J. Phys. Chem. C 2008, 112, 17883–17888
17883
Integrated Electrically Contacted Glucose Oxidase/Carbon Nanotube Electrodes for the Bioelectrocatalyzed Detection of Glucose Yi-Ming Yan, Ilina Baravik, Omer Yehezkeli, and Itamar Willner* Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel ReceiVed: June 26, 2008; ReVised Manuscript ReceiVed: August 27, 2008
Carbon nanotubes (CNTs) are coated with polyethylene imine (PEI) or poly(acrylic acid) (PAA). The polymerfunctionalized CNTs were deposited on glassy carbon electrodes and provided a support for the covalent binding of ferrocene derivatives 1 or 2 on the PEI or PAA polymers, respectively, and for the covalent association of the enzyme, glucose oxidase, on the polymers. The resulting integrated CNTs/ferrocene/enzyme assemblies exhibited electrical contact with the glassy carbon electrode. The systems were implemented for the bioelectrocatalyzed oxidation of glucose and for the development of amperometric glucose sensors. We demonstrate that the CNTs contribute to the effective bioelectrocatalyzed oxidation of glucose by the CNTs/ ferrocene/enzyme systems by providing high surface areas for the immobilization of the relay/enzyme unit and by providing enhanced charge transport between the enzyme redox center and the electrode that improve the electrical contacting efficiency. 1. Introduction The electrical contacting of redox enzymes with electrodes is one of the most challenging topics in bioelectrochemistry.1 Numerous methods to electrically contact redox enzymes with electrodes were reported, and these included the immobilization of the biocatalysts in redox-active polymer matrices2 or the tethering of redox-active relay units to enzyme thin-film assemblies associated with electrodes.3 The effectiveness of electrical contacting between the redox sites of the enzymes and the electrodes in these systems is, however, limited due to slow charge transport across the polymer/protein matrices and due to nonoptimal orientation of the enzyme redox centers in respect to the electrical contacting units and conducting supports. The reconstitution paradigm4 provides a versatile approach to structurally align the redox proteins in respect to the electrode. Indeed, effective electrical contacting of redox enzymes was accomplished by the reconstitution of enzymes on relay cofactor units,5 metal nanoparticle/cofactor,6 or carbon nanotube/cofactor7 monolayer-functionalized electrodes. For the generation of high current densities, the integration of the electrically contacted redox enzymes in high-surface-area thin film assemblies is essential. Carbon nanotubes (CNTs) provide a conducting, high surface area matrix, which could act as support for the electrical contacting of redox protein with electrodes. Different electrically contacted, enzyme-CNT hybrid configurations were assembled on electrodes. These include the reconstitution of enzymes on cofactor units tethered to the ends of cofactor-functionalized CNTs7 and the functionalization of the side walls of the CNTs with redox enzymes.8 For effective use of CNTs as supporting matrix for the immobilization of proteins, their solubilization in aqueous media is essential. The surface functionalization of CNTs with ionic or hydrophilic groups9 or the functionalization of CNTs with water-soluble polymers10 were used to enhance the solubility of CNTs in aqueous media. In the present study, we modify CNTs with the water soluble polymer polyethyleneimine, PEI, or poly(acrylic acid), PAA. The water-soluble * To whom correspondence should be addressed. E-mail: willnea@ vms.huji.ac.il. Phone: 972-2-6585272. Fax: 972-2-6527715.
CNTs include high densities of surface functionalities for the covalent immobilization of electron transfer mediating units (ferrocene) and for the attachment of the enzyme glucose oxidase. The enzyme-functionalized CNTs hybrid systems associated with the electrode provide effective charge transport matrices, high surface areas, and efficient electrical contacting features. 2. Experimental Section Chemicals. Single-walled CNTs (SWCNTs) with an average diameter of about 2 nm and a length of about 50 µm were purchased from Nanoport Co. Ltd. (Shenzhen, China). The SWCNT were purified by refluxing in 2.6 M HNO3 for 10 h, followed by their precipitation and rinsing with water. Glucose oxidase (GOx, E.C. 1.1.3.4 from Aspergillus niger, 210,000 U/g) was purchased from Sigma and used without further purification. N-(Ferrocenylmethyl) aminohexanoic acid (1) and methyloxyethylamine ferrocene (2) were synthesized as reported.11 PEI (MW ca. 25000) and PAA (MW ca. 30000, 40% in water) were purchased from Aldrich. All other chemicals, including 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), glutaric dialdehyde, β-D-(+)-glucose, and ethanol were purchased from Sigma and Aldrich and used as supplied. Ultrapure water from a Nanopure (Barnstead) source was used throughout the work. Modification of Electrodes. Glassy carbon electrodes (GC, 3-mm diameter) were purchased from Bioanalytical Systems Inc. (BAS, West Lafayette, IN). The electrodes were first polished with emery paper (#2000), 0.3 and 0.05 µm alumina slurry on a polishing cloth, then cleaned in a sonication bath for 10 min, and finally thoroughly rinsed with distilled water. 1.0 mg SWCNTs were dispersed in 1.0 mL of PAA (4% in water) or 1.0 mL of PEI (60 µg/mL) to yield a stable suspension. The suspensions were sonicated for 30 min and then centrifuged for separation. The separated SWCNTs were washed with water three times to remove any loosely adsorbed polymers on the surface. The resulting, polymer-wrapped, SWCNTs exhibited high solubility in aqueous solution. A 2.0 µL of the polymer-
10.1021/jp805637e CCC: $40.75 2008 American Chemical Society Published on Web 10/25/2008
17884 J. Phys. Chem. C, Vol. 112, No. 46, 2008 modified SWCNTs aqueous solution was deposited onto the GC electrodes to generate the SWCNT-polymer-modified GC electrodes. After drying in air, the GC-CNTs/PEI and GC-CNTs/ PAA electrodes were reacted with 50 mM N-(ferrocenylmethyl) aminohexanoic acid (1) or 50 mM Methyl-oxyethylamine ferrocene (2) solutions, respectively, in 0.1 M HEPES-buffer, pH ) 7.4, in the presence of 5 mM EDC for 2.0 h. Then, the electrodes were coated with 2 µL of 5 mg mL-1 GOx solution in 0.1 M phosphate buffer, pH ) 7.4 for 1.0 h. After washing, the electrodes were reacted with 10% (v/v) glutaric dialdehyde in 0.1 M phosphate buffer, pH ) 7.4, for 2 h at 4 °C. The resulting, cross-linked, integrated electrodes were used for the biocatalytic oxidation and detection of glucose. Electrochemical and Spectroscopic Measurements. A conventional three-electrode cell, consisting of the enzymeintegrated SWCNT-modified GC working electrode, a glassy carbon auxiliary electrode isolated by a glass frit, and a saturated calomel reference electrode (SCE) electrode connected to the working volume with a Luggin capillary, was used for the electrochemical measurements. All potentials are reported with respect to the SCE. Argon bubbling was used to remove oxygen from the solutions in the electrochemical cell. The cell was placed in a grounded Faraday cage. Cyclic voltammetry and impedance spectra were recorded using an Autolab electrochemical system (ECO Chemie, Netherland). Faradaic impedance measurements were recorded in the frequency range of 100 mHz to 10 kHz. The absorbance measurements were performed using a Shimadzu UV-2401PC UV-vis spectrophotometer. All measurements were carried out at room temperature. 3. Results and Discussion SWCNTs were modified with PEI. A thermogravimetric analysis (TGA) revealed that the coverage of the CNTs with PEI corresponded to 0.19 mg per 1.0 mg of CNTs. The resulting water-soluble CNTs were deposited on GC electrodes, and N-(ferrocenylmethyl)aminohexanoic acid (1) was covalently linked to the amino functionalities of the polymer coating, Scheme 1A. GOx was then deposited on the electrode surface, and the resulting film was cross-linked with glutaric dialdehyde to yield the integrated biocatalytic film on the electrode surface. After rinsing of the electrode, the enzyme layer deposited on the electrode is removable only by physical polishing of the electrode. The ferrocene units associated with the carbon nanotubes revealed a quasireversible redox wave at 0.32 V vs. SCE. The peak currents of the ferrocene units showed a linear dependence with scan rate, implying that the redox units are surface-confined. Coulometric analysis of the redox wave indicates a surface coverage of 7.1 × 10-10 moles · cm-2 of the ferrocene units (geometrical area of 0.07 cm-2). The surface coverage of the enzyme on the electrode was determined by assaying the enzyme activity of GOx associated with the electrode (see Supporting Information). The surface coverage was estimated to be 1.4 × 10-10 moles · cm-2. This value should be compared to the typical monolayer coverage of GOx on Au electrodes, 2 × 10-12 moles · cm-2, indicating that the procedure to assemble the enzyme electrodes leads, indeed, to a substantially higher surface coverage of the biocatalyst. Figure 1A shows the cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by the integrated GOx/Fc/ CNTs electrode. As the concentration of glucose increases, the electrocatalytic anodic currents are intensified. The saturation current, 20.8 µA, is observed at glucose concentrations higher than 80 mM. Knowing the surface coverage of the enzyme, and the saturation current, we estimated the turnover rate of electrons
Yan et al. between the redox centers of the enzyme and the electrode to be ca. 109.6 electrons s-1. Figure 1B depicts the derived calibration curve for analyzing glucose by the modified electrode. Control experiments revealed that the ferrocene units were essential to mediate the bioelectrocatalytic oxidation of glucose, and the immobilization of GOx on the PEI-modified CNTs electrode (without the ferrocene units) did not lead to any electrocatalytic anodic currents. To understand the function of the carbon nanotubes in the ferrocene-mediated bioelectrocatalyzed oxidation of glucose several complementary experiments were performed. The PEI was deposited directly on the GC electrode in the absence of the CNTs, and the ferrocene (1) and GOx were covalently tethered to the PEI, similar to the procedure used for the assembly of the enzyme/Fc/CNTs integrated electrode. Figure 2 depicts the cyclic voltammograms of the resulting integrated Fc/GOx electrode in the absence of glucose, curve a, and in the presence of glucose, curve b. A low-intensity electrocatalytic current is observed. Comparison of the magnitudes of the electrocatalytic anodic currents upon oxidation of glucose, 60 mM, by the GC-CNTs/PEI-Fc-GOx electrode and by the GCPEI-Fc-GOx reveals that the electrocatalytic current in the presence of the GC-CNTs/PEI-Fc-GOx electrode is ca. 50-fold higher. The active surface areas of the GC-CNTs/PEI elelctrode and that of the GC-PEI electrode were determined by recording the cyclic voltammograms of Fe(CN)63- at the presence of the two electrodes (see Figure S1 of Supporting Information). The surface area of the GC-CNTs/PEI corresponds to 0.27 cm2, which is 3.7-fold higher than the surface area of the GC-PEI electrode. The increased surface area of the GC-CNTs/PEI electrode allows, certainly, to immobilize a higher content of the ferrocene electron mediator units as well as the enzyme units. Coulometric analysis of the cyclic voltammogram of the Fc units linked to the GC-PEI electrode indicates a surface coverage of 1.6 × 10-10 moles · cm-2, a value that is ca. 4.5-fold lower than the surface coverage in the GC-CNTs/PEI system. Similarly, analysis of the activity of the enzyme linked to the GC-PEI-Fc system indicates that the content of the enzyme associated with the electrode is ca. 2.1 × 10-11 mol · cm-2, a value that is ca. 6.7-fold lower than the content of GOx associated with the GCCNTs/PEI-Fc-GOx system (for the determination of the loading of GOx on the GC electrode, see Figures S2-S4 of Supporting Information). Thus, we conclude that the coating of the CNTs with polyethyleneimine, PEI, provides a high surface area matrix for the immobilization of the electron-mediator (ferrocene) and enzyme (GOx). The association of the CNTs/PEI on the GC surface allows a ca. 7.0-fold increased content of the bioelectrocatalytically active ingredient on the electrode. Albeit the higher content of the Fc/GOx units on the GC-CNTs/PEI-FcGOx electrode contributes to the higher electrocatalytic anodic currents, as compared to the GC-PEI-Fc-GOx electrode, it is clear that this effect cannot be the only parameter that results in the very high electrocatalytic current in the GC-CNTs/PEIFc-GOx system. We observe a 50-fold higher electrocatalytic current in the GC-CNTs/PEI-Fc-GOx system as compared to the GC-PEI-Fc-GOx electrode, while the content of the bioelectrocatalytic components in the two systems differs by a factor of 7.0. We speculated that the high-surface area conductive array of CNTs might improve the charge transport at the GC-polymer interface, thereby enhancing the mediated electron transfer between the enzyme redox center and the electrode. The electron transfer resistances at electrode surfaces can be monitored by
Electrically Contacted Glucose Oxidase/CNT Electrodes
J. Phys. Chem. C, Vol. 112, No. 46, 2008 17885
SCHEME 1: (A) Assembly of the Integrated, Electrically Contacted, GC-CNTs/PEI-Fc-GOx Electrode; (B) Assembly of the Integrated, Electrically Contacted, GC-CNTs/PAA-Fc-GOx Electrode
impedance spectroscopy (IS).12 We thus probed the interfacial electron transfer resistances of different configurations of the GC electrode in the presence or absence of the CNTs. Curve a of Figure 3A shows the impedance spectrum of the GC-CNTs/ PEI-Fc-modified electrode (in the form of a Nyquist plot). A very small interfacial electron transfer resistance (diameter of the semicircle domain) corresponding to 280 Ω is observed. In turn, curve b of Figure 3A depicts the impedance spectrum of the GC-PEI-Fc-modified electrode. A substantially higher interfacial electron transfer resistance that corresponds to 1300 Ω is observed. Similarly, curve a of Figure 3B shows the impedance spectrum corresponding to the GC-CNTs/PEI-FcGOx electrode upon the bioelectrocatalyzed oxidation of glucose. The interfacial electron transfer resistance corresponds to 450 Ω. Curve b of Figure 3B depicts the impedance spectrum that corresponds to the GC-PEI-Fc-GOx electrode upon the bioelectrocatalytic oxidation of glucose. The interfacial electron
transfer resistance for the activation of the bioelectrocatalytic process is substantially higher and corresponds to 1700 Ω. Thus, we conclude that the charge transport at the CNTs-PEIfunctionalized electrode is substantially enhanced as compared to the GC-PEI-modified electrode that lacks the CNTs. The enhanced charge transport properties in the CNTs/PEI film facilitate the bioelectrocatalyzed oxidation of glucose, and this yields higher electrocatalytic currents. In fact, previous studies13 have similarly indicated that CNTs incorporated in polyaniline films facilitate the charge transport through the polymer film. A similar approach was implemented to coat the carbon nanotubes with the negatively charged PAA polymer. TGA indicated a PAA coverage of 0.16 mg per 1.0 mg of CNTs. The resulting CNTs/PAA composite was then used to assemble the integrated electrically contacted glucose oxidase electrode, Scheme 1B. The CNTs coated with PAA were deposited onto the GC electrode, and ferrocene methyl-oxyethylamine, 2, was
17886 J. Phys. Chem. C, Vol. 112, No. 46, 2008
Figure 1. (A) Cyclic voltammograms corresponding to the integrated, electrically contacted, GC-CNTs/PEI-Fc-GOx electrode in the presence of different concentration of glucose: (a) 0, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50, (g) 60, (h) 70, and (i) 80 mM. Data were recorded in 0.10 M phosphate buffer, pH 7.4, under Ar at room temperature, potential scan rate 10 mV s-1. (B) Calibration curve corresponding to the electrocatalytic currents measured at E ) 0.35 V at variable concentrations of glucose.
Figure 2. Cyclic voltammograms corresponding to the integrated, electrically contacted, GC/PEI-Fc-GOx electrode in the absence of glucose, (a), and the presence of glucose, 60 mM, (b). Data were recorded in 0.10 M phosphate buffer, pH 7.4, under Ar at room temperature. Potential scan rate was 10 mV s-1.
covalently linked to the carboxylic acid functionalities associated with the polymer coating. The biocatalyst, GOx, was subsequently deposited on the electrode and cross linked with glutaric dialdehyde to yield the integrated enzyme electrode, GC-CNTs/
Yan et al.
Figure 3. (A) Nyquist plots (Zim vs Zre) corresponding to the impedance spectra of (a) the GC-CNTs/PEI-Fc electrode and (b) the GC/PEI-Fc electrode. (B) Nyquist plots (Zim vs Zre) corresponding to the impedance spectra of (a) the GC-CNTs/PEI-Fc-GOx electrode, and (b) the GC/ PEI-Fc-GOx electrode, in the presence of 60 mM glucose in the solution. The impedance measurements were performed at a potential of 0.32 V vs Ag/AgCl, using an alternating voltage of 10 mV in the frequency range of 100 mHz to 50 kHz.
PAA-Fc-GOx. The peak current of the ferrocene groups revealed linear dependence as a function of scan rate, indicating that the ferrocene groups are, indeed, surface-confined. Coulometric analysis of the ferrocene redox wave indicated a surface coverage of 1.8 × 10-9 mol cm-2. The assay of the enzyme activity revealed a loading of the electrode that corresponded to 7.5 × 10-7 gr, a value that translates to a coverage of 7.7 × 10-11 mol cm-2 (see Supporting Information). Figure 4A shows the cyclic voltammograms of the modified electrode upon the bioelectrocatalyzed oxidation of different concentrations of glucose. Electrocatalytic anodic currents are observed at the redox potential of the ferrocene units, implying that the ferrocene units mediate the bioelectrocatalyzed oxidation of glucose. Indeed, the deposition of GOx on the CNTs/PAA in the absence of the ferrocene units did not yield any electrocatalytic currents. Figure 4B shows the derived calibration curve. Knowing the saturation current and the surface coverage of GOx on the electrode, the turnover rate of electrons between the redox center and the enzyme is estimated to be 50.6 electrons s-1. To characterize the functions of the CNTs in the bioelectrocatalyzed oxidation of glucose, we examined the bioelectrocatalytic oxidation of glucose by the deposition of PAA directly onto the GC electrode and the assembly of the integrated GCPAA-Fc-GOx electrode that lacks the CNTs. Figure 5 shows the cyclic voltammograms of the integrated GC-PAA-Fc-GOx
Electrically Contacted Glucose Oxidase/CNT Electrodes
Figure 4. (A) Cyclic voltammograms corresponding to the integrated, electrically contacted, GC-CNTs/PAA-Fc-GOx electrode in the presence of different concentration of glucose: (a) 0, (b) 10, (c) 20, (d) 30, (e) 40, (f) 50, (g) 60, (h) 70, and (i) 80 mM. Data were recorded in 0.10 M phosphate buffer, pH 7.4, under Ar at room temperature, with a potential scan rate of 10 mV s-1. (B) Calibration curve corresponding to the electrocatalytic currents measured at E ) 0.40 V at variable concentrations of glucose.
Figure 5. Cyclic voltammograms corresponding to the integrated, electrically contacted, GC/PAA-Fc-GOx electrode in the absence of glucose, (a), and the presence of glucose, 60 mM, (b). Data were recorded in 0.10 M phosphate buffer, pH 7.4, under Ar at room temperature, with a potential scan rate of 10 mV s-1.
electrode in the absence of glucose, curve a, and in the presence of glucose, 60 mM, curve b. A low magnitude electrocatalytic anodic current of ca. 0.15 µA at 0.35 V vs. SCE is observed.
J. Phys. Chem. C, Vol. 112, No. 46, 2008 17887
Figure 6. (A) Nyquist plots (Zim vs Zre) corresponding to the impedance spectra of (a) the GC-CNTs/PAA-Fc electrode and (b) the GC/PAAFc electrode. (B) Nyquist plots (Zim vs Zre) corresponding to the impedance spectra of (a) the GC-CNTs/PAA-Fc-GOx electrode and (b) the GC/PAA-Fc-GOx electrode, in the presence of 60 mM glucose in the solution. The impedance measurements were performed at a potential of 0.32 V vs Ag/AgCl, using an alternating voltage of 10 mV in the frequency range of 100 mHz to 50 kHz.
This value is ca. 18-fold lower than the electrocatalytic anodic current generated by the integrated GC-CNTs/PAA-Fc-GOx electrode. The lower electrocatalytic anodic currents generated by the GC-PAA-Fc-GOx are attributed, partially, to the lower coverage of the electron mediator and enzyme on the GC-PAAFc-GOx electrode. Coulometric analysis of the ferrocene units associated with PAA indicates a surface coverage of 2.1 × 10-10 mol cm-2, and assaying the activity of the enzyme linked to the PAA film reveals a loading of ca. 9.3 × 10-12 mol cm-2 (see Supporting Information). Thus, the loading of the electron mediator/enzyme components in the GC-PAA-Fc-GOx is ca. 8.0-fold lower than in the GC-CNTs/PAA-Fc-GOX system. Accordingly, the difference in the loading of Fc/enzyme cannot account, as a sole parameter, for the substantially higher current observed in the GC-CNTs/PAA-Fc-GOx system. As described before, we examined the electron transfer features at the different electrodes using impedance spectroscopy. Figure 6A shows the impedance spectrum of the GC-CNTs/PAA-Fc electrode, curve a, and the impedance spectrum of the Gc-PAA-Fc electrode that lacks the CNTs. While the interfacial electron transfer resistance of the GC-CNTs/PAA-Fc electrode is low, Ret ) 650 Ω, the interfacial electron transfer resistance of the GC-PAA-Fc electrode is substantially higher, Ret ) 2500 Ω. Similarly, Figure 6B depicts the impedance spectrum of the GC-CNTs/PAA-FcGOx electrode upon the bioelectrocatalyzed oxidation of
17888 J. Phys. Chem. C, Vol. 112, No. 46, 2008 glucose, curve a, and the impedance spectrum corresponding to the bioelectrocatalyzed oxidation of glucose by the GC-PAAFc-GOx electrode. The electron transfer resistance upon the bioelectrocatalyzed oxidation of glucose by the GC-CNTs/PAAFc-GOx is 800 Ω, and the interfacial electron transfer resistance increases to 3000 Ω for the bioelectrocatalyzed oxidation of glucose by the GC-PAA-Fc-GOx electrode. These results indicate that the CNTs also improve the electron transfer at the electrode surface, leading to the enhanced bioelectrocatalyzed oxidation of glucose. 4. Conclusions In conclusion, the present study has demonstrated the coating of CNTs with polyelectrolytes (PEI or PAA) and the use of the CNTs/polymer composites as matrices for the assembly of integrated, electrically contacted, electron-mediator/enzyme electrodes. The CNTs reveal two complementary functions in enhancing the resulting bioelectrocatalytic oxidation of glucose: (i) They provide a high surface area support for the immobilization of the electron relay/enzyme components. (ii) They facilitate electron transfer at the electrode surface, thus enhancing the electron transfer between the enzyme redox center and the electrode and the overall bioelectrocatalytic process. We found that the electrodes were stable, and the polymers were not removed from the CNTs within a time interval of 10 days. The bioelectrocatalytic activities of the polymer CNTs-functionalized electrodes lost
