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Electrodes modified with a siloxane copolymer containing interacting ferrocenes for determination of hydrogen peroxide and glucose
Sensors and Actuators B 88 (2003) 190–197
Electrodes modified with a siloxane copolymer containing interacting ferrocenes for determination of hydrogen peroxide and glucose M. Pilar Garcı´a Armadaa,*, Jose´ Losadaa, Isabel Cuadradob, Beatriz Alonsob, Blanca Gonza´lezb, Eulalia Ramı´rez-Olivab, Carmen M. Casadob a
Departamento de Ingenierı´a Quı´mica Industrial, Escuela Te´cnica Superior de Ingenieros Industriales, Universidad Polite´cnica de Madrid, 28006 Madrid, Spain b Departamento de Quı´mica Inorga´nica, Facultad de Ciencias, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 25 July 2002; received in revised form 22 September 2002; accepted 2 October 2002
Abstract The electrochemical characterization of a hydrogen peroxide sensor based on a ferrocene-containing polymer electrochemically deposited onto a platinum electrode is described. The redox polymer consists of a siloxane-based copolymer, with pendant electronically communicated ferrocenyl moieties. Amperometric biosensors for glucose were prepared by immobilization of glucose oxidase (Gox) onto these modified electrodes. The effects of substrate concentration, the thickness of the polymer layer, interferences and storage on the response of the sensors were investigated. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Sensor; Glucose; Ferrocene; Electronic communication; Modified electrodes; Hydrogen peroxide
1. Introduction The determination of hydrogen peroxide is of practical importance in clinical and environmental fields. Otherwise, the measurement of hydrogen peroxide is necessary for the development of many oxidase-based enzyme sensors [1]. A commonly used experimental principle is the electrochemical detection of hydrogen peroxide by its oxidation at a variety of electrodes. However, the major limitation of these techniques is the high operating potential required for detecting H2O2, which makes these devices susceptible to interfering substances [2]. In order to overcome these problems a number of approaches have been proposed. One of the methods employed in order to minimize the interferences and improve the sensitivity of electrochemical transduction is the use of mediators which are able to decrease the overvoltage of the redox reaction of hydrogen peroxide [3,4].
*
Corresponding author. Tel.: þ34-91-336-31-85; fax: þ34-91-336-30-09. E-mail address: [email protected] (M.P. Garcı´a Armada).
The redox polymer poly(vinylferricinium) has been reported as a useful electrocatalyst for H2O2 electrooxidation. Ferrocenium centers act as electron transfer mediators for the oxidation of the hydrogen peroxide, present in solution or formed as a consequence of an enzyme-catalyzed reaction [5]. Ferrocene and its derivatives have been shown to be used successfully as mediators in various enzyme electrodes [6,7]. Different methods have been studied for the immobilization of ferrocenes on electrode surfaces to produce chemically modified electrodes. Covalent binding to platinum or carbon is frequently used. In other approaches, the ferrocene moieties were part of a polymer or dendrimer backbone [8–12] and these materials were deposited onto a surface or incorporated to a carbon paste electrode. Thus, several research groups have investigated polymeric materials such as poly(pyrrole), poly(vinilpyridine), poly(siloxane) and poly(ethylene oxide) [13]. In the last years, we have studied redox-active organometallic dendritic and polymeric materials containing ferrocenyl moieties together with linear and cyclic siloxanes, cubic silsesquioxanes and silicon- and nitrogen-based dendrimers as frameworks [14,15]. In these multimetallic systems, the
0925-4005/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 2 ) 0 0 3 2 6 - X
M.P. Garcı´a Armada et al. / Sensors and Actuators B 88 (2003) 190–197
191
Scheme 1.
ferrocenyl moieties behave as independent, electronically isolated units, and the macromolecules undergo a simultaneous multielectron transfer at the same potential. In a recent paper, the synthesis and redox properties of novel ferrocene-based materials containing dendritic building-blocks with interacting ferrocenyl moieties grafted to multifunctional, flexible poly(methylsiloxane) backbones have been reported [16]. The combination of the architectural features of dendritic molecules together with the electronic communication between metal centers and the well-known, remarkable features and properties of poly(siloxanes) (such as chemical stability and low toxicity) is a promising strategy for controlling the physical and redox properties of the resulting hybrid macromolecules. In this study, we report the use of platinum electrodes modified with a siloxane-based copolymer (Scheme 1), containing pendant dendritic wedges possessing electronically communicated ferrocenyl moieties, for the enhanced electrocatalytic detection of hydrogen peroxide and the development of glucose biosensors based on glucose oxidase (Gox) immobilized onto these ferrocene modified electrodes.
2. Experimental 2.1. Reagents The polymer was synthesized according to the procedure described earlier [16]. Gox from Aspergillus niger (type VII, 185,000 u g1), bovine serum albumin (BSA), glutaraldehyde (25 wt.% solution in water) and glucose were supplied from Sigma. A solution of Nafion (5 wt.%) in mixed alcohols and water was obtained from Aldrich. Glucose solutions were allowed to reach mutarotational equilibrium at room temperature for 24 h before use. All other chemicals were of analytical grade and were used without further purification. All solutions were prepared with doubly distilled water.
2.2. Apparatus Electrochemical measurements were performed using an Ecochemie BV Autolab PGSTAT 12. All experiments were carried out in a conventional three-electrode cell at 20– 21 8C. A Pt disk of 3 mm diameter served as working electrode, a Pt wire as auxiliary electrode, and a saturated Calomel reference electrode (SCE) was employed. In stationary measurements, a Metrohm 628-10 rotating electrode was used. All amperometric measurements were performed in 0.1 M phosphate buffer with 0.1 M KCl (pH 7.0). In hydrogen peroxide determination, all solutions were deoxygenated by bubbling high-purity nitrogen for at least 15 min. The solutions for glucose measurements were air saturated. The background current was allowed to decay to a steady value before aliquots of stock hydrogen peroxide or glucose solution were added. 2.3. Electrode preparation The Pt disk electrode was polished using 0.1 mm alumina powder and rinsed with water in an ultrasonic bath. The electrode surface was then conditioned by cycling the potential between the limits for hydrogen and oxygen evolution in 0.5 M H2SO4 solution until well-defined cyclic voltammograms were obtained, and then rinsed with water. The polymer film was deposited on the above prepared surface from an electrolyte bath containing 1 mM in the redox-active species (ferrocene) and 0.1 M tetrabutylammonium hexafluorophosphate in dichloromethane, which was deaerated with nitrogen prior to electrooxidation, under potentiostatic control at a potential of þ0.9 V (versus SCE). The coated electrodes were rinsed with dichloromethane. Glucose sensors were constructed by cross-linking using a bifunctional group. The immobilization was carried out by combining 5 ml of glucose oxidase enzyme solution (10 mg ml1) and 5 ml of BSA solution, both in 0.1 M phosphate buffer (pH ¼ 7:0), and 5 ml of 25% glutaraldehyde
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[17], and applying 5 ml of this mixture to the polymer modified electrode surface. The prepared enzyme electrode was allowed to dry in air, and rinsed thoroughly with the buffer. For the construction of Nafion modified glucose electrodes, 5 ml of 1% Nafion solution in methanol was dropped onto Pt surface and allowed to dry in air for 15 min. The polymer and enzyme layers were further deposited in the same way as described above. The polymer modified and enzyme electrodes were stored at room temperature and at 4 8C, respectively, when not in use. The surface coverage of electroactive ferrocenyl sites in the film, G, was determined from the integrated charge of the cyclic voltammetric waves.
3. Results and discussion 3.1. Electrode characterization The cyclic voltammogram of a Pt electrode modified with a film of the copolymer is shown in Fig. 1. In CH2Cl2 solution, two well-defined, separated, reversible one-electron waves are observed, with potential values 1 E1=2 ¼ 0:42 V and 2 E1=2 ¼ 0:57 V [16]. This behavior is consistent with the existence of appreciable interactions between the two iron centers, which are linked together by a bridging silicon atom. The initial oxidation takes place at nonadjacent ferrocene sites, which makes the subsequent removal of electrons from the remaining ferrocenyl centers, adjacent to those already oxidized more difficult. Fig. 2A shows the steady-state cyclic voltammogram attained after successive scans in aqueous phosphate buffer between 0.1 and þ0.8 V potential limits. This voltammogram remains stable and unchanged after several hundreds of additional scans. Both peak currents and peak potentials in aqueous solution are affected regarding those found in
Fig. 2. (A) Steady-state cyclic voltammogram of siloxane-based copolymer platinum electrode in 0.1 deaerated phosphate buffer; scan rate: 50 mV s1. (B) Cyclic voltammograms of (1) platinum bare and (2) siloxane-based copolymer platinum (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) electrodes in presence of 5 mM hydrogen peroxide in deaerated 0.1 M phosphate buffer (pH 7.0). Scan rate: 50 mV s1.
CH2Cl2 solution. The electrochemical reactions of adjacent silicon-bridged ferrocenyl moieties give rise to broad oxidation and reduction redox waves (DEp ¼ 0:15 V) with the onset of the oxidation current occurring in the potential region þ0.2 to þ0.3 V. The electrochemical behavior of electrodes modified with films of this kind of polymers is dependent on the nature and concentration of the counter ions as has been previously reported by different authors for other ferrocenyl polymers [18,19]. When oxidized to the ferricinium state, anions will move to the electrode solution interface to counterbalance the positive charge of the copolymer film, provoking changes in the electroactivity of the ferrocene–ferricinium system, in the coated electrode, upon anion exchange. These changes most likely arise because of the effect of the anions from the phosphate buffer solution incorporated in the polymer structure on the rate of the charge transfer process [20]. 3.2. Hydrogen peroxide measurements
Fig. 1. Voltammetric response of a platinum-disk electrode modified with a film of the poly(ferrocenylsiloxane) copolymer, measured in CH2Cl2/ 0.1 M [Bu4N][PF6]. Scan rate: 100 mV s1.
Fig. 2B displays cyclic voltammograms for 5 mM hydrogen peroxide in aqueous phosphate buffer at bare and copolymer modified platinum electrodes. As can be seen at the modified electrode the oxidation potential of H2O2 is shifted cathodically by 200 mV clearly indicating a lowering of the activation energy for that reaction. The onset of potential of the current for oxidizing hydrogen peroxide
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on the modified electrode coincides with that for the oxidation of the ferrocene groups in the electrodeposited film on the surface electrode, although the magnitude of the anodic current on the copolymer modified electrode was very small in the absence of hydrogen peroxide. Thus, the ferrocenium sites act as mediators for the electrocatalytic oxidation of H2O2 according to H2 O2 þ 2Pol-Fcþ ! O2 þ 2Pol-Fc þ 2Hþ The voltammogram in Fig. 2B also shows an anodic shift of the potential together with an enhancement of the current in the cathodic response of the modified electrode. These results are indicative of electrocatalytic behavior. The electrocatalytic activity observed can be related with the possibility of the modified electrodes to carry out multielectronic transfers. The interaction of a substrate molecule, H2O2, concurrently with two redox groups in the polymer is favored by the cooperative effects of short distance and electronic communication between adjacent ferrocenyl groups within the film. This fact must facilitate the simultaneous transference of the two electrons necessary for the oxidation of H2O2. Fig. 3 compares the amperometric response at various fixed potentials of the modified electrode to successive additions of 0.1 mM H2O2. At applied potentials less than or equal to 200 mV, the cathodic response on H2O2 is detected indicating that electrochemical reduction of hydrogen peroxide takes place. By shifting the applied potential from 200 mV to less positive values, the cathodic current increases. On the other hand, at electrode potentials higher than 300 mV, the anodic response corresponding to the electrooxidation of hydrogen peroxide is observed, which increases on shifting the electrode potential to higher positive values.
Fig. 3. Response of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) to the batch addition of 0.1 mM hydrogen peroxide aliquots at several applied potentials, in deaerated 0.1 phosphate buffer (pH 7.0).
Figs. 4 and 5 present the calibration plots for hydrogen peroxide on the heteropolymer modified electrode at several fixed potential values. These plots indicate linearity up to a concentration of 2 mM.
Fig. 4. Hydrogen peroxide calibration plots from þ300 to þ500 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in 0.1 deaerated phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
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Fig. 5. Hydrogen peroxide calibration plots from þ200 to 50 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in deaerated 0.1 phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
A linear dependence between electrode response and substrate concentration could indicate that the chemical reduction of ferricinium sites in the polymer layer, by H2O2 is the rate-determining step and occurs slower than the electrochemical oxidation of the reduced ferrocene groups [4,21]. The maximal current was obtained at pH ¼ 7:0, however, the heteropolymer-based system exhibited a high activity over the 5–9 pH range. A significant effect of the polymer film thickness on the response of the heteropolymer electrode was detected. Fig. 6 shows the variation of the steady-state current for 1 mM H2O2 solutions at an operating potential of 400 mV.
Maximum response was exhibited with a polymer coverage of G ¼ 1:7 109 mol cm2. For thicker films (surface coverage G > 6:5 109 mol cm2) the response decreases to lower values. The decrease in the catalytic efficiency must be attributed to limitations due to charge propagation throughout the film. After addition of the H2O2 samples the time needed to attain the steady-state current was less than 15 s. The lower detection limits, calculated as twice the standard deviation of the background noise, were 0.1 mM for anodic measurements at 500 mV applied potential and 6.0 mM for cathodic measurements at applied potentials 0.0 and 50 mV.
Fig. 6. Effect of the polymer coverage on the response of the siloxane-based copolymer platinum electrode. Steady-state currents measured at 400 mV vs. SCE in 0.1 deaerated phosphate buffer (pH 7.0). Each value is the mean result for five electrodes.
M.P. Garcı´a Armada et al. / Sensors and Actuators B 88 (2003) 190–197
Fig. 7. Cyclic voltammograms of enzyme sensor (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness heteropolymer film) in absence (1) and presence (2) of 5 mM glucose in air saturated 0.1 M phosphate buffer (pH 7.0). Scan rate: 10 mV s1.
The effect of L-ascorbate, a typical electrochemical interferent in measuring H2O2, was examined. At an applied potential less than or equal to 0.0 V, the addition of 0.1 mM of ascorbic acid (AA) to a 1 mM H2O2 solution causes no noticeable decrease of the reductive current. 3.3. Glucose sensor characterization Fig. 7 shows cyclic voltammograms of the heteropolymer-enzyme electrode in the absence and presence of 5 mM of glucose taken at 10 mV s1 in phosphate buffer pH 7.0. The glucose response of the Pt-heteropolymer-Gox electrode was determined at several applied potentials (Fig. 8). In all cases, the time elapsed to reach the steady-state
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value of the current was less than 25 s. The anodic current increases continuously with increasing applied potentials and the resulting calibration plots indicate linearity up to 2 mM with sensitivities (slopes of the initial linear portions) of 1.21, 0.8 and 0.35 mA mM1 for 500, 400 and 300 mV applied potentials, respectively. The apparent Michaelis–Menten constants, KM, app, determined from the Linweaver–Burke plots are 12.6, 4.5 and 1.6 for applied potentials of 500, 400 and 300 mV, respectively. These results are characteristic of a process in which the rate of the substrate diffusion does not control the overall reaction rate and therefore, the apparent constant is dependent on the rate of electrolysis. In these cases, KM, app is intrinsically smaller than the Michaelis constant in homogeneous enzyme kinetics [10] and the linear range of the calibration curve does not extend beyond the KM values. As the working potential is increased the electrolysis rate becomes much faster and consequently KM, app increases. As seen above for hydrogen peroxide determination, the enzyme-heteropolymer electrode also exhibits cathodic activity. Fig. 9 shows cathodic responses of the enzyme electrode to glucose concentrations depending on operation potential. As expected from the voltammetric profiles improvements in the sensitivity were also observed for the cathodic detection of glucose at 200 and 100 mV, that yielded sensitivities of 34.4 and 217 nA mM1, respectively. However, the use of the cathodic region is limited by the cathodic reduction of molecular oxygen, which occurs interfering the glucose measurements at potentials lower than 100 mV. Therefore, the potential value of 100 mV should be considered as the lowest potential suitable for cathodic determination of glucose. In this case, the lower detection limits were 3.0 mM for anodic measurements at 500 and 400 mV applied potentials
Fig. 8. Glucose calibration plots from þ300 to þ500 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in air saturated 0.1 phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
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Fig. 9. Glucose calibration plots for þ200 and þ100 mV vs. SCE applied potential of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in 0.1 air saturated phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
and 23.6 mM for cathodic measurements at 100 mV applied potential. On other hand, in the available potential range, the sensor response was affected by the presence of ascorbic acid, which produced a substantial increase in the steady-state response, due to the direct or catalyzed electrooxidation of ascorbate ions. This interference is usually avoided by covering the electrode surface with Nafion. Since the Gox– BSA–glutaraldehyde polymer is soluble in methanol, we have deposited the electrocatalytic layer upon an electrode previously modified with an additional Nafion layer. Thus, the sensor response was unaffected by the presence of normal physiological ascorbic acid concentrations. In fact, the signal due to the addition of 0.1 mM AA to a 1mM glucose solution was practically cancelled. Comparing the response of a Nafion modified sensor to glucose concentrations, the amperometric signal decreases by ca. 50% related to the Nafion unmodified enzyme electrode. The stability of the glucose sensor was evaluated by repetitive measurements of its response to 5 mM glucose within a period of 12 h. The response remained unchanged during this period and the relative standard deviation of 50 measurements was about 1%. Only a 20% decrease of their initial glucose response was observed for an electrode stored at 4 8C in air for 8 weeks. Interestingly, after cycling between 0.0 and 0.8 V, the initial response was recovered.
4. Conclusions Electrodes modified with a polymer containing electronically communicated ferrocenyl moieties have shown to promote the redox reactions of hydrogen peroxide. The application of these modified electrodes for/in the amperometric determination of hydrogen peroxide has been demonstrated.
The redox polymer co-immobilized with Gox on platinum electrodes using glutaraldehyde and BSA has been used as an amperometric biosensor. The results obtained indicate that both anodic and cathodic operation mode may be used for glucose determinations with the copolymer modified electrode which acts as electrocatalyst in either oxidation or reduction of hydrogen peroxide arisen in the enzymecatalyzed reaction. The sensitivities and detection limits obtained were comparable or even better than other ferrocene-modified polymers mediated electrodes [5,8,12,17].
Acknowledgements The authors thank the Direccio´ n General de Ensen˜ anza Superior e Investigacio´ n Cientı´fica (project no. PB-97-0001) for financial support of this research.
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M.P. Garcı´a Armada et al. / Sensors and Actuators B 88 (2003) 190–197 [7] J.M. Dicks, W.J. Aston, G. Davis, A.P.F. Turner, Mediated amperometric biosensors for the D-galactose, glycolate and L-amino acids based on a ferrocene-modified carbon paste electrode, Anal. Chim. Acta 182 (1986) 103–112. [8] S.P. Hendry, M.F. Cardosi, A.P.F. Turner, E.W. Neuse, Polyferrocenes as mediators in amperometric biosensors for glucose, Anal. Chim. Acta 281 (1993) 453–459. [9] J. Losada, I. Cuadrado, M. Mora´ n, C.M. Casado, B. Alonso, M. Barranco, Ferrocenyl silicon-based dendrimers as mediators in amperometric biosensors, Anal. Chim. Acta 338 (1997) 191– 198. [10] Ch.J. Chen, Ch.C. Liu, R.F. Savinell, Polymeric redox mediator enzyme electrodes for anaerobic glucose monitoring, J. Electroanal. Chem. 348 (1993) 317–338. [11] L. Gorton, Carbon paste electrodes modified with enzymes, tissues, and cells, Electroanalysis 7 (1995) 23–45, and references cited therein. [12] S. Koide, K. Yokoyama, Electrochemical characterization of an enzyme electrode based on a ferrocene-containing redox polymer, J. Electroanal. Chem. 468 (1999) 193–201. [13] P.D. Hale, H.I. Lan, L.I. Boguslavsky, H.I. Karan, Y. Okamoto, T.A. Skotheim, Amperometric glucose sensors based on ferrocenemodified poly(ethylene oxide) and glucose oxidase, Anal. Chim. Acta 251 (1991) 121–128, and references cited therein. [14] C.M. Casado, I. Cuadrado, M. Mora´ n, B. Alonso, B. Garcı´a, B. Gonza´ lez, J. Losada, Redox-active ferrocenyl dendrimers and
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Electrodes modified with a siloxane copolymer containing interacting ferrocenes for determination of hydrogen peroxide and glucose M. Pilar Garcı´a Armadaa,*, Jose´ Losadaa, Isabel Cuadradob, Beatriz Alonsob, Blanca Gonza´lezb, Eulalia Ramı´rez-Olivab, Carmen M. Casadob a
Departamento de Ingenierı´a Quı´mica Industrial, Escuela Te´cnica Superior de Ingenieros Industriales, Universidad Polite´cnica de Madrid, 28006 Madrid, Spain b Departamento de Quı´mica Inorga´nica, Facultad de Ciencias, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received 25 July 2002; received in revised form 22 September 2002; accepted 2 October 2002
Abstract The electrochemical characterization of a hydrogen peroxide sensor based on a ferrocene-containing polymer electrochemically deposited onto a platinum electrode is described. The redox polymer consists of a siloxane-based copolymer, with pendant electronically communicated ferrocenyl moieties. Amperometric biosensors for glucose were prepared by immobilization of glucose oxidase (Gox) onto these modified electrodes. The effects of substrate concentration, the thickness of the polymer layer, interferences and storage on the response of the sensors were investigated. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Sensor; Glucose; Ferrocene; Electronic communication; Modified electrodes; Hydrogen peroxide
1. Introduction The determination of hydrogen peroxide is of practical importance in clinical and environmental fields. Otherwise, the measurement of hydrogen peroxide is necessary for the development of many oxidase-based enzyme sensors [1]. A commonly used experimental principle is the electrochemical detection of hydrogen peroxide by its oxidation at a variety of electrodes. However, the major limitation of these techniques is the high operating potential required for detecting H2O2, which makes these devices susceptible to interfering substances [2]. In order to overcome these problems a number of approaches have been proposed. One of the methods employed in order to minimize the interferences and improve the sensitivity of electrochemical transduction is the use of mediators which are able to decrease the overvoltage of the redox reaction of hydrogen peroxide [3,4].
*
Corresponding author. Tel.: þ34-91-336-31-85; fax: þ34-91-336-30-09. E-mail address: [email protected] (M.P. Garcı´a Armada).
The redox polymer poly(vinylferricinium) has been reported as a useful electrocatalyst for H2O2 electrooxidation. Ferrocenium centers act as electron transfer mediators for the oxidation of the hydrogen peroxide, present in solution or formed as a consequence of an enzyme-catalyzed reaction [5]. Ferrocene and its derivatives have been shown to be used successfully as mediators in various enzyme electrodes [6,7]. Different methods have been studied for the immobilization of ferrocenes on electrode surfaces to produce chemically modified electrodes. Covalent binding to platinum or carbon is frequently used. In other approaches, the ferrocene moieties were part of a polymer or dendrimer backbone [8–12] and these materials were deposited onto a surface or incorporated to a carbon paste electrode. Thus, several research groups have investigated polymeric materials such as poly(pyrrole), poly(vinilpyridine), poly(siloxane) and poly(ethylene oxide) [13]. In the last years, we have studied redox-active organometallic dendritic and polymeric materials containing ferrocenyl moieties together with linear and cyclic siloxanes, cubic silsesquioxanes and silicon- and nitrogen-based dendrimers as frameworks [14,15]. In these multimetallic systems, the
0925-4005/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 2 ) 0 0 3 2 6 - X
M.P. Garcı´a Armada et al. / Sensors and Actuators B 88 (2003) 190–197
191
Scheme 1.
ferrocenyl moieties behave as independent, electronically isolated units, and the macromolecules undergo a simultaneous multielectron transfer at the same potential. In a recent paper, the synthesis and redox properties of novel ferrocene-based materials containing dendritic building-blocks with interacting ferrocenyl moieties grafted to multifunctional, flexible poly(methylsiloxane) backbones have been reported [16]. The combination of the architectural features of dendritic molecules together with the electronic communication between metal centers and the well-known, remarkable features and properties of poly(siloxanes) (such as chemical stability and low toxicity) is a promising strategy for controlling the physical and redox properties of the resulting hybrid macromolecules. In this study, we report the use of platinum electrodes modified with a siloxane-based copolymer (Scheme 1), containing pendant dendritic wedges possessing electronically communicated ferrocenyl moieties, for the enhanced electrocatalytic detection of hydrogen peroxide and the development of glucose biosensors based on glucose oxidase (Gox) immobilized onto these ferrocene modified electrodes.
2. Experimental 2.1. Reagents The polymer was synthesized according to the procedure described earlier [16]. Gox from Aspergillus niger (type VII, 185,000 u g1), bovine serum albumin (BSA), glutaraldehyde (25 wt.% solution in water) and glucose were supplied from Sigma. A solution of Nafion (5 wt.%) in mixed alcohols and water was obtained from Aldrich. Glucose solutions were allowed to reach mutarotational equilibrium at room temperature for 24 h before use. All other chemicals were of analytical grade and were used without further purification. All solutions were prepared with doubly distilled water.
2.2. Apparatus Electrochemical measurements were performed using an Ecochemie BV Autolab PGSTAT 12. All experiments were carried out in a conventional three-electrode cell at 20– 21 8C. A Pt disk of 3 mm diameter served as working electrode, a Pt wire as auxiliary electrode, and a saturated Calomel reference electrode (SCE) was employed. In stationary measurements, a Metrohm 628-10 rotating electrode was used. All amperometric measurements were performed in 0.1 M phosphate buffer with 0.1 M KCl (pH 7.0). In hydrogen peroxide determination, all solutions were deoxygenated by bubbling high-purity nitrogen for at least 15 min. The solutions for glucose measurements were air saturated. The background current was allowed to decay to a steady value before aliquots of stock hydrogen peroxide or glucose solution were added. 2.3. Electrode preparation The Pt disk electrode was polished using 0.1 mm alumina powder and rinsed with water in an ultrasonic bath. The electrode surface was then conditioned by cycling the potential between the limits for hydrogen and oxygen evolution in 0.5 M H2SO4 solution until well-defined cyclic voltammograms were obtained, and then rinsed with water. The polymer film was deposited on the above prepared surface from an electrolyte bath containing 1 mM in the redox-active species (ferrocene) and 0.1 M tetrabutylammonium hexafluorophosphate in dichloromethane, which was deaerated with nitrogen prior to electrooxidation, under potentiostatic control at a potential of þ0.9 V (versus SCE). The coated electrodes were rinsed with dichloromethane. Glucose sensors were constructed by cross-linking using a bifunctional group. The immobilization was carried out by combining 5 ml of glucose oxidase enzyme solution (10 mg ml1) and 5 ml of BSA solution, both in 0.1 M phosphate buffer (pH ¼ 7:0), and 5 ml of 25% glutaraldehyde
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[17], and applying 5 ml of this mixture to the polymer modified electrode surface. The prepared enzyme electrode was allowed to dry in air, and rinsed thoroughly with the buffer. For the construction of Nafion modified glucose electrodes, 5 ml of 1% Nafion solution in methanol was dropped onto Pt surface and allowed to dry in air for 15 min. The polymer and enzyme layers were further deposited in the same way as described above. The polymer modified and enzyme electrodes were stored at room temperature and at 4 8C, respectively, when not in use. The surface coverage of electroactive ferrocenyl sites in the film, G, was determined from the integrated charge of the cyclic voltammetric waves.
3. Results and discussion 3.1. Electrode characterization The cyclic voltammogram of a Pt electrode modified with a film of the copolymer is shown in Fig. 1. In CH2Cl2 solution, two well-defined, separated, reversible one-electron waves are observed, with potential values 1 E1=2 ¼ 0:42 V and 2 E1=2 ¼ 0:57 V [16]. This behavior is consistent with the existence of appreciable interactions between the two iron centers, which are linked together by a bridging silicon atom. The initial oxidation takes place at nonadjacent ferrocene sites, which makes the subsequent removal of electrons from the remaining ferrocenyl centers, adjacent to those already oxidized more difficult. Fig. 2A shows the steady-state cyclic voltammogram attained after successive scans in aqueous phosphate buffer between 0.1 and þ0.8 V potential limits. This voltammogram remains stable and unchanged after several hundreds of additional scans. Both peak currents and peak potentials in aqueous solution are affected regarding those found in
Fig. 2. (A) Steady-state cyclic voltammogram of siloxane-based copolymer platinum electrode in 0.1 deaerated phosphate buffer; scan rate: 50 mV s1. (B) Cyclic voltammograms of (1) platinum bare and (2) siloxane-based copolymer platinum (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) electrodes in presence of 5 mM hydrogen peroxide in deaerated 0.1 M phosphate buffer (pH 7.0). Scan rate: 50 mV s1.
CH2Cl2 solution. The electrochemical reactions of adjacent silicon-bridged ferrocenyl moieties give rise to broad oxidation and reduction redox waves (DEp ¼ 0:15 V) with the onset of the oxidation current occurring in the potential region þ0.2 to þ0.3 V. The electrochemical behavior of electrodes modified with films of this kind of polymers is dependent on the nature and concentration of the counter ions as has been previously reported by different authors for other ferrocenyl polymers [18,19]. When oxidized to the ferricinium state, anions will move to the electrode solution interface to counterbalance the positive charge of the copolymer film, provoking changes in the electroactivity of the ferrocene–ferricinium system, in the coated electrode, upon anion exchange. These changes most likely arise because of the effect of the anions from the phosphate buffer solution incorporated in the polymer structure on the rate of the charge transfer process [20]. 3.2. Hydrogen peroxide measurements
Fig. 1. Voltammetric response of a platinum-disk electrode modified with a film of the poly(ferrocenylsiloxane) copolymer, measured in CH2Cl2/ 0.1 M [Bu4N][PF6]. Scan rate: 100 mV s1.
Fig. 2B displays cyclic voltammograms for 5 mM hydrogen peroxide in aqueous phosphate buffer at bare and copolymer modified platinum electrodes. As can be seen at the modified electrode the oxidation potential of H2O2 is shifted cathodically by 200 mV clearly indicating a lowering of the activation energy for that reaction. The onset of potential of the current for oxidizing hydrogen peroxide
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on the modified electrode coincides with that for the oxidation of the ferrocene groups in the electrodeposited film on the surface electrode, although the magnitude of the anodic current on the copolymer modified electrode was very small in the absence of hydrogen peroxide. Thus, the ferrocenium sites act as mediators for the electrocatalytic oxidation of H2O2 according to H2 O2 þ 2Pol-Fcþ ! O2 þ 2Pol-Fc þ 2Hþ The voltammogram in Fig. 2B also shows an anodic shift of the potential together with an enhancement of the current in the cathodic response of the modified electrode. These results are indicative of electrocatalytic behavior. The electrocatalytic activity observed can be related with the possibility of the modified electrodes to carry out multielectronic transfers. The interaction of a substrate molecule, H2O2, concurrently with two redox groups in the polymer is favored by the cooperative effects of short distance and electronic communication between adjacent ferrocenyl groups within the film. This fact must facilitate the simultaneous transference of the two electrons necessary for the oxidation of H2O2. Fig. 3 compares the amperometric response at various fixed potentials of the modified electrode to successive additions of 0.1 mM H2O2. At applied potentials less than or equal to 200 mV, the cathodic response on H2O2 is detected indicating that electrochemical reduction of hydrogen peroxide takes place. By shifting the applied potential from 200 mV to less positive values, the cathodic current increases. On the other hand, at electrode potentials higher than 300 mV, the anodic response corresponding to the electrooxidation of hydrogen peroxide is observed, which increases on shifting the electrode potential to higher positive values.
Fig. 3. Response of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) to the batch addition of 0.1 mM hydrogen peroxide aliquots at several applied potentials, in deaerated 0.1 phosphate buffer (pH 7.0).
Figs. 4 and 5 present the calibration plots for hydrogen peroxide on the heteropolymer modified electrode at several fixed potential values. These plots indicate linearity up to a concentration of 2 mM.
Fig. 4. Hydrogen peroxide calibration plots from þ300 to þ500 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in 0.1 deaerated phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
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Fig. 5. Hydrogen peroxide calibration plots from þ200 to 50 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in deaerated 0.1 phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
A linear dependence between electrode response and substrate concentration could indicate that the chemical reduction of ferricinium sites in the polymer layer, by H2O2 is the rate-determining step and occurs slower than the electrochemical oxidation of the reduced ferrocene groups [4,21]. The maximal current was obtained at pH ¼ 7:0, however, the heteropolymer-based system exhibited a high activity over the 5–9 pH range. A significant effect of the polymer film thickness on the response of the heteropolymer electrode was detected. Fig. 6 shows the variation of the steady-state current for 1 mM H2O2 solutions at an operating potential of 400 mV.
Maximum response was exhibited with a polymer coverage of G ¼ 1:7 109 mol cm2. For thicker films (surface coverage G > 6:5 109 mol cm2) the response decreases to lower values. The decrease in the catalytic efficiency must be attributed to limitations due to charge propagation throughout the film. After addition of the H2O2 samples the time needed to attain the steady-state current was less than 15 s. The lower detection limits, calculated as twice the standard deviation of the background noise, were 0.1 mM for anodic measurements at 500 mV applied potential and 6.0 mM for cathodic measurements at applied potentials 0.0 and 50 mV.
Fig. 6. Effect of the polymer coverage on the response of the siloxane-based copolymer platinum electrode. Steady-state currents measured at 400 mV vs. SCE in 0.1 deaerated phosphate buffer (pH 7.0). Each value is the mean result for five electrodes.
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Fig. 7. Cyclic voltammograms of enzyme sensor (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness heteropolymer film) in absence (1) and presence (2) of 5 mM glucose in air saturated 0.1 M phosphate buffer (pH 7.0). Scan rate: 10 mV s1.
The effect of L-ascorbate, a typical electrochemical interferent in measuring H2O2, was examined. At an applied potential less than or equal to 0.0 V, the addition of 0.1 mM of ascorbic acid (AA) to a 1 mM H2O2 solution causes no noticeable decrease of the reductive current. 3.3. Glucose sensor characterization Fig. 7 shows cyclic voltammograms of the heteropolymer-enzyme electrode in the absence and presence of 5 mM of glucose taken at 10 mV s1 in phosphate buffer pH 7.0. The glucose response of the Pt-heteropolymer-Gox electrode was determined at several applied potentials (Fig. 8). In all cases, the time elapsed to reach the steady-state
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value of the current was less than 25 s. The anodic current increases continuously with increasing applied potentials and the resulting calibration plots indicate linearity up to 2 mM with sensitivities (slopes of the initial linear portions) of 1.21, 0.8 and 0.35 mA mM1 for 500, 400 and 300 mV applied potentials, respectively. The apparent Michaelis–Menten constants, KM, app, determined from the Linweaver–Burke plots are 12.6, 4.5 and 1.6 for applied potentials of 500, 400 and 300 mV, respectively. These results are characteristic of a process in which the rate of the substrate diffusion does not control the overall reaction rate and therefore, the apparent constant is dependent on the rate of electrolysis. In these cases, KM, app is intrinsically smaller than the Michaelis constant in homogeneous enzyme kinetics [10] and the linear range of the calibration curve does not extend beyond the KM values. As the working potential is increased the electrolysis rate becomes much faster and consequently KM, app increases. As seen above for hydrogen peroxide determination, the enzyme-heteropolymer electrode also exhibits cathodic activity. Fig. 9 shows cathodic responses of the enzyme electrode to glucose concentrations depending on operation potential. As expected from the voltammetric profiles improvements in the sensitivity were also observed for the cathodic detection of glucose at 200 and 100 mV, that yielded sensitivities of 34.4 and 217 nA mM1, respectively. However, the use of the cathodic region is limited by the cathodic reduction of molecular oxygen, which occurs interfering the glucose measurements at potentials lower than 100 mV. Therefore, the potential value of 100 mV should be considered as the lowest potential suitable for cathodic determination of glucose. In this case, the lower detection limits were 3.0 mM for anodic measurements at 500 and 400 mV applied potentials
Fig. 8. Glucose calibration plots from þ300 to þ500 mV vs. SCE of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in air saturated 0.1 phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
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Fig. 9. Glucose calibration plots for þ200 and þ100 mV vs. SCE applied potential of siloxane-based copolymer platinum electrode (Pt electrode area ¼ 0:07 cm2 with 1:7 109 mol cm2 thickness film) in 0.1 air saturated phosphate buffer (pH 7.0). Each curve is the mean result for five electrodes.
and 23.6 mM for cathodic measurements at 100 mV applied potential. On other hand, in the available potential range, the sensor response was affected by the presence of ascorbic acid, which produced a substantial increase in the steady-state response, due to the direct or catalyzed electrooxidation of ascorbate ions. This interference is usually avoided by covering the electrode surface with Nafion. Since the Gox– BSA–glutaraldehyde polymer is soluble in methanol, we have deposited the electrocatalytic layer upon an electrode previously modified with an additional Nafion layer. Thus, the sensor response was unaffected by the presence of normal physiological ascorbic acid concentrations. In fact, the signal due to the addition of 0.1 mM AA to a 1mM glucose solution was practically cancelled. Comparing the response of a Nafion modified sensor to glucose concentrations, the amperometric signal decreases by ca. 50% related to the Nafion unmodified enzyme electrode. The stability of the glucose sensor was evaluated by repetitive measurements of its response to 5 mM glucose within a period of 12 h. The response remained unchanged during this period and the relative standard deviation of 50 measurements was about 1%. Only a 20% decrease of their initial glucose response was observed for an electrode stored at 4 8C in air for 8 weeks. Interestingly, after cycling between 0.0 and 0.8 V, the initial response was recovered.
4. Conclusions Electrodes modified with a polymer containing electronically communicated ferrocenyl moieties have shown to promote the redox reactions of hydrogen peroxide. The application of these modified electrodes for/in the amperometric determination of hydrogen peroxide has been demonstrated.
The redox polymer co-immobilized with Gox on platinum electrodes using glutaraldehyde and BSA has been used as an amperometric biosensor. The results obtained indicate that both anodic and cathodic operation mode may be used for glucose determinations with the copolymer modified electrode which acts as electrocatalyst in either oxidation or reduction of hydrogen peroxide arisen in the enzymecatalyzed reaction. The sensitivities and detection limits obtained were comparable or even better than other ferrocene-modified polymers mediated electrodes [5,8,12,17].
Acknowledgements The authors thank the Direccio´ n General de Ensen˜ anza Superior e Investigacio´ n Cientı´fica (project no. PB-97-0001) for financial support of this research.
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