Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands

Article abstract of DOI:10.1021/ja0749744

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.

Full text of DOI:10.1021/ja0749744

Published on Web 10/23/2007
Enhancing the Stability and Biological Functionalities of
Quantum Dots via Compact Multifunctional Ligands
Kimihiro Susumu,† H. Tetsuo Uyeda,†,§ Igor L. Medintz,‡ Thomas Pons,†,#
James B. Delehanty,‡ and Hedi Mattoussi*,†
Contribution from the DiVision of Optical Sciences and Center for Bio/Molecular Science and
Engineering, U.S. NaVal Research Laboratory, Washington, D.C. 20375
Received July 5, 2007; E-mail: hedi.mattoussi@nrl.navy.mil
Abstract: We have designed and synthesized a series of modular ligands based on poly(ethylene glycol)
(PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum
dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a
dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD
surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the
PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the
native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and
over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging
showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized
on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling
in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-
PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands
described here provide not only stable and highly water-soluble QDs but also simple and easy access to
various biological entities.
Introduction
successful integration in biotechnology necessitates the prepara-
tion of water-soluble QDs that are highly luminescent, stable
Semiconductor nanocrystals (quantum dots, QDs) and metallic
nanoparticles have attracted a tremendous interest in the past
two decades. This interest has been driven by both the potential
for use in developing a variety of technological applications,
ranging from electronic devices to biotechnology, and a strong
desire to better understand the fundamental properties of such
materials.1-5 Luminescent QDs (such as those made of CdSe-
ZnS core-shell nanocrystals), in particular, have generated an
increasing interest for use in developing bio-oriented applica-
tions. This interest is motivated by the QDs’ remarkable
photochemical stability and several unique properties, including
(i) size-dependent broad absorption, (ii) very high extinction
coefficients, (iii) readily size-tunable narrow emission, and (iv)
high fluorescence quantum yield.1,2,6 Such features offer several
advantages for use in biological assays and imaging. Their
over a broad range of biological conditions, and compatible with
simple conjugation techniques. High-quality QDs (nanocrystals
having low size distribution and high fluorescence quantum
yields) are synthesized using high-temperature solution reaction
from organometallic precursors and are capped with hydropho-
bic organic ligands made of a mixture of trioctylphosphine/
trioctylphosphine oxide (TOP/TOPO) and alkyl amines.6-8
Alternative approaches using co-precipitation in aqueous solu-
tion (and even in olive oil) have been reported, but additional
work is still needed to bring these materials to par with those
made using high-temperature reactions.9,10 QDs capped with the
native ligands are not soluble in aqueous solutions, and thus
further surface modification is required to make them biocom-
patible. Various methods have been reported for achieving
water-solubility of such materials,3,4 including silica coating,11,12
encapsulation of the native TOP/TOPO-capped QDs within
† Division of Optical Sciences.
‡ Center for Bio/Molecular Science and Engineering.
§ Present address: Promega Biosciences, Inc., 277 Granada Dr., San Luis
Obispo, CA 93401.
(6) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993,
115, 8706-8715.
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Lett. 2001, 1, 207-211.
# Present address: Ecole de Physique et Chimie Industrielle (ESPCI),
Laboratoire Photons et Matie`re, CNRS UPRA0005, 10 Rue Vauquelin,
75005 Paris, France.
(8) Clapp, A. R.; Goldman, E. R.; Mattoussi, H. Nat. Protocols 2006, 1, 1258-
1267.
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V.; Kornowski, A.; Eychmu¨ller, A.; Weller, H. J. Phys. Chem. B 2002,
106, 7177-7185.
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2005, 4, 435-446.
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3395.
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9
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J. AM. CHEM. SOC. 2007, 129, 13987-13996
13987

A R T I C L E S
Susumu et al.
amphiphilic polymer shells13 or lipid micelles,14 and exchanging
the native caps with hydrophilic ligands.15-18 Among those
reported strategies, cap exchange with bifunctional ligands is
relatively simple and has a strong potential for providing QDs
that are small in size. Compactness of hydrophilic QDs has
distinct advantages in targeted studies, including the ability to
realize efficient fluorescence resonance energy transfer (FRET)-
based sensing with minimal QD-to-acceptor dye ratios19,20 and
use in cellular uptake and imaging.3,21
Scheme 1. Modular Design of Hydrophilic Ligands with Terminal
Functional Groups Used in This Study
We have previously used dihydrolipoic acid (DHLA) as
surface ligands for cap exchange to prepare water-soluble QDs.16
The DHLA-capped QDs are easily dispersed in basic buffer
solutions and are stable over extended periods of time (a year
and longer). The enhanced stability is attributed to the bidentate
chelate effect afforded by the dithiol groups at the end of the
ligand, as compared to monothiol-terminated ligands.3,16 How-
ever, while the DHLA-capped QDs are stable in basic buffers,
they tend to slowly aggregate under acidic conditions. This
feature is attributed to the fact that steric stabilization of these
nanoparticles hinges on the deprotonation of the terminal
carboxyl groups of DHLA. In order to overcome the pH
limitation of DHLA-capped QDs, design of new (bidentate and
eventually multidentate) ligands is consequently needed. We
have utilized readily available thioctic acid and poly(ethylene
glycol)s (PEG) in simple esterification schemes, followed by
reduction of the 1,2-dithiolane to produce multigram quantities
of PEG-terminated dihydrolipoic acid (DHLA-PEG) capping
substrates.22 Promoting dispersion of inorganic nanoparticles in
water using PEG has been reported.23-26 The cap exchange
reactions of TOP/TOPO-capped QDs with these substrates
produced water-soluble nanocrystals that are stable over ex-
tended periods of time and over a relatively broad pH range,
from weakly acidic to strongly basic conditions. Though
compact and stable, these ligands still have not allowed easy
implementation of common QD bioconjugation techniques, such
as avidin-biotin binding and covalent conjugation chemistry
(via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cou-
pling) due to lack of accessible functionalities at their lateral
end.
synthesize a series of DHLA-PEG ligands having a variety of
functional end groups (biotin, amino, and carboxyl groups), as
shown in Scheme 1. Appending functions such as biotin at the
end of a PEG chain permits the use of the ubiquitous avidin-
biotin binding, whereas availability of amino and carboxyl
groups will allow the use of covalent reaction schemes to attach
QDs to a variety of bioreceptors.27 In our design, the ligands
are modular, and each has a central (tunable length) PEG
segment to promote hydrophilicity, a bidentate terminal group
for strong anchoring on the QD surface at one end, and a lateral
functional group that promotes biological linkage with target
biomolecules at the other end. Cap exchange reactions were
carried out with these new ligands, and preliminary assays of
the water-soluble QDs were performed to verify the enhanced
biocompatibility.
Experimental Section
1. Materials. All manipulations were carried out under nitrogen
(previously passed through a drying tower filled with anhydrous
CaSO4), unless otherwise stated. Air-sensitive solids were handled in
a MBraun Labmaster 130 glovebox (Stratham, NH). Poly(ethylene
glycol)s (molecular weight average 400 and 600), triphenylphosphine,
thioctic acid, 4-(N,N-dimethylamino)pyridine, N,N′-dicyclohexylcar-
bodiimide, and N-hydroxysuccinimide were purchased from Acros
Organics (Morris Plains, NJ). Sodium azide and biotin were purchased
from Alfa Aesar (Ward Hill, MA). Methanesulfonyl chloride was
purchased from GFS Chemicals (Powell, OH). Succinic anhydride was
purchased from Fluka (Milwaukee, WI). Sodium borohydride was
purchased from Strem Chemicals (Newburyport, MA). 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccin-
imide (sulfo-NHS) were purchased from Pierce Biotechnology (Rock-
ford, IL) and used as received. Lissamine rhodamine B ethylenediamine
dye was purchased from Invitrogen (Carlsbad, CA) and used as
received. All the other chemicals (including solvents and phosphate-
buffered saline, 0.138 M NaCl, 0.0027 M KCl, PBS) were purchased
from Sigma Aldrich and Acros Organics. Tetrahydrofuran (THF) and
pyridine were dried over CaH2. Deuterated solvents employed in all
NMR measurements were used as received.
In this study, we further developed the use of PEG attachment
onto thioctic acid (followed by ring opening) to design and
(13) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat.
Biotechnol. 2004, 22, 969-976.
(14) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.;
Libchaber, A. Science 2002, 298, 1759-1762.
(15) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018.
(16) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V.
C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-
12150.
(17) Wang, Y. A.; Li, J. J.; Chen, H.; Peng, X. J. Am. Chem. Soc. 2002, 124,
2293-2298.
(18) Wang, Q.; Xu, Y.; Zhao, X.; Chang, Y.; Liu, Y.; Jiang, L.; Sharma, J.;
Seo, D.-K.; Yan, H. J. Am. Chem. Soc. 2007, 129, 6380-6381.
(19) Clapp, A. R.; Medintz, I. L.; Mauro, J. M.; Fisher, B. R.; Bawendi, M. G.;
Mattoussi, H. J. Am. Chem. Soc. 2004, 126, 301-310.
(20) Clapp, A. R.; Medintz, I. L.; Mattoussi, H. ChemPhysChem 2006, 7, 47-
57.
Chemical shifts for 1H NMR spectra are reported relative to
tetramethylsilane (TMS) signal in the deuterated solvent (TMS, δ )
0.00 ppm). All J values are reported in hertz. Column chromatography
was performed using silica gel (Bodman Industries, Aston, PA; 60 Å,
230-400 mesh). The molar amounts of PEG400 derivatives were
calculated by assuming that the repeated number of PEG chain (n on
the chemical structures) is ∼8.
(21) Delehanty, J. B.; Medintz, I. L.; Pons, T.; Brunel, F. M.; Dawson, P. E.;
Mattoussi, H. Bioconjugate Chem. 2006, 17, 920-927.
(22) Uyeda, H. T.; Medintz, I. L.; Jaiswal, J. K.; Simon, S. M.; Mattoussi, H.
J. Am. Chem. Soc. 2005, 127, 3870-3878.
(23) Hong, R.; Fischer, N. O.; Verma, A.; Goodman, C. M.; Emrick, T.; Rotello,
V. M. J. Am. Chem. Soc. 2004, 126, 739-743.
(24) Charvet, N.; Reiss, P.; Roget, A.; Dupuis, A.; Gru¨nwald, D.; Carayon, S.;
Chandezon, F.; Livache, T. J. Mater. Chem. 2004, 14, 2638-2642.
(25) Dixit, V.; Van den Bossche, J.; Sherman, D. M.; Thompson, D. H.; Andres,
R. P. Bioconjugate Chem. 2006, 17, 603-609.
(26) Choi, J.-s.; Jun, Y.-w.; Yeon, S.-I.; Kim, H. C.; Shin, J.-S.; Cheon, J. J.
Am. Chem. Soc. 2006, 128, 15982-15983.
(27) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego,
1996.
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Enhancing Stability and Biological Functionality of QDs
A R T I C L E S
1
2. Instrumentation. H NMR spectra were recorded on a Bruker
pyridine (0.31 g, 2.5 × 10-3 mol), and CH2Cl2 (100 mL) were placed
in a 250-mL round-bottom flask, cooled to 0 °C. N,N′-Dicyclohexyl-
carbodiimide (2.52 g, 1.2 × 10-2 mol) was slowly added under N2
with stirring. The reaction mixture was stirred at 0 °C for 2 h, warmed
to room temperature, and further stirred overnight. The mixture was
filtered through Celite and rinsed with ethyl acetate. The filtrate was
evaporated, and the residue was chromatographed on silica gel with
CHCl3 and then 10:1 CH2Cl2:MeOH as the eluents; 6.37 g of material
was obtained (a yield of ∼92%). 1H NMR (400 MHz, CDCl3): δ 6.42
(br s, 1H), 3.6-3.9 (m), 3.56 (t, 2H, J ) 5.0 Hz), 3.46 (t, 2H, J ) 5.0
Hz), 3.40 (t, 2H, J ) 5.2 Hz), 3.08-3.23 (m, 2H), 2.46 (m, 1H), 2.20
(t, 2H, J ) 7.4 Hz), 1.91 (m, 1H), 1.7 (m, 4H), 1.47 (m, 2H). IR
SpectroSpin 400 MHz spectrometer. Electronic absorption spectra were
recorded using an HP 8453 diode array spectrophotometer (Agilent
Technologies, Santa Clara, CA). Fluorescence spectra were collected
using a Spex Fluorolog-3 spectrophotometer (Jobin Yvon Inc, Edison,
NJ) equipped with a red-sensitive R2658 Hamamatsu PMT detector.
The obtained fluorescence spectra were corrected (to account for the
wavelength dependence of the detection efficiency of the PMT) using
the spectral output of a calibrated light source supplied by the National
Bureau of Standards. FT-IR spectra were measured using a Nicolet
Nexus 870 FT-IR spectrometer (Thermo Fisher Scientific, Inc.,
Waltham, MA).
(neat): 3328, 3075, 2918, 2867, 2105, 1670, 1540, 1456, 1119 cm-1
.
3. Synthesis and Design. DHLA (1) and DHLA-PEG600 (2).
These compounds were synthesized by reduction of 1,2-dithiolane
groups of thiotic acid (TA) and TA-PEG600, respectively, with NaBH4
as previously reported.22 Even though the synthesis of TA-PEG600
was detailed in ref 22, the modified purification procedure we recently
developed expanded that scheme and significantly simplified the column
chromatography step. Briefly, for workup after DCC coupling, the
precipitate was filtered and the solvent evaporated. The residue was
mixed with saturated sodium bicarbonate (NaHCO3) solution and
extracted with ethyl acetate (EtOAc) until complete extraction of TA-
PEG600 from the aqueous layer. Importantly, excess unreacted PEG600
mostly stays in the saturated NaHCO3 solution, and TA-PEG600 and
TA-PEG600-TA (bis-TA-substituted PEG) are extracted with EtOAc.
Such selective extraction of the crude product makes the following
column chromatography purification much easier compared with the
previous procedure.22 The combined organic layers were dried over
Na2SO4 and filtered, and the solvent was evaporated. The crude product
was chromatographed on silica gel with CHCl3:MeOH (15:1), and a
pale yellow oil was obtained.
TA-PEG400-NH2 (4). TA-PEG400-N3 (3.40 g, ∼5.4 × 10-3
mol) was dissolved in THF (60 mL) in a 250-mL round-bottom flask.
Triphenylphosphine (2.00 g, 7.6 × 10-3 mol) was added, and the
reaction mixture was stirred at room temperature for 2 h under N2.
After H2O (3.0 mL, 0.17 mol) was added, the reaction mixture was
further stirred at room temperature for 3 days under N2. The solvent
was evaporated, and the residue was chromatographed on silica gel
with 10:1 CH2Cl2:MeOH and then 100:20:1 CH2Cl2:MeOH:Et3N as
1
the eluents; 2.46 g of product was measured (a yield of ∼76%). H
NMR (400 MHz, CDCl3): δ 6.4-6.8 (m, 1H), 3.4-3.8 (m), 3.4-3.5
(m, 2H), 3.06-3.22 (m, 2H), 2.87-2.99 (m, 2H), 2.5-2.8 (br s, 2H),
2.42-2.51 (m, 1H), 2.17-2.27 (m, 2H), 1.86-1.96 (m, 1H), 1.59-
1.78 (m, 4H), 1.39-1.55 (m, 2H). IR (neat): 3298, 3053, 2866, 1666,
1545, 1456, 1109 cm-1
.
Biotinyl-N-hydroxysuccinimide. d-Biotin (5.00 g, 2.05 × 10-2 mol)
and N-hydroxysuccinimide (2.36 g, 2.05 × 10-2 mol) were dissolved
in hot DMF (150 mL) in a 500-mL round-bottom flask with stirring.
N,N′-Dicyclohexylcarbodiimide (5.50 g, 2.67 × 10-2 mol) was added,
and the solution was stirred overnight at room temperature, during which
time a white precipitate was formed. The reaction mixture was filtered,
and the filtrate was evaporated and triturated with ether. The white
precipitate was filtered and washed with ether to give a white powder.
The yield was 6.99 g (∼100%). 1H NMR (400 MHz, 1 drop of DMSO-
d6 in CDCl3): δ 5.23 (s, 1H), 4.96 (s, 1H), 4.52 (m, 1H), 4.33 (m,
1H), 3.16 (m, 1H), 2.87-2.97 (m, 1H), 2.86 (s, 4H), 2.75 (d, 1H, J )
12.8 Hz), 2.58-2.70 (m, 2H), 1.6-1.9 (m, 4H), 1.5-1.6 (m, 2H). IR
(neat): 3226, 2941, 2918, 2876, 1820, 1789, 1746, 1731, 1708, 1470,
N3-PEG400-N3. Poly(ethylene glycol) (average MW 400) (72.4
g, ∼0.175 mol), THF (200 mL), and methanesulfonyl chloride (46.1
g, 0.402 mol) were placed in a 1-L round-bottom flask and cooled to
∼0 °C. Triethylamine (60.0 mL, 0.430 mol) was added dropwise over
30 min. The reaction mixture was gradually warmed to room temper-
ature and stirred overnight. The mixture was then diluted with H2O
(200 mL) and NaHCO3 (12.5 g, 0.149 mol). Sodium azide (31.0 g,
0.477 mol) was added, and the biphasic reaction mixture was heated
to distill off the THF and then refluxed overnight. After cooling, the
reaction mixture was extracted five times with CHCl3. The combined
organic layers were dried over MgSO4, filtered, and evaporated to give
a pale brown oil. The crude product was chromatographed on silica
gel with 20:1 CHCl3:MeOH as the eluent; the reaction yielded 73.13 g
(∼90%). 1H NMR (400 MHz, CDCl3): δ 3.62-3.71 (m), 3.40 (t, 4H,
1370, 1217, 1072 cm-1
.
TA-PEG400-Biotin (5). To a mixture of TA-PEG400-NH2 (0.22
g, 3.7 × 10-4 mol), biotinyl-N-hydroxysuccinimide (0.122 g, 3.6 ×
10-4 mol), and DMF (8.0 mL) was added triethylamine (0.24 mL, 1.7
× 10-3 mol) dropwise, and the reaction mixture was stirred at room
temperature for 18 h under N2. The reaction mixture was filtered, and
DMF was removed in vacuo. The residue was chromatographed on
silica gel with 8:1 CH2Cl2:MeOH as the eluent, to yield 0.264 g
J ) 5.0 Hz). IR (neat): 2868, 2103, 1453, 1115 cm-1
.
N3-PEG400-NH2. N3-PEG400-N3 (32.02 g, ∼6.89 × 10-2 mol)
and 0.7 M H3PO4 (150 mL, 0.105 mol) were placed in a 500-mL round-
bottom flask and cooled to 0 °C in an ice-bath with stirring. A solution
of triphenylphosphine (18.18 g, 6.93 × 10-2 mol) in ether (150 mL)
was added dropwise under N2 while temperature was maintained below
5 °C. The reaction mixture was then warmed to room temperature and
stirred under N2 for 20 h. The biphasic solution was separated, and the
aqueous layer was washed with ether (3 × 100 mL). KOH (30.0 g,
0.535 mol) was slowly added to the aqueous layer, which was cooled
to 0 °C with stirring overnight. After filtration, the filtrate was basified
with an additional 30.0 g of KOH and cooled using an ice-bath. The
mixture was extracted with CHCl3 (4 × 100 mL), the combined organic
layers were dried over MgSO4 and filtered, and the solvent was
evaporated. The crude product was chromatographed on silica gel with
5:1 CH2Cl2:MeOH as the eluent; 15.51 g of product was obtained (a
yield of ∼51%). 1H NMR (400 MHz, CDCl3): δ 3.6-3.9 (m), 3.52 (t,
2H, J ) 5.2 Hz), 3.40 (t, 2H, J ) 4.8 Hz), 2.87 (t, 2H, J ) 5.0 Hz).
1
(∼89%). H NMR (400 MHz, CDCl3): δ 6.85-7.05 (m, 1H), 6.4-
6.8 (m, 1H), 6.2-6.4 (m, 1H), 5.3-5.5 (m, 1H), 4.51 (m, 1H), 4.33
(m, 1H), 3.5-3.9 (m), 3.4-3.5 (m, 2H), 3.08-3.23 (m, 3H), 2.88-
2.96 (m, 1H), 2.75 (d, 1H, J ) 12.8 Hz), 2.42-2.51 (m, 1H), 2.16-
2.3 (m, 4H), 1.87-1.96 (m, 1H), 1.6-1.8 (m, 8H), 1.4-1.6 (m, 4H).
IR (neat): 3295, 3083, 2928, 2865, 1707, 1645, 1550, 1461, 1108 cm-1
.
DHLA-PEG400-Biotin (6). To a solution of TA-PEG400-Biotin
(0.56 g, ∼6.77 × 10-4 mol), EtOH (12 mL), and H2O (6.0 mL) was
added NaBH4 (0.132 g, 3.5 × 10-3 mol) in small portions, and the
reaction mixture was stirred at room temperature for 30 min under N2.
The reaction mixture was diluted with H2O and extracted with CHCl3
(four times). The combined organic layers were dried over MgSO4 and
filtered, and the solvent was evaporated. The residue was chromato-
graphed on silica gel with 10:1 CH2Cl2:MeOH as the eluent; 0.45 g of
product was collected (a yield of ∼80%). 1H NMR (400 MHz,
CDCl3): δ 6.6-6.8 (m, 1H), 6.3-6.6 (m, 1H), 5.52-5.7 (m, 1H), 4.8-
4.9 (m, 1H), 4.51 (m, 1H), 4.33 (m, 1H), 3.6-3.7 (m), 3.56 (t, 4H, J
) 4.6 Hz), 3.45 (m, 4H), 3.16 (m, 1H), 2.87-2.97 (m, 2H), 2.62-
IR (neat): 3379, 2868, 2107, 1598, 1455, 1119 cm-1
.
TA-PEG400-N3 (3). Thioctic acid (2.48 g, 1.2 × 10-2 mol), N3-
PEG400-NH2 (5.0 g, ∼1.1 × 10-2 mol), 4-(N,N-dimethylamino)-
9
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A R T I C L E S
Susumu et al.
2.79 (m, 3H), 2.16-2.28 (m, 4H), 1.86-1.96 (m, 1H), 1.4-1.8 (m,
13H), 1.36 (t, 1H, J ) 8.0 Hz), 1.32 (t, 1H, J ) 7.6 Hz). IR (neat):
3296, 3084, 2928, 2865, 2549, 1704, 1688, 1645, 1549, 1461, 1108
value measured, and data were reported as percentage binding of biotin-
coated QDs onto NeutrAvidin-functionalized wells.
5. Cellular Uptake of Quantum Dot-Bioconjugates and Imaging.
QDs emitting at 540 nm and capped with a mixture of DHLA-PEG600:
DHLA-PEG400-biotin (9:1 molar ratio) were utilized for these
experiments. One-to-one b-phycoerythrin-streptavidin conjugates (b-
PE, 1 mg/mL), Alexa Fluor 647 dye-labeled transferrin (AF647-TF),
and DAPI nuclear stain were obtained from Invitrogen (Carlsbad, CA).
COS-1 cell lines (ATCC, Manassas, VA) were cultured as previously
described.21 Cellular internalization experiments were performed in
sterile Lab-Tek chambered coverglass wells (Nunc, Rochester, NY).
Chambers were coated with 50 µg/mL fibronectin (Sigma-Aldrich) in
sodium bicarbonate buffer pH 8.5, and approximately 2 × 104 cells
were seeded into the wells and cultured overnight.
cm-1
.
TA-PEG400-COOH (7). A solution of TA-PEG400-NH2 (1.102
g, ∼1.83 × 10-3 mol) and succinic anhydride (0.313 g, 3.1 × 10-3
mol) in 15 mL of dry pyridine was stirred at room temperature for 26
h under N2. After the solvent was removed in vacuo, the residue was
chromatographed on silica gel with 10:1 CHCl3:MeOH as the eluent;
the reaction provided 0.718 g (a yield of ∼56%) of product. 1H NMR
(400 MHz, CDCl3): δ 6.78-6.98 (br s, 1H), 6.20-6.52 (m, 1H), 3.60-
3.74 (m), 3.52-3.60 (m, 5H), 3.45 (m, 4H), 3.08-3.22 (m, 2H), 2.67
(m, 2H), 2.55 (m, 2H), 2.42-2.51 (m, 1H), 2.21 (m, 2H), 1.86-1.96
(m, 1H), 1.60-1.78 (m, 4H), 1.39-1.55 (m, 2H). IR (neat): 3319,
3084, 2922, 2870, 1732, 1633, 1549, 1454, 1109 cm-1
.
The mixed-surface QD-b-PE conjugates used consist of QDs
assembled with b-PE (at 1:1 ratio on average) together with cell-
penetrating peptides (CPP) added at a ratio of 25 peptides per QD.
The CPP sequence (His8WGLA(Aib)SGR8, where Aib is R-ami-
noisobutyric acid) was appended with a terminal polyhistidine (His8)
tag to facilitate self-assembly on the QDs.21 QD-b-PE-CPP biocon-
jugates were diluted to 75 nM concentration in complete culture medium
containing the cells and incubated at 37 °C for 1 h as previously
described.21 AF647-TF endosome marker (at 40 µg/mL) was also added
during incubation. Excess unbound QD conjugates were removed by
washing with PBS. Cells were then fixed in 3.7% paraformaldehyde
at room temperature for 10 min, washed twice with PBS, and mounted
in ProLong Antifade mounting media containing DAPI dye to allow
for staining of the cell nuclei.21 Cultures were imaged using a total
internal reflection fluorescence microscope (Olympus IX-71), and when
necessary, split fluorescence images were collected and quantitated with
a DualView system (Optical Insights, Tucson, AZ) equipped with a
565-nm dichroic filter. QDs and b-PE were excited at 488 nm, separated
with the 565 nm dichroic filter, and then deconvoluted. DAPI
fluorescence was detected using a Xe lamp and a DAPI cube (D350/
50x for excitation, dichroic 400DCLP, D460/50m for detection). Finally,
AF647-TF was excited using the Xe lamp and fluorescence detected
using a Cy5 cube (excitation HQ620/60x, dichroic Q660LP, HQ700/
75m for detection). Cubes were purchased from Chroma Technology
(Rockingham, VT). Differential interference contrast (DIC) images were
collected using a bright light source.
6. EDC Coupling. QDs capped with a DHLA-PEG600:DHLA-
PEG400-COOH (19:1) mixture (1 nmol), Lissamine rhodamine B
ethylenediamine (15 nmol), sulfo-NHS (2.5 µmol), and different
amounts of EDC (0, 5, 20, and 50 µmol) were mixed with 0.1 M PBS
solution (pH ∼6.5) in Eppendorf tubes (0.5 mL in total), and each
reaction mixture was shaken for 2 h at room temperature. The reaction
mixture was loaded on a pre-equilibrated disposable PD-10 desalting
gel column (GE Healthcare, Piscataway, NJ) and eluted with the same
PBS solution to remove excess unreacted dyes and reagents. Fractions
of eluted solution were collected (∼1 mL each), and the absorption
spectrum was measured for each. These solutions/fractions were also
loaded onto a microtiter well plate, and the fluorescence spectra were
collected using a Tecan Safire plate reader (Tecan US, Research
Triangle Park, NC).
DHLA-PEG400-COOH (8). A solution of TA-PEG400-COOH
(0.502 g, ∼7.16 × 10-4 mol) in MeOH (10 mL) was cooled in an
ice-bath, and NaBH4 (0.132 g, 3.5 × 10-3 mol) in 4.0 mL of H2O was
added dropwise with stirring. The reaction mixture was gradually
warmed to room temperature and stirred for 18 h under N2. A 1 M
HCl solution was added until the reaction mixture reached pH ∼2,
and the reaction mixture was extracted with CHCl3 (four times). The
combined organic layers were dried over Na2SO4, filtered, and
evaporated. The residue was chromatographed on silica gel with 8:1
CHCl3:MeOH as the eluent; 0.38 g (a yield of ∼75%) of pure compound
1
8 was collected. H NMR (400 MHz, CDCl3): δ 6.8-7.0 (br s, 1H),
6.2-6.5 (m, 1H), 3.60-3.72 (m), 3.56 (t, 4H, J ) 4.8 Hz), 3.45 (m,
4H), 2.87-2.97 (m, 1H), 2.61-2.79 (m, 4H), 2.55 (m, 2H), 2.21 (m,
2H), 1.86-1.96 (m, 1H), 1.40-1.80 (m, 7H), 1.36 (t, 1H, J ) 8.0
Hz), 1.31 (d, 1H, J ) 7.6 Hz). IR (neat): 3319, 3082, 2924, 2854,
2553, 1734, 1649, 1547, 1462, 1111 cm-1
.
3. Quantum Dot Synthesis and Cap Exchange. CdSe-ZnS core-
shell QDs were synthesized using high-temperature reaction of orga-
nometallic precursors (e.g., trioctylphosphine selenium (TOP:Se) and
cadmium acetylacetonate) in a mixture of TOP/TOPO and hexadecy-
lamine, as described in the literature.6,28-30 The QDs are capped
primarily with TOP/TOPO ligands and, therefore, are not soluble in
aqueous media. Cap exchange of the TOP/TOPO-capped QDs with
the newly synthesized ligands was carried out following the procedures
described previously.22 Briefly, ∼50-300 mg of TOP/TOPO-capped
QDs were precipitated using EtOH or a mixture of methanol/butanol.
After centrifugation, the supernatant was discarded. To the precipitate
were added ∼0.5 mL in total of pure or mixed ligands and ∼0.5 mL
of EtOH. The mixture was then heated to 60-80 °C with stirring for
several hours. Once homogenized, the sample was then precipitated
out with hexane, EtOH, and CHCl3 mixtures and centrifuged. The
supernatant was discarded, and the precipitate was redispersed in water.
A few additional cleaning steps using an ultracentrifugal device were
used to remove excess ligands for the final dispersion. Concentrations
of QD solutions were estimated from size-dependent absorption cross
sections as previously described.31
4. Surface Binding Assay. The binding capacity of NeutrAvidin-
covered 96-well microtiter flat-bottom plates (Pierce Biotechnology)
is 60 pmol of biotin per well. A total of 100 pmol of QDs capped with
the desired ligand mixtures in 100 µL solutions were added to the well
and incubated overnight. After the initial fluorescence signal was
measured, the plates were washed three times with PBS buffer solution,
and the fluorescence was measured again. The remaining fluorescence
intensities following rinsing were compared (normalized) to the highest
Results and Discussion
Synthesis and Characterization. The chemical structures
and synthetic schemes of a series of DHLA and DHLA-PEG
capping substrates are summarized in Figure 1. The synthesis
of our modular DHLA-PEG-FN ligands (where FN designates
the terminal function of interest) was carried out following a
stepwise approach. Poly(ethylene glycol) of the desired length
(in our case here PEG400) was first transformed into diazide-
terminated PEG using a two-step reaction with methanesulfonyl
chloride and sodium azide. Monosubstitution of one azide into
(28) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468-471.
(29) Dabbousi, B. O.; Rodriguez-Viejo, J.; Mikulec, F. V.; Heine, J. R.;
Mattoussi, H.; Ober, R.; Jensen, K. F.; Bawendi, M. G. J. Phys. Chem. B
1997, 101, 9463-9475.
(30) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183-184.
(31) Leatherdale, C. A.; Woo, W.-K.; Mikulec, F. V.; Bawendi, M. G. J. Phys.
Chem. B 2002, 106, 7619-7622.
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Figure 1. Chemical structures and synthetic routes of the surface ligands used in this study: (a) DCC, DMAP, CH2Cl2; (b) PPh3, H2O, THF; (c) biotin
N-hydroxysuccinimide ester, Et3N, DMF; (d) NaBH4, EtOH, H2O; (e) succinic anhydride, pyridine; (f) NaBH4, EtOH, H2O.
an amine group in biphasic acidic solution32 was followed by
coupling to thioctic acid to provide azide-terminated TA-
PEG400 (compound 3). An additional modification of the azide
group into amine provided reactive amine-terminated TA-
PEG400 (compound 4), to which additional functions (including
biotin and carboxyl) can be attached. Biotin-terminated TA-
PEG (compound 5) was synthesized by coupling between 4 and
biotin N-hydroxysuccinimide ester. Alternatively, coupling
between 4 and succinic anhydride in pyridine provided the
carboxyl-terminated ligand (compound 7). As prepared, the TA-
terminated ligands do not interact with the QD surfaces and
thus cannot be used in cap exchange with TOP/TOPO-capped
QDs. A necessary transformation of the above TA-PEG-FN
into DHLA-PEG-FN can be carried out using simple ring-
opening of the terminal 1,2-dithiolane ring in the presence of
sodium borohydride (NaBH4). We limited our description to
using PEG with MW ∼400 (PEG400), but the method described
can be applied to shorter or longer segments. All the reaction
steps had relatively high yields, and each compound was simply
purified with silica gel column chromatography. All the
compounds were characterized by thin layer chromatography
(TLC) and 1H NMR, and when necessary, the new ligands were
further characterized by FT-IR.
Figure 2 shows 1H NMR spectra of five representative
compounds used in this study. The spectra of each PEG
derivative were basically superimposed of each component. All
these PEG compounds have large peaks around 3.6-3.7 ppm,
which are ascribed to CH2 groups of the repeated PEG chains.
For TA-PEG-NH2 (compound 4), most of the multiplet peaks
between 1.4 and 3.5 ppm are from thioctic acid. After coupling
between 4 and biotin N-hydroxysuccinimide ester, new distinct
multiplet peaks (at 2.75, ∼2.9, 4.33, and 4.51 ppm) appeared
in the NMR spectrum of 5, which were ascribed to biotin ring
protons. In addition, following reduction of the 1,2-dithiolane
group with NaBH4, new doublet and triplet peaks appeared at
1.32 and 1.36 ppm, respectively. These two peaks were assigned
as thiol protons of the DHLA unit (open dithiol).22 Though easily
reproducible for DHLA and DHLA-PEG molecules, the
successful ring-opening reaction in the presence of additional
end functions (e.g., biotin) was crucial to the use of these ligands
with QDs.
Cap Exchange. Water-soluble QDs functionalized with one
type or a mixture of the synthesized ligands was prepared via
cap exchange with the native caps (TOP/TOPO). QDs capped
with any of the DHLA-PEG400, DHLA-PEG600, DHLA-
PEG1000 ligands were dispersed well in aqueous buffers with
pH ranging between 5 and 10.22 When using the biotin- and
COOH-terminated ligands, cap exchange was carried out using
a mixture with either DHLA (1) or DHLA-PEG600 (2). This
is done to ensure that a controlled density of biotin or COOH
groups is available on each QD, which allows manipulation of
the reactivity of the QD and eventually control of the number
of biological receptors coupled to its surface. In this particular
study, we used a mixture of compounds 1 and 6 (4:1), mixtures
of compounds 2 and 6 (4:1 and 9:1), and a mixture of
compounds 2 and 8 (19:1) for the cap exchange. Ligand
mixtures are designated by their molar ratios. As expected, QDs
cap-exchanged with the new ligands were stable and aggregate-
free over extended periods of time (months) and over a relatively
broad pH range. Figure 3 shows solutions of QDs capped with
a mixture of compounds 2 and 8 (19:1) in water over the pH
range 5-11. The new water-soluble QDs with carboxyl groups
were stable and well-dispersed in a wide pH range, including
acidic conditions. This feature is quite different from what we
have observed for solutions of QDs capped with either DHLA
or mercaptoundecanoic acid (MUA). The DHLA-capped QD
solutions were well-dispersed in aqueous basic media but
showed progressive aggregation and eventual precipitation at
pH lower than 7 (Figure 3), indicating that deprotonation of
carboxyl groups is a crucial factor to solubilize those QDs in
aqueous solutions.3 In comparison, MUA-capped QDs were also
stable in basic buffer solution but only for a limited time (less
than one week), then aggregates built up in the solution (Figure
3B and Supporting Information); at pH < 7, aggregation
occurred within a few hours of sample preparation. The PEG
groups play a critical role in extending solubilization of QDs
having carboxyl-terminal groups over a broad pH range. The
stability of the QDs with PEG ligands even in acidic conditions
has a significant impact for aqueous conjugation chemistry and
(32) Schwabacher, A. W.; Lane, J. W.; Schiesher, M. W.; Leigh, K. M.; Johnson,
C. W. J. Org. Chem. 1998, 63, 1727-1729.
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Figure 2. 1H NMR spectra of compounds 4 (TA-PEG400-amine), 5 (TA-PEG400-biotin), and 6 (DHLA-PEG400-biotin) measured in CDCl3. The
spectra of thioctic acid and DHLA (1) are shown for comparison. The large peak at ∼1.75 ppm in the spectrum of 6 is attributed to water.
Figure 3. Luminescence image for a set of QD solutions in phosphate-buffered saline at various pH values. CdSe-ZnS samples emitting at 540 nm were
used and were excited with a hand-held UV lamp at 365 nm; the image was collected at room temperature. (A) QD with DHLA-PEG600:DHLA-
PEG400-COOH (19:1 molar ratio) at pH 5-11. (B) QD with DHLA at pH 6 and 9, and QD with MUA at pH 6 and 9. Images were collected 2 days after
sample preparation.
cell imaging studies. The PEG modules (PEG400 and 600) we
used to ensure water solubility of the QDs are relatively short.
Such compact size of surface ligands on QD is quite useful not
only for ensuring detectable interaction between QD and target
entities but also for efficient cell delivery and the following
imaging studies.3,21
FT-IR spectra were measured for QDs after cap exchange
reactions (using ligand mixtures) to verify that both ligands were
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Enhancing Stability and Biological Functionality of QDs
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Figure 5. Absorption and fluorescence (inset) spectra of QD with TOP/
TOPO ligands in toluene (black line) and QD with a mixture of DHLA-
PEG600 (2):DHLA-PEG400-biotin (6) (4:1 molar ratio) in H2O (broken
red line). λexc ) 400 nm.
Figure 4. FT-IR spectra of QDs with different surface ligands: (A)
DHLA-PEG600; (B) DHLA-PEG600:DHLA-PEG400-biotin (4:1 molar
ratio); (C) DHLA-PEG600:DHLA-PEG400-COOH (19:1 molar ratio).
(I) and (II) can be assigned as amide I and amide II bands, respectively.
indeed attached to the QD surface after cap exchange (Figure
4). Furthermore, data were collected using QDs having rather
low fractions of COOH- or biotin-terminated ligands, because
these are the samples that will eventually be employed in
designing practical assays; spectra for the pure ligands are
provided in the Supporting Information. The entire features of
DHLA-PEG600-capped QDs were essentially the same as the
spectrum of free DHLA-PEG600 ligand. The sharp peak at
1734 cm-1 was ascribed as the CdO stretch mode of the ester
group. The FT-IR spectra of QDs capped with DHLA-PEG600:
DHLA-PEG400-biotin (4:1) and QDs capped with DHLA-
PEG600:DHLA-PEG400-COOH (19:1) also have spectral
features similar to those of DHLA-PEG600-capped QDs. This
is not surprising since those QDs with mixed ligands still have
DHLA-PEG600 as the major surface ligand. However, Figure
4 also shows two additional distinct bands between 1500 and
1700 cm-1 (1666 and 1541 cm-1 for QD with DHLA-PEG600:
DHLA-PEG400-biotin (4:1), and 1666 and 1564 cm-1 for QD
with DHLA-PEG600:DHLA-PEG400-COOH (19:1)). Those
two bands can be assigned to amide I (CdO stretch) and amide
II (N-H bending) bands in ligands 6 and 8, respectively, and
are exclusively from either DHLA-PEG400-biotin or DHLA-
PEG400-COOH; DHLA-PEG600 does not have an amide
bond. The present FT-IR data qualitatively indicate that both
ligands are present on the QD surface after cap exchange with
a mixture of two ligands.
Figure 6. Surface binding assays of biotin-modified QDs to NeutrAvidin.
(Top) Schematic representation of the binding assay used; m and n designate
the number of ethylene oxide repeating units. (Bottom) Binding (%) of
QDs with different surface ligands: (A) DHLA (1), (B) DHLA-PEG600
(2), (C) 1:DHLA-PEG400-biotin (6) (4:1 molar ratio), and (D) 2:6 (4:1
molar ratio).
were essentially unchanged, though occasionally a few nanom-
eters red-shift of the lowest absorption maximum of the
hydrophilic QDs was measured compared with that of QDs
capped with TOP/TOPO ligands. The fluorescence spectra of
those QD solutions also showed similar trends (see Figure 5,
inset). These optical features indicate that cap exchange with
Optical Properties. Absorption and fluorescence spectra were
measured for QDs capped with the original TOP/TOPO ligands
and dispersed in toluene and for the hydrophilic QDs capped
with the new DHLA-PEG derivatives in H2O (Figure 5).
Absorption spectra measured before and after the cap exchange
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Figure 7. Cellular uptake of biotinylated 540-nm QD-b-phycoerythrin bioconjugates. (A) Schematic representation of the QD bioconjugate. Each QD is
conjugated to ∼1 b-PE-streptavidin and ∼25 CPPs on average. (B) Representative images of COS-1 cells incubated with ∼75 nM QD-CPPs (top), 75 nM
QD-b-PE (middle), and ∼75 nM QD-CPP-b-PE (bottom); QDs were capped with DHLA-PEG600:DHLA-PEG400-biotin (9:1). For each series, the
corresponding DIC, 540-nm QD, b-PE, AF647-TF, and DAPI fluorescence images are shown. Excitation and emission selections are described in the
Experimental Section.
hydrophilic ligands essentially has no effect on the spectroscopic
properties of the inorganic QD cores. The relative fluorescence
quantum yields measured for the water-soluble QDs were ∼50%
as compared to the native TOP/TOPO-capped QDs. Decrease
of the fluorescence quantum yields of QDs following transfer
into aqueous solutions has been commonly observed.11,22,33
The quantum yield values for our materials ranged from
10 to 30% for QDs capped with either pure or mixed surface
ligands.22
the QDs with the newly functionalized QDs. For example, the
binding properties of QDs capped with a mixture of DHLA-
PEG600 (2) and biotin-terminated DHLA-PEG400 (6) were
investigated for their ability to bind with NeutrAvidin-func-
tionalized substrates. Avidin (or streptavidin)-biotin binding
has one of the highest noncovalent affinities known (KD ≈ 1015
M-1) and has been widely used in a variety of biological
targeting studies.27 NeutrAvidin, which is a deglycosylated form
of avidin, has low nonspecific binding properties compared with
avidin or streptavidin, while it maintains the strong binding
affinity with biotin. Following incubation of the NeutrAvidin-
functionalized plate wells with the QD solutions, the wells were
rinsed three times with PBS buffer, and the fluorescence signals
were collected. Figure 6 shows the fluorescence intensities
Surface Binding Assay. Once cap exchange and dispersion
into aqueous solutions was achieved, we further carried out
targeted biological assays to demonstrate biocompatibility of
(33) Pinaud, F.; King, D.; Moore, H.-P.; Weiss, S. J. Am. Chem. Soc. 2004,
126, 6115-6123.
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Figure 8. (A) Luminescence image for solution fractions of 540-nm emitting QDs reacted with the dye after gel filtration: (i) no EDC, (ii) 0.01 M EDC,
(iii) 0.04 M EDC, (iv) 0.1 M EDC, and (v) Lissamine rhodamine B ethylenediamine only. Samples do not necessarily have the same QD and dye concentrations.
The low signal for sample (v) is due to poor excitation of the dye at 365 nm (provided by a UV lamp). (B) Absorption spectrum of a sample (with 0.1 M
EDC used) after gel filtration (blue line), together with those of pure QDs (green line) and dye in H2O (red line). (C) Composite emission spectrum of a
sample (with 0.1 M EDC used) after gel filtration (blue dot/line), together with the deconvoluted contributions of the QDs (green line) and dye (red line).
Fluorescence spectra were generated using 300 nm excitation.
measured for four different QD solutions: one capped with
DHLA (1), one capped with DHLA-PEG600 (2), one capped
with a mixture of 1 and DHLA-PEG400-biotin (6) (4:1), and
one capped with a mixture of 2 and 6 (4:1).
described,21 the QD-b-PE conjugates were also self-assembled
with cell-penetrating peptides (∼25 CPPs per QD) to mediate
cellular uptake (see Figure 7A). COS-1 cell cultures were
separately incubated with 540-nm QD-CPP, QD-b-PE, and
QD-CPP-b-PE assemblies. Each row of panels in Figure 7B
shows four representative images, consisting each of 540-nm
QDs, b-PE, AF647-transferrin, and DAPI emissions for the three
nanoassemblies used. In addition, DIC images of the cell cultures
are provided. The brightness of the b-PE is apparent in the
micrographs, even though a low ratio of 1 b-PE per QD was
used. We should note that, due to a slight leakage of the b-PE
emission into the QD channel, the nanocrystal contribution was
“corrected” using intensity ratios between the two channels
measured for b-PE alone (side-by-side fluorescence spectra of
QDs and b-PE are provided in the Supporting Information).
However, such correction is only partial, since the QD signal
is likely reduced while that of the protein is enhanced, due to
FRET interactions between QD and b-PE in the conjugate. The
patterns of staining from the QD, b-PE, and AF647-TF channels
can all be superimposed, confirming that the b-PE remains
attached to the QDs and essentially localized within endosomes.
It is also worth noting that attachment of the b-PE on the QD
surface did not preclude self-assembly of the smaller CPP nor
its availability to mediate cellular delivery.
The data clearly show that only wells incubated with QDs
capped with a mixture of biotin-terminated DHLA-PEG
produced large signal-to-background fluorescence ratios. This
result indicates that efficient and specific interactions between
the biotin groups on the QD surface and NeutrAvidin on the
substrate drive the surface capture of the QDs; control samples
using DHLA or DHLA-PEG600-capped QDs showed negli-
gible interactions with NeutrAvidin (see Figure 6). Also,
comparison of biotin-functionalized QDs with different hydro-
philic ligands (1:6 (4:1) and 2:6 (4:1)) indicates that long PEG
chains surrounding biotin ligand did not disrupt efficient binding
between biotin and NeutrAvidin. This promising preliminary
result was further complemented by cell-labeling assays.
Cellular Uptake of Quantum Dot-b-Phycoerythrin Con-
jugates. The ability of QDs functionalized with a mixture of
DHLA-PEG600:DHLA-PEG400-biotin and CPP to mediate
cellular uptake and delivery of specific protein-attached cargoes
was also investigated. QDs emitting at 540 nm, capped with a
DHLA-PEG600:DHLA-PEG400-biotin (9:1) mixture, were
exposed to monofunctionalized streptavidin-b-phycoerythrin (b-
PE) at a 1:1 molar ratio. This multisubunit phycobilin light-
harvesting protein has a molecular weight of ∼240 000 and ∼34
separate chromophores (open-chain tetrapyrroles) within its
structure, and its intense brightness has led to its use as a
fluorescent marker.34,35 Similar to what has been previously
EDC Coupling. In order to demonstrate easy access to
biochemical conjugation, the QDs with DHLA-PEG-COOH
ligands were tested for coupling to amine-terminated dyes using
EDC, which is commonly used for coupling of water-soluble
dyes to an array of biological molecules including peptides,
proteins, and antibodies by targeting amine and/or carboxyl
groups. In this study, QDs capped with DHLA-PEG600:
(34) Glazer, A. N. Methods Enzymol. 1988, 167, 291-303.
(35) Glazer, A. N. J. Appl. Phycol. 1994, 6, 105-112.
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DHLA-PEG400-COOH (19:1) were reacted with Lissamine
rhodamine B ethylenediamine in the presence of EDC and sulfo-
NHS in PBS buffer solution. After the reaction was complete,
excess unreacted dyes and reagents were removed by size
exclusion gel filtration.
can promote the compatibility of luminescent QDs with buffer
solutions and coupling to biomolecules. These ligands are
modular and consist of a short poly(ethylene glycol) (PEG)
segment, to which we attached an anchoring DHLA group on
one end to drive binding to the QD and a variety of biologically
reactive/relevant groups at the other end for attaching biore-
ceptors. Cap exchange with these hydrophilic ligands provided
QDs that are water-soluble over extended periods of time and
over a relatively broad pH range. Inserting a PEG segment
within the ligand allows us to attach a variety of functional
groups on the QDs without compromising water-solubility.
Furthermore, we find that the use of a mixed-ligand strategy,
achieved by simply varying the fraction of DHLA-PEG ligand
used in the mixture during cap exchange, is very effective in
fine-tuning the functionality of the QD surface. For example,
mixed-cap biotin-functionalized QDs showed strong and specific
interactions with NeutrAvidin in surface binding assays as well
as inside live cells. We also implemented EDC coupling to
attach COOH-functionalized QDs to amine-functionalized dyes.
This ubiquitous covalent coupling technique opens up easy
access to a variety of biological entities and offers additional
options for biological assay design. Further studies of these
surface-functionalized QDs and coupling with a variety of
biomolecules and their use in developing biological assays are
in progress.
No precipitation or aggregation buildup was observed either
during or after the reaction was complete. Absorption spectra
measured from the eluted solutions indicate that all the first
major fractions collected off the gel filtration column included
QDs. There is also a distinct change in the solution fluorescence
for reactions carried out in the presence of EDC (Figure 8A).
Both absorption and emission spectra collected from the
fractions containing QDs showed significant contributions (new
bands) at 571 nm for the absorption and 592 nm for the
fluorescence, identical to the absorption and fluorescence
maxima of the rhodamine dye alone (Figure 8B,C). In com-
parison, for the reaction without EDC (control experiment), the
first major fractions collected from the gel column contained
only unreacted QDs. Since the rhodamine dye has a very small
absorption at the excitation wavelength we used (at 300 nm),
we attribute the fluorescence contribution from the rhodamine
dye primarily to energy transfer from the bound and well-excited
QDs.18,19 Though the reaction conditions were not fully
optimized, the present result is promising and proves that we
can use our ligand design to covalently attach a variety of
biological entities and organic molecules to the QD surface in
aqueous media. Further optimization of the EDC couplings with
a variety of target molecules is one of our future goals. This
result contrasts with the macroscopic aggregation commonly
encountered when using QDs capped with DHLA or other short-
chain thiol-alkyl-COOH ligands.16 For these materials, the
EDC coupling involves reduction of the pH to ∼6 and activation
of the carboxyl groups, which in combination reduces the QDs
stability in water and often results in aggregate formation, though
fluorescence of the nanocrystals is usually preserved.
Acknowledgment. The authors acknowledge NRL, Office of
Naval Research, and the Army Research Office for financial
support. We thank Drs. Horn-Bond Lin and Charles D. Merritt
at NRL for assistance with the collection of the FTIR spectra
and the fluorescence images. We also acknowledge Prof. Philip
E. Dawson and his group (at the Scripps Research Institute, La
Jolla, CA) for assistance with the cell penetrating peptides used.
Supporting Information Available: Luminescence images of
solutions of DHLA- and MUA-capped green-emitting QDs, FT-
IR spectra of free ligands, and photoemission spectra of the
QDs and b-PE used in cell imaging. This material is available
free of charge via the Internet at http://pubs.acs.org.
Conclusions
In this study, we have successfully demonstrated a simple
and efficient synthetic procedure to prepare a series of new
capping ligands appended with terminal functional groups that
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13996 J. AM. CHEM. SOC. VOL. 129, NO. 45, 2007