Hydrogen Peroxide Biosensor Based on the Bioelectrocatalysis of Myoglobin Incorporated in Multi-Walled Carbon Nanotubes/Chitosan Composite Film

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1 Int. J. Electrochem. Sci., 3 () Hydrogen Peroxide Biosensor Bsed on the Bioelectroctlysis of Myoglobin Incorported in Multi-Wlled Crbon Nnotubes/Chitosn Composite Film Lin-Sheng Dun 1,, Qio Xu 1, Fen Xie 1, Sheng-Fu Wng 1,* 1 College of Chemistry nd Chemicl Engineering, Hubei University, Wuhn 3, P. R. Chin Deprtment of Chemistry nd Life Science, Hubei University of Eduction, Wuhn 35, P. R. Chin * E-mil: wngsf@hubu.edu.cn Received: 9 November 7 / Accepted: 9 November 7 / Online published: December 7 Myoglobin/multi-wlled crbon nnotubes/chitosn film (Mb/MWNTs/Cs) electrodes were fbricted bsed on cross-linking myoglobin (Mb) with multi-wlled crbon nnotubes/chitosn (MWNTs/Cs) composite film coted on glssy crbon electrode (GCE). Direct electrochemistry nd electroctlysis of Mb on the Mb/MWNTs/Cs/GCE were studied. The Mb/MWNTs/Cs/GCE exhibited excellent electroctlytic ctivity nd rpid response for H O in the bsence of meditor. The liner rnge of detection towrds H O ws from to mol L -1 by the method of mperometric i-t curve. The Mb/MWNTs/Cs/GCE hd good repetbility nd stbility for the determintion of H O. Keywords: Direct electrochemistry, Electroctlysis, Myoglobin, Chitosn, MWNTs 1. INTRODUCTION Crbon nnotubes (CNTs) hve ttrcted gret del of interest becuse of their dimensions nd structure-sensitive properties since CNTs were discovered by Iijim in 1991 [1]. CNTs show electricl properties s metl or semiconductors, depending on their size nd lttice helicity []. The subtle electronic properties suggest tht CNTs, when used s n electrode mteril in electrochemicl rections, hve the bility to promote electron-trnsfer rections. CNTs ws brodly explored s kind of mteril for electrode modifiction to investigte electroctlytic ctivity for some biomolecules including cytochrome c [3,], NADH [5], horserdish peroxidse [], hydrogen peroxide [7, ], nd ctecholmines such s dopmine [9] nd scorbic cid [1].

2 Int. J. Electrochem. Sci., Vol. 3, 119 Mb, kind of oxygen trnsporttion protein with the function to store nd trnsport oxygen, hs n ctive heme redox center. Although there is n electroctivity center in the Mb, its lrge sptil structure mkes the electroctivity unexposure. On the other hnd, electrode surfce could be pssivted with Mb bsorbing gretly, thus slows down the electron trnsference nd could not obtin good current response. So it is importnt to construct biofilm to keep the protein ctive nd fcilitte the electron trnsferring. In recent yers, mny studies focused on the direct electrochemistry of proteins which were incorported into the composites to investigte the stbility nd biocomptibility of films. These films contined wter-insoluble surfctnts [11,1], hydrogel polymers [13,1], polyelectrolyte- or cly-surfctnt composites [15], Cs [1] nd MWNTs. Herein, we developed composite biofilm, which contins MWNTs, Cs nd Mb, bsed on the ide tht the MWNTs could fcilitte the electron trnsfer nd Cs hd good biocomptibility. The preprtion of the modified electrodes is rther simple. The mixture of MWNTs nd Cs mkes the composite film posing the dvntges of fst electron trnsfer nd excellent bio-ffinity on the modified electrode. The excellent ctivity nd stbility of myoglobin sensor ws obtined. This film showed fvorble electroctlytic behvior towrd H O.. EXPERIMENTAL PART.1. Chemicls nd regents Horse hert Mb (MW 1.7 kd) ws from Sigm. Cs (MW, ) ws purchsed from Zhejing Jinke biochemicl limited compny. MWNTs crude mterils were purchsed from Huzhong Norml University (Wuhn, Chin). The MWNTs crude mterils were ultrsonic gittion in 3 mol L -1 HNO 3 for 1 h nd refluxed in 5 mol L -1 HCl for h t 11 o C. After cid tretment, the smples were clcined in sttic ir t 35 o C for h. 5 mg purified MWNTs were dispersed with the id of ultrsonic gittion in 1 ml of chitosn (7 mg ml -1 ) to give 5 mg ml -1 blck suspension. Other regents were of nlyticl grde. All solutions were prepred with double distilled wter... Apprtus Cyclic voltmmetry (CV) experiments were performed by using CHI A electrochemicl worksttion (CH Instrumentl, USA) coupled with conventionl three-electrode cell. The working electrode ws the Mb/MWNTs/Cs/GCE or bre GCE, the uxiliry electrode ws pltinum wire, nd the reference electrode ws sturted clomel electrode (SCE). All the potentils in this pper were given ginst the SCE..3. Preprtion of Mb/MWNTs/Cs/GCE The GCE ws crefully brded with emery pper, polished on chmois lether contining.5m lumin slurry, nd then wshed ultrsoniclly in wter, ethnol nd wter, respectively.

3 Int. J. Electrochem. Sci., Vol. 3, 1 Mb ws resolved in 1 mmol L -1 ph 7. PBS, then L Mb, 1 L MWNTs/Cs mixture (c Cs = 7 mg ml -1, c MWNTs = 5 mg ml -1 ) were mixed. The clened GCE ws coted by csting 3 L of the complex nd dried in the ir for h. Then the Mb/MWNTs/Cs/GCE ws prepred. 3. RESULTS AND DISCUSSION 3.1. UV-Vis spectr chrcteriztion of Mb/MWNTs/Cs film Shift of Fe /Fe Soret bsorb bnd in Mb could provide whether protein ws dentured or not [17]. When protein ws dentured, the Soret bsorb bnd would move or dispper. As shown in Fig. 1, the Soret bsorb bnd of Mb, Mb + Cs, Mb + MWNTs nd Mb + MWNTs + Cs lmost kept consistently, which indicted tht microenvironment ws little chnged round Mb d A c 1. b. 3 3 λ / nm Figure 1. UV-Vis spectr of protein-films on glss slides () Mb (b) Mb + Cs (c) Mb + MWNTs (d) Mb + MWNTs + Cs. 3.. Direct electrochemistry of Mb on Mb/MWNTs/Cs/GCE Fig. ws the CV of Mb on the Mb/MWNTs/Cs/GCE (Fig c) nd the Mb/Cs/GCE (Fig ) in the presence of ph 7. PBS. At bout -.35 V pir of stble nd qusi-reversible process ws involved. As cn be seen, t the sme window, the electro-response current of Mb on the Mb/MWNTs/Cs/GCE ws higher thn tht on the Mb/MWNTs/GCE or Mb/Cs/GCE. The reson for the better performnce of the MWNTs-modified GCE my be due to the nnometer dimensions of the CNTs, the electronic structure nd the topologicl defects present on the CNTs surfces [1]. Menwhile the CNTs incresed the effective re of the electrode, so the pek current incresed significntly. At the sme time, Cs with good comptibility mkes it possible to keep bioctivity of Mb.

4 Int. J. Electrochem. Sci., Vol. 3, b c i / 1 - A Figure. Cyclic voltmmogrms t. V s -1 ph 7. PBS for: () Mb/Cs/GCE, (b) Mb/MWNTs/GCE, (c) Mb/MWNTs/Cs/GCE. E / V 1 j 1 i /1 - A -1 i p / 1 - A E / V v / V s -1 Figure 3. Cyclic voltmmogrms of Mb/MWNTs/Cs/GCE in.1 mol L -1 PBS (ph 7.) t scn rtes of (from to j):.5,.1,.,.3,.,.5,.,.7,.,.9 V s -1. The reltionship between the pek currents (-i pc, b-i p ) nd scn rtes. i / 1 - A e E / V ph Figure. CV of Mb /MWNTs/Cs/GCE t different ph. Scn rte:.1 mv s -1. Plots of E vs. ph ( to e: 5.,., 7.,., nd 9.). E p / V

5 Int. J. Electrochem. Sci., Vol. 3, 1 From Fig. 3, it could be seen tht the pek currents on modified electrode were linerly with the scn rtes. This indictes tht surfce controlled process is involved on the Mb/MWNTs/Cs/GCE. Fig. is the plot of pek currents versus ph on the Mb/MWNTs/Cs/GCE. In ph 5.-9., Mb exhibited pir of irreversible redox pek current on the Mb/MWNTs/Cs/GCE, nd nodic nd cthodic pek potentils shifted positively with the increse of ph, which indicted tht the redox of Mb on the electrode ws relted with the combine or relese of proton Electrochemicl ctlysis of H O on Mb /MWNTs/Cs/GCE Seen in CV (Fig. 5), with the H O concentrtion incresed, oxidtion pek decresed grdully, nd reduction one incresed linerly with the H O concentrtion. When H O concentrtion rech to certin vlue, oxidtion pek disppered, nd reduction pek vlue strted to deprture the liner reltionship. Higher H O concentrtion would reduce bioctivity of enzyme, even denture it. In this condition, Michelis-Menten response hppened between electroctlysis reduction currents nd H O concentrtions. According to the Linewever-Burk eqution, Michelis-Menten constnt ws K m pp = mol L -1. Electroctlytic redox mechnism ws supposed to be follows [19]: MbFe 3+ + H O compound I + H O compound I + H + + e compound II compound II + H + + e MbFe e d c i / 1 - A b i / 1 - A E / V 1 1 c / 1 - mol L -1 Figure 5. Cyclic voltmmogrms t.1 V s -1 on Mb/MWNTs/Cs/GCE in ph 7. PBS contining ~e:, ,.7 1 -,.3 1 -, mol L -1 H O. The clibrtion plot of the ctlytic pek current to different concentrtions of H O. 3.. Amperometry i-t curve The electroctlytic reduction of hydrogen peroxide t Mb/MWNTs/Cs/GCE ws lso studied by mperometry i-t curve. The potentil dependence of mperometric signl ws tested in the rnge

6 Int. J. Electrochem. Sci., Vol. 3, 13 from to -. V. The stedy-stte reduction current incresed s the pplied potentil decresed from to -.3 V, which ws due to the incresed driving force for the fst reduction of H O t low potentil. The response pproched mximum t -.3 V, so we selected this vlue s the working potentil. Fig. illustrtes typicl mperometric response of the Mb sensor t -.3 V on successive step chnges of H O concentrtion under stirring. When the sme concentrtion nd volume of H O ws dded, the reductive current incresed steeply to rech stble vlue. The modified electrode chieved 95% of stedy-stte current within s. The current hd liner reltionship with the concentrtion of H O for the Mb/MWNTs/Cs/GCE. The reduction currents ws liner with H O concentrtions in the rnge of ~ mol L -1, the liner reltionship is i = c (mol L -1 ), R= i / 1 - A i / 1 - A t / s - 1 c / 1 - mol L -1 Figure. Typicl current-time response curve of the sensor upon successive dditions of 1 L every time mol L -1 H O to 7 ml ph 7. buffer t -.3V. The clibrtion plot of the ctlytic pek current to different concentrtions of H O. The developed biosensor displyed good stbiliztion nd reproducibility. The biosensor ws stored in PBS in refrigertor t o C when not in use. It retined 9% of its initil current response fter dys. In series of 5 sensors independently mde, reltive stndrd devition (RSD) of 5.1 % ws obtined for the individul current responses to the sme smple.. CONCLUSIONS A new mperometric biosensor for H O ws prepred bsed on cross linking Mb nd MWNTs/Cs composite film. Mb retined its originl conformtion well in MWNTs/Cs composite film which combined the utilities of MWNTs fcilitting the electron trnsfer, nd of Cs preserving bioctivity for enzyme on the modified electrode. The biosensor could be pplied for mperometric determintion of H O in the bsence of meditor. The mperometric experiments showed excellent electroctlyticl ctivity of the biosensor for H O.

7 Int. J. Electrochem. Sci., Vol. 3, 1 ACKNOWLEDGEMENT This work ws supported by the Ntionl Nture Science Foundtion of Chin (No ). References 1. S.Iijim, Nture 35 (1991) 5.. P.M.Ajyn, Chem. Rev. 99 (1999) J.J. Dvis, R.J. Coles, H. Allen, O. Hill, J. Electronl. Chem. (1997) 79.. J. Wng, M. Li, Z. Shi, N. Li, Z. Gu, Anl. Chem. 7 () M. Musmeh, J. Wng, A. Merkoci, Y. Lin, Electrochem. Comm. () 73.. L. Qin, Xiurong Yng, Tlnt () J. Wng, M. Musmeh, Y. Lin, J. Am. Chem. Soc. 15 (3).. J. Wng, M. Musmeh, Anl. Chem. 75 (3) K.B. Wu, J.J. Fei, S.H. Hu, Anl. Biochem. 31 (3) X.H. Zhng, S.F. Wng, Sens. Actutors, B 1 (5) A. E. F. Nssr, Z. Zhng, N. Hu, J. F. Rusling, T. F. Kumosinski, J. Phys. Chem. B 11 (1997). 1. J. Yng, N. Hu, Bioelectrochem. Bioenerg. (1999) N. Hu, J. F.Rusling, Lngmuir 13 (1997) J. Yng, N. Hu, J. F. Rusling, J. Electronl. Chem. 3(1999) H. Sun, H. M, N. Hu, Bioelectrochem. Bioenerg. 9 (1999)1. 1. He Hung, Nifei Hu, Yonghui Zeng, Gu Zhou. Anl.Biochem. 3 () P. George, G. I. H. Hnni, Biochem. J. 55(1953)3. 1. P.J. Britto, K.S.V. Snthnm, A. Rubio, J.A. Alonso, P.M. Ajyn, Adv. Mter. 11 (1999) Y.D. Zho, W.D. Zhng, H. Chen, Q.M. Luo, S.F.Y. Li, Sens. Actutors, B, Chem. 7 () 1. by ESG (

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