ZnTe
nanocrystal core is provided, comprising contacting a ZnTe core
with a Zn.sup.2+ salt and a Se.sup.0 precursor, at a temperature
sufficiently high to induce shell formation. The Se.sup.0 precursor
(e.g., R.sub.3PSe or Se.sup.o) and/or Zn.sup.2+ salt can,
optionally, be provided in a solvent. The solvent can comprise a
secondary or tertiary amine, and in certain embodiments, the
solvent does not include a primary amine. In some cases, the
Se.sup.0 precursor can be added after the Zn.sup.2+ salt. In
certain embodiments, the ZnTe nanocrystal core is heated in the
solvent with a Zn.sup.2+ salt before the Se.sup.0 precursor is
added. The Zn.sup.2+ salt can be the same or different than that
used to form the core and can include, e.g., an alkyl carboxylate
anion. The alkyl anion can comprise 4-24 carbon atoms. In some
cases, the alkyl group comprises at least one unsaturated group.
The Zn.sup.2+ salt can be, for example, ZnCl.sub.2, ZnCl(O.sub.2CR)
or Zn(O.sub.2CR).sub.2, wherein R is an alkyl group. The alkyl
group can include at least one unsaturated group. In some
embodiments, the Zn.sup.2+ salt comprises an oleate. The shelling
method can further comprise applying an outer shell of ZnS over the
ZnTe/ZnSe core/shell nanocrystal.
Methods of modifying the nanocrystals made by these methods, to
make them water-soluble and/or to adapt them to link to a target
molecule or an affinity molecule, and use in certain in vivo
applications are also provided by the disclosure, as are methods of
using these nanocrystals.
In one aspect, a method of detecting a target in a biological
sample is provided. The method includes contacting a biological
sample with a semiconductor nanocrystal, as provided herein, or a
composition including such nanocrystals, wherein the nanocrystal or
composition is non-toxic to cells or tissues; and detecting the
spectral (e.g., fluorescence) emission of the semiconductor
nanocrystal.
In another aspect, a method of detecting an interaction between a
compound and a biological target is provided. The method comprises
providing a non-toxic composition capable of a characteristic
spectral emission, the composition comprises a compound and a
semiconductor nanocrystal, as provided herein, associated with the
compound, wherein the composition is non-toxic to cells or tissues,
and wherein the emission provides information about a biological
state or event; allowing a sample comprising a biological target to
interact with the composition; and detecting interaction between
the compound and the biological target by monitoring the spectral
emission of the sample. The method can be used in various assays.
For example, the spectral emission can be associated with assays
selected from the group consisting of immunochemistry,
immunocytochemistry, immunobiology, or immunofluorescence assays;
DNA sequence analyses; fluorescence resonance energy transfer, flow
cytometry, or fluorescence activated cell sorting assays;
diagnotics in biological systems; in vivo imaging; and
high-throughput screening. The target can be any type of cell and
can be a dead, fixed, or live cell (e.g., a mammalian cell, a stem
cell, a cancer cell, or the like).
In yet another aspect, a method of imaging a tissue is provided.
The method includes contacting a tissue with a semiconductor
nanocrystal, as provided herein, or a composition including such
nanocrystals, wherein the nanocrystal or composition is non-toxic
to cells or tissues; and detecting the fluorescence emission of the
semiconductor nanocrystal. The method can be used to image tissue
in vivo (e.g., tumor tissue or retinal tissue).
In yet another aspect, a method of marking tissue during a surgical
procedure is provided. The method include contacting a tissue with
a semiconductor nanocrystal, as provided herein, or a composition
including such a nanocrystal, wherein the nanocrystal or
composition is non-toxic to cells or tissues; and detecting the
fluorescence emission of the semiconductor nanocrystal. In certain
embodiments, the method can be used to mark tumor tissue (e.g., as
part of a tumor resection surgery).
In yet another aspect, an electronic or photovoltaic device is
provided that includes a population of semiconductor nanocrystals,
as provided herein. Exemplary devices include electronic displays,
light emitting diodes, solar panels, and sensors.
BRIEF DESCRIPTION OF THE DRAWINGS
The following figures form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to
one or more of these figures in combination with the detailed
description of specific embodiments presented herein.
FIG. 1 shows how increasing reaction temperature improved the
morphology of ZnTe nanocrystal cores: (A) 290.degree. C., (B)
325.degree. C., and (C) 350.degree. C.
FIG. 2 shows a plot of HOMO energy for various nanocrystal core
materials relative to the oxygen acceptor level. Of the materials
shown, only ZnTe has a HOMO energy above the oxygen acceptor level.
ZnTe nanocrystal cores are, therefore, especially sensitive to
oxygen and particularly benefit from a protective shell.
FIG. 3 shows a series of absorption spectra for nanocrystals
prepared using two types of amine solvents after varying reaction
times. The three curves on the lower/left side correspond to
reaction using hexadecylamine (HDA) as solvent. The three upper
curves correspond to reactions using dioctylamine, rigorously
dried, as the solvent. Dioctylamine (DOA) is expected to reduce
amide formation, and thereby reduce generation of water in the
reaction mixture. The shift of the shoulders to longer wavelengths
demonstrates growth of nanocrystals. The dioctylamine reactions
were significantly faster. The results demonstrate that significant
improvements in the ZnTe core formation reaction can be achieved
when a secondary amine is used instead of the more conventional
primary amine as a solvent for the reaction.
FIG. 4 shows a plot of fluorescence emission as a function of
wavelength from the reaction mixture during growth of ZnSe shells
on ZnTe nanocrystals. As the reaction progressed, the fluorescence
emission increased sharply as nanocrystals formed and grew, and the
maximum wavelength increased as nanocrystals became larger, as
illustrated by increasing fluorescence intensity and red-shifting
of the fluorescence maximum.
FIG. 5 shows how pre-treatment of ZnTe cores with a zinc salt prior
to addition of a selenium precursor to the shell-forming reaction
improves the brightness of the nanocrystal products. Note the
difference in the two scales for the emission intensities from the
two reactions.
DETAILED DESCRIPTION OF THE INVENTION
While compositions and methods are described in terms of
"comprising" various components or steps (interpreted as meaning
"including, but not limited to"), the compositions and methods can
also "consist essentially of" or "consist of" the various
components and steps, unless otherwise clearly indicated herein.
Such terminology should be interpreted as defining essentially
closed-member groups.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of
ordinary skill in the art to which this invention belongs.
As used herein, "a" or "an" means "at least one" or "one or
more."
As used herein, `about` means that the numerical value is
approximate and small variations would not significantly affect the
practice of the invention. Where a numerical limitation is used,
unless indicated otherwise by the context, `about` means the
numerical value can vary by .+-.10% and remain within the scope of
the invention.
"Alkyl" as used in reference to alkyl phosphine, alkyl phosphine
oxide, alkylcarboxylate or alkylamine refers to a hydrocarbon group
having 1 to 24 carbon atoms, frequently between 4 and 24 carbon
atoms, or between 6 and 12 carbon atoms, or between 5 and 20 carbon
atoms, and which can be composed of straight chains, cyclics,
branched chains, or mixtures of these. The alkyl phosphine, alkyl
phosphine oxide, or alkylamine include embodiments having from one
to three alkyl groups on each phosphorus or nitrogen atom. In
preferred embodiments, the alkyl phosphine or alkyl phosphine oxide
has three alkyl groups on P, and the alkyl amine(s) have one alkyl
group on N. In some embodiments, the alkyl group contains an oxygen
atom in place of one carbon of a C.sub.4-C.sub.24 or a
C.sub.6-C.sub.12 alkyl group, provided the oxygen atom is not
attached to P or N of the alkyl phosphine, alkyl phosphine oxide,
or alkylamine. In some embodiments, the alkyl can be substituted by
1-3 substituents selected from halo and C.sub.1-C.sub.4 alkoxy. The
alkyl groups herein can also include one-two unsaturated bonds
(double bonds), provided those bonds do not include the carbon
directly attached to P or N in a phosphine, phosphonate,
phosphinate, phosphine oxide or amine.
Preferred alkyl phosphines include compounds of the formula
[(C.sub.4-C.sub.12).sub.3]P. Preferred alkyl phosphine oxides
include compounds of the formula [(C.sub.4-C.sub.12).sub.3]PO.
Preferred alkylamines include compounds of formula
(C.sub.4-C.sub.12).sub.2NH and (C.sub.4-C.sub.24)NH.sub.2, where
each C.sub.4-C.sub.12 or C.sub.4-C.sub.24 alkyl is a straight or
branched chain unsubstituted alkyl group. Preferred alkyl
phosphonic acids and alkyl phosphinic acids include those having
1-15 carbon atoms and preferably 2-12 carbon atoms or 3-8 carbon
atoms. Preferred alkyl carboxylates for use in the methods of the
invention include C.sub.5-C.sub.24 alkyl groups with an attached
carboxylic acid group, e.g., (C.sub.5-C.sub.24)alkyl-COOH, where
the alkyl can be straight chain, branched, cyclic or a combination
of these. In some embodiments, the alkyl carboxylate has at least
one double bond in its alkyl group.
"Hydrophobic" as used herein refers to a surface property of a
solid, or a bulk property of a liquid, where the solid or liquid
exhibits greater miscibility or solubility in a low-dielectric
medium than it does in a higher dielectric medium. A nanocrystal
that is soluble in organic solvents that are not miscible with
water, such as ethyl acetate, dichloromethane, MTBE, hexane, or
ether, is hydrophobic. By way of example only, nanocrystals that
are soluble in a hydrocarbon solvent such as decane or octadecene
and are insoluble in an alcohol such as methanol are
hydrophobic.
"Hydrophilic" as used herein refers to a surface property of a
solid, or a bulk property of a liquid, where the solid or liquid
exhibits greater miscibility or solubility in a high-dielectric
medium than it does in a lower dielectric medium. By way of
example, a material that is more soluble in methanol than in a
hydrocarbon solvent such as decane would be considered
hydrophilic.
"Growth medium" as used herein refers to a mixture of reagents
and/or solvents in which a nanocrystals is grown or in which a
shell is grown on a nanocrystals. These growth media are well known
in the art, and often include at least one metal, at least one
chalcogenide (a compound of S, Se, or Te), and one or more alkyl
phosphines, alkyl phosphine oxides, alkyl phosphonic acids, alkyl
phosphinic acids, alkyl carboxylic acids, or alkylamines.
"Coordinating solvents" as used herein refers to a solvent such as
TOP, TOPO, carboxylic acids, and amines, which are effective to
coordinate to the surface of a nanocrystal. `Coordinating solvents`
include phosphines, phosphine oxides, phosphonic acids, phosphinic
acids, amines, and carboxylic acids, which are often used in growth
media for nanocrystals, and which form a coating or layer on the
nanocrystal surface. They exclude hydrocarbon solvents such as
hexanes, toluene, hexadecane, octadecene, and the like, which do
not have heteroatoms that provide bonding pairs of electrons to
coordinate with the nanocrystal surface. Hydrocarbon solvents that
do not contain heteroatoms such as O, S, N or P to coordinate to a
nanocrystal surface are referred to herein as non-coordinating
solvents. Note that the term `solvent` is used in its ordinary way
in these terms and refers to a medium that supports, dissolves, or
disperses materials and reactions between them, but which does not
ordinarily participate in or become modified by the reactions of
the reactant materials.
"Luminescence" refers to the property of emitting electromagnetic
radiation from an object. Typically, the electromagnetic radiation
is in the range of UV to IR radiation and can refer to visible
electromagnetic radiation, for example light. Luminescence may
result when a system undergoes a transition from an excited state
to a lower energy state resulting in the release of a photon. The
transition responsible for luminescence can be stimulated through
the release of energy stored in the system chemically or
kinetically, or can be added to the system from an external source,
such as, for example by a photon or a chemical, thermal,
electrical, magnetic, electromagnetic, physical energy source, or
any other type of energy source capable of exciting the system. In
some embodiments, `luminescence` refers to fluorescence--emission
of a photon that is initiated by excitation with a photon of higher
energy (shorter wavelength) than the emitted photon.
"Exciting a system" or "exciting" or "excitation" refers to
inducing the energy state of a system into a higher state than that
of ground state. The term "excitation wavelength" refers to
electromagnetic energy which may have a shorter wavelength than
that of the emission wavelength that is used to excite the system.
The "energy states" of the system described herein can be
electronic, vibrational, rotational, or any combination thereof.
The term "emission peak" refers to the wavelength that has the
highest relative intensity within a characteristic emission
spectra.
A typical single-color preparation of nanoparticles has crystals
that are preferably of substantially identical size and shape.
Nanocrystals are typically thought of as being spherical or nearly
spherical in shape, but can actually be any shape. Alternatively,
the nanocrystals can be non-spherical in shape. For example, the
nanocrystal's shape can change towards oblate spheroids for redder
colors. It is preferred that at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%,
and ideally about 100% of the particles are of the same size. Size
deviation can be measured as root mean square ("rms") of the
diameter, with less than about 30% rms, preferably less than about
20% rms, more preferably less than about 10% rms. Size deviation
can be less than about 10% rms, less than about 9% rms, less than
about 8% rms, less than about 7% rms, less than about 6% rms, less
than about 5% rms, or ranges between any two of these values. Such
a collection of particles is sometimes referred to as being
"monodisperse". One of ordinary skill in the art will realize that
particular sizes of nanocrystals, such as of semiconductor
nanocrystals, are actually obtained as particle size
distributions.
"Nanoparticle" as used herein refers to any particle with at least
one major dimension in the nanosize range. Typically, a
nanoparticle has at least one major dimension ranging from about 1
to 1000 nm.
Examples of nanoparticles include a nanocrystal, such as a
core/shell nanocrystal, plus any tightly-associated organic coating
or other material that may be on the surface of the nanocrystal. A
nanoparticle can also include a bare core or core/shell
nanocrystal, as well as a core nanocrystal or a core/shell
nanocrystal having a layer of, e.g., TOPO or other material that is
not removed from the surface by ordinary solvation. A nanoparticle
may have a layer of ligands on its surface which may further be
cross-linked; and a nanoparticle may have other or additional
surface coatings that modify the properties of the particle, for
example, solubility in water or other solvents. Such layers on the
surface are included in the term `nanoparticle.`
"Nanocrystal" as used herein refers to a nanoparticle made out of
an inorganic substance that typically has an ordered crystalline
structure. It can refer to a nanocrystal having a crystalline core,
or to a core/shell nanocrystal, and may be 1-100 nm in its largest
dimension, preferably about 1 to 50 nm in its largest
dimension.
A core nanocrystal is a nanocrystal to which no shell has been
applied; typically it is a semiconductor nanocrystal, and typically
it is made of a single semiconductor material. It may be
homogeneous, or its composition may vary with depth inside the
nanocrystal. Many types of nanocrystals are known, and methods for
making a nanocrystal core and applying a shell to it are known in
the art. The nanocrystals provided herein are frequently bright
fluorescent nanocrystals, and the nanoparticles prepared from them
are typically also bright, e.g., having a quantum yield of at least
about 10%, sometimes at least 20%, sometimes at least 30%,
sometimes at least 40%, and sometimes at least 50% or greater.
Nanocrystals generally require a surface layer of ligands to
protect the nanocrystal from degradation in use or during
storage.
"Quantum dot" as used herein refers to a nanocrystalline particle
made from a material that in the bulk is a semiconductor or
insulating material, which has a tunable photophysical property in
the near ultraviolet (UV) to far infrared (IR) range.
"Water-soluble" is used herein to mean the item is soluble or
suspendable in an aqueous-based solution, such as in water or
water-based solutions or buffer solutions, including those used in
biological or molecular detection systems as known by those skilled
in the art. While water-soluble nanoparticles are not truly
`dissolved` in the sense that term is used to describe individually
solvated small molecules, they are solvated and suspended in
solvents that are compatible with their outer surface layer, thus a
nanoparticle that is readily dispersed in water is considered
water-soluble or water-dispersable. A water-soluble nanoparticle is
also considered hydrophilic, since its surface is compatible with
water and with water solubility.
"Hydrophobic nanoparticle" as used herein refers to a nanoparticle
that is readily dispersed in or dissolved in a water-immiscible
solvent like hexanes, toluene, and the like. Such nanoparticles are
generally not readily dispersed in water; rather, they clump or
precipitate from aqueous solutions.
Semiconductor nanocrystals can be 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 and 5,262,357, as well as International Patent
Publication No. WO 99/26299, published May 27, 1999. These methods
typically produce nanocrystals having a coating of hydrophobic
ligands on their surfaces which protect them from rapid
degradation. The nanocrystals are typically prepared in two steps
that produce two distinct layers, a core and a shell.
In some embodiments, the nanoparticle provided herein is a member
of a monodisperse population of nanoparticles of like composition.
Monodisperse means that the particles are similar in size, and fall
within about 30% of a particular mean dimension, preferably within
about 20%, more preferably less than about 10%. The monodisperse
particle population in some embodiments is characterized in that it
exhibits less than about 10% rms deviation in the diameter, or
largest dimension, of the core. In some embodiments, the
monodisperse particle population exhibits less than about 5% rms
deviation in the diameter, or largest dimension, of the core.
In some embodiments, a monodisperse population is produced by
making a single batch of nanocrystals all together, and they have
similar properties due to their production method. However, careful
control of reaction conditions permits a user of the methods to
produce nanocrystals of the invention consistently enough for
separate batches to form a monodisperse population; and a
monodisperse population can also be produced by careful control of
conditions using continuous flow production methods.
Nanocrystal sizes are typically from about 1 nm to about 100 nm in
diameter, sometimes from about 1 nm to about 50 nm in diameter, and
sometimes from about 1 nm to about 25 nm in diameter. For a
nanocrystal that is not substantially spherical, e.g. rod-shaped,
it may be from about 1 nm to about 100 nm, or from about 1 nm to
about 50 nm or 1 nm to about 25 nm in its smallest dimension.
Generally, a nanocrystal is a semiconductive particle, having a
diameter or largest dimension in the range of about 1 nm to about
100 nm, or in the range of about 2 nm to about 50 nm, and in
certain embodiments, in the range of about 2 nm to about 20 nm or
from about 2 to about 10 nm. More specific ranges of sizes include
about 0.5 nm to about 5 nm, about 1 nm to about 50 nm, and about 1
nm to about 20 nm Specific size examples include about 0.1 nm,
about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about
5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm,
about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm,
about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm,
about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm,
about 50 nm, and ranges between any two of these values. In some
embodiments, a core nanocrystal less than about 10 nm in diameter,
or less than about 7 nm in diameter, or less than about 5 nm in
diameter.
In some embodiments, provided herein is a nanocrystal core that is
less than about 10 nm in diameter, or less than about 7 nm in
diameter, or less than about 5 nm in diameter. Certain nanocrystal
cores are about 2-5 nm in diameter.
In some embodiments, provided herein is a core/shell nanocrystal
that is less than about 10 nm in diameter. In some embodiments the
core/shell nanocrystal is less than about 8 nm in diameter, or less
than about 6 nm in diameter.
In some embodiments, provided herein is a coated core/shell
nanocrystal that is less than about 20 nm in diameter. The coating
can comprise an organic material, such as a layer of TOP or TOPO or
other coordinating ligands that can make the nanocrystal or quantum
dot soluble in hydrophobic media, or a hydrophilic coating such as
an amphiphilic (AMP) coating as discussed in U.S. Pat. No.
7,108,915, which is incorporated herein by reference, which
provides a water-soluble or water dispersable composition.
The nanoparticles provided herein are generally fluorescent, due to
the presence of a fluorescent nanocrystal core. The nanoparticles
are often characterized by a fluorescence maximum in the visible
spectrum, and frequently the fluorescence of a monodisperse
population of ZnTe nanocrystals is characterized in that when
irradiated the population emits light for which the peak emission
is in the spectral range of from about 420 nm to about 670 nm.
Nanocrystals can be characterized by their percent quantum yield of
emitted light. For example, the quantum yield of the nanoparticles
provided herein can be greater than about 10%, greater than about
20%, greater than about 30%, greater than about 40%, greater than
about 50%, greater than about 60%, greater than about 70%, greater
than about 80%, greater than about 90%, and ranges between any two
of these values. The quantum yield is typically greater than about
30%, and preferably greater than 50% or greater than 70%.
It is well known that the color (emitted light) of the
semiconductor nanocrystal can be "tuned" by varying the size and
composition of the nanocrystal. Nanocrystals preferably absorb a
wide spectrum of wavelengths, and emit a narrow wavelength of
light. The excitation and emission wavelengths are typically
different, and non-overlapping. The nanoparticles of a monodisperse
population may be characterized in that they produce a fluorescence
emission having a relatively narrow wavelength band. Examples of
emission widths (FWHM) for the nanoparticles provided herein
include less than about 200 nm, less than about 175 nm, less than
about 150 nm, less than about 125 nm, less than about 100 nm, less
than about 75 nm, less than about 60 nm, less than about 50 nm,
less than about 40 nm, less than about 30 nm, less than about 20
nm, and less than about 10 nm. The width of emission is preferably
less than about 50 nm, and more preferably less than about 20 nm at
full width at half maximum of the emission band (FWHM). Particular
monodisperse populations of nanoparticles provided herein exhibit a
width (FWHM) of about 30 nm to about 50 nm. The emitted light
preferably has a symmetrical emission of wavelengths. The emission
maxima can generally be at any wavelength from about 200 nm to
about 2,000 nm. Examples of emission maxima include about 200 nm,
about 400 nm, about 600 nm, about 800 nm, about 1,000 nm, about
1,200 nm, about 1,400 nm, about 1,600 nm, about 1,800 nm, about
2,000 nm, and ranges between any two of these values. In certain
embodiments, the emission maxima ranges from about 425 nm to about
625 nm; or about 650 nm; or about 670 nm. In certain embodiments, a
green color is desirable, so a wavelength in the green region
(e.g., about 510 nm to about 500 nm) is selected.
The nanoparticles described herein may be a core/shell nanocrystal
having a nanocrystal core covered by a semiconductor shell. The
thickness of the shell can be adapted to provide desired particle
properties. The thickness of the shell can affect fluorescence
wavelength, quantum yield, fluorescence stability, and other
photostability characteristics.
In some embodiments, a core semiconductor nanocrystal is modified
to enhance the efficiency and stability of its fluorescence
emissions, prior to ligand modifications described herein, by
adding an overcoating layer or shell to the semiconductor
nanocrystal core. Having a shell may be preferred, because surface
defects at the surface of the semiconductor nanocrystal can result
in traps for electrons, or holes that degrade the electrical and
optical properties of the semiconductor nanocrystal core, or other
non-radiative energy loss mechanisms that either dissipate the
energy of an absorbed photon or at least affect the wavelength of
the fluorescence emission slightly, resulting in broadening of the
emission band. An insulating layer at the surface of the
semiconductor nanocrystal core can provide 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 processes.
Suitable materials for the shell 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 shell
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. The preparation of a coated
semiconductor nanocrystal may be found in, e.g., Dabbousi et al.
(1997) J. Phys. Chem. B 101:9463, Hines et al. (1996) J. Phys.
Chem. 100: 468-471, Peng et al. (1997) J. Am. Chem. Soc.
119:7019-7029, and Kuno et al. (1997) J. Phys. Chem. 106:9869. It
is also understood in the art that the actual fluorescence
wavelength for a particular nanocrystal core depends upon the size
of the core as well as its composition, so the categorizations
above are approximations, and nanocrystal cores described as
emitting in the visible or the near IR can actually emit at longer
or shorter wavelengths depending upon the size of the core.
In some embodiments, the metal atoms of a shell layer on a
nanocrystal core are selected from Cd, Zn, Ga and Mg. The second
element in these semiconductor shell layers can be selected from S,
Se, Te, P, As, N and Sb. ZnSe has been found to be a suitable shell
for the ZnTe cores provided herein; however, other materials, or
mixtures or alloys of ZnSe with other materials, may also be
used.
The nanocrystal can be of any suitable size; typically, it is sized
to provide fluorescence in the UV-visible portion of the
electromagnetic spectrum, since this range is convenient for use in
monitoring biological and biochemical events in relevant media. The
relationship between size and fluorescence wavelength is well
known, thus making nanoparticles smaller may require selecting a
particular material that gives a suitable wavelength at a small
size, such as ZnTe as the core of a core/shell nanocrystal.
Typically core/shell nanocrystals of interest are from about 1 nm
to about 100 nm in diameter, and sometimes from about 1 nm to about
50 nm, or from about 1 nm to about 25 nm. For a nanocrystal that is
not substantially spherical, e.g. rod-shaped, it may be from about
1 nm to about 100 nm, or from about 1 nm to about 50 nm, or from
about 1 nm to about 25 nm, in its smallest dimension.
The nanoparticles can have surface coatings adding various
functionalities. For example, the nanocrystals can be coated with
lipids, phospholipids, fatty acids, polynucleic acids, polyethylene
glycol, primary antibodies, secondary antibodies, antibody
fragments, protein or nucleic acid based aptamers, biotin,
streptavidin or other avidin derivatives, proteins, peptides, small
organic molecules, and organic or inorganic dyes. In certain
embodiments, ZnTe nanocrystals are treated with a coating
comprising a cationic polymer such as polyarginine. Certain
embodiments are directed towards nanoparticles coated with
phospholipids. Other semiconductor nanocrystal coating materials,
e.g., the AMP polymers discussed above also can be used.
Spectral characteristics of nanoparticles can generally be
monitored using any suitable light-measuring or light-accumulating
instrumentation. Examples of such instrumentation are CCD
(charge-coupled device) cameras, video devices, CIT imaging,
digital cameras mounted on a fluorescent microscope,
photomultipliers, fluorometers and luminometers, microscopes of
various configurations, and even the human eye. The emission can be
monitored continuously or at one or more discrete time points. The
photostability and sensitivity of nanoparticles allow recording of
changes in electrical potential over extended periods of time.
Additional methods of assaying the emission from the
nanoparticle(s) include measuring changes in light intensity, light
polarization, light absorption, color of the emission, emission
lifetime or half-life, or the "blinking" pattern.
Nanoparticles can be synthesized in shapes of different complexity
such as spheres, rods, discs, triangles, nanorings, nanoshells,
tetrapods, and so on. Each of these geometries have distinctive
properties: spatial distribution of the surface charge, orientation
dependence of polarization of the incident light wave, and spatial
extent of the electric field. In many embodiments, the nanocrystals
provided herein are roughly spherical. While methods for
synthesizing several different nanocrystal types are known, each
nanocrystal material and combination of materials can introduce
unexpected complications. Certain unexpected difficulties with
making ZnTe nanocrystal cores and applying semiconductor shells to
those cores have been identified and solutions to these problems
are provided herein, including improved methods specifically
adapted for use with ZnTe nanocrystal core production and
modification.
ZnTe Nanocrystal Cores
In one aspect, provided herein is a semiconductor nanocrystal that
comprises zinc and tellurium. In certain embodiments, the
nanocrystal consists primarily or essentially of zinc and tellurium
(ZnTe). Certain semiconductor nanocrystals provided herein include
a ZnTe core and a passivating shell. Described herein is a unique
set of reaction conditions for preparing ZnTe cores. The core can
be made to achieve suitable quantum yields. These synthesis
procedures should be conducted with special care to exclude
moisture and oxygen, thus solvents and reagents were carefully
purged of oxygen before mixing and all transformations should be
conducted under inert atmosphere throughout the process of forming
the ZnTe core and adding a shell to it.
The methods described herein address various insufficiencies in
current nanocrystal production methods and complications associated
with working with zinc and tellurium as starting materials. Efforts
to make high-quality ZnTe nanocrystal cores have focused on
substituting zinc and tellurium precursors in existing methods for
preparation of other chalcogenide-containing nanocrystals (e.g.,
CdSe). Although methods for producing CdSe cores are well known,
adapting those methods to make ZnSe or ZnTe cores has been
inefficient or ineffective. For example, reaction of Cd(TDPA) with
Se in TOP (TOPSe) is known to work well for preparation of CdSe
cores. However, when used to react Zn(TDPA) with (TMS).sub.2Se
under the same conditions, the synthesis provided polydisperse
particles in a very low yield. Thus, this product, produced by
substitution of the cadmium with zinc, is not acceptable for use in
core/shell nanocrystal preparation. The low particulate yield may
be due to insufficient nucleation. Use of a more reactive Zn
precursor, such as ZnEt.sub.2, can provide a better particulate
yield, and can produce high quality ZnSe nanocrystals with a
fluorescence maximum at about 420 nm. When used with tellurium in
TOP (TOPTe) in hexadecylamine as the solvent, the reaction with
ZnEt.sub.2 produces irregular particle shapes, which are not of
sufficient quality to use in shell-forming reactions. For some
applications, regular particle shapes (e.g., spherical particle
shapes) are preferred. Higher temperatures (350.degree. C.) can
produce more regular nanocrystal particle shapes, but the product
includes zinc particles along with nanocrystals, suggesting that
the diethyl zinc precursor is too reactive for these conditions.
FIG. 1 depicts the improved nanocrystal morphology associated with
increasing reaction temperature from 290.degree. C. to 325.degree.
C. and to 350.degree. C. Diethyl zinc can be replaced with various
alkyl carboxylic acid salts of zinc, e.g., Zn(O.sub.2CR).sub.2,
where R represents an alkyl group that can be saturated or
unsaturated. The identity of the R group can have a dramatic effect
on the quality of the nanocrystals produced. Where R is a saturated
alkyl group (e,g, stearate), the reaction produces little
nanocrystal formation. Unexpectedly, when the undecylenate salt (an
unsaturated alkyl carboxylic acid salt) is used rather than a
saturated alkyl group, good quality fluorescent nanocrystals can be
obtained.
While not being limited to a particular theory, it is believed that
the unsaturated alkyl carboxylic acid salt provides a weak
reductant to facilitate the core-forming reaction. This may be due
to mismatch between the oxidation states of the tellurium and zinc
precursors being used: the zinc undecylenate is in a +2 oxidation
state under the reaction conditions, but the tellurium precursor
R.sub.3PTe (a complex of trialkylphosphine and tellurium) provides
Te.sup.0 under the reaction conditions. It is believed that the
uncharged tellurium species cannot readily react with Zn.sup.+2,
and that reduction of either Te.sup.0 to Te.sup.2-, or of Zn.sup.+2
to Zn.sup.0 is required for reaction to occur. Undecylenate from
the Zn-containing precursor is believed to provide this reducing
capacity under the reaction conditions described herein, probably
by reducing Te.sup.0 to Te.sup.2-. Reaction of the `matched` active
species can then occur.
Two precursors are `matched` when they are in an oxidation state
that permits them undergo immediate reaction with each other. For
example, two neutral species, such as Te.sup.0 and Zn.sup.0, or two
ionic species having complementary charges, such as Te.sup.2-, and
Zn.sup.+2 are considered `matched.` Two precursors are considered
`mismatched` where they provide species that cannot react to form a
neutral species unless an electron transfer agent is present to
adjust the oxidation state of one of the precursors to provide
`matched` species capable of undergoing reaction. Frequently,
mismatched precursors are used where one precursor provides an
ionic species and the other precursor provides a non-ionic (i.e.,
neutral) species under the reaction conditions. For example,
Te.sup.0 and Zn.sup.2+ are mismatched precursors. Precursors that
provide two ionic species having the same charge (i.e., two cations
or two anions) would also be `mismatched.`
It is believed that use of a reductant that is sufficiently weak
(e.g., undecylenate) allows this reaction to occur at a slow and
controlled rate, suitable for promoting nanocrystal growth.
However, these conditions generally are not ideal for promoting
nucleation, because the reaction is too slow. Ideal nanocrystal
formation conditions typically involve rapid nucleation to provide
a large number of particles, then no further nucleation during the
growth stage. If all nucleation occurs quickly, the growth stage
can produce highly consistent particle size resulting in a
monodisperse particle distribution. If nucleation continues during
growth stage, a mixture of particle sizes is more likely to be
produced. The methods provided herein balance the need for rapid
nucleation and slow and controlled growth. An exemplary method to
make ZnTe nanocrystals achieves this balance by using a Zn.sup.2+
salt in combination with a Te.sup.0 precursor, such as R.sub.3PTe.
The reaction can be carried out in the presence of a weak
reductant. Optionally, the reductant can be present in a solvent
component such as an amine, a phosphine or an alkyl carboxylic
acid. In certain cases, a solvent is chosen that can function as
the weak reductant (e.g., an amine or phosphine). In some
embodiments, the weak reductant is provided by an unsaturated alkyl
group. For example, it can be provided by an unsaturated alkyl
carboxylic acid salt used as a zinc precursor. Representative zinc
precursors include, for example, Zn(O.sub.2CR).sub.2 and
Zn(O.sub.2CR)X, where each R is an unsaturated alkyl group having
4-24 carbons, and each X is another anion such as halide,
phosphonate, phosphinate, sulfonate, and the like. Certain
embodiments utilize an unsaturated alkyl group having 18 carbon
atoms. Exemplary zinc precursors include oleate or undecylenate
salts of Zn.sup.2+.
While the use of a weak reductant provides an advantageous means to
support nanocrystal growth, it does not necessarily provide ideal
conditions for nucleation. Ideally, nucleation occurs quickly and
essentially all at once, to produce a monodisperse population of
nanocrystals.
In order to further improve the preparation of ZnTe nanocrystals,
methods are provided for inducing nucleation as a rapid and
short-lived phase under the reaction conditions. An effective
method for inducing nucleation involves addition of a small amount
of a stronger reducing agent at the beginning of the reaction. The
quantity of the stronger reductant is sufficient to provide the
desired amount of nucleation, by reducing an appropriate amount of
the Te or Zn precursor. Generally, the amount of the strong
reducing agent can be about 0.1 equivalents to about 2 equivalents
of Zn.sup.2+, however, other amounts also can be used. In some
embodiments, the amount is less than about 1 equivalent of the
Zn.sup.2+ or, in some cases, less than about 2 equivalents of the
Zn.sup.2+. Other processes can utilize less than 0.2 equivalents of
Zn.sup.2+. A variety of chemical reducing agents can be used for
this purpose, including tertiary phosphines, secondary phosphines,
primary phosphines, amines, hydrazines, hydroxyphenyl compounds,
hydrogen, hydrides, metals, boranes, aldehydes, alcohols, thiols,
reducing halides, polyfunctional reductants, and mixtures thereof.
In a specific embodiment, lithium triethylborohydride is used. In
certain embodiments, the reductant is a cathode. Alternatively, in
order to induce nucleation, a small amount of `matched` precursors
can be used along with a mismatched pair in a larger amount,
suitable for supporting growth after nucleation has occurred. For
example, a small amount of a more reactive zinc precursor like
diethyl zinc can be used with R.sub.3PTe, in combination with a
larger amount of a Zn.sup.2+ precursor. The Zn.sup.0 precursor,
e.g. diethyl zinc, can be used in sufficient quantity to induce a
desired amount of nucleation. Once it has been consumed, which
occurs quickly at an appropriate reaction temperature, the
Zn.sup.2+ precursor is present in sufficient quantity for a desired
extent of growth, to produce a desired nanocrystal size. The
nanocrystal size can be determined readily during the growth phase
by monitoring the fluorescence wavelength.
In other embodiments, the method involves adding a weak reductant,
such as a phosphine, amine or alkene to the solvent to promote
nanocrystal growth. The amount of the weak reducing agent can be in
excess of the number of equivalents of the Zn.sup.2+.
Alternatively, an amount of a Te.sup.2- precursor can be added to
the solvent to initiate nanocrystal formation, wherein the amount
of the Te.sup.2- precursor is less than 1 equivalents of the
Zn.sup.2+.
A special problem arises with tellurium containing nanocrystals and
their production, because such nanocrystals tend to be extremely
sensitive to air and moisture. For example, as FIG. 2 illustrates,
ZnTe nanocrystals can react with oxygen when in its ground state,
i.e., it does not require photochemical activation for oxidation to
occur if oxygen is present. In addition, the Zn.sup.2+ precursor is
an especially `hard` Lewis acid, and interacts very strongly with
any moisture or hydroxide present, since oxygen is a `hard` Lewis
base. It is therefore important to take extra precautions to
exclude moisture from a ZnTe nanocrystal formation reaction.
The solvent for these core-forming reactions typically comprises an
amine with hexadecylamine as one typical example Amine solvents can
be difficult to purify sufficiently for the ZnTe nanocrystal
reactions, because of the especially high sensitivity the reaction
components and products exhibit toward moisture and air. While
nanocrystal preparations are typically done with purified solvents
and reagents, and under inert atmosphere, further special
precautions and steps are taken for purifying amine solvents used
to prepare ZnTe nanocrystals. The amine to be used as solvent for
these reactions is placed in a flask which is repeatedly evacuated
then filled with anhydrous inert atmosphere. Anhydrous NaOH or KOH,
having been dried under vacuum at over 100.degree. C., is then
added to the amine solvent, and the suspension is stirred for at
least 8 hours. The amine is filtered under inert atmosphere to
remove the solids, and the amine is then distilled under inert
atmosphere, and stored under an inert atmosphere.
It has been found that under the severe reaction conditions, e.g.,
temperatures of over 250.degree. C., and frequently 300.degree. C.
or higher, the amine solvent can react with carboxylic acids
present in the reaction mixture to form an amide. The formation of
water as a by-product and the consumption of the carboxylic acid
solvent by amide formation can be deleterious for reactions using
Zn, due to the high affinity Zn has for oxygen ligands. Thus, even
the purified amine can become a source of water under the reaction
conditions.
Methods are provided to avoid or at least significantly reduce the
presence of water in the reaction process and thereby increase the
product quality for ZnTe nanocrystals. The methods generally
utilize an amine solvent. Any type of amine solvent can be used,
however secondary and tertiary amines are generally preferred,
since they are less likely to result in amide formation. In
particular, secondary amines, R.sub.2NH, where each R is
independently a C.sub.4-C.sub.24 alkyl group, significantly enhance
the quality and yield of ZnTe nanocrystals using the methods
described herein. Preferably, the alkyl groups of the alkyl contain
a total of at least about 10 carbon atoms. One suitable choice for
the solvent is dioctylamine (R.sub.2NH, where each R is a C.sub.8
alkyl group, preferably n-octyl), which can be used successfully in
place of a primary amine such as hexadecylamine. FIG. 3
demonstrates how use of a secondary amine, in place of a primary
amine, improves the formation of ZnTe nanocrystal cores. Using
dioctylamine (DOA) dried and purified as described above, as
solvent for a reaction of zinc oleate with TOPTe produced ZnTe
nanocrystals having a fluorescence maximum emission at about 525 nm
wavelength. These nanocrystals were monodisperse as demonstrated by
a FWHM of under 50 nm for the population of nanocrystals produced.
The ZnTe nanocrystals were purified using conventional methods,
without exposure to moisture or air in order to protect their
photochemical activities.
Any of the methods provided herein can be employed in a flow
reaction system or in a batch process reaction.
Shell Formation
Especially in view of the water and moisture sensitivity of ZnTe
nanocrystals, it is often desirable to provide a shell of a
semiconductor material over the ZnTe nanocrystal. ZnTe core/shell
nanocrystals are useful in many typical `quantum dot` applications.
A number of candidates for shell material are known, as are methods
to apply the shell. However, application of ZnS shells using
standard protocols have failed to produce viable shells for ZnTe
nanocrystal cores.
Initial efforts at developing suitable shells for the ZnTe
nanocrystals described herein focused on modifying existing shell
reaction protocols and materials useful for well-known types of
core materials. For example, one process involves deposition of a
ZnS shell on ZnSe nanocrystals using diethyl zinc and a phosphinic
acid to produce a zinc phosphinate salt, which was then reacted
with the ZnSe cores and (TMS).sub.2S to form a thick protective ZnS
shell.
However, when the same conditions were used to apply a shell of ZnS
to a ZnTe nanocrystal core, the product was an irregular shell that
did not protect the nanocrystal's fluorescence. Without wishing to
be bound by theory, this suggested that lattice match between the
ZnS and ZnTe was insufficient to provide an epitaxial, protective
shell. Comparison of lattice parameters suggested ZnSe may be a
better match for a ZnTe nanocrystal. However, use of the same
conditions used for applying ZnS shells was unsuccessful. In
particular, when ZnTe cores were treated with a zinc phosphinate
salt in dioctylamine and warmed, the cores unexpectedly dissolved.
The addition of oleic acid, which is typically used to provide a
protective ligand for the nanocrystals, was likely too corrosive
with ZnTe nanocrystal cores, and destroyed the nanocrystals.
An acid-free medium has been found to minimize destruction of the
ZnTe nanocrystal cores. Such a medium can be formed using zinc
salts along with Se in TOP (TOPSe) in dioctylamine to form a shell
on the ZnTe cores. Various zinc salts can be used including, but
not limited to, phosphonate and phosphinate salts, zinc carboxylic
acid salts; and mixed salts having one carboxylic acid ligand and
one other ligand, such as halide.
TOPSe, which is typically added last, can be added quickly or
slowly. The rate of addition is not critical, because in the
absence of a strong reductant, zinc selenide shell formation is
preferred over nucleation.
The zinc precursor is preferably an unsaturated alkyl carboxylic
acid salt, such as oleate or undecylenate. For example, exemplary
zinc salts include Zn(oleate).sub.2, Zn(oleate)Cl,
Zn(undecylenate).sub.2 and Zn(undecylenate)Cl.
Also provided herein is a method for applying a shell of ZnSe to a
sensitive nanocrystal core such as ZnTe. The method involves use of
a zinc salt having at least one unsaturated carboxylate ligand on
the zinc (RCO.sub.2-, where R is a C.sub.4-C.sub.24 unsaturated
alkyl group, which can be straight-chain, branched chain, cyclic,
or a combination of these, and contains at least one, and
preferably only one, carbon-carbon double bond). In some
embodiments, this zinc salt is used in combination with a Se.sup.0
selenium precursor such as R.sub.3PSe to form a shell on a ZnTe
nanocrystal core. In some embodiments, the zinc salt is
Zn(O.sub.2CR).sub.2 or Zn(O.sub.2CR)Cl, and an amount of the zinc
salt that is sufficient to provide enough reduced selenium to
provide the desired amount of shell is used. Preferably, the
solvent is a secondary amine, such as a dialkylamineor a
trialkylamine, such as dioctylamine or other dialkylamines as
described above for use as the solvent in the ZnTe nanocrystal core
formation.
The zinc salt can be prepared in situ during the shell forming
reaction by using diethyl zinc or other dialkyl zinc species, and
allowing the dialkyl zinc to react with the unsaturated carboxylic
acid. The reaction of diethyl zinc with oleic acid, for example,
provides zinc oleate, and produces ethane as a byproduct.
Alternatively, using zinc chloride plus oleic acid forms the zinc
salt having at least one unsaturated carboxylic acid ligand.
The quality of the nanocrystal product can be further improved by
pre-treating the ZnTe cores with an amine solvent containing a zinc
salt prior to addition of the selenium precursor. The solvent can
include any type of amine. However, pre-treatment with secondary
amines (e.g., dialkylamine), in particular, can have certain
advantages. In one exemplary method, ZnTe cores are heated with
about 0.3 equivalents of the Zn salt (e.g., Zn(oleate)Cl) prior to
addition of the selenium precursor. Pretreatment significantly
improves the brightness of the nanoparticles. FIG. 5 illustrates
the improvement in nanocrystal quality when ZnTe cores are
pre-treated with a zinc salt (Zn(oleate)Cl) at elevated temperature
prior to adding the selenium precursor. It also was found that
using too much of the zinc salt for this pretreatment reduces the
brightness (quantum yield) of the nanocrystal product. The two
plots of FIG. 5 demonstrate that the order and amount of zinc salt
addition impacts the emission spectra of the resulting core/shell
nanoparticles. In one pre-treatment method, ZnTe cores were
dispersed in dioctylamine (DOA) to which was added 1.0 equivalent
of Zn(oleate)Cl. The reaction mixture was heated and then to it
added TOPSe and 0.3 equivalents of Zn(oleate)Cl. In another method,
ZnTe cores were dispersed in DOA to which was added 0.3 equivalents
Zn(oleate)Cl. The reaction mixture was heated and to it was added
TOPSe and 1.0 equivalent Zn(oleate)Cl. The pre-treated ZnTe cores
were treated with varying amounts of shell material. Referring to
the two plots of FIG. 5, each plot depicts three emission spectra
for cores treated with a full amount of shell material and 1/3 and
1/9 of this amount of shell material, where pre-treated ZnTe cores
not having a reduced amount of shell material yielded the most
intense emission.
Thus in another aspect, a method is provided for increasing the
brightness of a nanocrystal having a ZnTe core and a zinc
chalcogenide shell, comprising heating the ZnTe cores with an
amount of a zinc salt in a dialkylamine solvent prior to adding the
second (chalcogenide) shell precursor and remaining zinc salt. This
process can increase the brightness of core/shell nanocrystals by
over 100%, and as much as 200%, relative to a process where the
shell forming reaction is done by adding all of the zinc shell
precursor before the second shell precursor is added.
Nanocrystals made by this method exhibit high quantum yields, and
are stable in storage. Indeed, they can be treated with an
amphiphilic polymer coating to render them water soluble, and they
exhibit excellent stability in water. Alternatively, they can be
dispersed in a hydrophobic solvent such as hexanes without
significant loss of quantum yield for a period of at least about 3
weeks. These nanocrystals can also be surface-modified in other
ways, e.g. by PEGylation or conjugation to a bioaffinity molecule
to enhance their chemical stability. Although ZnTe nanocrystals
have relatively high chemical stability, their photostability is
lower, and may be improved by further treatment or additional
layers of shell material.
In certain embodiments, ZnTe containing nanocrystals are provided
that emit in the visible portion of the electromagnetic spectrum.
Certain ZnTe nanocrystals emit in the green portion of the
spectrum. For example, the conditions provided herein can yield
core/shell nanocrystals having a maximum fluorescence emission at
525 nm or above, and up to about 560 nm. For example, a desired
green color can be achieved with a nanocrystal having a ZnTe core
onto which is deposited a passivating shell material. Various shell
materials can be used with green emitting ZnTe nanocrystals. For
example, the shell may include one or more of ZnSe, ZnTe, ZnS, MgS,
MgSe, MgTe, BeSe, and BeTe. In certain embodiments, a ZnSe or ZnS
shell provides nanoparticles with particularly desirable
photoluminescence properties. The shell can be formed from a
combination (e.g., a mixture or alloy) of shell materials or can be
formed by depositing multiple layers onto the nanocrystal core.
Certain nanocrystal compositions provided herein include a ZnTe
core and a ZnSe shell. Another exemplary shell can include a layer
of ZnSe shell proximal to the ZnTe core and a shell layer of ZnS
deposited thereon. In one embodiment, a ZnTe nanocrystal having a
diameter of about 4 nm is combined with a shell of ZnSe about 1 nm
thick (making the ZnTe/ZnSe particle about 6 nm in diameter),
coated with an additional shell of ZnS that is about 1 nm thick. In
certain embodiments, the ZnSe and ZnS shells provide a red-shift
that moves the small ZnTe core to a wavelength of about 525 nm to
about 560 nm In other embodiments, ZnTe cores having ZnSe shells
exhibit an unexpected shift in peak emission wavelength to longer
wavelengths (e.g., from about 525 nm to about 600 nm or more) as a
function of shell thickness. As the thickness of the shell
increases, the peak emission wavelength of ZnTe/ZnSe nanoparticles
shifts dramatically to the red. For example, in some embodiments,
as the thickness of the shell increases, the peak emission
wavelength of ZnTe/ZnSe nanoparticles shifts to about 525 nm; or
about 550 nm; or about 575 nm; or about 600 nm. The effect of the
size of ZnTe/ZnSe nanoparticles on peak emission wavelength is
illustrated, for example, in FIG. 4.
Other methods for increasing photostability of the ZnTe/ZnSe
core/shell nanocrystals described herein include doping the ZnTe
core with Se before adding the ZnSe shell. Methods for doping an
additional material into a core nanocrystal are known in the art.
Other options include using a shell comprising MgX or BeX, wherein
X represents O, S or Se, in place of or in addition to the ZnSe
shell described above. Other methods include adding a coating of
phosphonic acid ligands to the nanocrystal. These ligands bind very
tightly, particularly to a Zn-containing semiconductor surface, and
thus provide extra protection for the surface.
Nanocrystals made by the described methods can be further modified
by modifications of the ligands present on the nanocrystal surface
as is known in the art. For example, the ligands on the surface of
the nanocrystal can be exchanged for other ligands to introduce new
properties such as water solubility to the nanocrystals. Methods
for making nanocrystals with water-solubilizing ligand coatings are
known in the art. For example, Adams, et al. (U.S. Pat. No.
6,649,138) provides methods to make water-soluble nanocrystals by
applying a coating of amphipathic polymeric material to the surface
of a hydrophobic nanocrystal. The methods start with a hydrophobic
nanocrystal, such as one described herein having a coating of
hydrophobic ligands, such as trialkyl phosphines, trialkyl
phosphine oxides, alkylamines, or alkylphosphonic acids. To this is
added an outer layer comprised of a multiply amphipathic dispersant
molecule comprising at least two hydrophobic domains and at least
two hydrophilic domains. In some embodiments, the amphiphilic
polymer comprises an acrylic acid or methacrylic acid polymer
having some acrylic acid groups converted into amides with
hydrophobic amine groups, such as monoalkyl amines or dialkylamines
having at least 4-12 carbons per alkyl group; and having some free
carboxylic acid groups to promote water solubility. These and other
suitable amphiphilic dispersants suitable for such use are
described at columns 14-18 of Adams et al, which is incorporated
herein by reference.
Thus in one aspect, nanocrystals include a coating of amphiphilic
dispersant (e.g., as described in U.S. Pat. No. 6,649,138). The
nanocrystals are thereby rendered water-soluble, making them
suitable for use in a variety of methods in which nanocrystals such
as quantum dots are known to be used. Another option for improving
the water-solubility of the nanocrystals described herein is
treatment with dihydrolipoic acid (DHLA). Certain embodiments
provide solubilized nanocrystals and methods of making them.
Other methods for rendering nanocrystals water-soluble are
described by Naasani, et al., in U.S. Pat. No. 6,955,855 and U.S.
Pat. No. 7,198,847. These methods involve coating the nanocrystal
with small water-solubilizing ligands, such as imidazole-containing
compounds like dipeptides. Suitable imidazole-containing compounds
are described at column 7 of the '855 Naasani patent.
By the term "imidazole-containing compound" is meant, for purposes
of the specification and claims to refer to a molecule that has at
least one imidazole group (e.g., imidazole ring) available for
binding a metal such as zinc or other metal cation, or substrate
containing such cation. Preferably, at least one imidazole moiety
is in a terminal position with respect to the structure of the
molecule. Imidazole ring nitrogens frequently serve as coordinating
ligand to operably bind a metal ion such as zinc or cadmium. In one
embodiment, the imidazole-containing compound comprises an amino
acid, or two or more amino acids joined together (e.g., known in
the art as "peptidyl" or "oligopeptide"), which may include, but is
not limited to, histidine, carnosine, anserine, baleine,
homocarnosine, 1-methylhistidine, 3-methylhistidine, imidazolysine,
imidazole-containing ornithine (e.g., 5-methylimidazolone),
imidazole-containing alanine (e.g.,
(beta)-(2-imidazolyl)-L(alpha)alanine), carcinine, histamine, and
the like Imidazole-containing amino acids may be synthesized using
methods known in the art (see, e.g., Stankova et al., 1999, J.
Peptide Sci. 5:392-398, the disclosure of which is herein
incorporated by reference).
By the term "amino acid" is meant, as known in the art and for
purposes of the specification and claims, to refer to a compound
containing at least one amino group and at least one carboxyl
group. As known in the art, an amino group may occur at the
position adjacent to a carboxyl group, or may occur at any location
along the amino acid molecule. In addition to at least one
imidazole moiety, the amino acid may further comprise one or more
additional reactive functionalities (e.g., amino, thiol, carboxyl,
carboxamide, and the like). The amino acid may be a naturally
occurring amino acid, a synthetic amino acid, a modified amino
acid, an amino acid derivative, an amino acid precursor, in D
(dextro) form, or in L (levo) form. Examples of derivatives may
include, but is not limited to, an N-methylated derivative, amide,
or ester, as known in the art, and where consistent with the
functions of the amino acid as a coating as described herein (e.g.,
imparts water-solubility, buffers sufficiently in a pH range
between about pH 6 and about pH 10, functions as a coat which can
increase fluorescence intensity, and has one or more reactive
functionalities that may be used to operably bind molecular probe).
One or more amino acids can be used. Histidine is an exemplary
imidazole-containing compound for coating the functionalized,
fluorescent nanocrystals described herein.
Ligands and coatings on the nanocrystals provided herein also can
be cross-linked to increase the stability of the nanocrystal
composition and improve its characteristics. The surface coating of
ligands on nanocrystals can be cross-linked using methods and
cross-linking agents described by Naasani. Exemplary cross-linking
agents include those described by Naasani, et al., including
tris(hydroxymethyl)phosphine (THP) and
tris(hydroxymethyl)phosphino-propionate (THPP). Also provided
herein are nanocrystals having water-solubilizing ligand coatings
that are cross-linked.
In certain embodiments, the semiconductor nanocrystals provided
herein can be a component in a composition. For example,
compositions are provided in which the semiconductor nanocrystals
are embedded in or applied to the surface of a solid or semi-solid
matrix (e.g., polymer matrix, bead, or resin). Alternatively, the
composition can be in the form of a liquid, gel, paste, cream,
patch, film, or a powder (e.g., lyophilized powder). Certain
compositions are provided that include one or more semiconductor
nanocrystals that emit light in a wavelength range that is
substantially non-absorbent to animal fluid, cells, or tissue; are
adapted for inserting into a mammalian body; or are formulated for
use in an in vitro biological assay or an in vivo assay. Also
provided are pharmaceutical compositions (e.g., non-toxic
compositions that are suitable for pharmaceutical use) that include
at least one semiconductor nanocrystal, as described herein.
Compositions can be formulated for various pharmaceutical uses,
including for ophthalmic and topical administration. The
pharmaceutical composition can further include a pharmaceutically
acceptable carrier for the nanocrystals (e.g., water, a saline
solution, or a buffer). Alternatively, the composition can be in
the form of a liquid, gel, paste, cream, patch, film, or a powder
(e.g., lyophilized powder).
In yet another aspect, kits containing ZnTe nanocrystals are
provided. The kits can be for pharmaceutical use or for use in a
biological assay. An exemplary kit for pharmaceutical use includes
a) one or more pharmaceutically acceptable containers; b) a
pharmaceutical composition as provided herein; and c) instructions
for use. Kits for biological assay can include, in addition to the
nanocrystals, other reagents, such as solvents, standards, buffers,
dyes, and the like.
Methods of Using the ZnTe Nanocrystals
ZnTe nanocrystals can be used in methods for tracking molecules
that are known in the art. For example, they can be linked to
various target molecules by known methods. Commonly, they are
linked to an affinity molecule or used in further transformations.
Such further transformations can be used to introduce onto the
surface of a nanocrystal a selected target (or cargo) molecule of
interest, such as an antibody or other specific affinity molecule.
Methods for attaching such affinity molecules to a fluorescent
carrier are known in the art and can readily be adapted for use in
the present methods: see, e.g., U.S. Pat. No. 6,423,551, which also
describes some bi-functional agents that can be used to link the
surface of a nanocrystal to a target molecule and to a nanocrystal
surface. These methods can also be used to introduce a number of,
or a layer of, functionalized molecules on the surface of a
nanocrystal, where the functionalized molecules can provide new
surface properties to the nanoparticle, such as
water-dispersability. In some embodiments, nanocrystals modified
for attachment of an affinity molecule are provided that can be
used to detect a desired target compound, cell or cellular
organelle.
The modified nanocrystals can be linked to an affinity molecule for
use in methods to track, identify, or localize molecules of
interest that the affinity molecule can bind to, demonstrating that
the molecule of interest is present and where it is distributed or
localized. The nanocrystals can also be used in binding experiments
to visualize distribution of molecules that the affinity molecule
recognizes. Selection of a suitable affinity molecule is within the
ordinary level of skill in the art once a target compound is
identified. For example, conventional methods can be used to
produce or identify an antibody suitable to specifically bind to a
target molecule of interest. The antibody can thus be linked to the
nanocrystal, which can then be used to identify the presence,
location, or movements of the target compound, using the
nanocrystal as a fluorescent label. In some embodiments, methods
are provided to identify or track a target molecule, by linking a
suitable affinity molecule that selectively binds to the target
molecule to a nanocrystal, and permitting the nanocrystal linked to
the affinity molecule to contact the target molecule. Tracking or
detection can be achieved by using conventional methods for
tracking a fluorescent labeled moiety, such as by use of a
fluorescence imaging system, microscope or camera.
In some embodiments, a functionalized nanocrystal is provided. The
nanocrystal can be linked to an affinity molecule selected to bind
specifically to a target molecule of interest. Optionally, the
nanocrystal linked to the affinity molecule can be bound to the
target molecule of interest to form a fluorescently labeled
complex. Target molecules of interest include proteins, enzymes,
receptors, nucleic acids, hormones, and cell surface antigens
characteristic of specific types of cells.
The ZnTe nanocrystals provided herein can be used in numerous
applications, such as flow cytometry, cellular imaging (including
live cell imaging and tracking of stem cells), and protein
blotting. In particular, these nanocrystals are particularly useful
in certain in vivo applications that involve visualizing a labeled
molecule and where toxicity concerns need to be minimized. For
example, ZnTe nanocrystals can be used in tracking stem cells, as
surgical markers, and in sentinel lymph node mapping. ZnTe
nanocrystals are sufficiently non-toxic to allow their use in
industrial applications where environmental disposal is
particularly problematic, such as lighting and display technology,
and in other high volume consumer applications, such as
electronics.
Certain nanocrystal compositions are particularly useful for
imaging in medical applications (e.g., screening assays or surgical
procedures) that require non-toxic materials which can be
visualized in the presence of body tissue and fluids. Certain ZnTe
nanocrystal compositions provided herein are particularly
appropriate for use in medical imaging. For example, ZnTe
nanocrystals that emit in the green region of the electromagnetic
spectrum (e.g., about 510-550 nm) are particularly useful in
ophthalmic applications. Since eye tissues are transparent to this
portion of the electromagnetic spectrum, green ZnTe nanocrystals
can be used for visualizing the blood vessels of the eye and for
evaluating certain eye diseases that can affect the retina. The
ZnTe nanocrystals provide an alternative to traditional fluorescein
angiography and provide further additional advantages. Since ZnTe
nanocrystals are far brighter than organic dyes such as
fluorescein, they can be used in minute quantities and can
facilitate imaging of single cell events (e.g., retinal cell
death), which is not feasible with existing dye-based angiography
procedures. In addition, standard instrumentation for performing
fluorescein angiograms can be used to detect the ZnTe nanocrystals
without modification.
A representative nanocrystal-based assay for ophthalmic screening
involves injecting a pharmaceutically acceptable ZnTe nanocrystal
composition (e.g., in the form of a saline solution), into a vein
in the arm or a patient. The nanocrystals travel to the blood
vessels inside the eye. Once within the blood vessels of the eye,
the nanocrystals can be imaged using a camera equipped with
appropriate filters as they circulate through the vessels in the
back of the eye. The images of the nanocrystals can be used to
assess whether there are any circulation problems, swelling,
leaking, or abnormality of the blood vessels.
Certain ZnTe nanocrystal materials provided herein can be used in
surgical procedure, such as, for example, as surgical markers. In
one representative method, tissue can be marked prior to a surgical
procedure. Certain portions of the tissue at the surgical site can
be treated with a pharmaceutically acceptable ZnTe-containing
composition. The composition can be formulated in various ways and
is typically formulated for direct topical application (e.g., as a
gel, paste, film, spray, powder, and the like). Once treated, the
marked tissue can be visualized using methods well known to those
in the art. In one exemplary method, a nanocrystal composition is
applied to the tumor tissue to mark the tumor tissue that is to be
removed during a tumor resection surgery. The marked tissue is
visualized prior to and after tumor resection. Any tumor tissue
remaining after excision (e.g., at the resection margin) can be
readily visualized using known methods.
The non-toxic ZnTe nanocrystals provided herein also are suitable
for use in industrial and consumer applications, especially where
manufacturing or disposal of large quantities of nanocrystals
(e.g., CdSe) is of concern. The described nanocrystals can be used,
for example, in electronic and photovoltaic devices, such as
displays, light emitting diodes (LED's), sensors, and solar panels.
Certain devices may require that the semiconductor nanocrystals be
embedded in or applied to the surface of a solid or semi-solid
matrix (e.g., polymer matrix, bead, or resin).
Other uses, adaptations and variations of the invention will be
apparent to those skilled in the art from the foregoing
description. The following examples are provided as further
guidance regarding the making and using of the methods of the
invention, and are not to be construed as limiting the scope of the
invention.
EXAMPLES
Example 1
Preparation of Znte Core Nanocrystals
This example describes an exemplary method for making ZnTe
nanocrystals of the invention. Dioctylamine is rigorously dried as
described herein, and is used as the solvent for ZnTe nanocrystal
core formation Zinc oleate is added, followed by tellurium in TOP,
which is referred to as TOPTe. This is heated at about 290.degree.
C. with a very small amount of lithium triethylborohydride,
sufficient to provide a desired amount of nucleation. Heating is
continued until the absorbance maximum for the ZnTe nanocrystals
reached the desired wavelength (color), e.g., about 425 nm to about
540 nm. The cores can be isolated by conventional methods.
Example 2
Addition of ZnSe Shell to ZnTe Cores
The ZnTe cores are treated as follows to grow a ZnSe shell on the
core. ZnTe cores are dispersed in dioctylamine, which has been
rigorously dried as described herein. Zn(oleate)Cl (0.3 equiv.) is
added, and the mixture is heated to about 300.degree. C. TOPSe and
Zn(oleate)Cl (about 1 equivalent) is then added, and heating is
continued until the desired fluorescent wavelength is reached,
indicating that the nanocrystals have reached a desired size.
Nanocrystals having a fluorescence maximum between about 525 nm and
about 560 nm can be produced, depending upon the size of the ZnTe
core and the thickness of the ZnSe shell. The core/shell
nanocrystals are isolated by conventional methods.
Example 3
Addition of ZnS Shell to ZnTe/ZnSe Core/Shells
ZnTe cores with a ZnSe shell were dispersed in dried dioctylamine.
Zn(oleate)Cl (0.3 equiv) was added and the mixture was heated to
about 250.degree. C. Zn(oleate)Cl and sulfur-oleylamine (about 1
equivalent) were added gradually over the course of 1-3 hours until
a desired shell thickness has been added. The mixture was cooled
and the nanocrystals were isolated by conventional methods.
All of the U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications
and non-patent publications referred to in this specification
and/or listed in the Application Data Sheet are incorporated herein
by reference, in their entirety. Aspects of the embodiments can be
modified, if necessary to employ concepts of the various patents,
applications and publications to provide yet further
embodiments.
These and other changes can be made to the embodiments in light of
the above-detailed description. In general, in the following
claims, the terms used should not be construed to limit the claims
to the specific embodiments disclosed in the specification and the
claims, but should be construed to include all possible embodiments
along with the full scope of equivalents to which such claims are
entitled. Accordingly, the claims are not limited by the
disclosure.
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