Voltammetric determination of epinephrine and xanthine based on sodium dodecyl sulphate assisted tungsten trioxide nanoparticles

Article abstract of DOI:10.1016/j.electacta.2017.03.098

This work reports the voltammetric determination of epinephrine (EP) and xanthine (XA) using gamma irradiated tungsten trioxide (WO₃) nanoparticles. WO₃ NPs synthesized by anionic surfactant sodium dodecyl sulphate (SDS) assisted template method was subjected to gamma rays under different doses (0-150 kGy). The experimental results revealed that the 50 kGy irradiation on SDS-WO₃ NPs led to significant changes in crystallite size and morphology which enriched its electrocatalytic activity greatly. The electrochemical behaviors of EP and XA at 50 kGy gamma irradiated SDS-WO₃ modified glassy carbon electrodes (GCE) were studied by cyclic voltammetry and differential pulse voltammetry. The outcomes confirmed that the proposed electrode demonstrate excellent electrocatalytic activity towardsthe oxidation of EP and XA in phosphate buffer solution (PBS, pH 7.0). The fabricated electrode possess lowest detection limit of 1.8?nM and 2.2?nM for EP and XA respectively with the very wide dynamic linear range of 9?nM to 1?mM. Finally, the modified sensor was successfully implemented to detect EP and XA in the human serum samples with excellent selectivity, good stability and reproducibility.

Full text of DOI:10.1016/j.electacta.2017.03.098

Accepted Manuscript
Title: Voltammetric determination of epinephrine and xanthine
based on sodium dodecyl sulphate assisted tungsten trioxide
nanoparticles
Authors: A.C. Anithaa, K. Asokan, C. Sekar
PII:
S0013-4686(17)30570-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.electacta.2017.03.098
EA 29132
To appear in:
Electrochimica Acta
Received date:
Revised date:
Accepted date:
24-11-2016
12-3-2017
14-3-2017
Please cite this article as: A.C.Anithaa, K.Asokan, C.Sekar, Voltammetric determination
ofepinephrineandxanthinebasedonsodiumdodecylsulphateassistedtungstentrioxide
nanoparticles, Electrochimica Actahttp://dx.doi.org/10.1016/j.electacta.2017.03.098
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<AT>Voltammetricdetermination ofepinephrine and xanthine based on sodium dodecyl sulphate
assisted tungsten trioxide nanoparticles
<AU>A.C. Anithaaa, K. Asokanb, C. Sekara,* ##Email##Sekar2025@gmail.com##/Email##
<AU>
<AFF>aDept. of Bioelectronics and Biosensors, Alagappa University, Karaikudi 630003, TN,
India
<AFF>bMaterials Science Division, Inter-University Accelerator Centre, New Delhi 110067,
India
<PA>Tel.: +91 9442563637.
<ABS-Head><ABS-HEAD>Graphical abstract
<ABS-P>► The gamma ray irradiated (50 kGy) sodium dodecyl sulphate (SDS) assisted
tungsten trioxide (WO3) nanoparticles with columnar polyhedron morphology acts as an
excellent electrocatalyst which remarkably manifested the simultaneous determination of
epinephrine and xanthine.
<ABS-HEAD>Highlights► A novel electrochemical sensor has been developed for the
simultaneous determination of epinephrine and xanthine based on gamma irradiated SDS-WO3
NPs for the first time. ► The fabricated sensor exhibits wide linear range (0.009-1000 µM), low
detection limit (1.8 nM for EP and 2.2 nM for XA), good stability and reproducibility. ► The
proposed method was successfully applied to the analysis of human serum samples with
satisfactory results.
<ABS-HEAD>ABSTRACT
<ABS-P>This work reports the voltammetric determination of epinephrine (EP) and xanthine
(XA) using gamma irradiatedtungsten trioxide (WO3) nanoparticles. WO3 NPs synthesized by
anionic surfactant sodium dodecyl sulphate (SDS) assisted template method was subjected to
gamma rays under different doses (0-150 kGy). The experimental results revealed that the 50
kGy irradiation on SDS-WO3 NPs led to significant changes in crystallite size and morphology
which enriched its electrocatalytic activity greatly. The electrochemical behaviors of EP and XA
at 50 kGy SDS-WO3 modified glassy carbon electrodes (GCE) were studied by cyclic
voltammetry and differential pulse voltammetry. The outcomes confirmed that the proposed
electrode demonstrateexcellent electrocatalytic activity towardsthe oxidation of EP and XA in
phosphate buffer solution (PBS, pH 7.0). The fabricated electrode possess lowest detection limit
of 1.8 nM and 2.2 nM for EP and XA respectively with the very wide dynamic linear range of 9
nM to 1 mM. Finally, the modified sensor was successfully implemented to detect EP and XA in
the human serum samples with excellent selectivity, good stability and reproducibility.
<KWD>Keywords: Tungsten trioxide; SDS; Gamma irradiation;Epinephrine; Xanthine;
Electrochemical sensor.
<H1>1. Introduction
Epinephrine (4-[(1R)-1-Hydroxy-2-(methylamino) ethyl]-1,2-benzenediol, EP) also known as
adrenaline, is an essentialendocrine hormone released from the adrenal glandwhich play vital
role on glycogen metabolism and nervous system [1,2]. In adults, the concentration of EP are
1

normally less than 10 ng/L, but may increase by 10-fold during exercise andby 50-fold in blood
and urine during stressdue to activation of the sympatho-adrenal system [3,4]. The concentration
of EP affects glycogenolysis in liver and skeletal muscle, lipolysis in adipose tissue, plasma
lactate andrate of heart contraction [5].Low levels of EPhave been found in patients with
Parkinson’sdisease. Due to such physiological effects, this vasopressor drug has been banned to
practice in games by world anti-doping agency and has medicalapplications for patients with
diseases such as cardiac arrest, asthma and sepsis in emergency cases [6].
Xanthine (3,7-dihydro-purine-2,6-dione, XA), an intermediate metabolite of purine nucleotide
and deoxynucleotide metabolism in humans,is formed after adenosine triphosphate
(ATP)decomposition [7,8].Abnormal concentration ofXA levelproduced from the degradation of
adenosine and inosine nucleosides causesLesch-Nyhan syndrome[9]. On the other
hand,decreased XA activity leads to xanthinuria, which is a rare genetic disorder that causes
health problems such as renal failure, kidneystone formation and urinary tract disease
[10,11].The physiological conversion of XA to uric acid (UA) by xanthine oxidase is of great
interest in the medical field, as this reaction is related to the formation of superoxide radical
anion, one of the reactive oxygen species involved in the cancer aging, neurodegenerative
diseases such as Parkinson’s diseaseand inflammation [12].Normally in our body fluids,
biomolecules such as ascorbic acid (AA), UA, EP and XA are usually coexisted and oxidized at
a potential close to each other at naked electrodes and interfere with each other for the
determination of any one of the analytes.Hence, there is a rising need for persistent development
of a novel, simple and sensitive analytical methodfordetermining EP and XA for both research
and clinical applications.Here, we report the construction of an electrochemical sensor using
gamma irradiated SDS assisted WO3NPs for the first time.
Nanostructured transition metal oxides (TMO) exhibit the development of new sensors due to
their chemical and physical properties leading to numerous potentialapplications. Tungsten
trioxide (WO3), an indirect n type semiconductor, exhibits fascinating electronic, structural and
mechanical properties with a wide range of applications in the area of gas sensors, photocatalysis
andcatalysis in electrochemical process and smart windows [13]. In order to improve the physic-
chemical properties of WO3-δ nanoparticles, different strategies such as microwave route [14],
surfactant mediated method [15,16] and precipitation method [17] have been adopted.Surfactant
assisted template synthesis has received considerable attention because the physical andchemical
properties of nanostructure materials are strongly related to morphology
controllability,simplicity, speed and ease of operation [18].Huo et al. have developed surfactant
templating strategies for the synthesis process in order to enhance the surface activity of the
materials [19]. Particularly anionic surfactant(Sodium dodecyl sulphate, SDS) employed
tosynthesize controlled production of WO3 NPs with different surface morphology dueto their
flexible processing chemistry with hydrophobic carbontail and a hydrophilic head made up of
sulfate group, inexpensive organic stabilizer, melting point (206° C),easily soluble in water
andbiocompatibility [20].The electrostatic interactions between surfactant molecules andcharged
orpolarized metal-oxy precursors generate metastable modified metal oxides [21].Furthermore to
enhance the properties of WO3 NPs, irradiation of WO3 with higher energeticionizing radiation
such as gamma irradiation produces significant changes in the microstructural and morphological
properties of WO3NPs and creates a wide variety of defects, which in turn modify
theirelectrochemical sensing properties [22]. Therefore, the investigation of damages or defects
and their effects on the sensing behavior of surfactant template on WO3 NPs plays a major role.
2

Earlier, we have successfully synthesized WO3 NPs by gamma irradiation method without the
surfactant template and fabricated electrochemical sensor for the detection of serotonin [23].
In the current work, we investigate about the impact of gamma ray irradiation (0-150 kGy)
on the morphological, optical and electrochemical behaviour of SDS assisted WO3NPs for
the first time. Cyclic voltammetry studies of 50 kGy GI SDS-WO3/GCE on EP and XA
oxidation exhibited a remarkable catalytic performance in 0.1 M phosphate buffer solution
(pH 7.0). The developed sensor exhibited wide linear dynamic concentration range, lowest
detection limit,long term stability and good reproducibility in response to EP and XA.
<H1>2. Experimental
<H2>2.1 Reagents
Epinephrine and Xanthine were purchased from Alfa Aesar, Mumbai. Tungstic acid, sodium
dodecyl sulphate, and 0.1 N sodium hydroxide solutions were purchased from Fischer Scientific,
Mumbai.
<H2>2.2. Apparatus
Powder X-ray diffraction (XRD) patterns were documented using a Bruker AXS D8, Germany
advanced diffractometer in the range of 20-80º. Energy bandgap values were calculated using
UV-Visible spectrophotometer (Thermoscientific-Evolution 201, USA). FESEM images and
EDAX spectra were performed using FEG QUANTA 250, Netherland.Electrochemical
experiments were executed using a CHI 609D electrochemical workstation (CHI, USA) in
conventionalthree-electrode cell system. Glassy carbon electrode (GCE) was used as the working
electrode, with a platinum wire as a counter electrode and Ag/AgCl as a reference electrode. The
differential pulse voltammetry (DPV) measurements were accomplished in 0.1 M PBS (pH 7.0)
in the potential region from 0 to 1.0 V with amplitude of 50 mV and pulse width of 0.06 s.
<H2>2.3. Synthesis of SDS assisted WO3 NPs
The precursor solution was prepared by dissolving 1 M of tungstic acid (H2WO4) in 10 mL
sodium hydroxide (NaOH) solution. This resulted in hydrated sodium tungstate solution due to
proton exchange protocol process [23]. Subsequently, 50 wt% of SDS was added to the precursor
solution to act as ananionic surfactant. In order to enrich the protonation process, HCl was
introduced into the solution to attain the pH value of 1. The final solution was exposed to
microwave (2.45 GHz) under the optimum power of 180 W for 15 min and dried at 100°C in
air.The above process was repeated without adding SDS under identical conditions. The presence
of SDS in the colloid limits the particle nucleation and growth which controls the size and
morphology of WO3 NPs. The resulted products were irradiated using Gamma Chamber 1200
(1.25 MeV 60Co source) at IUAC, New Delhi. WO3 NPs were exposed to different doses (50,
100 and 150 kGy) at room temperature and the activity at the time of exposure was 5.499
kGy/hr.
<H2>2.4 Fabrication of SDS-WO3 NPs modified GCE
GCEs were polished to a mirror-like surface with 0.30 and 0.50 μm alumina powder sequentially
and sonicated in ethanol and water respectively for 5 minutes. The pristine WO3NPs and gamma
irradiated SDS-WO3 NPs suspension were prepared by dispersing it (5 mg) in 1.0 mL of
3

deionised water. Then a known amount of WO3 suspension (10 μL) was dropcasted onto the
GCE surface and left to air-dry at room temperature overnight to obtain WO3 modified GCE.
<H1>3. Results and Discussion
<H2>3.1 Structural and optical characterization of gamma irradiated SDS-WO3 NPs
Powder XRD patterns of the as prepared and irradiated SDS-WO3 NPs at various dosages are
shown inFig. 1A. All the observed peaks of both non-irradiated and gamma irradiated SDS-WO3
NPs correspond to the monoclinic structure according to JCPDS data (720677). It can be noticed
that there is a significant increase in the diffraction peak intensity for the irradiated samples and
that the intensities increase with the increase in the gamma ray dosage levels. This could be
attributed to the localized heating occurrence during the exposure of gamma rays. The WO3 NPs
retained its phase even after intensive gamma irradiation, suggesting that the organic
additive(SDS) acts as the sizeand morphology controller without affecting the crystalline
structure of the WO3 NPs [24].
UV-Visible spectroscopy studies were performed on all the samples over the range of 200-800
nm, as shown in Fig. 1B. The broad absorption peak around 350 to 500 nm was observed in all
the samples. The gamma irradiated (50 kGy) NPs display hypochromic shift in the absorbance
spectra when compared to the unirradiated and other irradiated SDS-WO3 NPs.The band gap
energy (Eg) of non-irradiated SDS-WO3 NPs was found to be 3.13 eV which is the intrinsic
value for monoclinic WO3[14]. There is a slight decrease in Eg value for 50 kGy gamma
irradiated SDS-WO3 when compared to that of pristine SDS WO3 which could be attributed to
the induced structural disorder and formation of charge trapping of the radiolytic electrons or
holes. Contrary to this, the subsequent increase in doses (100 and 150 kGy) enhances the band
gap energy due to the aggregation of WO3 crystallites and localized heating caused by the
irradiation.
<Preferred positionof Fig. 1>
<H2>3.2 Morphological characterization of gamma irradiated SDS-WO3 NPs
Surface morphology of the pristine and gamma irradiated SDS-WO3 NPs were examined using
FESEM (Fig. 2). The pristine sample shows cauliflower like morphology with agglomerated
near spherical shaped nanoparticles. After gamma irradiation, the agglomerated particles tend to
fragmentand form columnar polyhedron like morphology. In particular, the edges of the
polyhedron were sharp and the facets were found to be dented for 50 kGyirradiation as shown in
Fig. 2(b).This unique morphology is expected to act as trapping centresfor the biomolecules. At
higher dosages (100 and 150 kGy), the edges of the polyhedrons were destroyed leading to the
spherical shape (Fig. 2 (c &d)).The high-magnification FESEM imagesshown in the inset ofFig.
2 revealed that the gamma irradiation on SDS-WO3 NPs plays a dynamic role on the shape and
size depending on the dosage level.Energy dispersive X-ray spectroscopy (EDX) analysis further
confirmed the oxygen loss during gamma irradiation with increasing dosages(see Fig. S1).
< Preferred position of Fig. 2>
4

<H2>3.3 Electrochemical behaviors of EPand XA using GI SDS-WO3 modified electrode
Cyclic voltammetry (CV) studies were carried out to understandthe electrochemical behavior of
EP and XA at different modified electrodes.Fig. 3A shows the CVs obtained for 10 µM EP at
bare and modified GCEs in 0.1 M PBS (pH 7) at scan rate of 50 mVs-1.At bare GCE, a broad
oxidation wave was observed for EP at about 0.33Vwith poorresponse current signifying slow
electron transfer kinetics at GCE. On the other hand, the peak potential was slightly reduced to
0.28 V without noticeable improvement in the peak current at pristine WO3/GCE. However,
under the identical conditions, large improvement in the redox peak current (nearly 3-fold)and
significant lowering of peak potential to 0.22 V were observed for the electrochemical oxidation
of EP at 50 kGy GI SDS-WO3 modified GCE surface, when compared to bare, pristine SDS-
WO3 and other gamma irradiated SDS-WO3 modified GCEs. The observed improvement in the
electrocatalytic activity of the gamma irradiated SDS-WO3 NPs for a particular dosage of 50
kGy could be attributed to large electroactive surface area and enhanced electron transfer
process. The electrochemical reaction mechanism of EP at 50 kGy GI SDS-WO3/GCE for the
first 3 cycles is shown in the inset of Fig.3A. In the first cycle, the prominent oxidation and
reduction peaks were observed at 0.22 and -0.20 V (curve c1), respectively corresponding to the
oxidation of EP to epinephrinequinone(EPQ) by the deprotonation process and subsequently,
EPQ cyclises to epinephrinechrome via 1,4-Micheal addition reaction at +0.20 V which is less
favorable [25].The prominent reduction peak at -0.20 V corresponds to the reduction of
epinephrinechrome to leucoepinephrinechrome. In the subsequent 2 cycles, a new oxidation
wave was appeared at -0.15 V attributed to the reversible oxidation ofleucoepinephrinechrome to
epinephrinechrome. The small oxidation peak at 0.12 V corresponds to the conversion of
leucoepinephrinechrome to5,6-dihydroxyl-N-methylindole which is converted to EPQ through
the formation of 5,6-diquinone-N-methylindole at 0.24 V. Thus, thecomplete electrochemical
behavior of EP could be detected reliably at the gamma irradiated (50 kGy) SDS-WO3/GCE
when compared to those of other modified electrodes.
Fig. 3B shows the CVs of 10 µM XAin 0.1 M PBS (pH 7) on bare and modified GCEs with
the scan rate of 50 mVs-1. The graph displays that the electro-oxidation of XA using the bare and
modified electrodes were asymmetric, indicative of an irreversible process, showing that the
anodic peaks in the forward scan have no corresponding discernible reduction peaks on the
reverse scan.Thus the electrochemical oxidation of XA proceeds by 2e-, 2H+ oxidation to form
UA at 0.72 V [7]. Clearly, there was a significant increase in the anodic peak current response
(5-fold) and lower overpotential(120 mV) were observed for 50 kGy SDS-WO3/GCE compared
to that of bare, pristine and other GI SDS-WO3/GCEs. The gamma irradiation with a particular
dosage of 50 kGy seems to enhance the electroactive surface area and electrical conductivity of
SDS-WO3, which allows two analyte molecules to be oxidized effectively.Therefore, this
5

proposed electrode (50 kGy GI SDS-WO3) is more appropriate for the simultaneous detection of
EP and XA.
< Preferred position of Fig. 3>
<H2>3.4 Effect of scan rate
The kinetics of the electrode reactions were investigated by studying the effects of scan rate on
the peak currents of EP. Fig. 4Ashows CVs of 10 µM EP on 50 kGy SDS-WO3/GCE at various
scan rates (10 – 500mVs-1). The oxidation peak currents increased with the increase in scan rate.
The plot of square of scan rate (ʋ1/2) vs. redox peak currents was linearly associated with
corresponding coefficients of 0.9993 and 0.9996 conforming to oxidation and reduction
respectively with the linear regression equations of
Ipa (µA) = - 3.212 - 71.353 ʋ1/2(mVs-1)1/2
Ipc (µA) = 0.615 + 49.913 ʋ1/2(mVs-1)1/2
This designates that the electrocatalytic reaction of EP at the modified electrode was a diffusion
controlled process. The diffusion coefficient (D) and electroactive area (A) have been estimated
using Randles–Sevcik equation which relates the peak current for an electron-transfer-controlled
process with the square root of the scan rate:
ip = 2.69* 105 n3/2 A D1/2 C υ1/2 (1)
where n represents number of electrons transferred and C is the concentration of the species.The
diffusion coefficient value (7.13 *10-5cm2s-1) was obtained from the slope of ʋ1/2 vs. Ipa. The
electroactive surface area values calculated from the above equation illustrates that the 50 kGyGI
SDS-WO3/GCE exhibits higher value (0.061 cm2) compared to pristine (0.026 cm2), 100 kGy
(0.038 cm2) and 150 kGy (0.031 cm2) irradiated SDS-WO3 electrode surfaces. Fig. 4Bshows the
variation of the peak potentials as a function of the scan rate in order to verify the
electrochemical mechanism. From the intercept of ʋ vs.Epa plot, the formal potential (E0) of EP
was deduced. From the plot (Epa - E0) vs.lnIpa (Fig. 4C), it waspossible to calculate the energy
transfer coefficient (α) value and is found to be as 0.47.The number of electrons (n) involved
during the oxidation of EP can be calculated from the slope (m) of lnʋ vs. Epa (Fig. 4D) and
substituted in the equation n = [RT/(1-α)mF] and established to be 1.92 (~2). This result was
proven by experimentation method and verified to be two electron processes. Further, the
heterogeneous rate constant (ko) value was calculated using Nicholson equation [26] reported for
reversible system.
1/2
RT

ΔE  k0
(2)




nFD
where ΔΕ represents potential difference,α is the electron transfer coefficient, and F is the
Faraday constant.The estimated ko values for the redox reaction of EP at 50 kGyGI SDS-
WO3/GCE is 21.7 s−1. The higher ko value for EP at 50 kGyGI SDS-WO3/GCE clearly indicates
6

that the redox reaction of EP was superior when compared to the other modified electrodes
which promotes the better electrocatalytic oxidation of EP.
Fig. 5A shows the CVs of 10 µM XA on 50 kGy gamma irradiated SDS-WO3/GCE at
various scan rates (10 – 500 mVs-1) in 0.1 M PBS (pH 7). The anodic peak current increases with
the increasing the scan rates and good linearity was established between Ipa and ʋ1/2according to
the linear equation Ipa (µA) = 9.531 – 83.55 ʋ1/2(mVs-1)1/2. The system features corresponding to
an adsorption controlled process.E0 was calculated from the intercept of ʋ vs. Epa plot (Fig.
5B).As shown in Fig. 5C, the plot (Epa–E0) vs. lnIpa, α valuewas found to be as 0.36. Based on
Laviron’s theory, from the slope of Epa against ln ν (Fig. 5D), the number of electrons involved
during XA oxidation calculated fromRT/αmF and estimated to be 1.85 (~2) and confirmed as
two electron process. The standard heterogeneous rate constant (k0) for the reaction was
calculated using the following Laviron equation for an irreversible electrode process [27]:



0 

2.303RT
nF
RTk
2.303RT
nF




Epa  E0 
log

log (3)








nF

The symbols have their usual meanings. The obtained ko value for XA at 50 kGy SDS-
WO3/GCE was estimated to be 10.5 s−1 which clearly showed that the electrocatlyticof XA was
faster than other modified GCE.
< Preferred position of Fig. 4>
< Preferred position of Fig. 5>
<H2>3.5 Effect of pH
Cyclic voltammograms were recorded to study the influence of pH on the electrocatalytic
behaviour of EP (10 µM) and XA (10 µM)at 50 kGySDS-WO3/GCE in 0.1 M PBS (pH 3.0 –
10.0). From Fig. 6A, it can be seen that the oxidation peak potential (Epa) of EP and
XAdecreases as pH increases demonstrating an intrusion of the protons in the oxidation of both
EP and XA. Linear relationship of Epa of EP and XA as function of pH with slopes of 62 and 55
mV/pH respectively were observed, indicating the electron transfer is preceded by a protonation
process with the same number of protons involved in the overall electrochemical reaction
mechanism. Fig. 6B shows the effect of pH on the oxidation peak currents (Ipa) of EP and XA at
the 50 kGy SDS-WO3/GCE investigated over the pH range from 3.0 to 10.0. The Ipa values of
both EP and XA increased with the increase in pH values and reached the maximum at pH 7.0.
7

With the further increase in pH (> 7.0), the Ipa values decreased. At lower pH values (< 7.0), the
concentration of H+ ions in the buffer is higher which suppresses the oxidation process resulting
in poor current responses. Similarly, the presence of excessive OH- ions at higher pH (> 7.0)
values diminishes the oxidation process. On the other hand, at neutral pH (7.0), oxidation of both
EP and XA was much higher due to unhindered electrocatalytic processes. As a result, the
oxidation peak currents of EP and XA were found to higher at pH 7.0. Thus, SDS-WO3 NPs
irradiated with gamma rays at lower dosage (50 kGy) effectively catalyzes both the EP and XA
as shown in Scheme 1. The gamma induced defects in the facets as seen in FE-SEM pictures
(inset of Fig. 2b) and oxygen deficiency caused by localized heating might act as trapping
centers leading to the improved electrocatalytic ability of gamma irradiated SDS-WO3 NPs.
< Preferred position of Fig. 6>
< Preferred position of Scheme 1>
<H2>3.6 Sensitivity and selectivity studies on the simultaneous determination
The simultaneous determination of EP and XA at the 50 kGyGI SDS-WO3/GCE, was carried out
by DPV in the potential range of 0 to 1 V in 0.1 M PBS (pH 7). As shown in Fig.7A, the
electrochemical response of EP and XA increases linearly with the increase of theirconcentration
over the wide range of 0.009 -1000 µM (Fig. 7B and C). The lowest detection limits were
deduced as 1.8 nM and 2.2 nM(S/N=3)for EP and XA respectively.At low concentration range,
the response current was due to theadsorptiontransport of EP and XA, whereas at high
concentration, the active sites were too fully occupied to further facilitate the transport of both
the analyte substrates. Therefore, the corresponding response signal was only due to the diffusion
transport according to Fick’s law [28] and the linear regression equations are tabulated in Table
1.Though there are large number of reports in the literature dealing with the individual
determination of EP and XA, simultaneous detection of EP and XA were not accomplished so
far. We have achieved for the first time, the simultaneous detection of EP and XA with the
lowest detection limits of 1.8 and 2.2 nM respectively over a very wide linear range of 9 nM to 1
mM for both the analytes. These results confirm the excellent catalytic activity of gamma
irradiated SDS-WO3 towards the detection of EP and XA with high sensitivity and selectivity. In
order to make a comparison with recently reported sensors [7, 10, 29-36], the characteristics of
different electrochemical sensors for EP and XA detection are summarized in Table 2. It can be
seen that the fabricated sensor offered simultaneous detection of EP and XA over very wide
linear ranges when compared to that of most sensors reported for detection of either EP or XA
individually. It was also noted that there is no report in the literature dealing with the
simultaneous detection of EP and XA.
Under the optimum experimental conditions, the influence of various interfering compounds had
been investigated on the simultaneous determination of EP and XA.Fig. 7D shows the DPVs for
the increment of 0.5 µM EP and 0.5 µM XA in the presence of 500 fold excess of ascorbic acid,
uric acid, folic acid, guanine, cysteine, ADP, ATP, glucose and 50-fold excess of dopamine,
tyrosine, serotonin andnorepinephrine in 0.1 M PBS (pH 7).No significant changes were
observed in the peak potentials and response current in the presence of the interfering mixtures
(signal change < 2%).No interference was observed for 500-fold excess of metal ions such as
Fe2+, Ca2+, Mg2+ and Na+ at 50 kGy gamma irradiated SDS-WO3 modified GCE by DPV method
(Fig. S2).This result indicates that the proposed electrode proved to be a highly selective sensor
8

for recognition of EP and XA in aqueous solutions. Thus, 50 kGy irradiated SDS-WO3 NPs is a
potential sensing element for construction of sensitive and selective electrochemical sensorsfor
real sample analysis.
< Preferred position of Fig. 7>
< Preferred position of Table 1>
< Preferred position of Table 2>
<H2>3.7 Stability and reproducibility
The stability of the modified electrode was examined over a period of a month by storing it in
0.1 M PBS (pH 7) at 4°C for every 2 days intervals. No significant changes were observed
during first two weeks and the response yielded over 97.8% of its initial value. Consequently, a
small decrease of the peak response current of EP and XA were observed with the relative
standard deviation (RSD) of 3.2%, which could be ascribed to the excellent stability of the 50
kGy GI SDS-WO3 electrode. The reproducibility of the modified electrode was investigated by a
series of ten repetitive measurements towards the oxidation of EP and XA mixture.Their
response peak current gave reproducible results with the RSD of 3.9%, indicating the excellent
reproducibility of the fabricated electrode.
<H2>3.8 Determination of EP and XA in real samples
The reliability and analytical utility of the proposed electrode was tested by the simultaneous
determination of EP and XA in human serum samples.The real samples were diluted 10-fold
with pH 7.0 phosphate buffer solution and the two biomolecules were determined simultaneously
by DPV method.The concentrations of EP and XA were determined using the standard addition
technique and the results are summarized in Table 2.In addition to testify the accuracy of this
method, the known concentration of EP and XA solution were spiked in the samples and the
DPV response signals were measured andreplicated. For the recovery, all the serum samples
were diluted 10 times with PBS (pH 7.0). It can be seen that all the spike recoveries values were
precise, which revealed that the fabricated sensor can be effectively utilized for the simultaneous
determination of EP and XA in real samples.
< Preferred position of Table 3>
<H1>4. Conclusions
In this paper, we demonstrated the application of gamma irradiation for inducing significant
changes on the morphological and optical properties of WO3 NPs. Among various dose levels
investigated, 50 kGyirradiated SDS-WO3 NPs modified GCE acts as an efficient
electrocatalystfor the simultaneous determination of EP and XA in the presence of several
potentially interfering substances in 0.1 M PBS (pH 7) by voltammetric method. These
biomolecules were effectively catalyzed at less positive potentials yielding higher peak current.
9

The kinetic parameters such as number of electrons transferred, electron transfer coefficient,
diffusion coefficient, heterogeneous electron transfer rate constant and pH dependence were
investigated. DPV studies exhibits the excellent sensitivity and selectivity towards the
simultaneous determination of EP and XA in the presence of 500-fold excess of the interfering
agents. The lowest detection limits of 1.8 nM and 2.2 nM(S/N =3)were achieved respectively for
EP and XA with dynamic linear range of 9 nM-1 mM for both the molecules.The practical
applicability of the fabricated electrode was successfully validated by measuring the
concentration of EP and XA in human serum samples.
Acknowledgments
This research was supported by the Inter University Accelerator Centre (IUAC) sponsored
project, New Delhi (IUAC-UFR-50312).The authors thank Prof. P. Manisankar and Dr. G. Ravi,
Alagappa University for providing FE-SEM and PowderXRD facility.
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<Figure>Fig. 1. Powder XRD patterns (A) and UV-visible spectra (B) of SDS-WO3 (curve
a), 50 kGy (curve b), 100 kGy (curve c), and 150 kGy gamma irradiated SDS-WO3 NPs
(curve d). Inset of Fig. 1B shows the plot of samples vs. energy bandgap.
<Figure>Fig. 2. FESEM images of (a) non-irradiated SDS-WO3, (b) 50 kGy, (c) 100 kGy
and (d) 150 kGy gamma irradiated SDS-WO3 NPs; Inset shows the corresponding higher
magnification.
<Figure>Fig. 3. Cyclic voltammetry recorded for the 10 μM EP (A) and 10 μM XA (B) at the
bare GCE (curve a), pristine SDS-WO3/GCE (curve b), 50 kGy SDS-WO3/GCE (curve c), 100
kGy SDS-WO3/GCE (curve d) and 150 kGy SDS-WO3/GCE (curve e) at a scan rate of 50 mV s-1
in 0.1 M PBS (pH 7.0). Inset of Fig.3A shows the CVs recorded for 10 μM EP at 50 kGy SDS-
WO3/GCE at first cycle (c1), second cycle (c2) and third cycle (c3).
12

<Figure>Fig. 4. (A) Cyclic voltammetry recorded for the 10 μM EP at 50 kGy gamma irradiated
SDS-WO3/GCE in 0.1 M PBS (pH 7.0) at the different scan rates (0.01-0.5 Vs-1). Inset shows the
calibration plots for the redox peak currents (Ipa) vs. the square root of scan rate (ʋ1/2); (B)
relationship between the peak potential (Epa) and scan rate (ʋ); (C) experimental variation of
peak current (ln Ipa) as a function of the difference of Epa – E0; (D) experimental variation of
scan rate (ln ʋ) vs. peak potential (Epa)
<Figure>Fig. 5. Cyclic voltammetry recorded for the 10 μM XA at 50 kGy SDS-WO3/GCE in 0.1
M PBS (pH 7.0) at the different scan rates (0.01-0.5 Vs-1). Inset shows the calibration plots for
the redox peak currents (Ipa) vs. the square root of scan rate (ʋ1/2); (B) relationship between the
peak potential (Epa) and scan rate (ʋ); (C) experimental variation of peak current (ln Ipa) as a
function of the difference of Epa – E0; (D) experimental variation of scan rate (ln ʋ) vs. peak
potential (Epa).
<Figure>Fig. 6. (A) Plot of oxidation peak potential of 10 μM EP and XA vs. pH (3.0 – 10.0);
(B) The corresponding oxidation peak current vs. pH (3.0 – 10.0) in PBS at a scan rate of 50
mVs-1 for 50 kGy SDS WO3/GCE.
<Figure>Fig. 7. (A) Differential pulse voltammograms of 50 kGy SDS-WO3/GCE in 0.1 M PBS
(pH 7.0) containing different concentrations of EP and XA (0.009-1000 µM); (B) and (C) show
the plots of the electrocatalytic oxidation peak current as a function of EP and XA concentration
respectively; (D) Selective response of 50 kGy SDS-WO3/GCE for the increment of 0.5 µM EP
and 0.5 µM XA with successive additions of interferents in the sequence of (a) ascorbic acid, (b)
dopamine, (c) tyrosine, (d) serotonin, (e) norepinephrine, (f) uric acid, (g) folic acid, (h) guanine,
(i) cysteine, (j) ADP, (k) ATP and (l) glucose in 0.1 M PBS (pH 7.0).
13

<Figure>Scheme 1 Electrocatalytic oxidation of epinephrine and xanthine at 50 kGy SDS-WO3
NPs.
<Table>Table 1 Analytical characteristics for simultaneous determination of EP and XA at the
50 kGy SDS-WO3 modified GC electrode.
Analyte Linear Range Linear regression equation
Correlation Detection
coefficient limit (nM)
RSD
%
(µM)
(Ipa: µA, C: µM )
EP
0.009-100
100-1000
0.009-100
100-1000
Ipa = 1.011 CEP – 0.663
Ipa = 0.061.011 CEP – 112.895
Ipa = 0.797 CXA – 0.518
Ipa = 0.054 CXA – 88.294
0.9995
0.9996
0.9992
0.9994
0.7
3.0
0.4
4.0
1.7
1.4
1.6
1.5
XA
<Table>Table 2 Comparison of the fabricated sensor with the EP and XA sensors recently
reported in the literature
Electrode
EP (µM)
LR
XA (µM)
LR
Ref.
LOD
LOD
p-ATT/GCE
2PHCM/CNPE
2,7-BFGPE
0.04-40
0.05-550
0.05-550
0.5-200
5-100
270 x 10-6
0.0096
0.027
-
-
-
-
-
-
-
-
-
-
[10]
[29]
[30]
[31]
[32]
SnO2/graphene/GCE
Gelatine- glutaraldhyde
P. chyrsosporium/Pt
CeO2 NCs /GCPE
Poly(ATD)/GCE
Co3O4/MWCNT/CS/GCE -
Poly(L-methionine)/GCE
Co doped CeO2 NPs/GCE -
50 kGy GI SDS-WO3 /
GCE
0.017
1.04
-
-
-
-
-
-
-
0.198-9.45
5-45
0.2-16
0.02-0.1
0.1-1000
0.009-1000 0.0022
0.0036
0.59
0.2
0.004
0.096
[33]
[34]
[35]
[7]
[36]
This work
-
0.009-1000 0.0018
2PHCM/CNPE: 2-(4-oxo-3-phenyl-3,4-dihydro-quinazolinyl)-N′-phenyl-hydrazinecarbothio-
amide modified carbon nanotube paste electrode
p-ATT: Poly (5-amino-1,3,4-thiadiazole-2-thiol)
2,7-BFGPE: 2,7-bis(ferrocenyl ethyl) fluoren-9-one and graphene nanosheets
14

<Table>Table 3 Simultaneous determination of EP and XA in human serum samples.
No. of EP
XA
EP
XA
Recovery (%)
RSD (%)
Exp
spiking spiking Found
Found
(nM)
EP
XA
EP
XA
2.4
(nM)
10
(nM)
10
(nM)
9.93
1
10.09
99.7
102.5
3.1
2
3
25
50
25
50
25.15
50.87
25.93
50.13
98.5
103.6
99.5
1.9
2.5
2.7
3.3
102.4
TDENDOFDOCTD
15