http://pubs.acs.

org/journal/acsodf Perspective

Controllable Synthesis of Bimetallic Nanostructures Using Biogenic

Reagents: A Green Perspective
Rufus Mart Ceasar R. Ramos and Michelle D. Regulacio*

Cite This: ACS Omega 2021, 6, 7212−7228 Read Online

ACCESS Metrics & More Article Recommendations

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: Bimetallic nanostructures are emerging as a significant class of metal

nanomaterials due to their exceptional properties that are useful in various areas of science
and technology. When used for catalysis and sensing applications, bimetallic nanostructures
have been noted to exhibit better performance relative to their monometallic counterparts
Downloaded via 61.0.86.219 on September 1, 2023 at 08:50:28 (UTC).

owing to synergistic effects. Furthermore, their dual metal composition and configuration can
be modulated to achieve optimal activity for the desired functions. However, as with other
nanostructured metals, bimetallic nanostructures are usually prepared through wet chemical
routes that involve the use of harsh reducing agents and hazardous stabilizing agents. In
response to intensifying concerns over the toxicity of chemicals used in nanomaterial synthesis,
the scientific community has increasingly turned its attention toward environmentally and
biologically compatible reagents that can enable green and sustainable nanofabrication
processes. This article aims to provide an evaluation of the green synthetic methods of
constructing bimetallic nanostructures, with emphasis on the use of biogenic resources (e.g.,
plant extracts, DNA, proteins) as safe and practical reagents. Special attention is devoted to biogenic synthetic protocols that
demonstrate controllable nanoscale features, such as size, composition, morphology, and configuration. The potential use of these
biogenically prepared bimetallic nanostructures as catalysts and sensors is also discussed. It is hoped that this article will serve as a
valuable reference on bimetallic nanostructures and will help fuel new ideas for the development of more eco-friendly strategies for
the controllable synthesis of various types of nanostructured bimetallic systems.

1. INTRODUCTION
The widespread use of bimetallic materials started way back in
the Bronze Age when it was discovered that combining Cu
with another metal can lead to considerable enhancement of
mechanical properties.1 With their superior durability,
bimetallic alloys (e.g., Cu−Sn alloy) have replaced stone as
the material of choice in the fabrication of tools and weapons
and as building and structural materials. In the succeeding
centuries, an extensive range of bimetallic systems has been
produced and this has led to the realization of new properties
and applications.2 With the advent of nanotechnology in the
present century, size and morphology have emerged as critical
parameters that can be manipulated when striving to improve Figure 1. Schematic representations of different configurations of
or modify the properties of metals to suit specific functions.3−6 bimetallic nanostructures.
For bimetallic systems, the nanostructure composition
(elemental ratio) and configuration (atom arrangement) are atoms of the two metals are uniformly distributed throughout
two additional parameters that can be adjusted to achieve the structure; the atom distribution can either be in an orderly
optimal performance for the intended applications.7−18
The different configurations that are possible for bimetallic
nanostructures can be categorized into two main types: (1) Received: February 6, 2021
alloy and (2) heterostructure. The distinction between them is Accepted: March 1, 2021
based on the relative arrangement of metal atoms in the Published: March 9, 2021
nanostructure, as schematically illustrated in Figure 1. Alloyed
bimetallic nanostructures can be further classified into
homogeneous and gradient alloys. In homogeneous alloys,
© 2021 The Authors. Published by
American Chemical Society https://dx.doi.org/10.1021/acsomega.1c00692
7212 ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

Table 1. Bimetallic Nanostructures with Controllable Features Synthesized Using Different Biogenic Reagents
biogenic reagent metal configuration morphology variable feature/s ref
plant extracts
Chinese wolfberry fruit Au−Ag alloy sphere composition, size 69
pomegranate seed Au−Ag alloy, core−shell sphere composition, configuration 70
Anacardium occidentale Au−Ag alloy, core−shell sphere composition, configuration 71
(cashew) leaf
Potamogeton pectinatus L. Au−Ag alloy sphere, polygon composition, size, morphology 72
(sago pondweed)
Cacumen platycladi leaf Au−Ag, Ag−Pd alloy sphere composition, size 73 and 74
C. platycladi leaf + ascorbic Au−Pd alloy flower, sphere composition, size, morphology 75
acid
C. platycladi leaf + ascorbic Au−Pd core−shell flower (Au core, Pd composition 76
acid petals)
Lantana camara flower Au−Pd alloy sphere, polygon composition, size, morphology 80
Schinus molle L. leaf Ag−Pt core−shell flower (Ag core, Pt composition 78
petals)
Cinnamomum camphora Pt−Pd alloy flower composition, size 79
leaf + ascorbic acid
nucleic acids
DNA Au−Ag heterodimer, snowman, sphere configuration, morphology, 83
core−shell assembly structure
DNA Au−Ag alloy, core−shell hollow cage, polyhedron configuration, morphology 84
DNA (A10, T10, G10, Pd−Au core−shell cubic core, varied shell shell morphology 85, 86
C10) morphology
DNA (T30) Au−Pd−Au core−multishell star shell thickness 87
proteins and peptides
bovine serum albumin Au−Ag, Au−Cu, Au− alloy sphere composition 94−97, 99, 100,
(BSA) Ni, Au−Pt and 101
lysozyme Au−Ag alloy sphere composition 103
peptide Au−Ag alloy sphere composition 104
glutathione (GSH) Au−Ag alloy sphere composition 105 and 106
carbohydrates
sucrose Au−Ag alloy sphere composition, size 107
starch + glucose Au−Ag alloy sphere composition 110
starch (degraded) Au−Ag alloy sphere composition 111
starch + ascorbic acid Pt−Cu alloy urchin composition 109
cellulose Ag−Pd alloy sphere composition 112
cellulose Au−Ag alloy sphere alloy configuration 113
(homogeneous, gradient)
xylan (hemicellulose) Au−Ag alloy, core−shell hollow sphere, solid configuration, morphology 114
sphere
other biomolecules
tannin (from bayberry) Au−Pd core−shell cube, sphere composition, shell morphology 116
lignin Au−Pd core−shell irregular composition 119

manner (i.e., intermetallic) or in a statistically random
manner.19,20 By contrast, gradient alloys have a composition-
ally graded structure, where atoms of one metal become
increasingly dominant toward the core, while atoms of the
other metal become more concentrated toward the surface.21
In the case of heterostructured bimetallic nanomaterials, there
is a clear segregation between the two metal components. One
of the most common examples is the core−shell architecture,
which is composed of a metal core that is entirely coated with a
different type of metal for its shell.22 A core−multishell (or
onion-like) structure is produced when the two metals form
alternating layers.23 There are cases where the metal core is
only partially enclosed by the other metal, such as in the core−
frame and core−satellite architectures.24,25 Another typical
example of a heterostructured configuration is the heterodimer
(or Janus) structure.26 In this mode of arrangement, the two
metal components are positioned adjacent to one another and
are bounded by an interface.
7213
When compared to their two constituent metals, bimetallic
nanostructures typically show superior physical and chemical
properties resulting from synergistic effects. These enhanced
properties and the ability to tune them through rational design
have stimulated scientists to dedicate much effort toward the
creation of a huge variety of bimetallic nanostructures with
applications that span a wide gamut of fields, including
catalysis, sensing, plasmonics, and biomedicine.7−26 Among the
different nanostructured bimetallic systems, the Au−Ag pairing
is one of the most extensively reported.25−28 Both Au and Ag
are recognized as strongly plasmonic metals but each has its
own merits and drawbacks.29 Au is chemically inert and
exhibits a stable plasmonic response, but its high cost has
become its major disadvantage. On the other hand, Ag is less
costly and is more plasmonically active than Au, but its inferior
stability limits its practical usability. Coupling these two metals
together to form bimetallic Au−Ag nanostructures is a strategy
that can be used to improve stability, reduce cost, and generate
a plasmonic response that is tunable over a broad range of
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

Figure 2. Phytosynthesis of alloyed Au−Ag nanoparticles using the Chinese wolfberry fruit extract. (a) Schematic depiction of the synthetic
protocol and the proposed formation mechanism. (b) TEM images and size distributions (insets) of the bimetallic nanoparticles synthesized using
a Au3+/Ag+ precursor molar ratio of 1:3.5 at different synthesis times (0.2, 5, and 60 min). Reproduced from ref 69. Copyright 2018. Published by
The Royal Society of Chemistry.

wavelengths.30,31 Other notable nanostructured bimetallic
systems that have elicited significant interests are Au−Pd,
Pt−Pd, and Pt−Cu.32−34
The synthesis of bimetallic nanostructures can be carried out
through physical top-down and chemical bottom-up methods.
Physical top-down techniques can produce nanostructures with
consistent size and shape, but they require the use of expensive
equipment with high maintenance costs (e.g., lithographic
tools).35−37 Chemical bottom-up routes are more economical.
They are normally done in solution (i.e., wet chemical
synthesis) and involve the reduction of metal ions (in the
form of soluble salt precursors) in the presence of reducing and
stabilizing agents.38−40 The reducing agent converts the metal
ions to their corresponding zerovalent metals, whereas the
stabilizing (or capping) agent protects the surface of the
resultant nanostructures and prevents their aggregation
through electrostatic and steric stabilization. In some cases,
the stabilizing agent also functions as a shape-directing agent.
Typical reagents that are used as reductants for the synthesis of
bimetallic nanostructures are sodium borohydride (NaBH4)
and hydrazine (N2H4).41 Meanwhile, common stabilizing
agents are cationic surfactants (e.g., cetyltrimethylammonium
bromide or CTAB) and long-chain organic ligands (e.g.,
oleylamine, trioctylphosphine).42−44 These chemicals have
been successfully employed in the creation of bimetallic
nanostructures with tunable features. The downside, however,
is that these are harsh and toxic substances.45 For instance,
CTAB has been revealed to be highly cytotoxic and this
7214
triggers concerns over the safety of CTAB-stabilized nanoma-
terials.46
Recent strategies in nanomaterial synthesis aim to minimize
negative environmental and health effects brought about by
harmful reagents. Central to this goal is the use of alternative
reagents that are environmentally and biologically compatible.
Biogenic entities, such as plant extracts and biomolecules (e.g.,
proteins, carbohydrates, nucleic acids), are regarded as ideal
green reagents because they are relatively safe, low-cost, and
convenient to use.45,47 In addition, they are multifunctional as
they can serve as reducing, capping, and shape-directing
agents.48 With the growing popularity of biogenic synthesis, it
has become the topic of several review articles published over
the past decade.47−55 Most of these publications are focused
on metal nanostructures that are monometallic, with no or very
limited discussion about bimetallic systems. There are a couple
of reviews centering on green approaches to bimetallic
nanostructures, but none of which paid particular attention
to controllable synthesis.56,57 Since the properties of bimetallic
nanostructures are largely determined by their size, composi-
tion, morphology, and configuration, it is essential to develop
synthetic methodologies that can allow for systematic
variations of these parameters. This Perspective devotes special
attention to biogenic synthetic protocols that are readily
amenable to adjustments to enable modulation of nanoscale
features. An organized summary of the different bimetallic
nanostructures that have been produced through controllable
synthesis using biogenic reagents is provided in Table 1.
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
2. BIOGENIC SYNTHESIS
2.1. Plant Extracts. Recent years have witnessed a deluge
of interest in the use of plant extracts as green reagents for the
synthesis of inorganic nanostructures.58−63 Termed as
phytosynthesis, this mode of nanomaterial fabrication presents
a safe, practical, and environmentally friendly approach, where
the various phytochemicals that exist in plant extracts mediate
in the formation and growth of the nanostructured materials.
In the phytosynthesis of metal nanostructures, the synthetic
protocol generally proceeds as follows. First, the extracts are
obtained in aqueous media from one or more plant parts, such
as leaves, fruits, flowers, seeds, and roots. The aqueous extracts
are then mixed with the metal salt precursors, and the reaction
is allowed to progress for a specified duration under mild
temperature conditions (≤100 °C). Reduction of the metal
ions and stabilization of the resulting metal nanostructures are
ascribed to the synergistic action of the different phytochem-
icals (e.g., phenolic acids, flavonoids, terpenoids, alkaloids,
carbohydrates) in the plant extracts due to the presence of
functional moieties such as COOH, CO, OH, and NH2.
Several reports have already been published on the
phytosynthesis of bimetallic Au−Ag nanostructures using
aqueous extracts from various plant sources, such as Piper
pedicellatum, Pulicaria undulata, Silybum marianum, and
Solidago canadensis.64−67 However, the Au−Ag nanostructures
that were produced in these works are polydisperse in size and
shape. Alti et al. obtained fairly monodisperse Au−Ag
nanospheres through the use of herbal leaf extracts from
fenugreek, coriander, and soybean, but precise control of
nanoscale features was not demonstrated.68 Meanwhile, high
monodispersity and controllable synthesis were both success-
fully achieved by Sun et al. in their phytosynthesis of alloyed
Au−Ag nanostructures using the extract derived from the
Chinese wolfberry fruit.69 The synthetic process and the
proposed mechanism are schematically shown in Figure 2a.
Transmission electron microscopy (TEM) imaging revealed
that their phytosynthesized nanostructures exhibit a well-
defined spherical morphology, with diameters and composi-
tions that can be modulated by simply altering the reaction
time (Figure 2b). In preparing these Au−Ag nanostructures, a
Au3+/Ag+ precursor molar ratio of 1:3.5 was used. By
prolonging the synthesis time from 0.2 to 60 min, the average
diameter was found to increase gradually from 5.1 to 14.8 nm.
Furthermore, the Ag mole fraction progressively increased with
time, yielding bimetallic compositions of Au 0.65 Ag 0.35 ,
Au0.50Ag0.50, and Au0.25Ag0.75, which correspond to synthesis
times of 0.2, 5, and 60 min, respectively. On the basis of these
results, it was proposed that nucleation occurs immediately
after the addition of the metal precursors into the boiling fruit
extract. The phytochemicals are able to reduce both Au3+ and
Ag+ ions, but since Au3+ has a higher reduction potential than
Ag+, the Au3+ ions are depleted first, resulting in bimetallic
seeds that are predominantly Au. As time progresses, the
remaining Ag+ ions are reduced continuously and the resulting
Ag atoms deposit onto the surface of the initially Au-rich
bimetallic seeds, consequently producing larger nanostructures
with increased Ag content. With further heating, interdiffusion
of Au and Ag atoms takes place, which eventually leads to Au−
Ag nanostructures that are homogeneously alloyed. It is worth
noting that the relative rate between the reduction of Ag+ and
the interdiffusion of atoms can influence the bimetallic
structure configuration. Conducting the synthesis at the
7215
Perspective
boiling temperature promoted interdiffusion; hence, the
interdiffusion rate is higher than the reduction rate. This is
favorable for the generation of a homogeneously alloyed
structure. If the reaction is performed at room temperature, the
interdiffusion of atoms can be stalled such that the reduction of
Ag+ can proceed at a faster rate than the diffusional exchange
between Au and Ag atoms. In this case, it is more likely that
the product would have a gradiently alloyed structure with a
Ag-rich surface and a Au-rich center.
The easiest way to manipulate the bimetallic composition is
to adjust the relative amounts of the starting metal precursors.
For instance, different Au3+/Ag+ (or HAuCl4/AgNO3)
precursor molar ratios have resulted in different Au−Ag
compositions as demonstrated by Kumari et al. in their
phytosynthesis of Au−Ag nanostructures using the pomegran-
ate seed extract.70 As expected, changing the Au3+/Ag+ ratio
from 1:1 to 1:5 led to an increase in the Ag mole fraction in the
resulting bimetallic nanostructures. A core−shell configuration
was observed when a Au3+/Ag+ ratio of 1:1 was used, but an
alloyed configuration was formed when the amount of Ag+ was
increased (i.e., a Au3+/Ag+ ratio of 1:2−1:5). Sheny et al.
similarly obtained different compositions and configurations by
varying the metal precursor ratio in their fabrication of Au−Ag
nanostructures using the cashew (A. occidentale) leaf extract.71
They also examined the use of the dried leaf powder of the
plant and concluded that it is more reliable and more
convenient to use than the aqueous leaf extract. Using the
aqueous extract of sago pondweed (P. pectinatus L.),
AbdelHamid et al. produced alloyed Au−Ag nanostructures
of different compositions by also changing the metal precursor
ratio.72 In addition, they have shown that altering the pH can
lead to different sizes and morphologies because pH conditions
can affect the reaction rate. For the 1:1 Au/Ag sample, assorted
polygonal shapes with an average size of 10.6 nm were
obtained at pH 4, whereas quasi-spheres measuring 6.6 nm
were mostly observed at pH 12. The reducing functionalities of
the phytochemicals in the extract are favorably activated under
alkaline conditions, leading to the more effective reduction of
the metal ions and faster nucleation rates. Consequently,
smaller-sized particles were produced in basic media.
The utility of the C. platycladi leaf extract as a biogenic
reagent has been reported by Sun and co-workers in their
phytosynthesis of bimetallic Au−Ag, Ag−Pd, and Au−Pd
nanostructures.73−76 Alloyed Au−Ag and Ag−Pd nanostruc-
tures that are predominantly sphere-like in shape were
generated when the metal precursors were simultaneously
reduced in the aqueous leaf extract at 90 °C.73,74 Interestingly,
they have created a flower-like morphology for their alloyed
Au−Pd nanostructures using ascorbic acid as a coreductant in
combination with the C. platycladi leaf extract.75 They noted
that a flower-like structure can only be achieved in the
presence of both reagents and when ascorbic acid is added
first; otherwise, a spherical morphology is obtained. Ascorbic
acid (or vitamin C) is a natural antioxidant that is present in
various fruits and vegetables, so it can also be classified as a
biogenic reagent. Aside from its ability to serve as a reductant,
it has been previously shown to participate in morphological
control, particularly in the formation of branched or dendritic
morphologies.48,77 Its ability to induce anisotropic branched
growth contributed significantly to the creation of nanoflowers.
In a separate publication, the group reported the fabrication of
Au−Pd nanoflowers with a core−shell configuration.76 This
type of structural configuration was attained through the
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
Figure 3. Biogenic synthesis of core−shell Au−Pd nanoflowers using the C. platycladi leaf extract and ascorbic acid. (a) Schematic diagram of the
proposed formation process. (b) Energy-dispersive X-ray elemental maps showing the distribution of Au and Pd in the nanoflowers. Reproduced
from ref 76. Copyright 2014. Published by MDPI.
successive reduction of the Au3+ and Pd2+ precursors (as
opposed to the simultaneous reduction that leads to an alloyed
configuration). A schematic depiction of the proposed
mechanism is presented in Figure 3a. Reduction of the Au3+
precursor by ascorbic acid was executed first to produce the Au
core. The Pd2+ precursor was then introduced through
controlled dropwise addition, and this was followed by the
addition of the leaf extract. These succeeding steps allowed for
the slow nucleation and growth of Pd on the surface of the
preformed Au core such that a core−shell architecture is
produced. The slow nucleation rate is necessary to discourage
the formation of separate Pd nanoparticles. Furthermore, the
process was performed at room temperature to inhibit the
interdiffusion of atoms between the Au core and the outer Pd
petals, as this can result in an alloyed configuration. The
distribution of Au and Pd atoms in the final product was
confirmed by the elemental maps that are displayed in Figure
3b.
The successive reduction technique was also employed in
the phytosynthesis of branched Ag−Pt nanoflowers with a
core−shell structure in the presence of the S. molle leaf
extract.78 Reduction of the Ag+ precursor was performed first
to generate the Ag seeds, and this was followed by the
reduction of the Pt4+ precursor to form the Pt petals. On the
other hand, alloyed Pt−Pd nanoflowers were synthesized
through simultaneous reduction of Pt2+ and Pd2+ precursors
using the C. camphora leaf extract with assistance from ascorbic
acid.79 Growth monitoring experiments revealed that small-
sized (≤5 nm) nanoparticles are formed first during the
concurrent reduction of the metal precursors. As the reaction
proceeds, these initial nanoparticles spontaneously aggregate
and grow to form the final nanoflowers. Varying sizes and alloy
compositions were obtained by adjusting the Pt2+/Pd2+
precursor molar ratio. The average size was found to increase
linearly with the increasing Pd mole fraction. Chowdhury et al.
described the phytosynthesis of alloyed Au−Pd nanostructures
with tunable shapes using the L. camara flower extract.80 The
shapes, which range from spherical to various polygonal
7216
Perspective
morphologies (e.g., triangular, hexagonal), were found to be
composition-dependent.
2.2. Biomolecules. 2.2.1. Nucleic Acids. With its easily
modifiable sequence-dependent structure, deoxyribonucleic
acid (DNA) has been extensively exploited as a programmable
biological template in the construction of metal nanostructures
with unique and complex morphologies.81,82 Although a
majority of the existing literature on DNA-mediated metal
nanostructure synthesis is focused on monometallics, there
have been some interesting studies over the past decade that
involves bimetallic systems. One example is the work on the
DNA-mediated synthesis of Au−Ag nanosnowmen that was
published by Lee and co-workers.83 Presynthesized DNA-
capped Au nanoparticles were used as the starting seeds, as
shown in the schematic diagram in Figure 4a. To create the
hybrid, the Ag component was grown through polymer-
assisted chemical reduction involving poly(vinylpyrrolidone)
(PVP), sodium ascorbate, and AgNO3 in the presence of salt
(NaCl). It was found that the salt concentration can strongly
affect the reaction kinetics because salts are able to increase the
DNA loading on the Au surface by reducing the repulsive
forces between the DNA strands. At high salt concentrations,
the DNA strands are densely and uniformly distributed around
the Au seed, which makes it difficult for the incoming Ag to
nucleate and grow on the Au surface. This results in slow
reaction kinetics, which favors multiple nucleation sites that
lead to a core−shell configuration. By contrast, when the salt
concentration is very low, the Ag precursors can readily
approach the sparsely and unevenly capped Au surface.
Consequently, Ag can rapidly nucleate and grow on one site,
yielding a heterodimer configuration with a shape that
resembles a snowman.
The Au−Ag nanosnowmen can be further utilized to
implement DNA-directed self-assembly as schematically
illustrated in Figure 4b.83 Because exposed DNA strands are
present on the Au head part of the nanosnowmen, oriented
assemblies were achieved via DNA hybridization, where the
DNA-capped Au heads are linked to nanostructures that are
capped with complementary DNA strands. Figure 4c shows the
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
Figure 4. DNA-directed synthesis and assembly of Au−Ag nano-
snowmen. Schematic illustration of the (a) synthesis of Au−Ag
nanostructures at varying salt concentrations and (b) directional
assembly of Au−Ag nanosnowmen. (c) TEM images of the different
assemblies achieved through DNA hybridization. Reproduced with
permission from ref 83. Copyright 2012 American Chemical Society.
TEM images of the various assemblies that were produced.
More recently, DNA-guided self-assembly of hollow Au−Ag
nanostructures into three-dimensional (3D) ordered super-
structures was reported by Sutter et al.84 First, they produced
core−shell Ag−Au nanostructures by immersing Ag polyhedral
seeds in an alkaline Au precursor solution. The Au shell was
then functionalized with thiolated DNA strands, and the
galvanic replacement was allowed to proceed by redispersion
in an aqueous Au precursor solution. This led to the
transformation of the initial core−shell structure into an
alloyed hollow (cage) architecture. Because the resultant
nanocages are DNA-terminated, the addition of suitable DNA
linkers enabled self-assembly via base pairing between self-
7217
Perspective
complementary recognition sequences, resulting in stable 3D
nanocage arrays.
Significant contributions to this area of study have been
made by Lu and his group with their demonstration of DNA-
directed morphological control in core−shell Pd−Au nano-
structures.85,86 They employed four different DNA strands that
are each made up of 10 identical deoxyribonucleotide units.
These four homo-oligomeric DNAs are designated as T10,
A10, C10, and G10, in accordance with their nucleobases,
which are thymine, adenine, cytosine, and guanine, respec-
tively. Starting with presynthesized Pd nanocubes as seeds, Au
was allowed to deposit onto the surface of these seeds in the
presence of DNAs to form core−shell nanostructures in a
kinetically controlled manner.85 Different shell morphologies
were produced as a result of the different binding affinities of
the DNA nucleobases. Figure 5 shows the schematics of the
proposed nucleobase-dependent formation mechanisms and
the scanning electron microscopy (SEM) images of the
resulting nanostructures over time. Shell formation generally
starts with the deposition of the incoming metal onto the
higher-energy {110} edges and {111} vertices of the Pd cubic
seeds, and this is followed by diffusion to the {100} faces. The
DNA binding affinity affects both the metal deposition and
diffusion rates. In the case of T10, which has a low binding
affinity to the Pd seed, its inability to substantially passivate the
seed surface encourages the deposition of the incoming Au
metal. Since deposition occurs at a much faster rate than
diffusion, shell growth is restricted to the edges and corners of
the cubic seed. For C10, which has a higher binding affinity,
diffusion is faster than deposition, and this allows for complete
coverage of the seed. Meanwhile, G10 has the tendency to
form secondary structures with the metal precursors, and
passivation of the seed surface by these secondary structures
permits deposition only in areas where these structures are
absent. Thus, the formation of clumpy islands is observed
initially. The succeeding diffusion event results in complete
coverage but with a markedly uneven shell. Finally, shell
growth in the presence of A10 follows a unique pathway due to
the high binding affinity of A10 to both the seed and the Au
precursor. In this case, Au crystallites are first formed
separately before depositing onto the Pd seed surface. Both
deposition and diffusion occur uniformly on all facets of the
seed, ultimately forming a rhombicuboctahedron morphology.
In their ensuing work, Lu et al. changed the Pd seed
morphology from a simple nanocube to a concave nanocube
with sites protruding in the ⟨110⟩ and ⟨111⟩ directions.86 The
deposition of Au was initially dictated by the seed’s high
surface energy. After the initial deposition, DNA-controlled
shell growth ensued, where different shell morphologies
develop due to the different binding affinities of the
nucleobases.
Bimetallic nanostars with a multilayered Au−Pd−Au
arrangement have been reported recently by Bhattarai and
Prozorov.87 Core−shell Au−Pd cubic seeds were synthesized
first, and these were then reacted with Au3+ ions in the
presence of ascorbic acid and T30 DNA strands (i.e., 30-mer
thymine-based oligonucleotide). Due to the weak binding
affinity of T30, the deposition of Au took place predominantly
on the high-energy vertices of the starting Au−Pd nanocubes.
The anisotropic growth of Au on the seed vertices eventually
yielded a star-like morphology.
2.2.2. Proteins. Proteins have been known to provide a
suitable structural framework for the synthesis and stabilization
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

Figure 5. DNA-mediated morphological control of core−shell Pd−Au nanostructures influenced by the nucleobase binding affinity. (a) Schematic
illustration of the proposed mechanisms of shell growth. (b) SEM images of the nanostructures obtained at different time points. Scale bars = 100
nm. Reproduced with permission from ref 85. Copyright 2016 American Chemical Society.

of ultrasmall metal particles termed metal nanoclusters, which
are composed of only a small number of metal atoms.88 Much
of the interest in metal nanoclusters is spurred by their strong
fluorescence that enables their use as fluorescent sensors.88,89
Proteins contain amino acid residues with functional groups
that can facilitate the coordination and reduction of metal ions,
as well as the encapsulation and stabilization of the resulting
nanoclusters. The protein-stabilized metal nanoclusters that are
produced are not only colloidally stable but are also
biocompatible, which is desirable if they are to be used for
biological applications.
Bovine serum albumin (BSA), a commercially available
globular blood protein derived from cows, has been
successfully utilized in the synthesis of Au nanoclusters.90−93
Adjusting the pH to 12 was found effective in activating the
reducing capability of the tyrosine residues (pKa ∼ 10) in the
protein.90 Furthermore, the sulfur-containing amino acid
residues (e.g., cysteine, methionine) in BSA were identified
as key players in the nucleation, growth, and stabilization of the
nanoclusters of Au.93 With the goal of achieving enhanced and
tunable emission properties, several groups have delved into
the synthesis of bimetallic Au−Ag nanoclusters using
BSA.94−97 In a typical synthesis, the Au3+ and Ag+ precursors
are incubated with BSA at the physiological temperature (37
°C) under alkaline conditions, and the reaction is allowed to
proceed for several hours (≥12 h).94,96 The reaction time can
7218
be dramatically reduced through heating. It is likely that the
denaturation of protein at elevated temperatures exposes more
of the protein’s reducing functionalities, and this leads to an
accelerated reaction rate. For instance, Zheng et al. were able
to produce BSA-stabilized alloyed Au−Ag nanoclusters within
3 min of microwave heating, as schematically depicted in
Figure 6.95 These bimetallic nanoclusters exhibit strong yellow
emission and display a good response to Hg2+ ions, making
them promising fluorescent probes for Hg2+ detection.
As the composition is known to affect the emission
wavelength, Sannigrahi et al. systematically varied the Au3+/
Ag+ precursor molar ratio in their BSA-assisted synthesis of
Au−Ag nanoclusters to obtain a series of samples with varying
alloy compositions.96 Through this, they were able to achieve a
composition-dependent tunable emission that extends to the
near-infrared (NIR) region. Mohanty et al. also prepared BSA-
stabilized Au−Ag nanoclusters with different alloy composi-
tions.97 Instead of using the typical protocol, where the Au3+
and Ag+ precursors (i.e., HAuCl4 and AgNO3) are con-
currently incubated with BSA, they explored two different
techniques that involve (1) cluster−cluster interaction and (2)
galvanic replacement reaction. In the cluster−cluster inter-
action approach, BSA-encapsulated monometallic nanoclusters
of Au and Ag are synthesized separately. They are later mixed
together and incubated to produce alloyed Au−Ag nano-
clusters in different ratios. The formation of bimetallic
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
Figure 6. Schematic depiction of the synthesis of BSA-stabilized Au−
Ag nanoclusters and their response to Hg2+. Adapted from ref 95.
Copyright 2015, with permission from Elsevier.
nanoclusters through this strategy is believed to occur via
interprotein metal transfer, where the BSA-stabilized Au and
Ag nanoclusters exchange metal atoms. This is feasible because
the secondary structure of protein changes with time and the
amino acid−metal interaction is dynamic.98 In the galvanic
replacement route, the Au3+ precursor is mixed with
presynthesized BSA-encapsulated Ag nanoclusters, and the
resulting mixture is stirred for several hours. In this case, the
formation of alloyed Au−Ag nanoclusters is ascribed to the
galvanic exchange reaction between Au3+ ions and Ag
nanoclusters, which is possible due to the higher standard
reduction potential of the AuCl4−/Au couple relative to that of
the Ag+/Ag pair. The galvanic nature of the reaction is
supported by the observed formation of AgCl crystallites.
In addition to Au−Ag, other examples of bimetallic systems
that have been prepared via BSA-mediated synthesis are Au−
Cu, Au−Ni, Au−Pt, and Pt−Bi.99−102 Meanwhile, lysozyme is
another protein aside from BSA that has been found useful in
the protein-directed synthesis of Au−Ag nanoclusters.103
Other related studies involve peptides with much shorter
amino acid chains than proteins.104,105 As an example, Wang et
al. made use of glutathione (GSH), a peptide comprised of
only three amino acids, to synthesize composition-tunable
Au−Ag nanoclusters via the galvanic replacement route.106
2.2.3. Carbohydrates. Carbohydrates (or saccharides) are
another class of biomolecules that can be used as green
reagents in the fabrication of inorganic nanostructures. Mono-,
di-, and polysaccharides have all been examined for their utility
in bimetallic nanostructure synthesis. Sun et al. used sucrose, a
dietary disaccharide found in many plants, as both the
reductant and surfactant in preparing monodisperse Au−Ag
Figure 7. Schematic illustration of the synthesis of cellulose-supported Au−Ag nanoparticles. Adapted from ref 113. Copyright 2020, with
permission from Elsevier.
7219
Perspective
nanospheres with different alloy compositions.107 They noted
that under hydrolysis conditions, sucrose exhibits a stronger
reducing ability as compared to glucose, fructose, and their
mixture. Mallikarjuna isolated polysaccharides from fenugreek
seeds for their synthesis of alloyed Au−Pd microspheres with a
highly porous architecture.108 It was found that these porous
microspheres (2−5 μm) are actually made up of much smaller
nanoparticles (∼5 nm) that are anchored to the polysaccharide
networks. On the other hand, an urchin-like morphology was
obtained by Devi et al. in their synthesis of alloyed Pt−Cu
nanostructures via coreduction of metal ions by ascorbic acid
in the starch matrix.109 The helical structure of the amylose
component of starch is believed to play an important role in
the growth and stabilization of the nanourchins. Raveendran et
al. made use of glucose and starch in the composition-tunable
synthesis of alloyed Au−Ag nanoparticles.110 Glucose acted as
the reductant, while starch provided the structural framework
for the stabilization of the nanoparticles. Meanwhile, Xia and
co-workers prepared their alloyed Au−Ag nanoparticles using
degradation products from Pueraria starch.111 Their reason for
degrading starch is to release the aldehyde groups of the
glucose units such that these functionalities will be accessible
for the reduction of the metal ions.
There have been a number of reports on the synthesis of
bimetallic nanostructures using cellulose and hemicellulose,
which are known scaffolding components of the plant cell wall.
For example, Li et al. used filter paper directly as cellulose to
synthesize alloyed Ag−Pd nanoparticles under hydrothermal
conditions at 200 °C.112 The high temperature facilitated the
hydrolysis of cellulose, which is necessary to expose the
reducing functional moieties that will enable the reduction of
the metal ions. They employed Ag2SO4 as the source of Ag as
well as of H2SO4, which can catalyze the cellulose hydrolysis.
In a more recent paper, Hu et al. reported that dissolving
cellulose (cotton linter pulp) in an aqueous NaOH/urea
solution can release the reducing functionalities at room
temperature conditions.113 This alkaline cellulose solution was
subsequently used in preparing their Au−Ag nanoparticles, as
schematically shown in Figure 7. By altering the addition order
of the Au3+ and Ag+ precursors into the cellulose solution, they
were able to tune the atom arrangement in their nanoparticles.
Simultaneous addition resulted in a homogeneously alloyed
configuration, whereas successive addition led to an inhomoge-
neous (gradient) alloy distribution. Xylan, a hemicellulosic
polysaccharide, was extracted from bagasse and then used by
Cai et al. in their synthesis of Au−Ag nanoparticles.114
Exploring two different protocols, they were able to produce
two types of configurations: (1) hollow alloy and (2) core−
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

Figure 8. Catalyst based on lignin-stabilized Au−Pd nanostructures. (a) Schematic diagram of the fabrication of the catalyst and its use in the
borohydride reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). (b−f) Time-dependent absorption spectra showing the borohydride
reduction of 4-NP to 4-AP in the presence of Au1.0Pd1.0, Au0.5Pd1.0, Au2.0Pd1.0, Au, and Pd nanoparticles. Reproduced with permission from ref 119.
Further permission related to the material excerpted should be directed to the ACS.

shell. The hollow alloy nanospheres were obtained as a result
of a galvanic exchange reaction between Au3+ ions and
presynthesized Ag nanoparticles at 80 °C. By contrast, the
core−shell Au−Ag nanospheres were produced through the
reaction of alkaline ammoniacal AgNO3 (known as Tollens’
reagent) and presynthesized Au nanoparticles under micro-
wave irradiation at 65 °C. Both reactions were performed in
the presence of xylan, which served dual roles as a stabilizer
and a reductant owing to its hydroxyl and reducing aldehyde
functionalities.
2.2.4. Other Biomolecules. Tannins are polyphenolic
biomolecules in plants that can serve as stabilizers in
nanomaterial synthesis due to their multiple hydroxyl groups.
Certain tannins can also function as mild reductants like in the
case of bayberry tannin (BT), which was utilized by Huang and
co-workers in reducing Au3+ ions to Au at ambient
conditions.115 Interestingly, they found that at high concen-
trations of BT, excess BT molecules self-aggregate to form a
dense supramolecular shell that coats the resultant Au
nanoparticles. Building on this work, the group exploited the
dense BT coating around Au nanoparticles for the reduction of
7220
Pd2+ ions to create bimetallic Au−Pd nanostructures.116 In
their synthesis, the Au3+ and Pd2+ ions are simultaneously
mixed with the BT solution, but only Au nanoparticles are
generated initially since Au3+ has a higher reduction potential
than Pd2+. The free Pd2+ ions are then adsorbed on the surface
and are slowly reduced by the BT coating, leading to core−
shell Au−Pd nanostructures. When the Pd2+/Au3+ precursor
molar ratio and the BT concentration used are high, sufficient
growth of the Pd shell takes place and this produces a thick
cubic Pd shell around the Au nanoparticles. On the contrary, a
thin spherical Pd shell is created at low concentrations of Pd2+
and BT. It is therefore possible to alter the Pd shell
morphology by adjusting the Pd2+/Au3+ precursor molar
ratio and the BT concentration.
Lignin, with its abundant hydroxyl and aldehyde moieties,
has also found utility as stabilizing and reducing agents in
metal nanomaterial synthesis.117,118 Typically found in wood
and barks, it is the second most abundant biopolymer (next to
cellulose) and is a major by-product of the paper and pulp
industry. The feasibility of using “waste” lignin recovered from
soda pulping effluents in preparing core−shell Au−Pd
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
nanostructures was examined by Han and co-workers.119
Synthesis was carried out through successive reduction of the
Au3+ and Pd2+ precursors in the presence of lignin under
microwave irradiation. They were able to prepare different
compositions by varying the Au3+/Pd2+ precursor molar ratio.
Furthermore, by coating a magnetic stir bar with the lignin-
stabilized nanostructures, they were able to fabricate a
heterogeneous catalyst that is reusable and easily recoverable.
A schematic representation of the fabrication process is
displayed in Figure 8a, and their use in catalysis is discussed
in the succeeding section.
3. APPLICATIONS IN CATALYSIS AND SENSING
3.1. Catalysis. Pioneering reports on the use of nanosized
metals in catalysis were published in the 1940s when it was
found that the rate of hydrogenation can be influenced by the
particle size of polymer-coated Pt and Pd nanoparticles.120 In
the late 1980s, another breakthrough was recorded with the
discovery of the enhanced catalytic activity of metal oxide-
supported Au nanoparticles for low-temperature oxidation of
CO.121 With the rising prominence of nanotechnology over
the past 3 decades, the potential of metal nanostructures as
heterogeneous catalysts have been tested in a wide range of
chemical transformations, and the nanostructure features have
been judiciously modified for the improvement of catalytic
performance. The construction of bimetallic nanostructures is
one way of enhancing the catalytic behavior as the synergistic
effects that result from combining two different metals can lead
to increased activity, selectivity, and stability.122 In general, the
superior performance of bimetallic nanostructures can be
attributed to one or a combination of the following effects: (1)
ensemble effect, where groups of dissimilar surface atoms take
on distinct mechanistic functionalities; (2) ligand effect, where
an electronic charge transfer between two dissimilar surface
atoms affects their electronic structure; and (3) geometric
effect, where the spatial arrangement of surface atoms is
influenced by strain, geometry, and size.7,123
One of the most common reactions that is used to evaluate
the catalytic activity of metal nanostructures is the reduction of
4-nitrophenol (4-NP) by NaBH4.124124 This reduction
reaction produces 4-aminophenol (4-AP), which is an
important intermediate in the synthesis of paracetamol. The
kinetics of this reaction can be easily monitored using a UV−
visible spectrophotometer. As the catalytic reduction proceeds,
the 400 nm peak that is associated with 4-NP disappears over
time, and this coincides with the appearance and gradual
increase in the intensity of the 300 nm absorption peak of 4-
AP. The lignin-stabilized Au−Pd nanostructure-coated mag-
netic stir bar described in the previous section was used as a
catalyst for this reaction, and the time-dependent absorption
spectra that were acquired for different compositions are
shown in Figure 8b−f.119 All of the bimetallic compositions
tested were able to catalyze the reaction at a faster rate as
compared to the monometallic counterparts (i.e., Au and Pd
nanostructures), with the 1:1 composition showing the highest
catalytic activity. The Au−Pd combination is believed to be
better than the single-component metals in facilitating the
transfer process of electrons from borohydride (BH4−) to the
catalyst surface, and this leads to faster reaction times for the
bimetallic catalysts. Similar results were obtained for the
starch-stabilized Au−Ag nanoparticles, which were found to be
better catalysts for the 4-NP reduction than the monometallic
Au and Ag nanoparticles.111 These bimetallic Au−Ag nano-
7221
Perspective
particles also performed better than the physical mixture of Au
and Ag nanoparticles, indicating the significance of creating a
bimetallic structure rather than simply mixing the two metals.
In a bimetallic system, charge transfer and mechanical strain
can result from the intimate interaction between the two
different metal components, and these can fundamentally alter
their electronic structure (i.e., shift the d band center), surface
properties, and catalytic activity.7,123
Bimetallic nanostructures have also been reported to
effectively catalyze the borohydride reduction of known
organic dye pollutants. For example, phytosynthesized Au−
Pd nanoparticles exhibited exceptional catalytic activity for the
reduction of methylene blue, with the reaction nearly
completed in less than a minute for certain alloy
compositions.80 On the other hand, phytosynthesized Au−
Ag nanoparticles displayed promising catalytic behavior for the
borohydride reduction of methyl orange.70 For bimetallic
systems that combine plasmonically active metals (e.g., Cu, Ag,
Au) and highly catalytic metals (e.g., Pt, Pd, Rh), the catalytic
activity can be promoted by coupling with light in a process
known as photocatalysis.10 For instance, the starch-stabilized
Pt−Cu nanourchins were shown to be capable of photo-
catalytically degrading malachite green under UV−visible light
irradiation.109 In this case, the plasmonic Cu component
endows the system with optical functionality, enabling it to
absorb light that can be used to induce dye degradation
reactions. Aside from the enhanced activity that is observed
when Pt is combined with Cu, an added benefit is the reduced
cost due to the lower loading of Pt, which is an expensive noble
metal.125 The branched structure of the nanourchins is also
believed to play a role in achieving remarkable catalytic
performance.109 This is because branched architectures possess
large specific surface area and high densities of corners and
edges, which can serve as catalytically active sites for chemical
reactions.126 Another example of a branched structure is the
flower morphology. Sun et al. compared the catalytic
performance of their phytosynthesized Au−Pd nanoflowers
with that of similar-sized Au−Pd nanospheres.75 They used the
oxidation of benzyl alcohol to benzaldehyde as the model
reaction for the evaluation of catalytic behavior. The Au−Pd
nanoflowers showed better catalytic properties than the Au−
Pd nanospheres in terms of catalytic activity and product
selectivity. Furthermore, the flower-shaped catalyst was found
to display excellent durability, which is a clear indication of its
reusability.
3.2. Sensing Applications. 3.2.1. Surface-Enhanced
Raman Scattering (SERS)-Based Detection. Raman spectros-
copy is a valuable characterization tool that can provide
vibrational fingerprints of molecules, enabling their detection
and quantification in a sample.127 It is a nondestructive
technique that is based on the inelastic scattering of photons
by molecular bond vibrations (i.e., the Raman effect).
However, for most molecules, the Raman effect is very weak,
and this makes it difficult to obtain a sizable signal intensity. It
was in the 1970s when it was discovered that the Raman
scattering signals can be dramatically enhanced, as observed for
pyridine that is adsorbed onto an electrochemically roughened
Ag electrode.128−130 This interesting phenomenon, which is
now known as the surface-enhanced Raman scattering (SERS)
effect, has grown into an active area of research that engages
the effort of several scientists worldwide. On average,
enhancement factors that are in the order of 104−106 have
been obtained when molecules are adsorbed onto an SERS-
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
Figure 9. SERS-based detection of 4-MBA using xylan-capped Au−Ag nanoparticles. (a) Schematic depiction of the SERS effect. (b) Raman
spectra of 20 mM 4-MBA on different nanoparticles: Au, Ag, Au@Ag core−shell, and hollow Au−Ag alloy. (c) Raman enhancement performance
of the different nanoparticles. Adapted with permission from ref 114. Copyright 2019 American Chemical Society.
active substrate.131 Two mechanisms are credited with
facilitating the SERS effect, and these are the electromagnetic
and the chemical (or charge transfer) enhancement mecha-
nisms.132 Of the two, the dominant contributor is the
electromagnetic enhancement mechanism, which associates
the SERS effect to the localized surface plasmon resonance
(LSPR) of roughened or nanostructured metals. The LSPR
originates from the light-induced excitation of the collective
oscillation of conduction electrons in the nanosized metal. This
produces strong local electromagnetic fields that are able to
substantially enhance the Raman scattering cross-sections of
adsorbed molecules.
Ag nanostructures are among the most commonly employed
SERS substrates due to the high LSPR sensitivity of Ag.132 The
downside is that Ag is prone to oxidation, which can
significantly lower the SERS efficiency. To improve its stability
toward oxidation, Ag is integrated with chemically robust
metals like Au to form bimetallic nanostructures. Cai et al.
demonstrated the use of their xylan-stabilized Au−Ag nano-
particles as SERS substrates for the detection of 4-
mercaptobenzoic acid (4-MBA).114 They examined two types
of Au−Ag configuration: (1) alloyed Au−Ag with a hollow
structure and (2) core−shell Au−Ag, where Au is coated with
Ag (designated as Au@Ag in Figure 9a). Both types displayed
higher SERS sensitivity than the monometallic Au and Ag
nanoparticles, with the core−shell sample giving the best
performance (Figure 9b,c). Their stability against oxidation
was tested by treating the samples with 1% H2O2 for 2 h. As
expected, the Raman signal intensity decreased less dramati-
cally for the bimetallic nanostructures as compared to the Ag
nanoparticles after H2O2 treatment due to improved stability
brought about by coupling. Xylan was also noted to contribute
to the stability of the nanoparticles as it was observed that the
xylan-capped Au−Ag nanoparticles performed better than the
7222
Perspective
uncapped ones. In addition, the xylan coating is thought to
promote the creation of hot spots between interconnected
nanoparticles that can amplify the Raman signals. To
demonstrate its potential for practical use, the xylan-capped
core−shell sample was further tested for the detection of
Sudan I, a food additive that is banned in many countries due
to its carcinogenicity. A limit of detection (LOD) as low as
0.126 ppm was obtained.
The core−shell Pd−Au nanostructures that were produced
through DNA-controlled growth were used as substrates for
detecting 4-mercaptobenzenethiol (4-MBT).86 Enhancement
factors in the order of 107 were observed. On the other hand,
the SERS activity of the phytosynthesized alloyed Au−Pd
nanoflowers was investigated using rhodamine 6G (R6G) as
the test analyte.75 Strong Raman signals were obtained at an
analyte concentration of 10−8 M, demonstrating high
sensitivity. To determine the impact of morphology on the
SERS effect, the activity of phytosynthesized Au−Pd nano-
spheres was also measured. The nanoflowers were found to
induce a much larger SERS enhancement relative to the
nanospheres, and this was ascribed to the hierarchical flower-
like architecture. Nanoflowers possess a large number of SERS-
active hot spots due to their sharp petal tips and nanoscale
gaps, which can concentrate the electromagnetic fields.127
These strong localized fields are crucial to the intensification of
Raman signals.
3.2.2. Fluorescence Sensing. Metal nanoclusters have been
attracting abundant attention as an emerging class of optical
sensors due to their intense and tunable fluorescence,
remarkable photostability, and good biocompatibility.88,89
Their ultrasmall dimensions are comparable to the Fermi
wavelength of electrons, placing them between single atoms
and larger nanoparticles.133,134 For this reason, their optical
properties originate from the molecular-like electronic
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

Figure 10. Fluorescence sensing of Pb2+ using BSA-stabilized Au−Ag nanoclusters. (a) Normalized fluorescence spectra of BSA-stabilized Au−Ag
nanoclusters with different alloy compositions. (b) Fluorescence intensity enhancement with increasing concentration of Pb2+. (c) Extent of
fluorescence intensity enhancement in the presence of different metal ions. Reproduced from ref 96. Copyright 2019. Published by The Royal
Society of Chemistry.

transitions between highest occupied molecular orbital
(HOMO)−lowest unoccupied molecular orbital (LUMO)
energy levels as opposed to the LSPR effect that is seen in
plasmonic metal nanoparticles. The emission wavelength of
metal nanoclusters can be tuned by altering the size of the
cluster. For bimetallic systems, adjusting the composition
presents an alternative route to fine-tune the emission
wavelength.
The fluorescence spectra of BSA-stabilized Au−Ag nano-
clusters with different alloy compositions are shown in Figure
10a.96 A systematic shifting of the emission peak position
toward the NIR region can be seen with the increasing Ag
mole fraction. The observed redshift is thought to arise from
charge transfer processes between the two metals and between
the metals and BSA. Among the different samples, the highest
fluorescence quantum yield was observed when the Au/Ag
molar ratio is 2.3:2.0, with an emission maximum at 718 nm.
This sample was employed as a fluorescent probe for the
detection of Pb2+, a highly toxic heavy metal ion that can
adversely affect almost any organ in the human body. The
addition of Pb2+ induced a significant increase in fluorescence
intensity at 718 nm (Figure 10b) with a detection limit of 96
nM. In comparison, the fluorescence intensity was practically
unaffected with the addition of other metal ions (Figure 10c).
This indicates good sensitivity and selectivity toward Pb2+.
Aside from Pb2+, two other heavy metal ions that are
7223
considered as major health risks due to their high toxicity
are Cd2+and Hg2+. Wang et al. used their BSA-stabilized Au−
Ni nanoclusters for the fluorimetric determination of these
ions.100 Interestingly, the sensing mechanism is different for
the two ions. The fluorescence intensity was considerably
enhanced when Cd2+ was added, but it was substantially
reduced in the presence of Hg 2+ . The Hg2+-induced
fluorescence quenching was also observed for BSA-stabilized
Au−Ag nanoclusters.94,95 The quenching of fluorescence is
attributed to the strong metallophilic interaction (i.e.,
amalgamation) between Hg2+ and Au+.135
Chen et al. demonstrated that through surface modification
with dopamine, BSA-capped Au−Cu nanoclusters can be used
as a fluorescent biosensor for glycine detection based on an
off/on fluorescence switching behavior.99 The fluorescence of
the presynthesized BSA-capped nanoclusters was switched off
after modification with dopamine as a result of a charge
transfer process. In the presence of glycine, this charge transfer
process was inhibited due to hydrogen bonding interactions,
thereby turning on the fluorescence. Meanwhile, Pang and Liu
reported the utility of lysozyme-stabilized Au−Ag nanoclusters
as a fluorescent probe for the detection of ascorbic acid.103 The
nanoclusters simply acted as a “turn on” sensor, where the
fluorescence intensity is enhanced by ascorbic acid. Zhang and
co-workers tested the versatility of their GSH-capped Au−Ag
nanoclusters using various metal ions, anions, and amino acids
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
as analytes.105 Their nanoclusters were found to respond as a
“turn off” probe for the detection of cupric ion, sulfide, iodide,
cysteine, and glutathione.
4. CONCLUSIONS AND OUTLOOK
Bimetallic nanostructures represent a promising class of metal
nanomaterials that can be leveraged for a multitude of uses
across a variety of fields, which include catalysis and sensing.
The synergistic effects that arise from the combination of two
distinct metals endow them with better catalytic and sensing
properties than their monometallic counterparts. Moreover,
the bimetallic composition and configuration serve as addi-
tional means, aside from size and shape, of attaining tunable
properties. With the significant advances in wet chemical
synthesis over the past decade, it has become easier to prepare
bimetallic nanostructures with tailorable nanoscale features.
However, the reagents that are typically used to achieve
controllable synthesis are harsh and toxic, prompting scientists
to increasingly turn to biogenic reagents such as plant extracts
and biomolecules, which are environmentally and biologically
compatible. An additional benefit is that they are multifunc-
tional as they can serve as reductants, surfactants, and
structure-directors. This allows for a relatively safe, facile,
and cost-effective synthesis.
The plant-mediated approach holds wide appeal because it is
convenient, rapid, and inexpensive. Given that the number of
plants that exist is practically countless, it is predicted that
more plant species will be explored for the phytosynthesis of
bimetallic nanostructures in the coming years. The majority of
previous work on this topic has focused only on establishing
the potential of extracts derived from specific plant parts in
generating the anticipated nanomaterial. More efforts should
be dedicated to identifying and quantifying the different
phytochemicals present in the plant extracts and understanding
the role of each constituent in the nanostructure formation
process. This could aid us in formulating the most suitable
mixture of biogenic reagents for the synthesis of bimetallic
nanostructures with tunable characteristics. When used for
catalysis, one cause of concern is the presence of biogenic
residues on the nanomaterial surfaces, as this can negatively
impact the catalytic performance. For instance, it has been
found that using coffee extract as the reductant creates a
persistent biogenic corona around the metal nanoparticle
surface, thereby blocking the material’s catalytically active
sites.136 To address this issue, efforts must be poured into
developing facile techniques that can facilitate the removal of
problematic adsorbates.
In many cases, the morphology and configuration have been
shown to play critical roles in the enhancement of the catalytic
and sensing performance. The use of nucleic acids or
oligonucleotides has been instrumental in the construction of
bimetallic nanostructures and assemblies with complex
configurations that would be difficult to fabricate through
other biogenic methods. Most of the studies about this have
chosen to employ homo-oligomeric DNAs as a way to
determine the influence of different nucleobases in morpho-
logical control. Future studies should venture into using
different sequence combinations as this could potentially
generate bimetallic nanostructures with more sophisticated
designs and advanced features. In the case of proteins, their
utility in preparing bimetallic nanostructures has yet to expand
to the creation of more refined morphologies. Previous work
has been limited to the synthesis and stabilization of small-
7224
Perspective
sized spherical bimetallic nanoclusters. With their highly
complex and dynamic conformations, there is no doubt that
proteins can also provide the structural framework that is
needed to direct the growth of bimetallic systems into unique
and intricate architectures. The challenge lies in the tedious
task of finding the optimal reaction conditions (e.g., pH,
temperature, concentration) that could facilitate the formation
of the desired morphology.
Despite the expanding literature on biogenic synthesis, there
remains much work to be done in terms of developing
methodologies that are not only green and controllable but
also reliable and scalable. A reliable synthetic procedure with
good reproducibility is often challenging to achieve because
the nucleation and growth processes are sensitive to
experimental conditions. In the case of phytosynthesis,
seasonal and environmental effects on the chemical contents
in plants can also lead to irreproducible data. Solving these
issues necessitates further investigations on the actual
formation mechanisms to better understand the extent of
influence of different experimental conditions on the
nanostructure formation processes. Scalability is another
issue that must be addressed if these nanomaterials are to be
used commercially as catalysts and sensors. The amounts of
samples that are produced in the lab are usually on the
milligram scale, which would not be able to meet the large
quantities of materials required for real-world applications.
Thus, further research should be directed toward the
development of biogenic protocols that can be readily scaled
up for mass production.
With Au being the most studied metal in nanomaterials
research, it is not surprising that it is contained in most of the
biogenically prepared bimetallic nanostructures that have been
reported thus far. The Au−Ag system, in particular, has been
receiving an enormous amount of attention due to its wide
utility. Future work should consider examining other bimetallic
systems as this could lead to interesting discoveries.
Considering the vast number of possible metal combinations,
computational tools can aid in deciding which systems are
worth exploring, especially in cases where specific properties
are desired or particular applications are targeted. Computa-
tional methods are also useful in the elucidation of formation
mechanisms and in the study of structure−property relation-
ships. As with other areas of research, experimental and
theoretical scientists should work together to realize further
advancements in the study of bimetallic nanostructures.
■
AUTHOR INFORMATION
Corresponding Author
Michelle D. Regulacio − Natural Sciences Research Institute
and Institute of Chemistry, University of the Philippines
Diliman, Quezon City 1101, Philippines; orcid.org/0000-
0002-8622-0965; Email: mdregulacio@up.edu.ph
Author
Rufus Mart Ceasar R. Ramos − Natural Sciences Research
Institute, University of the Philippines Diliman, Quezon City
1101, Philippines
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.1c00692
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
Author Contributions
This manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors gratefully acknowledge the Natural Sciences
Research Institute of the University of the Philippines Diliman
(Project CHE-21-1-04) for financial support.
■ REFERENCES
(1) Tylecote, R. F. A History of Metallurgy, 2nd ed.; Maney
Publishing for the Institute of Materials: London, 1992.
(2) Murty, B. S.; Yeh, J. W.; Ranganathan, S.; Bhattacharjee, P. P. A
Brief History of Alloys and the Birth of High-Entropy Alloys. High-
Entropy Alloys, 2nd ed.; Elsevier: Amsterdam, 2019; pp 1−12.
(3) Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Noble
metals on the nanoscale: optical and photothermal properties and
some applications in imaging, sensing, biology, and medicine. Acc.
Chem. Res. 2008, 41, 1578−1586.
(4) Tee, S. Y.; Ye, E. Recent Advancements in Coinage Metal
Nanostructures and Bio-applications. Mater. Adv. 2021,
DOI: 10.1039/D0MA00829J.
(5) Tao, A. R.; Habas, S.; Yang, P. Shape Control of Colloidal Metal
Nanocrystals. Small 2008, 4, 310−325.
(6) Hu, M.; Chen, J.; Li, Z.-Y.; Au, L.; Hartland, G. V.; Li, X.;
Marquez, M.; Xia, Y. Gold nanostructures: engineering their
plasmonic properties for biomedical applications. Chem. Soc. Rev.
2006, 35, 1084−1094.
(7) Gilroy, K. D.; Ruditskiy, A.; Peng, H.-C.; Qin, D.; Xia, Y.
Bimetallic Nanocrystals: Syntheses, Properties, and Applications.
Chem. Rev. 2016, 116, 10414−10472.
(8) Sankar, M.; Dimitratos, N.; Miedziak, P. J.; Wells, P. P.; Kiely, C.
J.; Hutchings, G. J. Designing bimetallic catalysts for a green and
sustainable future. Chem. Soc. Rev. 2012, 41, 8099−8139.
(9) Liu, X.; Astruc, D. From Galvanic to Anti-Galvanic Synthesis of
Bimetallic Nanoparticles and Applications in Catalysis, Sensing, and
Materials Science. Adv. Mater. 2017, 29, No. 1605305.
(10) Sytwu, K.; Vadai, M.; Dionne, J. A. Bimetallic nanostructures:
combining plasmonic and catalytic metals for photocatalysis. Adv.
Phys.: X 2019, 4, No. 1619480.
(11) Li, Y.-H.; Li, J.-Y.; Xu, Y.-J. Bimetallic nanoparticles as
cocatalysts for versatile photoredox catalysis. EnergyChem 2021, 3,
No. 100047.
(12) Min, Y.; Wang, Y. Manipulating Bimetallic Nanostructures
With Tunable Localized Surface Plasmon Resonance and Their
Applications for Sensing. Front. Chem. 2020, 8, No. 411.
(13) Mandal, R.; Baranwal, A.; Srivastava, A.; Chandra, P. Evolving
trends in bio/chemical sensor fabrication incorporating bimetallic
nanoparticles. Biosens. Bioelectron. 2018, 117, 546−561.
(14) DeSantis, C. J.; Weiner, R. G.; Radmilovic, A.; Bower, M. M.;
Skrabalak, S. E. Seeding Bimetallic Nanostructures as New Class of
Plasmonic Colloids. J. Phys. Chem. Lett. 2013, 4, 3072−3082.
(15) Huynh, K.-H.; Pham, X.-H.; Kim, J.; Lee, S. H.; Chang, H.;
Rho, W.-Y.; Jun, B.-H. Synthesis, Properties, and Biological
Applications of Metallic Alloy Nanoparticles. Int. J. Mol. Sci. 2020,
21, No. 5174.
(16) Srinoi, P.; Chen, Y.-T.; Vittur, V.; Marquez, M. D.; Lee, T. R.
Bimetallic Nanoparticles: Enhanced Magnetic and Optical Properties
for Emerging Biological Applications. Appl. Sci. 2018, 8, No. 1106.
(17) Sharma, G.; Kumar, A.; Sharma, S.; Naushad, M.; Dwivedi, R.
P.; Alothman, Z. A.; Mola, G. T. Novel development of nanoparticles
to bimetallic nanoparticles and their composites: A review. J. King
Saud Univ. Sci. 2019, 31, 257−269.
7225
Perspective
(18) Duan, S.; Wang, R. Bimetallic nanostructures with magnetic
and noble metals and their physicochemical applications. Prog. Nat.
Sci.: Mater. Int. 2013, 23, 113−126.
(19) Ferrando, R.; Jellinek, J.; Johnston, R. L. Nanoalloys: From
Theory to Applications of Alloy Clusters and Nanoparticles. Chem.
Rev. 2008, 108, 845−910.
(20) Zaleska-Medynska, A.; Marchelek, M.; Diak, M.; Grabowska, E.
Noble metal-based bimetallic nanoparticles: the effect of the structure
on the optical, catalytic and photocatalytic properties. Adv. Colloid
Interface Sci. 2016, 229, 80−107.
(21) Bonnemann, H.; Richards, R. M. Nanoscopic Metal Particles −
Synthetic Methods and Potential Applications. Eur. J. Inorg. Chem.
2001, 2001, 2455−2480.
(22) Toshima, N.; Yonezawa, T. Bimetallic nanoparticles − novel
materials for chemical and physical applications. New J. Chem. 1998,
22, 1179−1201.
(23) Rodríguez-González, B.; Burrows, A.; Watanabe, M.; Kiely, C.
J.; Marzán, L. M. L. Multishell bimetallic AuAg nanoparticles:
synthesis, structure and optical properties. J. Mater. Chem. 2005, 15,
1755−1759.
(24) Lu, N.; Wang, J.; Xie, S.; Brink, J.; McIlwrath, K.; Xia, Y.; Kim,
M. J. Aberration Corrected Electron Microscopy Study of Bimetallic
Pd−Pt Nanocrystal: Core−Shell Cubic and Core−Frame Concave
Structures. J. Phys. Chem. C 2014, 118, 28876−28882.
(25) Feng, J.; Wu, X.; Ma, W.; Kuang, H.; Xu, L.; Xu, C. A SERS
active bimetallic core−satellite nanostructure for the ultrasensitive
detection of Mucin-1. Chem. Commun. 2015, 51, 14761−14763.
(26) Song, Y.; Liu, K.; Chen, S. AgAu Bimetallic Janus Nanoparticles
and Their Electrocatalytic Activity for Oxygen Reduction in Alkaline
Media. Langmuir 2012, 28, 17143−17152.
(27) Mahmud, S.; Satter, S. S.; Singh, A. K.; Rahman, M. M.;
Mollah, M. Y. A.; Susan, M. A. B. H. Tailored Engineering of
Bimetallic Plasmonic Au@Ag Core@Shell Nanoparticles. ACS Omega
2019, 4, 18061−18075.
(28) Garcia-Leis, A.; Torregiani, A.; Garcia-Ramos, J. V.; Sanchez-
Cortez, S. Hollow Au/Ag nanostars displaying broad plasmonic
resonance and high surface-enhanced Raman sensitivity. Nanoscale
2015, 7, 13629−13637.
(29) Kang, H.; Buchman, J. T.; Rodriguez, R. S.; Ring, H. L.; He, J.;
Bantz, K. C.; Haynes, C. L. Stabilization of Silver and Gold
Nanoparticles: Preservation and Improvement of Plasmonic Func-
tionalities. Chem. Rev. 2019, 119, 664−699.
(30) Motl, N. E.; Smith, A. F.; DeSantis, C. J.; Skrabalak, S. E.
Engineering plasmonic metal colloid through composition and
structural design. Chem. Soc. Rev. 2014, 43, 3823−3834.
(31) Bai, Y.; Gao, C.; Yin, Y. Fully alloyed Ag/Au nanorods with
tunable surface plasmon resonance and high chemical stability.
Nanoscale 2017, 9, 14875−14880.
(32) Albrecht, W.; Bladt, E.; Vanrompay, H.; Smith, J. D.; Skrabalak,
S. E.; Bals, S. Thermal Stability of Gold/Palladium Octopods Studied
in Situ in 3D: Understanding Design Rules for Thermally Stable
Metal Nanoparticles. ACS Nano 2019, 13, 6522−6530.
(33) Fu, G.; Wu, K.; Lin, J.; Tang, Y.; Chen, Y.; Zhou, Y.; Lu, T.
One-Pot Water-Based Synthesis of Pt−Pd Alloy Nanoflowers and
Their Superior Electrocatalytic Activity for the Oxygen Reduction
Reaction and Remarkable Methanol-Tolerant Ability in Acid Media. J.
Phys. Chem. C 2013, 117, 9826−9834.
(34) Lu, L.; Chen, S.; Thota, S.; Wang, X.; Wang, Y.; Zou, S.; Fan, J.;
Zao, J. Composition Controllable Synthesis of PtCu Nanodendrites
with Efficient Electrocatalytic Activity for Methanol Oxidation
Induced by High Index Surface and Electronic Interaction. J. Phys.
Chem. C 2017, 121, 19796−19806.
(35) Yu, H.-D.; Regulacio, M. D.; Ye, E.; Han, M.-Y. Chemical
routes to top-down nanofabrication. Chem. Soc. Rev. 2013, 42, 6006−
6018.
(36) Xu, L.; Tan, L. S.; Hong, M. H. Tuning of localized surface
plasmon resonance of well-ordered Ag/Au bimetallic nanodot arrays
by laser interference lithography and thermal annealing. Appl. Opt.
2011, 50, G74−G79.
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
(37) Liu, F.-K.; Huang, P.-W.; Chang, Y.-C.; Ko, F.-H.; Chu, T.-T.
Combining Optical Lithography with Rapid Microwave Heating for
the Selective Growth of Au/Ag Bimetallic Core/Shell Structures on
Patterned Silicon Wafers. Langmuir 2005, 21, 2519−2525.
(38) Loza, K.; Heggen, M.; Epple, M. Synthesis, Structure,
Properties, and Applications of Bimetallic Nanoparticles of Noble
Metals. Adv. Funct. Mater. 2020, 30, No. 1909260.
(39) Bhol, P.; Bhavya, M. B.; Swain, S.; Saxena, M.; Samal, A. K.
Modern Chemical Routes for the Controlled Synthesis of Anisotropic
Bimetallic Nanostructures and Their Application in Catalysis. Front.
Chem. 2020, 8, No. 357.
(40) Banadaki, A. D.; Kajbafvala, A. Recent Advances in Facile
Synthesis of Bimetallic Nanostructures: An Overview. J. Nanomater.
2014, 2014, No. 985948.
(41) Dang-Bao, T.; Pla, D.; Favier, I.; Gómez, M. Bimetallic
Nanoparticles in Alternative Solvents for Catalytic Purposes. Catalysts
2017, 7, No. 207.
(42) Weiner, R. G.; Kunz, M. R.; Skrabalak, S. E. Seeding a New
Kind of Garden: Synthesis of Architecturally Defined Multimetallic
Nanostructures by Seed-Mediated Co-Reduction. Acc. Chem. Res.
2015, 48, 2688−2695.
(43) McGrath, A. J.; Chien, Y.-H.; Cheong, S.; Herman, D. A. J.;
Watt, J.; Henning, A. M.; Gloag, L.; Yeh, C.-H.; Tilley, R. D. Gold
over Branched Palladium Nanostructures for Photothermal Cancer
Therapy. ACS Nano 2015, 9, 12283−12291.
(44) Wang, S.; Doronkin, D. E.; Hähsler, M.; Huang, X.; Wang, D.;
Grunwaldt, J.-D.; Behrens, S. Palladium-Based Bimetallic Nanocrystal
Catalysts for the Direct Synthesis of Hydrogen Peroxide. Chem-
SusChem 2020, 13, 3243−3251.
(45) Duan, H.; Wuang, D.; Li, Y. Green chemistry for nanoparticle
synthesis. Chem. Soc. Rev. 2015, 44, 5778−5792.
(46) Zhang, Y.; Newton, B.; Lewis, E.; Fu, P. P.; Kafoury, R.; Ray, P.
C.; Yu, H. Cytotoxicity of organic surface coating agents used for
nanoparticles synthesis and stability. Toxicol. In Vitro 2015, 29, 762−
768.
(47) Bandala, E.; Stanisic, D.; Tasic, L. Biogenic nanomaterials for
photocatalytic degradation and water disinfection: a review. Environ.
Sci.: Water Res. Technol. 2020, 6, 3195−3213.
(48) Regulacio, M. D.; Yang, D.-P.; Ye, E. Toward greener methods
of producing branched metal nanostructures. CrystEngComm 2020,
22, 399−411.
(49) Singh, J.; Dutta, T.; Kim, K.-H.; Rawat, M.; Samdar, P.; Kumar,
P. ‘Green’ synthesis of metals and their oxide nanoparticles:
applications for environmental remediation. J. Nanobiotechnol. 2018,
16, No. 84.
(50) Qazi, F.; Hussain, Z.; Tahir, M. N. Advances in biogenic
synthesis of palladium nanoparticles. RSC Adv. 2016, 6, 60277−
60286.
(51) Faramarzi, M. A.; Sadighi, A. Insights into biogenic and
chemical production of inorganic nanomaterials and nanostructures.
Adv. Colloid Interface Sci. 2013, 189−190, 1−20.
(52) Huang, J.; Lin, L.; Sun, D.; Chen, H.; Yang, D.; Li, Q. Bio-
inspired synthesis of metal nanomaterials and applications. Chem. Soc.
Rev. 2015, 44, 6330−6374.
(53) Poulose, S.; Panda, T.; Nair, P. P.; Théodore, T. Biosynthesis of
Silver Nanoparticles. J. Nanosci. Nanotechnol. 2014, 14, 2038−2049.
(54) Varma, R. S. Greener and Sustainable Trends in Synthesis of
Organics and Nanomaterials. ACS Sustainable Chem. Eng. 2016, 4,
5866−5878.
(55) Mondal, P.; Anweshan, A.; Purkait, M. K. Green synthesis and
environmental application of iron-based nanomaterials and nano-
composites: A review. Chemosphere 2020, 259, No. 127509.
(56) Behera, A.; Mittu, B.; Padhi, S.; Patra, N.; Sing, J. Bimetallic
Nanoparticles: Green Synthesis, Applications, and Future Perspec-
tives. In Multifunctional Hybrid Nanomaterials for Sustainable Agrifood
and Ecosystems; Abd-Elsalam, K. A., Ed.; Elsevier: Amsterdam, 2020;
pp 639−682.
7226
Perspective
(57) Mazhar, T.; Shrivastava, V.; Tomar, R. S. Green Synthesis of
Bimetallic Nanoparticles and its Applications: A Review. J. Pharm. Sci.
Res. 2017, 9, 102−110.
(58) Dauthal, P.; Mukhopadhyay, M. Noble Metal Nanoparticles:
Plant-Mediated Synthesis, Mechanistic Aspects of Synthesis, and
Applications. Ind. Eng. Chem. Res. 2016, 55, 9557−9577.
(59) Akhtar, M. S.; Panwar, J.; Yun, Y.-S. Biogenic Synthesis of
Metallic Nanoparticles by Plant Extracts. ACS Sustainable Chem. Eng.
2013, 1, 591−602.
(60) Mohammadinejad, R.; Shavandi, A.; Raie, D. S.; Sangeetha, J.;
Soleimani, M.; Hahjibehzad, S. S.; Thangadurai, D.; Hospet, R.;
Popoola, J. O.; Arzani, A.; Gómez-Lim, M. A.; Iravani, S.; Varma, R. S.
Plant molecular farming: production of metallic nanoparticles and
therapeutic proteins using green factories. Green Chem. 2019, 21,
1845−1865.
(61) Vishnukumar, P.; Vivekhandhan, S.; Muthuramkumar, S. Plant-
Mediated Biogenic Synthesis of Palladium Nanoparticles: Recent
Trends and Emerging Opportunities. ChemBioEng Rev. 2017, 4, 18−
36.
(62) Iyer, R. I.; Panda, T. Biogenic synthesis of gold and silver
nanoparticles by seed plants. J. Nanosci. Nanotechnol. 2014, 14, 2024−
2037.
(63) Yang, D.-P.; Liu, X.; Teng, C. P.; Owh, C.; Win, K. Y.; Lin, M.;
Loh, X. J.; Wu, Y.-L.; Li, Z.; Ye, E. Unexpected formation of gold
nanoflowers by a green synthesis method as agents for a safe and
effective photothermal therapy. Nanoscale 2017, 9, 15753−15759.
(64) Tamuly, C.; Hazarika, M.; Borah, S. C.; Das, M. R.; Boruah, M.
P. In situ biosynthesis of Ag, Au and bimetallic nanoparticles using
Piper pedicellatum C.DC: Green chemistry approach. Colloids Surf., B
2013, 102, 627−634.
(65) Khan, M.; Al-Hamoud, K.; Liaqat, Z.; Shaik, M. R.; Adil, S. F.;
Kuniyil, M.; Alkathlan, H. Z.; Al-Warthan, A.; Siddiqui, M. R. H.;
Mondeshki, M.; Tremel, W.; Khan, M.; Tahir, M. N. Synthesis of Au,
Ag, and Au−Ag Bimetallic Nanoparticles Using Pulicaria undulata
Extract and Their Catalytic Activity for the Reduction of 4-
Nitrophenol. Nanomaterials 2020, 10, No. 1885.
(66) Gopalakrishnan, R.; Longanathan, B.; Raghu, K. Green
synthesis of Au−Ag bimetallic nanocomposites using Silybum
marianum seed extract and their application as a catalyst. RSC Adv.
2015, 5, 31691−31699.
(67) Elemike, E. E.; Onwudiwe, D. C.; Fayemi, O. E.; Botha, T. L.
Green synthesis and electrochemistry of Ag, Au, and Ag−Au
bimetallic nanoparticles using golden rod (Solidago canadensis) leaf
extract. Appl. Phys. A: Mater. Sci. Process. 2019, 125, 42.
(68) Alti, D.; Rao, M. V.; Rao, D. N.; Maurya, R.; Kalangi, S. K.
Gold−Silver Bimetallic Nanoparticles Reduced with Herbal Leaf
Extracts Induce ROS-Mediated Death in Both Promastigote and
Amastigote Stages of Leishmania donovani. ACS Omega 2020, 5,
16238−16245.
(69) Sun, L.; Yin, Y.; Lv, P.; Su, W.; Zhang, L. Green controllable
synthesis of Au−Ag alloy nanoparticles using Chinese wolfberry fruit
extract and their tunable photocatalytic activity. RSC Adv. 2018, 8,
3964−3973.
(70) Kumari, M. M.; Jacob, J.; Philip, D. Green synthesis and
applications of Au−Ag bimetallic nanoparticles. Spectrochim. Acta,
Part A 2015, 137, 185−192.
(71) Sheny, D. S.; Mathew, J.; Philip, D. Phytosynthesis of Au, Ag
and Au−Ag bimetallic nanoparticles using aqueous extract and dried
leaf of Anacardium occidentale. Spectrochim. Acta, Part A 2011, 79,
254−262.
(72) AbdelHamid, A. A.; Al-Ghobashy, M. A.; Fawzy, M.;
Mohamed, M. B.; Abdel-Mottaleb, M. S. A. Phytosynthesis of Au,
Ag, and Au−Ag Bimetallic Nanoparticles Using Aqueous Extract of
Sago Pondweed (Potamogeton pectinatus L.). ACS Sustainable Chem.
Eng. 2013, 1, 1520−1529.
(73) Zhang, G.; Du, M.; Li, Q.; Li, X.; Huang, J.; Jiang, X.; Sun, D.
Green synthesis of Au−Ag alloy nanoparticles using Cacumen
platycladi extract. RSC Adv. 2013, 3, 1878−1884.
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf
(74) Lu, F.; Sun, D.; Huang, J.; Du, M.; Yang, F.; Chen, H.; Hong,
Y.; Li, Q. Plant-Mediated Synthesis of Ag−Pd Alloy Nanoparticles
and Their Application as Catalyst toward Selective Hydrogenation.
ACS Sustainable Chem. Eng. 2014, 2, 1212−1218.
(75) Sun, D.; Zhang, G.; Jiang, X.; Huang, J.; Jing, X.; Zheng, Y.; He,
J.; Li, Q. Biogenic flower-shaped Au−Pd nanoparticles: synthesis,
SERS detection and catalysis towards benzyl alcohol oxidation. J.
Mater. Chem. A 2014, 2, 1767−1773.
(76) Sun, D.; Zhang, G.; Huang, J.; Wang, H.; Li, Q. Plant-Mediated
Fabrication and Surface Enhanced Raman Property of Flower-Like
Au@Pd Nanoparticles. Materials 2014, 7, 1360−1369.
(77) Wang, L.; Hu, C.; Nemoto, Y.; Tateyama, Y.; Yamauchi, Y. On
the Role of Ascorbic Acid in the Synthesis of Single-Crystal
Hyperbranched Platinum Nanostructures. Cryst. Growth Des. 2010,
10, 3454−3460.
(78) Mares-Briones, F.; Barragán-Mares, O.; López-Miranda, J. L.;
Esparza, R.; Rosas, G. Bimetallic Ag@Pt core-shell nanoparticles and
their catalytic activity by a green approach. Mater. Res. Express 2019,
6, No. 0850h8.
(79) Odoom-Wubah, T.; Li, Z.; Lin, Z.; Tang, T.; Sun, D.; Huang,
J.; Li, Q. Ascorbic acid assisted bio-synthesis of Pd-Pt nanoflowers
with enhanced electrochemical properties. Electrochim. Acta 2017,
228, 474−482.
(80) Chowdhury, R.; Mollick, M. M. R.; Biswas, Y.; Chattopadhyay,
D.; Rashid, M. H. Biogenic synthesis of shape-tunable Au-Pd alloy
nanoparticles with enhanced catalytic activities. J. Alloys Compd. 2018,
763, 399−408.
(81) Kumar, A.; Hwang, J.-H.; Kumar, S.; Nam, J.-M. Tuning and
assembling metal nanostructures with DNA. Chem. Commun. 2013,
49, 2597−2609.
(82) Satyavolu, N. S. R.; Loh, K. Y.; Tan, L. H.; Lu, Y. Discovery of
and Insights into DNA “Codes” for Tunable Morphologies of Metal
Nanoparticles. Small 2019, 15, No. 1900975.
(83) Lee, J.-H.; Kim, G.-H.; Nam, J.-M. Directional Synthesis and
Assembly of Bimetallic Nanosnowmen with DNA. J. Am. Chem. Soc.
2012, 134, 5456−5459.
(84) Sutter, E.; Zhang, B.; Sutter, P. DNA-Mediated Three-
Dimensional Assembly of Hollow Au−Ag Alloy Nanocages as
Plasmonic Crystals. ACS Appl. Nano Mater. 2020, 3, 8068−8074.
(85) Satyavolu, N. S. R.; Tan, L. H.; Lu, Y. DNA-Mediated
Morphological Control of Pd−Au Bimetallic Nanoparticles. J. Am.
Chem. Soc. 2016, 138, 16542−16548.
(86) Satyavolu, N. S. R.; Pishevareshafani, N.; Tan, L. H.; Lu, Y.
DNA-encoded morphological evolution of bimetallic Pd@Au core-
shell nanoparticles from a high-indexed core. Nano Res. 2018, 11,
4549−4561.
(87) Bhattarai, N.; Prozorov, T. Direct Observation of Early Stages
of Growth of Multilayered DNA-Templated Au-Pd-Au Core-Shell
Nanoparticles in Liquid Phase. Front. Bioeng. Biotechnol. 2019, 7,
No. 19.
(88) Chevrier, D. M.; Chatt, A.; Zhang, P. Properties and
applications of protein-stabilized fluorescent gold nanoclusters:
short review. J. Nanophotonics 2012, 6, No. 064504.
(89) Shellaiah, M.; Sun, K. W. Luminescent Metal Nanoclusters for
Potential Chemosensor Applications. Chemosensors 2017, 5, 36.
(90) Xie, J.; Zheng, Y.; Ying, J. Y. Protein-Directed Synthesis of
Highly Fluorescent Gold Nanoclusters. J. Am. Chem. Soc. 2009, 131,
888−889.
(91) Chevrier, D. M.; Thanthirige, V. D.; Luo, Z.; Driscoll, S.; Cho,
P.; MacDonald, M. A.; Yao, Q.; Guda, R.; Xie, J.; Johnson, E. R.;
Chatt, A.; Zheng, N.; Zhang, P. Structure and formation of highly
luminescent protein-stabilized gold clusters. Chem. Sci. 2018, 9,
2782−2790.
(92) Hu, D.; Sheng, Z.; Gong, P.; Zhang, P.; Cai, L. Highly selective
fluorescent sensors for Hg2+ based on bovine serum albumin-capped
gold nanoclusters. Analyst 2010, 135, 1411−1416.
(93) Hsu, Y.-C.; Hung, M.-J.; Chen, Y.-A.; Wang, T.-F.; Ou, Y.-R.;
Chen, S.-H. Identifying Reducing and Capping Sites of Protein-
Encapsulated Gold Nanoclusters. Molecules 2019, 24, No. 1630.
7227
Perspective
(94) Dai, R.; Deng, W.; Hu, P.; You, C.; Yang, L.; Jiang, X.; Xiong,
X.; Huang, K. One-pot synthesis of bovine serum albumin protected
gold/silver bimetallic nanoclusters for ratiometric and visual detection
of mercury. Microchem. J. 2018, 139, 1−8.
(95) Zheng, B.; Zheng, J.; Yu, T.; Sang, A.; Du, J.; Guo, Y.; Xiao, D.;
Choi, M. M. F. Fast microwave-assisted synthesis of AuAg bimetallic
nanoclusters with strong yellow emission and their response to
mercury(II) ions. Sens. Actuators, B 2015, 221, 386−392.
(96) Sannigrahi, A.; Chowdhury, S.; Nandi, I.; Sanyal, D.; Chall, S.;
Chattopadhyay, K. Development of a near infrared Au−Ag bimetallic
nanocluster for ultrasensitive detection of toxic Pb2+ ions in vitro and
inside cells. Nanoscale Adv. 2019, 1, 3660−3669.
(97) Mohanty, J. S.; Xavier, P. L.; Chaudhari, K.; Bootharaju, M. S.;
Goswami, N.; Pal, S. K.; Pradeep, T. Luminescent, bimetallic AuAg
alloy quantum clusters in protein templates. Nanoscale 2012, 4,
4255−4262.
(98) Chaudhari, K.; Xavier, P. L.; Pradeep, T. Understanding the
Evolution of Luminescent Gold Quantum Clusters in Protein
Templates. ACS Nano 2011, 5, 8816−8827.
(99) Chen, Z.; Ding, W.; Gu, Y.; Gao, S.; Yun, D.; Wang, C.; Li, W.;
Sun, F. Dopamine-Modified AuCu Bimetallic Nanoclusters as Charge
Transfer-Based Biosensors for Highly Sensitive Glycine Detection.
Langmuir 2020, 36, 13928−13936.
(100) Wang, Z.-X.; Guo, Y.-X.; Ding, S.-N. Fluorometric
determination of cadmium(II) and mercury(II) using nanoclusters
consisting of a gold-nickel alloy. Microchim. Acta 2015, 182, 2223−
2231.
(101) Ding, S.-N.; Guo, Y.-X. One-pot synthesis of dual-emitting
BSA−Pt−Au bimetallic nanoclusters for fluorescence ratiometric
detection of mercury ions and cysteine. Anal. Methods 2015, 7, 5787−
5790.
(102) Wu, G.-W.; Shen, Y.-M.; Shi, X.-Q.; Deng, H.-H.; Zheng, X.-
Q.; Peng, H.-P.; Liu, A.-L.; Xia, X.-H.; Chen, W. Bimetallic Bi/Pt
peroxidase mimic and its bioanalytical applications. Anal. Chim. Acta
2017, 971, 88−96.
(103) Pang, S.; Liu, S. Lysozyme-stabilized bimetallic gold/silver
nanoclusters as a turn-on fluorescent probe for determination of
ascorbic acid and acid phosphatase. Anal. Methods 2017, 9, 6713−
6718.
(104) Jia, M.; Mi, W.; Guo, S.; Yang, Q.-Z.; Jina, Y.; Shao, N.
Peptide-capped functionalized Ag/Au bimetal nanoclusters with
enhanced red fluorescence for lysosome-targeted imaging of
hypochlorite in living cells. Talanta 2020, 216, No. 120926.
(105) Zhang, J.; Yuan, Y.; Wang, Y.; Sun, F.; Liang, G.; Jiang, Z.; Yu,
S.-H. Microwave-assisted synthesis of photoluminescent glutathione-
capped Au/Ag nanoclusters: A unique sensor-on-a-nanoparticle for
metal ions, anions, and small molecules. Nano Res. 2015, 8, 2329−
2339.
(106) Wang, C.; Xu, L.; Xu, X.; Cheng, H.; Sun, H.; Lin, Q.; Zhang,
C. Near infrared Ag/Au alloy nanoclusters: Tunable photo-
luminescence and cellular imaging. J. Colloid Interface Sci. 2014,
416, 274−279.
(107) Sun, L.; Yin, Y.; Wang, F.; Su, W.; Zhang, L. Facile one-pot
green synthesis of Au−Ag alloy nanoparticles using sucrose and their
composition-dependent photocatalytic activity for the reduction of 4-
nitrophenol. Dalton Trans. 2018, 47, 4315−4324.
(108) Mallikarjuna, K.; Bathula, C.; Reddy, G. D.; Shrestha, N. K.;
et al. Au-Pd bimetallic nanoparticles embedded highly porous
Fenugreek polysaccharide based micro networks for catalytic
applications. Int. J. Biol. Macromol. 2019, 126, 352−358.
(109) Devi, H. S.; Singh, H. P.; Singh, T. D. Green synthesized
urchin like Pt/Cu bimetallic photonanocatalysts: Understanding
composition effect. Mater. Res. Bull. 2017, 86, 272−286.
(110) Raveendran, P.; Fu, J.; Wallen, S. L. A simple and “green”
method for the synthesis of Au, Ag, and Au−Ag alloy nanoparticles.
Green Chem. 2006, 8, 34−38.
(111) Xia, B.; He, F.; Li, L. Preparation of Bimetallic Nanoparticles
Using a Facile Green Synthesis Method and Their Application.
Langmuir 2013, 29, 4901−4907.
https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228
ACS Omega http://pubs.acs.org/journal/acsodf Perspective

(112) Li, X.; Odoom-Wubah, T.; Huang, J. Biosynthesis of Ag−Pd (132) Pérez-Jiménez, A. I.; Lyu, D.; Lu, Z.; Liu, G.; Ren, B. Surface-
bimetallic alloy nanoparticles through hydrolysis of cellulose triggered enhanced Raman spectroscopy: benefits, trade-offs and future
by silver sulfate. RSC Adv. 2018, 8, 30340−30345. developments. Chem. Sci. 2020, 11, 4563.
(113) Hu, X.; Xu, X.; Fu, F.; Yang, B.; Zhang, J.; Zhang, Y.; Touhid, (133) Zhang, L.; Wang, E. Metal nanoclusters: New fluorescent
S. S. B.; Liu, L.; Dong, Y.; Liu, X.; Yao, J. Synthesis of bimetallic silver- probes for sensors and bioimaging. Nano Today 2014, 9, 132−157.
gold nanoparticle composites using a cellulose dope: Tunable (134) Chakraborty, I.; Pradeep, T. Atomically Precise Clusters of
nanostructure and its biological activity. Carbohydr. Polym. 2020, Noble Metals: Emerging Link between Atoms and Nanoparticles.
248, No. 116777. Chem. Rev. 2017, 117, 8208−8271.
(114) Cai, J.; Li, Y.; Liu, C.; Wang, X. Green and Controllable (135) Xie, J.; Zheng, Y.; Ying, J. Y. Highly selective and ultrasensitive
Synthesis of Au−Ag Bimetal Nanoparticles by Xylan for Surface- detection of Hg2+ based on fluorescence quenching of Au nano-
Enhanced Raman Scattering. ACS Sustainable Chem. Eng. 2019, 7, clusters by Hg2+−Au+ interactions. Chem. Commun. 2010, 46, 961−
15154−15162. 963.
(115) Huang, X.; Wu, H.; Liao, X.; Shi, B. One-step, size-controlled (136) Metz, K. M.; Sanders, S. E.; Pender, J. P.; Dix, M. R.; Hinds,
synthesis of gold nanoparticles at room temperature using plant D. T.; Quinn, S. J.; Ward, A. D.; Duffy, P.; Cullen, R. J.; Colavita, P. E.
tannin. Green Chem. 2010, 12, 395−399. Green Synthesis of Metal Nanoparticles via Natural Extracts: The
(116) Huang, X.; Wu, H.; Pu, S.; Zhang, W.; Liao, X.; Shi, B. One- Biogenic Nanoparticle Corona and Its Effects on Reactivity. ACS
step room-temperature synthesis of Au@Pd core−shell nanoparticles Sustainable Chem. Eng. 2015, 3, 1610−1617.
with tunable structure using plant tannin as reductant and stabilizer.
Green Chem. 2011, 13, 950−957.
(117) Wang, B.; Yang, G.; Chen, J.; Fang, G. Green Synthesis and
Characterization of Gold Nanoparticles Using Lignin Nanoparticles.
Nanomaterials 2020, 10, No. 1869.
(118) Shen, Z.; Luo, Y.; Wang, Q.; Wang, X.; Sun, R. High-Value
Utilization of Lignin to Synthesize Ag Nanoparticles with Detection
Capacity For Hg2+. ACS Appl. Mater. Interfaces 2014, 6, 16147−
16155.
(119) Han, G.; Li, X.; Li, J.; Wang, X.; Zhang, Y. S.; Sun, R. Special
Magnetic Catalyst with Lignin-Reduced Au−Pd Nanoalloy. ACS
Omega 2017, 2, 4938−4945.
(120) Rampino, L. D.; Nord, F. F. Preparation of Palladium and
Platinum Synthetic High Polymer Catalysts and the Relationship
between Particle Size and Rate of Hydrogenation. J. Am. Chem. Soc.
1941, 63, 2745−2749.
(121) Haruta, M.; Tetsuhiko, K.; Hiroshi, S.; Nobumasa, Y. Novel
Gold Catalysts for the Oxidation of Carbon Monoxide at a
Temperature far Below 0 °C. Chem. Lett. 1987, 16, 405−408.
(122) Yan, Y.; Du, J. S.; Gilroy, K. D.; Yang, D.; Xia, Y.; Zhang, H.
Intermetallic Nanocrystals: Syntheses and Catalytic Applications. Adv.
Mater. 2017, 29, No. 1605997.
(123) Strasser, P.; Koh, S.; Anniyev, T.; Greeley, J.; More, K.; Yu, C.;
Liu, Z.; Kaya, S.; Nordlund, D.; Ogasawara, H.; Toney, M. F.;
Nilsson, A. Lattice-strain control of the activity in dealloyed core−
shell fuel cell catalysts. Nat. Chem. 2010, 2, 454−460.
(124) Berahim, N.; Basirun, W. J.; Leo, B. F.; Johan, M. R. Synthesis
of Bimetallic Gold-Silver (Au-Ag) Nanoparticles for the Catalytic
Reduction of 4-Nitrophenol to 4-Aminophenol. Catalysts 2018, 8,
No. 412.
(125) Shao, Q.; Wang, P.; Zhu, T.; Huang, X. Low Dimensional
Platinum-Based Bimetallic Nanostructures for Advanced Catalysis.
Acc. Chem. Res. 2019, 52, 3384−3396.
(126) Ye, E.; Regulacio, M. D.; Zhang, S.-Y.; Loh, X. J.; Han, M.-Y.
Anisotropically branched metal nanostructures. Chem. Soc. Rev. 2015,
44, 6001−6017.
(127) Chen, X.; Jiang, C.; Yu, S. Nanostructured materials for
applications in surface-enhanced Raman scattering. CrystEngComm
2014, 16, 9959−9973.
(128) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Raman
spectra of pyridine adsorbed at a silver electrode. Chem. Phys. Lett.
1974, 26, 163−166.
(129) Albrecht, M. G.; Creighton, J. A. Anomalously intense Raman
spectra of pyridine at a silver electrode. J. Am. Chem. Soc. 1977, 99,
5215−5217.
(130) Jeanmaire, D. L.; Van, R. P. Duyne. Surface raman
spectroelectrochemistry: Part I. Heterocyclic, aromatic, and aliphatic
amines adsorbed on the anodized silver electrode. J. Electroanal. Chem.
Interfacial Electrochem. 1977, 84, 1−20.
(131) Le Ru, E. C.; Blackie, E.; Meyer, M.; Etchegoin, P. G. Surface
Enhanced Raman Scattering Enhancement Factors: A Comprehensive
Study. J. Phys. Chem. C 2007, 111, 13794−13803.

7228 https://dx.doi.org/10.1021/acsomega.1c00692
ACS Omega 2021, 6, 7212−7228