Direct synthesis of graphene-chitosan composite and its application as an enzymeless methyl

Colloids and Surfaces B:Biointerfaces 96 (2012) 75–79Contents lists available at SciVerse ScienceDirectColloids and Surfaces B:Biointerfacesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r fbShort communicationDirect synthesis of graphene–chitosan composite and its application as an enzymeless methyl parathion sensorShanli Yang,Shenglian Luo ∗,Chengbin Liu,Wanzhi WeiState Key Laboratory of Chemo/Biosensing and Chemometrics,College of Chemistry and Chemical Engineering,Hunan University,Hunan,Changsha 410082,PR Chinaa r t i c l ei n f oArticle history:Received 11October 2011Received in revised form 14February 2012Accepted 13March 2012Available online 28 March 2012Keywords:One-step electrodeposition Graphene–chitosan filmOrganophosphorus pesticides Methyl parathiona b s t r a c tThis paper proposed a direct electrodeposition approach to synthesis of graphene–chitosan (GR–CS)composite onto glassy carbon electrode (GCE)under controlled potential.This direct electrodeposition approach for the construction of GR-based hybrid was environmentally friendly,which would not involve the chemical reduction of graphene oxide (GO)and therefore result in no further contamination.The whole procedure was simply and cost only several minutes.Moreover,Combining the advantages of GR (large surface-to-volume ratio and high conductivity)and CS (good and adsorption),GR–CS composite could be highly efficient to capture OPs and used as solid phase extraction (SPE).The GR–CS/GCE was used to detect organophosphate pesticides (OPs),using methyl parathion (MP)as a model analyte.The significantly redox response of MP on the GR–CS/GCE was proved.The linear range was wide from 4.0ng mL −1to 400ng mL −1,and a low detection limit of 0.8ng mL −1for MP was achieved.Moreover,the proposed sensor exhibited high reproducibility,long-time storage stability and satisfactory anti-interference ability.The proposed GR–CS/GCE opens new opportunity for green,fast,simple,and sensitive analysis of OP compounds.© 2012 Elsevier B.V. All rights reserved.1.IntroductionOrganophosphorus pesticides (OPs)have been widely used in agriculture for pest control and shown undoubted benefits.How-ever,due to their large persistence and high toxicity,the extensive use of OPs can cause environmental pollution and ecological prob-lem,and their presence in water and food poses hazard to human health [1–4].Determination of these toxic agents are important for public health and environmental security [5–7].A number of methods for OPs determination have been proposed [8–13],among which electrochemical technique,i.e.,solid phase extraction (SPE)of OPs combined with square-wave voltammetric analysis,has the advantages such as simplicity and convenience,and high sensitiv-ity.But electrochemical reactions of most of these pesticides show sluggish electrode kinetics,as they are very large organic molecules,resulting electrode fouling [14,15].This affects the performance of the electrode during electrochemical measurements.There-fore,it is important to discover appropriate electrode materials to improve the performance of the OPs sensing.Among the OPs,the nitro aromatic OPs,such as methyl parathion (O,O-dimethyl O-(4-nitrophenyl)phosphorothioate,MP)exhibits good redox activities at the electrode surface [3,14,16].∗Corresponding author.Tel.:+867318823805;fax:+867318823805.E-mail address:sllou@ (S.Luo).As a “rising star”material,graphene (GR)has attracted much attention since its first experimentally produced in 2004[17–20].GR possesses many novel properties,such as well bio-compatibility,large surface-to-volume ratio,excellent electrical conductivity,electron mobility and flexibility.Chitosan (CS)is a linear hydrophilic polysaccharide obtained by deacetylation of nature chitin.It is an attractive biocompatible,biodegradable,and nontoxic natural biopolymer that exhibits excellent film-forming ability.CS has been widely used to disperse nanomaterials and can provide a good biocompatible microenvironment to construct sen-sors.Due to the unique properties of GR and CS,it is believed that the GR–CS composite can be highly efficient to capture OPs and can be used as SPE.The GR–CS composite would be an excellent elec-trode material for OPs sensing,which can facilitate the enrichment of OPs,enhance the surface concentration,alleviate the fouling effect and improve the sensitivity because of the low electronic noise from thermal effect [19,21–23].For sensing applications,the methods for depositing active materials on electrodes are most crucial.So far,GR films on electrodes usually have been obtained by drop-casting solution-based GR obtained from chemical reduction of graphene oxide (GO)sheets [24].In that way,the thickness of the resulting films was hardly uniform and controllable,the sensor fabrica-tion was not so reproducible,and moreover,toxic chemicals are involved.Electrochemical reduction of GO to GR has drawn much attention.Recently,our group has reported that GR nanosheets could be electrodeposited onto glassy carbon electrode through0927-7765/$–see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.colsurfb.2012.03.00776S.Yang et al./Colloids and Surfaces B:Biointerfaces 96 (2012) 75–79Fig.1.(A)SEM image and (B)TEM image of the GR–CS composite electrodeposited on the GCE surface,(C)Raman spectra of the GR–CS composite.cyclic voltammetric reduction of GO,the electrochemical reduc-tion potential of GO was about −1.0V and the obtained GR coating was very stable [25].Herein,we prepare GR–CS composite on GCE directly from GO–CS dispersion by one-step electrodeposition under controlled potential.By combining the benefits of GR and CS,the obtained GR–CS film shows strong adsorption ability of OPs,and the goal was achieved for OPs sensing.To our best knowledge,this is the first report on a green and direct electrochemical synthesis of GR–CS composite and further utilizing GR–CS composite as SPE for electrochemical determination of OPs,using MP as a model analyte.2.Experimental2.1.ReagentsGO was purchased from Beijing University.CS from crab shells (85%deacetylated)was purchased from Sigma.All other chemicals were of analytic grade and were used without further purifica-tion.Double-distilled water was used throughout the experiments.0.1M,pH 5.2acetate buffer solution was used as the electrolyte.CS solution was prepared by dissolving CS solid in 0.10M acetic acid (HAc).2.2.Apparatus and instrumentationsAll the electrochemical measurements were performed on a CHI 660A electrochemical workstation (Shanghai Chenhua Instru-ment Company,China).A conventional three electrode system was used,which consisted of a modified or bare glassy carbon elec-trode (3mm in diameter)as working electrode,a platinum foil as counter electrode,and an Ag/AgCl (3M KCl)electrode as refer-ence electrode.All potentials were measured against Ag/AgCl.All experiments were done at room temperature (∼25◦C).The micrograph of GR–CS film was investigated by transmission electron microscopy (TEM,Philips EM400ST microscopy).2.3.One-step electrodeposition of GR–CS film onto glassy carbon electrodePrior to electrodeposition,the GCE was polished with 0.3␮m and 0.05␮m ␣-Al 2O 3powder until a mirror-shiny surface was obtained,then ultrasonicated in ethanol and twice-distilled water for 10min,respectively.The prepared GCE was dried with nitrogen gas and used for modification immediately.5mg of GO was dispersed in 5mL of 0.2%(w/t)CS solution with ultrasonication to form 1mg mL −1GO–CS solution.The prepared GCE was immersed into the GO–CS solution while stirring and aS.Yang et al./Colloids and Surfaces B:Biointerfaces96 (2012) 75–7977fixed potential of−1.0V was applied for400s.After the electrode-position,the GR–CS/GCE was obtained.The GR–CS/GCE was stored at4◦C in a refrigerator under dry conditions when not in use.For comparison,CS/GCE was obtained by electrodepositing CS onto the GCE under−1.0V for400s.2.4.Measurement procedureMPs were accumulated on the GR–CS/GCE at open circuit poten-tial by immersing the GR–CS/GCE into a5mL stirred0.1M acetate buffer solution(pH5.2)containing MP for180s.The electrode was removed,rinsed with MP free0.1M acetate buffer solution and transferred into a cell of0.1M acetate buffer solution for electrochemical measurements with cyclic voltammetry(CV)and square-wave voltammetry(SWV).CV measurements were per-formed from−800to400mV with a scan rate of100mV s−1.SWV measurements were performed from−400to200mV with a step potential of4mV,an amplitude of20mV,and a frequency of25Hz.2.5.Regeneration of electrode surface.After each stripping measurement,MP was removed from the electrode by stripping at+0.5V for180s in a5mL stirred0.1M acetate buffer solution(pH5.2).3.Results and discussion3.1.Characterization of GR–CS/GCEIt is well known that chemical reduction of GO sheets in aqueous solutions results in their irreversible agglomerate[26].Therefore,it is reasonable to speculate that when the GO sheets in direct contact with an electrode accept electrons to suffer from electrochemical reduction,the resulted GR sheets will also be insoluble,and thus directly attach to the electrode surface[25].Fig.1(A and B)are the SEM and TEM images of the GR–CS composite electrodeposited on the GCE surface,revealing the typical crumpled and wrinkled GR sheet structure on the rough surface of the CSfilm.The Raman spec-tra of GR–CS composite(Fig.1(C))shows a strong diamondoid(D) band at1290cm−1and a weak graphitic(G)band at1580cm−1, which are attributed to two vibration modes of GR.The D band associates with vibrations of carbon atoms with dangling bonds in plane terminations of disordered graphite and the G band corre-sponds to an E2g mode of graphite and is related to the vibration of sp2-bonded carbon atoms in a2D hexagonal lattice,such as in a graphite layer[27].The Raman result is also similar to the previous reports[28,29].All these results indicate that GR–CS nanocompos-ite has been prepared on electrode from GO–CS dispersion by direct one-step electrodeposition.3.2.Enhanced electrochemical sensing with GR–CS/GCEFig.2showed the cyclic voltammograms obtained by three dif-ferent modified GCEs in5mM[Fe(CN)6]3−/4−containing0.1M KCl at50mV s−1.As shown,a pair of well-defined redox peaks was observed for the bare GCE(Fig.2,line a),which was due to the reversible one-electron redox behavior of ferricyanide ion.After electrodepositing CS onto the bare GCE,a largely blocked interfa-cial charge transfer between the bare GCE and ferricyanide ion was observed(Fig.2,line b),due to CSfilm could act as a -pared with CS or even the bare GCE surface,increased redox peak currents were observed on GR–CS/GCE(Fig.2,line c),indicating that GR facilitates the conductivity and the electron transfer process.Fig.3(A)showed the CV responses of GR–CS/GCE(a)in blank 0.1M acetate buffer solution,and CS/GCE(b)and GR–CS/GCE(c) for150ng mL−1MP in0.1M acetate buffer solution.No peakswere Fig.2.Cyclic voltammograms of(a)Bare GCE,(b)CS/GCE and(c)GR–CS/GCE in 5mM[Fe(CN)6]3−/4−containing0.1M KCl at50mV s−1.observed on GR–CS/GCE in blank acetate buffer(curve a).A pair of redox peaks(E pa,96mV and E pc,37mV)and irreversible reduc-tion peak(E pc,−600mV)of MP were observed on the both CS/GCE (curve b)and GR–CS/GCE(curve c).The irreversible reduction peak corresponded to the reduction of the nitro group to the hydrox-ylamine group(reaction(1))and the reversible redox peaks were attributed to a two-electron-transfer process(reactions(2)and(3)), which was well consistent with the below reaction and literature [30].Compared with CS/GCE,the peak currents of MP increased obviously on the GR–CS/GCE.Based on the results,thefollowing Fig.3.(A)CV reponses and(B)SWV signals of GR–CS/GCE in blank0.1M acetate buffer solution(a),and CS/GCE(b)and GR–CS/GCE(c)for150ng mL−1MP in0.1M acetate buffer solution.78S.Yang et al./Colloids and Surfaces B:Biointerfaces 96 (2012) 75–79Fig.4.Effects of the electrodeposition time (A),electrodeposition potential (B),pH (C)and accumulation time (D)on the SWV peak current of GR–CS/GCE for 150ng mL −1MP in 0.1M acetate buffer solution.Step potential of 4mV,an amplitude of 20mV,and a frequency of 25Hz.conclusions can be drawn:first,as the good adsorption ability of CS,MP were strongly attached to CS film;second,the sensitivity of the GR–CS sensor towards MP was greatly improved due to the excellent electric conductivity and the large surface area ofGR.(1)(2)(3)Fig.3(B)showed a comparison of the SWV signals of GR–CS/GCE (a)in blank 0.1M acetate buffer solution,and CS/GCE (b)and GR–CS/GCE (c)for 150ng mL −1MP in 0.1M acetate buffer solution.No SWV peak was observed on GR–CS/GCE in blank acetate buffer (curve a).SWV peaks were observed on the both MP/CS/GCE and MP/GR–CS/GCE (curve b and curve c).In comparison,SWV peak on MP/GR–CS/GCE was much more sharper than on MP/CS/GCE at the potential range from −400to 200mV.Since SWV analysis hashigher sensitivity than cyclic voltammetry [27].SWV was used in following detection of MP.3.3.Optimization of the GR–CS/GCE electrodeposition conditionsThe deposition time and deposition potential have significant effects on the performance of the sensor.In Fig.4(A and B)depicted effects of the electrodepositing potential and electrodeposition time on the current response of GR–CS/GCE containing 150ng mL −1MP,respectively.As can be seen,the current response increased evidently as the electrodeposition time increased from 100to 400s,and no significant improvement of the current response was observed from 400to 900s.And the peak currents raised sharply with the decreased deposition potential,and the maximum responses are approached at −1.0V.Thus,400s and −1.0V were chosen as the optimal electrodeposition conditions for the sensor.3.4.Optimization of the GR–CS/GCE determination condition0.1M acetate buffer solution as supporting electrolyte with dif-ferent pH values in the range of 2.5–7.5were compared in respect of the response for 150ng mL −1MP on the GR–CS/GCE.As described Fig.4(C),the maximum current response of MP was approached at pH 5.2.Thus,pH 5.2was chosen as the optimal pH value.The accumulation time was also one of the most influential parameters in pesticide determination.As seen in Fig.4(D),the peak currents increased rapidly with an increase of accumulation time,and then tended to be stable after 180s,indicating that the adsorp-tion of MP onto the GR–CS/GCE reached saturation.Thus,180s was chosen as the optimal accumulation time.S.Yang et al./Colloids and Surfaces B:Biointerfaces96 (2012) 75–7979Fig.5.SWV peak current in0.1M acetate buffer solution containing different con-centrations of MP:400ng mL−1(a),350ng mL−1(b),300ng mL−1(c),250ng mL−1 (d),200ng mL−1(e),150ng mL−1(f),100ng mL−1(g),34ng mL−1(h),17ng mL−1 (i),8ng mL−1(j)and4ng mL−1(k).Step potential of4mV,an amplitude of20mV, and a frequency of25Hz.(Inset)The calibration curve of GR–CS/GCE to different concentration of MP in0.1M acetate buffer solution.(pH5.2.)Table1Effect of possible interferents on the detection of MP with GR–CS/GCE. Interferents Current ratio aNitrophenol 1.13Nitrobenzene0.98Trinitrotoluene0.83PO43−0.78SO42− 1.05NO3−0.92a Current ratio=I H+I/I H.I H+I,response current to MP(150ng mL−1)in the presence of interferents(5␮g mL−1);I H,response current to MP(150ng mL−1).3.5.Analytical performance of GR–CS/GCEFig.5displayed the SWV peak current of MP under the opti-mum conditions.The GR–CS/GCE displayed an expanded linear response range of4.0–400ng mL−1with a correlation coefficient of 0.998(Fig.5,inset).The detection limit was0.8ng mL−1(S/N=3). It was significantly lower than5.7ng mL−1at a ZrO2-nanoparticles modified carbon paste electrode[31],lower than1.2ng mL−1at bismuth-film-modified glassy carbon electrode and1.0ng mL−1at carbon nanotube/Au nanocomposite[16,32].Moreover,the detec-tion limit and linear response range of the proposed sensor were also comparable with those enzyme-based electrochemical sensors [12,33].The reproducibility and stability of the sensor were evaluated. 5electrodes modified identically were investigated in the current response for50ng mL−1MP.The relative standard deviation(R.S.D.) was5.8%.For10replicate measurements of one electrode yielded at50ng mL−1MP,the R.S.D.was2.43%.These experimental results confirmed that the results were highly reproducible.The stabil-ity of the electrode was checked by performing30consecutive potential scans in0.1M,pH5.2acetate buffer solution containing 150ng mL−1MP.Only5%of the current response was lost in the signal.The anti-interference ability of the proposed sensor have also been studied.As shown in Table1,no obvious interferences were observed from the other electroactive nitrophenyl derivatives such as nitrophenol,nitrobenzene,trinitrotoluene and other inorganic ions(PO43−,SO42−,NO3−).All these interferents have littler influ-ence on the MP determination.4.ConclusionIn this work,we have prepared GR–CSfilm on GCE directly from GO–CS dispersion by one-step electrodeposition under controlled potential.As GR-based composites have never been applied for the OPs sensing,the proposed GR–CS/GCE opens new opportunity for green,fast,simple,and sensitive analysis of OPs.Wide linear concentration range,low detection limit,excellent reproducibility, long-time storage stability,and satisfactory anti-interference abil-ity are achieved on the GR–CS/GCE,indicating GR-based composites a promising sensing platform for OPs determination.AcknowledgmentsThis work was supported by the National Science Foundation for Distinguished Young Scholars(No.507258250)and the National Science Foundation of China(No.50878079,51078129). 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