Universiti Kebangsaan Malaysia - Journal of Smart Sensor ......(Universiti Tun Hussein Onn Malaysia)...

Journal of Smart Sensor and Materials, 2019, 1-26

Journal of Smart Sensor and Materials E-ISSN xxxx-xxxx www.ukm.my/sensor

Issue: 1 Year: 2019

A Novel Electrochemical Detection Of Ochratoxin A In Cow Milk Using Nickel

Nanoparticle Modified Electrode

by Suleiman Salihu, Nor Azah Yusof, and Jaafar Abdullah

A Low-Cost Tracking System for Running Race Applications Based on Bluetooth

Low Energy Technology

by David Perez-Diaz-de-Cerio, Ángela Hernández-Solana, Antonio Valdovinos and

Jose Luis Valenzuela


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EDITORIAL BOARD Journal of Smart Sensor and Materials Chief Editor : Prof .Dr. Lee Yook Heng (Universiti Kebangsaan Malaysia) Editor (Physical Sensor) : Assoc. Prof Dr. Mohd Kamarulzaki Mustafa (Universiti Tun Hussein Onn Malaysia) Dr. Ruslinda A. Rahim (Universiti Malaysia Perlis) Editor (Chemical Sensor/Biosensor) : Prof. Dr. Nor Azah Yusof (Universiti Putra Malaysia) Assoc. Prof . Dr. Siti Aishah Hasbullah (Universiti Kebangsaan Malaysia) Dr. Jaafar Abdullah (Universiti Putra Malaysia) Dr. Tan Ling Ling (Universiti Kebangsaan Malaysia) Dr. Faridah Salam (Institut Penyelidikan dan Kemajuan Pertanian Malaysia) Dr. Sharina Abu Hanifah (Universiti Kebangsaan Malaysia) Dr. Jahwarhar Izuan Abdul Rashid (Universiti Pertahanan Nasional Malaysia) Editor (Sensing Materials): Dr. Zainiharyati Mohd Zain (Universiti Teknologi MARA) Assoc. Prof. Dr. Mohd Kamarulzaki Mustafa (Universiti Tun Hussein Onn Malaysia) Managing Editor : Dr. Tan Ling Ling (Universiti Kebangsaan Malaysia)

International Advisory Board Prof. Dr. Bambang Kuswandi, Universitas Jember, Indonesia Prof. Emeritus Fortunato B. Sevilla, University of Santo Tomas, Philippines

Committee 2018/2019 Malaysian Society for Sensor Technology Development

President : Assoc. Prof. Dr. Jaafar Abdullah Vice President : Prof. Dr. Nor Azah Yusof Secretary : Dr. Jahwarhar Izuan Abdul Rashid Assistant Secretary : Dr. Tan Ling Ling Treasurer Dr. Mohd Fairulnizal Md Noh Committee Assoc. Prof. Dr. Mohd Kamarulzaki Mustafa Assoc. Prof. Dr. Zainiharyati Mohd Zain Assoc. Prof. Dr Siti Aishah Hasbullah Dr. Sharina Abu Hanifah Dr. Nur Azura Mohd Said Dr. Nurul Huda Abd Karim

The Malaysian Society for Sensor Technology Development serves as a platform for all members and scientists to share their latest findings in sensor research and also to strengthen and promote sensor research and technology in Malaysia. Publisher: Malaysian Society for Sensor Technology Development

School of Chemical Sciences and Food Technology,

Faculty of Science and Technology,

University Kebangsaan Malaysia,

43600 UKM Bangi,

Selangor D. E., Malaysia.

https://www.researchgate.net/institution/Universitas_Jember


Copyright: Malaysian Society for Sensor Technology Development, 2019

Journal of Smart Sensor and Materials

Issue 1 CONTENTS

1-26 Contoh: A NOVEL ELECTROCHEMICAL DETECTION OF

OCHRATOXIN A IN COW MILK USING NICKEL NANOPARTICLE

MODIFIED ELECTRODE

by Suleiman Salihu, Nor Azah Yusof, and Jaafar Abdullah

27-45 Contoh: A Low-Cost Tracking System for Running Race

Applications Based on Bluetooth Low Energy Technology

by David Perez-Diaz-de-Cerio, Ángela Hernández-Solana, Antonio

Valdovinos and Jose Luis Valenzuela


Objectives Journal of Smart Sensor and Materials provides a forum for people working in the multidisciplinary fields of sensing technology, and publishes contributions describing original work in the experimental and theoretical fields, aimed at understanding sensing technology, related materials, design development, application of all sensors/biosensors, associated phenomena and applied systems. Scope The scope of Journal of Smart Sensor and Materials encompasses, but is not restricted to, the following areas:- •Sensing principles and mechanisms •Materials for Sensor Technology •Nanostructured materials •Synthetic organic chemistry •Synthetic inorganic chemistry •Polymer composites •New sensing transducers •Sensor fabrication technology •Actuators •Optical sensors •Electrochemical sensors •Chemical sensors •Biosensors •Physical sensors •Mass-sensitive devices •Gas sensors •Humidity sensors •Lab-on-a-chip •Sensor-array •Optoelectronic sensors •Mechanical sensors •Thermal sensors •Magnetic sensors •µTAS - Micro Total Analysis Systems •Remote Sensing •Pressure Sensing •Nuclear Sensing •Acoustic Sensing


A NOVEL ELECTROCHEMICAL DETECTION OF OCHRATOXIN A IN COW MILK

USING NICKEL NANOPARTICLE MODIFIED ELECTRODE

Suleiman Salihu1, Nor Azah Yusof

1,2, and Jaafar Abdullah

1,2

1,1,2Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400

Selangor, Malaysia.

**Corresponding author: email address: [email protected]

Abstract

A novel electrochemical sensor was fabricated for detection of ochratoxin A in food samples by

synthesized nickel nanoparticle using 3-aminopropyltriethoxysilane as a cross linker. The

fabricated nanocomposite was homogenized in dimethylformamide and drop casted on screen

printed electrode. The amine groups in APTES were used as growth point for the NiNP synthesis

through electrostatic attraction between the amine group (NH4+) and Ni(II) chloride

- while

sodium hydroxide acts as a reducing agent. Nickel nanoparticle has good properties of

conductivity and catalytic particles that is intensively exploited for electrode modification. The

Field emission scanning electron microscopy reveal that the formed nanoparticle are dominantly

spherical in shape and evenly distributed. Field emission scanning electron microscopy, energy

dispersive X-Ray, X-Ray diffraction and cyclic voltammetry were used to characterize the

synthesized nanoparticle. The results show that the synthesized nanoparticle induced a

remarkable synergetic effect for the oxidation of ochratoxin A. Effects of some parameters, such

as pH, buffer, scan rate, accumulation potential, accumulation time and amount of casted

nanoparticle, on the sensitivity of fabricated sensor were optimized. Under the optimum

conditions, there is a linear calibration ranges from 0.1–0.5 µM with equation of Ipa (µA) =

6.232 C (µM) + 0.9987, R2 = 0.998. The limit of detection and limit of quantitation were

calculated as 0.00041 µM and 0.00135 µM respectively. The fabricated electrochemical sensor

was successfully applied for determination of Ochratoxin A in cow milk samples and all results

compared with high performance liquid chromatography (HPLC) standard method.

mailto:[email protected]


Keywords: aminopropyltriethoxysilane; electrochemical sensor; Ochratoxin A; nickel (II)

chloride, Nickel nanoparticle

Introduction

The most conventional methods used for detecting toxins in foodstuffs are gas chromatography

with mass spectrometry (GC-MS) (Zhu et al., 2009, Mondello 2008), tandem mass spectrometry

(GC-MS/MS) (Mondello 2008), liquid chromatography with mass spectrometry (LC-MS) or

tandem mass spectrometry (LC-MS/MS) (Bruins et al., 1987). These analytical techniques have

the ability to detect target compounds down to the nanogram and microgram per liter. Traditional

methods for detecting antibiotics include microbiological inhibition tests, immunoassays, and

chemical physical methods such as gas/liquid chromatographic (GC/LC) (Mondello 2008, Bruins

et al., 1987) analysis and capillary electrophoresis (CE) (Tong et al., 2013). Ochratoxins belongs

to a group of mycotoxins produced as secondary metabolites by several fungi of the Aspergillus

or Penicillium families and are weak organic acids consisting of a derivative of an isocoumarin.

The family of ochratoxins consists of three members, A, B, and C which are slightly different

from each other in chemical structures. These differences, however, have marked effects on their

respective toxic potentials. Ochratoxin A (Fig.1) is the most abundant and hence the most

commonly detected member but is also the most toxic of the three (Van et al., 1965, Van der et

al., 1965). It is a potent toxin affecting mainly the kidney. As in other mycotoxins, ochratoxin A

can contaminate a wide variety of foods due to fungal infection in crops, in the field during

growth, at harvest time, in storage and in shipment under favourable environmental conditions

especially when they are not properly dried. Ochratoxin A may be present in a foodstuff even

when the visible mould is not seen.

Figure 1: Ochratoxin A


Ochratoxin A is found mainly in cereal and cereal products. This group of commodities has been

reported to be the main contributors to ochratoxin A exposure in exposure assessments carried

out by the European Commission (Jørgensen, 1997, Miraglia and Brera, 2002, ), accounting for

50% of total dietary exposure of ochratoxin A in European countries (SCOOP task 3.2.7, 2002).

Besides cereals and cereal products, ochratoxin A is also found in a range of other food

commodities, including coffee, cocoa, wine, beer, pulses, spices, dried fruits, grape juice, pig

kidney and other meat and meat products of non-ruminant animals exposed to feedstuffs

contaminated with this mycotoxin. Ruminant animals such as cows and sheep are generally

resistant to the effects of ochratoxin A due to hydrolysis to the non-toxic metabolites by protozoa

in the stomachs before absorption into the blood (Kiessling et.al., 1984). In ruminant animals like

cow, effective hydrolysis of ochratoxin A to the non-toxic ochratoxin alpha takes place in the

four stomachs in the presence of the ruminant protozoa (Kiessling et al., 1984) thus rendering the

species resistant to the effects of the toxin. Transfer to the milk has been demonstrated in rats,

rabbits and humans. In contrast, little ochratoxin A is transferred to the milk of ruminants, again

due to metabolism of this mycotoxin by the rumen microflora. The main target site of ochratoxin A

toxicity is the renal proximal tubule, where it exerts cytotoxic and carcinogenic effects. Ochratoxin A

has been reported to be an immune suppressor and affects the immune system in a number of

mammalian species. It was able to cause inhibition of protein biosynthesis and inhibition of

macrophage migration (Creppy et al., 1984). The European Commission’s Scientific Committee

for Food (SCF), after reviewing its opinion on Ochratoxin A, made its conclusion in 1998 that it

would be wise to reduce exposure to Ochratoxin A as much as possible, ensuring that exposures

are towards the lower end of the range of tolerable daily intakes which has been estimated by

other bodies, at a level below 5 ng/kg/day (De et al., 2016). Studies on levels of Ochratoxin A in

food, so far, have been conducted mainly in the Western part of the world. Consequentially,

international data accumulated at present are confined principally to the Western diet. Little is

known about levels of Ochratoxin A with regards to the rice-based Eastern diet pertaining to the

weather conditions in countries in the East.

Here we have reported a novel procedure for fabrication of NiNP/SPE for electrochemical sensor

by electrode deposition process using polyvinypyrollidone as a cross linker for detecting


Ochratoxin A in food. To the best of our knowledge, there is little documented information on

the detection of ochratoxin A in food samples using NiNP//SPE sensor.

Materials and Methods

All electrochemical measurements were carried out using a portable potentiostat (DropSens,

mStat 8000, Spain) electrochemical system. The surface of the working electrode, i.e. the screen-

printed carbon electrode (SPE, DropSens, Spain), was modified with NiNP/aptes and connected

to the potentiostat using a USB connection. All characterization studies on the morphology and

composition were investigated by field emission scanning electron microscopy-energy dispersive

spectroscopy (FESEM-EDS, JEOL, USA) A pH meter (Fisher Scientific, USA) was used to set

the pH values before each analysis. 3-aminopropyltriethoxysilane, and Nickel(II) chloride

hydrate (99.5%) were obtained from Aldrich (USA). All other chemicals utilized in the research

were of analytical grade and used as received.

A 0.05 M acetate buffer (pH 5.1) was prepared by dissolving the required amount of sodium

acetate in distilled and deionized water and the pH of the solutions was adjusted by addition of

drops of acetic acid. Stock solutions of Ochratoxin A 1 x 10-3

M were prepared in distilled and

deionized water daily. The working solutions for the voltammetric investigations were prepared

by dilution of the stock solution with aqueous buffer solutions. All stock solutions were kept in

the dark and were used within several hours to avoid decomposition.

All cyclic voltammetry (CV) measurements were performed in 1.00 mM K3Fe(CN)6 with 50 mM

KCl, while the differential pulse voltammetry (DPV) measurements were carried out in 0.01M

phosphate buffered saline (PBS) with pH 7.4. The prepared 0.5M of sulphuric acid (Sigma, St.

Louis, MO, USA) was used to activate the SPGE before modification.

Electrochemical measurement

All electrochemical experiments were performed with a three-electrode system at 25 °C. All

potentials were measured relative to the Ag/AgCl reference electrode. Cyclic voltammetric scans

were performed at the potential range from -0.6 to +0.6 V at scan rate of 0.1 V/s. The differential

pulse voltammetry measurements were performed at 0.6 V.


Results and Discussion

For the modified electrode, nickel nanoparticle was electrochemically deposited on the screen

printed carbon electrode surface by cyclic voltammetry. The nickel nanoparticle on modified

electrode were investigated using Zeta nanosizer, X-ray diffraction and FESEM analysis. The

DLS analysis using the zeta potential of the sample was determined with a Zetasizer Nano S90

(Malvern Instrument Ltd.) shows that the particle sizes in aqueous solutions are in nanosize

range. The pure nickel nanoparticle in solution give an average size value of approximately 15.1

nm. The DLS results places particles in nanorange while in solution, and nanopowders were

dried for X-ray diffraction as shown in Fig.2. The broad diffraction peaks are due to nanosize of

nanoparticle. Maximum intensity peaks (111) was used to estimate the crystalline size and it is

found to be 15 nm using Scherrer equation. The surface morphology of the unmodified and

modified screen printed carbon and the electrodeposited nickel nanoparticle on SPCE surface are

shown in Figure 3. The result in the FESEM image, Fig.3 indicated that nickel nanoparticle were

electrodeposited on the screen printed carbon electrode surface and were well distributed on the

surface with diameters in the range of 3 – 15 nm as confirmed by X-ray diffraction in Fig.2. The

shape of Ni particle is spherical and are linked together to form chains. X-ray diffraction analysis

was used to identify the phase and crystallinity of NP. The synthesized NiNP showed diffraction

peak at 2θ angles of 44.5o, 51.8

o and 76.4

o, corresponding to crystal planes of 111, 200 and 222,

indicating face-centered cubical structure (Sudhasree et al., 2014). FTIR spectroscopy is used to

study the interaction between different species and changes in chemical composition of the

mixture. The IR spectra of NiNP synthesized is recorded in the range of 400 - 4000 cm -1

. The IR

spectrum of NiNP synthesized shows the peaks at 3212.72, 1623.67, 1402.27, 1076.94, and 463

cm -1

correspond to O-H stretching which is the characteristic of Ni(OH)2, the bond in CHC

suggests cross-linking of C-C formation, CH bending, CO stretch, and NiO stretching,

respectively (Chanda et al., 2007, Sudhasree et al., 2014). The calculated size values for NiNP

by Scherrer formula (Coates, G. W. 2000)) at 2θ of 44.5º is a general approximate to those of

FESEM observation (Fig.3).


Figure 2: X-ray diffraction of Ni nanoparticle

Figure 3: The FESEM and EDX of NiNP.

Electrochemical behavior of the NiNP//SPCE

To study the electrochemical behavior, cyclic voltammograms of ochratoxin A using

NiNP/SPCE were observed. The Fig.4 shows cyclic voltammograms of the bare SPCE in 0.1 M

phosphate containing 10 µM ochratoxin A in the potential window ranging from 0.0 to 1.0 V

with scan rate of 0.1 V/s. As shown in Fig.4, a pair of redox peaks can be observed. with peak

currents of 0.5 mA and 10 mA because the effect of nickel nanoparticle on the SPCE exhibits

high electrocatalytic activity towards ochratoxin A detection.


-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6-30

-20

-10

0

10

20

30

Cur

rent

(uA

)

Potential (V)

a

b

a = BareSPCE

b = NiNP/SPCE/

Figure 4: Cyclic voltammograms of the NiNP/SPCE in the absence and presence of 10 µM

ochratoxin A in 0.1 M phosphate, at scan rate of 0.1 V/s

Fig.4 shows the cyclic voltammetry of modified and unmodified SPEs in 0.1 mM K3Fe(CN)6 and

0.1 M HCl, with redox peaks in the potential range of -0.6 to +0.6 V. The results shows lower

peak current for bare SPE (a) which is due to slow electron transfer property of the electrode.

The peak current increases from 0.15 µA to 10 µA as NiNP was used to modify the electrode (b),

which could be due to the effect of NiNP that enhanced properties of electrode.

It was observed that peak current of 10 µA obtained with each electrode increases with increase

in scan rate which is proportional to square of respective scan rates, and this suggest diffusion-

control process (Zhang et al., 2013a). Active surface area of bare and modified electrodes was

calculated according to Randles-Sevcik formula (Syrrokostas, et al., 2012b, Chethana, and Naik

2012, Du et al., 2014)

𝑖𝑝 = (2.69 × 105)𝑛3/2𝐴𝐷1/2𝐶𝑣1/2

Where n is number of electrons participating in the redox reaction, A is the surface area of

electrode (cm2), D is diffusion coefficient of molecules in solution (7.6 x10

-6 cm

2 s

-1 ), C is the


concentration of K3Fe(CN)6 (M) and v is scan rate (Vs-1

). From slope of plot of Ipc vs v1/2

, the

surface area of electrodes can be calculated from using the relation;

Aeff = 𝑆𝑙𝑜𝑝𝑒

2.69 𝑥 105 𝑛3/2 𝐷1/2 𝐶

As shown in Fig.4, the anodic current increases from bareSPE to modified NiNP on the surface

which suggest that the modifying materials was successfully deposited on the surface of the

SPCelectrode. The balanced redox reaction of K3[Fe(CN)6] in (Tris-HCl and PBS) at the surface

of the electrode is given below:

K3[Fe(CN)6] + 3HCl 3K+ + [Fe(CN)6]

3- + 3H

+ + 3Cl

-

3KCl + 3H+ + [Fe(CN)6]

3- + e

- [Fe(CN)6]

4- + 3H

+

The deposition of nickel nanoparticle on the NiNP/SPE surface induced a large reduction peak

current compared with bare SPE. There is migration of electron to the electrode and accepted,

which led to the reduction of the oxidation number from +3 to +2.

For bare SPE, the active surface area was calculated to be 0.034 cm2. While modification with

NiNP/SPE modified electrode has an increase in active surface area of 0.084 cm2

(Figures not

shown). This suggests that nanoparticles have brought about an increase in electro-active surface

area of SPE substrate.

To the electrochemical preparation of active and stable nickel nanoparticle on the electrode

surface were conditioned by potential cycling from 0.0 to 0.6 V at a scan rate of 0.1 V/s for 10

cycles.


The effect of supporting electrolytes

The supporting electrolyte plays vital role in electrochemical detection of Ochratoxin A. In this

study as in Fig.5, four different buffers such as, phosphate buffer, a mixture of monosodium

dihydrogen phosphate and disodium hydrogen phosphate, acetate buffer, a mixture of sodium

acetate and acetic acid, citrate buffer a mixture of sodium citrate and citric acid, and Britton

Robinson buffer a mixture of acetic, boric and phosphoric acid of equal strength (0.1 M, pH 5)

were investigated and Fig.5 show results of influence of each buffer solution towards

voltammetric oxidation of 10 µM Ochratoxin A. It was observed that best results with respect to

shape and sensitivity are obtained in phosphate buffer solution with peak current of 5.2 µA,

suggesting that it provided most favorable medium for voltammetric oxidation of Ochratoxin A.

(a) (b)

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

-40

-20

0

20

40

60

Cu

rre

nt (u

A)

Potential (V)

B

C

A

D

D

AcB

Figure 5: (a) CV and (b) Effect of supporting electrolytes on voltammetric oxidation of Ochratoxin A of

10 µM in 0.1 M pH 4.0 (A) Acetate buffer (B) Britton Robinson buffer (C) Citrate buffer (D) phosphate

buffer

0

1

2

3

4

5

6

A B C D

Cu

rre

nt

(µA

)

Supporting electrolytes


The effect of pH of supporting electrolytes were investigated. Both peak potential and peak

current directly depends on pH of buffer solution. Results of voltammetric response of 10 µM

Ochratoxin A at different pH range are presented in Fig.6. It was found that oxidation of

Ochratoxin A in all pH ranges displayed voltammetric signals. From results obtained, it shows

that as pH increases, peak current improved due to deprotonation or loss of electron (Bagheri et

al., 2014). So, the protonated Ochratoxin A cannot properly interact with working electrode

because of carboxylic groups that was attached to the surface of the NiNP, consequently a lower

oxidation current is observed at lower pH (Rezaei and Damiri, 2010). Thus, best results with

respect to sensitivity and shape of voltammogram is obtained at pH 6.0 with peak of 14.2 µA.

Therefore, 0.1 M phosphate buffer of pH 6.0 were chosen as optimum medium for voltammetric

oxidation of Ochratoxin A at NiNP/SPE.

2 4 6 8 10

4

6

8

10

12

14

16

Cu

rre

nt

(uA

)

pH

Figure 6: Effect of pH on voltammetric oxidation of Ochratoxin A at NiNP/SPE


The effect of accumulation potential on ochratoxin A

The detection potential is important for selectivity and sensitivity of ochratoxin A

electrochemical sensor. Results of study of effect of Eacc on voltammetric oxidation of ochratoxin

A shows that by varying Eacc from -0.6 to 0.4 V, in Fig.7 for Ochratoxin A detections, peak

current of 14.8 µA increased steadily by varying the potential between 0.2 to -0.4 V for Ochratoxin

A, due to reaction of their molecules which have more adsorptivity on surface of modified

electrode within these range of values, conversely at negative potentials lower than −0.4 V for

Ochratoxin A, peak current decreased, because at this potential, layer of nanocomposite on surface

of SPE is unstable and therefore, hydrogen bubbles are produced at the electrode which have

tendency to decrease sensitivity of electrode. Thus, the potential of -0.4 V was chosen as the

detection potential in this work.

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0

0

5

10

15

20

25

30

35

40

Cu

rre

nt

(uA

)

Potential (V)

Figure 7: Effect of potential accumulation of Ochratoxin A on electrode surface

The oxidation response of Ochratoxin A in connection with accumulation time in Fig.8 was

investigated using cyclic voltammetry. Results of the influence of accumulation time shows that

peak current increased with increase in accumulation time from 30 to 180 s as seen in Fig.8 for

Ochratoxin A, perhaps due to rapid adsorption of Ochratoxin A on surface of electrode.

However, as oxidation peak current reaches 14.8 µA, it become almost leveled off with further

increase in accumulation time beyond 180 s for Ochratoxin A. This is could be due to saturation

of Ochratoxin A on surface of NiNP electrode (Pumera, 2010). Thus, accumulation time of 180 s

was chosen as optimum value for the analysis.


Figure 8: Effect of accumulation time of Ochratoxin A on electrode surface

The Fig.9 below shows CV of 10 µM Ochratoxin A on electrode surface at different scan rate. It

was observed that as scan rate increases, oxidation peak current of Ochratoxin A also increases

linearly and peak potentials shifted to more positive potentials. A good linear relationship was

observed between oxidation peak current and scan rate (Fig. 10a) under equations of Ipa =

76.527v + 1.032; 𝑅2 = 0.9856. The linear correlation, 𝑅2 = 0.9856 suggest that

electrochemical oxidation of Ochratoxin A at NiNP is under adsorption control process. A plot

of log Ipavs log v, (Fig. 10b) also gives linear relationships that are expressed as log (Ipa)(µA) =

3.318 log v(mV/s) - 0.7568; 𝑅2 = 0.9986. is typical of adsorption control process also

confirmed that electrochemical oxidation of Ochratoxin A at NiNP is under adsorption control.

Thus, it is concluded that voltammetric Oxidation of Ochratoxin A at NiNP is under adsorption

control process.

0

2

4

6

8

10

12

14

16

0 50 100 150 200 250 300 350

Cu

rre

nt

(µA

)

taccumulation (s)


-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6

-20

-10

0

10

20

Cu

rren

t (u

A)

Potential (V)

100 mV/s

10 mV/s

Figure 9: Cyclic voltammograms for the oxidation of 10 µM Ochratoxin A on the surface of

NiNP in phosphate buffer (pH 6.0), accumulation potential -0.6 V and accumulation time

180 s.

0

1

2

3

4

5

6

7

8

0.01 0.03 0.05 0.07 0.09 0.11

Ipa

(µA

)

Scan rate (V/s)

(a) y = 76.527x + 1.032

R2 = 0.9856


Figure 10: (a) Relationship between anodic peak current and scan rate and (b) relationship

between log of peak current and log of scan rate on the voltammetric oxidation of 10 µM

Ochratoxin A

To achieve higher sensitivity and lower background current, 100 mV/s is chosen as optimum

scan rate and used in further analyses.

To achieve higher sensitivity and lower background current, 100 mV/s is chosen as optimum

scan rate and used in further analyses.

The number of electrons involve in electrochemical oxidation of Ochratoxin A at NiNP was

determined using equation below:

𝐼𝑝𝑎=𝑛𝐹𝑄𝑉

4𝑅𝑇

Where n is number of electrons transferred, F (C·mol−1

) is Faraday’s constant, Q (C) is quantity

of charge and v (V·s−1

) is scan rate. The value of n was estimated to be 0.798, which suggested

that one electron is involved in electro-oxidation reaction. Thus, it can also be concluded that

voltammetric reduction of Ochratoxin A, phosphate buffer (pH 6.0) at NiNP is accompanied by

loss of one electron.

0

0.5

1

1.5

2

2.5

3

0.1 0.15 0.2 0.25 0.3 0.35

Log

Ipa

Log v

(b) y = 3.318x + 2.031

R2 = 0.9986


It was also observed in scan rate studies that, as peak potential keeps shifting to more positive

values with every increase in scan rate, a plot of Ep vs log v yields a straight line with a slope that

can be expressed as 2.3 𝑅𝑇

(1−𝛼)𝑛𝐹 for oxidation peak current (Xu et al., 2018, Lawal, 2016), the

electron transfer coefficient (α) of electrochemical oxidation of Ochratoxin A at electrode

surface was estimated to be 0.621 which suggests high electron promotion process between

electrode surface and modifier towards oxidation of Ochratoxin A (Xu et al., 2018).

The sensitivity of NiNP towards detection of Ochratoxin A was investigated by differential pulse

voltammetry (DPV) under optimized experimental conditions for different concentration of

Ochratoxin A in Fig.11. A linear relationship do exist between peak current (Ipa) and Ochratoxin

A shows that peak height increases linearly with increase in concentration of Ochratoxin A.

Figure 11: Differential pulse voltammetry for detection of Ochratoxin A on the surface of

NiNP/SPE in phosphate buffer (pH 6.0), accumulation potential -0.6 V, accumulation time

180s.


Figure 12: Calibration curve of the peak current versus concentration of Ochratoxin A

under optimized experimental conditions.

Differential Pulse voltammograms of Ochratoxin A, Fig.11 clearly show that plot of peak current

versus concentration is linear for 0.01 – 0.5 µM of Ochratoxin A, the regression equation being

Ip(μA) = 6.232C + 0.9987 Ochratoxin A R2 = 0.9982), where C is µM concentration of

Ochratoxin A and Ip is peak current. The limit of detection was determined at 0.01 - 0.5 µM of

Ochratoxin A according to definition of YLOD = YB + 3σ (Gupta and Kumar 1999), and found

to be 0.00041 µM while LOQ is 0.00135 µM.

The limit of detection (LOD) of developed electrochemical sensors are calculated from a well

known equation (3σ/S) 10σ/S found in literature (Salih et al., 2017, Janati et al., 2012, Sun et al.,

2012, Lavudu et al., 2013a), where σ is standard deviation of measurements (n=5) and S is the

slope of linear regression equation of the plot of current versus log[Ochratoxin A]. The linear

regression equation of the plots Fig.12 represent y = mx + C (x is the concentration of

Ochratoxin A, y is the DPV peak current with nanoparticle.

Reproducibility of proposed sensor for Ochratoxin A detection was evaluated by determining

Ochratoxin A (10 µM) with seven different electrodes freshly prepared. They were used to

determined oxidation of anodic peak current, Ipa of 10 µM Ochratoxin A contained in 0.1M

PBS, with the aid of cyclic voltammetry. Results in for reproducibility show response with

0

2

4

6

8

10

12

14

0 0.5 1 1.5 2 2.5

Cu

rre

nt

(µA

)

Concentration (µM)

y = 6.232x + 0.9987

R2 = 0.9982

a


relative standard deviations of 1.92% using CV technique. The result indicates a satisfactorily

regeneration of the modification procedure in the preparation of NiNP/SPE with better

reproducibility and stability retaining 85% of its initial signal suggesting good stability at room

temperature for the period of 28 days and therefore can be used to determine Ochratoxin A. The

results obtained for interference (Table 1) under optimized conditions indicate that interference

species have less than 5% peak currents in each analysis.

Table 1: The effect of some co-existing compounds on the determination

of (10 µM) Ochratoxin A

Compounds Tolerancea

(mM)

Current change

%

Amoxicillin 50 +4.6

Penicillin G 50 +4.4

Thiamphenicol 50 +4.4

Sulfadiazine 100 +3.2

K+ 200 +4.4

Ca2+

200 -4.2

Na+ 200 +4.7

Mg(II) 200 -4.4

Zn(II) 200 -4.0

Mn(II) 200 -4.8

Fe(II) 200 -4.2


* aThe maximum mol ratio of each species that cause ≤5% change in the determination of

Ochratoxin A.

The recovery study of Ochratoxin A were measured by spiking Ochratoxin A free milk with

known amount of Ochratoxin A. The result shows drastic drop of Ochratoxin A recovery which

could be due to high adsorption of the Ochratoxin A at the surface of the electrode. The recovery

of the Ochratoxin A lies 72 to 99.2 %.

Detection of Ochratoxin A from real sample has been a crucial challenge in the development of

electrochemical sensor technique. Extraction was carried out according to (Tukiran et al., 2016,

Muhammad et al., 2018) method with little modification.

The applicability of the proposed sensor was evaluated by analyzing Ochratoxin A in fresh milk

samples supplied. There was no peak current observed in Fig 11 (a) with milk sample indicating

that there is no traces of Ochratoxin A in the milk sample supplied. The spiked sample of the

fresh milk with Ochratoxin A in Fig. 11 (b), shows current of 4.5 µA, 6.5 µA and 7.6 µA with

0.01 V, 0.02 V and 0.025 V while spiked sample with Ochratoxin A in Fig.11 (c) shows current

of 1.48 µA, 2.7 µA and 6.9 µA with potentials of -0.02 V, 0.023 V and 0.01 respectively,

confirming the presence of Ochratoxin A in the spiked sample as can be seen in Fig.11 (b) and

(c).

Conclusion

Applicability of the proposed NiNP electrochemical sensor for detection of ochratoxin A in food

samples was determined. The results obtained with the developed method were compared with

high performance liquid chromatographic (HPLC) technique. The electrodeposited nickel

nanoparticle on the SPCE were simple, rapid, cost effective and efficient. The proposed sensor

showed high sensitivity, low detection limit (LOD = 0.00041 μM, LOQ = 0.00135 µM S/N=7)

and wide concentration range (0.01 to 2.0 µM). The linearity and range are also similar as well

as their limit of detection and quantitation. These results suggest that the developed sensor can

serve as alternative means of detecting ochratoxin A in food samples

Acknowledgements


The authors acknowledge the Research grant No. 9443101 under Prof. Dr. Nor Azah Yusof,

Department of Chemistry, Faculty of Science, Universiti Putra Malaysia and also thank the

Faculty of Science.

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