Synthesis of compact multidentate ligands to prepare stable hydrophilic quantum dot fluorophores

Article abstract of DOI:10.1021/ja044031w

We describe a simple and versatile scheme to prepare an array of heterofunctional multidentate ligands that permit strong and stable interactions with colloidal semiconductor nanocrystals (quantum dots, QDs) and render them soluble in aqueous environments. These ligands were synthesized by reacting various chain length poly(ethylene glycols) with thioctic acid, followed by ring opening of the dithiolane moiety, creating a bidentate thiol motif with enhanced affinity for CdSe-ZnS core-shell QDs. Functionalization with these ligands permits processability of the nanocrystals not only in aqueous but also in many other polar solvents. These ligands provide a straightforward means of preparing QDs that exhibit greater resistance to environmental changes, making them more amenable for use in live cell imaging and other biotechnological applications.

Full text of DOI:10.1021/ja044031w

Published on Web 02/26/2005
Synthesis of Compact Multidentate Ligands to Prepare Stable
Hydrophilic Quantum Dot Fluorophores
†
‡
§
§
H. Tetsuo Uyeda, Igor L. Medintz, Jyoti K. Jaiswal, Sanford M. Simon, and
,
†
Hedi Mattoussi*
Contribution from the U.S. NaVal Research Laboratory, DiVision of Optical Sciences, Center for
Bio/Molecular Science and Engineering, Washington, D.C. 20375, and Laboratory of Cellular
Biophysics, The Rockefeller UniVersity, Box 304, 1230 York AVenue, New York, New York 10021
Received September 30, 2004; E-mail: hedimat@ccs.nrl.navy.mil
Abstract: We describe a simple and versatile scheme to prepare an array of heterofunctional multidentate
ligands that permit strong and stable interactions with colloidal semiconductor nanocrystals (quantum dots,
QDs) and render them soluble in aqueous environments. These ligands were synthesized by reacting various
chain length poly(ethylene glycols) with thioctic acid, followed by ring opening of the dithiolane moiety,
creating a bidentate thiol motif with enhanced affinity for CdSe-ZnS core-shell QDs. Functionalization
with these ligands permits processability of the nanocrystals not only in aqueous but also in many other
polar solvents. These ligands provide a straightforward means of preparing QDs that exhibit greater
resistance to environmental changes, making them more amenable for use in live cell imaging and other
biotechnological applications.
degradation, and readily tunable spectroscopic properties.7
-10,13,14
Introduction
Colloidal QDs (core and core-shell) are often prepared from
Semiconductor nanocrystals (quantum dots, QDs) have
organometallic precursors using high-temperature solution
generated a tremendous amount of interest in the past two
1
,22-25
decades.1
-5
chemistry routes.
As made, the inorganic materials are
This has been motivated by a desire to reach a
prepared in the presence of strongly coordinating organic
fundamental understanding of several of their unique properties
and by the wealth of potential applications involving the use of
these materials, ranging from electronic devices to in vivo
1,5,24,25
ligands.
The most widely used surface-capping materials
are ligand mixtures consisting of trioctylphosphine/trioctylphos-
phine oxide (TOP/TOPO) and long-chain alkyl-amines. Ex-
change of these ligands using postsynthetic processing tech-
niques permits tailoring the solubility of the nanocrystals in
various solvents with preservation of the optical properties.1
Since the publication of the first reports on the design of
hydrophilic QDs and the first proof of QD-protein conjugate
3-6
cellular imaging. Colloidal luminescent semiconductor nano-
crystals (luminescent QDs), in particular, those made of CdSe
7
-10
and CdTe, have been proposed as unique biomarkers.
They
,5
have the potential to overcome many of the limitations
encountered by conventional organic fluorophores and geneti-
cally engineered fluorescent proteins in a variety of biological
7
,8
1
0-21
formation, there have been a number of studies aimed at
applications.
Luminescent QDs exhibit high chemical
designing a variety of surface functionalization schemes to
stability, high photobleaching thresholds, resistance to photo-
(
13) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge,
N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41-46.
14) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol.
2003, 21, 47-51.
†
Division of Optical Sciences.
Center for Bio/Molecular Science and Engineering.
The Rockefeller University.
‡
(
§
(
1) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993,
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T.; Mauro, J. M. Anal. Chem. 2002, 74, 841-847.
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Mauro, J. M. Nat. Mater. 2003, 2, 630-638.
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3870
9
J. AM. CHEM. SOC. 2005, 127, 3870-3878
10.1021/ja044031w CCC: $30.25 © 2005 American Chemical Society

Multidentate Ligands for Preparing Stable QD Fluorophores
A R T I C L E S
render these inorganic fluorophores water-soluble and compat-
hydrophilic poly(ethylene glycol) chain with the solvent. This
presents an alternative mechanism for achieving water solubility
of the QDs that is less sensitive to pH. As many conventional
methods to prepare water-soluble QDs and final QD-biocon-
jugates necessitate the need for varying pH, these ligands
represent a potential means to achieve aqueous solubility of QDs
over a broad pH range based on the DHLA ligand motif. The
present approach also has the advantage of providing rather
compact hydrophilic QDs, which is particularly useful when
employing QDs in F o¨ rster Resonance Energy Transfer (FRET)
experiments since the efficiency of this process is strongly
dependent on the separation distance between donor and
ible with biological manipulations.9
,12,13,18,20,26
Many of the
reported schemes, although providing aqueous solubility and
coupling to biomolecules, often have several limitations which
include short-term stability, sensitivity to pH, as well as weak
7-13,18,20,26
and nonspecific ligand interactions with the QD surfaces.
We have previously developed a strategy based on utilizing
bidentate surface ligands composed of dihydrolipoic acid
(
DHLA) to render CdSe-ZnS core-shell nanocrystals water-
9,16
soluble and biocompatible.
DHLA ligands provide stable
interactions with QD surfaces due to the bidentate chelate effect
afforded by the dithiol groups.9 While homogeneous in basic
buffer solutions, QD dispersions prepared by utilizing this
method afford luminescent and functional materials for over a
,10
3
8,39
acceptor fluorophores.
Furthermore, using mixed surface
functionalities, these new ligands allow the design of nano-
crystals that are water-compatible, multifunctional, and less pH-
dependent. We also investigate the utility of QDs capped with
these ligands for use in in vitro bioassays and show that they
are better suited for intracellular imaging studies.
9
,10
year.
However, macroscopic aggregation is observed when
the local environment of these QDs is altered, such as when
placed in acidic solutions, mixed with cationic lipids, or directly
9,10,27,28
dispersed in the cytosol of cells.
These properties could
be attributed to loss of water compatibility once the carboxylic
acid end groups are no longer ionized. Furthermore, applying
the conjugation approach based on the use of 1-ethyl-3-(3-
Experimental Section
1
. Materials and Analysis. All syntheses were carried out under
2
9
dimethylaminopropyl) carbodiimide hydrochloride (EDC) to
DHLA, although simple in concept, is not reproducible with
mercapto acetic acid or DHLA and resulted in macroscopic
N
2
passed through an O scrubbing tower unless otherwise stated. Air-
2
sensitive materials were handled in an Mbraun M-150 glovebox, and
standard Schlenk techniques were used in manipulation of air-sensitive
solutions. All chemicals used in this work were obtained from Sigma-
Aldrich and used without further purification. Solvents were obtained
from Fisher Scientific and used as received. Chemical shifts for H
NMR spectra are relative to the residual protium (CDCl
9
,10
aggregates.
These limitations underscore the need for de-
veloping new approaches for surface functionalization of QDs
to improve their stability and broaden their resistance to
environmental changes. One of our long-term goals is to design
ligands that permit preparation of compact hydrophilic QDs
amenable to conjugation with a variety of biomolecules via
simple covalent and noncovalent binding strategies. These issues
are receiving considerable attention as they address many of
the problems currently associated with the design and use of
hydrophilic QDs and QD-bioconjugates.1
1
3
, δ ) 7.26
ppm; methanol-d , δ ) 4.87 ppm; or tetramethylsilane, δ ) 0.0 ppm).
4
All J values are reported in hertz. The number of attached protons is
found in parentheses, following the chemical shift value. Chromato-
graphic purification (silica gel 60 Å, 230-400 mesh, Bodman
Industries) of all newly synthesized compounds was accomplished on
the benchtop. Materials were visualized with I or ninhydrin spray on
2
8,26,30-37
aluminum-backed TLC plates. Absorption spectra were recorded on
an HP 8453 diode array spectrophotometer. Corrected photolumines-
cence spectra were recorded on a SPEX Fluorolog-3 spectrophotometer
fitted with a red-sensitive R2658 Hamamatsu PMT.
In this investigation, we report a general procedure for the
design and preparation of aqueous soluble CdSe-ZnS core-
shell nanoparticles featuring a variety of DHLA ligand deriva-
tives appended with poly(ethylene glycols) (PEG) of various
lengths to generate hydrophilic and biocompatible nanoparticles.
This approach, as demonstrated by our previous work, utilizes
the bidentate chelate interactions afforded by the dithiol domain
of the DHLA with the QD surface. However, the means for
achieving water solubility is governed not by the deprotonation
of the carboxylic acid moieties but by the interactions of the
2
. Synthesis and Design. Synthesis of Poly(ethylene glycol)-
Terminated Thioctic Acid Compounds. Poly(ethylene glycol) (PEG)
of the desired average molecular weight is first appended onto thioctic
acid (TA) using a dicyclohexylcarbodiimide (DCC)-mediated esteri-
fication reaction with a catalytic amount of 4-(dimethylamino)-pyridine
(DMAP). The same reaction was used for attaching tetra(ethylene
glycol), hexa(ethylene glycol), and various molecular weight PEGs onto
the thioctic acid (Figure 1). For brevity, we limit our description to the
preparation of three representative compounds, namely, 1, 6, and 7.
Additional information on the synthesis of compounds 2-5 and 8-11
is provided in the Supporting Information.
(
(
(
(
(
26) Potapova, I.; Mruk, R.; Prehl, S.; Zentel, R.; Basche, T.; Mews, A. J. Am.
Chem. Soc. 2003, 125, 320-321.
27) Voura, E. B.; Jaiswal, J. K.; Mattoussi, H.; Simon, S. M. Nat. Med. 2004,
1
0, 993-998.
5
-[1,2]-Dithiolan-3-yl-pentanoic Acid 2-{2-[2-(2-hydroxy-ethoxy)-
ethoxy]-ethoxy}-ethyl Ester [TA-TEG]: Compound 1. Thioctic acid
6.19 g, 30 mmol), tetra(ethylene glycol) (58 g, 300 mmol), 4-(dimeth-
ylamino)-pyridine (1.1 g, 9 mmol), and dichloromethane (300 mL) were
placed in a flask and degassed with a stream of N for 20 min. The
28) Jaiswal, J. K.; Goldman, E. R.; Mattoussi, H.; Simon, S. M. Nat. Methods
004, 1, 1-6.
29) Hermanson, G. T. Bioconjugate Techniques; Academic: San Diego, CA,
996.
30) Uyeda, H. T.; Medintz, I. L.; Mattoussi, H. Mater. Res. Soc. Symp. Proc.
2
(
1
2
004, 789, 111-116.
2
(
31) Skaff, H.; Emrick, T. Chem. Commun. 2003, 52-53.
reaction mixture was cooled to 0 °C in an ice bath, and a solution of
DCC (6.8 g, 33 mmol) in dichloromethane (20 mL) was added
dropwise. The reaction mixture was stirred at 0 °C for 1 h before it
was warmed to room temperature and stirred for 20 h. The precipitate
that formed was filtered over a plug of Celite and washed with brine
(
32) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello,
V. M. J. Am. Chem. Soc. 2004, 126, 739-743.
(
33) Nagasaki, Y.; Ishii, T.; Sunaga, Y.; Watanabe, Y.; Hidenori, O.; Kataoka,
K. Langmuir 2004, 20, 6396-6400.
(
34) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A.
L.; Keller, S.; Raedler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004, 4,
7
03-707.
(
(
(
35) Osaki, F.; Kanamori, T.; Sando, S.; Seara, T.; Aoyama, Y. J. Am. Chem.
Soc. 2004, 126, 6520-6521.
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Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301-310.
(39) Medintz, I. L.; Konnert, J. H.; Clapp, A. R.; Stanish, I.; Twigg, M. E.;
Mattoussi, H.; Mauro, J. M.; Deschamps, J. R. Proc. Natl. Acad. Sci. U.S.A.
2004, 101, 9612-9617.
36) Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. J. Am. Chem. Soc. 2004,
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26, 6115-6123.
37) Mattheakis, L. C.; Dias, J. M.; Choi, Y.-J.; Gong, J.; Bruchez, M. P.; Liu,
J.; Wang, E. Anal. Biochem. 2004, 327, 200-208.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 11, 2005 3871

A R T I C L E S
Uyeda et al.
6,8-Dimercapto-octanoic Acid 2-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-
ethoxy}-ethyl Ester [DHLA-TEG]: Compound 7. Compound 1
3.83 g, 10 mmol) was dissolved in 50 mL of 1:4 EtOH/water with
stirring. NaBH (416 mg, 11 mmol) was added and stirred for 60 min
or until the solution became colorless. The reaction mixture was diluted
with water (100 mL) and extracted with CHCl
(3 × 75 mL). The
combined organic phases were dried over magnesium sulfate (MgSO ),
(
4
3
4
filtered, and evaporated. The residue was purified by flash chroma-
tography (EtOAc/MeOH 95:5) and evaporated to give 3.4 g (88%) of
1
the desired product as a colorless oil. H NMR (400 MHz, CDCl
3
): δ
(ppm) 4.20 (t, J ) 4.8 Hz, 2H), 3.65 (m, 12H), 3.57 (t, J ) 4.5 Hz,
2
2
(
H), 2.88 (m, 1H), 2.66 (m, 2H), 2.49 (s, 1H), 2.32 (t, J ) 7.3 Hz,
H), 1.87 (m, 1H), 1.7-1.35 (m, 7H), 1.32 (t, J ) 8.0 Hz, 1H), 1.27
d, J ) 7.6 Hz, 1 H). 13C NMR (100 MHz, CDCl
): δ (ppm) 173.03,
3
7
2.18, 70.21, 70.10, 69.90, 68.73, 63.00, 61.18, 42.34, 38.89, 38.27,
3
3.56, 26.06, 24.10, 21.87.
Characterization of the ligand compounds 1-11 was accomplished
1
13
by H and C NMR spectroscopic techniques. The NMR data indicate
that when appending thioctic acid (TA) to the various poly(ethylene
glycols), the resulting spectrum is essentially a composite of the two
starting materials. Figure 2 shows the spectra of TA and compound 2
(TA-TEG); similar spectra were obtained for compounds 3-5. The
main contribution from the PEG moiety appears as a large broad
multiplet at 3.5-3.6 ppm with an additional two-proton triplet at 4.1
ppm. The absence of the carboxylic acid resonance (broad peak at 11
ppm) of compounds 2-5, but present in the thioctic acid spectrum, is
further evidence of successful coupling (see Supporting Information).
Figure 3 shows that the oxidized and reduced forms of related structures
(compounds 2 and 8) have very disparate spectra and are easily
distinguishable. The thiol protons of compound 8 are clearly discernible
as seen by the well-resolved triplet and doublet at 1.30 and 1.25 ppm,
respectively, with integrated intensities of one proton each. Furthermore,
new resonances are observed for compound 8 at 2.8 and 2.6 ppm but
are conspicuously absent from precursor compound 2, while resonances
at 3.0 and 2.3-2.35 ppm for 2 are absent in 8 (Figure 3). Similar
differences between the open and closed forms are observed for the
other compounds, namely, 3 versus 9, 4 versus 10, 5 versus 11, and
TA versus DHLA. It is these characteristic resonances that lead us to
unambiguously identify the compounds prepared in this investigation.
Starting from commercially available precursors, it is possible to quickly
and efficiently prepare large amounts of the poly(ethylene glycol)-
modified dihydrolipoic acid ligands.
Figure 1. Synthetic route to compounds 1-11. Reagents and conditions:
(i) DCC, DMAP, CH2Cl2, 0 °C to room temperature; (ii) NaBH4, EtOH/
H2O, rt; (iii) NaBH4, NaHCO3, H2O, 0 °C.
(
3 × 75 mL). The combined organic extracts were dried over MgSO
4
,
filtered, and evaporated to give a yellow oil. The crude product was
purified by flash chromatography (ethyl acetate/methanol 95:5) and
evaporated to give 7.1 g (62%) as a yellow oil. TLC (EtOAc/MeOH
1
9
2
:1) R
f
0.4. H NMR (400 MHz, CDCl
3
): δ (ppm) 4.23 (t, J ) 4.8 Hz,
H), 3.70 (m, 12H), 3.65 (m, 2H), 3.15 (m, 2H), 2.45 (m, 1H), 2.36 (t,
J ) 7.4 Hz, 2H), 2.28 (s, 1H), 1.92 (m, 1H), 1.69 (m, 4H), 1.48 (m,
1
3
2
7
2
3
H). C NMR (100 MHz, CDCl ): δ (ppm) 172.94, 72.17, 70.16,
0.05, 69.85, 68.68, 62.97, 61.14, 55.88, 39.78, 38.05, 34.13, 33.46,
8.24, 24.16.
We have also investigated the DCC-based esterification of dihydro-
lipoic acid with oligo- and poly(ethylene glycols). This route, however,
proved to be less efficient in that the products were isolated as mixtures
consisting of the reoxidized form of the ligand and desired product.
Thus, the higher yields and ease of separation using the procedure
outlined above provided the cleanest materials.
Synthesis of Poly(ethylene glycol)-Terminated Dihydrolipoic Acid
Compounds: Ring Opening. The ring opening of the dithiolane on
the various thioctic acid-terminated PEG ligands (compounds 1-5) and
thioctic acid was carried out using sodium borohydride as described in
Figure 1. While the conversion of thioctic acid (TA) to dihydrolipoic
acid (DHLA) (6) takes place in strictly aqueous solvent,9
,40,41
the
3
. Amylose Resin Assay of MBP-5HIS to QDs. Affinity columns
25 mm × 76 mm) of amylose resin (New England Biolabs, Beverly,
MA) were setup and equilibrated with 10 mM Na-tetraborate buffer
reduction of compounds 1-5 proceeded more cleanly when carried
out in a mixture of ethanol and water.
(
6
,8-Dimercapto-octanoic Acid [Dihydrolipoic Acid, DHLA]:
9
(
pH 9.55). Then, 100 pmol of CdSe-ZnS QDs capped with 6, 10,
9,40,41
Compound 6. Compound 6 was prepared as previously described,
and a 1:1 mixture of 6 and 10 was mixed with the equivalent of 20
with the following modifications. Thioctic acid (12.2 g, 59 mmol) was
dissolved in 250 mL of 0.25 M NaHCO and cooled to 0 °C in an ice
bath. NaBH (9.0 g, 238 mmol) was added slowly and the temperature
kept below 4 °C while stirring for an additional 2 h. The reaction
mixture was acidified with 6 M HCl to pH 1 and extracted with toluene
maltose binding proteins appended with an oligohistidine tail (MBP-
3
HIS) per QD in 200 µL aliquots of 10 mM borate buffer at pH 9.55
4
9,16
and allowed to self-assemble for 1 h.
Duplicates of each sample
tested were loaded onto the amylose resin pre-equilibrated in buffer.
After binding to the column, samples were washed with 1 mL of buffer,
eluted with 20 mM maltose in buffer, and the eluant captured. The
eluant was brought up to 500 µL with 20 mM maltose in buffer. A
quantity of 100 µL of all samples was then loaded onto a microtiter
well plate in triplicate, and the PL was measured on a Tecan Safire
plate reader (Tecan US, Research Triangle Park NC). By normalizing
the PL of the DHLA-capped QD self-assembly mixture to 100%, we
could estimate the percent of each QD sample binding to the column
(
3 × 75 mL). The combined organic phases were dried over MgSO
4
and filtered. Evaporation of the solvent yielded 12.0 g (98%) of the
desired product as a clear colorless oil.
(
40) Wagner, A. F.; Walton, E.; Boxer, G. E.; Pruss, M. P.; Holly, F. W.; Folkers,
K. J. Am. Chem. Soc. 1956, 78, 5079-5081.
(
41) Gunsalus, I. C.; Barton, L. S.; Gruber, W. J. Am. Chem. Soc. 1956, 78,
1
763-1766.
3872 J. AM. CHEM. SOC.
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VOL. 127, NO. 11, 2005

Multidentate Ligands for Preparing Stable QD Fluorophores
A R T I C L E S
1
1
Figure 2. H NMR spectra highlighting the coupling of tetraethylene glycol to thioctic acid: TA and compound 1. (Additional H NMR spectra for the
other PEG-terminated TA molecules are provided in the Supporting Information.)
1
Figure 3. Comparative H NMR spectra of the upfield region displaying the characteristic shifts resulting from the reduction of the dithiolane moiety;
compounds 2 and 8 are shown.
and being released with maltose relative to the solution containing
DHLA-capped QDs (Figure 4).
Results and Discussion
4
. Live Cell Imaging. HeLa and NRK cells were cultured in DMEM
1. Synthesis and Design. Utilizing an excess of the PEG in
the esterification reaction substantially minimizes formation of
the bisubstituted species and simplifies the chromatographic
purification of compounds 1-5. Prior to chromatography, much
of the unreacted glycol is removed by extraction with water,
thus greatly simplifying the purification process. Since the
bisubstituted species exhibits an Rf value larger than that of the
desired monosubstituted product, it is easily separable by flash
column chromatography. The subsequent ring opening process
is facile and can be monitored visually by following the gradual
disappearance of the yellow color of the precursor to a final
colorless solution (final product). Completion of the reaction is
supplemented with 10% fetal bovine serum (Invitrogen Life Technolo-
gies). Cells were grown on coverslips (Fisher Scientific) and imaged
in 10 mM Hanks buffered salt solution, Hepes, and 5% FBS, pH 7.4.
QDs capped with DHLA-PEG600 (compound 10) were diluted in
injection buffer (140 mM KCl/10 mM Hepes, pH 7.4) and microinjected
into cells using a Narashige microinjector (Japan). Cells were imaged
using an Olympus IX70 microscope equipped with a 75 W xenon lamp
source, a 480/40 nm excitation filter, and appropriate emission filters
(
Chroma Technologies Corp., Brattleboro, VT). Images were acquired
with an ORCA-ER camera (Hamamatsu Photonics). Image acquisition,
processing, and analysis were done using MetaMorph (Universal
Imaging, Downingtown, PA).
J. AM. CHEM. SOC.
9
VOL. 127, NO. 11, 2005 3873

A R T I C L E S
Uyeda et al.
Precipitated QDs (∼100-300 mg) obtained from centrifugation
in ethanol are dispersed in pure DHLA (∼1 mL) and heated to
∼
70 °C for ∼2-3 h until the solution becomes homogeneous.
Dimethyl formamide (5 mL) is added to the solution, and the
nanocrystals are precipitated with potassium tert-butoxide
(
∼400-600 mg) and centrifuged to produce a pellet of QDs
and excess potassium tert-butoxide. The supernatant, free of
QDs, is discarded, and the precipitate is dispersed in water and
purified using concentration/dilution with a membrane separation
filter (Millipore, Mw cutoff ∼50 000 Da) with water through
four cycles. However, achieving water compatibility via surface
ligand exchange of QDs with the various PEG-terminated
DHLA ligands (compounds 9-11) and mixtures of DHLA
(compound 6) with DHLA-PEG600 (compound 10) required
the use of a modified procedure outlined below. Mixtures of
DHLA (compound 6) with 9 or 11 proceeded with similar
results.
Surface ligand exchange of the native TOP/TOPO ligands
with compounds 7 and 8 resulted in dispersions of QDs that
were not soluble in water, however, readily formed homoge-
neous solutions in polar organic solvents, such as methanol,
ethanol, or DMF. It should be noted that QDs coated with TOP/
TOPO cannot be dispersed in these polar organics, thus
highlighting the ability of these DHLA-modified ligands to tailor
the solubility based on the poly(ethylene glycol) chain lengths.
While not dispersible in aqueous environments, QDs coated with
these ligands which are soluble in polar organics can find
potential utility in a variety of other applications, such as sol-
gel processing employed in the fabrication of thin solid films,
with high density of QDs for use in amplified stimulated
Figure 4. Amylose resin assay testing the ability of MBP-5HIS to attach
to QDs capped with DHLA (compound 6), DHLA-PEG600 (compound
1
0), and a 50:50 mixture of compounds 6 and 10. Even though they are
not as efficient as the pure DHLA capping, the mixed cap QDs conjugated
to MBP-5HIS showed efficient binding to amylose resin and release by
soluble maltose.
clearly reflected by the disparate NMR spectra of the oxidized
and reduced forms of the ligands (Figure 3).
The synthetic route described here, which begins by append-
ing the poly(ethylene glycol) chain of desired length onto
thioctic acid followed by reduction with NaBH4, provides a
simple, reproducible, and efficient scheme to prepare our desired
ligands. It is substantially better than the reverse route where
ring opening of thioctic acid is followed by coupling of the
poly(ethylene glycol). This route exhibits an overall lower yield
and a more difficult separation of the PEG-modified DHLA,
due to the formation of the reoxidized species and other side
products. This synthetic route provides a general and relatively
simple means to prepare large quantities of heterobifunctional
linkers based on poly(ethylene glycol) chains in which one end
is modified with thioctic acid/dihydrolipoic acid, thus maintain-
ing the bidentate surface functionality of the final compound.
It is then possible to modify the other end of the polymer chain
to provide other functional moieties for further covalent coupling
strategies.
4
3
emission (ASE) experiments.
Preparation of nanocrystals capped with compounds 9, 10,
and 11 was carried out as follows. TOP/TOPO-capped nano-
particles (100-300 mg) after size selective precipitation with
ethanol were suspended in 1-2 mL of surface-capping ligand
9
, 10, or 11, mixed with 1 mL of methanol, and heated to ∼60
°C overnight under N2 with stirring. The resulting homogeneous
sample was cooled to room temperature and diluted with
hexanes, ethanol, and chloroform to produce a monophasic
turbid solution (solvent-induced precipitation of the newly
capped QDs). The solution was centrifuged to produce a pellet
of nanoparticles, while the supernatant, free of color, was
discarded. The precipitate was dispersed in water, providing a
clear homogeneous dispersion of nanocrystals, and was purified
using four cycles of concentration/dilution with an Ultra-free
centrifugal filtration device (Millipore, Mw cutoff ∼50 000 Da).
These procedures permit elimination of soluble organics and
other materials from the solution and provide homogeneous
aggregate-free QD dispersions with concentrations ranging from
2
. Preparation of DHLA-PEG-Capped Hydrophilic CdSe-
ZnS Core-Shell QDs. Exchanging the native capping shell
TOP/TOPO/hexadecylamine) with our newly designed DHLA-
(
PEG ligand series to render the nanocrystals water-soluble was
carried out using a procedure similar to that for DHLA
functionalization described earlier with several modifica-
5
to 150 µM that were stored for further use. Homogeneous
aqueous dispersions of these hydrophilic QDs exhibited absorp-
tion and emission characteristics virtually identical to those of
the native materials with photoluminescence efficiencies of
9
,40,41
tions.
The nanocrystal fluorophores were synthesized using
known literature procedures.1
,2,22-24,42
Briefly, samples were
prepared utilizing a stepwise approach consisting of core
nanocrystal growth, overcoating with a thin layer of ZnS, size
selective precipitation, surface ligand exchange, and purification.
The use of DHLA in the ligand exchange process with the native
TOP/TOPO to generate stable, aggregate-free aqueous disper-
∼
25-30%, lower than what is measured in organic solutions
usually up to ∼60-80%).
. QDs Capped with Mixtures of DHLA and DHLA-PEG
(
3
ligands. Exchanging the native capping shell on the QDs with
mixtures of DHLA (compound 6) and one of the longer PEG-
9,15
sions of nanoparticles was carried out as described previously.
(
43) Petruska, M. A.; Bartko, A. P.; Klimov, V. I. J. Am. Chem. Soc. 2004,
(42) Reiss, P.; Bleuse, J.; Pron, A. Nano Lett. 2002, 2, 781-784.
126, 714-715.
3
874 J. AM. CHEM. SOC. VOL. 127, NO. 11, 2005
9

Multidentate Ligands for Preparing Stable QD Fluorophores
A R T I C L E S
Figure 5. Gel shift of CdSe-ZnS QDs coated with various ratios of 10 to
6
1
in 1% agarose gel buffered with Tris borate EDTA buffer. Lane 1, 1:0
0:6; lane 2, 1:1 10:6; lane 3, 3:1 10:6; lane 4, 10:1 10:6.
Figure 6. Spectra of TOP/TOPO-capped nanocrystals in toluene and after
surface ligand exchange with DHLA-PEG600 (compound 10) in water.
terminated ligands, namely, 9, 10, or 11, required addition of
10 mg of potassium tert-butoxide to achieve complete aqueous
∼
Surface ligand exchange of the QDs with compound 6, 9-11,
or any mixture of 6 and 10 to produce water-soluble QD
dispersions exhibited absorption profiles that were virtually
unchanged from the organic growth solutions. Presumably, the
length of the chain in the PEG is responsible for achieving
complete aqueous dissolution of the QDs as surface ligand
exchange using compounds 7 and 8 resulted in QDs that were
partially soluble in water. These results suggest that a minimum
poly(ethylene glycol) length is needed to achieve homogeneous
aqueous dispersions, which correspond to a chain length of
approximately 8 ethylene oxide units or a PEG with average
molecular weight of 400 when appended to dihydrolipoic acid.
It is important to note that the surface ligand exchange process
with either pure PEG-modified DHLA, DHLA, or a mixture of
the ligands is similar for all cases, and that the methodology is
quite general for this class of molecules.
dispersion of the sample, an amount much smaller than what
was required (∼400-600 mg) when using only DHLA.
Presumably, the base is needed for the ionization of the carboxyl
moieties in order to facilitate dispersion in water. Confirmation
of the cap exchange with a mixture of DHLA and compound
9
, 10, or 11 can also be obtained by investigating the effects of
surface charges using gel electrophoretic mobility shift. Figure
shows the gel mobility shift of a series of QD dispersions
5
capped with a mixture of DHLA (compound 6) and PEG-
modified DHLA compound 10. The extent of the mobility shift
is seen as a function of the molar ratio of the two compounds.
The molar ratios of compound 6 to compound 10 ranged from
1
:1, 1:3, and 1:10 during the surface ligand exchange process.
The higher the molar ratio of 6:10, the further the QDs migrated
to the positive electrode. As seen in lane 1 of Figure 5, QDs
coated with 100% of compound 10 show little or no shift from
the loading well under the applied voltage, due to the nonionized
nature of these QD systems. When 50% of compound 10 is
replaced with 6 (lane 2 in Figure 5), a substantial gel shift was
measured, which we attribute to the finite electrophoretic
mobility brought about by the presence of charged DHLA
ligands on the QD surface. Similarly, QDs coated with a mixture
of 6 and 10 but at lower concentrations of 6 (30% in lane 3 and
4. Optical Characterization. Estimates of the photoemission
quantum yields were obtained by comparing the integrated
emission from rhodamine 6G in ethanol (QY ) 0.90) or other
standards to that of the CdSe-ZnS nanoparticles whose
emission spectral profile closely matched that of the standard.
Concentrations were adjusted such that both reference and QD
samples gave optical densities of ∼0.05 at the excitation
wavelength, and solvent refractive indexes were taken into
45
1
0% in lane 4) resulted in smaller shifts relative to the case
account. Fluorescence spectra were collected between 450 and
700 nm at room temperature and integrated on a wavenumber
scale. The differences in the absorption and photoemission
spectral profiles of the QDs measured in toluene and QDs that
have been transferred into water using ligand 10 are negligible
with peak shifts of less than 3 nm in all cases (Figure 6). The
same trend is observed when compounds 9-11 or mixtures of
6 and any of these compounds are used, indicating that the
surface ligands have no effect on the electronic properties of
shown in lane 2. As the amount of charge is increased (with
increasing DHLA fraction), an increase of the mobility is also
observed, which tracks well in a linear fashion. This reflects a
lower electrophoretic mobility of the QDs, due to a smaller
density of surface charges when the fraction of 6 on the QD
surface is reduced. QDs capped with DHLA (compound 6)
4
4
showed an even more pronounced gel mobility shift.
(
44) Goldman, E. R.; Mattoussi, H.; Tran, P. T.; Anderson, G. P.; Mauro, J. M.
Mater. Res. Soc. Symp. Proc. 2001, 642, J281-J286.
(45) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991-1024.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 11, 2005 3875

A R T I C L E S
Uyeda et al.
It is important to note that compounds 1-5 and 7-11 could
also be used to functionalize other nanoparticles and surfaces,
4
7,48
such as those made of gold and silver.
In particular, due to
the strong affinity of thiols to gold surfaces, our designed ligands
could provide unique flexibility in preparing functionalized gold
surfaces and nanoparticles for use in targeted experiments.4
Whereas QDs coated with compound 6 resulted in aqueous
dispersions that were aggregate-free in the pH regime of 7-12,
the use of compound 10 in the surface ligand exchange process
resulted in aggregate-free solutions from pH ∼5 to 12 over at
least 1 year after preparation. Furthermore, NMR spectra of
DHLA-PEG ligands taken several months after preparation
showed no signs of degradation, indicating that the ligands
themselves are also stable. The gain in pH stability is substantial
7-49
Figure 7. (A) Luminescence image set of 555 nm emitting CdSe-ZnS
QD solutions in phosphate buffer saline at various pH values after 2 days
at room temperature: (vial 1) QDs coated with compound 5 at pH 5.8;
5
0
as it progresses into the acidic regime. By using mixtures of
ligands 6 and 10, for example, we have obtained nanoparticles
that were luminescent and homogeneous in the pH range of
(
8
vials 2-6) QDs coated with compound 10 at pH 5.8, 6.2, 6.6, 7.6, and
.0, respectively. (B) Same set as above with 590 nm emitting QDs. Samples
∼
6-12 and, thus, provided a rationale for exploring additional
were excited with a hand-held UV lamp at 365 nm.
properties conferred with these ligand mixtures. We have
previously demonstrated that QDs capped with DHLA (com-
pound 6) undergo electrostatic and metal-affinity-driven self-
assembly with maltose binding protein appended at its C-ter-
minal with a leucine zipper or an oligohistidine attachment
domain, MBP-zb or MBP-5HIS, respectively; the formed QD-
MBP conjugates were found to tightly bind to amylose resin
the inorganic QD core. However, although the absorption and
emission spectral properties of the organic soluble and aqueous
soluble QDs are nearly identical, there is a noticeable decrease
of the photoluminescence quantum yield for the QDs in aqueous
dispersions (Figure 6). The quantum yields are approximately
70-80% for the QDs measured in toluene or hexane solutions
9
,16
and elute with excess maltose. Figure 4 shows the results of
an amylose resin assay applied to three QD solutions incubated
with MBP-5HIS, one capped with DHLA (ligand 6), one capped
with DHLA-PEG600 (ligand 10), and one with a 1:1 mixture
of ligands 6 and 10, to verify the ability of MBP-5HIS to self-
assemble on these three variations of QD-ligand complexes.
The metal-affinity self-assembly process for QDs capped with
compound 6 is highly efficient, whereas for QDs coated with
and drop to 25-30% for the QDs coated with compound 9, 10,
or 11 and dispersed in water. This is not uncommon, as reported
quantum yields for QDs transferred into buffer solutions are
also smaller than those measured in organic solutions (∼10-
-10,36
3
0%).7
Concentrations were estimated from measurements
9,23
of the absorption spectra or using the size-dependent extinc-
tion coefficient at 350 nm, as done by Leatherdale et al.
4
6
Concentrations obtained from both methods were within ex-
perimental error.
1
0, nonspecific interactions may be responsible for the small
9
,16
QD-protein complex recovered (Figure 4).
The binding
5. Stability Measurements and Noncovalent Self-Assembly
efficiency of MBP-HIS to QDs capped with a mixture of DHLA
and DHLA-PEG600 (compound 10) is slightly lower than that
of QDs capped with DHLA; however, it is much higher than
what is measured for QDs capped with PEG-terminated DHLA.
We believe that the longer PEG molecules may prevent the
MBP-5HIS tail from interacting with the QD zinc and sulfur
with HIS-Terminated Proteins. The pH stability of aqueous
dispersions of QDs coated with DHLA-PEG compound 9, 10,
or 11 was investigated. Figure 7 shows a solution of QDs capped
with compound 6 at pH 5.8 side by side with solutions of QDs
capped with compound 10 in various PBS solutions whose pH
ranged from 5.8 to 8. Aqueous dispersions of QDs capped with
DHLA (compound 6) showed that at pHs lower than 7,
aggregation of the nanoparticles occurs, signifying the principal
role that the carboxylate functional group plays in determining
the solubility of the complex in aqueous solutions. This is clearly
seen in vial 1 of Figure 7A,B, where QDs capped with pure
DHLA were used. For QDs functionalized with ligand 10,
optically clear homogeneous solutions were obtained at pH
values of 5.8 and above (vials 2-6 of Figure 7A,B). QD samples
capped with ligand 9 or 11 exhibited similar pH stability trends.
Aqueous dispersions of QDs coated with mixtures of compounds
39
atoms. Nonetheless, this assay proves that the new PEG-
terminated ligands do not alter the ability of QDs partially
capped with DHLA to self-assemble with HIS-terminated
proteins. The self-assembly process for QDs coated with a 1:1
mixture of 6 and 10 is effective and represents a balance between
the ability to self-assemble protein onto the QD and maintain a
relatively broad usable pH range.
In addition to the pH stability, we investigated the behavior
of QDs capped with DHLA-PEG ligands in aqueous solutions
at various ionic strengths. In particular, the photoluminescence
properties of two sets of QDs dispersed in buffer solutions at
neutral pH were monitored as the concentration of NaCl was
increased from 0 to 1 M; one set of 510 nm emitting QDs capped
6
and 10 remained homogeneous at pH 5-7 after preparation;
however, aggregation build-up was observed after 1 day while
maintaining their luminescence when the fraction of DHLA in
the ligand mixture exceeds 50%, which underscores the
importance of maintaining relatively high pH conditions for QDs
that contain DHLA to remain in solution.
(
47) Zheng, M.; Huang X. J. Am. Chem. Soc. 2004, 126, 12047-12054.
(48) Querner, C.; Reiss, P.; Bleuse, J.; Pron, A. J. Am. Chem. Soc. 2004, 126,
11574-11582.
(
49) Charvet, N.; Reiss, P.; Roget, A.; Dupuis, A.; Gr u¨ nwald, D.; Carayon, S.;
Chandezon, F.; Livache, T. J. Mater. Chem. 2004, 14, 2638-2642.
50) QDs capped with compounds 6, 9, 10, and 11 or any mixtures of these are
still stable at pH values larger than 12. We did not explore that pH range
any further because they are not practically useful for biomotivated
applications.
(
(
46) Leatherdale, C. A.; Woo, W. K.; Mikulec, F. V.; Bawendi, M. G. J. Phys.
Chem. B. 2002, 106, 7619-7622.
3876 J. AM. CHEM. SOC.
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Multidentate Ligands for Preparing Stable QD Fluorophores
A R T I C L E S
Figure 8. (A) Effect of increasing ionic strength on the photoluminescence of 510 nm emitting CdSe-ZnS QDs coated with compound 11 ([) and 535 nm
emitting CdSe-ZnS QDs coated with compound 10 (2). Dispersions of QDs were placed in fluorescence cuvettes (1 cm optical path) containing an equal
concentration of QDs but various concentrations of sodium chloride. The resulting photoluminescence spectra were background-corrected, integrated, and
normalized to the emission form slat-free solution. The PL emission from QDs with no NaCl added was set to 100%. (B) Photoluminescence spectra of
535 nm emitting CdSe-ZnS QDs coated with compound 10 at three ionic strength concentrations. The 0.2 and 0.6 M solutions were normalized against the
0.1 M NaCl solution.
Figure 9. Distribution of DHLA-PEG600-capped QDs in live cells. Mammalian cells (Hela-A,E,F and NRK-B,C,D) were grown in culture media and
injected with a 10 µM QD solution. The images show epifluorescence images of cells 5 min after being injected with (A) DHLA-capped QDs, (B) QDs
capped with DHLA-PEG600 (compound 10), and (C) QDs capped with compound 11. (D) Epifluorescence image of cells 14 h after being microinjected
with QDs capped with DHLA-PEG600. (E and F) Bright field (left) and epifluorescence (right) images of cells injected with QDs coated with DHLA-
PEG600 (E) 5 min and (F) 35 min after being microinjected. Note that cells shown in (E) have different amounts of QDs, as judged by their fluorescence,
but within 30 min, they fuse together causing redistribution of their cytoplasm, thus the QD fluorescence.
with compound 10 (DHLA-PEG600) and one set of 535 nm
emitting QDs capped with compound 11 (DHLA-PEG1000),
each set having a fixed QD concentration, were studied. Over
the concentration range of NaCl used (0-1.0 M), we observed
little or no change in both the PL intensity and the characteristics
of the emission spectra for both cases (see Figure 8). These
experiments further highlight the stability of QDs capped with
these new ligands in high ionic strength conditions. Moreover,
the derived results are directly applicable to intracellular and
in vivo studies, where the ionic concentration is known to be
high. The data shown in Figure 8 are consistent with our
previous observations using DHLA-capped QDs electrostatically
self-assembled with engineered protein to form QD bioconju-
gates, where insignificant effects of salt concentration on the
6. Cellular Imaging Studies. One of the main limitations of
currently available QDs for intracellular imaging is their
tendency to aggregate in the cytosol and their sensitivity to pH
changes. The pH in the cytosol is close to 7.3, but it drops below
5 in some organelles. For example, dispersions of DHLA-capped
QDs are unstable at pH values below 7, and they aggregate
soon after they are introduced into the cytoplasm by microin-
jection (Figure 9A). Precipitation and aggregation of DHLA-
capped QDs in the cell cytoplasm is also observed using other
approaches, such as cationic lipid-based loading and scrape
27,28
loading.
In light of the enhanced pH stability of QDs capped
with the new DHLA-PEG ligands, we tested their use for
imaging the cytoplasm of live cells. The results presented here
were obtained using microinjection of QDs directly into the
cytoplasm, but other approaches for introducing QDs into live
cells yielded similar results. QDs capped with either compound
10 (Figure 9B) or 11 (Figure 9C) dispersed better than the
9
conjugates and their spectroscopic properties were observed.
The observations using QDs capped with PEG-terminated
ligands confirm and extend those results.
J. AM. CHEM. SOC.
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A R T I C L E S
Uyeda et al.
DHLA-capped QDs when injected into the cytosol. Over the
subsequent 12-24 h, a fraction of QDs slowly aggregated, as
assayed by the appearance of punctate QD fluorescence (Figure
mixed compositions of surface ligands with DHLA, we are able
to assess the degree of charge on the nanoparticle surface by
gel electrophoresis and to employ a metal-affinity-driven self-
assembly method to form QD-protein conjugates.
9
D). Microinjection of QDs capped with PEG-terminated
monothiol ligands showed homogeneous distribution in the
One limitation of using organic fluorophores in many cellular
applications is their sensitivity to the environment. For example,
across the compartments of a live cell, the concentrations of
protons can vary by almost three log units and the concentration
of calcium varies by five log units. Improving the stability of
these QDs and the ability to conjugate them to molecules will
make QDs more useful for numerous biological assays both in
vitro and in living cells. The use of QDs in living cells will be
further facilitated by the improved dispersion in the cytoplasm
of live cells versus QDs solubilized by a charge-driven mech-
anism, such as DHLA, mercapto acetic acid, or other mercapto
carbonic acids. These PEG-capped QDs have no obvious
cytotoxic effects on tissue culture cells observed over a two
day period. This should encourage their use for intracellular
labeling and long-term imaging. This method of QD solubili-
zation is an important step in creating all-purpose biocompatible
QDs.
51
cytoplasm of HeLa cells up to 2 h following reagent delivery.
Since poly(ethylene glycols) are capable of binding and fusing
5
2
lipid membranes, it is thus possible that the aggregates of
nanocrystals that appear upon longer incubation are due to
binding of the PEG-capped QDs to the membrane of the
intracellular organelles. However, from the present set of data,
we cannot unequivocally distinguish if the punctuates of
fluorescence are due to aggregation or to QDs binding to
membranes of the organelles present in the cytoplasm. In
agreement with the potential fusogenic role of PEG, we
occasionally observed that cells injected using a relatively high
concentration (>10 µM) of DHLA-PEG-capped QD solutions
fused together several minutes after loading (Figure 9E,F). When
monitored over a period of at least 2 days, the cells labeled
with DHLA-PEG-capped QDs were alive and showed no
obvious growth defects, compared to unlabeled cells. This
suggests that QDs prepared in this method could be useful for
long-term cellular imaging.
Acknowledgment. We thank K. Ward and A. Ervin at the
Office of Naval Research (ONR) for research support, Grant
N001404WX20270. We also thank DARPA for their financial
support. H.T.U. and I.L.M. acknowledge National Research
Council Fellowships through the Naval Research Laboratory.
J.K.J. and S.M.S. acknowledge the research grant from NSF
Conclusions
We have designed and synthesized a new series of organic
ligands that are capable of converting hydrophobic nanocrystals
to water-soluble QDs. This synthetic approach is based on a
simple modification of thioctic acid with various lengths of poly-
(BES-0119468 to S.M.S.).
(
ethylene glycols). Key features of the nanoparticles prepared
Supporting Information Available: Detailed synthesis of all
using these PEG-modified ligands include the ability to prepare
aqueous solutions of QDs that are aggregate-free and chemically
stable over long periods of time (several months). By using
the TA-PEG and DHLA-PEG ligands prepared along with the
corresponding NMR spectra. Chart of solubility for QDs capped
with DHLA-PEG ligands. This material is available free of
charge via the Internet at http://pubs.acs.org.
(
51) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. AdV. Mater. 2004, 16, 961-
66.
52) Lentz, B. R. Chem. Phys. Lipids 1994, 73, 91-106.
9
(
JA044031W
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