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Surface modification
of gold nanoparticles and nanoclusters
Master’s thesis
University of Jyväskylä
Department of Organic Chemisty
13.04.2016
Karolina Sokołowska
i
ABSTRACT
Gold nanoparticles are used in many beneficial technological applications in
biochemistry, medicine and electronics. Among them, monolayer protected gold
nanoclusters (MPCs) have received a significant attention in the scientific community
due to their well-defined atomic structure, which is important for fundamental studies of
nanoparticles properties and their functionalization. These particles, with a precise
number of atoms, exhibit size-dependent optical, chemical and electronic properties.
The thesis focuses on the structure, preparation, characterization, and properties of
MPCs.
For multifunctional applications, gold nanoparticles are an ideal class of compounds for
surface functionalization reactions. Incorporating various active groups into
nanoparticles’ surface opens new possibilities for broad applicability. The second part
of this thesis describes surface modification methods of gold nanoparticles and MPCs.
Typical surface modification methods are ligand exchange, chemical conjugation,
physical conjugation, and bioconjugation.
ii
PREFACE
The work presented in the thesis was carried out at Nanoscience Centre, Department of
Chemistry, University of Jyväskylä from May 2015 to November 2015.
I would like to thank my supervisors Tanja Lahtinen and Lauri Lehtovaara for
entrusting me with fascinating research topic. Their experience in the field and endless
new ideas in both theoretical and practical parts were conclusive for the success of the
work. I would also like to thank them for their guidance and for believing in me
throughout this project.
In addition, a special thank you is owed to my family and relatives for their endless
support, understanding and encouragement.
Jyväskylä, April 2016.
Karolina Sokołowska
iii
CONTENTS
ABSTRACT ...................................................................................................................... i
PREFACE ........................................................................................................................ii
CONTENTS ....................................................................................................................iii
ABBREVIATIONS ......................................................................................................... v
I LITERATURE PART .................................................................................................. 1
1 INTRODUCTION ........................................................................................................ 1
2 MONOLAYER-PROTECTED CLUSTERS ............................................................. 3
2.1 Synthetic methods ..................................................................................................... 4
2.1.1 Turkevich method .................................................................................................. 6
2.1.2 Brust-Schiffrin method .......................................................................................... 6
2.1.3 Modification of Brust-Schiffrin method ............................................................... 7
2.1.4 Other methods ........................................................................................................ 8
2.2 The Synthesis ............................................................................................................. 8
2.2.1 The Synthesis of Au144(SR)60 ................................................................................. 8
2.2.2 The Synthesis of Au25(SR)18................................................................................. 10
2.2.3 The synthesis of Au102(pMBA)44 ......................................................................... 11
2.2.4 Effect of the different synthetic parameters ...................................................... 12
2.3 Structure .................................................................................................................. 14
2.3.1 Isohedral core ....................................................................................................... 16
2.3.2 Decahedral core .................................................................................................... 18
2.3.3 Other structures ................................................................................................... 20
2.4 Unique properties of nanometre sized metal clusters .......................................... 21
2.4.1 Size dependent optical and electronic properties .............................................. 21
2.4.2 Chirality properties .............................................................................................. 23
2.4.3 Charge dependent properties .............................................................................. 24
2.4.4 Charge transfer properties .................................................................................. 25
2.4.5 Catalytic activity ................................................................................................... 26
2.5 Methods for detection and characterization of clusters ...................................... 27
2.5.1 Stability of the clusters......................................................................................... 27
2.5.2 Particle size and chemical composition .............................................................. 28
2.5.3 Determination of the molecular weight of clusters by ESI-MS and MALDIMS…….. ......................................................................................................................... 29
iv
2.5.4 Separation and purification of clusters by polyacrylamide gel electrophoresis
(PAGE) ........................................................................................................................... 30
2.5.5 Analysis of nanoparticle formation and morphology by nuclear magnetic
resonance (NMR) spectroscopy and fourier transform infrared (FT-IR)
spectroscopy ................................................................................................................... 30
3 SURFACE FUNCTIONALIZATION OF NANOPARTICLES AND
NANOCLUSTERS ........................................................................................................ 33
3.1 Ligand exchange ...................................................................................................... 36
3.1.1 Mechanism of ligand exchange ........................................................................... 37
3.1.2 Kinetics studies for ligand exchange on nanoparticles ..................................... 39
3.1.3 Effects of surface binding groups and head groups .......................................... 41
3.2 Chemical conjugation of gold nanoparticles ......................................................... 43
3.2.1 Coupling strategies ............................................................................................... 44
3.3 Physical conjugation of gold nanoparticles........................................................... 46
3.4 Bioconjugation of gold nanoparticles .................................................................... 49
4 CONCLUSION ........................................................................................................... 53
5 REFERENCES ........................................................................................................... 56
v
ABBREVIATIONS
AuNPs
gold nanoparticles
BPDT
biphenyl-4,4’ –dithiol
DCM
dichloromethane
DFT
density functional theory
DOSY
diffusion-ordered spectroscopy
FCC
face centered cubic
FTIR
Fourier transform infrared
HAuCl4
chloroauric acid
HOMO
highest occupied molecular orbital
LUMO
lowest unoccupied molecular orbital
MPCs
monolayer protected gold nanoclusters
MS
mass spectrometry
NaOH
sodium hydroxide
NH4OAc
ammonium acetate
NMR
nuclear magnetic resonance
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SPR
surface plasmon resonance
PET
phenylethanethiol
pMBA
para-mercaptobenzoic acid
TEM
transmission electron microscope
TGA
termogravimetric analysis
THF
tetrahydrofuran
TOABr
tetraoctylammonium bromide
TPDT
p-terphenyl-4,4” –dithiol
UV
ultraviolet
Vis
visible
XPS
x-ray Photoelectron Spectrometry
1
I LITERATURE PART
1 INTRODUCTION
Gold nanoparticles have been known for a long time, and they have an interesting
scientific history.1 First applications of gold nanoparticles took place over two thousand
years ago, when they were mainly used in aesthetic and medicine.2 Their colouring
properties in ceramics and fabrication of ruby glass are still utilized nowadays. The
scientific approach for studying nanoparticles was introduced by Faraday in the middle
of the 19th century. Faraday created a preparation of disperse gold colloids in a
solution.2 Since then the number of researches has increased exponentially.
Currently one of the main interest in nanoscience research are metallic nanoparticles.3,4
Among them, the nanometre-size gold nanoparticles are stable particles which are
widely employed in contemporary nanoscience studies.3 Therefore, many methods have
been developed to prepare particles with a specific size and purity.5 Phase solution
synthesis are practical method for preparation gold nanoparticles. They are easy to scale
up, and therefore a large scale production of particles is possible.5
Gold nanoparticles can be categorized into two size regimes. The first one is in the
range of subnanometre to 2 nm, and the second from 2 nm to 100 nm.2 In the early
works, particles were mainly called “colloidal golds” because of their size and the
arrangement of atoms.2 With the rise of knowledge, of particles sizes a term
“nanoparticles” was introduced, and it mainly referred to the particles in the size range
of 5-20 nm. The term “clusters” refers to smaller structures with defined numbers of
atoms.3 Over past few years, gold nanoparticles have attracted more and more attention
due to their unique properties, which enable scaling down electronic and optical
devices. Variations in electronic and optical properties hold a potential for a wide range
of applications including oscillators, transistors, sensors and switches.6 Their ability to
stabilize charge in their cores is considered an essential property for future electronics
applications.2 Additionally, they have shown a potential to be used in bioscience studies
as effective biosensors.7
The unusual physical and chemical properties of NPs differ from the corresponding
bulk materials and atoms; they rather behave like an intermediate of those.8 The
2
transition from bulk material to nanomaterial can lead to a number of changes in the
physical properties. Typical characteristics of gold nanoparticles include size-dependent
electronic, optical and chemical properties.5 As the particles get smaller the surface area
to volume ratio increases leading, to the dominance of numbers atoms which are on the
surface of the material. A main feature of nanoparticles compared to bulk properties is
that they exhibit a strong visible absorption in the optical spectrum, which is known as
located surface plasmon resonance.8 Moreover, their melting point is lower than that of
bulk materials and their charging can be quantized. The intense colour of gold
nanoparticles larger than 3 nm is caused by their surface plasmon resonance (SPR). The
plasmon band is sensitive to the size of particles and its intensity decreases as the core
size decreases, due to the loss of metallic character and the appearance of quantum size
effects.5
A crucial aspect of gold nanoparticles is their surface functionalization for
multifunctional applications.9 Surface modification reactions, where the bound ligand
can be conjugated or exchanged by the incoming molecule, is an important aspect of
gold nanoparticles. The reactions of this type are used to provide chemical
functionalities to the initially non-function nanoparticles by incorporating different
kinds of chemically active groups.9 Chemical functionality can be tailored by
introducing simple chemical groups, such as carboxylic acid, or by introducing
biomolecules, therapeutic molecules or other molecules of interest.7 In addition, most of
these properties are size-dependent and can be tuned by varying the size and shape of
gold nanoparticles.
Monolayer-protected clusters (MPCs) are a special type of nanoparticles that possess
high stability due to their protective coating formed by organic ligands. 10 These small
nanoparticles significantly differ from conventional plasmonic nanoparticles. The
electrons of metal atoms are cramped in molecular dimension and the discrete energy
level which provides various properties and therefore, they have become a fascinating
area of interest.11 The structure of MPCs is well-defined down to atomic scale which
allows direct comparison of theoretical and experimental work.12 This is crucial for
fundamental studies of the properties of nanoparticles and the mechanisms of their
functionalization.13 Therefore, noble metal clusters passivated by a monolayer of
thiolate ligands are the main focus of this thesis.
3
The next chapter of literature review discusses the area of MPCs from the physical,
chemical as well as biological point of view. For the electronics application, the most
interesting properties, are their unique electronic and optical properties such as
molecule-like energy gaps,11 high catalytic properties5 and strong photoluminescence3
are discussed. The preparation and characterization methods as well as the development
in understanding the cluster structure are also introduced.
In the last chapter of the thesis nanoparticles functionalization, with the main focus on
MPCs modification, is discussed.14 The small size, well-organised structure, in addition
to highly active surface area of MPCs enable various surface functionalization reactions
to
tune
nanoparticles’
properties,
providing
multifunctional
applications.9
Functionalized nanoclusters have already found a great practical interest in catalysis or
biosciences as an effective drugs deliverers or sensors.15,16 Because of their high surface
flexibility they can carry therapeutic chemical groups, immune-stabilisers or
translocating peptides.17 Therefore, for future applications, a fundamental understanding
of nanoparticles’ properties and modification techniques is required.
2 MONOLAYER-PROTECTED CLUSTERS
Monolayer-protected gold nanoclusters are a type of metal nanoparticles, typically
ranging from subnanometer to 2 nm in size, coated by a dense, monolayer of ligands
(e.g. thiols, phosphines, amines) (fig.1).3 The ligand layer protects the clusters from
aggregation, and influences the physical and chemical properties of the particles.6 The
interest in these clusters is due to their most typical metal–molecule interface which
turned out to be the most untypical. Additionally, the special combination of atomic
and electronic structures thus making them extremely stable.3
4
Figure 1. Schematic illustration of monolayer protected metal cluster.
In recent years among metal nanoparticles field, gold nanoclusters have become one of
the most studied metal nanoparticles in the nanoscience field. The number of researches
has increased exponentially, which opens up new, exciting opportunities for
fundamental studies and future applications. The break-thoughts of the field of MPCs
were given by Brust et al. in 1994.18 Their pioneering work was the preparation of
stable alkanethiolate protected gold clusters. After that Murray et al. introduced place
exchange reactions with another thiol ligands, which opened the new possibilities for
surface modification chemistry.19 Ligand exchange reaction, where the MPCs surface
bound thiol can be exchanged by other thiol is a powerful tool for introducing chemical
functionality to AuNPs (gold nanoparticles).20 Understanding kinetics and statistical
nature of ligand exchange reactions give rise to the new application paths.9
Nanoclusters have already found a great practical interest for applications in catalysis,
sensors and biochemistry.7 Nanoclusters are of great significance in catalysis due to
their large surface to volume ratio and high number of surface atoms.5 Their ability to
stabilize charge in the cores and to act as small capacitors is an important aspect in
electronic applications.5
2.1 Synthetic methods
One of the main challenges in the gold nanocluster field is to develop synthetic
chemistry routes which enable fabrication of monodisperse clusters.12 Control of
clusters fabrication is important in order to determine their structure and for
understanding their size-dependent properties. The size dependent properties of
nanoparticles require that the end product has a narrow size distribution. Therefore, it
still remains as a challenge in the synthetic chemistry since the current knowledge of the
5
kinetics of particle growth is quite limited. Nevertheless, a lot of work has been done
during past years, leading to remarkable progress in controlling the clusters with atomic
precision.
The nanoparticles and sub-nanometre clusters can be synthesised through “bottom-up”
and “top-down” approaches.21,22 For the top-down approach, the corresponding bulk
matter is subdivided into smaller pieces, yielding large distribution of sizes. Bottom-up
method of preparation gold nanoparticles results in defined building blocks. Generally,
the synthesis usually begins with the reduction of the metal precursor to atoms and in
the subsequent nucleation process metal clusters are formed.12 The particles are coated
with a stabilizing layer which inhibits the aggregation of the cluster core and terminates
the growth of particles.
Most of the recently used synthetic methods of nanoparticles are based on the bottomup synthetic strategies and are considered the best approach to produce size selected
clusters.21 Even though the synthetic methods have a lot of important advantages
obtaining a synthetic control still remains a challenge in MPC chemistry. Therefore,
often some additional methodology can be applied, such as the size focusing
processes.12 Those methods enable determination of the core and surface atom
rearrangement by effective control of the experimental parameters, permitting the most
stable clusters to survive the size focusing process.23 The methodologies are based on
stability of the different sized clusters. Among them, etching, aging, annealing or
ripening are based on “top-down” approaches. Currently all the size-focusing methods
are based on procedure where the smaller clusters are formed from larger ones.12A lot of
size focusing methods have been used to synthesis monodisperse metal clusters.
Although several methods for the preparation of hydrophilic and hydrophobic particles
have been published and the number is still increasing, only a few of them have shown
to be reliable and flexible to obtain a desired product. One of the most well-known
syntheses is Brust-Schiffrin method.18 The pioneering work included the preparation of
a stable monolayer protected cluster with alkane thiols. Another method is the older
Turkevich synthesis where the gold salt is reduced in hot aqueous solution by citrate,
producing water soluble particles.2 This method is considered a good one because of
high fraction of single size nanoparticles can be isolated from the synthesis. Therefore,
Brust-Schiffrin and Turkevich methods are widely known.
6
2.1.1 Turkevich method
In 1951 Turkevich et al. introduced an experimental method which involved the
reduction of tetrachloroaurate (HAuCl4) in hot aqueous solution using sodium citrate,
which acts as the reducing agent in this reaction.2 The citrate’s oxidation and
decarboxylation products stabilize the particles by terminating the growth and
preventing aggregation. The method produces water soluble particles ranging from 1520 nm and is still commonly used. They also studied the effect of reagent concentration
upon the nanoparticles’ size and distribution. It was found that by decreasing the
sodium citrate salt concentration and thus decreasing the number of stabilizing citrate
ions the larger particles were formed upon aggregation.
Even though the citrate reduction has a great number of advantages, such as nontoxicity water solubility, inexpensive reductant and low pollution level however, this
lack of stability restricts the variety of experimental conditions.2 Weak bonds between
citrate and gold particles make them unstable upon drying so large-scale manufacturing
cannot be achieved.
2.1.2 Brust-Schiffrin method
Extended stability of the particles was achieved by Brust et al. in 1994.18 They
investigated the method that included Faraday’s two-phase fabrication for gold
nanoparticles with self-assembly of thiolates on gold. Facile synthesis, simple handling,
and the rapidity of the biphasic method have had a considerable impact on the field. A
typical procedure involved transfer of gold ions from the aqueous phase by using
tetraoctylammonium bromine (TOABr) as a fast phase transfer reagent to the toluene,
and reduction of resulting polymeric gold-thiol complex with sodium borohydride
(NaBH4) in the presence of alkane thiol (fig.2). The reaction is usually completed just
after the addition of the reducing agent, due to a really high concentration of hydride in
the reducing agent which is typically NaBH4.
7
Figure 2. Syntheis of gold thiolate clusters via kinetic control.5
Originally, the Brust-Schiffrin method involved coating the gold with dodecanethiolate
as a stabilizing ligand in an equimolar ratio.18 The monolayer-protected particles are
extremely stable under drying conditions as well as in various solvents. The synthesis
gives stable, easy to isolate, purified thiolate-protected gold nanoparticles with a
diameter in the range of 1-3 nm. Another attractive feature of these nanoparticles is that
they can be used for further synthetic manipulation, including surface functionalization.
2.1.3 Modification of Brust-Schiffrin method
Discovery by Brust and Schiffrin opened up many possibilities of preparing
monodisperse nanoclusters. The method was modified, by optimizing the conditions, to
prepare self-assembled monolayers of thiols on a bulk gold surface.24 In 2004, Brust et
al. extended the synthesis to para-mercaptophenol – stabilized AuNPs,24 which rapidly
grow to different synthetic methods, which can be used to stabilize a variety of
functional thiols.24
Nowadays, a number of different particles with a precise formula Aun(SR)m can be
synthesised.12 Revised Brust-Schiffrin syntheses based on modification of reaction
conditions are currently available. The changes include ratio of thiol ligands to gold
halide salt, the number of used solvents and different gold precursor molecules. For
example, AuCl3 can be substituted in the place of AuCl4-.
The modified one-pot synthesis is carried out in polar solvents.4 In general, larger thiol
to gold ratios result is smaller average core sizes. The 3:1 thiol to gold ratio suggested
first by Schaaff et al.25 and verified by Goulet et al.26 leads to formation of particles
below 2 nm.27 Nevertheless, in the size range below 2 nm, the control of the sizes is
difficult to obtain by simply manipulating the thiol:Au ratio. Therefore, the synthesis
procedure is a combination of the initial synthesis and the post-synthesis treatments,
8
including various size-separation methods, such as chromatography, solvent
fractionation or fractional crystallization producing relatively monodisperse particles.
Based on these widely used methods, many types of synthesis have been developed.
Nowadays, the clusters with a defined formula can be isolated and modified by
changing the nature of ligands or reaction conditions.4
2.1.4 Other methods
In addition to thiol-based methods, the clusters can be synthesised with various ligands,
such as amines, phosphines or sulphides.12 The ligand plays an important role because it
influences the cluster structure, solubility, and chirality. Therefore, the ligand must be
chosen with respect to the desired properties.
The phosphine-stabilized cluster, known as a Schmid’s cluster (Au55(PPh3)12Cl6), had
long remained unique with a narrow size distribution (1.4 +/- 0.4 nm).2 The synthesis
was first introduced in 1981, and it involved the reduction of Ph3PAuCl by gaseous
B2H6 in hot toluene or benzene. The synthesis results in which Au55 cluster with a
stabilizing layer coordinated by PPh3 and Cl. The phosphine stabilized particles are
commercially available products can be used as bioconjugates.
Gold nanoparticles can be stabilized by other sulphur-containing ligands, including
xanthates, disulphides, dithiols, trithiols, and resorcinarene tetrathiols. However, the
binding affinity to the gold core is not as good as with thiols.2 Recently, the impact of
the presence of thiol and disulphide was studied on the size distribution of the gold
nanoclusters which were obtained by Shiffrin method. The results indicated that in the
presence of water, thiol is a better ligand than disulphide to produce small clusters.4 In
contrast, disulphide is more successful in the reactions without water.6
2.2 The Synthesis
2.2.1 The Synthesis of Au144(SR)60
Synthesis of Au144(SR)60 was first reported by Huifeng Qian et al. in 2009.10 They
developed a size focusing method without any post-synthesis treatments which turned
out to be difficult.3 The two-phase method involved the preparation of truly
monodisperse nanoparticles with the precise formula to be Au144(SCH2CH2Ph)60. In this
work, the first step involved the synthesis of the size focusing Au-cluster mixture by a
9
modified two-step Brust-Schiffrin method. High temperature and thiol concentration
were used to obtain monodispersity.
Even though, the method is comparatively facile and gives high yield (20%) and avoids
complicated size separation steps, the conditions of the reactions are relatively difficult
to handle. First, etching requires using high concentration of thiol, producing intense
odour. Second, due to the elevated temperature the method limits the use of low boiling
ligands.10,3
A simple and robust method was developed soon after the initial synthesis by the same
group under ambient conditions.28 Methanol was used as a solvent for the reaction and it
turned out that the size focusing process occurs after the initial formation of Au clusters,
preventing the growth of larger nanoparticles. In this one-pot synthesis, the gold salt
precursor is mixed with an excess of thiol and tetraoctylammonium bromide to form
Au(I)-SR polymers. Then, NaBH4 as a reduction agent, is rapidly added leading to
formation of two monodisperse sizes formation: Au144(SR)60 as a main product, and
Au25(SR)18 as a side product.28
Separation using different solvents has to be performed in order to remove the free
thiol residue, and evolve the core to a specific number of Au atoms. Au(I)-SR species,
which are poorly soluble, emerge as a white material and can be separated from the
desired product using dichloromethane (DCM). In addition, the main product Au144
can be easily isolated from Au25, due to a large solubility difference in acetone by
simple extraction.28 The synthesis is more convenient and simpler in comparison with
the previous two-step method. Moreover, the method’s versatility and applicability
enables it to be used with a wide range of thiols, including PhC 2H4SH and various
CnH2n+1SH (n= 4-8).
One-pot synthesis method of the pure Au144(SCH2Ph)60 nanocluster was recently
published by the Gao Li et al.29 In the synthesis the product can be obtained after
etching the reaction with polydispersed water-solvable Aun(SG)m through the
combination of ligand exchange and size focusing process. First, the synthesis includes
glutathione protected polydisperse cluster preparation, ranging from 400 nm to 1000
nm, by reducing Au(I)-SG in acetone. Subsequently, the size-mixed clusters react with
the excess of H-SCH2Ph ligand through ligand exchange process for 12h at 85 °C,
10
which leads to the polydispersed Au144(SCH2Ph)60 cluster. Then, particles are etched,
resulting in stable monodisperse clusters. The structure was determined by electrospray
ionization mass spectrometry (ESI-MS) and UV-vis spectroscopy.29 Even though the
method is not as convenient as the one described before, it still is based on ligand
exchange phenomena that will be discussed later in the thesis.
2.2.2 The Synthesis of Au25(SR)18
Au25(SR)18 cluster is the best known in the literature and the most extensively studied
MPC.12,3,30 Its small size and its extremely interesting properties, such as oxidation by
air3, photoluminescence properties3, high stability with different ligands3 and
unexpected reactivity with different types of salts30 have been the main target for
experimental investigation. However, the synthetic accessibility and isolation with good
and monodispersity have also played an important role.12 The identity of Au25 was
initially mislabelled as Au28(SG)18 and as Au38(SCH2CH2Ph)24.3 The correct assignment
of Au25(SR)18 was labelled by Tsukuda group by electrospray ionization mass
spectrometry (ESI-MS).30
Water-soluble glutathione-protected Au25(SG)18 nanoparticles were first synthesised by
Tsukuda et al.30 The synthesis involved mixing a gold salt precursor with glutathione
ligand while adding excess of aqueous sodium borohydride. The reaction was cooled
down to 0°C and conducted under vigorous stirring. The resulting polydisperse
precipitate is washed with methanol and size-fractionated by polyacrylamide gel. The
major drawback of the procedure is a relatively low yield, product polydispersity and
lengthy fractionation.
Thiolate protected Au25 was also synthesised through two-phase protocols including the
conversion of phosphine stabilized Au11(PPh3)8Cl3 cluster into thiolate protected
Au25(SG)18 via ligand exchange.14 Further improvements, used a modified version of
Brust-Shiffrin reaction for preparing functionalized thiol-capped Au25 nanocluster.32
The size-focusing was used in the growth process to evolve into a desired size of the
core. The method was also based on one-pot synthesis, which eliminated the phasetransfer agent and allows synthesis of Au25 nanoclusters with different capping thiols,
such as water-soluble and long chain thiols as well as thiols bearing a polymerizable
group.32
11
Low temperature and slow stirring conditions lead to a direct formation of Au25, thus
eliminating the formation of larger clusters.33 Moreover, it was observed that the careful
control of Au(I)-SR formation influences the product’s monodispersity. Surprisingly,
Au25 core framework is independent of surface thiolate ligands34,35. As an example, 2phenylethanethiol (-SCH2 CH2Ph), 1-dodecanethiol, 3-mercapto-2-butanol and 6mercapto-hexane (-SC12H25 ,-SC4 H10O, -SC6H13,), and bulky glutathione (glutathione,
N-acetyl-L-cystine, N-formyl-glutathione and N-acetyl-glutathione) produce the same
structure.34,35 The fluorescence properties of the MPC core, come not only from the
metal core but also from the protecting ligands. Therefore the ligands with electron rich
atoms such as –COOH or NH2 can considerably enhance fluorescence.
2.2.3 The synthesis of Au102(pMBA)44
The structure of Au102(pMBA)44 was first reported in 2007.36 However, the preparation
of Au102(pMBA)44 was obtained before as a minor component of the mixture; for the
first time the Kornberg group provided essentially pure material with a good yield. The
synthesis was based on a careful control of the ratio between the mixed water and
methanol in the presence of NaOH.37 The size control was achieved by the fractional
precipitation of clusters.
The preparation of water soluble Au102(pMBA)44 cluster is similar to the BrustSchiffrin method except that the phase transfer TOA+ ions are not needed because the
particles can be prepared in a water/methanol mixture.37 Three-to-one ratio of p-MBA
to gold is combined in water and 47% methanol resulting in the final gold concentration
of 3 mM. Following the procedure, the reduction agent NaBH4 was added in two to one
ratio of BH4- to gold and the reduction was allowed to proceed for five hours minimum
to as long as overnight. The monodispersity of the cluster was obtained by fractional
purification with methanol.37 Various analytical methods such as mass spectrometry
(MS),
UV-vis
spectroscopy,
Thermogravimetric
analysis
(TGA)
and
X-ray
Photoelectron Spectrometry (XPS) gave a consistent size with the X-ray crystal structure
measured for Au102(pMBA)44.24
Later, Salorinne et al. synthesised water soluble clusters with the core size of Au102
atoms, protected by p-MBA ligand.38 Using DOSY (Diffusion-ordered spectroscopy),
they studied, the hydrodynamic size of the cluster and found that the size of the cluster
depends on the size and nature of the counter ion of the deprotonated p-MBA ligand.
12
The experimental results were proven theoretically by DFT calculation which has
shown that the size and the choice of the counter ion affect the surface chemistry.
2.2.4 Effect of the different synthetic parameters
One of the most interesting aspects of metal clusters are their unique properties which
can be easily tailored, by using different thiol ligands with various chemical groups.27
Ligand plays an important role in MPC. It has a strong impact on nucleation and
chemical properties which directly influence the final size of the particles and the
solubility properties. The chemical group capped at the opposite end of the thiol ligand
can make the particle either hydrophilic or hydrophobic.27 The protecting ligand layer
keeps the particles from aggregation with each other, this enhances the stability of
clusters, which is strictly correlated with the surface charge. Because all thiols have
nearly the same affinity towards gold, it is worth mentioning that a place-exchange
reaction, where one protecting ligand is exchanged to another, is extremely important
for tuning the particles characteristics.27
The effect of size of the ligands on the nanoparticles’ core was studied by Tsukuda et
al.27 They proved that bulky glutathione ligand is effective for the synthesis of a wide
range of small clusters such as Au10, Au15, Au18, Au22 or Au25.30 Tsukuda et al.
demonstrated that if extremely bulky thiolate was applied it gave rise to the new surface
protecting motifs resulting in other Aun(SR)m sizes.39 Kauffman et al. approved this
finding by showing that under similar conditions the size of atomically precise cluster
was decreasing with increasing hindrance of methyl group.40
The resent studies have shown that all-thiolate capped Au25 cluster preserve the same
structure independent of the ligand type.35 The glutathione capped Au25 clusters were
studied by NMR and mass spectrometry and the results showed that the structure was
the same with the phenylethanethiolate protected Au25 clusters.35
The ligand effect occurred when Azubel et al.41 performed synthesis with
3-mercaptobenzoic acid (3-MBA). The well-known thiolate ligand in Au102(SR)44 was
para-mercaptobenzoic acid (pMBA). The small change of substituent position into 3MBA resulted in different size of uniform, water soluble, Au68 particles. Moreover, the
structure of the particle was determined, and it turned out to differs significantly from
that of Au102 species.41
13
Recently, a new approach was developed to induce size and structure transformation
and obtain new Aun(SR)m clusters.42 This approach utilizes ligand-exchange reactions
which enable control of size and structure under thermal conditions with addition of
large excess of thiol molecules. The transformation of Au25(SR)18 to Au28(SR′)20,
Au38(SR)24 to Au36(SR′)24, and Au144(SR)60 to Au133(SR′)52 was achieved. Moreover,
they confirmed that the incoming ligand is a key point in transformation chemistry, and
it should be significantly different than the original thiolate to induce the
transformation.42
Despite protecting group and its surface modification properties, the ratio between the
gold-ligand affect the final size of the gold core.23 The experimental observations have
shown that specific types of the ligands seem to be more effective in preparation of
certain core size. The final size seems to be affected by the amount of thiol that was
used. In a related work, Murray and co-workers synthesised different sizes of AuNPs
stabilized by hexanethiolate ligands by simply changing the mole ratio between ligand
and gold salt.43,44 Generally, they observed that when higher amounts of thiol were used
it gave smaller average core sizes.44 For instance, a thiol to gold ratio of 1:6 forms 4.4
nm diameter particles, whereas increasing the ratio of thiol to 3:1 leads to below 2 nm
sizes.
Schaaff et al.25 in their structural characterization studies generalized that low
temperatures, fast reductant addition and short reaction times give smaller size
particles.25 Subsequently, the nucleation and the growth process are likewise influenced
by the solution temperature which is directly correlated with the final size of the
nanoparticles. However, Sardar et al.23 investigated that the size can be reduced by
increasing temperature and thus reducing the reaction time. The rapid formation of
nuclei at higher temperature favours the nucleation and growth process leading to very
small 1.5 nm particles.23 On a note, different thiol protected clusters show different
thermal stability.23 The longer carbon chain indicates slightly higher stability.
Identification of the changes in reaction parameters and control of structural
characteristics at different stages of nanoparticles formation process, by manipulating
conditions to favour specific stages may provide an important insight into the stages of
nucleation and growth.
14
The two phase Brust-Shiffrin method of gold nanocluster synthesis opened new
possibilities of studying the mechanism of cluster formation. Even though the
mechanisms of the MPC synthesis has been studied widely, the pathways to the
formation of gold thiolate complexes from gold (III) chloride are not exactly
understood.27 Shortly, the synthesis including reduction of gold salt precursor (III) to
insoluble polymeric gold-thiol complex is accomplished by adding a specific amount of
thiol, followed by the reduction of the polymer and nanoparticles’ formation.28
Murray, in his studies assumed that the precursor species of the reaction was polymer
[AuI-SR]n which was formed upon the reduction of Au(III) to Au(I).1 Recently, Goulet
and Lenox26 showed, based on quantitative H1 NMR analyses of the two phase
synthesis, that the Au-(I) thiolate polymer is not the precursor of the reaction instead the
metal(I)-tetraoctylamonium complex halide is the relevant Au species under the
reduction with NaBH4. It was assumed that TOA+ ions affect the initial Au(I)-SR
polymer structure and modify the polymeric structure suitable for the formation of Au
clusters. Therefore, changing the reaction conditions, such as varying the ratio of thiol
to tetrachloroaurate, has an impact on ligand substitution causing changes in the core
size and structure.
More recently, Lauren et al. applied NMR techniques to study the noble metal
nanoparticles. They suggested that there are fundamental differences between the
formation pathways in the one-phase synthesis and the two-phase method.45 It was
experimentally shown that in the two-pot synthesis after the reduction of Au(III) to
Au(I) there was no evidence of metal-sulphur bond formation before addition of
NaBH4, instead TOA-[AuX2]- species were formed. Oppositely, one phase synthesis
which involved the same reagents, with the exception of phase transfer agent, the
metal–sulphur bond was observed before the introduction of NaBH4 indicating Au(I)thiolate formation which was consistent with the Murray results.1
2.3 Structure
It is widely known that the large metal nanocrystals have face centred cubic (fcc)
structures.46 The ligand packing and how the atoms are arranged in the metal core have
been intensively studied.46–51 In the MPCs X-ray and neutron diffraction techniques are
typical experimental methods to determine crystal structures of metal nanoparticles.52
The stability and chemical nature of clusters depends on cluster size and is associated
15
with the number of total valence electrons.53 In small clusters, the cluster is particularly
stable if the shell is fully filled with electrons.53 As the size increases the geometry of
the clusters becomes more relevant than the electron shell.53 The complete crystal
structure of the Aun(SR)m permits to better understand fundamental properties of the
cluster. Understanding the detailed information about how gold atoms and ligands
interact and are arranged in the cluster is highly crucial for future applications, including
signal transmittance properties, such as electron transport and electronic excitations.46
The determination of the exact structure via X-ray crystallography requires growing
single crystals which is challenging. Besides experimental determination of the
structure, DFT calculations have been commonly used to obtain more information about
clusters’ structures.54 It is worth pointing out that the electronic structure calculations
have been shown to estimate the structure successfully.55
Recently, significant progress has been achieved in the synthesis, crystal structure
determination and in the studies of physio-chemical properties of thiolate monolayerprotected gold nanoclusters. Due to high purity synthetic methods, a number of
monodisperse clusters have been obtained, including Au25, Au36, Au38, Au102 andAu144.
The seminal step in understanding the structure of thiolate–protected gold clusters was
the
structure
Au102(SR)44.24
determination
After
that,
geometry
the
of
structures
p-mercaptobenzoic
of
Au25
for
two
acid
protected,
redox
states,
Au25(SCH2CH2Ph)18- and Au25(SC2H4Ph)180, have also been determined.49,51 The crystal
structure was also supported experimentally with DFT calculations. In the recent
experimental structure determination, Qian et al. 56 and Lopez-Acevedo et al.57 obtained
the x-ray structure of a phenylethanethiolalte-protected Au38(SR)24 which was
additionally supported earlier by theoretical predictions by Jiang et al.58 and by Pei et
al.59 The nuances of these crystal structures led to theoretical prediction on the structure
of other nanoparticles including Au40(SR)24.
The abundance of different clusters indicated that certain sizes of clusters have unique
and exceptional stability. This unusual stability comes from the structure and it is
associated with the electronic shell structure.53 The shell structure is determined by the
numbers electrons. The identification of number of electrons corresponding to closed
shell in small clusters of sodium was done by Knight et al. in 1984.53
16
The structure of the Au core can be described as polyhedral geometry. Small MPCs
have icosahedral and decahedral cores.11 Both symmetries show a five-fold symmetry
axis and are constructed by regular polygonal faces. Icosahedral core is regular
polyhedron consisting of twelve vertices within each there of twenty triangular faces,
each for one vertex. The decahedral forms the junction of twelve regular pentagonal
faces and twenty vertices. Both structures are considered the most compact, symmetric
cores with complete steric protection.11
The structures of clusters differs from gold thiolate polymers made up of linear S-Au-S
bonds.50 The surface of gold atoms can bind two, one or zero sulphur atoms. The shorter
monomer units RS-Au-SR protect the Au144(SR)60 structure and longer dimer units RS(Au-SR)2 protect the Au25(SR)18. The protecting units are an important driving force to
understand the stability, chemistry and symmetry of clusters.50
2.3.1 Isohedral core
The Au25(SR)18 cluster has been the most extensively studied due to the availability of
high purity synthesis. After Zhu et al.33 group reported a high yield synthesis of Au25
clusters through kinetic control, the total structure of Au25(SCH2CH2Ph)18 was solved.49
The structure of Au25 is built up with icosahedral Au13 core which consists of one central
gold atom and twelve atoms on the vertices. The rest of the gold atoms form six -S-AuS-Au-S- units surrounding the Au13 core in the octahedral arrangement (fig. 3(1)). The
external gold atoms from the core were found to be bound to the sulphurs. The structure
exhibits unique bonding arrangement between eighteen thiolate ligands and the 24 gold
atoms (fig. 3(2)). The external gold atoms form six oligomers of –S-Au-S-Au-S- that
are capped by –SR ligands bridging between the gold atoms. 49
Surprisingly, Jin et al.4 found that the structure of Au25 turned out to be independent of
the surface thiolate ligand. All types of thiolate ligands exhibit the same UV-vis spectra,
indicating no changes in the core size. The second crystal structure of Au25 was
published by Murray et al.51 The crystal structure of the ionic form exhibits distortions
which are not observed in a neutral form. The distortions come from different motifs
bending of ligand and the ligands orientation. These structural differences are not only
caused by negative charge at the core cluster resulting from the presence of the TOA+
counter-ion. In the later work, positively charged Au25(SCH2CH2Ph)18+ was also
obtained.46,11 The anionic form can be easily oxidized to Au25(PET)180 and
17
Au25(PET)18+1. During the chemical oxidation or when the cluster is exposed to air, the
negative charge in Au13 core disappears without causing any destabilization in the
clusters. The most reasonable explanation comes with the fact that HOMO orbitals of
Au25 are located in the Au13 core and not in the surface Au-SR bonds.51,49
1
2
3
Figure 3. Core-shell structure of the Au25(PET)18 (1) space filling representation of
Au25(PET)18 nanoparticles. Au, orange; S, yellow; C, blue; H, white. (2) The view of
the Au13 core with six protecting RS–(AuSR)2 units (3) Close-up of the protecting RS–
(AuSR)2 unit.
Au144(SR)60 has unique structure and electronic properties, which can provide an
explanation for the stability and other properties.50 In 1996 Whetten and co-workers
identified the core cluster to be approximately Au-140 by laser desorption ionization
(LDI) mass spectrometry. Due to the fragmentation, the determination of the exact
molecular formula remained challenging. Tsukuda’s group by using the same
characterization technique determined the cluster to be Au144(SR)59. After that the
formula was redetermined by Murray et al. as Au144(SR)60.46 The one ligand difference
between those two formulas is perhaps due to the oxidation pre-treatment. In the
Murray’s work the cluster ionization was performed by formation of Cs+ adducts.46
18
Electronic structure calculations of Au144(SR)60 indicated that it was composed of
icosahedral Au114 core arranged into three concentric shells of 12, 42 and 60 atoms
(fig. 4).50 The core’s atom is surrounded by 30 equivalent RS-Au-SR units. The energy
binding of single unit to the core was calculated to be 2 eV. The two first shells of the
core consist of 54 atoms forming an icosahedral and 20 triangular faces. The third shell
is filled by three atoms in a bulk packing order in each of 20 triangular faces. An
interesting feature of this cluster is that it can appear in two enantiomeric isomers due
to the arrangement of the RS-Au-SR units.50 The crystal structure of Au144(SR)60
remains to be determined and will play a critical role for the future understanding of
optical properties of MPCs.
Figure 4. Core-shell structure of the Au144(PET)60. Au, orange; S, yellow; C, blue; H,
white.
2.3.2 Decahedral core
Au102(p-MBA)44 was the first reported structure for thiolate-capped nanoclusters by
Jadzinsky et al. in 2007.36 The arrangement of the atoms is similar to the Au144(SR)60
structure, however, it differed significantly from the standard model of geometries.50
The distances between surface atoms of the gold core are considered identical, which
was also observed in case of Au144.50 The protective layers of oligomers consist of short
units Au(SR)2
and long one Au2(SR)3. The core consists of Au79 and the shell
composed of a protective layer with composition Au23(pMBA)44 (fig. 5(1)). The central
gold atoms packed in a Marks decahedron are in a metallic state surrounded by 23
19
oxidized gold atoms. The 23 gold atoms belong to nineteen Au(SR)2 and two Au2(SR)3
oligomers, which are bound directly to the gold core by the thiols at both ends of the
oligomer (fig. 5(2,3)). The characteristic features of Au102 structure arise from the
“double anchoring” phenomena. Two gold atoms with two Au-S bonds are located at
the core-mantle interface. The arrangement of the atoms exhibits chirality arising from
the structure of the equatorial gold atoms and linked thiolates on the surface.36,11
1
2
3
Figure 5. Core-shell structure of the Au102(pMBA)44 (1) space filling representation of
Au102(pMBA)44 nanoparticle. Au, orange; S, yellow; C, blue; O, red; H, white. The
view of the Au79 core to nineteen Au(SR)2 and two Au2(SR)3 oligomers (2,3) Close-up
of the protecting Au2(SR)3 and Au(SR)2 oligomers.
Neqishi et al. has recently reported dedecanethiolate–protected Au130 nanocluster
synthesis following the modified Brust-Shiffrin method.3 However, the crystal structure
has not yet been obtained. The group proposed an elongated decahedral structure and
the chemical composition was revealed by the mass spectrometry studies. The X-ray
diffraction pattern of Au130(SC12H25)50 indicated that the core of Au102(SR)44 has similar
geometric structures. It was reported that the Au130 central core contains an additional
layer on Marks decahedral and consists of 105 gold atoms which are covered by 25
Au(SR)2 oligomers.3
20
2.3.3 Other structures
The high yielding synthesis60 of Au38(SCH2CH2Ph)24 has led to a successful
crystallization61 and structure determination56. The structure of Au38 significantly
deviates from a spherical and it is chiral due to the pair of enantiomeric clusters. Each
isomer contains a biicosahedral Au23 core and Au15(SR)24 shell. The shell consists of
three monomeric Au(SR)2 and six dimeric Au2(SR)3 oligomers.56 The arrangement of
the dimeric units on the bottom icosahedron is rotated relative to the top one making the
entire structure chiral. The DFT calculations by Lopez- Acevedo et al.57 showed good
agreement between the powder x-ray diffraction measurements. The structure of
Au40(SR)24 was first found in the size focusing intermediates of the Au38(SCH2CH2Ph)24
synthesis and separated by size exclusion chromatography. The structure hasn’t been
determined, either experimentally or by theoretical calculations.3 Surprisingly,
Au40(SR)24 does not exhibit as pronounced absorption peaks as Au38(SR)24 and their
optical spectra are significantly different. The differences are probably due to the
different structure assembly.
Li et al. synthesised Au99(SPh)42 through size-focusing method, and precise cluster mass
assignment and formula was obtained using ESI-MS.3 The same cluster was synthesised
with a SPh-Me ligand and consistent mass was obtained. Additionall confirmation was
obtained by thermogravimetric analysis (TGA). The UV-Vis spectrum of Au99(SR)42
indicated the absence of plasmon resonance band, therefore the cluster still remains in
the non-metallic regime.3
Zhu et al.62 first observed a 20-gold atom cluster protected by phenylethylthiolate (PET)
ligand in a size-controlled synthesis in 2009. The ultra-small structure of tertbutylbenzenethiolate protected Au20, Au20(SPh-tBu)16 was recently solved.63 The
structure features a vertex-sharing bitetrahedral Au7 kernel. Surprisingly, an octameric
ring Au8(SR)8 circles the Au7 kernel and interacts between each other through
Auring—Aukernel. The surface protecting octamer ring was observed for the first time in
nanoclusters and it might be common in smaller gold nanoclusters, such as Au18(SR)14
and Au15(SR)13. The interactions between the ring and the kernel7 make the structure
interesting compared with previously reported geometries. The gold atoms in the kernel
are not bonded to thiolate ligands from the ring therefore no covalent bonding
interaction Aukernel-S occurs. Additionally, the kernel is further protected by trimeric
staple −SR−Au−SR−Au−SR−Au−SR and two −SR−Au−SR−monomerics. However,
21
the theoretical structure prediction of Au20(SC2H4Ph)16 and Au20(SCH2Ph)16 by Jiang et
al.
64
and Zeng et al.63 differs from the experimentally determined crystal structure of
Au20(SPh-tBu)16. In the future, it remains to be found, whether the differences are caused
by ligands or by two isomeric forms of the core.3
2.4 Unique properties of nanometre sized metal clusters
The properties of nanoparticles dramatically change with decreasing core size.46 The
sub-nanometre gold nanoparticles exhibit discrete electronic structure which directly
influence on their unique optical and electronic properties which are different from large
nanoparticles.
Below, the most important properties of clusters, including optical,
catalytic, magnetic and capacitance charging energies are summarized.
2.4.1 Size dependent optical and electronic properties
A significant aspect of a smaller particles is their unique stability, which comes from
geometry effects and electronic properties of the clusters.65 The unusual stability and
abundance of clusters, derived from the geometric packing, number of electrons called
“magic numbers”, which indicate a stable cluster size. Based on the theory, the lowest
energy superatom orbital are mainly derived from gold 6s orbitals. If the numbers of
valence electrons correspond to the number of electron required to fill an electron shell
is 2, 8, 18, 34, 58, 92 or 138, then the cluster is considered as stable.53 This model is
often used to predict the stability of clusters.
The stability of thiol protected clusters is affected by the ligand layer and the electron
withdrawing nature of the thiol ligands.65 The divide and protect model may be used to
describe the stability of these clusters.11 However, it was also observed that for some of
them the rule couldn’t be applied, indicating that the geometric effects were more
important.66 Moreover, the size and geometry can be affected by the choice of the ligand
molecule.40
The optical and electronic properties of gold nanoparticles change dramatically as
function of size. The particles have a critical size for electronic state energy
quantization. The first size range is subnanometer particles with discrete electronic
orbitals and HOMO-LUMO energy gaps. The gaps can be quite large, for example, 1.3
eV for Au25(SR)18 and 0.9 eV for Au38 (SR)24. The second size range refers to larger
particles with surface plasmon resonance (SPR).11 The smaller clusters (>2 nm) do not
22
exhibit plasmon resonance due to the quantum effect. Therefore, the ultra-small clusters
display spectacular optical behaviour significantly different form the large plasmonic
particles.11
Molecular–like optical transition depends on the number of atoms forming the cluster.
The absorption spectra of very small metal clusters spectra show individual peaks that
give information about their electronic states. For example, Au25(SR)18 and Au38(SR)24
exhibit highly structured absorption bands. Absorption bands are due to a single
electron transition between quantized electronic stages.14,15,67
Recently, Mustalahti et al. studied the photodynamic properties of Au102(pMBA)44 by
using ultrafast time-resolved mid-IR spectroscopy and density functional theory
calculations in order to distinguish between molecular and metallic behavior.68
Interestingly, it was found that the cluster containing 102 atoms behaved like a small
molecule, which turned out to be in striking contrast to the Au144(SR)60 which showed
relaxation typical for metallic particles.
Au144(SR)60 is the smallest cluster to develop plasmonic response.69 Malola et al.
studied the optical properties and found out that the spectrum of the Au144 cluster is
rather featureless. Very weak but characteristic bands of Au144(SR)60 were observed at
around 540, 600 and 700 nm, appeared in the calculated spectrum as well.69 On the
contrary Weissker et al.70 demonstrated that the thiolate monolayer-protected gold
nanoclusters exhibit a broad spectrum of bands that were visible over the entire near-IR,
VIS and near UV-regions. The content of the spectra gave the information of the
quantum size effects, which helped to distinguish from bulk materials.70
Au25 exhibits interesting optical absorbance and fluorescence properties.34,67,8 The
luminescence properties of metal nanocluster come from the electronic transition
between unoccupied d bands. The absorption spectra of Au25 exhibit three transition
maxima at 670 nm, 450 nm and 400 nm called intraband, interband, and mix of
intraband and interband respectively. Intraband transition (sp->sp) is an excitation of
valence electron near the Fermi energy, which is rather low cost excitation. The
interband excitation occurs from d band to sp band.8 The electronic transition at 670 nm
corresponds to the HOMO-LUMO transition. According to the study, the absorption
bands are influenced by geometric and electronic interactions between the core and the
23
ligand resulting in a complex spectrum. Taken together, all three types of electron
transitions affect the optical absorption properties of the clusters, however in order to
fully understand the electron dynamic and properties more studies are needed on sizediscrete clusters.46
Similarly, fluorescence properties come from the metal core or the interaction between
metal core and surface ligands. The surface ligands with capability of donating electrons
to the metal core, enhance the fluorescence intensity.46,67 Recently, Hulkko et al. studied
spectroscopic properties of the Au102(pMBA)44.71 They found out the existence of
electronic band gap of 0.5 eV for Au102 which might indicate a possibility for
luminescence in IR region. Other photoluminescence observation has shown that
increasing electro-positivity of the metal core of Au25 cluster leads to a strong
fluorescence enhancement.46 Different charge states of Au25 nanoclusters display
various fluorescence contributions. Other studies were performed by Jin’s group
suggested that protecting ligand effects the fluorescence intensity.46 The charge
donating capability of ligands largely enhances the fluorescence of nanoclusters.
Therefore, the optical properties of subnanometre clusters can be tuned by controlling
the core size, charge state and the use of different stabilizing ligand layers. Decreasing
the core size of nanoclusters, the percentage of fraction of surface atoms increases,
affecting the optical properties of gold nanoclusters.7
2.4.2 Chirality properties
The optical properties of clusters were first observed in glutathione (GSH) protected
Au25 nanoclusters.4,5 The distinct circular dichroism properties were predicted to
originate from the inherent properties of the cluster or the ligand-core interactions.
Later, the same gold structure capped with different types of thiolate ligands was
studied by Wu and co-workers. Surprisingly, the obtained 1D and 2D NMR spectra
indicated no chirality from Au25(SCH2CH2Ph)18 structure.67 The results suggested that
the chirality of metal nanoclusters arise directly from the glutathione induced chiral
field, contrary to previous expectations about metal core and the ligands themselves.
The recently reported structure of Au102(pMBA)44 nanoclusters also exhibit chirality.4
Chirality can be achieved either by using chiral molecules as a protecting ligands
directly in the synthesis of metal clusters or by surface functionalization methods such
24
as ligand exchange.5 The enantiomeric studies on different sizes of optically active Dand L-penicillamine-capped gold nanoclusters pointed out that the optical activity
increases with the core size decreasing. Further, the studies indicated that gold
nanoclusters also have well-defined stereostructures, which is similar to chiral
molecules.5 The strong optical activity of the nanoclusters were explained by the chiral
core model.5
2.4.3 Charge dependent properties
Besides electronic structure, the stability of thiolate protected gold clusters is correlated
with the thiolate ligand layer, preventing it from aggregation. Generally, there are two
factors that can influence clusters stability: electronic shell closing and geometric shell
closing. However, Tsukuda et al. concluded that electronic shell closing is not a main
issue in cluster stability.5 For example, the [Au25(SC6)18]q (q = 1, 0, 1+) clusters charge
states can be easily changed by the redox reactions. Therefore, it was concluded that the
cluster stability is attributed with geometric structure. On the other hand, the structure
of Au25 is an open structure, having incomplete second shell and the number of valence
electron of anionic Au25 cluster seems to fit with the superatom model, which can
explain the stability.
The core of the particles can either possess excess or deficiency of the electrons. The
charging may come from the ligand and its charged groups, such as carboxylic acid or
amine functionalities.1,5 The charged particles suspended in the electrolyte solution are
surrounded by an ionic cloud termed the electrical double layer.
The stability of water soluble charged stabilized particles protected by carboxylic acid
or amine terminated ligands depends on their structure and pH of the system.3 Longer
ligand chains have a significantly higher stability than the shorter ones, preventing them
from aggregation. The electrolyte enhances stability and enables modification of the
system. It was observed that citrate protected clusters were detached when the solvent
was removed, leading to irreversible aggregation.2 At low pH, carboxylic acid-stabilised
nanoparticles agglomerate due to protonation and hydrogen bonding, making them
soluble in basic conditions. Whereas at high pH, the carboxylic acid groups deprotonate
and stabilise the particle dispersion through electrostatic repulsion. The electrical double
layers surrounding the particles prevent them from approaching each other and
agglomerating by attractive van der Waals forces.
25
Among the charge states of Au25 the magnetic properties were also found.5,67 The
magnetic properties were studied by Negishi group, however the unknown structure of
cluster made impossible to develop studies further. Nowadays, the magnetic properties
of Au25 can be easily tuned by controlling the charge state of the particles. 67 The charge
neutral Au25(SR)180 cluster exhibits magnetism arising from the unpaired HOMO
electron. The oxidation mechanism of Au25(SR)18- is a one electron transfer mechanism
whereas cluster reduction is composed of several steps. Reduction mechanism is
followed by a reversible desorption of thiolate anion after which the neutral cluster is
capable of accepting another electron.13 The desorption of thiolate anion is known as a
one electron transfer reaction. Findings are significantly different compared to the
previous ones that showed the magnetism of gold originates from the particle surface
via charge transfer in the Au-S bond.67
Figure 6. Reversible conversion between the neutral and anionic Au25(SR)18
nanoclusters.5
2.4.4 Charge transfer properties
Physical properties of nanoparticles strongly depend on the particle size, shape and the
nature of the surrounding ligand.69 The thiolate-stabilized cluster can act as an electron
donor or acceptor. The addition or removal of an electron creates an energy barrier that
needs to be overcome before the second electron is added or removed.5
A quantization effects appear as the particles size approaches molecular behaviour,
resulting in a HOMO-LUMO gap between the valence band and the conduction band.5
As the size is reduced the metal-like electron band converts to discrete energy levels
analogous to molecular orbitals. In electrochemical experiments the gap between
highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital
(LUMO) is seen as a voltage gap between the first electron addition and removal. The
spacing between energy level results from the capacitive charging. Each cluster has its
26
own charge state which changes with the core size and strongly depends on
monodispersity of the sample. Ramakrishna et al. studied two photon absorption of
Au25(SR)18 cluster.3 The fast electron transfer was observed in both neutral and anionic
form of the cluster. The photooxidation occurs near the HOMO-LUMO gap and was
assigned to the core-to-surface relaxation.
Thiolate stabilized clusters are commonly used for electrochemical studies of gold
nanoparticles because their size significantly affect the capacitance of the cluster. For
highly polar ligands discrete charging cannot be resolved due to too large dielectric
constant of the ligand. In water, the increase in ionic strength would collapse the charge,
making the electrostatically stabilised MPCs more susceptible to changes in salt
concentration.
2.4.5 Catalytic activity
Among noble metals, gold has rather poor activity for reactive molecule adsorption.4,5,72
The filled d band of gold leads to higher activation barriers compared to other transition
metals with half-filled d bands. It was investigated that the clean bulk gold surface is
quite inert for the O2 adsorption.4,5 However, this changes when the dimensions of the
gold catalyst approach nanoscale. Gold clusters were found to be reactive with oxygen
at the room temperature.4 The unusual catalytic properties of gold nanoparticles arise
from the fact that oxygen binds more strongly at the defects, such as steps and edges on
the particle surface, which consequently increase as the size of particle decreases.72
Another factor is the Au-interface which may induce strain influencing the surface
reactivity.4,5 Last years, gold nanoclusters have been studied as an active catalyst for
oxygen electro reduction and CO oxidation.
Herzing et al. found that the catalytic activity for CO oxidation came from ten atom
gold nanoclusters, which clearly suggested that metal clusters represent good class of
materials for catalysis.4 Further theoretical studies proved that the smaller clusters are
more favourable for O2 adsorption onto cluster surface, and therefore, more reactive.4
The
studies
demonstrated
that
~1.5
nm
gold
clusters
surrounded
by
Polyvinylpyrrolidone (PVP) showed higher catalytic activity than larger clusters
stabilized by poly(allylamine) (PAA). Chen et al. also studied the size effect on the
catalytic activity.4,5 They prepared a wide range of gold clusters ranging from 11 to 140
gold atoms and evaluated the size effect on the electrocatalytic activity for oxygen
27
reduction. Again, the results showed that electrocatalytic activity increases as the core
size decreases. More recently, Gao et al. studied the catalytic activity for 12 different
clusters from 16 to 35 gold atoms and concluded that the anionic clusters can adsorb O 2
and CO more strongly than a neutral one.4
2.5 Methods for detection and characterization of clusters
Because of the electronic, optical and catalytic activities of metal nanoclusters depend
on their size, morphology, composition and surface properties, various techniques have
been applied to characterize them. A detailed structural characterization enables to
understand the structure-property relationships which provide the basis for cluster
structural optimization for different applications. For instance, it was found that the size
of the gold core has an influence on the electrocatalytic activities of particles.72 The
main aim of nanoparticle characterization is the determination of particle size and
monodispersity.
Nanoparticle characterization is based on the determination of particle mass, diameter or
the analysis of the exact cluster composition. To evolve the core to a specific number of
Au atoms post-synthesis treatments may be applied.2 Fractions with different sizes may
be separated via centrifugation methods with different rotational speeds and times.73
Currently used purification methods of nanocluster samples are focused on removing
unreacted reducing agents and free ligands. The impurities may be removed by
washings, centrifugation, extraction or reprecipitaton.73 Nevertheless, the purification of
distribution of crude metal nanoparticles may be more challenging and other techniques
need to be utilized. Separation using different solvents, high pressure liquid
chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE) are commonly
used techniques to improve monodispersity.7 Purification techniques of clusters enable
easier characterization with respect to mass, diameter, composition and structure.
2.5.1 Stability of the clusters
To examine the stability of clusters, UV-vis spectroscopy is a powerful tool. UV-vis
absorption involves electronic transitions from valence-orbital of the clusters. The
technique enables to monitor changes of the metallic core, including oxidation state and
the number of metal atoms in the cluster.7,74 Therefore, UV-Vis absorption spectroscopy
is a powerful tool to identify clusters size. Moreover, as the method can be used to
detect changes in the core, therefore can be used to examine stability of clusters over the
28
time. Gold clusters are rather stable in a solution or in a powder form, indicating no
changes in the absorbance peaks. The UV-vis spectroscopy was also used to confirm the
stability of Aun(SG)m clusters when exposed to air.75 For metal nanoparticles, position
and absorption of peaks are well-defined by Mie theory, which is used to define
polydispersity and concentration of particles.6 However, in more rigorous analysis
particles shape and capping ligands should be considered. The choice of the medium
can cause shifts in the spectra. Spectrum of large gold nanoparticles are dominated by
plasmon peaks at around 420 nm, 520 nm and 600 nm, whereas the subnanometre
nanoparticles exhibit molecular-like optical transitions with absorbance bands due to the
quasi-continuous electronic band structure.46 UV-vis absorption spectroscopy has been
a convenient and powerful tool to study size-dependent optical properties and the
electronic state structure of gold nanoparticles.74
2.5.2 Particle size and chemical composition
Particle diameter measurements can be done using transmission electron microscopy
(TEM) and x-ray spectroscopic techniques, including x-ray absorption spectroscopy
(XAS) and x-ray photoelectron spectroscopy (XPS). Thermogravimetric analysis (TGA)
and elemental analysis (EA) can be used to obtain the ratio between metal part and
organic thiolate layer part.52 The x-ray diffraction analysis of crystals resulted in a
determination of a particle with 102 gold atoms and 44 p-mercaptobenzoic acids
(pMBA).36 This finding was very important because was the first crystal structure of
thiolate-protected nanoclusters which was solved. The mentioned techniques are the
best known analytical methods to gain chemical composition and cluster size.
The transmission electron microscope (TEM) enables seeing nanoparticle morphology
and precise size.23,75 Nowadays, TEM analyses with a nanometre resolution are used in
measuring particle sizes larger than 1nm, and studying the morphology of particles. Due
to the large scattering cross section of the metal atoms the high quality images can be
taken. However, performing TEM measurements sometimes can be challenging because
migration of particles and coalescence of the particles may occur.73 It is worth noting
that TEM measurements have usually small uncertainty. Damages of particles may
influence the imaging due to the electron beam heating. TEM can usually confirm that
the particles are smaller than 2 nm. However, for the particles around 1 nm it is hard to
distinguish the cluster size, even by phase-contrast high-resolution TEM (HR-TEM).31
However, HR-TEM is often used as a complementary tool to evaluate the crystalline
29
structure and the size of particles. TEM sample preparation of gold nanoclusters
requires diluted solution to avoid cluster aggregation and consequently provide to
imaging difficulties. Densely distributed metal clusters can quickly agglomerate under
electron beam exposition. Besides HR-TEM, scanning transmission electron
microscopy (STEM), atomic force microscopy (AFM) and scanning tunnelling
microscopy (STM) have been used to study metal nanoclusters.73
X-ray absorption spectroscopy (XAS) is highly correlated with the regular UV-vis
measurements. The main difference lies in transitions’ level. XAS involved core-level
electronic transition to unoccupied valence states thus making it useful for the studies of
electronic properties of absorbing atoms.52 Zhang52 was studied X-ray absorption
spectroscopy of Au144, Au38, Au36, Au25, Au24Pt, and Au19 clusters. He reported that in
order to understand their-structure relationship X-ray structure methods play an
important role. The spectra can also provide reliable information about local structure
of absorbing atoms and size information which is directly related to the number of
neighbouring atoms. The quantitative information can be obtained by fitting with
theoretical
framework.
X-ray
photoelectron
spectroscopy
(XPS)
provides
complementary information to the XAS technique. This analytical method found
broader applications in material characterization. It gives straightforward information
about the chemical composition of materials from the peak position and shapes.
2.5.3 Determination of the molecular weight of clusters by ESI-MS and MALDIMS
As described above, it has been challenging to determine the exact size of clusters only
by using microscopic methods due to the inaccuracy of measurements. Weis et al.
realized that mass spectrometry methods provide reliable information of clusters,
including the number of metal atoms and the surface protecting ligands.76 They were the
first to use laser-desorption/ionization and time-of-flight MS to analyze gold
nanoclusters. Nowadays, the molecular weight of monolayer protected clusters can be
determined by choosing a suitable mass spectrometer.73 A variety of mass spectrometry
methods have been applied to the study of nanoparticles. For example, Laser desorption
ionization (LDI) mass spectrometry analysis were used to confirm the same structure of
Au25(SR)18 cluster, regardless of the types of thiols.35 The molecular formula of
Au68(SR)34 was assigned by matrix assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry. The research reported that the stability of the cluster
30
was attributed to the 34-electron count shell closure.77 Nevertheless, the analyses are
often challenging due to fragmentation of clusters and their high molecular weight. The
problems can be overcome by using soft ionization techniques such as electrospray
ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) techniques.
Both techniques are in favor of the analysis of such systems because they result in no or
little fragmentation. Additionally, ESI-MS allows the mass determination of intact
clusters and can produce multiply charged ions which enable the high molecular weight
determination.
2.5.4 Separation and purification of clusters by polyacrylamide gel electrophoresis
(PAGE)
Despite all the available syntheses of monodisperse sub-nanometre metal clusters, still
purification of core sizes remain challenging. One of purification method,
polyacrylamide gel electrophoresis (PAGE), has been widely used to separate different
core sizes, even smaller than 2 nm.78,79 PAGE is based on the migration of charged
molecules and separating them due to the mass difference. The charged molecules
migrate in response to electric field created between a cathode and an anode. Rate of
migration depends on the charge, shape and size of the molecule and also on the
properties of the medium where the molecules are moving. The sample is run in a
polyacrylamide matrix, which is the most common matrix to separate molecules by size.
Porous structure of the matrix acts as a sieve by retarding the movement of large
macromolecules. The method is used to separate and purify water-soluble gold
nanoclusters protected by various ligands, including glutathione or pMBA. The method
turned out to be a convenient separation technique of gold nanoparticles due to their
colour which made them directly visible by the eye. The separation of Au:SG
glutathione clusters was reported by Negishi et al.80 They succeeded in separating series
of cluster sizes into the fractions by high-resolution PAGE analysis. Of a note, the
preparation of polyacrylamide is a crucial step in separation of clusters. The
concentration affects the separation process.
2.5.5 Analysis of nanoparticle formation and morphology by nuclear magnetic
resonance (NMR) spectroscopy and fourier transform infrared (FT-IR)
spectroscopy
Nuclear magnetic resonance (NMR) is a characterization tool that requires little
perturbation of the analysed system, giving information and details about the chemical
environment of the nuclei.81,45 NMR has been widely used in analysis of chemical
31
structure, reactions and dynamics. Cluster properties can be investigated by proton (1H)
and carbon (13C) NMR, which provide information about the properties of cluster and
surface structure.45 On the other hand, diffusion-ordered NMR spectroscopy (DOSY)
was recently used to study hydrodynamic size of Au102(pMBA)44 cluster.38 The
versatility of the method and chemical resolution enable characterization of formation
of noble metal nanoparticles. Different chemical shifts of free ligands and ones bound to
the gold core, enable verification of purity of the synthesised nanoclusters.81 Studies
showed that when protecting ligands were bound to the nanoparticle’s surface, spectral
broadening occurred. The reason of this broadening is probably ligand-core interface
which results in rapid spin relaxation by dipolar interactions.38 Another factor that may
cause the spectral changes is associated with different surface sites for ligand binding.
Recently, Lauren et al. has used NMR techniques and reported that NMR provides
crucial insight into nanoparticles formation, morphology and properties.45 They focused
on using NMR techniques that are generally accessible to the synthetic nanochemistry
community. Following a formation of nanoparticles, requires a method that can capture
the chemical and physical transformation in real-time with sufficient resolution. Broad
range of NMR techniques, as well as their combination with other analytical methods,
can be used to study nanoparticle formation and structure. The chirality of Au38(SR)24
and Au25(SR)18 was also studied by 1H NMR. For Au38(SCH2CH2Ph)24 clusters,
especially for a-CH2, the chirality shift was observed for each terminal proton,
indicating that Au38 has chiral properties, whereas for Au25 cluster no chirality was
observed.64 Moreover, the crystal structure of Au25 protected by different ligands was
studied using 1D and 2D NMR, and other analyses.46 On the other hand, the technique
was used to study the mechanism of metal nanoclusters. Contrary to the previous
assumptions, it was concluded that Au(I)–and Au(III)–tetraalkylammonium complexes
are the relevant species in the reaction mechanism.45
Another technique to study the surface structure of synthesised metal nanoclusters is
detecting molecular vibrations by FTIR spectroscopy.82 The formation mechanism of
metal clusters can be studied by comparing the metal particle and the free organic
protecting ligand. Thiol protected nanoparticles are formed through Au-S bond,
therefore the peak associated with S-H vibrational stretching disappears when the S-H
group is attached to the metal cluster surface, indicating the formation of metal-sulphur
bond. The FTIR spectroscopy was used to study the differences between Au25, Au38
and Au144 2-phenylethylthiol (PET) protected gold clusters. The studies were performed
32
in order to eludicate how the ligand binds to the metal core and what are the differences
between clusters. It turned out that besides small perturbation, the IR spectra were very
similar when comparing to bare ligand. 82
33
3 SURFACE FUNCTIONALIZATION OF NANOPARTICLES AND
NANOCLUSTERS
The chemical properties of the gold nanoparticles mainly depend on the ligands that are
present on the external and internal structure.20 The modification of ligands enables
possible applications by providing a new functionality by improving their inherent
characteristics. Modification methods can control physico-chemical properties of
nanoparticles, such as preventing aggregation, improving stability and making them
compatible with biological environment.20 This can be done to avoid compatibility
problems between two insoluble phases and to expand the mechanical properties.
Furthermore, nanoparticles can accommodate multiple functional groups which tune
their properties nearly endlessly.7 The synthesis methods have been continuously
evolving, leading to a control of the shape and size improvements.
There are three primary methods to introduce a functional groups to the surface of noble
nanoparticles (fig.7).20,83 The first one, direct synthesis, is based on introducing the
whole functional ligand in one step (method 1). The bifunctional organic compound
reacts with the metal particle by attaching one of its functional groups to the
nanoparticle surface, generating a gold nanoparticle surrounded by dense protecting
ligand layer with a desired functionality. The second method is the ligand exchange
method, where the initial ligands undergo substitution by incoming ligand bearing
desired functionality. The last method is based on conjugation where, the bifunctional
compound reacts with a metal particle, while, the other group acts as a coupling site
(method 3). The next step is based on chemical converting of the coupling site to gain
the final functionality. The main drawback of the first method may be the
incompatibility of functional groups, which can react with the surface.20
34
Figure 7. Three ways to functionalize particles. Method 1 (top) Nanoparticle reacts
directly with the ligands with the Z functionality; method 2: substitution (exchange) of
initial ligands in order to obtain desired functionality; method 3 (bottom): a ligand with
a Y functionality reacts directly with the nanoparticle and acts as a coupling site in the
second step to obtain another functionality Z.20
The choice of stabilizing ligand mainly depends on the size of particle and the solvent.
The capping ligands that permit introducing various functional groups are usually
thiols.20,84 It was found that thiols form dense packing ligand layer which stabilizes the
core of particles. Water soluble particles, containing polar ligand molecules were found
to stabilize the particles for a longer time.83 The most common groups that are used to
functionalize metal nanoparticles are: COOH, NH2, OH, and long alkyl chains.20 They
can ensure compatibility and stability with the environment of the nanoparticles, and
can be used as a base for further chemical reactions once attached to the particle
surface.20 Functionalized trialkoxysilanes groups are commonly used as coupling sites.
Many groups can be attached to alkoxysilanes, such as epoxy, amino, vinyl, sulphurcontaining, and phosphonic acid groups. Chlorohydrogenosilanes, (H(CH3)2SiCl) are
also powerful agents, which are reactive towards alkynes, alkenes and triazonium
salts.20
The main drawback of the direct synthesis is the limitation of suitable types of capping
ligands and functional groups. Ligand exchange reactions and modification methods
have become necessary in order to adapt these materials for different applications.7
35
Metal nanoparticles can be modified in various ways by ligand exchange, chemical,
physical and bio-conjugation reactions.7 The strategy to functionalize and modify
ligands depends on the structure of particle surfaces and their interactions with ligands.
To overcome some of the synthetic difficulties, ligand exchange can be applied.14
Recently, alkane thiols used as capping ligands have been the ligands of choice for
exchange reactions.81,14,85 However, gold nanoparticles can also be modified by
disulphides, amines, nitriles, phosphines and carboxylic acids.
Many chemical approaches have been used to conjugate nanometre sized gold clusters
with biomolecules of interest. Proteins, nucleic acids, lipids, biologically active small
molecules, therapeutic molecules, targeting moieties, and contrast agents can be
modified as example for biological applications. A main aim of conjugation strategies is
to bind a targeting moiety without losing its functionality after attachment to the surface
of nanoparticle.86 Conjugation of multifunctional groups on the particles surface allow
constructing of nanomaterials which can be used for detection of the biological
structures, and functioning of biomolecules and organelles, and also for targeted
imaging and treatment of cancers. The functional group at the end of the ligands can be
easily tailored, enabling the nanoparticles to interact with organic molecules, other
nanoparticles, and polymers. Consequently, the possibilities of surface modification
synthesis are endless and are extremely important for future applications.7
Traditionally, there are three chemical approaches for conjugating the molecules into
gold nanoparticles.87 The conjugation methods can be categorized based on the nature
of the interaction between the biomolecule and gold particles. The molecules can be
covalently attached, adsorbed, or conjugated with biomolecules to the surface. The first
approach, where chemical conjugation, is based on covalent bonding of the molecule to
the nanoparticle surface. Another approach called encapsulation is based on weak noncovalent interactions. Encapsulation is non-specific bonding, based on electrostatic
interactions effected by medium.87
The conjugation of nanoparticles with the biomolecules of interest is another strategy
that has been developed.7,15 Biological interactions are based on both chemical and
physical interactions which make them highly selective. Therefore, combination of
interactions with gold nanoparticles, makes them extremely useful as sensors for
detecting biological molecules.88 Various functional groups on nanoparticle’s surface
36
allow conjugation with peptides, ligands, antibodies, genes, drugs, and therefore
constructing multifunctional nanomaterials. Due to their unique properties, nanoclusters
are of considerable interest because of their unlimited variety of potential applications in
various fields, such as optics, biosensing, electronics, nanotechnology, catalysis and
DNA or drug delivery.7 Consequently, the studies of chemical surface modification
have become an important issue towards nanotechnology applications.
3.1 Ligand exchange
Ligand exchange reactions are versatile methods to control the composition of organic
layer on the nanoparticle surface. The concept of ligand exchange reaction is very
simple and includes mixing nanoparticles with the free ligands, resulting in the
replacement of outgoing ligands with an incoming one (fig. 8).13 Such an exchange is
usually applied to transfer the NPs from aqueous solution to organic phase, providing
new properties and stability. Typically, the hydrophobic ligands are replaced by
hydrophilic ones that bind strongly to surface of nanoparticles.83 After the exchange of
ligands they may be conjugated with various biomolecules and used as catalyst,
biosensor, or for electronics application.7
Figure 8. Schematic diagram of a ligand exchange reactions of nanoparticles.
The main challenges of nanoparticle synthesis are restrictions of medium which the
particle remains stable, and the lack of functionalities of the ligands.13 The end group of
ligands, such as thiols, amines or phosphines are electron donating groups which can
undergo several binding and unbinding processes that may lead to aggregation.
Therefore, the molecules bound to the nanoparticle’s surface are not only responsible of
the growth control but also prevent them from aggregation.83 The ligands with two
functional head groups can be used to ensure strong binding affinity and to induce
37
stability and dispersion of particles in both aqueous and organic solution. Exchanged
ligands can be used for targeting agents, drug deliveries, and to make possible to modify
nanoparticles with ligands that cannot be introduced during nanoparticles synthesis.13
Although the concept of ligand exchange is simple the researches has proved that the
mechanism is extremely variable and it strongly depends on bonding strength, binding
sites, nanoparticle size, and the ligand chain length.13 For this reason it remains
particularly challenging to obtain uniform results and compare them from one
experiment to another, as the nanoparticle systems and ligands are all different. For the
ligand exchange reaction, the ratio of incoming to outgoing ligands is extremely
important and too high concentration may decompose the nanocluster, whereas too low
results in incomplete exchange.4 Despite the significant progress that has been made
towards surface functionalization of gold clusters, most of the synthetic methods still
struggle with the wide size distributions and low yield of functional nanoparticles.
Recently, Qian et al. reported a large scale synthesis of monodisperse Au38 clusters.61
The water soluble glutathione stabilized clusters were mixed with organic phase of 1dodecanethiol (C12SH), resulting in Au38(SC12H25)24 nanoclusters. The ligand exchange
process lead to etching of Au glutathione clusters. Transfer to organic phase indicated
that the gold cores were capped with a hydrophobic monolayer.4
3.1.1 Mechanism of ligand exchange
As mentioned before, the atomic structure of nanoparticles plays a crucial role in ligand
exchange synthesis. Ackerson et al. studied the structure of Au25(SC2H4Ph)16(p-BBT)2
and Au102(p-MBA)40(p-BBT)4 clusters (p-BBT = p-bromobenzenethiol, p-MBA = pmercaptobenzoic acid) and calculated the sites where the reaction is more likely to take
place.14,85 Recently, Millstone and co-workers perfomed a quantitative analysis of
thiolated ligand exchange on gold nanoparticles by H1NMR spectroscopy.81 They found
that the ligand addition mechanism is influenced not only by the functional group
interacting with nanoparticle’s surface, but also by intermolecular interactions within
the ligand shell. More densely packed ligands which are formed by molecules
containing strong intermolecular interactions were found to be passive on ligand
exchange modification.
Depending on size of the core of the particle, solvent type, and the ligand molecule,
different attractive interactions have impact on stability of particles.83 The ligands
38
molecule can be bound to the nanoparticles’ surface by electrostatic attraction,
chemisorption, or hydrophobic interaction. Typically, these interactions localized to the
head group of the ligand molecule.83 In organic solvents, the hydrophobic ligand
molecule bound to nanoparticle surface undergo dynamic binding and unbinding
processes, leading to aggregation. In the aqueous solution, the interactions are similar,
however, the particles are additionally stabilized by electrostatic repulsion.
General distinction of ligand exchange processes was first suggested by Langford and
Gray. Ligand exchange process with similar to associative (SN2), similar to dissociative
(SN1) and a interchange (SN2/SN1) mechanisms.89In the associative one, the ligand
exchange is driven by the introduced free ligand, while in the dissociative the exchange
in controlled by detachment of ligand already bound to the nanoparticles. The kinetics
of ligand exchange can be classified in a three steps process. The first step includes
rapid exchange, followed by slower ligand exchange and concluding with ligand
rearrangement on the particle surface.
Ligand exchange reaction on thiol monolayer-protected gold nanoparticles was first
introduced by Murray et al.88 Many studies have been done to try to fully understand
the reaction mechanism.90,91 Murray et al. in their work used nuclear magnetic
resonance and mass spectrometry which provided information on the rates of ligand
exchange reaction and the chemical composition of thiol functionalized Au38 and Au140
with p-substituted arylthiols. 90,91 They found that molecules exchange first at defects in
the ligand shell or at corners and edges of the core crystal. Moreover, the exchange
kinetics on the surface was described as an “SN-2 type” mechanism where the initially
bound ligands are replaced with the incoming one. This means that the ligand exchange
is an associative mechanism, depending upon the incoming ligand. The method has
been further extended to the preparation of water soluble gold nanoparticles with the
functional groups including carboxyl acids, alkyl halides, amines, azides, maleimides,
phenols, alcohols, carbohydrates, amphiphilic polymers, amino acids, nucleic acids,
peptides and proteins.
Caragheorgheopol and Chechik conducted a series of experiments under strictly
controlled reaction condition and concluded that the kinetics of thiol exchange with
thiol protected gold nanoparticles strongly depend on their morphology. 13 The surface
of nanoparticles consists of two sites (two defect and one non-defect site). The vertexes
39
and edges are classified as defect sites, whereas terraces are classified as non-defect
sites (fig. 9). Each of the binding sites possesses a different electron density and steric
accessibility leading to different exchange kinetics. It is assumed that the vertexes and
edges have a higher reactivity than terrace sites, and are thus responsible for initial rapid
exchange. The rate of ligand exchange is dependent on the ratio between different
binding sites. Further, the reactivity of nanoparticles depends on the surface curvature
and the reaction rate may differ with differently sized particles.84,91
Recently, kinetic study on the adsorption process of dodecanethiol ligands of gold
nanocrystals surface was reported.84 The study concluded that the adsorption process
occurred via two-steps process. At the beginning of ligand exchange thiol molecules are
adsorbed at corner and edges sites.84 After that, the reaction rate is slowed significantly.
They explained, that during the second step the surface structure may be reorganised
into more ordered ligand shell, which resulted in slow ligand adsorption. 84
Figure 9. Schematic diagram representing the nanoparticles’ surface sites.92
3.1.2 Kinetics studies for ligand exchange on nanoparticles
Understanding the mechanism of ligand exchange is crucial in order to modify the
ligand shell and its kinetics.84 Chemical reaction kinetics determine the rate of chemical
processes which are usually broken down into several steps yielding the products.
Additionally, kinetcs gives information about the reaction mechanism, with variables
such as pressure, temperature, activation energy, surface area and reactant concentration
which directly influence reaction rate. In the past, ligand exchange kinetics of aromatic
thiolate ligands on gold nanoparticles were investigated by using NMR whereas the
kinetics of short-chain thiolates, amines, and disulfides were studied by EPR
spectroscopy.93 Highly reactive ligand thiols require faster kinetics approaches such as
fluorescence or optical spectroscopies.94
40
The kinetics of the exchange of 2-phenylethanethiolate on the Au38 and Au40 by chiral
BINAS (1,1’-binaphthyl-2,2’-dithiol) were studied by using circular dichroism (CD)
spectroscopy.95 It was shown that the ligands were only partially exchanged by BINAS,
and each BINAS ligand replaces two PhCH2CH2S. The reason was assumed to be the
nature of BINAS ligand, and its steric hindrance, as well as the binding sites in gold
clusters. They explained that the changes in reactivity between Au38 and Au40 are
caused by the different ratio of defect to non-defect sites. The comparison of both
exchange reactions shows that Au40 undergoes the exchange much faster and to a higher
extent.95 However, the kinetics of both reactions slow drastically after a few hours even
in a huge excess of incoming molecules.96 More recently, the studies on absorption
kinetics of dodecanethiol ligands on cluster’s surface were reported.5 The most dramatic
changes were observed within the first reaction minutes, indicating that the reaction
occurred initially more preferably on the edge and corner sites.
Furthermore, Murray et al. studied kinetics of the exchange of phenylethanethiolate
ligands of monolayer-protected gold cluster of Au38(SC2Ph)24 and Au140(SC2Ph)53 by
para-substituted arylthiols.91 They assumed that the reactivity of clusters at the initial
stage is almost independent of their size. The places for initial ligand exchange such as
vertices and edges, are common for these species. However, the later stages of ligand
exchange are much slower because larger terraces are present with the increasing size.
While several authors observed an increase in the ligand exchange rate of gold
nanoparticles with a decreasing particle size5,91, the other results were quite contrar.97
Further, the kinetics were followed during the period of rapid exchange and the data
indicated that the mechanism occurred via first-order-rate. However, the reaction started
varying linearly with an incoming arylthiol concentration indicating a second-order
reaction. The results were consistent with ligand exchange being an associative process.
Further studies on ligand exchange reaction, using a high resolution separation method
indicated that the reaction started to occur preferably at thiolates rather than disulfides
or diselenides.9 The studies have shown that disulphides exchange at a much lower rate
than thiol ligands.13 However, density density theory structure investigation showed that
the framework structure of the selenolate protected gold cluster is similar to the thiolate
gold cluster. The studies also indicated that the formation of selenolate protected
clusters is thermodynamically more favorable than thiolate protected clusters.89
41
Similar results were obtained by Graf et al., who studied the effect of multivalence and
nanoparticles’ size on the binding kinetics of thiol ligands on gold nanoparticles.94 The
monovalent, divalent and trivalent ligands were explored and the results showed that
with the increasing valence, the rate of reaction was decreased. The particle size
dependence was also studied by the same group, and it was shown that the exchange
rate increases with the particle size.54 In addition, the effect of the length of ligand chain
was investigated.98 It was concluded that longer chains as well as the bulkiness indicate
much slower reaction rate comparing to shorter ligands.98 Interestingly, it was found
that charged ligands require shorter exchange time than uncharged ones. This may be
explained by the increased solubility of the particle in the aqueous layer. Furthermore,
the exchange reaction of phenylethanethiolate ligands on gold clusters by parasubstituted aryl thiols was studied.91 At the initial stage the rate of reaction increased
rapidly and after the equilibrium was reached it slowed down. They concluded that
thiols with an electron donating substituent at the end group of the ligand usually
increase the rate.
3.1.3 Effects of surface binding groups and head groups
One of the major factors of surface modification reactions is the ligand being used. The
properties and stability of nanoparticles can be enhanced by the use of different
functionalized ligands.99
Various ligands have a different binding affinity on the surface of gold.84 The gold
surface composition and structure of particles are crucial for the selection of a new
ligand. Metal nanoparticles can be modified by thiols, disulfides, amines, nitriles,
carboxylic acids and phosphines. Most of the studies involve thiol or phosphine as
headgroups.4 In 2005, Hutchison et al. exchanged all the phosphine ligands by using
variable alkyl or arylthiols -functionalized ligands. The synthesis turned out to be easy
to prepare a large variety of functional groups. Chen et al. found out that the surface
ligand field exhibits a strong effect on the electronic energy structure of nanoparticles.
They exchanged phosphine stabilized ligands with dodecanethiol protected Au11 clusters
and observed photoluminescence from the thiol protected clusters.4
Modification of metal nanoparticles by using organosulfur compounds is a well-known
method for introducing ligands into the core.84 Sulphur groups have strong affinity to
various metals, including gold. The modification reaction usually takes place rather
42
quickly because the sulphur group often adsorbs spontaneously. In addition to the
compounds with only one thiol group, the compounds with more than one thiol group
are often used. Recently, multi-thiols compounds have been of particular interest
because of the enhanced bond strength to metal cations. Disulfides also result in better
stabilization comparing to their monovalent derivatives. The first functional group
facilitates the surface binding, whereas the other initiates the designed chemical
reactions. The thiol-thiol exchange reactions mostly take place in excess of incoming
ligand. The exchange may occur completely or partially depending on the reaction time,
amount of ligand and the ligand itself. The partial exchanged gold’s surface is
composed of both the previous and the incoming ligand in a certain ratio.20 The weak
Van der Waals interactions are the main driving force in the surface modification
reactions thus the stronger binding is necessary between gold and the ligand. Therefore,
gold particles usually require sulphur as a binding group due to its strong affinity
towards gold.
The surface of particles can be modified by the use of amines.83 The interaction between
amino group and metal surface is weaker than that of thiolate groups, which has an
impact on their size. The adsorption of long chain ammonium ions is commonly used
because of their amphiphilic properties. The hydrophobic particles can be tuned into
hydrophilic which is useful for bio-applications. Shorter chain ammonium ions have
been used to stabilize transition metal nanoparticles.20 Other molecules including
fluorescent dyes, drugs, protein, peptides, antibodies or other molecules can also be
attached to amine/carboxyl groups. The advantage of using biomolecules such peptides
over other surface ligands is that they offer solubilisation and biofunctionalization
simultaneously. Other studies used amines to functionalize gold structure because of
their weak covalent bonding with gold. They reported that the amine-gold interaction is
much weaker comparing to the sulphur-gold interaction. They also claimed that the
alcohol group does not exhibit any interaction with the Au0 surface.100
Even though the phosphine stabilized metal nanoparticles have been widely studied, the
synthesis process involves several steps, in addition to a challenging purification.4
Moreover, phosphine interaction with nanoparticles is very weak, which makes them
unstable, with a tendency to decompose even at ambient conditions. Due to their high
exchange probability such an exchange usually proceeds completely in a way that every
phosphine ligand becomes exchanged. Phosphine ligands can be exchanged with thiols
43
resulting
in
increased
stability
triphenylphosphines protecting
of
particles.
Hutchison
et
al.
replaced
Au11 cluster through the exchange reaction and
obtained an alkanethiol protected cluster.71 Recently, Shischibu et al. also used a
phosphine-stabilized Au11 clusters to obtain glutathione (GSH) protected Au25
nanoclusters under optimized conditions.4 Amine protected clusters can be synthesised
by ligand exchange reaction with the original phosphine stabilized ligands.
Nevertheless, the results showed that less stable particles were obtained. Interestingly,
the growth of particles was observed after the exchange reaction.20
Nanoparticles can be modified with organic or inorganic molecules depending on their
solubility. The hydroxyl group appears to be commonly used capping group because it
can easily react with the carboxyl group or with various silane groups.101 Organic
soluble particles have to be first modified with functional groups such as mercapto or
hydroxyl groups for further conjugation reactions. A great number of ligand exchange
reactions on alkane thiol protected gold nanoparticles have been introduced by Murray
et al.93 After that the reactions were extended to the preparation of water-soluble
particles containing various functional groups. Small molecules with functional head
groups such as thiols, carboxyl acids, alkyl halides, amines, azides, maleimides,
phenols, alcohols, amino acids, nucleic acids, peptides and phosphine groups have been
reported as a good candidates for generating water soluble particles. Nevertheless,
electrostatic interaction in solution stabilizes nanoparticles capped with small
molecules, hence they become dependent on solution conditions, sometimes causing
aggregation. Therefore, polymeric ligands are an alternative way to overcome poor
stability of nanoparticles. Among them, poly(ethylene glycol) (PEG) offers good
stability and water solubility due to the hydrogen bonding of ether group in the back
bone of PEG chain.7
3.2 Chemical conjugation of gold nanoparticles
Figure 10. Chemical conjugation of gold nanoparticles.102
44
Chemical conjugation is based on the ligand modification strategy, where the ligand
molecule stabilizing the particle is modified (fig. 10). For example, the polarity of
hydrophilic nanoparticles stabilized by mercaptobenzoic acid (pMBA) can be changed
by modification with hydrophobic molecule by using carboxylic terminal group, by
formation of complex on nanoparticles surface or by covalent attachment of ligand.83
Ligand addition may be efficient phase transfer concept, however, some of the systems
may not be compatible with each other, leading to restrictions.
In the chemical conjugations covalent bonding is the most widely used interaction,
yielding a stable and specific conjugation of molecules with NPs.7,83 The molecules
with the functional groups capable of covalent bonding result in higher stability. The
functional groups on nanoparticles surface such as amides, thiols and carboxylic acids
can be terminated with molecules through various coupling methods. Generally,
covalent gold-molecule coupling ensure a good control of the particle size distribution,
stability, solubility, and stable linkage. On the contrary, covalent bonding is challenging
in producing site specific linkage and controlling stoichiometry of conjugated
nanoparticles and molecules.
3.2.1 Coupling strategies
The most common coupling method, for immobilizing molecules with various
functionalized head groups, is the amine group (-NH2) because its stability.20,83
Therefore, the amide linkages are highly attractive for covalent coupling conjugation
strategies. Generally, any primary or secondary amino group can be coupled using
carbodiimide coupling chemistry. For example, amine-terminated nucleic acids, small
molecules with amine groups, and various proteins (enzymes and antibodies) can be
coupled to carboxylic group functionalized NPs. In addition, amine-functionalized gold
nanoparticles can be conjugated to a carboxylic acid bearing molecule or material by
using the same method. In addition, the nucleophile character of the amine group allows
reacting with some other functional groups such as aldehydes, thiols, isocynates and
epoxides. In addition, nanoparticles stabilized by amine group can be conjugated by
amide bond with various crosslinker molecules such as SMCC (succinimidyl-4-(Nmaleimidomethyl) cyclohexane-1-carboxylate). The group from linker molecule can be
converted to maleimides that are reactive towards thiols.83
45
Commonly used conjugation methods are based on thiolate groups of cytosine amino
residue which coordinate proteins or peptides to noble metal nanoparticles.20,83 The thiol
group selectively conjugates with primary amine groups, which provides various
conjugation possibilities with multiple types of thiolated biomolecules, peptides and
proteins with free or reduced cysteine and residue thiol-terminated DNA. Other typical
thiol-reactive functional groups include maleimides, iodoacetamides and disulfides.7
Thiolates undergo Michael addition reaction with maleimides to form succinimidyl
thioethers. At high concentration the selectivity of the reagents of iodoacemides and
maleimides is relatively low. In contrast, disulphide reagents react selectively with
thiols. Disulphides, are susceptible to reduction by a biological reducing agents, hence
they are used for reversible coupling of NPs.102
Esterification method can be also used for functionalization and bioconjugation of gold
nanoparticles.7 Typically, the reaction takes place between alkyl halogen-functionalized
nanoparticles with phenols or carboxylic acids in a mild environments. Gold
nanoparticles functionalized with carboxylic acids can also form ester linkage by
reacting with phenols or molecules with hydroxyl functionalized molecules. This
approach enables to conjugate various carbohydrates and polymers groups to the surface
of gold nanoparticles.7
Conjugation of monodisperse nanoparticles to large bionanomolecules is a new tool for
tracking and bioimaging biological systems.7 Conjugation methods with biological
materials can be used to study pathogenesis of virus infection by tracking the virus in
tissues and cells. Recently, Marjomäki et al. described a procedure of site-specific
covalent conjugation of monodisperse gold nanocluster to viral surface (fig. 11).15
Water soluble Au102(pMBA)44 clusters were functionalized with maleimide linkers that
bind
covalently via an ester bond. The functionalized gold nanoclusters were
conjugated to target cysteine of viral capsid proteins via Michel addition reaction.
Additionally, it was also confirmed that the labelling procedure didn’t compromise the
infectivity of the virus.
46
Figure 11. The maleimide functionalized Au102(pMBA)44 clusters and site-specific
conjugation to enteroviruses. Adapted from Ref. 15.15
Click chemistry is another highly versatile and practical approach for conjugation of
gold nanoparticles. Typically, brominated alkane thiol capped gold nanoparticles upon
treatment with sodium azide give an azide 1,2,3 triazole ring which can be conjugated
by click chemistry to any molecules with the alkyne group.7,103 This conjugation
chemistry gives the possibility to introduce multiple functional groups into NPs. A
consequence of a click chemistry strategy is that it requires a special modification and
preparation of alkyne-functional bioactive species and an azide, often resulting in low
yield.20
3.3 Physical conjugation of gold nanoparticles
Figure 12. Physical conjugation of gold nanoparticles.104
In contrast to various phase-transfer ligand exchange and modification approaches
addition of amphiphilic coating layer that adsorbs by hydrophobic interaction to the
hydrophobic ligand molecules of the nanoparticles does not depend on the core material
and ligand type.83 Addition of the molecular layer on the particles’ surface is an addition
of an external surface of the NPs shell without causing any changes of the initial ligands
(fig. 12).83 The idea is based on the addition of a hydrophobic layer of polymer and
encapsulating it into the initial shell of NPs. This approach enables to transfer
hydrophobic nanoparticles to water, and particles from aqueous phase to organic
47
solvents.83 Taking advantage of using the polymer coating method, various NPs can be
transferred into water with one amphiphilic polymer. One of the most known surfactant
are quaternary ammonium salts.83 This molecule can transfer hydrophilic nanoparticles
to the organic phase by adsorbing electrostatically onto the negatively charged surface
of the nanoparticles. On the contrary, CTAB can be used to transfer hydrophobic
particles to the aqueous phase by hydrophobic interactions. Additionally, they appear to
be stable over wide pH range and high salt concentration.
Physical conjugation strategies use noncovalent binding in a combination of
hydrophobic and electrostatic interactions of the gold surface and molecules.99
Examples of physical conjugation include adsorption of proteins on nanoparticles
surface105 or assembly of therapeutic agents onto nanoparticles.86 Nanoparticles coated
with hydrophobic layer can adsorb hydrophobic anticancer drugs and the drugs may
then be released inside cells.86 Even though this type of conjugation has several
advantages, such as the lack of modification step and simple rapid binding, it is
extremely difficult to control the orientation of the bound ligand. Therefore, this binding
it is non-specific and is not appropriate for immobilizing targeting moieties.
Gold nanoparticles were successfully adapted as powerful sensor based on electrostatic
properties of single and double-stranded DNA.106 DNA sequencing and sensing is of
great importance for pathogen detection and biomedical fields. It was found that single
and double stranded oligonucleotides have a different affinity towards the nanoparticle’s
surface. By adsorption of single stranded DNA, the nanoparticles are stabilized, which
prevents them from aggregation. The colour of nanoparticles depends on SPR and their
aggregation state, therefore electrostatic properties of single-stranded DNA and doublestranded DNA can be used to design a simple colorimetric hybridization assay. More
interestingly, the studies indicated that the various sizes and shapes of gold particles are
essential for enhancing the sensitivity of the SPR biosensor applications.106
Flexible surface chemistry, rather simple synthesis, and a large surface area of
nanoparticles makes them ideal candidates for the intracellular delivery of genes,
antibacterial drugs, or particular anticancer drugs. These molecules can be either
covalently or non-covalently conjugated. The release of covalently bonded molecule
from nanoparticle’s surface can be accomplished by photo-uncaging or redox reactions.5
In contrast to covalent conjugations, non-covalent ones are released as the solution
48
condition changes or by the photothermal effect. The functionalization of polycationic
molecules such as polylysines, polyarginices, polyaminoesters, polyethyleneamines or
amphiphilic thiols on nanoparticle’s surface enables them to attach negatively charged
DNA or RNA molecules to the surface of nanoparticles and also renders intracellular
delivery.
Adsorption and self-assembly are straight forward of surface modification of
nanoparticles, providing good hydrophilicity and stability in suspension.
Direct
adsorption is considered a relatively strong, non-covalent interaction of certain
biological molecules to NPs.88 Small molecules or even polymers can attach to the
surface of particles through adsorption or exchange with original ligands under mild
conditions. Nevertheless, a lot of effort has been applied to direct the self-assembly on
nanoparticles, especially with biomolecules or other templating agents.
Recently, Ackerson et al. reported the assembly, of gold nanoclusters mediated by
gold-ligand interactions with nonthiolate ligands.107 They investigated the dynamics and
the
nature
of
Au20(SC2H4Ph)15-diglyme
into
Au20(SC2H4Ph)15-diglyme-
Au20(SC2H4Ph)15 assembly and tried to understand the generality of the electron sharing
among neighbouring particles. The assembly arises from the attractive forces between
the gold nanoclusters and diethylene glycol dimethyl ether (diglyme), only in the
presence of diglyme as a cosolvent. Similar assembly of nanoclusters was observed for
p-mercaptobenzoic acid and glutathione protected gold nanoclusters. The results
indicated that synthesis of nanoclusters in the presence of diglyme result in a system
that merges the solvent and clusters together. Understanding the nature and dynamic of
these assemblies may open a avenue to their fundamental properties which may find
applications in sensing.107
Breaking the interaction symmetry in gold nanoparticles by placing a known number of
other molecules in their ligand shell would open the avenue for potential applications.
Stellacci et al. reported creative method to obtain anisotropic assembly of gold
nanoparticles.108 The topological nature of monolayer protected gold nanoparticles was
used to functionalize them at two diametrically opposed points. The synthesised
particles worked as divalent building blocks which could produce self-standing films,
by reacting with complementary divalent molecules. The carried out tests confirmed
that van der Waals interactions between ligand shell of the particles and interchain
49
molecules morphology were stable and mechanically strong.108 Generally, selfassembly of metal nanoparticles driven by the interaction of surface ligands such as
proteins, DNA or multivalent thiolates, is a method capable of making one, two or three
dimensional nanoparticle structures. The major driving forces for self-assembly include
electrostatic interactions, capillary forces, surface tension, hydrophobic interaction, and
bio-specific recognition. Host-guest interactions are common for biological systems,
however, the non-biological molecules can be also stabilized by these weak interactions.
Biomolecules and different chemicals can be adsorbed to nanoparticle’s surface via an
electrostatic interaction.20 The negatively charged molecules such as nucleic acids can
be attached to the positively charged surface of nanoparticles. Further, the proteins
which have a natural positive or negative charge domains can be adsorbed to NPs with
negative or positive charges.109 As the electrostatic interaction is the main driving force
of the conjugation, the attachment is simply dependent on the surface charge domain.
There are, however, lots of drawbacks which need to be considered before testing this
approach. Due to a high degree of non-specific binding the target biomolecules can lose
their biological function.16 Therefore, poor control over the site of modification,
sensitiveness to pH and salt concentration, often require a strict physiological conditions
or engineering of proteins in some cases.86
3.4 Bioconjugation of gold nanoparticles
Bioconjugation is an extension of previously described approaches of chemical and
physical conjugation (fig. 13).20 It’s an effective approach to introduce extra
functionality into nanoparticles by using various functional molecules, including small
molecules like lipids, vitamins, sugars, peptides, and larger ones such as protein,
polymers, enzymes, RNA and DNA.7,83 Biological processes are often based on highly
specific interactions between biomolecules. These interactions include receptor and
target interaction, antibody-antigen interaction, enzyme and substrate interaction, and
complementary base pair of nucleic acid.7 They can react directly and rigidly to specific
sites of different kinds of nanoparticles. Taking advantages of those bioconjugate
interactions of nanomaterials formulation is essential for future applications.20 The large
surface area of nanoparticles enables to conjugate them with various sensors, contrast
agents, targeting molecules, drugs and genes.83 For example, nanoparticles that are
bioconjugated with DNA have been used to specifically recognize target genetic
materials.86 Their tunable optical properties make them ideal for the selective and
50
sensitive detection of analytes87. Additionally, non-toxic nature, good water solubility,
high biocompatibility and well-defined surface chemistry make them promising
bioimaging materials. Even though the coupling strategies are highly efficient and
specific, it still remains challenging to control them fully.83 The main challenge is to
control the orientation of the biomolecules attached to NPs.
Figure 13. Examples of nanoparticle bioconjugation protocols.104
The use of cross-linking agents is a common conjugation strategy. Crosslinking
reagents are molecules that contain two or more functional groups which control the
binding orientation, therefore, are capable of attaching to specific functional groups on
another molecule. They can have different chemical reactivity and properties that affect
their behavior depending on the application. Common linker chemistry is based on the
reactions
between
sulfhydryl-containing
biomolecules
and
amine-modified
nanoparticles. Commercially available N-hydroxysuccinimide (NHS) ester and 3-sulfoNHS ester derivatives of organic dyes, biotin can be conjugated to primary or secondary
amine functionalized nanoparticles through the amide bond. The reaction mechanism
can form covalent complexes between ligands and nanoparticles. Nanoparticles
51
functionalized with carbonyl group can be covalently conjugated to amine group
through amide linkage.86 Carbodiimide chemistry employs mild reaction condition and
is effective for the attachment of molecules bearing single amine group. Moreover, it
gives versatility to bioconjugation for a wide variety of protein and enzyme
molecules.110 However, it remains difficult to control the binding orientation, leading to
loss of functionality of the targeting ligands.86
Nanoparticles can be conjugated to antibodies by using variety of methods. 16 One of the
most common biological interactions is to use biotinylated gold nanoparticles and
attaching them to avidin/streptavidin through strong coupling between biotin and avidin.
The specific and strong bonding between biotin-avidin has allowed this approach to be
used in many other application.83 Similarly, fluorescent dyes, drugs, amino groups in
antibodies, proteins, peptides or DNAs can be biotinylated and further attached in one
of the free biotin binding pockets in the streptavidin-functionalized gold nanoparticle.7
Recently, the coupling of protein to gold nanoparticles was studied by using strong
binding between biotin and streptavidin in comparison to carbodiimide chemistry.110
First, the gold particles and catalase were biotinylated and then coupled together by
using a streptavidin crosslinker.110 The biotin-streptavidin binding required two-step
synthesis procedure and the enzyme bound particle turned out to be stable to be used for
further studies.
The selectivity and sensitivity response of gold nanoparticles to the biological
environment plays a crucial role for the biomedical applications. Therefore, conjugtion
strategies can be used to connect of biologically active molecules, such as
oligonucleotides, DNA hairpins, peptides, antibodies, proteins, fluorescent proteins and
organic dyes, and many other biological specimens to the surface of gold nanoparticles
for construction of biosensors.7 Previously mentioned, site-specific conjugation of
malemide functionalized Au102(pMBA)44 cluster to enteroviruses was recently published
by Marjomäki et al.15 This covalent bioconjugation of gold particle to viral surface is of
great importance tool for tracking of biological systems, for example, in understanding
the pathogenesis of virus infection.
Surface plasmon resonance properties of gold nanomaterial are extensively applied in
biological applications especially in a large variety of light-based techniques. Therefore,
they can be conveniently adapted as powerful sensors of DNA hybridization processes,
52
pathogens, protein-protein interactions, antigens, and various toxic materials. For
example, the blue shift in the SPR band is observed when the gold nanoparticles
functionalized with oligonucleotides sense complementary DNA strands. Surface
plasmon resonance properties are used when sensing of human chorionic gonadotropin
(b-hCG). Antibody-functionalized gold nanoparticles are exploited in pregnancy tests
exhibiting such a shift in the SPR band. Moreover, gold nanoparticles have been used to
develop assays for cholera cells detection by using the SPR sensor technique. The
information of biological interaction can be obtained by carefully monitoring SPR
coupling characteristic. They have also received great attention because of detection of
the DNA, aptamers or oligonucleotides with the use of functionalized AuNPs are
straightforward and simple.106
Additionally, the fluorescence properties of dye-functionalized oligonucleotides to
complementary DNA strands have found various applications in biosensing.
Nanoparticles functionalized with amine groups can be conjugated with fluorescence
dyes.86 Recently, Liu group reported the successful application of Au nanoclusters as a
fluorescent sensor in the detection of cyniade with high selectivity and sensitivity.4
Furthermore, the fluorescent clusters can be applied to the detection of heavy ions in the
environment. The enhancement in the light scattering intensity of nanoparticleaggregates via protein-protein interaction is utilized in pharmacology to sense molecules
such as adenosine, urinary, immunoglobulins and toxic substances including arsenic or
pathogenic bacteria and viruses.
In addition to fluorescence properties, green fluorescent protein (GFP) functionalized
nanoparticles are used cells as a biosensing method for the detection and labeling of
cancer cells and microorganism. Wu et al. applied gold nanoparticles with near-infrared
(NIR) emission in tumor fluorescence imaging in vivo.86 The imaging signal could be
well distinguished from the background and the studies confirmed no potential toxicity
to the body. Moreover, a functionalized gold nanoparticle with the proper molecule such
as oligonucleotide or dye is also useful for Raman- or SERS-based detection of
pathogens or complementary DNA strands.
Gu et al. described efficient method for cancer cell imaging which was based on
fluorescence dye-modified chitosan-coated magnetic nanoparticles.86 Aldehyde
functionalized nanoparticles were recently used to synthesize tumor-targeted
53
multifunctional viral nanoparticles using hydrazone ligation reaction.86 Moreover, the
ligation methods enable introduction of different peptides. The hydrazide functionalized
nanoparticles can react directly with anticancer drugs containing carbonyl group.86
4 CONCLUSION
A typical gold nanoparticle is a metallic particle surrounded by a dense, protecting
ligand layer whose diameter is in nanoscale.3 Metal NPs have become important
scientific tool, due to their special advantages, in many fields such as electronics,
photochemical, biomedicine and chemistry. Their extremely small size, stability, high
surface area, non-cytotoxicity and tuneable optical, chemical and physical properties
have found wide spread applications in imaging, target drug delivery, therapeutics and
diagnosis. During recent years, tremendous attention has been paid towards controlling
the size and the surface chemistry of gold nanoparticles, mainly because all their unique
properties are influenced by their diameter and protecting ligand layer.12 One of the
main advantages of gold nanoparticles is that these particles are biocompatible and can
be conjugated with small biomolecules, such as enzymes, proteins, carboxylic acids
amino acids and DNA, resulting in endless functionalities.
The particular interest in this thesis was in nanoclusters that can be categorized by size
with diameter below 2 nm, and containing less than 200 atoms.3 These, monolayer
protected gold nanoclusters are interesting because they possess unique chemical and
physical properties which can be tuned by changing their size. Moreover, metal
nanoclusters form a very important intermediate size regime between bulk materials and
discrete atoms. Typically, they are composed of definite number of atoms and possess
stable structure.3 The reduced size of metal structures has strong implications on their
properties. The presence of quantum effects contribute for the appearance of new
electronic, optical and chemical properties such as photoluminescence, magnetism and
catalytic activity.3
Monolayer protected nanoclusters can be prepared by the chemical reduction of metal
salt in the presence of the protecting ligand. The precise control and stability of the
clusters are the most critical factors. The ligand which adsorb on the nanoparticles’
surface prevents them from aggregation and increase the stability. Besides, the ligand
54
layer allows introducing of functionalities by various surface functionalization
reactions.
Most of the particles can be synthesised in the organic solution. Therefore, surface
modification reactions are of great importance because they can possibly provide new
properties or functionality to the particles. In these cases, ligand exchange or ligand
conjugation strategy can be applied. Introducing different molecules, compounds, and
bridging groups to the nanoparticle’s surface enable large number of possibilities with a
variety of functionalities. Conjugation of nanoparticles with biomolecules allows them
to interact nanoparticles specifically with biological systems. For example,
nanoparticles can be site-specifically conjugated with biomolecules by molecular
recognition.17 By choosing appropriate modification strategy molecules, surfaces, cells
or biomolecules can be tuned, bringing new functionalities and unique properties.
For that reason, modified particles need to be characterized by using various analytical
tools, depending on the nature of particles. Surface modification reactions often directly
influence particle’s physical and chemical properties, therefore transmission electron
microscopy (TEM) can be used as preliminary tool to confirm particle size and samples’
morphology. The optical properties of gold clusters can be studied using UV-vis
spectroscopy. It is a valuable tool to study nanoparticles size, agglomeration state and
concentration.
Generally, the properties of nanoparticles have proved to be promising in labelling and
imaging, sensing and detection and as an active elements for optical sensitizing,
delivery vehicles and heat mediation.17 Additionally, due to the well-defined structure
of monolayer protected gold nanoclusters (MPC), functionalization surface can be
examined with atomic precision. Several approaches have been used to modify MPC,
such as dynamic linking of atomically precise Au20(SR)16 clusters via weakly
interacting diglyme molecules107, and self-assembly of gold nanoclusters driven by
interaction of surface ligands and proteins111. Ligand exchange synthesis have been
utilized
to develop a large scale, facile synthesis of Au38(SC12H25)24 cluster.61 It
involved glutathione capped nanoclusters as starting material, which further were
exchanged with HSC12H25 under etching procedure. Shischibu et al. also used a
phosphine stabilized Au11 cluster to obtain glutathione (GSH) protected Au25
nanoclusters
under
optimized
conditions.4
Moreover,
the
conversion
of
55
Au38(SC2H4Ph)24 to Au36(SPh-tBu)24 was the first example of
size transformation
caused by the thiol-to-thiol ligand exchange.3 Monolayer protected gold nanoparticles
were also successfully adapted in conjugation method with biological material which
enable to study pathogenesis of virus infection by tracking the virus in tissues and
cells.15
The chemistry of surface monolayer protected nanoclusters is a versatile and rapidly
developing field. However still not too many functionalization studies exist so far.17
Several major issues need to be worked out for future applications. The main challenge
remains in developing a suitable strategy that enables chemical control in surface
modification and bioconjugation strategies of gold nanoparticles. Fundamental
understanding of the molecular mechanism of the growth of nanoclusters and the
principle of size control would lead to investigation of their properties and also for
crystallization trials.3 Structure determination and characterization methods of clusters
may help to understand the relationship between the structure and clusters properties.
Moreover, from the atomic packing structure the electronic properties of clusters can be
evaluated. The evaluation of the cluster’s behaviour would lead to a fundamental insight
into their property-structure correlation, providing new principles for the design of
functional nanoparticles.
56
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