Complexation of calix[4]arene protected gold nanoparticles with pyridinium and bipyridinium compounds

Article abstract of DOI:10.1039/c2ra21761a

Calix[4]arene and calix[4]arene/alkanethiol protected gold nanoparticles with narrow size distributions were synthesised and characterized with NMR, thermogravimetric analysis (TGA) and a transmission electron microscope (TEM). NMR and light scattering (LS) were used to study the complexation of the nanoparticles with a pyridinium modified polyethylene oxide (Pyr-PEO2k-Pyr) complexant and a small 16 carbon pyridinium compound (Pyr-C16). Results give clear evidence of complexation induced aggregation of the nanoparticles as pyridinium proton signals shift upon changing the host: guest ratio and LS shows a change from a narrow size distribution into a broad one. The addition of alkanethiols with longer dimensions than that of the calixarene derivative and the type of the complexant can be used to tune the complexation. The studies also provide evidence of induced fit complexation into calix[4]arene cavities and solution phase interdigitation (secondary monolayer formation) when the nanoparticles are complexed with Pyr-C16.

Full text of DOI:10.1039/c2ra21761a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Complexation of calix[4]arene protected gold
nanoparticles with pyridinium and bipyridinium
compounds
Cite this: RSC Advances, 2013, 3, 733
Petri M. S. Pulkkinen, Szymon Wiktorowicz, Vladimir Aseyev and Heikki Tenhu*
Calix[4]arene and calix[4]arene/alkanethiol protected gold nanoparticles with narrow size distributions
were synthesised and characterized with NMR, thermogravimetric analysis (TGA) and a transmission
electron microscope (TEM). NMR and light scattering (LS) were used to study the complexation of the
nanoparticles with a pyridinium modified polyethylene oxide (Pyr-PEO2k-Pyr) complexant and a small 16
carbon pyridinium compound (Pyr-C16). Results give clear evidence of complexation induced aggregation
of the nanoparticles as pyridinium proton signals shift upon changing the host : guest ratio and LS shows
a change from a narrow size distribution into a broad one. The addition of alkanethiols with longer
dimensions than that of the calixarene derivative and the type of the complexant can be used to tune the
complexation. The studies also provide evidence of induced fit complexation into calix[4]arene cavities and
solution phase interdigitation (secondary monolayer formation) when the nanoparticles are complexed
with Pyr-C16.
Received 19th June 2012,
Accepted 3rd November 2012
DOI: 10.1039/c2ra21761a
www.rsc.org/advances
1
0
et al. employed a well known NMR ppm shift method to
establish the guest–host interactions between nanoparticles
carrying disulfide modified tetraphosphonate cavitands and
Introduction
Nanoscale gold atom clusters, gold nanoparticles, have gained
steadfast interest from the scientific community since original
work of Brust et al. in the nineties. Gold nanoparticles can be
regarded as fascinating nanotechnological objects with many
optical, electrical and catalytic properties that are tuneable
with the size of the particles due to the quantum size effect.
Calixarenes are cyclic basket-like compounds known to be
important in supramolecular chemistry, mainly due to their
ability to engage in the guest–host interactions. The proper-
ties of calixarenes can be tuned by modification of the upper
and the lower rim, this offers a wide range of interesting
functionalities and possible applications.
In the past, gold nanoparticles have been complexed with
numerous different compounds, such as DNA and cyclodex-
11
pyridinium modified short polyethylene oxide polymers. Ha
et al. have also worked extensively with calixarene nanoparti-
1
6,12–14
cles.
The development of future nanotechnological devices via
bottom-up methodology is dependent on the targeting of
nano-sized active components to the required location in the
device. Nanoparticles inheriting the guest–host interactions
of the cavitands attached to their surface may reveal answers
to such problems in the future.
2
2
3
The aim of this study is to examine the aggregation
behavior of calixarene derivatized gold nanoparticles with
pyridinium compounds using proton NMR and light scatter-
ing methods. A combination of such methods has not been
used in the literature to quantitatively link the complexation of
the metal surface bound calixarene with the aggregation
behavior of the nanoparticles. The NMR methodology provides
important information about the mechanism of the complexa-
tion, while light scattering allows the observation and
characterization of the formed aggregates. Knowledge of the
complexation behavior is expanded by experimentation with a
polymeric complexant carrying pyridinium moieties on both
ends. The behavior with a small organic compound carrying a
single pyridinium group is also examined. Finally, a novel
method of fine-tuning the complexation with addition of
alkanethiols with varying aliphatic tail lengths is presented.
4
trin, to name just a few. Calix[4]arene protected nanoparticles
are of particular interest for nanotechnology applications due
to the fact that calix[4]arenes are among the smallest known
basket-like organic compounds with a complexation ability.
Although gold nanoparticles and calixarenes have been
known for decades, increased interest towards the scientific
field of calixarene-modified gold nanoparticles has surfaced
5
relatively recently, the main interest focusing on various
6
7
8
catalysis, sensor or molecular recognition systems. In an
UV-visible spectrometry study, Ciesa et al. observed aggrega-
tion of such nanoparticles via small organic ligands. Dionisio
9
Laboratory of Polymer Chemistry, Department of Chemistry, University of Helsinki,
P.O.Box 55, 00014 Helsinki, Finland. E-mail: heikki.tenhu@helsinki.fi
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 733–742 | 733

View Article Online
Paper
RSC Advances
temperature and poured into 3% HCl (30 ml per eq. of t-butyl
calixarene) and stirred for 30 min. The organic part was
Experimental
Materials and methods. Cetyl pyridinium chloride (Pyr-C16)
and other chemicals were purchased from Aldrich and Fluka.
Solvents were distilled and reactants dried in a vacuum
2 4
extracted with water and dried over Na SO . The crude product
was dried and MeOH was added. The precipitate was collected
by filtration. The purity was improved by recrystallization from
1
5
desiccator prior use. The starting materials t-butyl calixarene
a chloroform/MeOH mixture. Typical yield was around y70%.
1
6
and 1-tosyloxyundec-10-ene were synthesised as described
elsewhere.
1
3
H NMR (500 MHz, CDCl ) d 10.23 (4H, s), 7.08 (8H, d), 6.76
(4H, t), 4.30 (4H, broad), 3.57 (4H, broad).
The synthesis of Pyr-PEO2k-Pyr was adapted and combined
Synthesis of Calix1 was conducted by modifying an existing
1
6,17
from multiple references
and is shown in Fig. 1.
16
literature method. 1 eq. of Calix0 was mixed with 10 eq. of
In all synthesis phases, thorough vacuum drying was
performed for the hygroscopic PEO2k (polyethylene oxide,
MW 2000) polymer or its derivatives. 1 eq. PEO2k was mixed
2 3
K CO in acetonitrile (15 ml per eq. of starting calixarene). 2
eq. of 1-tosyloxyundec-10-ene was added after stirring the
mixture for 1 h at room temperature. The reaction was run for
under a N
2
atmosphere at room temperature with 20 eq.
3
days under reflux conditions. The reaction mixture was dried
in a vacuum, dissolved in dichloromethane and then extracted
with 1 M HCl and water. The product was dried with Na SO
4-toluenesulfonyl chloride and 40 eq. triethylamine in dichlor-
omethane (50 ml per eq. of PEO2k). The mixture was stirred
for 3 days and the product was purified by precipitation from
THF into diethyl ether. Further purification was performed by
passing the product through a long plug of silica (75 : 25
2
4
and evaporated to dryness. The crude product was purified
using column chromatography (SiO , CH Cl : cyclohexane
2
2
2
f
75 : 25, R 75%). Yield was typically y50%. NMR showed full
CH Cl : cyclohexane) and dried. Yield: 60%. 1 eq. of the
2
2
substitution of the two aromatic hydroxyl groups per calixar-
1
tosylated PEO2k was dissolved in DMF (15 ml per eq. of
tosylated PEO2k) with 8 eq. of NaBr. The mixture was stirred at
ene. H NMR (300 MHz, CDCl ) d 8.25 (2H, s), 7.07 (4H, d), 6.93
3
(
(
4H, d), 6.80–6.63 (4H, m), 5.82 (2H, quin.), 4.98 (2H, d), 4.33
4H, d), 4.01 (4H, t), 3.38 (4H, d), 2.07 (8H, m), 1.71 (8H, m),
40 uC for 24 h. The polymer was purified by precipitation from
THF into cold ether. Yield: 75%. Finally, 1 eq. of the
brominated PEO2k was mixed with 15 eq. of pyridine in
acetonitrile (30 ml per eq. of brominated PEO2k) and refluxed
for two days. The product was precipitated from MeOH into
cold ether and twice from MeOH into hexane. Yield: 90%.
NMR and elemental analysis both indicated 90% pyridinium
terminal group substitution. MALDI-TOF analysis indicated a
1
.5–1.3 (24H, m).
Synthesis of Calix2 was conducted by following an existing
8
literature method. Briefly, 1 eq. of Calix1 and 4 eq. of
thioacetic acid were dissolved in toluene (25 ml per eq. of
Calix1) under an argon flow. After 30 min of argon bubbling, a
catalytic amount of AIBN was added and the mixture was
refluxed overnight. Solvent was evaporated and the crude
product was dissolved in CH Cl and extracted with a
2
1
n
M difference of 169 g mol between the starting PEO2k and
2
2
the product Pyr-PEO2k-Pyr, matching the molar masses of two
concentrated NaHCO3 solution and water, and then dried
over Na SO . The product was thoroughly dried and dispersed
pyridinium groups. The polymer polydispersity, 1.03,
2
4
1
remained unchanged. H NMR (500 MHz, CDCl
3
) d 9.59 (4H,
in ethanol (poor solubility, 15 ml per eq. Calix1). Concentrated
MeOH/MeONa solution was added under vigorous stirring and
the mixture was further stirred at room temperature for 24 h.
Solvent was evaporated, the solid residue was dissolved in
CH Cl and extracted with 2 M HCl and water. After Na SO
4
d), 8.56 (2H, t), 8.12 (4H, t), 5.27 (4H, t), 4.10 (4H, t), 3.70–3.50
(180H, m).
The calixarene lower rim modification procedure is shown
in Fig. 2.
Synthesis of Calix0. 1 eq. t-butyl calixarene was mixed with
eq. of phenol and 8 eq. of AlCl in 15 ml of toluene per eq. of
2
2
2
drying and thorough drying in a vacuum, the product was
recrystallized from a chloroform/MeOH mixture. Common
reaction yields were y60%. NMR data showed full disappear-
ance of the double bond signals. Integration indicated full
6
3
t-butyl calixarene under a nitrogen flow. The reaction was run
overnight at 60 uC. The resulting mixture was cooled to room
Fig. 1 The reaction pathway to Pyr-PEO2k-Pyr.
7
34 | RSC Adv., 2013, 3, 733–742
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
Fig. 2 The reaction pathway to thiol modified calix[4]arene.
1
addition of the SH groups into the double bonds. H NMR (500
MHz, CDCl ) d 8.24 (2H, s), 7.06 (4H, d), 6.93 (4H, d), 6.80–6.63
4H, m), 4.34 (4H, d), 4.01 (4H, t), 3.39 (4H, d), 4.01 (4H, t), 2.70
was added as a single fast injection into the vigorously stirred
mixture. The mixture was stirred for 2 h, after which the
toluene phase was extracted with 0.1 M H SO , 1 M Na CO
3
(
(
2
4
2
3
4H, m), 2.07 (4H, m), 1.71–1.28 (28H, m).
and water. The toluene phase was dried with Na
filtered.
2
SO
4
and
Synthesis of AuNPs (reaction shown in Fig. 3) was
conducted as described in the literature. The synthesis was
conducted with 300 mg (0.88 mmol) of HAuCl 6 3H O.
Briefly, 3.5 eq. of tetraoctylammoniumbromide (TOAB) was
dissolved in toluene (90 ml per eq. of HAuCl ). 1 eq. of HAuCl
4
in water (20 ml per eq. of HAuCl ) was added and the mixture
1
8
4
0.6 eq. per HAuCl of Calix2 was added and the mixture was
stirred for one week at room temperature in order to obtain
maximal ligand exchange from TOAB into the thiol modified
calixarene. In order to obtain gold nanoparticles with
calixarene, calixarene/butanethiol and calixane/dodecanethiol
protection, the resulting mixture was divided into three flasks.
4
2
4
4
was stirred vigorously. After all the gold was transferred to
toluene, 14 eq. of NaBH in water (20 ml per eq. of HAuCl )
Butanethiol or dodecanethiol were added (1 eq. per HAuCl
present in the flask) to the corresponding flasks and stirred for
h at room temperature. All the samples containing gold
4
4
4
1
nanoparticles protected by calixarene, calixarene/butanethiol
and calixarene/dodecanethiol ligands were purified through
the same method presented here. NPs were precipitated by
addition of ethanol and filtered onto a glass filtration frit.
Copious amounts of ethanol and acetone were poured through
the filter. Particles were collected from the frit with chloroform
and purified and fractionated using a centrifuge. Methanol
was added to the nanoparticles dispersed in chloroform
(40 : 60 MeOH : chloroform) and centrifugation at 3773
relative centrifugal force (RCF) for 15 min precipitated the
particles. The supernatant was removed, the precipitate was
again dissolved in chloroform and centrifugation at 340 RCF
for 10 min was performed. The small precipitate consisting of
larger gold particles and aggregates was discarded and the
supernatant was mixed with methanol. Centrifugation at 8720
RCF for 20 min precipitated the particles of the desired size
Fig. 3 The synthetic pathway to calixarene (C-AuNP), calixarene/butanethiol (C/
B-AuNP) and calixarene/dodecanethiol (C/D-AuNP) protected nanoparticles.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 733–742 | 735

View Article Online
Paper
RSC Advances
range, which were collected. The particles were analyzed by
NMR and were observed to be spectroscopically pure (Fig. 4).
After purification, the gold yield was around 50%.
studies, the amount of calixarene per each nanoparticle was
determined. An accurately weighted amount of nanoparticles
were dispersed into 1 ml of deuterated chloroform. A
concentrated solution of the complexant was prepared
separately by accurately measuring a known amount of the
substance and dissolving it into a known volume of deuterated
chloroform. The amount of the complexant solution required
to achieve a desired calixarene : complexant ratio was calcu-
lated using the TGA data. A required amount of the complex-
ant solution was added into the gold nanoparticle NMR
sample tube using a micropipette. The tube was shaken for a
few minutes to mix the contents. After measuring the NMR
spectrum for one ratio, another calculated dose of the
complexant solution was added to the same NMR tube. This
cycle was repeated until the NMR spectra were recorded for all
calixarene : complexant ratios ranging from 6.0 to 0.25. The
NMR spectra of the aromatic areas of pyridinium and
calixarene were of particular interest, because these signals
are known to shift their positions upon complexation, i.e.,
alterations in their electronic environment.
Characterization
UV-vis measurements were performed using a Shimadzu UV-
1601PC spectrometer with quartz cuvettes. Samples were
diluted until a satisfactory absorption intensity (absorbance
y0.5) was obtained.
Thermogravimetry (TGA) measurements were done under a
flowing nitrogen atmosphere using Mettler-Toledo TGA850
equipment with STARe software. The temperature range was
2
1
2
5–800 uC and a heating rate of 10 uC min was used. TGA
results were used to estimate the amount of calixarene on the
particles. This data was used to calculate the calixarene and
complexant amounts to be used in the NMR studies.
For transmission electron microscopy (TEM), diluted
2
1
dispersions of nanoparticles (y1 mg ml ) were dried onto
a holey carbon grid, copper mesh TEM grids, which were then
observed using bright-field TEM on a FEI Tecnai 12 transmis-
sion electron microscope. Images were analyzed with the
ImageJ software. Over one thousand nanoparticles per sample
were included in the size distribution determination.
For the estimation of the dimensions of the compounds,
computational visualization was performed using Accelrys
Material Studio 4.2 software. Energy minimization and
molecular dynamics methods were combined with the
Discover program using a PCFF force field. The structures
were minimized with a conjugated gradient method and
subjected to a 50 ps dynamics run at 298 K with a 1 fs time
step. The radius of gyration for the PEO2k and aliphatic chain
lengths for Pyr-C16 and Calix2 were estimated. The alkyl
chains in Calix2 were assumed to have an all-trans chain
conformation due to the confinement to the gold surface. For
the PEO2k chain, the hydrodynamic radius was estimated
using the obtained radius of gyration assuming a monodis-
NMR studies for reaction products were performed either
on a Bruker 500 MHz or a Varian 300 MHz NMR system. The
mixed monolayers were detached from the gold nanoparticle
1
9,20
surface by using the iodine death reaction.
Iodine was
added to the NMR tube containing the nanoparticles and the
tube was shaken for several minutes. After allowing the tube to
stand for a few hours, the NMR spectrum was recorded and it
was observed that the broad signals from the nanoparticle
bound ligands were replaced with sharp signals originating
from the free ligands. The complexation experiments were
performed on the higher resolution Bruker 500 MHz system.
All proton spectra measurements were done in deuterated
chloroform, 100 pulses, 5 s relaxation delays. Using the TGA
2
1
perse random coil in a good solvent. The obtained data were
used only for a rough comparison with the experimental data.
Methodological aspects of dynamic light scattering (DLS)
2
2,23
can be found elsewhere.
DLS measurements were con-
ducted using a Brookhaven Instruments BI-200SM goni-
ometer, a BIC-TurboCorr digital pseudo-cross-correlator, and
a BI-CrossCorr detector, including two BIC-DS1 detectors.
Pseudo-cross-correlation functions of the scattered light
intensity were collected at 90u scattering angle with the self-
beating scheme. A BIC Mini-L30 diode laser operating at the
o
wavelength of l = 637 nm and the power of 30 mW was used
as a light source. DLS studies were performed at 20 uC. For
each sample, 4–5 correlation functions recorded during 40
min were averaged and then analyzed with an inverse Laplace
transform program CONTIN.
Results and discussion
Calixarenes with aliphatic thiols attached to two of the four
hydroxyl groups were synthesised. Disubstituted calixarene
was preferred over a tetrasubstituted one because the previous
work by Gutsche and Bauer indicates that unsubstituted OH-
1
Fig. 4 H NMR spectrum of the purified C-AuNPs. Inset shows region 5–3 ppm
in more detail.
7
36 | RSC Adv., 2013, 3, 733–742
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
2
4
groups play a significant role in the complexation behavior.
It is known that free OH-groups improve the complexation
ability the calixarenes.
Calixarene protected gold nanoparticles were synthesised
and characterized with thermogravimetric analysis, NMR
studies and transmission electron microscopy. The complexa-
tion of the nanoparticles with pyridinium compounds were
1
studied with H NMR and light scattering techniques. In this
manuscript we denote the resulting products as calixarene
only (C-AuNP), calixarene/dodecanethiol (C/D-AuNP) and
calixarene/butanethiol (C/B-AuNP) protected nanoparticles.
TGA measurements are shown in Fig. 5, where it can be
seen that the thermolysis weight losses were found to be
26.5%, 25.6% and 24.6% (¡0.33) for C-AuNP, C/D-AuNP and
C/B-AuNP, respectively. The reaction time for the alkanethiol
addition was only 1 hour and the mass loss difference between
the nanoparticles is low. However, the small decreasing trend
from C-AuNP to C/B-AuNP suggests that at least some ligand
exchange has taken place. Because the mass loss differences
are very small (less than 2% at the most), the ligand exchange
effect to the complexation ability is most likely to be also low.
Therefore the alkanethiol modified particles can effectively be
considered as C-AuNPs, but with small amounts of ligand
exchanged butanethiol or dodecanethiol added to the surface.
The effect of the minor ligand exchange is considered further
in the later sections.
Fig. 6 Transmission electron microscopy size distribution analysis. The number
of particles included in the size determination is shown in each distribution
graph. a) C-AuNP, b) C/B-AuNP and c) C/D-AuNP. d) A microscopy image of
C-AuNP.
were identified as Calix57Au635
Calix50Dod Au604 nanoparticles.
More detailed data for the nanoparticles is shown in
,
Calix50Bt Au629 and
7
A representative TEM image is shown in Fig. 6, with size
distributions of the different samples. Particles were reason-
ably monodisperse, with diameters varying between 1.5 and
6
2
5
Table 1. Data for previously prepared dodecanethiol pro-
tected nanoparticles (Dod-AuNP) synthesised in a similar
fashion is shown for comparison. The dodecanethiol footprint
4.5 nm. The mean diameter was 3.06, 3.10 and 3.11 nm (¡0.7)
for C/D-AuNP, C/B-AuNP and C-AuNP, respectively. The result
shows that alkanethiol addition had no significant effect on
the particle sizes within the experimental error. The size
distributions also seem to be almost identical.
For the NMR characterization studies, iodine was used to
detach the ligands from the nanoparticles, and integration of
2
26
size is in line with the literature data (21 Å ). The area of the
2
7
2
calix[4]arene cavity is known to be 27 Å . The Calix2 ligands
on the C-AuNP surface exhibit roughly a 2.2 times larger
footprint area than dodecanethiol protected Dod-AuNPs. The
Calix2 footprint area on the NP surface is also 1.7 times larger
than the calixarene cavity area. This indicates that the Calix2
aliphatic chains spread away from each other and from the
calixarene ring due to steric effects and that the gold surface is
much less densely packed with thiolates in the case of Calix2
than in the dodecanethiol case.
2
the overlapping calixarene/alkanethiol –CH –S and calixarene
aromatic proton signals showed that the molar composition
was 13% of butanethiol and 10% of dodecanethiol in C/B-
AuNP and C/D-AuNPs, respectively. Using TEM, TGA and NMR
data, full characterization of the nanoparticle composition was
conducted: the average C-AuNP, C/B-AuNP and C/D-AuNPs
As shown by NMR and TGA sections, some ligand exchange
has occurred, a small number of calixarenes have been
removed and replaced with alkanethiols. The extent of the
exchange is, however, so low that any effect on the complexa-
tion experiments remain negligible.
During the NMR complexation studies, the NMR signals of
calixarene bound onto the nanoparticle surface were broa-
dened due to the motional restriction of the molecules on the
surface. However, as seen from the NMR (Fig. 4), there are two
obvious doublet signals at 4.4 and 3.4 ppm. These correspond
to the methylene bridges between the aromatic rings. It is well
known that the symmetry of these signals is broken upon the
3
loss of the cone conformation. The symmetrical splitting is
Fig. 5 TGA data for the synthesised nanoparticles, C-AuNP (red), C/B-AuNP
clear evidence that the calixarenes attached to the nanoparti-
cles are indeed in the cone conformation.
(
blue) and C/D-AuNP (green).
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 733–742 | 737

View Article Online
Paper
RSC Advances
Table 1 Nanoparticle data. Diameter, gold atoms per cluster, the number of
ligand molecules (L) per gold nanoparticle
2
2
NP
Diam. (nm) Au (cluster) L (cluster) L (nm ) LF (Å )
C-AuNP
C/B-AuNP
C/D-AuNP 3.06
Dod-AuNP 3.00
3.11
3.10
635
629
605
570
57
2.18
2.23
2.22
4.55
45.8
44.9
45.1
22.0
a
50 + 7b
50 + 6
c
110
a
b
Calixarenes + butanethiol. Calixarenes + dodecanethiol, L per
2
2
nm and ligand molecule footprint (LF) in Å . All the results were
calculated using TGA, NMR and TEM data. Truncated octahedron
morphology (circumradius fit for the Archimedean solid and
nanoparticle radius observed with TEM) and gold number density of
2
3
28,29
c
5
9 nm were assumed
in the calculations. Ref 25.
Fig. 7 NMR proton spectra for C-AuNP/Pyr-C16 complexes. From bottom up:
Complexation experiments were performed by mixing
pure complexant and mixtures with an increasing calixarene : complexant ratio.
different complexants (Pyr-PEO2k-Pyr and Pyr-C16) first with
free unbound calixarene and then with calixarene bound on
nanoparticles (C-AuNP, C/B-AuNP and C/D-AuNP). In each
experiment, pyridinium proton signals shifted from the
position characteristic for the pure complexant as the
calixarene : complexant ratio was increased. The various
host–guest pairs are summarized in Table 2. As an example,
Fig. 7 shows the NMR spectra for C-AuNPs mixed with varying
amount of Pyr-C16. A significant change in the chemical shift
of pyridinium was observed. The change is most considerable
for the c-signal and smallest for the a-signals. The observed
changes show the orientation of the pyridinium group in the
complex; the c-proton is deepest inside the calixarene cavity
and thus experiences the most significant alteration in its
only for a-protons. For this sample, the b- and c-proton signals
changed so strongly that they overlapped with other signals
and were therefore not reliably observable. As seen in Fig. 7,
the complexed pyridinium signals are not separate from the
free pyridinium signals, which indicates a dynamic guest–host
equilibrium between the free and bound pyridinium.
Observing the peak position change as a function of host :
guest ratio enables the determination of a saturation point.
Once host cavities have been saturated, the observed complex
signals should move more rapidly towards the free guest signal
position upon further addition of free guest. This is shown
schematically in Fig. 9. In Fig. 8 the calixarene : complexant
ratio where the slope changes was taken as the saturation ratio
and the results are summarized in Table 2. Generally, the ppm
shift is always stronger for Pyr-C16 than for Pyr-PEO2k-Pyr.
This is rationalized by the steric inhibition of pyridiniums and
calixarene cavities by the larger polymer coil. Calixarenes
bound to NPs exhibit lower shifts than their free calixarene
counterparts, which is due to the close proximity of the
calixarene rings on the metal surface in the NP case; one guest
complexed by a cavity inhibits the complexation of other
guests into the neighbouring cavities. For the PEO2k with
pyridinium groups at both ends, the complexation ratio is
roughly half of the corresponding Pyr-C16 experiment. This is
attributed to the relatively low concentration (Ctot y 5 mg
1
0
electronic environment.
The peak multiplets also show interesting changes; at low
calixarene : complexant ratios, the multiplets are clearly
visible, but with increasing the ratio the multiplets disappear
due to peak broadening. The broadening is attributed to the
interaction of the complexant with the calixarene that has
been attached to a gold nanoparticle, i.e. the motion of the
3
0
pyridinium is restricted by the complexation.
Fig. 8 summarizes the changes in the chemical shifts for all
samples. Fig. 8a shows full calixarene : complexant ratio range
Table 2 Complexation result summary for the hosts and guests used.
Broadening: the host : guest ratio range in which the multiplicity broadening
occurs. Pure calixarene was omitted due to no bulky gold nanoparticles being
present. CCR: calculated complexation ratio (calixarenes per complexant), from
the change of the slopes seen in Fig. 8. TCR: theoretical complexation ratio
assuming that all the calixarenes present would be fully complexed. C% actual
complexation percentage
21
ml ) of the studied system (low amount of complexation
induced aggregation of nanoparticles), the random coiling of
the polymer (pyridinium hidden inside the coil), and
polymer’s larger steric blockade of the neighbouring calixar-
ene cavities. Interestingly, the host : guest ratio in which the
multiplicity broadening occurs, coincides with the calculated
saturation ratio. This supports the validity of the measure-
ments, that at low calixarene : complexant ratios the popula-
tion of free complexant compounds is high and thus
contributes to the visibility of the signal multiplicities. At a
high host : guest ratio, most of the pyridiniums are com-
plexed, which suppresses the multiplets due to the broadening
that occurs to compounds bound to a stationary surface. The
Host
Guest
Broadening CCR
TCR
C%
a
Free calixarene
Free calixarene
C-AuNP
C-AuNP
C/B AuNP
Pyr-C16
Pyr-PEO2k-Pyr
Pyr-C16
Pyr-PEO2k-Pyr
Pyr-PEO2k-Pyr
Pyr-PEO2k-Pyr
—
—
0.66
0.62
0.69
0.72
0.74
0.58
1
2
1
2
2
2
66
31
69
36
37
29
a
0.5–1.0
0.5–1.0
0.5–1.0
0.5–1.0
C/D AuNP
a
Not determined.
7
38 | RSC Adv., 2013, 3, 733–742
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
Fig. 8 Calixarene induced change in the pyridinium proton signals compared to the corresponding pure complexant signal position. a) Free calixarene : Pyr-C16; b)
free calixarene : Pyr-PEO2k-Pyr; c) C-AuNP : Pyr-C16; d) C-AuNP : Pyr-PEO2k-Pyr; e) C/B-AuNP : Pyr-PEO2k-Pyr; f) C/D-AuNP : Pyr-PEO2k-Pyr. Blue squares, green
triangles and red diamonds denote a-, b- and c-protons, respectively.
broadening range for the free calixarene complexation is not
shown in Table 2 because there is no gold nanoparticle to
induce the broadening.
(enhanced resonance stabilization) hydroxyl substituted aro-
matic ring. The proximity of the pyridinium moiety causes
significant but opposite changes in the ether substituted
aromatics. The data suggest that the calixarene cavity changes
its shape upon complexation. This observation is in line with
the previously detected induced fit behavior for calix[8]ar-
enes, and suggests that even the smallest of the calixarene
family, calix[4]arene, bound to a nanoparticle surface can
undergo an induced fit complexation.
Estimation of dimensions of the compounds indicated that
Calix2 is y1.7 nm in length. When attached to the gold core of
1.55 nm radius, the radius for the whole protected nanopar-
ticle was y3.2 nm. The Pyr-C16 chain length was estimated to
According to Table 1 and 2, for C-AuNP and Pyr-C16, on
average, 68% of the cavities of a single particle are filled at the
saturation host : guest ratio. For C-AuNP and Pyr-PEO2k-Pyr,
the corresponding scenario is 37%. The results also show that
the calculated complexation ratio is almost the same for both
C-AuNP and C/B-AuNP. This clearly shows that the addition of
butanethiol on the nanoparticles has a negligible effect on the
complexation ability of the nanoparticles. In contrast, the C/D-
AuNP complexation ratio is lower than the C-AuNP. Therefore,
addition of longer chain dodecanethiol has a detrimental
effect on the complexation ability. A likely cause is that the
long aliphatic chain inhibits complexation sterically, i.e., the
pyridinium cation cannot fully reach the electron-rich ring due
to nearby long alkanethiol chains. This suggests that the
addition of alkanethiols of varying chain length can be used to
fine-tune the sensitivity of metal surface-bound calixarenes. C/
D-AuNP and C/B-AuNP showed a similar extent of ligand
exchange upon alkanethiol addition, as discussed in the TGA
and NMR sections. Since C-AuNP (no alkanethiol) and C/B-
AuNP (butanethiol added) have almost identical complexation
ratios, the observed ligand exchange effect does not signifi-
cantly affect the complexation ability.
NMR experiments also provided details on the changes in
the chemical environment in the calixarene ring itself. Fig. 10
shows the aromatic region of the free calix[4]arene when
complexed with Pyr-PEO2k-Pyr. The effects were observable for
all complexants, but barely visible for calixarenes bound to
metal nanoparticles. Interesting opposing effects are observed
for aromatic protons upon the guest addition. Signals from the
aromatics with hydroxyl substituents show a downfield
change, which is attributed to the positively charged pyridi-
nium ring interacting with the relatively electron rich
1
4
h
be 2.0 nm and the R for the PEO2k chain was estimated to be
y4.6 nm. Assuming a full and dense complexation layer on
the nanoparticle calixarene cavities, the expected radii would
be y3.3, y5.3 and y12.5 nm for C-AuNP, C-AuNP + Pyr-C16
and C-AuNP + Pyr-PEO2k-Pyr cases, respectively. Here we do
not take into account the case where nanoparticles aggregate
via complexation.
Light scattering studies were performed in order to
estimate the size of the calixarene protected gold particles in
chloroform and to investigate possible effects of the pyridi-
nium compounds on their sizes and size distributions.
Solutions studied by other techniques in this work were dark
red due to the light absorption, practically within the whole
range of the visible light spectrum. For LS studies, nanopar-
ticle samples were diluted with chloroform down to 0.2 mg
2
1
ml
0
and a red laser (l = 637) was used to minimize the
absorption effect. The studies were performed for samples
with a Calix2 : complexant ratio of 0.25, to ensure maximal
calixarene cavity saturation.
Fig. 11 shows the distributions of the hydrodynamic radii
h h
(R ). C-AuNP (Fig. 11a) shows particles with R ranging from 2
to 20 nm with a mean of 7.6 nm. This suggests that in low
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 733–742 | 739

View Article Online
Paper
RSC Advances
Fig. 10 Calixarene aromatic region for free Calix2 when complexed with Pyr-
PEO2k-Pyr. From top to bottom, pure free Calix2 and decreasing Calix2 : Pyr-
PEO2k-Pyr ratio. Arrows show how the signal positions change with increasing
amount of complexant.
intensity overcomes the contribution of smaller but more
numerous scatterers.
Upon addition of Pyr-C16 or Pyr-PEO2k-Pyr, a drastic
increase in the particle size is observed. The upper limit of
h
the R distribution range increases up to 400 nm and 110 nm
for Pyr-C16 and Pyr-PEO2k-Pyr, respectively. A significant
portion of the observed scatterers are still of a similar size to
the C-AuNP sample, but larger scatterers emerge in addition.
h
The observed R is significantly larger than would be expected
from the size estimation for individual particles in dispersions
of low concentration: the broad distribution indicates the
presence of larger aggregates, formed by coupling of several
nanoparticles. These aggregates are also visible in the UV-Vis
spectra as a slight red shift in the surface plasmon resonance
(SPR) band, Fig. 12. NMR complexation results combined with
the observed emergence of the larger scatterers suggest that
upon Pyr-PEO2k-Pyr addition (Fig. 11c), the nanoparticles are
Fig. 9 NMR spectra of the complexes (green) and free Pyr-C16 complexant
(
red). a) At a high calixarene : complexant ratio (4.0) most of the Pyr-C16 is
complexed with the calixarene cavities. Signals from the complexed pyridinium
are broadened. b) Until the saturation calixarene : complexant ratio (0.5–1.0),
the population of free complexants increases together with the bound
complexant. At the saturation point the signal broadening is overcome by the
emerging sharp multiplets of the free complexants. c) After passing the
saturation ratio towards low (0.25) calixarene : complexant ratios, further
addition of complexant increases only the free complexant population. Signals
move closer to those of the free complexant and clear sharp multiplets of the
free Pyr-C16 become more discernible.
concentrations, C-AuNP can undergo minor aggregation. The
distributions shown in Fig. 11 are intensity weighted and the
relative contribution of the larger species to the overall
h
Fig. 11 Distribution of the hydrodynamic radii (R ): a) C-AuNPs, b) C-AuNP with
h
Pyr-C16 and c) C-AuNP with Pyr-PEO2k-Pyr. Mean and peak values of R are
shown in the inset.
7
40 | RSC Adv., 2013, 3, 733–742
This journal is ß The Royal Society of Chemistry 2013

View Article Online
RSC Advances
Paper
possible with other complexed pyridinium aliphatic chains or
into the protecting calixarene shell itself due to the strong
curvature of the surface. For the Pyr-PEO2k-Pyr complexant,
the particles are aggregated by mediating polymer chains. In
low concentrations, intra-nanoparticle polymer loops are very
probable, but due to the dynamic character of the guest–host
complexation, the chains are likely to realign themselves when
two particles are in close proximity. Upon higher concentra-
tions, the effect of polymer chain entanglement is expected to
become more prominent. The proposed complexation inter-
actions for both Pyr-C16 (a) and Pyr-PEO2k-Pyr (b) are shown
as a summary in Fig. 13. Since the solutions were studied at
low concentration, the formation of even larger aggregates was
not observed, but would be expected in solutions of higher
concentrations. A high concentration regime can not be
studied by LS, however, and this will be the subject of further
studies.
Fig. 12 UV-Vis spectra. From top to bottom: C-AuNPs (red), C-AuNPs + Pyr-C16
(
green), CnAuNPs + Pyr-PEO2k-Pyr (blue). SPR absorption maximas are 516, 518
0
and 522 nm, respectively. l = 637 nm, the incident light used in the LS
experiments.
linked together with bifunctional polymers. Interestingly, Pyr-
C16 addition (Fig. 11b) shows an even larger dispersity
shoulder in the size distribution. The expected result would
be individual nanoparticles coated with Pyr-C16, because the
compound has only one pyridinium moiety. In this case, a
similar size distribution, but of a slightly larger size, as for the
pure C-AuNP sample would be observed. Obviously this is not
the case, and the nanoparticles are clearly aggregated by the
action of Pyr-C16.
Conclusions
Calixarene protected gold nanoparticles with narrow size
distribution and a polymeric complexant Pyr-PEO2k-Pyr were
synthesised and characterized. The complexation-induced
aggregation of the calix[4]arene and calix[4]arene/alkanethiol
protected gold nanoparticles were studied with TEM, TGA, UV-
Vis, NMR and LS to yield new quantitative information about
the complexation phenomenon and links the complexation of
the calixarenes to the aggregation of the nanoparticles.
NMR studies showed a significant shift of the pyridinium
signals upon complexant addition, broadening of the complex-
ant signal multiplicities upon saturation calixarene : complex-
ant ratios and evidence of induced fit coupling for
calix[4]arene. Plotting of signal shifts against the calixarene:-
complexant ratio allowed the estimation of the saturation
point of the complexant addition. The smaller complexant,
Pyr-C16, showed higher complexation ability than polymeric
Pyr-PEO2k-Pyr. Also free calixarenes complexed more strongly
than their metal bound counterparts.
Interdigitation is a phenomenon that is observed when
alkanethiol-protected nanoparticles are dried or cast into a
3
1
film, first observed by Terrill et al. Interdigitation is also
known to occur in solution when the nanoparticle alkanethiol
shell is penetrated by an aliphatic chain with a polar group at
3
2
the end. This description is analogous with the Pyr-C16
complexant. Therefore, the observed aggregation upon the Pyr-
C16 addition is attributed to the interdigitation of the added
pyridinium aliphatic chains into the gold nanoparticle
protective shell, forming a secondary monolayer, which
facilitates the aggregation due to the propensity of the
pyridinium moieties to interact with a calixarene cavity of a
neighbouring metal nanoparticle. Interdigitation could be
AuNPs complexed with calixarenes were quickly treated
with alkanethiols. Just minute amounts of aliphatic com-
Fig. 13 Proposed aggregation of C-AuNP via complexation of a) Pyr-C16 or b) Pyr-PEO2k-Pyr with the calixarene cavity.
This journal is ß The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 733–742 | 741

View Article Online
Paper
RSC Advances
pounds bind to the particle surfaces and a small amount of
ligand exchange occurs but that has no significant effect on
the complexation ability. Addition of short alkanethiol
9 F. Ciesa, A. Plech, C. Mattioli, L. Pescatori, A. Arduini,
A. Pochini, F. Rossi and A. Secchi, J. Phys. Chem. C, 2010,
1
14, 13601–13607.
0 S. Ishihara and S. Takeoka, Tetrahedron Lett., 2006, 47,
81–184.
1
1
(butanethiol) among the calixarenes on the gold surface
1
showed no impact on the complexation ability, but the longer
dodecanethiol clearly interfered with it, suggesting the
possibility of tuning the calixarene complexation ability or
selectivity.
1 M. Dionisio, F. Maffei, E. Rampazzo, L. Prodi, A. Pucci,
G. Ruggeri and E. Dalcanale, Chem. Commun., 2011, 47,
6596–6598.
12 J.-M. Ha, A. Katz, A. B. Drapailo and V. I. Kalchenko, J. Phys.
Light scattering experiments, combined with TGA and
TEM, showed interesting results for C-AuNP nanoparticles. In
the TEM images, the average diameter of C-AuNP was found to
be 3.11 nm, with y60 calixarene groups attached per gold
nanoparticle. Complexation studies showed that at the
saturation ratio, Pyr-C16 pyridinium groups, on average,
complexed with 69% of these cavities. For polymeric Pyr-
PEO2k-Pyr the corresponding value was 36%. Light scattering
studies, confirmed with UV-Vis spectroscopy, showed that
addition of Pyr-PEO2k-Pyr induced the aggregation of the
particles. However, even larger aggregates were formed when
C-AuNP was complexed with Pyr-C16. This was rationalized by
assuming solution phase interdigitation of polar Pyr-C16
molecules, either into metal nanoparticle bound calixarene
shell itself or other Pyr-C16 aliphatic chains attached to the
electron rich calixarene cavity.
Chem. C, 2009, 113, 1137–1142.
13 J.-M. Ha, A. Solovyov and A. Katz, Langmuir, 2009, 25,
153–158.
14 J.-M. Ha, A. Solovyov and A. Katz, J. Phys. Chem. C, 2010,
114, 16060–16070.
1
1
5 C. D. Gutsche and M. Iqbal, Org. Synth., 1989, 68, 234–236.
6 R. M ´e tivier, I. Leray, B. Lebeau and B. Valeur, J. Mater.
Chem., 2005, 15, 2965–2973.
7 J. Gong, V. G. Vaidyanathan, X. Yu, T. W. Kensler, L.
A. Peterson and S. J. Sturla, J. Am. Chem. Soc., 2007, 129,
1
2101–2111.
1
8 M. Brust, D. Bethell, C. J. Kiely and D. J. Schiffrin,
Langmuir, 1998, 14, 5425–5429.
19 A. C. Templeton, M. J. Hostetler, C. T. Kraft and R.
W. Murray, J. Am. Chem. Soc., 1998, 120, 1906–1911.
20 Z. Tang, D. A. Robinson, N. Bokossa, B. Xu, S. Wang and
G. Wang, J. Am. Chem. Soc., 2011, 133, 16037–16044.
2
2
2
1 W. Burchard, Advances in Polymer Science, Springer Verlag,
Berlin Heidelberg, 1999, vol. 143, pp. 113–194.
2 B. Chu, Laser Light Scattering: Basic Principles and Practice,
Academic Press, Boston, 1991, pp. 4–318.
3 W. Sch ¨a rtl, Light Scattering from Polymer Solutions and
Nanoparticle Dispersions, Springer Verlag, Berlin
Heidelberg, 2010, pp. 1–183.
Acknowledgements
We would like to thank the FUNMAT Center of Excellence for
Functional Materials and the Academy of Finland (project no.
127329) for financial support. We also thank Janne
2
2
4 L. J. Bauer and C. D. Gutsche, J. Am. Chem. Soc., 1985, 107,
Ruokolainen and Jani Seitsonen for assistance with TEM
measurements. Sami Hietala is thanked for assistance with the
Material Studio software.
6063–6069.
5 A. M ¨a ¨a tt ¨a nen, P. Ihalainen, P. Pulkkinen, S. Wang,
H. Tenhu and J. Peltonen, ACS Appl. Mater. Interfaces,
2
012, 4, 955–964.
6 R. G. Shimmin, A. B. Schoch and P. V. Braun, Langmuir,
004, 20, 5613–5620.
7 O. Kuchma, Y. Zub and A. Dabrowski, Colloid J., 2006, 68,
21–728.
8 R. L. Whetten, J. T. Khoury, M. M. Alvarez, S. Murthy,
I. Vezmar, Z. L. Wang, P. W. Stephens, C. L. Cleveland, W.
D. Luedtke and U. Landman, Adv. Mater., 1996, 8, 428–433.
2
2
2
2
References
1
M. Brust, M. Walker, D. Bethell, D. J. Schiffrin and
R. Whyman, J. Chem. Soc., Chem. Commun., 1994, 801–802.
G. Ozin and A. Arsenault, Nanochemistry: A chemical
approach to nanomaterials, Royal Society of Chemistry,
Cambridge, 2005, pp. 265–319.
7
2
29 M. K. Corbierre, N. S. Cameron and R. B. Lennox,
Langmuir, 2004, 20, 2867–2873.
3
C. D. Gutsche, Calixarenes, Royal Society of Chemistry,
Cambridge, 1989, pp.149–181.
30 M. J. Hostetler, J. E. Wingate, C.-J. Zhong, J. E. Harris, R.
W. Vachet, M. R. Clark, J. D. Londono, S. J. Green, J.
J. Stokes, G. D. Wignall, G. L. Glish, M. D. Porter, N.
D. Evans and R. W. Murray, Langmuir, 1998, 14, 17–30.
31 R. H. Terrill, T. A. Postlethwaite, C. Chen, C.-D. Poon,
A. Terzis, A. Chen, J. E. Hutchison, M. R. Clark and
G. Wignall, J. Am. Chem. Soc., 1995, 117, 12537–12548.
32 A. Swami, A. Kumar and M. Sastry, Langmuir, 2003, 19,
1168–1172.
4
5
6
M.-C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293–346.
A. Wei, Chem. Commun., 2006, 1581–1591.
J.-M. Ha, A. Solovyov and A. Katz, Langmuir, 2009, 25,
1
0548–10553.
C. Han, L. Zeng, H. Li and G. Xie, Sens. Actuators, B, 2009,
37, 704–709.
7
8
1
A. Arduini, D. Demuru, A. Pochini and A. Secchi, Chem.
Commun., 2005, 645–647.
7
42 | RSC Adv., 2013, 3, 733–742
This journal is ß The Royal Society of Chemistry 2013