Novel enzymatic synthesis of core/shell AgNP/AuNC bimetallic nanostructure and its catalytic applications

Article abstract of DOI:10.1016/j.molliq.2020.112463

Herein, we report the enzyme-mediated synthesis of bimetallic nanostructure consisting of gold nanoclusters shell- silver nanoparticles core (AuNCs-AgNPs) for catalytic reduction applications. For the synthesis of core AgNPs, thiocholine (TCh), an enzymatic product from the acetylcholinesterase (AChE), and acetylthiocholine (ATCh) interaction was used as a stabilizing agent. The AuNCs-AgNPs was prepared using TCh as a reducing as well as stabilizing agent. The synthesized AuNCs-AgNPs were characterized with help of UV–Vis spectroscopy, dynamic light scattering (DLS) analyses and High-Resolution Transmission Electron Microscopy (HR-TEM). The HR-TEM micrographs confirmed that the formation and arrangement of AuNCs on the core AgNPs, resulting in AuNCs-AgNP bimetallic structures. The catalytic reduction activity of thus prepared AgNPs and AuNCs-AgNPs was investigated against methyl orange (MO) and potassium ferricyanide K₃[Fe(CN)₆] in the presence of sodium borohydride (NaBH₄). The rate constants (k), for MO and K₃[Fe(CN)₆] catalytic reduction in the presence of AuNCs -AgNPs were measured to be 2.3 × 10^{? 2} s^{? 1} and 1.219 × 10^{? 1} s^{? 1} respectively.

Full text of DOI:10.1016/j.molliq.2020.112463

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Novel enzymatic synthesis of core/shell AgNP/AuNC bimetallic
nanostructure and its catalytic applications
A. Vinotha Alex, N. Chandrasekaran, Amitava Mukherjee
PII:
S0167-7322(19)36137-9
DOI:
https://doi.org/10.1016/j.molliq.2020.112463
MOLLIQ 112463
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
5 November 2019
29 December 2019
4 January 2020
Please cite this article as: A. Vinotha Alex, N. Chandrasekaran and A. Mukherjee, Novel
enzymatic synthesis of core/shell AgNP/AuNC bimetallic nanostructure and its catalytic
applications,
Journal
of
Molecular
Liquids(2020),
https://doi.org/10.1016/
j.molliq.2020.112463
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Novel enzymatic synthesis of core/shell AgNP/AuNC bimetallic
nanostructure and its catalytic applications
Vinotha Alex A, N. Chandrasekaran, Amitava Mukherjee*
Centre for Nanobiotechnology, Vellore Institute of Technology, Vellore, India.
*Corresponding author
Dr Amitava Mukherjee
Senior Professor & Deputy Director
Centre for Nanobiotechnology
Vellore Institute of Technology, Vellore – 632014, India.
Email: amit.mookerjea@gmail.com, amitav@vit.ac.in
Phone: 91 416 2202620
Fax: 91-416-2243
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Abstract
Herein, we report the enzyme-mediated synthesis of bimetallic nanostructure consisting of gold
nanoclusters shell- silver nanoparticles core (AuNCs -AgNPs) for catalytic reduction
applications. For the synthesis of core AgNPs, thiocholine (TCh), an enzymatic product from the
acetylcholinesterase (AChE), and acetylthiocholine (ATCh) interaction was used as a stabilizing
agent. The AuNCs -AgNPs was prepared using TCh as a reducing as well as stabilizing agent.
The synthesized AuNCs-AgNPs were characterized with help of UV-Vis spectroscopy, dynamic
light scattering (DLS) analyses and High-Resolution Transmission Electron Microscopy (HR-
TEM). The HR-TEM micrographs confirmed that the formation and arrangement of AuNCs on
the core AgNPs, resulting in AuNCs-AgNP bimetallic structures. The catalytic reduction activity
of thus prepared AgNPs and AuNCs-AgNPs was investigated against methyl orange (MO) and
potassium ferricyanide K3[Fe(CN)6] in the presence of sodium borohydride (NaBH4). The rate
constants (k), for MO and K3[Fe(CN)6] catalytic reduction in the presence of AuNCs -AgNPs
were measured to be 2.3 x 10-2 s-1 and 1.219 x 10-1 s-1 respectively.
Keywords: Core AgNPs, AuNCs-AgNPs; Enzymatic synthesis; catalytic reduction; Methyl
orange; Potassium ferricyanide.
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1. Introduction
Owing to unique physicochemical and optical properties along with high surface area to
volume ratio, metal nanoparticles have considerable use in variety of applications namely,
sensing, therapeutics, catalysis, etc., Especially, silver (Ag) and gold (Au) nanoparticles (NPs),
have been widely used in biomedical field because of their biocompatibility and antibiotic
behavior [1]. Unlike other metal NPs, AgNPs and AuNPs involve simple preparation procedure
and are considerably less toxic[2, 3]. On the other hand, Au and AgNPs can also be used as an
effective catalyst in many reactions including reduction /degradation of chemical pollutants and
oxidation of CO, glucose and alcohol due to their improved surface area, the occurrence of more
reactive sites and enhanced reaction selectivity[4]. In recent past, the synergistic effects of
individual metal nanoparticles in the form of bimetallic nanostructures have been explored, to
enhance the overall properties of bimetallic nanoparticles. Considering the high oxidation
potential of Ag, it is necessary to form Au shell around AgNPs, which has relatively lower
oxidation potential. This in turn increases the stability of core AgNPs and offers the combined
effect of both Ag and AuNPs[5].
Further, several parameters like shape, size, and composition of core and shell determine
the overall catalytic performance of the nanomaterials. For instance, spherical nanoparticles
possess better catalytic reduction potency than that of nanorods. Because, the absence of sharp
edges and corners in the spherical particles, the interacting reactant molecules can be completely
exposed to the reaction sites. In case of size, the large surface area of ultrasmall nanoparticles
promotes catalytic activity. Hence, smaller nanoclusters are preferred for catalytic
applications[6].
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Among various methods to synthesize nanostructures, the chemical reduction approach is
highly favored, because of the simple and cost-effective steps with increased output. Recent
concerns on the environmental safety of nanoparticle, an eco-friendly and swift-alternative
synthesis process are required. Due to its surplus availability, cost-effectiveness, non-toxic, eco-
friendly nature and many advantages over chemical and physical methods, researchers have
focused on bio-based approaches[7]. Among the bio-based reactants, enzymes such as α-
amylase, extracellular laccase, keratinase, nitrate reductase, cellulose, fungal xylanases,
acetylcholinesterase and horse radish peroxidase were used for the synthesis of monometallic
nanoparticles for various applications[8]. This offers a green synthetic process to prepare the
nanostructures. A detailed analysis of prior art reveals that the enzymatic synthesis of the
bimetallic core/shell nanostructures is yet to be explored.
The discharge of organic dyes and toxic compounds into water resources plays a major
role in water contamination. In the current study, methyl orange (MO) and potassium
ferricyanide (K3[Fe(CN)6]) were selected as model pollutants to investigate the catalytic property
of prepared bimetallic nanocatalyst. Generally, K3[Fe(CN)6] is a well-known toxicant and
carcinogenic contaminant, therefore it is necessary to reduce the toxicity by converting the
[Fe(CN)6]3- to [Fe(CN)6]. Moreover, various industries generate abundant organic dyes and
utilize them for coloring their products. Especially, the organic dyes having N=N groups like,
MO are highly carcinogenic and toxic, which causes severe threat to living beings. Considering
the demerits associated with conventional methods such as time-consuming, difficult handling,
etc., the harmless, effective and simple strategies are developed to degrade the pollutants in the
water system. Hence, the catalytic reduction is the simple and effective method was used to
eliminate the contaminants from the wastewater system[9].
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In recent times, Veerakumar et al.[10] have investigated catalytic reduction against
K3[Fe(CN)6] using porous Carbon-supported Ruthenium in the presence of NaBH4 as a reducing
agent. Similarly, Miao et al.[11] have studied the catalytic reduction activity with gold nanostars
with magnetic cores, and Ahsan et al.[12] studied the reductive degradation of C@Co against
MO. However, these processes involved harmful reagents and tedious preparation steps for the
nanocatalyst. Thus, a less toxic and simpler procedure to synthesize the nanocatalyst is essential.
So far, no prior study reported catalytic reduction of contaminants using enzymatically
synthesized green nanocatalysts having core/shell AgNP/AuNCs bimetallic structures.
The present research work proposes a novel green synthesis approach for bimetallic
nanostructures (AuNCs-AgNPs) using an enzymatic reaction mediated product, namely,
thiocholine, a non-toxic reducing and stabilizing agent. In this, TCh capped AgNPs was used as
the core for preparing AuNCs-AgNPs. The prepared nanomaterials were thoroughly
characterized by UV-Visible spectroscopy, High Resolution–Transmission Electron Microscopy
(HR-TEM), Energy Dispersive X-ray (EDX), Dynamic Light Scattering (DLS), and Zeta
potential analyses. The obtained core AgNPs and bimetallic nanostructure AuNCs-AgNPs were
investigated for catalytic reduction activity towards MO, and K3[Fe(CN)6]. The use of green
synthesized bimetallic nanostructures resulted significantly enhanced catalytic activity with
improved rate constants (k) and reduction percentage in case of both the pollutants.
2. Materials and Methods
2.1 Materials
Silver nitrate (AgNO3), hydrogen tetrachloroaurate(III) trihydrate (HAuCl4.3H2O) and
sodium borohydride (NaBH4) were procured from SRL Pvt. Ltd (India). Acetylcholinesterase
(AChE, from Electrophorus electricus), Methyl orange and Potassium ferricyanide (III) were
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acquired from Sigma Aldrich, India. The above chemicals were of analytical grade and used
without further purification. Acetylthiocholine iodide (ATChI) was purchased from HiMedia
Laboratories Pvt. Ltd., India and the iodide ions were removed using AgNO3. The excess Ag+
ions were eliminated from the medium by treating with NaCl[13]. Using phosphate-buffered
saline (pH 7 – 7.4) required AChE and ATCh stock solutions were prepared and stored at 4°C
until use. All the glassware were cleaned with Aqua Regia solution (HCl: HNO3 =3:1) followed
by rinsing with Milli-Q water (18.2 MΩ.cm resistivity) from Cascada Bio water (Pall
Corporation, USA) then dried under hot air oven.
2.2 Synthesis of core and bimetallic NPs
2.2.1. Synthesis of core Ag nanoparticles
The synthesis of TCh stabilized AgNPs was carried out according to the method
previously described by our group with few modifications [14]. Briefly, 40 µL of AChE (ranging
from 9 to 15 mU) was added to 80 µL of ATCh (1mM) and incubated at room temperature (RT)
for 10 minutes. To this mixture, 400 µL of AgNO3 (0.1 mM) and 80 µL of NaBH4 (1mM) were
added subsequently and mixed thoroughly. Yellow coloration was resulted proving the formation
of AgNPs, thus the suspension was stored under ambient conditions and used as a core for
preparing AuNCs -AgNPs.
2.2.2. Bimetallic AuNCs-AgNPs synthesis
To synthesis bimetallic AuNCs-AgNPs, 300 µL of HAuCl4 (0.161 mM) was added to
500 µL of core AgNPs followed by the subsequent addition of 300 µL of AChE (821 mU) and
ATCh (0.003 mM) and incubated at 300 K for 10 min. Production of AuNCs-AgNPs was
verified using spectroscopic and microscopic characterization.
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2.3 Catalytic reduction activity of AgNPs and AuNCs-AgNPs
Three sets of catalytic reduction experiments were conducted for AgNPs, AuNCs-
AgNPs, and NaBH4 against MO and K3[Fe(CN)6], respectively. Among the sets, core AgNPs
was represented as test 1, as-synthesized AuNCs-AgNPs was used and represented as test 2, and
the NaBH4 was considered as control. Herein, AgNPs and AuNCs-AgNPs were used as a
catalyst and the NaBH4 as a reducing agent. In test 1, 2 mL of MO (0.166 mM) or 2 mL of
K3[Fe(CN)6] (5 mM), and 1 mL of NaBH4 (25 mM ) were added and mixed, to which 50 µL of
AgNPs catalyst was added. Whereas, in test 2, 50 µL of AuNCs-AgNPs was introduced instead
of core AgNPs. In the control set, neither core AgNPs nor AuNCs-AgNPs was added. Finally,
absorbance was measured against time at a fixed wavelength, respectively for MO and
K3[Fe(CN)6] using the kinetic mode in UV-2600 UV–visible spectrophotometer.
2.4 Response Surface Methodology optimization
To troubleshoot the week points and process complications in bimetallic nanostructure
synthesis, Box–Behnken experimental design (BBD) under RSM was employed, wherein the
specific and combinational interaction of experimental variables on response was implemented
to obtain optimized conditions. To assess the impact of interaction parameters on bimetallic
nanostructure synthesis, three independent factors at two levels each, namely HAuCl4 (mM),
ATCh (mM) and AChE (mU) were chosen (Table 1). The mean experimental data from triplicate
experiments were fed and analyzed using the Design-Expert, Trial version 7.1.5 (Stat-Ease, MN,
USA).
2.5 Instrumentation
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The morphology, elemental composition and lattice parameters of synthesized AgNPs
and AuNCs-AgNPs were determined using high-resolution transmission electron microscope
(HR-TEM) (JEOL JEM 2100, Japan) at an operating voltage of 200 kV. The samples were drop
coated onto the carbon-coated copper grids and dried at 45 ºC before the TEM analysis. The
mean hydrodynamic size and zeta potentials of core AgNPs during pre- and post-AuNCs
formation were measured adopting 90 Plus Particle Analyzer (Brookhaven Instruments
Corporation, USA). Using UV-2600 UV–visible spectrophotometer (Shimadzu, Tokyo, Japan)
the absorption spectra of AgNPs of pre- and post-AuNCs formation was recorded in the range
between 200–800 nm.
3. Results and Discussion
3.1 Effect of AChE concentration on core AgNPs preparation
It is well known that the enzymatic reaction between ATCh (substrate) and AChE
(enzyme) generates the product TCh. The presence of the thiol group in TCh can prevent the
nanoparticles from aggregation [14]. Thus, the product TCh was used as a stabilizing agent for
the preparation of core AgNPs while NaBH4 as a reducing agent. The stability of core AgNPs
was monitored with an increase in SPR intensity at 417 nm (Fig. 1). To study optimal production
of TCh related with strong stabilization of AgNPs, the AChE concentration was varied from 6
mU mL-1 to 15 mU mL-1 with constant AgNO3 (0.1 mM), ATCh (1 mM) and NaBH4 (1 mM)
concentrations. The broadening in the SPR peak and the formation of the secondary peak at
longer wavelength was observed in the AChE concentrations ranging from 6 mU mL-1 to 13 mU
mL-1, demonstrating AgNPs destabilization. As represented in Figure 1, an increment in peak
intensity was observed at 14 mU mL-1 concentration of AChE, suggesting that the core AgNPs
were well stabilized with TCh. The absorbance started decreasing at 15 mU mL-1 AChE
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concentration. Based on these results, AChE (14 mU mL-1), AgNO3 (0.1 mM), ATCh (1 mM)
and NaBH4 (1 mM) concentrations were selected for core AgNPs synthesis which resulted in the
highly stable AgNPs with a sharp SPR band at 417 nm (Fig. 1).
Fig. 1. Effect of AChE concentration on AgNPs formation
3.2 BBD optimization and statistical analysis for the synthesis of bimetallic AuNCs-AgNPs
For the formation of AuNCs shell over the core AgNPs, the reaction concentrations of
HAuCl4, AChE and ATCh were optimized using RSM technique. Herein, box behnken design
was chosen because it requires less number of runs (17 runs with five center points) compared to
composite central and full factorial designs. Three factors namely, the concentration of HAuCl4
(A), AChE (B) and ATCh (C) were selected as independent variables at two levels (low [-1] and
high [+1]) for optimization process (Table 1). All the 17 recommended experimental runs were
performed in triplicate as shown in Table 2, while the resulting SPR peak shift confirmed the
formation of bimetallic nanostructure and used as a response for the mathematical modeling. The
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quadratic polynomial model was chosen for data-fitting and statistical analysis. The obtained F-
value 197.19, P-value <0.0001 from the ANOVA indicates that the analyzed model is highly
significant. The fit statistics (Table 3) explains that there is only <0.2 difference between
predicted- and an adjusted-R² value indicating a reasonable agreement [15, 16]. The obtained F-
value (104.56) discloses that the Lack of Fit is significant.
Factors
Name
HAuCl4
AChE
ATCh
Units
Minimum
Maximum
0.5
mM
mU mL-1 100
0.1
A
B
C
1000
mM 0.001
0.1
Table 1. Three Independent variables and two levels used in BBD design
AChE
Standard
order
1
Run HAuCl4 (mM)
ATCh (mM) Actual value
Predicted value
(mU mL-1)
12
8
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
100
100
1000
1000
550
550
550
550
100
1000
100
1000
550
550
550
550
550
0.0505
0.0505
0.0505
0.0505
0.001
0.001
0.1
432.5
542.5
427
540.5
427.5
524.5
419.5
548
429.25
539.75
429.75
543.75
430.13
526.63
417.38
545.38
524.13
517.13
517.88
529.38
507
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
9
3
16
15
4
2
0.1
14
13
1
7
5
0.001
0.001
0.1
523.5
522.5
512.5
530
0.1
0.0505
0.0505
0.0505
0.0505
0.0505
506.5
506.5
507
508
507
6
507
507
507
507
10
11
17
Table 2. The experimental runs for BBD design along with obtained response values
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As observed in Table 3, the value of adequate precision (signal to noise ratio) was found
to be 40.456, indicating that the analysis was desirable. The positive coefficients of HAuCl4,
HAuCl4*AChE, HAuCl4*ATCh, AChE*ATCh, AChE2 and ATCh2 in equation 1 denotes the
2
combined effect on SPR peak shift, while negative coefficients of AChE, ATCh, and HAuCl4
signifies the opposite effect on SPR peak. The second-order equation for the response peak shift
is specified below:
+388.22198 + 712.29482 HAuCl4 - 0.067600 AChE -
515.75094 ATCh + 0.009722 HAuCl4 * AChE + 795.45455
HAuCl4 * ATCh + 0.207632 AChE * ATCh -795.31250
HAuCl4² + 0.000052 AChE² + 1913.07009 ATCh²
Wavelength =
……….. (eq. 1)
Quadratic
4.13
Source
St.dev
R2
0.9961
0.9910
0.9379
40.456
197.19
< 0.0001
104.56
0.003
Adjusted R2
Predicted R2
Adequate precision
F-value
P-value
Lack of fit F-value
Lack of fit P-value
Table 3. The obtained statistical values of the model through ANOVA
The interaction between all the factors on the response (peak shift) was examined by
keeping one factor at fixed while varying the other two factors (Fig. 2). It is further observed that
the SPR peak shift is directly proportional to the HAuCl4 concentration due to the increase in the
thickness of Au shell on Ag core surface (Fig. 2 and Fig. S1, Supplementary Information). The
optimized parameters for AuNCs-AgNPs synthesis were identified by the numerical desirability
function. Accordingly, the optimized conditions with the highest desirable and lowest AChE and
ATCh concentrations were selected for AuNCs-AgNPs fabrication. The obtained concentration
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for each factor namely, HAuCl4, AChE, and ATCh was 0.161 mM, 821 mU, and 0.003 mM,
respectively. Using these concentrations the fabrication of AuNCs-AgNPs was further carried
out and confirmed by various characterization techniques.
Fig. 2. The 3D plots for the interaction between factors
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3.3 Mechanism of formation of AuNCs on core AgNPs
In the present study, the bimetallic nanostructure was successfully prepared, wherein the
core AgNPs was synthesized from Ag precursor salt AgNO3 using NaBH4 (1 mM) at minimal
concentration (1 mM) as a reducing agent. Herein, thiocholine enzymatically generated from the
reaction between AChE (14 mU mL-1) and ATCh (1 mM), acted as the non-toxic stabilizing
agent [14]. For the development of bimetallic nanostructures, an aqueous suspension of precursor
i.e., HAuCl4 (0.161 mM) was transferred to the seed, i.e. core AgNPs. To this, AChE (821 mU)
and ATCh (0.003 mM) were subsequently introduced, leading to the production of TCh (acts as
both reductant and stabilizer). In the presence of TCh, Au3+ reduced to Au0 and the
heterogeneous nucleation of AuNCs occurred on the surface of core AgNPs resulting in the
formation of the bimetallic nanostructure. The strong affinity of Au atoms towards the terminal
+
sulfhydryl group of TCh (HS–(CH2)2–N(CH3)3 ) facilitates the formation of AuNCs on Ag core.
The presence of positively charged TCh around AuNCs prevents further ripening of the NCs to
AuNPs[17, 18] [17, 19-24] (Fig 3).
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Fig. 3. Schematic representation for the mechanism of core AgNPs and AuNCs-AgNPs
formation
3.4 Characterization of core AgNPs and AuNCs-AgNPs:
Fig. 4a shows the UV visible absorption spectra of TCh stabilized core AgNPs and
AuNCs-AgNPs. The formation of the core AgNPs is confirmed by a sharp SPR band at 417 nm,
whereas AuNCs-AgNPs showed characteristic absorbance maxima at 455 nm. During the
bimetallic nanostructure formation, the decline in the absorbance at 417 nm corresponding to the
core AgNPs and concomitant redshift of SPR band to 455 nm (Au SPR region) was noted
confirming the AuNCs formation on AgNPs (Fig. 4a). However, a unique and prominent Au
peak between 500-530 nm region was not observed. This could be due to the low absorption
capacity of AuNCs with a size <3nm. Rather a broad SPR peak between 300 -700 nm with an
absorbance maximum at 455 nm appeared denoting AuNCs-AgNPs formation (Fig. 4a) [25-31].
To prove that the AuNCs - AgNPs formation was possible only in the presence of TCh, various
combinations of HAuCl4, AgNPs, AChE, and ATCh were prepared and monitored under UV
visible spectra (Fig. 4b). Except the combinations containing AChE and ATCh, no other
combination could produce TCh and hence the peak shift was not noted. Similarly, there was no
SPR band shift during the individual interactions of ATCh, AChE, and HAuCl4, respectively
with AgNPs denoting the complete absence of HAuCl4 reduction. On the other hand, the
combinations of HAuCl4 +AChE + AgNPs and HAuCl4 +ATCh + AgNPs showed negligible
redshift due to changes in the dielectric constant of the stabilizing layer on the AgNPs leading to
slight aggregation.
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Fig. 4. UV-Vis absorption spectra for synthesized nanomaterials. (a) core AgNPs and bimetallic
AuNCs-AgNPs, (b) Effect of individual and combinations of all factors on core AgNPs SPR
band
The morphology, elemental composition and crystalline structure of the synthesized
AgNPs and AuNCs-AgNPs were determined using HR-TEM equipped with EDX. The HR-TEM
micrographs and the size distribution histogram of core AgNPs and AuNCs-AgNPs are presented
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in figures 5 (a, b, d, e, and g). Under TEM, core AgNPs was found to be spherical with well-
dispersed particles ranging from 2-20 nm (Fig. 5a).
Fig. 5. HR-TEM and EDX analysis of mono and bimetallic nanoparticles. (a-c) HR-TEM, size
histogram and EDX analysis images of core AgNPs, (d-f and h) HR-TEM, SAED and EDX
analysis images of bimetallic AuNCs-AgNPs, (g) size histogram of AuNCs
The particle size histogram represented from 100 discrete, randomly chosen nanoparticles
signifies the average size of synthesized AgNPs is 11.27±3.63 nm (Fig. 5b). While the AuNCs-
AgNPs (Fig. 5d) showed spherical shape, polydisperse particles with the size range of 50-60 nm.
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Here, we observed an increase in the core AgNPs size with AuNCs (1.5±0.62 nm) (Fig. 5g)
capping on their surface. The increase in the size of the core AgNPs might be attributed to the
presence of large amounts of reducing moieties leading to AgNPs aggregation[32, 33].
Additionally, the presence of larger sized core AgNPs in the images might also be due to the
drying process involved in sample preparation which might have promoted a nonuniformed
particle deposition or aggregation[34, 35]. Interestingly, the formation of individual AuNCs
(Fig.5e) with a size of less than 2 nm indicates the presence of a negligible quantity of bimetallic
alloy in the solution.
The lattice parameters of AuNCs-AgNPs were determined using HRTEM- SAED
pattern. The observed d-spacing values from the bimetallic nanostructure depicted highly
structured orientation and crystalline nature of Ag and Au atoms in the sample (Fig. 5f). The
calculated distance between the fringes were found to be 0.12nm, 0.111nm, 0.26nm
corresponding to (311), (222), (111) lattice planes for the fcc Ag nanocrystal whereas the d
spacing 0.23nm, 0.20nm, 0.141nm and 0.115nm were analogous to reflections at (111), (200),
(220) and (311) lattice planes of Au crystal lattice, respectively. The d spacing values closely
corroborated with the previously reported fringe distances [36-39]. The elemental composition of
the synthesized nanoparticles was examined with help of EDX analyses. The resulting EDX
spectra of core AgNPs (Fig. 5c) and AuNCs-AgNPs (Fig. 5f) exhibited a sharp peak at 3KeV,
and 2.1 KeV and 3KeV confirming the formation of AgNPs [40] and presence of Au and Ag,
respectively. Additional EDX peaks corresponding to C, O, Na were also detected, and this could
be attributed to the elements present in AChE, ATCh and other derivatives.
The mean particle size, PDI and zeta potential of thiocholine capped core AgNPs and
AuNCs -AgNPs were found to be 101 nm, 0.334 and 71mV, and 179 nm, 0.259 and 49mV,
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respectively. Herein, the observed mean hydrodynamic diameter values were found to be higher
than those in the TEM images. This could be due to the presence of the solvation layer around
the nanoparticles[41, 42]. The presence of cationic groups on thiocholine (quaternary ammonium-
terminated thiol) on core AgNPs and AuNCs-AgNPs provided stability to the particles via
electrostatic repulsion[43]. This was also confirmed from highly positive zeta potential values
(greater than +25 mV) in both cases indicating a high degree of stability in aqueous media [44].
High degree of colloidal stability would facilitate their further application for catalytic reduction
of environmental contaminants in the aqueous medium.
3.5 Evaluation of catalytic reduction activity of synthesized nanostructures
3.5.1 Catalytic reduction of MO
The catalytic reduction of MO was monitored using the time-dependent decrease in
absorbance at 470ꢀnm. Under the optimum conditions, the catalytic reduction of MO (0.166 mM)
was carried out with thiocholine capped nanostructures, i.e., core AgNPs and AuNCs -AgNPs
using NaBH4 as reductant. In this method, the AuNCs-AgNPs catalyzed the reduction of MO by
NaBH4 efficiently i.e., the initial absorbance declined to zero in a very short time interval (160s),
whereas the AgNPs with NaBH4 achieved complete reduction of MO dye in nearly 30 min, while
the NaBH4 alone could not achieve complete reduction even after 1 day. At 160s, the reducing
agent NaBH4 (25ꢀmM) alone and with the catalyst AgNPs could reduce 33% and 55.25% MO
(0.166 mM) in 160s signifying that the reduction is kinetically quite slower than that of
bimetallic nanostructure (Fig. 6a). During the reduction reactions, the peak centered at 470nm
was decreased, and a new peak at 290nm was produced due to the hydrazine derivatives which
confirms the reduction of MO (Fig. S2)[45, 46]. Further, the MO reduction percent was
calculated using the following formula:
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Reduction (%) = (Ao – At/Ao) x100
…………(eq. 2)
where Ao is the initial absorbance of MO at 470nm and At is the absorbance at time t in
the catalytic reaction. Figure. 6b reveals reaction kinetics plot of -ln(Ct/Co) vs time for the
catalytic reduction of MO by NaBH4 alone and in the presence of bimetallic AuNCs-AgNPs and
core AgNPs. The pseudo first-order kinetics derived from Langmuir–Hinshelwood model was
used to analyze the catalytic reduction ability of nanocatalysts by calculating rate constants k
(1/s) according to the following first-order rate equation:
-ln (Ct/Co) = kt
…………(eq. 3)
where Ct and Co are the concentration at time t and initial concentration of MO,
respectively. The first-order rate constants (k) were obtained by calculating the slopes of each
plot from the linear regression graph. The rate constant k for MO reduction were found to be
kAuNCs -AgNPs = 2.3 x 10-2 s-1, kAgNPs= 2 x 10-3 s-1, kNaBH4 = 7 x 10-6 s-1 and the k value signifies the
catalytic reduction activity of the nanocatalyst. Herein, the obtained rate constant values
represent that the MO reduction catalyzed by bimetallic nanoparticles (AuNCs-AgNPs) was
faster than monometallic (AgNPs), which is faster than NaBH4 alone. This indicates there is
increased relaying of electrons from BH−4 ions to reactant dye molecules in the presence of
AuNCs-AgNPs. Moreover, the reductive catalytic degradation of AuNCs-AgNPs towards MO
was much found superior to previously reported nanomaterials (Table 4).
Rate constant value
Nanocatalyst
Condition
Time
References
(k)
Ag@Fe core/Shell
NPs
NaBH4
14 min
4 min
1.6×10−3 s−1
1.08954 min−1
2.03×10-2 min-1
[47]
Nanosheet CuSbOS
bimetal oxysulfide
NaBH4
halogen lamp
illumination
[48]
[49]
C60-AuNPs-TiO2
160 min
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PtNPs
PdNPs
AuNPs
AgNPs
1.25x10-2 min-1
6.7x10-3 min-1
9.0x10-5 min-1
7.0x10-5 min-1
formic acid
10 min
[50]
Current
study
Current
study
TCh stabilized AgNPs
AuNCs -AgNPs
NaBH4
NaBH4
160 s
160 s
2x10-3 s-1
2.3x10-2 s-1
Table 4. Comparison table of core AgNPs and AuNCs-AgNPs with other nanocatalysts utilized
for the reduction of methyl orange (MO)
3.5.2 Catalytic reduction of K3[Fe(CN)6]
The catalytic reduction of K3[Fe(CN)6] has been evaluated using AuNCs-AgNPs and
AgNPs as a catalyst and NaBH4 as a reducing agent. In this experiment, the nanostructured
particles assisted the reduction of K3[Fe(CN)6] by NaBH4 was estimated by observing the
concentration of [Fe(CN)6]3− using the time-dependent decrement of absorbance value at
wavelength 420nm. Generally, the catalyst can significantly increase the reducing efficacy of a
reducing agent[10]. When the AuNCs-AgNPs was added in the reducing reaction, K3[Fe(CN)6]
absorbance value (3.5) at 420 nm was rapidly decreased to zero within 40 s due to the formation
of [Fe(CN)6]4−. The rapid decrease in absorbance validates the complete reduction of Fe(III) ions
to Fe(II), resulting in a visible color change from lime yellow to colorless confirms it further.
Similarly, the reducing action of the core AgNPs was evaluated in the presence of NaBH4, which
resulted in a decrease in the absorbance value. However, the catalytic activity of monometallic
AgNPs was less pronounced compared to bimetallic AuNCs-AgNPs particles. It is important to
mention that the NaBH4 alone showed no significant decrease in absorbance. The reduction rate
(%) of NaBH4 in the presence of catalysts was calculated as per (eq. 2). At 40s, 99.74% of
K3[Fe(CN)6] reduction was observed with AuNCs-AgNPs catalyst, whereas AgNPs catalyst
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showed 84.48% reduction. In contrast, NaBH4 without catalyst offered only 6.6% reduction (Fig.
6c). The -ln(Ct/Co) vs time plot reveals that the reduction follows pseudo first-order kinetics. The
obtained k values for K3[Fe(CN)6] reduction were kAuNCs -AgNPs = 1.219 x 10-1 s-1, kAgNPs = 3.2 x
10-2 s-1, kNaBH4 = 2.21 x 10-4 s-1, which denotes that the AuNCs-AgNPs exhibited faster catalytic
activity than that of core AgNPs and NaBH4 (Fig. 6d).
Fig. 6. Mono and bimetallic particles mediated reduction of MO and K3[Fe(CN)6]. (a,b) the plot
of reduction percentage (%) and –ln(Ct/Co) Vs reaction time for MO, (c,d) the plot of reduction
percentage (%) and –ln(Ct/Co) Vs reaction time for K3[Fe(CN)6]
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Nanocatalyst
Condition
Time
Rate constant value (k)
References
[51]
carboxymethylated gum
kondagogu capped
AgNPs
NaBH4
NaBH4
NaBH4
15 min
0.415 min-1
Ag Cu NPs
210 s
250 s
75 s
18.2×10-3 s -1
0.025 s-1
[52]
Au/SiO2 capsules
Au/ TiO2 capsules
[53]
0.063 s-1
NaBH4
NaBH4
NaBH4
NaBH4
45 s
0.1011 s−1
[10]
Ru@PDC
Graphene-PDA-Pd
TCh stabilized AgNPs
AuNCs-AgNPs
1000s
40 s
2.330×10-2 s-1
3.2 x 10-2 s-1
1.219 x 10-1 s-1
[54]
Current study
Current study
40 s
Table 5. Comparison table of core AgNPs and AuNCs-AgNPs with other nanocatalysts utilized
for the reduction of K3[Fe(CN)6]
3.5.3 Mechanism of catalytic reduction of MO and K3Fe(CN)6
Towards the catalytic reduction experiment, NaBH4 was selected as the reducing agent
because of the high electron injection capability of BH-4 ions[45]. Certainly, the electrons of
BH-4 ions required a catalyst surface and assistance for the rapid electron transfer from BH-4
ions to the reactant molecules[55]. Because of the high surface area to volume ratio and rich
active centers of nanoparticles showed significant catalytic properties by means of migration of
reactant molecules to the nanocatalyst surface followed by electron relay effect by the
nanocatalyst from borohydride ions (an electron donor) to reactant dye molecules (an electron
acceptor) [56-57]. Therefore in the present study, mono and bimetallic nanostructures were
prepared and tested for catalytic reduction against MO and K3[Fe(CN)6], wherein, the individual
AuNCs formation and distribution on the core AgNPs surface showed a faster catalytic reduction
of both MO and K3Fe(CN)6. This could be due to their smaller size, and the larger numbers of
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low coordination sites (edges and sharp corners) of AuNCs. This would mean an increase in the
surface area to volume ratio and the number of contact sites, which facilitates the interaction of
reactant molecules. The AuNCs-AgNPs catalyst successfully transfers the electrons from BH-4
ions to –N=N– linkage of MO and thus promotes rapid MO reduction leading to the
discoloration. In a similar fashion, AuNCs-AgNPs catalyzed the reduction of K3 [Fe (CN)6] to
K4[Fe(CN)6] by means of NaBH4 effectively in the reaction medium as represented in schematic
diagram (Fig.7) [12, 20, 58-61].
Fig. 7. Schematic representation of core AgNPs and AuNCs-AgNPs mediated MO and
K3[Fe(CN)6] catalytic reduction
4. Conclusion
In conclusion, we successfully synthesized the bimetallic nanostructure, wherein the core
AgNPs was synthesized from AgNO3 with a low concentration of NaBH4 and TCh, a stabilizing
agent. To the core AgNPs, HAuCl4, AChE, and ATCh were added, and enzymatically produced
TCh reduced Au3+ to Au0 which further heterogeneously nucleated on the surface of core AgNPs
to form AuNCs-AgNPs. Thereupon, catalytic reduction activity was performed for monometallic
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AgNPs and AuNCs-AgNPs nanoparticles with NaBH4 and NaBH4 alone towards MO and
[K3Fe(CN)6]. The results revealed that AuNCs-AgNPs exhibited considerably faster catalytic
activity for the reduction of MO and K3[Fe(CN)6] with NaBH4 as compared to the core AgNPs
and NaBH4. Based on the present study, it is concluded that the enzymatically produced TCh
mediated formation of bimetallic nanoparticles can be a novel, non-toxic, efficient nanocatalyst
for catalytic reductive applications.
Conflict of interest
There are no conflicts to declare.
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