Sensors and Actuators B 257 (2018) 220–227

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Research paper

Real-time tracking and quantification of endogenous hydrogen

peroxide production in living cells using graphenated carbon
nanotubes supported Prussian blue cubes
T.S.T. Balamurugan a , Veerappan Mani a,b,∗ , Chang-Che Hsieh b , Sheng-Tung Huang a,b,∗ ,
Tie-Kun Peng a,b , Hsin-Yi Lin a,b
a
Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei, 106,
Taiwan
b
Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, No.1, Section 3, Chung-Hsiao East Road, Taipei, 106, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Recent years have seen a growing interest towards real-time tracking and quantification of reactive
Received 26 April 2017 oxygen species such as, H2 O2 released in living cells, as they are potential biomarkers for pathogenesis.
Received in revised form 24 October 2017 Herein, we described a rapid and sensitive enzymeless H2 O2 assay using Prussian blue microcubes (PB
Accepted 25 October 2017
MCs) decorated graphenated carbon nanotubes (g-CNTs) modified electrode for the tracking of in-vivo
Available online 26 October 2017
H2 O2 production in mammalian cells. The hydrothermally prepared PB MCs were blended with g-CNTs to
yield 3D hierarchical network of PB MCs/g-CNTs nanocomposite, suitable for efficient electrocatalysis. The
Keywords:
g-CNTs/PB MCs film modified electrode was fabricated which displayed excellent electrocatalytic activity
Reactive oxygen species
Signal transduction molecules
to H2 O2 reduction at minimized overpotential. A straightforward amperometric sensor was constructed
Oxidative stress that displayed working range of 25 nM–1598 ␮M, and detection limit of 13 nM. The described non-
2D layered materials enzymatic H2 O2 assay is rapid, sensitive, selective, durable, reproducible and robust. The method was
Nanotechnology successful in sensing in-vivo H2 O2 production from Raw 264.7 cells indicating its potential in biochemical
Modified electrode and clinical applications.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction majorly produced in mitochondria and playing vital functions in

Reactive oxygen species (ROS) are highly reactive byproduct
from aerobic metabolism that must be systematically removed
from cells in order to prevent damage to nucleic acids and pro-
teins [1,2]. Despite having better metabolic pathways to eliminate
the formation of ROS, the cells prefer to choose a pathway that
involves the deliberate production of ROS in first place before ROS
get removed in second place [3,4]. Later, it was discovered that
ROS molecules are not just an inevitable toxic byproduct, rather
their intracellular production in optimum levels is vital to facili-
tate many biological roles [5,6]. Nevertheless, high levels of ROS
are linked to many pathological conditions [6]. H2 O2 , ‘a necessary
evil for cell signaling’ is a well-known representative of ROS that
∗ Corresponding authors at: Department of Chemical Engineering and Biotech-
nology, National Taipei University of Technology, No.1, Section 3,Chung-Hsiao East
Road, Taipei, 106, Taiwan.
E-mail addresses: veera.678@gmail.com (V. Mani), ws75624@ntut.edu.tw
(S.-T. Huang).
proteins metabolism, cell apoptosis, cellular proliferation, regulat-
ing DNA damage, tyrosine phosphorylation etc., [2,7]. However,
its overproduction and accumulation in body can cause severe
damages to the cells that can lead to oxidative stress, neurode-
generation, Alzheimer’s disease, autoimmune diseases, Parkinson’s
disease, cancer, cardiovascular disease etc. The trace levels of H2 O2
in biological system holds the key between life and death as a sig-
nificant marker for cancer; hence, accurate quantification of H2 O2
in living system is of great importance [2,8,9]. Besides, H2 O2 is a
substrate of horseradish peroxidase (HRP), a popular tag enzyme
employed in molecular biology. Moreover, it is widely used as
oxidizing and bleaching agents in chemical and pharmaceutical
industries owing to its antiseptic and anti-bacterial properties [10].
Despite the fact that H2 O2 is linked to various physiological and
pathological processes, a large part of the underlying mechanisms
remains unknown. The lack of reliable sensors that can track and
quantify endogenous H2 O2 in real-time is key limiting step in
understanding its functions. As the amount of endogenous H2 O2
production is normally in the nanomolar range, developing a sen-
sor with such low sensitivity is challenging [11,12]. The presence

https://doi.org/10.1016/j.snb.2017.10.151
0925-4005/© 2017 Elsevier B.V. All rights reserved.
 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227
of other electroactive compounds in biological medium is impos-
ing interference issue that has to be tackled in the sensor design.
Certainty, rapid and sensitive determination of H2 O2 that allows
quantitative and dynamic assessment in live cells is extremely
important.
In electrochemical sensor design, H2 O2 can be directly reduced
at bare electrodes; but usually it requires high overpotential
that causes severe interference from other biological electroactive
species that leads false positive results [13]. Studies revealed that
nanomaterials have the ability to catalyze the H2 O2 reduction at
minimized overpotential, limits surface fouling, improve selectiv-
ity, and amplify signal sensitivity. Therefore, recently increasing
interest has been focused on the development of nanomate-
rials modified electrodes for H2 O2 sensing which includes, Au
nanoparticles/nitrogen-doped graphene quantum dots [13], MnO2
nanowires/graphene paper [14], Pt nanoparticles/porous graphene
[15], 3D graphene/Fe3 O4 quantum dots [16], MoS2 –Au nanorod
hybrid [17], and graphene−Pt nanocomposites [18]. Recently, Maji
et al., described a cancer cell detection through H2 O2 sensing
using 2D nanohybrid system [19]. Chang et al., described a sensor
for evaluation of oxidative stress of tumor cells elicited by H2 O2
that was based on Layer-by-layer assembly of graphene, Au and
poly(toluidine blue O) films [20]. Irrespective of the reported sen-
sors, the development of robust sensing device for accurate and
reliable quantification of H2 O2 in mammalian cells holds great sig-
nificance form analytical science perspective.
Prussian blue (PB), is a renowned electrocatalyst for H2 O2 and
termed as ‘artificial peroxidase’. However, PB suffers from low
electrochemical stability and it requires acidic conditions [21].
To address these issues, supporting materials have been devel-
oped. Particularly, graphene and carbon nanotubes (CNTs) based
nanocomposites are the critically acclaimed supports attributed
to their large surface area [22–28]. Indeed, the hybridization of
1D CNTs with 2D graphene generates a 3D hierarchical nanohy-
brid network, termed as graphenated CNTs (g-CNTs) which was
revealed to hold synergic properties of CNTs and graphene [29].
Stoner et al. found that the g-CNTs possess both high charge den-
sity and large surface area [30]. Several previous reports and our
own recent reports revealed that g-CNTs could be better support
over either graphene or CNTs [29,31–33]. Remarkably, g-CNTs were
reported to have highest edge density per unit nominal area among
all the other carbon nanostructures (graphite, MWCNTs, SWCNTs,
graphene, activated carbon, aligned CNT, HOPG and bamboo CNTs).
Herein, we are reporting the preparation of a robust nanocom-
posite composed of PB microcubes (PB MCs) supported on g-CNTs
for real-time tracking and quantification of H2 O2 in living cells.
The g-CNTs were prepared via one-step wet-chemical method,
while PB MCs were prepared via hydrothermal method. The g-
CNTs/PB MC/GCE catalyzes the H2 O2 reduction at less overpotential
away from common biological interfering region, which makes this
method applicable in biological analysis. We configured a sensing
platform that can be used to track the amount of H2 O2 production
in living mammalian cells.
2. Experimental
2.1. Chemicals and apparatus
Graphite (powder, <20 ␮m), carbon nanotubes (CNTs)
(bundled >95%, O.D × I.D × length = 7–15 × 3–6 × 0.5–200 ␮m),
polyvinylpyrrolidone (PVP), Potassium hexacyanoferrate(II), Fer-
rric chloride, and all other reagents and solvents were purchased
from Sigma-Aldrich and used as received. All the electrochemical
measurements were carried out using CHI 612d electrochemical
workstation (CH Instruments, Inc., U.S.A) at ambient temperature.
The electrochemical studies were performed in a conventional
221
three electrode cell using nanocomposite modified glassy carbon
electrode (GCE) (Bioanalytical Systems, Inc., USA) as a working
electrode (area = 0.071 cm2 ), saturated Ag/AgCl (saturated KCl) as
a reference electrode and Pt wire as a counter electrode. In the
case of amperometry studies, the electrode area was 0.21 cm2 .The
supporting electrolyte used for the electrochemical studies was
0.1 M phosphate buffer (PB), pH 6.8. Scanning electron microscopy
(SEM) and Transmission electron microscopy (TEM) were per-
formed using Hitachi S–3000 H scanning electron microscope and
Hitachi H-7000 transmission electron microscope, respectively.
Powder X-ray diffraction (XRD) studies were performed in a
XPERT-PRO (PANalytical B.V., The Netherlands) diffractometer
using Cu K␣ radiation (k = 1.54 Å). FTIR spectra were carried out
using a Perkin-Elmer IR spectrometer. Electrochemical impedance
spectroscopy (EIS) studies were carried out using EIM6ex Zahner
(Kronach, Germany).
2.2. Preparation of PB MCs and g-CNTs/PB MCs
PB microcubes were prepared by a hydrothermal method. About
15.2 g of PVP and 0.44 g of potassium hexacyanoferrate (II) were dis-
solved in 200 mL HCl solution (0.1 M) via stir using magnetic stirrer.
When the solution became clear, it was transferred to a Teflon-lined
stainless steel autoclave, sealed and heated at 180 ◦ C for 24 h. The
resultant product, PB MCs were collected by filtration, washed with
distilled water and ethanol, and overnight dried under vacuum.
Graphite oxide was prepared from graphite by Hummers
method and exfoliated to graphene oxide (GO) via ultrasonic
agitation for 2 h [34]. Then, the aqueous dispersion of GO was
subjected to centrifugation for 30 min at 4000 rpm to remove unex-
foliated graphite oxide. Next, 15 mg CNTs were added into 20 mL
GO (1 mg mL−1 ) (GO/CNTs; w/w = 1/0.75) and ultrasonicated for
2 h at room temperature. The unstabilized CNTs and excess GO
were removed by subjecting two successive centrifugation cycles
at 8000 and 12000 rpm (30 min each), respectively [35]. 25 mg GO-
CNTs powder was re-dispersed in 100 mL water (1 mg mL−1 ) via
ultrasonication for 30 min and subsequently mixed with hydrazine
(1 mL, 32.1 mmol), refluxed at 90 ◦ C for 12 h, filtered, washed with
copious amount of water and ethanol and dried overnight to yield
the desired product g-CNTs [36]. About 1 mg mL−1 of g-CNTs dis-
persion was prepared in DMF and mixed with 1 mg mL−1 of PB MCs
and the whole solution was ultrasonicated for 30 min to obtain
g-CNTs/PB MCs.
2.3. Fabrication of g-CNTs/PB MCs film modified GCE
GCE surface was polished with 0.05 ␮m alumina slurry and
Buehler polishing kit, washed with water and dried at ambient
temperature. 5 ␮L of g-CNTs/PB MCs was drop casted on the pre-
cleaned GCE and dried. As control, g-CNTs/GCE and PB MCs/GCE
were also prepared.
2.4. Real time tracking and quantification of H2 O2 in RAW 264.7
cells
Raw 264.7 cells were obtained from Tissue Engineering and Cell
Culture Laboratory, which were grown in 5% CO2 in 12.5 cm2 con-
tainer with Dulbecco’s modified Eagle’s medium (DMEM) along
with 1% antibiotics and 10% (v/v) fetal bovine serum (FBS) at 37 ◦ C.
After growing to 90% confluence, the cells were scraped with cell
scraper and collected by centrifugation (1500 rpm, 5 min), then
washed with phosphate buffer saline (PBS) three times. The num-
ber of the cells was counted by a hemocytometer. A pellet of 2 × 106
cells was used for each experiment. Cells were transferred to an
electrochemical cell with 0.1 M PB, pH = 6.8 as supporting elec-
trolyte.
222 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227
Fig. 1. SEM TEM images of g-CNTs (A), PB MCs (B), and g-CNTs/PB MCs (C). TEM images of g-CNTs (D), PB MCs (E), and g-CNTs/PB MCs (F).
3. Results and discussion
3.1. Characterizations of the nanocomposite
The morphology and structure of PB MCs, g-CNTs and g-CNTs/PB
MCs were examined through SEM and TEM. The SEM and TEM
images of g-CNTs presented typical 3D morphology consisted of
tubular networks of CNTs and thin sheets of graphene (Fig. 1A, D)
[37]. The SEM and TEM image of PB MCs displayed cubes struc-
tured microparticles with high crystalline state and the size was
in micrometer range (Fig. 1B, E). The SEM and TEM images of
g-CNTs/PB MCs displayed 3D porous structure in which, PB MCs
were supported on the interconnected networks of g-CNTs hybrid
(Fig. 1C, F).
Fig. 2A displayed the XRD patterns of g-CNTs (a), PB MCs (b)
and g-CNTs/PB MCs (c). The XRD curve of g-CNTs featured with a
characteristic sharp peak at 24.9◦ (001) [37]. The XRD profile of
PB MCs displayed significant diffraction peaks at 17.5◦ (200), 24.7◦
(220), 29.9◦ (222), 35.4◦ (400), 39.4◦ (420), 43.5◦ (442), 50.8◦ (440),
54.4◦ (442), and 57.6◦ (622), and these peaks and their positions
were consistent with those of standard PB crystal lattice (JCPDS no.
73-0689). The XRD pattern of g-CNTs/PB MCs featured with all the
aforementioned peaks of g-CNTs and PB MCs and the peak positions
were not altered describing that the crystal structures of individual
components were not altered during composite formation. FT-IR
spectra of g-CNTs (a), PB MCs (b) and g-CNTs/PB MCs (c) were dis-
played in Fig. 2B. The FTIR spectrum of g-CNTs showed two notable
peaks at wavenumbers of 3741 and 1685 which were assigned
to the stretching vibrations of–OH, and C C [38]. There might be
residual oxygen functionalities located on the sheets of graphene
that contributed to the hydroxyl group vibrations. The spectrum
of g-CNTs/PB MCs featured with additional peak at 1352 cm−1 that
originated from the incorporated PB MCs.
3.2. Impedance and electrochemical study of PB MCs/g-CNTs
Fig. 3A displayed the EIS curves obtained at g-CNTs (a), PB
MCs (b), and g-CNTs/PB MCs (c) film modified GCEs in 0.1 M KCl
containing 5 mM Fe(CN)6 3−/4− . Randles equivalent circuit model
that was used to fit the experimental data was given as inset to
Fig. 3A, wherein the parameters Rs , Rct Cdl and W were represent-
ing electrolyte resistance, charge transfer resistance, double layer
capacitance and Warburg impedance, respectively. EIS measure-
ments were represented as Nyquist plots, in which semicircles
designates the parallel combination Rct and Cdl at electrode sur-
face resulting from electrode impedance. The semicircle diameter
of the curves was in the following order of PB MCs (175.2 ) > g-
CNTs/PB MCs (59.8 ) > g-CNTs (28.5 ). The Rct value of g-CNTs/PB
MCs was about three folds lower than the Rct of PB MCs indicating
the improved conductivity of PB MCs after its incorporation with
g-CNTs as composite.
The cyclic voltammograms (CVs) of PB MCs (a), and g-CNTs/PB
MCs (b) films modified GCEs over a potential range of −0.15 V to
0.50 V at a scan rate of 50 mV s−1 were illustrated in Fig. 3B. Both PB
MCs and g-CNTs/PB MCs displayed characteristic redox couple of PB
(FeIII/FeII) at formal potential (E0 ) of + 0.20 V (vs. Ag/AgCl), ascribed
to the reversible conversion of Prussian white/Prussian blue [39].
The redox peak currents observed at g-CNTs/PB MCs/GCE was com-
paratively higher than that of PB MCs. As the scan rate increased, the
redox peak currents were increased indicating surface-controlled
electrochemical process (Fig. 3C). Next, the electrochemical sta-
bility was investigated by performing 200 cyclic voltammetric
cycles on PB MCs/GCE (Figure not shown), and g-CNTs/PB MCs/GCE
(Fig. S1) in 0.1 M KOH. About 91.2% and 45.1% of initial peak
currents (cathodic peak) were retained from 1st to 200th cycles
indicating that g-CNTs significantly enhanced the electrochemical
stability of PB. The electrochemically effective surface areas of PB
MCs/GCE, and g-CNTs/PB MCs/GCE were calculated to be 0.115 and,
0.152 cm2 , respectively. Evidently, the electrochemical active area
of g-CNTs/PB MCs film modified electrode was higher than that of
PB film modified electrode alone.
3.3. Electrocatalysis of H2 O2 using g-CNTs/PB MCs
The electrochemical behavior of g-CNTs/PB MCs and control
electrodes were measured using cyclic voltammogram (CV) stud-
ies. Fig. 4A displayed the CVs recorded at unmodified (a), PB MCs
(b), g-CNTs (c), and g-CNTs/PB MCs (d) films modified GCEs in
 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227 223

Fig. 2. XRD patterns (A), and FT-IR spectra (B) of g-CNTs (a), PB MCs (b), and g-CNTs/PB MCs (c).

Fig. 3. (A) EIS spectra of g-CNTs (a), PB MCs (b), and g-CNTs/PB MCs (c). Inset: Randles equivalent circuit model, Rs , Rct Cdl and W were electrolyte resistance, charge transfer
resistance, double layer capacitance and Warburg impedance, respectively. (B) CVs obtained at PB MCs (a), and g-CNTs/PB MCs (b) film modified GCEs in 0.1 M KOH at a scan
rate of 50 mV s−1 . (C) CVs of g-CNTs/PB MCs/GCE in 0.1 M KOH at different scan rates (a to j; 0.25–25 mV s−1 ).

PB (pH 6.8) containing 1 mM H2 O2 measured at a scan rate of
50 mV s−1 in the potential range of + 0.70 to −0.70 V. The GCE mod-
ified with g-CNTs/PB MCs showed superior electrocatalytic ability
to H2 O2 reduction evident for the enhanced Ipc and minimized
overpotential (+ 0.34 V, Ag/AgCl). In fact, the reduction peak cur-
rent obtained at g-CNTs/PB MCs was 5, and 3.2 fold higher than
those obtained at g-CNTs and PB MCs films modified GCEs, respec-
tively. The overpotential of H2 O2 reduction at g-CNTs/PB MCs/GCE,
PB/GCE were of + 0.20 V, −0.227 and −0.288 V, respectively, while
the onset potentials of those were 0.345 V, 0.242 V and −0.050 V,
respectively. Remarkably, the overpotential of H2 O2 reduction at g-
CNTs/PB MCs/GCE was 427 and 488 mV lower than that observed
at PB MCs and g-CNTs modified GCEs, respectively. Besides, the
onset potential of H2 O2 reduction at g-CNTs/PB MCs was consider-
ably lowered than those of previously reported graphene or CNTs
supported PB composites [25,27,40–42]. The lower working poten-
224 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227
Fig. 4. (A) CVs obtained at unmodified (a), PB MCs (b), g-CNTs (c) and g-CNTs/PB MCs (d), films modified GCEs in PB (pH 6.8) containing 1 mM H2 O2 , scan rate = 50 mVs−1 .
(B) CVs obtained at GCE/g-CNTs/PB MCs in PB (pH 6.8) containing different concentrations of H2 O2 (a = 0, b = 2, c = 4, d = 6, e = 8, f = 10, g = 12, h = 14, i = 16, j = 18, k = 20 mM).
Inset = current vs. [H2 O2 ] (C) CVs obtained at GCE/g-CNTs/PB MCs in PB (pH 6.8) containing 1 mM H2 O2 at different scan rates (a to j; 0.01–0.1 Vs−1 ). Inset: plot of current/␮A
vs. (scan rate)1/2 /(V.s−1 )1/2.
tial observed at g-CNTs/PB MCs modified electrode is useful to
minimize the response of possible interferences in living cell anal-
ysis. Fig. 4B displayed the voltammograms of g-CNTs/PB MCs/GCE
towards various concentrations of H2 O2 . The value of Ipa increases
linearly as the concentration of H2 O2 increases indicating that this
electrode can be suitable to fabricate H2 O2 sensor. The effect of
scan rate towards H2 O2 reduction was studied by applying differ-
ent scan rates (Fig. 4C). The value of Ipa was linearly related to the
square root of scan rate indicated the efficient diffusion controlled
process.
3.4. Amperometric determination of H2 O2
Fig. 5A displayed amperometric response of the g-CNTs/PB MCs
film modified electrode upon successive injections of H2 O2 into PB
(pH 6.8) at regular intervals of 50 s. The electrode rotation speed
was 1300 RPM and the applied potential (Eapp ) was − 0.05 V. Well-
defined and rapid responses were obtained. As displayed in the
enlarged view of amperogram, the steady-state current has reached
in less than 5s. The plot between current signal and concentration of
H2 O2 exhibited good linearity and the corresponding linear regres-
sion equation can be expressed as, I (␮A) = 0.279 [H2 O2 ]/␮M + 7.9
(Fig. 5B). The working linear range was 0.025–1598 ␮M and the
sensitivity was 1.328 ␮A␮M−1 cm−2 . The limit of detection (LOD)
was calculated to be 13 nM. The LOD of the sensor was calculated
by using the formula, LOD = 3 sb /S (where, sb = standard deviation
of blank signals and S = sensitivity). The sensor parameters includ-
ing linear range, LOD and sensitivity were compared with existing
H2 O2 sensors in a Table 1. As shown in the table, g-CNTs/PB MCs
modified electrode displayed either comparable or superior perfor-
mance over previous modifiers. Many of the reported sensors are
inadequate for monitoring cellular levels of H2 O2 ; however, our
electrode able to attain detection limit in nanomolar level, which
has high significance in fabricating biosensors for H2 O2 quantifica-
tion in living cells (Table 1).
3.5. Selectivity, durability, operational stability and
reproducibility
The selectivity of the sensor was evaluated by performing the
interference experiments in presence of likely interfering agents.
Fig. 5C displayed the amperometric response of g-CNTs/PB MCs
film modified electrode towards 1 ␮M of H2 O2 and 0.5 mM of
dopamine, uric acid, ascorbic acid and folic acid. The modified elec-
trode responded rapidly to H2 O2 ; however, it was insensitive to
the other species investigated. Thus, the electrode specifically rec-
ognizes and sense H2 O2 in the pool of other biological analytes. It
is widely accepted that the polycrystalline structure of PB allows
small H2 O2 molecules to pass through; however, it screens other
species such as dopamine and ascorbic acid [51,52]. Particularly,
negatively charged surface of g-CNTs have the ability to repel neg-
atively charged ascorbate anions [53].
In order to evaluate storage stability of the sensor, its per-
formance (in terms of Ipa ) was monitored every day towards
determination of 1 ␮M H2 O2 (Fig. S2). The electrode was stored
at 4 ◦ C when not in use. The electrode retained more than 95% of
its initial current after one week of its continuous use, which val-
idated excellent storage stability of the electrode. The operational
stabilities of g-CNTs/PB MCs and PB MCs films modified electrodes
were investigated under hydrodynamic condition (Electrode rota-
tion speed = 1300 RPM) in PB (pH 6.8) containing 1 ␮M H2 O2 upto
2500 s. At PB MCs film, the current response was not stable and
46.1% of signal was lost after 2500 s indicating poor stability. On
the other hand, g-CNTs/PB MCs film modified electrode displayed
stable current response upto 2500 s and retains 91.5% of initial
amperometric signal revealing excellent operational stability. To
perform reproducibility studies, measurements at six individual
electrodes were recorded in PB (pH 6.8) towards 1 ␮M H2 O2 ; the
relative standard deviation (R.S.D) was 1.86% signifying acceptable
reproducibility.
3.6. Real time tracking and quantification of H2 O2 in RAW 264.7
cells
The real-time practical utility of g-CNTs/PB MCs film modified
electrode incorporated sensor was demonstrated in RAW 264.7
mamallian cells grown in cell-culture laboratory. Lipopolysaccha-
ride (LPS, 97%), a known stimulant for H2 O2 production in living
cells was used.
The grown RAW 264.7 cells (2 × 106 cells) were transferred to
an electrochemical cell and amperometric experiments were per-
formed by applying a potential of − 0.05 V (vs. Ag/AgCl) (Fig. 6).
A steady current background was obtained and no current change
was observed in the absence of LPS injection (curve a). A sudden
increase in amperometric current was observed immediately after
injection of 10 ␮g mL−1 LPS into the electrolyte (curve b). Further-
more, the successive injections of LPS induced observable increase
in current. Here, LPS stimulated the RAW 264.7 cells to produce
H2 O2 ; the as-produced H2 O2 was detected as a measurable cur-
rent signal by g-CNTs/PB MCs film modified electrode. The current
change was about 0.08 ␮A (average of three readings), equivalent
to 0.286 ␮M of H2 O2 in cell solution. The amount of H2 O2 released
from each cell was calculated to be 16.1 × 10−14 mol, which is con-
sistent with previous reports showing that the g-CNTs/PB film is
capable to quantify in-vivo release of H2 O2 in living cells [50,54,55].
 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227 225

Fig. 5. (A) Amperometric response of g-CNTs/PB MCs film modified electrode for each sequential addition of H2 O2 into PB (pH 6.8). Electrode rotation speed = 1300 RPM and
applied potential = − 0.05 V, (B) Current (␮A) vs. [H2 O2 ] (␮M). (C) Amperometric response of g-CNTs/PB MCs towards 1 ␮M of H2 O2 (a) and 0.5 mM of dopamine (b), uric acid
(c), ascorbic acid (d), and folic acid (e).

Table 1
Comparison of electroanalytical parameters at g-CNTs/PB MCs nanocomposite modified electrode with previously reported modifiers.

Electrode Linear range (␮M) Detection limit (␮M) Sensitivity (␮A␮M−1 cm−2 ) Ref.

ZnO nanosheets/Cytochrome c 1–1000 0.8 0.002 [43]

Graphene-carbon nanotubes 20–2100 9.4 0.03291 [44]
Pt nanoparticles/porous graphene 1–1477 0.50 0.341 [15]
Pd nanowire 53–4400 13.3 0.368 [45]
Nanowires of diphenylalanine and graphene/Hemoglobin/Nafion 0.5–500 0.1 – [46]
Fe3 O4 quantum dots/3D graphene 0.8–334.4 0.078 0.27415 [16]
MoS2 nanosheet–Au nanorod/Catalase 0.5–200 0.10 0.187 [17]
Cube-like CoSn(OH)6 nanostructure 4–400 1 0.01935 [47]
PB/chitosan-graphene 10–400 0.213 0.8164 [40]
MoS2 nanoparticles 0.005−0.10 0.0025 2.58 [48]
Polydopamine/PB coated microelectrode 1–140 0.4 – [49]
Graphene oxide/PB 5–1000 0.8 – [50]
g-CNTs/PB MCs 0.025–1598 0.013 1.328 This work

ments [31,60,61]. The described non-enzymatic H2 O2 tackled many

of the limitations of previous methods. Particularly, the limit of
detection obtained at g-CNTs/PB MCs is in sub-nanomolar level,
which is highly beneficial for the construction of sensitive biosensor
for H2 O2 monitoring in living cells, thus the described nanocompos-
ite holds promising applicability in biomedical applications.

4. Conclusions

An enzymeless electrochemical H2 O2 sensor assay plat-

Fig. 6. Amperometric response of g-CNTs/PB MCs film modified electrode in PB (pH
6.8) containing RAW 264.7 cells without LPS (curve a) and with continuous injections
of 10 ␮g mL−1 LPS (curve b). Eapp = − 0.05 V.
Although, several analytical methods including fluorescence,
spectrometry, chromatography and electrochemical methods were
developed for H2 O2 detection, electrochemical instruments are
more preferable as they are low-cost, simple, easy-to-operate, fast,
and miniaturizable [56–59]. Enzymatic H2 O2 biosensors encounter
important drawbacks such as, enzyme instability on solid elec-
trodes, tedious immobilization procedures, denaturation, lack of
durability, and intolerance to non-physiological chemical environ-
form incorporated with g-CNTs/PB MCs modified electrode was
described for the continuous tracking and quantification of H2 O2
that released by living cells (Raw 264.7). The synthesis of nanocom-
posite and fabrication of electrode are straightforward. The
structure and elemental composition were analyzed. The described
amperometric sensor showed detection limit in sub-nanomolar
range and displayed excellent selectivity, reproducibility and dura-
bility. The overpotential of the electrode was greatly minimized
which helped to attain good selectivity. The outstanding charac-
teristics of the nanomaterial significantly increased the sensitivity
of the assay, as a result the method was successfully quantified the
trace amounts of H2 O2 with good sensitivity. The future work will
be focused on the fabrication of g-CNTs/PB MCs based stretchable
sensor device for H2 O2 tracking in living cells.
Acknowledgements
This work was supported by the Ministry of Science and Tech-
nology, Taiwan (Republic of China). (MOST 105-2113-M-027-003;
MOST 105-2622-M-027 −001 −CC3).
226 T.S.T. Balamurugan et al. / Sensors and Actuators B 257 (2018) 220–227
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.snb.2017.10.151.
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227
Biographies
T.S.T. Balamurugan received his B.S degree from Department of Chemistry, St.
Xavier’s college, M.S University, Tamilnadu, India and M. S degree from School of
Chemistry, Bharathidasan University, Tamil Nadu, India. Currently he is studying
Ph.D. degree in Department of Chemical Engineering and Biotechnology, National
Taipei University of Technology, Taiwan.
Veerappan Mani received PhD from Department of Chemical Engineering and
Biotechnology, National Taipei University of Technology, Taipei, Taiwan in 2014 and
currently he is a research assistant professor. His research interest is mainly focusing
on the synthesis of two-dimensional layered nanomaterials, nanocomposites and
nanohybrids for sensors, biosensors, and energy applications. He published over 62
peer-reviewed scientific publications in well-reputed SCI journals and presented
numerous papers in national and international level conferences.
Chang Che Hsieh received his B.S degree from Oriental Institute of Technology,
Taiwan. Currently he is doing M.S. in Graduate Institute of Biomedical and Biochem-
ical Engineering, National Taipei University of Technology, Taiwan.
Sheng-Tung Huang is currently a professor at the Department of Chemical Engi-
neering and Biotechnology, National Taipei University of Technology, Taiwan. His
research interests include organochemical, medicinal, biochemical, biosensor, sen-
sor and green energy researches.
Tie-Kun Peng received his B.S degree from Chung Yuan Christian University, Taiwan.
Currently he is doing M.S. in Graduate Institute of Biomedical and Biochemical
Engineering, National Taipei University of Technology, Taiwan.
Hsin-Yi Lin is currenly an assistant professor at the Department of Chemical Engi-
neering and Biotechnology, National Taipei University of Technology, Taiwan. Her
research interests include tissue engineering, mammalian cell culture and analysis,
biomaterials development and analysis