Biosensors and Bioelectronics 97 (2017) 352–359

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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

Core-shell iron oxide-layered double hydroxide: High electrochemical MARK

sensing performance of H2O2 biomarker in live cancer cells with plasma
therapeutics
Muhammad Asif, Hongwei Liu, Ayesha Aziz, Haitao Wang, Zhengyun Wang, Muhammad Ajmal,
⁎ ⁎
Fei Xiao , Hongfang Liu
Key Laboratory for Large-Format Battery Materials and System, Ministry of Education, Hubei Key Laboratory of Material Chemistry and Service Failure,
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, PR China

A R T I C L E I N F O A BS T RAC T

Keywords: In this work, we develop a new type of multifunctional core-shell nanomaterial by controllable integration of
Fe3O4 nanospheres CuAl layered double hydroxides (LDHs) over the surface of iron oxides (Fe3O4) nanospheres (NSs) to fabricate
p-type layered double hydroxide (Fe3O4@CuAl NSs) hybrid material with interior tunability of LDH phase and explore its practical application in
Electrochemical biosensor ultrasensitive detection of emerging biomarker, i.e., H2O2 as cancer diagnostic probe. In addition, atmospheric
Hydrogen peroxide detection
pressure plasmas (APPs) have also been used as potential therapeutic approach for cancer treatment. Due to the
Live cells
synergistic combination of p-type semiconductive channels of LDHs with multi-functional properties, unique
morphology and abundant surface active sites, the Fe3O4@CuAl NSs modified electrode exhibited attractive
electrocatalytic activity towards H2O2 reduction. Under the optimized conditions, the proposed biosensor
demonstrated striking electrochemical sensing performances to H2O2 including linear range as broad as 8
orders of magnitude, low real detection limit of 1 nM (S/N = 3), high sensitivity, good reproducibility and long-
term stability. Arising from the superb efficiency, the electrochemical biosensor has been used for in vitro
determination of H2O2 concentrations in human urine and serum samples prior to and following the intake of
coffee, and real-time monitoring of H2O2 efflux from different cancer cell lines in normal state and after plasma
treatment. We believe that this novel nano-platform of structurally integrated core-shell nanohybrid materials
combined with APPs will enhance diagnostic as well as therapeutic window for cancer diseases.

1. Introduction Considering the practical significance of H2O2 in biological systems,

precise detection of cellular H2O2 is critically essential to avoid lethal
Hydrogen peroxide (H2O2), with the ongoing need of gigantic attacks of oxidative stress, neurodegenerative disorders, tumor growth
investigation, has emerged as rapidly increasing current research (Yu et al., 2015), and further offering reliable diagnosis of destructive
themes and fascinated great enthusiasm of researchers due to its physiological conditions.
extreme biological significance. It is worth mentioning that H2O2 plays To date, among the various developed analytical techniques,
a vital role in cell proliferation, cell death, response to chronic diseases electrochemical transducer based biosensors have attracted consider-
and intracellular signaling transduction (Ai et al., 2009; Maji et al., able attention because of their high sensitivity, selectivity, portability
2014). A key feature of studying H2O2 in living organisms is to ensure and aptitude for real-time in vitro and in vivo tracking of H2O2 efflux
that its concentration in cellular compartments should be in physiolo- (Luo et al., 2009; Yin et al., 2014). The natural enzyme based
gical range of 1 nM to 0.5 µM in all mammals (Weinstain et al., 2014; electrochemical biosensors are found to be more reliable, sensitive
Zhu et al., 2016). Recent biomarker based cancer diagnosis has drawn and quick responsive for detection of H2O2 than conventional calori-
extensive attention of scientists because of their high reliability, metric method (Ding et al., 2010) owing to profound specificity of
sensitivity and simplicity. Studies have profiled the cellular functions enzymes for catalytic H2O2 reduction (Li et al., 2011; Wu et al., 2011).
associated with endogenous H2O2 efflux in living cells are emerging However, the enzyme modified electrode suffers from instability,
recognized biomarker for cancer diagnostics, principally endowing the complication in immobilization and high cost (Xu et al., 2015; Yuan
measurement facilities with conventional assays (Xi et al., 2016). et al., 2014), these natural enzymes are not readily available to be used

⁎
Corresponding authors.
E-mail addresses: [email protected] (F. Xiao), [email protected] (H. Liu).

http://dx.doi.org/10.1016/j.bios.2017.05.057
Received 16 March 2017; Received in revised form 12 May 2017; Accepted 31 May 2017
Available online 03 June 2017
0956-5663/ © 2017 Elsevier B.V. All rights reserved.
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

under such electrochemical conditions for detection of H2O2. highest current of around 300 mA to investigate the role of charged
Therefore, the development of non-enzymatic H2O2 biosensors based species in cancer therapy. This elegant approach involves indirect
on direct electrocatalytic redox of H2O2 is highly desirable. Particularly, plasma therapy of cancer along with plasma treated solutions contain-
several non-noble metals, such as Cu, Mn, Fe and their oxides with ing cancer cells which is termed as plasma activated medium (PAM).
unique nanostructures have exhibited large surface area, quick electron With high therapeutic efficacy, plasma treatment of cancer would
transfer, enhanced electrocatalytic abilities and high stability (Ganesan contribute efficiently to enhance therapeutic window.
and Lee, 2005), which have been considered to be even superior to the
rare, and expensive noble metals, and hold great promise in H2O2 2. Experimental section
electrocatalysis systems.
Layered double hydroxides (LDHs), a class of inorganic materials 2.1. Fabrication of nanospheres
with brucite type layered structure and intriguing properties, have been
extensively explored in catalysis, cancer therapy (Senapati et al., 2016), Core-shell Fe3O4@CuAl NSs were synthesized by a facile hydro-
separation and sensors due to their distinctive features of typical thermal and co-precipitation method as shown in Fig. 1A. In the first
memory effect, excellent thermal stability and inherent electrocatalytic step, Fe3O4 NSs were synthesized by means of modified solvothermal
capabilities (Asif et al., 2015). This is originated from the p-type approach. Typically, 2.7 g of FeCl3·6H2O were dissolved in 100 ml of
semiconductive channels of LDHs, which consist of high ordered ethylene glycol under vigorous stirring along with the addition of 7.2 g
alternative configuration of M2+ and M3+ cations and charge compli- sodium acetate. The resulting mixture was kept in Teflon-lined stain-
mentary anions in the host layers. In virtue of topotactic transforma- less-steel autoclave and heated up to 200 °C. After proceeding 8 h of
tion, large -OH surface functional groups and excessive positive charge reaction, the autoclave was allowed to cool down naturally at room
on LDHs, LDHs ensure marvelous intercalation features to form temperature. The resulting black product was separated and sequen-
multilamellar hierarchical morphology. Especially, biocompatible tially washed with ethanol several times and dried at 60 °C under
non-noble metal nanoparticles like Fe3O4 (Williams et al., 2016) vacuum overnight.
intercalated LDH materials have drawn considerable interest of Second, 50 mg of Fe3O4 NSs were thoroughly dispersed in 50 ml of
researchers to explore them as an alternative electrocatalyst in the water/methanol (Vwater/Vmethanol = 1:1) solution with ultrasonic agita-
field of electrochemistry (Wang et al., 2012). tion for 20 min to make it homogeneous. An alkaline solution of 50 ml
Herein, we report structurally integrated core-shell Fe3O4@CuAl (Vwater/Vmethanol = 1:1) containing 0.30 g Na2CO3 and 0.46 g NaOH
nanospheres (NSs) with Fe3O4 NSs as the core and CuAl LDHs as the was added drop wise to the above solution under vigorously mechanical
shell, and explore their practical application as dedicated electrocata- stirring to obtain pH 10. Another 50 ml (Vwater/Vmethanol = 1:1) mixed
lyst for H2O2 reduction and its application in sensitive and selective suspension of 4.5 mM Cu(NO3)3·3H2O and 1.5 mM Al(NO3)3·9H2O
detection of H2O2 in different real samples. A series of Fe3O4@CuAl was added dropwise to the above suspension under vigorous stirring
NSs have been synthesized by facile hydrothermal and co-precipitation while keeping pH constant at 10 by simultaneously adding same
approaches, which offer the benefits of inexpensive, simple processing, alkaline solution. The resulting suspension was aged under strong
controlled size and enhanced loading capacity. The as-obtained hier- mechanical stirring at 65 ◦C in water bath for 24 h. The as-obtained
archical assembly exhibits enhanced catalytic ability for the reduction product was separated by magnet, washed several time with deionized
of H2O2 by interfacial collaboration between Fe3O4@CuAl and the water of pH 7 and kept at 60 ◦C overnight Fe3O4@CuAl NSs. Similarly,
fractional charge transfer between p-type conductive LDHs and Fe3O4 CuAl LDHs was also synthesized using same concentrations of
at nanoscale interface. Benefits from large specific surface area, charge Cu(NO3)3·3H2O and Al(NO3)3·9H2O. Finally, 5 µL of the as-prepared
transfer aptitude and availability of gigantic surface active sites, Fe3O4@CuAl NSs suspension (3 mg ml-1) was casted on the surface of
Fe3O4@CuAl NSs demonstrated excellent electrochemical sensing thoroughly polished GCE and dried at ambient temperature, and
performance for H2O2 detection, such as broad linear range, very low Fe3O4@CuAl NSs modified GCE (Fe3O4@CuAl/GCE) was obtained.
real detection limit of 1 nM. The practical application of Fe3O4@CuAl Fe3O4 NSs/GCE and CuAl/GCE were also prepared through the same
NSs used in modifying electrode for in vitro sensitive detection of the procedure.
endogenous H2O2 in human serum, urine as well as cellular H2O2
triggered from different cancer cells. 2.2. In-vitro plasma treatment of living cells
Cancer has become growing cause of mortalities worldwide (Chen
et al., 2016; Xi et al., 2016, 2015). Despite of countless struggles, the To explore the therapeutic effects of charged particles, different
art of potential early cancer detection and therapeutic modalities bear cancer cells in 6-well plate were treated directly by plasma plume. A
several limitations in clinical examinations, histopathology, imaging copper wire of 2 mm diameter inserted in 4 cm long quartz tube with
mammography and terrible side effects of therapeutic anticancer drugs one end closed was used as high voltage (HV) electrode. Then this HV
as well as their high cost (Cardoso et al., 2016; Liu et al., 2016). An electrode was inserted in 6 mm diameter hallow barrel of syringe along
accumulated exposure to radiations led by mammography and its with injecting helium gas in hallow barrel as working gas and HV
insufficient sensitivity, driving the patients into histopathology which is electrode pulsed dc voltage was applied to generate plasma. The
consequently an invasive approach, when the disease is grown generated plasma plume could approximately be 6 cm in length. The
(Yahalom et al., 2013). Therefore, substantial advances in technologies cells were placed under plasma plume with adjustable distance of 2 cm
for non-invasive, economical and reliable early cancer screening, between plasma jet nozzle and upper surface of medium in 6-well plate
prognostic evidences and effective therapeutic options are urgently and irradiated for 1 min and 2 min. The cell cultured plate was kept in
needed. incubator for 24 h at 37 °C after plasma treatment (Kaushik et al.,
The goal of our study is not only focused on developing novel 2016).
material based biosensing platform for cancer diagnosis, but also on
uncovering a challenging therapeutic approach for in vitro treatment of 3. Results and discussion
cancer. Considering the enhanced plasma chemistry of atmospheric
pressure plasmas (APPs), a brand new plasma medicines have been 3.1. Characterization of Fe3O4@CuAl NSs
extensively studied for novel biological applications such as steriliza-
tion, inactivation of bacteria (Von Woedtke et al., 2014), wound healing The specific surface area of as-prepared Fe3O4@CuAl NSs along
(Keidar, 2012), and cancer therapy (Tanaka et al., 2015). In this study, with percent loading of copper was measured by Brunauer-Emmett-
we also have established a plasma source with plasma plume carrying a Teller (BET) method, which shows that the surface area increases as Cu

353
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

Fig. 1. (A) Flow chart of the synthesis mechanism of Fe3O4@CuAl NSs. (B) Schematic representation of experimental setup for direct irradiation of cancer cells (a), photograph of
plasma jet device for direct irradiation of cancer cells (b).

content increases in Fe3O4@CuAl NSs and attains the maximum the flowerlike Fe3O4@CuAl NSs with typical core-shell structure
surface area of 50.66 m2 g−1 at an intermediate loading of 18.96% of constructed by a compact core of 300 nm in diameter and low density
copper (Supporting Table S1). Possibly, higher content of Cu results in horizontally oriented LDH shell, and thickness of shell is around 40 nm
weak crystallization of LDH materials on the surface of core due to the (Fig. 2G). The HRTEM image in Fig. 2H shows that LDH phase has a
formation of Cu oxides such as Cu2O, which caused the decrease of lattice fringe with d-spacing of 0.25 nm, which is well matched with
surface area (Asif et al., 2017). The variation in surface area of various (111) crystal plane of XRD and can be indexed to CuO with monolithic
samples is possibly due to different stacking modes of CuAl LDH layers. texture (Ko et al., 2014).
The morphology and analytical performances of Fe3O4@CuAl NSs XPS characterization was further employed to investigate the
with 18.96% of copper loading have further been investigated. The element composition, and metallic configuration of Fe3O4@CuAl NSs.
concentration ratio of metallic salts and Fe3O4 NSs were adjusted to get A wide scan spectrum as presented in Fig. 3A shows the distinct peaks
different morphologies and size of LDH shell. At low concentration assigned to Cu 2p, Fe 2p and Al 2p along with two predominant peaks
ratio, LDH shell established an even distribution of very small CuAl of carbon and oxygen corresponding to charge complementary hydro-
platelets on Fe3O4 core (Fig. S1A). By increasing the ratio of metallic xyl and carbonate ions existing in between LDH layers. Fig. 3B exhibits
salts and Fe3O4 NSs, a very thick layer of LDH shell was obtained due the core level spectrum of Fe 2p, which is further deconvoluted into
to the agglomeration of CuAl platelets, which entirely covers the whole couple of peaks Fe 2p3/2 and Fe 2p1/2 at 710.46 eV and 724.31 eV,
surface of Fe3O4 NSs. After certain intermediate ratio of metallic salts respectively. Moreover, Fe 2p spectrum of Fe3O4@CuAl NSs shows a
and Fe3O4 NSs, the entire core of Fe3O4 NSs is seemed to be buried weaker peak centered at 710.46 eV correspond to Fe 2p3/2 for Fe3+
completely in LDH platelets like shells (Fig. S1B). Fig. 2 shows the SEM species as compared to Fe3O4, most probably due to its less intensity as
and TEM images of as-synthesized Fe3O4 and Fe3O4@CuAl core-shell well as horizontal intercalation of platelet like multilamellar shell of
nanospheres. It is observed that Fe3O4 particles show smoothed CuAl LDH on the surface of Fe3O4 core (Song et al., 2010). Both
surface, nearly monodispersed spherical structure and uniform size samples contain mixed phase of Fe2+ and Fe3+ because two deconvo-
with mean diameter of 300 nm (Fig. 2A, B and E). After being coated by luted peaks of Fe 2p3/2 appeared at 710.2 eV and 712.1 eV (Wang et al.,
CuAl LDH, the surface of Fe3O4@CuAl NSs becomes rough as 2016). The interaction between core and shell influenced the oxidation
compared with pristine Fe3O4, which is due to the assembly of almost states of metals which arises the possibility of Cu-O-Fe linkages. Fig. 3C
horizontally along with vertically oriented platelets like LDH shells shows the core fitting peaks of Cu 2p3/2 and Cu 2p1/2 originated at
(Fig. 2C). The higher magnification confirms the hydrotalcite like 932.1 eV, 952.4 eV and 934.2 eV, 954.3 eV, corresponding to Cu+ and
typical stacked layers of CuAl LDHs (Yan et al., 2015), (Fig. 2D). The Cu2+ species. Two satellite peaks at around 742 eV and 762 eV are the
unique preparation process by adjusting pH at 10 enriches the surface fingerprint of CuO phase with d9 electronic configuration (Al-Gaashani
of Fe3O4 with negative charge and particular stirring speed, which et al., 2011; Shen et al., 2015).
facilitates the growth of LDH layers on surface of Fe3O4 NSs to Fig. 3D exhibits the typical XRD pattern of Fe3O4, CuAl LDH and
construct core-shell morphology. The TEM image in Fig. 2F presents Fe3O4@CuAl NSs. After in situ growth of LDH shell on Fe3O4 core, the

354
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

Fig. 2. (A) and (B) SEM images of Fe3O4 nanospheres, (C) and (D) SEM images of Fe3O4@CuAl NSs with different magnification. (E) TEM images of Fe3O4 nanospheres, (F) and (G)
Fe3O4@CuAl NSs with different magnification. (H) HRTEM image of the CuAl LDH shell with fringe spacing of 0.25 nm.

superimposition reflections of both phases have been shown by XRD layered rhombohedral structure of LDH (Han et al., 2015). The
pattern. The typical crystal reflections of (003), (006) and (009) appear crystallographic plane of (111) at 38.90° collapsed with a typical
at 2θ value of 11.2°, 23.1° and 34°, respectively, which are attributed to LDH plane of (015), can be attributed to CuO phase in LDHs. The

Fig. 3. (A) Wide scan XPS survey spectra, (B) Fe 2p Core level XPS spectra, (C) Cu 2p Core level XPS spectra of Fe3O4@CuAl NSs and Fe3O4 NSs, (D) XRD patterns of Fe3O4@CuAl NSs,
CuAl LDHs and Fe3O4 NSs (from upper to lower).

355
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

crystal peaks of (220), (311), (400), (511) and (440) at 2θ value of

30.2°, 35.52°, 43.13°, 57.2° and 62.52° respectively are credited to
Fe3O4 phase.

3.2. Electrochemical properties of Fe3O4@CuAl/GCE

To investigate interfacial properties of electrode surface modified by

Fe3O4, CuAl LDH and Fe3O4@CuAl NSs, EIS has been performed with
[Fe(CN)6]3‑/4 redox probe, as shown in Fig. 4A. And the inset is the
Randle equivalence circuit, where Rs, Rct, Cdl and W are the resistance
of solution, charge transfer resistance, double layer capacitance, and
Warburg constant, respectively. The Fe3O4 NSs modified GCE (Fe3O4/
GCE) shows a small Rct value of 708 Ω due to its good conductivity as
compared with Rct value of 1224 Ω corresponding to Fe3O4@CuAl/
GCE. The increased Rct of Fe3O4@CuAl/GCE is due to deposition of
semiconductive CuAl LDH shell on Fe3O4 NS core, which hinders the
fast electron transfer of interface to some extent. The electron transfer
resistance of CuAl LDH modified GCE (CuAl/GCE) increased drama-
tically to 23,280 Ω because of semiconductive properties of LDH
materials, but still less than bare GCE with Rct of 3096 Ω, indicating
fractional charge transfer ability due to the presence of holes within the
layers of LDHs. These evidences indicate enhanced the electron
transfer ability of Fe3O4@CuAl NSs owing to the synergistic effect of
the Fe3O4 core wrapped by CuAl LDHs.
To evaluate electrocatalytic performances of Fe3O4@CuAl/GCE to
H2O2, the cyclic voltammetric (CV) measurements were performed in
the absence and presence of 1 mM H2O2 in deoxygenated 0.1 M PBS
(pH 7.4) (Fig. 4B). In the absence of H2O2, a pair of minor peaks which
can be credited to iron phosphate redox of iron ions combined with
phosphate ions in PBS (Zhang et al., 2008). When H2O2 was added,
Fe3O4@CuAl/GCE exhibited an increased reduction peak at the
potential of −0.70 V. By augmenting H2O2 concentrations in PBS, the
reduction peak current densities also increased linearly (Fig. 4B inset).
Electrocatalytic properties of as-prepared nonomaterial with varied
loading of Cu in shell and CuAl LDH alone were also measured to
compare their potential in H2O2 detection. Benefited from the syner-
gistic effect of Fe3O4 NS core and CuAl LDH shell, the Fe3O4@CuAl/
GCE exhibits enhanced the reduction current density at lower over-
potential as compared to CuAl/GCE, as shown in Fig. 4C. Moreover, by
increasing the deposition of CuAl LDH shell upto transitional composi-
tion of 18.96% copper loading on Fe3O4 NSs, the reduction peak
current is increasing while reduction potential shifts negatively.
Beyond intermediate composition, further increase in copper loading
buried Fe3O4 core completely and resulted in lower reduction peak
current with more negative potential (Fig. S2A). Unlike the CV curves
for CuAl LDH and Fe3O4@CuAl NSs, Fe3O4 NSs catalyst has redox
peaks which are attributed to the Fe(II)/Fe(III) redox couple.
Fig. 4. (A) Nyquist plots of Fe3O4@CuAl/GCE, CuAl/GCE, Fe3O4/GCE and bare GCE in
Compared with Fe3O4 NSs, Fe3O4@CuAl NSs shows well-defined
0.1 M KCl consisting of 1.0 mM K3Fe(CN)6 and 1.0 mM K4Fe(CN)6. Frequency range:
reduction peak which is due to the charge transfer from Fe to CuAl 0.1–105 Hz. Inset is the equivalent circuit. (B) Typical CV curves of Fe3O4@CuAl/GCE in
LDH layers, consequently causes the slight modification in electronic N2 saturated 0.1 M PBS in the absence (black line) and presence (red line) of 1 mM
structure of CuAl LDH in Fe3O4@CuAl NSs improving electrocatalysis H2O2. Inset is the CV curves of Fe3O4@CuAl/GCE in 0.1 M PBS with different
significantly. Therefore, synergistic effect is proposed to arise from the concentrations of H2O2. (C) CV curves of different nanohybrid electrodes in the presence
of 1 mM H2O2 in 0.1 M PBS solution.
partial electron transfer from Fe3O4 to CuAl LDH at nanoscale inter-
face enhancing catalytic activity of as-prepared Fe3O4@CuAl NSs. The
outstanding sensing performances of Fe3O4@CuAl NSs with 18.96% and activated on active sites of highly rough surface created by the shell
copper loading have been shown due to large surface area facilitating of LDH layers on Fe3O4@CuAl NSs as well as fast electron transfer
more surface active sites, the synergetic effect of Fe3O4 NSs and CuAl kinetics. The real time detection limit with signal-to-noise factor of 3
LDHs and unique core-shell morphology which endows maximum (S/N = 3) has also been evaluated by as-synthesized Fe3O4@CuAl/GCE,
interfacial effect between Fe3O4 core and CuAl shell. which is as low as 1 nM (Fig. 5C). These findings are superior than
Amperometric measurements were applied to evaluate the sensi- most of the H2O2 sensors reported in the literature published over the
tivity of Fe3O4@CuAl NSs upon successive additions of H2O2 concen- last year as depicted by Table S2. A broad linear range of reduction
tration in stirring 0.1 M PBS (pH 7.4) at an applied potential of current density versus H2O2 concentration is presented by Fig. 5D. The
−0.70 V, as depicted by Fig. 5A and B. A rapid response was observed curve exhibits three different linear responses in the range of 3–
with the addition of 3 nM up to 10.05 mM H2O2, which achieve the 108 nM, 0.1–1.05 µM and 1.14–10052 µM with R2 values of 0.995,
steady-state current of ∼95% within 3 s. This quick response of 0.999 and 0.997 respectively. The phenomenon of reduction kinetics of
electrode can be credited to the fact that H2O2 being rapidly diffused catalytic processes is controlled by H2O2 absorption at extreme low

356
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

Fig. 5. (A) Amperometric responses of Fe3O4@CuAl/GCE with successive additions of different H2O2 concentrations in N2 saturated 0.1 M PBS at −0.70 V. Inset is amperometric
responses to 10 and 20 nM H2O2. (B) The closed look responses of Fe3O4@CuAl/GCE to several micromolars of H2O2. (C) Current responses for determining detection limit. (D) The
calibration plot of current density vs. H2O2 concentration. (E) Anti-interference property with 1 mM addition of each electroactive species. (F) Time dependence responses with 1 mM
H2O2. Inset is the current responses of six different electrodes to 1 mM H2O2.

concentration and by activation of H2O2 at high concentration, which is and D. BEL-7402 cell line is the fifth common malignancy, owing to
a rate-determining step, whereas in middle region the reduction notoriously high chemoresistive and scarcity of effective therapy, it is
current is controlled by combined effect of absorption and activation ranked as third public issue of cancer related death (Zhang et al.,
of H2O2 on electrode surface (Zhu et al., 2014). 2014). The mouse liver cancer cell line H-22 is one of the common
The selectivity of proposed biosensor against potential interfering malignancies, at most five years of survival after its diagnosis,
substance such as UA, AA, DA, glucose, nitrite has been evaluated at an threatening seriously global public health with high mortality and
applied potential of −0.70 V. As presented by Fig. 5E, the interferences mostly diagnosed after getting an advanced stage (Yang et al., 2014).
upon the addition of 1 mM of interfering species were negligible at an The only way to improve resectability as well as survival rate and get rid
applied detection potential, while the reduction current of 1 mM H2O2 of these lethal attacks is their early diagnosis. With marvelous
reached its extreme value demonstrating preferable selectivity of sensitivity achieved the practical application of as synthesized
resultant biosensor for the detection of H2O2. This excellent anti- Fe3O4@CuAl NSs modified electrode has been explored for real-time
interfering ability of Fe3O4@CuAl/GCE is due to negative operating tracking of H2O2 released by different living cells and therapeutic
potential that limits the oxidation of electroactive substances. The efficacy of plasma medicines has also been assessed in cancer treatment
stability of Fe3O4@CuAl/GCE stored at room temperature was further by the proposed sensing platform. The endogenous H2O2 released was
investigated by recording the current responses of modified electrode triggered by the addition of 0.1 mM fMLP. Upon the addition of 10 µL
to the reduction of 1 mM H2O2 which retained 95% of its initial current fMLP to the test wells, the significant reduction current densities of
value up to 25 days of post preparation (Fig. 5F). To evaluate 0.121 mA cm−2 and 0.0891 mA cm−2 were observed in the presence of
reproducibility of biosensor, six different glassy carbon electrodes were BEL-7402 and H-22 cells, respectively, as presented by Fig. 6B and E.
modified similarly with Fe3O4@CuAl NSs suspension and current Furthermore, current densities gradually decreased to a background
responses of these electrodes were recorded with 1 mM H2O2 providing level upon addition of catalase, indicating that released H2O2 is
relative standard deviation (RSD) value less than 5%. A reproducible decomposed or diffused away from electrode in electrolytic solution.
current with RSD less than 4% has also been observed for six successive The control experiment without cells did not generate any detectable
assays (Fig. 5F inset). These results authenticate the superb stability current signal, further suggesting that the triggered H2O2 efflux was
and reproducibility of proposed biosensor. due to stimulation of cells by fMLP (Fig. 6G).
Because of enhanced plasma chemistry, the therapeutic efficacy of
PAM has been evaluated by detecting extracellular H2O2 released upon
3.3. In vitro tracking of H2O2 in biotic fluids being stimulated by adding 10 µL of fMLP with proposed biosensor
after plasma treatment. The significant decrease in current densities of
The life-threatening public health problem due to leading cause of 0.0334 mA cm−2, 0.0088 mA cm−2 and 0.0196 mA cm−2,
cancer mortalities worldwide, the precise monitoring and potential −2
0.0068 mA cm was observed for H-22 and BEL-7402 cell lines after
therapy of such malignancy have attained wealth of attention. In this being irradiated with plasma plumes for 1 min and 2 min, respectively
work, two different living cancer cell lines, i.e., mouse hepatoma cell (Fig. 6C and F), which decreased 72.39%, 92.15% and 78%, 91% in
line H-22 and human hepatocellular carcinoma cell line BEL-7402 are comparison to that without treatment (Fig. 6H). As manifested by
chosen for real-time monitoring of H2O2 flux after being stimulated. plethora of plasma therapy experiments, that plasma consisting of OH,
And the bright-field images of these living cells are shown in Fig. 6A

357
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

Fig. 6. Bright-field images of (A) H-22 cells, and (D) BEL-7402 cells. Amperometric responses of Fe3O4@CuAl/GCE upon the addition of 10 µL of fMLP in the tested wells with (B) H-
22 cells, (E) BEL-7402 cells, and (G) without cells before plasma treatment, and (C) H-22 cells, and (F) BEL-7402 cells after 1 min (upper) and 2 min (lower) of plasma treatment,
respectively. (H) Corresponding histogram of decreasing amperometric responses after plasma therapy. Amperometric responses of Fe3O4@CuAl/GCE upon an aliquot addition of (I)
serum, and (J) and (K) urine prior and following to the intake of coffee by successive addition of 20 μM and 30 μM H2O2, respectively.

NO and NO2 species interact with liquid to generate numerous ROS et al., 2009). Ultimately, endogenous amount of H2O2 from biofluids has
and RNS like H2O2, NO2−, NO3− which cause imbalancement of been measured by as-synthesized biosensor based on Fe3O4@CuAl/GCE as
oxidants and antioxidants in living cells (Yan et al., 2012). The presented by Fig. 6I-K. The aliquots of serum and urine samples were
increased oxidative stress due to excessive production of H2O2 results inoculated into 15 ml of 0.1 M stirring PBS (pH 7.4) with subsequently
in effective lipid peroxidation and ultimately triggers the cell death. spiking of some standard H2O2 concentrations. The ubiquitous amount of
Moreover, these species after diffusion into cells further interact with H2O2 excreted was determined by using regression equation and found the
protein, DNA, RNA and switch on signal transduction as well as gene results for serum 12 µM, urine before 35.4 µM and after coffee intake
expression leading to induce cell death and change of cellular mor- 51.1 µM respectively. Considerable rise in urinary H2O2 level one hour after
phology (Tanaka et al., 2015). Therefore, most of living cancer cells die the intake of coffee has been noticed. Coffee beans are rich in hydroxy
as exposure time of plasma plumes increased from 1 to 2 min resulting hydroquinone and polyphenols like chlorogenic acid, which is one possible
in decrease of current densities caused by less secretion of H2O2 from cause of upsurge in urinary H2O2 concentration subsequently after coffee
cells. All these findings demonstrate the potential use of proposed intake (Chatterjee and Chen, 2012). It can be concluded that these
biosensor for cancer diagnosis and enhanced therapeutic efficacy of biological molecules are first absorbed into the body and then autoxidize
plasma medicines for cancer treatments as well. upon exposure to oxygen, generating H2O2 after being excreted through
Additionally, H2O2 is a pathogenic factor for various diseases like urine. These results substantially demonstrate the reliable application of
kidney failure, parasitic infection and diabetes, so it is also necessary to Fe3O4@CuAl NSs for cancer detection along with determination of
monitor its exact level in human serum and urine samples (Chandramathi ubiquitous H2O2 in biotic fluids.

358
M. Asif et al. Biosensors and Bioelectronics 97 (2017) 352–359

4. Conclusions Chandramathi, S., Suresh, K., Anita, Z.B., Kuppusamy, U.R., 2009. Parasitology 136,
359–363.
Chatterjee, S., Chen, A., 2012. Biosens. Bioelectron. 35 (1), 302–307.
In summary, a novel hierarchical core-shell structured nanosphere Chen, M., Hou, C., Huo, D., Bao, J., Fa, H., Shen, C., 2016. Biosens. Bioelectron.
hybrid material has been fabricated with controlled integration of p- Ding, N., Yan, N., Ren, C., Chen, X., 2010. Anal. Chem. 82, 5897–5899.
Ganesan, R., Lee, J.S., 2005. Angew. Chem. Int. Ed. 44, 6557–6560.
type semiconductive nanosheets of CuAl LDHs functionalized on Fe3O4 Han, X., Fang, K., Zhou, J., Zhao, L., Sun, Y., 2015. J. Colloid Interface Sci.
NS core via facile, robust co-precipitation and hydrothermal approach. Kaushik, N.K., Kaushik, N., Yoo, K.C., Uddin, N., Kim, J.S., Lee, S.J., Choi, E.H., 2016.
Arising from the drastic interfacial conduction, the synergistic effect Biomaterials 87, 118–130.
Keidar, M., 2012. Cold atmosphere plasma in cancer therapy. APS Meeting Abstracts,
derived from extraordinary intercalation features of LDHs shell and 2005.
Fe3O4 NS core, and the abundant charge carriers, the resultant core- Ko, Y.H., Nagaraju, G., Lee, S.H., Yu, J.S., 2014. Mater. Lett. 117, 217–220.
shell Fe3O4@CuAl NSs hybrid material exhibits enhanced electrocata- Li, C., Zhang, H., Wu, P., Gong, Z., Xu, G., Cai, C., 2011. Analyst 136, 1116–1123.
Liu, C.J., Yu, S.L., Liu, Y.P., Dai, X.J., Wu, Y., Li, R.J., Tao, J.C., 2016a. Eur. J. Med.
lytic properties, The Fe3O4@CuAl NSs hybrid material based electro-
Chem. 115, 26–40.
chemical biosensor has been deviced for quantification of H2O2 under Luo, Y., Tian, Y., Rui, Q., 2009. Chem. Commun. 21, 3014–3016.
biological environment which has exhibited high performance towards Maji, S.K., Sreejith, S., Mandal, A.K., Ma, X., Zhao, Y., 2014. ACS Appl. Mater. Interfaces
sensitive and specific H2O2 detection. The practical application of 6, 13648–13656.
Senapati, S., Thakur, R., Verma, S.P., Duggal, S., Mishra, D.P., Das, P., Shripathi, T.,
biosensor has been explored in detecting endogenous H2O2 concentra- Kumar, M., Rana, D., Maiti, P., 2016. J. Control Release 224, 186–198.
tions in human serum and urine as well as in vitro detection of cancer Shen, W., Yang, C., Bao, X., Sun, L., Wang, N., Tang, J., Chen, W., Yang, R., 2015. Mater.
and evaluating therapeutic efficacy of plasma irradiated medium in Sci. Eng. B 200, 1–8.
Song, S., Yang, H., Rao, R., Liu, H., Zhang, A., 2010. Appl. Catal. A: Gen. 375, 265–271.
cancer therapy. Thus, our findings hold promising application potential Tanaka, H., Mizuno, M., Ishikawa, K., Kondo, H., Takeda, K., Hashizume, H., Nakamura,
in biosensing platform, pathological diagnostics and effectiveness of K., Utsumi, F., Kajiyama, H., Kano, H., Okazaki, Y., Toyokuni, S., Akiyama, Si,
plasma in cancer therapy. Maruyama, S., Yamada, S., Kodera, Y., Kaneko, H., Terasaki, H., Hara, H., Adachi,
T., Iida, M., Yajima, I., Kato, M., Kikkawa, F., Hori, M., 2015. Clin. Plasma Med. 3,
72–76.
Notes Von Woedtke, T., Metelmann, H.R., Weltmann, K.D., 2014. Contrib. Plasma. Phys. 54,
104–117.
Wang, Y., Peng, W., Liu, L., Gao, F., Li, M., 2012. Electrochim. Acta 70, 193–198.
The authors declare no competing financial interest.
Wang, Z., Chen, M., Shu, J., Li, Y., 2016b. J. Alloy. Compd. 682, 432–440.
Weinstain, R., Savariar, E.N., Felsen, C.N., Tsien, R.Y., 2014. J. Am. Chem. Soc. 136,
Acknowledgments 874–877.
Williams, M.J., Sánchez, E., Aluri, E.R., Douglas, F.J., MacLaren, D.A., Collins, O.M.,
Cussen, E.J., Budge, J.D., Sanders, L.C., Michaelis, M., 2016. RSC Adv. 6,
National Natural Science Foundation of China (No. 51572094), the 83520–83528.
Innovation Foundation of Huazhong University of Science (Nos. Wu, P., Cai, Z., Chen, J., Zhang, H., Cai, C., 2011. Biosens. Bioelectron. 26, 4012–4017.
2015TS150 and 2015ZZGH010) and Technology and the Key Xi, J., Xie, C., Zhang, Y., Wang, L., Xiao, J., Duan, X., Ren, J., Xiao, F., Wang, S., 2016.
ACS Appl. Mater. Interfaces 8, 22563–22573.
Laboratory for Large-Format Battery Materials and System, ministry Xi, J., Zhang, Y., Wang, N., Wang, L., Zhang, Z., Xiao, F., Wang, S., 2015. ACS Appl.
of Education and Fundamental Research Funds for the Central Mater. Interfaces 7, 5583–5590.
Universities (No. 2014QN110), We acknowledge the support of the Xu, Z., Yang, L., Xu, C., 2015. Anal. Chem. 87, 3438–3444.
Yahalom, G., Weiss, D., Novikov, I., Bevers, T.B., Radvanyi, L.G., Liu, M., Piura, B.,
Analytical and Testing Center of the Huazhong University of Science Iacobelli, S., Sandri, M.T., Cassano, E., 2013. Biomark. Cancer 5, 71.
and Technology for SEM and XPS measurements. Yan, L.G., Yang, K., Shan, R.R., Yan, T., Wei, J., Yu, S.J., Yu, H.Q., Du, B., 2015. J.
Colloid Interface Sci. 448, 508–516.
Yan, X., Xiong, Z., Zou, F., Zhao, S., Lu, X., Yang, G., He, G., Ostrikov, K.K., 2012. Plasma
Appendix A. Supplementary material Process. Polym. 9, 59–66.
Yang, J., Li, X., Xue, Y., Wang, N., Liu, W., 2014. Int. J. Biol. Macromol. 64, 276–280.
Supplementary data associated with this article can be found in the Yin, X., Guo, M., Xia, Y., Huang, W., Li, Z., 2014. J. Electroanal. Chem. 720–721, 19–23.
Yu, C., Wang, L., Li, W., Zhu, C., Bao, N., Gu, H., 2015. Sens. Actuators B: Chem. 211,
online version at http://dx.doi.org/10.1016/j.bios.2017.05.057. 17–24.
Yuan, M., Liu, A., Zhao, M., Dong, W., Zhao, T., Wang, J., Tang, W., 2014. Sens.
References Actuators. B: Chem. 190, 707–714.
Zhang, L., Zhai, Y., Gao, N., Wen, D., Dong, S., 2008. Electrochem. Commun. 10,
1524–1526.
Ai, F., Chen, H., Zhang, S.H., Liu, S.Y., Wei, F., Dong, X.Y., Cheng, J.K., Huang, W.H., Zhang, Z., Zhang, C., Ding, Y., Zhao, Q., Yang, L., Ling, J., Liu, L., Ji, H., Zhang, Y., 2014.
2009. Anal. Chem. 81, 8453–8458. Food Chem. Toxicol. 63, 38–47.
Al-Gaashani, R., Radiman, S., Tabet, N., Razak Daud, A., 2011. J. Alloy. Compd. 509, Zhu, H., Sigdel, A., Zhang, S., Su, D., Xi, Z., Li, Q., Sun, S., 2014. Angew. Chem. Int. Ed.
8761–8769. 126, 12716–12720.
Asif, M., Aziz, A., Dao, A.Q., Hakeem, A., Wang, H., Dong, S., Zhang, G., Xiao, F., Liu, H., Zhu, L., Zhang, Y., Xu, P., Wen, W., Li, X., Xu, J., 2016. Biosens. Bioelectron. 80,
2015. Anal. Chim. Acta 898, 34–41. 601–606.
Asif, M., Haitao, W., Shuang, D., Aziz, A., Zhang, G., Xiao, F., Liu, H., 2017. Sens
Actuators B: Chem. 239, 243–252.
Cardoso, A.R., Moreira, F.T., Fernandes, R., Sales, M.G., 2016. Biosens. Bioelectron. 80,
621–630.

359