Facile synthesis of macroporous Ag and CuO monoliths as an efficient nonenzymatic electrochemical sensor and antimicrobial agent

Article abstract of DOI:10.1016/j.jssc.2019.03.002

An efficient, green, acid free, reliable synthetic approach has been explored for the synthesis of macroporous silver and copper oxides monoliths. The concise tuning of macroporous materials using surfactants and structural directing agents like TiO₂ nanoparticles, and carbon nanotubes produces the monolith with a random network of pores, high porosity and surface area. The FESEM and BET analysis demonstrated the pore sizes of 40–500 nm and surface area in the range 40–114 m²/g. The XRD, EDAX and TGA analysis confirms the purity and crystallinity of monoliths. A comparative account of electrochemical sensing and antimicrobial applications has been developed. The electrochemical characterization reveals that Ag/TiO₂ and CuO/TiO₂ show excellent electrochemical properties and thus utilized as a nonenzymatic electrode material for simultaneous determination of Levodopa (L-dopa) and ascorbic acid (AA). The results show Ag/TiO₂/GCE show excellent sensing ability with a low detection limit as 0.034 μg mL^?1 and 0.014 μg mL^?1 in the concentration range of 0.1–20.0 μg mL^?1 and 0.01–2.0 μg mL^?1 for L-dopa and AA respectively. The synthesized monoliths have been tested for antimicrobial susceptibility against the 8 g-positive and gram-negative strains. The macroporous Ag/CNT exhibited better antimicrobial results against strains of Klebsiellapneumoniae, Listeria monocytogenes, Vibriovulnificus and Bacillus subtilis.

Full text of DOI:10.1016/j.jssc.2019.03.002

Accepted Manuscript
Facile synthesis of macroporous Ag and CuO monoliths as an efficient nonenzymatic
electrochemical sensor and antimicrobial agent
Devendra Ahirwar, Mustri Bano, Imran Khan, Surendra Singh Gound, Mehraj Ud Din
Sheikh, Rajesh Mondal, Farid Khan
PII:
S0022-4596(19)30108-2
https://doi.org/10.1016/j.jssc.2019.03.002
YJSSC 20646
DOI:
Reference:
To appear in: Journal of Solid State Chemistry
Received Date: 25 September 2018
Revised Date: 24 February 2019
Accepted Date: 1 March 2019
Please cite this article as: D. Ahirwar, M. Bano, I. Khan, S.S. Gound, M.U.D. Sheikh, R. Mondal,
F. Khan, Facile synthesis of macroporous Ag and CuO monoliths as an efficient nonenzymatic
electrochemical sensor and antimicrobial agent, Journal of Solid State Chemistry (2019), doi: https://
doi.org/10.1016/j.jssc.2019.03.002.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Facile synthesis of macroporous Ag and CuO monoliths as an efficient nonenzymatic
electrochemical sensor and antimicrobial agent
1
1
1
2
Devendra Ahirwar , Mustri Bano , Imran Khan , Surendra Singh Gound , Mehraj Ud Din
1
2
1
Sheikh , Rajesh Mondal , Farid Khan*
1
Nanomaterials Discovery Laboratory, Department of Chemistry,
Dr. Harisingh Gour University (A Central University), Sagar (M.P) 470003, India.
2
Molecular Pathogenesis Laboratory, Department of Microbiology
Dr. Harisingh Gour University (A Central University), Sagar (M.P) 470003, India.
*
Corresponding author: - Prof. Farid Khan;
Nanomaterials Discovery Laboratory,
Department of chemistry,
Dr. Harisingh Gour University (A Central University), Sagar (M.P) 470003, India.
Email id: faridkhan58@yahoo.com
Phone: - +91-9425626334
1

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Abstract
An efficient, green, acid free, reliable synthetic approach has been explored for the synthesis of
macroporous silver and copper oxides monoliths. The concise tuning of macroporous materials
using surfactants and structural directing agents like TiO nanoparticles, and carbon nanotubes
2
produces the monolith with a random network of pores, high porosity and surface area. The
FESEM and BET analysis demonstrated the pore sizes of 40-500 nm and surface area in the
2
range 40 to 114 m /g. The XRD, EDAX and TGA analysis confirms the purity and crystallinity
of monoliths. A comparative account of electrochemical sensing and antimicrobial applications
has been developed. The electrochemical characterization reveals that Ag/TiO and CuO/TiO2
2
show excellent electrochemical properties and thus utilized as a nonenzymatic electrode material
for simultaneous determination of Levodopa (L-dopa) and ascorbic acid (AA). The results show
−
1
Ag/TiO /GCE show excellent sensing ability with a low detection limit as 0.034 µg mL and
2
−
1
−1
−1
0
.014 µg mL in the concentration range of 0.1-20.0 µg mL and 0.01-2.0 µg mL for L-dopa
and AA respectively. The synthesized monoliths have been tested for antimicrobial susceptibility
against the eight gram-positive and gram-negative strains. The macroporous Ag/CNT exhibited
better antimicrobial results against strains of Klebsiellapneumoniae, Listeria
monocytogenes, Vibriovulnificus and Bacillus subtilis.
Keywords: Macroporous Ag; Macroporous CuO; Simultaneous electrochemical sensing;
nonenzymatic electrode material; Antimicrobial activity;
2

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1
. Introduction
Metallic macroporous nanomaterials of silver (Ag) and copper oxide (CuO) monoliths are
dynamic and highly efficient promising materials for diverse electrochemical and biomedical
applications e.g. chemical sensing, energy devices, supercapacitors, and antimicrobial activity
photocatalysis and widely used as heterogeneous catalysis environmentally benign [1-9]. Among
the various noble metals and nanoporous-metal oxides i.e. Au and CuO have drawn attention due
to its unique properties of conductivity, biocompatibility, chemical stability and mechanical
flexibility [11]. The sensing and antimicrobial capability of Nanoporous materials have been
generally evolved since last few years due to their high surface areas, external porosity and
degree of crystallinity. Various techniques have been reported for the synthesis of porous Ag and
CuO nanomaterials like electrochemical, sonochemical, solvothermal, microwave,
photochemical, radiation assisted process [5-10, 12-16]. Although the development of reliable,
green, economic and size controllable synthesis scheme of porous noble metal (micro-, meso-
and macro-structured) nanomaterials is still a challenging task. The surface properties of
nanoporous materials have been varied with varying porosity, pore size, composition and pore
size distribution.
The present study is focused on the synthesis of macroporous Ag and CuO monoliths
using Tween-80 as a reducing agent via a modified sol-gel route without using an acidic or basic
medium i.e. “green synthesis approach”. The present synthesis protocol is controllable and does
not use high temperature, pressure, and toxic chemicals. The titanium dioxide nanoparticles
(
TiO ) and carbon nanotubes (CNTs) were used as structural directing agents to produces the
2
monolith having high porosity and surface area with a three-dimensional random network of
pores. The CNTs and TiO help to increase the structural and physical properties like electrical
2
conductivity, chemical stability, surface area [4, 17-20]. Although synthesized monolith has high
surface/volume ratio, high mechanical strength and chemically variable morphologies.
The synthesized macroporous Ag and CuO and their monoliths (Ag/CNT, Ag/TiO2,
CuO/CNT and CuO/TiO ) were tested for their electrochemical and antimicrobial properties. The
2
monoliths were used to fabricate the modified electrode and their sensing ability was
investigated for the simultaneous determination of L-dopa and AA. The electrocatalytic
behaviour was analysed using cyclic voltammetry (CV) and differential pulse voltammetry
(DPV). L-dopa is a naturally producing dietary supplement and psychoactive drug. It is produced
from the amino acids like L-phenylalanine and L-tyrosine in the brain and mammalian body. L-
dopa is used as therapeutic drug in the treatment of Parkinson’s disease and used by the brain to
3

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generate dopamine which balanced the deficiency of dopamine in the organism and decreases the
symptoms of Parkinson’s disease [21-23]. AA is generally found in the extracellular fluid of the
central nervous system and serum and plays a vital role in the metabolic reaction. It works as an
antioxidant and essential nutrient for human being [24-25]. AA is recommended as effective
preservative and antioxidant compound for food beverages. It has medicinal importance to
increase high density lipoprotein formation, regulate cholesterol and triglyceride formation,
lowers blood sugar and insulin [26].
Since the last decades, several reports show nanoparticles of Ag and Cu and their
nanocomposite compounds are effective antimicrobial agents [2-6, 27-32].The comparative bank
of antibacterial effects of the synthesized Ag and CuO monoliths was created by analyzing them
against eight group of bacterial isolates (gram negative and gram-positive bacteria) namely,
Escherichia coli (MTCC 9537), Salmonella enterica (MTCC 734,), Staphylococcus aureus
(
MTCC 1144), Listeria monocytogenes (MTCC 1143), Klebsiellapneumoniae (MTCC 109),
Clostridiumsporogenes (MTCC 1349), Vibriovulnificus (MTCC 1145) and Bacillus subtilis
MTCC 1272). Their activity can be enhanced by modification of surface morphology. Due to
(
the growing bacterial resistance against diverse group of antibiotics, researchers from worldwide
gained interest to look for newer antimicrobial agents. The metal nanoparticles may become the
solution for development of microbial resistant compound.
2
. Material and Chemicals
Tween-80 (molecular weight = 1310 Daltons, non-ionic surfactant), titanium dioxide
nanoparticles (TiO NPs with purity 99.5% and average particle size 21 nm), structure directing
2
6
agents such as dextran (2×10 M) and carbon nanotubes (CNT) were purchased from Sigma
Aldrich (St Lois, U.S.A.). Silver nitrate and Copper nitrate trihydrate were purchased from BDH
(British drug house limited, England) and used as a precursor. All the experimental solutions
were prepared using double distilled water.
3
3
. Experimental
.1 Synthesis of hierarchically macroporous silver monoliths
In a typical synthesis, 2.0 g of silver nitrate (50 wt %) in 2.0 g of double distilled water
(50 wt %) was added to 2.0 g of soft template Tween-80 (14.81 wt %) in 11.50 g of double
distilled (85.19 wt %) and stirred vigorously. A paste of dextran prepared by dissolving 1g
6
dextran (33.33 wt %, Mw = 2x10 Daltons) in 2 g distilled water (66.33 Wt %) was added to
Ag/Tween-80.The resulting gel (Ag/Tween-80/dextran) was stirred at room temperature for 1 h
4

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to form the paste which gradually became dark in colour. Similarly, Ag/CNT/Tween-80/dextran
and Ag/TiO Tween-80/dextran monoliths were prepared using the above protocol by adding 4.0
2
/
g dextran (57.14 wt %) in 3.0 g of ultrapure water (42.14 wt %), 0.10 g of single-wall carbon
nano tubes (SWCNT) and 0.25 g of TiO NPs separately to the Ag/Tween-80/dextran gel. The
2
resulting gels were aged for 48 h at room temperature and then calcined at 400 °C for 2 h at a
heating rate of 2 °C/min followed by cooling at a rate of 2 °C/min to room temperature in an
Elite thermal system furnace. Finally, after calcination Ag/Tween-80/dextran, Ag/CNT/Tween-
8
0/dextran and Ag/TiO Tween-80/dextran was converted into macroporous Ag, Ag/CNT, and
2/
Ag/TiO respectively.
2
3
.2. Synthesis of hierarchically macro/mesostructured porous copper-oxide monoliths
Macroporous CuO monoliths were prepared by dissolving 2.0 g of copper(II) nitrate
trihydrate in 2.0 g of double distilled (50 wt %) and 2.0 g of Tween-80 (14.81 wt %) in 11.5 g of
double distilled (85.19 wt %) at 25 °C. A paste of dextran prepared by dissolving 1 g dextran
6
(
33.33 wt %, Mw = 2×10 Daltons) in 2 g distilled water (66.33 wt %) was added to
CuO/Tween-80.The gel was heated for 1 h at 55 °C on a magnetic stirrer to form a paste which
gradually became light blue in colour. The resulting gel (CuO/Tween-80/dextran) was aged for
_1
2
-3 days at room temperature and then calcined at 400 °C for 2 h at a heating rate of 1 °C min
_
1
followed by cooling at a rate of 1 °C min to room temperature in an Elite thermal system
furnace. Similarly, CuO/CNT/Tween-80/dextran and CuO/TiO /Tween-80/dextran monoliths
2
were prepared using the above protocol by adding 4.0 g dextran (57.14 wt%) in 3.0 g of ultrapure
water (42.14 wt%), 0.10 g of single-wall carbon nanotubes (SWCNT) and 0.25 g of TiO NPs
2
separately to the CuO/Tween-80/dextran gel. The resulting gels were calcined at 400 °C with the
same heating and cooling rate. Finally, after calcination CuO/Tween-80/dextran, CuO/CNT
Tween-80/dextran, and CuO/TiO /Tween-80/dextran were converted into macroporous CuO,
2
CuO/CNT, and CuO/TiO respectively. The systematic pathway of synthesis of macroporous
2
Silver and CuO showed in Scheme 1.
4
. Structural characterization
The structural studies i.e. phase purity and composition of macroporous silver and copper
monoliths were examined by using Bruker D-8 Advance powder X-ray diffractometer (PXRD)
-1
equipped with Cu Kα (λ = 1.5418 Å) with scan rate 2° min (steps: 0.02°) and diffraction angle
range 10-90°. The X-ray patterns obtained for macroporous Ag (Fig. 1a), Ag/CNT (Fig. 1b) and
Ag/TiO (Fig. 1c) showed reflection peaks at d spacings of 2.37, 2.06, 1.45, 1.23 and 1.18 Å,
2
5

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which indexed to lattice planes as (111), (200), (220), (311), and (222) respectively (JCPDS No.
4
.783). These lattice planes correspond to face centered cubic unit cell structures associated with
Fm3m symmetry. The sharp and intense peaks in XRD patterns are evident for high crystallinity
of the synthesized sample. In Fig. 1c, small noises like peaks of TiO nanoparticles reveal that
2
TiO NPs are buried inside the macroporous Ag monolith. The PXRD patterns of macroporous
2
CuO (Fig. 1d), CuO/CNT (Fig. 1e) and CuO/TiO (Fig. 1f) show clear resolved peaks with
2
reflections at d spacings of 2.75, 2.52, 2.32, 1.86,1.71, 1.58, 1.50, 1.41, 1.40, 1.30, 1.26, and 1.16
Å which correspond to lattice planes as (110), (002), (200), (-202), (020), (202), (-113), (022), (-
3
11), (311), (004), and (312) represents base-centered monoclinic unit cell (tenorite) crystal
(JCPDS no. 04.783). The sharp XRD pattern without impurity peaks reveals the formation of
pure macroporous CuO nanomaterials. In the case of CuO/TiO monoliths (Fig. 1f), small noise
2
like peaks of TiO nanoparticles confirms the encapsulation of CuO monoliths.
2
The surface morphology of macroporous CuO, CuO/TiO , CuO/CNT, Ag, Ag/TiO , and
2
2
Ag/CNT monoliths were examined by using NOVA NANOSEM 450 field emission scanning
electron microscope (FESEM) equipped with energy dispersive X-ray spectroscopy (EDAX).
The overview of FESEM images (Fig. 2) clearly depicted the random network of pores in
macroporous Ag and CuO and their monoliths (Fig. 2a and 2d). The pores are distributed in the
range of 40-500 nm in the porous monoliths. The addition of TiO nanoparticles and CNT
2
increases the pore size and surface area. The addition of dextran as a structural directing agent
helps to increase the porosity of the material. The CNTs are tubular in nature with intertwined,
twisted tubes with an average diameter of 5-10 nm, well mixed in Ag and CuO monoliths (Fig.
2
c and 2f). The TiO nanoparticles were distributed and stacked on surface of CuO and Ag
2
macroporous materials (Fig. 2b and 2e). The Fig. 2g, 2h and 2i exhibited the EDAX analysis to
ensure the presence of pure Ag monolith, and TiO nanoparticles encapsulated CuO and Ag
2
monoliths respectively.
The N sorption isotherms of the as-synthesized macroporous monoliths were analyzed
2
by using AutoSorb 1Q instrument (Quantachrome instrument Inc.). The Bruauer- Emmett-Teller
2
(
BET) surface area was determined by physisorption of N at 77 K while Helium gas was used
2
for degassing of samples. Among the series of as-synthesized monoliths, exhibits a sorption
isotherm (Fig. 3a and f) of the IUPAC classification, featuring the macroporous characteristics
of Ag and CuO materials. In the sorption isotherms when P/P is above 0.8 or close to 1, the
0
macroporous Ag (a), Ag/CNT (b), Ag/TiO (c), CuO (d), CuO/CNT (e) and CuO/TiO (f) show a
2
2
rise in values (Fig. 3 a-f), revealing the existence of pores which is consistent agreement with
FESEM results. The surface area and pore diameter of the as-prepared monoliths show a
6

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dramatic increase with the variation of encapsulating materials. The pore size distributions of all
samples were calculated by BJH method. The encapsulation of TiO nanoparticles and CNT in
2
Ag and CuO greatly increases such parameters, as shown in Table 1.
The thermal studies of as-synthesized monoliths was carried out in Perkin elmer thermal
-1
analyzer using an alumina reference crucible at a heating rate of 2 °C min . The TGA curves
Fig. 4) of macroporous Ag and CuO revealed an initial weight loss of 8-15 % from room
(
temperature to 150-175 °C due to the removal of moisture. Similarly, in Ag and CuO monoliths,
a weight drop of 25-40 % was observed between 190-360 °C due to the decomposition of
AgNO , Tween-80 and dextran and Cu(NO ) .3H O in respective monoliths. Consecutively 13
3
3 2
2
%, 12 %, 12 %, 11 %, 18 %, and 21 % mass of macroporous Ag (Fig. 4a), Ag/CNT (Fig. 4b),
Ag/TiO (Fig. 4c), CuO (Fig. 4d), CuO/CNT (Fig. 4e) and CuO/TiO (Fig. 4f) respectively were
2
2
remains to observe.
5
. Electrochemical analysis
The electrochemical analyses were performed in metrohm autolab B.V. PGSTAT128N
controlled with software NOVA version 1.10.1.9. A three-electrode system was used namely,
saturated calomel electrode (SCE) as reference electrode and platinum wire as a counter
electrode and bare and modified glassy carbon electrode (GCE, a diameter of 3.0 mm) were
served as working electrode, respectively. Prior to the analysis, analyte was purged with pure
nitrogen for 150 s, while stirring the solution for removal of oxygen.
5
.1. Fabrication of modified electrode
For the fabrication of modified electrode, namely, macroporous Ag/GCE, Ag/CNT/GCE,
Ag/TiO /GCE, macroporous CuO/GCE, CuO/CNT/GCE, and CuO/TiO /GCE, the individual
2
2
dispersion of all synthesized monoliths (Macroporous Ag, Ag/CNT and Ag/TiO , macroporous
2
CuO, CuO/CNT, and CuO/TiO monoliths) were prepared. For which 5 mg synthesized
2
monoliths were the dispersed separately in 10 mL of ethanol by ultra-sonication. The 10 µL of
dispersed mixture was cast over the polished GCE surface and the solvent was allowed to
evaporate at room temperature. Prior to the electrode modification, electrode surface was
polished with alumina powders (0.05 µm and 0.1 µm) and then cleaned by sonication in 0.1 M
HCl for 10 minutes and dried at room temperature. The electrochemical activation of modified
GCEs was performed by using cyclic potential sweeps in the range of –1.0 to +2.0 V in 0.1 M
−
1
nitric acid solution at the scan rate of 50 mVs to obtain a steady voltammogram. After each
7

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electrochemical measurements, the electrode surface was cleaned by rinsing it with distil water
and then cyclic sweeps in the reverse direction (1.0 V to 0.0 V) in phosphate buffer.
5
.2. Electrochemical properties of Ag and CuO monoliths modified GCEs
The electrochemical properties of all Ag and CuO monoliths modified GCEs was
evaluated by performing cyclic voltammetry using 1.0 mM K Fe(CN) probe with 0.1 M KCl
3
6
-1
solution at the scan rate of 50 mVs (Fig. 5a). The well defined quasi-reversible peak was
3
-/4-
observed represents one electron redox behavior of Fe(CN)6
ions. The electrochemical
surface area was calculated by recording cyclic voltammetric measurements at variable scan
rates and by applying Randles-Sevick equation [10]:
ꢅ
ꢉ/ꢊ
ꢄ
ꢉ/ꢊ
ꢀꢁꢂ
= 0.4463 ꢃ ꢈ ꢋꢌ/ꢊꢍꢎꢏꢎ ꢐꢑꢉ/ꢊ
(1)
ꢆꢇ
Where I is anodic peak current, n is the number of electrons transferred, A is surface area of
pa
0
2
3-/4-
the electrode (cm ), D is diffusion coefficient, C is concentration of Fe(CN)
and ʋ is the
0
6
-1
-1
-1
scan rate, R is molar gas constant (8.314 JK mol ) and F is Faraday’s constant (96480 C mol ).
-6
2 -1
For 1.0 mM K Fe(CN) in 0.1 M KCl at temperature, T = 298 K, n = 1 and D = 7.6×10 cm s .
3
6
0
1
/2
The surface area was calculated from the slope obtained from the plot of I versus ν . In the
pa
2
present study, the surface area of the b-GCE was calculated as 0.0460 cm and for macroporous
Ag/GCE, macroporous CuO/GCE, Ag/CNT/GCE, CuO/CNT/GCE, Ag/TiO /GCE, and
2
2
2
2
2
2
CuO/TiO /GCE found to be 0.157 cm , 0.235 cm , 0.437 cm , 0.552 cm , 0.812 cm and 0.977
2
2
cm respectively. The ∆E (anodic and cathodic peak separation) for b-GCE was calculated to be
P
0
.541 V. After being modified with macroporous CuO and macroporous Ag, ∆E was decreased
P
3
-/4-
to 0.485 V and 0.445 V respectively. The peak current for Fe(CN)6
ions was also increases by
4
3 % and 64 % respectively. The subsequent modification of GCEs with CuO/CNT, Ag/CNT,
CuO/TiO and Ag/TiO monoliths, decrease the value of ∆E as 0.384 V, 0.307 V, 0.238 V and
2
2
P
0
.205 V respectively with increase in peak currents by 86 %, 98 %, 112 %, and 140 %
respectively. The obtained values indicate the high conductivity of Ag/TiO /GCE and
2
Cu/TiO /GCE with relatively high electrochemical surface area, favours the faster and easier
2
charge transfer on the electrode surface.
5
.3. Simultaneous voltammetric determination of L-dopa and Ascorbic acid
The electrocatalyzed nonenzymatic voltammetric behaviour of L-dopa and AA on bare
and monoliths modified GCEs was investigated by recording cyclic voltammograms (CVs). The
-6
CV curves of 1.0 × 10 M AA in 0.02 M phosphate buffer solution (pH 7.0) show a well defined
8

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irreversible, single anodic peak at 0.550 V during the potential sweep of -1.0 V to 1.0 V (Fig.
5
b). This peak is attributed to the oxidation of AA, with no peak observed in the cathodic scan,
-6
indicating the irreversible nature of the oxidation process. Similarly 1.0 × 10 M L-dopa in 0.02
M phosphate buffer solution (pH 7.0) was analyzed with CV sweeps in the potential range of -
1
.6 V to 0.3 V (Fig. 5c). A well defined reversible peaks, anodic peak at -0.540 V, and the
cathodic peak at -0.200 V were obtained, attributed to the complete redox behaviour of L-dopa.
The significant higher peak intensity was observed at Ag/TiO /GCE, and CuO/TiO /GCE than
2
2
macroporous Ag/GCE, macroporous CuO/GCE, Ag/CNT/GCE, CuO/CNT/GCE and b-GCE.
This confirms the increased electrocatalytic behavior towards L-dopa and AA. The efficiency of
electrodes for simultaneous analysis of L-dopa and AA were tested with CVs. The CV of 10.0 ×
-6
-6
1
0 M L-dopa and 1.0 × 10 M AA in phosphate buffer (pH 7.0) was recorded at the scan rate of
-1
5
0 mVs in the potential range of -1.0 V to 1.0 V (Fig. 5d). The concentration of AA influences
peak generation of L-dopa, therefore 10 times higher concentration was taken for obtaining sharp
and clear voltammogram. Ag/TiO /GCE and CuO/TiO /GCE show the enhanced peak intensity
2
2
of these biomolecules analyte, possibly due to the increase in electron transfer rate, high
electroactive surface area and excellent electrode conductivity. The quantification efficiency of
Ag/TiO /GCE and CuO/TiO /GCE for simultaneous nonenzymatic voltammetric sensing of L-
2
2
dopa and AA was evaluated by recording differential pulse voltammograms (DPVs) at the
variable concentration in phosphate buffer (pH 7.0), as shown in Fig. 6a and 6b. The
-1
incorporated potential range of -1.0 V to 1.25 V at the scan rate of 50 mVs . The best linear
−
1
correlation was found in the concentration range of 0.1 – 20.0 µg mL for L-dopa and 0.01 – 2.0
−
1
µg mL for AA. The linear dependence of peak current intensity on the concentration of analyte
2
was clearly depicted by linear regression equation and correlation coefficient (R ) expressed in
Table 2. The analytical figures of merit for simultaneous determination i.e. limit of detection
(
LOD = 3 s/m, where s is the standard deviation of the intercept (n=5), m is the slope of the
regression line [10]) and limit of quantification (LOQ = 10 s/m) are summarized in Table 2. The
RSD for five measurements at each concentration (n = 5) were ranged from 0.021 % to 0.148.
%
5
.4. Stability, reproducibility, and repeatability
The repeatability of Ag/TiO /GCE and CuO/TiO /GCE was evaluated by analyzing peak
2
2
-6
currents of 20 successive DPV measurements in a mixture solution of 10.0 × 10 M L-dopa and
-6
1
.0 × 10 M AA in phosphate buffer (pH 7.0). The %RSD for L-dopa and AA were obtained to
be 0.052 and 0.086 at Ag/TiO /GCE while 0.085 and 0.015 at CuO/TiO /GCE respectively
2
2
indicate that electrode surface was not foul by the oxidation products. In order to analyze the
9

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reproducibility, five Ag/TiO /GCE and CuO/TiO /GCE was prepared under the same conditions
2
2
and used to record DP voltammogram of the same samples. The % RSD of peak current and
peak potential obtained from the average of five replicated analysis are 0.310 % and 0.410 % for
Ag/TiO /GCE respectively and 0.562 % and 0.382 % for CuO/TiO /GCE. The stability of
2
2
Ag/TiO /GCE and CuO/TiO /GCE was investigated over a period of one month by storing them
2
2
in 0.02 M phosphate buffer (pH 7.0). The current response of L-dopa and AA was decreased
slightly by 2.1 % and 2.5 % respectively in first 18 days and after that sensor retained 94 % - 90
%
of its initial peak response for remaining days. Therefore, the result manifests that the
modified electrode exhibited good fabrication reproducibility, repeatability and stability which
makes the best candidate to detect L-dopa, and AA concentration in the practical clinical
analysis.
6
. Antimicrobial activity
To evaluate the antibacterial activity of synthesized monoliths, eight pathogenic bacterial
strains have been chosen e.g. Escherichia coli (MTCC 9537), Salmonella enterica (MTCC 734),
Staphylococcus aureus (MTCC 1144), Listeria monocytogenes (MTCC 1143),
Klebsiellapneumoniae (MTCC 109), Clostridium sporogenes (MTCC 1349),
Vibriovulnificus (MTCC 1145), and Bacillus subtilis (MTCC 1272) obtained from microbial
type culture collection (MTCC) from the Institute of microbial technology (IMTECH),
Chandigarh, India. Muller-Hinton agar (MHA) medium (Himedia, Mumbai) was used for
cultivation. The constituents of the media include-300 gms/liter beef infusion, 1.5 gms/liter
soluble starch, 17.5 gms/liter casein acid hydrolysate, 17 gms/liter Agar-Agar and suspended in
one liter of double distilled water, and pH 7.2 ±0.1. The antimicrobial activity of synthesized
different macroporous monoliths (CuO, Cu/TiO , CuO/CNT, Ag, Ag/TiO and Ag/CNT)
2
2
-1
dissolved (100 ꢀg ml ) in double distilled sterile water was tested against above mentioned
pathogens by Kirby-Baur disc diffusion for standard or as a control activity and used well
diffusion method for macroporous materials described in CLSI guidelines-2017. Kanamycin disc
(30mcg/disc) was used as a positive control. The diameter of the zone of inhibition was
measured after incubation for overnight at 37 °C. Experiments were done in triplicates and
repeated three times and the mean results are represented in Table 3. The best antibacterial
results were obtained for Klebsiellapneumoniae (MTCC 109), Listeria monocytogenes
(
MTCC 1143), Vibriovulnificus (MTCC 1145) and Bacillus subtilis (MTCC 1272) out of 8
pathogens, as shown in Fig. 7. The Fig. 8 showed the graphical representation of growth
-1
inhibition at wells loaded with 100 ꢀg mL concentrations of macroporous Ag and CuO
1
0

ACCEPTED MANUSCRIPT
monoliths as compared to positive control Kanamycin disc (30 mcg/disc). Their activity can be
enhanced by concise tuning of surface area and surface morphology. In this comparative study,
the results show that macroporous Ag combined with CNT exhibited better antimicrobial
susceptibility against the gram-positive and gram-negative strains tested. In case of macroporous
CuO, higher concentrations were required to achieve high antibacterial activity. However,
macroporous Ag and CuO are small enough to disrupt bacterial cell membranes and gain entry in
order to disrupt enzyme function. Likewise, these monoliths have been shown to attach to the
bacterial cell surface and penetrate inside, where intracellular targets, including respiratory
enzymes, are disrupted.
6
.1. Mechanisms of antibacterial effects of macroporous silver materials
In the literature, there are various theories and mechanisms for antimicrobial action of
+
silver and copper nanomaterials. The macroporous Ag/CNT releases the Ag ions, which
interacts with the bacterial cell membrane to causes chemical and structural changes. The
permeation of cell membrane and disrupts of the cell wall causes cell death. In another
+
bactericidal mechanism, the released Ag ions and Ag/CNT gets accumulated and aggregated
near the cell wall, consequently forms free radicals and reactive oxygen which have the ability
to break the cell membrane and leads to cell death [28-29]. The plausible mechanism of
antibacterial activity of Ag/CNT monolith is illustrated in Fig. 9. The similar mechanism also
+
2+
occurs with macroporous CuO monoliths [30]. The Ag and Cu ions generate active free
+
radicals and reactive oxygen that are responsible for damaging the bacterial cells. The Ag and
2
+
Cu ions also have the ability to interact with the thiol (-SH) group of DNA, proteins, and
enzymes and prevented the DNA replication leading to enhanced pyrimidine dimerization. The
highly stable interaction of ions with soft bases of DNA damages the bacterial cell [4]. In CNT
supported macroporous materials, CNT provides the physical surface to Ag and Cu ions for
interaction with the cell membrane [40-41]. The surface area, the pore volume of Ag and strong
metal-carbon adhesion with CNT make a significant factor for their antibacterial property.
The present study evaluated the antimicrobial behaviour of porous Ag and CuO
monoliths by encapsulations of CNT and TiO . The antibacterial results reveal that Ag/CNT and
2
CuO/CNT showed better activity than Ag/TiO and CuO/TiO despite high surface area
2
2
materials, because of needle-like structural morphology of CNT attached. The CNTs penetrate
the bacterial cell membrane and finally burst the cell. Here parallel and synergistic effect of
CNTs works with CuO and Ag for exhibiting better antibacterial activity. However in case of
TiO NPs encapsulated with Ag and CuO which have spherical in shape, stacked and trapped in
2
1
1

ACCEPTED MANUSCRIPT
the monoliths do not perform such antibacterial activity as free CNTs encapsulating monolith
did. [31-35]. In terms of the antimicrobial property of CNTs, the tendency to translocate cellular
barriers i.e. plasma membrane and nano-needle piercing of pathogen cell, by phenomenon is
great considerable properties described [34, 35]. Numerous laboratories use various types of
CNTs based nano-materials have now reported their cellular uptake by a wide range of cells [34,
3
6–39]. This study solves the problem of wastage of costly nanomaterials and helps to reduce the
amount of antimicrobial agent with encapsulating the high coast TiO2 NPs and CNTs in CuO
and Ag monoliths such as TiO NPs and CNTs by encapsulating porous monolith with the lesser
2
amount.
Conclusion
An efficient, green, acid-free, reliable synthetic approach for fabrication of macroporous
Ag and CuO monoliths have been explored. The procedure has a higher ability for concise tuning
of macroporous materials by using different surfactants and structural directing agents. The
synthesized monoliths were crystalline containing random network of pores which increases
their porosity and surface area. The comparative study of electrochemical sensing and the
antimicrobial study show the macroporous Ag/TiO and CuO/TiO monoliths show excellent
2
2
electrochemical properties. The fabricated monoliths modified electrodes were efficaciously
applied for nonenzymatic simultaneous determination of L-dopa and AA. The results show
Ag/TiO /GCE show excellent sensing ability in comparison to other monolith modified
2
electrode. It reports selective recognition, sensitive and precise determination with low detection
limit. The as-synthesized macroporous Ag and CuO monoliths have found to be important
antimicrobial agents. Their activity can be enhanced by concise tuning of surface morphology.
However, macroporous Ag/CNT exhibited better the antimicrobial susceptibility against the
gram-positive and gram-negative bacterial strains tested. The development of the non-toxic, eco-
friendly and non-enzymatic bio molecule sensing material and antimicrobial agents such as
macroporous metals and metal oxides will serve as an alternative to emergent sensing devices
and traditional antibiotics, might be promising for future of medicine, pharmaceutics and clinical
analysis.
Acknowledgement
The authors acknowledge Department of Science & Technology, New Delhi for financial
support, Sophisticated Instrumentation Centre and Department of Chemistry, Dr. Harisingh Gour
Vishwavidyalaya (A Central University) Sagar, India for providing instrumentation facility.
1
2

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Ethical Approval was not required.
There is no conflict of interest to all authors in this research article.
The International Standard Randomized Controlled Trial Number (ISRCTN) or
ClinicalTrials.gov registration number is "Not applicable".
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Table 1: N adsorption-desorption results (surface area, pore volume and average pore
2
diameter) of macroporous (1) Ag, (2) Ag/CNT, (3) Ag/TiO , (4) CuO, (5) CuO/CNT, and (6)
2
CuO/TiO2.
BET surface area
Average pore
S. No.
Sample
2
-1
(
m g )
diameter (nm)
1
2
3
4
5
6
Ag
40.175
42.442
51.531
62.178
100.706
114.068
46
56
Ag/CNT
Ag/TiO2
CuO
70
62
CuO/CNT
CuO/TiO2
95
100
Table 2: Analytical figure of merits for simultaneous voltammetric sensing of L-dopa and
AA using differential pulse voltammetry.
Linear
LOD
(µg
LOQ Mean
range
µg
Linear regression
equation
Analyte
L-dopa
AA
Electrode
(µg
%
(
−1
−1
mL ) mL )
RSD
−
1
mL )
-
1
Ip (µA) = 5.4843 (µg mL )
Ag/TiO /GCE
0.034
0.061
0.014
0.030
0.114
0.203
0.048
0.102
0.021
0.131
0.060
0.148
2
2
0
.01 -
.0
LD + 2.9810; R = 0.988
-1
2
Ip (µA) = 4.2665 (µg mL )
CuO/TiO /GCE
2
2
LD + 2.7503; R = 0.980
-1
Ip (µA) = 7.1053 (µg mL )
Ag/TiO /GCE
2
2
0
.1-
AA + 8.9031; R = 0.985
-1
2
0.0
Ip (µA) = 5.5003 (µg mL )
AA + 7.73563, R = 0.985
CuO/TiO /GCE
2

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-1
Table 3: The zone of inhibition (mm) from wells loaded with 100 µg mL a = CuO; b =
Cu/TiO ; c = CuO/CNT; d = Ag; e = Ag/TiO ; f = Ag/CNT and K 30 = Kanamycin disc (30
2
2
mcg/disc).
Zone of inhibition (mm)
S. No.
Pathogen
a
b
c
d
e
f
K30
1
2
3
4
5
6
7
8
MTCC 109
MTCC 734
MTCC 1143
MTCC 1144
MTCC 1145
MTCC 1272
MTCC 1349
MTCC 9537
10
12
13
13
12
10
12
12
12
14
15
15
14
12
13
14
16
19
20
20
18
16
17
14
14
14
12
14
13
12
13
13
12
14
14
12
15
16
18
16
19
18
20
19
21
22
20
16
18
20
21
19
23
23
19
18

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Scheme 1. A schematic protocol for the synthesis of macroporous monoliths.

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Fig. 1 P-XRD patterns of macroporous (a) Ag, (b) Ag/CNT, (c) Ag/TiO , (d) CuO, (e) CuO/CNT
2
2
and (f) CuO/TiO .

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Fig. 2 SEM micrograph of (a) CuO, (b) CuO/TiO , (c) CuO/CNT, the black coloured circle showing
2
the morphology of CNTs encapsulated in CuO (d) Ag, (e) Ag/TiO and (f) Ag/CNT; EDAX analysis
2
of (g) Ag, (h) CuO/TiO and (i) Ag/TiO monolith.
2
2

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Fig. 3 N adsorption-desorption isotherm of macroporous (a) Ag, (b) Ag/CNT, (c) Ag/TiO , (d)
2
2
CuO, (e) CuO/CNT and (f) CuO/TiO
respectively.
2
, where A and D represents adsorption and desorption curves

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Fig. 4 TGA of macroporous (a) Ag, (b) Ag/CNT, (c) Ag/TiO , (d) CuO, (e) CuO/CNT and (f)
2
CuO/TiO before calculations.
2

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(a)
(b)
(c)
(d)
3
-/4-
-6
-6
Fig. 5 (a-d) CV of (a) 1.0 mM Fe(CN)
in 0.1 M KCl (b) 1.0×10 M Ascorbic acid, (c) 1.0×10
6
-6
-6
M Levodopa and (d) 10.0 × 10 M Levodopa and 1.0 × 10 M Ascorbic acid in phosphate buffer
pH 7) at bare and monoliths modified GCEs.
(

ACCEPTED MANUSCRIPT
(a)
(b)
Fig. 6 DPV of simultaneous addition of different concentration of Ascorbic acid (0.01, 0.05, 0.1, 0.2,
−
1
0
2
.4, 0.6, 0.8, 1.0, 1.5 and 2.0) µg mL and Levodopa (0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0 and
−
1
0.0) µg mL on (a) Ag/TiO /GCE and (b) CuO/TiO /GCE.
2
2
Fig. 7 Shows representative images of plates Klebsiellapneumoniae (MTCC 109), Listeria
monocytogenes (MTCC 1143), Vibriovulnificus (MTCC 1145) and Bacillus subtilis (MTCC
-1
1
272) and the zone of inhibition (mm) from wells loaded with 100 µg ml a = CuO; b = Cu/TiO ; c
2
=
2
CuO/CNT; d = Ag; e = Ag/TiO ; f = Ag/CNT and g = Kanamycin disc (30 mcg/ disc).

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-1
2
Fig. 8 The average zone of inhibition from wells loaded with 100 µg ml of CuO; Cu/TiO ;
CuO/CNT; Ag; Ag/TiO ; Ag/CNT and K 30 (Kanamycin disc 30 mcg/ disc).
2
Fig. 9 Systematic mechanisms for bactericidal effects of macroporous Ag/CNT material.

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Highlights:
•
•
•
An efficient, green, acid free, reliable synthetic approach has been explored for the
synthesis of macroporous silver and copper oxides monoliths.
A comparative account of electrochemical sensing property and antimicrobial
applications of synthesized monoliths has been developed.
Efficacious nonenzymatic simultaneous determination of levodopa and ascorbic acid on
Ag/TiO and CuO/TiO monoliths modified electrodes shows selective recognition,
2
2
sensitive and precise determination with low detection limit.
The synthesized monoliths have also been tested for antimicrobial susceptibility against
the eight gram-positive and gram-negative strains for making better candidate of
antimicrobial agents.
•