Preparation of peptide-functionalized gold nanoparticles using one pot EDC/sulfo-NHS coupling.
ABSTRACT Although carbodiimides and succinimides are broadly employed for the formation of amide bonds (i.e., in amino acid coupling), their use in the coupling of peptides to water-soluble carboxylic-terminated colloidal gold nanoparticles remains challenging. In this article, we present an optimization study for the successful coupling of the KPQPRPLS peptide to spherical and rodlike colloidal gold nanoparticles. We show that the concentration, reaction time, and chemical environment are all critical to achieving the formation of robust, peptide-coated colloidal nanoparticles. Agarose gel electrophoresis was used for the characterization of conjugates.
Published:July 05, 2011
r2011 American Chemical Society
dx.doi.org/10.1021/la2022177|Langmuir 2011, 27, 10119–10123
Preparation of Peptide-Functionalized Gold Nanoparticles Using One
Pot EDC/Sulfo-NHS Coupling
Dorota Bartczak and Antonios G. Kanaras*
School of Physics and Astronomy, University of Southampton, Southampton SO17 1BJ, U.K.
S Supporting Information
Applications of nanotechnology in biomedical and physical
sciences require sophisticated nanoparticles with precisely de-
fined chemical composition, size, shape, and functionality.1?4In
particular,understandingthenanoparticle surface chemistryisof
critical importance to obtaining stable and functional nanopar-
ticles. A popular type of surface capping ligand, frequently
utilized for the preparation of biocompatible gold nanoparticles,
is based on thiol-containing oligoethylene glycols with a termi-
nated amine or carboxylic group [i.e., monocarboxy(1-mercap-
toundec-11-yl) hexa(ethylene glycol) (OEG)].5?7The thiol
group of OEG binds to the gold surface, the ethylene glycol unit
imparts hydrophilic character to the ligand, and the terminal
carboxy or amine group serves as a binding site to conjugate
functional biomolecules such as peptides8,9and antibodies.10
The coupling of biomolecules to inorganic colloidal nanoparti-
cles is quite challenging because one needs to consider not
only the several chemical parameters that affect the kinetics of
the reaction but also the robustness of the final product
There are various methods available for cross-linking biomo-
lecules to nanoparticles.11Among the most commonly used
strategies are so-called click chemistry12,13and EDC/sulfo-NHS
EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hy-
drochloride] is one of the most commonly used carbodiimides,
which catalyzes the formation of amide bonds between carboxy
in water and the ease of removal of the byproduct. However,
sulfo-NHS (N-hydroxy sulfosuccinimide) is used to increase the
stability of active intermediates in coupling reactions via the
formation of active ester functional groups with carboxylates.
Several reports demonstrate the successful coupling of biomole-
cules to water-soluble nanoparticles using EDC or its derivatives
as the main coupling reagent.15?20For example, Nie and co-
workers showed the coupling of proteins to mercaptoacetic
the successful conjugation of antibodies to carboxy-terminated
PEG dithiol-coated gold NPs.22However, the employment of
EDC to bind biomolecules efficiently to water-soluble colloidal
nanoparticles remains a challenge. Quite often, the biomolecules
do not bind efficiently to the particles or the particle?biomolecule
conjugates are not stable.
the appropriate chemical conditions for the successful coupling
of the KPQPRPLS peptide to carboxy-terminated oligoethylene
glycol gold nanoparticles (OEG NPs). The KPQPRPLS?nano-
particle conjugates are of critical importance to the activation of
angiogenic genes, as has been shown in earlier studies.8,9A
number of experimental parameters are explored, including the
variation of reagent concentration, the reaction time, and the
type of nanocrystal morphology.
Synthesis of Peptide-OEG NPs. Spherical gold NPs were pre-
pared according to the well-established citrate reduction method23and
then stabilized withbis(p-sulfonatophenyl)phenyl phosphine dehydrate
dipotassium salt (BSPP).24,25Gold nanorods (NR) were synthesized
following the procedure by El-Sayed and co-workers.26Both types of
gold NPs were functionalized with OEG. In detail, an aqueous solution
of OEG (5 mg/mL, 200 μL) was added to a solution of gold spheres
(10 mL, 5 nM) or gold nanorods (5 mL, OD 0.5) while stirring. The
4 ?C. Then, the particles were purified by centrifugation (three times,
16.400 rpm, 15 min) and redispersed in 0.01 M sodium borate buffer at
pH 9. Prior to the coupling reactions, the particles were characterized
using a nanozetasizer (Supporting Information).
EDC/s-NHS Coupling Reactions. The KPQPRPLS peptide was
June 13, 2011
July 5, 2011
ABSTRACT: Although carbodiimides and succinimides are broadly employed for the formation of
amide bonds (i.e., in amino acid coupling), their use in the coupling of peptides to water-soluble
carboxylic-terminated colloidal gold nanoparticles remains challenging. In this article, we present an
optimization study for the successful coupling of the KPQPRPLS peptide to spherical and rodlike
colloidal gold nanoparticles. We show that the concentration, reaction time, and chemical environment
are all critical to achieving the formation of robust, peptide-coated colloidal nanoparticles. Agarose gel
electrophoresis was used for the characterization of conjugates.
dx.doi.org/10.1021/la2022177 |Langmuir 2011, 27, 10119–10123
to carboxylic acids. In a typical reaction, a solution of the peptide
(10 μL, 1 mg/mL, MW = 922.1, in 0.01 M sodium borate buffer,
pH 9)was added to OEG NPs (0.5 mL, 1.5 nM for spherical or OD =
solution, aqueous solutions of coupling reagents EDC (5 μL, 0.2 M)
and sulfo-NHS (10 μL, 0.2 M) were introduced simultaneously.
Then, the reaction mixture was stirred for 24 h at room temperature.
The particles were purified by centrifugation/decantation (three
times, 16.400 rpm, 15 min) and redispersed in tris-borate-EDTA
buffer (0.5? TBE) at pH 9.
For the different types of experiments, the following reaction param-
eters were varied one at a time while keeping the rest of the parameters
the same as in the typical reaction.
(a) The amounts of EDC and sulfo-NHS were varied in the reaction
mixture while keeping the molar ratio between them constant
at 1:2 (EDC/sulfo-NHS). The added volumes were kept the same
as in the typical reaction. The minimum used quantity of EDC/
20 mmol, respectively.
(b) The amount of peptide added to the reaction mixture was varied
while keeping the volume of introduced solution the same as in the
typical reaction. The minimum introduced quantity of the peptide
was 10.85 nmoles, and the maximum was 10.85 μmoles.
(c) The concentration of sodium borate buffer was varied from 0.1 to
0.001 M, and the concentrations of all other reactants were kept
constant, as in the typical reaction.
(d) The reaction time, measured after all reagents were added to the
reaction mixture, was varied between 24 and 48 h.
(e) Two shapes of OEG NPs, spherical and rodlike, were used
in EDC/sulfo-NHS coupling reactions. This was to investigate
if morphologically different NPs can still be successfully conjugated
to the peptide using the typical reaction conditions.
Gel Electrophoresis. Gel electrophoresis was employed to deter-
mine the variations in charge and size as previously reported for gold
particles of different functionalities.27,28A horizontal agarose gel system
was used in all experiments. The agarose gel (0.75%) was prepared by
dissolving agarose (0.45 g, Sigma-Aldrich) in 0.5? TBE (60 mL, pH 8).
Liquid agarose was poured into a gel tray (10 ? 7 cm2, Bio-Rad) fixed
within the gel caster (Bio-Rad). Next, a teeth comb (Bio-Rad) was
placed in the middle slot, and the gel was left to cool and solidify for
30 min at room temperature. The gel caster was leveled, ensuring the
formation of an evenly thick (1 cm) matrix with identical wells (well
capacity 41.6 μL). The gel was placed in a mini-sub cell GT base (Bio-
Rad) along with TBE buffer (0.5?) for submersion of the gel beneath
5 mm of liquid. The colloids (16 μL, 50 nM spherical or OD = 10 for
nanorods, in 0.5? TBE buffer) were mixed with glycerol (4 μL, 30%, in
10 V (steady current) per 1 cm of gel. Digital images of gels were taken
with a Cannon Power Shot A480 digital camera.
’RESULTS AND DISCUSSION
Water-soluble carbodiimides, such as EDC, are some of the
most popular reagents used in cross-linking reactions. EDC
reacts with carboxylic acids and forms reactive o-acylisourea
intermediates, which are then attacked by a nucleophile (i.e., a
Scheme 1. Schematic Representation of Amide Bond Formation between the KPQPRPLS Peptide (Blue) and OEG NPs (Red
Shape) Using EDC (Red) and Sulfo-NHS (Green)
dx.doi.org/10.1021/la2022177 |Langmuir 2011, 27, 10119–10123
primary amine) to create an amide bond (Scheme 1). However,
o-acylisourea intermediates are labile in the presence of polar
solvents and must react immediately after they have been
dissolved in water. The reason is that oxygen atoms, which
intermediate and releasing isourea, thus inactivating EDC. To
with sulfo-NHS. Sulfo-NHS forms an active ester with the
carboxylic acid attached to the nanoparticle surface. This type
of intermediate ester is very hydrophilic and stable and hydro-
lyzes relatively slowly in water, offering an extra advantage for
coupling reactions. In the presence of amine nucleophiles, the
sulfo-NHS ester is rapidly hydrolyzed, allowing the formation of
an amide bond.
Although the EDC/sulfo-NHS coupling reaction can be
performed in several steps, in our experiments we chose to add
all of the reagents in one step. The successful coupling, for the
different reaction parameters, was evaluated by gel electrophor-
esis. All conjugation reactions were performed under alkaline
conditions to reduce the possibility of peptide polymerization
when the peptide was present in excess. In the first set of
experiments, the concentrations of EDC and sulfo-NHS were
varied, and the concentration of the peptide was kept steady. As
can be seen in Figure 1A, a mobility trend (V shape) is observed
for lanes 4A?8A, indicating that different coupling efficiencies
depend on the experimental conditions. The electrophoretic
mobility of batch 6A is significantly lower than those of the
the nanoparticles. When a higher number of peptides is asso-
gel matrix because of their larger hydrodynamic size and their
less-negative charge (because the peptide used in our experi-
ments has a slightly positive charge). Figure 1B shows the
variations in the electrophoretic mobilities of Pep-OEG NPs
when the concentrations of the coupling reagents are kept fixed
and the concentration of the peptide is modified. Again, it is
evident that the most effective coupling conditions are observed
for lane 5B. When a large amount of peptide is introduced
(lane 3B), most of the particles have a very low mobility on the
gel. This could be due to particle agglomeration caused by peptide
cross-linking. In any case, from the results presented in Figure 1,
it is clear that the best conditions to achieve the conjugation of the
peptide to the particles are met for a concentration of 1 mmole of
EDC, 2 mmoles of sulfo-NHS, and 1.09 μmoles of peptide.
Another very interesting observation in Figure 1A is that the
degree of coupling seems to correlate in an unexpected way with
the concentration of the coupling reagents used in every experi-
ment. If we assume the ideal coupling conditions (lane 6A) to be a
increasing or decreasing the concentrations of EDC and sulfo-NHS.
One may speculate that the lower the concentration of coupling
reagents, the lower the efficiency of coupling (lanes 4A and 5A).
The explanation of the lower coupling efficiency at higher
concentrations of EDC and sulfo-NHS (lanes 7A and 8A) does
not seem to be very obvious. With more coupling agents, it
of EDC and sulfo-NHS may promote the polymerization be-
tween the highly concentrated active species (peptides), thus
reducing the quantity of peptides available for coupling to the
The last observation in Figure 1 is that the individual reagents
(peptides, EDC, and sulfo-NHS) do not influence the nanopar-
ticle mobility in the gel (lanes 2A and 3A) unless they are all
present in the reaction mixture.
Moreover, similar mobility is observed for samples that
contain different amount of peptides but not coupling reagents
(lanes 4B, 6B, and 8B). This means that for the given experi-
mental conditions the nanoparticles remain inert to a large
amount of the peptide.
To realize further the optimum coupling conditions, we
investigated the influence of the buffer concentration in the
Figure 1. EDC/sulfo-NHS coupling reaction of the peptide (KPQPRPLS) to OEG NPs under different experimental conditions. (A) The peptide
concentration and the ratio between EDC and sulfo-NHS are kept constant while changing the moles of coupling reagents. (B) The peptide
concentration is varied, while the concentrations of coupling agents are kept fixed. Black arrows indicate the most efficient coupling conditions.
dx.doi.org/10.1021/la2022177 |Langmuir 2011, 27, 10119–10123
as in the typical coupling reaction. The agarose gel in Figure 2
shows that for all samples the particles are delayed in the matrix,
thus indicating a successful coupling (lanes 2C?5C). However,
in lower buffer concentrations, a lower nanoparticle mobility is
observed in the gel (lanes 6C and 7C), suggesting a more
efficient coupling of the peptides to the particles. For a smaller
buffer concentration (lane 8C), some particles do not run in the
gel, indicating a degree of agglomeration. The variation in
particle mobility in lane 8C may be due to the lower capacity
of the borate buffer. It is difficult to realize how sudden pH
variations, as the reaction proceeds, could affect the degree of
coupling. However, there is a strong possibility that sudden
charge changes could cause the agglomeration of the colloids.
To evaluate if the incubation time affects the degree of
coupling, we performed two independent reactions where all
of the experimental conditions were kept constant and only the
incubation times were varied to 24 or 48 h. Figure 3 shows in
both cases the particle delay on the gel (lanes 3D and 6D),
indicating a successful coupling. However, as expected, lanes 1D
and 4D show that plain OEG NPs are stable at different reaction
times; when the particles are mixed with the coupling reagents
but no peptide, the results vary (lanes 2D and 5D). Specifically,
when particles and coupling reagents (without peptide) are
incubated together for 48 h the sample smears on the gel,
indicating a variation in charge and/or the agglomeration of
particles. This observation suggests that the coupling reaction
takes place in the first 24 h.
In the final experiment, we investigated if the chosen coupling
conditions are suitable for the conjugation of the peptides to
short aspect ratio gold nanorods. This is an essential experiment
reaction (physicochemical characteristics of the particles in
Supporting Information). Figure 4 shows the gel electrophoresis
of spherical and rodlike gold nanoparticles when the typical
experimental coupling conditions were applied. It is evident that
in both cases the peptide-coated nanoparticle samples are
delayed in the gel matrix, thus indicating a successful coupling.
Therefore, it was concluded that the experimental conditions
chosen for the coupling of the peptide to 16 nm spherical
nanoparticles can be employed for the conjugation of the same
peptide to gold nanorods with dimensions of 16 ? 47 nm2
(nanozetasizing measurements in Supporting Information).
to OEG NPs when the borate buffer concentration is varied. All of the
other experimental conditions are kept fixed. Black arrows indicate the
most efficient coupling conditions.
OEG NPs, with varying incubation time. All other experimental condi-
and 5D are particles incubated with the coupling reagents but without
The black arrows indicate the successful coupling.
OEG NPs, when the shape of the nanoparticles is varied. Lanes 1E and
3E are untreated OEG NPs; lanes 2E and 4E are particles incubated
under the optimized coupling conditions. Black arrows indicate the
dx.doi.org/10.1021/la2022177 |Langmuir 2011, 27, 10119–10123
This work provides insight into the chemical conditions re-
quired for the conjugation of the KPQPRPLS peptide to nano-
degree of peptide coupling as well as the nanoparticle stability.
Beyond the importance of the KPQPRPLS-nanoparticle conju-
gates as highlighted in earlier work,8,9we believe that our observa-
tionswill be the cornerstone for more in-depth studies concerning
tion of conditions to retain the stability of colloidal nanoparticles.
tics of the particles. This material is available free of charge via
the Internet at http://pubs.acs.org.
The University of Southampton (NanoUSRG) and the Royal
K. thanks the Research Council UK (RCUK) for a Roberts
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