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Effects of protecting groups on luminescent metal

nanoclusters: spectroscopic signatures
Cite this: Chem. Commun., 2022,
58, 29 and applications
Subhajit Chakraborty and Saptarshi Mukherjee *
Published on 12 November 2021. Downloaded on 1/20/2023 7:08:58 AM.

Luminescent metal nanoclusters (NCs) have been established as next-generation fluorophores. Their
biocompatible and non-toxic nature, along with excellent chemical- and photo-stability, enables them
to find applications in multi-disciplinary areas. However, preparing NCs which are stable is always
challenging, primarily owing to their small size and propensity to self-aggregate. In this review, we
highlight a holistic approach as to how ligands and templates can monitor the stability of NCs, tune their
spectroscopic signatures, and alter their applications. The role of small molecules of a large ligand in the
Received 24th September 2021, preparation of NCs and their associated limitations are also discussed. We have summarized how these
Accepted 12th November 2021 NCs can be utilized in sensing several metal ions, pH, viscosity and temperature of many systems which
DOI: 10.1039/d1cc05396e have biological relevance. Additionally, these luminescent metal NCs find usage in cell-imaging,
discriminating between cancerous and non-cancerous cell lines and also targeting specific organelles
rsc.li/chemcomm within the cellular environment.

Introduction
Recently, ultra-small-sized metal nanoclusters (NCs) have
drawn considerable research interest owing to their unique
size-dependent characteristics consequent to the quantum
Department of Chemistry, Indian Institute of Science Education and Research
Bhopal, Bhopal Bypass Road, Bhopal 462 066, Madhya Pradesh, India. confinement effect.1–14 These metal NCs are characterized by
E-mail: [email protected] easy synthetic protocols, high photostability, water solubility,

Subhajit Chakraborty completed Professor Saptarshi Mukherjee

his BSc with Chemistry majors received his BSc and MSc degrees
from the Ramakrishna Mission from Jadavpur University, Kolkata,
Vivekananda Centenary, Kolkata, India. He carried out his PhD under
India, and received his MSc degree the supervision of Professor Kankan
from the Presidency University, Bhattacharyya at IACS, Kolkata,
Kolkata. He has joined PhD and post-doctoral research with
program in the Department of Professor H. Peter Lu at the
Chemistry, IISER Bhopal, under Bowling Green State University,
the supervision of Professor Ohio. He joined the Department of
Saptarshi Mukherjee. He is Chemistry, IISER Bhopal, in 2008.
primarily working on luminescent His research interests include
Subhajit Chakraborty metal nanoclusters, and their Saptarshi Mukherjee luminescent metal nanoclusters,
applications in biological systems protein unfolding and refolding
using spectroscopic and microscopic techniques. He loves to paint dynamics, and surface chemistry using spectroscopic and microscopic
and write in his leisure time. approaches. He is the recipient of INSA Young Scientists Award, CRSI
Bronze Medal, CRSI Young Scientist Award and is a Senior Editor of
Chemical Physics Impact. He is also a founding member of the Indian
National Young Academy of Science.

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catalytic properties, biocompatibility, non-toxicity, etc., which model, which connects the Fermi energy of the particular metal
enable them as next-generation fluorophores.15–30 Generally, (EFermi), the corresponding emission energy (Eem) and the
metal NCs are very robust to photon irradiation; they are much number of atoms in a given cluster–core (N), is as follows:16
more photostable as compared to commercial organic dyes.13,15
This thereby enables them to be used in single-molecule Eem = EFermi/N0.3 (1)
spectroscopic investigations where photobleaching is one of The most unique feature of metal NCs is their size-
the challenges that need to be overcome. Additionally, these dependent luminescence properties. By tuning the size, one
metal NCs are generally non-toxic and biocompatible.13,19,29 can regulate the photophysical characteristics of metal NCs.43
This feature of metal NCs differentiates them from analogous However, various other factors like capping ligand and the
quantum dots (QDs) which are generally far more toxic. Due to environment around the cluster core have an enormous influence
these interesting features, metal NCs can be utilized for a on the photophysical properties of metal NCs (will be discussed
plethora of applications in interdisciplinary research fields later).43 Thus, the optical properties of metal NCs do not follow
and thereby can act as a replacement for organic dyes and a linear relationship with the size of the NC alone.43,44 Due to the
quantum dots (QDs). However, despite these advantages, there presence of free electrons and their collective oscillations, NPs
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are some bottlenecks related to metal NCs which require display characteristic surface plasmon resonance (SPR) bands in
further exploration and refinements. One of the major the UV-Vis absorption profile. This feature differentiates them
challenges is related to the stability of these metal NCs after from metal NCs. The formation of discrete energy levels due
their formation. Lack of proper surface protection can lead to to the reduction in size during the formation of metal NCs
the oxidation of metal atoms present in the core of NCs which facilitates the one-electron transition in between quantized
results in their degradation. Often, larger nanoparticles are energy states. Consequently, a stepwise optical absorption
generated along with NCs which are mostly non-fluorescent. results in a continuous absorption signature. Interestingly, the
Consequently, an inherent heterogeneity appears within the concept of enhancement of luminescence intensity through
system. For this reason, the selection of capping ligands and aggregation has been explained in recent times. In 2012, Xie
the optimization of synthetic protocols must always be properly and co-workers reported the formation of bright emitting AuNCs
addressed. Another shortcoming of these metal NCs is their and their luminescence properties have been ascribed to the
low quantum yields (QY) as compared to those of QDs and concept of aggregation-induced emission (AIE).45 In contrast to
conventional fluorophores. Although investigations are going larger sized non-luminating NPs, AIE provides the attachment of
on to circumvent this aspect, it requires more exploration so the neighbouring protecting ligands of the individual cluster–
that these metal NCs can be used in applications that require cores with one another.7,46 Thus, the AIE phenomenon is
greater photoluminescence intensities. associated with the large Stokes shift and long excited-state
The optical properties of larger metal nanoparticles (NPs) lifetime decay components.46
are strongly dependent on the free electron density and their There are various ways by which NCs can be synthesized,
sizes, which can be explained by the Mie theory.31–33 The Mie and such protocols are well-documented.47,48 Generally, two
theory can still provide an understanding for the particles such standard approaches are followed: (i) top-down approach
having a size comparable to the electron mean free path.31,34 and (ii) bottom-up approach (Scheme 1). The top-down
However, interestingly, when the particle size approaches the approach involves some dry and wet grinding/etching
Fermi wavelength of the conduction electron (i.e., formation of processes. Additionally, the yields of the final products are
NCs), the optical properties become drastically different from generally low which thereby limits this approach toward the
the larger metal NPs as the continuous electronic band structure formation of stable and cost-effective NCs. Hence, the favoured
is converted into quantized discrete energy levels.35–39 approach is the bottom-up approach, which is a combination of
Consequently, these NCs become luminescent and act like reduction of metal ions to their zero-valent oxidation states
molecular species. According to the Drude model,1 the free followed by the aggregation of the reduced metal atoms and
electrons of the metal atoms (valence s-orbital for noble metals) encapsulation by ligands which leads to the formation of stable
become delocalized over the cluster core. The total Hamilto- metal NCs.12 In general, this process involves two steps: (i) bio-
nian of the metal NCs may be expressed as a combination mineralization;12,49 reduction of metal ions in the presence of
of the noninteracting Hamiltonians of the free electrons.1 reducing agent(s) (sometimes the ligand itself can serve this
This concept is based on the ‘‘one-particle approximation’’ purpose) which produces polydispersed cluster–cores50 and (ii)
and is related to the particle-in-a-box model which explains subsequent nucleation; formation of molecularly pure cluster–
the behaviour of free electrons in metals.1 According to Hooke’s cores. Very recently, Xie’s and Ying’s groups well described the
law, a linear restoring force when applied on free electrons 2e reduction process in a stepwise manner for the growth of
makes the quantized energy levels behave as three-dimensional cluster–cores of AuNCs containing Au0.51–53 Sometimes the pH
harmonic oscillators.40–42 Using the concept of free electrons of the medium activates the ligand molecule or particularly, the
based on the Drude theory, the Jellium model has been functional group of the ligand molecule by deprotonation to
introduced to estimate the cluster–core composition which exhibit the reducing ability.13,15
actually governs the luminescence properties of the said metal To avoid the aggregation of tiny clusters once formed and
NC.16 A mathematical expression of the most accepted Jellium the subsequent formation of bigger-sized NPs, the ligands play

30 | Chem. Commun., 2022, 58, 29–47 This journal is © The Royal Society of Chemistry 2022
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ChemComm Feature Article

Fig. 1 (A) Theoretical optical curves of Au25(SePh)18 and Au25(SC2H4Ph)18

nanoclusters as marked in the figure. Comparison of the component of KS
orbitals of Au25(SePh)18 (B) and Au25(SC2H4Ph)18 (C) nanoclusters.
Reproduced with permission from ref. 61. Copyright 2014, The Royal
Society of Chemistry.

In a seminal work, Yang and Chen demonstrated that the

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Scheme 1 Schematic representation of the two different ways by which

NCs can be prepared: (A) top-down approach and (B) bottom-up increment of particle band gap energy (from 1.4 to 1.8 eV)
approach. can be achieved for gold particles by altering the capping ligand
from triphenylphosphines to alkanethiolates.60 Zhu and
co-workers very nicely demonstrated that selenolate capped
a pivotal role by protecting the zero-valent cluster cores.54,55 Not
Au25(SePh)18 NCs exhibited totally different electronic
only the protection and surface stabilization of the cluster–core,
distribution and optical properties in comparison to its thiolate
but also the photophysical and morphological properties of
counterpart (Fig. 1), Au25(SCH2CH2Ph)18, despite having a
NCs are also highly influenced by the capping agents.56,57
similar icosahedral Au13 core and semi-ring motifs (Au2(SeR)3
To date, a large variety of ligands have been used for the
or Au2(SR)3) (Fig. 2).61 Thus, the rational use of ligands can alter
facile preparation of noble metal (Au, Ag and Cu) NCs, such
the HOMO–LUMO energy gap of the molecule.
as protein, peptides, polymers, DNA, small molecules, etc.
Also, the facile binding affinity (between the electron-
(Scheme 2).8
donating atom of the ligand and metal ions/atoms) leads to
In 1978, Bartlett and co-workers prepared 0.8 nm sized
the selectivity of ligands toward the formation of NCs. For
gold nanoclusters (AuNCs) using an amino-substituted triaryl-
example, the soft–soft interaction between the S atom and Au
phosphine ligand (Ar3P)7Au11X3).58 They observed that
atom leads to the formation of highly stable AuNCs.62,63 Using
when the substituent, ‘‘X’’ was iodine, the clusters so formed
Raman spectroscopy, Duguid et al. monitored the interaction of
were unstable and had a tendency to undergo oxidative
DNA and a series of divalent metal atoms and demonstrated
decomposition.58 However, Hutchison and co-workers demon-
that the nitrogen centers of the nucleobases are more
strated that stable AuNCs having a similar core composition
favourable toward bond formation with metal ions.64 In two
(i.e., Au11) can be prepared when alkanethiols are used as
separate studies, Ono et al. and Ritchie et al. had opined that the
a capping agent instead of triarylphosphine ligands.59
cytosine-rich (C-rich) sequence of DNA promotes the easy formation
of AgNCs as cytosine has a high affinity to bind with Ag+ ions.65,66

Fig. 2 Crystal structures of Au25(SePh)18 (A) and Au25(SC2H4Ph)18 (B)

nanoclusters. (C) The Au13 core of Au25(SePh)18. (D) and (E) The distor-
tions of Au2Se3 and Au2S3 motifs, respectively, in the sh plane along x–y
(color labels: yellow = Au, violet = Se, orange = S, for clarity C and H atoms
Scheme 2 Representation of different ligands used for the preparation of are omitted). Reproduced with permission from ref. 61. Copyright 2014,
NCs. The Royal Society of Chemistry.

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they have direct relevance to biomedical investigations.67

Discriminating cancerous and non-cancerous cells is extremely
important for the early detection of diseases. It has been
reported that AuNCs, templated by the globular protein
(human serum albumin, HSA; bovine serum albumin, BSA)
can be effectively utilized to differentiate between cancerous
and non-cancerous cell lines.62,63 The uptake of these AuNCs is
much more in the case of cancerous cells as compared to the
non-cancerous ones. Additionally, targeting specific organelles
within a cell is also important from the perspective of drug
delivery and subsequent release. The associated dynamics
Fig. 3 The plots represent the Au L3-edge WT-EXAFS for (A)
inside a cell are primarily controlled by the microenvironment
Au18(SC6H11)14 in toluene and (B) Au18(SG)14 in water. Reproduced with
permission from ref. 57. Copyright 2018, American Chemical Society.
viscosity, polarity, pH, etc. To have an in-depth understanding
of the dynamics inside a cell and probe the microenvironment
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in and around an organelle, luminescent metal NCs are widely

In a recent study, Chevrier et al. investigated how the used.75–78 The fact that these NCs are photostable, non-toxic
template influences the structure and properties of Au18(SR)14 and biocompatible in nature enables them to be efficiently
NCs (where, –SR = cyclohexanethiolate ligand and glutathione used in single-molecule spectroscopic studies and for
ligand) using synchrotron X-ray spectroscopy and quantum bioimaging investigations.62,63,79–81 For most of the cases,
mechanics/molecular mechanics (QM/MM) simulations specific targeting of organelles within a cell can be realized
(Fig. 3).57 This report clearly demonstrated that encapsulation by rational use of ligands.67 It is pertinent to be mentioned that
by glutathione can provide more stability through compact the translational diffusion of NCs is not similar in every cell
surface construction with suitable rigidity (Fig. 4). Also, the due to the different microenvironment and system-specific
enhanced photoluminescence and biocompatibility of these localization of NCs inside the cell lines.62,81 Bhattacharyya
AuNCs have been explained based on the above-mentioned and co-workers studied the translational relaxation of AuNCs
concept.57 using fluorescence correlation spectroscopy (FCS) (Fig. 5).62
Noble metal NCs have been widely used in sensing They demonstrated that the AuNCs follow 2D diffusion in the
applications due to their selective and sensitive detection non-cancerous MCF10A cell lines (i.e., diffusion through the
ability towards various chemical entities.63,67–74 This property cell membrane only), whereas the same set of NCs experienced
of these next-generation fluorophores enables them to be used a combination of 2D and 3D diffusion in the cancerous MCF7
for both in vitro and in vivo sensing. It is always challenging to cell lines (i.e., localization both in the membrane and the
sense chemical entities inside complex biological systems as cytoplasm, Fig. 5).62
Additionally, NCs are the suitable choice for the sensitive
detection of pH,82–84 various biomolecules such as proteins,85
nucleic acids,86,87 small biomolecules67 and even viruses.74,88
Triulzi et al. were the first to report the detection of human IgG
using PAMAM coated AuNCs which were used as a fluorescence
probe.89 In another study, Chen et al. observed the rapid
detection ability of GSH templated AuNCs for the detection
of glutathione S-transferase (GST)-tagged proteins.90 Using
DNA-templated AgNCs, Martinez and co-workers reported the
detection of a specific protein, thrombin, with an impressive
detecting limit (1 nM).91 Digestion of proteins is mainly
controlled by the protease enzyme which basically breaks the
peptide bonds of the proteins. Wang et al. developed AuNCs
using BSA as a capping ligand and demonstrated the sensitive
nanoscale detection of protease (detection limit of 1 ng mL1)
by turn-off emission signal phenomena.92 In another work,
Fig. 4 (A) Simulated structures of Au18(SC6H11)14 in the gas phase and
Zhu and co-workers prepared DNA-protected AgNCs which can
toluene phase, respectively, obtained from QM and QM/MM simulations. act as a sentient towards the determination of adenosine
(B) Simulated structures of Au18(SG)14 in the gas phase and water phase, deaminase (ADA).93
respectively obtained from QM and QM/MM simulations. Color labels: pink Detecting toxins within biological systems and that too at
atoms = Au, yellow sticks = S, and cyan sticks = C. In (A), the toluene
low concentrations will certainly be useful. Nair et al. strategically
molecules of the first solvation shell are represented in black (carbon) and
white (hydrogen). In (B), the water molecules of the first solvation shell are
prepared urease encapsulated NIR emitting AuNCs and demon-
shown in red (oxygen) and white (hydrogen). Reproduced with permission strated that these AuNCs can be utilized to selectively detect urea
from ref. 57. Copyright 2018, American Chemical Society. in human blood and cow milk samples.94 Xie et al. developed GSH

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Fig. 6 Detection of hROS using the crown nanoparticle assembly. The

system consists of a core dye encapsulated silica particle and multiple
satellite glutathione-templated AuNCs. Reproduced with permission from
ref. 97. Copyright 2013, American Chemical Society.

We have demonstrated that HSA templated AuNCs can show

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ultra-sensitive and selective detection of Hg2+ ions in vitro as

well as in vivo environments.63 In a previous study from our
group, Ghosh et al. demonstrated that HSA-templated Ag9:HSA
NCs can sense Co2+ ions by a turn-off luminescence
mechanism.99 Interestingly, these quenched NCs can again
detect Zn2+ ions in solution by restoring the luminescence
intensity (turn-on luminescence mechanism).99 Chang and
co-workers100 developed DNA-templated AgNCs for the selective
detection of Cu2+ ions in Montana soil (SRM 2710) and pond
water. GSH-capped CuNCs displayed the selective sensing
ability towards Fe3+ ions with a low limit of detection (LOD)
of B25 nM.101 Cytidine-stabilized AuNCs have the ability for
the selective detection of Ag+ ions in solution by turn-on
Fig. 5 Confocal images of breast cells stained with BSA-templated
AuNCs. The degree of staining is much more for cancerous cells as fluorescence,102 whereas Pb2+ ions can also be detected by
compared to non-cancerous cells. (A) Normal breast cell (MCF10A) and GSH-templated AgNCs with a LOD of B0.2 nM.103 Additionally,
(B) breast cancer cell (MCF7). (C) Normalized FCS traces of these AuNCs in metal NCs can serve as efficient biomarkers.19,63,81 The easy
bulk water (black), inside the cell membrane of MCF10A (blue), inside the encapsulation of NCs inside the cell lines and their bio-imaging
cytoplasm of MCF7 (green), and in the cell membrane of MCF7 (red).
abilities have been well-documented.19,63,81,93 Sun et al.
Reproduced with permission from ref. 62. Copyright 2014, American
Chemical Society. fabricated NIR luminescent AuNCs using GSH as a capping
and reducing agent and demonstrated that further conjugation
of folic acid rendered these NCs as efficient biomarkers through
stabilized AgNCs for the sensitive and specific detection of encapsulation inside HeLa cell lines.104 A ligand comprising 18
cysteine (Cys), a sulfur-containing amino acid with the detection peptides has been used to prepare AuNCs characterized by
limit of o3 nM.95 It is already established that a certain level of bright emission at the NIR region (1100–1350 nm). These NCs
reactive oxygen species (ROS) is important for the normal were subsequently utilized for in vivo brain vessel imaging and
functioning of a cell. However, the generation of excessive ROS tumour metastasis.105 Recently, Xie and co-workers developed
inside the cell can easily affect this severely which may, in turn, cyclodextrin (CD) templated AuNCs exhibiting NIR-II emission
lead to permanent damage of the cell. We have recently developed (lem = 1050 nm) which have thereby been used as a NIR-II
a potential antimicrobial agent (tyrosine templated silver nano- imaging probe for in vivo investiogations.106
materials, AgNMs) which can show the prompt killing of cell lines In our research work, we have used various kinds of ligands,
by creating a high level of ROS inside the cells.96 On the other such as proteins (human serum albumin, HSA), tripeptides
hand, detection and quantification of ROS is equally important (reduced glutathione, GSH), small molecules (tyrosine, Tyr),
for therapeutic applications. Chen et al. developed a dual emissive etc., to prepare several metal NCs.63,72,80,96,99,107 We have
AuNC decorated silica nanosensor for the efficient detection of prepared AgNCs having two different compositions (Ag9:HSA
highly ROS (hROS) inside the live HeLa cell lines (Fig. 6).97 and Ag14:HSA) using HSA as a template and for the first time
Additionally, NCs can be utilized for the efficient detection demonstrated that NCs can be toggled/interconverted using
of various inorganic cations and anions. In a recent work, Nain simple redox chemistry.107,108 Using the same template HSA,
et al. synthesized AuNCs, AgNCs and CuNCs using a mixture of we have also reported that CuNCs can exhibit thermal sensitivity
BSA and thiosalicylic acid (TSA) as the encapsulating ligands.98 and act as a nanothermometer. Additionally, these CuNCs
Interestingly, they have shown that each of these NCs can serve as a FRET pair with the standard dye Coumarin-153,
selectively detect a metal ion(s); AuNCs, AgNCs, and CuNCs characterized by an efficiency of energy transfer of B85%.109
can separately detect Hg2+, As3+ and Cr6+ ions, respectively.98 We have also prepared CuNCs using GSH as a capping agent.104

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These CuNCs act as a biomarker for cancerous cells and also more robust and exhibits greater photo-, thermal- and chemical
selectively detected Fe3+ ions in solution and the protein stability.63,109
haemoglobin, which in turn can lead to the early detection of We have prepared blue light-emitting CuNCs using the
cyanosis.104 In our investigations on HSA-capped AgNCs and globular protein HSA as a capping as well as reducing
CuNCs, no external reducing agent was used. This was achieved agent.109 HSA is the most abundant unglycosylated monomeric
by keeping the pH of the medium greater than the pKa of circulatory protein found in the human blood plasma and is
tyrosine.15 In these studies, we had proposed that the 18 tyrosine produced in the liver.111,112 The average molecular mass of HSA
amino acid residues inherently present within the protein matrix is 66.4 kDa and this protein consists of 585 amino acid residues
provide the necessary reducing environment (at a pH B 11, i.e., in a single poly-peptide chain.113 Ghosh et al. proposed that the
greater than 10.46, which is the pKa of tyrosine15,107) and result Tyr amino acid residues which are present in the HSA scaffolds
in the formation of stable NCs. To substantiate our proposal, we are actually responsible for the formation of NCs.109 Bao and
developed a unique strategy of using bare L-tyrosine as a capping co-workers also reported that the Tyr act at the final step in
agent outside the protein environment to prepare stable NCs.110 reducing Au+ ions to Au atoms during the preparation of AuNCs
These investigations not only highlight the role of a small using BSA as a protective ligand.114 In these synthetic protocols,
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molecule of a large ligand in preparing stable NCs, but also the pH of the reaction medium is maintained at B11 which
clearly demonstrate the advantages of having larger templates as renders the Tyr to behave as a reducing agent as the pKa of Tyr
compared to smaller ones as the former reduces the extent of is 10.46.15,107 These CuNCs exhibited a strong luminescence
heterogeneity within the system.110 This feature article high- intensity centered at B414 nm upon excitation at 330 nm.
lights the various fundamental aspects and related applications However, this spectroscopic signature was almost completely
of luminescent metal NCs. The field of cluster chemistry is lost when these CuNCs were treated with a strong oxidant,
developing extremely rapidly and encompasses various other H2O2. Here, H2O2 oxidizes the Cu0 from the cluster–core to Cu+
interdisciplinary research objectives. Since the properties of or Cu2+ which eventually results in the diminution of lumines-
luminescent metal NCs greatly depend on the choice of ligands cence intensity.109 The time-resolved decay transients of these
used, we have in detail delineated the effect of ligands on the CuNCs exhibited a bi-exponential decay [1.10 ns (30%) and
spectroscopic signatures of these NCs. Besides the fundamental 3.40 ns (70%)]. This bi-exponential nature of the decay transi-
physics/chemistry involved with their preparation, metal NCs ent has been attributed to the electronic transitions involved
have been established as next-generation fluorophores owing to between the filled d orbitals and the vacant sp conduction
their tunable size-dependent luminescence properties. Herein, band. The longer component of 3.40 ns has been ascribed to
we have discussed how these NCs can be used in bio-medics, the transition between Cu and the ligand (here HSA), while
sensing metal ions with unprecedented limits of detection, Cu–Cu interactions account for the shorter component of
sensing the pH and viscosity of cellular environments, and 1.10 ns.109 Through time-dependent emission profiles, it has
serving as bio-markers in differentiating both cancerous and been demonstrated that the formation of stable CuNCs is a
non-cancerous cell lines. slow process with the entrapped Cu2+ ions getting stabilized
through electrostatic interactions involving several functional
groups and the bulky structure of the protein. Besides showing
Effects of protecting ligands thermal sensitivity and reversibility, these CuNCs demon-
strated temperature-dependent FRET. The CuNCs served as
As mentioned earlier, for the preparation of stable metal NCs, the donor while the acceptor was Coumarin153, non-
generally, the ‘‘bottom-up’’ approach is preferred. In this covalently attached to the protein matrix (Scheme 3). The
process, reduction of metal ions to zero-valent metal atoms protein, HSA, is a heart-shaped molecule having three domains,
followed by cluster–core formation and encapsulation by I, II and III, each having two sub-domains, A and B.113 Mole-
ligands are the key factors that lead to the formation of NCs. cular docking study suggested that the probe, Coumarin153,
Generally, the cluster–core, comprising metal atoms in their gets attached at the subdomain IB of HSA (probable location
zero-valent oxidation states, has a strong propensity to self- being near the Cys-34 amino acid residue of the protein) and
aggregate owing to a very high surface energy. This in turn leads attains stabilization through hydrophobic and electrostatic
to the formation of larger-sized nanoparticles. However, to interactions.109 The distance between the donor (CuNCs) and
restrict further aggregation (i.e., to prevent the formation of the acceptor (Coumarin153) estimated from molecular docking
nanoparticles from NCs), ligands play a vital role and offer the was B20 Å, while that estimated from FRET-based assay was B
necessary surface stability.54,55 Not only the formation, but also 19 Å (Fig. 7).109
the long-time stability, photophysical properties, etc. can be In a different work, Anand et al. prepared two AgNCs having
regulated by capping agents.63,109,110 In general, the compactness different compositions, but using the common capping agent/
around the cluster–core can provide extra surface stability to template, i.e., HSA.107 For the preparation of both these AgNCs,
NCs.63,109 Recently, small molecules (like thiolates, amino acids, the pH of the medium was maintained greater than 10.46, i.e.,
etc.) have been widely used for the preparation of stable NCs.101,110 the pKa of Tyr,107 and the temperature was maintained at 37 1C.
However, macromolecular ligands generally provide extra surface However, the time taken for synthesis vastly differed; Ag9:HSA
rigidity that is required to ensure the formation of a NC, which is was prepared in 10 hrs and no external reducing agent was

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Scheme 3 (A) Schematic representation displaying the phenomenon of

FRET (donor: CuNCs; acceptor: Coumarin153) and nanothermometric
Scheme 4 Schematic representation showing the formation and inter-
behaviour exhibited by HSA-templated CuNCs. Reproduced with permis-
conversion of the Ag9:HSA and Ag14:HSA NCs using redox chemistry. The
sion from ref. 109. Copyright 2015, American Chemical Society.
two NCs were prepared independently using two different synthetic
protocols. Reproduced with permission from ref. 107. Copyright 2012,
American Chemical Society.

Fig. 7 (A) Molecular docking results representing the minimum-energy

conformations of HSA upon binding with Coumarin153 (non-covalently
attached to subdomain IB of HSA) and the plausible location of the binding
of the fluorophore, Coumarin153. (B) Zoomed-in view displaying the
several amino acid residues which are present in the immediate
vicinity of Coumarin153 and the estimated distance between the acceptor
(Coumarin153) and Cys-34 (20.2 Å). Reproduced with permission from
ref. 109. Copyright 2015, American Chemical Society.
Fig. 8 Normalized fluorescence excitation spectra of the two HSA-
templated AgNCs. The similar nature of these excitation spectra corrobo-
used, while the other NC, Ag14:HSA, was prepared within
rates the fact that even after inter-conversion (from Ag9:HSA to Ag14:HSA
2–3 minutes as in this case, and a strong reductant, NaBH4, and vice-versa), both the NCs retain their spectroscopic signatures
was used (Scheme 4). Since the composition differed, so did signifying that upon inter-conversion, there are no major structural
their spectroscopic signatures; while Ag9:HSA (QY B 16%) alterations. Reproduced with permission from ref. 107. Copyright 2012,
emitted at 460 nm, Ag14:HSA (QY B 11%) exhibited a strong American Chemical Society.

luminescence at 620 nm.107 The most interesting aspect of this

investigation was the interconvertibility among these NCs
(Fig. 8). Using simple redox chemistry, it has been demonstrated has been reported for DNA-coated AgNCs by Shao and
that these NCs can be toggled retaining almost all their photo- co-workers (Fig. 9B).116 Previously, this phenomenon was
physical properties (Scheme 4). These results highlight the role ascribed to the formation of heterogeneity (i.e., the presence
played by a bulky ligand. HSA provides the necessary protective of NCs with different sizes) in the system.117,118 However,
environment as the cluster–cores do not get destroyed when a Zhang and co-workers first explained this observation based
strong reductant (NaBH4) and an oxidant (H2O2) were used to on the surface plasmon and emitter coupling.115 In these
carry out the toggling between the two NCs.107 AgNCs, the luminescence was controlled by the coupling
Besides surface stabilization, ligands can also influence between ligand-to-metalmetal charge transfer (LMMCT), i.e.,
the photophysical properties of NCs. Zhang and co-workers from the complex Ag(I)–carboxylate to the core Ag atoms
prepared carboxylate-protected AgNCs which showed (Fig. 9C). Coupling between the surface plasmon and emitter
excitation-dependent emission (Fig. 9A).115 Similar observation can generate a new energy state which can be mathematically

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Fig. 9 (A) Excitation-dependent emission exhibited by carboxylate-

templated AgNCs using PMAA as a scaffold, owing to a strong coupling
between the surface plasmon and the emitter. (B) Excitation-dependent Fig. 10 (A) Photographic images of (c/n/a)-BSA-templated AuNCs. The
emission profiles of the DNA-templated AgNCs. The samples were excited left panel represents the photographs when viewed under ordinary
from 440 nm to 650 nm, with an interval of 10 or 20 nm. (C) Structural daylight while the right panel represents the same when viewed under a
illustrations of luminescent Ag–carboxylate with Ag+–carboxylate 365 nm UV lamp. (B) Luminescence profiles of different BSA-templated
complex shell. (D) Emission spectra of Tyr-templated AgNCs as a function AuNCs as marked in the figure. (C) Cell viability assay of HeLa cells when
of excitation wavelengths as marked in the figure. (A) and (C) Reproduced incubated with (0 mg mL1, 250 mg mL1, 500 mg mL1 and 750 mg mL1)
with permission from ref. 115. Copyright 2014, American Chemical Society. (c/n/a)-BSA-templated AuNCs for 4 hours. (D) Uptake efficiency of (c/n/a)-
(B) Reproduced with permission from ref. 116. Copyright 2015, American BSA-templated AuNCs after incubation with HeLa for 4 hours. (E) Pictorial
Chemical Society. (D) Reproduced with permission from ref. 96. Copyright representation of the cellular uptake of BSA-templated AuNCs with low
2021, American Chemical Society. and high charge densities. Reproduced with permission from ref. 119.
Copyright 2020, The Royal Society of Chemistry.

expressed as follows:116
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi from normal BSA (nBSA): one having more negative net charges
EPL ðlÞ þ E0 ½EPL ðlÞ  E0 2 (aBSA) and another one having more positive net charges
EL ðlÞ ¼  D2 þ (2)
2 4 (cBSA). Zeta-potential study confirmed the successful transfor-
mation of these two types of BSA molecules.119 Interestingly,
Here, EPL(l) represents the uncoupled surface plasmon the spectroscopic signatures of these (a/n/c)-BSA-templated
energy and l is the excitation wavelength, with EPL and l AuNCs were very different (Fig. 10A). The following order was
expressed in eV and nm, respectively. E0 denotes the transition observed both in terms of luminescence intensities (all being
energy of the ligand to metal charge transfer (LMCT) and D is red-emitting) and excited-state lifetimes: (cBSA capped AuNCs)
the energy related to the interactions between the photons and 4 (nBSA capped AuNCs) 4 (aBSA capped AuNCs) (Fig. 10B).
electrons.116 This coupling process can also influence the QY Generally, the formation of AuNCs is initiated by the
and excited-state lifetime of NCs. On the other hand, when we complex formation between Au(I) and the ligand (in this case,
prepared AgNCs using L-Tyr as a template, no such excitation- ligands are cBSA and aBSA molecules). It is expected that this
dependent emission profiles were observed (Fig. 9D).96 In this complex formation could be more facile for the positively
case, the emission wavelength did not alter with a change in charged amino groups as the salt of Au is in AuCl4 form in
excitation wavelength, and only a change in emission intensity the solution phase. Also, the emission maximum for cBSA
was obtained, as expected. Thus, it can be rationalized that the capped AuNCs was observed to be more blue-shifted as
template can play a pivotal role in facilitating the plasmon- compared to the aBSA capped AuNCs (Fig. 10B). Based on the
emitter coupling phenomenon. Jellium model, one can expect that the number of metal atoms
The surface charge of any particular ligand also has a comprising one NC will be more in the case of aBSA capped
profound influence on the properties of metal NCs.119 Shang AuNCs as compared to the cBSA capped AuNCs. In fact, the TEM
and co-workers strategically engineered the surface charged micrographs reported by Shang and co-workers also support the
groups of BSA molecules for the preparation of different same conjecture.119 Additionally, these two AuNCs demonstrated
AuNCs.119 They designed two different types of BSA molecules different cellular uptake and viabilities (Fig. 10C–E).119 Inductively

36 | Chem. Commun., 2022, 58, 29–47 This journal is © The Royal Society of Chemistry 2022
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coupled plasma-optical emission spectrometry (ICP-OES) becomes an issue when such small molecules are used, recently,
suggested that the encapsulation of these AuNCs inside cell lines this topic has garnered enough scientific interest.101,120 We have
was around 2.5-fold higher for the aBSA capped AuNCs compared already discussed that the small moiety of large molecules may
to the other two AuNCs (Fig. 10D). In this regard, the non-specific be responsible for the formation of NCs, such as the Tyr
interaction of negatively charged (z = 31.6 mV) AuNCs with molecules present inside the serum albumin proteins act as a
positive sites of the cell membrane favoured the endocytosis of reducing agent of metal ions.63,107,109,114 Thus, to understand
AuNCs (Fig. 10E).119 the role of small ligands and the subsequent fundamental
In another work, Song et al. demonstrated that the capping aspects related to the preparation of NCs, in two different
ligand can also regulate the electrochemical properties of NCs independent studies, we have recently investigated the role
despite their similar core crystal structures.61 Crystal structure played by the common template, Tyr, in the generation of AgNCs
determination revealed that the icosahedral Au13 core and and CuNCs.96,110 For both cases, we have developed optimized
semi-ring Au2(XR)3 motifs (where, X = Se and S) are common and easy one-pot synthetic protocols. We have thoroughly
for both AuNCs. They have determined the electrochemical investigated the role of Tyr in the formation of stable CuNCs
properties of selenolate-templated Au25 clusters using square using various experimental techniques as well as theoretical
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wave voltammogram (SWV) experiments and outlined the approaches.110 The as-prepared CuNCs displayed blue lumines-
differences with the previously reported thiolate-capped Au25 cence (at 415 nm) upon excitation at 315 nm. The spectroscopic
clusters.61 While the Au25(SePh)18 NCs displayed four reduction signatures of these CuNCs were in excellent agreement with
peaks (R1, R2, R3 and R4), the Au25(SCH2CH2Ph)18 NCs those reported for the other CuNCs prepared using the large
exhibited only two reduction peaks (R1 and R2) (Fig. 11).61 molecule, HSA, as the template.76,87 This observation supported
Also, thiolate protected AuNCs were characterized by a greater the argument/conjecture regarding the role of Tyr towards the
electrochemical HOMO–LUMO energy gap (B1.62 V) as com- formation of CuNCs. However, the luminescence signal obtained
pared to selenolate protected AuNCs (B1.24 V). Furthermore, from NCs when HSA was used as a template was far more
the gap between two successive oxidation states (O1 and O2) intense than what was observed when Tyr was used instead
was calculated to be B0.70 V for the Au25(SePh)18 NCs and (Fig. 12).109,110 Additionally, the cluster–core comprised 12 Cu
B0.30 V for the Au25(SC2H4Ph)18 NCs.61 This difference was atoms (using HSA as a template)109 against 9 Cu atoms (when
explained based on the different dielectric environments Tyr was used as the template).110 These minor discrepancies
experienced by both the cluster–cores owing to the highlight the fact that the macromolecular structure of HSA
different templates used. Thus again, the role played by a provided extra surface stability and compactness in contrast to
ligand in controlling the electrochemical properties of a NC is the small molecule, Tyr. Hence, it can be rationalized that
exemplified. besides the role played in the formation of NCs, large molecules
So far, we have discussed how NCs can be prepared using can provide extra surface stability to the cluster–core.
relatively large molecules as templates. However, the situation Although only Tyr can lead to the formation of NCs, it must
becomes far more challenging when small molecules are used be mentioned that such systems are not devoid of hetero-
instead to prepare stable NCs. Although difficult in terms of geneity. When HSA or BSA is used for the generation of NCs,
synthetic protocols, and many-a-times stability after formation generally, a narrow distribution in terms of cluster size is
obtained.63,109,114 However, when we synthesized CuNCs using
Tyr as the template, a Tamarix dioica leaf-like fibrillar pattern
was observed from the TEM imaging experiments (Fig. 13A).110
Based on several control experiments, we concluded that the
origin of fibrillar-like structures was the Tyr molecule itself
(Fig. 13B). The TEM images displayed that the as-synthesized
system consists of an aggregated fibrillar structure along
with tiny CuNCs.110 It is already reported that in an alkaline
medium, Tyr exists as a di-Tyr molecule.121,122 Our results
suggested that during synthesis, the formation of di-Tyr is very
rapid in nature (as compared to the formation of CuNCs) as the
pH of the reaction medium was alkaline. This rapid formation
of di-Tyr in an alkaline medium was well-corroborated by our
UV-Vis absorption kinetics study.110
The associated thermodynamics due to this transformation
(from Tyr to di-Tyr) was also studied using the isothermal
Fig. 11 Square-wave voltammograms (SWVs) of (A) Au25(SePh)18 and (B) titration calorimetry (ITC) technique. From the ITC results, we
Au25(SCH2CH2Ph)18 NCs. The experiment was carried out in CH2Cl2
obtained the evidence of di-Tyr formation in the presence of the
containing 0.1 M Bu4NPF6 having a concentration of NCs as 3 mg mL1.
Here the asterisk (*) indicates the wave for incompletely removed O2.
added alkali (NaOH), along with the generation of hydrophobic
Reproduced with permission from ref. 61. Copyright 2014, The Royal interactions (characterized by DH 4 0, DS 4 0).110,123 To support
Society of Chemistry. this result, we have also performed molecular docking studies

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hydrophobic interaction is one of the major factors for fibril

formation.110 Thus, we concluded that the p–p interaction of the
Tyr or di-Tyr molecules in an alkaline medium facilitated
fibrillar-like structure formation during the synthesis of CuNCs.
Su and co-workers also reported the presence of di-Tyr inside the
BSA molecule after the preparation of AuNCs (Fig. 14E).122
Although the di-Tyr molecules were also present within the
system, the bulky nature of the BSA molecule provided the
necessary encapsulation to AuNCs once formed, and hence,
the aspect of heterogeneity has not been reported.122 Using the
same template Tyr, we have observed a different type of hetero-
geneity in the case of AgNCs.96 The time required for the
formation of AgNCs was less than that required for CuNCs.
Interestingly, in this study, besides the stable Tyr-templated
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AgNCs, the system was having larger-sized nanoparticles which

Fig. 12 Excitation-dependent emission profiles of (A) HSA-capped

CuNCs and (B) Tyr-capped CuNCs. The greater luminescence intensities
obtained for HSA-capped CuNCs can be readily seen from the figures.
Reproduced with permission from ref. 109 and 110. Copyright 2015 and
2021, American Chemical Society.

Fig. 14 (A) and (C) The best-docked structures displaying the distance of
one Tyr molecule bound to another Tyr molecule and one di-Tyr molecule
Fig. 13 (A): (i–iii) TEM images of the fibrillar-like pattern formed by Tyr
interacting with another di-Tyr molecule, respectively. (B) and (D) The
ligands during the preparation of Tyr-templated CuNCs. These fibrillar
best-docked structures demonstrating the angle of Tyr bound to another
structures were formed along with smaller sized CuNCs. (B): (iv–vi) TEM
Tyr molecule and di-Tyr interacting with another di-Tyr molecule,
images displaying the aggregated Tyr molecules (due to the formation of
respectively. All these data suffice the pre-requisites for obtaining p–p
di-Tyr) in the presence of NaOH and in the absence of Cu2+. Reproduced
interactions present in these systems. Reproduced with permission from
with permission from ref. 110. Copyright 2021, American Chemical Society.
ref. 110. Copyright 2021, American Chemical Society. (E) Schematic
representation of the formation of di-Tyr residues inside the protein
matrix. Step (i) represents the protein oxidation via the formation of AuNCs
inside the protein scaffolds. Step (ii) represents the subsequent removal of
and found that the distance and angle between two p clouds AuNCs by the cysteamine-induced etching process, thereby leading to
satisfied the pre-requisites for p–p interactions (distance: o5 Å the luminescence properties of di-Tyr. Reproduced with permission from
and angle: o451) (Fig. 14A–D).124 It is also well known that this ref. 122. Copyright 2015, American Chemical Society.

38 | Chem. Commun., 2022, 58, 29–47 This journal is © The Royal Society of Chemistry 2022
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were non-luminescent.96 Once smaller-sized NCs were formed Owing to their photostable and biocompatible nature,
within a very short period, they were not protected instantly by luminescent metal NCs are widely used for bio-marking
the small template and hence self-aggregated to form larger- studies.62,63,67,126 For targeting the point of interest within a cell,
sized nanoparticles. Thus, the role played by a ligand is again intercellular localization studies are extremely important.
established as in this case we obtained a dual-component system Convertible organelle-targeting properties can be achieved by a
using a small molecule as a template.76 controlled design of ligand exchange method.127 Recently, Yang
The effect of surface rigidity on the luminescence properties et al. efficiently demonstrated such a ligand exchange strategy
of AuNCs was demonstrated by Pyo et al.125 In this study, the that empowers AuNCs to specifically target mitochondria instead
surface of the already prepared AuNCs using glutathione as a of lysosomes which were initially targeted prior to ligand
template was more rigidified by using tetraoctylammonium exchange.75 This study demonstrated that hydrosoluble glu-
(TOA) cations (Fig. 15A(i–ii)). This led to further enhancement tathione (–SG) templated Au18SG14 NCs can accumulate
of the QY of glutathione-capped AuNCs as compared to what mainly in lysosomes inside the cell lines. Using a suitable ligand
was observed without the addition of TOA (Fig. 15A(iii)).125 exchange engineering, Au18SG12MTPB2 NCs were prepared
Similarly, by monitoring the luminescence intensities of (where MTPB = (4-mercaptobutyl)triphenylphosphonium
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HSA-templated AuNCs, we observed that with a decrease in bromide).75 Interestingly, these Au18SG12MTPB2 NCs were more
temperature, the luminescence intensity of AuNCs increases inclined to encapsulate in the mitochondrial site within the cell
and vice-versa (Fig. 15B(i)).63 Upon decreasing the temperature, lines investigated.75 Additionally, it was further demonstrated
the protein achieves thermal compactness, and hence, the that upon photoexcitation at 638 nm, the Au18SG12MTPB2 NCs
wrapping of the ligand around the cluster–core gets augmented. can induce reduced cytotoxicity (due to the generation of lesser
A blue shift in the emission maximum, which reflects a higher singlet oxygen, 1O2) compared to Au18SG14 NCs (Scheme 5).
order of hydrophobic environment in and around the fluoro-
phore (here AuNCs), was also observed (Fig. 15B(ii)), which
further delineates our temperature-dependent observation.63 Sensing
Due to their high QYs, organic dyes and quantum dots (QDs)
were the most popular fluorescence probes for sensing
applications.128–136 However, tedious synthetic protocols,
environmentally hazardous nature, low biocompatibility, poor
water solubility, lack of cost-effective production and, most
importantly, toxicity limit their usage and hence alternative
solutions to circumvent these limitations are surely needed.
Hence, owing to their remarkable properties as discussed
earlier, luminescent metal NCs are widely used for sensing
purposes.62,63,79–81 These NCs, which thereby serve as next-
generation fluorophores, have been utilized for sensing appli-
cations in various inter-disciplinary research fields like in vitro

Fig. 15 (A): (i) Schematic illustration of the binding of TOA to Au22(SG)18

NCs (color label: Au = blue; S = green). (ii) Photographic images of
Au22(SG)18 in water and TOA–Au22 NCs in toluene when viewed under a
365 nm irradiation. (iii) Luminescence spectral profiles of Au22(SG)18 NCs in
water and TOA–Au22 in toluene. The luminescence intensities have been
compared with a standard fluorophore, Rhodamine B (RhB, QY = 31%),
with the same optical density. Reproduced with permission from ref. 125.
Copyright 2015, American Chemical Society (B): (i) Variation in the max-
imum luminescence intensity as a function of temperature for the HSA-
templated AuNCs, as marked in the figure. (ii) Variation in the emission Scheme 5 Switching of intracellular targeting organelles (from lyso-
wavelength maximum as a function of temperature for the HSA-templated somes to mitochondria) by Au18SG12MTPB2 NCs and demonstration of
AuNCs, as marked in the figure. Reproduced with permission from ref. 63. photodynamic therapy (PDT) toward cancer cells. Reproduced with per-
Copyright 2021, The Royal Society of Chemistry. mission from ref. 75. Copyright 2018, The Royal Society of Chemistry.

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and in vivo sensing of cations, anions, pH, temperature,

viscosity, biomolecules, etc.19,63,67,80,86,106

Inorganic ion sensing

From environmental pollution to industrial purposes, the
detection of ions (especially metal ions) is of utmost impor-
tance as it has direct relevance to health issues. Although some
instrumental techniques (e.g., ICP-OES, ICP-MS, AAS, AFS, and
some X-ray based techniques) are being used extensively for the
detection of elements, such approaches are not user-friendly
because of the long analysis period, high power consumption,
expensive instrumentation, complex analysis procedures, need
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for large-scale samples, limitation in in vivo detection, etc.137,138

In this context, metal NCs become a natural choice, especially
for the detection of ions based on their luminescent signatures.
Metal NCs serve as optical sensors and can be categorized as
‘‘turn-on’’ and ‘‘turn-off’’ luminescence probes. The efficiency
of detection, which is normally quantified from the limit of
detection (LOD), categorizes these NCs accordingly.
Xie et al. first reported the detection of Hg2+ ions in solution
using red emissive AuNCs.139 Recently, we have reported HSA
templated AuNCs as a selective sensor for Hg2+ ions with very
high sensitivity.63 These AuNCs were robust in nature against
thermal and chemical perturbations except for Hg2+ ions,
whereby the luminescence intensity gets severely quenched in
the presence of these Hg2+ ions (Fig. 16A).63 The optimum QY
(410%) and very high photostability (the AuNCs were stable
more than a year) make these NCs suitable sensors (LOD B
1 nM). This is the first ever approach to sense Hg2+ ions with
HSA-templated AuNCs using the FCS technique.63 This single
molecular study demonstrated the ultra-sensitive nature of
AuNCs towards Hg2+ detection with an extremely low LOD
value (B0.01 nM) which is much lower than the international
standards set for Hg2+ levels in drinking water (10 nM) according
to the U.S. EPA.140 We also studied the effect of interaction of
AuNCs with Hg2+ when this ion was bound to a biomolecule,
insulin. To this effect, we have fabricated a model system by
Fig. 16 (A) Modulations in the emission spectra of the AuNCs with the
mixing insulin and Hg2+ forming the insulin–Hg (I–H) complex. increasing concentrations of Hg2+. The gradual quenching of the red
We have concluded that these AuNCs can also sense Hg2+ with emission peak (lem = 660 nm) is due to the step-wise dissociation of the
almost similar efficiency as observed in an aqueous medium.63 parent NC and the simultaneous generation of blue-emitting NCs
From our FCS investigations, the step-wise decrement of (lem B500 nm) of smaller size observed at the higher concentrations of
Hg2+ ions. (B) Auto correlation curves obtained from the FCS measurements
correlation signal and the diffusion time were demonstrated
for AuNCs with the increasing concentrations of Hg2+ ions. (C) Histogram
which correlate our observation based on the quenching of displaying the selectivity of these AuNCs in sensing Hg2+ ions among the
luminescence intensity owing to the gradual destruction of the several other ions which were also screened. (D) Photographic images
cluster–core (Fig. 16B). The selectivity toward Hg2+ ion detection displaying the change in color of the AuNC solutions in the presence of
and the visible color change of the AuNCs upon interaction with increasing concentrations of Hg2+ ions, when viewed under ordinary and
UV light. (E) Confocal images of the MDA-MB-231 (breast cancer) cell lines
the Hg2+ ions are represented in Fig. 16C and D, respectively.
when incubated with Hg2+ ions in the presence of AuNCs. Reproduced with
This interaction of AuNCs and Hg2+ can be rationalized permission from ref. 63. Copyright 2021, The Royal Society of Chemistry.
by the concept of metallophillic interaction (d10–d10)
phenomenon.63,139,141 Also, the higher reduction potential of
Hg2+/Hg0 (Ered = 0.86 V) as compared to other heavy metals was Hg2+ ions inside the breast cancer cell lines (MDA-MB-231)
the important factor that led to the interaction of Hg2+ with Au (Fig. 16E).63
cluster–cores. Most interestingly, bio-imaging study as a In another work from our group, we demonstrated the
function of time showed that these AuNCs can also sense the photo-switch nature of HSA templated Ag9:HSA NCs for the

40 | Chem. Commun., 2022, 58, 29–47 This journal is © The Royal Society of Chemistry 2022
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label-free detection of Co2+ and Zn2+ ions in the aqueous Recently, Pradeep and co-workers studied the effect of heavy
phase.99 Ag9:HSA NCs showed a ‘‘turn-off’ signal in the metal ions on the luminescence signatures of BSA-coated Au38
presence of Co2+ ions; the luminescence intensity of Ag9:HSA NCs.144 This report demonstrated that these AuNCs can act as a
NCs was reduced by 74% in the presence of 1 mM Co2+ ions fluorescent chemodosimeter for selectively sensing Cd2+ and
(Fig. 17A). The LOD was calculated to be 100 nM. Most inter- Pb2+ through the formation of cluster aggregates and subse-
estingly, these quenched NCs regained their luminescence quent augmentation of luminescence intensity (Fig. 18A).144
intensity, i.e., exhibited a ‘‘turn-on’’ signal in the presence of The DLS study showed that the aggregation phenomenon gets
Zn2+ ions, characterized by a LOD of 50 nM (Fig. 17A).99 This initiated beyond 500 ppb concentrations of both metal ions.144
sensing mechanism of these Ag9:HSA NCs based on ‘‘turn-on’’ Shen et al. designed nanospheres and nanovesicles containing
and ‘‘turn-off’’ luminescence intensities make them behave as a self-assembled Ag6 NCs using polyethyleneimine (PEI) as a
label-free photoswitch (Fig. 17B) which can be used to detect template.145 They examined the sensing ability of these nano-
pollutants in water from several sources.99 aggregates and reported their superior sensing ability towards
He and co-workers developed thymine-rich (T-rich) DNA the detection of Al3+ ions (by monitoring the quenching of
strand templated yellow luminating CuNCs which can efficiently emission signal of AgNCs, Fig. 18B). In a seminal work,
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detect Mn2+ ions in the aqueous phase by the ‘‘turn-on’’ signal Ngeontae and co-workers reported the detection of Al3+ ions
with a detection limit of 10 mM.142 In an earlier study, Lan et al. in solution with a LOD of 26.7 nM using the concept of ‘‘turn-
demonstrated an interesting application of DNA-capped AgNCs on’’ luminescence signal of cysteamine-capped CuNCs.146
which were used to detect Cu2+ ions in Montana soil (SRM 2710) Das et al. prepared a relatively small molecule, tripeptide
and pond water with a very low LOD of 8 nM in water.100 glutathione-templated blue-emissive CuNCs, as a selective
In another study having direct application in pharmaceutical sensor for the detection of Fe3+ ions and estimated the LOD
industries, Kim and co-workers143 successfully sensed the to be B25 nM.101 Addition of 1 mM Fe3+ to CuNCs resulted in
presence of Ag+ (as silver sulfadiazine) in a dermatological burn an 80% reduction of the luminescence signal of the CuNCs
ointment, Silmazinss. In this study, Cytosine12 (Cyt12) DNA (Fig. 19). Low concentrations of Fe3+ ions led to complex
chain was used for the preparation of red luminating AgNCs formation with the CuNCs through the mechanism of static
which were induced to form a dimer in the presence of Ag+ ions quenching. However, further addition of Fe3+ ions resulted in a
(the monomeric AgNCs were anchored by a bridge formation) drastic decrease in the luminescence intensity involving the
and the detection of Ag+ ions were demonstrated by a visual dynamic quenching phenomenon followed by electron transfer
color change of the sample from red to green.143 between the CuNCs and Fe3+ ions.101 Additionally, this work
also delineated how these CuNCs can sense Fe3+ ions present in
the methemoglobin of human blood, thereby proving how
these can be used for the early detection of cyanosis.101

Fig. 17 (A) Histograms demonstrating the selectivity of Ag9:HSA NCs in Fig. 18 (A) Schematic representation demonstrating the augmentation in
detecting Co2+ and Zn2+ ions in solution by ‘‘turn-off’’ and ‘‘turn-on’’ luminescence intensity of Au38 NCs in the presence of Cd2+ and Pb2+ ions.
signals, respectively. (B) Schematic illustration of the mechanism of ‘‘on’’ Reproduced with permission from ref. 144. Copyright 2019, The American
and ‘‘off’’ luminescence features of the Ag9:HSA NCs which make them Chemical Society. (B) Selective detection of Al3+ ions by Ag6 NCs.
behave as a label-free photoswitch. Reproduced with permission from ref. Reproduced with permission from ref. 145. Copyright 2018, The American
99. Copyright 2014, The American Chemical Society. Chemical Society.

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Table 1 pH values estimated from different water samples using SF-

templated CuNCs (designated as pHa) and compared with those recorded
using a conventional pH meter (designated as pHb). Reproduced with
permission from ref. 153. Copyright 2016, The Royal Society of Chemistry

Sample pHa pHb Relative error (%)

Tap water 1 7.72 7.76 0.52
Tap water 2 7.77 7.84 0.89
Tap water 3 7.76 7.79 0.39
River water 1 8.06 8.00 0.75
River water 2 8.02 7.97 0.63
River water 3 8.10 8.04 0.74

Fig. 19 Selective detection of Fe3+ ions in solution by glutathione-

templated CuNCs. The process was characterized by a LOD of B25 nM. of HSA-templated AuNCs inside two complex systems: a pluronic
Reproduced with permission from ref. 101. Copyright 2015, The American co-polymer hydrogel, F127 (Fig. 20A); living cervical cancerous
Published on 12 November 2021. Downloaded on 1/20/2023 7:08:58 AM.

Chemical Society. cell lines, HeLa cells.80 Using the conventional method,132 we
have prepared the F127 hydrogel with the incorporation of
AuNCs inside the gel network.80 Anisotropy data revealed that
Not only the cations, several reports also demonstrated the
the rotational time-scale inside the gel was almost similar to that
detection of anions by luminescent metal NCs. Chang and co-
of bulk water (Fig. 20B). From our translational dynamics
workers synthesized CuNCs using thiosalicylic acid (TA) as a
measurements, we concluded that AuNCs were sensing
capping agent.147 This system acts as a dual sensing probe for
B12 to 18 times more viscosity inside the F127 hydrogel
the selective detection of nitrite (NO2) and cyanide (CN)
compared to water.80 From our FCS and time-resolved aniso-
anions and interestingly, it has been demonstrated that the
tropy experiments, we concluded that although the AuNCs were
sensing was controlled by the pH of the medium. An alkaline
actually localized at the corona region of the hydrogel network,
medium favours the detection of CN, whereas NO2 sensing
they were more exposed to the void or ‘‘water pool’’ region
was prominent only under acidic conditions.147 Tai et al.
(Fig. 20A).80 We have extended this work to more relevant
utilized cysteamine stabilized CuNCs for the selective detection
applications by monitoring similar dynamics of the AuNCs
of I ions in the aqueous phase as well as in human urine.148
inside the living HeLa cells and finally compared the results
Li et al. fabricated CuNCs which displayed an elevation of
with the hydrogel system. We also concluded that the viscosity
luminescence signal through an aggregation-induced process
of the micro-environment of the HeLa cell lines around the
in the presence of S2 ions with a detection limit of 42 nM.149
localization site of AuNCs to be B23 cP (Fig. 20C), which is close
to the value obtained inside the F127 hydrogel (B12–18 cP).80
pH, viscosity and temperature sensing Previously, Bhattacharyya and co-workers also studied the
translational dynamics of BSA-coated AuNCs inside the cancer-
pH, viscosity and temperature play significant roles in governing ous MCF7 and non-cancerous MCF10A cell lines.62 Feng and
the dynamics and associated structure-function inter-relations co-workers, using the concept of aggregation induced emission,
in complex biological systems.150–152 Zhang and co-workers demonstrated the ‘‘turn-on’’ sensing ability with an increase of
prepared stable CuNCs using natural silk fibroin (SF) as a ligand system viscosity of GSH-protected AuNCs and also used these
precursor and demonstrated the pH sensitivity and reversibility NCs for the inter-cellular imaging of the A549 (lung cancer) cell
of these CuNCs displaying a linear relationship in the pH range lines.156
of 6.08–10.05.153 The pH of sample water collected from different In another study from our group, we have prepared CuNCs
tap waters and rivers was examined using these CuNCs, and using HSA as a template (partially discussed earlier) and
when compared with commercial pH meters, the results showed that these NCs can act as temperature sensors.109 With
obtained were in good agreement (Table 1).153 Huang and co- an increase in temperature, the luminescence intensity of
workers154 synthesized trypsin-templated CuNCs and reported CuNCs reduced gradually and vice-versa. The elevation of
the linear nature of sensing over a pH range of 2.02–12.14. In temperature resulted in the unfolding of the protein around
another study, Bonanno et al. reported that b-nicotinamide the core of CuNCs, i.e., decrease of rigidity and thus, the
adenine dinucleotide phosphate (NADP) templated dual stability of the surface diminishes. Furthermore, abatement
emissive AuNCs can efficiently sense very acidic pH (0.6– of temperature enabled the cluster–core surface to gain stability
2.7).155 As Helicobacter pylori (micro-organism) and several through the refolding of the protein and consequently, led to
enteric pathogens favour acidic conditions and even gastric juice an increase of emission intensity of CuNCs (Fig. 20D). Most
possesses a pH of B1.0 to 3.5, this investigation could be interestingly, this temperature-dependent luminescence
relevant for further applications in estimating the pH of such property was reversible in nature and thus, this feature
biological systems. rendered these CuNCs as efficient temperature detectors
To understand the microenvironmental viscosity, we have (served as nano-thermometric probes).109 Similarly, Tseng
performed rotational and translational dynamics measurements and co-workers demonstrated that BSA-coated AuNCs can

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Fig. 21 (A) Confocal images of live lung cells stained by in situ generated
Au-NCs: (i) blank A549, (ii) 200 mM [pmim][AuCl4], (iii) 600 mM [pmim]
[AuCl4] and (iv) WI38 at 600 mM [pmim][AuCl4]. The scale bar corresponds
Fig. 20 (A) Pictorial illustration of the micelle and gel network formed by a to 4 mm. (B) Fluorescence spectra recorded by a spectrograph attached to
triblock copolymer, pluronic F127, and the localization of HSA-templated the confocal microscope of the in situ generated AuNCs in live lung cells
AuNCs inside the gel. The red spheres and the blue spheres denote the (lex = 405 nm): A549 (green) and WI38 (black) at 600 mm [pmim][AuCl4].
Au23 and Au9 NCs, respectively. (B) Fluorescence anisotropy decay of Reproduced with permission from ref. 136. Copyright 2016, John Wiley
AuNCs in bulk water (Green), inside the F127 gel (red) and inside the HeLa and Sons. (C) Confocal image of the MDA-MB-231 (breast cancer) cell line
cell (blue) (lex = 405 nm, lem = 473 nm for both water and F127 gel and incubated with 30 mM AuNCs. (D) Confocal image of the MCF10A (normal
lem = 510 nm for the HeLa cell). (C) FCS trace with best fit of AuNCs breast cell) cell line treated with AuNCs. Reproduced with permission from
incorporated inside the HeLa cell lines, lem = 510 nm. Reproduced ref. 63. Copyright 2021, The Royal Society of Chemistry.
with permission from ref. 80. Copyright 2020, John Wiley and Sons.
(D) PL reversibility exhibited by HSA-templated CuNCs as a function of
temperature. The protein unfolds upon increasing the temperature and a
inside the MDA-MB-231 cancerous cell lines, but our AuNCs is
reverse phenomenon is observed when the temperature is ramped down.
Reproduced with permission from ref. 109. Copyright 2015, The American red-luminating in nature (lem = 660 nm, Au25) before endocytosis
Chemical Society. inside the cell lines.63 In contrast, we did not get any fluorescence
image after the incubation of AuNCs with normal cell lines
MCF10A.63 These results can thus help us in the discrimination
display temperature sensing ability with reversibility in the of cell lines (cancerous and non-cancerous) by luminescent
range of 21–41 1C.157 Huang et al. also observed the reversible AuNCs (Fig. 21C and D). Besides this, the etching of relatively
and linear relationship between temperature and emission larger Au25 NCs (to relatively smaller Au13 NCs) during inter-
intensity in the range of 20–65 1C using lipoic acid-protected cellular incorporation become understandable.
Au/Ag bimetallic NCs.158 Xie and co-workers displayed the sensing ability of nucleic
acid stabilized AgNCs towards adenosine as well as the RNA of
Sensing of biological systems let-7a.160 For both cases, the ‘‘turn-on’’ fluorescence signal
With the advancement of bio-imaging techniques and single strategy was used with the help of the photoinduced electron
molecular studies, the detection of biomolecular as well as transfer (PET) mechanism.160 Recently, Pramanik et al. utilized
biological systems became growing interest for researchers. GSH-stabilized CuNCs to discriminate single nucleotide mis-
Chattoraj et al. reported the in-situ generation of AuNCs matches (SNM) in a 20 base pair long double-stranded DNA.161
inside live cells by incorporating HAuCl4 and the imidazolium- This report demonstrated that CuNCs could differentiate
based room-temperature ionic liquid (RTIL, [pmim][Br]) between well-matched DNA sequences and sequences containing
conjugate inside the A549, MCF7, and HCT116 cancerous cell single mismatched base pairs formed by cytosine.161 The calorimetric
lines as well as WI38 and MCF10A normal cell lines (Fig. 21A).159 and spectroscopic results revealed that tripeptide function-
They observed that the localization of evolved AuNCs was 20– alized CuNCs possess a stronger binding affinity towards
40 times higher in cancerous cell lines as compared to the cytosine-thymine mismatched (CT MM) DNA. They also showed
normal cell lines (Fig. 21B).159 The higher concentration of that CuNCs could be utilized as luminescent probes to monitor
GSH inside the cancerous cell lines facilitated the high number the molecular diffusion of SNM DNA using FCS.161 The equili-
of AuNC generation. The emission signal, peaked at 490– brium binding constants derived from the association and dis-
530 nm, indicates the formation of the Au8-13 cluster–core sociation rate constants suggested that CuNCs interact with CT
composition inside the cell lines. This observation is highly MM DNA with a B3 times higher binding strength than that of
corroborated with our data where we got the emission signal the WM DNA-CuNC system at single molecular resolution.161
of HSA-templated AuNCs around 510 nm after incorporation Hosseini and co-workers reported the ‘‘turn-on’’ detection of

This journal is © The Royal Society of Chemistry 2022 Chem. Commun., 2022, 58, 29–47 | 43
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sequence-specific microRNA-155 (miRNA-155) by DNA-RNA het- control their applications. A major section of this review deals
eroduplex formation.162 Luminescent CuNCs have also been used with the sensing capabilities of these NCs. Although there is a
for the discrimination of nucleosides therein.162 Ni and co- plethora of reports that deal with the modifications of ligands
workers prepared CuNCs in the presence of citric acid with four used in the preparation of NCs which in turn control the
different nucleosides as templates (adenosine (A), cytidine (C), application aspects, yet a more cumulative and comprehensive
guanosine (G) and thymidine (T)) and demonstrated that T failed understanding is required. In this feature article, we have
to produce CuNCs whereas C produced most intensely luminating summarized how and why these can be used to detect metal
CuNCs, but A-templated CuNCs possessed the highest QY (1.34%) ions with selectivity and unprecedented limit of detection.
as compared to other templates.120 In an interesting work, Bonis- Metal ions which are detrimental to human health (like heavy
O’Donnell and co-workers developed DNA-capped AgNCs for the metals, mercury, etc.) can be easily detected by these NCs.
selective detection of a neurotransmitter, dopamine, over other The fact that these NCs are photostable enables them to be
neurotransmitters under in vitro conditions.163 Verma and used in single molecular investigations. We have showed how
co-workers demonstrated the specificity of 8-mercapto-9- they can be used to sense the pH, viscosity and temperature of
propyladenine stabilized AuNCs towards the nuclei of several cell several biological systems along with their ability to discriminate
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lines via macropinocytosis pathway encapsulation.164 In a very cancerous and non-cancerous cell lines and specifically target
recent review, Li et al. thoroughly discussed the role of AgNCs for certain organelles within a cellular environment.
being a potential sensor towards the detection of various Although the field related to NCs has seen a huge upsurge
viruses.74 For example, Zhang et al. demonstrated the prominent recently, more in-depth investigations are needed to realize
detection of H5N1 gene using two DNA strands165 and Shen et al. their applications in biomedical research. The future of these
reported the possibility for the detection of Norovirus RNA by next-generation fluorophores will be directed towards the
using GCC-loop containing DNA as the template.166 In an development of catalysis, solar cells, safe and pure drinking
important study, Han et al. demonstrated the detection of three water, and investigations which will have direct physiological
viral DNAs (HIV, H1N1 and H5N1) using AgNCs as a sensor with and biomedical relevance such as targeted drug delivery and
the detection limits of 3.53 nM, 0.12 nM, and 3.95 nM, early detection of diseases.
respectively.167

Author contributions
Conclusions and future outlook SC collated all the data; SM conceptualized the review; both SC
and SM wrote the entire review article.
This review deals with the fundamental aspects of luminescent
metal NCs. These small-sized metal-aggregates in their zero-
valent oxidation states are non-toxic in nature and exhibit high Conflicts of interest
cell viability. Thus, these can be safely used in deciphering There are no conflicts to declare.
dynamics inside biological self-assemblies.
However, the propensity of these NCs to undergo oxidation
and self-aggregation to form larger nanoparticles makes the Acknowledgements
synthetic protocols and optimization processes more tedious
SC thanks IISER Bhopal for the fellowship and SM thanks SERB
and challenging. On the other hand, since the associated ligand
(CRG/2018/000760) for the financial support. SM thanks all the
covers the core of the metal NCs, they can interact with the
past and present members of the group. The support from
system and thus, their various applications can be regulated.168
IISER Bhopal, CIF IISER Bhopal and the Departmental TEM
Beside this, the stability, luminescence intensity and other
facility (FIST grant) is deeply acknowledged.
photophysical properties of the metal NCs are also influenced
by the compactness, surface coverage and functional groups
inherently present within the ligands.168 The solubility of the References
NCs in the solvent is highly influenced by the capping ligands.
1 J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem.,
The hydrophilic ligands (having groups such as hydroxyl, 2007, 58, 409.
carboxyl, amine, etc.) facilitate the aqueous phase solubility of 2 L. A. Peyser, A. E. Vinson, A. P. Bartko and R. M. Dickson, Science,
the NCs. This enables the development of newer templates that 2001, 291, 103.
3 C. T. Campbell, S. C. Parker and D. E. Starr, Science, 2002, 298, 811.
can be used for the preparation of water-soluble NCs which 4 L. Shang, S. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401.
can have varied applications, especially related to biological 5 K. Zheng, V. Fung, X. Yuan, D. Jiang and J. Xie, J. Am. Chem. Soc.,
systems. Thus, the role of ligands and templates becomes 2019, 141(48), 18977.
6 Z. Wu, Q. Yao, O. Jin, H. Chai, N. Ding, W. Xu, S. Zang and J. Xie,
extremely critical in the development of NCs. Herein, we have Angew. Chem., Int. Ed., 2020, 59, 9934.
systematically highlighted the role of such ligands/templates 7 T. Kawawaki, Y. Kataoka, M. Hirata, Y. Akinaga, R. Takahata,
in developing NCs which are not only stable but also have K. Wakamatsu, Y. Fujiki, M. Kataoka, S. Kikkawa, A. S. Alotabi,
S. Hossain, D. J. Osborn, T. Teranishi, G. G. Andersson,
characteristic spectroscopic signatures. These ligands help in G. F. Metha, S. Yamazoe and Y. Negishi, Angew. Chem., Int. Ed.,
tuning the morphological aspects of these NCs and this in turn 2021, 60, 21340.

44 | Chem. Commun., 2022, 58, 29–47 This journal is © The Royal Society of Chemistry 2022
 View Article Online

ChemComm Feature Article

8 H. Yu, B. Rao, W. Jiang, S. Yang and M. Zhu, Coord. Chem. Rev., 46 Z. Wu, Q. Yao, S.-Q. Zang and J. Xie, Natl. Sci. Rev., 2021, 8, nwaa208.
2019, 378, 595. 47 Z. Wang, X. Pan, S. Qian, G. Yang, F. Du and X. Yuan, Coord. Chem.
9 D. Bera and N. Goswami, J. Phys. Chem. Lett., 2021, 12, 9033. Rev., 2021, 438, 213900.
10 W.-M. He, Z. Zhou, Z. Han, S. Li, Z. Zhou, L.-F. Ma and S.-Q. Zang, 48 L. Zhu, M. Gharib, C. Becker, Y. Zeng, A. R. Ziefuß, L. Chen,
Angew. Chem., Int. Ed., 2021, 60, 8505. A. M. Alkilany, C. Rehbock, S. Barcikowski, W. J. Parak and
11 M. R. Narouz, K. M. Osten, P. J. Unsworth, R. W. Y. Man, I. Chakraborty, J. Chem. Educ., 2020, 97, 239.
K. Salorinne, S. Takano, R. Tomihara, S. Kaappa, S. Malola, 49 R. Jin, C. Zeng, M. Zhou and Y. Chen, Chem. Rev., 2016, 116, 10346.
C.-T. Dinh, J. D. Padmos, K. Ayoo, P. J. Garrett, M. Nambo, 50 Z. Liu, Z. Wu, Q. Yao, Y. Cao, O. J. H. Chai and J. Xie, Nano Today,
J. H. Horton, E. H. Sargent, H. Häkkinen, T. Tsukuda and 2021, 36, 101053.
C. M. Crudden, Nat. Chem., 2019, 11, 419. 51 Q. Yao, X. Yuan, T. Chen, D. T. Leong and J. Xie, Adv. Mater., 2018,
12 E. Khatun and T. Pradeep, ACS Omega, 2021, 6, 1. 30, 1802751.
13 K. Bhattacharyya and S. Mukherjee, Bull. Chem. Soc. Jpn., 2018, 52 Q. Yao, T. Chen, X. Yuan and J. Xie, Acc. Chem. Res., 2018, 51, 1338.
91, 447. 53 X. Yuan, L. L. Chng, J. Yang and J. Y. Ying, Adv. Mater., 2020,
14 Y. Cao, S. Malola, M. F. Matus, T. Chen, Q. Yao, R. Shi, 32, 1906063.
H. Häkkinen and J. Xie, Chem, 2021, 7, 2227. 54 X. S. Shen, G. Z. Wang, X. Hong and W. Zhu, Phys. Chem. Chem.
15 J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888. Phys., 2009, 11, 7450.
16 J. Zheng, C. W. Zhang and R. M. Dickson, Phys. Rev. Lett., 2004, 55 P. Born and T. Kraus, Phys. Rev. E, 2013, 87, 062313.
93, 077402. 56 E. S. Shibu, M. A. H. Muhammed, T. Tsukuda and T. Pradeep,
Published on 12 November 2021. Downloaded on 1/20/2023 7:08:58 AM.

17 A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, J. Phys. Chem. C, 2008, 112, 12168.
T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology, 2010, 57 D. M. Chevrier, L. Raich, C. Rovira, A. Das, Z. Luo, Q. Yao, A. Chatt,
21, 055103. J. Xie, R. Jin, J. Akola and P. Zhang, J. Am. Chem. Soc., 2018,
18 Q. Yao, Z. Wu, Z. Liu, Y. Lin, X. Yuan and J. Xie, Chem. Sci., 2021, 140, 15430.
12, 99. 58 P. A. Bartlett, B. Bauer and S. J. Singer, J. Am. Chem. Soc., 1978,
19 I. Zare, D. M. Chevrier, A. Cifuentes-Rius, N. Moradi, Y. Xianyu, 100, 5085.
S. Ghosh, L. Trapiella-Alfonso, Y. Tian, A. Shourangiz-Haghighi, 59 G. H. Woehrle, M. G. Warner and J. E. Hutchison, J. Phys. Chem. B,
S. Mukherjee, K. Fan and M. R. Hamblin, Mater. Today, 2021, DOI: 2002, 106, 9979.
10.1016/j.mattod.2020.10.027. 60 Y. Yang and S. Chen, Nano Lett., 2003, 3, 75.
20 X.-D. Zhang, J. Chen, Z. Luo, D. Wu, X. Shen, S.-S. Song, Y.-M. Sun, 61 Y. Song, J. Zhong, S. Yang, S. Wang, T. Cao, J. Zhang, P. Li, D. Hu,
P.-X. Liu, J. Zhao, S. Huo, S. Fan, F. Fan, X.-J. Liang and J. Xie, Adv. Y. Pei and M. Zhu, Nanoscale, 2014, 6, 13977.
Healthcare Mater., 2014, 3, 133. 62 S. Chattoraj and K. Bhattacharyya, J. Phys. Chem. C, 2014,
21 X. Yuan, Z. Luo, Y. Yu, Q. Yao and J. Xie, Chem. – Asian J., 2013, 118, 22339.
8, 858. 63 S. Chakraborty, A. Nandy, S. Ghosh, N. K. Das, S. Parveen, S. Datta
22 S. Hasegawa, S. Takano, K. Harano and T. Tsukuda, JACS Au, 2021, and S. Mukherjee, Analyst, 2021, 146, 1455.
1, 660. 64 J. Duguid, V. A. Bloomfield, J. Benevides and G. J. Thomas, Jr.,
23 K. Koyasu and T. Tsukuda, J. Chem. Phys., 2021, 154, 140901. Biophys. J., 1993, 65, 1916.
24 Y. Song, H. Abroshan, J. Chai, X. Kang, H. Kim, M. Zhu and R. Jin, 65 A. Ono, S. Cao, H. Togashi, M. Tashiro, T. Fujimoto, T. Machinami,
Chem. Mater., 2017, 29, 3055. S. Oda, Y. Miyake, I. Okamotoa and Y. Tanaka, Chem. Commun.,
25 Q. Li, S. Wang, K. Kirschbaum, K. Lambright, A. Das and R. Jin, 2008, 4825.
Chem. Commun., 2016, 52, 5194. 66 C. M. Ritchie, K. R. Johnsen, J. R. Kiser, Y. Antoku, R. M. Dickson
26 T. Udayabhaskararao and T. Pradeep, J. Phys. Chem. Lett., 2013, and J. T. Petty, J. Phys. Chem. C, 2007, 111, 175.
4, 1553. 67 Y. Xiao, Z. Wu, Q. Yao and J. Xie, Aggregate, 2021, 2, 114.
27 Y. Du, H. Sheng, D. Astruc and M. Zhu, Chem. Rev., 2020, 120, 526. 68 Y. Zhang, C. Zhang, C. Xu, X. Wang, C. Liu, G. I. N. Waterhouse,
28 R. Jin, G. Li, S. Sharma, Y. Li and X. Du, Chem. Rev., 2021, 121, 567. Y. Wang and H. Yin, Talanta, 2019, 200, 432.
29 H. Haidari, Z. Kopecki, R. Bright, A. J. Cowin, S. Garg, N. Goswami 69 H. Zhang, Z. Zhao, P. R. McGonigal, R. Ye, S. Liu, J. W. Y. Lam,
and K. Vasilev, ACS Appl. Mater. Interfaces, 2020, 12, 41011. R. T. K. Kwok, W. Z. Yuan, J. Xie, A. L. Rogach and B. Z. Tang,
30 M. Cao, R. Pang, Q.-Y. Wang, Z. Han, Z.-Y. Wang, X.-Y. Dong, Mater. Today, 2020, 32, 275.
S.-F. Li, S.-Q. Zang and T. C. W. Mak, J. Am. Chem. Soc., 2019, 70 M. Shellaiah and K. Sun, Chemosensors, 2017, 5, 3667.
141, 14505. 71 I. Chakraborty and T. Pradeep, Chem. Rev., 2017, 117, 8208.
31 U. Kreibig and M. Vollmer, Optical Properties of Metal Clusters, 72 N. K. Das and S. Mukherjee, Phys. Sci. Rev., 2018, 3, 20170081, DOI:
Springer, Berlin, 1995. 10.1515/psr-2017-0081.
32 Y. A. Eremin, SCATTERING|Scattering Theory, Encyclopedia of 73 Q. Wang, S. Liu and Z. Tang, J. Mater. Chem. B, 2021, 9, 8174.
Modern Optics, 2005, 326–330. 74 D. Li, H. Chen, X. Gao, X. Mei and L. Yang, ACS Sens., 2021, 6, 613.
33 Y. Tang, X. Yu, J. Xu, B. Audit and S. Zhang, Noble Metal-Metal 75 Y. Yang, S. Wang, S. Chen, Y. Shen and M. Zhu, Chem. Commun.,
Oxide Hybrid Nanoparticles, 2019, 431–455. 2018, 54, 9222.
34 N. W. Ashcroft and N. D. Mermin, Solid State Physics, Holt/Rinehart 76 S. Kundu, M. Ghosh and N. Sarkar, Langmuir, 2021, 37, 9281.
& Winston, New York, 1976. 77 S. Behzadi, V. Serpooshan, W. Tao, M. A. Hamaly,
35 S. Link, A. Beeby, S. FitzGerald, M. A. El-Sayed, T. G. Schaaff and M. Y. Alkawareek, E. C. Dreaden, D. Brown, A. M. Alkilany,
R. L. Whetten, J. Phys. Chem. B, 2002, 106, 3410. O. C. Farokhzad and M. Mahmoudi, Chem. Soc. Rev., 2017,
36 C. Felix, C. Sieber, W. Harbich, J. Buttet, I. Rabin, W. Schulze and 46, 4218.
G. Ertl, Phys. Rev. Lett., 2001, 86, 2992. 78 T. Pan, W. Song, H. Gao, T. Li, X. Cao, S. Zhong and Y. Wan, ACS
37 X. Du and R. Jin, Dalton Trans., 2020, 49, 10701. Appl. Mater. Interfaces, 2016, 8, 19217.
38 R. S. Dhayal, W. E. van Zyl and C. W. Liu, Dalton Trans., 2019, 79 N. K. Das, S. Chakraborty, M. Mukherjee and S. Mukherjee, Chem.
48, 3531. Phys. Chem., 2018, 19, 2218.
39 B. Nieto-Ortega and T. Bürgi, Acc. Chem. Res., 2018, 51, 2811. 80 S. Chakraborty, S. Nandi, K. Bhattacharyya and S. Mukherjee,
40 W. A. de Heer, K. Selby, V. V. Kresin, J. Masui, M. Vollmer, Chem. Phys. Chem., 2020, 21, 406.
A. Chatelain and W. D. Knight, Phys. Rev. Lett., 1987, 59, 1805. 81 S. Nandi, S. Ghosh and K. Bhattacharyya, J. Phys. Chem. B, 2018,
41 W. A. de Heer, Rev. Mod. Phys., 1993, 65, 611. 122, 3023.
42 R. L. Johnston, Atomic and Molecular Clusters, Taylor & Francis, 82 M. Shamsipur, A. Barati and Z. Nematifar, J. Photochem. Photobio.
London, 2002. C, 2019, 39, 76.
43 X. Kang and M. Zhu, Chem. Soc. Rev., 2019, 48, 2422. 83 R. Ali, S. M. Saleh and S. M. Aly, Microchim. Acta, 2017, 184, 3309.
44 X. Yuan, N. Goswami, W. Chen, Q. Yao and J. Xie, Chem. Commun., 84 H. Xiong, W. Wang, J. Liang, W. Wen, X. Zhang and S. Wang, Sens.
2016, 52, 5234. Actuators, B, 2017, 239, 988.
45 Z. Luo, X. Yuan, Y. Yu, Q. Zhang, D. T. Leong, J. Y. Lee and J. Xie, 85 M. V. Romeo, E. Lopez-Martinez, J. Berganza-Granda, F. Goni-de-
J. Am. Chem. Soc., 2012, 134, 16662. Cerio and A. L. Cortajarena, Nanoscale Adv., 2021, 3, 1331.

This journal is © The Royal Society of Chemistry 2022 Chem. Commun., 2022, 58, 29–47 | 45
 View Article Online

Feature Article ChemComm

86 L. Zhang and E. Wang, Nano Today, 2014, 9, 132. 126 R. R. Nasaruddin, T. Chen, Q. Yao, S. Zang and J. Xie, Coord. Chem.
87 B. Wang, M. Zhao, M. Mehdi, G. Wang, P. Gao and K.-Q. Zhang, Rev., 2021, 426, 213540.
Mater. Chem. Front., 2019, 3, 1722. 127 G. H. Woehrle and J. E. Hutchison, Inorg. Chem., 2005, 44,
88 Y. Zhang, C. Zhu, L. Zhang, C. Tan, J. Yang, B. Chen, L. Wang and 6149.
H. Zhang, Small, 2015, 11, 1385. 128 M. Li, T. Chen, J. J. Gooding and J. Liu, ACS Sens., 2019, 4, 1732.
89 R. C. Triulzi, M. Micic, S. Giordani, M. Serry, W.-A. Chiou and 129 J. Goicoechea F. J. Arregui and I. R. Matias, in Quantum Dots for
R. M. Leblanc, Chem. Commun., 2006, 5068. Sensing, ed. F. Arregui, Sensors Based on Nanostructured Materials,
90 C.-T. Chen, W.-J. Chen, C.-Z. Liu, L.-Y. Chang and Y.-C. Chen, Springer, Boston, MA., 2009.
Chem. Commun., 2009, 7515. 130 N. Chaniotakis and R. Buiculescu, Nanosensors for Chemical and
91 J. Sharma, H.-C. Yeh, H. Yoo, J. H. Werner and J. S. Martinez, Chem. Biological Applications, Woodhead publishing, 2014, 267.
Commun., 2011, 47, 2294. 131 M. Ganesan and P. Nagaraaj, Anal. Methods, 2020, 12, 4254.
92 Y. Wang, Y. Wang, F. Zhou, P. Kim and Y. Xia, Small, 2012, 8, 3769. 132 S. Chakraborty, S. Nandi, K. Bhattacharyya and S. Mukherjee,
93 K. Zhang, K. Wang, M. Xie, X. Zhu, L. Xu, R. Yang, B. Huang and Chem. Phys. Chem., 2019, 20, 3221.
X. Zhu, Biosens. Bioelectron., 2014, 52, 124. 133 K. R. A. S. Sandanayake and I. O. Sutherland, Sens. Actuators, B,
94 L. V. Nair, D. S. Philips, R. S. Jayasree and A. Ajayaghosh, Small, 1993, 11, 331.
2013, 9, 2673. 134 N. K. Das, S. Ghosh, S. Jaiswal, A. Tewary and S. Mukherjee, Chem.
95 X. Yuan, Y. Tay, X. Dou, Z. Luo, D. T. Leong and J. Xie, Anal. Chem., Phys. Lett., 2017, 682, 147.
2013, 85, 1913. 135 U. Pramanik, S. Chakraborty, K. Bhattacharyya and S. Mukherjee,
Published on 12 November 2021. Downloaded on 1/20/2023 7:08:58 AM.

96 S. Chakraborty, P. Sagarika, S. Rai, C. Sahi and S. Mukherjee, ACS Chem. Phys. Lett., 2021, 762, 138105.
Appl. Mater. Interfaces, 2021, 13, 36938. 136 S. Pramanik, A. Nandy, S. Chakraborty, U. Pramanik, S. Nandi and
97 T. Chen, Y. Hu, Y. Cen, X. Chu and Y. Lu, J. Am. Chem. Soc., 2013, S. Mukherjee, J. Phys. Chem. Lett., 2020, 11, 2436.
135, 11595. 137 B. Wei and L. Yang, Microchem. J., 2010, 94, 99.
98 A. Nain, Y.-T. Tseng, Y.-S. Lin, S.-C. Wei, R. P. Mandal, 138 E. Peňa-Vázquez, J. Villanueva-Alonso and P. Bermejo-Barrera,
B. Unnikrishnan, C.-C. Huang, F.-G. Tseng and H.-T. Chang, Sens. J. Anal. At. Spectrom., 2007, 22, 642.
Actuators, B, 2020, 321, 128539. 139 J. Xie, Y. Zheng and J. Y. Ying, Chem. Commun., 2010, 46, 961.
99 S. Ghosh, U. Anand and S. Mukherjee, Anal. Chem., 2014, 140 L. Shang, L. Yang, F. Stockmar, R. Popescu, V. Trouillet, M. Bruns,
86, 3188. D. Gerthsen and G. U. Nienhaus, Nanoscale, 2012, 4, 4155.
100 G.-Y. Lan, C.-C. Huang and H.-T. Chang, Chem. Commun., 2010, 141 C. Guo and J. Irudayaraj, Anal. Chem., 2011, 83, 2883.
46, 1257. 142 B. Han, R. Xiang, X. Hou, M. Yu, T. Peng, Y. Li and G. He, Anal.
101 N. K. Das, S. Ghosh, A. Priya, S. Datta and S. Mukherjee, J. Phys. Methods, 2017, 9, 2590.
Chem. C, 2015, 119, 24657. 143 J. Lee, J. Park, H. H. Lee, H. Park, H. I. Kim and W. J. Kim, Biosens.
102 Y. Zhang, H. Jiang and X. Wang, Anal. Chim. Acta, 2015, 870, 1. Bioelectron., 2015, 68, 642.
103 C. Wanga, J. Wub, K. Jiangb, M. G. Humphrey and C. Zhang, Sens. 144 J. S. Mohanty, K. Chaudhari, C. Sudhakar and T. Pradeep, J. Phys.
Actuators, B, 2017, 238, 1136. Chem. C, 2019, 123, 28969.
104 Y. Sun, J. Wu, C. Wang, Y. Zhao and Q. Lin, New J. Chem., 2017, 145 J. Shen, Z. Wang, D. Sun, C. Xia, S. Yuan, P. Sun and X. Xin, ACS
41, 5412. Appl. Mater. Interfaces, 2018, 10, 3955.
105 H. Liu, G. Hong, Z. Luo, J. Chen, J. Chang, M. Gong, H. He, J. Yang, 146 C. Boonmee, V. Promarak, T. Tuntulani and W. Ngeontae, Talanta,
X. Yuan, L. Li, X. Mu, J. Wang, W. Mi, J. Luo, J. Xie and X.-D. Zhang, 2018, 178, 796.
Adv. Mater., 2019, 31, 1901015. 147 J. Cang, C.-W. Wang, P.-C. Chen, Y.-J. Lin, Y.-C. Li and H.-T. Chang,
106 X. Song, W. Zhu, X. Ge, R. Li, S. Li, X. Chen, J. Song, J. Xie, X. Chen Anal. Methods, 2017, 9, 5254.
and H. Yang, Angew. Chem., Int. Ed., 2021, 60, 1306. 148 Y.-T. Tai, T. Simon, Y.-Y. Chu and F.-H. Ko, Sens. Bio-Sens. Res.,
107 U. Anand, S. Ghosh and S. Mukherjee, J. Phys. Chem. Lett., 2012, 2020, 27, 100319.
3, 3605. 149 Z. Li, S. Guo and C. Lu, Analyst, 2015, 140, 2719.
108 I. Chakraborty and T. Pradeep, Nanoscale, 2014, 6, 14190. 150 E. Nader, S. Skinner, M. Romana, R. Fort, N. Lemonne, N. Guillot,
109 S. Ghosh, N. K. Das, U. Anand and S. Mukherjee, J. Phys. Chem. A. Gauthier, S. Antoine-Jonville, C. Renoux, M.-D. Hardy-
Lett., 2015, 6, 1293. Dessources, E. Stauffer, P. Joly, Y. Bertrand and P. Connes, Front.
110 S. Chakraborty and S. Mukherjee, J. Phys. Chem. Lett., 2021, Physiol., 2019, 10, 1329.
12, 3266. 151 A. Cuecas, J. Cruces, J. F. Galisteo-López, X. Peng and
111 T. Peter Jr, All about Albumin: Biochemistry, Genetics and Medical J. M. Gonzalez, Biophys. J., 2016, 111, 875.
Applications, 1996, Academic Press. 152 A. Curnutt, K. Smith, E. Darrow and K. B. Walters, Sci. Rep., 2020,
112 S. Naveenraj and S. Anandan, J. Photochem. Photobiol., C, 2013, 10, 8760.
14, 53. 153 G. Zhang, T. Xu, H. Du, Y. Qiao, X. Guo, L. Shi, Y. Zhang, S. Shuang,
113 X. He and D. C. Carter, Nature, 1992, 358, 209. C. Donga and H. Ma, J. Mater. Chem. C, 2016, 4, 3540.
114 Y. Xu, S. Palchoudhury, Y. Qin, T. Macher and Y. Bao, Langmuir, 154 W. Wang, F. Leng, L. Zhan, Y. Chang, X. Xi Yang, J. Lana and
2012, 28, 8767. C. Z. Huang, Analyst, 2014, 139, 2990.
115 Y. Chen, T. Yang, H. Pan, Y. Yuan, L. Chen, M. Liu, K. Zhang, 155 A. Bonanno, I. Pérez-Herráez, E. Zaballos-Garcı́a and J. Pérez-
S. Zhang, P. Wu and J. Xu, J. Am. Chem. Soc., 2014, 136, 1686. Prieto, Chem. Commun., 2020, 56, 587.
116 X. Liu, R. Hu, Z. Gao and N. Shao, Langmuir, 2015, 31, 5859. 156 S. Pan, J. Zhou, W. Liu, Y. Ye, G. Chen, J. Xu, Z. Qian, J. Chen and
117 L. Shang and S. J. Dong, Chem. Commun., 2008, 1088. H. Feng, Analyst, 2019, 144, 4483.
118 K. Morishita, J. L. MacLean, B. Liu, H. Jiang and J. Liu, Nanoscale, 157 Y.-T. Wu, C. Shanmugam, W.-B. Tseng, M.-M. Hiseh and W.-
2013, 5, 2840. L. Tseng, Nanoscale, 2016, 8, 11210.
119 W. Zhong, M. Wen, J. Xu, H. Wang, L.-L. Tana and L. Shang, Chem. 158 H. Huang, H. Li, J.-J. Feng and A.-J. Wang, Sens. Actuators, B, 2016,
Commun., 2020, 56, 11414. 223, 550.
120 Y. Wang, T. Chen, Q. Zhuang and Y. Ni, ACS Appl. Mater. Interfaces, 159 S. Chattoraj, Md. A. Amin, S. Mohapatra, S. Ghosh and
2017, 9, 32135. K. Bhattacharyya, Chem. Phys. Chem., 2016, 17, 61.
121 C. Giulivi, N. Traaseth and K. Davies, Amino Acids, 2003, 25, 227. 160 K. Zhang, K. Wang, X. Zhu, Y. Gao and M. Xie, Chem. Commun.,
122 L. Su, T. Shu, J. Wang, Z. Zhang and X. Zhang, J. Phys. Chem. C, 2014, 50, 14221.
2015, 119, 12065. 161 S. Pramanik, L. Khamari, S. Nandi and S. Mukherjee, J. Phys. Chem.
123 P. D. Ross and S. Subramanian, Biochemistry, 1981, 20, 3096, and C, 2019, 123, 29047.
references therein. 162 Y.-S. Borgheia, M. Hosseinia and M. R. Ganjali, Clin. Chim. Acta,
124 Y. An, J. W. G. Bloom and S. E. Wheeler, J. Phys. Chem. B, 2015, 2018, 483, 119.
119, 14441. 163 J. T. D. Bonis-O’Donnell, A. Thakrar, J. W. Hirschberg, D. Vong,
125 K. Pyo, V. D. Thanthirige, K. Kwak, P. Pandurangan, B. N. Queenan, D. K. Fygenson and S. Pennathur, ACS Chem.
G. Ramakrishna and D. Lee, J. Am. Chem. Soc., 2015, 137, 8244. Neurosci., 2018, 9, 849.

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164 V. Venkatesh, A. Shukla, S. Sivakumar and S. Verma, ACS Appl. 166 F. Shen, Y. Cheng, Y. Xie, H. Yu, W. Yao, H. W. Li, Y. Guo and
Mater. Interfaces, 2014, 6, 2185. H. Qian, Anal. Biochem., 2019, 583, 113365.
165 Y. Zhang, F. Mu, Y. Duan, Q. Li, Y. Pan, H. Du, P. He, X. Shen, 167 D. Han and C. Wei, Talanta, 2018, 181, 24.
Z. Luo, C. Zhu and L. Wang, ACS Appl. Mater. Interfaces, 2020, 168 B. Zhang, J. Chen, Y. Cao, O. J. H. Chai and J. Xie, Small, 2021,
12, 48357. 17, 2004381.
Published on 12 November 2021. Downloaded on 1/20/2023 7:08:58 AM.

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