Enhanced Charge Transport in Enzyme-Wired Organometallic Block

Article pubs.acs.org/Macromolecules

Enhanced Charge Transport in Enzyme-Wired Organometallic Block Copolymers for Bioenergy and Biosensors Joungphil Lee,† Hyungmin Ahn,† Ilyoung Choi,‡ Markus Boese,§ and Moon Jeong Park*,†,‡ †

Department of Chemistry and ‡Division of Advanced Materials Science (WCU), Pohang University of Science and Technology (POSTECH), Pohang, Korea 790-784 § National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, University of California, Berkeley, Berkeley, California 94720, United States ABSTRACT: Wiring of glucose oxidase (GOx) onto electrode surface was successfully achieved by cross-linked networks of organometallic block copolymers comprising electroactive ferrocene moieties and chemically cross-linkable diene groups, poly(ferrocenyldimethylsilaneb-isoprene)s (PFS−PIs). Different nanoscale morphologies of PFS−PIs, i.e., bicontinuous structure, nanowires, and nanoparticles, have been derived by varying molecular weights and casting solvents. Upon examining catalytic current responses of the GOx integrated PFS−PI systems, notably, the morphology of PFS−PI is found out to be a crucial parameter in determining the efficiency of electron transfer. For example, the use of bicontinuous PFS−PI confirms 2−50 times improved catalytic current densities, compared with the values of other morphologies; the maximum catalytic current of glucose oxidation was 0.7 mA/cm2 at 70 mM glucose concentration. The biosensing ability of the fabricated electrode with structural optimization was also exploited, and good sensitivity is obtained at the physiological concentration of glucose in blood.

■

With “wiring” point of view, carbon nanotubes (CNTs) have been received great interests21,22 owing to their large specific surface area23,24 and excellent electrochemical properties.25−27 One drawback of applying CNTs to the large area electrodes is the difficulty in preventing CNT aggregations if chemical modification of the CNTs or preorganization of CNT arrays is not carried out.12,21 Concurrently with the development of the CNT-based mediator systems, substantial efforts have been also paid to redox-active polymers. A number of organometallic polymers were identified as effective materials transferring the electrons as a result of redox reactions.28,29 However, although the kinds of redox polymers and their electrochemical properties are wellestablished,7,13,30,31 there is an open question about how the morphology of redox polymers makes effect on the electronmediating properties. Herein, we have explored the electrochemical properties of enzyme integrated redox polymers by varying the self-assembled structures of the redox polymers in nanometer scales. In present study, a glucose oxidase (GOx) is employed as a model enzyme29 and ferrocene (Fc)-containing organometallic block copolymers with Os-reactive diene groups, poly(ferrocenyldimethylsilane-b-isoprene) (PFS−PI) copolymers, are used as electron mediators. Our efforts are inspired by

INTRODUCTION The development of efficient enzymatic biofuel cells is a subject of considerable studies in past decades for potential applications such as biomedical devices,1−3 microchip systems,4,5 and portable electronics.6 Since biofuel cells produce electrical power upon consuming ranges of biomass such as alcohols and glucose, their renewable nature has been highlighted. One of the key challenges in advancing the technology lies in power densities of biofuel cells. Limitations have been arisen from the buried redox active sites within enzyme structures, which result in poor interplay between redox reactions and electron transfer. In fact, the low power density of enzymatic biofuel cells significantly hinders the development of power systems for miniaturized biomedical devices.7 Several different approaches to enhance the power density of biofuel cells are proposed.8,9 One of widely studied methodology is the use of electron mediators to help electron transfer in the systems by shuttling electrons between enzyme catalytic centers and the current collector. Various materials possessing the ability of electron mediation have been extensively reported in recent years.9−18 Example includes nanocarbons,10−12 redox active polymers,13−15 cofactor relay,16 and metal nanoparticles.17,18 To attain more stable and higher current densities of biofuel cells in the presence of electron mediators, the essential requirement is the immobilization of enzymes to the electrodes. Consequently, significant amounts of studies on the electron mediators are concerned with the electrical contact of enzymes to the electrode surfaces with covalently attached mediators.19,20 © 2012 American Chemical Society

Received: January 19, 2012 Revised: March 6, 2012 Published: March 21, 2012 3121

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

the pioneering studies of Manners et al.32,33 where the synthesis and self-assembly nature of Fc containing block copolymers are discovered. The Fc moieties packed within the self-assembled structures are particularly aimed at improving the electron transfer rate between the flavin adenine dinucleotide (FAD) cofactor in GOx and an electrode surface.34

■

EXPERIMENTAL SECTION

Synthesis of Organometallic Block Copolymers. A ferrocenecontaining monomer, 1,1′-dimethylsilylferrocenophane monomer, was first synthesized by coupling lithiated ferrocene with silane according to the methods described by Manners et al.33 The synthesized monomer was purified by repeated sublime/recrystallization processes under vacuum. Poly(ferrocenyldimethylsilane-b-isoprene) (PFS−PI) copolymers with different molecular weights were synthesized by sequential anionic polymerization.35 The PI precursor synthesis was performed in purified benzene as a solvent and sec-butyllithium as an initiator. An aliquot of the living PI precursor was isolated in the glovebox for molecular weight determination. The aliquot was terminated using methanol. If the molecular weights of the PI precursor were close to target values, a preweighed amount of the purified 1,1′-dimethylsilylferrocenophane monomer was added to the reactor in the glovebox. The reactor was then returned to the vacuum line and was thoroughly degassed. A small quantity of purified THF was distilled into the reactors to speed up the polymerization of the 1,1′-dimethylsilylferrocenophane monomer. As the 1,1′-dimethylsilylferrocenophane monomer polymerized, the color of the reaction solution changed from red to brown. The polymerization proceeded for at least 6 h and then was terminated in the glovebox with isopropanol. Upon termination, the color of the solution changed from brown to red. The PFS−PI copolymers were isolated by precipitation in hexane. The molecular weights and molecular weight distribution of PFS−PI copolymers were characterized by combining 1H nuclear magnetic resonance (NMR, Bruker AVB-300) spectroscopy and gel permeation chromatography (GPC, Waters Breeze 2 HPLC). The 1H NMR spectra were taken by dissolving the copolymers in deuterated choloroform. For GPC experiments, THF was used as an eluent, and the results were calibrated with respect to polystyrene standards (Polymer Standard Service). Molecular weights of the PFS−PI coplymers were determined as 10.4−8.0 and 69.0−92.0 kg/mol where the polydispersity indices were less than 1.08. The molecular structure of PFS−PI copolymers is shown in Figure 1a. Electrode Preparation. Glucose oxidase from Aspergillus niger (∼200 units/mg, Lot # 001330219) is purchased from Sigma-Aldrich. GOx is deposited on porous carbon electrode by dropping aliquots of 60 μM GOx stock solution until targeted mass of 2 mg is attained. The GOx-deposited electrode is then completely dried under Ar blanket for 2 days. Homogeneous 0.1 wt % PFS−PI solutions prepared by different solvent mixtures are immediately drop-coated onto the dried electrode by varying the thickness of the PFS−PI layer. The thickness of electrode comprising the GOx and PFS−PI was determined using a XL30S FEG Philips scanning electron microscope (SEM). After complete evaporation of solvents in vacuum oven, the electrodes were exposed to OsO4 vapor for 3 h. Note that the OsO4 staining procedures are dangerous, and careful cautions are required. After Os decoration, the electrodes were stored in PBS buffer solution before use. FT-IR Spectroscopy. Infrared spectra were recorded at room temperature using a Vertex 70 FT-IR spectrometer. Before the measurements, 30 min of equilibration time was allowed under N2 gas purge, and 32 accumulations were signal-averaged at a resolution of 1 cm−1. Baseline corrected infrared spectra were obtained for the PFS−PI copolymers on silicon wafers in transmission mode. Morphology Characterization. Surface morphologies of fabricated electrodes were determined by AFM (Digital Instruments, Nanoscope III) in tapping mode. All measurements were made with a phase contrast value of 10°. Samples for transmission electron microscopy (TEM) experiments were prepared by drop-coating of 0.1 wt % PFS−PI copolymers in various solvent mixtures. All imaging have been performed before OsO4 decoration due to the enough electron

Figure 1. (a) Molecular structure of PFS−PI. TEM images of PFS−PI (10.4−8.0 kg/mol); (b) in toluene/hexane (20/80 vol %), (c) EELS image of (b), and (d) in THF/hexane (20/80 vol %). (e) X-ray diffraction patterns of bicontinuous, nanowire PFS−PI copolymers and PFS homopolymer. TEM images of PFS−PI (69.0−92.0 kg/mol); (f) in THF/hexane (20/80 vol %) and (g) in THF/hexane (25/75 vol %). All TEM samples are prepared by drop-coating on Formvar-coated Cu grids. contrast between Fe-rich domains and PI domains using a Zeiss LIBRA 200FE microscope operating at 200 kV equipped with a cold stage (−160 °C) and an Omega energy filter. Fe elemental mapping was obtained using energy filtered imaging with the three-window method at the Fe L edge of 709 eV energy loss. Focused ion beam etched TEM (cross-sectional FIB-TEM) samples were prepared with a FEI Strata 235 dual beam FIB system operated at 30 kV. For the FIBTEM imaging, surface protection of samples was accomplished by exposure to RuO4 vapor for 10 h. Imaging of etched samples was performed with a JEOL 3010 microscope operated at 200 kV. Catalytic Activity Characterization. Cyclic voltammetry (CV) measurements were recorded using a EG&G PAR 273A in a threeelectrode cell with a platinum gauze as counter electrode and Ag/ AgCl/(KCl saturated) as reference electrode. The working electrode was a porous carbon electrode. All measurements were performed in phosphate buffered saline (PBS) (1× PBS in 1 L of deionized water; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 2.0 mM 3122

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

Figure 2. Fabrication process of functional electrodes comprised of GOx and PFS−PI mediators. For brevity, we only show representative graphics for the case of nanowire morphology. KH2PO4, adjusted to pH 7.4) at 25 °C and ambient atmosphere. The conductivities of PFS−PI films were measured using ac impedance spectroscopy in the presence of PBS buffer solution using a home-built two-electrode cell with 1 cm × 1 cm Pt working/counter electrodes. Data were collected using a 1260 Solatron impedance analyzer operating over a frequency range of 1−100 000 Hz.

that slight increase in THF amounts to 25 vol % results in short and relatively nonuniform nanowires, as given in Figure 1g. This implies that the nanoparticle morphology is only stable at the narrow window of THF/hexane compositions. It is worthwhile to mention here that the nanowire morphology of PFS−PI block copolymers is well-discovered by Manners et al.32 while the bicontinuous and nanoparticle morphologies were first discovered in the present study. The nanostructured PFS−PI organometallic polymers are then utilized to transfer electrons from redox reactions of GOx/ glucose to electrodes. Figure 2 illustrates the fabrication process of the electrode comprised of GOx and PFS−PI mediators. GOx is deposited on porous carbon electrode by dropping aliquots of 60 μM GOx stock solution until targeted mass (2 mg) is attained. The GOx deposited electrode is then completely dried under Ar blanket for 2 days, followed by dropcoating of homogeneous 0.1 wt % PFS−PI solutions onto the dried electrode by varying the thickness of the PFS−PI layer. The maximum catalytic current is observed for GOx concentration of 30 wt %. At very high enzyme contents above 40 wt %, strong precipitation of GOx was observed. In present study, PI was particularly chosen as a supporting matrix since the diene groups in PI chains allow us to introduce cross-linking points. The chemical cross-linking has been carried out by exposing the electrodes to OsO4 vapor for 3 h in which the OsO4 moieties simultaneously form covalent bonds to hydrocarbon groups in close proximity; hereafter, the OsO4 cross-linked PFS−PI is referred to as PFS−PIOS. It should be noted here that no noticeable changes in GOx activity owing to the exposure to OsO4 vapor was confirmed by monitoring the catalytic activity of GOx as a function of exposure time. In addition, without Os decoration, the film becomes unstable within the physiological environments. As a final step, activation of Fc moieties is carried out by applying anodic potential sweep. The activation process prior to the addition of glucose helps to obtain improved current values when glucose is added. Because the Fc moieties will be positively charged after activation, it is expected to enhance association with the negatively charged GOx. The covalent cross-linking reaction between OsO4 and PI chains and the chemical stability of the PFS−PIOS electrode under various solution conditions are confirmed by FT-IR experiments. As shown in Figure 3a, the IR peak intensities from 2800 to 3100 cm−1 corresponding to characteristic peaks

■

RESULTS Nanoscale Morphologies of PFS−PI Redox Polymers. Figure 1a shows the chemical structure of PFS−PI copolymers where x and y indicate degree of polymerization of each blocks. We begin by describing the morphologies of PFS−PI copolymers. Figures 1b−d,f,g show the representative TEM images obtained from solution-cast PFS−PI copolymers. We found that PFS−PI copolymers offer various nanostructures when molecular weights and/or solvent compositions are altered. For example, a low molecular weight PFS−PI (10.4− 8.0 kg/mol) in toluene/hexane (20/80 vol %) mixtures yields a bicontinuous structure with spacings of ca. 100 nm, as shown in Figures 1b,c. Figure 1c is obtained by Fe elemental map to confirm that the dark region in Figure 1b is Fc-rich area. When THF/hexane (20/80 vol %) mixture is employed, in contrast, distinctly different morphology of nanowires with the lengths of several micrometers is seen, as shown in Figure 1d. The diameter of the nanowires is 20 nm, which only reflects the size of PFS domains. It should be noted here that the solubility parameter of PFS (18.6 MPa1/2) is quite close to the values of toluene (18.3 MPa1/2) and THF (18.5 MPa1/2) while the values of n-hexane (14.9 MPa1/2) and PI chains (16.2 MPa1/2) are relatively close.36 This implies that the dissimilar morphologies depending on the choice of solvents are not originated from the solubility issues. Instead, it is found that the crystalline characteristics of PFS are significantly affected by the choice of the core-filling solvents. In Figure 1e, we compared X-ray diffraction (XRD) patterns of bicontinuous PFS−PI (toluene, amorphous) and nanowire PFS−PI (THF, crystalline). For a reference, a XRD profile of PFS homopolymer (melt processed) with molecular weight of 8.1 kg/mol is also given in the Figure 1e. While keeping the THF/hexane (20/80 vol %) mixtures, the use of large molecular weight PFS−PI (69.0−92.0 kg/mol) results in fairly monodisperse nanoparticles with size of 55 nm (the darker phase is corresponding to core PFS domains and PI shell was not visible), as shown in Figure 1f. We note in passing 3123

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

Figure 3. FT-IR data of PFS−PI electrodes (a) before/after OsO4 staining and (b) under various experimental conditions. Figure 4. (a) FIB-TEM images of the GOx integrated PFS−PIOS nanowire electrode and (b) SEM images of the GOx incorporated bicontinuous PFS−PIOS electrode; cross-sectional view and top view.

of polyisoprene were significantly reduced, as a result of Os staining. The reaction yield was ∼70% at ca. 200 nm thick PFS−PI film. By examining the low wavenumber region, we found the appearance of new peaks at 993 and 645 cm−1, owing to the development of C−O and Os−O bonds, respectively.37 The absence of OsO peaks signals that most OsO4 takes part in cross-linking reaction, which is in good agreement with the results in ref 38. Upon exposing the PFS−PIOS to various experimental conditions, the IR data given in Figure 3b indicate no detectable degradation of the PFS−PIOS electrodes in both PBS buffer solutions (our experimental condition) and 10 wt % H2O2 containing PBS buffer solutions (severe condition). Note that all IR measurements were carried out under N2-filled conditions, and the low wavenumber regions of Figures 3a,b are intentionally magnified to clarify the ester peaks. The structural analysis of the fabricated electrodes is elucidated by combining focused ion beam technique (FIB), TEM, and SEM. Figure 4a presents the FIB-TEM images of the GOx integrated PFS−PIOS nanowire electrode. To minimize the sample damage during milling process, Ru is deposited on the surface of the electrode. As clearly seen from Figure 4a, the electrode comprises layers of PFS−PIOS mediators and GOx with some intermixing and interpenetration of these materials. The thickness of PFS−PIOS layer at the top surface of the electrode is ∼100 nm. The cross-sectional view of nanowires with 20 nm diameter is shown in the upper right inset image. The intermixed/interpenetrated layers are also revealed, as shown in the inset boxes, where the GOx is detected as 40− 80 nm sized aggregates. It should be noted here that the GOx was extremely unstable under the electron beam even in the presence of cold stage (−160 °C). Typical example of GOx degradation is shown inside the inset box with different contrast, as indicated in the Figure 4a. The structure of GOx integrated bicontinuous PFS−PIOS electrode was investigated by SEM experiments. As shown in Figure 4b, the top view of the fabricated electrode is well agreed with the TEM image seen in Figure 1b. Upon examining the

cross-sectional structure of the electrode, it is found that the electrode is porous that glucose has access to the enzymes, yet it provides a protective cage for immobilizing the GOx without affecting biological function. Note that the current collector is intentionally delaminated with an aid of liquid nitrogen to examine the topology of bottom side of the electrode. As can be seen from Figure 4b, the bicontinuous morphology was again revealed, which leads us to conclude that PFS−PIOS mediators exist at both air surface and the substrate. Morphology-Sensitive Redox Properties of the GOx Integrated PFS−PIOS Electrodes. To demonstrate the efficiency of GOx wiring onto the electrode, assisted by the networks of PFS−PIOS mediators, we first carried out cyclic voltammetry (CV) measurements of the fabricated electrode. The concentration of the Fc moiety was fixed as 2 mM and a low scan rate of 20 mV was used. As shown in Figure 5a, in the absence of GOx, well-defined current responses upon electrochemical cycling of the Fc moieties are detected at 420 and 550 mV. Note that the standard reduction potential of Fc+ (versus SHE) is known as E° = 400 mV. When GOx is incorporated into PFS−PIOS nanowires, however, distinctly difference redox responses are seen. New anodic and cathodic waves at 390 and −80 mV were detected. The shift of oxidation peak from 550 to 390 mV reflects the change in solvation of the Fc moieties due to the polar environment provided by GOx.39 The disappearance of Fc reduction peak at 420 mV implies that Fc+ is clearly used up in the catalytic regeneration of GO(FAD): GO(FADH2) + 2Fc+ → GO(FAD) + 2Fc° + 2H +

(1)

where Fc regenerated in the catalytic cycle is again oxidized to Fc+ at the anode. In the absence of glucose, the NaH2PO4 in PBS buffer solution presumably acts as proton donor, as described in the literature.40,41 Such characteristic change in electrochemical response of the electrode indicates that catalytically active GOx has 3124

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

Figure 5. (a) CVs of electrodes with PFS−PIOS nanowires in the absence/presence of GOx. CVs of electrodes with (b) GOx/PFS−PIOS nanowires and (c) GOx/bicontinuous PFS−PIOS as a function of glucose concentration. The inset plots in (b) and (c) show the current values at anodic peaks. (d) CVs of the electrodes with various PFS−PIOS morphologies at 8 mM glucose. The inset AFM images show the surface topology of the electrodes. A scan rate of 20 mV/s is used.

been successfully embedded into the network of PFS−PIOS, yielding the effective communication with Fc sites. To explore the morphology effects on catalytic responses of the electrodes, CVs of electrodes with different morphologies were recorded as a function of glucose concentration. As given in eq2, FAD is reduced to FADH2 by glucose and Fc+ is reduced to Fc° in the catalytic regeneration of GO(FAD).

mediators so that less than 2% of the peak current was lost over 75 consecutive redox cycles. This implies that no considerable leaching of GOx occurs from the fabricated anodes. Interestingly, when the morphology effect was further exploited by employing nanoparticle-forming PFS−PIOS, fairly negligible redox activity is seen, signaling insufficient communication between Fc moieties and GOx due to the isolated spherical PFS domains. On the basis of the insignificant catalytic responses of PFS−PIOS nanoparticles, we can infer that the Os moieties in PI chains do not play a role in electron mediation due to the loosely packed characteristics. Figure 5d summaries the morphology effects on the current density of GOx/PFS−PIOS electrodes. We plotted only one representative set of CV data at glucose concentration of 8 mM. Among nanowire, nanoparticle, and bicontinuous morphology, the GOx confined by bicontinuous PFS exhibits the best activity for the catalytic reactions. This was also demonstrated from the lower potential (−150 mV) for the regeneration of FAD with bicontinuous PFS−PIOS than the value (−80 mV) of PFS−PIOS nanowire. In Figure 6, we provided schematic diagram summarizing Fcmediated catalysis of glucose. The morphology of PFS−PI electron mediators (drawn as a green layer in the scheme) was varied as bicontinuous, nanowire, and nanoparticle, as confirmed by AFM experiments on the electrodes in tapping mode. Typical examples of AFM images representing the surface topology of fabricated electrodes are given in Figure 6. It has been revealed that the morphology of PFS−PI electron mediators is an important parameter in determining the charge transfer rate of GOx-wired electrodes. It is worthwhile to compare the catalytic properties of GOx integrated nanostructured PFS−PIOS polymers with those of

When the PFS−PIOS nanowire is utilized, as shown in Figure 5b, even with 1.3 mM of glucose, 40% increment in peak currents is seen, reflecting Fc-mediated catalysis of glucose. With the increase in the amount of glucose, catalytic currents gradually increase until the values level off at 60 mM of glucose. In the inset figure of Figure 5b, we plot the anodic current at 390 mV as a function of glucose concentration. The inset AFM image shows the surface topology of the fabricated electrode. What is surprising is that the redox process of the electrodes appears to be dependent on the morphology of PFS−PIOS copolymers. When the morphology of PFS−PIOS is switched to bicontinuous structure, as shown in Figure 5c, analogous redox waves to the case of nanowire are seen. However, almost 2 times increase in the peak current is detected at the same level of glucose concentration, and the current at 390 mV reaches to high and stable value of 700 μA/cm2 at 70 mM glucose, as shown in the inset plot of Figure 5c. The inset AFM image shows the surface topology of the electrode. It should be noted here that the electrodes were very stable regardless of morphologies of PFS−PIOS 3125

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

morphologies were measured at 25 °C in PBS buffer solution to elucidate the morphology effects, which should be a key ingredient in modeling how electron transfer reactions couple to GOx and substrate. Interestingly, the conductivities of activated PFS−PIOS films diverge as 1.34 × 10−5 (bicontinuous) and 4.03 × 10−6 S/cm (nanowire). Consequently, the enhanced catalytic response of GOx integrated bicontinuous PFS−PIOS electrode can be rationalized by the better connectivity of the bicontinuous structure as well as larger contact area between PFS domains and entrapped GOx. From the data obtained so far, there is no doubt about the role of morphologies of the redox copolymers on determining the electron-mediating ability of functional electrodes. Our results are intriguing since while a number of publications described the successful use of Fc-based redox polymers,28−31 none of them demonstrated that the electrode properties are strongly affected by self-assembled nanostructures of the redox polymers. We hope to conclude our paper by exploring the biosensing ability of the electrodes. The biocatalytic activity of the electrodes was evaluated upon the injection of glucose with different concentrations. Because the physiological concentration of glucose in blood is between 5 and 10 mM, it is essential to obtain sufficient electrical signal at low amount of glucose. The electrode prepared with the bicontinuous PFS− PIOS was tested and the significantly decreased amount of GOx (0.2 mg) was employed to evaluate the sensitivity of the fabricated electrodes. The current responses at anodic wave around 400 mV vs Ag/AgCl were recorded with time. As shown in Figure 7, the addition of glucose to the physiological

Figure 6. Schematic diagram illustrating Fc-mediated catalysis of glucose and AFM results of electrodes in tapping mode representing nanostructures of PFS−PI electron mediators (drawn as a green layer in the scheme).

GOx-wired other redox polymers reported widely in the literature. In Table 1, we listed anodic current densities of other systems by focusing on Os- and Fc-based redox polymers. All polymers in the literature possess the redox active sites in pendant groups, and self-assembled morphologies were not identified. As seen from the table, the anode comprising bicontinuous PFS−PIOS indicates considerably improved current densities despite rather small amounts of GOx in the electrode. This could be intimately related to the enhanced charge transport rate in the nanostructured PFS−PIOS. Table 1. Anodic Current Densities of GOx Integrated Redox Polymers Reported in the Literature and Observed in Present Study redox polymera

GOx activity (U)

GOx amounts applied (mg)

glucose concn (mM)

pH

Os

b

b

15

5

Os

150

4

15

7

Os

b

b

15

7

Fc

150

150

100

7

Fc/CNT Fc

150 200

b 2

40 60

5 7

15

max current density 360 μA/cm2 200 μA/cm2 11 μA/mm2 3000 μA/cm2 40 μAc 664 μA/cm2 d

ref 42 43 44 45 46 present study

Figure 7. Current responses of a glucose sensor composed of GOx and bicontinuous PFS−PIOS with the injection of glucose; the arrows indicate the injections of glucose.

455 μA/cm2 d

environments causes a step changes in the observed current with a final steady state value proportional to the concentration of glucose, as a result of continuous electron transfer from the enzyme to the Fc units. In particular, even with fairly small amount of glucose, 1.3 mM, a rapid and obvious current response was revealed by a discontinuous increase in peak current from 7 to 11 μA. Approximately 35 min of equilibrium is allowed at each glucose concentration and the electrode exhibited excellent stability. The strategy demonstrated here is believed to be generally applicable for the development of other kinds of enzymatic

a

Only redox active moieties are marked. bThe information is unknown in the literature. cArea of the electrode is unknown. dCurrent densities were obtained with bicontinuous PFS−PIOS.

■

DISCUSSION

Then, what would be the origin of the higher current values with bicontinuous PFS−PIOS than those with other morphologies? The conductivities of PFS−PIOS films with different 3126

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

(3) Hansen, B. J.; Liu, Y.; Yang, R.; Wang, Z. L. ACS Nano 2010, 4 (7), 3647. (4) Chen, T.; Barton, S. C.; Binyamin, G.; Gao, Z.; Zhang, Y.; Kim, H. H.; Heller. J. Am. Chem. Soc. 2001, 123, 8630. (5) Moore, C. M.; Minteer, S. D.; Martin, R. S. Lab Chip 2005, 5 (2), 218. (6) Heller, A. Phys. Chem. Chem. Phys. 2004, 6 (2), 209. (7) Mano, N.; Soukharev, V.; Heller, A. J. Phys. Chem. B 2006, 110 (23), 11180. (8) Blanford, C. F.; Heath, R. S.; Armstrong, F. A. Chem. Commun. 2007, 17, 1710. (9) Deng, L.; Wang, F.; Chen, H.; Shang, L.; Wang, L.; Wang, T.; Dong, S. Biosens. Bioelectron. 2008, 24, 329. (10) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Adv. Mater. 2006, 18 (19), 2639. (11) Zeng, G.; Xing, Y.; Gao, J.; Wang, Z.; Zhang, X. Langmuir 2010, 26 (18), 15022. (12) Miyake, T.; Yoshino, S.; Yamada, T.; Hata, K.; Nishizawa, M. J. Am. Chem. Soc. 2011, 133 (13), 5129. (13) Gallaway, J. W.; Barton, S. C. J. Am. Chem. Soc. 2008, 130 (26), 8527. (14) Scodeller, P.; Carballo, R.; Szamocki, R.; Levin, L.; Forchiassin, F.; Calvo, E. J. J. Am. Chem. Soc. 2010, 132 (32), 11132/. (15) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125, 4951. (16) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124 (10), 2120. (17) Scodeller, P.; Flexer, V.; Szamocki, R.; Calvo, E. J.; Tognalli, N.; Troiani, H.; Fainstein. J. Am. Chem. Soc. 2008, 130 (38), 12690. (18) Yehezkeli, O.; Tel-Vered, R.; Raichlin, S.; Willner, I. ACS Nano 2011, 5 (3), 2385. (19) Carturan, G.; Toso, R. D.; Boninsegna, S.; Monte, R. D. J. Mater. Chem. 2004, 14, 2087. (20) Lim, J.; Malati, P.; Bonet, F.; Dunn, B. J. Electrochem. Soc. 2007, 154, A140. (21) Gao, F.; Viry, L.; Maugey, M.; Poulin, P.; Mano, N. Nat. Commun. 2010, 1, 2. (22) Yan, Y.; Zheng, W.; Zhang, M.; Wang, L.; Su, L.; Mao, L. Langmuir 2005, 21, 6560. (23) Girishkumar, G.; Vinodgopal, K.; Kamat, P. V. J. Phys. Chem. B 2004, 108, 19960. (24) Ning, J. W.; Zhang, J. J.; Pan, Y. B.; Guo, J. K. J. Mater. Sci. Lett. 2003, 22, 1019. (25) Lyons, M. E. G.; Keeley, G. P. Chem. Commun. 2008, 22, 2529. (26) Gallaway, J.; Wheeldon, I.; Rincon, R.; Atanassov, P.; Banta, S.; Barton, S. C. Biosens. Bioelectron. 2008, 23, 1229. (27) Hirsch, A. Angew. Chem., Int. Ed. 2002, 41, 1853. (28) Abd-EI-Aziz, A. S.; Manners, I. Frontiers in Transition MetalContaining Polymers; John Wiley & Sons, Inc.: Hoboken, NJ, 2007. (29) Wang, J. Chem. Rev. 2008, 108 (2), 814. (30) Ammam, M.; Fransaer, J. Biosens. Bioelectron. 2010, 25, 1474. (31) Barton, S. C.; Kim, H. H.; Binyamin, G.; Zhang, Y. C.; Heller, A. J. Phys. Chem. B 2001, 105, 11917. (32) Wang, X.; Liu, K.; Arsenault, A. C.; Rider, D. A.; Ozin, G. A.; Winnik, M. A.; Manners, I. J. Am. Chem. Soc. 2007, 129 (17), 5630. (33) Ni, Y. Z.; Rulkens, R.; Manners, I. J. Am. Chem. Soc. 1996, 118 (17), 4102. (34) Gallardo, B. S.; Gupta, V. K.; Eagerton, F. D.; Jong, L. I.; Craig, V. S.; Shah, R. R.; Abbott, N. L. Science 1999, 283, 57. (35) Rulkens, R.; Lough, A. J.; Manners, I. J. Am. Chem. Soc. 1994, 116, 797. (36) Muthukumar, M.; Ober, C. K.; Thomas, E. L. Science 1997, 277, 1225. (37) Brumley, W. C.; Hinkcley, C. C. J. Inorg. Nucl. Chem 1980, 42, 1277−1284. (38) Ribbe, A. E.; Bodycomb, J.; Hashimoto, T. Macromolecules 1999, 32 (9), 3154−3156. (39) Ju, H.; Leech, D. Phys. Chem. Chem. Phys. 1999, 1 (7), 1549. (40) Ma, M.; Bong, D. Langmuir 2011, 27, 8841−8853.

biofuel cells and could prove to be a new and facile route to nanostructured enzymatic biofuel cells. In particular, efficient electron transfer between enzymes and redox polymers as a result of structural optimization will open the way to design sensors for other biomass such as alcohol and amino acids. The investigation on the power density of biofuel cell by combining cathode enzyme is currently under way.

■

CONCLUSIONS The catalytic activities of functional electrodes comprising glucose oxidase and cross-linkable, nanostructured ferrocenecontaining organometallic block copolymers were investigated. Unlike non-microphase-separated redox polymers, the use of nanostructured PFS−PI block copolymers allow us to control electron transfer rate. The improved stability of the enzyme integrated electrode by osmium decoration is found out where dienes in PI chains are selectively cross-linked. We have demonstrated that morphologies of organometallic polymers play a key role in determining the electrochemical properties of enzyme integrated electrodes. In other words, the different morphologies of the same organometallic polymer yield significant discrepancy in current densities of the fabricated electrodes at the same glucose concentration. The utilization of bicontinuous structure forming PFS-b-PIOS reveals remarkable enhancement in catalytic currents and good glucose sensitivities at low glucose concentrations. These results were rationalized by better connectivity in bicontinuous structure than other morphologies as well as large contact area of PFS domains with glucose oxidase. Our results are the first experimental evidence on the adjustable catalytic current densities of enzyme-wired organometallic polymer electrodes with the successive tuning of their morphologies.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by Basic Science Research Program (Project No. 2011-0004375) and Midcareer Researcher Program (Project No. 2011-0015343) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. We also acknowledge WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (Project No. R31-10059). XRD measurements were conducted on the beamline 1W1A under the approval of the Beijing Synchrotron Radiation Facility and the support of PAL through the abroad beamtime program of Synchrotron Radiation Facility Project under MEST. We gratefully acknowledge Prof. Andrew M. Minor for providing access to the TEM instrument of the National Center for Electron Microscopy, Lawrence Berkeley Lab, which is supported by the U.S. Department of Energy under Contract DE-AC02-05CH11231. Prof. M. J. Park acknowledges a Chong-Am Science Fellowship.

■

REFERENCES

(1) Willner, I. Science 2002, 298, 2407. (2) Barton, S. C.; Gallaway, J.; Atanassov, P. Chem. Rev. 2004, 104, 4867. 3127

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128

Macromolecules

Article

(41) Alvarez-Pez, J. M.; Ballesteros, L.; Talavera, E.; Yguerabide, J. J. Phys. Chem. A 2001, 105, 6320−6332. (42) Mano, N.; Mao, F.; Shin, W.; Chena, T.; Heller, A. Chem. Commun. 2003, 518. (43) Kim, H. H.; Mano, N.; Zhang, X. C.; Heller, A. J. Electrochem. Soc. 2003, 150, A209. (44) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2002, 124, 12962. (45) Tamaki, T.; Yamaguchi, T. Ind. Eng. Chem. Res. 2006, 45, 3050. (46) Tan, Y. M.; Xie, Q. J.; Huang, J. H.; Duan, W. S.; Ma, M.; Yao, S. Z. Electroanalysis 2008, 20, 1599.

3128

dx.doi.org/10.1021/ma300155u | Macromolecules 2012, 45, 3121−3128