Reduced Graphene Oxide/Poly(Methylene Blue)Composite Modified Electrode for the Detection ofAcrylamide

ZHOU Ying,LI Ruichun,WANG Tingting,SHUANG Shaomin

(School of Chemistry and Chemical Engineering,Shanxi University,Taiyuan 030006,China)

Abstract:Acrylamide is a product of food heat processing,which is reproductively toxic,neurotoxic and carcinogenic.Here,using a three-electrode system of the electrochemical workstation,the electrochemical reduction of graphene oxide and the oxidation of methylene blue were carried out simultaneously on the surface of glassy carbon electrode.The reduced graphene oxide/poly(methy‐lene blue)(RGO/PMB)complex was synthesized on the electrode surface and a sensitive electrochemical sensor for acrylamide was constructed.In the optimal case,the detection limit of the proposed sensor was up to 1.67×10−9mol/L and the detection range was 5×10−9-2.5×10−5mol/L.The results showed that the sensor had good selectivity,stability and reproducibility,and could be used for the determination of acrylamide in actual potato chip samples.

Key words:acrylamide;electrochemical sensor;reduced graphene oxide;poly(methylene blue)

1 Introduction

Acrylamide is a product of the Maillard reac‐tion that occurs when foods richly containing starch and aspartic acid are heated,baked and fried above 120℃(mostly at 150℃−180℃)[1].Although acrylamide is also widely used in industry,it can cause damage to humans to a certain extent[2].Its toxicity mainly includes neurotoxicity,reproductive toxicity,and potential carcinogenicity[3].It was clas‐sified as a class 2A carcinogen by the International Agency for Research on Cancer(IARC)in 1994[4].In 2002,researchers from the Swedish National Food Administration(NFA)and Stockholm Univer‐sity first announced the presence of high content acrylamide in potato chips,toast and coffee[5].In 2015,the European Food and Safety Authority(EF‐SA)announced that the maximum amount of acryl‐amide that had the lowest risk of cancer was 170 μg·kg−1body weight[6].The United States and the European Union have recently taken measures to re‐duce acrylamide intake[7].Therefore,the detection ofacrylamide is very indispensable to human health.However,currently,the detection of acryl‐amide is facing the challenge of measuring lower concentration with a simpler,time-saving and laborsaving method.The traditional detection methods include chromatography[8],fluorescence[9-11],capillary electrophoresis[12],enzyme-linked immunosorbent as‐say[13]and so on.These methods exhibit high sensi‐tivity,favorable selectivity and excellent accuracy,but all of them involve expensive equipment,profes‐sional operators and a complex sample pretreatment process.In comparison,electrochemical methods have attracted much attention because of the sensi‐tive current response,simple operation,economy and effectiveness.Recently,a little distinct work for the detection of acrylamide has been done.For instance,the Wulandari group used wet-chemical seeding method to prepare the PtNPs-modified bo‐ron-doped diamond electrode[14];the Wu group modi‐fied SnO2-SiC hollow sphere nanochains/gold nano‐rod on the electrode surface with chitosan to con‐struct an immunosensor[15];and the Mersal group de‐veloped a molecular biomimetic sensor[16].Though these methods have merits,each of them has a com‐plex preparation process for electrode modification materials.

Graphene is a two-dimensional carbon material with a honeycomb shape.It is extensively used as an electrochemical modification material because of its good electrical conductivity[17-18].Reduced gra‐phene oxide(RGO)can be obtained by reducing graphene oxide(GO)which has rich oxygen-con‐taining functional groups,so it has properties simi‐lar to graphene.In contrast,reduced graphene oxide has some residual oxygen atoms and heteroatomic,and therefore has more excellent performance[19].The methods to obtain RGO include ultra-high vacu‐um heating[20],chemical treatment with hydra‐zine[21-22],UV lamp irradiation[23]and electrochemical methods[24].Recently,conductive polymers,especial‐ly electroactive polymers,have also attached much attention because they can be used to develop novel porous polymer materials with excellent electrical,optical,biodegradable and renewable properties[25].Methylene blue(MB)is a phenothiazine redox dye.Poly(methylene blue)(PMB)film with good catalyt‐ic activity,high conductivity,stability and good bio‐compatibility is obtained by the polymerization of MB,which can further improve the conductivity and is widely used in the field of electrochemis‐try[26].It can be used for electrocatalytic oxidation of nicotinamide adenine dinucleotide(NADH)[27];achieve indirectly electrochemical sensing and scav‐enging of OH radicals[28];and detect acrylamide sen‐sitively[29].Some groups also hybrid PMB with met‐al-organic frameworks(MOFs)[30],carbon nano‐tubes[31],metal nanoparticles[32]and other composite materials for electrochemical sensing.

In this work,we delicately prepared an electro‐chemical sensor for the determination of acrylamide via electrochemical polymerization of graphene ox‐ide and methylene blue simultaneously.The results indicated that the facilitated sensors were sensitive,rapid,cost-effective and could be used for the detec‐tion of acrylamide in commercial potato chip sam‐ples.

2 Experimental

2.1 Reagents and apparatus

Reagents:Graphite powder was ordered from Shanghai Macklin Reagent Company.Sodium ni‐trate,sulfuric acid(98%),potassium permanganate,hydrogen peroxide(30%),hydrochloric acid(5%),acrylamide,glutamic acid,arginine,isoleucine,tyro‐sine,CaCl2,NaCl,methylene blue(MB),acetoni‐trile,n-hexane,Na2HPO4•12H2O,NaH2PO4•2H2O,KCl,potassium ferrocyanide were purchased from Shanghai Aladdin ReagentCompany.Phosphate buffer solutions with various pH values were pre‐pared from reserve standard solutions of Na2HPO4and NaH2PO4.All reagents were analytically pure and used without further purification.

Apparatus:PHS-3C pH meter(Thunder Mag‐netic Instrument Company,Shanghai,China)was used to measure the pH of the solution.A threeelectrode system(glassy carbon electrode as work‐ing electrode,SCE electrode as the reference elec‐trode,platinum wire electrode as the counter elec‐trode)was used for electrochemical detection by CHI660E electrochemical workstation(CHI Instru‐ment Company,Shanghai,China).Field emission scanning electron microscope(FESEM)(SU8020,Japan)was used to study the morphology of the modified electrode surface.

2.2 Preparation of GO

GO was prepared through modified Hummer's method[33].Firstly,the mixture of 3 g graphite pow‐der,3 g sodium nitrate and 75 mL of sulfuric acid(98%)was added in a round-bottom flask,then stirred in an ice bath(0℃)for 2 h.Secondly,un‐der stirring conditions,9 g potassium permanganate was slowly added to the mixture and continuously stirred at room temperature for 2 h.Then the mix‐ture was heated to 98℃ and stirred for 8 h.Third‐ly,the obtained suspension was cooled to room tem‐perature,100 mL water and 30 mL hydrogen perox‐ide(30%)were added,and the GO layer was re‐moved by ultrasound.Finally,the obtained GO sus‐pension was centrifuged and the precipitation was thoroughly cleaned with hydrochloric acid(5%)and deionized water.The precipitation was placed in a vacuum oven to dry for 12 h to obtain GO powder.

2.3 Preparation of RGO/PMB modified elec‐trodes

The modified electrodes were prepared by elec‐trochemicalpolymerization graphene oxide and methylene blue composite.GO(2 mg/mL)were drop-coated on the surface of the polishing glassy carbon electrode and dried naturally.The electrodes were immersed in 0.1 mol/L phosphate buffer solu‐tion(pH=7)containing 0.03 mmol/L methylene blue and scanned for 15 cycles at a rate of 50 mV·s-1in the range from−1.5 V to 1.3 V to complete the preparation of RGO/PMB modified electrodes.As a control,RGO modified electrodes were pre‐pared by direct electrochemical reduction of GO in 0.1 mol/L PBS,and PMB modified electrodes were prepared by soaking bare glassy carbon electrode in 0.1 mol/L PBS(pH=7)containing 0.03 mmol/L MB.

2.4 Preparation of real potato chips sample

Firstly,the potato chip sample prepared in ad‐vance was ground to powder,and 2.0 g sample powder and 10 mL acetonitrile were evenly mixed in a 50 mL centrifuge tube,which was oscillated for 10 min,centrifuged at 10 000 rpm for 5 min,and the supernatant was acquired.Continuously,10 mL acetonitrile was added into the residue and repeated the above operation three times to obtain the ex‐tract.Secondly,the extract was mixed with 20 mL n-hexane and shook for 5 min.The upper oily liq‐uid was removed by standing,and n-hexane was added to the lower liquid three times to obtain the lower liquid.Finally,5 mL deionized water was added to the lower liquid and mixed evenly.The mixture was placed in a 50 mL round-bottom flask,distilled for 2 h at 85℃ and cooled to room tem‐perature.The liquid in the flask was stored 4℃as the sample for later use.

3 Results and discussions

3.1 The construction of the RGO/PMB sensor and the principle of acrylamide detection

As shown in Scheme 1(a),the preparation of the modified electrode by drop-coating GO on the glassy carbon electrode,followed by immersing GCE into 0.1 mol/L(pH=7)PBS containing 0.03 mmol/L MB.The reduction of GO and oxidation of MB were carried out simultaneously through electro‐chemical polymerization to prepare the RGO/PMB complex modified electrode.The modified elec‐trode showed an excellent DPV electrochemical re‐sponse(black line)and displayed a significant de‐crease(red line)in the presence of acrylamide.This may be due to the reaction between acrylamide and MB at the electrode-electrolyte interface.Acryl‐amide is a non-electroactive substance,which hin‐ders the electron transfer at the electrode-electrolyte interface.Thus,the modified electrode could be used for acrylamide detection.The possible reaction between MB and acrylamide on the modified elec‐trode was shown in Scheme 1(b).

As shown in Fig.1(a),continuous potential cy‐cling was carried out for the electrochemical polym‐erization of MB at a rate of 50 mV/s at a voltage range from−1.5 V to 1.3 V.In the first cycle(black line),it could be seen that two oxidation peaks appeared at about−0.2 V and 1.1 V,corre‐sponding to the monomer oxidation of MB and the irreversible oxidation of MB radical cations,respec‐tively.With the cycles increasing,another wide and quasi-reversible feature oxidation peak appeared at about 0.6 V,and the peak potential exhibited a posi‐tive shift compared with the monomer peak.This suggested the formation of PMB.The formation of radical cation proceeded at a higher potential owing to the presence of the tertiary amino substituent in MB rings.The formation of radical cation triggered the reaction with MB monomer near the electrodeelectrolyte interface,which led to the polymeriza‐tion of MB.Due to the electronegativity of the car‐bon atom in the vicinity of the amino group,the formed unstable radical cation was covalently bound to another aromatic ring of the monomer through the ortho position of the amino group.The electro‐chemical polymerization of MB was carried out by direct π-π stacking or nitrogen bridge.

Scheme 1 Schematic diagram of RGO/PMB modified electrode(a);The principle of acrylamide sensing(b)

Fig.1(b)shows the electrochemical polymer‐ization process of RGO/PMB with the potential cy‐cling increasing.Similar to the process of PMB in Fig.1(a),two pairs of redox peaks appeared in the first cycle(black line).Two significant oxidation peaks located at−0.1 V and 1.2 V were attributed to the oxidation of MB monomer and the oxidation of MB radical cations,respectively.The reduction peaks located at about−0.25 V and −0.84 V cor‐responded to the reduction of GO.With the in‐crease of the scanning cycle,an increasing oxidation peak with a positive potential shift appeared at about 0.4 V,which indicated the formation of PMB film.At the same time,it could be seen that the re‐duction peak located at−0.25 V also had a current increasing trend,which indicated that the reduction of GO and oxidation of MB were carried out simul‐taneously in the process of electrochemical polymer‐ization.By comparing the current response intensi‐ty in the two figures,it also could be found that RGO/PMB modified electrode was about 4 times than PMB modified electrode.It indicated that the existence of RGO accelerated the electron transfer rate at the electrode-electrolyte interface and promot‐ed the current response.

Fig.1 Electrochemical polymerization of PMB(a)and RGO/PMB(b)

3.2 The characterization of RGO/PMB modified electrode

The formation of the modified electrode was characterized by electrochemical impedance spec‐troscopy.In Fig.2,EIS Nyquist plots of the differ‐ent modified electrodes were analyzed by the Ran‐dles equivalent circuit.In a typical Nyquist dia‐gram,the diameter of the semicircular domain repre‐sents the charge transfer resistance.The larger the diameter is corresponding to greater the impedance of electron transfer.As shown in Fig.2,RGO modified electrode had a semicircle domain with a diameter of about 270.5 Ω in the high-frequency re‐gion,which indicated that the lower electron trans‐fer efficiency between the electrode and the electro‐lyte and the larger impedance.However,the semi‐circle domain wholly disappeared when PMB and RGO/PMB modified the electrode,which suggested that the porous structure formed during the forma‐tion of PMB film and indicated that electron trans‐fer was quick and diffusion-controlled.In addition,the slope of RGO/PMB was greater than PMB,which indicated that the RGO/PMB modified elec‐trode had a higher electron transfer rate and meant that the presence of RGO accelerated the electron transfer rate of the electrode-electrolyte interface and promoted the electrochemical response.

Fig.2 EIS plot for RGO;PMB;RGO/PMB composite modi‐fied electrodes in 0.1 mol/L KCl solution containing 5 mmol/L[Fe(CN)6]3－/4－

To further verify the formation of RGO/PMB modified electrode,scanning electron microscope al‐so was used.As shown in Fig.3(a),the morpholo‐gy of GO showed a thin folded sheet structure.The SEM image of the RGO/PMB showed that the lumpy structure adhered together located at the fold‐ed layered structure surface(Fig.3(b)),which was considered as the formation of PMB.This unique structure could provide more active sites for the re‐actants.

Fig.3 SEM images of GO(a)and RGO/PMB(b)

3.3 Influence of scan rate

The electrochemical response of acrylamide on RGO/PMB modified electrodes at different scan rates was evaluated by CV.As shown in Fig.4(a),the electrochemical response increased with the scan rate increasing at the range of 200 mV·s－1−800 mV·s－1.Fig.4(b)shows the linear relationship between peak current and scanning rate.The linear equation is as follows:Ipa(μA)=0.111 67v1/2(mVs－1)1/2+87.003 3,R2=0.988 37;Ipc(μA)= −0.151 73v1/2(mV·s－1)1/2−97.760 44,R2=0.989 78,which in‐dicates that the electrochemical behavior of acryl‐amide on RGO/PMB modified electrode is affected by diffusion.

Fig.4 Cyclicvoltammogram of RGO/PMB in the presence of 2.5×10−7mol/L acrylamide at different scan rates(200 mV·s−1−800 mV·s−1)(a);and the linear relationship of anode and cathode versus the scan rate(b)

3.4 Electrochemical sensing of acrylamide

Fig.5(a)showed the electrochemical response of the RGO/PMB modified electrode with different concentrations of acrylamide.And the electrochemi‐cal response decreased significantly with the acryl‐amide concentration increasing.This result is con‐sistent with the detection principle.As shown in Fig.5(b),a calibration plot of the peak current and the logarithm concentration ofacrylamide was shown,and a linear relationship ranged from 5.0×10－9mol/L−2.5×10－5mol/L was as follows:IDPV(μA)=70.04 − 7.30logC(M),R2=0.993 5.Ac‐cording to the LOD=3σ/k,where σ is the standard deviation of the blank sample detection value and k is the slope,the limit of detection(LOD)is calculat‐ed to be 1.67×10－9mol/L.

Fig.5 DPV response of RGO/PMB modified electrode with acrylamide range from a to o(5.0×10－9mol/L to 2.5×10－5mol/L)in PBS(a);and the linear calibration between the current responses and the logarithm concentration of acrylamide(b)

In order to evaluate the electrochemical perfor‐mance of the constructed RGO/PMB sensor for acrylamide detection,the linear range and LOD val‐ues of other electrochemical sensors reported were listed in Table 1.The results suggested that RGO/PMB modified electrode had the lowest detection limit and a wide linear range.Due to its simple op‐eration and high sensitivity,this sensor had great po‐tential for practical acrylamide detection.

Table 1 Comparison of acrylamide sensors reported in the other literature with RGO/PMB

Table 2 Assay Results of acrylamide for the real potato chips using the proposed platform

3.5 Stability,selectivity and reproducibility of RGO/PMB modified electrode

The stability was examined by placing RGO/PMB modified electrode in a refrigerator at 4℃and testing the DPV response of 2.5×10－7mol/L acrylamide solution in PBS every two days.As shown in Fig.6(a),the DPV response value re‐mained 96.25% after two weeks,which proved that the sensor had good stability.Selectivity is critical for sensors due to actual samples containing other chemicals.Fig.6(b)showed the DPV response of blank,acrylamide,glutamate,arginine,isoleucine,tyrosine,Ca2+,Na+,Cl-at the same concentration.Compared with the blank control,the DPV signal of acrylamide only showed a significant decline,which supposed that this sensor had a good anti-interfer‐ence ability for acrylamide detection.The reproduc‐ibility of the sensor was studied by using the same modified RGO/PMB electrode to detect 2.5×10－7mol/L acrylamide 10 times under the same condi‐tions.The DPV results showed that the peak shape remained unchanged and the peak current changed slightly(the relative standard deviation was less than 3.0%),indicating that the constructed sensor had good reproducibility.

Fig.6 Stability assays of RGO/PMB modified electrode with 2.5×10-7mol/L acrylamide(the modified electrodes were placed in a refrigerator of 4℃and tested every two days.)(a);DPV signals of 2.5×10-7mol/L acrylamide and same concentration reference solution(b)

3.6 Analysis of real samples

For exploring the practical application of the sensor,real acrylamide samples were extracted from commercially available potato chips and the results were listed in Table 2.The experiments were car‐ried out using the standard dosing method by spik‐ing acrylamide solution with known concentration into potato chip extract and diluting with PBS.The results showed that the recovery rate was in the range of 97.6%−102.4% and the relative standard deviation(RSD)was less than 2.02%,which indi‐cated the good applicability of this sensor for acryl‐amide detection in actual potato chip samples.

4 Conclusion

In summary,we delicately designed an RGO/PMB electrochemical sensor for acrylamide detec‐tion.The modified electrode was prepared by elec‐trochemical polymerization of graphene oxide and methylene blue simultaneously. Electrochemical sensing of acrylamide was carried out by interaction between acrylamide and PMB.The facilitated sen‐sor showed excellent sensing performance in the range of 5×10－9mol/L−2.5×10－5mol/L,and the detection limit was as low as 1.67×10－9mol/L.Compared with the electrochemical sensors report‐ed,RGO/PMB sensor had the advantages of simple operation,good stability and high sensitivity.In or‐der to explore the practical application of this sen‐sor,we took commercially available potato chips as an example,and the results supposed that RGO/PMB had great potential to be applied to the detec‐tion of acrylamide in actual samples.