Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18

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Journal of the Taiwan Institute of Chemical Engineers

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

Low energy nitrogen ion beam implanted tungsten trioxide thin films
modified indium tin oxide electrode based acetylcholine sensor
A.C. Anithaa a, K. Asokan b, C. Sekar a,∗
a
Department of Bioelectronics and Biosensors, Alagappa University, Karaikudi, 630 003, TN, India
b
Materials Science Division, Inter-University Accelerator Centre, New Delhi, 110067, India

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

Article history: This paper reports the fabrication of non-enzymatic acetylcholine (ACh) electrochemical sensor based on
Received 7 September 2017 low energy nitrogen ion (100 keV) implanted tungsten trioxide (WO3 ) thin films modified indium tin
Revised 2 January 2018
oxide (ITO) electrode. The WO3 thin films deposited on ITO by spin coating technique was subjected
Accepted 3 January 2018
to nitrogen ion implantation with the optimum fluence of 1 × 1015 ions/cm2 . The implanted electrode
Available online 17 January 2018
was characterized by X-ray diffraction, field emission scanning electron microscopy (FESEM), photolumi-
Keywords: nescence spectra, Hall Effect measurements, cyclic voltammetry and amperometry. The implanted WO3
Low energy ion beam implantation modified ITO electrode induces desirable changes in the crystal structure, surface morphology and elec-
WO3 thin films tron mobility. Compared with pristine WO3 /ITO, nitrogen ion implanted WO3 /ITO displayed improved
Acetylcholine electrical conductivity and electrocatalytic activity towards ACh oxidation in 0.1 M potassium hydroxide
Non-enzymatic solution (KOH). Amperometric measurements showed that the fabricated electrode had a wide linear
Electrochemical sensor
response range of 0.1–80 0 0 μM, higher sensitivity of 140.57 ± 0.62 mA/M/cm2 and the lowest detection
limit of 28 nM, which are superior among the non-enzymatic ACh electrochemical sensors. The proposed
sensor exhibits an excellent anti-interferential ability, good stability and reproducibility and successfully
applied for the determination of ACh levels in human serum samples.
© 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction mass spectrometry [4], high performance liquid chromatogra-

Acetylcholine (ACh) was the first discovered neurotransmitter
which can be found in the central nervous system, neuromuscu-
lar junctions, spinal cord and preganglionic and motor neurons [1].
It acts as a key link in the communication between neurons in
the spinal cord and nerve skeletal junctions in vertebrates, and
also plays an important role in transmitting signals in the brain.
In peripheral nervous system, ACh binds to acetylcholine recep-
tors (AChR) and regulates muscle contraction whereas in the cen-
tral nervous system, it plays a crucial role in the processes related
to behavioural activities such as arousal, attention, learning and
memory [2]. Abnormal levels of ACh are associated with nerve dis-
orders including Parkinson’s disease, Alzheimer’s disease, progres-
sive dementia, Schizophrenia and motor dysfunction [3]. Therefore,
in vitro and in vivo determination of ACh in biological samples is
of considerable interest in neuronal cholinergic system research.
Various methods had been reported for ACh detection which
includes matrix-assisted laser desorption ionization time-of-flight
∗
Corresponding author.
E-mail address: Sekar2025@alagappauniversity.ac.in (C. Sekar).
phy coupled to post-column chemiluminescence detection [5],
gas chromatography mass spectrometry [6], capillary zone elec-
trophoresis [7] and enzymatic electrochemical biosensors [8]. Over
the past decade, development of electrochemical non-enzymatic
ACh biosensors has risen at considerable rate for ACh detection.
The advantages include simple design, cost effectiveness, good
stability and effective enzyme-like catalysis against temperature
and pH [9]. Nanostructured metal oxides have recently aroused
tremendous interests in a broad range of technological applications
because of their significant size and shape-dependent properties,
attractive nanomorphologies, functional biocompatibility, superior
electron-transfer kinetics, non-toxic properties and high biological
activity leading to enhanced sensing characteristics [10]. Among
various nanostructured oxides of metals, tungsten trioxide (WO3 ),
an n-type indirect semiconductor with wide band gap (Eg ∼ 2.5
– 3.6 eV), possesses distinctive features of polymorphic structural
flexibility, high specific capacitance, high surface-to-volume ratio,
high chemical stability, environmental compatibility and high reac-
tivity [11,12]. Here, we propose a new strategy of using low energy
nitrogen ion implantation on surfactant assisted WO3 thin films
modified indium tin oxide (ITO) for an efficient electrocatalytic ox-
idation towards ACh.

https://doi.org/10.1016/j.jtice.2018.01.001
1876-1070/© 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
12 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18
Ion implantation has been employed to modify the surface
chemistry and to regulate the depth of penetration by enhanc-
ing the physic-chemical properties of semiconducting nanostruc-
tures [13]. Several approaches for physic-chemical properties have
been exploited using microwave irradiation, gamma rays, ultravio-
let (UV) light, visible light, electron beams and ion beams. We have
previously studied the effects of microwave, gamma ray and swift
heavy ion irradiation on WO3 NPs and investigated their sensing
performances by fabricating electrochemical sensors for the de-
tection of L-dopa, dopamine, serotonin, epinephrine, xanthine and
guanine, respectively [14–18]. Low energy ion beam (LEIB) implan-
tation is found to be an effective tool to incorporate foreign ions
intrinsically into the host lattice which modifies the surface com-
position and consequently the properties of host counterparts [19].
Zhang et al., observed the effect of nitrogen ion implantation on
tungsten surfaces leading to the formation of the Wx (O,N) and
WN which are not thermodynamically favoured. Meanwhile, sta-
ble WO3 is also formed at the near-surface region of the chamber
[20]. As reported by Livraghi et al., ion doping is an attractive ap-
proach to heal the oxygen vacant sites in a TiO2 lattice and N-ion
carriers are of particular interest for DSSC applications [21]. Sud-
hagar et al. examined the nitrogen ion implanted TiO2 photoan-
odes to fabricate quantum-dot sensitized electrochemical cells [22].
Diwald et al. investigated the influence of nitrogen ion implanta-
tion on the photoactivity of the rutile TiO2 single crystal surface
[23]. Accordingly, we expect ion-doped metal oxides band bend-
ing due to modification in the electron concentration, morphology
and favourable for efficient charge separation at interfacial electron
transfer sites. Nevertheless, to the best of our knowledge, nitrogen
ion implanted on WO3 /ITO electrode has not been used for sensor
applications.
In the present study, WO3 thin films were deposited on
ITO substrate by spin coating method and were implanted with
100 keV nitrogen ions under optimum fluence of 1 × 1015 ions/cm2 .
The effect of LEIB on the morphological, optical, electrical and elec-
trochemical behaviour of implanted WO3 thin films has been in-
vestigated. The electrochemical kinetics of the fabricated electrode
was studied and finally applied to the determination of ACh by
voltammetric and amperometric methods.
2. Experimental
2.1. Reagents
Acetylcholine chloride was purchased from Alfa Aesar, and the
stock solution was prepared by dissolving 9.08 mg of acetylcholine
chloride in 50 mL of de-ionized water (18 MΩ/cm). Tungstic acid,
ethylene diamine tetra acetic acid and sodium hydroxide solutions
were purchased from Fischer Scientific. ITO plates were purchased
from Techinstro Technologies Limited. The solution of phosphate
buffer (PBS, 0.1 M) was prepared by mixing Na2 HPO4 and NaH2 PO4 .
2.2. Apparatus
Powder X-ray diffraction (XRD) patterns were recorded using
a Bruker AXS D8 advanced diffractometer in the range of 20–
80°. FESEM image and EDAX spectra were recorded using FEG
QUANTA 250. Photoluminescence studies of the films were done
by Optistat Oxford Instruments (Varian Cary Eclipse). The electri-
cal properties of the films have been performed by Ecopia HMS
30 0 0 van der Pauw Hall effect measurement system at room tem-
perature. Electrochemical experiments were accomplished using a
CHI 609D electrochemical workstation (CHI, USA) with a standard
three-electrode cell. An indium tin oxide (ITO) substrate was used
as the working electrode (working area of 1 × 1 cm2 ), a Pt wire as
a counter electrode and Ag/AgCl as a reference electrode.
2.3. Low energy ion beam implantation on WO3 thin films
The WO3 NPs obtained from the previous procedure [16] were
dispersed in de-ionized water solution (5 mg/ml) and sonicated for
1 h. Subsequently, the homogeneous suspension was coated on
ITO substrate. A simple droplet technique was adopted to make
WO3 /ITO electrode, where 100 μl of this spreading solution was
dispensed onto the ITO electrode and a standard spinner (Spin
NXG-P1) was employed to spin the electrode at different speeds
ranging from 100 to 10 0 0 rpm for 200 s. The resulting thin films
were dried at 60 °C for 1 h. The WO3 thin films on ITO electrode
have been implanted with nitrogen ion using the low-energy ion
beam facility (LEIBF) at Inter University Accelerator Centre, New
Delhi. The ion energy and beam current of nitrogen ion beams
were set to 100 keV and 1 μA, respectively and implanted with the
optimized fluence of 1 × 1015 ions/cm2 . Ion beam was allowed to
incident on the scanned area of 1 × 1 cm2 .
2.4. Sensing system
The activation of modified electrodes were performed by shift-
ing the prepared electrodes to the electrolyte (0.1 M KOH), and
subjecting it to 100 cyclic voltammetry (CV) cycles across the po-
tential range −0.3 to 0.9 V (vs. Ag/AgCl), at a scan rate of 50 mV/s.
CV measurements were recorded for 500 μM of ACh at the scan
rate of 50 mV/s and the response current was documented. Am-
perometric measurements were conducted for the quantification
of ACh by the nitrogen ion implanted WO3 /ITO at + 0.595 V (vs.
Ag/AgCl) in 10 mL of stirred alkaline solution at the rotation rate of
400 rpm. The geometric surface area of the thin film is 0.4325 cm2 .
3. Results and discussion
3.1. Physical characterization of nitrogen ion implanted WO3 thin
films
The XRD patterns of WO3 thin films coated on ITO electrodes
before and after nitrogen ion implantation are shown in Fig. 1A.
The pristine WO3 displays low intensity diffraction peaks suggest-
ing that the film is of poor crystalline quality. Upon nitrogen ion
implantation, the peak intensity increases and the peak widths
get tapered indicating that the induced grain growth occurred
due to localized heat generation which melt the particle surface
through ion-matter interactions. The diffraction peaks of both pris-
tine and implanted WO3 could be well-indexed to intrinsic mono-
clinic structure of WO3 (JCPDS No. 72–0677). However, there exists
trivial change in the lattice parameters of the implanted WO3 /ITO,
implying a slight lattice distortion in the W-O framework after
nitrogen ion implantation. The average crystallite size of pristine
WO3 and the implanted WO3 were found to be 35 nm and 53 nm,
respectively. The significant increase in crystallite size confirms the
grain growth during ion implantation.
Fig. 1B illustrates the room temperature photoluminescence
(PL) spectra of pristine and nitrogen ion implanted WO3 thin films.
The high intensity peaks at 420 nm and 488 nm are associated with
oxygen vacancies and defect concentration, respectively [24]. Com-
pared to pristine, increment in peak intensity occurs at nitrogen
ion implanted WO3 thin films which could be attributed to the fast
nucleation growth of the WO3 octahedral group with high crys-
tallinity and higher recombination rate.
Surface morphology of the implanted WO3 thin films were ex-
amined using FESEM. The pristine WO3 film shows agglomerated
clusters with near spherical shapes (Fig. 1C). After the exposure of
nitrogen ion implantation, the spherical WO3 crystallites were con-
verted into bean-like morphology as shown in Fig. 1D. It is worthy
 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18 13

Fig. 1. (A) XRD patterns and (B) PL spectra of pristine WO3 thin films (curve a) and nitrogen ion (1 × 1015 ions/cm2 ) implanted WO3 thin films (curve b) coated on ITO
substrates; FESEM images of pristine WO3 /ITO (C) and nitrogen ion implanted WO3 /ITO (D).

Table 1 fect results correlate well with PL spectra result for the same sam-
Electrical properties of the WO3 thin films before and after nitrogen ion implanta-
ple.
tion.

Samples Carrier concentration Mobility Conductivity

(cm−3 ) (×102 cm2 /Vs)
Pristine WO3 /ITO (1.65 ± 0.02) x 1015 3 ± 0.4
Nitrogen ion implanted (92 ± 2.3) x 1015 58 ± 2.2
WO3 /ITO
to note that the ion implantation gives rise to inter-planar neck-
ing defects and detrimental damage which might act as trapping
centers for the biomolecules detection. The energy dispersive X-
ray spectroscopy (EDX) analysis confirmed the oxygen loss during
nitrogen ion implantation (see Fig. S1).
Table 1 shows the Hall Effect measurements of the WO3 thin
films before and after implantation. It can be noticed that the car-
rier concentration and mobility are superior for the nitrogen ion
implanted WO3 when compared with pristine WO3 thin films. The
increase in carrier concentration and mobility of the implanted
WO3 thin films suggest the substitution of nitrogen ions on O sites
which increases the conductivity of the thin films [25]. The Hall Ef-
(−1 /cm) 3.2. Electrochemical characterization of nitrogen ion implanted WO3
thin films
1.02 ± 0.02
44 ± 0.5
Fig. 2A shows the cyclic voltammograms (CV) of 0.1 M KCl so-
lution containing 1 mM [Fe (CN)6 ]3−/4− obtained at the bare ITO
(curve a), pristine WO3 /ITO (curve b) and nitrogen ion implanted
WO3 /ITO (curve c) with the scan rate of 50 mV/s. The implanted
WO3 thin films showed a positive peak potential shift and a
decrease in redox peak currents (Ip ), which could be attributed
to the electrostatic repulsion between the negatively charged
[Fe(CN)6 ]3-/4− anion and the negatively charged nirogen ion im-
planted WO3 electrode surface. In order to confirm this, we have
carried out CV studies at WO3 modified ITO in 0.1 M KCl contain-
ing positively charged redox probe 1 mM [Ru (NH3 )6 ]2+/3+ with the
scan rate of 50 mV/s (Fig. 2B). The [Ru (NH3 )6 ]2+/3+ marker shows
a remarkable enhancement in electron transfer rate at the im-
planted WO3 /ITO compared to bare ITO and pristine WO3 /ITO. This
is expected because the electrostatic attraction between positively
charged [Ru (NH3 )6 ]2+/3+ and the negatively charged nitrogen
14 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18

Fig. 2. Cyclic voltammograms measured at a scan rate of 50 mV/s in 0.1 M KCl solution containing 1 mM each (A) [Fe(CN)6 ]3−/4− and (B) [Ru(NH3 )6 ]2+/3+ at the bare ITO
(curve a), pristine WO3 /ITO (curve b) and nitrogen ion implanted WO3 /ITO (curve c).

ion implanted WO3 /ITO electrode surface facilitates the diffusion

of [Ru (NH3 )6 ]2+/3+ ions to the electrode surface. These results are
in good agreement with the PL and Hall Effect studies and con-
firm that the implanted WO3 modified ITO electrode demonstrate
selectivity by charge exclusion.
Fig. S2 demonstrates the Nyquist plots of bare ITO, pristine
WO3 /ITO and implanted WO3 /ITO in 0.1 M KCl solution contain-
ing 1 mM [Fe(CN)6 ]3-/4− (Fig. S2A) and 1 mM [Ru(NH3 )6 ]2+/3+ (Fig.
S2B). The insert displayed the equivalent circuit (Randles model)
used to fit Nyquist diagrams for both the electrolytes. In the case
of [Fe(CN)6 ]3-/4− , the Rct value was found to be higher for im-
planted WO3 /ITO (1606 Ω/cm2 ) when compared to that of bare ITO
(253 Ω/cm2 ) and pristine WO3 /ITO (734 Ω/cm2 ). Contrary to this,
the Rct values for [Ru(NH3 )6 ]2+/3+ was found to be lower for im-
planted WO3 /ITO (108 Ω/cm2 ) when compared to that of bare ITO
(9688 Ω/cm2 ) and pristine WO3 /ITO (7394 Ω/cm2 ). At the nitrogen
implanted WO3 /ITO electrode, the electron transfer of Fe(CN)6 3-/4−
ions is completely blocked, whereas no such blocking effects oc-
cur for Ru(NH3 )6 3+/4+ ions. This distinguished difference in elec-
trochemistry between Fe(CN)6 3-/4− and Ru(NH3 )6 3+/4+ at nitrogen Fig. 3. Cyclic voltammograms recorded for the 500 μM ACh at the bare ITO (curve
a), pristine WO3 /ITO (curve b), and nitrogen ion implanted WO3 /ITO (curve c) at a
implanted WO3 /ITO electrode might be due to the electrostatic ef-
scan rate of 50 mV/s.
fects of the carrying charges of the electrochemical probes and the
surface charges of the nitrogen ions and WO3 electrode. hydrolyzed in base aqueous solution to produce acetic acid an-
ion and 2-Hydroxy-N,N,N-trimethylethan-1-aminium cation, then
3.3. Electrocatalytic oxidation of ACh at nitrogen implanted WO3 /ITO WO3 gets oxidized to form WOH(OH)2 /WO(OH) catalytic system
electrodes with adsorbed choline molecules. Subsequently, the alcohol group
of choline get oxidized to the carboxylic acid by WO(OH)/WO2 . At
Cyclic voltammograms (CV) recorded for 500 μM ACh at bare the electrode-electrolyte interface, availability of more OH- ions fa-
ITO (curve a), pristine WO3 /ITO (curve b) and implanted WO3 /ITO cilitates ACh hydrolysis which enhances choline content resulting
(curve c) in 0.1 M KOH solution at a scan rate of 50 mV/s are in higher response current at the electrode surface. Hence, we have
shown in Fig. 3. In case of bare ITO (curve a), there is no re- chosen nitrogen ion implanted WO3 /ITO electrode as sensing ele-
sponse for ACh. For the pristine WO3 /ITO (curve b), a minimal re- ment for further investigations.
sponse current was observed for ACh oxidation and reduction in
alkaline solution. This is in accordance with the typical CV of ACh 3.4. Effect of scan rate
oxidation on the Ni-based electrode in alkaline solution [26]. In-
terestingly, nitrogen implanted WO3 modified ITO displayed a sig- Fig. 4A shows CVs of implanted WO3 /ITO recorded at different
nificant improvement towards the electro-oxidation of ACh with scan rates in the range of 10–500 mV/s. The redox peak current
an enhanced peak current (Ipa = 25 μA) which is five times com- linearly increases with the increase in scan rate. The observed lin-
pared to that of bare and pristine modified WO3 electrodes. This ear relationship between the plot of square root of the scan rate
interesting phenomenon can be attributed to the abundant nega- (ʋ1/2 ) vs. the redox peak currents (Fig. 4B) indicates that the elec-
tive surface charges on nitrogen implanted WO3 surface which at- trocatalytic process was controlled by ACh diffusion in 0.1 M KOH
tracts positively charged ACh due to high degree of porosity and solution with corresponding coefficients of 0.9991 and 0.9994 con-
diffusion paths for the analyte to permeate into the nanostruc- forming to oxidation and reduction, respectively;
ture. Based on these results, the possible mechanism of ACh oxi-
Ipa (μA ) = −4.891 − 0.1452 υ 1/2 (Vs−1 )1/2
dation [27] on the fabricated electrode can be depicted as shown
in Scheme 1. The mechanism of electrocatalytic behaviour of ACh
at implanted WO3 /ITO has been described as follows; ACh gets Ipc (μA ) = 4.573 + 0.1119 υ 1/2 (Vs−1 )1/2
 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18 15

Scheme 1. Electrocatalytic oxidation of ACh at nitrogen ion implanted WO3 thin film.

Fig. 4. Cyclic voltammograms (A) of nitrogen ion implanted WO3 /ITO in the presence of 500 μM ACh in 0.1 M KOH at various scan rates; the numbers 1–10 correspond to
10, 25, 50, 100, 150, 200, 300, 400 and 500 mV/s; (B) the calibration plots for the redox peak currents (Ipa ) vs. the square root of scan rate (ʋ1/2 ); (C) Variation of the anodic
peak potential vs. natural logarithm of scan rate (ν ); and (D) Variation of anodic peak potential vs. scan rate.

The electrocatalytic reaction of ACh at the modified electrode ing equation [29]:
was a diffusion controlled process. The values of the diffusion co-  RT 
efficient and electroactive area can be estimated by using Randles– Ep = ln ν + constant (7)
Sevick equation [28], 2α F

  Based on this Eq. (7), the electron-transfer coefficient for the

Ipa = 2.995 ∗ 105 α 1 / 2 n 3 / 2 A D 1 / 2C υ 1 / 2
Where Ipa is the anodic peak current, D is the diffusion coeffi-
cient, and C is the bulk concentration of ACh. From the slope of
ʋ1/2 vs. Ipa , the diffusion coefficient of ACh was obtained to be 1.45
∗ 10−4 cm2 s−1 . The value of the electron-transfer coefficient (α )
can be found based on the dependency of anodic peak potential
vs. the natural logarithm of the scan rate (Fig. 4C) and the follow-
(6) electro-oxidation of ACh on the implanted WO3 /ITO surface was
obtained as 0.67. By substituting the values of D and α in Eq. (6),
the electroactive surface area (0.89 cm2 ) calculated for nitrogen ion
implanted WO3 /ITO is higher compared to pristine (0.26 cm2 ) WO3
modified ITO electrodes. From Fig. 4D, the formal potential (E0 )
was deduced from the intercept of the variation of peak poten-
tial as a function of the scan rate plot. Further, the electron trans-
fer rate constant (ks ) value calculated using Eq. (8) from Laviron
16 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18

Fig. 5. (A) Amperometric response of nitrogen ion implanted WO3 /ITO in 0.1 M KOH containing different concentrations of ACh (0.1–80 0 0 μM) added in steps of (a) 0.1 μM,
(b) 10 μM, (c) 100 μM and (d) 10 0 0 μM. The inset shows a magnified view of the response for ACh from 0.1 to 80 μM; (B) Calibration plots of the peak current as a function
of ACh concentration within the range of 0.1–10 0 0 μM and 10 0 0–80 0 0 μM.

Table 2
Analytical parameters of ACh determination were compared with different modified electrodes.

Electrode E(V) Linear range (μM) Sensitivity (mA/M/cm2 ) Detection limit (μM) Ref.

Cu NPs 0.65 120–2680 – 39 [31]

Ni microspheres /C microparticles / Nafion 0.67 0.24–828 48.58 ± 0.52 0.049 [32]
NiAl-LDH/ ordered mesoporous carbon 0.6 2–4922 – 0.042 [33]
Nickel oxide nanostructure – 250–5880 392.4 ± 2.39 26.7 [34]
NiAl-LDH/CD composites 0.65 5–6885 133.2 ± 0.03 1.7 [35]
Nitrogen ion implanted WO3 /ITO 0.595 0.1–80 0 0 140.57 ± 0.62 0.028 This work

Cu – Copper, C–Carbon, LDH – Layered double hydroxides, CD – Carbon dots.

equation [30] was found to be 50.8/s for nitrogen ion implanted ACh electrochemical sensors [31–35]. It can be noticed that the
WO3 /ITO. fabricated sensor offers lower oxidation potential, wider linear
 RT  α nF E
range and lowest detection limit than all the other reported sen-
lnks = α ln (1 − α ) + (1 − α )l nα − l n − (1 − α ) sors. Compared with pristine WO3 /ITO, the low energy ion beam
nF ν RT implanted WO3 surface has higher crystallinity and unique mor-
(8) phology which effectively increases the electrode-electrolyte con-
tact area leading to fast transfer kinetics, enhanced electroconduc-
These results demonstrate that the fabricated electrode has
tivity, good biocompatibility and improved affinity towards ACh.
superior electron transfer kinetics which promotes the electro-
oxidation of ACh.
3.6. Selective detection of ACh at nitrogen ion implanted WO3 / ITO
electrode
3.5. Sensitivity studies of ACh determination

In order to study the selectivity of the nitrogen ion implanted

Amperometry method was employed for electrochemical deter-
WO3 /ITO towards the detection of ACh, amperometric measure-
mination of ACh in 0.1 M KOH solution at +0.595 V (vs. Ag/AgCl).
ments were carried out in the presence of several potential phys-
Fig. 5A shows the results for the nitrogen ion implanted WO3 /ITO
iological interferents such as ascorbic acid (AA), uric acid (UA),
electrode under optimized conditions for the successive increment
epinephrine (EP), dopamine (DA), norepinephrine (NEP), serotonin
of ACh concentrations. The inset in Fig. 5A shows a magnified view
(5-HT) and tyrosine (Ty). It was found that a 500-fold excess of AA
of the typical current-time response for early additions of ACh.
and UA, 10-fold excess of DA, EP, NEP and 5-HT and 100-fold ex-
The linear relationship between the calibrations curves of ACh con-
cess of tyrosine, cysteine, melatonin, folic acid and glucose did not
centration as a function of the corresponding catalytic currents is
interfere with the measurement of 5 μM ACh as shown in Fig. 6.
shown in Fig. 5B. The nitrogen ion implanted WO3 modified ITO
Successively, the addition of 10 0 0-fold excess of metal ions such
electrode exhibited a linear response over a very wide range of
as Na+ , Fe2+ and K+ also did not interfere with the ACh oxida-
0.1 to 80 0 0 μM ACh. The corresponding linear regression equations
tion potential. No chemical interference was observed for all the
are
  potentially interfering target compounds due to the fact that the
IACh (μA ) = 1.9659 + 0.0608 CACh (0.1 − 10 0 0 μM ) R2 = 0.9985 diffusion of these molecules in the alkaline solution are rejected
by the modified WO3 /ITO electrode surface.
 
IACh (μA )=55.4542+0.0 075 CACh (10 0 0−80 0 0 μM ) R2 = 0.9988
3.7. Stability and reproducibility of nitrogen ion implanted WO3 / ITO
The lowest limit of detection (LOD) is estimated to be 28 nM electrode
(average) for ACh at a signal/noise ratio of 3, and the sensitivity is
140.57 ± 0.62 mA/M/cm2 . Table 2 summarizes the present results in The reproducibility a study of the fabricated sensor was car-
comparison with those of other previously reported non-enzymatic ried out on five replicated WO3 /ITO electrodes prepared under the
 A.C. Anithaa et al. / Journal of the Taiwan Institute of Chemical Engineers 84 (2018) 11–18
Fig. 6. Amperometric response of the nitrogen ion implanted WO3 /ITO for the ad-
dition of 5 μM ACh with successive additions of interferents in the sequence of (a)
Ascorbic acid (b) uric acid, (c) dopamine, (d) norepinephrine, (e) epinephrine, (f)
cysteine, (g) serotonin, (h) L-tyrosine, (i) folic acid, (j) glucose, (k) melatonin, (l)
Na+ , (m) Fe2+ and (n) K+ ions in 0.1 M KOH.
same conditions. These five tests yielded a relative standard devi-
ation of 4.1% for the determination of ACh. In order to verify the
stability of the nitrogen ion implanted WO3 /ITO electrode by CV in
0.1 M KOH solution containing 500 μM ACh, 75 continuous cycles
were carried out at a scan rate of 50 mV/s and the results shows
only slight change in the oxidation peak current (<1.3%) shown
in Fig. S3. The long-term stability of the modified electrode was
validated by recording the CV response of 500 μM ACh in 0.1 M
KOH solution for a period of 3 months. The peak potential of ACh
oxidation remained almost the same and the current signals re-
tained 98.2% of its initial response. Thus, the proposed sensor held
satisfactory performances in terms of both stability and repeata-
bility.
3.8. Practical applications of the fabricated electrode
The normal ACh concentration range in mammalian nervous
system is 20–60 μM, which is well within the linear response
range of the prepared electrochemical sensor [36]. To confirm the
practical usefulness of the proposed method, the nitrogen ion im-
planted WO3 /ITO sensor was applied to detect ACh in serum sam-
ples by amperometry measurements. Samples were obtained from
healthy volunteers and stored frozen. Before spiking with ACh,
the serum samples were diluted (1:10) with the supporting elec-
trolyte and filtrated through a 5 μm filter to produce protein-free
human serum. The observed oxidation current was compared with
that of known concentration of commercial ACh and the con-
tent was calculated to be 22.3 μM for 70 μl. Subsequent measure-
ments were carried out by adding known amount of ACh to 5.5 ml
of serum sample in 0.1 M KOH solution. The protein-free spiked
serum solutions were directly transferred into the electrochemi-
cal cell, and the amperometric measurements were analyzed by
using the standard addition procedure [17] to confirm the accu-
racy of this method. The results of ACh determination by proposed
amperometric method and referenced radioimmunoassay method
[37] are reported in Table 3. The high recovery percentage reveals
that the modified electrode has an excellent capability for accurate
determination of ACh in serum samples which could be used for
clinical diagnosis.
17
Table 3
Determination of ACh in human serum samples based on nitrogen ion implanted
WO3 /ITO.
Sample Added Founda (μM) Recovery Reference
(μM) (%) methodb [37]
Human serum 0 22.3 (±0.3) – 456 pg/ml
5 28.2(±0.1) 99.6 –
10 34.1(±0.3) 97.3 –
15 38.4(±0.2) 98.4 –
a
Average of five determinations at optimum conditions.
b
Reference method compared: Radioimmunoassay.
4. Conclusions
In summary, 100 keV nitrogen ion implantation process has
been adopted for the alteration of spin coated WO3 thin films on
ITO electrode. This implantation causes significant changes on the
morphological, optical, electrical and electrochemical properties of
WO3 thin films. A systematic study revealed that the optimum flu-
ence of 1 × 1015 nitrogen ions/cm2 on WO3 /ITO films is more suit-
able for electrocatalytic detection of ACh in the presence of sev-
eral interfering physiological biomolecules. Remarkably, the lowest
detection limit of 28 nM has been obtained for ACh over a wide
dynamic range of 0.1 to 80 0 0 μM for N ion implanted WO3 /ITO
in potassium hydroxide solution. The major improvements in the
electrocatalytic activity of the fabricated electrode towards the ox-
idation of ACh have been attributed to the low energy ion beam
induced improvement in crystallinity, high surface density defects
and higher conductivity. It is demonstrated that the proposed pro-
cess can be used for the determination of ACh in human blood
samples with high sensitivity, stability, reproducibility, and selec-
tivity.
Acknowledgements
Authors ACA & CS acknowledge the Department of Science and
Technology (DST-SERB –SB/S2/LOP-027/2013) for the financial as-
sistance. This research was also partly supported by the Inter Uni-
versity Accelerator Centre (IUAC) sponsored project, New Delhi
(IUAC-UFR-50312).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.jtice.2018.01.001.
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