Article

Cite This: ACS Appl. Nano Mater. 2019, 2, 5049−5060 www.acsanm.org

ZnCo2O4 Nanoflowers Grown on Co3O4 Nanowire-Decorated Cu

Foams for in Situ Profiling of H2O2 in Live Cells and Biological Media
Veerappan Mani,*,†,‡ Shanthi Selvaraj,§,∥ Tie-Kun Peng,†,‡ Hsin-Yi Lin,†,‡ Nithiya Jeromiyas,†,‡
Hiroya Ikeda,∥ Yasuhiro Hayakawa,∥ Suru Ponnusamy,§ Chellamuthu Muthamizhchelvan,*,§
and Sheng-Tung Huang*,†,‡
†
Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan, Republic of
China
‡
Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao
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East Road, Taipei 106, Taiwan, Republic of China

§
Centre for Nanoscience and Nanotechnology, Department of Physics and Nanotechnology, SRM Institute of Science and
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Technology, Kattankulathur, Tamil Nadu, India

∥
Research Institute of Electronics, Shizuoka University, 3-5-1 Johoku, Naka-ku, Hamamatsu, Japan

ABSTRACT: A robust real-time quantification method is essential to

understand the physiological roles of endogenous H2O2 in biological
systems. For this purpose, we described a binary transition-metal oxide
(TMO)-based nanointerface, i.e., spinal zinc cobaltite/cobalt oxide
ternary nanoarrays (ZnCo2O4/Co3O4) on a Cu foam (CF). The
ZnCo2O4/Co3O4/CF facilitates H2O2 reduction at a minimized over-
potential (−0.10 V vs Ag/AgCl). which is several millivolts away from
the voltammetric regions of common biological and oxygen interferences,
making the electrode highly selective in the presence of 5-fold excess
concentrations of biological species. In the presence of ZnCo2O4, the
electrocatalytic capability of Co3O4 has increased significantly by
enlarging the electrochemical active area of the electrode (0.538 cm2).
A substantial improvement in the stability (97.24%) and reproducibility
(relative standard deviation = 3.14%) are attained because the direct
growth of nanomaterials is generated on CF in close proximity with the electrode surface and strengthens the affinity. The
modified electrode endows ultrasensitivity (detection limit = 1 nM) and quantifies the amount of H2O2 released from
mammalian cells (8.7 × 10−14 mol). Binary TMOs hold promise in tailoring a reliable H2O2-detecting interface for real-time, in
vivo applications.
KEYWORDS: binary transition-metal oxides, interface tailoring, electrocatalysis, electroanalytical chemistry, endogenous chemicals

1. INTRODUCTION cost, portable, and simple, electrochemical techniques stand

In biological systems, the endogenous production of hydrogen
peroxide (H2O2) plays a paramount role in regulating
innumerable signal transduction processes in cellular reac-
tions.1 The normal extracellular levels of H2O2 are found to be
in the range of 0.001−0.1 μM.2 On the other hand, its
overproduction and accumulation in the cellular compartments
can cause severe damage to cells, leading to oxidative stress.3 In
recent years, oxidative stress has gained greater attention in the
biomedicine field owing to its strong affiliation with neuro-
degenerative and cardiovascular diseases.4 Thus, H2O2 is a
protagonist at normal levels, but it can be transformed into an
antagonist at excess levels. The investigation of H2O2-engaged
physiological, physiopathological, and pharmacological pro-
cesses requires robust analytical tools of very fine scale and is
reliable for real-time in situ applications.5,6 Therefore,
developing such an analytical tool has attracted great attention
of the analytical community.2,7,8 Being easy-to-operate, low-
out against other analytical methods.9−11 In the design of
electrochemical sensors, interface tailoring using a suitable
electrocatalytic material is a convincing strategy.12−14 How-
ever, the major challenges are poor sensitivity, lack of
selectivity, and electrode fouling.15,16 Recently, nonenzymatic
H2O2 sensors based on metal oxides are emerging as a robust
alternative for H2O2 biosensors;17,18 however, many of them
are not amendable for continuous in vivo detection mainly
because of the intricate nature of the biological systems.19,20
Thorough literature checks indicate that transition-metal
oxides (TMOs) of Co and Zn are state-of-the-art electro-
catalysts for H2O2 reduction. In addition to their excellent
catalytic properties, Co and Zn TMOs are low-cost and
Received: May 24, 2019
Accepted: August 1, 2019
Published: August 1, 2019

© 2019 American Chemical Society 5049 DOI: 10.1021/acsanm.9b00969

ACS Appl. Nano Mater. 2019, 2, 5049−5060
ACS Applied Nano Materials
abundant.21 Some of the previously reported Co3O4-based
materials for H2O2 sensing are graphene oxide−Co3O4/
nafion,22 Au@C−Co3O4 heterostructures,23 Co3O4 spinel
nanoparticles/graphite electrodes,24 urchin-like Co3O4 nano-
structures,25 vertically aligned Co3O4 nanowalls,26 and Co3O4
nanowires/N-doped C foam.27 Even though Co3O4 has been
regarded as a potential electrocatalytic material for H2O2, its
sensitivity is limited by its poor electrical conductivity, lower
surface area, and hostile stability. Recently, ZnCo2O4, a type of
mixed binary TMO, has attracted greater research interest in
electrochemical applications owing to its multiple redox
reactions, enlarged electrocatalytic sites, mixed-valence states,
and high electrical conductivity.28 In fact, ZnCo2O4 displayed a
better electrochemical performance than Co3O4 in terms of
enlarged specific area, improved conductivity, high porosity,
high ion permeability, high adsorption performance, and low
cost.29 ZnCo2O4 has a cubic spinel crystal structure similar to
that of Co3O4, in which Zn2+ atoms substitute Co2+, where the
Zn ions occupy tetrahedral sites and the trivalent Co ions
occupy octahedral sites, exposing a brilliant electrocatalytic
activity over Co3O4.29 To obtain better electrochemical
performances, various morphology-oriented nanostructured
ZnCo2O4 structures have been studied.30 In addition,
developing porous-based electrode materials is highly recom-
mended in order to achieve excellent electrocatalytic activity
because of its enlarged specific area, well-distributed active
porous sites for the diffusion of analyte molecules through
channel walls, efficient electrolyte ion contacts, and electro-
active sites for analyte diffusion.31 Hence, it is extremely
endorsed to fabricate porous nanostructures for excellent
electrocatalytic performances. The hybridization of ZnCo2O4
with other metal oxides is capable of producing materials with
synergic electrochemical performances that are useful in the
fabrication of high-performance batteries and supercapaci-
tors.32,33 Although the applicability of ZnCo2O4 has been
demonstrated in electrochemical energy applications, its use in
electrochemical sensors has scarcely been reported29 and the
ZnCo2O4/Co3O4 heterostructure has never been reported for
a H2O2 sensor.
Here, we describe a ZnCo2O4/Co3O4 nanostructure grown
on a Cu foam (CF) for real-time monitoring of H2O2
production in live cells (Figure 1). Initially, Co3O4 nanowires
Figure 1. Two-step hydrothermal growth of ZnCo2O4/Co3O4
nanoarrays on CF.
were grown on a CF, and then ZnCo2O4 nanoflowers were
developed on Co3O4/CF, bestowing a porous ZnCo2O4/
Co3O4/CF hybrid electrode. The interactions at the interface
between ZnCo2O4 and Co3O4 are expected to offer unique
electronic states and chemical properties. Moreover, the CF
surface can offer significantly increased current densities by
several orders of magnitude for the same materials at the solid
state, beneficial for enriching the analytical sensitivity. In
addition, the direct growth of nanostructures on a CF is
generated in close proximity with the electrode surface, helping
to improve the stability.
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2. EXPERIMENTAL SECTION
2.1. Materials and Techniques. All reagents including cobalt
nitrate hexahydrate [Co(NO3)2·6H2O], ammonium fluoride (NH4F),
urea [Co(NH2)2], and zinc nitrate hexahydrate [Zn(NO3)2·6H2O]
were of analytical grade, were purchased from Wako Chemicals,
Japan, and were used as received without further purification. Cu
foam (CF) was obtained from Sigma-Aldrich. The electrochemical
measurements were performed using a CHI 1205A workstation. A
human blood sample was collected from a healthy human, and bovine
serum isolated from cows was used for real sample analyses.
Amperometric measurements were performed using an AFMSRX
analytical rotator (PINE instruments, USA). Field-emission scanning
electron microscopy (FESEM), energy-dispersive X-ray (EDX), and
elemental mapping characterizations were performed using a JSM-
7001F microscope (JEOL, Japan). Powder X-ray diffraction (XRD)
studies were performed using a RINT-2200 diffractometer (Rigaku,
Japan; Cu Kα radiation, λ = 1.54178 Å). Raman spectra were acquired
using a NRS-7100 spectrometer with a laser excitation wavelength of
532 nm (spot size 1 μm). X-ray photoelectron spectroscopy (XPS)
spectra were obtained by using an ESCA 3100 spectrometer
(Shimadzu, Japan).
2.2. Hydrothermal Growth of Co3O4/CF-Step 1. CF (2 cm × 2
cm) was precleaned with a 3 M HCl solution for 15 min to remove
the surface impurities and then rinsed with deionized (DI) water and
ethanol. In a typical synthesis, 2 mmol of Co(NO3)2·6H2O, 4 mmol
of NH4F, and 10 mmol of Co(NH2)2 were dissolved in 50 mL of DI
water and stirred for 20 min to form a homogeneous solution. The
solution was transferred into an 80 mL Teflon-lined stainless steel
autoclave along with precleaned CF, maintained at 150 °C for 5 h,
and then cooled to room temperature. The obtained Co3O4/CF was
rinsed with DI water and ethanol several times and dried at 80 °C
overnight. Finally, the samples were calcined at 200 °C for 5 h in air.
2.3. Hydrothermal Synthesis of ZnCo2O4/Co3O4/CF-Step 2.
ZnCo2O4/Co3O4/CF was fabricated through a hydrothermal method.
A total of 3 mmol of Zn(NO3)2·6H2O, 6 mmol of Co(NO3)2·6H2O,
10 mmol of NH4F, and 15 mmol of Co(NH2)2 were dissolved in 80
mL of DI water and stirred for 30 min to yield a homogeneous
solution. The above solution with the as-synthesized Co3O4/CF was
transferred to a 100 mL Teflon-lined stainless steel autoclave,
maintained at 140 °C for 7 h, and cooled to room temperature. The
obtained ZnCo2O4/Co3O4/CF was rinsed with DI water and ethanol
several times and dried at 80 °C overnight. Finally, the samples were
calcined at 350 °C for 5 h in air.
2.4. Electrochemical Studies. The electrochemical studies were
carried out in a conventional three-electrode cell using ZnCo2O4/
Co3O4/CF as a working electrode, Ag/AgCl (saturated KCl) as a
reference electrode, Pt wire as a counter electrode, and 0.1 M
phosphate-buffered saline (PBS; pH 7.4) as a supporting electrolyte.
The working area was fixed at 0.25 sq. cm2 (0.5 cm × 0.5 cm) using
an insulating tape. A total of 2 mg (sq. cm)−2 was found to be the
optimum for maximum sensing performance, and this condition is
sustained in all of the experiments (explained in Figure 6B).
2.5. RAW 264.7 Cell Cultivation for Living Cell Studies. RAW
264.7 cells were grown in 5% CO2 in a 12.5 cm2 container with
Dulbecco’s modified Eagle’s medium (DMEM) along with 1%
antibiotics [100 U mL−1 penicillin and 100 μg mL−1 streptomycin
(LOT198 9515; Gibco, NY)] and 10% (v/v) fetal bovine serum
(FBS) at 37 °C. After being grown to 90% confluence, the cells were
scraped with a cell scraper and collected by centrifugation for 15 min
at 1600 rpm. The grown cells were washed with PBS several times.
The number of cells was counted using a hemocytometer. A pellet of
1 × 106 cells was used for each electrochemical experiment. After
counting, the cells were transferred to an electrochemical cell, in vivo
stimulated to produce H2O2, and subsequently tracked and quantified
by ZnCo2O4/Co3O4/CF.
3. RESULTS AND DISCUSSION
3.1. Characterization of ZnCo2O4/Co3O4/CF. The XRD
patterns of Co3O4/CF and ZnCo2O4/Co3O4/CF are pre-
DOI: 10.1021/acsanm.9b00969
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Figure 2. (A) XRD curves of Co3O4/CF (blue line) and ZnCo2O4/Co3O4/CF (green line). Raman spectra of Co3O4/CF (B) and ZnCo2O4/
Co3O4/CF (C). FESEM images of Co3O4/CF (D) and ZnCo2O4/Co3O4/CF (E). STEM images of Co3O4 nanowires (F and G) and ZnCo2O4
nanoflowers (H and I).
sented in Figure 2A. Conferring to the literature, the XRD peak
positions of the CF substrate peaks are symbolized.34 From the
XRD pattern of Co3O4/CF, the dominant diffraction crystal
planes of (311) and (400), along with the other planes (220),
(222), (422), (440), and (531), can be indexed to the cubic
spinel structure of Co3O4 (JCPDS 42-1467).35 From the XRD
pattern of ZnCo2O4/Co3O4/CF, the dominant diffraction
crystal planes of (311) and (400) with additional crystal planes
of (222), (511), (400), and (533) can be indexed to the
crystalline cubic spinel ZnCo2O4 nanostructure (JCPDS 23-
1390), which formed over Co3O4/CF.36
Parts B and C of Figure 2 present the Raman spectral curves
of Co3O4/CF and ZnCo2O4/Co3O4/CF. For both cases, the
characteristic Raman peaks observed at 270 and 315 cm−1 were
assigned to the Cu substrate.37 In the Raman spectrum of
Co3O4/CF, the signals at 180, 478, 514, 612, and 663 cm−1
were assigned to F12g, Eg, F22g, F32g, and A1g, which exhibit the
Co−O vibrational mode of the Co3O4 nanostructures.38 For
the Raman spectrum of ZnCo2O4/Co3O4/CF, the five
characteristic Raman peaks spotted at 175, 474, 510, 614,
and 679 cm−1 were attributed to the F12g, Eg, F22g, F32g, and A1g
characteristic modes of the ZnCo2O4 nanostructures.39
Next, FESEM analysis (Figure 2D,E) was performed to
analyze the structural morphology of Co3 O4 /CF and
ZnCo2O4/Co3O4/CF. It is clearly seen that Co3O4 nanowires
are formed in a vertical axis, over the entire surface of the CF
substrate in a large scale, with an average length of 1 μm and a
diameter of 100 nm, during the step 1 hydrothermal method,
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as shown in Figure 2D. High-magnification FESEM analysis
(Figure 2D, inset) further clarified the uniform distribution of
Co3O4 nanowires over the CF substrate. To fabricate
ZnCo2O4/Co3O4/CF, the ZnCo2O4 nanoflowers were formed
over the as-synthesized Co3O4/CF, using the step 2 hydro-
thermal method. From Figure 2E, it is confirmed that the
ZnCo2O4 nanoflowers are also formed in a large scale on the
surface of the Co3O4/CF substrate. High-magnification
FESEM analysis (Figure 2E, inset) exposed the complete
structure of one single ZnCo2O4 nanoflower over the substrate.
The diameter of each nanoflower is about 5 μm. Each
ZnCo2O4 nanoflower is composed of 20−40 nanosheets,
which are aligned as a petallike structure, with thicknesses of
50−100 nm and facing the core region.
Parts F−I of Figure 2 show the typical scanning transmission
electron microscopy (STEM) analysis of an individual Co3O4
nanowire and a ZnCo2O4 nanoflower. As shown in Figure
2F,G, the Co3O4 nanowire is fully composed of nanocrystalline
structure, in which the dominant diffraction fringes to an
interplanar distance of 0.24 nm can be indexed to the (311)
spinel Co3O4.40 Additionally, the interplanar distances of 0.28
and 0.47 nm are also identified, which can be indexed to (220)
and (111) of spinel Co3O4.35 Parts H and I of Figure 2 show
the STEM analysis of a ZnCo2O4 nanoflower. It is clearly seen
that the ZnCo2O4 nanoflower is comprised of a large number
of agglomerated ultrathin nanosheets. Each nanosheet is fully
constructed by an irregular porous assembly, over the entire
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ACS Applied Nano Materials
Figure 3. EDX profile of ZnCo2O4/Co3O4/CF (A), EDX quantitative results (B), and mappings of ZnCo2O4/Co3O4/CF (C−G).
sheet surface.41 It is further clarified that the STEM results are
in good agreement with the XRD and FESEM results.
Figure 3 portrays the EDX spectra and mapping analysis of
ZnCo2O4/Co3O4/CF. Parts A and B of Figure 3 confirm the
presence of Zn, Co, O, and Cu from the obtained energy
signal. The as-synthesized ZnCo2O4/Co3O4/CF could be
further explored by mapping analysis. Figure 3C epitomizes
the magnified view of ZnCo2O4/Co3O4/CF. The correspond-
ing elemental mappings of Zn, Co, and O in the CF substrate
are evenly distributed, as shown in Figure 3D−G.
XPS measurement analyses were done to further explore the
structure and composition of ZnCo2O4/Co3O4/CF, as shown
in Figure 4. The Zn 2p electronic configuration is shown in
Figure 4A. The peaks achieved at 1025.3 and 1048.3 eV were
assigned to Zn 2p3/2 and Zn 2p1/2, in which the energy-level
difference of 23.0 eV verified the existence of Zn in the 2+
oxidation state.42 From Figure 4B, the electronic config-
urations of Co 2p identified at 780.8 eV (Co3+) and 797.1 eV
(Co 2+ ) were allocated to Co 2p 3/2 and Co 2p 1/2 ,
respectively.43 The O 1s spectrum was obtained at 531.8 eV,
as shown in Figure 4C. The Cu 2p spectrum of the substrate
peaks identified at 933.1 and 953.9 eV were assigned to Cu
2p3/2 and Cu 2p 1/2, as revealed in Figure 4D, respectively.44
The electrochemical behavior of ZnCo2O4/Co3O4/CF was
investigated by cyclic voltammetry (CV), and the correspond-
ing CV curves in a potential range of 0 to +0.68 V at different
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scan rates are given Figure 5A. In all cases, the CV curves
displayed well-defined redox peaks, which confirms the typical
pseudocapacitive behavior of ZnCo2O4/Co3O4 governed by
faradaic reactions.45 Clear redox peaks were spotted that
should have originated from faradic redox reactions assigned to
Co2+/Co3+, Co3+/Co4+ redox couples. However, the peaks are
not clear, and it was expanded that they can be attributed to
the overlapping Zn2+/Zn3+ reaction; a similar behavior was
observed in previous works.46 When the scan rate was
increased from 5 to 100 mV s−1, the corresponding current
was enhanced, while the shape of the CV curves remained
largely unchanged, except for the shifts of the peak positions.
The redox reactions of ZnCo2O4 and Co3O4 in the alkaline
electrolyte are based on eqs 1−3.31,47,48
ZnCo2O4 + 2OH− ↔ Zn(OH)2 + 2CoO2 + 2e− (1)
− −
Co3O4 + OH + H 2O ↔ 3CoOOH + e (2)
− −
CoOOH + OH ↔ CoO2 + H 2O + e (3)
Using the Randles−Sevcik equation, the electrochemical
active surface areas of unmodified CF, Co3O4/CF (b),
ZnCo2O4/CF (c), and ZnCo2O4/Co3O4/CF have been
found to be 0.205, 0.293, 0.361, and 0.538 cm2, respectively;
i.e., the active area of ZnCo2O4/Co3O4/CF is the maximum,
and it was 2.63-, 1.84-, and 1.49-fold as high as the unmodified
CF, Co3O4/CF, and ZnCo2O4/CF, respectively. The stabilities
DOI: 10.1021/acsanm.9b00969
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Figure 4. XPS spectra of ZnCo2O4/Co3O4/CF: (A) Zn 2p; (B) Co 2p; (C) O 1s; (D) Cu 2p.

Figure 5. (A) CV curves of ZnCo2O4/Co3O4/CF at different scan rates in 0.1 M KOH as a supporting electrolyte. (B) Cyclic stabilities of various
modified electrodes in 0.1 M KOH for 50 continuous cycles. Scan rate = 50 mV s−1.

of CF, Co3O4/CF, ZnCo2O4/CF, and ZnCo2O4/Co3O4/CF,
upon potential ramping of 100 continuous cycles in 0.1 M
KOH, were performed. After being subjected to 50 continuous
cycles, all of the electrodes retained more than 95% of the
initial peak currents, suggesting good stability of the electrode
and a strong attachment of the material to the electrode surface
(Figure 5B). The direct growth of nanostructures on CF is
generated in close proximity with the electrode surface, which
affords good stability and reproducibility to the electrode.
Moreover, the strong affinity between the electrode and
material can minimize the phase potential differences that can
decrease the required applied potential.
3.2. Electrocatalytic Activity of ZnCo2O4/Co3O4/CF
toward H2O2 Reduction. Figure 6A displays the CV curves
of ZnCo2O4/Co3O4/CF recorded at a scan rate of 50 mV s−1
in the presence and absence of 100 μM H2O2. In the presence
of H2O2, a noticeable reduction peak and shoulder were
spotted at −0.12 and −0.25 V, respectively, which are
characteristic peaks expected for H2O2 reduction that can be
correlated with eqs 4 and 5.49 As per the previous literature,
plausible mechanisms for the electrochemical reduction of
H2O2 can be expressed as eqs 4−6.50−52
H 2O2 + e− ↔ OHad + OH− (4)
OHad + e− ↔ OH− (5)
2OH− + 2H+ ↔ 2H 2O (6)

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Figure 6. (A) CV curves of ZnCo2O4/Co3O4/CF in the absence and presence of 100 μM H2O2 suspended in 0.1 M PBS (pH 7.4). Scan rate =
0.05 V s−1. (B) CV curves obtained as unmodified CF (a), Co3O4/CF (b), ZnCo2O4/CF(c), and ZnCo2O4/Co3O4/CF (d) in 0.1 M PBS (pH 7.4)
containing 100 μM H2O2. (Inset) ZnCo2O4/Co3O4 weight optimization: H2O2 sensing current (μA/cm2) versus weight of ZnCo2O4/Co3O4
(grams). Experimental conditions: 100 μM H2O2 + 0.1 M PBS (pH 7.4). (C) CV curves of ZnCo2O4/Co3O4/CF at various H2O2 concentrations
(a, 0.1 mM; b, 0.2 mM; c, 0.3 mM; d, 0.4 mM; e, 0.5 mM). (D) Corresponding linear regression plot. (E) CV curves of ZnCo2O4/Co3O4/CF at
different scan rates (a−j: 0.02−0.2 V) toward 100 μM H2O2 suspended in 0.1 M PBS (pH 7.4). (F) Plot of the square root of the scan rate [(mV
s−1)1/2] versus peak current (μA).

Figure 6B displays the CV curves recorded at a scan rate of
50 mV s−1 for bare CF (a), Co3O4/CF (b), ZnCo2O4/CF (c),
and ZnCo2O4/Co3O4/CF (d) in a PBS (pH 7.4) in the
presence of 100 μM H2O2. CV curves of ZnCo2O4/Co3O4/CF
in the absence and presence of 100 μM H2O2 suspended in 0.1
M PBS (pH 7.4). The comparative trend of the electrocatalytic
ability among the electrode was ZnCo2O4/Co3O4/CF >
Co3O4/CF > ZnCo2O4/CF > bare CF. The current signals
obtained for H2O2 reduction at ZnCo2O4/Co3O4/CF were
about 10.93-, 2.36-, and 3.52-fold as high as the currents
obtained at CF, Co3O4/CF, and ZnCo2O4/CF, respectively.
The overpotentials observed at ZnCo2O4/Co3O4/CF were
381, 336, and 142 mV lower than those obtained at CF,
Co3O4/CF, and ZnCo2O4/CF, respectively. Thus, ZnCo2O4/
Co3O4/CF acts as a good electrocatalyst for the H2O2
reduction reaction and also facilitates electron transfer to
superior rates. The excellent synergy between ZnCo2O4 and
Co3O4, 3D hierarchical network, porous structure, large surface
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area, and excellent electronic properties of the nanointerface
are the reasons for the superior electrocatalytic properties.
Also, the direct growth of ZnCo2O4/Co3O4 on CF can
generate an intimate contact with the electrode surface to
lower the charge-transfer resistance.
For the observed minimized overpotential, a possible
explanation can be speculated based on the difference in the
driving forces before and after nanomaterial modification. In
general, the driving force for an electrode reaction comes from
the energy difference between the energy of the highest
occupied molecular orbital (HOMO) of the electrode and that
of the lowest unoccupied molecular orbital (LUMO) of the
analyte.53 The energy difference between the HOMO of the
unmodified Cu electrode and the LUMO of H2O2 is small,
which causes insufficient driving force, which is incapable of
igniting the reaction at a low reduction potential. In order to
drive the reaction, we should supply extra energy, i.e., >−0.4 V
vs Ag/AgCl, to modulate the energy of the electrons in the
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Figure 7. (A) H2O2 sensing performance of ZnCo2O4/Co3O4/CF for 12 continuous days (stability test). (B) Repeatability of ZnCo2O4/Co3O4/
CF for 10 measurements on a single electrode. (C) Reproducibility of ZnCo2O4/Co3O4/CF for 7 measurements using 7 modified electrodes. For
stability, repeatability, and reproducibility experiments, CV curves were recorded at a scan rate of 0.05 V s−1 in 0.1 M PBS (pH 7.4) suspended with
0.1 mM H2O2.
electrode. Interestingly, the modification of ZnCo2O4/Co3O4
on the Cu electrode alters the interfacial electrochemical
potential difference between ZnCo2O4/Co3O4/Cu and H2O2.
In other words, the electrode modification increases the energy
gap between the HOMO of the electrode and the LUMO of
H2O2 and induces a greater driving force that settles a facile
transfer of electrons at a minimized overpotential.54
Various amounts of ZnCo2O4/Co3O4 were deposited on
CF, and their H2O2 sensing aptitudes were tested (Figure 6B,
inset). The current signal was increased linearly from 0 to 2 mg
(sq. cm)−2, reached maxima at 2 mg (sq. cm)−2, and declined
from 2 to 2.5 mg (sq. cm)−2. It was found that less than 2 mg
(sq. cm)−2 is not sufficient, while more than 2 mg (sq. cm)−2 is
too much, which thickens the film and hinders electron
transfer. Thus, 2 mg (sq. cm)−2 of ZnCo2O4/Co3O4 was found
to be the optimum for a H2O2 sensor. Figure 6C presents the
CV curves obtained at ZnCo2O4/Co3O4/CF in PBS (pH 7.4)
in the presence of varying concentrations of H2O2. The current
was linearly increased with a concentration of H2O2, which is
implicit evidence for the good antiinterference ability of the
modifier. The plot between them displayed good linearity
(Figure 6D). A consistent linear increase in the peak currents
upon successive injections of increasing amounts of H2O2 was
witnessed, suggesting a good antifouling property of the film.
In order to investigate the antifouling ability of ZnCo2O4/
Co3O4/CF, cyclic ramping of 50 continuous CV curves was
inflicted on the electrode in PBS (pH 7.4) containing 100 μM
H2O2. The variation of the peak current at the 50th and 1st
cycles was estimated to be just 4.5%. In other words, 95.5% the
initial peak currents were retained after 50 continuous
voltammetric cycles were imposed, endorsing negligible
electrode fouling. The effect of the scan rate toward
electrocatalytic H2O2 reduction was studied by applying
different scan rates from 20 to 200 mV s−1 (Figure 6E). The
plot between the cathodic peak current and the square root of
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the scan rate exhibits good linearity, suggesting a diffusion-
controlled process (Figure 6F).
Next, the stability, repeatability, and reproducibility of the
ZnCo2O4/Co3O4/CF electrode have been investigated in
order to understand the electrode reliability as a sensor device
for live cells. The voltammetric data of ZnCo2O4/Co3O4/CF
toward H2O2 reduction were recorded every day (Figure 7A).
The electrode retained more than 97.24% of its initial current
up to 12 days of continuous use. For repeatability, 10 repetitive
measurements were recorded using ZnCo2O4/Co3O4/CF and
the relative standard deviation (RSD) of 10 such readings was
3.82% (Figure 7B). The RSD of the reproducibility was 3.14%
for 7 individual electrodes (Figure 7C). Thus, the electrode
held excellent stability, repeatability, and reproducibility,
suggesting a strong interaction between ZnCo2O4/Co3O4
and CF. Commonly, most of the existing modified electrodes
for H2O2 analysis are fabricated by the drop-casting method, in
which thin films are prepared by casting a relevant liquid
dispersion on the electrode surface (e.g., glassy carbon
electrode), followed by solvent evaporation. However, the
stability and uniformity of such thin films are not appropriate
for producing high-stability sensors. On the other hand, the
direct growth of nanostructures on the CF substrate can
enforce a superior intimate contact between the material and
CF; apparently this offers better stability than drop-casting.
Moreover, the solvothermal synthetical mechanism involves
the formation of nuclei and the growth of interfaces with CF,
which also strengthen the surface−material interaction.
3.3. Amperometric Quantification of H2O2. Figure 8A
displays the amperometric response of ZnCo2O4/Co3O4/CF
upon successive injections of H2O2 into PBS (pH 7.4) at a
regular 50 s time interval. An optimized electrode potential was
−0.10 V vs Ag/AgCl (Figure 8A, inset). Deoxygenation of the
electrolyte was not necessary because the operating potential
was located away from the common oxygen interference
potential region. Well-defined and prompt responses were
DOI: 10.1021/acsanm.9b00969
ACS Appl. Nano Mater. 2019, 2, 5049−5060
ACS Applied Nano Materials Article

Figure 8. (A) Amperometric response of ZnCo2O4/Co3O4/CF for each sequential addition of H2O2 into PBS (pH 7.4). Electrode potential =
−0.10 V. (B) Linear regression plot: response current (μA) versus [H2O2] (μM). (C) Bar diagram for the selectivity test; amperometric response
of ZnCo2O4/Co3O4/CF toward 50 μM H2O2 and 250 μM dopamine, uric acid, ascorbic acid, glucose, cysteine, glutathione, glycine, tryptophan,
and lactose. (D) Endogenous H2O2 production and its electrocatalytic detection. (E) Amperometric response of ZnCo2O4/Co3O4/CF at an
electrode potential of −0.10 V vs Ag/AgCl for monitoring of endogenous H2O2 released from RAW 264.7 cells via continuous injections of 10 μg
mL−1 LPS (curve c), cells without LPS stimulation (curve a), and no cells stimulated with LPS stimulation (curve b). (F) Quantitative cell viability
results.

obtained for each aliquot of H2O2, as shown in Figure 8A. A
steady-state current was attained in less than 4 s, thus offering a
rapid response time. A good linear relationship was observed in
a linear range of 10 nM to 750 μM. Thanks to its wide working
linear range, the sensor can be used to monitor H2O2 in both
normal and cancer cells. The linear regression equation is I
(μA) = 0.8162[H2O2] (μM) + 3.187 (Figure 8B). The limit of
detection (LOD) was calculated to be 1 nM (S/N = 3), which
covers the physiological levels of H2O2. The sensitivity was
3.26 μA μM−1 cm−2. The amperometric curve of ZnCo2O4/
Co3O4/CF toward H2O2 displayed good operational stability
because the steady-state current can be maintained up to 5000
s (figure not shown). The linear range, LOD, and sensitivity
are compared in Table 1 with those of previous reports;
evidently, ZnCo2O4/Co3O4/CF exhibited a superior electro-
5056
analytical performance over many of the reported electrodes.
Graphene nanocomposites are the common recent kinds of
H2O2 sensors; however, the synthesis of high-quality graphene
requires time-consuming procedures involving toxic oxidizing
and reducing agents. The preparation of ZnCo2O4/Co3O4/CF
involves a fast and facile hydrothermal route and cheaper
precursors and yields a high-quality uniform nanostructure.
Previously, we reported a NiCo2S4/CoS2 heterostructure for
endogenous H2O2 detection; however, it requires a higher
overpotential of −0.62 V vs Ag/AgCl, in contrast to ZnCo2O4/
Co3O4/CF, which requires just −0.10 V vs Ag/AgCl.
Next, the selectivity of the modified electrode was evaluated
by monitoring of its sensing performance in the presence of
likely biological interfering agents. The amperometric
responses of ZnCo2O4/Co3O4/CF toward 50 μM H2O2 and
DOI: 10.1021/acsanm.9b00969
ACS Appl. Nano Mater. 2019, 2, 5049−5060
ACS Applied Nano Materials Article

Table 1. Comparison of the H2O2 Sensing Parameters of Table 2. Real Sample Analyses of H2O2 Using ZnCo2O4/
ZnCo2O4/Co3O4/CF with Those of Previously Reported Co3O4/CF
Modified Electrodesa
added found recovery RSD fluorimetric
linear range detection sample (nM) (nM) (%) (%)a H2O2 assay
electrode (μM) limit (μM) ref human 50 47.9 95.9 4.3 48.4
Fe3O4@Ag nanocomposite 0.5−20 0.16 55 blood
Co3O4 nanowires/3D N-doped C 10−1400 1.4 27 100 97.3 97.3 4.5 98.2
foam FBS 50 48.8 97.4 3.8 49.2
graphene oxide/cobalt oxide 450−1250 0.015 21 100 97.51 97.5 3.5 98.9
polyhedra DMEM 50 47.8 95.6 3.5 48.7
CuxO@zeolitic imidazolate 1.5−21442 0.15 56 100 96.8 96.8 3.9 98.8
framework
RAW 50 47.5 95.0 3.8 47.8
Cu/Cu2O/FTO 0.2−2000 0.04 57 264.7
ZnO nanosheets/cytochrome c 1−1000 0.8 58 100 96.4 96.4 4.3 98.1
MnO2/graphene paper electrode 100−45400 10 59 a
RSD = relative standard deviation of three individual measurements.
ZnMn2O4 microspheres/GCE 20−15000 0.13 60
Cu2O/graphene 30−7800 20.8 61
Au nanoparticles/N-doped 0.25−13327 0.12 62 the selectivity of the method not only in laboratory samples
graphene quantum dots but also in real samples.
nano-Au, Pt alloy/reduced 5−4000 0.008 63 3.5. Real-Time Quantification of H2O2 in Living Cells.
graphene oxide The practical feasibility of ZnCo2O4/Co3O4/CF for H2O2
MoS2−Au nanorod/catalase/GCE 0.5−200 0.10 64 quantification was demonstrated in RAW 264.7 cells (Figure
Mn2O3 nanoparticle hollow 0.10−126.5 0.07 65 8D). First, the cells were cultivated in a cell-culture laboratory
spheres
by following reported procedures and transferred into an
polydopamine/Prussian blue@ 1−140 0.4 66
microelectrode electrochemical cell. The electrochemical cell was maintained
ZnCo2O4/Co3O4/CF 0.010−750 0.001 this to comprise 1 × 106 cells suspended in 10 mL of the medium
work (pH 7.4). A sharp rise in the amperometric current was
a
Here, GCE stands for glassy carbon electrode. observed immediately after the injection of 10 μg mL−1
lipopolysaccharide (LPS) into the electrolyte (curve c, Figure
8E). A steady-state current was reached in less than 5 s. A
250 μM dopamine, uric acid, ascorbic acid, glucose, further increase in the current was observed upon consecutive
glutathione, glycine, tryptophan, and lactose were recorded, injections of LPS. However, only a steady background current
and the results are presented as a bar diagram in Figure 8C. (nonfaradaic charging current) was observed for the control
ZnCo2O4/Co3O4/CF displayed a good response to H2O2; experiments: (curve a) broth containing living cells without
however, it was insensitive to other compounds. The current LPS induction and (curve b) broth not containing living cells
contribution of the interfering compounds to the total current but stimulated with LPS. From this result, we infer that the
signal was less than 10%, suggesting an acceptable specificity of injection of LPS induced the cells to produce H 2 O 2
the electrode in recognizing H2O2. Some of the enzyme- endogenously, the as-produced H2O2 was sensitively detected
functionalized reports are also compared in Table 1. Despite at ZnCo2O4/Co3O4/CF via electrocatalytic reduction, and the
the good selectivity of enzymes, they are fragile, expensive, resulting faradaic current was recorded as an amperometric
unstable, and nonrobust. Alternatively, ZnCo2O4/Co3O4/CF- signal. The amount of in vivo produced H2O2 was quantified
based enzymeless electrocatalytic sensors are highly robust and from the average current change for three simulations. The
cheaper without compromising selectivity (Figure 8C). amount of H2O2 released from each cell was calculated to be
3.4. Real Sample Analyses. The complete investigation of 8.7 × 10−14 mol, which is in good agreement with previous
ZnCo2O4/Co3O4/CF for electrocatalytic H2O2 sensing con- reports.9,67
firms its outstanding analytical capability in terms of The amount of H2O2 released by RAW 264.7 cells was also
ultrasensitivity, high selectivity, appreciable stability, satisfac- quantified by a standard fluorimetric H2O2 assay kit. An
tory reproducibility, and excellent antifouling abilities, and, excitation wavelength (λex) of 640 nm and an emission
hence, the electrode may be suitable for practical in situ wavelength (λem) of 680 nm were used, and the kit’s standard
analyses. The practical applicability of ZnCo2O4/Co3O4/CF procedure was adopted. The fluorimetric technique found the
was tested in human whole blood, FBS, DMEM, and RAW amount of H2O2 to be 9 × 10−14 mol, which is close to the
264.7 cells. Whole samples were taken for analyses without any value reported by a ZnCo2O4/Co3O4/CF-based electro-
pretreatments and analyzed via amperometry. These media are catalytic sensor, thus validating the accuracy of our method.
found to be H2O2-free. Then known amounts of H2O2 were Although modified electrodes have been reported for
spiked and analyzed. The found and recovery values were endogenous H2O2 detection, binary TMOs directly deposited
calculated via the standard addition method and found to be in on conducting substrates are scarcely reported. Our present
the acceptable range of 95−100% (Table 2). The results are study indicates that ZnCo2O4 belonging to the group of binary
also authenticated by a standard fluorimetric H2O2 assay kit TMOs is a versatile electrocatalyst for real-time continuous
(Sigma). The real sample analyses suggest that ZnCo2O4/ monitoring of endogenous H2O2 produced in live cells. Next,
Co3O4/CF holds excellent practical feasibility in major the cell viability was tested by incubating ZnCo2O4/Co3O4/CF
biological fluids. Irrespective of the presence of other biological in a broth solution containing RAW 264.7 cells. The
compounds in the aforementioned biological fluids, our incubation period was 2 h. The response currents after each
electrocatalytic sensing method is capable of accurate 30 min were recorded and used to calculate the cell viability
quantification of H2O2, which adds additional evidence for (Figure 8F). About 95.9% of the cell viability was retained after
5057 DOI: 10.1021/acsanm.9b00969
ACS Appl. Nano Mater. 2019, 2, 5049−5060
ACS Applied Nano Materials
2 h of continuous incubation, indicating good biocompatibility
and cell viability.
4. CONCLUSIONS
In summary, an ultrasensitive, highly selective, stable, and
reproducible electrode was devised for enzymeless H2O2 assay
by virtue of binary TMOs. The successful growth of ZnCo2O4/
Co3O4 on CF was verified by state-of-the-art high-vacuum
surface characterization techniques. The electrochemical
characterization results revealed the excellent electrochemical,
electrocatalytic, and surface properties of the modified
electrode. The electrode attained a fast response time, a low
detection limit, and a wide linear range, which are comparable
to those of the existing methods. The method is practically
feasible in human blood, FBS, DMEM, and RAW 264.7 cells.
ZnCo2O4/Co3O4/CF successfully traced the continuous
production of in situ H2O2 and accurately quantified the
amount. ZnCo2O4/Co3O4/CF holds promise in electro-
catalytic sensing applications.
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: veera.678@gmail.com (V.M.).
*E-mail: selvancm@gmail.com (C.M.).
*E-mail: ws75624@ntut.edu.tw (S.-T.H.).
ORCID
Veerappan Mani: 0000-0002-0756-7398
Sheng-Tung Huang: 0000-0003-0214-6436
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors are grateful for financial support from the Ministry
of Science and Technology, Taiwan (Grants 106-2113-M-027-
004 and 107-2113-M-027-007).
■
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5060 DOI: 10.1021/acsanm.9b00969

ACS Appl. Nano Mater. 2019, 2, 5049−5060