Research ArticleEnhancement of Electrochemical Performance ofBilirubin Oxidase Modified Gas Diffusion Biocathode byPorphyrin 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 sameersinghalcfdrccom

Received 16 December 2017 Revised 12 March 2018 Accepted 28 March 2018 Published 3 June 2018

Academic Editor Ramasamy Karvembu

Copyright copy 2018 Erica Pinchon et al This is an open access article distributed under the Creative Commons Attribution Licensewhich 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 biocathodesto enhance oxygen reduction Biocathodes modified with natural substrates specific for MCO enzymes demonstrated drasticimprovement for oxygen reduction Performance of 1-pyrenebutanoic acid succinimidyl ester (PBSE) and 25-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde (Di-Carb) oriented bilirubin oxidase (BOx) modified gas diffusion biocathode has been highlyimproved by incorporating hematin a porphyrin precursor as electron transfer enhancement moiety Hematinmodified electrodesdemonstrated direct electron transfer reaction of BOx exhibiting largerO

2reduction in current density in phosphate buffer solution

(pH 70) without the need of a mediator A remarkable improvement in the catalytic currents with 25-fold increase compared tonon-hematin modified oriented BOx electrodes was achieved Moreover a mediatorless and compartmentless glucoseO

2biofuel

cell based on DET-type bioelectrocatalysis via the BOx cathode and the glucose dehydrogenase (GDH) anode demonstrated peakpower densities of 1mWcm2 at pH 70 with 100mM glucose10mM NAD fuel The maximum current density of 16mAcm2 andthe maximum power density of 04mWcm2 were achieved at 300mV with nonmodified BOx cathode while 35mAcm2 and11mWcm2 of current and power density were achieved with hematin modified cathode The performance improved 24 timeswhich attributes to the hematin acting as a natural precursor and activator for BOx activity enhancement

1 Introduction

Biofuel cell technology has received much attention asenergy harvesting devices for powering portable devicesand microscale electronic systems Enzymes functionalizedwith nanomaterials such as carbon nanotubes graphene andnanoparticles provide extremely powerful platforms for widerange of biofuel cell applications that are capable of operatingindependently over a prolonged period of time without theneed of external recharging or refueling of devices Enzymessuch as multicopper oxidases (MCOs) belonging to oxidore-ductase family can reduce oxygen into water performingoxygen reduction reaction (ORR)These reactions have beenextensively studied and described in previous literature [1ndash4] The most common ldquoBluerdquo MCO enzymes are ascorbateoxidase (AOx) laccase (Lac) and bilirubin oxidase (BOx)that can act as excellent biocathode materials for biofuel cell

applications [5 6] These enzymes are more active and selec-tive than the state-of-the-art electrocatalyst platinumbecauseBOx can promote a four-electron reduction of oxygen [7 8]leading directly to water rather than production of significantamount of hydrogen peroxide (H

2O2- 2-electron exchange)

[9] It has been shown previously that BOx demonstratedhigh electrocatalytic oxygen reduction and low overpotentialnecessary to catalyze the reaction and a turnover rate of 07O2per Cusdotsminus1 while Pt catalysts turnover rate is three times

lower at overpotential of 350mV [2 10]Moreover BOx enzymes are well known to perform

ORR when immobilized onto the surface of solid supportsEnhanced activity has been observed when functionalizedwith nanomaterials since nanomaterials have been consid-ered as excellent scaffolding structures for immobilization ofthe enzymes without sacrificing their bioactivity The ORR ismainly dependent on the structure of BOx which was well

HindawiAdvances in Physical ChemistryVolume 2018 Article ID 4712547 9 pageshttpsdoiorg10115520184712547

2 Advances in Physical Chemistry

studied and reported previously [11 12] It contains three dif-ferent copper centers (T1 T2 and T3) with overall four cop-per ions that catalyze the oxidation of bilirubin to biliverdin[13] thereby reducing molecular oxygen to water The mech-anism of electron transfer involves the T1 site of MCOacting as the primary electron acceptor from the substratevia an intramolecular electron transfer (IET) to the T2T3cluster site which converts molecular oxygen to water Recentresearch unveiled whether the ORR in BOx is a four-electrontransfer or a two-electron transfer with a hydrogen peroxideintermediate [7 14] Much research has also been devoted inunderstanding the BOx direct electrochemistry (DET) andelectron transfer via mediation by redox mediators [15ndash17]Nevertheless the use of these enzymes as biocatalysts has notyet been generally adopted for commercial purposes

One persistent challenge is maintaining the catalyticactivity of the enzyme and improving the performance oftenwhen immobilized on a solid support [18 19] Enzymes mayadsorb successfully and however tend to denature renderingsome of the immobilized enzyme inactive and ineffective[20ndash22] To overcome these challenges enzymes that catalyzeelectron transfer reactions must be entrapped in hydrogelsor stabilized with the use of orienting agents Orientingagents promote correct and proper allocation of enzymes onthe surface of the electrode to obtain high current densityThe lone copper on BOx should not be no more than 1-2 nm from the electrode surface to avoid interfacial electrontransfer being the rate-limiting step in oxygen reductionelectrocatalysis [23 24]

Recent studies have demonstrated a trend in surfacemodifications at biointerface level for MCOs based biocath-ode that incorporates the use of aromatic hydrophobic andhydrophilic molecules in order to suitably orient these redoxenzymes with the T1 copper site immobilized on carbonnanotube sidewalls to enhance the oxygen electroreduction[25] These molecules are natural substrates that are spe-cific for the MCO enzymes for oxygen reduction Laccasewas initially studied by the Armstrong group where thehydrophobic pocket of laccase interaction with polycyclicaromatic compounds such as anthracene resulted in remark-able enhancement of electrocatalytic currents The aromaticcompounds structure is very similar to the natural substrateof Lac (phenols) and the strong hydrophobic interactionshave been reported to promote the apt orientation forthe DET [26] Similar strategy was extended to the BOxenzymes where the studies show that the substrate-pocketdid not exhibit hydrophobic interactions but electrostaticinteractions which are an efficient way to achieve directwiring of BOx [27] Along this line different literature reportshave focused on incorporating specific substrates of BOxsuch as bilirubin [28] quinones [29] and syringaldazine[30] towards appropriate orientation that can be convenientfor DET-type electrocatalysis Our group has previouslyreported on crosslinking the enzyme to the electrode withorienting agents two bilirubin functional analogues pyrrole-2-carboxaldehyde and 25- dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde for enzyme orientation and 1-pyrenebutanoicacid succinimidyl ester (PBSE) as the tethering agent Thesecompounds were chosen because they each contain a pyrrole

moiety functionalized with a carbonyl group Thus theelectronegative N-atom from the pyrrole moiety and the O-atom from the aldehyde group can act as hydrogen bondacceptors and the H-atom as a hydrogen bond donor [29]Subsequently we reported on the utilization of syringaldazine(Syr) for enzyme orientation of both Laccase and BOx thatdemonstrated approximately 6 and 9 times increase in cur-rent density respectively compared to physically adsorbedand randomly oriented Lac cathodes [30]

Our present study follows from observations that theactivity and the performance of the oriented BOx werefurther enhanced from incorporating a porphyrin precur-sor solutionmdashhematinmdashon the biocathode Hematin is aferriprotoporphyrin-IX with a hydroxide ion bound to theferric ion formed when hemin (ferriprotoporphyrin-IX witha chloride ion bound to ferric ion) is treated with strongNaOH solution Hemin is an active center of family ofhemeprotein such as b-type cytochromes peroxidase myo-globin and hemoglobin The first electrochemical behaviorof hemin was studied in 1968 on a platinum electrodeby coulostatic method [31] Hemin adsorption on graphiteelectrode demonstrated fast electron transfer that can exceedmonolayer coverage with high amount of active species Sev-eral literature studies show that hemin modified electrodeswere extended in the catalysis and reduction of hydrogenperoxide [32ndash34] oxygen [35] and superoxide [31] Utiliza-tion of hemin protoporphyrin derivatives as pretreatmentof the surface for improving the activity of BOx on cathodeelectrode has also been reported [36]

Herein we demonstrate the enhancement of BOx airbreathing cathode performance via the modification of theelectrode with hematin We also investigated the capabilityand effects of hematin versus hemin to promote an efficientelectron transfer mechanisms for oxygen reduction Wefurther constructed a mediatorless and compartmentlessglucoseO

2DET-type biofuel cell to investigate the cell

performance

2 Materials and MethodologyHemin 1-pyrenebutanoic acid succinimidyl ester (PBSE)was obtained from Setareh Biotech LLC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)was obtained fromTCI America andN-hydroxysuccinimideplusmn 98 (NHS) was obtained from Alfa Aaesar Hematin(945) was obtained from Chem-Impex Intrsquol IncNAD-dependent glucose dehydrogenase (GDH fromPseudomonas sp EC 11147) and glucose were obtainedfrom Sigma Aldrich and used as received BOx was obtainedfrom Myrothecium verrucaria (EC 1335) Amano EnzymeUSA 50 Co Ltd Multiwalled nanotubes (MWNTs)paper (Buckeye Composites) MWNTs (119889 = 20ndash30 nm 119871= 10ndash30 120583m) and single walled nanotubes (SWNTs) (99purity) were obtained from cheaptubescom

3 Electrode Preparations31 Hemin and Hematin Modified Electrodes Air breathingcathodeswere fabricated as in the previously described proce-dure with slight modification [37] Briefly teflonized carbon

Advances in Physical Chemistry 3

Nickel mesh

GDL consisting of teflonized carbon

Catalyst layer consisting of HematinBOxCNT

ink on buckypaper

Oxygen (2)

（+

（2O

(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 XC35and XC50) and MWNTs paper (Buckeye Composites) werehydraulically pressed for 5 minutes at 500 psi Figure 1 showsthe schematic illustration of fabrication of gas diffusion layer(GDL) cathode (a) and image of the biofuel cell (b) A10mM hemin solution was prepared by mixing hemin intoDMSO A 10mM hematin solution was prepared by mixinghemin into 20mM sodium hydroxide (NaOH) solution The20mM NaOH solution was also used to prepare cathodeswith NaOH only modification The as-prepared solutionswere sonicated for 1 hour and 285120583L of the mixture wasdeposited onto the buckeye paper layer (2 cm2) of the pressedair breathing cathode The electrodes were left to dry for 4hours at room temperature prior to being stored overnight at4∘C

32 Hemin and Hematin Modified Electrodes with BOxFurther investigation was carried out with BOx immobilizedon hemin and hematin modified cathodes Preparation ofBOx inkwas carried out as follows 2 wtMWNT ink solutionwas prepared by dissolving 4 1 ratio of water to ethanol(by weight) and added to 100mg MWNTs (10ndash20 nm) Theprepared mixture was subjected to sonication for 1 hourin ice bath vortexing every 20min interval time 5 120583L of300mM Di-Carb (in DMSO) and 170 120583L of stock solutionof 2 MWNT ink were mixed vortexed and incubated for1 hour This was followed by addition of 5 120583L of 300mMPBSE (in DMSO) and 20 uL of water and left for incubationfor 1 hour To this BOx (8mg 16 unitsmL) was weighedand added to MWNT ink and incubated again for 1 hourat room temperature Later 200120583L of the ink was thendeposited on the prewetted buckeye paper of air breathingcathode modified with hemin and hematin respectivelyControl electrodeswere prepared by drop casting the ink ontounmodified air breathing cathodes Following ink depositiona chemical deposition of tetramethyl orthosilicate (TMOS)was performed by sealing cathodes in a Petri dish containingsmall caps filled with water and TMOS The Petri dishremained sealed for 5min before discarding the TMOSCathodes were then stored at 4∘C overnight

33 HematinEDC-NHS Coupling Modified Electrodes withBOx The air breathing BOxhematin electrodes weresuccessively modified with additional components 1-(3-dimethylaminopropyl)-31015840ethylcarbodiimide hydrochloride(EDC) and N-hydroxysuccinimide (NHS) Hematin solution(10mM) was prepared as described in previous section After1-hour sonication 98mg of EDC and NHS was added andincubated to 285120583L hematin solution BOx ink describedearlier was immobilized onto the modified hematinEDC-NHS air breathing cathodes were left for 4 hours at roomtemperature prior to being stored overnight at 4∘C Figure 2shows the schematic representation of stepwise procedure ofhematin modification and BOx deposition on GDL cathodeOxygen enters the cathode through the teflonized carbonGDLand is reduced toH

2Oat the hematin-BOx catalyst layer

34 Electrochemical Testing All electrochemical experimentswere performed with a VMP3 potentiostat (Biologic) Aconventional three-electrode system was used in the mea-surements with AgAgCl as a reference electrode a Pt wireas the auxiliary electrode and a bare or modified electrode asworking electrode Cyclic voltammetry was carried out andpotentiostatic polarization curves were obtained to charac-terize half-cell testing All tests were conducted in 245mMpH 70 PBS buffer at room temperature

35 Complete Fuel Cell Testing Glucose dehydrogenase(GDH Toyobo) based anodes were prepared for completefuel cell testing Polymethylene green (PMG) was electrode-posited onto (electrode area 73 cm2) carbon felt electrodesfollowing a modified version of a previously describedprocedure [30] A single wall nanotube (SWNT-PEI) inksolution containing GDH was drop-casted on top of thePMG treated carbon felt electrodes The anodes were storedovernight at 4∘C prior to testing The GDH anodes werepaired with 9 cm2 (73 cm2 working area) hematin modifiedBOx or unmodified BOx cathodes for complete fuel celltestingThe fuel cells were testedwith 100mMglucose10mMnicotinamide adenine dinucleotide (NAD) in 245mM PBSbuffer pH 70 A Constant Load Discharge (CLD) technique

4 Advances in Physical Chemistry

EDC-NHS couplingDry 4 hrs

Hematin

MWCNT

PBSE

DMY-carb

BOx

Schematic illustration of hematinmodified BOx enzyme constructedon the gas diffusion layer electrode

Figure 2 Schematic illustration of stepwise procedure of hematin modified BOx on gas diffusion layer cathode

was employed to generate power and current density curvesLater discharge curves were generated by applying a constantload of 30mA to the fuel cell The cells were filled withfresh fuel before each subsequent discharge Preparation ofBOx ink for complete fuel cell testing was described inSupplementary File

4 Results and Discussion41 Electrochemical Testing and Characterization of Heminand Hematin Electrodes To better realize the functiontowards the enhancement of BOx cathode hematin airbreathing cathodes were prepared without BOx to determineif the electrodes were independently capable of catalyzing theORR This test was compared simultaneously with hemin airbreathing cathode without BOx The step potential studieswere conducted to evaluate the electrodes under quiescentwith no oxygen saturation conditions At 50mV the currentdensity for the hemin cathode was 04mAcm2 (Figure 3)while current density of 005mAcm2 was observed forhematinThe results showed that hemin demonstrated highercurrent density compared to hematin suggesting that hemincan serve as a catalyst for ORR Several studies have identifiedhemin as a DET-type electrocatalyst for ORR Ma et al haveshown that hemin modified PAMAMMWCNT nanocom-posite films on glassy carbon electrodes can act as both anelectron conductor and catalyticmediator for L-tyrosine [38]Others have identified hemin as an electrocatalyst for oxygenreduction and superoxide detection biosensors Thereforethe increase in current density was predictable howeververy little increase in current density was observed for thehematin electrodes This suggests that hematin without the

250

200

150

100

50

0

minus5000 02 04 06

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

HeminHematin

Figure 3 Representative step potential curves for hemin (blacksquare) and hematin (red circle) air breathing cathodes tested with245mM PBS pH 70

BOx does not act as an electrocatalyst by itself and cannotassist as a catalytic mediator for ORR reduction Thereforewe may assume that hematin adsorbed onto BOx cathodesmay enhance DET catalysis by acting as a natural substrate orprecursor for the BOx enzyme

A cyclic voltammetry (CV) study was conducted tocompare the electrochemical behavior of hematin modifiedair breathing cathodes with bare and EDCNHS-hematin

Advances in Physical Chemistry 5

modified cathodes Figure 1 (Supplementary) shows that thehematin modified air breathing cathodes had a reductionpeak at around 300mV when compared to a bare air breath-ing cathodeThepeak current densitywas 3mAcm2 while noclear oxidation peaks have been observed Since no distinctreversible redox reaction was observed for the hematinmodified air breathing cathodes hematin most likely actsas a natural substrate and a precursor for oxygen reductionreaction (ORR) at the BOx cathode In literature hematinhas been identified as a potential alternative to horseradishperoxidase (HRP) for H

2O2detection [39] and catalysis of

phenol compounds [40] With further addition of crosslink-ing couple EDCNHS the reduction peak for the hematinmodified electrodes has become less distinct A high scanrate of 250mVs was employed for the CV study to test thestability of the EDCNHS-hematin air breathing cathodesFigure 2 (Supplementary) compares cycle 2 to cycle 20 for theair breathing cathodes modified with EDCNHS-hematinThere was little variation observed between the magnitude ofcycles 2 and 20 Shrinking CV curves would suggest that theEDCNHS-hematin was being stripped from the electrodesurfaceThe result of this study suggests that the air breathingcathodes modified with hematinEDCNHS were stable Thestability of the EDCNHS-hematin electrodes wasmost likelydue to the covalent attachment of the hematin to BOx viathe EDCNHS crosslinker Hematin contains two carboxylicacid moieties which allows the hematin to react with EDCand NHS further stabilizes the EDC-hematin intermediateand allows the intermediate product to form a covalentattachment to the BOx EDC and NHS promote the reactionof carboxylic groups of hematin with amino groups of theBOx enzyme

42 Electrochemical Testing and Characterization of HeminBOx and HematinBOx Electrodes We further examined theeffect of hematin hemin and nonmodified BOx catalyzedoxygen reduction A comparison of the current densitiesfrom potentiostatic polarization curves is shown in Figure 4The current density from the electrode containing BOx andhemin was 0007mAcm2 The current density for BOx onlywas 032mAcm2 and the current density in the presenceof hematin modified was 07mAcm2 For all electrodes thecurrent densities generated at 300mV versus sat AgAgClconcluded that hematin led to a significant increase in BOxperformance The polarization experiments with hematindemonstrated more than 2 times increase in the maximumcurrent densities compared to only BOx cathodes with noenhancements (Figure 4) Hematin is a ferriprotoporphyrin-IX with a hydroxide ion bound to the ferric ion formed whenhemin (ferriprotoporphyrin-IX with a chloride ion bound toferric ion) is treated with strong NaOH solution As men-tioned previously hemin has been studied as direct electrontransfer moiety for oxygen reductionTherefore the functionof hematin as an enhancement agent was compared withhemin and ferricyanide as electron transfer mediators (datanot shown) Many such hemin related compounds have beenidentified in literature Catechol hydroquinone pyrogalloland bilirubin which are catalytically oxidized by BOx wereemployed inmodification of biocathodes [28] Recent studies

650

600

550

500

450

400

350

300

250minus01 00 01 02 03 04 05 06 07 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

BOxHeminBOxHematinBOx

Figure 4 Representative step potential curves for BOx (blacksquare) hemin-BOx (red circle) and hematin-BOx (blue triangle)air breathing cathodes tested in 245mM PBS pH 70

have focused on incorporating specific substrates of BOx suchas bilirubin quinones syringaldazine and protoporphyrinderivatives such as protoporphyrin IX iron (III) (PPFeIII)protoporphyrin IX dimethyl ester (PPDE) and octaethylpor-phyrin (OEP) towards appropriate orientation that can beconvenient for DET-type electrocatalysis [36] However nostudy has been previously done on hematin as substrate forBOxTherefore hematinwas further tested as a precursor andpremodifier in the enhancement of BOx enzyme

To demonstrate that improved performance was dueto the addition of hematin BOx electrodes were testedwith only NaOH modification NaOH-hematin and NaOH-EDCNHS-hematin Based on the results (Figure 5) treat-ing the BOx electrode with NaOH led to enhanced airbreathing cathode performance However performance washigher when the electrodes were modified with hemin inNaOH Increased current densities achieved with NaOHmodification were most likely due to the electrode surfacebecoming more hydrophilic which would create a morefavorable environment for the ORR taking place at theelectrode surfaceThehighest current densities were achievedwhen the crosslinking couple EDCNHS were added to thehematin solution Current densities greater than 08mAcm2were achieved by modifying the electrode surface with theEDCNHS-hematin solution All BOx cathode types weretested in triplicate The EDCNHS BOx air breathing cath-odes performed better than the BOx only (no enhancements)cathodes at 300mV but performed significantly less than theNaOH NaOH-hematin and NaOH (EDCNHS)-hematin-BOx cathodes The NaOH (EDCNHS)-hematin-BOx cath-odes remained the highest performing cathodes

43 BOx Loading Optimization High enzyme loadings arerequired for the optimal fuel cell performance However

6 Advances in Physical Chemistry

650

600

550

500

450

400

350

300

25000 1002 04 06 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

NaOHNaOH-Hematin

NaOH (EDCNHS)-HematinNo enhancement

Figure 5 Representative step potential curves for BOx air breathingcathodes modified with NaOH only (black square) NaOH-hematin(red circle) and NaOH (EDCNHS)-hematin (blue triangle) or noenhancement (green inverted triangle) tested in 245mM PBS pH70

600

550

500

450

400

350

300

250

Pote

ntia

l (m

V) v

s A

gA

gCl

Current Density (mA＝Ｇ2)00 10 12 1402minus02 04 06 08

1mg＝Ｇ2 BOx4mg＝Ｇ2 BOx7mg＝Ｇ2 BOx

10mg＝Ｇ2 BOx14mg＝Ｇ2 BOx

Figure 6 Representative step potential curves for BOx air breathingcathodes prepared with different amounts of BOx ranging from1mgcm2 to 14mgcm2 tested in 245mM PBS pH 70

when the enzyme loadings are too high substrate diffusionand product inhibition can limit the rate of overall processesTo minimize the mass diffusion limitations the BOx loadingon hematin modified air breathing cathodes was optimizedThe enzyme loading ranging from 1mgcm2 to 14mgcm2was tested (Figure 6) The results show increase in currentdensities with increase in BOx loading on the cathodeSince the initial rate of reaction of the enzyme correspondsto the activity and amount of enzyme loaded the current

BOx Cathode Performance at 300 mV14

13

12

11

1

09

08

07

Curr

ent D

ensit

y (m

Ac

m2)

BOx Loading (mg＝Ｇ2)05 25 45 65 85 105

y = 0046x + 07991

R2 = 098

Figure 7 Linear regression plot for current density as a function ofBOx loading

600

500

400

300

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

00 10 12 14 1602 04 06 08

Current Density (mA＝Ｇ2)

10mg＝Ｇ2 BOx (hematin modification)14mg＝Ｇ2 BOx (no hematin modification)

Figure 8 Representative step potential curves for optimized BOxair breathing cathodes prepared without hematin (black square) orwith hematin (red circle) tested in 245mM PBS pH 70

increased until optimal loading of 10mgcm2 has reachedand decreased when the electrode surface was loaded with14mgcm2 enzymeThis could be due to diffusion limitationswhere part of the immobilized enzyme may be not beingavailable for catalysis Figure 7 shows the linear dependenceof current density at 300mV with the BOx loading rangingfrom 1mgcm2 to 10mgcm2 It is to be noted that non-hematin-BOx cathodes performed optimally at 14mgcm2BOx loading and by employing hematin BOx loading wasreduced by 29 At 300mV the optimized hematin-BOxcathode performed twice better than the non-hematin-BOxcathode (Figure 8)

44 Complete Fuel Cell Testing A DET-type biofuel cellwithout separators was constructed The biocathode was anair breathing hematinBOx and the bioanodewasGDHPMGcarbon felt electrodes Complete fuel cell testing was con-ducted with 100mM glucose10mM NAD fuel in 245mMPBS buffer pH 70 under quiescent conditions at roomtemperature The current densities dependence of the cellpower density is shown in Figure 9(a)while Figure 9(b) shows

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

Advances in Physical Chemistry 3

Nickel mesh

GDL consisting of teflonized carbon

Catalyst layer consisting of HematinBOxCNT

ink on buckypaper

Oxygen (2)

（+

（2O

(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 XC35and XC50) and MWNTs paper (Buckeye Composites) werehydraulically pressed for 5 minutes at 500 psi Figure 1 showsthe schematic illustration of fabrication of gas diffusion layer(GDL) cathode (a) and image of the biofuel cell (b) A10mM hemin solution was prepared by mixing hemin intoDMSO A 10mM hematin solution was prepared by mixinghemin into 20mM sodium hydroxide (NaOH) solution The20mM NaOH solution was also used to prepare cathodeswith NaOH only modification The as-prepared solutionswere sonicated for 1 hour and 285120583L of the mixture wasdeposited onto the buckeye paper layer (2 cm2) of the pressedair breathing cathode The electrodes were left to dry for 4hours at room temperature prior to being stored overnight at4∘C

32 Hemin and Hematin Modified Electrodes with BOxFurther investigation was carried out with BOx immobilizedon hemin and hematin modified cathodes Preparation ofBOx inkwas carried out as follows 2 wtMWNT ink solutionwas prepared by dissolving 4 1 ratio of water to ethanol(by weight) and added to 100mg MWNTs (10ndash20 nm) Theprepared mixture was subjected to sonication for 1 hourin ice bath vortexing every 20min interval time 5 120583L of300mM Di-Carb (in DMSO) and 170 120583L of stock solutionof 2 MWNT ink were mixed vortexed and incubated for1 hour This was followed by addition of 5 120583L of 300mMPBSE (in DMSO) and 20 uL of water and left for incubationfor 1 hour To this BOx (8mg 16 unitsmL) was weighedand added to MWNT ink and incubated again for 1 hourat room temperature Later 200120583L of the ink was thendeposited on the prewetted buckeye paper of air breathingcathode modified with hemin and hematin respectivelyControl electrodeswere prepared by drop casting the ink ontounmodified air breathing cathodes Following ink depositiona chemical deposition of tetramethyl orthosilicate (TMOS)was performed by sealing cathodes in a Petri dish containingsmall caps filled with water and TMOS The Petri dishremained sealed for 5min before discarding the TMOSCathodes were then stored at 4∘C overnight

33 HematinEDC-NHS Coupling Modified Electrodes withBOx The air breathing BOxhematin electrodes weresuccessively modified with additional components 1-(3-dimethylaminopropyl)-31015840ethylcarbodiimide hydrochloride(EDC) and N-hydroxysuccinimide (NHS) Hematin solution(10mM) was prepared as described in previous section After1-hour sonication 98mg of EDC and NHS was added andincubated to 285120583L hematin solution BOx ink describedearlier was immobilized onto the modified hematinEDC-NHS air breathing cathodes were left for 4 hours at roomtemperature prior to being stored overnight at 4∘C Figure 2shows the schematic representation of stepwise procedure ofhematin modification and BOx deposition on GDL cathodeOxygen enters the cathode through the teflonized carbonGDLand is reduced toH

2Oat the hematin-BOx catalyst layer

34 Electrochemical Testing All electrochemical experimentswere performed with a VMP3 potentiostat (Biologic) Aconventional three-electrode system was used in the mea-surements with AgAgCl as a reference electrode a Pt wireas the auxiliary electrode and a bare or modified electrode asworking electrode Cyclic voltammetry was carried out andpotentiostatic polarization curves were obtained to charac-terize half-cell testing All tests were conducted in 245mMpH 70 PBS buffer at room temperature

35 Complete Fuel Cell Testing Glucose dehydrogenase(GDH Toyobo) based anodes were prepared for completefuel cell testing Polymethylene green (PMG) was electrode-posited onto (electrode area 73 cm2) carbon felt electrodesfollowing a modified version of a previously describedprocedure [30] A single wall nanotube (SWNT-PEI) inksolution containing GDH was drop-casted on top of thePMG treated carbon felt electrodes The anodes were storedovernight at 4∘C prior to testing The GDH anodes werepaired with 9 cm2 (73 cm2 working area) hematin modifiedBOx or unmodified BOx cathodes for complete fuel celltestingThe fuel cells were testedwith 100mMglucose10mMnicotinamide adenine dinucleotide (NAD) in 245mM PBSbuffer pH 70 A Constant Load Discharge (CLD) technique

4 Advances in Physical Chemistry

EDC-NHS couplingDry 4 hrs

Hematin

MWCNT

PBSE

DMY-carb

BOx

Schematic illustration of hematinmodified BOx enzyme constructedon the gas diffusion layer electrode

Figure 2 Schematic illustration of stepwise procedure of hematin modified BOx on gas diffusion layer cathode

was employed to generate power and current density curvesLater discharge curves were generated by applying a constantload of 30mA to the fuel cell The cells were filled withfresh fuel before each subsequent discharge Preparation ofBOx ink for complete fuel cell testing was described inSupplementary File

4 Results and Discussion41 Electrochemical Testing and Characterization of Heminand Hematin Electrodes To better realize the functiontowards the enhancement of BOx cathode hematin airbreathing cathodes were prepared without BOx to determineif the electrodes were independently capable of catalyzing theORR This test was compared simultaneously with hemin airbreathing cathode without BOx The step potential studieswere conducted to evaluate the electrodes under quiescentwith no oxygen saturation conditions At 50mV the currentdensity for the hemin cathode was 04mAcm2 (Figure 3)while current density of 005mAcm2 was observed forhematinThe results showed that hemin demonstrated highercurrent density compared to hematin suggesting that hemincan serve as a catalyst for ORR Several studies have identifiedhemin as a DET-type electrocatalyst for ORR Ma et al haveshown that hemin modified PAMAMMWCNT nanocom-posite films on glassy carbon electrodes can act as both anelectron conductor and catalyticmediator for L-tyrosine [38]Others have identified hemin as an electrocatalyst for oxygenreduction and superoxide detection biosensors Thereforethe increase in current density was predictable howeververy little increase in current density was observed for thehematin electrodes This suggests that hematin without the

250

200

150

100

50

0

minus5000 02 04 06

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

HeminHematin

Figure 3 Representative step potential curves for hemin (blacksquare) and hematin (red circle) air breathing cathodes tested with245mM PBS pH 70

BOx does not act as an electrocatalyst by itself and cannotassist as a catalytic mediator for ORR reduction Thereforewe may assume that hematin adsorbed onto BOx cathodesmay enhance DET catalysis by acting as a natural substrate orprecursor for the BOx enzyme

A cyclic voltammetry (CV) study was conducted tocompare the electrochemical behavior of hematin modifiedair breathing cathodes with bare and EDCNHS-hematin

Advances in Physical Chemistry 5

modified cathodes Figure 1 (Supplementary) shows that thehematin modified air breathing cathodes had a reductionpeak at around 300mV when compared to a bare air breath-ing cathodeThepeak current densitywas 3mAcm2 while noclear oxidation peaks have been observed Since no distinctreversible redox reaction was observed for the hematinmodified air breathing cathodes hematin most likely actsas a natural substrate and a precursor for oxygen reductionreaction (ORR) at the BOx cathode In literature hematinhas been identified as a potential alternative to horseradishperoxidase (HRP) for H

2O2detection [39] and catalysis of

phenol compounds [40] With further addition of crosslink-ing couple EDCNHS the reduction peak for the hematinmodified electrodes has become less distinct A high scanrate of 250mVs was employed for the CV study to test thestability of the EDCNHS-hematin air breathing cathodesFigure 2 (Supplementary) compares cycle 2 to cycle 20 for theair breathing cathodes modified with EDCNHS-hematinThere was little variation observed between the magnitude ofcycles 2 and 20 Shrinking CV curves would suggest that theEDCNHS-hematin was being stripped from the electrodesurfaceThe result of this study suggests that the air breathingcathodes modified with hematinEDCNHS were stable Thestability of the EDCNHS-hematin electrodes wasmost likelydue to the covalent attachment of the hematin to BOx viathe EDCNHS crosslinker Hematin contains two carboxylicacid moieties which allows the hematin to react with EDCand NHS further stabilizes the EDC-hematin intermediateand allows the intermediate product to form a covalentattachment to the BOx EDC and NHS promote the reactionof carboxylic groups of hematin with amino groups of theBOx enzyme

42 Electrochemical Testing and Characterization of HeminBOx and HematinBOx Electrodes We further examined theeffect of hematin hemin and nonmodified BOx catalyzedoxygen reduction A comparison of the current densitiesfrom potentiostatic polarization curves is shown in Figure 4The current density from the electrode containing BOx andhemin was 0007mAcm2 The current density for BOx onlywas 032mAcm2 and the current density in the presenceof hematin modified was 07mAcm2 For all electrodes thecurrent densities generated at 300mV versus sat AgAgClconcluded that hematin led to a significant increase in BOxperformance The polarization experiments with hematindemonstrated more than 2 times increase in the maximumcurrent densities compared to only BOx cathodes with noenhancements (Figure 4) Hematin is a ferriprotoporphyrin-IX with a hydroxide ion bound to the ferric ion formed whenhemin (ferriprotoporphyrin-IX with a chloride ion bound toferric ion) is treated with strong NaOH solution As men-tioned previously hemin has been studied as direct electrontransfer moiety for oxygen reductionTherefore the functionof hematin as an enhancement agent was compared withhemin and ferricyanide as electron transfer mediators (datanot shown) Many such hemin related compounds have beenidentified in literature Catechol hydroquinone pyrogalloland bilirubin which are catalytically oxidized by BOx wereemployed inmodification of biocathodes [28] Recent studies

650

600

550

500

450

400

350

300

250minus01 00 01 02 03 04 05 06 07 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

BOxHeminBOxHematinBOx

Figure 4 Representative step potential curves for BOx (blacksquare) hemin-BOx (red circle) and hematin-BOx (blue triangle)air breathing cathodes tested in 245mM PBS pH 70

have focused on incorporating specific substrates of BOx suchas bilirubin quinones syringaldazine and protoporphyrinderivatives such as protoporphyrin IX iron (III) (PPFeIII)protoporphyrin IX dimethyl ester (PPDE) and octaethylpor-phyrin (OEP) towards appropriate orientation that can beconvenient for DET-type electrocatalysis [36] However nostudy has been previously done on hematin as substrate forBOxTherefore hematinwas further tested as a precursor andpremodifier in the enhancement of BOx enzyme

To demonstrate that improved performance was dueto the addition of hematin BOx electrodes were testedwith only NaOH modification NaOH-hematin and NaOH-EDCNHS-hematin Based on the results (Figure 5) treat-ing the BOx electrode with NaOH led to enhanced airbreathing cathode performance However performance washigher when the electrodes were modified with hemin inNaOH Increased current densities achieved with NaOHmodification were most likely due to the electrode surfacebecoming more hydrophilic which would create a morefavorable environment for the ORR taking place at theelectrode surfaceThehighest current densities were achievedwhen the crosslinking couple EDCNHS were added to thehematin solution Current densities greater than 08mAcm2were achieved by modifying the electrode surface with theEDCNHS-hematin solution All BOx cathode types weretested in triplicate The EDCNHS BOx air breathing cath-odes performed better than the BOx only (no enhancements)cathodes at 300mV but performed significantly less than theNaOH NaOH-hematin and NaOH (EDCNHS)-hematin-BOx cathodes The NaOH (EDCNHS)-hematin-BOx cath-odes remained the highest performing cathodes

43 BOx Loading Optimization High enzyme loadings arerequired for the optimal fuel cell performance However

6 Advances in Physical Chemistry

650

600

550

500

450

400

350

300

25000 1002 04 06 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

NaOHNaOH-Hematin

NaOH (EDCNHS)-HematinNo enhancement

Figure 5 Representative step potential curves for BOx air breathingcathodes modified with NaOH only (black square) NaOH-hematin(red circle) and NaOH (EDCNHS)-hematin (blue triangle) or noenhancement (green inverted triangle) tested in 245mM PBS pH70

600

550

500

450

400

350

300

250

Pote

ntia

l (m

V) v

s A

gA

gCl

Current Density (mA＝Ｇ2)00 10 12 1402minus02 04 06 08

1mg＝Ｇ2 BOx4mg＝Ｇ2 BOx7mg＝Ｇ2 BOx

10mg＝Ｇ2 BOx14mg＝Ｇ2 BOx

Figure 6 Representative step potential curves for BOx air breathingcathodes prepared with different amounts of BOx ranging from1mgcm2 to 14mgcm2 tested in 245mM PBS pH 70

when the enzyme loadings are too high substrate diffusionand product inhibition can limit the rate of overall processesTo minimize the mass diffusion limitations the BOx loadingon hematin modified air breathing cathodes was optimizedThe enzyme loading ranging from 1mgcm2 to 14mgcm2was tested (Figure 6) The results show increase in currentdensities with increase in BOx loading on the cathodeSince the initial rate of reaction of the enzyme correspondsto the activity and amount of enzyme loaded the current

BOx Cathode Performance at 300 mV14

13

12

11

1

09

08

07

Curr

ent D

ensit

y (m

Ac

m2)

BOx Loading (mg＝Ｇ2)05 25 45 65 85 105

y = 0046x + 07991

R2 = 098

Figure 7 Linear regression plot for current density as a function ofBOx loading

600

500

400

300

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

00 10 12 14 1602 04 06 08

Current Density (mA＝Ｇ2)

10mg＝Ｇ2 BOx (hematin modification)14mg＝Ｇ2 BOx (no hematin modification)

Figure 8 Representative step potential curves for optimized BOxair breathing cathodes prepared without hematin (black square) orwith hematin (red circle) tested in 245mM PBS pH 70

increased until optimal loading of 10mgcm2 has reachedand decreased when the electrode surface was loaded with14mgcm2 enzymeThis could be due to diffusion limitationswhere part of the immobilized enzyme may be not beingavailable for catalysis Figure 7 shows the linear dependenceof current density at 300mV with the BOx loading rangingfrom 1mgcm2 to 10mgcm2 It is to be noted that non-hematin-BOx cathodes performed optimally at 14mgcm2BOx loading and by employing hematin BOx loading wasreduced by 29 At 300mV the optimized hematin-BOxcathode performed twice better than the non-hematin-BOxcathode (Figure 8)

44 Complete Fuel Cell Testing A DET-type biofuel cellwithout separators was constructed The biocathode was anair breathing hematinBOx and the bioanodewasGDHPMGcarbon felt electrodes Complete fuel cell testing was con-ducted with 100mM glucose10mM NAD fuel in 245mMPBS buffer pH 70 under quiescent conditions at roomtemperature The current densities dependence of the cellpower density is shown in Figure 9(a)while Figure 9(b) shows

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

4 Advances in Physical Chemistry

EDC-NHS couplingDry 4 hrs

Hematin

MWCNT

PBSE

DMY-carb

BOx

Schematic illustration of hematinmodified BOx enzyme constructedon the gas diffusion layer electrode

Figure 2 Schematic illustration of stepwise procedure of hematin modified BOx on gas diffusion layer cathode

was employed to generate power and current density curvesLater discharge curves were generated by applying a constantload of 30mA to the fuel cell The cells were filled withfresh fuel before each subsequent discharge Preparation ofBOx ink for complete fuel cell testing was described inSupplementary File

4 Results and Discussion41 Electrochemical Testing and Characterization of Heminand Hematin Electrodes To better realize the functiontowards the enhancement of BOx cathode hematin airbreathing cathodes were prepared without BOx to determineif the electrodes were independently capable of catalyzing theORR This test was compared simultaneously with hemin airbreathing cathode without BOx The step potential studieswere conducted to evaluate the electrodes under quiescentwith no oxygen saturation conditions At 50mV the currentdensity for the hemin cathode was 04mAcm2 (Figure 3)while current density of 005mAcm2 was observed forhematinThe results showed that hemin demonstrated highercurrent density compared to hematin suggesting that hemincan serve as a catalyst for ORR Several studies have identifiedhemin as a DET-type electrocatalyst for ORR Ma et al haveshown that hemin modified PAMAMMWCNT nanocom-posite films on glassy carbon electrodes can act as both anelectron conductor and catalyticmediator for L-tyrosine [38]Others have identified hemin as an electrocatalyst for oxygenreduction and superoxide detection biosensors Thereforethe increase in current density was predictable howeververy little increase in current density was observed for thehematin electrodes This suggests that hematin without the

250

200

150

100

50

0

minus5000 02 04 06

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

HeminHematin

Figure 3 Representative step potential curves for hemin (blacksquare) and hematin (red circle) air breathing cathodes tested with245mM PBS pH 70

BOx does not act as an electrocatalyst by itself and cannotassist as a catalytic mediator for ORR reduction Thereforewe may assume that hematin adsorbed onto BOx cathodesmay enhance DET catalysis by acting as a natural substrate orprecursor for the BOx enzyme

A cyclic voltammetry (CV) study was conducted tocompare the electrochemical behavior of hematin modifiedair breathing cathodes with bare and EDCNHS-hematin

Advances in Physical Chemistry 5

modified cathodes Figure 1 (Supplementary) shows that thehematin modified air breathing cathodes had a reductionpeak at around 300mV when compared to a bare air breath-ing cathodeThepeak current densitywas 3mAcm2 while noclear oxidation peaks have been observed Since no distinctreversible redox reaction was observed for the hematinmodified air breathing cathodes hematin most likely actsas a natural substrate and a precursor for oxygen reductionreaction (ORR) at the BOx cathode In literature hematinhas been identified as a potential alternative to horseradishperoxidase (HRP) for H

2O2detection [39] and catalysis of

phenol compounds [40] With further addition of crosslink-ing couple EDCNHS the reduction peak for the hematinmodified electrodes has become less distinct A high scanrate of 250mVs was employed for the CV study to test thestability of the EDCNHS-hematin air breathing cathodesFigure 2 (Supplementary) compares cycle 2 to cycle 20 for theair breathing cathodes modified with EDCNHS-hematinThere was little variation observed between the magnitude ofcycles 2 and 20 Shrinking CV curves would suggest that theEDCNHS-hematin was being stripped from the electrodesurfaceThe result of this study suggests that the air breathingcathodes modified with hematinEDCNHS were stable Thestability of the EDCNHS-hematin electrodes wasmost likelydue to the covalent attachment of the hematin to BOx viathe EDCNHS crosslinker Hematin contains two carboxylicacid moieties which allows the hematin to react with EDCand NHS further stabilizes the EDC-hematin intermediateand allows the intermediate product to form a covalentattachment to the BOx EDC and NHS promote the reactionof carboxylic groups of hematin with amino groups of theBOx enzyme

42 Electrochemical Testing and Characterization of HeminBOx and HematinBOx Electrodes We further examined theeffect of hematin hemin and nonmodified BOx catalyzedoxygen reduction A comparison of the current densitiesfrom potentiostatic polarization curves is shown in Figure 4The current density from the electrode containing BOx andhemin was 0007mAcm2 The current density for BOx onlywas 032mAcm2 and the current density in the presenceof hematin modified was 07mAcm2 For all electrodes thecurrent densities generated at 300mV versus sat AgAgClconcluded that hematin led to a significant increase in BOxperformance The polarization experiments with hematindemonstrated more than 2 times increase in the maximumcurrent densities compared to only BOx cathodes with noenhancements (Figure 4) Hematin is a ferriprotoporphyrin-IX with a hydroxide ion bound to the ferric ion formed whenhemin (ferriprotoporphyrin-IX with a chloride ion bound toferric ion) is treated with strong NaOH solution As men-tioned previously hemin has been studied as direct electrontransfer moiety for oxygen reductionTherefore the functionof hematin as an enhancement agent was compared withhemin and ferricyanide as electron transfer mediators (datanot shown) Many such hemin related compounds have beenidentified in literature Catechol hydroquinone pyrogalloland bilirubin which are catalytically oxidized by BOx wereemployed inmodification of biocathodes [28] Recent studies

650

600

550

500

450

400

350

300

250minus01 00 01 02 03 04 05 06 07 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

BOxHeminBOxHematinBOx

Figure 4 Representative step potential curves for BOx (blacksquare) hemin-BOx (red circle) and hematin-BOx (blue triangle)air breathing cathodes tested in 245mM PBS pH 70

have focused on incorporating specific substrates of BOx suchas bilirubin quinones syringaldazine and protoporphyrinderivatives such as protoporphyrin IX iron (III) (PPFeIII)protoporphyrin IX dimethyl ester (PPDE) and octaethylpor-phyrin (OEP) towards appropriate orientation that can beconvenient for DET-type electrocatalysis [36] However nostudy has been previously done on hematin as substrate forBOxTherefore hematinwas further tested as a precursor andpremodifier in the enhancement of BOx enzyme

To demonstrate that improved performance was dueto the addition of hematin BOx electrodes were testedwith only NaOH modification NaOH-hematin and NaOH-EDCNHS-hematin Based on the results (Figure 5) treat-ing the BOx electrode with NaOH led to enhanced airbreathing cathode performance However performance washigher when the electrodes were modified with hemin inNaOH Increased current densities achieved with NaOHmodification were most likely due to the electrode surfacebecoming more hydrophilic which would create a morefavorable environment for the ORR taking place at theelectrode surfaceThehighest current densities were achievedwhen the crosslinking couple EDCNHS were added to thehematin solution Current densities greater than 08mAcm2were achieved by modifying the electrode surface with theEDCNHS-hematin solution All BOx cathode types weretested in triplicate The EDCNHS BOx air breathing cath-odes performed better than the BOx only (no enhancements)cathodes at 300mV but performed significantly less than theNaOH NaOH-hematin and NaOH (EDCNHS)-hematin-BOx cathodes The NaOH (EDCNHS)-hematin-BOx cath-odes remained the highest performing cathodes

43 BOx Loading Optimization High enzyme loadings arerequired for the optimal fuel cell performance However

6 Advances in Physical Chemistry

650

600

550

500

450

400

350

300

25000 1002 04 06 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

NaOHNaOH-Hematin

NaOH (EDCNHS)-HematinNo enhancement

Figure 5 Representative step potential curves for BOx air breathingcathodes modified with NaOH only (black square) NaOH-hematin(red circle) and NaOH (EDCNHS)-hematin (blue triangle) or noenhancement (green inverted triangle) tested in 245mM PBS pH70

600

550

500

450

400

350

300

250

Pote

ntia

l (m

V) v

s A

gA

gCl

Current Density (mA＝Ｇ2)00 10 12 1402minus02 04 06 08

1mg＝Ｇ2 BOx4mg＝Ｇ2 BOx7mg＝Ｇ2 BOx

10mg＝Ｇ2 BOx14mg＝Ｇ2 BOx

Figure 6 Representative step potential curves for BOx air breathingcathodes prepared with different amounts of BOx ranging from1mgcm2 to 14mgcm2 tested in 245mM PBS pH 70

when the enzyme loadings are too high substrate diffusionand product inhibition can limit the rate of overall processesTo minimize the mass diffusion limitations the BOx loadingon hematin modified air breathing cathodes was optimizedThe enzyme loading ranging from 1mgcm2 to 14mgcm2was tested (Figure 6) The results show increase in currentdensities with increase in BOx loading on the cathodeSince the initial rate of reaction of the enzyme correspondsto the activity and amount of enzyme loaded the current

BOx Cathode Performance at 300 mV14

13

12

11

1

09

08

07

Curr

ent D

ensit

y (m

Ac

m2)

BOx Loading (mg＝Ｇ2)05 25 45 65 85 105

y = 0046x + 07991

R2 = 098

Figure 7 Linear regression plot for current density as a function ofBOx loading

600

500

400

300

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

00 10 12 14 1602 04 06 08

Current Density (mA＝Ｇ2)

10mg＝Ｇ2 BOx (hematin modification)14mg＝Ｇ2 BOx (no hematin modification)

Figure 8 Representative step potential curves for optimized BOxair breathing cathodes prepared without hematin (black square) orwith hematin (red circle) tested in 245mM PBS pH 70

increased until optimal loading of 10mgcm2 has reachedand decreased when the electrode surface was loaded with14mgcm2 enzymeThis could be due to diffusion limitationswhere part of the immobilized enzyme may be not beingavailable for catalysis Figure 7 shows the linear dependenceof current density at 300mV with the BOx loading rangingfrom 1mgcm2 to 10mgcm2 It is to be noted that non-hematin-BOx cathodes performed optimally at 14mgcm2BOx loading and by employing hematin BOx loading wasreduced by 29 At 300mV the optimized hematin-BOxcathode performed twice better than the non-hematin-BOxcathode (Figure 8)

44 Complete Fuel Cell Testing A DET-type biofuel cellwithout separators was constructed The biocathode was anair breathing hematinBOx and the bioanodewasGDHPMGcarbon felt electrodes Complete fuel cell testing was con-ducted with 100mM glucose10mM NAD fuel in 245mMPBS buffer pH 70 under quiescent conditions at roomtemperature The current densities dependence of the cellpower density is shown in Figure 9(a)while Figure 9(b) shows

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

Advances in Physical Chemistry 5

modified cathodes Figure 1 (Supplementary) shows that thehematin modified air breathing cathodes had a reductionpeak at around 300mV when compared to a bare air breath-ing cathodeThepeak current densitywas 3mAcm2 while noclear oxidation peaks have been observed Since no distinctreversible redox reaction was observed for the hematinmodified air breathing cathodes hematin most likely actsas a natural substrate and a precursor for oxygen reductionreaction (ORR) at the BOx cathode In literature hematinhas been identified as a potential alternative to horseradishperoxidase (HRP) for H

2O2detection [39] and catalysis of

phenol compounds [40] With further addition of crosslink-ing couple EDCNHS the reduction peak for the hematinmodified electrodes has become less distinct A high scanrate of 250mVs was employed for the CV study to test thestability of the EDCNHS-hematin air breathing cathodesFigure 2 (Supplementary) compares cycle 2 to cycle 20 for theair breathing cathodes modified with EDCNHS-hematinThere was little variation observed between the magnitude ofcycles 2 and 20 Shrinking CV curves would suggest that theEDCNHS-hematin was being stripped from the electrodesurfaceThe result of this study suggests that the air breathingcathodes modified with hematinEDCNHS were stable Thestability of the EDCNHS-hematin electrodes wasmost likelydue to the covalent attachment of the hematin to BOx viathe EDCNHS crosslinker Hematin contains two carboxylicacid moieties which allows the hematin to react with EDCand NHS further stabilizes the EDC-hematin intermediateand allows the intermediate product to form a covalentattachment to the BOx EDC and NHS promote the reactionof carboxylic groups of hematin with amino groups of theBOx enzyme

42 Electrochemical Testing and Characterization of HeminBOx and HematinBOx Electrodes We further examined theeffect of hematin hemin and nonmodified BOx catalyzedoxygen reduction A comparison of the current densitiesfrom potentiostatic polarization curves is shown in Figure 4The current density from the electrode containing BOx andhemin was 0007mAcm2 The current density for BOx onlywas 032mAcm2 and the current density in the presenceof hematin modified was 07mAcm2 For all electrodes thecurrent densities generated at 300mV versus sat AgAgClconcluded that hematin led to a significant increase in BOxperformance The polarization experiments with hematindemonstrated more than 2 times increase in the maximumcurrent densities compared to only BOx cathodes with noenhancements (Figure 4) Hematin is a ferriprotoporphyrin-IX with a hydroxide ion bound to the ferric ion formed whenhemin (ferriprotoporphyrin-IX with a chloride ion bound toferric ion) is treated with strong NaOH solution As men-tioned previously hemin has been studied as direct electrontransfer moiety for oxygen reductionTherefore the functionof hematin as an enhancement agent was compared withhemin and ferricyanide as electron transfer mediators (datanot shown) Many such hemin related compounds have beenidentified in literature Catechol hydroquinone pyrogalloland bilirubin which are catalytically oxidized by BOx wereemployed inmodification of biocathodes [28] Recent studies

650

600

550

500

450

400

350

300

250minus01 00 01 02 03 04 05 06 07 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

BOxHeminBOxHematinBOx

Figure 4 Representative step potential curves for BOx (blacksquare) hemin-BOx (red circle) and hematin-BOx (blue triangle)air breathing cathodes tested in 245mM PBS pH 70

have focused on incorporating specific substrates of BOx suchas bilirubin quinones syringaldazine and protoporphyrinderivatives such as protoporphyrin IX iron (III) (PPFeIII)protoporphyrin IX dimethyl ester (PPDE) and octaethylpor-phyrin (OEP) towards appropriate orientation that can beconvenient for DET-type electrocatalysis [36] However nostudy has been previously done on hematin as substrate forBOxTherefore hematinwas further tested as a precursor andpremodifier in the enhancement of BOx enzyme

To demonstrate that improved performance was dueto the addition of hematin BOx electrodes were testedwith only NaOH modification NaOH-hematin and NaOH-EDCNHS-hematin Based on the results (Figure 5) treat-ing the BOx electrode with NaOH led to enhanced airbreathing cathode performance However performance washigher when the electrodes were modified with hemin inNaOH Increased current densities achieved with NaOHmodification were most likely due to the electrode surfacebecoming more hydrophilic which would create a morefavorable environment for the ORR taking place at theelectrode surfaceThehighest current densities were achievedwhen the crosslinking couple EDCNHS were added to thehematin solution Current densities greater than 08mAcm2were achieved by modifying the electrode surface with theEDCNHS-hematin solution All BOx cathode types weretested in triplicate The EDCNHS BOx air breathing cath-odes performed better than the BOx only (no enhancements)cathodes at 300mV but performed significantly less than theNaOH NaOH-hematin and NaOH (EDCNHS)-hematin-BOx cathodes The NaOH (EDCNHS)-hematin-BOx cath-odes remained the highest performing cathodes

43 BOx Loading Optimization High enzyme loadings arerequired for the optimal fuel cell performance However

6 Advances in Physical Chemistry

650

600

550

500

450

400

350

300

25000 1002 04 06 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

NaOHNaOH-Hematin

NaOH (EDCNHS)-HematinNo enhancement

Figure 5 Representative step potential curves for BOx air breathingcathodes modified with NaOH only (black square) NaOH-hematin(red circle) and NaOH (EDCNHS)-hematin (blue triangle) or noenhancement (green inverted triangle) tested in 245mM PBS pH70

600

550

500

450

400

350

300

250

Pote

ntia

l (m

V) v

s A

gA

gCl

Current Density (mA＝Ｇ2)00 10 12 1402minus02 04 06 08

1mg＝Ｇ2 BOx4mg＝Ｇ2 BOx7mg＝Ｇ2 BOx

10mg＝Ｇ2 BOx14mg＝Ｇ2 BOx

Figure 6 Representative step potential curves for BOx air breathingcathodes prepared with different amounts of BOx ranging from1mgcm2 to 14mgcm2 tested in 245mM PBS pH 70

when the enzyme loadings are too high substrate diffusionand product inhibition can limit the rate of overall processesTo minimize the mass diffusion limitations the BOx loadingon hematin modified air breathing cathodes was optimizedThe enzyme loading ranging from 1mgcm2 to 14mgcm2was tested (Figure 6) The results show increase in currentdensities with increase in BOx loading on the cathodeSince the initial rate of reaction of the enzyme correspondsto the activity and amount of enzyme loaded the current

BOx Cathode Performance at 300 mV14

13

12

11

1

09

08

07

Curr

ent D

ensit

y (m

Ac

m2)

BOx Loading (mg＝Ｇ2)05 25 45 65 85 105

y = 0046x + 07991

R2 = 098

Figure 7 Linear regression plot for current density as a function ofBOx loading

600

500

400

300

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

00 10 12 14 1602 04 06 08

Current Density (mA＝Ｇ2)

10mg＝Ｇ2 BOx (hematin modification)14mg＝Ｇ2 BOx (no hematin modification)

Figure 8 Representative step potential curves for optimized BOxair breathing cathodes prepared without hematin (black square) orwith hematin (red circle) tested in 245mM PBS pH 70

increased until optimal loading of 10mgcm2 has reachedand decreased when the electrode surface was loaded with14mgcm2 enzymeThis could be due to diffusion limitationswhere part of the immobilized enzyme may be not beingavailable for catalysis Figure 7 shows the linear dependenceof current density at 300mV with the BOx loading rangingfrom 1mgcm2 to 10mgcm2 It is to be noted that non-hematin-BOx cathodes performed optimally at 14mgcm2BOx loading and by employing hematin BOx loading wasreduced by 29 At 300mV the optimized hematin-BOxcathode performed twice better than the non-hematin-BOxcathode (Figure 8)

44 Complete Fuel Cell Testing A DET-type biofuel cellwithout separators was constructed The biocathode was anair breathing hematinBOx and the bioanodewasGDHPMGcarbon felt electrodes Complete fuel cell testing was con-ducted with 100mM glucose10mM NAD fuel in 245mMPBS buffer pH 70 under quiescent conditions at roomtemperature The current densities dependence of the cellpower density is shown in Figure 9(a)while Figure 9(b) shows

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

6 Advances in Physical Chemistry

650

600

550

500

450

400

350

300

25000 1002 04 06 08

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

Current Density (mA＝Ｇ2)

NaOHNaOH-Hematin

NaOH (EDCNHS)-HematinNo enhancement

Figure 5 Representative step potential curves for BOx air breathingcathodes modified with NaOH only (black square) NaOH-hematin(red circle) and NaOH (EDCNHS)-hematin (blue triangle) or noenhancement (green inverted triangle) tested in 245mM PBS pH70

600

550

500

450

400

350

300

250

Pote

ntia

l (m

V) v

s A

gA

gCl

Current Density (mA＝Ｇ2)00 10 12 1402minus02 04 06 08

1mg＝Ｇ2 BOx4mg＝Ｇ2 BOx7mg＝Ｇ2 BOx

10mg＝Ｇ2 BOx14mg＝Ｇ2 BOx

Figure 6 Representative step potential curves for BOx air breathingcathodes prepared with different amounts of BOx ranging from1mgcm2 to 14mgcm2 tested in 245mM PBS pH 70

when the enzyme loadings are too high substrate diffusionand product inhibition can limit the rate of overall processesTo minimize the mass diffusion limitations the BOx loadingon hematin modified air breathing cathodes was optimizedThe enzyme loading ranging from 1mgcm2 to 14mgcm2was tested (Figure 6) The results show increase in currentdensities with increase in BOx loading on the cathodeSince the initial rate of reaction of the enzyme correspondsto the activity and amount of enzyme loaded the current

BOx Cathode Performance at 300 mV14

13

12

11

1

09

08

07

Curr

ent D

ensit

y (m

Ac

m2)

BOx Loading (mg＝Ｇ2)05 25 45 65 85 105

y = 0046x + 07991

R2 = 098

Figure 7 Linear regression plot for current density as a function ofBOx loading

600

500

400

300

Pote

ntia

l (m

V) v

s sa

t A

gA

gCl

00 10 12 14 1602 04 06 08

Current Density (mA＝Ｇ2)

10mg＝Ｇ2 BOx (hematin modification)14mg＝Ｇ2 BOx (no hematin modification)

Figure 8 Representative step potential curves for optimized BOxair breathing cathodes prepared without hematin (black square) orwith hematin (red circle) tested in 245mM PBS pH 70

increased until optimal loading of 10mgcm2 has reachedand decreased when the electrode surface was loaded with14mgcm2 enzymeThis could be due to diffusion limitationswhere part of the immobilized enzyme may be not beingavailable for catalysis Figure 7 shows the linear dependenceof current density at 300mV with the BOx loading rangingfrom 1mgcm2 to 10mgcm2 It is to be noted that non-hematin-BOx cathodes performed optimally at 14mgcm2BOx loading and by employing hematin BOx loading wasreduced by 29 At 300mV the optimized hematin-BOxcathode performed twice better than the non-hematin-BOxcathode (Figure 8)

44 Complete Fuel Cell Testing A DET-type biofuel cellwithout separators was constructed The biocathode was anair breathing hematinBOx and the bioanodewasGDHPMGcarbon felt electrodes Complete fuel cell testing was con-ducted with 100mM glucose10mM NAD fuel in 245mMPBS buffer pH 70 under quiescent conditions at roomtemperature The current densities dependence of the cellpower density is shown in Figure 9(a)while Figure 9(b) shows

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

Advances in Physical Chemistry 7

0 1 2 3 4 5

Standard BOxGDH Fuel CellHematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Pow

er D

ensit

y (m

W＝Ｇ

2)

00

10

12

14

02

minus02

04

06

08

(a)

700

600

500

400

300

200

100

0

minus1 0 1 2 3 4 5 6 7

Pote

ntia

l (m

V)

Hematin-BOxGDH Fuel Cell

Current Density (mA＝Ｇ2)

Standard BOxGDH Fuel Cell

(b)

Figure 9 Representative power density (a) and current density (b) curves for optimizedBOx air breathing cathodes preparedwithout hematin(black square) or with hematin (red circle) paired with GDH anodes tested in 100mM glucose

the maximum current densities for the hematin modifiedBOx cathodes The open circuit potential of 07 V wasobtained The maximum current density was 66mAcm2and maximum power density was 11mWcm2 at 300mV ofthe fuel cell On the other hand with non-hematin modifiedcathode the maximum current density of 25mAcm2 andmaximum power density of 02mWcm2 were obtainedFrom the results the determining factor for the high currentdensity is controlled by the BOx cathode The performanceof the hematin-BOx cathodes was 24 times higher thanthe performance for the BOx cathodes without hematinmodification Constant current load discharge studies of thehematinBOx cathode and GDH anodes based biofuel cellswere further conducted with 50mM glucose10mM NADfuel in 245mM PBS buffer pH 70 A constant current loadof 30mA was applied to each fuel cell until the potentialreached 350mV With injection of fresh fuel the fuel cellwas tested under repeated discharge cycles A total of 4discharges were performed consecutively with 10-minuterecovery periods between discharges as shown in Figure 3(Supplementary)The average runtime varied between 076 hand 10 h with average capacity ranging from 126mAhg to167mAhgThe capacity of the BOxGDH fuel cells decreasedby less than 25 after being discharged to 350mV multipletimes The results suggest that the fuel cells can withstandmultiple discharges (with fresh fuel) without experiencing asignificant loss in capacity These results also indicate thatthere was minimal leaching out of adsorbed functionalitiesof anode and cathode biomaterials

5 Conclusion

In this study direct interaction of BOx on hematin mod-ified MWCNT is capable of increasing the current density

driven by a combination of synchronous effect of naturalsubstrate and favorable orientation for BOx With hematinbased electrode surface modification the optimized loadingof BOx was drastically decreased by 286 and an averagepeak power density above 1mWcm2 was achieved Usinghematin it was investigated that the assembly of hematinwith EDCNHS resulted in significant increase of currentdensity compared to the non-hematin modified electrodeThe hematin modified electrode showed remarkable stabilitywhen subjected to more than 15 cycles of cyclic voltammetryThe current density as high as 66mAcm2 at 300mV andpower density of 11mWcm2 were obtained with GDHO

2

biofuel cell

Disclosure

The content of this manuscript does not necessarily reflectthe position or the policy of the Government and no officialendorsement should be inferred Abstract on this work wassubmitted at 233rd ECS Meeting

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This researchwas supported by theUS ArmyResearchOffice(STTR Contract W911NF-13-C-0015)

Supplementary Materials

Supplementary materials contain the procedure for prepa-ration of bilirubin oxidase (BOx) ink for complete fuel

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

8 Advances in Physical Chemistry

cell testing The cyclic voltammetry studies for testing thebare hematin modified and EDCNHS-hematin modifiedelectrode are shown in Supplemental Figure 1 and showed thestability of hematin modified electrode (Supplemental Figure2) Recycling and discharge studies under constant currentload of GDHBOx fuel cell were shown in SupplementalFigure 3 (Supplementary Materials)

References

[1] N Mano and L Edembe ldquoBilirubin oxidases in bioelectro-chemistry Features and recent findingsrdquoBiosensors andBioelec-tronics vol 50 pp 478ndash485 2013

[2] N Mano J L Fernandez Y Kim W Shin A J Bard andA Heller ldquoOxygen Is Electroreduced to Water on a ldquoWiredrdquoEnzyme Electrode at a Lesser Overpotential than on PlatinumrdquoJournal of the American Chemical Society vol 125 no 50 pp15290-15291 2003

[3] N Mano H-H Kim and A Heller ldquoOn the relationshipbetween the characteristics of bilirubin oxidases and O

2cath-

odes based on their ldquowiringrdquordquoThe Journal of Physical ChemistryB vol 106 no 34 pp 8842ndash8848 2002

[4] N Mano V Soukharev and A Heller ldquoA laccase-wiring redoxhydrogel for efficient catalysis of O

2electroreductionrdquo The

Journal of Physical Chemistry B vol 110 no 23 pp 11180ndash111872006

[5] E I Solomon A J Augustine and J Yoon ldquoO2Reduction to

H2O by the multicopper oxidasesrdquo Dalton Transactions no 30

pp 3921ndash3932 2008[6] E I Solomon U M Sundaram and T E Machonkin ldquoMulti-

copper oxidases and oxygenasesrdquoChemical Reviews vol 96 no7 pp 2563ndash2606 1996

[7] S Brocato C Lau and P Atanassov ldquoMechanistic study ofdirect electron transfer in bilirubin oxidaserdquo ElectrochimicaActa vol 61 pp 44ndash49 2012

[8] D Ivnitski K Artyushkova and P Atanassov ldquoSurface charac-terization and direct electrochemistry of redox copper centersof bilirubin oxidase from fungi Myrothecium verrucariardquo Bio-electrochemistry vol 74 no 1 pp 101ndash110 2008

[9] R D Milton F Giroud A E Thumser S D Minteer and RC T Slade ldquoBilirubin oxidase bioelectrocatalytic cathodes Theimpact of hydrogen peroxiderdquo Chemical Communications vol50 no 1 pp 94ndash96 2014

[10] C C L McCrory X Ottenwaelder T D P Stack and C E DChidsey ldquoKinetic andmechanistic studies of the electrocatalyticreduction of O

2to H2O with mononuclear Cu complexes

of substituted 110-phenanthrolinesrdquo The Journal of PhysicalChemistry A vol 111 no 49 pp 12641ndash12650 2007

[11] B T Doumas B Perry B Jendrzejczak and L Davis ldquoMea-surement of direct bilirubin by use of bilirubin oxidaserdquoClinicalChemistry vol 33 no 8 pp 1349ndash1353 1987

[12] E I Solomon R K Szilagyi S DeBeer George and LBasumallick ldquoElectronic Structures of Metal Sites in Proteinsand Models Contributions to Function in Blue Copper Pro-teinsrdquo Chemical Reviews vol 104 no 2 pp 419ndash458 2004

[13] S Murao and N Tanaka ldquoA New Enzyme ldquoBilirubin OxidaserdquoProduced by Myrothecium verrucaria MT-1rdquo Agricultural andBiological Chemistry vol 45 no 10 pp 2383-2384 1981

[14] L Quintanar C Stoj A B Taylor P J Hart D J Kosman andE I Solomon ldquoShall we dance How a multicopper oxidase

chooses its electron transfer partnerrdquo Accounts of ChemicalResearch vol 40 no 6 pp 445ndash452 2007

[15] P Ramırez N Mano R Andreu et al ldquoDirect electron transferfrom graphite and functionalized gold electrodes to T1 andT2T3 copper centers of bilirubin oxidaserdquo Biochimica et Bio-physica Acta (BBA) - Bioenergetics vol 1777 no 10 pp 1364ndash1369 2008

[16] S Shleev A El Kasmi T Ruzgas and L Gorton ldquoDirectheterogeneous electron transfer reactions of bilirubin oxidaseat a spectrographic graphite electroderdquo Electrochemistry Com-munications vol 6 no 9 pp 934ndash939 2004

[17] S Tsujimura K Kano and T Ikeda ldquoBilirubin oxidase inmultiple layers catalyzes four-electron reduction of dioxygento water without redox mediatorsrdquo Journal of ElectroanalyticalChemistry vol 576 no 1 pp 113ndash120 2005

[18] M Rabe D Verdes and S Seeger ldquoUnderstanding proteinadsorption phenomena at solid surfacesrdquo Advances in Colloidand Interface Science vol 162 no 1-2 pp 87ndash106 2011

[19] M Rasmussen S Abdellaoui and S D Minteer ldquoEnzymaticbiofuel cells 30 years of critical advancementsrdquo Biosensors andBioelectronics vol 76 pp 91ndash102 2016

[20] W Norde ldquoAdsorption of proteins from solution at the solid-liquid interfacerdquo Advances in Colloid and Interface Science vol25 no C pp 267ndash340 1986

[21] W Norde and T Zoungrana ldquoSurface-induced changes in thestructure and activity of enzymes physically immobilized atsolidliquid interfacesrdquoBiotechnology andApplied Biochemistryvol 28 no 2 pp 133ndash143 1998

[22] M van der Veen M C Stuart and W Norde ldquoSpreading ofproteins and its effect on adsorption and desorption kineticsrdquoColloids and Surfaces B Biointerfaces vol 54 no 2 pp 136ndash1422007

[23] C C Page C C Moser X Chen and P L Dutton ldquoNaturalengineering principles of electron tunnelling in biologicaloxidation-reductionrdquoNature vol 402 no 6757 pp 47ndash52 1999

[24] H B Gray and J R Winkler ldquoLong-range electron transferrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 102 no 10 pp 3534ndash3539 2005

[25] N Lalaoui A L Goff M Holzinger M Mermoux and SCosnier ldquoWiring laccase on covalently modified grapheneCarbon nanotube assemblies for the direct bio-electrocatalyticreduction of oxygenrdquo Chemistry - A European Journal vol 21no 8 pp 3198ndash3201 2015

[26] C F Blanford C E Foster R S Heath and F A ArmstrongldquoEfficient electrocatalytic oxygen reduction by the rsquobluersquo copperoxidase laccase directly attached to chemically modified car-bonsrdquo Faraday Discussions vol 140 pp 319ndash335 2008

[27] L Dos Santos V Climent C F Blanford and F A ArmstrongldquoMechanistic studies of the rsquobluersquo Cu enzyme bilirubin oxidaseas a highly efficient electrocatalyst for the oxygen reductionreactionrdquo Physical Chemistry Chemical Physics vol 12 no 42pp 13962ndash13974 2010

[28] K So S Kawai Y Hamano et al ldquoImprovement of a directelectron transfer-type fructosedioxygen biofuel cell with asubstrate-modified biocathoderdquo Physical Chemistry ChemicalPhysics vol 16 no 10 pp 4823ndash4829 2014

[29] R J Lopez S Babanova Y Ulyanova S Singhal and PAtanassov ldquoImproved interfacial electron transfer in modifiedbilirubin oxidase biocathodesrdquo ChemElectroChem vol 1 no 1Article ID CELC201300085 2014

Advances in Physical Chemistry 9

[30] Y Ulyanova S Babanova E Pinchon I Matanovic S Singhaland P Atanassov ldquoEffect of enzymatic orientation throughthe use of syringaldazine molecules on multiple multi-copperoxidase enzymesrdquo Physical Chemistry Chemical Physics vol 16no 26 pp 13367ndash13375 2014

[31] J Chen U Wollenberger F Lisdat B Ge and F W SchellerldquoSuperoxide sensor based on hemin modified electroderdquo Sen-sors andActuators B Chemical vol 70 no 1-3 pp 115ndash120 2000

[32] J Chen L Zhao H Bai and G Shi ldquoElectrochemical detectionof dioxygen and hydrogen peroxide by hemin immobilizedon chemically converted graphenerdquo Journal of ElectroanalyticalChemistry vol 657 no 1-2 pp 34ndash38 2011

[33] G-X Wang Y Zhou M Wang W-J Bao K Wang and X-H Xia ldquoStructure orientation of hemin self-assembly layerdetermining the direct electron transfer reactionrdquo ChemicalCommunications vol 51 no 4 pp 689ndash692 2015

[34] Y Guo J Li and S Dong ldquoHemin functionalized graphenenanosheets-based dual biosensor platforms for hydrogen per-oxide and glucoserdquo Sensors and Actuators B Chemical vol 160no 1 pp 295ndash300 2011

[35] S Antoniadou A D Jannakoudakis and E TheodoridouldquoElectrocatalytic reactions on carbon fibre electrodes modifiedby hemine II Electro-oxidation of hydrazinerdquo Synthetic Metalsvol 30 no 3 pp 295ndash304 1989

[36] N Lalaoui A Le GoffM Holzinger and S Cosnier ldquoFully Ori-ented Bilirubin Oxidase on Porphyrin-Functionalized CarbonNanotube Electrodes for Electrocatalytic Oxygen ReductionrdquoChemistry - A European Journal vol 21 no 47 pp 16868ndash168732015

[37] S Babanova K Artyushkova Y Ulyanova S Singhal and PAtanassov ldquoDesign of experiments and principal componentanalysis as approaches for enhancing performance of gas-diffusional air-breathing bilirubin oxidase cathoderdquo Journal ofPower Sources vol 245 pp 389ndash397 2014

[38] Q Ma S Ai H Yin Q Chen and T Tang ldquoTowards theconception of an amperometric sensor of l-tyrosine based onHeminPAMAMMWCNT modified glassy carbon electroderdquoElectrochimica Acta vol 55 no 22 pp 6687ndash6694 2010

[39] S Pirillo F S Garcıa Einschlag E H Rueda and M LFerreira ldquoHorseradish peroxidase and hematin as biocatalystsfor alizarin degradation using hydrogen peroxiderdquo Industrial ampEngineering Chemistry Research vol 49 no 15 pp 6745ndash67522010

[40] J A Akkara J Wang D-P Yang and K E GonsalvesldquoHematin-catalyzed polymerization of phenol compoundsrdquoMacromolecules vol 33 no 7 pp 2377ndash2382 2000

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

International Journal ofInternational Journal ofPhotoenergy

Hindawiwwwhindawicom Volume 2018

Journal of

Chemistry

Hindawiwwwhindawicom Volume 2018

Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

Journal of

SpectroscopyAnalytical ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

MaterialsJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International Electrochemistry

International Journal of

Hindawiwwwhindawicom Volume 2018

Na

nom

ate

ria

ls

Hindawiwwwhindawicom Volume 2018

Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

International Journal ofInternational Journal ofPhotoenergy

Hindawiwwwhindawicom Volume 2018

Journal of

Chemistry

Hindawiwwwhindawicom Volume 2018

Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

Journal of

SpectroscopyAnalytical ChemistryInternational Journal of

Hindawiwwwhindawicom Volume 2018

MaterialsJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

BioMed Research International Electrochemistry

International Journal of

Hindawiwwwhindawicom Volume 2018

Na

nom

ate

ria

ls

Hindawiwwwhindawicom Volume 2018

Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom