PH-Responsive quantum dots via an albumin polymer surface coating

Article abstract of DOI:10.1021/ja909570v

Multifunctional peptide?polymer hybrid materials have been applied as efficient and biocompatible quantum-dot coating materials. Significant pH responsiveness (e.g., an influence of the pH on the quantum yields of the peptide?polymer/QDs) was found and is attributed to conformational rearrangements of the peptide backbone.

Full text of DOI:10.1021/ja909570v

Published on Web 03/19/2010
pH-Responsive Quantum Dots via an Albumin Polymer Surface Coating
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†
‡
‡
†
Yuzhou Wu, Sabyasachi Chakrabortty, Radu A. Gropeanu, Joerg Wilhelmi, Yang Xu,
†
†
‡
,†,§
,†,‡
Kai Shih Er, Seah Ling Kuan, Kaloian Koynov, Yinthai Chan,*
and Tanja Weil*
Department of Chemistry, National UniVersity of Singapore, 3 Science DriVe 3, Singapore 117543, Max Planck
Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany, and Institute of Research and
Engineering, A*STAR, Singapore 117602
Received November 18, 2009; E-mail: chmchany@nus.edu.sg; chmweilt@nus.edu.sg
Quantum dots (QDs) have many unique features, such as high
resistance to photobleaching and a wide excitation range, which
make them promising as fluorophores for biomedical diagnostic
HSA, giving dHSA (2). Michael addition of 2 with PEO-5000
carrying a single maleimide group yielded dHSA-PEO (4), which
was very soluble in aqueous solution. N-(2-Aminoethyl)maleimide
trifluoroacetate salt (3) was applied in order to react with the
remaining thiol groups to improve the stability and shelf life of 4.
Subsequently, the condensation reaction between the free amino
groups of the lysine side chains of 4 and thioctic acid groups
activated via N-hydroxysuccinimide (NHS) ester 5 in dimethylfor-
1
applications. Conventional as-synthesized QDs are hydrophobic.
Facilitating their long-term, aggregation-free use in an aqueous
environment via surface coating with suitable hydrophilic substit-
2
uents represents a key challenge. It was previously reported that
ligands with more than one anchor group interacting with the QD
surface (i.e., polydentate ligands) showed improved stability and
3
mamide in the presence of NaHCO resulted in the formation of
multifunctional dHSA-PEO-TA hybrid 6.
3–5
reduced ligand loss and that capping groups such as dihydrolipoic
5
–9
acid (DHLA) were determined to be particularly effective.
Following this motif, we have synthesized a novel peptide-polymer
hybrid material consisting of a biocompatible peptide backbone of
defined chain length having poly(ethylene oxide) (PEO) side chains
to reduce nonspecific interactions as well as a high number of thiol
(
originating from thioctic acid, TA) and amine anchor groups
interacting with the QD surface. This peptide-polymer hybrid was
derived from human serum albumin (HSA) utilizing reaction
sequences that facilitated the stabilization of linear denatured human
serum albumins (dHSAs) in aqueous solution. In contrast to dHSA,
the linearized multifunctional albumin peptide-polymer hybrids
described herein represent stable macromolecules that are particu-
larly suitable as surface ligands for QDs, as they impart biocom-
patibility, high stability in an aqueous environment, and multiple
functional groups facilitating further modifications for various
applications. In comparison with other polymers, the peptide-polymer
hybrids reported in this work benefit from a precisely known length
of the main chain and a distinct number of functional groups at
defined locations (e.g., 100 carboxylic acid groups of glutamate
and aspartate side chains) that allow for further chemical modifica-
tions. We demonstrate that unlike previously reported fluorescence
Figure 1. Synthesis of polypeptide hybrids 4 and 6 and an illustration of
a QD coated with dHSA-PEO-TA hybrid. While it was possible for 6
and consequently 7 to include more than one QD, such occurrences were
rare, and the majority of conjugates contained a single QD.
10–12
resonanceenergytransfer(FRET)-basedQDpH-sensingschemes,
SDS-PAGE revealed a significantly increased molecular weight
for 4 in the range 150-210 kDa after attachment of the PEO chains.
An additional slight increase in the molecular weight was found
after conjugation of the thioctic acid groups to 4 [Figure S3 in the
SupportingInformation(SI)].FigureS7showsatypicalMALDI-TOF
mass spectrum of 6 with an average molecular weight of ∼200
kDa, which corresponds to the SDS-PAGE result (Figure S3).
Furthermore, the polymer dispersity of 6 was investigated by gel-
permeation chromatography (GPC). A sharp signal indicative of a
narrow size distribution and low dispersity was detected (Figure
S6). However, because of the unique architecture of 6, no GPC
standard with a similar structure and polarity could be identified,
thus preventing size estimation via GPC. In this context, commonly
used standards such as PEO of different chain lengths and Dextran
were not applicable. In order to assess the number of TA groups
in 6, 4,4′-dithiodipyridine (DPS) was applied. This reagent is
reduced in the presence of free thiol groups, forming 4-thiopyridone
with a characteristic absorbance maximum at 325 nm (Scheme S1
our peptide-polymer hybrid-coated ODs have a direct optical
response to changes in pH. This work presents a facile route to
biocompatible, multivalent surface coatings on QDs that may
contribute to the development of QD-based pH sensors. QD-based
pH sensors would potentially be able to give better fluorescence
signals in comparison with conventional organic-based fluorescent
pH sensors, especially in cases where it is necessary to use two-
photon excitation for imaging and where continuous monitoring
of the pH and therefore continuous excitation is required.
A schematic of the synthesis of dHSA-PEO-TA hybrids (6)
as surface ligands for QDs is given in Figure 1. First, HSA (1)
was dissolved in a urea/phosphate buffer (pH 7.4). The reducing
agent tris(2-carboxyethyl)phosphine hydrochloride was then applied
to reduce the 34 disulfide bridges within the tertiary structure of
†
National University of Singapore.
‡
Max Planck Institute for Polymer Research.
§
A*STAR.
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012 9 J. AM. CHEM. SOC. 2010, 132, 5012–5014
10.1021/ja909570v  2010 American Chemical Society

C O M M U N I C A T I O N S
and Figure S5). A good correlation between the TA concentration
and the absorption of 4-thiopyridone was obtained, allowing a
quantification of the free thiol groups of 6.
Significant changes in emission intensity were observed as the pH
was varied, as depicted in Figure 2a,b.
As the pH changed from 2 to 12, the QY of 7 increased nearly
1
0-fold (Table 1). Within the physiological pH range 5-9, where
Three different batches of 6 were investigated, and an average
the most significant increases in QYs were found, we verified that
these changes were fully reversible, as shown in Figure 2c.
Additionally, no discernible shifts in the peak emission were
observed at the different pH values. These findings collectively
suggest that neither shell degradation and hole trapping from
thiolate formation nor aggregation through irreversible ligand loss
could explain the changes in QY.
2
2 ( 5 TA groups (corresponding to ∼44 thiol groups) was very
reproducibly obtained. In addition, ∼40 amino groups of lysine
residues are also available to interact with the QD surface. Coupling
of the dHSA-PEO-TA hybrids 6 to hydrophobic as-synthesized
core-shell CdSe-CdZnS QDs proceeded via reduction of the
1
4
15
4
disulfide group of the TA substituents in 6 with NaBH under argon
to afford DHLA followed by addition of processed QDs dispersed
in a minimum amount of tetrahydrofuran under vigorous stirring.
The solution was stirred overnight, and the unreacted QDs were
filtered out. The coated QDs were homogeneously dispersed in
water (Figure S4). The emission spectrum of QDs coated with 6
revealed a slight bathochromic shift relative to the as-synthesized
QDs in hexane, consistent with previous reports on ligand exchange
1
3
with thioalkyl acids. The quantum yield (QY) of the as-
synthesized core-shell QDs decreased from ∼48% in hexane to
∼
16% on average in water, which is typical of QD ligand-exchange
4,8
processes. Fluorescence correlation spectroscopy (FCS) measure-
ments afforded a hydrodynamic diameter of ∼34 nm, consistent
with the diameter of ∼30 nm obtained using dynamic light
scattering (DLS) measurements (see the SI). The narrow hydro-
dynamic size dispersion from the DLS measurements suggested
that a statistical distribution of QDs per polymer was unlikely, and
transmission electron microscopy (TEM) of the QD-polymer
conjugates revealed that the vast majority of conjugates contained
a single QD (see Figure 4 and the SI). We found that the albumin
peptide-polymer-coated QDs 7 were stable in water under ambient
conditions for months with respect to aggregation (according to
DLS) as well as QY.
Figure 3. Changes in the secondary structure of 6 at different pH values.
The changes in the PL efficiency at different pH may broadly
be attributed to corresponding changes in surface passivation of
the QD. We hypothesized that structural changes in the dHSA-
PEO-TA hybrids 6 at different pH may influence their ability to
interact with the QD surface. In order to verify this assertion, the
secondary structure of 6 was investigated via circular dichroism
(
CD) at different pH values, as summarized in Figure 3 and Table
. At acidic pH (pH 3-5), 6 displayed a significantly larger R-helix
content, which represents an opposite trend relative to the secondary
1
1
6
structure changes of native albumin proteins at low pH, again
highlighting the unique structural characteristics of 6. With increas-
ing pH, ꢀ-sheet and random coil structural elements became more
predominant. The larger content of structurally more rigid R-helix
elements at low pH likely imposes restrictions on the ability of the
polymer backbone to conform to the surface of the QD and allow
for more ligand-QD interactions. Additionally, we expect a decline
in the number of thiol-QD binding interactions in accordance with
previously observed dissociation of thiolate ligands from QDs at
1
7
low pH, leading to less surface passivation and low QYs.
However, with increasing pH and in particular above pH 7, more
ꢀ-sheet as well as flexible random coil structural elements occur,
which can facilitate anchoring of more of the thiol and amine groups
on the polymer backbone to the QD surface, thus leading to
increased QYs. These dramatic changes of the secondary structure
may thus play a significant role in the observed trends in pH
responsiveness of the coated QDs 7. The exact identities of the
resultant trapped states formed or removed at different pH,
appropriately probed with fast time-resolved techniques, was beyond
the scope of this work and will be reported at a later time.
DLS and TEM were used to investigate the average diameter d
of 7 in solution (Figure 4b) and on a surface (Figure 4a). It was
found that the samples contained mostly single, isolated QDs. The
average diameter of 7 was 29.4 ( 3.9 nm with a narrow size
distribution (Figure 4b). A few clusters were detected by TEM;
they may have formed during the solvent evaporation process. The
Figure 2. (a) Emission intensity of 7 at different pH (λex ) 254 nm). (b)
Integrated emission intensities vs pH based on four consecutive measure-
ments. (c) Reversibility of the pH responsiveness between pH 5 and 9 based
on three independent measurements. The emission intensity was recovered
after increasing the pH from 5 to 9 as well as decreasing the pH from 9 to
5
.
When the stability of 7 at different pH values was assessed,
surprisingly strong optical responses to changes in pH were
observed. A series of universal buffer solutions with pH ranging
systematically from 3 to 12 were prepared as described in the SI,
and the fluorescence intensities of mixtures of 7 in buffers of
different pH were monitored with a fluorescence microplate reader.
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C O M M U N I C A T I O N S
d values of 7 at different pH were also investigated using DLS.
Only a slight increase in d between pH 3 and 9 was detected.
Therefore, the altered emission intensity is attributed to passivation
events on the QD surface, and pH-induced aggregate formation is
unlikely.
multivalent interactions with QDs and their subsequent stabilization
in water. The resulting peptide-polymer-coated QDs displayed
acceptable QYs of up to 26% and remained aggregation-free in
aqueous solution for several months. Interestingly, a significant
influence of the pH on the QY of the coaated QDs was found,
which was fully reversible at physiological pH. These changes were
attributed to conformational rearrangements of the peptide backbone
of the dHSA-PEO-TA hybrids 6, which likely influenced the
amount of surface passivation by the functional groups on the
polymerbrushmoiety.Furtherdevelopmentofthesepeptide-polymer-
coated QDs into biocompatible pH sensors is currently ongoing.
Acknowledgment. This work was supported by NUS Startup
Grants WBS-R143-000-393-646 and WBS-R143-000-367-133.
Supporting Information Available: Experimental details regarding
the synthesis and characterization techniques used for structures 4, 6,
and 7 and the core-shell CdSe-CdZnS QDs. This material is available
free of charge via the Internet at http://pubs.acs.org.
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