1 Synthesis of gold nanostructures

Optical and biological properties of noble metal colloids result from a delicate bal-
ance between material, size, shape, and dispersion of the nanostructures (see
Chapter 2) [1]. The interest in tuning the behaviors of nanomaterials has encouraged
the exploration of many synthetic processes in the last two decades, in order to pro-
duce nanoparticles with peculiar features set for specific applications [2]. In this
chapter, the most common protocols to produce gold nanoparticles (AuNPs) are dis-
cussed and a practical guide to the synthetic processes is reported at the end of the
chapter. In this new edition, a section on gold–silica hybrid nanomaterials has
been added due to their potentiality for the clinical translation of noble metals to
the clinical practice [3].
In general, nanostructures are produced by two approaches: bottom-up and top-
down [4]. In the first approach, the synthesis begins from the interaction of metal ions,
in order to build a more complex structure, while in the latter the bulk material is grad-
ually eroded by physic-chemical mechanisms until the desired size and shape is
achieved. Nanostructures for biological applications are usually produced by employ-
ing the bottom-up approach, in which the lower control over the final product is bal-
anced by the amount/batch [5]. In the bottom-up approach, synthesis are based on the
reduction of salts of the metal of interest (the precursor) in the presence of reducing
and surfactant agents in aqueous or organic media. By changing some key variables,
among which the reactants, their relative molar concentrations, the reaction tempera-
ture, or the stirring velocity, the nucleation and growth processes can be controlled,
achieving colloids with the desired properties (Figure 1.1).
The control on the temporal separation of these two effects is critical to obtain
AuNPs with a narrow size-dispersion (Figure 1.1). In the following, the synthetic
strategies are named as usually accepted.

1.1 Gold nanospheres (AuNSs)

1.1.1 Turkevich method

The method proposed by Turkevich is based on the reduction of tetrachloroauric

acid (HAuCl4) with sodium citrate in water at 90–100 °C [7]. This is the most com-
mon process to synthesize AuNSs due to its simple and environmental benign pro-
cedure. Furthermore, the size of the nanospheres can be readily tuned from 10
to 150 nm by varying the molar ratio of citrate to HAuCl4. The experimental proto-
cols are based on a rapid addition of sodium citrate solution to a hot (90–100 °C),
aqueous solution of HAuCl4. In the redox reaction, sodium citrate acts both as a
reducing/capping and buffering agent. In particular, a study by Ji and coworkers

https://doi.org/10.1515/9781501511455-001
2 1 Synthesis of gold nanostructures

Au+3 Reducing agent

+
Reduction

Au atom

Au atom
Au+3
Nucleation by collision
between ions, atoms
Au+3
Au cluster and clusters

Au atom

Au nucleus

Size-controlled growth Shape-controlled growth

A
I n
s i
o s
t Nanorod Hollow particle Triangular particle o Successive
r Au/Au+3 t controlled
o r growth
p o
i p
c i
Faceted particle Nanocube Nanocage c
g
r Au/Au+3 g
o r
w o
t w
h t
Nanobelt Branched particle Nanokite
h

Figure 1.1: Formation mechanism of gold nanoparticles (AuNPs) with various particle sizes and
shapes by chemical reduction method. Reproduced with permission from D.T. Nguyen, D.J. Kim and
K.S. Kim, Micron, 2011, 42, 207. ©2011, Elsevier [6].

highlighted the fact that citrate species buffer the pH of the HAuCl4 solution from
pH ≈ 2 to higher values (even neutral), depending on the amount of citrate added
[8]. Accordingly to the pH of the reaction solution, AuCl4− ions [acid dissociation
constant (pKa) 3.3] are hydrolyzed to several kind of auric precursor ions, such as:
 1.1 Gold nanospheres (AuNSs) 3

AuCl3(OH)− (pKa 6.2), AuCl2(OH)2− (pKa 7.1), AuCl(OH)3− (pKa 8.1), and Au(OH)4−
(pKa 12.9). Their reactivity decreases as in the following sequence: AuCl4− > AuCl3
(OH)− > AuCl2(OH)2− > AuCl(OH)3− > Au(OH)4− [7, 9]. In the first step of the reaction
(Figure 1.2), sodium citrate is oxidized to sodium acetone dicarboxylate (SADC)
while any precursor of HAuCl4 is reduced to AuCl by pH-dependent kinetics.

Figure 1.2: Hypothesis of the reactions involved in the formation of colloidal gold in the citrate
methods. Reproduced with permission from H.B. Xia, S.O. Bai, J. Hartmann and D.Y. Wang,
Langmuir, 2010, 26, 3585. ©2010, American Chemical Society [10].
4 1 Synthesis of gold nanostructures

The nucleation process (formation of gold clusters) is caused by the formation of AuCl/
SADC macromolecular complexes and, subsequently, the growing steps to AuNSs are
catalyzed by gold clusters themselves, which support the dismutation of AuCl on their
surfaces (Figure 1.2) [11]. It is important to remember here that a dismutation reaction
occurs when the same ion acts as oxidant and reducing agent. The coagulation of mac-
romolecular SADC/AuCl complexes is induced by concentration fluctuation in agree-
ment with LaMer theory [12]. Matijevic and coworkers have demonstrated that rapid
coagulation favors the formation of monodisperse spherical particles [13]. Thus, a rapid
formation of a large amount of SADS support the formation of nanoparticles with a
narrower size distribution. On the other hand, SADC can also readily decompose to ac-
etone at high temperatures (>90 °C), especially at neutral to basic pH [14]. Acetone can
reduce the HAuCl4 precursor ions to AuCl, leading to a secondary nucleation that re-
sults in a broadening of the size distribution of the colloid. The secondary nucleation
can be decreased by fast oxidation of citrate to SADC at a lower pH using silver ions
under ultraviolet (UV)-light irradiation [15]. Interestingly, silver ions also foster the for-
mation of very spherical nanostructures (Figure 1.3(c)). For example, Xia and col-
leagues [9] improved the Turkevich method by adding a catalytic quantity of Ag+ ions
during the reaction, obtaining quasispherical nanoparticles [10]. Theoretical calcula-
tions have demonstrated that different gold facets caused peculiar deposition potential
for Ag: 0.12, 0.17, and 0.28 eV for (111), (100), and (110) facets, respectively [16]. This
suggests that Ag atoms produced by citrate reduction of Ag+ ions deposit preferentially
on the (110) and (100) facets of AuNSs. Then, the Ag layer is oxidized and replaced by
gold ions. Overall, the deposition of silver significantly decreases the growth rate of
AuNSs on the (110) and (100) facets, promoting the formation of spheres. The presence
of weak ligands such as citrate does not interfere with the deposition and decomposi-
tion of Ag+ on AuNSs, enabling the reshaping of the polycrystalline nanoparticles to
quasispherical geometries [11].
In summary, because of the many interdependent variables involved in the re-
action process, AuNSs obtained by the Turkevich method (Figure 1.3(a)) usually
have a broad distribution of size and shape. Indeed, considering that both the nu-
cleation and crystal growth of AuNSs are very fast at high temperatures (less than
10 min at 100 °C), the buffering effect of citrate, and the inhomogeneous nucleation
caused by potential nonuniform mixing, a temporal overlap between nucleation
and crystal growth may happen, broadening the final size distribution of the col-
loid. For example, despite the several improvements to the Turkevich method,
AuNSs are generally produced with a diameter in the range of 12–60 nm with a rela-
tive size distribution of 13%–16% and usually with a nonuniform and irregular
shape (such as quasispheres, ellipsoids, and triangles) [10, 17–19]. On the other
hand, it is useful to remember that if geometrical uniformity is not a key-point for
the investigation or the final application, the Turkevich method is the most easy and
fast process to produce high throughput water-soluble AuNSs.
 1.1 Gold nanospheres (AuNSs) 5

(a) (b)

200 nm 50 nm

(c) (d)

200 nm
200 nm

Figure 1.3: (a) Transmission electron microscopy (TEM) image of gold nanospheres synthesized by
the Turkevich method. Average sphere diameter of 20 nm, scale bar 200 nm. (b) TEM image of gold
nanospheres synthesized by the Brust method. Average sphere diameter of 10 nm, scale bar
50 nm. (c) Scanning electron microscopy (SEM) image of gold nanospheres synthesized by the Xia
method. Average sphere diameter of 15 nm, scale bar 200 nm. (d) TEM images of gold nanospheres
synthesized by the Zhong method. Average sphere diameter of 30 nm, scale bar 200 nm.

1.1.2 Zhong method

The Zhong method yields water-soluble AuNPs with size in the range of 10–100 nm
diameter and dispersion down to 2–5% (Figure 1.3(d)) [20]. It is a two-step reaction
that involves the synthesis of gold seeds followed by a seeded growth process in
aqueous solutions using sodium acrylate as a reducing and capping agent. In the
first step, like the Turkevich method, HAuCl4 gold precursors are reduced by sodium
acrylate at 100 °C, yielding 15 nm diameter gold seeds. The major by-products of
the oxidation of sodium acrylate (Wacker reaction) are CH3COCO2H (pyruvic acid
or 2-oxopropanoic acid) and CHOCH2CO2H (3-oxopropanoic acid) [21]. The second
step involves a seeded and aggregative growth mechanism, as demonstrated by
Njoki and coworkers [22]. The growth of larger and monodisperse particles arise
from the reduction of Au(III) on the surface of Au seeds by a mix of acrylic acid
and sodium acrylate agents. The reaction occurs at room temperature (20 ± 0.5 °C)
6 1 Synthesis of gold nanostructures

in about three days under control of the pH and the reaction temperature. The size-
dispersion and the shape-uniformity are controlled by the Ostwald ripening
(Figure 1.4), a mechanism often considered to be the general driving force for the
growth of particles. In the Ostwald ripening, smaller particles dissolve and recrys-
tallize onto larger particles, triggering the growth of larger particles from smaller
ones [23].

Figure 1.4: Illustration of the seeded and aggregative growth mechanism for the controlled growth
of gold nanoparticles. In the central picture the coalescence of the smaller particles on the larger
one is shown. Reproduced with permission of P.N. Njoki, I-I.S. Lim, D. Mott, H-Y. Park, B. Khan,
S. Mishra, R. Sujakumar, J. Luo and C-J. Zhong, Journal of Physical Chemistry C, 2007, 111, 14664.
©2007, American Chemical Society [20].

Using a spherical model for AuNSs with an initial seed radius r (cm) and a growth
thickness d (cm), in order to produce particles of total radius r + d (cm), the mass
balance between the grown seeds and the concentration C of AuCl4− (mM) is shown
in eq. (1.1):


4π 4π 3
N× ðr + dÞ3 − r ×ρ=C×V ×M (1:1)
3 3

where N is the number of AuNPs in the total volume, ρ is the density of bulk gold
(18.9 g/cm3), M is the molecular weight of Au (197 g/mol), C is the concentration of
Au precursor (mol/l), and V is the total reaction volume (L) [24]. From eq. (1.1) is
derived the d/r expression of eq. (1.2):
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
d 3 C×V
= 1 + 2.49 × −1 (1:2)
r N × r3

The desired monodispersed AuNSs can be achieved by using the eq. (1.2) to finely
control the molar ratio of the reactants. In summary, the Zhong method shows some
peculiar interesting features: (i) very narrow size-dispersion, (ii) high throughput
production, (iii) reproducibility, and (iv) good control over the geometry of AuNSs.
On the other hand, the reaction is time-consuming and the success of the process is
strictly related to the control of the reaction temperature.
 1.2 Gold nanoparticles 7

1.1.3 Brust method

The strategy proposed by Brust (Brust method) consists of growing the metallic nano-
spheres with the simultaneous attachment of self-assembled thiol monolayers on the
growing nuclei [25]. In order to allow the surface reaction during metal nucleation
and growth, the particles are produced in a two-phase system. In this method, AuCl4−
is transferred from an aqueous solution to toluene using tetraoctylammonium bro-
mide as the phase-transfer reagent and reduced with aqueous sodium borohydride in
the presence of dodecanethiol (C12H25SH). Upon addition of the reducing agent, the
organic phase changes color from orange to deep brown within a few seconds. The
overall reaction is summarized in eq. (1.3):

AuCl4 − ðaqÞ + NðC8 H17 Þ4 + ðC6 H5 MeÞ ! NðC8 H17 Þ4 + AuCl4 − ðC6 H5 MeÞ

mAuCl4 − ðC6 H5 MeÞ + nC12 H25 SHðC6 H5 MeÞ + 3 me − ! 4 mCl − ðaqÞ + (1:3)

ðAum ÞðC12 H25 SHÞn ðC6 H5 MeÞ

where the source of electrons is BH4−. The result of the reaction is determined by the
ratio of thiol to gold, that is, the ratio n/m. This single reaction yields a surface-
functionalized gold colloid in the range of 2–8 nm diameter with a dispersion of about
4–6% (Figure 1.3(b)). The kinetics of AuNSs growth is determined by the thiol surface
coverage, and, as usual in two-phase reaction, AuNSs size can be mainly controlled by
the reaction conditions. The major drawback of this reaction relies on the difficulty of
growing uniform and regular-shape nanostructures with diameter >25 nm. Also, this
reaction is performed in organic media, resulting in AuNSs that requires further steps
to obtain water-soluble nanoparticles useful for bioapplications [26].

1.2 Gold nanoparticles

1.2.1 Gold Nanorods

The first approach to the synthesis of gold nanorods, based on an electrochemical

route within reverse micelles, was introduced by Wang and coworkers (Figure 1.5) [27].
The synthesis is carried out in an electrochemical cell with two 3.0 cm × 1.0 cm ×
0.05 cm electrodes: the sacrificial anode is formed by a plate of gold, and the cath-
ode is formed by a plate of platinum. The electrodes are immersed in an electrolytic
solution containing a mix of two cationic surfactants, hexadecyltrimethylammo-
nium bromide (CTAB), and a small amount of a more hydrophobic cationic surfac-
tant, tetradodecylammonium bromide, which acts as a rod-inducing cosurfactant.
CTAB is used to both support the electrolyte solution and as a stabilizer for the final
colloid, in order to prevent aggregation phenomena. An appropriate amount of
8 1 Synthesis of gold nanostructures

(a) (b)
VA

Figure 1.5: (a) Schematic diagram of the set-up for preparation of gold nanorods via the
electrochemical method containing: (i) VA (volt-ampere), power supply, (ii) G, glass
electrochemical cell, (iii) T, Teflon spacer, (iv) S, electrode holder, (v) U, ultrasonic cleaner, (vi) A,
anode, (vii) C, cathode. (b) TEM micrographs of Au nanorods with different aspect ratios: 2.7 (top)
and 6.1 (bottom). Scale bars represent 50 nm. Reproduced with permission of J. Pérez-Juste,
I. Pastoriza-Santos, L.M. Liz-Marzán and P. Mulvaney, Coordination Chemistry Reviews, 2005, 249,
1870. ©2005, Elsevier [28].

acetone and cyclohexane are usually added to the electrolytic solution in order to
promote the inclusion of the cosurfactant into the CTAB micelles, which support
their elongation [29]. A typical electrolysis process (Figure 1.5) is conducted at a cur-
rent intensity of 3 mA for 30′. During the synthesis, the anode in gold is consumed,
to form AuBr4−. Anions are complexed by the cationic surfactants and migrate to
the cathode where the reduction to metal gold nanorods occurs. At present, it is un-
clear whether nucleation occurs on the cathode surface or within the micelles. A
key factor to control the aspect ratio of the Au nanorods is the presence of a silver
plate inside the electrolytic solution, which is gradually immersed behind the cath-
ode. The redox reaction between gold ions generated from the anode and silver
metal leads to the formation of silver ions. Wang and coworkers have found that
the concentration of silver ions and their release rate control the length of the nano-
rods [27]. The complete mechanism, as well as the role of the silver ions, is still
unknown.
Nowadays, the seeded growth method is the most used process to synthesize
gold nanorods in water. In this two-step method, the primary nuclei are prepared
by borohydride reduction of HAuCl4 in the presence of CTAB as capping agent, in
order to form 3.5 nm diameter gold seeds [30]. In addition, the growing step is car-
ried out in aqueous media and in presence of CTAB. It is worth to notice that
 1.2 Gold nanoparticles 9

potential secondary nucleation during the growing stage is inhibited by carefully

controlling the growth conditions, and in particular by using ascorbic acid as a
weak reducing agent. Indeed, ascorbic acid cannot reduce gold salts in the presence
of micelles if gold seeds are not present. Interestingly, the aspect ratio of gold nano-
rods is easily tunable by controlling the growth conditions (Figure 1.6).

100 nm

Figure 1.6: TEM images of gold nanorods indicate an average length and width of 48.1 ± 5.5 nm and
14.3 ± 2.2 nm, respectively. Reproduced with permission of N.J. Durr, T. Larson, D.K. Smith,
B.A. Korgel, K. Sokolov and A. Ben-Yakar, Nano Letters, 2007, 7, 941. ©2007, American Chemical
Society [31].

Aspect ratio as well as the monodispersity and reaction yield are dependent on sev-
eral factors, among which the stability of the seed, the reaction temperature, the con-
ductance of the water, and the kind/concentration of the surfactant. Furthermore,
the addition of AgNO3 to the growing solution influences the mechanism of nano-
structure formation, and thus the yield, the aspect ratio, and also the crystal structure
of the resulting gold nanorods [28]. In this regard, a very interesting guide on “how
to produce the desired nanorods” comprehensive of many tips and tricks was re-
cently reported by Scarabelli et al. [32].
The mechanism of formation of rod-shaped nanoparticles in aqueous surfactant
media still remains mostly unclear [28]. Based on the idea that CTAB absorbs onto
gold nanorods in a bilayer moiety, with the trimethylammonium head-groups of the
first monolayer on the surface of the nanoparticle, it was proposed that the CTAB
head-group preferentially binds to the crystallographic faces of the sides of the rods
rather than the faces at the tips [33, 34]. Thus, the syntheses of gold nanorods are
governed by preferential adsorption of CTAB to different crystal faces during the
growth, rather than acting as a soft micellar template [34].
Gao and coworkers reported on the geometrical dependence of the rods from
CTAB analogues, in which the length of the hydrocarbon tails was varied keeping the
head-group and the counterion constant [35]. The length of the surfactant tail resulted
10 1 Synthesis of gold nanostructures

critical to control the length of the nanorods and the reaction yield, that is, the shorter
chain lengths produce shorter nanorods in low yields and vice versa. The seeds are
another fundamental parameter to take into account. For example, the yield of nano-
rods prepared by CTAB capped seeds is higher than from citrate-stabilized seeds.
Moreover, the lower the amount of seed added, the higher the aspect ratio of the
nanorods formed. It is important to remember here that an increase in the aspect
ratio of nanorods implies a red-shift of the localized surface plasmon resonances
(LSPRs). In summary, the general observations over gold nanorods production are as
follows [28]: (i) the yield of rods improves with increasing colloidal stability of the
seeds, thus dimers or aggregates are not precursors of rods, (ii) the length of the sur-
factant tail increases the yield and the aspect ratio of the nanorods, (iii) an increase
in the ionic strength produces a decrease in the yield of rods, (iv) the aspect ratio can
be controlled through seeds to HAuCl4 ratio in the growing solution, that is, an in-
crease in the amount of seeds solution means a decrease in the aspect ratio of the
rods, (v) both AuCl4− and AuCl2− are quantitatively adsorbed to CTAB [36], and (vi) the
higher the curvature of the gold surface is, the faster is the rate of growth. The redox
reactions between ascorbic acid and HAuCl4 in the presence of CTAB (and the subse-
quent dismutation of Au+) can be described by eqs. (1.4)–(1.7):

AuCl4 − + 2 e − $ AuCl2 − + 2 Cl − (1:4)

3 AuCl2 − $ AuCl4 − + 2 Au0 + 2 Cl − (1:5)

AuCl2 − + e − $ Au0 + 2 Cl − (1:6)

AuCl2 − ðCTABÞ + Aum $ Aum + 1 ðCTABÞ + CTAB + 2 Cl − (1:7)

Ascorbic acid reduces Au(III) to Au(I) in the presence of CTAB (eq. (1.4)). However,
the dismutation of AuCl2− does not occur (eq. (1.5)) in the absence of gold seeds.
Consequently, the reduction of Au(I) should proceed through electron transfer at
the surface of the electron-rich gold seeds (eq. (1.6)). Then the total redox process
can be described by eq. (1.7).
The presence of silver nitrate allows better control of the shape of gold nano-
rods, even if the mechanism by which Ag+ ions modify the metal nanoparticle
shape is not fully understood. It has been hypothesized that Ag+ adsorbs on the
particle surface in the form of AgBr (thanks to the bromide species related to CTAB)
and hinders the growth of the AgBr-passivated crystal facets [37]. Moreover, the re-
duction process of silver ions due to ascorbic acid is prevented due to the experi-
mental conditions (pH~2.8 and room temperature) [38]. However, there is a critical
silver ion concentration, above which the aspect ratio of the nanorods decreases
again [33].
 1.2 Gold nanoparticles 11

1.2.2 Gold nanocubes and polyhedral nanocrystals

Despite their very interesting behavior, only few synthetic processes were explored
for the synthesis of gold nanocrystals with many edges in the size range of 20–80
nm [39]. The two main reactions are proposed by Wu and coworkers and by Seo
and coworkers [40, 41].
The method proposed by Wu and coworkers is a versatile process similar to the
synthesis of gold nanorods. The process is, such as in the case of rods, a two-step
seeding growth method, and the main reactants are a gold precursor (HAuCl4), ascor-
bic acid as reducing agent, and, as usual, hexadecyltrimethylammonium chloride
(CTAC) as a capping agent. By changing the reactants’ concentrations (Figure 1.7),
they were able to produce nanocrystals with a well-controlled size and morphology:
cubes, bipyramids, truncated cubes, stars, and rhombic dodecahedrons.

(a) (b) (c)

50 nm 50 nm 50 nm

(d) (e) (f)

50 nm 50 nm 50 nm

Figure 1.7: SEM images of the gold nanocrystals synthesized with shape evolution from truncated
cubic to rhombic dodecahedral structures by increasing the amount of ascorbic acid added. The
nanocrystals are: (a) truncated cubes, (b) cubes, (c) type I transitional product, (d) trisoctahedra,
(e) type II transitional product, and (f) rhombic dodecahedra. Reproduced from H.L. Wu, C.H. Kuo
and M.H. Huang, Langmuir, 2010, 26, 14, 12307. ©2010, American Chemical Society [40].

In particular, the control of the size of the nanostructures is related to the volume
of the seed solution added to the growth solution. Usually, the increase in the seed
concentration reflects a decrease in size. On the other hand, the geometry is tuned
12 1 Synthesis of gold nanostructures

by a synergistic effect in the growing solution between silver ions, bromide ions,
and concentration of ascorbic acid [42]. In particular, by changing the concentra-
tion of the latter (Figure 1.7), the kinetics and the thermodynamics of the reaction
are well-controlled because ascorbic acid promotes the growing process on specific
crystal facet of the gold seed. Moreover, the concentration of bromide ions in the
growing solution (Figure 1.8) also acts as a shape-controller [43].

100 nm 100 nm 100 nm

Polyhedral nanocrystals Nanostars Penta-branched nanocrystals

Without Br– ions 0.01 M of Br– ions 0.10 M of Br– ions

Concentration of Br– ions

Figure 1.8: Shape evolutions from polyhedral gold nanocrystals to nanostars and penta-branched
nanocrystals with increasing bromide ion concentration in the reaction solution. For the synthesis of
gold nanostars, 100 μl of 0.01 M AgNO3 was also added. Reproduced with permission of H-L. Wu, C-H.
Chen and M.H. Huang, Chemistry of Materials, 2008, 21, 110. ©2008, American Chemical Society [43].

For this reason, Wu and colleagues have used as a capping agent CTAC in the pres-
ence of NaBr (instead of CTAB), in order to strictly control the Br− to Ag+ ratio [43].
On one hand, as discussed in Section 1.2.1, silver ions are critical to the promotion
of the development of edges because Ag+ adsorbs on the nanostructure’s surface in
the form of AgBr or AgCl, and hinders the growth of the Ag+-passivated crystal fac-
ets [37]. On the other hand, the authors report that a better control in Br− ions re-
sults in a fine tuning of the shape, due to the intrinsic control performed on the
passivated facets [40].
Remarkably, the control over the reaction conditions and the concentration of
the reactive (ascorbic acids, silver ions, seeds, CTAB, and gold precursors) may result
in the production of exotic geometries, among which triangular, pod-shape, and
“branched” AuNPs (Figures 1.7 and 1.8) [40, 42, 44, 45]. These particular geometries
 1.2 Gold nanoparticles 13

are not discussed here because the synthetic processes show very slight variations
with respect to the rod- and cube-shape synthesis.
Seo and coworkers proposed another way for the synthesis of gold cubes, octa-
hedrons, and cuboctrahedrons, which use a polyol route at high temperature in the
presence of silver ions [41]. In this process, the diol is used both as a solvent and as
a reducing agent, in the presence of polyvinylpyrrolidone (PVP, a surface regulating
polymer). The use of the diol is also required due to its high boiling temperature
(for 1,5-pentanediol, PD, 197 °C). The high temperature: (i) promotes the formation
of more thermodynamically stable structures (Figure 1.9) resulting in uniform sin-
gle-crystalline products, and (ii) reduces the formation of less stable twinned par-
ticles such as decahedra and rods.
The reaction is a multi-step process in which the gold precursor is reduced from
Au(III) to Au(I) and eventually to Au0, while PD is oxidized. The high temperature in-
creases the kinetics of the gold nanocrystals formation, and the various gold intermedi-
ates generated during the reaction do not affect the final product morphology. On the
other hand, PVP concentration affects the nanocrystal size, still preserving the shape.
This behavior is probably due to the nonselective PVP binding to the surface of the
gold seeds, which results in a reduced crystal growth along all directions.
As in the case of gold nanorods, the introduction of AgNO3 may suppress the
growth of a certain face of the seeds, and greatly influence the shape and surface
structure of the final nanocrystals. The nanocrystal shape changed from octahedral
to truncated octahedral, cuboctahedral, cubic, and spherical by increasing the
amount of AgNO3 (Figure 1.10) [41].
Regarding the surface structure (pointing out that {hkl} describes the orienta-
tion of the planes in a crystal by Miller indices), an octahedron has only {111} faces
exposed to the surface. The {100} surface fraction continuously increases by in-
creasing the AgNO3 amount up to complete {100} coverage, that is, a cube. Overall,
Ag species generated from AgNO3 enhance the selective growth of {100} and/or sup-
press the growth of {111}. X-ray photoelectron spectroscopy analysis of the gold
cubes indicates that the silver species participate in the redox reaction of the gold
precursors that occurred on the seed surface. Ag+ is readily reduced to Ag0 by PD at
high temperatures, and Ag0 is oxidized again to Ag+ by a galvanic exchange reac-
tion with AuCl4−. If the silver concentration is too high and exceeds the selective
deposition condition, seed growth is completely restricted along all directions, lead-
ing to small spherical particles. Furthermore, particle edge sharpening occurred
concomitantly with particle growth (Figure 1.11). Overall, the total reaction is a
rapid seed formation, followed by an edge sharpening, and shape/size focusing by
subsequent Ostwald ripening [46].
14 1 Synthesis of gold nanostructures

(a) (b)

(c) (d)

(e) (f)

Figure 1.9: Gold nanocubes. (a) Ideal cube structure. (b, d) SEM images of the cubes. (c, d) SEM
images of cubes 45° tilted. (e, f) TEM images of the cubes. Inset in (f) is a diffraction pattern. The
bars indicate 500 nm (b, c), 200 nm (d, e), and 100 nm (f). Reproduced with permission of D. Seo,
J.C. Park and H. Song, Journal of the American Chemical Society, 2006, 128, 14863. ©2006,
American Chemical Society [41].
 1.2 Gold nanoparticles 15

Truncated Higher
Octahedron octahedron Cuboctahedron Cube polygon

AgNO3
amount
(equiv) 1/600 1/500 1/400 1/200 1/60
Surface exclusively dominantly {100} + exclusively mixture
structure {111} {111} {111} {100}

Figure 1.10: Polyhedral structures of AuNPs with respect to the amount of AgNO3 added in to the
reaction mixture. Reproduced with permission of D. Seo, J.C. Park and H. Song, Journal of the
American Chemical Society, 2006, 128, 14863. ©2006, American Chemical Society [41].

HAuCl4

Rapid seed Shape and size

Edge sharpening
generation focusing

Figures 1.11: Proposed process for the polyol-route formation of gold nanocubes. Reproduced with
permission of D. Seo, J.C. Park and H. Song, Journal of the American Chemical Society, 2006, 128,
14863. ©2006, American Chemical Society [41].

1.2.3 Gold nanocages

Xia and coworkers proposed a polyol synthetic route in which the preparation of gold
nanocages is linked to the synthesis of high quality silver nanocubes that serve as
sacrificial templates during a galvanic replacement reaction (Figure 1.12) [47].
The polyol reduction is a robust route to produce high-quality silver nanocubes
in large quantities. In a typical polyol synthesis, ethylene glycol (EG) acts as both
the solvent and reducing agent as in eq. (1.8) [48]:

2 Ag + + HOCH2 CHO + H2 O ! 2 Ag + HOCH2 COOH + 2 H + (1:8)

where the glycolaldehyde (HOCH2CHO) is formed due to the oxidation of EG by oxy-

gen from the air. The reduction typically generates seeds with different structures:
single-crystal, single-twinned, and multiple-twinned. These structures can further
grow into cubes, bipyramids, and pentagonal wires, respectively [49]. In order to
produce silver nanocubes in high yields, the general approaches are to: (i) remove
the twinned seeds by oxidative etching, or (ii) perform a fast reduction through
16 1 Synthesis of gold nanostructures

{111}
(1) (2) (3) (4) (5) (6) (7)
{100} {100}

Ag
Au/Ag
Au

Figure 1.12: Schematic illustration summarizing how hollow nanostructures with various porosities
evolve from Ag nanocubes with increasing amounts of HAuCl4 solution added to the reaction. The
major steps include the (1) initiation of replacement reaction at a specific site with the highest
surface energy; (2) continuation of the replacement reaction between Ag and HAuCl4 and the
formation of a partially hollow nanostructure; (3) formation of nanoboxes with a uniform, smooth,
homogeneous wall composed of Au/Ag alloy; (4) initiation of dealloying and corner reconstruction
of the Au/Ag nanobox; and (5, 6) continuation of dealloying and formation of an Au
nanocage–nanobox with pores in the walls. The cross-sectional views correspond to the plane
along dashed lines. Note that the size of the initial Ag nanocubes will influence the amount of
HAuCl4 solution necessary to achieve a particular morphology. Reproduced with permission of
S.E. Skrabalak, L. Au, X.D. Li and Y. Xia, Nature Protocols, 2007, 2, 2182. ©2007, Nature [48].

in situ formation or direct addition of external clusters. In the first approach, oxida-
tive etchants (e.g., Cl− ions combined with O2) are introduced to selectively elimi-
nate the twinned seeds due to the high reactivity of defects on their surface. The
remaining single-crystal seeds can lead to the growth of nanocubes with the assis-
tance of PVP, which selectively binds to the {100} facets of the silver nanostruc-
tures. The oxidative etching is typically a slow process and the synthesis usually
takes more than 10 h. The second approach is based on the formation of Ag2S clus-
ters through the addition of Na2S directly in the reaction solution during the nucle-
ation of Ag atoms. Sodium sulfide significantly increases the rate of the reduction
process. Thus, the rapid reduction effectively limits the formation of twinned seeds,
supporting the formation of thermodynamically favored single-crystal seeds, that
is, the growth of nanocubes. At the very beginning, AgNO3 was used as a precursor
for silver ions. On the other hand, silver nitrate resulted in low yield production of
silver nanocubes, because the reaction is highly sensitive to many conditions, in-
cluding salt impurities and the amount of oxygen [47]. The nitrate group is also an
oxidizing agent that may cause etching of the seeds, leading to poor reproducibility.
Moon and coworkers improved the reproducibility of the polyol synthesis of Ag
nanocubes by employing silver trifluoroacetate (CF3COOAg) as precursor instead of
AgNO3 [50]. Interestingly, the total reaction time to produce nanocubes in the range
of 30–70 nm is, not comprising the preheating of EG to 150 °C and the addition of
all the reactants, about 1 h.
Gold nanocages (Figure 1.13) are prepared via the galvanic replacement reaction
(eq. (1.9)) between Ag cubes (as sacrificial templates) and HAuCl4, which is driven by
the difference in electrochemical potential between Ag/Ag+ (0.80 ) and Au/AuCl4−
(1.00 V) [48]:
 1.2 Gold nanoparticles 17

Figure 1.13: A SEM image (insert: TEM image) of Au nanocages prepared through the galvanic
replacement reaction between Ag nanocubes and HAuCl4 aqueous solution (scale bars are 100 nm
for both images). Reproduced by permission of S.E. Skrabalak, L. Au, X. D. Li and Y. Xia, Nature
Protocols, 2007, 2, 2182. ©2007, Nature [48].

3 Ags + AuCl4 − ! Aus + 3 Ag + + 4 Cl − (1:9)

To maintain epitaxial growth for the Au atoms on the Ag nanocubes, Xia and cow-
orkers performed the reaction at 100 °C, in order to avoid any precipitation of AgCl
(solubility constant at 100 °C, Ksp100 = 1 × 10−6) that is formed during the galvanic
replacement. Once cooled down to room temperature, the AgCl solid is generally
redissolved by addition of a saturated sodium chloride (NaCl) solution, which pro-
motes the formation of a soluble coordination complex with chloride.

1.2.4 Gold nanoshell

Gold nanoshells are produced in two different types, accordingly to their final ap-
plication: hollow or filled with other materials. For the first type, the shell is built
on a sacrificial core that is subsequently removed, while the latter type has a core/
shell moiety. In both cases, the core can be composed by nanoscale materials such
as silver or cadmium sulfide colloids, silica beads, polymers, or metal-oxides such
as magnetite [51].
18 1 Synthesis of gold nanostructures

In a typical procedure, the surfaces of the templates are coated with thin layers of
the desired material (or its precursor) to form core-shell nanostructure (Figure 1.14).

Figure 1.14: (a–f) TEM images of nanoshell growth on 120 nm diameter silica nanoparticle. (a) Initial
gold colloid-decorated silica nanoparticle. (b–e) Gradual growth and coalescence of gold colloid on
silica nanoparticle surface. (f) Completed growth of metallic nanoshell. Reproduced by permission
of S.J. Oldenburg, R.D. Averitt, S.L. Westcott and N.J. Halas, Chemical Physics Letters, 1998, 288,
243. ©1998, Elsevier [52].

An example of this strategy was proposed by Oldenburg and coworkers [52]. They syn-
thesized silica/gold core/shell nanoparticles. Monodisperse silica nanoparticles were
produced by the Stöber method as dielectric cores. A layer of organosilane molecules
(3-aminopropyltriethoxysilane, APTES) were then adsorbed on to those templates.
APTES bonded to the surface of the silica nanoparticles, thanks to the silane groups,
 1.2 Gold nanoparticles 19

while the amine groups point outward as a new termination of the nanoparticle sur-
face. Subsequently, modified silica nanoparticles were coated by gold clusters (1–2 nm
in diameter). Gold clusters were adsorbed on the surface of silica nanoparticles due to
ionic interactions in a number most likely limited by interparticle Coulomb repulsion
(Figure 1.14). A subsequent sodium borohydride reduction of a gold precursor, such as
chloroauric acid, in the presence of potassium carbonate resulted in the growth of a
gold shell around the silica core, due to the catalytic effect of gold nucleation sites on
the silica nanoparticle surface (Figure 1.14).
In order to obtain hollow nanostructures from the core/shell nanoparticles, the
template-core is generally removed by calcination at high temperature in air or by se-
lective etching in an appropriate solvent [52]. It is important to notice that the size and
shape of the shell is determined by the geometry of the templates. The major problem
associated with the shell growing on templates is the polycrystalline nature of the pro-
duced nanoshell. It is worth to notice that in some cases the nanoshell is made of dis-
crete gold colloids (or domains) characterized by poor connections among themselves
(Figure 1.14). Sun and coworkers avoided this problem by developing a new process
based on template-engaged replacement reactions, which generate hollow nanostruc-
tures of noble metals with defined geometry and homogeneous, highly crystalline
walls [53].
The key reaction involved in this process is the galvanic replacement previously
discussed in Section 1.2.3 for gold nanocages (eq. (1.9)). Briefly, silver nanostructures
can be oxidized by HAuCl4 because the standard reduction potential of the AuCl4−/
Au pair (0.99 V, versus standard hydrogen electrode (SHE)) is higher than that of the
Ag+/Ag pair (0.80 V, versus SHE). The metallic gold produced by the galvanic reac-
tion is confined to the vicinity of the template surface, and it nucleates and grows
into very small particles until it evolves into a thin shell around the silver template
(Figure 1.15).
Nanoshells produced by this process usually have an incomplete structure at
the initial stage. Indeed, Sun and coworkers found that both HAuCl4 and AgCl
can continuously diffuse across gold layer until the silver template has been
completely consumed. When the reaction is refluxed at 100 °C for a certain time,
the gold shells can reconstruct their walls into highly crystalline structures due
to the Ostwald ripening process. At the same time, the surfaces of these hollow
structures are smoothened, and any holes in the incomplete shells seem closed
to form seamless shells (Figure 1.15). These gold shells have a morphology simi-
lar to that of the silver templates, and their void sizes are mainly determined by
the size of the templates. On the basis of the stoichiometric relationship shown
in eq. (1.9), the wall thickness of each gold nanoshell should be approximately
one-tenth of the lateral dimension of the corresponding silver template. As for
the synthesis of gold nanocages (Section 1.2.3), AgCl produced in this replace-
ment reaction is completely soluble in water under experimental conditions [53].
20 1 Synthesis of gold nanostructures

(a)

Ag

AuCl4– Ag+ Cl–

Au (b)
3 Ag + AuCl4– 3 Ag++ 4 Cl–+ Au

Ag

(c)

Au

Figure 1.15: Schematic illustration of the experimental procedure that generates nanoscale shells
of gold from silver templates with various morphologies. The reaction scheme is: (a) addition of
HAuCl4 to a dispersion of silver nanoparticles and initiation of the replacement reaction, (b) the
continued replacement reaction of HAuCl4 with the silver nanoparticles; (c) Depletion of silver and
annealing of the resultant shells to generate smooth hollow structures. Note that the shape of
each silver nanoparticle is essentially preserved in this template-engaged reaction. Reproduced by
permission of Y. Sun, B.T. Mayers and Y. Xia, Nano Letters, 2002, 2, 481. ©2002, American
Chemical Society [53].

It is important to notice that despite the potential of this process to produce

fine-tuned and good crystallinity nanoshells, there are two important drawbacks:
(i) nanostructures are only hollow, and (ii) the metal precursor must be reducible
by the Ag+/Ag pair.

1.2.5 Hybrid gold nanostructures

Very recently, disassembling noble metal nanoplatforms have attracted a growing

interest due to the possibility to combine theranostics features and efficient excre-
tion of the building blocks. A breakthrough in this topic was the introduction of the
ultrasmall-in-nano approach [2]. Ultrasmall-in-nano consists in the design of biode-
gradable nanocapsules comprising excretable ultrasmall nanoparticles (USNPs).
The key-advances of this approach are as follows: (i) USNPs mimic the light-matter
interactions of bigger nanoparticles when strictly packed together [54], (ii) the size
of the nanocapsules promotes accumulation in the target and cellular uptake [55],
 1.3 Methods 21

and (iii) the degradation products of the nanocapsules are quickly excretable or can
be reused from organisms for physiological functions [56]. One of the most promis-
ing material developed by the ultrasmall-in-nano approach is the family of passion
fruit-like nanoarchitectures (NAs) [54, 57, 58]. Their massive production, reproduc-
ibility, versatility, potential applications, biosafety, and biokinetics were already
demonstrated [56, 58–63].
Gold USNPs with a size of 2.8 ± 0.4 nm are usually synthesized in aqueous
environment in the presence of poly(sodium 4-styrene sulfonate) (PSS) and as-
sembled in controlled aggregates through ionic interactions with cationic poly
(L-lysine) (PL) [54]. The aggregates are then employed as templates in order to
grow silica shells of about 20 nm in thickness by employing a modified Stöber
method, obtaining NAs of about 100 nm in diameter. It is useful to remember
here the mechanism of the standard Stöber synthesis [64]. The introduction of
tetraethoxysilane (TEOS) into the reaction medium is followed by its hydrolysis
reactions, resulting in orthosilicic acid. The polymerization of orthosilicic acid
occurs when the concentration exceeds the saturation limit in ethanol (about
0.02–0.03%) [65]. The process yields, in sequence, low- to high-molecular pol-
ymers and, due to their condensation, particles of 1–2 nm in diameter. Then
nuclei increase in size following a LaMer growth pattern until their diameter
reaches a critical value of 5–7 nm, which results in their aggregation to form
solid silica nanoparticles [65]. This process continues until the concentration
of orthosilicic acid in the reaction medium exceeds the saturation limit. A sim-
ilar mechanism also happens in water solutions, but usually with longer time-
frame [66]. In the case of NAs, the composition of the silica shell around gold
arrays is related to the simultaneous presence of both amines from PL and ar-
omatic moieties from PSS [58]. Indeed, aromatic additives have been demon-
strated to be required for the composition of hollow silica microspheres [67].
TEOS is hydrolyzed to orthosilicic acid and partially adsorbed into gold USNPs
polymeric arrays during its polymerization. Then, silica nuclei of 1–2 nm read-
ily formed in solution, aggregate on the external surface of the arrays and the
remaining orthosilicic acid continues to form a complete shell until its satura-
tion limit is reached or the reaction interrupted. Interestingly, the silica shell
of the NAs does not show gold USNPs inclusion. NAs produced by the stan-
dard synthetic approach usually show a diameter of 107.6 ± 16.1 nm, a wall
thickness of 18.9 ± 2.2 nm, and a gold content of 5.9 ± 1.3% w/w.

1.3 Methods

All the following synthetic processes were personally tested by the author.
22 1 Synthesis of gold nanostructures

1.3.1 Nanoparticle characterization

The basic characterizations for geometry and stability of AuNPs are UV-Vis spec-
tra, electron microscopy, dynamic light scattering (DLS), and electrophoresis [68].
The extinction spectra of colloids are usually collected using a standard UV-Vis
double-beam spectrophotometer. Spectra have important contributions from both
absorption and scattering of light with respect to the ones collected from solutions
of organic molecules, where the absorbance dominates [69]. The spectral features
are linked to the size of the structures and to the peculiar interactions between
light and nanomaterials (see Chapter 2). In general, the principal band in AuNPs
spectra (extinction band) is the sum of absorption and scattering, due to the stim-
ulation of the LSPR, while the rising of background at short wavelength results
from Rayleigh scattering, an elastic phenomenon whose probability increases
with the frequency of light. These two effects produce the final shape of the typical
spectrum of colloids. The shape, width, and intensity of the plasmon band can
give preliminary information over the shape and dispersion of AuNPs solutions
and, if the molar extinction coefficient is known, on their concentration [70].
Electron microscopies, such as scanning electron microscopy (SEM) or trans-
mission electron microscopy (TEM) are employed to precisely determine the shape
and the size of nanoparticles. These techniques are usually quite expensive, rela-
tively time-consuming, and require immobilization of the samples on suitable sup-
ports. For SEM analysis, a drop of the colloid has to be dried on a silicon chip to be
imaged. For TEM analysis, the sample has to be dripped on a suitable TEM grid and
imaged, for aqueous samples, after at least half an hour. Lots of software, such as
the freeware ImageJ, can be employed to analyze images and evaluate, usually on
300–500 nanoparticles, the average size and the sphericity (if required) of the
nanostructures.
DLS measurements provide nanoparticles hydrodynamic diameter and zeta (ζ)
potential. In both cases, the light of a laser scattered from the particles in solution
is measured. For particle size measurements, the scattered light is usually collected
at 13° or 173° and the intensity fluctuations due to Brownian motion are determined.
Brownian motion is the stochastic movement of particles caused by random colli-
sions of the particles in the media. Smaller particles move faster than bigger ones
and the relationship between the size of a particle and its diffusivity due to
Brownian motion is governed by the Stokes–Einstein equation (see Chapter 2). As
the particles move, the constructive and destructive interference of the scattered
light will cause fluctuations in the scattered intensity. By the analysis of the time
correlation in intensity fluctuations, it is possible to obtain the size distribution of
AuNPs in the colloid.
The ζ-potential is a measure of the charge of nanoparticles. It can be considered
the potential difference between the dispersion medium and the external part of the
stationary layer of fluid complexed around the nanoparticle. In particular, this
 1.3 Methods 23

value gives an indication of the potential stability of the colloidal system (normally
colloids with values more positive than +30 mV or more negative than −30 mV are
considered stable) and it is usually dependent on the pH of the solution. The ζ-
potential is calculated by determining the electrophoretic mobility and, then, apply-
ing the Henry equation. The electrophoretic mobility is obtained by performing an
electrophoresis experiment on the liquid sample in a standard capillary cuvette and
measuring the velocity of the particles using a laser Doppler velocimetry (LDV).
Generally, an LDV collects the light scattered at 13° and combines it with the refer-
ence beam. This produces a fluctuating intensity signal where the rate of fluctua-
tion is proportional to the speed of the particles and, through the Henry equation,
to the ζ-potential value. Thus, DLS measurements provide indication about superfi-
cial changes on nanoparticles since a substitution in the coating or a reaction on
the metal surface change the total charge and/or the hydrodynamic radius of the
nanomaterial. Usually, DLS measurements are performed on 1 ml of colloidal solu-
tions buffered at specific pH and salt concentrations.
Gel-electrophoresis is an inexpensive and fast technique to measure the size/
charge ratio and the stability of nanoparticles [71, 72]. When an electric field is ap-
plied across an agarose gel, charged particles are attracted toward the electrode of
the opposite charge, and reach a limit drift velocity proportional to the electric field
and the nanoparticle motility because of the friction of the gel mesh. Thus, particles
with the same size and charge move with the same constant velocity, and the col-
loid, if stable, will be separated in different bands. Indeed, if the sample is not sta-
ble, nanoparticles aggregate and may run in a smear. If samples run in bands, the
difference in their retention time can be determined because it is inversely propor-
tional to the ratio of the charge and hydrodynamic radius (inclusive of the solvation
sphere). An increase of the radius usually results from volume increases due to a
coating substitution or to the conjugation of the nanoparticle to (bio)molecules
(dyes or proteins). In general, the larger the molecule the larger and slower will be
the nanosystem. Concerning charge variation, the total charge of functionalized
nanostructures at a given pH depends on the protonation state of the superficial
groups and of the conjugates. Finally, electrophoretic analysis is usually performed
on 0.6–2% agarose gels in TBE (trizma/borate/ethylenediaminetetraacetic acid)
buffer 0.5× and applying at the electrodes a potential difference of 90 V for 30–90
min, depending on the size and geometry of the nanoparticles.

1.3.2 Turkevich method

In the standard procedure to obtain a water colloid of AuNPs with an average diam-
eter of 20 nm, a solution of trisodium citrate (50 ml, 2.2 mM) was heated to boiling
point and a solution of HAuCl4 (1 ml, 25 mM) was added rapidly [7]. In about 25 s
the boiling solution turns from a light yellow to faintly blue. After an additional
24 1 Synthesis of gold nanostructures

70 s (approximately) the blue color suddenly changes into a brilliant red, indicating
the formation of spherical AuNPs. The solution was further refluxed for 15 min with
stirring to promote the monodispersion of the samples.

1.3.3 Brust method

An aqueous solution of hydrogen tetrachloroaurate (30 ml, 30 mM) was mixed with
a solution of tetraoctylammonium bromide in toluene (80 ml, 50 mM) [25]. The two-
phase mixture was vigorously stirred until all the tetrachloroaurate was transferred
into the organic layer and dodecanethiol (170 mg) was then added to the organic
phase. A freshly prepared aqueous solution of sodium borohydride (25 ml, 0.4 M)
was slowly added with vigorous stirring. After further stirring for 3 h the organic
phase was separated, evaporated to 10 ml in a rotary evaporator and mixed with
400 ml ethanol to remove excess thiol. The mixture was kept for 4 h at −18 °C and
the dark brown precipitate was filtered off and washed with ethanol. The crude
product was dissolved in 10 ml of toluene and again precipitated with 400 ml etha-
nol, obtaining a black solid of AuNSs with an average diameter of 10 nm.

1.3.4 Xia method

An HAuCl4 aqueous solution (1 ml, 0.5 wt%) and an aqueous solution of AgNO3
(42.5 μl, 0.1 wt%) were added to an aqueous solution of citrate (1.5 ml, 1% wt) with
stirring [10]. Note that adding the citrate solution to the HAuCl4/AgNO3 mixture solu-
tion may cause its color to change from light yellow to orange, leading to black. This
mixture was incubated for 5 min before quickly adding it to a solution of citrate
(50 ml, 1% wt) at 100 °C with vigorous stirring. The color of the reaction solution
changed quickly from colorless, to greyish blue, to purple, and finally to ruby red
within less than 1 min at a citrate concentration ranging from 0.297 to 0.00610 wt%.
The reaction solution was further refluxed for 1 h under stirring to form uniform
quasi-spherical 15 nm diameter AuNSs.

1.3.5 Zhong method

Gold seeds were prepared by refluxing for 30 min an aqueous solution of HAuCl4
(100 ml, 1 mM) with sodium acrylate (24 mM) [20]. The reaction produced a deep-
red solution (typical color for spheres with diameter from 10 to 20 nm). For
the second step all the seed solution was mixed in a solution of HAuCl4 (15 ml,
25 mM) and diluted in MilliQ water (845 ml). Acrylic acid solution (40 ml, 0.5 M)
was added, and the mixture was adjusted to pH 7. After 3 days at 22 °C with
 1.3 Methods 25

continuous stirring, the reaction produced a wine-red solution of 30 ± 1.7 nm

AuNSs at a 0.5 nM (2.92 × 1011 NPs/ml) concentration.

1.3.6 Gold nanorods

Au nanorods were synthesized using the silver ion-assisted seed-mediated method

[73]. Typically, the seed solution was prepared by the addition of HAuCl4 (0.25 ml,
0.01 M) to CTAB solution (10 ml, 0.1 M) in a 15 ml plastic tube with gentle mixing
(CTAB solution must be pre-prepared at 60 °C). A freshly prepared, ice-cold NaBH4
solution (0.6 ml, 0.01 M) was then quickly injected into the mixture, followed by
rapid inversion for 2 min. The seed solution was kept at room temperature for at least
2 h before use. To grow Au nanorods, HAuCl4 (2.0 ml, 0.01 M) and AgNO3 (0.4 ml,
0.01 M) were mixed with CTAB (40 ml, 0.1 M) in a 50 ml plastic tube. Hydrochloric
acid (0.8 ml, 1.0 M) was then added to adjust the pH of the solution to 1–2, followed
by the addition of ascorbic acid (0.32 ml, 0.1 M). Finally, the seed solution (0.096 ml)
was injected into the growth solution. The solution was gently mixed for 10 s and left
undisturbed at room temperature for at least 6 h before its pH was adjusted to 3–4 by
the addition of NaOH (0.8 ml, 1.0 M) for the storage of the final colloid.

1.3.7 Gold nanocubes

Gold nanocubes were grown following a two-step method [39]. In the first step, gold
seeds were prepared by the addition of a freshly prepared, ice-cold aqueous NaBH4 so-
lution (0.6 ml, 0.01 M) into an aqueous mixture (10 ml) composed of HAuCl4 (0.01 M)
and CTAB (0.1 M), followed by rapid mixing for 2 min (CTAB solution must be pre-
prepared at 60 °C). The resultant brownish seed solution was kept at room temperature
for 1 h before use in order to decompose the excess borohydride. The growth solution
was prepared by the sequential addition of CTAB (6.4 ml, 0.1 M), HAuCl4 (0.8 ml,
0.01 M), and ascorbic acid (3.8 ml, 0.1 M) into water (32 ml). The CTAB-stabilized seed
solution was diluted 10 times with water. The diluted seed solution was then added (in
a range from 0.02 to 0.8 ml) into the growth solution. The resultant mixture was mixed
by gentle inversion for 10 s and then left undisturbed overnight. The average edge
length of the Au nanocubes produced was from 45 to 70 nm.

1.3.8 Gold nanocubes and polyhedrons

The gold seed solution was prepared with the same procedure described in
Section 1.3.6 for the synthesis of gold nanorods, while the growing process is ex-
plained in Wu and coworkers [40]. Note that the final morphology (cubes,
26 1 Synthesis of gold nanostructures

bipyramids, truncated cubes, stars, and rhombic dodecahedral) is decided by the

amount of ascorbic acid added to the growing solution [40, 43]. Two vials were la-
beled A and B, and a growth solution was prepared in each of the two vials. First,
CTAC surfactant (0.32 g, final concentration 0.10 M) was added. Depending on the
morphology of gold nanocrystals to be synthesized, slightly different volumes of de-
ionized water (9.640–9.565 ml) were added to each vial. The vials were then kept in
a water bath at 30 °C. To both vials were added HAuCl4 solution (250 μl, 0.01 M),
AgNO3 (100 μl, 0.01 M), and sodium bromide (NaBr, 10 μl, 0.01). Finally, ascorbic
acid (75–150 μl, 0.04 M) was introduced. For example, for the synthesis of gold
nanocubes, 90 μl of ascorbic acid was used, whereas 150 μl of ascorbic acid was
added for the growth of rhombic dodecahedra. The total solution volume in each
vial was 10 ml. The color of the solution turned colorless after the addition of ascor-
bic acid, indicating the reduction of Au3+ to Au+ species. Next, 25 μl of the seed so-
lution was added to the solution in vial A with shaking until the solution color
turned light pink (∼5 s). Then 25 μl of the solution in vial A was transferred to vial B
with thorough mixing for ∼10 s. The solution in vial B was left undisturbed for
15 min for particle growth and the resulting nanostructures purified by 10 min
cycles of centrifugation at 3,000 rpm. The gold nanocubes and rhombic dodecahe-
dra have average sizes of 72 and 74 nm, respectively. To make smaller gold nano-
cubes and rhombic dodecahedra, the volume of the seed solution used needs to be
varied (e.g., for the preparation of gold nanocubes with average sizes of 40 nm,
65 μl of the seed solution were added to the growth solution in vial A).

1.3.9 Gold nanocages

The reaction procedure is a two-step method in which the first reaction serves to
produce the silver nanocubes (the sacrificial templates), and in the second gold
nanocages are produced through the galvanic replacement reaction [48].
A solution of EG (6 ml) was warmed at 150 °C for 1 h in order to allow the water
vapor to escape. Then, 80 μl of a 3 mM EG solution of Na2S were added maintaining
the heating. After 10 min the following were added to the solution: (i) 1.5 ml of a
0.67 mM EG solution of PVP 30k, and (ii) 0.5 ml of 0.28 M of EG solution of silver
nitrate. The solution turned to a green-ochre color in 15–20 min and the reaction was
quenched by stopping the heating, in order to reach room temperature. Acetone
(15 ml) was added and the solution centrifugated (2000 × g for 30 min). The precipi-
tate was dispersed in milliQ water and purified by three cycles of precipitation/redis-
persion in milliQ water by centrifugation (10 min at 9,000 rpm). At the final step the
precipitate was dissolved in 4 ml of milliQ water.
An aqueous solution of PVP (5 ml, 9 mM) was mixed with 100 µl of the as-
synthesized silver nanocubes colloid, and heated to 100 °C. After 10 min, an aque-
ous solution of gold tetrachloroaurate (0.1 M) was added at a constant rate of
 1.3 Methods 27

0.75 ml/min. By adding the gold salt, the solution varies its color from ocher to yel-
low/blue. Remember that the gold precursor volume could vary from 3 to 8 ml ac-
cording to the type of cages desired. After further 10 min at 100 °C, the solution was
left to reach room temperature, and then NaCl was added till saturation. In order to
purify the structures, the solution was centrifuged at 2,000 rpm for 30 min. The pre-
cipitate (a mix of gold nanocages and NaCl) was dispersed in water, and purified by
five cycles of centrifugation/redispersion in water.

1.3.10 Passion fruit-like nanoarchitectures (NAs)

The following protocol is standardized for the production of 1.5 mg NAs in about
4 h. The protocol can be scaled-up to 20 mg [54, 61, 62].
Synthesis of AuNPs: Ultrasmall AuNPs with a diameter of approximately 3 nm
were prepared according to the following procedure. To 20 mL of milliQ water were
added 10 μL of poly(sodium 4-styrene sulfonate) (70 kDa, 30% aqueous solution,
PSS) and 200 μL of HAuCl4 aqueous solution (10 mg/mL). During vigorously stir-
ring, 200 μL of sodium borohydride (8 mg/mL in milliQ water) was added quickly,
and the mixture was vigorously stirred for 2 min. After the addition of NaBH4, the
solution underwent some color changes until becoming brilliant orange. Before its
use the solution was aged for 10 min and employed without further purification.
Synthesis of AuNPs arrays: About 20 mL of AuNPs solution was added to a
50 mL round-bottomed flask, followed by 200 μL water solution of poly(L-lysine)
hydrobromide 15–30 kDa (PL, 20 mg/mL), and the mixture was allowed to stir for
20 min at room temperature. The as-synthesized gold aggregates were collected by
centrifugation (13,400 rpm for 3 min), suspended in 2 mL of milliQ water and soni-
cated for maximum 4 min.
Synthesis of NAs: About 70 mL of absolute ethanol followed by 2.4 mL of ammo-
nium hydroxide solution (30% in water), and 40 μL of tetraethyl orthosilicate (TEOS,
98%) were added in two 50 mL plastic Falcon tubes. The solution was allowed to stir
for 5 min at RT. About 2 mL of the AuNPs arrays previously prepared were added to
the Falcon (1 mL each) and the solution was allowed to gently shake for further 3 h.
The as-synthesized NAs were collected by 30 min centrifugation at 4,000 rpm,
washed twice with ethanol to remove unreacted precursors and suspended in 1 mL of
ethanol. A short spin centrifugation was employed to separate the structures over
150 nm from the supernatant, which was recovered as a pink-iridescent solution. The
solution containing about 1.5 mg of NAs was stored at −20 °C until the employment.
It remains usually stable for at least 1 year. Product recovery: (i) 2 min centrifugation
at 13400 rpm, (ii) remove the colorless supernatant, and (iii) add the solvent of inter-
est. The solubility of NAs in water, buffers, and physiological fluids is tested for up to
60 mg/mL.
28 1 Synthesis of gold nanostructures

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