Sensing and Bio-Sensing Research 4 (2015) 16–22

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Sensing and Bio-Sensing Research

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

Development of melamine sensor based on ionicliquid/nanoparticles/chitosan with modified gold electrode fordetermination of melamine in milk product

http://dx.doi.org/10.1016/j.sbsr.2015.02.0032214-1804/� 2015 The Authors. Published by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author. Tel.: +60 88320000x8467 (O); fax: +60 88 320993.E-mail address: shafiqpab@ums.edu.my (S. Siddiquee).

Kobun Rovina a, Shafiquzzaman Siddiquee a,⇑, Nyet Kui Wong b

a Biotechnology Research Institute, Universiti Malaysia Sabah, Jln UMS, 88400 Kota Kinabalu, Sabah, Malaysiab Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jln UMS, 88400 Kota Kinabalu, Sabah, Malaysia

a r t i c l e i n f o

Keywords:

MelamineCalcium oxide nanoparticlesElectrochemical biosensorCyclic voltammetry

a b s t r a c t

The illegal adulteration of infant milk powder with melamine in China has resulted in chronic kidney andurinary tract failure in September 2008. To date, several analytical techniques have been used for thedetermination of melamine, although these techniques are complicated, time consuming and costly. Inthis study, we developed a novel electrochemical biosensor method based on the modification of goldelectrode with chitosan, calcium oxide nanoparticles and an ionic liquid for the determination in thepresence of melamine in milk products. The electrochemical behaviour of the modified gold electrodewas studied by using cyclic voltammetry and differential pulse voltammetry in the presence of methy-lene blue used as a redox indicator. The morphological characteristics of nanomaterials were observedunder scanning electron microscope. Under optimal conditions, differential pulse voltammetry wasdetected at different concentrations of melamine from 9.6 � 10�3 to 9.6 � 10�15 M, with a detection limitof 9.6 � 10�16 M. The developed melamine sensor method is a very simple and fast procedure for analys-ing melamine level in milk and milk products.

� 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Melamine (2,4,6-triamino-1,3,5-triazine) is a kind of triazineanalogue together with three amino groups (pKa = 5.1).Melamine tends to hydrolyse under strong acid or alkali conditionsresulting in the formation of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), ammeline (4,6-diamino-2-hydroxy-1,3,5-triazine) andammelide (6-amino-2,4-dihydroxy-1,3,5-triazine) [1–4]. It has achemical compound that has been industrially used to synthesisemelamine–formaldehyde resins (MFR) for the production oflaminates, glues, dinnerware, adhesives, moulding compounds,coatings, as well as flame-retardant materials since 1950s [5].

Melamine’s high-rich nitrogen content is about 66% by mass.When, it is illegally added into dairy products by unethical com-pany to obtain incorrectly high readout of apparent protein con-tent, it is measured by the conventional standard Kjeldahl test[6]. In March 2007, melamine came to public attention in NorthAmerica when there are two cases reported involving the additionof melamine and its analogues in pet foods. Consequently, a petfood manufacturer alerted the US Food and Drug administration

to animal deaths associated with the deaths of dogs and cats inthe US that appeared in certain batches of their pet food. In the fol-lowing months, consumers and veterinarians again reported moreillnesses and deaths potentially associated with pet food [7–9]. Inearly September 2008, about 294,000 children from China are diag-nosed with urinary tract stone with 50,000 infants being hospita-lised and resulting in six babies’ death due to the melaminecontamination in milk products [10,11]. High concentration inges-tion of melamine has proven to be toxic to humans and found inthe formation of insoluble melamine cyanurate crystals in kidneyscausing renal failure. For this reason, there is increasing demand tofind effective and reliable method for the analysis of melamine inmilk and other food products.

During the past decades, several analytical methods have beenapplied for the determination of melamine in food products suchas high-performance liquid chromatography (HPLC) [12,13], gaschromatography–mass chromatography (GC–MS) and liquid chro-matography–tandem mass chromatography (LC–MS–MS) [14–16].However, most of these methods are complicated preconcentra-tion, time-consuming steps and required expensive instruments.Advances in chemistry, physics, biochemistry and molecular biol-ogy have led to the development of an electrochemical sensor,which can able to detect a wide range of biological elements due

K. Rovina et al. / Sensing and Bio-Sensing Research 4 (2015) 16–22 17

to their simplicity, low cost, sensitivity, selectivity, and possibilityfor miniaturization, portability and integration in automateddevices [17]. Thus, nanomaterials and nanocomposite membranebased developed-electrochemical biosensor are needed in a smallamount of sample for rapid detection of melamine.

Chitosan (CHIT) is an interesting natural biopolymer that con-tains reactive amino and hydroxyl functional groups. It has beenwidely used for immobilization due to high ease of use andabsorption, excellent film-forming ability, high permeability, highheat-stability, mechanical strength, non-toxicity, biocompatibility,low cost and easy availability. Moreover, chemical modification ofthe amino groups of CHIT has provided hydrophilic environmentfor the biomolecules. It is soluble in diverse acids that the inter-action with polyanions to form complexes and gels [18–21].However, hybrid materials based on CHIT have been used indevelopment of conducting polymers, carbon nanotubes, redoxmediators, metal nanoparticles and oxide agents, due to excellentproperties of individual components and outstanding synergisticeffects simultaneously for electrochemical biosensing platforms[22,23].

With advent of nanotechnology, nanoparticles (NPs) haveattracted much attention due to their novel optical, electrical,and mechanical properties. Amongst various NPs, calciumoxide nanoparticles (CaONPs) have obtained considerableattention because they are non-corrosive, non-explosive, non-volatile, high basicity and eco-friendly. CaONPs have onlyrequired mild reaction conditions to produce high yields ofproducts in short reaction times when compared to traditionalcatalysts [24–27].

Ionic liquids (ILs) are salts with melting point below 100 �C.they have been used during the past decade due to their uniqueproperties, such as high chemical and thermal stability, relativelyhigh ionic conductivity, low vapour pressure, wide liquid rangeand large electrochemical windows [28,29]. ILs are usually usedin homogeneous catalytic reactions because they high catalyticactivity and good selectivity. It is not only used for supporting elec-trolyte, but also used as the modifier in chemically modified elec-trodes in the biosensor field [30].

Taking advantages of the above characteristics, the main aim ofthis study was to develop an electrochemical biosensor methodbased on CHIT nanocomposite film, CaO nanoparticles and ionicliquid (1-ethyl-3-methylimidazolium trifluoromethanesulfonate([EMIM][Otf]) in the presence of methylene blue used as a redoxindicator for the determination of melamine (Fig. 1). Different mor-phological characteristics of nanomaterials and interaction withmelamine were observed by using SEM. The experimental parame-ters were optimised such as pH, interaction time and scan rate.Subsequently, the developed method was successfully applied forthe determination of melamine in milk samples with satisfactoryresults.

2. Experimental

2.1. Chemical and materials

Melamine, chitosan (CHIT) nanocomposite membrane, methy-lene blue (MB), Tris–HCl and acetic acid were purchased fromSigma–Aldrich (USA). The MB (1 mM) solution was prepared in50 mM Tris–HCl, 20 mM NaCl buffer solutions (pH 7.0) and storedat room temperature. [EMIM][Otf] and CaONPs (<100 nm) wereobtained from Biosensors and Bioelectronics Laboratory,Department of Chemistry, Faculty of Science, Universiti PutraMalaysia. Other chemicals used in the experiments were of analyti-cal reagent grade. All aqueous solutions were prepared with deion-ized water and were carried out at room temperature condition of25 ± 0.1 �C.

2.2. Apparatus and equipments

All electrochemical experiments were conducted using a lAutolab potentiostat/galvanostat (PGSTAT) driven with NOVAAutolab 1.8 software package in conjunction with the conventionalthree electrode system and a personal computer for data process-ing. Three electrode system was used in the measurement whichcomposed of 3 mm gold electrode (AuE) used as working electrode,Ag|AgCl|KCl as the reference electrode and the platinum (Pt) wireas counter electrode, respectively. The morphological characteris-tics of nanomaterials and nanocomposite membrane wereobserved under Hitachi S-3400N scanning electron microscope(SEM).

2.3. Preparation of CHIT/CaONPs/[EMIM][Otf]

A 2% of CHIT solution was prepared by dissolving the CHIT pow-der in 1% acetic acid and then stirred for at least 4 h until all dis-solved. Then, CaONPs were added into the 2% CHIT solution withmass ratio of 2:5 (CaONPs:CHIT). After that, the mixture was soni-cated for 20 min then it was stirred for 8 h for highly dispersed col-loidal suspension. A 3% (v/v) [EMIM][Otf] was dispersed in theCHIT/CaONP mixture and again sonicated for 3 h to produce aCHIT/CaONP/[EMIM][Otf] homogenous suspension.

2.4. Electrochemical measurements

Before modification, pre-treatment of AuE was performedaccording to Siddiquee et al. [31] procedure with some modifica-tions. Firstly, the AuE was polished with 3 lM aluminium slurryfor 2 min. After that, the AuE was sonicated for 2 min and rinsedwith deionized water for about 2 min. Later, 10 lL of the CHIT/CaONPs/[EMIM][Otf] suspensions was casted onto the AuE andthen dried at room temperature for at least 4 h in order to obtaina uniform coated membrane of CHIT/CaONPs/[EMIM][Otf]/AuE.After successful immobilization, the surface of the modified AuEwas soaked in 0.001 M of MB for 2 min followed by washing withTris–HCl buffer (pH 7.0) to remove unspecific physically adsorp-tion. The electrochemical measurements of the modified AuE andbare AuE were conducted in 50 mM Tris–HCl, 20 mM NaCl buffersolutions (pH 7.0) containing 1.0 mM of MB using CV and DPVmethods.

2.5. Detection of melamine in the milk sample

The milk sample was bought from local supermarket in KotaKinabalu, Sabah, Malaysia. The milk sample was pretreated accord-ing to Cao et al. [32] procedure. In a 50 mL falcon tube, 5 g milksample was first mixed with 5 mL of 0.4 M trichloroacetic acidand 40 mL of methanol solution. The mixture was sonicated for15 min and then shaken for 10 min. After that, the mixture wascentrifuged at 10,000 rpm for 10 min, and the supernatant was fil-trated using filter paper. Later on, the filtrate was condensed toreach a total volume of 5 mL and filtered through a 0.45 lm filtermembrane to obtain the samples for analysis. Finally, the pre-treated sample was carried out under the optimal conditions usingDPV experiment.

3. Results and discussion

3.1. Morphology of the modified AuE

The SEM images of CHIT/CaONPs/[EMIM][Otf] nanocompositeare shown in Fig. 2. The formation of homogeneous nanocompositeshowed that CHIT and CaONPs were uniformly integrated with

Fig. 1. Schematic representation of the sensing processes for determination of melamine.

Fig. 2. SEM images of (a) CHIT, (b) CHIT/CaONPs, (c) CHIT/CaONPs/[EMIM][Otf] and (d) CHIT/CaONPs/[EMIM][Otf]/melamine.

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[EMIM][Otf]. Initially, the SEM images of CHIT, CHIT/CaONPs andCHIT/CaONPs/[EMIM][Otf] are characterised by the surface mor-phologies structure of the nanomaterials as shown in Fig. 2. TheCaONPs were found well distributed and attached to the circumfer-ence of the CHIT membrane, and making the surroundings of theparticles appear brighter under SEM images (Fig. 2b). On the other

hand, CHIT/CaONPs/[EMIM][Otf] showed homogenous surfacewith uniform granular porous morphology attributed to thehomogeneous dispersion of [EMIM][Otf] in CHIT network due tothe electrostatic interaction (Fig. 2c). From the pattern of results,it can be concluded that the addition of CaONPs in the nanocom-posite membranes lead to increase the pore size. Besides, CHIT

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can provide a better performance in terms of sensitivity and stabil-ity of the modification. The surface morphological characteristicswere changed after adsorption of melamine onto CHIT/CaONPs/[EMIM][Otf] (Fig. 2d). Based on this result, the CaONPs were suc-cessfully dispersed and melamine was effectively adsorbed ontoCHIT/CaONPs/[EMIM][Otf].

3.2. Optimum condition for melamine determination

3.2.1. Effect of methylene blueMB is a heterocyclic aromatic chemical compound and used as a

redox indicator to increase the electron transfer in electrochemicalsensor. With and without MB, the CV peak currents of bare AuE areshown in Fig. 3. The oxidation peak current was found to be0.44 � 10�3 A without MB (Fig. 3a) and 1.06 � 10�3 A with MB(Fig. 3b), respectively, with the potential volt of 1.3. MB was abun-dantly adsorbed on the surface of the modified AuE by the electro-static interaction between MB and modified AuE. Accumulation ofMB acting as electro-active substance successfully increased thepeak current response, which can be used for electrochemical sens-ing of melamine. Hence, MB was accumulated onto the surfacemodified AuE for further experiments carried out.

3.2.2. Effect of pHElectrochemical biosensor provided a good performance with

different optimisation parameters such as the effects of pH, inter-action times of melamine and effects of scan rate in the presenceof MB. The MB covalently attached with melamine provided a sen-sitive electrochemical indicator in melamine electrochemistrymeasurements. The pH of the analytical buffer had a great effecton electrochemical behaviour of the sensor. The redox responseof melamine with the MB was pH dependent, the interaction of3.3 � 10�4 M concentration melamine in Tris–HCl buffer solutionswith different pH ranges (6.0, 6.5, 7.0, 7.5, 8.0, 8.5) and investigatedby using the CV method. Fig. 4 shows the effects of pH on the peakcurrents and found that the current responses increased with themaximum level from pH 6.0 to 7.0 after that started to decreaseuntil 8.5. According to Xie and Cai [33] proteins (melamine)adsorbed H+ or OH� with different pH values, which led to the dif-ferent electrochemical responses. By considering the response andthe activity of sensor, pH 7.0 was chosen as the optimum value formelamine measurement.

Fig. 3. Cyclic voltammetry of AuE without MB (a) and AuE with MB (b) in Tris–HClcontaining 3.3 � 10�4 M melamine.

3.2.3. Effect of interaction timeThe interaction times of melamine influenced the performance

of the modified AuE in the presence of MB. Fig. 5 shows the inter-action between AuE and melamine in aqueous solution when start-ing the electrochemical experiment. The result found rapidlyincreased the peak current response within 30 s when comparedto the uses other times, revealing that the interaction of melaminedecreased and MB was saturated on the surface of the modifiedAuE. Thus, 30 s was selected as an optimum interaction time forthe detection of melamine.

3.3. Electrochemical characteristics on electrode surface

Cyclic voltammetry (CV) is an effective and convenient methodfor the probing feature of the modified electrode surface. In thisstudy, CV was used for the observation of electrochemical beha-viours with MB after that each assemble step is shown in Fig. 6.The presence of MB induced clearly increased the oxidation peakcurrents of melamine with AuE (Fig. 6b). After the pretreatedAuE was modified with CHIT by adsorption of CaONPs, the peakcurrent was increased gradually indicating that the introductionof the CHIT/CaONPs played a vital role in the increase of the elec-troactive surface area and provided the conducting bridges of theelectron transfer within MB (Fig. 6c). After that, [EMIM][Otf] wasadded to the CHIT/CaONPs with the presence of MB. The peak cur-rents of the modified electrode (CHIT/CaONPs/[EMIM][Otf]) werequite similar to those of CHIT/CaONPs (Fig. 6d), but the potentialdifference between the two electropolymerization processes wasthe peak current. It can be seen that the peak currents increasedin the presence of [EMIM][Otf] due to the positive charges of theionic liquid acts as a supporting electrolyte. The combinations of[EMIM][Otf] and CaONPs enhanced the electron transfer in theelectrochemical reaction for determination in melamine.

3.4. Electrochemical determination of melamine

Under the optimal experimental conditions, electrochemicalresponse studies were conducted with the modified AuE at differ-ent concentrations of melamine by using the DPV method. Fig. 7shows that the peak current increases with the increase concentra-tions of melamine. The oxidation peak currents were found to beproportional to the melamine at different concentrations in rangeof 9.6 � 10�15–9.6 � 10�3 M. The detection limit was calculatedas 9.6 � 10�16 M (n = 5). The fabricated CHIT/CaONPs/[EMIM][Otf]showed good electrocatalytic behaviours towards the oxidationof melamine with the enhanced oxidation peak current anddecrease the peak-to-peak separation. The performances of theconstructed melamine biosensor and the other electrochemicalbiosensors based on the nanoparticles and ionic liquid were com-pared and the results are summarized as shown in Table 1. It canbe seen that the developed melamine biosensor has a low detec-tion limit and a wider linear range at different concentrations ofmelamine. The linear regression equation for the calibration plotwas calculated to be y = 0.4184x + 8.0117 (R2 = 0.9933), where xis the concentration of melamine and y is the oxidation current.

3.5. Storage stability and reproducibility of the modified electrode

The stability of the [EMIM][Otf]/CaONPs/CHIT/AuE was exam-ined. After keeping the modified AuE for 1 week, the currentresponse of the electrode did not remarkably change (Fig. S1).Thus, it was showed a good stability of the [EMIM][Otf]/CaONPs/CHIT/AuE with relative standard deviation of 3.34%. The repro-ducibility of the current response for the [EMIM][Otf]/CaONPs/CHIT/AuE was examined in the presence of 1.0 mM MB solutioncontaining 9.6 � 10�4 M of melamine (Fig. S2). It was found that

Fig. 4. Cylic voltammetry and relationship between potential current with different pH of Tris–HCl buffer using MB in solution containing 3.3 � 10�4 M melamine.

Fig. 5. Cylic voltammetry and relationship between potential currents at different interaction times (a–e: the time is 5, 10, 50, 40 and 30 s) in pH 7.0 Tris–HCl buffer in thepresence of 3.3 � 10�4 M melamine.

Fig. 6. Cylic voltammetry of different surface electrodes measured in the presenceof pH 7.0 Tris–HCl buffer: (a) bare AuE, (b) AuE/MB, (c) AuE/CHIT/CaONPs/MB and(d) AuE/CHIT/CaONPs/[EMIM][Otf]/MB with 3.4 � 10�4 M melamine.

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the oxidation peak current remained the same with a relativestandard deviation of 2.47% for 5 repetitive measurements,indicating that the electrode has good reproducibility. Theseresults indicate that the modified electrode shows good stabilityand reproducibility.

3.6. Analytical application in milk

In order to evaluate the performance of the proposed methodfor real sample analysis, the method was applied to the analysisof melamine in the milk powder bought from the local supermar-ket, Kota Kinabalu, Sabah, Malaysia. The powdered milk samplewas pretreated according to Cao et al. [32] procedures. DPV experi-ments were carried out under optimal conditions: analytical buf-fer, pH 7.0 and the accumulation time of 30 s. Experiments wereconducted in parallel 5 times. The average potential current at�0.183 V was obtained as 6.52 � 10�6 A with a recovery rate of102%. The amount of melamine in the tested fresh milk samplemet with the standard requirement.

Fig. 7. (a) Differential pulse voltammetry of different concentrations of melaminein pH 7.0 Tris–HCl buffer. (a–g: the concentration of melamine is 0 M (control),9.6 � 10�4, 9.6 � 10�7, 9.6 � 10�9, 9.6 � 10�11, 5.0 � 10�13 and 9.6 � 10�15 M). (b)Linear relationship between oxidation peak currents obtained by differential pulsevoltammetry and the logarithm of melamine concentrations.

Table 1Comparison of the electrochemical sensor techniques to determine melamine indifferent food products.

Methods Samples Limit ofdetection

References

Gold electrode modified byCHIT/CaONPs/[EMIM][Otf]

Milk powder 9.6 � 10�16 M This work

Glassy carbon electrode bychemical coupling ofhorseradish peroxidase

Infant formulapowder and fishfeed samples

1.0 � 10�11 M [34]

Indium tin oxide electrodemodified by gold NPs

Liquid milk 0.13 ppb [35]

Graphite–epoxy compositeelectrode by usingbismuthyl chloride

Fresh milk 2.5 x 10�12 M [36]

Screen printed carbonelectrode modified bypolymer film

Dairy productsand pet foods

98.3 ppb [37]

Gold electrode modified byoligonucleotide (d(T)20)film

Milk 9.6 � 10�9 M [32]

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4. Conclusion

Melamine biosensor is developed based on the modified([EMIM][Otf]/CaONPs/CHIT) gold electrode. The modified goldelectrode has increased the surface area of the electrode andincreased the efficiency of the immobilization process in melamine

and enhanced the detection sensitivity level. This developed mela-mine biosensor can be applied for the determination of melaminewith a dynamic concentration in the range of 9.6 � 10�15–3.3 � 10�3 M, with detection limit of 9.6 � 10�16 M. It can beapplied for the determination of melamine in milk products; thus,this method may have a potential application in food inspection.So, these results suggested that the developed melamine biosensoroffers a simple, fast, good selectivity, high sensitivity, a wide detec-tion range and convenient method for applying in food researchlaboratories.

Conflict of interest

The authors confirm that there is no conflict of interest todeclare.

Acknowledgement

The authors would like to thank the Ministry of Science,Technology and Innovation (MOSTI), Malaysia for their supportunder research project No. SCF0090-IND-2013.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.sbsr.2015.02.003.

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