Synthesis of fluorescent gold nanoclusters for biological applications

Dinesh Mishra

Introduction
The synthesis of colloidally stable nanocrystals (such as plasmonic nanoparticles and semi-
conducting quantum dots) have been pursued for several decades due to their potential applications
in therapeutics.1,2 To achieve these goals, much effort has been devoted to engineer the properties
of these materials so that they behave in desired way in living systems. These properties are size,
shape, stability, biocompatibility and selectivity.3 It is believed that these advances in
nanomedicine will improve and replace our conventional diagnoses and therapies.4 A special type
of nanomaterials based on gold with average size below 2 nm have been studied actively over the
last decade due to their interesting optical and electronic properties arising from the ultra-small
size. These nanomaterials are known as gold nanoclusters (AuNCs). Although thiolated AuNCs
have been well known for more than a decade, their application in biological sensing was hindered
by their poor luminescent property. In the last few years, significant progress has been made in the
preparation of stable, highly luminescent, water dispersible and biocompatible AuNCs.5 As a result
of these advances, AuNCs (and other metal based clusters, such as silver, copper, etc.) are gaining
recognition as useful tools in analytical applications.

AuNCs consist of a few to hundreds of atoms with size ≤2 nm, which is comparable to the fermi
wavelength of the electrons. Due to their small size, there is strong quantum confinement effect,
which results in the formation of discrete energy levels. Thus, nanoclusters exhibit molecule-like
properties.6 Unlike larger gold nanoparticles (AuNPs), which have strong optical absorption due
to surface plasmon resonance (SPR) arising from collective oscillation of the conduction electrons,
the AuNCs have distinct optical absorption features arising from the electron transition between
different electronic states. It is also known that the optical absorption of AuNCs is also influenced
by their size, surface ligands, pH, temperature and oxidation state.
 Peptides/ Proteins

Nucleic Acids e-
Photo Photo  2 nm
excitation emission

Polymers

Molecular scale ligands

Figure 1. Schematic representation of a ligand-stabilized nanocluster and the photo-luminescence

in AuNCs.

The most intriguing feature of AuNCs is their photo-luminescence. Luminescent AuNCs

are being actively pursued for potential applications in biological sensing and imaging, particularly
because of their small size, low cytotoxicity and multi-color emission.5,7-9 The mechanism of
photo-luminescence in AuNCs is still not completely understood. The basic model used to explain
fluorescence in AuNCs is the excitation of the electron in the highest occupied molecular orbital
(HOMO) to higher energy level, followed by the radiative recombination of the excited state
singlet electron back to the ground state. Although this model works for several AuNCs reported,
it is however insufficient to account for the observed optical properties of several AuNCs, whose
optical absorption and emission are independent of their sizes. This indicates that some other
mechanisms of photo-luminescence may exist. It is suggested that the ligand to metal charge
transfer (LMCT) at the ligand-cluster interface or ligand to metal-metal charge transfer (LMMCT)
might also be responsible for the fluorescence in AuNCs.10 This kind of interaction is indicated by
a large stokes shift along with a relatively long average fluorescence life-time.
Synthesis of luminescent metallic nanoclusters

Several studies have reported the synthesis of luminescent gold nanoclusters with multi-color
emission. These methods can be broadly classified into two categories: (i) reduction of Au3+ in
presence of ligands or templates (e.g. proteins) and (ii) etching of large size gold nanoparticles
(AuNPs) by thiol compounds. The properties of these nanoclusters are dependent on the growth
conditions and the ligands used. Different kinds of molecules which have high binding affinity to
the metal atoms are used to stabilize noble metal nanoclusters. Molecular scale ligands include
thiols (phenylethanethiol, mercaptobenzoic acid, mercaptopropanoic acid, etc.), phosphines (e.g.
triaryl phosphine) and amines. Macromolecules such as proteins, peptides, polymers and nucleic
acids are also used in the template mediated synthesis of gold nanoclusters with multi-color
emission. Other important factors that determine the properties of the nanoclusters are: ratio of
metal to ligand, pH, temperature, time and reducing agent. By varying above mentioned
parameters, it is possible to control the size, surface properties and optical properties of
nanoclusters.

1) Chemical reduction

Several stable and atomically precise gold nanoclusters can be prepared via reduction of Au 3+ to
metallic Au in presence of capping ligands. Thiols are widely used capping ligands because of the
strong Au-S bonding and common reducing agent is sodium borohydride.11,12 The particle size of
the nanoclusters can also be controlled by varying the ratio of metal to thiolates. Usually, greater
than 1 molar excess of thiols is required for the formation of nanoclusters. Both non-fluorescent
and fluorescent thiol-protected gold nanoclusters have been prepared using borohydride reduction
method. However, sodium borohydride is a strong reducing agent, which makes it harder to control
the growth of the particles during synthesis. Almost all of the fluorescent gold nanoclusters
prepared by borohydride reduction have optical emission limited between red to near IR region of
the spectrum. Thiols, besides acting as capping ligands are also known to be mild reducing agents.
The reducing property of thiols have been exploited recently to obtain multi-color emitting AuNCs
which contain metallic Au in the core surrounded by a monolayer of Au(I)-thiolate
complexes.9,13,14 Polymers (e.g. PAMAM dendrimers) biomolecules (e.g. Bovine Serum Albumin,
nucleic acids) can also act as scaffolds which help in the nucleation and growth of luminescent
gold nanoclusters.15,16

2) Photo-reduction

To avoid using harsh reducing agent such as sodium borohydride, which can alter the properties
of certain sensitive functional groups in the capping ligands and template biomolecules (peptides
or proteins), photo-reduction strategy has been introduced. Using polymers as ligand scaffolds,
several highly luminescent AuNCs have been prepared by photo-reduction.17,18 As in chemical
reduction, here also the size of the nanoclusters is controlled by varying the molar ratio of Au ions
to the polymer.

3) Etching
Etching is a top-down process, which involves the core reduction of slightly bigger sized gold
nanoparticles (AuNPs) in presence of excess of ligands. AuNPs with size 2-5 nm can be chemically
etched by thiols in alkaline conditions to form AuNCs. AuNCs with various color emission have
been prepared by etching AuNPs using various thiol ligands.19-21 Additionally, several non-
fluorescent AuNCs can also be further etched in presence of thiols to form highly luminescent
AuNCs.

Bioconjugation

AuNCs with reactive functional groups such as carboxyl, amine, alcohol, thiol and azide
can be covalently conjugated with various molecules which can be delivered to specific cellular
sites. Thus, AuNCs can act as vehicles for transporting drugs, biomolecules across cell membrane
and their delivery can be probed by tracking the luminescence of the AuNCs. Several covalent
conjugation strategies are already well explored using AuNPs and quantum dots as the platforms.
Several conjugation techniques such as EDC/NHS coupling, CDI coupling, maleimide coupling,
click reactions, etc. are highly efficient for the covalent linkage of commonly used functional
groups. AuNCs, due to their surface and composition dependent photo-physical properties,
sometimes lead to change in the optical absorption and emission during conjugation process, such
as blue or red-shift and photoluminescence quenching. Despite these difficulties, significant
progress has been made in the development of facile conjugation strategies giving a robust cluster-
bioconjugate. Besides covalent conjugation, certain peptides and proteins can be directly adsorbed
on the cluster surface through electrostatic interaction between the oppositely charged groups on
the cluster surface and the biomolecule.

COOH
NH2
FG OH
SH
N3

Drug molecule,
Peptide,
AuNC FG Protein, etc.

Bioconjugate
Chemistry

Figure 2. Schematic representation of bioconjugation of AuNCs with functional molecules.

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