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Iron(III) protoporphyrin IX—single-wall carbon nanotubes modified electrodes for hydrogen peroxide and nitrite detection
Electrochimica Acta 51 (2006) 6435–6441
Iron(III) protoporphyrin IX—single-wall carbon nanotubes modified electrodes for hydrogen peroxide and nitrite detection Graziella L. Turdean a,b,∗,1 , Ionel Catalin Popescu a , Antonella Curulli c , Giuseppe Palleschi b a
b
“Babes Bolyai” University, Physical Chemistry Department, Arany Janos 11, 400 028 Cluj-Napoca, Romania Dipartimento di Scienze e Technologie Chimiche, Universita di Roma “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy c Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) CNR Division 2, Via del Castro Laurenziano 7, 00161 Rome, Italy Received 14 February 2006; received in revised form 12 April 2006; accepted 18 April 2006 Available online 5 June 2006
Abstract Iron(III) protoporphyrin IX (Fe(III)P), adsorbed either on single-walled carbon nanotubes (SWCNT) or on hydroxyl-functionalized SWCNT (SWCNT-OH), was incorporated within a Nafion matrix immobilized on the surface of a graphite electrode. From cyclic voltammetric measurements, performed under different experimental conditions (pH and potential scan rate), it was established that the Fe(III)P/Fe(II)P redox couple involves 1e− /1H+ . The heterogeneous electron transfer process occurred faster when Fe(III)P was adsorbed on SWCNT-OH (∼11 s−1 ) than on SWCNT (∼4.9 s−1 ). Both the SWCNT-Fe(III)P- and SWCNT-OH-Fe(III)P-modified graphite electrodes exhibit electrocatalytic activity for H2 O2 and nitrite reduction. The modified electrodes sensitivities were found varying in the following sequences: SSWCNT-OH-Fe(III)P = 2.45 mA/ M ≈ SSWCNT-Fe(III)P = 2.95 mA/M > SFe(III)P = 1.34 mA/M for H2 O2 , and SSWCNT-Fe(III)P = 3.54 mA/M > SFe(III)P = 1.44 mA/M > SSWCNT-OH-Fe(III)P = 0.81 mA/M for NO2 − . © 2006 Elsevier Ltd. All rights reserved. Keywords: Hemin; Single-wall carbon nanotubes; H2 O2 ; Nitrite reduction
1. Introduction The discovery of fullerenes in 1985 and the invention of carbon nanotubes (CNT) in 1991 opened new perspectives on carbon materials based on flat graphite-like hexagonal layers [1]. Their nanometer dimension [2] and their interesting physicochemical properties, such as excellent electrical conductivity, high chemical stability and remarkable mechanical strength [3,4], recommended them as promising materials for various applications. The carbon nanotubes were found to have two types of structures: single-wall CNT (SWCNT) and multi-wall CNT (MWCNT) [5]. Due to the above mentioned properties and diversity, CNTs of various lengths and internal diameters have allowed a wide range of practical applications in electroanalytical chemistry and nanotechnology (i.e., probes for scanning microscopy [5], electron field emission sources, actu-
∗ 1
Corresponding author. Tel.: +402 64595872; fax: +402 64 590818. E-mail address: [email protected] (G.L. Turdean). ISE member.
0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2006.04.028
ators, nanoelectronic devices, batteries, nanotube-reinforced materials, hydrogen storage materials [6] and chemical sensors [4,5,7]). In the domain of electroanalytical chemistry, the subtle electronic behavior of CNT, even if not yet fully characterized electrochemically, is associated with several other characteristics [8]: (i) CNT are conducting and relatively stable and thus can be used as advanced electrode materials; (ii) CNT can be functionalized with various chemical groups, facilitating the immobilization of biomolecules; (iii) CNT have a high surface area to weight ratio, favorable to both charge transfer and biomolecule immobilization. Recently, it has been demonstrated that CNTs can decrease the electroreduction potential for various redox substrates and, at the same time, increase the reaction rate, improving the selectivity and sensitivity of amperometric detection. For this reason, CNTs have been used as redox catalysts for electron transfer processes involving various electrochemical systems, as for example: calixarene [9], nitrite [10], ferrocene [9], catecholamine [11], H2 O2 [12], NADH [13], norepinephrine [14], dopamine [15], epinephrine, ascorbic acid [5,9], hemin
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[16], hemoglobin [9,17], myoglobin [18], cytochrome c [9,19,20], glucose oxidase [3,21–23] and peroxidases [17,24,25]. The study of the direct heterogeneous electron transfer process between electrodes and biomolecules (redox proteins, oxidoreductases, etc.) is a convenient and informative means to understand the kinetics and thermodynamics of biological redox processes. In this context, hemin (iron(III) protoporphyrin IX), which has a good stability in solution, a low molecular weight, and is relatively inexpensive [26], plays an important role: (i) as model molecule for understanding the redox activity of heme-proteins (e.g. b-cytochromes, peroxidases, catalases) [27,28] and oxygen-carrying proteins (myoglobin and hemoglobin [29]); (ii) as mimetic compound for peroxidases [26] or nitrite reductase [30]); (iii) as mediator, in conditions close to the native biological environment and for amperometric detection of various species, as for example: O2 [31,32], H2 O2 [26], NO/NO2 − [30,33], superoxide [27], tryptophan and its derivatives [34]. Taking advantage of the fact that hemin has a porphyrin ring, promoting its adsorption on carbonaceous materials, it was immobilized on a CNT surface for O2 detection [35]. Unfortunately, it was reported that when dispersed in aqueous media nanotubes aggregate easily because of the substantial van der Waals attractions [36]. Consequently, in order to improve the dispersion of CNTs in water, which is an important condition for their use in biomedical application, several approaches were proposed: polymer wrapping [37,38]; CNT coating with surfactants [37,38]; CNT functionalization with –OH [39] or –COOH groups [19,24]. Taking into account all these considerations, the aim of this paper was to obtain modified electrodes for H2 O2 and NO2 − electrocatalytic reduction, based on the immobilization of hemin on SWCNT and its further incorporation within a Nafion matrix, which was deposited on the surface of a graphite electrode. Using the rate constants for the heterogeneous electron transfer process, estimated with the Laviron approach [40], a quantitative evaluation of the influence of SWCNT functionalization on the electron transfer process between hemin and the graphite electrode was performed. For the first time, to our best knowledge, the electrocatalytic activities of the hemin-SWCNT- and hemin-SWCNT-OH-modified graphite electrodes towards two important biological analytes, H2 O2 and nitrite, have been quantitatively evaluated. 2. Materials 2.1. Chemicals Single-walled carbon nanotubes (SWCNT) (CarboLex AP-grade, 1.2–1.5 nm diameter) were acquired from (Aldrich, USA). The iron(III) protoporphyrin IX chloride (Fe(III)P), 2amino-2-hydroxymethyl-1,3-propanediol(tris-(hydroxymethyl) amino methane) (TRIS), Nafion solution (5% in methanol with equivalent weight of about 1100) and potassium bromide were purchased from Fluka, Switzerland. KOH for CNT functionalization was from Carlo Erba (Italy). The sodium dodecyl
sulphate (SDS) and the K3 [Fe(CN)6 ] were from Sigma, Italy. The hydrogen peroxide stock solution (30%, w/v) was from J.T. Baker, Italy and the sodium nitrite from Merck, Germany. A phosphate buffer solution (PBS; pH 7, ionic strength 0.2 M) containing 0.05 M KBr was prepared from 0.05 M KH2 PO4, 0.05 M K2 HPO4 . Also, a 0.05 M Tris–HCl containing 0.05 M KBr solution was employed as supporting electrolyte. In order to perform the pH-dependent experiments, the pH of PBS was adjusted with HCl or KOH. The buffer solutions were prepared using distilled-deionized water and were kept refrigerated to minimize bacterial growth. The 5 mM Fe(III)P solution was prepared by dissolving its chloride salt in a 0.05 M Tris–HCl buffer, containing 0.05 M KBr (pH 8). All chemicals were of analytical grade and were used without further purification. 2.2. Apparatus Cyclic voltammetric investigations were carried out using a trace analyzer (model AMEL 433, Amel, Milan, Italy). All electrochemical measurements were done using a standard single-compartment three electrode cell equipped with a platinum counter electrode, an Ag|AgCl, KClsat reference electrode (Amel, 805/CPG/6) and a disc working electrode made from spectral graphite (3 mm diameter) (RingsdorffWerke, Gmbh, Bonn-Bad Godesberg, Germany). Prior to experiments the buffer solutions were purged with high-purity nitrogen (Linde, Italy) for at least 15 min and a nitrogen environment was then kept over the solution in the cell during measurements. All experiments were performed at room temperature. 2.3. Preparation of G/SWCNT–Fe(III)P-Nafion electrode The graphite electrode was wet polished with Carbimet paper PSA (600 grit) (Buehler, USA) and rinsed with distilled water. The 0.010 g of SWCNT were dispersed in 50 ml aqueous solution of 1 wt% SDS. After homogenization and ultrasonication, the suspension of SWCNT obtained was ultracentrifugated at 120,000 G for 4 h. For all further experiments only the upper layer of the stable SWCNT suspension was used. Functionalized SWCNT (SWCNT-OH) were obtained by mixing 0.01 g SWCNT with 50 ml of 10 M KOH for 1 h. The suspension obtained was heated for 1 h at 100 ◦ C and then dried in a stove. After washing with water, the dried SWCNT-OH were treated as described above. SWCNT of 500 l or SWCNT-OH suspensions were mixed thoroughly with 5 mM Fe(III)P solution. Then, 2 l of the resulting mixture were cast onto the surface of the graphite electrode and allowed to dry at ambient temperature. This operation was repeated successively three times. Finally, 2 l of 5% Nafion was dropped on the modified electrode surface. The electrodes were stored at 4 ◦ C. The surface coverage with hemin (Γ , mol cm−2 ) was estimated by integrating the peak surface area (Q) under its reduction peak recorded at low potential scan rate, and assuming that the redox process involves one electron.
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3. Results and discussions 3.1. The SWCNT functionalization Among the techniques known to improve the SWCNT dispersion in aqueous solution, two approaches were compared in the present work: (i) SWCNT coating with a surfactant; (ii) the SWCNT functionalization with –OH groups by treatment with KOH at room temperature and the subsequent coating with a surfactant. The first method was chosen because the thermodynamic tendency toward bundling is overcome in the presence of surfactants [16,36,41]. The second method is supposed to induce a simple solid-phase mechano-chemical exfoliation of carbon nanotubes, producing bundles of SWCNT functionalized with –OH groups to various degrees and having a higher solubility in water [39]. FTIR spectra, recorded for SWCNT treated by 10 M KOH, showed a broad band centered between 3100 and 3400 cm−1 [39,42], which is characteristic to the stretching vibration of –OH groups (Fig. 4 in reference [43]). It was supposed that the alkali treatment introduced the –OH groups on the already existing defects, without breaking the graphite structure nor cutting the tube [43]. 3.2. Electrochemical behavior of hemin-modified electrodes Fig. 1 shows the cyclic voltammograms recorded in phosphate buffer (pH 7) at G/SWCNT-Nafion and at G/SWCNTFe(III)P-Nafion electrodes, prepared with non-functionalized and functionalized SWCNT. As expected, in the absence of
Fig. 1. Cyclic voltammograms of hemin-modified graphite electrodes incorporated in SWCNT (. . .) and SWCNT–OH (– – –). Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; potential scan rate, 50 mV s−1 ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution. The voltammetric response of G/SWCNT-Nafion electrode (—) was included for comparison.
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hemin, no redox peaks were observed for either electrode in the potential range investigated. By contrast, a pair of welldefined redox peaks was clearly seen when the hemin was present on the electrode surface. The pair peaks was attributed to a quasi-reversible monoelectron transfer (EFWHM = 197 mV for G/SWCNT-Fe(III)P-Nafion and 177 mV for G/SWCNT-OHFe(III)P-Nafion), involving the Fe(III)/Fe(II) redox couple coordinated in the porphyrinic ring [44]. For both modified electrodes investigated, the value of EFWHM was higher than the theoretical value (90.5/n mV) corresponding to a surface confined redox couple [40,42], indicating either a certain non-uniformity of the adsorbed redox couples, or the existence of repulsive interactions between the active redox centers [45]. The slight decrease of the peak split observed for G/SWCNT-OH-Fe(III)P-Nafion (Ep = 35 mV) modified electrode in comparison with the peak split corresponding to G/SWCNT-Fe(III)P-Nafion (Ep = 55 mV) electrodes was attributed to a beneficial effect due to the presence of –OH groups. It can be suggested that the –OH groups increase the van der Waals interactions between hemin and SWCNT, favoring its adsorption on the electrode material [37,39]. The formal potential (E◦ ; defined as the average value of the anodic and cathodic peak potentials) was not affected by SWCNT functionalization: −0.500 V versus Ag/AgCl, KClsat for G/SWCNT-OH-Fe(III)P-Nafion and −0.515 V versus Ag/AgCl, KClsat for G/SWCNT-Fe(III)P-Nafion. However, in both cases the E◦ value was more negative than that observed for hemin immobilized on MWCNT deposited on a glassy carbon electrode covered with Ta (E◦ = −0.34 V versus Ag/Ag, 3 M KCl) [35]. This difference probably reflects the specific influence of the immobilization matrix on the redox behavior of hemin. Over a relatively wide range of scan potential (5–2000 mV s−1 ) the anodic and cathodic peak currents increase linearly with the potential scan rate (v), indicating a surface confined redox couple [44]. This conclusion is also supported by the slope of the log I versus log v dependence, which was found to be very close to 1, for both electrodes investigated (results not shown). The Fe(IIIP/Fe(II)P redox couple bound in the porphyrinic ring exhibits a quasi-reversible behavior as shown by a Ipa /Ipc ratio very close to 1 [46] and by the slight increase of Ep with increase in the potential scan rate. The decrease of the peak intensities observed when hemin was immobilized on SWCNT-OH, in comparison with those recorded for hemin immobilized on SWCNT (see Figs. 1 and 2), could be the result of a decrease of the hemin redox activity induced by the increase of the matrix tightness due to the presence of –OH groups in the local microenvironment. This is consistent with the deviation from the linear correlation, which in the case of the SWCNT-OH based matrix, appears at higher potential scan rate (Fig. 2). This behavior indicates the lesser contribution of diffusion to the global process of charge transfer in the more compact matrix (SWCNT-OH) in comparison with loose matrix (SWCNT) [47]. In order to perform a quantitative evaluation of the matrix permeability variation as a function of SWCNT functionalization, cyclic voltammograms were recorded for different potential
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G.L. Turdean et al. / Electrochimica Acta 51 (2006) 6435–6441 Table 1 Rate constant for the heterogeneous electron transfer at G/SWCNT-Fe(III)PNafion electrodes Type of electrode
ks (s−1 )
G/SWCNT-Fe(III)P-Nafion G/SWCNT-OH-Fe(III)P-Nafion
4.9 ± 0.5 11 ± 0.8
Experimental conditions (pH 7, see Fig. 2).
Fig. 2. Dependence of the current peak intensity on the potential scan rate for G/SWCNT-Fe(III)P-Nafion (䊉) and G/SWCNT-OH-Fe(III)P-Nafion () electrodes. Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution.
scan rates at G/SWCNT-OH-Fe(III)P-Nafion and G/SWCNTFe(III)P-Nafion electrodes, brought in contact with a 5 mM K3 [Fe(CN)6 ] solution, containing 1 M KCl. The voltammetric wave corresponding to the [Fe(CN)6 ]3− /[Fe(CN)6 ]4− couple does not overlap with that for Fe(III)P/Fe(II)P in hemin, and the corresponding anodic and cathodic peak currents obey the following equation: Ip = (2.69 × 105 )n2/3 AD1/2 v1/2 Co
(1)
where n is the number of electrons, A the active surface area (cm2 ), D the diffusion coefficient (cm2 /s), v the scan rate and Co is the concentration in bulk solution (mol/cm3 ). Assuming that for the SWCNT matrix the K3 [Fe(CN)6 ] diffusion coefficient is identical with that reported in solution (i.e., D = 5.9 × 10−5 cm2 /s [35]) and that the active surface (0.048 cm2 ) remains the same for both modified electrodes investigated, the permeability in the case of the SWCNT-OH matrix was found to be lower by a factor of 1.7. This result proved an additional confirmation of the previous experimental observations, pointing out that the SWCNT-OH matrix is more compact than the SWCNT one. Using Laviron’s treatment describing the voltammetric response of adsorbed species when Ep < 200/n mV and α = 0.5 [40], the rate constants for the heterogeneous electron transfer process corresponding to the Fe(IIIP/Fe(II)P redox couple were estimated for both modified electrodes (Table 1). Irrespective of the CNT type, the ks values were found to be higher than the values reported either for hemoglobin adsorbed on CNT (0.062 s−1 [48]) and for hemin immobilized on MWCNT (2.9 s−1 [35]) or the value obtained for a HRP-modified glassy carbon electrode (3.4 s−1 for HRP-C/GC [49]). On the other
hand, they were much lower than the values observed for a hemin monolayer deposited on glassy carbon (4.9 × 103 s−1 , [27]) or on a basal plane pyrolytic graphite electrode (160 s−1 , [27]). Additionally, it is worth to mention that the presence of –OH groups on the CNT surface has a beneficial effect on the charge transfer rate between hemin and the graphite electrode. It is known that the solution pH modulates the accessibility of water to the heme and the protonation of the heme is iron-bound proximal histidine and/or the distal histidine in the heme pocket [16,50]. Accordingly, the solution pH influences the redox potential of hemin: a pH increase leads to a negative shift of the standard formal potential (E◦ ; results not shown). The E◦ versus pH dependencies, investigated in the pH range from 6.5 to 8.5, were linear with the following slopes: −56.2 mV/pH (R = 0.945, n = 6) for G/SWCNT-Fe(III)P-Nafion electrode, and −57.7 mV/pH (R = 0.990, n = 5) for G/SWCNT-OH-Fe(III)PNafion electrode. These results, together with the EFWHM values, point to a 1e− /1H+ process, in accordance with the overall Eq. (2) [51,52]: (H2 O)(OH)Fe(III)P + e− + H+ ↔ (H2 O)2 Fe(II)P
(2)
The stability of the modified electrodes was evaluated under potentiodynamic conditions, by continuously cycling the electrode potential between 0 and −0.95 V (at 50 mV s−1 ) when the electrodes were in contact with a phosphate buffer solution (pH 7). Assuming that the time evolution of the surface coverage for the electrodes investigated obeys a first order kinetic [47], the plot Γ cat versus time allowed the estimation of the rate constants characterizing the activation/deactivation of the response process (Fig. 3). Both electrodes showed good short-term stability and this was not influenced by the SWCNT functionalization. 3.3. Electrocatalytic behavior of G/SWCNT-Fe(III)P-Nafion electrode 3.3.1. Hydrogen peroxide reduction Hydrogen peroxide is the product of the reactions catalyzed by a large number of oxidases and, consequently, the determination of hydrogen peroxide (H2 O2 ) is of considerable importance in clinical, food, pharmaceutical and environmental analysis [42,51]. The cyclic voltamogramms recorded in the presence of H2 O2 at G/SWCNT-Fe(III)P-Nafion and G/SWCNT-OH-Fe(III)PNafion electrodes (Fig. 4A) show that hemin immobilized in the SWCNT-OH matrix exhibits a higher electrocatalytic activity for H2 O2 reduction than when it was incorporated in the SWCNT matrix. The two supplementary waves, observed at −0.1 and
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Fig. 3. The stability of the voltammetric response of G/SWCNT-Fe(III)PNafion (䊉) and G/SWCNT-OH-Fe(III)P-Nafion () electrodes, observed during repetitive potential cycling between 0 and −0.950 V vs. Ag/AgCl, KClsat . Experimental conditions: potential scan rate, 50 mV s−1 ; starting potential 0 V vs. Ag/AgCl, KClsat ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution.
−0.33 V versus Ag/AgCl, KClsat , were attributed to the oxygen electro-reduction, mediated by hemin [35]. A Michaelis–Menten type dependence between the corrected current difference and H2 O2 concentration was found for all hemin-modified electrodes (Fig. 4A, inset). The SWCNTs presence in the modified electrode matrix increases the maximum catalytic current, the highest sensitivity (S; estimated as Imax /KM ratio) being observed in the case of SWCNT-OH (SSWCNT-OH-Fe(III)P = 2.45 mA/M ≈ SSWCNT-Fe(III)P = 2.95 mA/ M > SFe(III)P = 1.34 mA/M).
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3.3.2. Nitrite reduction Nitrate and nitrite reduction has gained renewed attention in view of its relevance to monitoring of pollution due to the excessive use of fertilizers, detergents, industrial processes and food technologies. Also, the control of water quality is important in order to avoid contamination of food produced when water is used as a raw material. Electrochemical reduction can be advantageously applied to the treatment of industrial wastewater, whereby nitrate species are transformed into harmless reduction products on various cathodic materials and from solutions of different compositions [53]. However, the electrochemical reactions of interest have been found to proceed at potentials substantially more negative than their thermodynamic values and with low current density, providing evidence that their energies of activation are very high. It is well-known that appropriately modifying the traditional electrode surface opportunely is an effective way of enhancing its electrocatalytic activity. Consequently, in recent years, some new electrocatalytic systems for nitrite determination were developed using electrodes modified with: copper–thallium composite film [54]; tetrakis (N-methylpyridinium) Ni(II) porphyrin [55]; tetrakis(3methoxy-4-hydroxyphenyl)Ni(II) porphyrin [56]; hemoglobin [30,33,57]; hemin, myoglobin [30]; Os(bpy)3 2+ [58]. As can be seen from Fig. 4B, the presence of immobilized hemin allows the NO2 − electroreduction, revealed by the appearance of a new cathodic wave, situated at approximately −1.1 V versus Ag/AgCl, KClsat . This result is in agreement with that reported for graphite electrodes modified with hemoglobin adsorbed onto mesoporous WO3 [57]. Similarly to H2 O2 reduction on hemin-modified electrode, a Michealis–Menten dependence was observed for NO2 − electroreduction, when the cathodic current (corrected for the corresponding baseline) was plotted against the NO2 − concentration (Fig. 4B, inset). However, it is interesting to note that contrary to the behavior noticed for H2 O2 electrocatalytic reduction, the sensitivity (S; estimated as Imax /KM ratio) for the NO2 − electroreduction observed in the case of G/SWCNT-Fe(III)P-
Fig. 4. Electrocatalytic reduction of 10 mM H2 O2 (A) and 10 mM NO2 − (B) at G/SWCNT-Fe(III)P-Nafion (. . .) and G/SWCNT-OH-Fe(III)P-Nafion (– – –) electrodes. Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; potential scan rate, 20 mV s−1 ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution. For comparison the voltammetric response of G/SWCNT-OH-Fe(III)P-Nafion (—) electrode in buffer solution was included. The dependence of the corrected current difference on the H2 O2 (A) and NO2 − (B) concentration for G/SWCNT-Fe(III)P-Nafion (䊉), G/SWCNT-OH-Fe(III)P-Nafion () and G/Fe(III)P-Nafion () electrodes is presented in the insets.
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Nafion electrodes [SSWCNT-Fe(III)P ] was higher than that observed for G/SWCNT-OH-Fe(III)P-Nafion electrodes [SSWCNT-OH-Fe(III)P ]. Thus, the following sequence of sensitivity decreasing was obtained: SSWCNT-Fe(III)P = 3.54 mA/M > SFe(III)P = 1.44 mA/M > SSWCNT-OH-Fe(III)P = 0.81 mA/M. 4. Conclusions This study provides a new example of hemin immobilization on simple and –OH functionalized SWCNTs incorporated within a Nafion matrix, which was deposited on a graphite electrode by a simple and reproducible drop-coating method. As shown by potential scan rate and pH experiments, the immobilized hemin retain its normal voltammetric response, corresponding to a quasi-reversible, 1e− /1H+ process in both investigated matrices. Greater reversibility was noticed in the case of –OH functionalized SWCNT, expressed as a significant increase in the rate constant for the heterogeneous electron transfer process and by a slight decrease in the peak potential split. For the first time, to our knowledge, the electrocatalytic activities of the hemin-SWCNT- and hemin-SWCNT-OH-modified graphite electrodes towards H2 O2 and nitrite electroreduction, were quantitatively evaluated. It was found that only for the H2 O2 electrocatalytic reduction was the presence of SWCNTOH beneficial for the hemin catalytic activity. Thus, the use of –OH functionalized SWCNTs as electrode material opens a simple and elegant way to construct new amperometric biosensors for detection the small signal of molecules and to investigate the direct electron transfer for redox proteins incorporating a hemin-like active center (as for example, b-cytochromes, peroxidases, myoglobin and hemoglobin).
[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32]
Acknowledgements G.L.T. acknowledges the financial support for the postdoctoral fellowship from NATO-CNR Italy. The work was supported by Grants Program A-55/375/2004, A-85/375/2005 from CNCSIS—Romanian Ministry of National Education. A special thanks to Adriana Coppe (University “La Sapienza”) for the preparation of the –OH functionalised SWCNT. A.C. and G.P. thank the Consiglio Nazionale delle Ricerche (C.N.R.) Target Project MADESS II subproject: sensors and the MIUR project FIRB 2001 for financial support.
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Iron(III) protoporphyrin IX—single-wall carbon nanotubes modified electrodes for hydrogen peroxide and nitrite detection Graziella L. Turdean a,b,∗,1 , Ionel Catalin Popescu a , Antonella Curulli c , Giuseppe Palleschi b a
b
“Babes Bolyai” University, Physical Chemistry Department, Arany Janos 11, 400 028 Cluj-Napoca, Romania Dipartimento di Scienze e Technologie Chimiche, Universita di Roma “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Rome, Italy c Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) CNR Division 2, Via del Castro Laurenziano 7, 00161 Rome, Italy Received 14 February 2006; received in revised form 12 April 2006; accepted 18 April 2006 Available online 5 June 2006
Abstract Iron(III) protoporphyrin IX (Fe(III)P), adsorbed either on single-walled carbon nanotubes (SWCNT) or on hydroxyl-functionalized SWCNT (SWCNT-OH), was incorporated within a Nafion matrix immobilized on the surface of a graphite electrode. From cyclic voltammetric measurements, performed under different experimental conditions (pH and potential scan rate), it was established that the Fe(III)P/Fe(II)P redox couple involves 1e− /1H+ . The heterogeneous electron transfer process occurred faster when Fe(III)P was adsorbed on SWCNT-OH (∼11 s−1 ) than on SWCNT (∼4.9 s−1 ). Both the SWCNT-Fe(III)P- and SWCNT-OH-Fe(III)P-modified graphite electrodes exhibit electrocatalytic activity for H2 O2 and nitrite reduction. The modified electrodes sensitivities were found varying in the following sequences: SSWCNT-OH-Fe(III)P = 2.45 mA/ M ≈ SSWCNT-Fe(III)P = 2.95 mA/M > SFe(III)P = 1.34 mA/M for H2 O2 , and SSWCNT-Fe(III)P = 3.54 mA/M > SFe(III)P = 1.44 mA/M > SSWCNT-OH-Fe(III)P = 0.81 mA/M for NO2 − . © 2006 Elsevier Ltd. All rights reserved. Keywords: Hemin; Single-wall carbon nanotubes; H2 O2 ; Nitrite reduction
1. Introduction The discovery of fullerenes in 1985 and the invention of carbon nanotubes (CNT) in 1991 opened new perspectives on carbon materials based on flat graphite-like hexagonal layers [1]. Their nanometer dimension [2] and their interesting physicochemical properties, such as excellent electrical conductivity, high chemical stability and remarkable mechanical strength [3,4], recommended them as promising materials for various applications. The carbon nanotubes were found to have two types of structures: single-wall CNT (SWCNT) and multi-wall CNT (MWCNT) [5]. Due to the above mentioned properties and diversity, CNTs of various lengths and internal diameters have allowed a wide range of practical applications in electroanalytical chemistry and nanotechnology (i.e., probes for scanning microscopy [5], electron field emission sources, actu-
∗ 1
Corresponding author. Tel.: +402 64595872; fax: +402 64 590818. E-mail address: [email protected] (G.L. Turdean). ISE member.
0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2006.04.028
ators, nanoelectronic devices, batteries, nanotube-reinforced materials, hydrogen storage materials [6] and chemical sensors [4,5,7]). In the domain of electroanalytical chemistry, the subtle electronic behavior of CNT, even if not yet fully characterized electrochemically, is associated with several other characteristics [8]: (i) CNT are conducting and relatively stable and thus can be used as advanced electrode materials; (ii) CNT can be functionalized with various chemical groups, facilitating the immobilization of biomolecules; (iii) CNT have a high surface area to weight ratio, favorable to both charge transfer and biomolecule immobilization. Recently, it has been demonstrated that CNTs can decrease the electroreduction potential for various redox substrates and, at the same time, increase the reaction rate, improving the selectivity and sensitivity of amperometric detection. For this reason, CNTs have been used as redox catalysts for electron transfer processes involving various electrochemical systems, as for example: calixarene [9], nitrite [10], ferrocene [9], catecholamine [11], H2 O2 [12], NADH [13], norepinephrine [14], dopamine [15], epinephrine, ascorbic acid [5,9], hemin
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[16], hemoglobin [9,17], myoglobin [18], cytochrome c [9,19,20], glucose oxidase [3,21–23] and peroxidases [17,24,25]. The study of the direct heterogeneous electron transfer process between electrodes and biomolecules (redox proteins, oxidoreductases, etc.) is a convenient and informative means to understand the kinetics and thermodynamics of biological redox processes. In this context, hemin (iron(III) protoporphyrin IX), which has a good stability in solution, a low molecular weight, and is relatively inexpensive [26], plays an important role: (i) as model molecule for understanding the redox activity of heme-proteins (e.g. b-cytochromes, peroxidases, catalases) [27,28] and oxygen-carrying proteins (myoglobin and hemoglobin [29]); (ii) as mimetic compound for peroxidases [26] or nitrite reductase [30]); (iii) as mediator, in conditions close to the native biological environment and for amperometric detection of various species, as for example: O2 [31,32], H2 O2 [26], NO/NO2 − [30,33], superoxide [27], tryptophan and its derivatives [34]. Taking advantage of the fact that hemin has a porphyrin ring, promoting its adsorption on carbonaceous materials, it was immobilized on a CNT surface for O2 detection [35]. Unfortunately, it was reported that when dispersed in aqueous media nanotubes aggregate easily because of the substantial van der Waals attractions [36]. Consequently, in order to improve the dispersion of CNTs in water, which is an important condition for their use in biomedical application, several approaches were proposed: polymer wrapping [37,38]; CNT coating with surfactants [37,38]; CNT functionalization with –OH [39] or –COOH groups [19,24]. Taking into account all these considerations, the aim of this paper was to obtain modified electrodes for H2 O2 and NO2 − electrocatalytic reduction, based on the immobilization of hemin on SWCNT and its further incorporation within a Nafion matrix, which was deposited on the surface of a graphite electrode. Using the rate constants for the heterogeneous electron transfer process, estimated with the Laviron approach [40], a quantitative evaluation of the influence of SWCNT functionalization on the electron transfer process between hemin and the graphite electrode was performed. For the first time, to our best knowledge, the electrocatalytic activities of the hemin-SWCNT- and hemin-SWCNT-OH-modified graphite electrodes towards two important biological analytes, H2 O2 and nitrite, have been quantitatively evaluated. 2. Materials 2.1. Chemicals Single-walled carbon nanotubes (SWCNT) (CarboLex AP-grade, 1.2–1.5 nm diameter) were acquired from (Aldrich, USA). The iron(III) protoporphyrin IX chloride (Fe(III)P), 2amino-2-hydroxymethyl-1,3-propanediol(tris-(hydroxymethyl) amino methane) (TRIS), Nafion solution (5% in methanol with equivalent weight of about 1100) and potassium bromide were purchased from Fluka, Switzerland. KOH for CNT functionalization was from Carlo Erba (Italy). The sodium dodecyl
sulphate (SDS) and the K3 [Fe(CN)6 ] were from Sigma, Italy. The hydrogen peroxide stock solution (30%, w/v) was from J.T. Baker, Italy and the sodium nitrite from Merck, Germany. A phosphate buffer solution (PBS; pH 7, ionic strength 0.2 M) containing 0.05 M KBr was prepared from 0.05 M KH2 PO4, 0.05 M K2 HPO4 . Also, a 0.05 M Tris–HCl containing 0.05 M KBr solution was employed as supporting electrolyte. In order to perform the pH-dependent experiments, the pH of PBS was adjusted with HCl or KOH. The buffer solutions were prepared using distilled-deionized water and were kept refrigerated to minimize bacterial growth. The 5 mM Fe(III)P solution was prepared by dissolving its chloride salt in a 0.05 M Tris–HCl buffer, containing 0.05 M KBr (pH 8). All chemicals were of analytical grade and were used without further purification. 2.2. Apparatus Cyclic voltammetric investigations were carried out using a trace analyzer (model AMEL 433, Amel, Milan, Italy). All electrochemical measurements were done using a standard single-compartment three electrode cell equipped with a platinum counter electrode, an Ag|AgCl, KClsat reference electrode (Amel, 805/CPG/6) and a disc working electrode made from spectral graphite (3 mm diameter) (RingsdorffWerke, Gmbh, Bonn-Bad Godesberg, Germany). Prior to experiments the buffer solutions were purged with high-purity nitrogen (Linde, Italy) for at least 15 min and a nitrogen environment was then kept over the solution in the cell during measurements. All experiments were performed at room temperature. 2.3. Preparation of G/SWCNT–Fe(III)P-Nafion electrode The graphite electrode was wet polished with Carbimet paper PSA (600 grit) (Buehler, USA) and rinsed with distilled water. The 0.010 g of SWCNT were dispersed in 50 ml aqueous solution of 1 wt% SDS. After homogenization and ultrasonication, the suspension of SWCNT obtained was ultracentrifugated at 120,000 G for 4 h. For all further experiments only the upper layer of the stable SWCNT suspension was used. Functionalized SWCNT (SWCNT-OH) were obtained by mixing 0.01 g SWCNT with 50 ml of 10 M KOH for 1 h. The suspension obtained was heated for 1 h at 100 ◦ C and then dried in a stove. After washing with water, the dried SWCNT-OH were treated as described above. SWCNT of 500 l or SWCNT-OH suspensions were mixed thoroughly with 5 mM Fe(III)P solution. Then, 2 l of the resulting mixture were cast onto the surface of the graphite electrode and allowed to dry at ambient temperature. This operation was repeated successively three times. Finally, 2 l of 5% Nafion was dropped on the modified electrode surface. The electrodes were stored at 4 ◦ C. The surface coverage with hemin (Γ , mol cm−2 ) was estimated by integrating the peak surface area (Q) under its reduction peak recorded at low potential scan rate, and assuming that the redox process involves one electron.
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3. Results and discussions 3.1. The SWCNT functionalization Among the techniques known to improve the SWCNT dispersion in aqueous solution, two approaches were compared in the present work: (i) SWCNT coating with a surfactant; (ii) the SWCNT functionalization with –OH groups by treatment with KOH at room temperature and the subsequent coating with a surfactant. The first method was chosen because the thermodynamic tendency toward bundling is overcome in the presence of surfactants [16,36,41]. The second method is supposed to induce a simple solid-phase mechano-chemical exfoliation of carbon nanotubes, producing bundles of SWCNT functionalized with –OH groups to various degrees and having a higher solubility in water [39]. FTIR spectra, recorded for SWCNT treated by 10 M KOH, showed a broad band centered between 3100 and 3400 cm−1 [39,42], which is characteristic to the stretching vibration of –OH groups (Fig. 4 in reference [43]). It was supposed that the alkali treatment introduced the –OH groups on the already existing defects, without breaking the graphite structure nor cutting the tube [43]. 3.2. Electrochemical behavior of hemin-modified electrodes Fig. 1 shows the cyclic voltammograms recorded in phosphate buffer (pH 7) at G/SWCNT-Nafion and at G/SWCNTFe(III)P-Nafion electrodes, prepared with non-functionalized and functionalized SWCNT. As expected, in the absence of
Fig. 1. Cyclic voltammograms of hemin-modified graphite electrodes incorporated in SWCNT (. . .) and SWCNT–OH (– – –). Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; potential scan rate, 50 mV s−1 ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution. The voltammetric response of G/SWCNT-Nafion electrode (—) was included for comparison.
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hemin, no redox peaks were observed for either electrode in the potential range investigated. By contrast, a pair of welldefined redox peaks was clearly seen when the hemin was present on the electrode surface. The pair peaks was attributed to a quasi-reversible monoelectron transfer (EFWHM = 197 mV for G/SWCNT-Fe(III)P-Nafion and 177 mV for G/SWCNT-OHFe(III)P-Nafion), involving the Fe(III)/Fe(II) redox couple coordinated in the porphyrinic ring [44]. For both modified electrodes investigated, the value of EFWHM was higher than the theoretical value (90.5/n mV) corresponding to a surface confined redox couple [40,42], indicating either a certain non-uniformity of the adsorbed redox couples, or the existence of repulsive interactions between the active redox centers [45]. The slight decrease of the peak split observed for G/SWCNT-OH-Fe(III)P-Nafion (Ep = 35 mV) modified electrode in comparison with the peak split corresponding to G/SWCNT-Fe(III)P-Nafion (Ep = 55 mV) electrodes was attributed to a beneficial effect due to the presence of –OH groups. It can be suggested that the –OH groups increase the van der Waals interactions between hemin and SWCNT, favoring its adsorption on the electrode material [37,39]. The formal potential (E◦ ; defined as the average value of the anodic and cathodic peak potentials) was not affected by SWCNT functionalization: −0.500 V versus Ag/AgCl, KClsat for G/SWCNT-OH-Fe(III)P-Nafion and −0.515 V versus Ag/AgCl, KClsat for G/SWCNT-Fe(III)P-Nafion. However, in both cases the E◦ value was more negative than that observed for hemin immobilized on MWCNT deposited on a glassy carbon electrode covered with Ta (E◦ = −0.34 V versus Ag/Ag, 3 M KCl) [35]. This difference probably reflects the specific influence of the immobilization matrix on the redox behavior of hemin. Over a relatively wide range of scan potential (5–2000 mV s−1 ) the anodic and cathodic peak currents increase linearly with the potential scan rate (v), indicating a surface confined redox couple [44]. This conclusion is also supported by the slope of the log I versus log v dependence, which was found to be very close to 1, for both electrodes investigated (results not shown). The Fe(IIIP/Fe(II)P redox couple bound in the porphyrinic ring exhibits a quasi-reversible behavior as shown by a Ipa /Ipc ratio very close to 1 [46] and by the slight increase of Ep with increase in the potential scan rate. The decrease of the peak intensities observed when hemin was immobilized on SWCNT-OH, in comparison with those recorded for hemin immobilized on SWCNT (see Figs. 1 and 2), could be the result of a decrease of the hemin redox activity induced by the increase of the matrix tightness due to the presence of –OH groups in the local microenvironment. This is consistent with the deviation from the linear correlation, which in the case of the SWCNT-OH based matrix, appears at higher potential scan rate (Fig. 2). This behavior indicates the lesser contribution of diffusion to the global process of charge transfer in the more compact matrix (SWCNT-OH) in comparison with loose matrix (SWCNT) [47]. In order to perform a quantitative evaluation of the matrix permeability variation as a function of SWCNT functionalization, cyclic voltammograms were recorded for different potential
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G.L. Turdean et al. / Electrochimica Acta 51 (2006) 6435–6441 Table 1 Rate constant for the heterogeneous electron transfer at G/SWCNT-Fe(III)PNafion electrodes Type of electrode
ks (s−1 )
G/SWCNT-Fe(III)P-Nafion G/SWCNT-OH-Fe(III)P-Nafion
4.9 ± 0.5 11 ± 0.8
Experimental conditions (pH 7, see Fig. 2).
Fig. 2. Dependence of the current peak intensity on the potential scan rate for G/SWCNT-Fe(III)P-Nafion (䊉) and G/SWCNT-OH-Fe(III)P-Nafion () electrodes. Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution.
scan rates at G/SWCNT-OH-Fe(III)P-Nafion and G/SWCNTFe(III)P-Nafion electrodes, brought in contact with a 5 mM K3 [Fe(CN)6 ] solution, containing 1 M KCl. The voltammetric wave corresponding to the [Fe(CN)6 ]3− /[Fe(CN)6 ]4− couple does not overlap with that for Fe(III)P/Fe(II)P in hemin, and the corresponding anodic and cathodic peak currents obey the following equation: Ip = (2.69 × 105 )n2/3 AD1/2 v1/2 Co
(1)
where n is the number of electrons, A the active surface area (cm2 ), D the diffusion coefficient (cm2 /s), v the scan rate and Co is the concentration in bulk solution (mol/cm3 ). Assuming that for the SWCNT matrix the K3 [Fe(CN)6 ] diffusion coefficient is identical with that reported in solution (i.e., D = 5.9 × 10−5 cm2 /s [35]) and that the active surface (0.048 cm2 ) remains the same for both modified electrodes investigated, the permeability in the case of the SWCNT-OH matrix was found to be lower by a factor of 1.7. This result proved an additional confirmation of the previous experimental observations, pointing out that the SWCNT-OH matrix is more compact than the SWCNT one. Using Laviron’s treatment describing the voltammetric response of adsorbed species when Ep < 200/n mV and α = 0.5 [40], the rate constants for the heterogeneous electron transfer process corresponding to the Fe(IIIP/Fe(II)P redox couple were estimated for both modified electrodes (Table 1). Irrespective of the CNT type, the ks values were found to be higher than the values reported either for hemoglobin adsorbed on CNT (0.062 s−1 [48]) and for hemin immobilized on MWCNT (2.9 s−1 [35]) or the value obtained for a HRP-modified glassy carbon electrode (3.4 s−1 for HRP-C/GC [49]). On the other
hand, they were much lower than the values observed for a hemin monolayer deposited on glassy carbon (4.9 × 103 s−1 , [27]) or on a basal plane pyrolytic graphite electrode (160 s−1 , [27]). Additionally, it is worth to mention that the presence of –OH groups on the CNT surface has a beneficial effect on the charge transfer rate between hemin and the graphite electrode. It is known that the solution pH modulates the accessibility of water to the heme and the protonation of the heme is iron-bound proximal histidine and/or the distal histidine in the heme pocket [16,50]. Accordingly, the solution pH influences the redox potential of hemin: a pH increase leads to a negative shift of the standard formal potential (E◦ ; results not shown). The E◦ versus pH dependencies, investigated in the pH range from 6.5 to 8.5, were linear with the following slopes: −56.2 mV/pH (R = 0.945, n = 6) for G/SWCNT-Fe(III)P-Nafion electrode, and −57.7 mV/pH (R = 0.990, n = 5) for G/SWCNT-OH-Fe(III)PNafion electrode. These results, together with the EFWHM values, point to a 1e− /1H+ process, in accordance with the overall Eq. (2) [51,52]: (H2 O)(OH)Fe(III)P + e− + H+ ↔ (H2 O)2 Fe(II)P
(2)
The stability of the modified electrodes was evaluated under potentiodynamic conditions, by continuously cycling the electrode potential between 0 and −0.95 V (at 50 mV s−1 ) when the electrodes were in contact with a phosphate buffer solution (pH 7). Assuming that the time evolution of the surface coverage for the electrodes investigated obeys a first order kinetic [47], the plot Γ cat versus time allowed the estimation of the rate constants characterizing the activation/deactivation of the response process (Fig. 3). Both electrodes showed good short-term stability and this was not influenced by the SWCNT functionalization. 3.3. Electrocatalytic behavior of G/SWCNT-Fe(III)P-Nafion electrode 3.3.1. Hydrogen peroxide reduction Hydrogen peroxide is the product of the reactions catalyzed by a large number of oxidases and, consequently, the determination of hydrogen peroxide (H2 O2 ) is of considerable importance in clinical, food, pharmaceutical and environmental analysis [42,51]. The cyclic voltamogramms recorded in the presence of H2 O2 at G/SWCNT-Fe(III)P-Nafion and G/SWCNT-OH-Fe(III)PNafion electrodes (Fig. 4A) show that hemin immobilized in the SWCNT-OH matrix exhibits a higher electrocatalytic activity for H2 O2 reduction than when it was incorporated in the SWCNT matrix. The two supplementary waves, observed at −0.1 and
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Fig. 3. The stability of the voltammetric response of G/SWCNT-Fe(III)PNafion (䊉) and G/SWCNT-OH-Fe(III)P-Nafion () electrodes, observed during repetitive potential cycling between 0 and −0.950 V vs. Ag/AgCl, KClsat . Experimental conditions: potential scan rate, 50 mV s−1 ; starting potential 0 V vs. Ag/AgCl, KClsat ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution.
−0.33 V versus Ag/AgCl, KClsat , were attributed to the oxygen electro-reduction, mediated by hemin [35]. A Michaelis–Menten type dependence between the corrected current difference and H2 O2 concentration was found for all hemin-modified electrodes (Fig. 4A, inset). The SWCNTs presence in the modified electrode matrix increases the maximum catalytic current, the highest sensitivity (S; estimated as Imax /KM ratio) being observed in the case of SWCNT-OH (SSWCNT-OH-Fe(III)P = 2.45 mA/M ≈ SSWCNT-Fe(III)P = 2.95 mA/ M > SFe(III)P = 1.34 mA/M).
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3.3.2. Nitrite reduction Nitrate and nitrite reduction has gained renewed attention in view of its relevance to monitoring of pollution due to the excessive use of fertilizers, detergents, industrial processes and food technologies. Also, the control of water quality is important in order to avoid contamination of food produced when water is used as a raw material. Electrochemical reduction can be advantageously applied to the treatment of industrial wastewater, whereby nitrate species are transformed into harmless reduction products on various cathodic materials and from solutions of different compositions [53]. However, the electrochemical reactions of interest have been found to proceed at potentials substantially more negative than their thermodynamic values and with low current density, providing evidence that their energies of activation are very high. It is well-known that appropriately modifying the traditional electrode surface opportunely is an effective way of enhancing its electrocatalytic activity. Consequently, in recent years, some new electrocatalytic systems for nitrite determination were developed using electrodes modified with: copper–thallium composite film [54]; tetrakis (N-methylpyridinium) Ni(II) porphyrin [55]; tetrakis(3methoxy-4-hydroxyphenyl)Ni(II) porphyrin [56]; hemoglobin [30,33,57]; hemin, myoglobin [30]; Os(bpy)3 2+ [58]. As can be seen from Fig. 4B, the presence of immobilized hemin allows the NO2 − electroreduction, revealed by the appearance of a new cathodic wave, situated at approximately −1.1 V versus Ag/AgCl, KClsat . This result is in agreement with that reported for graphite electrodes modified with hemoglobin adsorbed onto mesoporous WO3 [57]. Similarly to H2 O2 reduction on hemin-modified electrode, a Michealis–Menten dependence was observed for NO2 − electroreduction, when the cathodic current (corrected for the corresponding baseline) was plotted against the NO2 − concentration (Fig. 4B, inset). However, it is interesting to note that contrary to the behavior noticed for H2 O2 electrocatalytic reduction, the sensitivity (S; estimated as Imax /KM ratio) for the NO2 − electroreduction observed in the case of G/SWCNT-Fe(III)P-
Fig. 4. Electrocatalytic reduction of 10 mM H2 O2 (A) and 10 mM NO2 − (B) at G/SWCNT-Fe(III)P-Nafion (. . .) and G/SWCNT-OH-Fe(III)P-Nafion (– – –) electrodes. Experimental conditions: starting potential, 0 V vs. Ag/AgCl, KClsat ; potential scan rate, 20 mV s−1 ; supporting electrolyte, 0.1 M phosphate buffer (pH 7); N2 saturated solution. For comparison the voltammetric response of G/SWCNT-OH-Fe(III)P-Nafion (—) electrode in buffer solution was included. The dependence of the corrected current difference on the H2 O2 (A) and NO2 − (B) concentration for G/SWCNT-Fe(III)P-Nafion (䊉), G/SWCNT-OH-Fe(III)P-Nafion () and G/Fe(III)P-Nafion () electrodes is presented in the insets.
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Nafion electrodes [SSWCNT-Fe(III)P ] was higher than that observed for G/SWCNT-OH-Fe(III)P-Nafion electrodes [SSWCNT-OH-Fe(III)P ]. Thus, the following sequence of sensitivity decreasing was obtained: SSWCNT-Fe(III)P = 3.54 mA/M > SFe(III)P = 1.44 mA/M > SSWCNT-OH-Fe(III)P = 0.81 mA/M. 4. Conclusions This study provides a new example of hemin immobilization on simple and –OH functionalized SWCNTs incorporated within a Nafion matrix, which was deposited on a graphite electrode by a simple and reproducible drop-coating method. As shown by potential scan rate and pH experiments, the immobilized hemin retain its normal voltammetric response, corresponding to a quasi-reversible, 1e− /1H+ process in both investigated matrices. Greater reversibility was noticed in the case of –OH functionalized SWCNT, expressed as a significant increase in the rate constant for the heterogeneous electron transfer process and by a slight decrease in the peak potential split. For the first time, to our knowledge, the electrocatalytic activities of the hemin-SWCNT- and hemin-SWCNT-OH-modified graphite electrodes towards H2 O2 and nitrite electroreduction, were quantitatively evaluated. It was found that only for the H2 O2 electrocatalytic reduction was the presence of SWCNTOH beneficial for the hemin catalytic activity. Thus, the use of –OH functionalized SWCNTs as electrode material opens a simple and elegant way to construct new amperometric biosensors for detection the small signal of molecules and to investigate the direct electron transfer for redox proteins incorporating a hemin-like active center (as for example, b-cytochromes, peroxidases, myoglobin and hemoglobin).
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Acknowledgements G.L.T. acknowledges the financial support for the postdoctoral fellowship from NATO-CNR Italy. The work was supported by Grants Program A-55/375/2004, A-85/375/2005 from CNCSIS—Romanian Ministry of National Education. A special thanks to Adriana Coppe (University “La Sapienza”) for the preparation of the –OH functionalised SWCNT. A.C. and G.P. thank the Consiglio Nazionale delle Ricerche (C.N.R.) Target Project MADESS II subproject: sensors and the MIUR project FIRB 2001 for financial support.
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