Environmentally sustainable route to SiO2@Au-Ag nanocomposites for biomedical and catalytic applications

Article abstract of DOI:10.1039/c8ra04502j

A facile, sustainable, operationally simple and mild method for the synthesis of SiO₂@Au-Ag nanocomposites (NCs) using Nephrolepis cordifolia tuber extract is described and its catalytic, antibacterial and cytotoxic properties were investigated. The fabricated SiO₂@Au-Ag NCs were well characterized by UV-visible spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FT-IR) spectroscopy, powder X-ray diffraction (XRD), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) to determine the optical activity, size and morphology, elemental composition, functional groups present, crystallinity, thermal stability and chemical state respectively. The obtained SiO₂@Au-Ag NCs exhibited spherical shape SiO₂ decorated with Au and Ag nanoparticles. The diameter of the SiO₂ nanoparticles ranges from 200-246 with average 3 nm diameter of Au and Ag NPs. Synthetic utility of this protocol has been demonstrated by exploring its effective catalytic activities for the solvent-free amidation of carboxylic acid with a primary amine with excellent yields. Moreover, the synthesized nanocomposite exhibited as noticeable antibacterial effect against Gram negative and Gram positive bacteria and better bio-compatibility against human keratinocytes. Thus, additive free SiO₂@Au-Ag NCs display the potential for catalysis and biomedical applications.

Full text of DOI:10.1039/c8ra04502j

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Environmentally sustainable route to SiO2@Au–Ag
nanocomposites for biomedical and catalytic
Cite this: RSC Adv., 2018, 8, 31311
applications†
ab
Kanti Sapkota,ab Prerna Chaudharyab and Sung Soo Han
*
A facile, sustainable, operationally simple and mild method for the synthesis of SiO2@Au–Ag nanocomposites
(NCs) using Nephrolepis cordifolia tuber extract is described and its catalytic, antibacterial and cytotoxic
properties were investigated. The fabricated SiO2@Au–Ag NCs were well characterized by UV-visible
spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), Fourier transform
infrared (FT-IR) spectroscopy, powder X-ray diffraction (XRD), thermogravimetric analysis (TGA) and X-ray
photoelectron spectroscopy (XPS) to determine the optical activity, size and morphology, elemental
composition, functional groups present, crystallinity, thermal stability and chemical state respectively. The
obtained SiO2@Au–Ag NCs exhibited spherical shape SiO2 decorated with Au and Ag nanoparticles. The
diameter of the SiO2 nanoparticles ranges from 200–246 with average 3 nm diameter of Au and Ag NPs.
Synthetic utility of this protocol has been demonstrated by exploring its effective catalytic activities for the
solvent-free amidation of carboxylic acid with a primary amine with excellent yields. Moreover, the
synthesized nanocomposite exhibited as noticeable antibacterial effect against Gram negative and Gram
positive bacteria and better bio-compatibility against human keratinocytes. Thus, additive free SiO2@Au–Ag
NCs display the potential for catalysis and biomedical applications.
Received 26th May 2018
Accepted 30th August 2018
DOI: 10.1039/c8ra04502j
rsc.li/rsc-advances
system, easy surface modication, biocompatibility and chemical
stability.13–17 In addition, nanostructured Au and Ag materials are
Introduction
highly applicable in interfacial science and (bio)analytical chem-
istry because their optical properties are appropriate for applica-
tions of surface-sensitive techniques like surface enhanced Raman
(SER) spectroscopy.18–25 Moreover, over the past few years, silver
and gold nanoparticles have been increasingly used due to their
powerful optical, electrical and microbial properties in various
areas of research as biological sensors, catalysis, drug delivery
vehicles, and anti-microbial agents as well as having low cytotoxic
effects on mammalian cells.26–31
Recent advances in the synthesis of novel nanostructures with
metal nanoparticles (Ag, Au, Fe, Cu, Ru, Pd, etc.) deposited on
metallic oxide surfaces have gained substantial interest in nano-
technology and material science because of their signicant
applications in diverse elds such as biomedical, catalysis, bio-
sensing, information storage, solar cells, optical and many
more.1–7 Particularly important are silica-metal nanocomposites
because of their broad applications in catalysis and biomedical
elds.8,9 For example, silica based nanocomposites like SiO2–Au,
SiO2–Ag and SiO2–Au/Ag display noteworthy advantages in various
elds of nanomedicine such as bioimaging, photothermal therapy,
drug delivery, biosensing and catalysis due to their highly attrac-
tive properties such as tunable porous structure, remarkable
plasmonic properties, unique composition, biocompatibility and
physiochemical properties.10–12 Therefore, the silica-metal nano-
structure is a widely used material due to its unique properties like
optical transparency, porous structure, ability to functionalize pore
Due to the wide applications and usefulness in various elds,
silica-metal nanocomposites have been synthesized by various
research groups. For example, b-cyclodextrin (b-CD) coated
SiO2@Au@Ag nanoparticles has been synthesized in a single-
step reaction using b-CD as both the reducing and stabilizing
agents for the surface-enhanced Raman scattering (SERS)
detection of polychlorinated biphenyls (PCBs).32 Recently, silica
coated Au/Ag nanomaterials have been reported for photo-
thermal and photodynamic cancer therapy.33 Moreover, Au, Ag,
Pd/SiO2 mesoporous nanocomposites has been synthesized by
sonochemical approach.34 Monodisperse a-Fe2O3@SiO2@Au
core/shell nanocomposite spheres has been synthesized by sol–
aSchool of Chemical Engineering, Yeungnam University, 280 Daehak-Ro, Gyeongsan,
Gyeongbuk 38541, Republic of Korea
bDepartment of Nano, Medical & Polymer Materials, College of Engineering, Yeungnam
University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk 38541, Republic of Korea. E-mail:
sshan@yu.ac.kr; Fax: +82-53-810-4686; Tel: +82-53-810-2773
gel process based on the modied Stober method.35 Gold/silver
¨
bi-metal nanodots on silica spheres prepared by N-[3-(trime-
thoxysilyl)propyl]diethylenetriamine and modied strobe
method respectively and exhibited antimicrobial properties.36
† Electronic supplementary information (ESI) available: 1H NMR and 13C NMR of
the synthesized compounds. See DOI: 10.1039/c8ra04502j
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In addition, highly sensitive surface-enhanced Raman scat- and ammonium hydroxide (NH4OH) were purchased from
tering (SERS) probes based on SiO2@Au@Ag nanoparticles Sigma-Aldrich. The fresh tubers of Nephrolepis cordifolia (L.) C.
(NPs) and bifunctional Ag@SiO2/Au NPs for probing sequential Presl was obtained from Kaski district of Nepal. Human kerati-
catalytic reactions have been recently reported.37,38
nocyte cells [CCD 1102 KERTr (ATCC/CRL-2310)] were obtained
Though there are several reports for the synthesis of SiO2– from the American Type Culture Collection (ATCC). Dulbecco's
metal nanomaterials, most of the reported methods utilize medium, Phosphate Buffered Saline (PBS), fetal bovine serum
chemical approaches for the synthesis of such nanoparticles. (FBS), dimethyl sulphoxide (DMSO) was purchased from Sigma-
However, these chemical based methods are toxic, hazardous Aldrich (St Louis, MO, USA). Penicillin, streptomycin, trypsin/
and have side effects for health and environment. Moreover, EDTA and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
using toxic and harmful chemicals for the fabrication of metal bromide (MTT) were also procured from Sigma-Aldrich. The
nanoparticles raises the environmental pollution and toxicity bacterial strains of Staphylococcus aureus, (KCTC-1916) and
levels. Therefore development of environmental friendly, clean Escherichia coli (KCTC-1924) were obtained from Korean Collec-
and green approach is necessary for the synthesis of metal tion for Type Cultures (KCTC). Muller–Hinton broth media for
nanoparticles to minimize the environmental impact and bacterial culture and agar were procured from Becton, Dickinson
hazardous chemicals.39–42 In order to fulll the need for devel- and Company (Sparks, MD 21152 USA). Firstly, the used glass-
oping environmentally benign methods, plant materials can be ware was washed and rinsed with double distilled water and then
used as green material that can be easily available at low cost, dried in an oven. Double distilled water was used throughout the
easy to handle, renewable and more importantly non-toxic and experiments. All the obtained chemicals were used as received
environmental friendly for the preparation of number of metal without further purication. The tubers of Nephrolepis cordifolia
and metal oxide nanoparticles.43–46 Very recently, we have re- was washed with double distilled water and dried at room
ported Au–Ag@AgCl nanocomposites using AgNO3 and HAuCl4 temperature. The 5 grams of tubers were cut into small pieces
precursor and the tuber extract of Nephrolepis cordifolia as and 100 mL of distilled water was added. Then, the mixture was
a reducing and stabilizing agents.47
boiled for 10 minutes. The resultant solution was centrifuged at
As a continuous effort in developing environmentally benign 4000 rpm for 10 minutes and ltered.
method for the synthesis of metal nanomaterials, herein, we
disclose the synthesis of SiO2@Au–Ag nanocomposites through
a facile and green technique using tuber extract of Nephrolepis
cordifolia which acts as both reducing and capping agents
without using external additives (Scheme 1). Then, synthesized
SiO2@Au–Ag NCs were checked for its catalytic potential for the
solvent-free amidation of carboxylic acid with primary amine.
Furthermore, antibacterial and cytotoxic activities were inves-
tigated using the synthesized SiO2@Au–Ag NCs.
Synthesis of SiO2 NPs
Firstly 100 mL of prepared tuber extract of Nephrolepis cordifolia
was mixed to 100 mL of ethanol in a 500 mL round bottom ask.
Then the mixture was stirred using a magnetic bar for 30 min at
30 ꢀC. Aer 30 min, 6 mL of tetraethyl orthosilicate (TEOS)
added to the extract quickly by dropwise and the pH was
adjusted 12.0 using ammonium hydroxide. The resultant
mixture was vigorously stirred using a magnetic bar for 18 h and
reuxed for 3 h. The presence of white precipitate indicated the
formation of silica nanoparticles. Then the reaction was
stopped and washed the solution with ethanol 4 times by
centrifugation at 10 000 rpm, at 4 ꢀC for 20 min (using a Labo-
Gene 1580R centrifuge: Gyrozen). ꢀFinally SiO2 nanoparticles
was dried in a vacuum oven at 60 C for 3 h and collected in
a sterile airtight vial.
Experimental
Materials and methods
Tetraethyl orthosilicate (TEOS; purity 99.9%), silver nitrate
(AgNO3, 99%), gold(III) chloride trihydrate (HAuCl4$3H2O, 99.9%)
Synthesis of SiO2@Au–Ag nanocomposites (NCs)
The prepared 100 mg of SiO2 nanoparticles (NPs) were
dispersed in 100 mL double distilled water and sonicated for
1 h. Then, 10 mL of freshly prepared Nephrolepis cordifolia tuber
extract solution was mixed with 100 mL of 2 mmol AgNO3
aqueous solution and mixed with dispersed SiO2 NPs solution
and sonicated for 30 min. Subsequently, 100 mL of aqueous
solution of 2 mmol HAuClO4 and 10 mL of tuber extract was
added to the dispersed solution. The resultant mixture of
solutions was sonicated for 1 h. Then the brownish red color
solution indicated the formation of SiO2@Au–Ag nano-
composites (NCs). The prepared NCs were washed and isolated
by centrifugation at 10 000 rpm, at 4 ꢀC for 20 min and the
process was repeated four times and dried in a vacuum oven at
Scheme 1 Green synthesis of recyclable SiO2@Au–Ag NCs and their
diverse catalytic and biomedical applications.
ꢀ
60 C for 5 h.
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Characterization of NCs
Antibacterial activity of SiO2@Au–Ag NCs
The surface plasmon resonance was performed in order to The antibacterial activities of the green synthesized nano-
identify the formation of nanocomposites using a quartz composites were evaluated against Gram-positive (S. aureus
cuvette by a UV-vis spectrophotometer (Shimadzu UV-2600). KCTC-1916) as well as Gram-negative (E. coli KCTC-1924)
The powder X-ray diffraction (XRD) was recorded to conrm bacterium using a modied disc diffusion method.48 Briey,
the crystallinity of the SiO2@Au–Ag NCs using a PANalytical 3–4 colonies of each bacterium were inoculated in 10 mL of
X'Pert PRO MPD unit operating at voltage 40 kV and 30 mA with Mueller–Hinton (M–H) broth medium and the bacteria were
Cu Ka as the X-ray source (l ¼ 1.5406 A) with scanning 2q angle grown at 37 ꢀC in a shaking incubator at 200 rpm until the
˚
range, 20–90ꢀ, at rate of 1.2ꢀ minꢁ1. Fourier transform infrared optical density (O.D) was reached 0.8 at 600 nm. From this
(FT-IR) spectra were recorded using a Perkin-Elmer FT-IR bacterial suspension, 100 ml was spread over M–H agar plates.
spectrometer in transmittance mode between the wave- Various concentrations of nanoparticles (50–200 mg mLꢁ1) were
number range 400–4000 cmꢁ1. For transmission electron added to the sterilized 6 mm lter discs (Whatman, GE
microscopy (FE-TEM) sample was prepared by dipping the Healthcare UK Limited) placed on the M–H agar plates. Ampi-
copper grid twice into the diluted and 2 h sonicated homoge- cillin (200 mg Lꢁ1) was used as reference positive control for the
nised NCs sample solution. Then air dried grid was analysed to experiments. All the plates were incubated at 37 ꢀC for 24 h and
identify the size and shape of the synthesized NCs using eld the diameters of zone of inhibition were measured. All the
emission transmission electron microscopy (FE-TEM, FEI Tec- experiments were performed in triplicates and mean diameter
nai F20). Similarly, the morphology of the synthesized NCs was was calculated.
characterized by eld emission scanning electron microscopy
(Fe-SEM, S4800, Hitachi, Ltd.) with an acceleration voltage of
In vitro cytotoxicity assay
5.0 kV. The elemental analysis of prepared NCs was performed
The cytotoxic effect of SiO2, SiO2@Au, SiO2@Ag and SiO2@Au–
using high-angle annular dark-eld scanning TEM energy-
Ag nanocomposites was analyzed using a standard MTT (3-(4,5-
dispersive X-ray spectroscopy (HAADF-STEM-EDS) at an accel-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
erating voltage of 200 kV with a point resolution of 0.24 nm, a Cs
Human keratinocyte cells were used to assess the cytotoxic
and Cc of 1.2 mm each with a focal length of 1.7 mm, and by
effects of the nanoparticles. Briey, keratinocyte cells [CCD
a Genesis liquid nitrogen cooled EDS detector.
1102 KERTr (ATCC/CRL-2310)] were grown in Dulbecco's
X-ray photoelectron spectroscopy (XPS) was carried out to
modied Eagle's (DMEM) medium supplemented with 10%
determine the surface chemistry and electronic states of
fetal bovine serum along with 100 U mLꢁ1 penicillin–strepto-
prepared NCs using a Thermo Scientic K-Alpha system an Al K-
mycin in a humidied environment containing 5% CO2/95% air
alpha X-ray source with an ion source energy region of 100 V to 3
keV. Zeta potential of NCs was recorded using Zetasizer Nano ZS
(Malvern Instrument). The sample was prepared in distilled
water and sonicated for 30 min. The prepared sample was
at 37 ꢀC. Aer reaching the conuence, cells were passaged and
seeded in a 96 well plate at a density of 1 ꢂ 105 cells per well for
overnight. Next day, the cells were treated with various
concentrations (50–500 mg mLꢁ1) of nanoparticles in triplicates
equilibrated for 2 min in a disposable cell and the measurement
and kept for 24–96 h at 37 ꢀC in a humidied environment.
ꢀ
was conducted three times at 25 C. Thermogravimetric anal-
Untreated cells were used as a control.
yses (TGA) was carried out by observing the percentage of mass
Aer treatment, old media was removed and each well was
loss using a differential scanning calorimeter (TG-DTA, SDT-
washed with PBS followed by incubation with 50 ml of MTT
(5 mg mLꢁ1 stock) for 4 h. The purple colored formazan crystal
formed, were dissolved in 200 ml DMSO. The absorbance was
taken at 570 nm using a multi well ELISA plate reader (BioTek
Instruments, Inc., USA). The cell viability were measured using
the formula-
ꢀ
Q600 V20.5 Build 15) in the temperature range 30 to 800 C at
a heating rate of 10 C minꢁ1 in N2 atmosphere.
ꢀ
Catalytic activity
The synthesized SiO2@Au–Ag NCs were used as excellent cata-
lyst for the solvent-free amidation of carboxylic acid with
primary amine.
Cell viability ¼ (O.D of experiment/O.D. of control) ꢂ 100
All the readings were taken in triplicates and a graph
showing percentage cell viability was plotted against nano-
particles concentration.
General procedure for the solvent-free syntheses of amide
derivatives 3a–3d
2 mol% SiO2@Au–Ag NCs were added to a stirred solution of
amine 1 (1.0 mmol) and carboxylic acid 2 (5.0 mmol). The
reaction mixture was heated at 100 ꢀC for 8 h and the progress of
the reaction was monitored by TLC. Aer reaction completion,
Results and discussion
Characterization of SiO2@Au–Ag NCs
SiO2@Au–Ag NCs were recovered via hot ltration as residue Firstly, formation of brownish red color resultant solution
and the ltrate was subjected to silica gel column chromatog- indicated the formation of SiO2@Au–Ag nanocomposites. Fig. 1
raphy to isolate the pure products 3a–3d.
illustrates the UV spectra of SiO2, SiO2@Ag, SiO2@Au and
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Fig. 3 (a) FT-IR spectra and (b) TGA and DSC curve of SiO2@Au–Ag
NCs.
Fig. 1 UV-vis spectra of SiO2, SiO2@Ag, SiO2@Au and SiO2@Au–Ag.
The inset images show: (a) plant extract, (b) SiO2, (c) SiO2@Ag, (d)
SiO2@Au and (e) SiO2@Au–Ag NCs.
corresponds to O–H stretching of alcohols or phenols and
aromatic C–H stretching. The absorption peaks at wave number
1633 cmꢁ1 assigned to asymmetric stretching of COOꢁ and
1449 cmꢁ1 are attributed to the symmetric stretching. The FTIR
absorption band observed at around 1016 cmꢁ1 corresponds to
asymmetric vibration of Si–O–Si.49
A number of bioactive molecules present in the plant mate-
rials play a vital role as a reducing and capping agents for the
formation of nanocomposites. The reducing phytochemicals
which bind and capped the metal ion to form the stable
nanoparticles as mechanism presented in Fig. 4. Therefore, the
organic molecules which played the main role for formation of
SiO2@Au–Ag nanocomposites are phytol, ethyl palmitate, b-
ionone, ethyl linolenate diterpene alcohol, eugenol, anethol
and a-cadinol which have been extracted from Nephrolepis
cordifolia.50
SiO2@Au–Ag nanocomposites. The prepared SiO2@Au–Ag
nanocomposites showed the characteristic surface plasmon
resonance (SPR) bands at 415 nm and 530 nm which can be
attributed to the SPR of Au–Ag formed on the surface of SiO2
where SiO2@Au NCs was observed at 540 nm. The strong
absorption band of SiO2@Ag NCs was observed at 420 nm
which ascribed to the formation of Ag on the surface of SiO2.
The observed characteristic SPR peaks and color change of the
NCs indicated the formation of SiO2@Au–Ag nanocomposites.
Fig. 2 shows the powder X-ray diffraction pattern of
SiO2@Au–Ag synthesized from Nephrolepis cordifolia with
intense characteristic peaks. In Fig. 2, the XRD peaks of Neph-
rolepis cordifolia mediated SiO2@Au–Ag observed at 2q value of
38.2ꢀ, 44.4ꢀ, 64.6ꢀ, 77.6ꢀ and 81.7ꢀ were indexed to the reection
planes (111), (200), (220), (311), and (222) respectively, due to
the face-centred cubic phase of silver (JCPDS no. 04-0784) and
cubic gold (JCPDS no. 04-0783), which conrmed the crystalline
nature of Ag and Au metal species, whereas SiO2 NPs didn't
show any phase pattern because of amorphous in phase. The
diffraction pattern of the silica nanoparticles were studied
around 21ꢀ at 2q value.
Thermal stability of SiO2@Au–Ag NCs prepared from tuber
extract of Nephrolepis cordifolia was examined by DSC-TGA as
illustrated in Fig. 3b. The sample of prepared NCs was suꢀbjected
at temperature starting from room temperature to 900 C with
ramp rate 10 ꢀC minꢁ1 under nitrogen atmosphere. The
decomposition of mass was recorded in three successive steps.
The rst step of mass loss was found to be 8.01% around
temperature 100 ꢀC due to the moisture and some volatile
compounds contains in the sample. Secondly, 5.17% and nally
Fig. 3a shows the FT-IR spectra of synthesized nano-
composites which analyze functional groups present in the
Nephrolepis cordifolia mediated SiO2@Au–Ag NCs as both
capping agents as well as reducing agents which are responsible
for the fabrication of nanocomposites. As presented in Fig. 3a,
the absorption band at 3219 cmꢁ1 of obtained SiO2@Au–Ag NCs
ꢀ
2.1% mass loss at the decomposition temperature 430 C and
Fig. 2 XRD patterns of the SiO2, SiO2@Ag, SiO2@Au and SiO2@Au–Ag
NCs. Inset shows the magnified view of SiO2@Au–Ag, SiO2@Au and Fig. 4 Proposed mechanisms for the green synthesis of SiO2@Au–Ag
SiO2@Ag.
NCs.
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beyond 900 ꢀC were examined respectively due to the loss of
capping agents and other bioactive molecule present on the
Nephrolepis cordifolia mediated NCs. Thus, the total percentage
of mass degradation (15.19%) was observed in TGA graph. The
remaining percentage of mass (84.81%) of sample indicated the
existence of Si, O, Au and Ag in biosynthesized SiO2@Au–Ag
NCs.
TEM and SEM analysis revealed the morphology and size of
the SiO2@Au–Ag NCs prepared from Nephrolepis cordifolia tuber
(Fig. 5). Fig. 5a–f displays TEM images and HR-TEM of
SiO2@Au–Ag NCs which conrmed the spherical shape of Au
and Ag nanoparticles decorated on the porous spherical shape
silica nanoparticles. TEM micrographs investigated the poly-
dispersed silica nanoparticles with size ranges 200–246 nm.
HRTEM image clearly showed the well dispersed silver–gold
nano particles on the porous silica nanoparticles (Fig. 5b–f).
Similarly, SEM analysis displayed the spherical morphology of
the synthesized NCs and the Au and Ag NPs could not be clearly
visible on the surface of SiO2 due to the very small size of Au and
Ag NPs (3 nm) in low resolution SEM (Fig. 5g–i). The formation
of SiO2@Au–Ag system could be due to the strong interaction of
Au–Ag metals with the support material SiO2. In general, SiO2 is
regarded as an inert support and does not show signicant
metal support interaction (SMSI). However, in Pt–SiO2 system,
SMSI effect has been demonstrated.51 Moreover, a covalent
interaction has been reported between metal atoms and surface
anions in metal–SiO2 system.52,53 Fig. 6a–e display HAADF-STEM
images of Si, O, Au and Ag present in the SiO2@Au–Ag NCs
using the high-angle annular dark-eld imaging with scanning
transmission electron microscopy (HAADF-STEM). Fig. 6f shows
the SAED pattern of the SiO2@Au–Ag NCs. Furthermore, energy-
dispersive X-ray spectroscopy (EDS) analysis revealed the
elemental composition of synthesized nanocomposites which
conrmed the presence of Si, O, Au and Ag element in the
SiO2Au@Ag NCs (Fig. 6g).
Fig. 6 HAADF-STEM micrographs at 100 nm (a–e), (f) SAED pattern
and (g) EDS elemental mapping of Si, O, Au and Ag in SiO2@Au–Ag
NCs.
the silica, gold and silver along with carbon elements present in
the obtained SiO2@Au–Ag NCs (Fig. S1, ESI†). The spectrum of
carbon element present in the gure indicated the presence
organic compound in plant mediated SiO2@Au–Ag NCs. As
presented in Fig. 7a, the single Si 2p spectrum at 103.5 eV
attributed to the binding energy of Si in SiO2.35 Similarly, the
XPS Au 4f spectra displayed the double bands at 84.6 eV and
88.4 eV binding energies for Au 4f7/2 and Au 4f5/2 respectively in
agreement with the characteristic binding energy of Au0
(Fig. 7c).54 In Fig. 7d, Ag 4d XPS band showed two peaks at the
binding energies of 368.8 eV and 374.8 eV which belong to Ag
3d5/2 and Ag 3d3/2 along with spin splitting of 6.0 eV respectively
which conrmed the presence of Ag0.55,56 From the XPS analysis,
the surface atomic percentage of Si, O, Au and Ag was found to
be 30.07%, 68.49%, 0.22% and 1.21% respectively.
XPS survey spectra of synthesized nanocomposites which
conrmed the chemical state of the NCs and further claried
Stability analysis of SiO2@Au–Ag
The stability of green synthesized SiO2@Au–Ag NCs using
Nephrolepis cordifolia was evaluated as shown in Fig. 8. Firstly,
Fig. 7 XPS spectra of SiO2@Au–Ag NCs (a) Si 2p-electrons of silica
Fig. 5 TEM images of SiO2@Au–Ag NCs at scale bar (a) 50 nm, (b) containing NCs (b) O 1s-electrons of oxygen containing NCs (c) Au 4f-
10 nm (c) 10 nm (d) 5 nm (e) 5 nm (f) 5 nm and SEM images at (g) electrons of gold containing NCs and (d) Ag 3d-electrons of silver
400 nm (h) 100 nm and (i) 500 nm.
containing NCs.
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Table
2
SiO2@Au–Ag NCs catalyzed solvent free amidation of
carboxylic acid with primary aminea
Fig. 8 (a) Zeta potential of the NCs. and (b) UV-vis spectra in various
conditions (after 3 months, pH 6.0, pH 7.4, pH 8.0, 10% NaCl and after
autoclave).
a
Reaction conditions: amine 1 (1.0 mmol), carboxylic acid 2 (5 mmol)
the prepared NCs was analysed by observing zeta potential
distribution and the value was obtained 41.1 mV. The higher
negative zeta potential value of Nephrolepis cordifolia stabilized
SiO2@Au–Ag NCs conrmed the formation of stable NCs
(Fig. 8a).47,57 Furthermore, the surface plasmon resonance (SPR)
spectra of prepared SiO2@Au–Ag NCs was investigated using
spectrophotometry in various conditions such as in different
pH conditions (at pH 8, 7.4, and 6) along with using 10% NaCl
and aer autoclave the sample at 121 ꢀC for 60 min. The Fig. 8b
displays the SPR peaks which showed nearly same wave length
(ꢃ425 nm) in every condition, which further conrmed the
stability of green synthesized SiO2@Au–Ag NCs in biological
uids.
and SiO2@Au–Ag NCs (2.0 mol%).
aniline (1b) or 2-chloro aniline (1c) and aniline (2a) with acetic
acid (2a) in the presence 2 mol% SiO2@Au–Ag NCs at 100 ꢀC for
8 h provided the desired products 3b and 3c in 98% and 95%
yields respectively (entry 2 and 3, Table 2). Moreover, treatment
of aniline (1a) with triuoroacetic acid (2b) in the presence
2 mol% SiO2@Au–Ag NCs at 100 ꢀC for 8 h provided amide 3d in
95% yield (entry 4, Table 2). These results demonstrate the
efficient catalytic activities of the synthesized SiO2@Au–Ag NCs
for the solvent-free amidation of carboxylic acid with primary
amine in good yield. The structure of compounds 3a–3d was
1
identied by H and 13C NMR and by direct comparison with
reported data (data and spectra of the synthesized compounds
are presented in the ESI†).
Catalytic activity
The optimization of reaction condition for the solvent free
amidation of carboxylic acid is presented in Table 1. The
highest yield of the product 3a was obtained using SiO2@Au–Ag
NCs compared to other catalysts such as SiO2, SiO2@Ag,
SiO2@Au. In absence of catalyst, the yield was even lower. Based
on the optimization study, catalytic activities of the synthesized
SiO2@Au–Ag NCs for the solvent-free amidation of carboxylic
acid with primary amine is summarized in Table 2. Reaction of
aniline (1a) with acetic ꢀacid (2a) in the presence 2 mol%
SiO2@Au–Ag NCs at 100 C for 8 h provided amide 3a in 97%
yield (entry 1, Table 2). Similarly, reaction between 4-methoxy
Comparative study of the SiO2@Au–Ag NCs with other
catalysts
The enhanced catalytic performance of the synthesized
SiO2@Au–Ag NCs can be evaluated by comparing the observed
result with that of other reported catalysts. The results are
outlined in Table 3.58–63 We compared the results of solvent free
amidation of carboxylic acid with primary amine in the pres-
ence of various catalysts with respect to the solvent, tempera-
ture and product yield. Eventhough, the reported methods have
their own advantages, the amidation of carboxylic acid in the
presence of the biosynthesized SiO2@Au–Ag NCs were carried
out rapidly with high yields and low loading of the catalyst.
Table 1 Optimization of the reaction conditions for the synthesis of
3aa
Antimicrobial activity of SiO2@Au–Ag NCs
The antibacterial activity of synthesized nanoparticles was
investigated against Staphylococcus aureus and Escherichia coli
bacteria. The bactericidal effects of different nanoparticles as
depicted by the zone of inhibition are shown in the Fig. 9. From
the results, different patterns of zone of inhibition were
observed. As shown in the gure, SiO2@Au–Ag nanocomposites
were found to be most effective against both Gram-positive as
well as Gram-negative bacterium at all the concentrations; on
the other hand SiO2 nanoparticles were inert for both bacterial
species at all the tested particle concentrations (Fig. 9).
SiO2@Au nanoparticles were also found to be inactive for Gram-
positive bacterium at all the concentrations used, whereas for
Gram-negative bacterium, only at higher concentration (200 mg
Entry
Catalyst (2 mol%)
Conditions
Yieldb (%)
1
2
3
4
5
SiO2
8 h, 100 ꢀC
8 h, 100 ꢀC
8 h, 100 ꢀC
8 h, 100 ꢀC
8 h, 100 ꢀC
56
65
67
97
32
SiO2@Ag
SiO2@Au
SiO2@Au–Ag
—
a
Reaction conditions: amine 1a (1.0 mmol), carboxylic acid 2a (5
mmol). b Isolated yield.
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Table 3 Comparative catalytic activity of SiO2@Au–Ag with other catalysts
Entry
1
Catalyst
Temperature
100 ꢀ
Yield (%)
90
Ref.
58
Thiamine
C
hydrochloride
PANI emarldine
Nb2O5
2
3
4
5
6
7
Dioxane, reux
Toluene, reux
Ultrasonic, rt
87
80
94
93
87
97
59
60
61
62
rGO-SO3H
Yttria-zirconia
Starbon-400-SO3H
SiO2@Au–Ag NCs
125 ꢀ
130 ꢀ
100 ꢀ
C
C
C
63
Present work
respectively, whereas for S. aureus it was measured 6.5 and
12 mm at 150 and 200 mg mLꢁ1 concentrations respectively,
Table 2 (Fig. 9). Silver nanoparticles bind to the thiol groups
present on the bacterial membrane and create the pores on the
cell membrane. As a result there is a complete damage of the
membrane which leads to the death of the bacterium.66 In case
of SiO2@Au–Ag nanoparticles, a clearance zone of 7, 13, 18 and
21 mm was measured against E. coli at 50 mg mLꢁ1, 100 mg
mLꢁ1, 150 mg mLꢁ1 and 200 mg mLꢁ1 concentrations respec-
tively as shown in Table 2. A diameter of 23 mm was measured
for positive control ampicillin (200 mg mLꢁ1), which was
comparable to the nanoparticles concentration. However, zone
of inhibition of 7, 10, 13 and 14 mm was observed against Gram
positive bacteria S. aureus at 50 mg mLꢁ1, 100 mg mLꢁ1, 150 mg
mLꢁ1 and 200 mg mLꢁ1 concentrations respectively (Table 4 and
Fig. 9).
The highest antibacterial activities displayed by SiO2@Au–Ag
nanocomposites might be due to the synergistic effect of Au–Ag
nanoparticles. When particles are alone, they are likely to be
aggregate and their surface gets oxidized, which in turn reduce
their antibacterial performances, on the other hand together
they tend to be more stable with higher antimicrobial activi-
ties.67 The Gram-positive bacterium were less sensitive to the
nanoparticles in comparison to the Gram-negative bacterium,
and only a higher concentrations of nanoparticles could inhibit
the growth of Gram-positive bacterium. This might be due to
the presence of thick, lipoteichoic acid containing peptido-
glycan cell wall around the Gram-positive bacterial membrane,
which was difficult to cross by nanoparticles at lower
concentrations.68
Fig. 9 Images of comparative antibacterial studies of different nano-
particles SiO2, SiO2@Au, SiO2@Ag and SiO2@Au–Ag at different
concentration 50 mg mLꢁ1, 100 mg mLꢁ1, 150 mg mLꢁ1, 200 mg mLꢁ1
against bacteria E. coli and S. aureus with respect to positive control of
ampicillin (200 mg mLꢁ1).
mLꢁ1), an inhibition zone of 10 mm could be seen (Fig. 9).
Although the exact mechanism of antibacterial effect of gold
nanoparticles is not fully understood but it is assumed that Au
NPs binds to the cell wall of the bacterium and suppressed
bacterial growth at the rst stage of their reproduction.64 Due to
their well-developed surface chemistry and the smaller size,
gold nanoparticles can interact with the microorganisms with
ease and cause destructive effects on them.65
SiO2@Ag nanoparticles showed better antibacterial effects
than the SiO2@Au nanoparticles and were able to hinder the
growth of both bacterial species. This difference might be due
to the strong antibacterial properties of silver. In case of
SiO2@Ag nanoparticles, the inhibition diameter for E. coli was
measured 0, 7, 11 and 18 mm at a particle concentration of 50
mg mLꢁ1, 100 mg mLꢁ1, 150 mg mLꢁ1 and 200 mg mLꢁ1
Table 4 Antibacterial activity of Nephrolepis cordifolia mediated SiO2, SiO2@Au, SiO2@Ag and SiO2@Au–Ag NCs using disc diffusion method
Zone of inhibition (ZOI) in mm
Concentration (mg mLꢁ1
)
SiO2
SiO2@Au
SiO2@Ag
SiO2@Au–Ag
Gram –ve bacteria E. coli
(KCTC-1924)
50
100
150
200
50
100
150
200
—
—
—
—
—
—
—
—
—
—
—
10
—
—
—
—
—
—
7
11
—
—
6.5
12
7
13
18
21
7
10
13
14
For positive control ampicillin
(200 mg mLꢁ1) ZOI ¼ 23 mm
Gram +ve bacteria S. aureus
(KCTC-1916)
For positive control ampicillin
(200 mg mLꢁ1) ZOI ¼ 18 mm
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Analysis of cytotoxic effects of nanocomposites
The cytotoxic effect of SiO2, SiO2@Au, SiO2@Ag and SiO2@Au–Ag
nanocomposites on human keratinocyte cells was studied using
the MTT assay (Fig. 10). MTT assay helps in determination of cell
viabilities in vitro conditions. The dehydrogenase enzymes present
in the metabolically active cells convert yellow colored MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) salt solu-
tion to an insoluble violet-blue formazan which can be dissolved in
organic solvents like DMSO and analyzed spectrophotometrically.
The number of viable cells is directly proportional to the intensity
of formazan crystal produced.69 The viability of the cells depends
on various factors like, concentration, shape, size and incubation
period of the nanoparticles.70 The results obtained showed
concentration dependent cytotoxic effects on keratinocytes aer
exposure of the cells to the various concentrations (50–500 mg
mLꢁ1) of nanoparticles. With the increase in the exposure time of
nanoparticles from 24–96 hours, cytotoxicity was also increased. As
shown in the Fig. 10, SiO2 nanoparticles were most toxic to the
cells, and in 96 hours at a concentration of 500 mg mLꢁ1, almost
40% of the cells were dead. It is assumed that the SiO2 nano-
particles interact with the macromolecules of the cells which
activate the apoptotic and p53 pathway leading to the generation of
reactive oxygen species (ROS) and nally causing the death of the
cells.71 SiO2@Ag nanoparticles also induced cellular toxicity to the
keratinocyte cells in a concentration and time dependent manner.
Aer exposure of keratinocyte cells to SiO2@Ag nanoparticles at
a concentration of 500 mg mLꢁ1 for 96 hours, the cell viability was
reduced to 75% (Fig. 10). It is supposed that SiO2@Ag nano-
particles bind to the cell membranes which disturb membrane
permeability by increasing the ionic concentration and the oxida-
tive stress causes the cell death.72 SiO2@Au nanoparticles were not
very much toxic to the keratinocyte cell and longer exposure of the
Fig. 11 Representative images from independent experiments when
human keratinocyte cells were treated with biosynthesized nano-
particles at 500 mg mLꢁ1 concentration and cells stained with DAPI
after 96 hours, (a) control cells (b) cells treated with SiO2 (c) SiO2@Ag
(d) SiO2@Au and (e) SiO2@Au–Ag NCs.
cells with higher concentrations of nanoparticles could exerts their
cytotoxic effect (Fig. 10). Conceptually, the cellular uptake of
nanoparticles increases with the increase in their concentration
and treatment time, which cause higher cytotoxic effect on the
cells.73 Maximum viability, was observed in the cells which were
treated with SiO2@Au–Ag nanocomposites. From Fig. 10, it can be
seen that only the highest concentration 500 mg mLꢁ1 of particle
induced the cell death and at this concentration 95% of the cells
were viable, however the lower concentrations of nanoparticles did
not exert any toxic effect on the cells. The expected reason might be
the tight binding of the gold and silver cations on to the surface of
silica nanoparticles, which caused a slow release of Ag and Au ions
causing higher cell viabilities.74 These results demonstrate that the
Table 5 Recyclability of the SiO2@Au–Ag NCsa
Entry
Cycle
Yieldb (%)
1
2
3
4
5
First
97
95
93
92
90
Second
Third
Fourth
Fih
Fig. 10 Cytotoxic effect of different nanoparticles SiO2, SiO2@Au.
SiO2@Ag and SiO2@Au–Ag with different concentrations 50 mg mLꢁ1
,
a
Reaction conditions: amine 1a (1.0 mmol), acid 2a (5.0 mmol),
SiO2@Au–Ag nanocatalyst (2 mol%). b Isolated yield.
150 mg mLꢁ1, 300 mg mLꢁ1 and 500 mg mLꢁ1 on human keratinocytes.
31318 | RSC Adv., 2018, 8, 31311–31321
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Conclusions
An efficient, external additive-free and green approach for the
facile synthesis of SiO2@Au–Ag NCs using tuber extract of
Nephrolepis cordifolia was successfully developed. The
morphology and size of the synthesized Au/Ag nanocomposites
with an average diameter of 3 nm decorated in silica nano-
particles with highly dispersed manner were investigated by
transmission electron microscopy (TEM) analysis. The crystal-
linity and functional group present in the plant mediated NCs
were conrmed by X-ray diffraction (XRD) and Fourier trans-
form infrared (FT-IR) analysis. Chemical composition and
oxidation state of prepared NCs were conrmed by XPS and EDS
analysis. Synthetic functions of fabricated nanocatalyst were
established by solvent-free amidation of carboxylic acid with
primary amine. In addition, the synthesized nanocomposites
demonstrated efficient catalytic activity along with good recy-
clability. Furthermore, the prepared nanocomposites showed
potent bactericidal activities and good biocompatibility.
Therefore, the green synthesized SiO2@Au–Ag nanocomposites
could be used as cost effective, sustainable, additive free,
effective catalyst in organic synthesis as well as safe and
biocompatible NCs for in vivo bio-medical applications.
Fig. 12 XPS spectra of SiO2@Au–Ag NCs after recycle (a) Si 2p-elec-
trons of silica (b) O 1s-electrons of oxygen (c) Au 4f-electrons of gold
(d) Ag 3d-electrons of silver of SiO2@Au–Ag NCs.
synthesized nanocomposite is safe and biocompatible for poten-
tial bio-medical applications.
The cytotoxic effect of nanoparticles on human keratinocyte
cells was further evaluated by staining with DAPI (40,6-
diamidino-2-phenylindole). Briey, 1 ꢂ 103 cells keratinocyte
cells were seeded into culture plates and were incubated in
ꢀ
a CO2 incubator at 37 C for 24 hours. We can correlate these
results with that of MTT assay, where similar kind of effects
were observed. Both MTT and cell staining experiments showed
a concentration dependent cell death of keratinocyte cells aer
treatment with the biosynthesized nanoparticles (Fig. 11).
Conflicts of interest
The authors declare no competing nancial interest.
Acknowledgements
Recyclability of catalyst
This research was fully supported from 2016 Yeungnam
University Research Grant. The authors would like to acknowl-
edge Basic Science Research Programme through the National
Research Foundation of Korea (NRF) funded by the Ministry of
SiO2@Au–Ag NCs fabricated by Nephrolepis cordifolia was recy-
cled and reprocessed ve times without loss of any chemical
compositions and catalytic activities (Table 5). Aer the
completion of each reaction, the nanocatalyst was recovered by
washing with ethyl acetate followed by hot water ltration for
four times. Then the recycled nanocatalyst was dried in
a vacuum oven for 4 h at 70 ꢀC. In order to investigate the
stability of the recycled nanocatalysts, XRD and XPS analysis
was further performed. The XPS and XRD results showed the
similar surface chemical compositions and crystallinity of the
synthesized SiO2@Au–Ag nanocomposites before and aer
catalysis process without any alterations (Fig. S1, ESI,† Fig. 12
and 13).
Education,
Science
and
Technology
(NRF-
2017R1D1A3B03031234).
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