Strong polyelectrolyte quantum dot surface for stable bioconjugation and layer-by-layer assembly applications

Article abstract of DOI:10.1039/c0cc04524a

A series of quantum dot (QD) ligands are reported that can make strong polyelectrolyte QD surfaces with sulfonates or quaternary ammoniums, which can endow QDs with excellent colloidal stability independent of the pH and ionic strength, minimal hydrodynamic size, and can be exploited to achieve stable and flexible bioconjugations and layer-by-layer assembly.

Full text of DOI:10.1039/c0cc04524a

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Strong polyelectrolyte quantum dot surface for stable bioconjugation and
layer-by-layer assembly applicationsw
Ho Jin,a Jutaek Nam,a Joonhyuck Park,b Sungho Jung,a Kyuhyun Im,c Jaehyun Hur,c
Jong-Jin Park,c Jong-Min Kimc and Sungjee Kim*ab
Received 20th October 2010, Accepted 4th November 2010
DOI: 10.1039/c0cc04524a
A series of quantum dot (QD) ligands are reported that can
make strong polyelectrolyte QD surfaces with sulfonates or
quaternary ammoniums, which can endow QDs with excellent
colloidal stability independent of the pH and ionic strength,
minimal hydrodynamic size, and can be exploited to achieve
stable and flexible bioconjugations and layer-by-layer assembly.
Quantum dots (QDs) are resistant to photo-bleaching, have
narrow emission profiles and large extinction coefficients, and
can be multiplexed by single excitation.1,2 Highly crystalline and
bright QDs are typically obtained in hydrophobic solutions using
solvothermal methods. Transferring the as-synthesized QDs to
various aqueous media while maintaining the colloidal stability is
of great importance for biological applications of QDs.
Attaching small molecules to the QD surface to replace the
native hydrophobic coating has been a successful strategy.3,4 It
adds smaller volume around the inorganic QD cores and
maintains smaller overall hydrodynamic size (HDS) than do
encapsulation methods that use polymers or lipids.5,6 For the
surface exchanging molecules, QD-anchoring dithiol moieties
such as dihydrolipoic acid (DHLA) have been widely used.7–12
DHLA is more stable than monodentate ligands,7 and the
synthetic procedures are relatively simpler than required for
polydentate ligands.8 DHLA can render QDs in various aqueous
media and also allow conjugations by carbodiimide coupling.
However, the conjugation often causes QDs to aggregate.7,13
Electrostatic attraction can be used to circumvent the necessity
of the covalent linkages.7 Novel carbodiimide coupling reagents
were recently reported that were designed neutral to minimize
the aggregation.14 DHLA-derived QD surface molecules that
contain polyethylene glycol (PEG) moieties were reported
to stably conjugate biomolecules, but at the cost of con-
siderable increase in HDS due to the PEG.10–12 Here, we
report a series of QD surface ligands that have strongly
charged functional groups such as sulfonate or quaternary
ammonium (Scheme 1, and ESIw). CdSe/CdS/ZnS core-shell
Scheme 1 (a) Synthesis of quantum dot surface ligands that contain
sulfonate ion (1) and quaternary ammonium ion (2). (b) Scheme for
quantum dot surface ligand exchange. Initial surface ligands can be
replaced by 1, 2, or the mixture of 1 and dihydrolipoic acid.
QDs were synthesized following procedures previously
described.15,16 N,N0-dicyclohexylcarbodiimide/N-hydroxysuccin-
imide (DCC/NHS) coupling was used to derivatise DHLA.
This synthesis is flexible and can be easily extended to other
functional groups such as alkynes, azides, or other amines.
Derivatization was followed by reduction using NaBH4 to
cleave the disulfide bond and to expose the dimercapto
anchoring group. The as-synthesized QDs were transferred
from chloroform to phosphate-buffered saline (PBS) using
two-phase exchange.4 The transfer did not affect the QDs’
absorption and emission profiles (Fig. S5, ESIw). Typical
photoluminescence quantum yield of the QDs in PBS was
B20%. The exchange causes QD surfaces to be decorated by
strong polyelectrolytes that retain their charges regardless of
the environment pH or salt concentration. During gel electro-
phoresis, QDs decorated by sulfonate move in the opposite
direction than QDs decorated by quaternary ammonium ions
(Fig. S7, ESIw). As a control to the strong positive or negative
polyelectrolyte QDs, weak polyelectrolyte QDs were prepared
that have carboxyl acid or primary amine groups (ESIw).
The colloidal stabilities of the QDs decorated with strong and
weak polyelectrolytes were studied using dynamic light scattering
by measuring the effect of pH on HDS because increase in HDS
indicates loss of colloidal stability (Fig. 1a). Carboxylate QDs
started to lose colloidal stability and to aggregate when pH o 7,
and primary amine QDs tended to lose colloidal stability at
a Department of Chemistry, Pohang University of Science and
Technology, Kyungbuk, 790784, South Korea
b School of Interdisciplinary Bioscience and Bioengineering,
Pohang University of Science and Technology, Pohang,
790-784, South Korea
c Samsung Advanced Institute of Technology, Mt. 14-1,
Nongseo-Dong, Giheung-Gu, Yongin-Si, Gyeonggi-Do 446-712,
South Korea. E-mail: sungjee@postech.ac.kr; Fax: +82 54-2791484;
Tel: +82 54-2792108
w Electronic supplementary information (ESI) available: Synthesis of
sulfonate, quaternary ammonium and primary amine surface ligands,
NMR data and mass spectrometer data. Synthesis of CdSe/CdS/ZnS
and some spectral data. See DOI: 10.1039/c0cc04524a
c
1758 Chem. Commun., 2011, 47, 1758–1760
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Fig. 1 Colloidal stability test of quantum dots that were surface-
modified by different functional groups of sulfonate (black filled
circles), carboxylate (black empty circles), quaternary ammonium
(red filled circles), and primary amine (red empty circles). Hydro-
dynamic size measurements by dynamic light scattering vs. pH (a) and
NaCl concentrations (b).
high pH. In contrast, strong polyelectrolyte QDs (both sulfonate
and quaternary ammonium QDs) show remarkable stability over
the entire pH range. Strong polyelectrolyte QDs also show better
colloidal stability than do weak polyelectrolyte QDs at high salt
concentrations (Fig. 1b). Strong polyelectrolyte QDs do not lose
colloidal stability even in saturated NaCl solution, whereas weak
polyelectrolyte QDs start to aggregate at [NaCl] > 1 M. QDs
with primary amines are less stable in a high salt environment
than are those with carboxylic acids, presumably due to the
strong hydrogen interaction.17
Fig. 2 Stability tests of quantum dots that are modified by a mixture of
two different surface ligands that have carboxylate or sulfonate func-
tional group. The ratio between the carboxylate ligand and sulfonate
ligand varies as 1 : 0 (black), 10: 1 (red), 1 : 1 (blue), 1 : 10 (green), and
0 : 1 (orange). Hydrodynamic size measurements by dynamic light
scattering at different pH (a) and at different salt concentrations (b).
(c) Gel electrophoresis of quantum dots that are surface modified by a
mixture of the carboxylate ligand and the sulfate ligand with the ratio of
1 : 0. 10: 1, 1 : 1, 1 : 10, and 0 : 1 (from left to right) (2% agarose gel,
pH 6.0 MES buffer). Positive and negative signs show the positions of
the electrodes. The arrow indicates the starting line. Cuvettes of aqueous
quantum dots solutions under room light (left) and under UV (right).
Quantum dots that are surface modified by a mixture of the carboxylate
ligand and the sulfonate ligand using the ratio of 1 : 0. 10: 1, 1 : 1, 1 : 10,
and 0 :1 (from left to right) are dispersed at different pH conditions
(d) and at different salt concentrations (e).
For many applications including bioconjugation, the QD
surface is desired to have more than one functional group: one
as the conjugation site, and the other to maintain colloidal
stability. A mixed surface ligand system was investigated using
carboxylates and sulfonates on the QD surface. The nominal
mole ratio (carboxylate : sulfonate) with which the ligands
were introduced for ligand exchange is used for the notation.
This ratio is considered to reflect the actual ligand populations
on QD surface, because both ligands have the identical dithiol
for the QD anchoring. Regardless of the ligand ratios, QDs
retain identical absorption profiles as before the exchange
(Fig. S8, ESIw). Colloidal stability of QDs with different ligand
ratio was studied while varying pH or [NaCl] (Fig. 2). As the
ratio of sulfonate increases, the colloidal stability of the QDs
increased. The ratio of 1 : 1 has colloidal stability that is
comparable to pure sulfonate QDs (Fig. 2a and b). This is
also consistent with the gel electrophoresis result that 1 : 1 QDs
show band position and tailing that are similar to those of pure
sulfonate QD (Fig. 2c). Gel electrophoresis was performed in
slightly acidic condition (pH 6) using relatively thick 2%
agarose gel. Carboxylate QDs moved less than sulfonate
QDs and had long tailing, due to the colloidal instability.
Colloidal stability tests (Fig. 2a and b) were further visualized
by juxtaposing QD samples with different ratios (Fig. 2d and e).
At pH 7.4, all samples were well dispersed and showed
yellow band edge emission under UV. At pH 6.0, the pure
carboxylate QD sample (1 : 0 ratio) fluoresced orange under
UV. This red-shift in emission is due to the energy transfers in
QD aggregates. The 10 : 1 ratio sample did not aggregate at
pH 6.0. At pH 5.0, both 10 : 1 and 1 : 0 samples aggregated. At
pH 3.4, 1 : 0 and 10 : 1 samples precipitated. This result is in
agreement with those of the salt concentration experiment, in
which the emissions of 1 : 0 (carboxylate only) and 10 : 1
samples red-shifted at high [NaCl].
QD probes are desired to have conjugation sites that can be
post-modified for specific interactions with desired targets,
while maintaining good colloidal stability. We have demon-
strated that 1 : 1 carboxylate and sulfonate QDs are colloidally
very stable over a broad pH range and even under high [NaCl].
The carboxylic acid can be used to provide conjugation sites,
and the sulfonates to guarantee colloidal stability. The 1 : 1
mixed-ligand QDs were conjugated to streptavidin (SA) using
typical carbodiimide coupling. Specific binding of the SA–QD
conjugates was confirmed using biotinylated polystyrene
microbeads (Fig. 3a). The fluorescence signal-to-noise ratio
was > 400 times higher than that of a control sample which
was co-incubated with unconjugated QDs.
Primary amine is another popular conjugation site. Analogous
to the mixed ligand system of carboxylate and sulfonate, a
mixed ligand system with primary amine and tertiary amine
was also demonstrated (ESIw). Regardless of the mixing ratio,
the positively charged mixed ligand QDs retained identical
absorption and emission profiles after the ligand exchange
(Fig. S9, ESIw). Their colloidal stability at basic pH increased
as the ratio of tertiary amines increased (Fig. 3b). Conjugation
capability of primary amines in the positively charged mixed
ligand system was demonstrated using bead assays which was
used for Fig. 3a (Fig. 3c).
c
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diameter of 6.5 nm as determined by transmission electron
microscopy. Refractive index of the QD film was determined
as 1.385 by ellipsometry. Based on the inorganic QD volume,
surface organic layer and overall refractive index, the QD
volume fraction was estimated as 21% (ESIw). This high QD
fraction could be obtained because QDs decorated with strong
polyelectrolytes were used without any other polymers. For
example, QD volume fraction could not exceed 15% when
QDs decorated by mercaptoacetic acids were LbL assembled
with positive polyelectrolyte polymers.21 High QD volume
fraction can be potentially very useful for many optoelectronic
applications such as low-threshold QD lasers.22
Fig. 3 (a and c) Transmission (left) and fluorescence (right) micro-
scope images of biotinylated polystyrene beads after co-incubation
with streptavidin conjugated quantum dots (top) and with unconjugated
quantum dots (bottom). The conjugates were prepared by mixed
ligand systems of carboxylate and sulfonate (a), and by the mixed
ligand system with primary amine and tertiary amine (b). Scale bar:
10 m. (c) Stability test of quantum dots of mixed surface ligands with
primary amines and tertiary amines. Samples prepared by different
ligand ratios are noted as 10 : 0 (black), 10 : 1 (red), 1 : 1 (blue), 1 : 10
(green), and 0 : 10 (pink).
In conclusion, we report strong polyelectrolyte QD surfaces
and the applications thereof. By using in combination with
other functional group surface ligands, they can be flexibly
applied for conjugations while retaining the good colloidal
stability. Oppositely charged strong polyelectrolyte QDs were
exploited for LbL assembly, demonstrating highly QD-loaded
composite films. This QD surface chemistry is expected to be
exploited for many future applications including bioimaging
probes, diagnostic assays, and QD light emitting devices.
This work was supported by KOSEF grant funded by MOST
R0A-2008-000-20114-0(2008), Priority Research Centers
Program through NRF 2009-0094037, Basic Science Research
Program 20090090412 and 20090090897.
Layer-by-layer (LbL) assembly is a simple yet versatile process
that can control the deposition thickness with sub-nanometre
control over a very large area.18 QDs have been used for LbL
assembly, mostly in conjunction with oppositely-charged
polyelectrolyte polymers.19,20 We present the first report
(to the best of our knowledge) of LbL assembly of QDs by
strong polyelectrolyte surfaces. QDs were deposited LbL by
alternating dipping into negative sulfonate QD solution with
dipping into positive quaternary ammonium QD solution
(Fig. 4a). As the dipping cycle was repeated, QD thickness
increased linearly (Fig. 4b). After six cycles, the LbL-assembled
QD substrate fluoresced uniformly bright under UV (Fig. 4c,d).
The film thickness was 27.6 nm with a root-mean-square
surface roughness of 13.2 nm (atomic force microscopy,
Fig. 4e,f). The QDs were approximately spherical with the
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c
1760 Chem. Commun., 2011, 47, 1758–1760
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