Gold nanoparticle size controlled by polymeric Au(I) thiolate precursor size

Article abstract of DOI:10.1021/ja076333e

We developed a method in preparing size-controllable gold nanoparticles (Au NPs, 2-6 nm) capped with glutathione by varying the pH (between 5.5 and 8.0) of the solution before reduction. This method is based on the formation of polymeric nanoparticle precursors, Au(I)-glutathione polymers, which change size and density depending on the pH. Dynamic light scattering, size exclusion chromatography, and UV-vis spectroscopy results suggest that lower pH values favor larger and denser polymeric precursors and higher pH values favor smaller and less dense precursors. Consequently, the larger precursors led to the formation of larger Au NPs, whereas smaller precursors led to the formation of smaller Au NPs. Using this strategy, Au NPs functionalized with nickel(II) nitriloacetate (Ni-NTA) group were prepared by a mixed-ligand approach. These Ni-NTA functionalized Au NPs exhibited specific binding to 6x-histidine-tagged Adenovirus serotype 12 knob proteins, demonstrating their utility in biomolecular labeling applications.

Full text of DOI:10.1021/ja076333e

Published on Web 12/23/2007
Gold Nanoparticle Size Controlled by Polymeric Au(I) Thiolate
Precursor Size
Raymond P. Brin˜as, Minghui Hu, Luping Qian, Elena S. Lymar, and
James F. Hainfeld*
Biology Department, BrookhaVen National Laboratory, Upton, New York 11973
Received August 22, 2007; E-mail: Hainfeld@bnl.gov
Abstract: We developed a method in preparing size-controllable gold nanoparticles (Au NPs, 2-6 nm)
capped with glutathione by varying the pH (between 5.5 and 8.0) of the solution before reduction. This
method is based on the formation of polymeric nanoparticle precursors, Au(I)-glutathione polymers, which
change size and density depending on the pH. Dynamic light scattering, size exclusion chromatography,
and UV-vis spectroscopy results suggest that lower pH values favor larger and denser polymeric precursors
and higher pH values favor smaller and less dense precursors. Consequently, the larger precursors led to
the formation of larger Au NPs, whereas smaller precursors led to the formation of smaller Au NPs. Using
this strategy, Au NPs functionalized with nickel(II) nitriloacetate (Ni-NTA) group were prepared by a mixed-
ligand approach. These Ni-NTA functionalized Au NPs exhibited specific binding to 6×-histidine-tagged
Adenovirus serotype 12 knob proteins, demonstrating their utility in biomolecular labeling applications.
Introduction
Formation of Au NPs via the reduction of HAuCl4 in the
presence of the thiol ligand has been a widely used technique
in preparing ligand-stabilized Au NPs.14 Schiffrin and co-
workers proposed a two-step mechanism for the formation of
Au NP from HAuCl4.15 The first step involves the conversion
of Au(III) salt to the Au(I) state (as thiolate salt). The Au(I)
thiolate at this point is in a polymeric form. The second step
involves the reductive decomposition of the polymeric Au(I)
thiolate to form the NP. Although this method has been
extensively used, its mechanistic details have not been eluci-
dated.14 One of the most intriguing steps is the formation of
polymeric Au(I) thiolate or the NP precursor. Exploration of
this step could lead to clues on how to control the size of the
resulting NPs, since the properties of the polymeric precursor
are dependent on several factors, such as the nature of ligand,
ionic strength, solvent conditions, and pH. The variation in the
polymeric structure by the nature of the ligand has been well
documented in related compounds, such as gold based antiar-
thritic agents.16-18 For example, Myocrisin assumes a small
cyclic structure (tetrameric) due to the repulsive force brought
about by the carboxylate groups in adjacent ligands.16
Controlling the size of nanoparticles (NPs) has always been
one of the challenges in colloidal science. Changing the size of
NPs can result in modulation of their physical and chemical
properties. Au NPs have been a good candidate for applications
in drug delivery,1 cell imaging,2 and immobilization of proteins
for conformational studies,3 among others. Since the discovery
of various reducing agents for the gold compounds to form the
gold nanoparticles, like sodium citrate, sodium borohydride,
phosphorus, alcohols, and tannic acid/citrate mixtures, the
synthesis and applications of Au NPs of different sizes have
flourished.4 More recently, several techniques that focus on the
size control via the reduction of tetrachloroauric acid (HAuCl4)
were introduced. These include varying the gold-to-thiol ratio,5
varying the reaction temperature,6 varying the pH by varying
the concentration of sodium citrate (reducing agent),7 sonochem-
ical8 and sonoelectrochemical methods,9 seeding methods,10 the
use of mixed reverse micelles,11 photochemical methods,12 and
the use of polymeric ligands such as block copolymers.13
(1) Salem, A. K.; Searson, P. C.; Leong, K. W. Nature Mater. 2003, 2, 668-
671.
(2) Shukla, S.; Priscilla, A.; Banerjee, M.; Bhonde, R. R.; Ghatak, J.; Satyam,
P. V.; Sastry, M. Chem. Mater. 2005, 17, 5000-5005.
(3) Kra¨mer, S.; Xie, H.; Gaff, J.; Williamson, J. R.; Tkachenko, A. G.; Nouri,
N.; Feldheim, D. A.; Feldheim, D. L. J. Am. Chem. Soc. 2004, 126, 5388-
5395.
Choosing the right ligand for NP synthesis is key in forming
Au NPs with desirable properties. In this study, we chose the
naturally occurring peptide ligand, glutathione (GSH). GSH is
(4) Handley, D. A. In Colloidal Gold: Principles, Methods, and Applications;
Hayat, M. A., Ed.; Academic Press, Inc.: San Diego, 1989; Vol. 1, pp
13-30.
(11) Chiang, C.-L. J. Colloid Interface Sci. 2001, 239, 334-341.
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2005, 123, 184701.
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2007, 129, 13939-13948.
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2001, 3, 257-261.
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4943-4945.
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Am. Chem. Soc. 2000, 122, 7146-7147.
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a ubiquitous tripeptide (γ-Glu-Cys-Gly), which acts primarily
as a reducing agent in biological systems. Because of the
favorable properties such as the presence of thiol, carboxylic
acid, and amino groups, water solubility at relevant biological
pH, biological compatibility, and ease of functionalization, GSH
is a very attractive ligand in making water-soluble NPs for
biological applications. The presence of carboxylic acid and
amino groups on GSH ligand complexed with Au(I) has the
potential advantage of being a pH-sensitive compound, which
can adopt different conformational states and sizes depending
on the pH of solution.
The first reported use of GSH as a ligand for Au clusters
was by Hainfeld and co-workers19 and further studied by
Whetten and co-workers in 1998.20,21 Whetten et al. synthesized
the Au NPs via sodium borohydride reduction of the mixture
of tetrachloroauric acid and GSH in methanol-water (2:3) and
obtained Au NPs with most abundant component having a
diameter of 0.9 nm. Tsukuda and co-workers conducted a more
extensive electrophoretic separations and high-resolution mass
spectrometric analyses of the Au NPs, prepared in a similar
manner as that of the Whetten’s study, to reveal the varied size
composition of the NP mixture (i.e., Au10(SG)10, Au15(SG)13,
Au18(SG)14...) depending on the reaction temperature (low or
room temperature).22,23 In both studies, the size of the NPs
formed was between 0.8 and 1.1 nm. Although the preparation
of the Au NPs was well-established and the composition of the
Au NPs was extensively characterized, the issues of size
controllability and further functionalization of the Au NPs to
suit particular applications, such as biomolecular labeling, were
not addressed in these studies. These issues are very important
especially for the utility of the Au NPs in applications that
require functionality and size control. Herein, we report a
technique for which the size of the Au NPs coated with GSH
can be controlled by varying the size of the nanoparticle
precursor [Au(I)SG] through pH control. The size of the final
NPs can be varied from 2 to 6 nm. Furthermore, functional-
ization of Au NPs coated with GSH is demonstrated by the
introduction of nitrilotriacetic acid (NTA) moiety by mixing
another ligand (i.e., NTA-lysine modified ligand) with GSH.
To demonstrate the utility of the size-controllable Ni-NTA-
functionalized Au NP in the 2-6 nm size regime, a biomolecule,
adenovirus serotype 12 (Ad12) knob expressed with 6×-
histidine (His) tags, is labeled with 4.4 nm Au NP and analyzed
by size exclusion chromatography (SEC) and transmission
electron microscopy (TEM).
Figure 1. Crystal structure of Ad12 knob protein showing the top view
(A) and the side view (B) orientations (ref 24). The arrows in B indicate
the N-termini where His-tags were placed.
are, therefore, three His-tags in close proximity. Our synthesized
Ni-NTA functionalized Au NPs show highly specific binding
to His tags on Ad12 knob proteins. Labeling of biomolecules
with Au NPs is attracting more interests in applications like
high-resolution structural analysis,25 medical imaging,26 and
molecular sensing.27 These applications require functionalized
Au NPs with physical/chemical stability, water solubility,
biological compatibility, and site-specific binding.28,29
Ad12 knob protein is a globular knob-like domain located at
the ends of trimeric fibers protruding from the adenovirus capsid.
It acts as the initiating point for virus infection upon interactions
with specific receptors on host cell surfaces.24 The Ad12 knob
proteins exist as a trimer with a total molecular weight (MW)
of about 60 kDa (Figure 1A). It has C3 symmetry with the His-
tagged N-termini clustered near each other (Figure 1B). There
Experimental Section
General Remarks. HAuCl4‚XH2O was purchased from Strem
Chemicals and used as received. Reduced glutathione (GSH), sodium
(19) Hainfeld, J. F.; Leone, R. D.; Furuya, F. R.; Powell, R. D. U.S. Patent
5,521,289, 1996.
(25) Rice, S.; Lin, A. W.; Safer, D.; Hart, C. L.; Naberk, N.; Carragher, B. O.;
Cain, S. M.; Pechatnikova, E.; Wilson-Kubalek, E. M.; Whittaker, M.; PateI,
E.; Cookek, R.; Taylor, E. W.; Milligan, R. A.; Vale, R. D. Nature 1999,
402.
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R. L. J. Phys. Chem. B 1998, 102, 10643-10646.
(21) Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630-2641.
(22) Negishi, Y.; Takasugi, Y.; Sato, S.; Yao, H.; Kimura, K.; Tsukuda, T. J.
Am. Chem. Soc. 2004, 126, 6518-6519.
(26) Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M.; Smilowitz, H. M. Br. J.
Radiol. 2006, 79, 248-253.
(27) Aubin, M. E.; Hamad-Schifferli, K. Langmuir 2005, 21, 12080-12084.
(28) Hainfeld, J. F.; Powell, R. D. J. Histochem. Cytochem. 2000, 48, 471-
480.
(23) Negishi, Y.; Nobusada, K.; Tsukuda, T. J. Am. Chem. Soc. 2005, 127,
5261-5270.
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Science 1999, 286, 1579-1583.
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techniques and applications; CRC Press: Boca Raton, FL, 2005.
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Scheme 1. Synthesis of Lys-NTA-SH
containing 10 mM phosphate buffered saline (PBS, 150 mM NaCl)
was added an aqueous solution of Ni-NTA functionalized Au NPs (40
µL,1 µM) with an average size of 4.4 nm. The mixed solution was
incubated at rt for 3 min. The crude mixture was purified by SEC
(Superdex 200, Eluant: 10 mM PBS, 140 mM NaCl, pH 7.4) and the
labeled fractions were collected and analyzed by TEM.
Dynamic Light Scattering Measurements for the Polymeric Au-
(I)SG. Aqueous solutions of HAuCl4 (10 mg/mL) and GSH (6 mg/
mL) were filtered separately through syringe filters (0.22 µm PVDF
membrane, Millipore) prior to mixing. To a stirred solution of HAuCl4
(0.5 mL) was added GSH solution (4.5 mL). The resulting mixture
was stirred for 1-2 min. The pH of the mixture was adjusted to 4.92
by adding 1 M NaOH. The mixture was filtered through a syringe filter
(0.45 µm PVDF membrane, Millipore) before measuring the DLS.
Mixtures with pH values of 5.08, 5.55, 6.16, 7.25, and 8.11 were also
prepared and analyzed using the same procedure as above.
borohydride, N6-carbobenzyloxy-L-lysine, and 4-butyrothiolactone were
purchased from Sigma and used as received. UV-vis spectra were
recorded on Agilent 8453 spectrophotometer. TEM specimens were
prepared by evaporating sample solutions on plasma-treated Cu mesh
grids covered with carbon thin films. NanoW (Nanoprobes, Inc.)
solution was used for negative stain samples. TEM images were taken
using a JEM 1200CX (JEOL) operating at 120 kV at low dose (<10
e/Å2) with a 1k × 1k CCD camera (Gatan). The sizes of Au NPs were
analyzed with TEM images using ImageJ (NIH). The dynamic light
scattering (DLS) data were recorded on a Precision Detectors PD 2000
DLS Plus with CoolBatch 40T. The hydrodynamic radii of polymeric
Au(I)SG solutions were analyzed using Precision Deconvolve 32
software.
Size Exclusion Chromatographic Analysis of the Polymeric Au-
(I)SG at Different pH Values. The polymeric Au(I)SG samples at
different pH values (4.8, 6.4, and 7.9) were prepared in a similar manner
as described above for the DLS samples, but without filtering the
solutions. The polymeric solutions (200 µL per injection) were analyzed
by SEC-HPLC using Superose 6 column and either 10 mM PBS with
150 mM NaCl (pH 6.4 or 7.9) or 10 mM acetate buffer with 150 mM
NaCl (pH 4.8) as the eluting solvent.
General Procedure for the Preparation of Au NP coated with
GSH. An aqueous solution of tetrachloroauric acid (HAuCl4‚XH2O, 1
mL, 0.025 M) was mixed with an aqueous solution of GSH (7.8 mL,
0.019 M). The pH of the resulting mixture was adjusted between 5.3
and 8.0 using 1 M NaOH. A freshly prepared NaBH4 solution (2 mg/
mL in water, 10-fold molar excess) was added with stirring. The mixture
was allowed to react overnight at room temperature. The excess
reactants were removed by size exclusion chromatography (GH-25,
water) and the gold cluster fraction was collected and concentrated by
centrifugal filtration (molecular weight cutoff or MWCO 50 kDa,
Millipore Amico Ultra).
Synthesis of Lys-NTA-SH. Lys-NTA-SH ((1S)-N-[5-[(4-mercap-
tobutanoyl)amino]-1-carboxypentyl]iminodiacetic acid) was synthesized
using a modified literature procedure.30-32 Briefly, N6-carbobenzyloxy-
L-lysine (Lys(Z)OH) was reacted with bromoacetic acid under basic
conditions to form Lys(Z)-NTA (Scheme 1). Lys(Z)-NTA was then
deprotected by hydrogenolysis in 210 mL of 20:1 THF-water mixture
(instead of the reported 20:1 methanol-water mixture30,31) under 1 atm
of H2 for 20 h to give Lys-NTA. Reaction of Lys-NTA with
4-butyrothiolactone gave the desired product Lys-NTA-SH in 61%
yield (1.0 g).
Representative Procedure for the Preparation of Au-GSH/Lys-
NiNTA. A solution of Lys-NTA-SH (8 mg, 0.022 mmol) in water
(1.3 mL) was mixed with a solution of GSH (20 mg, 0.066 mmol) in
water (3.4 mL). The mixed solution was added to a solution of hydrogen
tetrachloroaurate (HAuCl4, 5 mg, 0.015 mmol) in water (0.5 mL),
followed by adjusting the pH to 6.0-6.5 with 1 M NaOH. A solution
of sodium borohydride (5.6 mg, 0.15 mmol) in water (2.8 mL) was
added with vigorous stirring for 10 min at rt and the mixture was
allowed to react overnight. The Au NPs were purified by gel filtration
chromatography (GH-25, water), followed by centrifugal concentration
with Centri-prep filter (MWCO 30kDa, Millipore Amico Ultra). The
resulting solution was passed through a cation exchange column (CM-
Sepharose, Pharmacia) pretreated with 0.05 M NiCl2 solution. The
colored band was collected and used for the next step.
Results and Discussion
The Au NPs were synthesized employing a three-step
procedure (Scheme 2A). The first step involves the reaction
between the GSH and HAuCl4 (6:1 molar ratio), the second
step involves the adjustment of pH of the mixture from the first
step. Last, the Au NPs (1) are formed by addition of NaBH4.
This method can be extended to the synthesis of Au NPs with
mixed ligands (Scheme 2B). For example, a mixture of GSH
and Lys-NTA-SH in the 3:1 molar ratio was used to prepare
NTA-functionalized Au NP (2) used in this study for biomo-
lecular labeling.
Au NPs coated with GSH (1) were synthesized at various
pH values of the pre-borohydride reduced solutions (mixture
of HAuCl4 and ligand). The final Au NPs are stable and can be
stored at 4 °C for 3 months without aggregation. The change
in size of the Au NPs as the pH changes was monitored using
UV-vis spectroscopy. There was a blue shift in the conspicuous
surface plasmon resonance band from 532 to 507 nm as the pH
value was increased from 5.5 to 8.0 (see Figure S1, Supporting
Information), indicating that the Au NP size decreases as the
pH increases. The exact sizes of 1 were analyzed from the TEM
micrographs (Figure 2), and plotted with the pH values as shown
in Figure 3. The sizes decreased from 6 to 2 nm with standard
deviations of 20-30% as the pH values increased from 5.3 to
8.0.
The same trend is followed when a mixed ligand of 3:1 molar
ratio of GSH/Lys-NTA-SH is used, as shown by hollow
squares at pH 6 and 8 (Figure 3). Combining GSH with Lys-
NTA-SH to form a mixed-ligand Au NP (2) was done in order
to functionalize the Au NP and to use as a site-specific labeling
agent for electron microscopy applications. A mixture of 3:1
molar ratio of GSH to Lys-NTA-SH proved to be the optimal
condition wherein stable clusters with spherical shapes were
formed and the size dependence on pH was maintained. This
indicates the controllability of the size even in the presence of
another ligand by varying the pH of the prereduction solution.
The effect of the excess GSH ligand was determined by
Labeling of Histidine-Tagged Ad12 Knob Proteins. To a solution
of His-tagged Ad12 knob proteins (24 µL, 1 mg/mL) at pH 7.5
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Scheme 2. Synthesis of Au NPsa
a (A) Au NP coated with GSH only; (B) Au NP coated with 3:1 mixture of GSH and Lys-NTA-SH. Reaction conditions: (i) HauCl4, (ii) adjust pH,
(iii) NaBH4.
Figure 3. Size dependence of the AuNP 1 with pH as measured by EM.
Solid squares correspond to the average sizes of Au NP capped with GSH,
whereas open squares represent the average sizes of Au NP 2. The error
bars indicate the standard deviations. About one hundred particles were
analyzed for each point.
with water to remove the excess GSH. Dilution of the retentate
to the original volume and subsequent reduction with sodium
borohydride gave a spectrum similar to that of the “unfiltered”
solution (Figure S2, Supporting Information). This indicates that
any excess GSH has negligible effect on the size of the resulting
Au NP.
The formation of nanoparticle 1 follows the general mech-
anism proposed previously for the formation of Au NP from
Au3+ and thiols.15,20,33 In this mechanism (Scheme 3), Au3+ is
reduced by GSH first to Au+1. The Au+1 then forms a polymeric
structure ([Au(I)SG]n), where Au+1 ions have a coordination
number of 2 and are bridged by the thiolate sulfur atom of
GSH.16,34 Addition of sodium borohydride solution decomposes
the polymeric structure to form the Au NP.
The second equation is the key step in this study because the
size of the polymer was found to be pH dependent. The
Figure 2. TEM micrographs of the Au NP 1 synthesized at different pH
values; in parentheses are the average sizes in nm. (a) 5.5 (6.3), (b) 6.1
(5.4), (c) 6.3 (4.7), (d) 6.5 (6.0), (e) 6.8 (3.6), (f) 7.0 (3.3), (g) 7.5 (2.6), (h)
8 (2.2). All of the micrographs are in the same scale.
performing control experiments. Before borohydride reduction,
the mixture of HAuCl4 and GSH (1:6 molar ratio, pH 8) was
filtered using Centricon Ultrafilter (MWCO 5kDa) and washed
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L.; Cullen, W. G.; First, P. N. J. Phys. Chem. B 1997, 101, 7885-7891.
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A R T I C L E S
Scheme 3. Proposed Mechanism for the Formation of Au NP
from Au3+ and GSH
polymeric structure of Au(I) thiolates, including Au(I) SG and
gold-based antiarthritic agents, has been established and exten-
sively studied in the past using Mo¨ssbauer spectroscopic
techniques.34 Although the X-ray structures of some Au(I)
thiolates have been solved, that of [Au(I) SG]polymer is yet to
be determined. It is likely that the Au(I)SG polymers exist in
random coils consistent with these precedent data.
To further characterize the structure the precursor Au(I)SG
polymers, their relative sizes and spectroscopic behavior were
studied at different pH values. The presence of polymeric
structures with different sizes at different pH values was
followed by DLS. The results of a mixture of HAuCl4 and GSH
(1:6 molar ratio) at various pH values (Figure 4A) reveal that
the average hydrodynamic radius of the polymeric gold species
decreases with increasing pH values. SEC analysis of the
polymeric precursors at different pH values confirmed the DLS
results (Figure 4B). At low pH (4.8), the mixture is composed
of predominantly larger polymeric structures as indicated by
the presence of a faster-moving peak with a retention time of
22.9 min, which could be attributed to larger polymers. At
moderate pH (6.4), the mixture is possibly composed of a
combination of larger and smaller polymers, as evident in the
broad peak centered at 23.4 min. Last, at high pH (7.9), a slower-
moving peak (retention time of 23.7 min) could be attributed
to smaller polymers. The excess GSH apparently had no effect
on the size of the polymeric precursors. A 1:6 mixture of
HAuCl4 and GSH at pH 8 was filtered using Centricon Utrafilter
(MWCO 5kDa) and washed with water to remove excess GSH.
The hydrodynamic radius of the original solution (24.5 ( 2.8
nm) and the filtered one gave a similar size (26.5 ( 2.7 nm),
indicating that GSH has no effect on the size of the polymers.
Figure 4. Studies on the dependence of polymeric Au(I)SG precursor size
on pH. A. Hydrodynamic radius at different pH values of the polymeric
Au(I)SG precursors measured using DLS. Error bars are standard deviation
of three trials. B. The size exclusion chromatograms of the polymeric Au-
(I)SG precursors at pH 4.8, 6.4, and 7.9 with retention times of 22.9, 23.4,
and 23.7 min, respectively. The precursors were separated using Superose
6 column with 10 mM phosphate or acetate buffered saline as the elution
buffer.
The structural changes in the polymeric precursors can be
studied by analyzing the interaction of adjacent Au(I) ions using
UV-vis spectroscopy. A UV-vis spectroscopic investigation
of the mixture of tetrachloroauric acid and GSH in 1:6 molar
ratio at different pH values showed an absorption at ∼320 nm
(shoulder) that diminishes in intensity as the pH increases
(Figure 5). This band could be assigned to the dσ* f pσ transition
similar to that observed for diphosphine bridged binuclear Au-
(I) complexes,35-39 which is an indication of metal-metal
Figure 5. UV-vis spectra of the polymeric Au(I)SG precursor at different
pH values.
interaction. At higher pH values (6-8), the dσ* f pσ absorption
band is shifted and considerably smaller but still monotonically
changes with pH. At lower pH values (4.72-5.57), the higher
intensity of the band suggests the increased presence of metal-
metal interactions. The pH range going from 4.7 to 6.0 gave
the highest decrease in the intensity of the metal-metal
interaction band. In terms of the Au-S-Au angle (æ), the UV-
vis data indicates that a smaller angle is favored at lower pH
and a larger angle at higher pH (Scheme 4). This also implies
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Scheme 4. Illustration of the Dependence of the Au-S-Au Angles and the Corresponding Intramolecular Forces of the NP Precursor on pH
Scheme 5. Ionization of GSHa
a The pK1, pK2, pK3, and pK4 values are 2.12, 3.53, 8.66, and 9.62, respectively (ref 40).
Scheme 6. Schematic Representation of the Proposed Mechanism of the Formation of Size-Controllable Au NPs Capped with GSH
that denser polymers are formed at low pH and less dense at
high pH.
one thiol group). The first (pK1 ) 2.12)40 and second (pK2 )
3.53)40 ionizations can be ignored because their pKa values are
below the lowest pH value used in this study. Also, the
ionization of the thiol group (pK4 ) 9.62)40 can be ignored
because its pKa is above the highest pH value and coordination
with Au(I) converted the thiol into the thiolate form. Thus, the
major contributing species in the pH range of interest are species
C and D. At low pH (4.8), species C predominates over D and
so the net charge on the GSH ligands is about -1. On the other
On the basis of the UV-vis data and results from the DLS
and SEC studies, we hypothesize that at lower pH the polymeric
precursors have larger and denser structures, whereas at higher
pH the polymeric precursors assume less dense and smaller
structures. This could be a consequence of the strength of
repulsive electrostatic forces acting between neighboring GSH
ligands (Scheme 4). GSH can have four ionization forms
(Scheme 5, A-E), because it has four ionizable functional
groups (one ammonium group, two carboxylic acid groups, and
(40) Pirie, N. W.; Pinhey, K. G. J. Biol. Chem. 1929, 321-333.
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980 J. AM. CHEM. SOC. VOL. 130, NO. 3, 2008

Size-Controllable Gold Nanoparticles
A R T I C L E S
Scheme 7. Binding Reaction between a His-Tagged Protein and the Au NP Functionalized with NiNTA
hand, at high pH (8.0), species D predominates over species C
and the net charge of the ligand approaches -2. At higher pH
values, the monomeric units (Au(I)SG) are negatively charged
and so the growth of the polymer is limited by the stronger
repulsive interaction; hence, only smaller and denser precursors
are formed. At lower pH values, the monomeric units are able
to approach a growing polymer due to the less repulsive force
and so the precursors formed are larger and less dense.
Higher density of the Au(I)SG polymeric structures at lower
pH values is also predicted (Scheme 4). The larger polymeric
precursors at low pH lead to the formation of fewer nuclei for
borohydride reduction to NPs (Scheme 6). Fewer nuclei which
have more Au ions in close proximity result in larger Au NPs.
This is consistent with the common principle that fewer nuclei
lead to larger particles, since during the growth phase Au(I) is
preferentially reduced onto an existing Au(0) surface rather than
independently forming a new particle. Conversely, higher pH
values favor a lower degree of polymerization of Au(I)SG.
Repulsive negative charges increase on the GSH ligand at higher
pH values due the deprotonation of the ammonium group (pKa
8.66),40 thus favoring smaller polymeric structures or possibly
monomeric Au(I)SG units. This results in the formation of more
nuclei, hence leading to smaller NPs (Scheme 6).
Au NP through a ligand exchange mechanism (Scheme 7). Two
water molecules are expelled in the process to maintain the
octahedral coordination. Labeling of His-tagged Ad12 knob
proteins with 4.4 nm Ni-NTA functionalized Au NPs was
confirmed by SEC (see Figure S3 and note, Supporting
Information for details of labeling experiment) and TEM
analyses. The TEM micrograph of the labeled-protein fraction
shows that there is indeed labeling of the Ad12 proteins as
evident in the formation of core-shell structures (Figure 6A),
This strategy gives an insight into the possibility of tailoring
the desired properties such as size, solubility, and functionality
of the NPs based on the structure of the ligand and its Au(I)
thiolate form. For the size controllability, the structure of the
ligand is key. By virtue of its carboxylic acid groups and amino
group, GSH responds to the change in pH by changing its
conformation due to the repulsive or attractive forces. The size
controllability could be potentially extended to the use of ligands
that can change conformation upon changing the temperature,
or upon exposure to radiation, etc.
Because of the better visibility of larger gold nanoparticles
(>2 nm) in EM, the 4.4 nm Au NPs functionalized with Ni-
NTA were used to label His-tagged Ad12 knob protein. The
Au NPs used in this investigation were prepared from Au NPs
capped with GSH/Lys-NTA-SH mixture (3:1 molar ratio), which
was complexed with Ni2+ ion by passing through an ion
exchange column pretreated with a NiCl2 solution.
The binding of the Ni-NTA-functionalized Au NP with a His-
tagged protein is governed by the formation of stable coordinate
bonds between two imidazoles from two histidines of the His-
tagged protein and the Ni2+ center of the Ni-NTA group of the
Figure 6. (A) TEM micrograph of His-tagged Ad12 knob proteins labeled
with Au NP coated with GSH/Lys-Ni-NTA-SH (negatively stained). (Inset)
Higher magnification image of the core-shell structure formed between
the knob proteins and Au NP. Arrows indicate the position of the knob
proteins around the black dot (Au NP). (B) TEM micrograph of the mixture
of non-His-tagged Ad12 knob proteins and Au NPs. Both images are shown
in the same scale.
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A R T I C L E S
Brin˜as et al.
which is a consequence of the multiple Ni-NTA functional
groups on the surface of each Au NP. The number of knob
proteins on the surface of Au NP is limited by steric hindrance.
It is also worth mentioning that the binding efficiency of this
labeling is very high because all of the Au NPs were
encapsulated by the knob proteins.
To discount the possibility of nonspecific binding in the
labeling experiment, a control experiment was done using Ad12
knob complexes without histidine tags. The absence of core-
shell NP-protein structures in the control experiment (Figure
6B) indicates that Ad12 knob complexes without histidine tags
do not bind to the Ni-NTA Au NP. This shows that binding
of Ni-NTA Au NP to Ad12 knob protein is highly specific for
the presence of histidine tag on the protein. Specific binding of
NPs to biomolecules has proven to be an indispensable attribute
in applications such as electrochemistry,41 carbohydrate-protein
recognition,42 and DNA-based diagnostic applications.43
the formation of larger and denser polymeric precursors because
of the weaker repulsive electrostatic forces, present between
adjacent ligands. On the other hand, smaller and less dense
polymeric precursors were present at higher pH values due to
the stronger repulsive electrostatic forces of the carboxylate
groups between adjacent ligands. The larger precursors lead to
the formation of larger NPs, whereas smaller precursors lead
to the formation of smaller NPs.
The NPs were functionalized by mixing together two types
of ligands, GSH and modified lysine bearing the Ni-NTA
moiety and a thiol linker. This approach paves the way to
preparing Au NPs of various sizes and various functionalities
without destroying the desirable properties of solubility and
stability. The Ni-NTA functionalized Au NPs were shown to
have specific binding to His-tagged Ad12 knob proteins. This
labeling technique can have potential biological application in
high-resolution structural analysis, medical imaging, drug
delivery, molecular sensing, and scaffold-directed assembly
where binding specificity and size of NPs are important.
Conclusion
In conclusion, we have shown that GSH-capped Au NPs of
various sizes, in the 2-6 nm size regime, can be prepared by
controlling the pH. The size controllability has been attributed
to the dependence of the size of the Au NP precursor, Au(I)-
SG, on pH. The ligand, GSH, plays an important role in the
size dependence on pH. Not only does it impart water-solubility
to the resulting NPs, but also modulates the size of the NP
precursors through the attractive or repulsive forces acting on
adjacent ligands at certain pH values. Lower pH values favored
Acknowledgment. We would like to thank Yanbiao Zhang
and Paul I. Freimuth for providing the protein samples. We are
grateful to Walter Mangel and Vito Graziano for use of their
DLS machine. This work was supported by BNL LDRD Grant
04-055, DOE Grant 06742, and NIH Grants P41EB002181 and
R01RR017545.
Supporting Information Available: UV-vis spectra of the
Au NPs prepared at different prereduction pH values, the UV-
vis spectra of the Au NPs prepared in the presence and absence
of excess GSH during reduction, and the chromatographic data
of the Au(I)SG polymers at different pH values. This material
is available free of charge via the Internet at http://pubs.acs.org.
(41) Kerman, K.; Chikae, M.; Yamamura, S.; Tamiya, E. Anal. Chim. Acta 2007,
588, 26-33.
(42) Lin, P.-C.; Ueng, S.-H.; Yu, S.-C.; Jan, M.-D.; Adak, A. K.; Yu, C.-C.;
Lin, C.-C. Org. Lett. 2007, 9, 2131-2134.
(43) Storhoff, J. J.; Marla, S. S.; Bao, P.; Hagenow, S.; Mehta, H.; Lucas, A.;
Garimella, V.; Patno, T.; Buckingham, W.; Cork, W.; Muller, U. R. Biosens.
Bioelectron. 2004, 19, 875-883.
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