BuckypaperâBilirubin Oxidase Biointerface for Electrocatalytic

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Buckypaper-Bilirubin Oxidase Biointerface for Electrocatalytic Applications: Buckypaper Thickness Charuksha Walgama, Anuruddha Pathiranage, Mayowa Akinwale, Roberto Montealegre, Jinesh Niroula, Elena Echeverria, David McIlroy, Tres A Harriman, Don A. Lucca, and Sadagopan Krishnan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00189 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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ACS Applied Bio Materials

Charuksha Walgama, a,‡ Anuruddha Pathiranage,a Mayowa Akinwale, a Roberto Montealegre,a Jinesh Niroula,a Elena Echeverria,b David N. McIlroy,b Tres A. Harriman,b Don A. Lucca,c and Sadagopan Krishnan a,* a

Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078, United States. Department of Physics, Oklahoma State University, Stillwater, Oklahoma 74078, United States. c School of Mechanical and Aerospace Engineering, Oklahoma State University, Stillwater, Oklahoma 74078, United States. b

ABSTRACT: Electrode materials play an important role on the electrocatalytic properties of immobilized biocatalysts. In this regard, achieving direct electronic communication between electrode and redox sites of biocatalysts eliminates the need for additional electron transfer mediators for biocatalytic applications in fuel cells and other electrochemical energy devices. In order to increase electrocatalytic currents and power in fuel cells and metal-air batteries, conductive carbon nanostructures modified large surface area electrodes are quite useful. Among various electrode materials, freestanding buckypapers made from carbon nanotubes have gained significance as they do not require a solid support material and thus facilitate miniaturization. In this report, we present the effect of buckypaper (BP) thickness on the electrocatalytic properties of bilirubin oxidase (BOD) enzyme. In this study, we prepared BPs of varying thicknesses ranging from 87 micron, the minimum thickness for suitable handling with good stability in aqueous experiments, to 380 micron. BOD was adsorbed overnight onto the BPs, mostly via hydrophobic and pi-pi interactions since the nanotubes used were as produced and not chemically functionalized; and additionally via possible intercalation of BOD onto the nanotubes’ multi cylindrical network. We determined that the lower range BP thickness (< 220 micron) exhibited better sigmoidal shaped electrocatalytic currents than the higher BP thickness based BOD biofilms with larger capacitive currents. Oxygen reduction current density of up to 3 mA cm–2 is achieved without use of any redox mediators or tedious electrode modifications. Using the 87 micron thick BP as the representative case, we were able to obtain distinguishable peaks for all Cu sites of BOD, and assigned their types, T1, T2, and T3, based on peak-width at half-maximum in anaerobic cyclic voltammograms. Our peak assignment is further supported by the appearance of dual electrocatalytic reduction waves at higher scan rate region (> 10 mV s -1) in oxygen-saturated buffer, which is identified to be driven by an ~ 3.5-times faster electron transfer rate from the buckypaper to the T2/T3 center than the T1 Cu site. Findings from this study are significant for designing enzyme electrocatalytic systems and biosensors in general, and fuel cells and aerobic energy storage devices in particular, where the cathodic oxygen reduction current is often inadequate. KEYWORDS: Buckypaper thickness, Bilirubin oxidase, Copper sites, Direct electron transfer, Enzyme Electrocatalysis

1. INTRODUCTION Delivering electrons from nanostructure-modified electrode surfaces to redox enzymes is a fundamental requirement for driving electrocatalytic reactions that are useful for synthesis, sensors, energy, catalysis, and designing of bioelectronic devices.1-5 Thermodynamic and kinetic properties of biological redox processes depend on the heterogeneous direct electron transfer (DET) reactions between a redox enzyme and the electrode surface in contact with the enzyme. 6 The DET properties enable the enzyme molecules to function at high turnover rates under ambient conditions in simple aqueous solutions. Among different nanomaterials, carbon nanotubes (CNTs) exhibit excellent electronic, optical, and mechanical properties because of their unique molecular and morphological features.7-9 Buckypaper (BP) made out of CNTs is an emerging class of free standing electrodes (i.e., no solid support material is required) for electrocatalytic and fuel cell applications.10 -12 Among numerous electrocatalytic enzymatic reactions, the four-electron oxygen reduction is the most important cathodic reaction in aerobic energy storage devices such as fuel cells and metal-air batteries. Bilirubin oxidase (BOD) is a multicopper oxidase and is the most efficient electrocatalyst known to date

for catalyzing the kinetically challenging four-electron reduction of molecular oxygen to water under neutral conditions.13 Heller et al. reported that BOD (with its abundant copper (Cu) centers) wired onto electrodes enabled oxygen reduction in buffer electrolytes at lower overpotentials and higher current densities than a Pt metal catalyst.14 Consequently, studying the electrochemical properties of BOD has attracted considerable attention for its applications as a biocathode in biological fuel cells. 15-17 The catalytic center of BOD contains four Cu atoms that are spectroscopically categorized into three types, namely, T1, T2, and T3.13 The crystal structure of BOD present in the fungus Myrothecium verrucaria shows that the T2 Cu atom and the two T3 Cu atoms are arranged as a trinuclear cluster (TNC) located at a distance of approximately 13 Å from the T1 Cu atom. Similar to other multicopper oxidases, in the natural catalytic reaction, the electrons released in the oxidation of the bilirubin substrate are accepted by the T1 center and then transferred to TNC to aid reduction of oxygen to water.18 In a BOD film immobilized on an electrode, both the T1 and TNC centers are located at a reasonable distance (< 14 Å) from the protein surface to facilitate the tunneling of electrons from the solid electrode support under an applied electrochemical

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driving force (i.e., potential). Shleev et al. described the direct electron transfer properties of BOD under both aerobic and anaerobic conditions on graphite and metal electrode surfaces.1922 Another report confirmed the redox potentials of the T1 center and TNC of BOD by spectroelectrochemical redox titrations.23 Chemical modifications to orient BOD on the electrode surface has also been achieved.24,25 DET of T1 and combined TNC Cu sites with the electrode in anaerobic conditions has also been reported.26 Similarly, buckypaper-based bioelectrodes featuring BOD for mediated electrocatalysis have been demonstrated. However, the effect of bucky paper thickness in achieving electrochemical separation of all three Cu sites of BOD and additionally obtain insights into direct electron transfer and electrocatalytic properties have not been reported yet. In this study, we prepared flexible, freestanding, and aqueous solution stable BPs of different thicknesses using multiwalled carbon nanotubes (MWNTs) by the vacuum filtration method,27 and immobilized the BOD molecules by simple adsorption. We are able to obtain the DET features of the T1, T2, and T3 Cu sites from an adsorbed film of BOD on a freestanding BP strip of 87-µm thickness. Further, the electrocatalytic features are discussed with an increase in buckypaper thickness. 2. EXPERIMENTAL SECTION 2.1 Materials and Methods. MWNTs was a gift from SWeNT Inc. (Norman, OK, USA) [SWeNT SMW200 Lot#SMW200-L7, ≥ 98% carbon basis, O.D. × I.D. × L = (10 nm ± 1 nm) × (4.5 nm ± 0.5 nm) × (3–6 µm), 6–8 tube walls]. BOD (from Myrothecium verrucaria) was purchased from Amano Enzyme Inc. (Nagoya, Aichi, Japan) and used as received. Cyclic voltammetry studies were carried out in a CH Instrument 6017E electrochemical analyzer (Austin, TX, USA). Measurements were made on a standard three-electrode cell consisting of an Ag/AgCl reference electrode (1 M KCl, CH Instruments), a Pt-mesh counter electrode, and a BP working electrode adsorbed with a film of BOD. The capacitance values of bare BPs with different thicknesses in the absence of enzyme were calculated using the capacitive currents measured for increasing scan rates. The usability of the prepared paper as an electrode was tested using phosphate buffer solution containing a mixture of Fe(CN)6–3/–4 (10 mM each). Amperometry experiment of the BP/BOD electrodes to assess electrocatalytic stability was carried out at an applied potential of +0.5 V (vs. Ag/AgCl reference) in an oxygen-purged phosphate buffer, pH 6.5 at 25 °C. The measured Ag/AgCl potential scale was converted to standard hydrogen electrode (SHE) using the following conversion: ESHE = EAg/AgCl + 0.206 V at 25 oC. 2.2 Preparation of BP/BOD electrodes. MWNTs were dispersed by ultrasonication in dimethylformamide solvent for 4 h at room temperature. The dispersed MWNTs were vacuum filtered using a PTFE filter (Sigma, pore size 0.45 μm, diameter 47 mm) to obtain disks of BPs of various thicknesses by controlling the amount of MWNTs used.27 They were further dried using physical methods (compression and heat pressing using a blow drier) to remove any residual solvents. The prepared BPs from MWNT suspensions were characterized for capacitance, morphological features, and BOD adsorption for electrochemical measurements.

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Freshly prepared BPs were washed three times using ethanol followed by ultrapure water and then incubated in potassium phosphate pH 6.5 buffer for a day before use. Each paper was dried and cut into rectangular strips (0.4 cm x 2.5 cm) for electrochemical studies. The strips were incubated in 10 mg mL–1 of BOD solution overnight at 4 ºC, rinsed three times with potassium phosphate pH 6.5 buffer, and attached to a three-electrode cell for voltammetric measurements. Voltammetry in pure argon atmosphere was used for direct electrochemistry and electron transfer kinetics of T1, T2, and T3 Cu centers of BOD. Magnetically stirred buffer solution with saturated oxygen was employed for electrocatalytic studies of oxygen reduction and identification of the electrocatalytic roles of Cu T1 and combined T2/T3 TNC of BOD. 2.3 Spectroscopic characterization. UV-vis spectroscopy (Thermo Scientific, Model: Evolution 60S) was used to estimate the amount of adsorbed BOD molecules onto the BP strips. For this, the difference in absorbance of free BOD solution (600 nm) before and after adsorption onto BPs of various thicknesses was used. Raman spectroscopy experiments were performed using a WITec alpha-300 confocal Raman microscope. A 100X/0.9 NA objective was utilized to focus laser light (532 nm Nd:YAG) on the surface of the specimen. The scattered light was collected using the same objective and was focused on a 100 μm optical fiber acting as the confocal pinhole. The light was then dispersed by a 0.3m monochromator that utilized a 600 groove/mm grating and was detected by a thermoelectrically cooled CCD camera. Five collections were performed on each sample and the presented results are the average of the five replicates. X-ray photoelectron spectroscopy (XPS) was conducted using a dual anode X-ray lamp (XR 04-548, Physical Electronics Inc., Chanhassen, MN) under high vacuum conditions (~ 10-10 Torr), and an EA 125 hemispherical energy analyzer (resolution 0.02 eV) was used to acquire the XPS spectra. An Al-Kα line (1486.6 eV) was used as the X-ray source. The X-ray was set at an incident angle of 54.7ᵒ. The relative atomic concentrations were calculated by the equation,28 𝑎𝑡. % 𝑖 =

𝐴𝑖 𝐹𝑖 𝐴𝑖 𝑖𝐹 𝑖

Where, Ai is the area of the ith element and Fi is the relative sensitivity factor (RSF). Fi accounts for the binding energy of each element and its cross section. The RSF values for C 1s, N 1s, and O 1s core level elements are 1.0, 1.8, and 2.93. 2.4 Microscopic characterization. The surface morphologies of BPs before and after immobilization of BOD were characterized by scanning electron microscopy (SEM, Model: FEI Quanta 600FE, accelerating voltage of 20 kV). The images were obtained using FEIxT Microscope Control Software.

3. RESULTS 3.1 Buckypaper preparation and characterization.

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Figure S1A shows the sequential vacuum filtration (Fig. S1Aa), pressing and heat gun treatment (Fig. S1A-b), and washing steps (Fig. S1A-c) for the preparation of BPs. The thickness of the BP disk was controlled by the amount of MWNT used in the starting suspension (Table 1). Nitrogen dried disks of various thicknesses were cut into rectangular strips [0.4 cm wide x 2.5 cm length (~ 1.4 cm was submerged in the electrolyte solution)] for use in electrochemical studies (Fig. S1A-d, e). Fig. S1-A (g, f) represents the flexible nature of the prepared BPs.

Table 1. Physical and electronic properties of prepared BPs.

Amount of MWNT used for BP preparation/mg 20 30 40 50 75 100

Thickness of BPs/ m (av.±STDEV) 87±3 170±4 190±4 220±7 320±10 380±8

Capacitance (mF/cm2)

Conductivity (S m-1)

69±3 71±2 72±4 132±5 224±2 279±2

8227 ± 275 3293 ± 61 2843 ± 54 2511 ± 51 808 ± 30 683 ± 11

Measured conductivity values for various thicknesses of BPs were in the range of 700-8000 S m-1, which is in the usual range for BPs, and is influenced by the type and electronic properties of nanotubes used.27,29 The trend of decreasing conductivity with increasing BP thickness may be attributed to residual, entrapped non-conductive dimethylformamide (solvent) molecules and thicker BPs approaching bulk material properties. The attachment of BOD by adsorption on to an 87 um BP (representative case) decreased the conductivity from 8227 ± 275 to 6940 ± 152 S m-1 confirming the formation of an adsorbed insulating protein layer around the BP surface. Raman spectra (Figure S2 and Table S1) can provide insight into the overall crystal quality and amount of defects in the BPs. Since all spectra have a distinct G′ band, they are not highly damaged and the measured ratio of intensities of the D to G Raman bands (ID/IG) provides a qualitative measure of the amount of crystal defects.3 0 Ultrasonication resulted in an increase in the ID/IG indicating an increase in defects compared to the as received MWNT powder. However, smaller ID/IG values from the BPs compared to the as received MWNTs indicate that fewer defects were present after creating the BPs and that the preparation process did not result in a detrimental morphological change. Figure S1-B shows the reversible cyclic voltammograms (CVs) of a bare BP strip at different scan rates in 10 mM Fe(CN)6–3/–4 solution in pH 6.5 phosphate buffer. Electrochemical behavior of the BP for ferri/ferrocyanide redox pair displayed a linear relationship between the square root of scan rate and peak current (Fig. S1-B inset) as would be expected for a diffusion controlled redox process from the dissolved redox probe, as per the Randles–Sevcik equation given below:31

𝑖𝑝 = (2.69 × 105 )𝑛3/2 𝐴𝐷1/2 𝐶𝜈1/2 where ip is the peak current in ampere, n is the number of electrons transferred in the redox reaction (n = 1 for the ferri/ferrocyanide redox process), A is the geometric electrode area in cm2, D is the diffusion coefficient of the redox probe in solution in cm2 s–1, C is the bulk concentration of Fe(CN)6–3/–4 solution in mol cm–3, and  is the scan rate in V s–1 at 25º C. 3.2 Background capacitive cyclic voltammograms of BP strips and determination of their capacitance values. Figure S3 shows the representative CVs with increase in scan rates for a BP strip of thickness 87 µm. Capacitance values were calculated from the slopes of the linear plots of double-layer charging current density (J) versus scan rate for BPs of different thicknesses as shown in Figure S4. Table 1 presents the capacitance values, amount of MWNT used for the preparation of BPs, and their measured average thicknesses (N = 10 replicates) 3.3 Adsorption of BOD onto BP strips of various thicknesses. BOD exhibits an intense absorption band at 600 nm due to the charge transfer process from S– (Cys) to Cu2+ (T1).32 Using this property, we confirmed the adsorption of BOD onto BP strips by measuring the difference in absorbance of the free BOD solution at 600 nm before and after immobilization onto BP strips of varying thicknesses (Figure 1-A to C). Figure 1-A shows the characteristic blue color of prepared BOD solution. Figure 1-B shows the notable disappearance of the blue color after overnight incubation with a BP strip of 87 m thickness. Figure 1C shows the decrease in absorbance values of BOD in solution after adsorption onto BPs of increasing thicknesses prepared in this study. Results reveal greater amount of BOD immobilization on the thicker BPs (Figure 1-C (inset)). We found that our as purchased BOD corresponded to 8% w/w of BOD with respect to the dry sample powder (i.e., 10 mg powder yielded 0.8 mg of adsorbed BOD with a complete disappearance of blue color in the free enzyme solution after adsorption onto thicker BP strips).



Figure 1. Photographs of BOD solution (A) before, and (B) after overnight incubation with BP (87 m thickness). (C) Absorbance spectra of BOD solutions before and after overnight incubations with BP strips of varying thicknesses (dimension: 0.4 cm x 2.5 cm). Inset shows the adsorbed BOD amounts versus the BPs of different thicknesses. 3.4 X-Ray Photoelectron Spectroscopy (XPS). XPS characterization was used to obtain elemental composition of a bare BP (87 m thickness used) and that after adsorbing a BOD biofilm (Figure 2-a and b, respectively). On the bare BP surface, the characteristics carbon and oxygenated groups from the multiwalled carbon nanotubes of BP are evident (Figure 2a). Following the adsorption of BOD, appearance of N element from the aminoacids of the enzyme can be observed (Figure 2b). Table 2 presents the relative elemental composition of the bare BP and BOD adsorbed BP. In addition to the unique appearance of nitrogen, we observed a 3-fold increase in oxygen content from aminoacids for the BOD enzyme adsorbed BP over the bare BP alone. The percentage of carbon slightly decreased due to the BOD enzyme adsorption onto the bare BP of predominantly carbonaceous surface.

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In addition to the UV-vis absorbance and XPS based confirmation of BOD adsorption onto BPs, we performed SEM imaging of the biofilms. Figures S5-A, B and S5-C, D show the scanning electron micrographs of BP before and after adsorption with BOD, respectively (1 µm and 500 nm resolutions shown for each case). A typical MWNT network is visible on the bare BP surface. The tubes thickened, flattened, and exhibited a coated layer morphology following the immobilization of BOD, which confirmed the successful localization of the enzyme on the nanotube network. 3.6 Direct electrochemistry of BOD attached to BP strips of various thicknesses and peak assignments. Among the tested BPs with different thicknesses (87-380 µm), multiple DET features to Cu centers were observable when using BPs of thicknesses within the range 87–220 µm. We identified that the increase in capacitance for BP with thickness beyond 220 µm led to diminished intensities of all redox peaks in the CVs and made it difficult to distinguish the peaks of T2 and T3 Cu atoms (Figure S6). For subsequent studies to obtain the detailed voltammetric characterization of non-turnover BOD electrochemistry, we chose 87 µm thick BP as the representative case (data presented with this thickness are for N=5 replicates). Figure 3 shows the CV of a BOD film adsorbed overnight onto a BP strip of 87 µm thickness under argon atmosphere in pH 6.5 phosphate buffer, 10 mV s–1 scan rate (red curve). The CV obtained after subtracting the background non-faradaic capacitive currents is shown in the same figure (black curve) to visualize the peaks with a better clarity. Only BP strips in the absence of an adsorbed film of BOD did not show any redox peaks in the argon-saturated buffer (Figures S3 and S6). The non-linear response of capacitance with BP increasing thickness indicates the characteristics surface heterogeneity of the nanotube network.

Figure 2. XPS survey spectra of (a) bare 87 m BP, and (b) after adsorbing a BOD biofilm for overnight at 4 ºC followed by a water rinse to remove weakly and unbound BOD . C 1s ( ~ 285 eV), N 1s (~ 400 eV), and O 1s (~ 533 eV) were measured.

Table 2. XPS results on elemental concentrations. Elemental Concentration (%) C1s N1s O1s Bare 87 m buckypaper BOD adsorbed buckypaper

96.0±0.4

-

4.0±0.4

80.7±0.8

6.9±0.7

12.4±0.5

3.5 SEM characterization.

Figure 3. Cyclic voltammogram of a BOD film adsorbed onto a BP strip of 87 m thickness (as acquired, red curve), and the background subtracted voltammogram (black curve) at 10 mV s–1 scan rate in an anaerobic phosphate buffer, pH 6.5, argon atmosphere, 25 °C. The peak-width at half maximum (PWHM) for an ideal oneelectron film voltammetry is 90.6 mV.31 The PWHM values measured from the reduction peaks of BOD on an 87 µm-thick BP for the T1, T2, and T3 Cu sites were 115 (±12), 90 (±4), and


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49 (±3) mV, respectively (Figure 3) indicating a non-ideal voltammetric behavior of the designed BOD films, which is similar to other enzyme films on electrodes.33-35 These PWHM values are in accordance with a one-electron transfer at the T1 Cu site (E°′ 0.92 V), a one-electron transfer at the T2 Cu site (E°′ 0.58 V), and a two-electron transfer at the T3 Cu site (E°′ 0.42 V), respectively. Based on the PWHM values, the observed three redox pairs with formal potentials (E°′) of 0.92 (±0.02), 0.58 (±0.01), and 0.42 (±0.01) V vs. SHE, are assigned to T1, T2, and T3 Cu sites, respectively. These formal potentials are the result of the characteristics BP material property as the electrode.

Formation of the up-hill catalytic feature with increasing scan rate is presented in Figure 5. This result can be explained by the relatively slower heterogeneous electron receiving rate of the T1 Cu center (0.07 s-1) from the BP strip, intramolecular electron transfer (IET) process from the T1 to TNC, and the subsequent catalytic oxygen reduction at the TNC (T2, 0.25 s-1/T3, 0.11 s-1) (Table S2). At smaller scan rates, the T1 Cu center has sufficient time to receive electrons from the BP strip and shuttle them to the TNC for oxygen reduction. Whereas, at higher scan rates, the electron mediation by the T1 site and the rate of IET process is slower than the direct electron transfer to

3.7 Electroactive amounts. The electroactive surface coverage (Γ in mol cm–2) was calculated using the following equation:31 Γ = 𝑄/𝑛𝐹𝐴 where Q is the Faradaic charge (area under the reduction peaks used), n is the number of electrons (n = 1 for T1 and T2, and n = 2 for T3), and A is the geometric area of the BP. Estimation of the electrochemically active T1, T2, and T3 species yielded values of 5.5 (±0.7), 3.8 (±0.4), and 1.0 (±0.2) nmol, respectively, per cm2 BP geometric area. Thus, the uncommon nature of the CV peak shapes seen in Fig. 1 is attributed to the differences in the extent that each Cu site is electroactively connected with the nanotube network of BP. 3.8 Direct electron transfer rates. We have determined the direct electron transfer rate constants (ks) for the T1, T2, and T3 Cu sites using Laviron’s method36 as 0.07±0.01, 0.25±0.02, and 0.11±0.02 s-1, respectively. Figure S7 shows the CVs with increase in scan rates for a BOD film adsorbed on an 87-µm BP strip chosen as the representative case. The trumpet plots representing the separation of the oxidation and reduction peak potentials with logarithm of scan rate for all three Cu sites are presented in Figure S8. Table S2 provides a comparative account of the electron transfer rates of BOD Cu sites from this study and the previously reported T1 and that of combined T2/T3 ET rates on various electrode materials. Based on this comparison, we concluded that the observed separation of the individual T1, T2, and T3 peaks was the result of measureable and distinguishable sluggish electron transfer rates between the freestanding BP material with a nanotube network and their different extent of interactions with the three BOD Cu sites. 3.9 Electrochemical oxygen reduction by the BP/BOD biocathode. The oxygen reduction currents catalyzed by the BP/BOD biocathode were tested in an oxygen-saturated buffer under magnetically stirred solution conditions. Figure 4 shows the voltammograms of a BOD film adsorbed onto an 87-µm BP strip (orange and red curves). The catalytic waves showed a single onset potential at 1 mV s–1 scan rate but two onset potentials at 10 mV s–1. The dominant onset potential of 0.9 V vs. SHE at all scan rates confirms the direct electronic communication to the thermodynamically favored T1 site of BOD for oxygen reduction. The second onset potential of 0.55 V vs. SHE at higher scan rates is attributed to the TNC-catalyzed oxygen reduction.

Figure 4. Electrocatalytic voltammograms of a BOD film adsorbed onto an 87-µm BP strip at 10 mV s–1 (orange) or 1 mV s–1 (red) scan rate, and that of a bare BP strip with no adsorbed enzyme (dashed lines) at 1 and 10 mV s-1 scan rates in a stirred oxygen-saturated phosphate buffer, pH 6.5, 25 °C. the T2/T3 sites of TNC. This phenomenon is supported by the appearance of the up-hill catalytic wave at higher scan rates (≥ 10 mV s–1) near the formal potential of the TNC to uptake electrons directly from the BP without depending on the T1 center.

Figure 5. Electrocatalytic voltammograms of BOD films adsorbed onto an 87-µm BP strip with increasing scan rate in



stirred oxygen saturated phosphate buffer, pH 6.5, 25 °C. Arrows indicate the additional up-hill catalytic waves appearing at higher scan rates, ≥ 10 mV s–1. 3.10 Electrochemical oxygen reduction by the BP/BOD biocathode of various thicknesses. Figure 6 shows the oxygen reduction voltammograms of BP/BOD biocathodes of different BP thicknesses. The BP thickness of ~ 200 m displayed a large current density (~ 3 mA cm–2) with narrower double layer capacitive currents than other higher thicknesses. Our data show that the greater background capacitance at higher thicknesses can act as a barrier (Figure S6 data) for direct electrocatalytic oxygen reduction (i.e., thickness > 200-m). In Table S3, we present a comparative current density data with representative prior BP/BOD reports, which confirms the high catalytic current achieved in the present work.

Figure 6. Cyclic voltammograms of BOD films adsorbed on BPs of different thicknesses at 1 mV s–1 in oxygen-purged phosphate buffer, pH 6.5, 25 °C.

4. DISCUSSION The background subtracted cyclic voltammogram (Figure 3) indicates that the anodic current passed from the TNC center in the reverse scan is higher than the respective cathodic currents in the forward scan (i.e., QA > QC). Similar, incomplete reversible peak intensities has been discussed for both BOD and laccase enzymes on gold and carbon nanocomposites,37-3 9 and they were attributed to an intramolecular oxidation process from the T1 to TNC site and the associated diminished peak intensity of the T1 site in the reverse scan. The designed buckypaper material follows a similar trend. Atanassov et al. reported three individual peaks for T1, T2 and T3 copper sites of BOD on a carbon screen-printed electrode.4 0 They assigned the redox potentials of T1, T2 and T3 centers as 0.752, 0.423, and 0.591 V, respectively (vs. SHE at pH 6.8). Additionally, they investigated the direct electrochemistry of two other MCOs, laccase from Trametes versicolor and ascorbate oxidase from Cucurbita sp. in the same study. Collectively all three enzymes showed discrete redox activities in potential regions of low (0.21-0.51 V), mid (0.51-0.81 V) and high (0.811.01 V) vs. SHE. In another study, Tkac et al. identified three distinct cathodic redox peaks at 0.699, 0.580, and 0.409 V with a single anodic peak at 0.445 V (vs SHE at pH 6) for BOD on KetjenBlack na-

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noparticle, single walled carbon nanotubes, and chitosan modified glassy carbon electrodes and the order of the peak assignment was similar to Atanassov et al. Using a series of control studies, they proposed that tyrosine redox chemistry could interfere with the redox potential of the T2 site.38 Recently, Le Goff et al. observed a similar behavior for bilirubin oxidase from Magnaporthe orizae (MoBOD) on functionalized MWNT electrodes.9 In the present report, we would like to base the measured PWHM data to assign the number of electrons accordingly as one each for T1 and T2 sites and two for the T3 site with their respective positions of average E°′ values observed at 0.92, 0.58, and 0.42 V vs. SHE, respectively. The significant positive shift on the T1 formal potential compared to prior reports is attributed to the characteristic property of the designed freestanding BP as the electrode material. We propose hydrophobic, pipi, and intercalation as feasible major forces of interactions between BP and the BOD moiety around all Cu centers due to the use of unfunctionalized as produced nanotubes and electrostatic interactions to a certain extent due to the presence of polar edges and surface defects on nanotubes. Multiple heterogeneous electron transfer signals have also been observed in the case of laccase immobilized on modified gold,37,41-42 and carbon electrodes.43 These noteworthy electrochemical studies and the BOD crystal structure elucidations 18 reported by Cracknell et al. provided clear evidence that both the T1 and TNC sites were located within reasonable distances (< 14 Å)44 from the protein surface to facilitate electron transfer. In our study, BOD was adsorbed onto BP strips with no control placed over the orientations of BOD. Hence, we believe that random orientations of the enzyme could indeed expose varying extents of T1, T2, and T3 sites based on their relative interactions with the nanotube network; which are hydrophobic and pipi stacking due to the unfunctionalized MWNTs used for the BP preparation.45,46 The average dimension of the MWNTs used was (10 nm ± 1 nm) × (4.5 nm ± 0.5 nm) × (3–6 µm) (o.d. × i.d. × l), and the diameter of the BOD molecule is ~ 5 nm,47 which suggests the possibility for intercalation of BOD with the freestanding MWNT network. By identifying the significance of BP thickness, we determined that the direct electrochemistry of the BP/BOD films under anaerobic conditions offered the possibility to access the electron relay to all Cu sites. The bare BP strip with no adsorbed BOD exhibited a non-enzymatic electrocatalytic current of 0.4 mA at 0.2 V vs. SHE (dashed line CVs in Figure 4), which is at a much more negative potential than the BOD catalyzed currents. It has been shown that the presence of oxidized carbon species and trace metal impurities in the MWNTs could contribute to the non-enzymatic oxygen reduction.48 The observed dual onset electrocatalytic waves supported the existence of biologically active BOD molecules, and the 3.5times larger heterogeneous electron transfer rate of the T2 center than the T1 center in enabling the faster scan rate dependent appearance of the second catalytic wave (Figure 5). Shleev and coworkers previously suggested the existence for a TNC catalytic intermediate with the redox potential closer to 400 mV vs. SHE and an intramolecular electron transfer from T1 to TNC. This electron transfer was considered the rate limiting process in the catalytic cycle under alkaline conditions.49


Page 7 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In some studies, the base electrodes were modified with aromatic amines, porphyrins,5 0 surfactants,51 pyrenes,52 syringaldazine,53 bilirubin,54 and many other chemicals that could form π–π stacks with the basal planes of MWNTs to act as tethering or orienting agents for BOD.15 Additionally, redox mediators were used to enhance the catalytic current density in prior studies. In contrast, we implemented a simple adsorption process to construct the BP-based BOD biocathode and achieved high current densities in the present work (Table S3). The stability of the BP/BOD bioelectrode was investigated by amperometric i-t curve monitoring the oxygen reduction current density for ~18 h under saturated oxygen conditions (Figure S9). We observed that 75% of the initial current density was retained after 3 h and about 50% of the current was retained after 10 h of continuous catalytic reaction. These catalytic stability levels are appreciable for a BOD film simply adsorbed onto a BP strip and suggest the existence of reasonably strong non-covalent interactions between BOD and BP.

In this study, we presented the DET features of all Cu sites of BOD from Myrothecium verrucaria accessed by immobilizing the enzyme onto a nanostructured MWNT network of a BP strip. The multiple redox peaks observed under anaerobic conditions were assigned to heterogeneous electron transfers from the BP to the T1, T2, and T3 Cu sites of BOD based on PWHM values. The BP/BOD biocathode exhibited high current densities from the four-electron reduction of molecular oxygen to water in the absence of a mediator or chemical modification of electrode surface. Scan-rate dependent dual onsets of electrocatalytic oxygen reduction waves confirmed the existence of two electron transfer pathways between BOD and the BP strip when the IET rate (T1 to TNC) becomes a limiting factor. We believe the findings in this article have considerable significance for fuel cell applications and metal-air batteries. In addition, accessing electrochemical and catalytic properties of often-buried inaccessible redox centers of many metalloenzymes can be achieved by the freestanding BP design of optimal thickness. As a result, successful design of efficient new electrocatalytic biosystems is possible.

5. CONCLUSIONS

Supporting Information. Figures S1-S9 and Tables S1-S3. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

‡Present

address: Department of Chemistry, The University of Texas at Austin, Austin, TX 78712, United States.

*[email protected]

We thank the College of Arts and Sciences, Oklahoma State University for financial support.

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