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Modification of carbon nanotubes with redox hydrogel: Improvement of amperometric sensing sensitivity for redox enzymes
Biosensors and Bioelectronics 24 (2009) 1723–1729
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Modification of carbon nanotubes with redox hydrogel: Improvement of amperometric sensing sensitivity for redox enzymes Hui-Fang Cui a , Jian-Shan Ye b , Wei-De Zhang b , Fwu-Shan Sheu c,d,∗ a
Department of Bioengineering, Zhengzhou University, 100# Science Avenue, Zhengzhou, 450001, PR China School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510641, PR China c Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore d Nanoscience and Nanotechnology Initiative, National University of Singapore, 10 Kent Ridge Crescent, 117576, Singapore b
a r t i c l e
i n f o
Article history: Received 16 June 2008 Received in revised form 1 September 2008 Accepted 1 September 2008 Available online 12 September 2008 Keywords: Redox polymer Carbon nanotubes Redox enzymes Enzyme activity Amperometry Electrochemical sensor
a b s t r a c t This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4 -dimethylbpy)2 Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)6 3− solution and electrochemical impedance measurements in an equimolar Fe(CN)6 3−/4− solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15 . The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Redox enzymes contain redox centers, such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and heme as a prosthetic group, which can be electro-oxidized and electroreduced. For example, glucose oxidase (GOD) is a FAD containing glycoprotein which catalyzes the oxidation of glucose using molecular oxygen with the formation of gluconolactone (further hydrolysed to gluconic acid) and H2 O2 as products. Lactate oxidase (E.C.1.1.3.2) (LOD) is a member of the family of FMN-containing flavoproteins that catalyze the oxidation of lactate using molecular oxygen with the formation of pyruvate and H2 O2 as products (Yorita et al., 1997). Measurement of the activity of redox enzymes is of importance in clinical, pharmaceutical, therapeutic, analytical, chemical, food, and beverage researches and industries.
∗ Corresponding author at: Nanoscience and Nanotechnology Initiative, National University of Singapore, 10 Kent Ridge Crescent, 117576, Singapore. Tel.: +65 6516 2857; fax: +65 6779 2486. E-mail address: [email protected] (F.-S. Sheu).
Most of the assays for enzyme activity and for kinetic studies of enzyme reactions involve the use of auxiliary enzymes to generate easily detectable species such as NAD(P)+ or chromophores which absorb strongly either in ultraviolet or visible regions of spectrophotometers (Karmali et al., 2004; Bergmeyer, 1974). These linked enzyme assays present some disadvantages since the presence of other substances and enzymes in the reaction mixture may affect the kinetic behavior of the enzyme that is being investigated (Karmali et al., 2004). On the other hand, the assay conditions (i.e., temperature, pH, and ionic strength) may not be the same for different enzymes involved in the assay system (Schindler and Lendl, 1999). Therefore, it is of great interests to devise novel enzyme assays that are specific and fast and do not require auxiliary enzymes. Recently, Karmali et al. reported the use of Fourier transform infrared (FTIR) spectroscopy as a simple and direct assay method for the activity of GOD (Karmali et al., 2004). However, this method can only be applied to the enzymes for which the substrate and the product have quite distinguishable frequencies of IR absorbing. In this paper, we report a simple and direct assay method that may be applicable to universal redox enzymes. It uses redox polymer modified carbon nanotube (CNT) electrode to directly detect the activity of redox enzymes. GOD and LOD were
0956-5663/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.09.002
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used as model redox enzymes to investigate and demonstrate this direct assay method. Redox polymers are soluble polymers complexed with redox moieties. When crosslinked on electrodes, the redox polymers become insoluble, but swell in water to form three-dimensional redox hydrogels (Gregg and Heller, 1991). Upon swelling, the mobility of their segments is increased, increasing in the frequency of electron-transferring collisions between the tethered redox centers, and the electronic conductivity of the redox hydrogels. Redox polymers, such as poly(vinylimidazole) (PVI) complexed with [Os(bpy)2 Cl]+ (termed PVI-Os) [bpy: the short name for (2,2 bipyridine)] (Ohara et al., 1993; Alpeeva et al., 2005), or with Os(4,4 -dimethylbpy)2 Cl (termed PVI-dmeOs) (Ohara et al., 1994; Mikeladze et al., 2002) have been immobilized on different kinds of conventional electrodes, such as vitreous carbon, gold, and glassy carbon (GC) electrodes (Ohara et al., 1994, 1993; Gao et al., 2002; Alpeeva et al., 2005; Mikeladze et al., 2002). While the redox potential of PVI-Os is about 200 mV, that of PVI-dmeOs is reduced to 95 mV (versus saturated calomel electrode) (Ohara et al., 1994). When redox polymers are co-immobilized with redox enzymes on electrodes, the redox centers of the enzymes can be “wired” by the redox centers tethered on the mobile redox polymer backbones to the electrodes, therefore can also be electro-oxidized and electroreduced (Ohara et al., 1994, 1993; Gao et al., 2002). Due to its low redox potential, when co-immobilized with enzymes, PVI-dmeOs has been demonstrated to result in low oxidation potentials for redox enzymes (Ohara et al., 1994). CNTs are hollow carbon tubes made of a single or several to tens of concentrically arranged cylindrical graphite layers, capped by fullerenic hemispheres, which are referred to as single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) respectively (Little, 2003). CNTs have become the subject of intense investigations since their first discovery by Iijima in 1991 (Iijima, 1991). Such considerable interests were inspired by their unique structures, remarkable electrical (Merkoci et al., 2005) and mechanical (Treacy et al., 1996) properties, high chemical stability, and potentials in applications in many fields. CNTs, especially well-aligned MWCNTs grown on conductive Ta substrates have been demonstrated very good electrode materials for electrochemical biosensors (Cui et al., 2005, 2006, 2007). Two methods have been used to immobilize and crosslink redox polymers onto electrodes. One is to crosslink the redox polymers with poly-(ethylene glycol) diglycidyl ether, a crosslinking agent (Ohara et al., 1994, 1993). The other one is to electrodeposit redox polymers onto vitreous carbon by coordinative crosslinking (Gao et al., 2002). In this study, the redox polymer PVI-dmeOs was electrodeposited on, the first time, CNTs and the redox hydrogelmodified CNT electrodes (PVI-dmeOs/CNTs) were found exhibiting strong responses to redox enzymes in electrolyte solutions containing the corresponding substrate of the measured redox enzyme. Well-aligned MWCNTs grown on conductive Ta substrates were used as the CNT electrode material for the PVI-dmeOs deposition. The mechanisms of the strong response to redox enzymes at the PVI-dmeOs modified CNT electrodes are explored in this paper.
2. Experimental 2.1. Instrument and reagents The morphologies of modified MWCNTs were observed by scanning electron microscopy (SEM) (JEOL JSM 6700F, operated at 5 kV). Static water contact angles on MWCNTs were measured with VCA Optima surface analysis system (AST Products Inc., USA) using sessile drop method in air atmosphere. The size of the water drop for
the measurement was 1.0 L. Electrochemical measurements were performed using BAS 100B electrochemical analyzer equipped with a BAS 100B cell stand (Bioanalytical Systems Inc., Indiana, USA) in a three-electrode system, including a working electrode (MWCNTs, GC, or PVI-dmeOs/CNTs electrode), a platinum counter electrode and a 3 M KCl–Ag/AgCl reference electrode, at room temperature (≈25 ◦ C). All potentials were quoted versus this reference electrode. Ta plates were obtained from Goodfellow Cambridge Ltd. (Huntingdon, England). All chemicals were purchased from Sigma–Aldrich Co. (Saint Louis, MO, USA) and were used without further purifications. Purified nitrogen gas was purchased from Singapore Oxygen Air Liquid Pte Ltd. Phosphate buffer solution (PBS) contains: 8 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2 HPO4 , and 0.24 g L−1 KH2 PO4 with pH 7.4. Deionized water obtained from a Millipore water system was used throughout the experiment. 2.2. Methods 2.2.1. Synthesis of well-aligned MWCNTs and construction of MWCNT electrodes MWCNTs were synthesized on small Ta plates (with size about 3 mm × 3 mm) by chemical vapor deposition with ethylenediamine as a precursor (Zhang et al., 2002). The Ta plate with MWCNTs grown on was connected to the surface of a GC electrode using conductive silver paint (Structure probe Inc., USA). The edge of the Ta plate and GC electrode was insulated by pasting with nail enamel (Maybelline, NY, USA). The as-constructed MWCNTs electrode was used as working electrode and for the subsequent modification with redox polymer PVI-dmeOs. 2.2.2. Synthesis of redox polymer: poly(vinylimidazole) complexes of Os(4,4 -dimethylbpy)2 Cl Poly(1-vinylimidazole) (PVI) was prepared by a procedure similar to that reported by Heller and his coworkers (Ohara et al., 1993). Os(4,4 -dimethylbpy)2 Cl2 was synthesized following the procedure reported by Kober et al. (1988). PVI-dmeOs was synthesized from the PVI and Os(4,4 -dimethylbpy)2 Cl2 products obtained above by a procedure similar to that of Heller and his coworkers (Ohara et al., 1994). These synthetic procedures are briefly described in Supplementary Materials. 2.2.3. Electrodeposition of PVI-dmeOs on MWCNT electrodes Crosslinked films of PVI-dmeOs were irreversibly electrodeposited on MWCNT electrodes from a 1.5 mg mL−1 PVI-dmeOs solution supported by PBS. The electrodeposition was achieved by applying repetitive potential step cycles between +0.8 V (2 s) and −0.3 V (2 s) (Figure S-1 in Supplementary Materials). While the application of the negative potential would produce crosslinking reductive condition for the electrodeposition of PVI-dmeOs, the purpose of the oxidative half cycle is to oxidize the electrode surface so to increase the surface density of the redox polymer prior to its crosslinking (Gao et al., 2002). The PVI-dmeOs electrodeposited CNT electrodes were washed with water and dried in the air. 2.2.4. Investigation of PVI-dmeOs/CNT electrodes for redox enzyme activity measurement LOD and GOD were used as model redox enzymes to investigate the measurement of redox enzyme activity at PVI-dmeOs/CNT electrodes by using amperometric technique. By holding the electrode potential at +0.3 V, the response to redox enzymes was monitored by recording the current changes with time upon injecting the redox enzyme of interests into a neutral PBS containing the substrate of the corresponding enzyme. To minimize diffusion effects, during amperometric recording, the PBS solution was stirred with a magnetic stirring bar at a speed of 150 rpm on the BAS 100B cell
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Fig. 1. CVs in de-aerated PBS at a bare MWCNT electrode (a) and at the MWCNT electrode electrodeposited with PVI-dmeOs by applying 50 (b), 100 (c), and 350 (d) potential step cycles from a 1.5 mg mL−1 PVI-dmeOs solution supported by PBS. Inset shows the relationship between the potential cycle number of electrodeposition and the cathodic peak current of CVs at the corresponding PVI-dmeOs/CNT electrode in de-aerated PBS. Scan rate of CVs: 50 mV s−1 . Effective surface area of the bare MWCNTs: 0.073 cm2 .
stand. To investigate the response specificity of the PVI-dmeOs/CNT electrodes to redox enzymes, a mixed enzyme substrate was contained in PBS. The performance of the PVI-dmeOs/CNT electrode on redox enzyme activity measurement was compared to that of bare CNT electrode and PVI-dmeOs/GC electrode. To explore the mechanisms of sensing sensitivity enhancement to redox enzymes at the PVI-dmeOs/CNT electrode, the experiments of amperometric responses to H2 O2 , the reaction product of redox enzymes and their substrates, were performed in PBS solution at a stirring speed of 150 rpm. 3. Results and discussion
Fig. 2. (A and B) CVs in 5 mM K3 [FeCN6 ] supported by 1 M KCl at the scan rate of 20 mV s−1 (a), 100 mV s−1 (b), 300 mV s−1 (c), 500 mV s−1 (d), and 700 mV s−1 (e) at a Ta-supported well-aligned MWCNT electrode before (A) and after (B) the electrodeposition of PVI-dmeOs film. (C) The separations of peak potentials (Ep ) of CVs under different scan rates in 5 mM K3 [FeCN6 ] supported by 1 M KCl solution, and (D) impedance spectra in 0.1 M KCl solution containing equimolar Fe(CN)6 3−/4− (10 mM/10 mM) at the Ta-supported well-aligned MWCNT electrode before (filled triangle) and after (non-filled circle) the electrodeposition of PVI-dmeOs film. Effective surface area of bare MWCNTs: 0.073 cm2 (for A, B, and C), 0.088 cm2 (for D). Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
As shown in the inset of Fig. 1, the redox peak current of PVI-dmeOs on PVI-dmeOs/CNT electrodes increases monotonically with the applied electrodeposition potential cycle numbers, when the number is smaller than 300 cycles. Above this number, the redox peak current and the redox peak area (data not shown) reaches plateau, indicating that the amount of PVI-dmeOs saturated at 300 potential cycles of electrodeposition. The amount of redox centers (1.16 × 10−10 mol) on the saturated PVI-dmeOs was estimated from the integration of the cathodic peak area (1.12 × 10−5 C) using Faraday’s law (Eq. (1)).
3.1. Electrodeposition of PVI-dmeOs film on MWCNT electrodes Fig. 1 illustrates the cyclic voltammograms (CVs) in de-aerated PBS at a bare MWCNT electrode and at PVI-dmeOs/CNT electrodes prepared by electrodepositing PVI-dmeOs on the MWCNT electrode from a 1.5 mg mL−1 PVI-dmeOs solution by applying different cycle numbers of potential steps between +0.8 V (2 s) and −0.3 V (2 s). The CV of the bare CNT electrode exhibits a small reductive peak at −60 mV. This peak is from the reduction of trace oxygen remained in the de-aerated PBS. With the electrodeposition of PVI-dmeOs, a pair of redox waves appears at +119 mV (Epc) and +134 mV (Epa), respectively, corresponding to the single electron process of the redox centers on PVI-dmeOs. Further experimental results provide supporting evidence showing that the redox reaction of PVI-dmeOs is a surface electrochemical process (please refer to Figure S-3 for a detailed description in Supplementary Materials). The formal potential of the redox reaction of PVI-dmeOs on PVI-dmeOs/CNT electrode estimated by (Epc + Epa)/2 is +127 mV, which is similar to that on PVI-dmeOs/GC (+128.5 mV, Figure S3 in Supplementary Materials), suggesting that the formal potential of electrodeposited PVI-dmeOs does not obviously depend on the characteristics of specific electrode surfaces.
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N=
Q nF
(1)
In Eq. (1), Q, n, F, and N represents redox charge (C), electrons per molecule oxidized or reduced (single electron for the redox center of PVI-dmeOs), Faraday constant (96485.4 C mol−1 ) and the moles of oxidized or reduced molecules. The value of the redox centers of the saturated PVI-dmeOs per unit area of CNTs (1.59 × 10−9 mol cm−2 ) was calculated from this amount and the effective surface area of the MWCNT electrode (0.073 cm2 ). Effective surface areas of MWCNT electrodes were obtained by running CVs of 5 mM K3 [Fe(CN)6 ] in 1 M KCl at various scan rates (the detailed method is described in Supplementary Materials). The successful electrodeposition of PVI-dmeOs on MWCNT electrodes indicates that the oxidative half potential cycles (+0.8 V) can oxidize the CNTs surface and accumulate PVI-dmeOs molecules on the CNTs surface to the density that is high enough for the crosslinking of PVI-dmeOs to the CNT surface. The electrodeposited redox polymer layer was very stable and can be stored in the air and in aqueous solutions without obvious decrease of redox peak current. In view of storage and stabilities of usage, the electrodeposited redox polymer in the present study shows advantages than the
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Fig. 3. SEM images of bare well-aligned MWCNTs (A and B) and well-aligned MWCNTs electrodeposited with PVI-dmeOs (C and D). C and D represents newly prepared PVI-dmeOs/CNTs and PVI-dmeOs/CNTs that have been used for electrochemical enzyme sensing, respectively. Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
other method of immobilizing redox polymers on electrodes by using crosslinking agents (Ohara et al., 1994). 3.2. Electrochemical characterization of PVI-dmeOs/CNT electrodes In order to investigate the electron-transfer properties of the MWCNTs with and without PVI-dmeOs film, K3 [Fe(CN)6 ] was used as a probe in CV experiments. Fig. 2A and B shows the typical CVs of 1 M KCl solution containing 5 mM K3 [Fe(CN)6 ] at a MWCNT electrode before (A) and after (B) the electrodeposition of PVI-dmeOs. At the bare CNT electrode, a symmetric pair of current responses from the redox reaction of Fe(CN)6 3− were observed. In contrast, two pairs of redox peaks were observed at the PVI-dmeOs/CNT electrode. The redox pair at the formal potential of about +80 mV are resulted from the redox reaction of the PVI-dmeOs film, and the redox pair at a higher formal potential (around +268 mV) are assigned to the redox reaction of Fe(CN)6 3−/4− . For the redox reaction of the PVI-dmeOs film, the formal potential in the K3 [Fe(CN)6 ] solution shifted negatively for about 47 mV as compared to that in neutral PBS (at about +127 mV, as shown in Fig. 1). The reason for this negative shift is not clear at this moment. For the redox reaction of Fe(CN)6 3−/4− , the reductive peak current at the PVI-dmeOs/CNT electrode is higher than the oxidative peak current, possibly due to an electrocatalytic reduction of Fe(CN)6 3− by PVI-dmeOs at the cathodic scanning. The separations of peak potentials (Ep ) under different scan rates at bare CNTs and PVI-dmeOs/CNTs are shown in Fig. 3C. At bare CNTs (filled triangle), Ep is around 59 mV at all
sweep rates (10–700 mV s−1 ), suggesting that MWCNT electrode is ideally reversible for Fe(CN)6 3− . On the other hand, Ep of PVIdmeOs/CNT electrode (non-filled circle) increases gradually from 61 to 100 mV with the increase of scan rates from 10 to 800 mV s−1 , indicating a quasi-reversible Fe(CN)6 3− redox reaction at the PVIdmeOs/CNT electrode. This result suggests that the frequency of electron-transferring collisions between the tethered redox centers on the redox polymer is not high enough to result in a highly electronic conductive redox hydrogel. Increasing the content of redox centers on the redox polymer or decreasing the degree of polymer crosslinking may increase the conductivity of the electrodeposited redox hydrogel. Electrochemical impedance spectroscopy (EIS) experiments of 0.1 M KCl solution containing equimolar Fe(CN)63−/4− (10 mM/10 mM) with AC from 0.1 Hz to 100 KHz were also performed to investigate the electron-transfer properties of the MWCNTs with and without PVI-dmeOs film (Fig. 2D). The results of EIS are consistent with those of CVs in 1 M KCl solution containing 5 mM K3 [Fe(CN)6 ]. The Nyquist complex plane plot of bare MWCNT electrode (filled triangle) exhibits an almost straight line that is characteristic of a diffusion limiting step of an electrochemical process (Ren and Pickup, 1997), while the plot of PVI-dmeOs/CNT electrode is characterized by part of a single semicircle at high frequency domain and a straight line at low frequency domain. The membrane resistance of PVI-dmeOs/CNT, which can be determined from the semicircle diameter in the Nyquist complex plane plot, is about 15 , suggesting that the PVI-dmeOs thin film is conductive, but the conductivity is not as high as CNTs or metals.
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The conductivity of the electrodeposited PVI-dmeOs film may be further improved by optimizing the content of redox centers on the redox polymer and/or the electrochemical crosslinking conditions for electrodepositing PVI-dmeOs on CNTs. 3.3. Surface morphology and hydrophilicity of PVI-dmeOs/CNT electrodes The surface morphologies of modified MWCNTs were observed by SEM. As shown in Fig. 3, deposition of PVI-dmeOs did not cause obvious surface morphology changes to the well-aligned CNTs, indicating that the electrodeposited PVI-dmeOs film was very thin. Although a very thin film was formed on the surface of CNTs, it causes a significant change to the surface hydrophilicity. Upon the PVI-dmeOs film was electrodeposited on the surface of CNTs, the static water contact angle changed from 117.7◦ to 26.7◦ (Figure S-4 in Supplementary Materials), indicating that the CNT surface changed from hydrophobic to hydrophilic. This result is quite expectable, considering the hydrophilic properties of redox polymers. The production of hydrophilic CNTs without impairing their physical properties could expand their applications in many fields, for example, biomaterials, composites, and biosensors (Wang et al., 2003). 3.4. Amperometric measurement of redox enzyme activities Amperometric technique is widely used for electrochemical sensing due to its simplicity, sensitivity, fastness and directness. These features are also important and useful for measuring enzyme activities. Except these features, an ideal assay for enzyme activity also requires specificity with the obviation of using auxiliary enzymes. Fig. 4A demonstrates the amperometric curves in response to LOD at PVI-dmeOs/CNT (a), bare CNT (b), and PVIdmeOs/GC (c) electrodes (potential was hold at +0.3 V) in neutral PBS containing 12.5 mM LA. The values of current density were obtained by dividing recorded amperometric current with the effective surface area of MWCNT electrodes. Upon injecting LOD into the bulk PBS solution, a strong oxidation current appeared rapidly at the PVI-dmeOs/CNT electrode (Curve a in Fig. 4A). The response intensity at the PVI-dmeOs/CNT electrode was much stronger than that at the bare CNT electrode (Curve b in Fig. 4A). The PVI-dmeOs/GC electrode did not exhibit obvious response to the injections of LOD (Curve c in Fig. 4A). These results indicate that CNTs can enhance the amperometric response to LOD, and the PVI-dmeOs thin film on the CNT surface can further enhance the response intensity. CNTs have been reported to enhance the electrochemical reactivity of redox proteins with the redox active center not only close to the surface of the protein such as horseradish peroxidase (Zhao et al., 2002a), but also embedded deeply within the glycoprotein such as GOD (Guiseppi-Elie et al., 2002; Zhao et al., 2002b). This enhanced electrochemical reactivity of GOD was assumed due to the position of tubular fibrils within tunneling distance of the FAD cofactor with little consequence to denaturation (Guiseppi-Elie et al., 2002). In addition, CNTs can catalyze the electro-oxidation of H2 O2 (Tsai et al., 2006), the reaction product of redox enzymes (LOD and GOD) and their substrates. The enhancement of amperometric response to LOD by CNTs may be attributed to a combinatory effect of these two factors. The mechanisms for the enhancement of LOD response by the PVI-dmeOs thin film will be explored in the next section. Although the amperometric response to LOD was fast and the response sensitivity was high at the PVI-dmeOs/CNT electrode, it took a long time to reach equilibrium at both PVI-dmeOs/CNT and bare CNT electrodes, suggesting a still not very fast electrontransfer process of redox enzymes at the electrode surface. As
Fig. 4. (A) Amperometric response curves to LOD at (a) PVI-dmeOs/CNT, (b) bare CNT, and (c) PVI-dmeOs/GC electrodes in PBS solution containing 12.5 mM LA. (d) In (A): amperometric response curve to the interference of GOD at the PVI-dmeOs/CNT electrode in the LA/PBS solution. Arrows position the enzyme injection time point. (B) Amperometric response sensitivity to LOD at PVI-dmeOs/CNT, bare CNT, and PVIdmeOs/GC electrodes in the LA/PBS solution. Error bars represent mean ± S.E.M. (n = 3). Values marked with ‘*’ are statistically different with p < 0.05 (Student’s ttest). The applied potential for amperometry: +0.3 V. Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
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shown in Fig. 4A, the oxidation current did not reach plateau even after 1200 s. of LOD injection at the PVI-dmeOs/CNT electrode. By optimizing the content of redox centers on the redox polymer and the electrochemical crosslinking conditions for PVI-dmeOs electrodeposition, the conductivity of the electrodeposited PVI-dmeOs film, and the amperometric equilibration time in response to LOD may be adjusted. Based on the amperometric response to 0.9 Unit LOD in 4 mL PBS solution containing 12.5 mM LA, LOD response sensitivities at different kinds of electrodes were statistically compared. The results are shown in Fig. 4B. At the PVI-dmeOs/CNT electrode, the LOD response sensitivity (0.178 ± 0.049) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ] is significantly higher than that at the bare CNT electrode (0.0347 ± 0.0039) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ] and that at the PVI-dmeOs/GC electrode (3.41 × 10−4 ± 2.28 × 10−4 ) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ]. In addition to the high response sensitivity, the PVI-dmeOs/CNT electrode also exhibits high sensing specificity to LOD in the presence of LA in the solution (substrate specificity of LA to LOD). As shown at Curve d in Fig. 4A, the PVI-dmeOs/CNT electrode did not respond to the interference of GOD in the PBS solution containing LA. Similar results were found for the sensing of GOD in PBS solution containing glucose (Figure S5 in Supplementary Materials), suggesting that PVI-dmeOs/CNTs may be developed as a simple, sensitive, specific, and direct biosensor for universal redox enzymes. 3.5. Exploration of the mechanisms of response enhancement to redox enzymes by PVI-dmeOs film at PVI-dmeOs/CNT electrodes In the presence of enzyme substrate in PBS solution, the enhancement of sensing sensitivity to redox enzymes by the PVIdmeOs thin film at the PVI-dmeOs/CNT electrodes may be resulted from three plausible mechanisms: (1) the centers of redox enzymes may be wired by the redox centers tethered on the mobile redox polymer backbones to the electrodes, so that the redox enzymes can be directly electro-oxidized; (2) the electrodeposited PVIdmeOs film can promote the electro-oxidation of H2 O2 , the product of redox enzymes; and (3) redox enzymes may be more readily adsorbed on the surface of PVI-dmeOs/CNTs than on the surface of bare CNTs. The modes of protein adsorption on solid surfaces mainly derive from hydrophobic and electrostatic interactions. Upon the electrodeposition of PVI-dmeOs on the surface of CNTs, the surface was changed from hydrophobic to hydrophilic. Therefore, the force of hydrophobic interaction between redox enzymes and the CNT surface should be much reduced with the deposition of PVI-dmeOs thin film. Although the introduction of the cationic PVI-dmeOs film to the surface of CNTs may enhance the electrostatic adsorption of redox enzymes to the electrode surface, the overall amount of redox enzymes adsorbed could have been compromised by the reduced hydrophobic force. As shown in Fig. 3, the SEM images of PVI-dmeOs/CNTs that have been used for electrochemical enzyme sensing (D) do not show obvious differences to those of the newly prepared PVI-dmeOs/CNTs (C), suggesting no severe enzyme adsorption on the surface of PVI-dmeOs/CNTs. Hence, the third mechanism may not be a dominant one. Although no obvious enzyme adsorption on the electrode surface was observed after detection, dynamic contacts between carbon nanotube electrodes and redox enzymes could happen, in dynamic form of adsorption–desorption. To verify the second mechanism, amperometric experiments in response to H2 O2 were performed at the applied potential of +0.3 and −0.2 V at both PVI-dmeOs/CNT and bare CNT electrodes. Fig. 5A illustrates the amperometric curves at the applied potential of +0.3 V. Amperometric response sensitivities were determined from
Fig. 5. (A) Amperometric response curves to the step increase of 4.4 mM H2 O2 at (a) bare CNT, and (b) PVI-dmeOs/CNT electrodes in PBS solution at the applied potential of +0.3 V. Arrows position the H2 O2 injection time point. (B) Amperometric response sensitivity to H2 O2 at PVI-dmeOs/CNT (grey bar) and bare CNT electrodes (black bar) in PBS solution at the applied potential of +0.3 and −0.2 V. Error bars represent mean ± S.E.M. (n = 3). The values marked with ‘***’ are statistically different with p < 0.001 (Student’s t-test). Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
the slopes of the calibration curves of amperometric current (oxidation or reduction, depending on the applied potentials) versus the H2 O2 concentration. The statistic results of amperometric response sensitivities are shown in Fig. 5B. The sensing sensitivity to H2 O2 at the PVI-dmeOs/CNT electrode is significantly smaller than that at the bare CNT electrode at +0.3 V. In contrast, at the applied potential of −0.2 V, the sensing sensitivity to H2 O2 at the PVI-dmeOs/CNT electrode has no significant differences to that at the bare CNT electrode [p = 0.53 (Student’s t-test)]. These results indicate that the electrodeposited PVI-dmeOs film does not promote the electrooxidation of H2 O2 , thus not supporting a contribution of the second mechanism. The decrease of sensing sensitivity to H2 O2 at +0.3 V by
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electrodeposition and the amount of electrodeposited PVI-dmeOs can be saturated. This thin film of PVI-dmeOs changes the surface of CNTs from hydrophobic to hydrophilic without changing the structure of CNTs, which is a desirable property for many applications, such as biomaterials, biosensors, and high-performance polymer composites. More importantly, the redox polymer PVIdmeOs modified CNT electrodes exhibited direct, sensitive, specific and fast amperometric responses to model redox enzymes (LOD and GOD) in electrolyte solution containing the corresponding substrate of the measured redox enzyme. Both the CNT substrate and the thin film of PVI-dmeOs enhance the sensing sensitivity of the redox enzymes at the PVI-dmeOs modified CNT electrodes. This redox hydrogel-modified CNT electrode constitutes the first step towards the development of simple, sensitive, specific, fast, and direct amperometric sensors for universal redox enzymes. Acknowledgments This work was supported by Academic Research Grant of the National University of Singapore R-398-000-024-112 to FSS, and by Academic Research Grant of Talent Introducing Program of Zhengzhou University to HFC. Fig. 6. CVs at a PVI-dmeOs/CNT electrode in de-aerated neutral PBS solution containing 12.5 mM LA (dashed line) or containing 12.5 mM LA + 0.375 U mL−1 LOD (solid line). Effective surface area of bare MWCNTs: 0.073 cm2 . Scan rate of CVs: 50 mV s−1 . Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
the PVI-dmeOs thin film may be caused by an adsorption of O2 , the electro-oxidation product of H2 O2 , on the PVI-dmeOs thin film. The adsorbed O2 may inhibit the adsorption of H2 O2 to the electrode surface for electrochemical reaction. The contribution from the first mechanism was supported by the CV experiments of a PVI-dmeOs/CNT electrode in neutral PBS solution containing only LA or containing both LA and LOD (Fig. 6). An obvious oxidation peak typically representing the electro-oxidation of wired redox centers of redox enzymes was observed with the addition of LOD (solid line). Thus, it can be concluded that the wiring of the redox centers of redox enzymes by the redox centers tethered on the mobile redox polymer backbones mainly contributes to the enhancement of sensing sensitivity to redox enzymes at the PVI-dmeOs/CNT electrodes as compared to bare CNT electrodes. The dramatic wiring of redox enzymes by redox polymers deposited on the surface of well-aligned MWCNTs may be due to the nanometer size, the one-dimensionality, and the high specific surface area of CNTs. Firstly, because the tubular fibrils of CNTs may be positioned within or close to the tunneling distance of the redox centers of redox enzymes (Guiseppi-Elie et al., 2002), the many mobile redox centers of the thin redox hydrogel on the tubular fibrils could also be positioned within the tunneling distance of the redox centers of redox enzymes, increasing the chances to wire redox enzymes. Secondly, the nanotubular morphology and the high specific surface area of CNTs should result in electrodeposited redox polymers with more accessible redox centers, further increasing the chances to wire redox enzymes. 4. Conclusions The present study demonstrates that redox polymer PVI-dmeOs can be stably immobilized on the surface of MWCNT electrodes by
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2008.09.002. References Alpeeva, I.S., Vilkanauskyte, A., Ngounou, B., Csoregi, E., Sakharov, I.Y., Gonchar, M., Schuhmann, W., 2005. Microchim. Acta 152, 21–27. Bergmeyer, H.U., 1974. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 1. Academic Press, New York, pp. 457–460. Cui, H.F., Ye, J.S., Zhang, W.D., Wang, J., Sheu, F.S., 2005. J. Electroanal. Chem. 577, 295–302. Cui, H.F., Ye, J.S., Liu, X., Zhang, W.D., Sheu, F.S., 2006. Nanotechnology 17, 2334–2339. Cui, H.F., Ye, J.S., Zhang, W.D., Li, C.M., Luong, J.H.T., Sheu, F.S., 2007. Anal. Chim. Acta 594, 175–183. Gao, Z.Q., Binyamin, G., Kim, H.H., Barton, S.C., Zhang, Y.C., Heller, A., 2002. Angew. Chem. Int. Ed. 41, 810–813. Gregg, B.A., Heller, A., 1991. J. Phys. Chem. 95, 5970–5975. Guiseppi-Elie, A., Lei, C.H., Baughman, R.H., 2002. Nanotechnology 13, 559– 564. Iijima, S., 1991. Nature 354, 56–58. Karmali, K., Karmali, A., Teixeira, A., Marcelo Curto, M.J., 2004. Anal. Biochem. 333, 320–327. Kober, E.M., Caspar, J.V., Sullivan, B.P., Meyer, T., 1988. J. Inorg. Chem. 27, 4587–4598. Little, R.B., 2003. J. Cluster Sci. 14, 135–185. Merkoci, A., Pumera, M., Llopis, X., Perez, B., del Valle, M., Alegret, S., 2005. TrAC, Trends Anal. Chem. 24, 826–838. Mikeladze, E., Schulte, A., Mosbach, M., Blochl, A., Csoregi, E., Solomonia, R., Schuhmann, W., 2002. Electroanalysis 14, 393–399. Ohara, T.J., Rajagopalan, R., Heller, A., 1993. Anal. Chem. 65, 3512–3517. Ohara, T.J., Rajagopalan, R., Heller, A., 1994. Anal. Chem. 66, 2451–2457. Ren, X.M., Pickup, P.G., 1997. J. Electroanal. Chem. 420, 251–257. Schindler, R., Lendl, B., 1999. Anal. Chim. Acta 391, 19–28. Treacy, M.M.J., Ebbesen, T.W., Gibson, J.M., 1996. Nature 381, 678–680. Tsai, Y.C., Li, S.C., Liao, S.W., 2006. Biosens. Bioelectron. 22, 495–500. Wang, J., Musameh, M., Lin, Y.H., 2003. J. Am. Chem. Soc. 125, 2408–2409. Yorita, K., Janko, K., Aki, K., Ghisla, S., Palfey, B.A., Massey, V., 1997. Proc. Natl. Acad. Sci. U. S. A. 94, 9590–9595. Zhang, W.D., Wen, Y., Lin, S.M., Tjiu, W.C., Xu, G.Q., Gan, L.M., 2002. Carbon 40, 1981–1989. Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., Li, S.F.Y., 2002a. Sens. Actuators, B 6355, 1–5. Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., 2002b. Anal. Sci. 18, 939–941.
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Modification of carbon nanotubes with redox hydrogel: Improvement of amperometric sensing sensitivity for redox enzymes Hui-Fang Cui a , Jian-Shan Ye b , Wei-De Zhang b , Fwu-Shan Sheu c,d,∗ a
Department of Bioengineering, Zhengzhou University, 100# Science Avenue, Zhengzhou, 450001, PR China School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510641, PR China c Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore d Nanoscience and Nanotechnology Initiative, National University of Singapore, 10 Kent Ridge Crescent, 117576, Singapore b
a r t i c l e
i n f o
Article history: Received 16 June 2008 Received in revised form 1 September 2008 Accepted 1 September 2008 Available online 12 September 2008 Keywords: Redox polymer Carbon nanotubes Redox enzymes Enzyme activity Amperometry Electrochemical sensor
a b s t r a c t This study demonstrated that redox hydrogel-modified carbon nanotube (CNT) electrodes can be developed as an amperometric sensor that are sensitive, specific and fast and do not require auxiliary enzymes. A redox polymer, poly(vinylimidazole) complexed with Os(4,4 -dimethylbpy)2 Cl (PVI-dmeOs) was electrodeposited on Ta-supported multi-walled CNTs. The resulted PVI-dmeOs thin film did not change the surface morphology of the CNTs, but turned the CNT surface from hydrophobic to hydrophilic, as studied by scanning electron microscopy (SEM) and static water contact angle measurements. Cyclic voltammetry measurements in a Fe(CN)6 3− solution and electrochemical impedance measurements in an equimolar Fe(CN)6 3−/4− solution demonstrated that the PVI-dmeOs hydrogel thin film was electronic conductive with a resistance of about 15 . The PVI-dmeOs/CNT electrodes sensed rapidly, sensitively and specifically to model redox enzymes (glucose oxidase (GOD) and lactate oxidase (LOD)) in amperometric experiments in electrolyte solutions containing the substrates of the measured redox enzymes. Both the CNT substrate and the thin PVI-dmeOs film enhanced the sensing sensitivities. Exploration of the mechanisms suggests that the PVI-dmeOs film may enhance the sensing sensitivities by wiring the enzyme molecules through the redox centers tethered on the mobile redox polymer backbones to the CNT electrodes. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.
1. Introduction Redox enzymes contain redox centers, such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and heme as a prosthetic group, which can be electro-oxidized and electroreduced. For example, glucose oxidase (GOD) is a FAD containing glycoprotein which catalyzes the oxidation of glucose using molecular oxygen with the formation of gluconolactone (further hydrolysed to gluconic acid) and H2 O2 as products. Lactate oxidase (E.C.1.1.3.2) (LOD) is a member of the family of FMN-containing flavoproteins that catalyze the oxidation of lactate using molecular oxygen with the formation of pyruvate and H2 O2 as products (Yorita et al., 1997). Measurement of the activity of redox enzymes is of importance in clinical, pharmaceutical, therapeutic, analytical, chemical, food, and beverage researches and industries.
∗ Corresponding author at: Nanoscience and Nanotechnology Initiative, National University of Singapore, 10 Kent Ridge Crescent, 117576, Singapore. Tel.: +65 6516 2857; fax: +65 6779 2486. E-mail address: [email protected] (F.-S. Sheu).
Most of the assays for enzyme activity and for kinetic studies of enzyme reactions involve the use of auxiliary enzymes to generate easily detectable species such as NAD(P)+ or chromophores which absorb strongly either in ultraviolet or visible regions of spectrophotometers (Karmali et al., 2004; Bergmeyer, 1974). These linked enzyme assays present some disadvantages since the presence of other substances and enzymes in the reaction mixture may affect the kinetic behavior of the enzyme that is being investigated (Karmali et al., 2004). On the other hand, the assay conditions (i.e., temperature, pH, and ionic strength) may not be the same for different enzymes involved in the assay system (Schindler and Lendl, 1999). Therefore, it is of great interests to devise novel enzyme assays that are specific and fast and do not require auxiliary enzymes. Recently, Karmali et al. reported the use of Fourier transform infrared (FTIR) spectroscopy as a simple and direct assay method for the activity of GOD (Karmali et al., 2004). However, this method can only be applied to the enzymes for which the substrate and the product have quite distinguishable frequencies of IR absorbing. In this paper, we report a simple and direct assay method that may be applicable to universal redox enzymes. It uses redox polymer modified carbon nanotube (CNT) electrode to directly detect the activity of redox enzymes. GOD and LOD were
0956-5663/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.09.002
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used as model redox enzymes to investigate and demonstrate this direct assay method. Redox polymers are soluble polymers complexed with redox moieties. When crosslinked on electrodes, the redox polymers become insoluble, but swell in water to form three-dimensional redox hydrogels (Gregg and Heller, 1991). Upon swelling, the mobility of their segments is increased, increasing in the frequency of electron-transferring collisions between the tethered redox centers, and the electronic conductivity of the redox hydrogels. Redox polymers, such as poly(vinylimidazole) (PVI) complexed with [Os(bpy)2 Cl]+ (termed PVI-Os) [bpy: the short name for (2,2 bipyridine)] (Ohara et al., 1993; Alpeeva et al., 2005), or with Os(4,4 -dimethylbpy)2 Cl (termed PVI-dmeOs) (Ohara et al., 1994; Mikeladze et al., 2002) have been immobilized on different kinds of conventional electrodes, such as vitreous carbon, gold, and glassy carbon (GC) electrodes (Ohara et al., 1994, 1993; Gao et al., 2002; Alpeeva et al., 2005; Mikeladze et al., 2002). While the redox potential of PVI-Os is about 200 mV, that of PVI-dmeOs is reduced to 95 mV (versus saturated calomel electrode) (Ohara et al., 1994). When redox polymers are co-immobilized with redox enzymes on electrodes, the redox centers of the enzymes can be “wired” by the redox centers tethered on the mobile redox polymer backbones to the electrodes, therefore can also be electro-oxidized and electroreduced (Ohara et al., 1994, 1993; Gao et al., 2002). Due to its low redox potential, when co-immobilized with enzymes, PVI-dmeOs has been demonstrated to result in low oxidation potentials for redox enzymes (Ohara et al., 1994). CNTs are hollow carbon tubes made of a single or several to tens of concentrically arranged cylindrical graphite layers, capped by fullerenic hemispheres, which are referred to as single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) respectively (Little, 2003). CNTs have become the subject of intense investigations since their first discovery by Iijima in 1991 (Iijima, 1991). Such considerable interests were inspired by their unique structures, remarkable electrical (Merkoci et al., 2005) and mechanical (Treacy et al., 1996) properties, high chemical stability, and potentials in applications in many fields. CNTs, especially well-aligned MWCNTs grown on conductive Ta substrates have been demonstrated very good electrode materials for electrochemical biosensors (Cui et al., 2005, 2006, 2007). Two methods have been used to immobilize and crosslink redox polymers onto electrodes. One is to crosslink the redox polymers with poly-(ethylene glycol) diglycidyl ether, a crosslinking agent (Ohara et al., 1994, 1993). The other one is to electrodeposit redox polymers onto vitreous carbon by coordinative crosslinking (Gao et al., 2002). In this study, the redox polymer PVI-dmeOs was electrodeposited on, the first time, CNTs and the redox hydrogelmodified CNT electrodes (PVI-dmeOs/CNTs) were found exhibiting strong responses to redox enzymes in electrolyte solutions containing the corresponding substrate of the measured redox enzyme. Well-aligned MWCNTs grown on conductive Ta substrates were used as the CNT electrode material for the PVI-dmeOs deposition. The mechanisms of the strong response to redox enzymes at the PVI-dmeOs modified CNT electrodes are explored in this paper.
2. Experimental 2.1. Instrument and reagents The morphologies of modified MWCNTs were observed by scanning electron microscopy (SEM) (JEOL JSM 6700F, operated at 5 kV). Static water contact angles on MWCNTs were measured with VCA Optima surface analysis system (AST Products Inc., USA) using sessile drop method in air atmosphere. The size of the water drop for
the measurement was 1.0 L. Electrochemical measurements were performed using BAS 100B electrochemical analyzer equipped with a BAS 100B cell stand (Bioanalytical Systems Inc., Indiana, USA) in a three-electrode system, including a working electrode (MWCNTs, GC, or PVI-dmeOs/CNTs electrode), a platinum counter electrode and a 3 M KCl–Ag/AgCl reference electrode, at room temperature (≈25 ◦ C). All potentials were quoted versus this reference electrode. Ta plates were obtained from Goodfellow Cambridge Ltd. (Huntingdon, England). All chemicals were purchased from Sigma–Aldrich Co. (Saint Louis, MO, USA) and were used without further purifications. Purified nitrogen gas was purchased from Singapore Oxygen Air Liquid Pte Ltd. Phosphate buffer solution (PBS) contains: 8 g L−1 NaCl, 0.2 g L−1 KCl, 1.44 g L−1 Na2 HPO4 , and 0.24 g L−1 KH2 PO4 with pH 7.4. Deionized water obtained from a Millipore water system was used throughout the experiment. 2.2. Methods 2.2.1. Synthesis of well-aligned MWCNTs and construction of MWCNT electrodes MWCNTs were synthesized on small Ta plates (with size about 3 mm × 3 mm) by chemical vapor deposition with ethylenediamine as a precursor (Zhang et al., 2002). The Ta plate with MWCNTs grown on was connected to the surface of a GC electrode using conductive silver paint (Structure probe Inc., USA). The edge of the Ta plate and GC electrode was insulated by pasting with nail enamel (Maybelline, NY, USA). The as-constructed MWCNTs electrode was used as working electrode and for the subsequent modification with redox polymer PVI-dmeOs. 2.2.2. Synthesis of redox polymer: poly(vinylimidazole) complexes of Os(4,4 -dimethylbpy)2 Cl Poly(1-vinylimidazole) (PVI) was prepared by a procedure similar to that reported by Heller and his coworkers (Ohara et al., 1993). Os(4,4 -dimethylbpy)2 Cl2 was synthesized following the procedure reported by Kober et al. (1988). PVI-dmeOs was synthesized from the PVI and Os(4,4 -dimethylbpy)2 Cl2 products obtained above by a procedure similar to that of Heller and his coworkers (Ohara et al., 1994). These synthetic procedures are briefly described in Supplementary Materials. 2.2.3. Electrodeposition of PVI-dmeOs on MWCNT electrodes Crosslinked films of PVI-dmeOs were irreversibly electrodeposited on MWCNT electrodes from a 1.5 mg mL−1 PVI-dmeOs solution supported by PBS. The electrodeposition was achieved by applying repetitive potential step cycles between +0.8 V (2 s) and −0.3 V (2 s) (Figure S-1 in Supplementary Materials). While the application of the negative potential would produce crosslinking reductive condition for the electrodeposition of PVI-dmeOs, the purpose of the oxidative half cycle is to oxidize the electrode surface so to increase the surface density of the redox polymer prior to its crosslinking (Gao et al., 2002). The PVI-dmeOs electrodeposited CNT electrodes were washed with water and dried in the air. 2.2.4. Investigation of PVI-dmeOs/CNT electrodes for redox enzyme activity measurement LOD and GOD were used as model redox enzymes to investigate the measurement of redox enzyme activity at PVI-dmeOs/CNT electrodes by using amperometric technique. By holding the electrode potential at +0.3 V, the response to redox enzymes was monitored by recording the current changes with time upon injecting the redox enzyme of interests into a neutral PBS containing the substrate of the corresponding enzyme. To minimize diffusion effects, during amperometric recording, the PBS solution was stirred with a magnetic stirring bar at a speed of 150 rpm on the BAS 100B cell
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Fig. 1. CVs in de-aerated PBS at a bare MWCNT electrode (a) and at the MWCNT electrode electrodeposited with PVI-dmeOs by applying 50 (b), 100 (c), and 350 (d) potential step cycles from a 1.5 mg mL−1 PVI-dmeOs solution supported by PBS. Inset shows the relationship between the potential cycle number of electrodeposition and the cathodic peak current of CVs at the corresponding PVI-dmeOs/CNT electrode in de-aerated PBS. Scan rate of CVs: 50 mV s−1 . Effective surface area of the bare MWCNTs: 0.073 cm2 .
stand. To investigate the response specificity of the PVI-dmeOs/CNT electrodes to redox enzymes, a mixed enzyme substrate was contained in PBS. The performance of the PVI-dmeOs/CNT electrode on redox enzyme activity measurement was compared to that of bare CNT electrode and PVI-dmeOs/GC electrode. To explore the mechanisms of sensing sensitivity enhancement to redox enzymes at the PVI-dmeOs/CNT electrode, the experiments of amperometric responses to H2 O2 , the reaction product of redox enzymes and their substrates, were performed in PBS solution at a stirring speed of 150 rpm. 3. Results and discussion
Fig. 2. (A and B) CVs in 5 mM K3 [FeCN6 ] supported by 1 M KCl at the scan rate of 20 mV s−1 (a), 100 mV s−1 (b), 300 mV s−1 (c), 500 mV s−1 (d), and 700 mV s−1 (e) at a Ta-supported well-aligned MWCNT electrode before (A) and after (B) the electrodeposition of PVI-dmeOs film. (C) The separations of peak potentials (Ep ) of CVs under different scan rates in 5 mM K3 [FeCN6 ] supported by 1 M KCl solution, and (D) impedance spectra in 0.1 M KCl solution containing equimolar Fe(CN)6 3−/4− (10 mM/10 mM) at the Ta-supported well-aligned MWCNT electrode before (filled triangle) and after (non-filled circle) the electrodeposition of PVI-dmeOs film. Effective surface area of bare MWCNTs: 0.073 cm2 (for A, B, and C), 0.088 cm2 (for D). Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
As shown in the inset of Fig. 1, the redox peak current of PVI-dmeOs on PVI-dmeOs/CNT electrodes increases monotonically with the applied electrodeposition potential cycle numbers, when the number is smaller than 300 cycles. Above this number, the redox peak current and the redox peak area (data not shown) reaches plateau, indicating that the amount of PVI-dmeOs saturated at 300 potential cycles of electrodeposition. The amount of redox centers (1.16 × 10−10 mol) on the saturated PVI-dmeOs was estimated from the integration of the cathodic peak area (1.12 × 10−5 C) using Faraday’s law (Eq. (1)).
3.1. Electrodeposition of PVI-dmeOs film on MWCNT electrodes Fig. 1 illustrates the cyclic voltammograms (CVs) in de-aerated PBS at a bare MWCNT electrode and at PVI-dmeOs/CNT electrodes prepared by electrodepositing PVI-dmeOs on the MWCNT electrode from a 1.5 mg mL−1 PVI-dmeOs solution by applying different cycle numbers of potential steps between +0.8 V (2 s) and −0.3 V (2 s). The CV of the bare CNT electrode exhibits a small reductive peak at −60 mV. This peak is from the reduction of trace oxygen remained in the de-aerated PBS. With the electrodeposition of PVI-dmeOs, a pair of redox waves appears at +119 mV (Epc) and +134 mV (Epa), respectively, corresponding to the single electron process of the redox centers on PVI-dmeOs. Further experimental results provide supporting evidence showing that the redox reaction of PVI-dmeOs is a surface electrochemical process (please refer to Figure S-3 for a detailed description in Supplementary Materials). The formal potential of the redox reaction of PVI-dmeOs on PVI-dmeOs/CNT electrode estimated by (Epc + Epa)/2 is +127 mV, which is similar to that on PVI-dmeOs/GC (+128.5 mV, Figure S3 in Supplementary Materials), suggesting that the formal potential of electrodeposited PVI-dmeOs does not obviously depend on the characteristics of specific electrode surfaces.
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N=
Q nF
(1)
In Eq. (1), Q, n, F, and N represents redox charge (C), electrons per molecule oxidized or reduced (single electron for the redox center of PVI-dmeOs), Faraday constant (96485.4 C mol−1 ) and the moles of oxidized or reduced molecules. The value of the redox centers of the saturated PVI-dmeOs per unit area of CNTs (1.59 × 10−9 mol cm−2 ) was calculated from this amount and the effective surface area of the MWCNT electrode (0.073 cm2 ). Effective surface areas of MWCNT electrodes were obtained by running CVs of 5 mM K3 [Fe(CN)6 ] in 1 M KCl at various scan rates (the detailed method is described in Supplementary Materials). The successful electrodeposition of PVI-dmeOs on MWCNT electrodes indicates that the oxidative half potential cycles (+0.8 V) can oxidize the CNTs surface and accumulate PVI-dmeOs molecules on the CNTs surface to the density that is high enough for the crosslinking of PVI-dmeOs to the CNT surface. The electrodeposited redox polymer layer was very stable and can be stored in the air and in aqueous solutions without obvious decrease of redox peak current. In view of storage and stabilities of usage, the electrodeposited redox polymer in the present study shows advantages than the
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Fig. 3. SEM images of bare well-aligned MWCNTs (A and B) and well-aligned MWCNTs electrodeposited with PVI-dmeOs (C and D). C and D represents newly prepared PVI-dmeOs/CNTs and PVI-dmeOs/CNTs that have been used for electrochemical enzyme sensing, respectively. Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
other method of immobilizing redox polymers on electrodes by using crosslinking agents (Ohara et al., 1994). 3.2. Electrochemical characterization of PVI-dmeOs/CNT electrodes In order to investigate the electron-transfer properties of the MWCNTs with and without PVI-dmeOs film, K3 [Fe(CN)6 ] was used as a probe in CV experiments. Fig. 2A and B shows the typical CVs of 1 M KCl solution containing 5 mM K3 [Fe(CN)6 ] at a MWCNT electrode before (A) and after (B) the electrodeposition of PVI-dmeOs. At the bare CNT electrode, a symmetric pair of current responses from the redox reaction of Fe(CN)6 3− were observed. In contrast, two pairs of redox peaks were observed at the PVI-dmeOs/CNT electrode. The redox pair at the formal potential of about +80 mV are resulted from the redox reaction of the PVI-dmeOs film, and the redox pair at a higher formal potential (around +268 mV) are assigned to the redox reaction of Fe(CN)6 3−/4− . For the redox reaction of the PVI-dmeOs film, the formal potential in the K3 [Fe(CN)6 ] solution shifted negatively for about 47 mV as compared to that in neutral PBS (at about +127 mV, as shown in Fig. 1). The reason for this negative shift is not clear at this moment. For the redox reaction of Fe(CN)6 3−/4− , the reductive peak current at the PVI-dmeOs/CNT electrode is higher than the oxidative peak current, possibly due to an electrocatalytic reduction of Fe(CN)6 3− by PVI-dmeOs at the cathodic scanning. The separations of peak potentials (Ep ) under different scan rates at bare CNTs and PVI-dmeOs/CNTs are shown in Fig. 3C. At bare CNTs (filled triangle), Ep is around 59 mV at all
sweep rates (10–700 mV s−1 ), suggesting that MWCNT electrode is ideally reversible for Fe(CN)6 3− . On the other hand, Ep of PVIdmeOs/CNT electrode (non-filled circle) increases gradually from 61 to 100 mV with the increase of scan rates from 10 to 800 mV s−1 , indicating a quasi-reversible Fe(CN)6 3− redox reaction at the PVIdmeOs/CNT electrode. This result suggests that the frequency of electron-transferring collisions between the tethered redox centers on the redox polymer is not high enough to result in a highly electronic conductive redox hydrogel. Increasing the content of redox centers on the redox polymer or decreasing the degree of polymer crosslinking may increase the conductivity of the electrodeposited redox hydrogel. Electrochemical impedance spectroscopy (EIS) experiments of 0.1 M KCl solution containing equimolar Fe(CN)63−/4− (10 mM/10 mM) with AC from 0.1 Hz to 100 KHz were also performed to investigate the electron-transfer properties of the MWCNTs with and without PVI-dmeOs film (Fig. 2D). The results of EIS are consistent with those of CVs in 1 M KCl solution containing 5 mM K3 [Fe(CN)6 ]. The Nyquist complex plane plot of bare MWCNT electrode (filled triangle) exhibits an almost straight line that is characteristic of a diffusion limiting step of an electrochemical process (Ren and Pickup, 1997), while the plot of PVI-dmeOs/CNT electrode is characterized by part of a single semicircle at high frequency domain and a straight line at low frequency domain. The membrane resistance of PVI-dmeOs/CNT, which can be determined from the semicircle diameter in the Nyquist complex plane plot, is about 15 , suggesting that the PVI-dmeOs thin film is conductive, but the conductivity is not as high as CNTs or metals.
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The conductivity of the electrodeposited PVI-dmeOs film may be further improved by optimizing the content of redox centers on the redox polymer and/or the electrochemical crosslinking conditions for electrodepositing PVI-dmeOs on CNTs. 3.3. Surface morphology and hydrophilicity of PVI-dmeOs/CNT electrodes The surface morphologies of modified MWCNTs were observed by SEM. As shown in Fig. 3, deposition of PVI-dmeOs did not cause obvious surface morphology changes to the well-aligned CNTs, indicating that the electrodeposited PVI-dmeOs film was very thin. Although a very thin film was formed on the surface of CNTs, it causes a significant change to the surface hydrophilicity. Upon the PVI-dmeOs film was electrodeposited on the surface of CNTs, the static water contact angle changed from 117.7◦ to 26.7◦ (Figure S-4 in Supplementary Materials), indicating that the CNT surface changed from hydrophobic to hydrophilic. This result is quite expectable, considering the hydrophilic properties of redox polymers. The production of hydrophilic CNTs without impairing their physical properties could expand their applications in many fields, for example, biomaterials, composites, and biosensors (Wang et al., 2003). 3.4. Amperometric measurement of redox enzyme activities Amperometric technique is widely used for electrochemical sensing due to its simplicity, sensitivity, fastness and directness. These features are also important and useful for measuring enzyme activities. Except these features, an ideal assay for enzyme activity also requires specificity with the obviation of using auxiliary enzymes. Fig. 4A demonstrates the amperometric curves in response to LOD at PVI-dmeOs/CNT (a), bare CNT (b), and PVIdmeOs/GC (c) electrodes (potential was hold at +0.3 V) in neutral PBS containing 12.5 mM LA. The values of current density were obtained by dividing recorded amperometric current with the effective surface area of MWCNT electrodes. Upon injecting LOD into the bulk PBS solution, a strong oxidation current appeared rapidly at the PVI-dmeOs/CNT electrode (Curve a in Fig. 4A). The response intensity at the PVI-dmeOs/CNT electrode was much stronger than that at the bare CNT electrode (Curve b in Fig. 4A). The PVI-dmeOs/GC electrode did not exhibit obvious response to the injections of LOD (Curve c in Fig. 4A). These results indicate that CNTs can enhance the amperometric response to LOD, and the PVI-dmeOs thin film on the CNT surface can further enhance the response intensity. CNTs have been reported to enhance the electrochemical reactivity of redox proteins with the redox active center not only close to the surface of the protein such as horseradish peroxidase (Zhao et al., 2002a), but also embedded deeply within the glycoprotein such as GOD (Guiseppi-Elie et al., 2002; Zhao et al., 2002b). This enhanced electrochemical reactivity of GOD was assumed due to the position of tubular fibrils within tunneling distance of the FAD cofactor with little consequence to denaturation (Guiseppi-Elie et al., 2002). In addition, CNTs can catalyze the electro-oxidation of H2 O2 (Tsai et al., 2006), the reaction product of redox enzymes (LOD and GOD) and their substrates. The enhancement of amperometric response to LOD by CNTs may be attributed to a combinatory effect of these two factors. The mechanisms for the enhancement of LOD response by the PVI-dmeOs thin film will be explored in the next section. Although the amperometric response to LOD was fast and the response sensitivity was high at the PVI-dmeOs/CNT electrode, it took a long time to reach equilibrium at both PVI-dmeOs/CNT and bare CNT electrodes, suggesting a still not very fast electrontransfer process of redox enzymes at the electrode surface. As
Fig. 4. (A) Amperometric response curves to LOD at (a) PVI-dmeOs/CNT, (b) bare CNT, and (c) PVI-dmeOs/GC electrodes in PBS solution containing 12.5 mM LA. (d) In (A): amperometric response curve to the interference of GOD at the PVI-dmeOs/CNT electrode in the LA/PBS solution. Arrows position the enzyme injection time point. (B) Amperometric response sensitivity to LOD at PVI-dmeOs/CNT, bare CNT, and PVIdmeOs/GC electrodes in the LA/PBS solution. Error bars represent mean ± S.E.M. (n = 3). Values marked with ‘*’ are statistically different with p < 0.05 (Student’s ttest). The applied potential for amperometry: +0.3 V. Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
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shown in Fig. 4A, the oxidation current did not reach plateau even after 1200 s. of LOD injection at the PVI-dmeOs/CNT electrode. By optimizing the content of redox centers on the redox polymer and the electrochemical crosslinking conditions for PVI-dmeOs electrodeposition, the conductivity of the electrodeposited PVI-dmeOs film, and the amperometric equilibration time in response to LOD may be adjusted. Based on the amperometric response to 0.9 Unit LOD in 4 mL PBS solution containing 12.5 mM LA, LOD response sensitivities at different kinds of electrodes were statistically compared. The results are shown in Fig. 4B. At the PVI-dmeOs/CNT electrode, the LOD response sensitivity (0.178 ± 0.049) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ] is significantly higher than that at the bare CNT electrode (0.0347 ± 0.0039) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ] and that at the PVI-dmeOs/GC electrode (3.41 × 10−4 ± 2.28 × 10−4 ) (mean ± S.E.M.) [A (U L−1 )−1 cm−2 ]. In addition to the high response sensitivity, the PVI-dmeOs/CNT electrode also exhibits high sensing specificity to LOD in the presence of LA in the solution (substrate specificity of LA to LOD). As shown at Curve d in Fig. 4A, the PVI-dmeOs/CNT electrode did not respond to the interference of GOD in the PBS solution containing LA. Similar results were found for the sensing of GOD in PBS solution containing glucose (Figure S5 in Supplementary Materials), suggesting that PVI-dmeOs/CNTs may be developed as a simple, sensitive, specific, and direct biosensor for universal redox enzymes. 3.5. Exploration of the mechanisms of response enhancement to redox enzymes by PVI-dmeOs film at PVI-dmeOs/CNT electrodes In the presence of enzyme substrate in PBS solution, the enhancement of sensing sensitivity to redox enzymes by the PVIdmeOs thin film at the PVI-dmeOs/CNT electrodes may be resulted from three plausible mechanisms: (1) the centers of redox enzymes may be wired by the redox centers tethered on the mobile redox polymer backbones to the electrodes, so that the redox enzymes can be directly electro-oxidized; (2) the electrodeposited PVIdmeOs film can promote the electro-oxidation of H2 O2 , the product of redox enzymes; and (3) redox enzymes may be more readily adsorbed on the surface of PVI-dmeOs/CNTs than on the surface of bare CNTs. The modes of protein adsorption on solid surfaces mainly derive from hydrophobic and electrostatic interactions. Upon the electrodeposition of PVI-dmeOs on the surface of CNTs, the surface was changed from hydrophobic to hydrophilic. Therefore, the force of hydrophobic interaction between redox enzymes and the CNT surface should be much reduced with the deposition of PVI-dmeOs thin film. Although the introduction of the cationic PVI-dmeOs film to the surface of CNTs may enhance the electrostatic adsorption of redox enzymes to the electrode surface, the overall amount of redox enzymes adsorbed could have been compromised by the reduced hydrophobic force. As shown in Fig. 3, the SEM images of PVI-dmeOs/CNTs that have been used for electrochemical enzyme sensing (D) do not show obvious differences to those of the newly prepared PVI-dmeOs/CNTs (C), suggesting no severe enzyme adsorption on the surface of PVI-dmeOs/CNTs. Hence, the third mechanism may not be a dominant one. Although no obvious enzyme adsorption on the electrode surface was observed after detection, dynamic contacts between carbon nanotube electrodes and redox enzymes could happen, in dynamic form of adsorption–desorption. To verify the second mechanism, amperometric experiments in response to H2 O2 were performed at the applied potential of +0.3 and −0.2 V at both PVI-dmeOs/CNT and bare CNT electrodes. Fig. 5A illustrates the amperometric curves at the applied potential of +0.3 V. Amperometric response sensitivities were determined from
Fig. 5. (A) Amperometric response curves to the step increase of 4.4 mM H2 O2 at (a) bare CNT, and (b) PVI-dmeOs/CNT electrodes in PBS solution at the applied potential of +0.3 V. Arrows position the H2 O2 injection time point. (B) Amperometric response sensitivity to H2 O2 at PVI-dmeOs/CNT (grey bar) and bare CNT electrodes (black bar) in PBS solution at the applied potential of +0.3 and −0.2 V. Error bars represent mean ± S.E.M. (n = 3). The values marked with ‘***’ are statistically different with p < 0.001 (Student’s t-test). Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
the slopes of the calibration curves of amperometric current (oxidation or reduction, depending on the applied potentials) versus the H2 O2 concentration. The statistic results of amperometric response sensitivities are shown in Fig. 5B. The sensing sensitivity to H2 O2 at the PVI-dmeOs/CNT electrode is significantly smaller than that at the bare CNT electrode at +0.3 V. In contrast, at the applied potential of −0.2 V, the sensing sensitivity to H2 O2 at the PVI-dmeOs/CNT electrode has no significant differences to that at the bare CNT electrode [p = 0.53 (Student’s t-test)]. These results indicate that the electrodeposited PVI-dmeOs film does not promote the electrooxidation of H2 O2 , thus not supporting a contribution of the second mechanism. The decrease of sensing sensitivity to H2 O2 at +0.3 V by
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electrodeposition and the amount of electrodeposited PVI-dmeOs can be saturated. This thin film of PVI-dmeOs changes the surface of CNTs from hydrophobic to hydrophilic without changing the structure of CNTs, which is a desirable property for many applications, such as biomaterials, biosensors, and high-performance polymer composites. More importantly, the redox polymer PVIdmeOs modified CNT electrodes exhibited direct, sensitive, specific and fast amperometric responses to model redox enzymes (LOD and GOD) in electrolyte solution containing the corresponding substrate of the measured redox enzyme. Both the CNT substrate and the thin film of PVI-dmeOs enhance the sensing sensitivity of the redox enzymes at the PVI-dmeOs modified CNT electrodes. This redox hydrogel-modified CNT electrode constitutes the first step towards the development of simple, sensitive, specific, fast, and direct amperometric sensors for universal redox enzymes. Acknowledgments This work was supported by Academic Research Grant of the National University of Singapore R-398-000-024-112 to FSS, and by Academic Research Grant of Talent Introducing Program of Zhengzhou University to HFC. Fig. 6. CVs at a PVI-dmeOs/CNT electrode in de-aerated neutral PBS solution containing 12.5 mM LA (dashed line) or containing 12.5 mM LA + 0.375 U mL−1 LOD (solid line). Effective surface area of bare MWCNTs: 0.073 cm2 . Scan rate of CVs: 50 mV s−1 . Potential cycle number for electrodepositing PVI-dmeOs on CNTs: 350.
the PVI-dmeOs thin film may be caused by an adsorption of O2 , the electro-oxidation product of H2 O2 , on the PVI-dmeOs thin film. The adsorbed O2 may inhibit the adsorption of H2 O2 to the electrode surface for electrochemical reaction. The contribution from the first mechanism was supported by the CV experiments of a PVI-dmeOs/CNT electrode in neutral PBS solution containing only LA or containing both LA and LOD (Fig. 6). An obvious oxidation peak typically representing the electro-oxidation of wired redox centers of redox enzymes was observed with the addition of LOD (solid line). Thus, it can be concluded that the wiring of the redox centers of redox enzymes by the redox centers tethered on the mobile redox polymer backbones mainly contributes to the enhancement of sensing sensitivity to redox enzymes at the PVI-dmeOs/CNT electrodes as compared to bare CNT electrodes. The dramatic wiring of redox enzymes by redox polymers deposited on the surface of well-aligned MWCNTs may be due to the nanometer size, the one-dimensionality, and the high specific surface area of CNTs. Firstly, because the tubular fibrils of CNTs may be positioned within or close to the tunneling distance of the redox centers of redox enzymes (Guiseppi-Elie et al., 2002), the many mobile redox centers of the thin redox hydrogel on the tubular fibrils could also be positioned within the tunneling distance of the redox centers of redox enzymes, increasing the chances to wire redox enzymes. Secondly, the nanotubular morphology and the high specific surface area of CNTs should result in electrodeposited redox polymers with more accessible redox centers, further increasing the chances to wire redox enzymes. 4. Conclusions The present study demonstrates that redox polymer PVI-dmeOs can be stably immobilized on the surface of MWCNT electrodes by
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2008.09.002. References Alpeeva, I.S., Vilkanauskyte, A., Ngounou, B., Csoregi, E., Sakharov, I.Y., Gonchar, M., Schuhmann, W., 2005. Microchim. Acta 152, 21–27. Bergmeyer, H.U., 1974. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 1. Academic Press, New York, pp. 457–460. Cui, H.F., Ye, J.S., Zhang, W.D., Wang, J., Sheu, F.S., 2005. J. Electroanal. Chem. 577, 295–302. Cui, H.F., Ye, J.S., Liu, X., Zhang, W.D., Sheu, F.S., 2006. Nanotechnology 17, 2334–2339. Cui, H.F., Ye, J.S., Zhang, W.D., Li, C.M., Luong, J.H.T., Sheu, F.S., 2007. Anal. Chim. Acta 594, 175–183. Gao, Z.Q., Binyamin, G., Kim, H.H., Barton, S.C., Zhang, Y.C., Heller, A., 2002. Angew. Chem. Int. Ed. 41, 810–813. Gregg, B.A., Heller, A., 1991. J. Phys. Chem. 95, 5970–5975. Guiseppi-Elie, A., Lei, C.H., Baughman, R.H., 2002. Nanotechnology 13, 559– 564. Iijima, S., 1991. Nature 354, 56–58. Karmali, K., Karmali, A., Teixeira, A., Marcelo Curto, M.J., 2004. Anal. Biochem. 333, 320–327. Kober, E.M., Caspar, J.V., Sullivan, B.P., Meyer, T., 1988. J. Inorg. Chem. 27, 4587–4598. Little, R.B., 2003. J. Cluster Sci. 14, 135–185. Merkoci, A., Pumera, M., Llopis, X., Perez, B., del Valle, M., Alegret, S., 2005. TrAC, Trends Anal. Chem. 24, 826–838. Mikeladze, E., Schulte, A., Mosbach, M., Blochl, A., Csoregi, E., Solomonia, R., Schuhmann, W., 2002. Electroanalysis 14, 393–399. Ohara, T.J., Rajagopalan, R., Heller, A., 1993. Anal. Chem. 65, 3512–3517. Ohara, T.J., Rajagopalan, R., Heller, A., 1994. Anal. Chem. 66, 2451–2457. Ren, X.M., Pickup, P.G., 1997. J. Electroanal. Chem. 420, 251–257. Schindler, R., Lendl, B., 1999. Anal. Chim. Acta 391, 19–28. Treacy, M.M.J., Ebbesen, T.W., Gibson, J.M., 1996. Nature 381, 678–680. Tsai, Y.C., Li, S.C., Liao, S.W., 2006. Biosens. Bioelectron. 22, 495–500. Wang, J., Musameh, M., Lin, Y.H., 2003. J. Am. Chem. Soc. 125, 2408–2409. Yorita, K., Janko, K., Aki, K., Ghisla, S., Palfey, B.A., Massey, V., 1997. Proc. Natl. Acad. Sci. U. S. A. 94, 9590–9595. Zhang, W.D., Wen, Y., Lin, S.M., Tjiu, W.C., Xu, G.Q., Gan, L.M., 2002. Carbon 40, 1981–1989. Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., Li, S.F.Y., 2002a. Sens. Actuators, B 6355, 1–5. Zhao, Y.D., Zhang, W.D., Chen, H., Luo, Q.M., 2002b. Anal. Sci. 18, 939–941.