U.S. patent application number 11/589078 was filed with the patent office on 2007-02-22 for stable, water-soluble quantum dot, method of preparation and conjugates thereof.
Invention is credited to Warren Chan, Hans Fischer, Wen Jiang, Sawitra Mardyani.
Application Number | 20070042576 11/589078 |
Document ID | / |
Family ID | 35311253 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042576 |
Kind Code |
A1 |
Chan; Warren ; et
al. |
February 22, 2007 |
Stable, water-soluble quantum dot, method of preparation and
conjugates thereof
Abstract
A method for manufacturing powdered quantum dots comprising the
steps of: a) reacting quantum dots comprising a core, a cap and a
first ligand associated with the outer surfaces thereof with a
second ligand, the second ligand displacing the first ligand and
attaching to the outer surfaces of the quantum dots, b) isolating
the quantum dots having the attached second ligand from the
reaction mixture, c) reacting the isolated quantum dots having the
attached second ligand with a small organic molecule whereby the
small organic molecule attaches to the second ligand, d) reacting
the quantum dots having the attached small organic molecule with a
cross-linking agent to cross-link the small organic molecule
attached to the second ligand with an adjacent second ligand
attached to the surfaces of the quantum dots, e) isolating the
quantum dots formed in step (d); and f) drying the isolated quantum
dots to form powdered quantum dots. The invention includes the
quantum dots.
Inventors: |
Chan; Warren; (Toronto,
CA) ; Fischer; Hans; (Toronto, CA) ; Mardyani;
Sawitra; (North York, CA) ; Jiang; Wen;
(Toronto, CA) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
35311253 |
Appl. No.: |
11/589078 |
Filed: |
October 30, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11116454 |
Apr 28, 2005 |
7151047 |
|
|
11589078 |
Oct 30, 2006 |
|
|
|
60565903 |
Apr 28, 2004 |
|
|
|
Current U.S.
Class: |
438/497 ;
257/E29.069; 438/962; 977/773; 977/774; 977/775 |
Current CPC
Class: |
Y10S 438/962 20130101;
C09K 11/02 20130101; Y10S 977/775 20130101; Y10S 977/774 20130101;
Y10S 977/773 20130101; B82Y 20/00 20130101; B82Y 10/00 20130101;
C09K 11/883 20130101 |
Class at
Publication: |
438/497 ;
438/962; 977/773; 977/774; 977/775; 257/E29.069 |
International
Class: |
H01G 9/20 20060101
H01G009/20 |
Claims
1. A method for manufacturing powdered quantum dots comprising the
steps of: a) reacting quantum dots comprising a core, a cap and a
first ligand associated with the outer surfaces thereof with a
second ligand, the second ligand displacing the first ligand and
attaching to the outer surfaces of the quantum dots, b) isolating
the quantum dots having the attached second ligand from the
reaction mixture, c) reacting the isolated quantum dots having the
attached second ligand with a small organic molecule whereby the
small organic molecule attaches to the second ligand, d) reacting
the quantum dots having the attached small organic molecule with a
cross-linking agent to cross-link the small organic molecule
attached to the second ligand with an adjacent second ligand
attached to the surfaces of the quantum dots, e) isolating the
quantum dots formed in step (d); and f) drying the isolated quantum
dots to form stable, oxidation-resistant, powdered quantum
dots.
2. The method of claim 1, wherein the second ligand is a compound
having a formula ROC--(CH.sub.2).sub.n--COOH, where R is a thiol,
and n is a whole integer in the range of 8 to 13.
3. The method of claim 1, wherein the second ligand comprises a
hydrophilic moiety.
4. The method of claim 3, wherein the second ligand is
mercaptoundecanoic acid.
5. The method of claim 1, wherein the small organic molecule is
selected from molecules containing at least two amino groups and
one carboxylic acid group.
6. The method of claim 5, wherein the organic molecule is an amino
acid.
7. The method of claim 6, wherein the amino acid is lysine.
8. The method of claim 5, wherein the small organic molecule is a
compound having a formula R.sub.1OC--(CH.sub.2).sub.nCOR.sub.2
where each of R.sub.1 and R.sub.2 is an amine group and n is a
whole integer in the range of 8 to 13.
9. The method of claim 1, wherein the second ligand is supplied in
a molar excess of at least about 4,000 compared to the quantum dot
having an attached first ligand population.
10. The method of claim 1 further comprising the steps of: a)
dissolving the powdered quantum dots in an aqueous solution; and b)
contacting the dissolved quantum dots with a biomolecule whereby
the quantum dot and the biomolecule form a conjugate.
11. The method of claim 10, wherein the biomolecule is selected
from the group comprising: a protein or an antigenically reactive
fragment thereof, an antibody or an antigenically reactive fragment
thereof and a nucleic acid.
12. A method of detecting a biomolecule in a sample comprising the
steps of: a) contacting the sample with the conjugate prepared by
the method of claim 10, wherein the biomolecule to be detected
specifically binds to the biomolecule conjugated to the quantum
dots; and b) detecting luminescence, wherein the detection of
luminescence is indicative of the presence of the biomolecule in
the sample.
13. The method of claim 11, wherein the biomolecule to be detected
is selected from the group comprising: a protein, an antigenically
reactive fragment of the protein, an antibody, an antigenically
reactive fragment of the antibody and a nucleic acid.
14. A water-soluble powder comprising quantum dots manufactured
according to the method of claim 1.
15. The method of claim 2 further comprising the steps of: a)
dissolving the powdered quantum dots in an aqueous solution; and b)
contacting the dissolved quantum dots with a biomolecule whereby
the quantum dot and the biomolecule form a conjugate.
16. The method of claim 15, wherein the biomolecule is selected
from the group comprising a protein or an antigenically reactive
fragment thereof, an antibody or an antigenically reactive fragment
thereof and a nucleic acid.
17. A method of detecting a biomolecule in a sample comprising the
steps of: a) contacting the sample with a conjugate prepared by the
method of claim 15, whereby the biomolecule to be detected
specifically binds to the biomolecule conjugated to the quantum
dot; and b) detecting luminescence, wherein the detection of
luminescence is indicative of the presence of the biomolecule in
the sample.
18. A quantum dot comprising: a core, a cap, a ligand attached to
the outer surface of the cap, said ligand being a compound having a
formula ROC--(CH.sub.2), --COOH, where R is an organic molecule
with a hydrophilic moiety and n is a whole integer in the range of
8 to 13; and, a cross-linker comprising a molecule having a formula
R.sub.1OC--(CH.sub.2).sub.n--COR.sub.2 where each of R.sub.1 and
R.sub.2 is an amine group and n is a whole integer in the range of
8 to 13, said cross-linker joining two adjacent ligands.
19. A quantum dot of claim 18 further comprising a biomolecule
conjugated to an exposed polar group on the cross-linker.
20. The quantum dot of claim 18 that remains monodispersed in an
aqueous solution for at least 10 days.
Description
RELATED APPLICATION DATA
[0001] This application claims the benefit of U.S. patent
application Ser. No. 11/116,454 filed Apr. 28, 2005 which claims
the benefit of U.S. Provisional Patent Application No. 60/565,903
filed Apr. 28, 2004, both of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of production of a
water-soluble quantum dot, and the quantum dot thereby
produced.
BACKGROUND OF THE INVENTION
[0003] Semiconductor nanocrystals, often referred to as quantum
dots (QDs), offer a viable alternative to presently used isotopic
and non-isotopic detection systems for use in biomolecular research
protocols and assays, as well as for clinical and diagnostic
assays. The goal of these systems is the detection and reporting of
a specific molecule that is indicative of the presence of a certain
molecular, cellular or organismal entity, or of the occurrence of a
particular molecular event, such as the transcription of a
particular gene or the production of a particular protein within an
organism. While isotopic detection systems offer a high degree of
sensitivity, there are inherent problems associated with their use
such as safety and disposal concerns, half-life of the isotope and
where very low levels of the target molecule are present, the
length of film exposure time (e.g. up to 7 days) required in order
to detect a signal. While non-isotopic systems offer a safety
advantage, the fluorescent reporter molecules are often susceptible
to rapid fading (i.e. photobleaching). As well, while many of the
currently available non-isotopic detection systems are highly
sensitive, these systems rely upon the use of a secondary-antibody
detection regimen wherein the actual detection is of a fluorescent
or chromatogenic agent linked to a secondary-antibody targeted
against a primary-antibody that binds to an antigen linked to a
molecular probe against the target molecule. Successful utilization
of such systems requires the use of expensive reagents that often
exhibit only a limited storage or shelf-life, and further requires
a user to perform a number of procedural steps, the
less-than-optimal performance of which may lead to a false-negative
result.
[0004] Interest from the medical and research communities regarding
quantum dots stems largely from the unique optical and electrical
properties that are associated with QDs. In comparison to organic
fluorophores, certain types of QDs possess up to twenty times
greater luminescence, are highly resistant to photobleaching,
exhibit narrow spectral linewidths, and are size and
materials-tuneable so as to be excitable using only a single
wavelength. Problematic, however, is the fact that in order for QDs
to be used in the context of a biological setting, for example,
imaging and detection of and within live cells, the QD must possess
a coating that makes the QD bio-compatible with biological systems,
such as being aqueously soluble, and at the same time does not
lessen the stability of the QD under physiological conditions.
Overcoming this problem is exasperated by the fact that QDs are
generally synthesized in an organic solvent as the hydrophobic
solvent ligands act as stabilizing agents for QD nucleation and
growth, and inhibit the aggregation of the QDs during their
synthesis.
[0005] In terms of their basic structure, the synthesis of a QD
comprising an inner nanoparticle-sized semiconductor "core"
together with an outer semiconductor "cap" that is of a different
material than the core and which binds to the core is a process
that is well known in the art (U.S. Pat. Nos. 6,468,808 and
6,699,723). Usually, the QD core is selected from a combination of
Group IIB-VIB, Group IIIB-VB or Group IVB-IVB elements from the
periodic table, while the cap is selected from a material that, in
combination with the core, results in a luminescent quantum dot.
The cap is selected to passivate the core by having a higher band
gap than the core, and as such, the cap is preferred to be a
semi-conducting material from the Group IIB-VIB combination of
elements from the periodic table.
[0006] The luminescent properties of QDs result from quantum size
confinement, which occurs when metal and semiconductor core
particles are smaller than their exciton Bohr radii, about 1 to 5
nm (Alivisatos, Science, 271, 933-37 (1996); Alivisatos, J. Phys.
Chem., 100, 13226-39 (1996); Brus, Appl. Phys., A 53, 465-74
(1991). It is known that an improvement in the QD luminescence
results from the capping of a size-tunable lower band gap core
particle with a higher band gap shell. For example, CdSe quantum
dots passivated with a ZnS layer are strongly luminescent (35 to
50% quantum yield (QY)) at room temperature, and their emission
wavelength can be tuned from blue to red by changing the particle
size. Moreover, the ZnS capping protects the core surface and leads
to greater stability of the quantum dot (Hines et al., J. Phys.
Chem., 100, 468-471 (1996); and Dabbousi et al., J. Phys. Chem. B
101, 9463-75 (1997)). Despite having these greater luminescent
capacities, such capped QDs are not water-soluble and are thus not
suitable for use in biological systems.
[0007] To date, numerous attempts have been made to produce a QD
that has a bio-compatible surface that does not promote
non-specific binding of the QD to molecules, does not cause an
abatement of the optical properties of the QD, nor increase the
size of the QD, nor negate the ability of the QD to be further
coated with a desired molecule(s) of choice, but allows for the
large-scale and cost effective production of the QD. QDs have been
provided that have their surface modified through the addition of
amphiphilic polymers, phospholipids, dendrimers, oligomeric
ligands, biofunctional molecules such as deoxyribonucleic acid
(DNA), and genetically-modified proteins (Chan and Nei, Science,
281, 2016-2018 (1998); Bruchez et al., Science, 281, 2013-2016
(1998); Mattoussi et al., J. Am. Chem. Soc., 125, 12142-12150
(2000); Kim and Bawendi, J. Am. Chem. Soc., 125, 14652-14653
(2003); Dubertret et al., Science, 298, 1759-1762 (2002); Wang et
al., J. Am. Chem. Soc., 124, 2293 (2002); Wu et al., Nature
Biotechnology, 21, 41-46 (2003); Guo et al., J. Am. Chem. Soc.,
125, 3901 (2003)). While such modifications impart water solubility
to the QD, such surface modifications do not allow cost-effective,
commercial scale production. In an effort to provide a thin, secure
organic shell around a QD without increasing the diameter of the QD
so as to render the QD inaccessible to target systems or limit the
number of QDs that can be attached to a target, Kim and Bawendi (J.
Am. Chem. Soc., 125, 14652-14653 (2003)) have succeeded in
surrounding QDs with an oligomeric phosphine shell. Problematic,
however, is that the approach put forward by Kim and Chan requires
the complex synthesis of a stabilizing and interfacing
oligophosphine ligand, thereby severely limiting the potential for
the large scale production of such QDs.
[0008] It would be thus advantageous to provide a QD that has a
coating that would allow for the QD to be used in conjunction with
biological systems. Any coating that is provided should allow for
the maintenance of long-term monodispersity of the QDs in an
aqueous environment, not promote non-specific binding of the QD to
other molecules, not detract from the optical properties of the QD
when compared to the organic solvent soluble counterpart of the
coated QD, maintain the small size of the QD, allow for the QD to
be further coated with biomolecules of a range of types, and allow
for the QD to be produced on a commercial scale in a cost-effective
manner.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method for manufacturing
powdered quantum dots comprising the steps of: a) reacting quantum
dots comprising a core, a cap and a first ligand associated with
the outer surfaces thereof with a second ligand, the second ligand
displacing the first ligand and attaching to the outer surfaces of
the quantum dots, b) isolating the quantum dots having the attached
second ligand from the reaction mixture, c) reacting the isolated
quantum dots having the attached second ligand with a small organic
molecule whereby the small organic molecule attaches to the second
ligand, d) reacting the quantum dots having the attached small
organic molecule with a cross-linking agent to cross-link the small
organic molecule attached to the second ligand with an adjacent
second ligand attached to the surfaces of the quantum dots, e)
isolating the quantum dots formed in step (d); and f) drying the
isolated quantum dots to form powdered quantum dots.
[0010] Other and further advantages and features of the invention
will be apparent to those skilled in the art from the following
detailed description thereof, taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The invention will now be described in more detail, by way
of example only, with reference to the accompanying drawings, in
which like numbers refer to like elements, wherein:
[0012] FIG. 1 is a schematic diagram generally showing a method of
preparing a coated QD of the present invention, together with a
photograph of a 400 mg sample of a quantum dot preparation;
[0013] FIG. 2 is a graphical representation showing aggregation
stability and optical properties of quantum dots prepared in
accordance with the method of the present invention;
[0014] FIG. 3 is a light micrograph showing a monodispersion of a
population of quantum dots produced in accordance with the method
of the present invention;
[0015] FIG. 4 is a graphical representation showing optical
properties of quantum dots produced in accordance with the method
of the present invention; and
[0016] FIG. 5 is a light micrograph showing a population of
mammalian culture cells with endocytosed protein-conjugated quantum
dots produced according to the method of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] The present invention provides a method for large-scale
production of water-soluable semiconductor nanocrystals,
alternatively referred to as quantum dots, in a cost-effective
manner. More particularly, the present invention provides a method
of producing water-soluble quantum dots wherein the quantum dots so
produced are supplied to a user as a powder. The present invention
also provides for a method of producing a quantum dot in a powdered
form wherein the quantum dot can thereafter be conjugated to a
biomolecule selected from a range of different biomolecule species.
As well, the present invention provides a method of detecting a
biomolecule in a sample through the use of a probe
molecule-luminescent reporter molecule construct comprising a
quantum dot conjugated to the probe molecule.
Definitions
[0018] The term "quantum dot" will be understood to mean a
water-soluble luminescent semiconductor nanocrystal, which comprise
a core, a cap and a hydrophilic attachment group.
[0019] The term "core" will be understood to mean a
nanoparticle-sized semiconductor. While any core of the IIB-VIB,
IIIB-VB or IVB--IVB semiconductors can be used in the context of
the present invention, the core must be such that, upon combination
with a cap, a luminescent quantum dot results. A IIB-VIB
semiconductor is a compound that contains at least one element from
Group IIB and at least one element from Group VIB of the Periodic
Table; a IIIB-VB semi conductor is a compound that contains at
least one element from Group IIIB and at least one element from
Group VIB of the Periodic Table, and so on. The core may be a
IIB-VIB, IIIB-VB or IVB-IVB semiconductor that ranges in size from
about 1 nm to about 10 nm. In one form the core is a IIB-VIB
semiconductor and ranges in size from about 2 nm to about 5 nm.
Examples include a core that is CdS or CdSe.
[0020] The term "cap" will be understood to mean a semiconductor
that differs from the semiconductor of the core and which binds to
the core, thereby forming a surface layer on the core. The cap must
be such that, upon combination with a given semiconductor core, a
luminescent quantum dot results. The cap should passivate the core
by having a higher band gap than the core. In this regard, the cap
may be a IIB-VIB semiconductor of high band gap. In particular, the
cap may be ZnS or CdS. In particular forms of the invention, the
cap is ZnS when the core is CdSe or CdS and the cap is CdS when the
core is CdSe.
[0021] The term "first ligand" is used to describe a passivating
organic layer present on the surface of the quantum dot comprised
of the organic solvent in which the quantum dot is prepared. The
first ligand is displaced as described below to provide an outer
coating that renders the quantum dot in a state for processing
according to the method of the present invention. In one embodiment
of the present invention, the first ligand is any molecule that is
hydrophobic (e.g., trioctylphosphine oxide (TOPO), octylamine, or
lipid-type molecules).
[0022] The term "second ligand" encompasses ligands which are used
to displace the first ligand from the surface of the quantum dot.
More specifically, the second ligand can be any organic group that
can be attached, such as by any stable physical or chemical
association, to the surface of the cap of the luminescent
semiconductor quantum dot and can render the quantum dot
water-soluble without rendering the quantum dot non-luminescent.
Accordingly, the second ligand may comprise a hydrophilic moiety.
In one embodiment, the second ligand enables the quantum dot to
remain in solution for at least about one hour. In another
embodiment, the second ligand enables the quantum dot to remain in
solution for at least about one day. In yet another embodiment, the
second ligand allows the quantum dot to remain in solution for at
least about one week. The second ligand may also allow the quantum
dot to remain in solution indefinitely. Desirably, the second
ligand is attached to the cap by covalent bonding and is attached
to the cap in such a manner that the hydrophilic moiety is exposed.
The second ligand may be attached to the quantum dot via a sulfur
atom. The second ligand may be an organic group comprising a sulfur
atom and at least one hydrophilic attachment group. A suitable
hydrophilic attachment group includes, for example, a carboxylic
acid or salt thereof, a sulfonic acid group or salt thereof, a
sulfamic acid group or salt thereof, an amino substituent, a
quaternary ammonium salt, or a hydroxyl. The organic group of the
hydrophilic attachment group of the present invention may be a
C.sub.8-C.sub.13 alkyl group or an aryl group. C.sub.8-C.sub.13
alkyl groups have been quite useful and so has the C.sub.10 alkyl
group. Therefore, specifically the second ligand of the present
invention may be a thiolcarboxylic acid, or the second ligand may
be mercaptoundecanoic acid (MUA).
[0023] The term "cross-linking agent" is used to describe a
compound that is capable of forming a chemical bond between
molecular groups on similar or dis-similar molecules so as to
covalently bond together the molecules. In the present invention, a
suitable cross-linking agent is one that couples amines to carboxyl
groups, for example N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
(EDC), and dicyclohexylcarbodiimide (DCC).
[0024] The term "small organic molecule" is used to describe an
organic compound either synthesized in the laboratory or found in
nature. Typically, a small organic molecule is characterized in
that it contains several carbon-carbon bonds, and has a molecular
weight of less than 1500 grams/Mol. In the present invention, a
small organic molecule can be an amino acid, such as a basic amino
acid, and more particularly the amino acid lysine. As well, in the
present invention, a small molecule can also be a substituted
dicarboxylic acid, such as diaminopimelic acid. The small organic
molecule interacts with the carboxylic acid group provided at the
exposed end of each second ligand to result in the formation of an
amide bond between adjacent second ligand molecules attached to the
cap of the quantum dot while at the same time leaving exposed a
polar group such as a carboxylic acid group if the small organic
molecule in question is lysine.
[0025] The term "biomolecule" is used to describe a synthetic or
naturally occurring protein, glycoprotein, lipoprotein, amino acid,
nucleic acid, nucleotide, carbohydrate, sugar, lipid, fatty acid
and the like.
[0026] The term "conjugate" is used to describe the quantum dot
described above and a biomolecule wherein the biomolecule is
attached to the quantum dot either directly or indirectly by any
suitable means. The biomolecule can be attached to the quantum dot
by being covalently bonded to the exposed polar group of the small
organic molecule, for example, to the carboxyl group of the lysine
that cross-links together the second ligand molecules. Indirect
attachment of the biomolecule can occur through the use of a
"linker" molecule, so long as the linker does not negatively affect
the luminescence of the quantum dot or the function of the
biomolecule. It is preferred that the linker be one that is
bio-compatible. Common molecular linkers known in the art include a
primary amine, a thiol, streptavidin, neutravidin, biotin, or a
like molecule. In the context of the present example of the
invention, a suitable linker is EDC.
EXAMPLE
[0027] The following example is included to illustrate the present
invention, and should not be used to limit the claims in any way.
The parts and percentages are by weight unless otherwise
indicated.
Preparation of Quantum Dots (ODs) Coated with the Second Ligand
[0028] To obtain a quantity of water-soluble quantum dots for
subsequent utilization in a cross-linking procedure, one gram of
mercaptoundecanoic acid (MUA) (Aldrich, 95%) was added to a 3-neck
flask and melted at 65.degree. C. under argon to provide a liquid
MUA solution. The molecules of MUA function as the second ligand
coating the cap of the QD on displacing the first ligand from the
cap of the QD. Quantum dots having a core comprising CdSe and a cap
comprising ZnS were prepared using a known organometallic
procedure. See the following references for a description of this
procedure.
[0029] Hines, M. A., Guyot-Sionnest, P. "Synthesis of strongly
luminescing ZnS-capped CdSe nanocrystals" J. Phys. Chem. B, 100,
468-471 (1996); Peng, X. G., Schlamp, M. C., Kadavanich, A. V.,
Alivistos, A. P. "Epitaxial growth of highly luminescent CdSE/CdS
core/shell nanocrystals with photostability and electronic
accessibility" J. Am. Chem. Soc., 199, 7019-7029 (1997); Dabbousi,
B. O. et al. "(CdSe)ZnS core-shell quantum dots: synthesis and
characterization of a size series of highly luminescent
nanocrystallites" J. Phys. Chem. B, 101, 9463-9475 (1997).
[0030] Quantum dots are commercially available from, for example,
Quantum Dot Corporation and Evident Technologies.
[0031] In the present example, these quantum dots were provided
with a coating of trioctylphosphine oxide (TOPO) as the first
ligand. The molar concentration of these QDs was determined using
the molar absorptivity value from the published report by Yu et al.
(Chem. Mater., 2003, 15, 2854-2860). A quantity less than about 100
mg of the TOPO-coated QDs were injected into the MUA-solution. This
can be done either in a Schlenk Line system or in air. The quantity
of MUA to TOPO-coated QDs was such that the MUA was in
approximately 8,000 times molar excess to the TOPO-coated QDs so as
to adequately coat yellow-emitting QDs (Rem=580 nm) with MUA. A
person of skill in the art will understand that the concentration
of MUA to TOPO-coated QDs will have to be adjusted for different
sizes of QDs in order to achieve optimal results. Following
injection of the QDs, the temperature of the solution was raised to
80.degree. C. overnight with continuous stirring. After two hours
of 80.degree. C. incubation, 25 mL of dimethyl sulfoxide (DMSO)
(EMD, 99.9%) was injected into the 3-neck flask, whereupon the
solution became optically clear. This solution was stirred for a
further two hours, followed by cooling to room temperature
whereupon chloroform was added to precipitate out the QDs. Any kind
of highly nonpolar solvent can be used in place of chloroform.
Precipitated QDs were centrifuged at 3,700 RPM to separate them
from unbound MUA that had not become attached to the surface of any
given QD on displacement of the TOPO coating. Thereafter,
MUA-coated QDs were redissolved in DMSO for a subsequent
cross-linking step.
Cross-Linking of the Second Ligand on the Surface of the QD
[0032] A method according to the present invention of preparing a
quantum dot having a cross-linked ligand present on its surface is
generally illustrated in the schematic diagram of FIG. 1, while a
population of such dots is shown in the photograph provided as an
inset in FIG. 1.
[0033] Prior to undertaking a cross-linking of the second ligand
molecules that were attached to the cap of the QD, the following
solutions were prepared: solution (A) comprised DL-lysine (Aldrich,
98%) dissolved in phosphate buffer saline (PBS) (10 mM, pH=7.4),
resulting in a concentration of about 16,000 lysine molecules/QD,
while solution (B) comprised dicylcohexylcarbodiimide (DCC)
(Aldrich, 99%) dissolved in DMSO at 5 times the concentration of
lysine. Solutions A and B>1 mL were directly added to MUA-coated
QDs and the resultant solution, which became cloudy immediately
upon mixing, was stirred for 2 hours at room temperature. Large
aggregations of QDs began to form in the solution after
approximately 30 minutes of stirring, such large aggregations being
indicative of the cross-linking of the second ligand on the surface
of the QDs and the QDs began to precipitate from the solution.
Aggregated QDs were recovered by centrifugation at 3700 RPM for 5
minutes, followed by washing twice with tetrahydrofuran (THF) to
remove MUA molecules that were weakly attached to the QDs.
Recovered, washed QDs were re-dissolved in distilled water and
dialyzed overnight using a membrane dialysis having a pore size of
12 to 14 kDa and made of regenerated cellulose to remove
uncross-linked MUA against distilled water. As MUA is insoluble in
distilled water, that which was desorbed from the surface of the
QDs appeared as a white precipitate inside the dialysis tube, and
was removed using a syringe filter (Sigma, 0.22 .mu.m pore
diameter). For final recovery, the QDs having cross-linked second
ligand on their surface were precipitated from the aqueous solution
with the addition of THF or excess salt (>500 mM) and recovered
by centrifugation at 3000 RPM, for five minutes. Recovered,
cross-linked QDs were washed once with THF, re-centrifuged, and
dried overnight to a powder in a fume hood at room temperature. It
is also possible to take this aqueous solution of quantum dots and
place it in a lyophilizer for preparation of powdered quantum dots.
The resultant powdered cross-linked QDs were stored at room
temperature in air (short term) or under nitrogen for long-term
storage (>1 year). Using an initial quantity of TOPO-coated QDs
as described, a per batch quantity of approximately 400 mg of
powdered QDs were prepared using the method as described. A person
of skill in the art will, of course, appreciate that the method of
the present invention allows for the production of various sizes
and quantities of powdered QDs depending upon the amount of
TOPO-coated QDs that are utilized as starting material, and that
larger quantities than those as described can be prepared.
[0034] Further cross-linking of the QDs can be accomplished by
incubating the QDs in PBS (10 mM, pH=7.4) in the presence of excess
lysine and cross-linking agent
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC)
(Sigma-Aldrich).
[0035] It is predictable that other types of small organic
molecules, such as diaminopimelic acid (Sigma-Aldrich), can be used
to cross-link adjacent MUA molecules present on the surface of the
QD via the carboxylic acid group on the MUA, thereby forming a
stable coating or shell on the QD. It is believed that most types
of molecules that contain at least 2 primary amino groups and 1
carboxylic acid can be used.
Quality Assessment of Powdered QDs
[0036] Powdered quantum dots produced in accordance with the method
of the present invention were assessed for their ability to
maintain their luminescent property and to remain in a
monodispersed state upon being re-dissolved in an aqueous solution
under a variety of conditions. Samples of QDs were re-dissolved in
water, the pH of which was adjusted through the drop-wise addition
of NaOH or HCl (greater than 100 mg can be dissolved in 1 mL of
distilled water) and monitored with a pH meter, and as shown for
the two samples presented in FIG. 2A, the fluorescence of the
re-dissolved QDs did not fluctuate greatly over the pH range of
about 4 to 12. Lack of fluorescence observed at a pH of less than 4
could possibly be attributed to an acid etching effect upon the QDs
or a breakdown of the QDs under highly acidic conditions.
[0037] Quantum dots produced in accordance with the method of the
present invention could be subjected to various ranges of
temperatures, for example, those commonly used in conjunction with
the performance of a polymerase chain reaction, or in
cell-incubation studies, or under the elevated (up to 70.degree.
C.) temperature conditions found in DNA hybridization experiments.
To assess whether quantum dots manufactured according to the method
of the present invention could remain luminescent over a varying
temperature range (25.degree. C. to 70.degree. C.), aliquots of the
powdered QDs were dissolved in water (1 mg/mL) and heated to
varying temperatures and the fluorescence measured using a
spectrofluorimeter (Fluoromax, Jobin-Yvon, .lamda..sub.ex=350 nm,
.lamda..sub.em=580 nm). Referring to FIG. 2B, the quantum yield of
the dissolved quantum dots decreased in a linear relationship the
increased temperatures to which the dots were exposed. Effects of
increased temperature exposure were not permanent, however, as the
quantum yield of the dissolved quantum dots returned to original
temperature upon cooling of the dots.
[0038] Quantum dots, produced in a powdered format in accordance
with the method of the present invention, retained the ability to
remain in a monodispersed state after being re-dissolved in an
aqueous solution for an extended period of time. Referring to FIG.
3, a quantity (10 mg/mL) of QDs in powdered form was dissolved in
10 mM phosphate buffered saline (PBS) of pH 7.4, and after 10 days
in solution, an aliquot of the dissolved QDs were spread on a glass
cover slip and imaged using an epifluorescence microscope (Olympus,
100.times., N.A.=1.4, 100 W Hg lamp, .lamda..sub.ex=530+/-30 nm,
.lamda..sub.em=610+/-40 nm). As shown in FIG. 3, the dissolved QDs
were present as individually fluorescing entities as opposed to an
aggregate of dots. The monodispersity of the powdered form of the
quantum dots dissolved in various saline (NaCl) concentrations was
also confirmed by epifluorescence microcopy (which is single
quantum dot image analysis).
[0039] To confirm that the quantum dots produced using the method
of the present invention did not have altered absorbance and
emission characteristics, samples of the powdered form of the
quantum dots were tested. As indicated in FIG. 4, when compared to
quantum dots having the TOPO ligand on the surface of the dot,
quantum dots having the cross-linked MUA ligand on their surface
exhibited no observable change in either the absorbance or emission
spectra. The quantum dots were examined under epifluorescence
imaging, fluorescence spectroscopy, and absorbance
spectroscopy.
[0040] Quantum dots produced in accordance with the method of the
present invention can be conjugated to various biomolecules, such
as proteins or antibodies. The QDs can be conjugated to any
biomolecule containing primary amino functional groups. Depending
on the biomolecule with which the quantum dot is conjugated, the
resulting conjugate can be used as a probe to detect the presence
of a biomolecule that may be present within a sample, for example,
to detect whether a specific protein or nucleic acid is present in
a protein or nucleic acid sample that has been isolated from an
organism or group of organisms and electrophoresed through an
acrylamide or agarose gel. To demonstrate the ability of the
quantum dots having a cross-linked ligand on their surface to form
a conjugate with a biomolecule, a stock solution was prepared by
dissolving 10 mg/ml of the powdered quantum dots in double
distilled water. 15 .mu.l of the stock solution was mixed with 20
.mu.l of a 10 mg/ml solution of the protein transferrin
(Sigma-Aldrich) in PBS (10 mM, pH=7.4). To conjugate the protein to
the quantum dot, 10 .mu.l of 50 mM stock solution (dissolved double
distilled water) of the cross-linking agent EDC was added to
quantum dot--protein mixture and the mixture shaken at room
temperature for two hours to allow the conjugation reaction to
occur. The entire volume of QD-transferrin conjugate was then
transferred to a culture of HeLa cells (30-50% confluence, cells
were grown at 37.degree. C. and 5% CO.sub.2 in a 15 mm.times.100 mm
tissue culture dish in the presence of Dulbecco Minimum Essential
Media (Gibco) supplemented with 10% Fetal Bovine Serum (Sigma), 1%
penicillin (Sigma), and 1% amphotericin B (Sigma)) and incubated
overnight in 37.degree. C. Cells were washed repeatedly after the
overnight incubation, and thereafter observed microscopically. For
controls, cells were incubated with either a solution of
unconjugated-QDs, or tranferrin/QD without EDC to HeLa cell
cultures. Referring to FIG. 5, when subjected to excitatory
radiation (100 W Hg excitation, emission filters 610+/-40, and QDs
(.lamda..sub.ex=350 nm, .lamda..sub.em=580 nm)) HeLa cells
incubated in the presence of the QD-transferrin conjugate exhibited
a fluorescent pattern consistent with having endocytosed the
QD-transferrin conjugate (micrograph A), while control cells
exhibited low autofluorescence (micrograph B).
[0041] This concludes the description of a presently preferred
embodiment of the invention. The foregoing description has been
presented for the purpose of illustration and is not intended to be
exhaustive or to limit the invention to the precise form disclosed.
Many modifications and variations are possible in light of the
above teaching and will be apparent to those skilled in the art. It
is intended the scope of the invention be limited not by this
description but by the claims that follow.
* * * * *