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/lp/hindawi-publishing-corporation/enhancement-of-electrochemical-performance-of-bilirubin-oxidase-Bm1lXvgo0z
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- Hindawi Publishing Corporation
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- Copyright © 2018 Erica Pinchon et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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- 1687-7985
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- 1687-7993
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- 10.1155/2018/4712547
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Abstract
Hindawi Advances in Physical Chemistry Volume 2018, Article ID 4712547, 9 pages https://doi.org/10.1155/2018/4712547 Research Article Enhancement of Electrochemical Performance of Bilirubin Oxidase Modified Gas Diffusion Biocathode by Porphyrin Precursor Erica Pinchon, Mary Arugula, Kapil Pant, and Sameer Singhal CFD Research Corporation, 701 McMillian Way, Suite D, Huntsville, AL 35806, USA Correspondence should be addressed to Sameer Singhal; sameer.singhal@cfdrc.com Received 16 December 2017; Revised 12 March 2018; Accepted 28 March 2018; Published 3 June 2018 Academic Editor: Ramasamy Karvembu Copyright © 2018 Erica Pinchon et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Recent studies have focused on tailoring the catalytic currents of multicopper oxidase (MCO) enzymes-based biocathodes to enhance oxygen reduction. Biocathodes modified with natural substrates specific for MCO enzymes demonstrated drastic improvement for oxygen reduction. Performance of 1-pyrenebutanoic acid, succinimidyl ester (PBSE), and 2,5-dimethyl-1- phenyl-1H-pyrrole-3-carbaldehyde (Di-Carb) oriented bilirubin oxidase (BOx) modified gas diffusion biocathode has been highly improved by incorporating hematin, a porphyrin precursor as electron transfer enhancement moiety. Hematin modified electrodes demonstrated direct electron transfer reaction of BOx exhibiting larger O reduction in current density in phosphate bueff r solution (pH 7.0) without the need of a mediator. A remarkable improvement in the catalytic currents with 2.5-fold increase compared to non-hematin modified oriented BOx electrodes was achieved. Moreover, a mediatorless and compartmentless glucose/O biofuel cell based on DET-type bioelectrocatalysis via the BOx cathode and the glucose dehydrogenase (GDH) anode demonstrated peak 2 2 power densities of 1 mW/cm at pH 7.0 with 100 mM glucose/10 mM NAD fuel. The maximum current density of 1.6 mA/cm and 2 2 the maximum power density of 0.4 mW/cm were achieved at 300 mV with nonmodified BOx cathode, while 3.5 mA/cm and 1.1 mW/cm of current and power density were achieved with hematin modified cathode. eTh performance improved 2.4 times which attributes to the hematin acting as a natural precursor and activator for BOx activity enhancement. 1. Introduction applications [5, 6]. These enzymes are more active and selec- tive than the state-of-the-art electrocatalyst platinum because Biofuel cell technology has received much attention as BOx can promote a four-electron reduction of oxygen [7, 8], energy harvesting devices for powering portable devices leading directly to water rather than production of significant and microscale electronic systems. Enzymes functionalized amount of hydrogen peroxide (H O -2-electronexchange) 2 2 with nanomaterials such as carbon nanotubes, graphene, and [9]. It has been shown previously that BOx demonstrated nanoparticles provide extremely powerful platforms for wide high electrocatalytic oxygen reduction and low overpotential range of biofuel cell applications that are capable of operating necessarytocatalyzethe reaction andaturnover rateof 0.7 −1 independently over a prolonged period of time, without the O per Cu⋅s ,whilePt catalyststurnoverrateisthree times need of external recharging or refueling of devices. Enzymes lower at overpotential of 350 mV [2, 10]. such as multicopper oxidases (MCOs) belonging to oxidore- Moreover, BOx enzymes are well known to perform ductase family can reduce oxygen into water performing ORR when immobilized onto the surface of solid supports. oxygen reduction reaction (ORR). es Th e reactions have been Enhanced activity has been observed when functionalized extensively studied and described in previous literature [1– with nanomaterials, since nanomaterials have been consid- 4]. eTh most common “Blue” MCO enzymes are ascorbate ered as excellent scao ff lding structures for immobilization of oxidase (AOx), laccase (Lac), and bilirubin oxidase (BOx) the enzymes without sacricfi ing their bioactivity. eTh ORR is that can act as excellent biocathode materials for biofuel cell mainly dependent on the structure of BOx which was well 2 Advances in Physical Chemistry studied and reported previously [11, 12]. It contains three dif- moiety functionalized with a carbonyl group. us, Th the ferent copper centers (T1, T2, and T3) with overall four cop- electronegative N-atom from the pyrrole moiety and the O- per ions that catalyze the oxidation of bilirubin to biliverdin atom from the aldehyde group can act as hydrogen bond [13], thereby reducing molecular oxygen to water. eTh mech- acceptors and the H-atom as a hydrogen bond donor [29]. anismofelectrontransferinvolvestheT1 site of MCO Subsequently, we reported on the utilization of syringaldazine acting as the primary electron acceptor from the substrate (Syr), for enzyme orientation, of both Laccase and BOx that via an intramolecular electron transfer (IET) to the T2/T3 demonstrated approximately 6 and 9 times increase in cur- cluster site which converts molecular oxygen to water. Recent rent density, respectively, compared to physically adsorbed research unveiled whether the ORR in BOx is a four-electron and randomly oriented Lac cathodes [30]. transfer or a two-electron transfer with a hydrogen peroxide Our present study follows from observations that the intermediate [7, 14]. Much research has also been devoted in activity and the performance of the oriented BOx were understanding the BOx direct electrochemistry (DET) and further enhanced from incorporating a porphyrin precur- electron transfer via mediation by redox mediators [15–17]. sor solution—hematin—on the biocathode. Hematin is a Nevertheless, the use of these enzymes as biocatalysts has not ferriprotoporphyrin-IX with a hydroxide ion bound to the yet been generally adopted for commercial purposes. ferric ion formed when hemin (ferriprotoporphyrin-IX with One persistent challenge is maintaining the catalytic a chloride ion bound to ferric ion) is treated with strong activity of the enzyme and improving the performance oeft n NaOH solution. Hemin is an active center of family of when immobilized on a solid support [18, 19]. Enzymes may hemeprotein, such as b-type cytochromes, peroxidase, myo- adsorb successfully and however tend to denature rendering globin, and hemoglobin. eTh first electrochemical behavior some of the immobilized enzyme inactive and ineffective ofheminwasstudiedin1968onaplatinum electrode [20–22]. To overcome these challenges, enzymes that catalyze by coulostatic method [31]. Hemin adsorption on graphite electron transfer reactions must be entrapped in hydrogels electrode demonstrated fast electron transfer that can exceed or stabilized with the use of orienting agents. Orienting monolayer coverage with high amount of active species. Sev- agents promote correct and proper allocation of enzymes on eral literature studies show that hemin modified electrodes the surface of the electrode to obtain high current density. were extended in the catalysis and reduction of hydrogen The lone copper on BOx should not be no more than 1- peroxide [32–34], oxygen [35], and superoxide [31]. Utiliza- 2nmfromtheelectrodesurface to avoidinterfacial electron tion of hemin, protoporphyrin derivatives as pretreatment transfer being the rate-limiting step in oxygen reduction of the surface for improving the activity of BOx on cathode electrocatalysis [23, 24]. electrode has also been reported [36]. Recent studies have demonstrated a trend in surface Herein, we demonstrate the enhancement of BOx air modifications at biointerface level for MCOs based biocath- breathing cathode performance via the modicfi ation of the odethatincorporatesthe useofaromatic, hydrophobic,and electrodewithhematin.Wealsoinvestigatedthe capability hydrophilic molecules in order to suitably orient these redox and effects of hematin versus hemin to promote an ecffi ient enzymes with the T1 copper site immobilized on carbon electron transfer mechanisms for oxygen reduction. We nanotube sidewalls to enhance the oxygen electroreduction further constructed a mediatorless and compartmentless [25]. es Th e molecules are natural substrates that are spe- glucose/O DET-type biofuel cell to investigate the cell cific for the MCO enzymes for oxygen reduction. Laccase performance. was initially studied by the Armstrong group where the hydrophobic pocket of laccase interaction with polycyclic 2. Materials and Methodology aromatic compounds such as anthracene resulted in remark- Hemin, 1-pyrenebutanoic acid, succinimidyl ester (PBSE) able enhancement of electrocatalytic currents. The aromatic was obtained from Setareh Biotech LLC; 1-ethyl-3-(3- compounds structure is very similar to the natural substrate dimethylaminopropyl) carbodiimide hydrochloride (EDC) of Lac (phenols), and the strong hydrophobic interactions was obtained from TCI America; and N-hydroxysuccinimide have been reported to promote the apt orientation for ± 98% (NHS) was obtained from Alfa Aaesar. Hematin the DET [26]. Similar strategy was extended to the BOx (94.5%) was obtained from Chem-Impex Int’l Inc. enzymes where the studies show that the substrate-pocket NAD-dependent glucose dehydrogenase (GDH, from did not exhibit hydrophobic interactions but electrostatic Pseudomonas sp., E.C. 1.1.1.47) and glucose were obtained interactions, which are an efficient way to achieve direct fromSigmaAldrich andusedasreceived; BOxwasobtained wiring of BOx [27]. Along this line, different literature reports from Myrothecium verrucaria (EC 1.3.3.5.), Amano Enzyme have focused on incorporating specific substrates of BOx U.S.A. 50 Co., Ltd. Multiwalled nanotubes (MWNTs) such as bilirubin [28], quinones [29], and syringaldazine paper (Buckeye Composites), MWNTs (𝑑 = 20–30 nm, 𝐿 [30] towards appropriate orientation that can be convenient =10–30𝜇m), and single walled nanotubes (SWNTs) (99% for DET-type electrocatalysis. Our group has previously purity) were obtained from cheaptubes.com. reported on crosslinking the enzyme to the electrode with orienting agents, two bilirubin functional analogues, pyrrole- 3. Electrode Preparations 2-carboxaldehyde and 2,5- dimethyl-1-phenyl-1H-pyrrole-3- carbaldehyde, for enzyme orientation and 1-pyrenebutanoic 3.1. Hemin and Hematin Modiefi d Electrodes. Air breathing acid, succinimidyl ester (PBSE) as the tethering agent. These cathodes were fabricated as in the previously described proce- compounds were chosen because they each contain a pyrrole dure with slight modification [37]. Briefly, teflonized carbon Advances in Physical Chemistry 3 Oxygen (／ ) Nickel mesh GDL consisting of teflonized carbon （ O Catalyst layer consisting of Hematin/BOx/CNT ink on buckypaper (a) (b) Figure 1: (a) Schematics of hematin-BOx air breathing cathode and (b) image of the biofuel cell. black powder (35% teflonization and 50% teflonization, XC35 3.3. Hematin/EDC-NHS Coupling Modiefi d Electrodes with and XC50) and MWNTs paper (Buckeye Composites) were BOx. The air breathing BOx/hematin electrodes were hydraulically pressed for 5 minutes at 500 psi. Figure 1 shows successively modified with additional components: 1-(3- the schematic illustration of fabrication of gas diffusion layer dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride (GDL) cathode (a) and image of the biofuel cell (b). A (EDC) and N-hydroxysuccinimide (NHS). Hematin solution 10 mM hemin solution was prepared by mixing hemin into (10 mM) was prepared as described in previous section. After DMSO. A 10 mM hematin solution was prepared by mixing 1-hour sonication, 9.8 mg of EDC and NHS was added and hemininto20mMsodiumhydroxide (NaOH) solution.The incubated to 285𝜇L hematin solution. BOx ink described 20 mM NaOH solution was also used to prepare cathodes earlier was immobilized onto the modified hematin/EDC- with NaOH only modification. The as-prepared solutions NHS air breathing cathodes were left for 4 hours at room were sonicated for 1 hour and 285𝜇Lofthemixturewas temperature prior to being stored overnight at 4 C. Figure 2 shows the schematic representation of stepwise procedure of deposited onto the buckeye paper layer (2 cm )ofthe pressed hematin modica fi tion and BOx deposition on GDL cathode. air breathing cathode. eTh electrodes were left to dry for 4 Oxygen enters the cathode through the teflonized carbon hours at room temperature prior to being stored overnight at GDLandisreducedtoH O at the hematin-BOx catalyst layer. 4 C. 3.4. Electrochemical Testing. All electrochemical experiments 3.2. Hemin and Hematin Modied fi Electrodes with BOx. were performed with a VMP3 potentiostat (Biologic). A Further investigation was carried out with BOx immobilized conventional three-electrode system was used in the mea- on hemin and hematin modified cathodes. Preparation of surements with Ag/AgCl as a reference electrode, a Pt wire BOxinkwascarriedoutasfollows:2 wt%MWNTinksolution as theauxiliaryelectrode,andabareormodiefi delectrode as was prepared by dissolving 4 : 1 ratio of water to ethanol working electrode. Cyclic voltammetry was carried out and (by weight) and added to 100 mg MWNTs (10–20 nm). eTh potentiostatic polarization curves were obtained to charac- prepared mixture was subjected to sonication for 1 hour terize half-cell testing. All tests were conducted in 245 mM, in ice bath vortexing every 20 min interval time. 5𝜇Lof pH 7.0 PBS buffer at room temperature. 300 mM Di-Carb (in DMSO) and 170𝜇Lofstock solution of 2% MWNT ink were mixed, vortexed, and incubated for 3.5. Complete Fuel Cell Testing. Glucose dehydrogenase 1 hour. This was followed by addition of 5 𝜇Lof300mM (GDH, Toyobo) based anodes were prepared for complete PBSE (in DMSO) and 20 uL of water and left for incubation fuel cell testing. Polymethylene green (PMG) was electrode- for 1 hour. To this, BOx (8 mg, 16 units/mL) was weighed posited onto (electrode area 7.3 cm ) carbon felt electrodes and added to MWNT ink and incubated again for 1 hour following a modified version of a previously described at room temperature. Later 200𝜇Loftheinkwas then procedure [30]. A single wall nanotube (SWNT-PEI) ink deposited on the prewetted buckeye paper of air breathing solution containing GDH was drop-casted on top of the cathode modied fi with hemin and hematin, respectively. PMG treated carbon felt electrodes. eTh anodes were stored Control electrodes were prepared by drop casting the ink onto overnight at 4 C prior to testing. The GDH anodes were unmodified air breathing cathodes. Following ink deposition, 2 2 a chemical deposition of tetramethyl orthosilicate (TMOS) paired with 9 cm (7.3 cm working area) hematin modified was performed by sealing cathodes in a Petri dish containing BOx or unmodified BOx cathodes for complete fuel cell small caps filled with water and TMOS. eTh Petri dish testing. The fuel cells were tested with 100 mM glucose/10 mM remained sealed for 5 min before discarding the TMOS. nicotinamide adenine dinucleotide (NAD) in 245 mM PBS Cathodes were then stored at 4 Covernight. buer ff pH 7.0. A Constant Load Discharge (CLD) technique 4 Advances in Physical Chemistry Dry 4 hrs EDC-NHS coupling Schematic illustration of hematin modified BOx enzyme constructed Hematin on the gas diffusion layer electrode. DMY-carb MWCNT BOx PBSE Figure 2: Schematic illustration of stepwise procedure of hematin modified BOx on gas diffusion layer cathode. was employed to generate power and current density curves. Later, discharge curves were generated by applying a constant load of 3.0 mA to the fuel cell. eTh cells were filled with fresh fuel before each subsequent discharge. Preparation of BOx ink for complete fuel cell testing was described in Supplementary File. 4. Results and Discussion 4.1. Electrochemical Testing and Characterization of Hemin and Hematin Electrodes. To better realize the function towards the enhancement of BOx cathode, hematin air breathing cathodes were prepared without BOx to determine if the electrodes were independently capable of catalyzing the ORR. This test was compared simultaneously with hemin air −50 0.0 0.2 0.4 0.6 breathing cathode without BOx. eTh step potential studies were conducted to evaluate the electrodes under quiescent Current Density (mA/＝Ｇ ) with no oxygen saturation conditions. At 50 mV, the current Hemin density for the hemin cathode was 0.4 mA/cm (Figure 3) Hematin while current density of 0.05 mA/cm was observed for Figure 3: Representative step potential curves for hemin (black hematin. The results showed that hemin demonstrated higher square) and hematin (red circle) air breathing cathodes tested with current density compared to hematin suggesting that hemin 245 mM PBS, pH 7.0. can serve as a catalyst for ORR. Several studies have identified hemin as a DET-type electrocatalyst for ORR. Ma et al. have shown that hemin modified PAMAM/MWCNT nanocom- BOx does not act as an electrocatalyst by itself and cannot posite films on glassy carbon electrodes can act as both an assist as a catalytic mediator for ORR reduction. eTh refore, electron conductor and catalytic mediator for L-tyrosine [38]. we may assume that hematin adsorbed onto BOx cathodes Others have identified hemin as an electrocatalyst for oxygen may enhance DET catalysis by acting as a natural substrate or reduction and superoxide detection biosensors. er Th efore, precursor for the BOx enzyme. the increase in current density was predictable; however A cyclic voltammetry (CV) study was conducted to very little increase in current density was observed for the compare the electrochemical behavior of hematin modified hematin electrodes. This suggests that hematin without the air breathing cathodes with bare and EDC/NHS-hematin Potential (mV) vs. sat. Ag/AgCl Advances in Physical Chemistry 5 modified cathodes. Figure 1 (Supplementary) shows that the hematin modiefi d air breathing cathodes had a reduction peak at around 300 mV when compared to a bare air breath- ing cathode. eTh peak current density was 3 mA/cm while no clear oxidation peaks have been observed. Since no distinct reversible redox reaction was observed for the hematin modified air breathing cathodes, hematin most likely acts as a natural substrate and a precursor for oxygen reduction reaction (ORR) at the BOx cathode. In literature, hematin has been identified as a potential alternative to horseradish peroxidase (HRP) for H O detection [39] and catalysis of 2 2 phenol compounds [40]. With further addition of crosslink- ing couple EDC/NHS, the reduction peak for the hematin modified electrodes has become less distinct. A high scan rate of 250 mV/s was employed for the CV study to test the 250 −0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 stability of the EDC/NHS-hematin air breathing cathodes. Current Density (mA/＝Ｇ ) Figure 2 (Supplementary) compares cycle 2 to cycle 20 for the air breathing cathodes modified with EDC/NHS-hematin. BOx eTh re was little variation observed between the magnitude of Hemin/BOx cycles 2 and 20. Shrinking CV curves would suggest that the Hematin/BOx Figure 4: Representative step potential curves for BOx (black EDC/NHS-hematin was being stripped from the electrode square), hemin-BOx (red circle), and hematin-BOx (blue triangle) surface. The result of this study suggests that the air breathing air breathing cathodes tested in 245 mM PBS, pH 7.0. cathodes modified with hematin/EDC/NHS were stable. The stability of the EDC/NHS-hematin electrodes was most likely due to the covalent attachment of the hematin to BOx via have focused on incorporating specific substrates of BOx such the EDC/NHS crosslinker. Hematin contains two carboxylic as bilirubin, quinones, syringaldazine, and protoporphyrin acid moieties, which allows the hematin to react with EDC derivatives such as protoporphyrin IX iron (III) (PPFeIII), and NHS further stabilizes the EDC-hematin intermediate protoporphyrin IX dimethyl ester (PPDE), and octaethylpor- and allows the intermediate product to form a covalent phyrin (OEP) towards appropriate orientation that can be attachment to the BOx. EDC and NHS promote the reaction convenient for DET-type electrocatalysis [36]. However no of carboxylic groups of hematin with amino groups of the study has been previously done on hematin as substrate for BOx enzyme. BOx. Therefore, hematin was further tested as a precursor and 4.2. Electrochemical Testing and Characterization of Hemin/ premodifier in the enhancement of BOx enzyme. BOx and Hematin/BOx Electrodes. We further examined the To demonstrate that improved performance was due effect of hematin, hemin, and nonmodified BOx catalyzed to the addition of hematin, BOx electrodes were tested oxygen reduction. A comparison of the current densities with only NaOH modicfi ation, NaOH-hematin, and NaOH- from potentiostatic polarization curves is shown in Figure 4. EDC/NHS-hematin. Based on the results (Figure 5), treat- The current density from the electrode containing BOx and ingtheBOxelectrode with NaOH ledtoenhancedair hemin was 0.007 mA/cm .Thecurrent density forBOx only breathing cathode performance. However, performance was was 0.32 mA/cm , and the current density in the presence higher when the electrodes were modified with hemin in NaOH. Increased current densities achieved with NaOH of hematin modiefi d was 0.7 mA/cm . For all electrodes the modification were most likely due to the electrode surface current densities generated at 300 mV versus sat Ag/AgCl becoming more hydrophilic, which would create a more concluded that hematin led to a significant increase in BOx favorable environment for the ORR taking place at the performance. The polarization experiments with hematin electrode surface. eTh highest current densities were achieved demonstratedmorethan2timesincreaseinthe maximum when the crosslinking couple EDC/NHS were added to the current densities compared to only BOx cathodes with no hematin solution. Current densities greater than 0.8 mA/cm enhancements (Figure 4). Hematin is a ferriprotoporphyrin- IX with a hydroxide ion bound to the ferric ion formed when were achieved by modifying the electrode surface with the EDC/NHS-hematin solution. All BOx cathode types were hemin (ferriprotoporphyrin-IX with a chloride ion bound to ferric ion) is treated with strong NaOH solution. As men- tested in triplicate. eTh EDC/NHS BOx air breathing cath- tioned previously, hemin has been studied as direct electron odes performed better than the BOx only (no enhancements) cathodes at 300 mV but performed significantly less than the transfer moiety for oxygen reduction. er Th efore, the function of hematin as an enhancement agent was compared with NaOH, NaOH-hematin, and NaOH (EDC/NHS)-hematin- BOx cathodes. eTh NaOH (EDC/NHS)-hematin-BOx cath- heminandferricyanideaselectrontransfermediators(data odes remained the highest performing cathodes. not shown). Many such hemin related compounds have been identified in literature. Catechol, hydroquinone, pyrogallol, and bilirubin which are catalytically oxidized by BOx were 4.3. BOx Loading Optimization. High enzyme loadings are employed in modification of biocathodes [28]. Recent studies required for the optimal fuel cell performance. However, Potential (mV) vs. sat. Ag/AgCl 6 Advances in Physical Chemistry 650 BOx Cathode Performance at 300 mV 1.4 1.3 1.2 1.1 0.9 500 y = 0.046x + 0.7991 0.8 2 R = 0.98 0.7 0.5 2.5 4.5 6.5 8.5 10.5 BOx Loading (mg/＝Ｇ ) Figure 7: Linear regression plot for current density as a function of 350 BOx loading. 0.0 0.2 0.4 0.6 0.8 1.0 Current Density (mA/＝Ｇ ) NaOH NaOH (EDC/NHS)-Hematin NaOH-Hematin No enhancement Figure 5: Representative step potential curves for BOx air breathing cathodes modified with NaOH only (black square), NaOH-hematin (red circle), and NaOH (EDC/NHS)-hematin (blue triangle) or no enhancement (green inverted triangle) tested in 245 mM PBS, pH 7.0. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Current Density (mA/＝Ｇ ) 14 mg/＝Ｇ BOx (no hematin modification) 10 mg/＝Ｇ BOx (hematin modification) Figure 8: Representative step potential curves for optimized BOx air breathing cathodes prepared without hematin (black square) or with hematin (red circle) tested in 245 mM PBS, pH 7.0. increased until optimal loading of 10 mg/cm has reached and decreased when the electrode surface was loaded with −0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 14 mg/cm enzyme.This couldbedue todiffusion limitations Current Density (mA/＝Ｇ ) wherepartoftheimmobilizedenzymemaybe notbeing 2 2 available for catalysis. Figure 7 shows the linear dependence 1 mg/＝Ｇ BOx 10 mg/＝Ｇ BOx 4 mg/＝Ｇ BOx 14 mg/＝Ｇ BOx of current density at 300 mV with the BOx loading ranging 2 2 7 mg/＝Ｇ BOx from 1 mg/cm to 10 mg/cm .Itistobenoted that non- Figure 6: Representative step potential curves for BOx air breathing hematin-BOx cathodes performed optimally at 14 mg/cm cathodes prepared with different amounts of BOx ranging from BOx loading and by employing hematin, BOx loading was 2 2 1mg/cm to 14 mg/cm tested in245mMPBS,pH7.0. reduced by 29%. At 300 mV, the optimized hematin-BOx cathode performed twice better than the non-hematin-BOx cathode (Figure 8). when the enzyme loadings are too high, substrate diffusion andproduct inhibition canlimit therateofoverall processes. 4.4. Complete Fuel Cell Testing. A DET-type biofuel cell To minimize the mass diffusion limitations, the BOx loading without separators was constructed. eTh biocathode was an on hematin modified air breathing cathodes was optimized. air breathing hematin/BOx and the bioanode was GDH/PMG 2 2 The enzyme loading ranging from 1 mg/cm to 14 mg/cm carbon felt electrodes. Complete fuel cell testing was con- was tested (Figure 6). eTh results show increase in current ducted with100mMglucose/10mMNAD fuelin 245mM densities with increase in BOx loading on the cathode. PBS buffer pH 7.0 under quiescent conditions at room Since the initial rate of reaction of the enzyme corresponds temperature. The current densities dependence of the cell to the activity and amount of enzyme loaded, the current power density is shown in Figure 9(a) while Figure 9(b) shows Potential (mV) vs. Ag/AgCl Potential (mV) vs. sat. Ag/AgCl Potential (mV) vs. sat. Ag/AgCl Current Density (mA/cm2) Advances in Physical Chemistry 7 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 −0.2 012 3 4 5 −1 01234567 Current Density (mA/＝Ｇ ) Current Density (mA/＝Ｇ ) Standard BOx/GDH Fuel Cell Standard BOx/GDH Fuel Cell Hematin-BOx/GDH Fuel Cell Hematin-BOx/GDH Fuel Cell (a) (b) Figure 9: Representative power density (a) and current density (b) curves for optimized BOx air breathing cathodes prepared without hematin (black square) or with hematin (red circle) paired with GDH anodes tested in 100 mM glucose. the maximum current densities for the hematin modified driven by a combination of synchronous effect of natural BOxcathodes.Theopencircuit potentialof0.7Vwas substrate and favorable orientation for BOx. With hematin obtained. eTh maximum current density was 6.6 mA/cm based electrode surface modification the optimized loading of BOx was drastically decreased by 28.6%, and an average and maximum power density was 1.1 mW/cm at 300 mV of the fuel cell. On the other hand, with non-hematin modiefi d peak power density above 1 mW/cm was achieved. Using cathode the maximum current density of 2.5 mA/cm and hematin, it was investigated that the assembly of hematin with EDC/NHS resulted in significant increase of current maximum power density of 0.2 mW/cm were obtained. density compared to the non-hematin modified electrode. From the results the determining factor for the high current eTh hematin modified electrode showed remarkable stability density is controlled by the BOx cathode. The performance whensubjectedtomorethan15cyclesofcyclicvoltammetry. of the hematin-BOx cathodes was 2.4 times higher than the performance for the BOx cathodes without hematin The current density as high as 6.6 mA/cm at 300 mV and modification. Constant current load discharge studies of the power density of 1.1 mW/cm were obtained with GDH/O hematin/BOx cathode and GDH anodes based biofuel cells biofuel cell. were further conducted with 50 mM glucose/10 mM NAD fuel in 245 mM PBS bueff r pH 7.0. A constant current load Disclosure of 3.0 mA was applied to each fuel cell until the potential The content of this manuscript does not necessarily reflect reached 350 mV. With injection of fresh fuel, the fuel cell thepositionorthe policy oftheGovernment, andnoocffi ial was tested under repeated discharge cycles. A total of 4 endorsement should be inferred. Abstract on this work was discharges were performed consecutively with 10-minute rd submitted at 233 ECS Meeting. recovery periods between discharges as shown in Figure 3 (Supplementary). The average runtime varied between 0.76 h Conflicts of Interest and 1.0 h with average capacity ranging from 126 mAh/g to 167 mAh/g. eTh capacity of the BOx/GDH fuel cells decreased The authors declare no conflicts of interest. by less than 25% aeft r being discharged to 350 mV multiple times. eTh results suggest that the fuel cells can withstand multiple discharges (with fresh fuel) without experiencing a Acknowledgments significant loss in capacity. es Th e results also indicate that ThisresearchwassupportedbytheU.S.ArmyResearchOcffi e there was minimal leaching out of adsorbed functionalities (STTR Contract W911NF-13-C-0015). of anode and cathode biomaterials. 5. Conclusion Supplementary Materials In this study, direct interaction of BOx on hematin mod- Supplementary materials contain the procedure for prepa- ified MWCNT is capable of increasing the current density ration of bilirubin oxidase (BOx) ink for complete fuel Power Density (mW/＝Ｇ ) Potential (mV) 8 Advances in Physical Chemistry cell testing. The cyclic voltammetry studies for testing the chooses its electron transfer partner,” Accounts of Chemical Research,vol.40,no.6,pp. 445–452, 2007. bare, hematin modified, and EDC/NHS-hematin modified electrodeareshowninSupplementalFigure1andshowedthe [15] P. Ram´ırez, N. Mano, R. Andreu et al., “Direct electron transfer stability of hematin modified electrode (Supplemental Figure from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase,” Biochimica et Bio- 2). 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