Subscriber access provided by Universiteit Utrecht
Theoretical Modelling, Facile Fabrication and Experimental Study of Optimally Bound Bilirubin Oxidase on Palladium Nanoparticles for Enhanced Oxygen Reduction Reaction Kee Chun Poon, Xiaohua Ma, Desmond Chun Long Tan, Haibin Su, and Hirotaka Sato ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00640 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Theoretical Modelling, Facile Fabrication and Experimental Study of Optimally Bound Bilirubin Oxidase on Palladium Nanoparticles for Enhanced Oxygen Reduction Reaction Kee Chun Poon,†‡ Xiaohua Ma,§‡ Desmond C.L. Tan,† Haibin Su,*§∥ and Hirotaka Sato*† †School of Mechanical & Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 §Institute of Advanced Study, Division of Material Science, School of Materials Science & Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 ∥Department
of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong
Kong 999077 ABSTRACT: This paper presents an optimally bound bilirubin oxidase (BOD) (Myrothecium verrucaria) on palladium nanoparticles (Pd NPs) for enhanced oxygen reduction reaction (ORR). Theoretical modelling of BOD on Pd demonstrated that Pd has strong preferential binding to BOD via T1 copper (Cu) site due to its high adsorption energy. This preferential binding was accompanied by a reduction in distance between the Cu active sites and Pd which would result in an increase in electron transfer rate (kcat) and an enhancement in catalytic activity of BOD. Inspired by the computational results, a biocathode comprising of carbon nanotube (CNT), Pd NPs and BOD (CNT-Pd-BOD) was facilely fabricated using an electroless deposition method. The CNT-Pd-BOD biocathode exhibited higher catalytic activity (1.52 times) and kcat (1.71 times) when compared to CNT-BOD only biocathode. These results demonstrate of Pd NPs as suitable substrate for preferential binding with BOD to increase catalytic activity.
KEYWORDS: palladium, bilirubin oxidase, force field molecular dynamics, oxygen reduction reaction, biofuel cell Bio-fuel cell has attracted much interest over the years as sustainable and renewable source of power for implanted system in living organisms.1-2 In particular, oxygen reduction reaction (ORR) which is the main cathodic reaction in bio-fuel cell, is of great importance as oxygen is the most frequently used reactant.3-4 Currently, bilirubin oxidase (BOD) (Myrothecium verrucaria) is one of the most commonly used enzyme for ORR due to its high catalytic activity at near neutral pH as well as greater tolerance to enzyme deactivation by ions.5 However, the problem with using BOD or enzymes in general is the poor electron transfer between the substrate and active site mostly due to suboptimal binding of the enzyme to substrate which in turn affects the catalytic activity.6-8 Over recent years, researchers have achieved some success in overcoming this poor electron transfer problem by using redox mediators such as 2,2'-Azinobis(3ethylbenzothiazoline-6-sulfonate) (ABTS).3, 9-10 However, these mediators gave rise to other difficulties such as voltage loss due to potential difference between the mediator and enzyme as well as leaching of the mediator.5 Furthermore, these mediators are typically hazardous in nature which severely limits its implantation applications.5, 11
Other research groups have begun exploring alternatives such as using metals (eg. Au, Pd) to improve catalytic performance of BOD. Gutierrez-Sanchez et al. have reported success in modifying the Au electrode surface to achieve different BOD binding (with T1 Cu being the optimal with highest catalytic activity) although the BOD deactivates over time.7 Also, Wen et al. have achieved combining the high catalytic current of Pd-Pt aerogel together with the positive onset potential of BOD although the overall catalytic current was still low.12 Therefore, drawing inspiration from these papers where it is possible to use metals to enhance enzymatic catalytic activity, this research aims to use Pd NPs to such an effect. Theoretical investigation was first conducted to demonstrate that Pd NPs were able to bind optimally to BOD. Based on these results, a biocathode comprising of carbon nanotube (CNT), Pd NPs and BOD (CNT-PdBOD) was facilely fabricated. Lastly, the proposed biocathode was tested experimentally and proven to enhance the BOD catalytic activity by increasing the heterogeneous electron transfer rate. In this work, computational modelling was first conducted to determine if Pd NPs would be a suitable substrate to achieve optimal binding with BOD. BOD is
ACS Paragon Plus Environment
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 11
Figure 2. (a) SEM image of CNT-Pd composite and (b) the EDS of the Pd NPs on the CNT.
ent support substrates. This BOD modulation can be viewed in Figure S1. Figure 1. Computational modelling of (a) BOD T1 orientation on C, (b) BOD T2/T3 orientation on C, (c) BOD T1 orientation on Pd and (d) BOD T2/T3 orientation on Pd.
Table 1. Computational calculations of adsorption energies and distance between Cu active sites (T1, T2/T3) and support substrate. Adsorbed Orientation
T1-Substrate Distance (Å)
T2-Substrate Distance (Å)
Adsorption Energy -1 (kcal mol )
T1 on Pd
5.74
23.24
-179.1
T2/T3 on Pd
51.13
35.97
-106.4
T1 on C
6.20
24.11
-59.3
T2/T3 on C
52.70
37.17
-18.7
known to contain four copper (Cu) active sites mainly Type 1 (T1), Type 2 (T2) and Type 3 (T3).13-14 Typically in BOD, the T1 Cu site is bonded nearest and receives the electrons from the substrate and transfers these electrons through a histidine-cysteine bridge to the trinuclear T2/T3 Cu clusters where ORR takes place.5, 15 This has been previously reported to be the optimal binding conformation of BOD to the substrate to achieve high catalytic activity.14, 16 The distance of the T1 Cu to the substrate and the T2/T3 Cu were reported to be 7-8 Å and 12-14 Å respectively.17 This distance, which is affected by the adsorption energies between BOD and the substrate, can influence the catalytic activity of BOD with shorter distance improving electron transfer rate which in turn increases the catalytic activity.18-20 Therefore, molecular dynamics simulations were conducted to calculate the adsorption energies of BOD onto two different substrate materials which are Pd and carbon (C). These adsorption energies were shown to have a significant impact on the distance between the Cu active sites and the substrate materials. It is important to note that the above mentioned calculation of adsorption energies and difference in distance were a result of BOD modulation due to interaction between BOD and differ-
Figure 1 shows the different orientation of BOD adsorbed onto two different support substrates, Pd and C. The Pd was modelled using Pd (111) surface, which is the most dominant facet of Pd on C support material, while the C modelled was sp2 C, analogous to CNT which is commonly used as a substrate for BOD due to its high conductive nature.21-23 Figure 1a and 1b illustrates the BOD bonded to C while Figure 1c and 1d demonstrates BOD bonded to Pd. For Figure 1a, the T1 Cu active site is close to C (T1 BOD-C) while for Figure 1b it is the T2/T3 Cu cluster which is bound to the C (T2/T3 BOD-C). This is similar for the Pd with Figure 1c showing the T1 Cu active site bonded to Pd (T1 BOD-Pd) while Figure 1d is the T2/T3 Cu cluster bonded to Pd (T2/T3 BOD-Pd). By using this model, the adsorption energies as well as the distance between the T1 and T2/T3 to the substrates were calculated and presented in Table 1. Table 1 shows that regardless of the adsorption orientation of BOD, Pd had higher adsorption energies to BOD compared to C. Both the adsorption energies for T1 BODPd and T2/T3 BOD-Pd were -179.1 kcal mol-1 and -106.4 kcal mol-1 which were higher than that of their counterparts T1 BOD-C and T2/T3 BOD-C -59.3 kcal mol-1 and 18.7 kcal mol-1. In fact, even the adsorption energy for T2/T3 BOD-Pd (previously reported to be suboptimal binding) is still higher than that of T1 BOD-C (optimal binding). This would mean that irrespective of the orientation of BOD, Pd would bind stronger to BOD than C. The effect of these adsorption energies on the distance between Cu active sites and substrates were also illustrated in Table 1. It is observed in Table 1 that at the corresponding binding (T1 or T2/T3) to the substrates, higher adsorption energies would have the shorter distance between Cu active sites and substrates. For the T1 orientated binding, T1 BOD-Pd (T1-5.74 Å, T2/T3-23.24 Å), with the higher adsorption energy, had the shorter distance between the Cu active sites and the substrate compared to T1 BOD-C (T1-6.20 Å, T2/T3-24.11 Å). This trend is also reflected for the T2/T3 orientated binding, with T2/T3 BOD-Pd (T1-51.13 Å, T2/T3-35.97 Å) which had the higher adsorption energy having the lesser distance between Cu active sites and substrate compared to T2/T3 BOD-C (T152.70 Å, T2/T3-37.17 Å).
2 ACS Paragon Plus Environment
Page 3 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Figure 3. Full CV of CNT-Pd-BOD and CNT-BOD biocathode. Measurements were conducted in O2-saturated 0.1 M -1 pH 6.5 PBS solution. Scan rate: 20 mV s
Overall, these results strongly suggest that Pd compared to C was able to achieve stronger binding (higher adsorption energies) which would result in shorter binding distance and would likely improve the electron transfer rate and catalytic activity of BOD. Furthermore, by using Pd as a substrate material, the optimal T1 binding should be achieved due to it having the highest adsorption energy. Lastly, this high adsorption energy of T1 BOD to Pd would imply that this bonding is stable. Based on the results of the computational studies, this report facilely fabricated a CNT-Pd-BOD biocathode for ORR. Firstly, CNT is used as a base support material and is casted onto the electrode. Next, Pd NPs are coated onto the CNT using a facile stepwise electroless deposition method (developed previously) and act as the binding sites for the BOD. This facile fabrication of the Pd NPs required no harsh conditions and could be completed within 10 minutes. Lastly, BOD is casted onto the CNT-Pd composite material to complete the mediatorless CNTPd-BOD biocathode. It is worthwhile to note that CNT serves an additional purpose. The secondary function of CNT is to hold and prevent the leaching of the BOD. This is explained from the cyclic voltammetry (CV) and UV-Vis results that without CNT (Pd NPs on bare glassy carbon electrode) BOD was leached from the surface during the washing process and there was no oxygen reduction reaction (ORR). (Figure S2) The CNT-Pd composite material was observed and characterized under scanning electron microscopy (SEM). It is shown from Figure 2 that CNT coated on the electrode surface was as expected and observed to be similar to a 3D network. Also from Figure 2, the Pd NPs (mean 12 nm) were observed to be well dispersed and coated on the CNT. These Pd NPs were also previously characterized to be crystalline.24 The catalytic activity of the CNT-Pd-BOD biocathode for ORR were analyzed using CV. Figure 3 shows the CVs of the CNT-Pd-BOD and CNT-BOD biocathodes. From Figure 3, it is observed that there are two discrete ORR
Figure 4. CV of CNT-Pd-BOD and CNT-BOD only biocathodes normalized by BOD loading. Measurements were conducted in O2-saturated 0.1 M pH 6.5 PBS solution. Scan -1 rate: 20 mV s
peaks for the CNT-Pd-BOD while the CNT-BOD only has one ORR peak. The first peak which occurs in the range of 0.3 V to 0.6 V and is seen in both CNT-Pd-BOD and CNTBOD is the ORR due to BOD bound by T1 to the substrate. The onset potential of 0.55 V and the potential range corresponds to the literature value for the onset potential and potential range of T1 BOD binding.7, 16, 25 This means that both CNT-BOD and more importantly CNT-Pd-BOD are bound by the optimal T1 BOD substrate binding which is in agreement with the earlier computation results. The second peak which occurs in the range of 0 V to 0.3 V and is seen exclusively in the CNT-Pd-BOD is the ORR peak of the Pd NPs. This is because Pd itself is able to undergo ORR albeit at a lower potential range.12 (Figure S3) Therefore, using the CNT-Pd-BOD provides the additional benefit of increased current at 0 V to 0.3 V compared to using CNT-BOD only. In order to evaluate only the effect of Pd NPs on the ORR capabilities of BOD, only potential range of 0.3 V to 0.6 V is compared. Figure 4 shows the CVs of CNT-PdBOD and CNT-BOD from the range of 0.3 V to 0.6 V normalized by BOD loading (obtained from UV-vis results shown in Figure S5 - S7). By normalizing by BOD loading, it eliminates the possibility of the current increment as a result of different amounts of BOD loading. Figure 4 illustrates that at every potential within the range of 0.3 V to 0.6 V, CNT-Pd-BOD has a higher current compared to CNT-BOD. In fact, the maximum current for CNT-Pd-BOD was 1.02 mA µg-1 while for CNT-BOD it was 0.67 mA µg-1. Therefore Pd NPs is shown to be able to enhance the catalytic current of BOD ORR by 1.52 times. These tallies and strongly supports the computational results that Pd NPs is able to enhanced the catalytic activity of BOD ORR due to optimal T1 binding as well as the strong adsorption energy that minimizes the distance between Cu active sites to the substrate and improves the electron transfer rate. Lastly, it is interesting to note that BOD T1 binding to Pd was stable with subsequent scan
3 ACS Paragon Plus Environment
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
showing little to no drop in catalytic current unlike previously reported results (Figure S4).7 To further confirm the computational results, the electron transfer rate (kcat) for both CNT-Pd-BOD and CNTBOD were calculated using the following formula:9, 12, 26-27 = × × ᴦ where j is the maximum catalytic current achieved, n is the number of electrons involved in the reaction, F is Farady's constant and ᴦ is the surface coverage of the enzyme. The kcat values for CNT-Pd-BOD were determined to be 148 ± 22 s-1 while for CNT-BOD it was 100 ± 13 s-1. This shows that CNT-Pd-BOD had faster electron transfer rate compared to CNT-BOD (1.48 times). In fact, the electron transfer rate of CNT-Pd-BOD was faster (1.30 times) compared to previously reported literature.7 Furthermore, the well-established distance dependent electron transfer rate by Gray et al was used to analyze the electron transfer rate between BOD and the substrate.28-29 Using the computational (distance) and experiment (kcat) results, the β value of our system was found to be 0.78 Å-1 which is in good agreement with that of literature (β = 0.8 Å-1).30 This conclusively shows that the variation of distance of the judiciously chosen substrate greatly improves the electron transfer rate of this bio-fuel cell system. In addition, MET/DET experiment was also conducted.16, 31-32 The CVs of CNT-Pd-BOD and CNT-BOD before and after the addition of ABTS can be seen in Figure S8. The MET/DET ratio for CNT-Pd-BOD was 1.53 ± 0.05 while it was 1.83 ± 0.11 for CNT-BOD. This shows that although the addition of ABTS resulted in an increment of catalytic activity (most probably due to unoptimally bound BOD trapped in CNT matrix), the increment was less pronounced for CNT-PdBOD than for CNT-BOD. These results strongly suggest that there are more optimally bound BOD in CNT-PdBOD compared to that in CNT-BOD. Therefore, these results conclusively proves that the computational studies and experimental results are in agreement with each other and that by using Pd NPs as a substrate, the catalytic activity of BOD ORR is enhanced due to shorter distance to active sites which in turn increases the electron transfer rate. In summary, this report demonstrated using both computational and experimental studies, Pd NPs as a suitable substrate for enhancing the ORR capabilities of BOD by binding preferentially to it. These results are the first known demonstration of Pd NPs binding optimally to BOD and enhancing its catalytic activity. This enhancement was proven by computational calculation to be due to preferred adsorption of T1 Cu active site of BOD to the Pd NPs which is indicated by the high adsorption energy between the BOD and Pd NPs. This in turn reduced the distance between Pd NPs and the Cu active sites of BOD and therefore improved the electron transfer rate. These computational results were supported by the experi-
Page 4 of 11
mental studies which showed that the facile fabrication of CNT-Pd-BOD biocathode had higher catalytic activity (1.52 times) compared to that of CNT-BOD only. This was determined to be a result of CNT-Pd-BOD having a faster electron transfer rate (1.71 times) than CNT-BOD only. These results opens up many new research possibilities in overcoming the electron transfer problems in enzymes and also future studies on the structures and mechanisms on enzymes as well.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] *E-mail:
[email protected] Author Contributions ‡These authors contributed equally.
Funding Sources Nanyang Assistant Professorship (NAP, M4080740) Singapore Ministry of Education (MOE2013-T2-2-049)
Notes The authors declare no competing financial interests.
ASSOCIATED CONTENT Supporting Information The experimental procedures are included in the Supporting Information along with the CV of Pd-BOD (Figure S2) and UV-Vis results (Figure S5 - S7) as well as CV of CNT-Pd only (Figure S3). The Supporting Information is available free of charge via the Internet at http://pubs.acs.org
ABBREVIATIONS Pd, palladium; NPs, nanoparticles; BOD, bilirubin oxidase; copper, Cu; C, carbon; ORR, oxygen reduction reaction; PBS, phosphate buffer solution; CNT, carbon nanotubes; kcat, electron transfer rate
ACKNOWLEDGMENT This study was financially supported by the Nanyang Assistant Professorship (NAP, M4080740) and Singapore Ministry of Education (MOE2013-T2-2-049). The authors appreciate Ms. Koh Joo Luang, Ms. Yong Mei Yoke and Mr. Leong Kwok Phui at Material & Chemical Laboratories at MAE, NTU, for their continuous support and effort to set up and maintain an excellent experimental environment.
REFERENCES (1) Rasmussen, M.; Ritzmann, R. E.; Lee, I.; Pollack, A. J.; Scherson, D. An Implantable Biofuel Cell for a Live Insect. J. Am. Chem. Soc. 2012, 134, 1458-1460. (2) Halamkova, L.; Halamek, J.; Bocharova, V.; Szczupak, A.; Alfonta, L.; Katz, E. Implanted Biofuel Cell Operating in a Living Snail. J. Am. Chem. Soc. 2012, 134, 5040-5043. (3) Cooney, M. J.; Svoboda, V.; Lau, C.; Martin, G.; Minteer, S. D. Enzyme catalysed biofuel cells. Energy Environ. Sci. 2008, 1, 320-337.
4 ACS Paragon Plus Environment
Page 5 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
(4) Calabrese Barton, S.; Gallaway, J.; Atanassov, P. Enzymatic Biofuel Cells for Implantable and Microscale Devices. Chem. Rev. 2004, 104, 4867-4886. (5) Falk, M.; Blum, Z.; Shleev, S. Direct electron transfer based enzymatic fuel cells. Electrochim. Acta 2012, 82, 191-202. (6) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis. Chem. Rev. 2008, 108, 2439-2461. (7) Gutierrez-Sanchez, C.; Ciaccafava, A.; Blanchard, P. Y.; Monsalve, K.; Giudici-Orticoni, M. T.; Lecomte, S.; Lojou, E. Efficiency of Enzymatic O2 Reduction by Myrothecium verrucaria Bilirubin Oxidase Probed by Surface Plasmon Resonance, PMIRRAS, and Electrochemistry. ACS Catal. 2016, 6, 5482-5492. (8) Christenson, A.; Shleev, S.; Mano, N.; Heller, A.; Gorton, L. Redox potentials of the blue copper sites of bilirubin oxidases. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 1634-1641. (9) Trifonov, A.; Herkendell, K.; Tel-Vered, R.; Yehezkeli, O.; Woerner, M.; Willner, I. Enzyme-Capped Relay-Functionalized Mesoporous Carbon Nanoparticles: Effective Bioelectrocatalytic Matrices for Sensing and Biofuel Cell Applications. ACS Nano 2013, 7, 11358-11368. (10) Palmore, G. T. R.; Kim, H.-H. Electro-enzymatic reduction of dioxygen to water in the cathode compartment of a biofuel cell. J. Electroanal. Chem. 1999, 464, 110-117. (11) Ramanavicius, A.; Kausaite, A.; Ramanaviciene, A. Enzymatic biofuel cell based on anode and cathode powered by ethanol. Biosens. Bioelectron. 2008, 24, 761-766. (12) Wen, D.; Liu, W.; Herrmann, A.-K.; Eychmüller, A. A Membraneless Glucose/O2 Biofuel Cell Based on Pd Aerogels. Chem. - Eur. J. 2014, 20, 4380-4385. (13) Mano, N.; Edembe, L. Bilirubin oxidases in bioelectrochemistry: Features and recent findings. Biosens. Bioelectron. 2013, 50, 478-485. (14) Ramírez, P.; Mano, N.; Andreu, R.; Ruzgas, T.; Heller, A.; Gorton, L.; Shleev, S. Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase. Biochim. Biophys. Acta, Bioenerg. 2008, 1777, 1364-1369. (15) Lopez, R. J.; Babanova, S.; Ulyanova, Y.; Singhal, S.; Atanassov, P. Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Biocathodes. ChemElectroChem 2014, 1, 241248. (16) Lalaoui, N.; Holzinger, M.; Le Goff, A.; Cosnier, S. Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme. Chem. - Eur. J. 2016, 22, 10494-10500. (17) dos Santos, L.; Climent, V.; Blanford, C. F.; Armstrong, F. A. Mechanistic studies of the 'blue' Cu enzyme, bilirubin oxidase, as a highly efficient electrocatalyst for the oxygen reduction reaction. PCCP 2010, 12, 13962-13974. (18) Freire, R. S.; Pessoa, C. A.; Mello, L. D.; Kubota, L. T. Direct electron transfer: an approach for electrochemical biosensors with higher selectivity and sensitivity. J. Braz. Chem. Soc. 2003, 14, 230-243. (19) Matanovic, I.; Babanova, S.; Chavez, M. S.; Atanassov, P. Protein-Support Interactions for Rationally Designed Bilirubin Oxidase Based Cathode: A Computational Study. J. Phys. Chem. B 2016, 120, 3634-3641. (20) Kim, J.; Jia, H.; Wang, P. Challenges in biocatalysis for enzyme-based biofuel cells. Biotechnol. Adv. 2006, 24, 296-308.
(21) Holzinger, M.; Le Goff, A.; Cosnier, S. Carbon nanotube/enzyme biofuel cells. Electrochim. Acta 2012, 82, 179190. (22) Zebda, A.; Gondran, C.; Le Goff, A.; Holzinger, M.; Cinquin, P.; Cosnier, S. Mediatorless high-power glucose biofuel cells based on compressed carbon nanotube-enzyme electrodes. Nat. Commun. 2011, 2, 370. (23) Prasad, K. P.; Chen, Y.; Chen, P. Three-Dimensional Graphene-Carbon Nanotube Hybrid for High-Performance Enzymatic Biofuel Cells. ACS Appl. Mater. Interfaces 2014, 6, 3387-3393. (24) Poon, K. C.; Tan, D. C. L.; Vo, T. D. T.; Khezri, B.; Su, H.; Webster, R. D.; Sato, H. Newly Developed Stepwise Electroless Deposition Enables a Remarkably Facile Synthesis of Highly Active and Stable Amorphous Pd Nanoparticle Electrocatalysts for Oxygen Reduction Reaction. J. Am. Chem. Soc. 2014, 136, 5217-5220. (25) de Poulpiquet, A.; Kjaergaard, C. H.; Rouhana, J.; Mazurenko, I.; Infossi, P.; Gounel, S.; Gadiou, R.; GiudiciOrticoni, M. T.; Solomon, E. I.; Mano, N.; Lojou, E. Mechanism of Chloride Inhibition of Bilirubin Oxidases and Its Dependence on Potential and pH. ACS Catal. 2017, 7, 3916-3923. (26) Trifonov, A.; Tel-Vered, R.; Fadeev, M.; Willner, I. Electrically Contacted Bienzyme-Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and MultifuelDriven Biofuel Cells. Adv. Energy Mater. 2015, 5, 1401853-n/a. (27) McArdle, T.; McNamara, T. P.; Fei, F.; Singh, K.; Blanford, C. F. Optimizing the Mass-Specific Activity of Bilirubin Oxidase Adlayers through Combined Electrochemical Quartz Crystal Microbalance and Dual Polarization Interferometry Analyses. ACS Appl. Mater. Interfaces 2015, 7, 25270-25280. (28) Gray, H. B.; Winkler, J. R. Electron flow through metalloproteins. Biochim. Biophys. Acta, Bioenerg. 2010, 1797, 1563-1572. (29) Langen, R.; Chang, I.; Germanas, J.; Richards, J.; Winkler; Gray, H. Electron tunneling in proteins: coupling through a beta strand. Science 1995, 268, 1733-1735. (30) Winkler, J. R. J. R. Electron tunneling pathways in proteins. Curr. Opin. Chem. Biol. 2000, 4, 192-198. (31) Lalaoui, N.; Le Goff, A.; Holzinger, M.; Cosnier, S. Fully Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon Nanotube Electrodes for Electrocatalytic Oxygen Reduction. Chem. - Eur. J. 2015, 21, 16868-16873. (32) Mazurenko, I.; Monsalve, K.; Rouhana, J.; Parent, P.; Laffon, C.; Goff, A. L.; Szunerits, S.; Boukherroub, R.; GiudiciOrticoni, M.-T.; Mano, N.; Lojou, E. How the Intricate Interactions between Carbon Nanotubes and Two Bilirubin Oxidases Control Direct and Mediated O2 Reduction. ACS Appl. Mater. Interfaces 2016, 8, 23074-23085.
5 ACS Paragon Plus Environment
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Paragon Plus Environment
Page 6 of 11
6
Page 7 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Figure 1. Computational modelling of (a) BOD T1 orienta-tion on C, (b) BOD T2/T3 orientation on C, (c) BOD T1 orientation on Pd and (d) BOD T2/T3 orientation on Pd. 72x51mm (300 x 300 DPI)
ACS Paragon Plus Environment
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 2. (a) SEM image of CNT-Pd composite and (b) the EDS of the Pd NPs on the CNT. 88x34mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 8 of 11
Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Figure 3. Full CV of CNT-Pd-BOD and CNT-BOD biocath-ode. Measurements were conducted in O2-saturated 0.1 M pH 6.5 PBS solution. Scan rate: 20 mV s-1 68x41mm (300 x 300 DPI)
ACS Paragon Plus Environment
ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 4. CV of CNT-Pd-BOD and CNT-BOD only biocath-odes normalized by BOD loading. Measurements were conducted in O2-saturated 0.1 M pH 6.5 PBS solution. Scan rate: 20 mV s-1 48x29mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 10 of 11
Page 11 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Catalysis
Table 1. Computational calculations of adsorption energies and distance between Cu active sites (T1, T2/T3) and support substrate. 37x20mm (300 x 300 DPI)
ACS Paragon Plus Environment
