Analytical & Bioanalytical Electrochemistry

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1 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, Anlyticl & Bionlyticl Electrochemistry Full Pper 211 by CEE Three-Dimensionlly Microporous Polypyrrole Film s n Efficient Mtrix for Enzyme Immobiliztion Jing Qin 1, Lei Peng 2, Ull Wollenberger 2, Frieder W. Scheller 2 nd Songqin Liu 1,* 1 School of Chemistry nd Chemicl Engineering, Southest University, Nnjing, , P. R. Chin 2 Deprtment of Anlyticl Biochemistry, Institute of Biochemistry nd Biology, University of Potsdm, Krl-Liebknecht-Strsse 24-25, Potsdm, Germny *Corresponding uthor, Tel: ; Fx: E-Mil: liusq@seu.edu.cn Received: 27 Jnury 211/ Accepted: 7 June 211 / Published online: 2 June 211 Abstrct- The development of new porous mterils hs opened unprecedented opportunities for wide rnge of pplictions. In the present work, three-dimensionlly interconnected porous polypyrrole structure ws proposed for biosensing. Silic nnoprticles with good monodispersion nd uniformity of the spheres synthesized by improved Stöber methods ws used s templte for porous polypyrrole electrodeposition. Voltmmetric nd electrochemicl impednce mesurements showed tht the polypyrrole 3D-network rrys provided excellent mtrices for the immobiliztion of horserdish peroxidse. The immobilized horserdish peroxidse on these polypyrrole 3D-network rrys retined its bioctivity effectively nd displyed pir of redox peks with forml potentil of V nd n excellent electroctlytic response towrd the reduction of hydrogen peroxide (H 2 O 2 ). This llowed the detection of H 2 O 2 concentrtion with liner rnge from 1 M to 7.94 M. The detection limit ws found to be.22 M t signl-to-noise rtio of 3. This method provided n lterntive wy for the biosensor construction. Keywords- Silic Templte, 3D-Network Arrys, Horserdish Peroxidse, Porous Polypyrrole

2 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, INTRODUCTION Current studies on the direct electron trnsfer between immobilized redox-proteins nd electrode surfce hs received wide ttention due to their distinguished pplictions in biologicl science, environmentl science, energy science nd nlyticl chemistry [1-3]. Erly reports demonstrted tht the redox protein immobilized on biocomptible electrode surfce exhibited enhnced electrochemicl ctivity, which llowed the electrochemicl mesurement of its substrte with higher sensitivity nd better selectivity [4,5]. The most populr mterils for protein immobiliztion re polycrylmide hydrogel [6] surfctnt [7,8] DNA [9] nd series of nnostructured mterils such s clcium phosphte,1 colloidl gold [11,12] cly [13] ZnO [14] grpheme [15,16] TiO 2 [17] nd crbon nnotubes [18,19]. These mterils fcilitted direct chemiclly reversible electron exchnge between redox proteins nd electrodes, nd re thus being widely used in sensor construction. The development of new porous mterils hs opened unprecedented opportunities for wide rnge of pplictions [2,21] Severl chemicl preprtions of ordered micro- nd mcroporous mterils bsed on colloidl crystl templtes hve been described [22-3]. These methods use close-pcked rrys of monodisperse spheres s templtes for the formtion of three-dimensionlly ordered micro- or mcroporous structures in rnge of mterils, such s silic [22,23] metls [24,25] crbon [26,27] nd polymers [28-3]. One of the chrcteristics of porous mterils is their high surfce-re/volume rtio, fvoring thereby the interction with externl regents[31] which could llow immobiliztion of suitble proteins likely to induce novel properties nd pve the wy to be exploited in biosensing pplictions [32,33]. For exmple, three-dimensionl (3D) porous chitosn membrne mteril prepred by electrodeposition of chitosn-encpsulted silic nnoprticle on n ITO electrode, followed by removl of silic nnoprticles with HF solution, ws used s mtrix for heptitis B surfce ntibody immobiliztion. It ws shown tht the 3D chitosn porous structure possesses high surfce re, good mechnicl stbility, good biocomptibility nd incresed protein loding. These benefits llowed the resultnt immunosensor to hve wide liner rnge nd low detection limit [34]. With clcium crbonte microsphere s crrier for the loding of cdmium telluride quntum dots, Zhu nd co-workers [35] developed protein-cdte-cco 3D rchitecture for electrochemistry biosensing. The unique properties of CCO 3, such s good biocmptibility nd its chnnel-like structure, llowed the formtion of thick nd uniform quntum-dot shell, which in turn improved the soluble stbility of the spheres nd contributed to fst nd effective direct electron trnsfer between the protein redox center nd the mcroscopic electrode. The present work ws lso motivted by the very promising pplictions of 3D structure mterils in protein immobiliztion for electrochemicl biosensing. In order to chieve the 3D porous interconnected polypyrrole structure, silic nnoprticles with good

3 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, monodispersion nd uniformity of spheres, synthesized by improved Stöber methods[36] were chosen s templtes. The porous polypyrrole film on the gold electrode surfce provided excellent mtrices for the immobiliztion of horserdish peroxidse (HRP). The immobilized HRP not only retined its bioctivity but lso showed high ffinity for H 2 O 2 reduction, which produced novel H 2 O 2 biosensor. Scheme 1. Schemtic representtion of the fbriction procedure for 3D porous- Ppy/Au nd HRP immobiliztion 2. MATERIALS AND METHODS 2.1. Mterils Horserdish peroxidse (HRP, EC , RZ 3.2, 29 purpurogllin units per mg solid, from horserdish), chitosn (CS, from crb shells, minimum 85% decetylted), nd pyrrole were obtined from Sigmldrich..1 M ph 7. phosphte buffer solution (PBS) ws prepred by mixture of NH 2 PO 4 nd N 2 HPO 4 stock solution, nd ws modulted by H 3 PO 4 nd NOH. 1 mg/ml HRP ws prepred by.1 M ph 7. PBS, 1% CS solution ws prepred by 1% cetic cid. All other chemicls were nlyticl grde regents. All the solutions were prepred with doubly distilled wter nd stored in refrigertor t 4 o C when not in use Apprtus Cyclic voltmmetry(cv) nd mperometric mesurement were conducted with threeelectrode system comprised of the modified gold electrodes s working electrode, n Ag/AgCl (1 M KCl) s reference electrode, nd pltinum wire uxiliry electrode. The electrodes were connected to computer-controlled CHI75A electrochemicl nlyzer. All electrochemicl experiments were crried out in 1 ml of.1 mol L -1 phosphte buffer (ph 7., prt from the ph prmeter experiments) t room temperture. H 2 O 2 ws dded to

4 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, PBS while being stirred. All experimentl solutions were thoroughly deoxygented by bubbling nitrogen through the solution for t lest 1 min. Electrochemicl impednce spectroscopy (EIS) ws performed with Gmery Instruments (Reference 6, USA) in 5 mm [Fe(CN) 6 ] 3-/4- mixture with.1 M KCl s supporting electrolyte. An lternting current voltge of 5. mv ws used, within the frequency rnge of.1-1 khz Preprtion of the 3D Ppy modified Electrode The monodispersed SiO 2 spheres with dimeters of 18 3 nm were synthesized with n improved Stöber seed-growth method, which we previous reported [36]. The silic nnosphere templtes were built by dispersing of dilute suspension of SiO 2 nnospheres onto gold disc electrode surfce. In this cse, the gold disc electrode ws polished before ech experiment with 1.,.3 nd.5 μm lumin powder, respectively, nd followed by thorough wsh with cetone nd doubly distilled wter. Au electrode ws dipped into dilute silic suspension in ethnol for 1 s, nd then Au electrode ws put out nd llowed to evporte the solvent nturlly. After tht, the resultnt Au electrode ws dipped into.1 M KCl solution (djust ph to 2. with HCl) tht contined.5 M pyrrole for 2 min. Polypyrrole ws deposited into the void spces of such templte through CV, t the potentil window of to.7 V (vs. Ag/AgCl). After deposition, the silic templte ws etched by queous HF (5%) for bout 5 min to leve behind n ordered porous polypyrrole frmework, donted s porous-ppy/au. Finlly, the electrode ws immersed in PBS for t lest 3 min for the following use Immobiliztion of HRP The immobiliztion of HRP on porous-ppy/au ws performed through n electrochemicl co-deposition process. The Ppy/Au ws dipped in HRP/CS solution t -2.5 V for 3 s. HRP/CS solution ws prepred by mixture tht involved equl volumes of 1 mg/ml HRP solution in.1 M ph 7. PBS nd 1% CS solution in 1% cetic cid (1:1, v/v). After deposition the electrode ws removed nd rinsed with wter to obtin HRP/CS modified electrode (HRP/CS-porous-Ppy/Au). In contrst, CS- porous-ppy/au, the porous- Ppy/Au deposited in CS solution in the bsence of HRP, were used for control mesurements. 3. RESULTS AND DISCUSSION 3.1. The synthesis of the porous-ppy/au The porous-ppy/au ws prepred bsed on the silic nnosphere templte. The responses of the electrochemicl probe to the resultnt porous-ppy film were checked by CV, s shown in Fig. 1. When the Ppy ws electrodeposited into the void spces of the templte, the resultnt silic-ppy/au (Fig. 1, curve b) displyed much lower signls in [Fe(CN) 6 ] 3-/4-

5 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, contining solution thn tht of the bre gold electrode (Fig. 1, curve ). This indicted the formtion of silic nnospheres doped Ppy lyer on the electrode surfce, which cn effectively block electron trnsfer between the electroctive species nd electrode. This would hve produced poor electrochemicl response [37]. After the removl of silic nnosphere templtes with 5% HF solution, the porous-ppy/au (Fig. 1, curve c) displyed lrge response to the [Fe(CN) 6 ] 3- probe. Both the node nd node pek current were much lrger thn tht of silic nnospheres doped Ppy film, but still smller thn tht of the bre electrode. This observtion suggested the formtion of the porous Ppy film on the electrode surfce, which prtly hindered the electron trnsfer between [Fe(CN) 6 ] 3-/4- species nd the electrode. In ddition, the node nd node pek current of the silic-ppy/au decresed with n increse of the deposition cycles from 3 to 9, which relted to the mount of Ppy coted onto silic templte (dt not shown). When the pyrrole ws deposited on the Au electrode for 9 cycles, both the pek currents of silic-ppy/au reched minimum vlue (Fig. 1, curve c). This low vlue indicted the formtion of dense Ppy film on gold electrode, which effectively blocked the electron trnsfer between [Fe(CN) 6 ] 3-/4- species nd the electrode. The CV of silic-ppy/au hs the sme shpe s the ones deposited for 9 nd 12 cycles. Despite this, no chnges were observed fter the removl of silic templte with queous HF for 12 cycles, which suggested tht the Ppy film ws too thick for HF to efficiently remove from porous polypyrrole frmework on gold. Therefore, the pyrrole deposited for 9 cycles ws selected in the following study. The formtion of the porous Ppy film, fter being removed from the silic templte, ws illustrted by the re-coupling tests. When the porous-ppy/au ws gin soked in silic nnosphere suspension for 1 s, the Au electrode ws lso put out nd evportion of the solvent ws llowed. The electrode displyed similr response to silic-ppy/au (Fig. 1, curve b nd d), which indicted the re-coupling of silic nnospheres into the pore of the porous- Ppy/Au. The slight decrese of the electrochemicl response to [Fe(CN) 6 ]3-/4- for silic nnospheres re-coupled electrode compred to tht of silic-ppy/au suggested the recoupling process llowed more silic nnoprticles to be ttched on the electrode. Some silic nnoprticles were ble to ttch in the hole nd some ws covered on the surfce. The formtion of the porous Ppy film, fter being removed from the silic templte, ws lso supported by the electrochemicl impednce spectr. The impednce mesurements were crried out in.1 M KCl tht contined 5 mm [Fe(CN) 6 ] 3 /4 nd t open circuit potentil. Fig.2 presents the Nyquist plots of the different electrodes. After deposition of Ppy for 9 cycles, the chrge-trnsfer resistnce (Rct) of the bre gold electrode (Fig. 2, curve ) ws much smller thn tht of the silic-ppy/au (Fig. 2, curve b). The increse of impednce from to of the silic-ppy/au reltve to the bre gold electrode ws due to the formtion of Ppy film on gold surfce. The cot of Ppy blocked the diffusion of redox species

6 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, to the electrode surfce, which mde the redox process more difficult nd cused the impednce to increse. After immersion of the silic-ppy/au in HF solution for 5 min, the impednce decresed from to due to the relese of silic nnospheres from the silic-ppy film. However, the impednce ws still greter thn tht of bre gold electrode, which indicted tht the retention of Ppy film on the gold surfce. After second immersion of this electrode in silic suspension for 1 s, the impednce (Fig. 2, curve d) begn to increse from to This ws slightly lrger thn in Fig. 2, curve b, which indicted the presence of re-coupled silic nnospheres on the Ppy film. Repeted experiments produced very similr results. These results indicted the formtion of porous Ppy film on gold nd tht the chnges in impednce were due to the reversible coupling nd relese of silic nnospheres. 6 Current / 3 c b d E / V (vs. Ag/AgCl) Fig. 1. The cyclic voltmmogrms of () bre Au, (b) silic-ppy/au, (c) porous- Ppy/Au, nd (d) re-coupled silic on porous-ppy/au in.1 M KCl tht contined 5. mm [Fe(CN) 6 ] 3-/4- t 1 mvs -1. The Ppy ws electrodeposited in.1 M ph 2. KCl+HCl+.5 M pyrrole solution through 9 cycles of CV

7 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, d -Z''/ 5 c b Z'/ Fig. 2. EIS of () bre Au, (b) silic-ppy/au, (c) porous-ppy/au, nd (d) re-coupling of silic nnoprticles on porous-ppy/au in.1 M KCl tht contined 5. mm [Fe(CN) 6 ] 3-/ Direct Electrochemistry of immobilized HRP on porous-ppy/au The immobiliztion of HRP on porous-ppy/au ws performed through n electrochemicl co-deposition process; the Ppy/Au ws dipped in HRP/CS solution t -2.5 V for 3 s. At the pplied potentil of -2.5 V, the H + in HRP/CS colloidl solution could be reduced to H 2, which would result in grdul increse in ph vlue t the electrode surfce. Since the solubility of CS is ph dependent, when the ph ws higher thn the pk of CS (bout 6.3), the dissolved CS would flocculte to form n insoluble hydrogel network due to deprotontion of its mine groups. As result, CS hydrogel incorported with HRP ws electrodeposited on the cthode surfce. The co-immobiliztion of HRP with CS on porous-ppy/au could be demonstrted by comprison of the impednce of HRP/CS-porous-Ppy/Au with CS-porous-Ppy/Au, s shown in Fig. 3A. The impednce of HRP/CS-porous-Ppy/Au ws much lrger thn tht of CSporous-Ppy/Au. The increse of impednce from to indicted the incorportion of the insultion of the protein shell into CS film. The HRP/CS-porous-Ppy/Au ws chrcterized by CV. Fig. 3B showed the typicl CVs obtined from porous-ppy/au (Fig. 3B, curve ), HRP/CS/Au (Fig. 3B, curve b), nd HRPporous-Ppy/Au (Fig. 3B, curve c) in.1 M ph 7. PBS t 1 mv s -1, respectively. HRPporous-Ppy/Au exhibited cthodic pek t mv, with the corresponding nodic pek on the reverse scn, t mv. No pek ws observed t either bre gold or porous-

8 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, Ppy/Au, which suggested tht the response of HRP-porous-Ppy/Au is ttributed to the redox of the electroctive center of HRP on electrode surfce [39]. The surfce concentrtion of electroctive HRP (Γ*) for porous-ppy film ws deduced from the following eqution [4]: Γ*=Q/nFA Where Q is the chrge, the electron trnsfer number is n = 1, F is the Frdy constnt, nd A denotes the effective surfce re of the working electrode. From the integrtion of the nodic pek of the sensor, the surfce concentrtion of the ctive HRP on HRP/CS-porous- Ppy/Au surfces were clculted to be mol cm -2. The HRP/CS-porous-Ppy/Au collected higher concentrtion of the electroctive HRP, which ws bout 6 times the monolyer coverge of HRP ( mol cm -2 ) on 3-mercpto-propionic cid modified gold electrode [41]. This suggested tht multiple lyers of HRP were coted on the electrode. Compring this clculted vlue with those reported for HRP surfce concentrtion in other immobilized mtrices, such s DNA ( mol cm -2 ), mol cm -2 for multilyer of HRP on ZnO nnorods.43 Porous-Ppy mtrix ws more efficient for HRP immobiliztion A b 3 2 B -Z''/ 1 5 Current/ 1-1 b c Z'/ Potentil/V Fig. 3. EIS (A) of CS-p-Ppy/Au () nd HRP/CS-porous-Ppy/Au (b) in.1 M, KCl tht contined 5. mm [Fe(CN)6] 3-/4-, nd CVs (B) of porous-ppy/au (), HRP/cs/Au (b), nd HRP/CS-porous-Ppy/Au(c) in.1 M, PBS (ph 7.) with scn rte 1 mv s -1

9 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, As shown in Fig. 4, the cthodic nd nodic pek current (I pc ) incresed grdully when the scn rte ws ccelerted from 1 to 1 mv s -1. Insert of Fig. 4 plotted the I pc versus the scn rte nd reveled their liner reltionship. This indicted tht the redox rection is surfce process nd the electrons esily trnsfer between HRP nd porous-ppy/au electrode [43]. It proved tht the HRP ws successfully immobilized on the porous-ppy/au nd tht it retined its bioctivity perfectly. The kinetics of the direct electron trnsfer ws nlyzed using the model of Lviron. Suppose tht the chrge trnsfer coefficient,, is between.3 nd.7, the electron trnsfer rtes, ks, cn be estimted with the formul k s =mnfv/rt when the pek-to-pek seprtion is less thn 2 mv [44] where m is prmeter relted to the pek-to-pek seprtion. The pek-to-pek seprtions of 74.3 mv t 1 mv s -1 produced the k s vlue of 1.49 s -1, which is close to the reported vlue from some better biosensors constructed by crbon nnotubes [45]. This vlue ws lrger thn tht obtined t HRP Au SPCE (.75±.4 s -1 ) [46] HRP/DNA/PG electrode (1.13 s-1) [47] nd close to tht obtined t ZnO-GNPs-Nfion-HRP/GCE (1.94 s -1 ) [48] suggesting the HRP immobilized on porous- Ppy film hs perfect ctlytic ctivity, nd tht the electrons could esily trnsfer from HRP to electrode. Ppy nd chitosn composite film is n effective biocomptible mteril to retin the bioctivity of HRP. Current/ Pek current/ A Scn rte/mv s Poentil/ V n Fig. 4. Cyclic voltmmogrms of HRP/CS-porous-Ppy/Au t severl scn rtes: (from to n: 1, 5, 1, 15, 2, 25, 3, 4, 5, 6, 7, 8, 9, nd 1 mvs -1 ). Insert is the reltionship between peks current nd scn rtes

10 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, Response of the HRP/CS-porous-Ppy/Au to H 2 O 2 Upon ddition of H 2 O 2 to PBS, the shpe of cyclic voltmmogrm of HRP-porous- Ppy/Au for direct electron trnsfer of HRP chnged drmticlly with n increse of reduction current (Fig. 5). Menwhile, no obvious chnge ws observed t bre gold nd porous-ppy/au (dte not shown). The incresed reduction pek nd the decresed oxidtion pek current of HRP t HRP-porous-Ppy/Au displyed the obvious electroctlytic behvior of immobilized HRP to the reduction of H 2 O 2. It demonstrted tht the HRP immobilized on the porous-ppy/au could mintin its ctivity nd ctlyzed H 2 O 2 reduction effectively. Fig. 6 illustrted the chronomperometric response of porous-ppy/au (curve ), HRP/CSporous-Ppy(n=6)/Au (curve b), nd HRP/CS-porous-Ppy(n=9)/Au(curve c) with successive dditions of H 2 O 2 to.1 M ph 7. PBS. Upon ddition, the sensor chieved 95% of the stedy-stte current in 5 s. The liner response rnge of the HRP/CS-porous-Ppy(n=9)/Au to H 2 O 2 concentrtion is from 1 M to 7.94 mm with correltion coefficient of.9982 nd detection limit of M, t signl- to-noise rtio of 3. While the liner response rnge of the HRP/CS-porous-Ppy (n=6) /Au to H 2 O 2 concentrtion is from 1 M to 5.77 mm, with correltion coefficient of.9939 nd detection limit of M t signl- to-noise rtio of Current 4 2 b Potentil/ V Fig. 5. Cyclic voltmmogrms of the HRP/CS-porous-Ppy/Au in the bsence (), nd presence of M (b) H 2 O 2 When the concentrtion of H 2 O 2 is higher thn 7.94 mm, pltform ws observed, which displyed chrcteristic of the Michelis Menten kinetic mechnism. The pprent Michelis Menten constnt (K ppm), reflection of both the enzymtic ffinity nd the rtio

11 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, of microscopic kinetic constnts, could be obtined from the electrochemicl version of the Linewever Burk eqution[49]: 1/I ss =1/I mx +K ppm/i mx C Here I ss, I mx nd C represented the stedy current, mximum current nd H 2 O 2 concentrtion, respectively. It is well known tht smller Kppm implied higher ctlytic ctivity of the immobilized enzyme. Bsed on the Linewever-Burk eqution, the K ppm of HRP/CS-porous-Ppy(n=6) nd HRP/CS-porous-Ppy(n=9) were clculted to be 58.6 μm nd 29.4 μm, respectively. This results ws close to the reported vlue of some better biosensors constructed from silichydroxyptite (HAp) hybrid film-modified glssy crbon electrode (silic/hrp-hap/gce) [39].This vlue ws much lower thn.159 mm for C 16 -C 12 -C 16 -OMIMPF 6 -HRP/GCE,5 nd 1.8 mm for ntive HRP in solution [51]. As is well-known, the lower pprent Kppm reflected the better ffinity effect of enzyme to ctlyze trget, thus, the result demonstrted tht the s immobilized HRP hd the mximl ctlysis to H 2 O 2 rection. Current/ Current/ b CH 2 O 2 /mm c b Time/s Fig. 6. Typicl stedy-stte response of porous-ppy/au(), HRP/CS-porous-Ppy 3.4. Reproducibility nd stbility of the biosensor The mesurement repetbility of the HRP/CS-porous-Ppy (n=9)/au electrode ws investigted in.1 M PBS with the sme electrode. The reltive stndrd devitions (RSD) were 6.5% for 6 successive ssys in the presence of.1 mm H 2 O 2. The fbriction

12 Anl. Bionl. Electrochem., Vol. 3, No. 3, 211, reproducibility for three HRP electrodes gve RSD of 5.1% for typicl stedy-stte response t.1 mm H 2 O 2. The good reproducibility my be due to the fct tht the 3D structure ws consistent nd HRP molecules were ttched firmly onto the pores of polypyrrole. The stbility of the HRP/CS-porous-Ppy (n=9)/au ws investigted when the current response of.1 mm H 2 O 2 ws mesured every 3 dys. The results showed tht the cthodic pek current only decresed bout 8.7% fter storge t 4 o C in PBS for 2 weeks. Good long-term stbility could be ttributed to the strong interction between HRP nd chitosn embedded in the polypyrrole network, which prevented the loss of enzymes. Porous polypyrrole-chitosn composite mtrix could hve provided biocomptible microenvironment which cted to prevent the desorption of enzyme. 4. CONCLUSIONS We hve developed n innovtive method for the preprtion of n enzyme biosensor for H 2 O 2, through 3D interconnected structures of porous polypyrrole film for the immobiliztion of HRP, to fbricte new third-genertion H 2 O 2 biosensor. The lrge surfce re of the electrodes could be potentilly exploited to increse the mount of enzyme molecules loded on the electrode surfce, through the formtion of multiple coverge of HRP, vi the 3D network. The porous polypyrrole film effectively mintined the bioctivity of HRP. The direct electron trnsfer between HRP nd electrode surfce ws lso chieved. Furthermore, the resultnt HRP-porous-Ppy/Au electrode showed electrochemicl ctivity in the reduction of H 2 O 2 without the id of ny electron meditor. The proposed biosensor exhibited fst mperometric response, wide liner rnge, nd high sensitivity nd stbility. Acknowledgments We thnk the support from DAAD supported project PPP VR Chin, the Ntionl Nturl Science Foundtion of Chin (Grnt Nos , ), the Specilized Reserch Funds for the Doctorl Progrm of Higher Eduction ( ). REFERENCES [1] X. D. Zeng, X. F. Li, X. Y. Liu, Y. Liu, S. L. Luo, B. Kong, S. L. Yng, nd W. Z. Wei, Biosens. Bioelectron. 25 (29) 896. [2] T. F. Pulo, I. C. N. Diógenes, nd H. D. Abruñ. Lngmuir 211, In press, DOI: 1.121/l1355x. [3] N. J. Yng, R. Hoffmnn, W. Smirnov, A. Kriele, nd C. E. Nebel, Electrochem. Commun. 12 (21) 1218.

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