How the Intricate Interactions between Carbon Nanotubes and Two

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How the intricate interactions between carbon nanotubes and two bilirubin oxidases control direct and mediated O2 reduction Ievgen Mazurenko, Karen Monsalve, Jad Rouhana, Philippe Parent, Carine Laffon, Alan Le Goff, Sabine Szunerits, Rabah Boukherroub, Marie-Thérese Giudici-Orticoni, Nicolas Mano, and Elisabeth Lojou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07355 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 18, 2016

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ACS Applied Materials & Interfaces

How the intricate interactions between carbon nanotubes and two bilirubin oxidases control direct and mediated O2 reduction Ievgen Mazurenko,(a) Karen Monsalve,(a) Jad Rouhana,(b) Philippe Parent,(c) Carine Laffon,(c) Alan Le Goff,(d) Sabine Szunerits,(e) Rabah Boukherroub,(e) Marie-Thérèse Giudici-Orticoni,(a) Nicolas Mano,(b) Elisabeth Lojou*(a) (a) Aix Marseille Univ, CNRS, BIP, Bioénergétique et Ingénierie des Protéines UMR7281, 31 chemin Joseph Aiguier 13402 Marseille Cedex 20, France (b) Centre de Recherche Paul Pascal, UPR 8641, CNRS, Bordeaux University, 33600 Pessac, France (c) Aix Marseille Univ, CNRS, CINaM UMR 7325, 13288 Marseille, France (d) Univ. Grenoble Alpes, DCM UMR 5250, 38000 Grenoble, France (e) Institute of Electronics, Microelectronics and Nanotechnology (IEMN, UMR CNRS 8520), Université Lille 1, Cité Scientifique, Avenue Poincaré – BP60069, 59652 Villeneuve d’Ascq, France

KEYWORDS:

Bioelectrocatalysis,

carbon

nanotubes,

bilirubin

oxidase,

electrostatic

interactions, Direct Electron Transfer, Mediated Electron Transfer.

ACS Paragon Plus Environment

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ABSTRACT. Due to the lack of a valid approach in the design of electrochemical interfaces modified with enzymes for efficient catalysis, many oxidoreductases are still not addressed by electrochemistry. We report in this work an in-depth study of the interactions between two different bilirubin oxidases, (from the fungus Myrothecium verrucaria and from the bacterium Bacillus pumilus), catalysts of oxygen reduction, and carbon nanotubes bearing various surface charges (pristine, carboxylic- and pyrene-methylamine-functionnalized). The surface charges and dipole moment of the enzymes as well as the surface state of the nanomaterials are characterized as a function of pH. An original electrochemical approach allows determining the best interface for direct or mediated electron transfer processes as a function of enzyme, nanomaterial type and adsorption conditions. We correlate these experimental results to theoretically determined voltammetric curves. Such an integrative study suggests strategies for designing efficient bioelectrochemical interfaces toward the elaboration of biodevices such as enzymatic fuel cells for sustainable electricity production.


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Introduction Metalloenzymes that compose energy metabolic chains in microorganisms are very efficient and specific catalysts that allow the conversion of a large panel of substrates. Providing their activity is maintained once immobilized on solid supports, they can especially act as biocatalysts in biosensors,1,2 biodevices for CO2 reduction,3 water splitting

4,5

or sustainable energy

production using enzymatic fuel cells (EFCs). Among EFCs, H2/O2 EFCs, which usually use hydrogenase at the anode and multicopper bilirubin oxidase (BOD) at the cathode,6–12 have showed a marked increase in performances during the last five years. Although the power densities and open circuit voltage (OCV) delivered by these EFCs have reached more than 1 mW/cm2 and 1.1 V respectively, further increase of their performances is needed to be competitive with conventional fuel cells. This issue is often addressed by the modification of electrochemical interfaces by conductive nanomaterials to enhance the surface-to-volume ratio, hence consequently increasing the density of incorporated enzymes.13–16 In the case of BODs this has included the use of metal nanoparticles,17–19 and carbon nanomaterials.20–30 Thanks to the confinement into 3D network such nanomaterials are also expected to increase the direct electrical connection (DET) of enzymes. An additional advantage of nanomaterials, which is somehow less considered, relies on their surface chemistry that can be tuned to induce chemical functions having influence on DET of adsorbed enzyme molecules. The intrinsic amino acid composition of enzyme helps in the structural organization of in vivo respiratory pathways and serves for pre-orientation of physiological partners allowing electron transfer.31 When an electrode takes the role of the partner, the amino acid composition of the enzyme results in mixed interactions with the solid surface that most probably involve


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electrostatic, hydrophobic and van der Waals forces32. As a consequence, the enzyme may adopt a preferred orientation on a surface as a function of both enzyme structure and surface composition. The orientation adopted by the molecule upon its adsorption controls the DET process. It is generally accepted that the distance between the electron relay closest to the enzyme surface and the electrode should not exceed 1.5 nm to allow efficient electron tunneling.13,33 In most BODs, the mononuclear T1 copper center located near the surface of the protein was proposed to be the site interacting with the electrode.34,35 Some molecular key parameters for proper orientation of BOD from Myrothecium verrucaria (Mv BOD) were defined on various self-assembled monolayers on gold electrodes.36,37 Based on the crystallographic structure, it was proposed that carboxylic functionalities on the electrode would efficiently connect Mv BOD through the T1.37,38 The chemical forces underlying enzyme orientation in case of non-specific chemisorption on carbon nanomaterials remain however less clear, although it was proposed that both electrostatic and π-π interactions play a role.25,27 Nevertheless, the rational design of electrodes suitable for a specific enzyme, which would take into account both the enzyme structure and the surface chemistry of the electrode in various environmental conditions still requires a deep understanding of the interactions involved. We are currently studying a [NiFe]-hydrogenase which is an attractive biocatalyst presenting outstanding properties such as O2-, CO- and temperature tolerance.39 To take advantage from faster kinetics at elevated temperature, the use of thermostable biocathodes is essential if we envision their use in H2/O2 EFCs.21 While Mv BOD is not stable at temperatures above 35°C,40 we recently identified a thermostable BOD in Bacillus pumilus bacterium (Bp BOD).41 Bp BOD was successfully incorporated into redox hydrogels, exhibiting high mediated current densities


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(MET).42–44 Attempts to directly connect Bp BOD were made by Tsujimura et al. who reported direct current of O2 reduction in mesoporous cryogel and Ketjenblack, although a slow interfacial electron transfer was denoted and attributed to a buried T1 center.45 We immobilized Bp BOD into hydrophobic carbon nanofiber networks21,46 and non-functionalized carbon nanotubes (CNTs).10 However, preliminary studies based on an enzymatic model constructed from the sequences underlined a strong dipole moment pointing opposite to the T1, that is difficult to correlate with the DET-favorable orientation on the non-charged nanomaterials. These apparent discrepancies highlight the requirement for the determination of the molecular basis for an efficient immobilization of the thermostable Bp BOD. To gain insight in the interactions that drive the interfacial electron transfer between enzymes and electrodes, we undertake in this work an integrative study of the immobilization of Mv BOD and Bp BOD on three types of CNTs presenting different surface chemistry. Pristine (i.e. bearing no significant charge), COOH-functionalized (bearing negative charge) and pyrenemethylaminefunctionalized (bearing positive charge) CNTs were used. The interactions between the CNTsurfaces and enzymes are explored by modifying the solution pH during enzyme adsorption. This modulation of pH modifies both the electrode surface charge as a function of the type of CNTs, and the enzyme surface charge as a function of BODs. The DET and MET processes are analyzed experimentally and theoretically, and the key parameters controlling the orientation process of the BODs are determined. This study allows the characterization of the surface chemistry suitable for efficient catalysis using the thermostable Bp BOD but also Mv BOD, and more generally provides the tools to rationalize electrochemical interfaces for enzymatic catalysis.


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Experimental section. Reagents. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), N-Methyl-2pyrrolidone (NMP) and dimethylformamide (DMF) were purchased from Sigma-Aldrich. 1pyrenemethylamine hydrochloride (PyrNH2) was from Aldrich. Citrate buffer solutions were prepared by mixing Na2HPO4 and citric acid with appropriate ratio to obtain pH in the range 3 – 7.8 and a final buffer concentration of 0.1 M (PBS-Cit buffer). Enzymes. Mv BOD was a gift from Amano Enzyme Inc. (Japan). Bp BOD was produced, purified and tested as previously described.47 Carbon nanotubes. Multi-walled carbon nanotubes (MWCNT) (1.5 µm x 9.5 nm, >95% purity) produced by catalytic chemical vapor deposition (CCVD) were obtained from Nanocyl SA (Belgium). MWCNT dispersions (1 mg.mL-1) were prepared by sonication in NMP for 4 hours. These dispersions were centrifuged at 13 000 rpm for 2 min and the supernatant was used for electrode modification. Carboxyl-functionalized multi-walled carbon nanotubes (MWCNTCOOH) (1-5 µm x 15±5 nm, >95% purity, 2-7% COOH-groups) were purchased from NanoLab Inc. (USA). MWCNT-COOH dispersions (1 mg.mL-1) were prepared by sonication for 1 hour in Milli-Q water. Material Characterization. Zeta-potential measurements were made with Zetasizer Nano ZSP (Malvern Instruments, UK) in 0.1 M PBS-Cit buffer using folded disposable cell. Contact angle measurements were performed by depositing a drop of 5 µL of Milli-Q water on the CNTmodified surface. The image of the drop was captured by digital camera and analyzed using standard half-angle method. X-ray photoelectron spectroscopy (XPS) on the CNT deposited onto Au-substrate was carried out using a Resolve 120 hemispherical electron analyzer (PSP Vacuum


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Ltd.) with a pass energy of 20 eV and a unmonochromatized X-ray source (Mg-Kα at 1253.6 eV, 120 W, PSP Vacuum Ltd.). Electrochemistry. All electrochemical measurements were performed in the standard 3electrode cell comprising 3 mm diameter working pyrolytic graphite electrode (PG-electrode) modified with different CNTs, Ag/AgCl (sat. NaCl) reference electrode with double-junction and Pt-wire auxiliary electrode. All the potentials are referred to an Ag/AgCl reference electrode. The cell was controlled by Autolab M101 (Metrohm, Switzerland) or BioLogic SP 150 (BioLogic, France) potentiostats. It was thermostated and the oxygen was constantly purged through the solution unless otherwise specified. To ensure the reproducibility of CNT-deposit, the electrode capacitance was measured by performing cyclic voltammetry (CV) in the range 0.1-0.4 V and retrieving charging current at different scan rates from 35 to 200 mV s-1. The value of the capacitance was determined as a slope of the current versus the scan rate dependence. Only the electrodes whose capacitance deviation did not exceed 10% from the average for a given type of CNTs were used. The enzymatic activity was tested by cyclic voltammetry in the O2-saturated PBS-Cit buffer (0.1 M) at one unique pH 6 in the range 0 – 0.65 V at 5 mV s-1. All current densities were reported taking into account the geometric surface area of the PGelectrode. The relative errors of current measurements have been estimated for each series on the basis of triplicate electrode modification in the same conditions. Electrode modification. PG-electrode was polished by wet emery paper (1200) and cleaned by sonication in 30% ethanol solution. After drying, a dispersion of 5 µL of MWCNT or MWCNT-COOH was deposited on the PG surface and dried at 60°C. This operation was repeated two more times to get 3 layers of CNTs (denoted hereafter as PG-MWCNT and PGMWCNT-COOH electrodes). To functionalize MWCNTs by π-π stacking with PyrNH2, the PG-


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MWCNT electrode was immersed into 20 mM PyrNH2 solution in DMF overnight at 4°C and washed with water before use (denoted hereafter as PG-MWCNT-PyrNH2 electrode). In all cases CNT-films showed good adhesion and were mechanically stable on the electrode surface, as attested by the stability of the catalytic signal along voltametry cycling. The adsorption of the enzymes was performed during potentiodynamic cycling of the electrode between 0 and 0.65 V at different pHs. This allowed us to follow in-situ the adsorption process and to minimize the influence of extreme pHs (pH 3 and 4) on the enzyme stability by making the time of adsorption as short as possible. Briefly, an electrode was put into the cell containing a buffer constantly purged with O2 flow at 25°C. The first two blank cycles were recorded in the absence of enzyme and then 1 nmol (200 nM final concentration) of enzyme was added into the cell while continuing cycling. The increment of enzymatic current for oxygen reduction with each cycle was observed as more and more enzymes were adsorbing on the electrode, and participating in the catalysis (see Figure S1). Once a steady state of adsorption has been reached (i.e. the reduction current stopped growing with each cycle), the electrode was removed from solution, gently washed with buffer and transferred in an enzyme-free PBS-Cit buffer (0.1 M) pH 6 for further experiments. pH 6 was chosen because it was previously used for designing the H2/O2 EFCs.7 No or very small current loss was observed upon the transfer of BOD-modified electrode (Figure S1). Molecular Modelling. All modelling studies were performed on the monomer from Mv BOD (2XLL) and on a model for Bp BOD built by homology to 1GSK (68% identity - Clustal 2.148) using UCSF chimera49 molecular modeling package and Modeller software.50 Structures were prepared and charges were assigned to each atom as function of pH by applying AMBER force field using PDB2PQR and PROPKA servers.51 Dipole moments were determined from the pqr


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output files using the Protein Dipole Moments Server.52 Electrostatic charges at the surface of proteins and dipole moments were illustrated using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.). “Surface closest to the T1” is the protein surface interacting with the electrode for the optimal orientation of the enzyme i.e. for which the distance between the T1 copper and the surface electrode is the smallest (Figure S2). Protein and surface near T1 charges were calculated from the protonated state of ionisable residues in the 1-13 pH range determined using PHEMTO server.53 Electrochemical data fitting. The formalism (1) developed by Armstrong and coworkers54,55 was used to fit the enzymatic curves: =

where

( )

(

) ( )

& = exp (( / )(! − !#$(%) )),

(1)

*+

& ' = exp ((−' / )(!#$(%) − !( /) ( )),

*+

& is the redox potential of T1 Cu, !( /) ( is the equilibrium , = (-'. + -'0 )/-+. . !#$(%)

potential of O2/H2O redox couple, n1 and n2 are the numbers of electrons transferred in the electrochemical and enzymatic reactions respectively, and βd0 is the dispersion parameter. The parameter p describes how proficient the enzymatic electrocatalysis relative to interfacial transfer rate is. In case of the irreversible catalysis by BOD, it becomes , = -'0 /-+. , where k2c is the turnover frequency of BOD and kmax is the interfacial electron transfer constant in case of the best orientation. The background was subtracted from the cyclic voltammograms prior to the fitting and the forward demi-cycles (from 0.65 to 0 V) were used for the fitting using Origin 8.5 software. The potential window was restrained to the range of potentials 0.2 – 0.65 V for Mv BOD and 0.1 - 0.6 V for Bp BOD in which the mass transfer limitation can be neglected (the onset potential of Bp


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BOD is ca. 100 mV lower than Mv BOD). The CV were recorded at a unique pH (pH 6), hence & of T1 remained the same for each enzyme whatever the conditions of the redox potential !#$(%)

adsorption. Furthermore, we assumed that the parameter p is constant for each enzyme/surface pair, independently from the pH of adsorption, since kmax in the best orientation depends only on the CNT surface. Therefore, a shared fit of enzymatic curves was performed by forcing p and & !#$(%) to be the same for each enzyme/CNT pair. The fit was not done for the Bp BOD on

MWCNT-COOH because the DET current was too low and hardly distinguishable from the background to obtain reliable information.

Results and discussion CNTs surface composition and charge. For this study three different types of CNTs were used to provide neutral, negative and positive interfaces. Pristine MWCNT are composed of graphite-like carbons and are supposed to bring no significant charges, except in the places of structure defects. MWCNT-COOH, obtained by strong oxidizing treatment of pristine CNTs show an overall negative surface charge due to induced defects and oxygen functionalities on the CNT walls. Adsorption of an amine-functionalized pyrene derivative on pristine MWCNT provides positive charged structures. Pyrene is interacting in this case strongly with the CNTsurface due to the π-π stacking interaction so no desorption happens upon immersion of the CNTs into the solution.56 The amine-group of pyrene derivative is expected to contribute to the positive charging of the MWCNT-PyrNH2 in a wide pH range.


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The three types of CNTs were analyzed by water contact angle measurements (Figure S3). MWCNTs appear to be highly hydrophobic (contact angle around 120°), while MWCNT-COOH are super hydrophilic (contact angle < 10°). MWCNT-PyrNH2 have a contact angle of 80° The CNTs were characterized by XPS to obtain quantitative information about the purity and functional groups present on their surface. The spectra of all CNTs present well-defined peaks of carbon (C1s at 285 eV) and oxygen (O1s at 533 eV) (Figure 1). Small amounts of nitrogen (N1s at 400 eV) are also detected on all samples. The Au4f and Au4d lines (85 eV and 334-353 eV, respectively) observed on the MWCNT-COOH sample originate from the gold substrate and are due to the inhomogeneity of the deposit (Figure 1, red curve). No other element is detected (within their detection limit of XPS; 0.1-1% at.), indicating the high purity of all CNTs and the absence of noticeable amounts of metallic nanoparticles derived from CNT synthesis. The results of the deconvolution of C1s and O1s peaks (Figure S4) allowing discrimination of different functional groups on the surface are presented in the Table 1. Non-functionalized MWCNTs contain 5.0 at.% of oxygen that may appear on the defect places in the structure of CNTs, notably at the tubes ends.57,58 The deconvolution of the O1s band shows that the oxidative functions mostly consist in ether-type C-O-C (3.2 at.%) and carbonyl C=O (1.3 at.%). The analysis of the C1s band shows that this includes few carboxyl groups (ca. 0.2 at.%). The presence of ionizable carboxylic groups suggests that the charge of MWCNT surface may vary as a function of pH. Therefore, they should not be considered as completely neutral and hydrophobic. As expected, oxidized MWCNT-COOH contain three times more oxygen (14.8 at.%) than pristine MWCNT, divided almost equally between ether-type (5.6 at.%) and carbonyl bonds (5.6


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at.%). The latter is mostly a part of carboxylic groups (4.8 at.%), confirming the high degree of oxidation. Surprisingly, MWCNT contain a higher amount of oxygen (8.0 at.%) after functionalization with PyrNH2 than pristine MWCNT (5.0 at.%). Unlike the case of pristine MWCNT, significant amount of oxygen in MWCNT-PyrNH2 is present in the form of carboxylic groups (2.5 at.%). This difference could arise from prolonged sonication of MWCNT-PyrNH2 resulting in partial oxidation of CNTs.59 An increase of nitrogen content from 0.1 to 1.3 at.% upon modification of MWCNT with PyrNH2 confirms the presence of strongly adsorbed pyrene-derivatives. However, the origin of 1.7 at.% of nitrogen in MWCNT-COOH remains less clear. It might be attributed to the oxidative treatment of CNTs with HNO3/H2SO4 mixture during the fabrication process.57 Thus, although the modification of MWCNT with PyrNH2 is confirmed by the presence of nitrogen, the amount of carboxylic groups on the surface remains almost two times higher leading to a mixed behavior of CNT surface. Although providing the electric double-layer charge rather than absolute values of surface charge, zeta potential measurement gives an indication of the sign of average surface charge.60 The zeta potential values of CNT-dispersions measured at various pHs are showed in Figure 2. These values are in agreement with XPS data, confirming the presence of carboxylic groups on all kinds of CNTs since the most rapid change of the charge occurs at pH 4-5, i.e. near the pKa (≈ 4.5) of COOH-groups. MWCNT are thus almost uncharged at pH 3 and 4 but they demonstrate a slight negative charge (i.e. -10 mV) in the range of pH 5-8 due to deprotonation of carboxylic groups (Figure 2, black curve). On the other hand, MWCNT-COOH have high negative zetapotentials around -50 mV over all the range of pHs studied, confirming the presence of numerous


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acidic groups on the surface (Figure 2, red curve). Their negative charge at pH as low as pH 3 suggests also the presence of some stronger acidic functionalities than COOH-groups that remain deprotonated even at low pH. These functionalities might have been introduced on the stage of acid mixture treatment during the fabrication of MWCNT-COOH. MWCNT-PyrNH2 show a positive charge of up to 20 mV only in the acidic region (pH 3-5), while they bear a negative charge comparable with pristine MWCNT at higher pHs (Figure 2, blue curve). In accordance with the XPS results, this reflects simultaneous presence of amine and carboxylic groups on the CNT surface with a ratio of 1:2. This leads to the emergence of the positive charge only when both functionalities are protonated (i.e. below the pKa of carboxylic groups, ca. 4.5), while the average surface charge appears negative at higher pH due to the predominance of deprotonated carboxylic groups over protonated amine groups. Molecular charge and dipole moments of the BODs. Both BODs belong to the same MCO family but Mv BOD is obtained from a fungus while Bp BOD from a bacterium. While being of approximately the same size, the two BODs have significant differences in the sequences (only 36 % of homology) and, therefore dissimilar distribution of amino acids on their surfaces.41 To correlate the electroenzymatic activity of the two enzymes to their specific interaction with the different CNT-based surfaces, the electrostatic properties of the enzyme molecules were calculated on the basis of their 3D-structures (see Molecular Modelling).38 Figures 3a and b show the values and the directions of dipole moments of the two BODs at different pHs. Bp BOD displays a dipole moment 2-3 times higher than Mv BOD on the whole pH range studied. Thus, its interaction with the electrode surface should be more sensitive to the CNT charge. Moreover, the directions of dipoles for the two BODs are completely opposite. The dipole moment of Mv BOD points mostly in the direction of the T1 copper center, meaning that the


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surface in the vicinity of the T1 center is likely to have a net positive charge, at least in the pH range 5-8 (Figure 3a). For pH 3 and 4, the dipole moment direction deviates from the T1 copper center direction and its value is also twice less. On the contrary, Bp BOD has the dipole moment directed towards the opposite side of the T1 copper in all the pH range studied (Figure 3b), with values in the same order except for pH 3. This argues for a rather negative charge of the part of the surface near the T1 copper center of this enzyme. Further evidence of the difference in electrostatic properties can be obtained by calculating the net molecular charge adopted by Mv BOD (black curve) and Bp BOD (red curve) at different pHs taking into account all the pKa of ionizable amino acids for the whole enzyme molecule (Figure 3c) or only the amino acids near the T1 copper center (Figure 3d, see detailed explanation in Molecular modeling and in Figure S2). The behaviors of the net molecular charge versus pH for both BODs are comparable pointing out a similar ratio between acidic and basic amino acids for the two enzymes (Figure 3c). The isoelectric points derived from the calculation are 4.4 for Mv BOD and 5.2 for Bp BOD, in good agreement with the values determined experimentally.61 The distribution of the amino acids is however specific for each enzyme. In accordance with dipole moments, the two BODs demonstrate substantial differences when only the part of the surface in the vicinity of the T1 Cu was taken into account for the charge calculations (Figure 3d). The environment of the T1 of Mv BOD is charged positively in the pH range from 3 to 8, while the vicinity of the T1 Cu of Bp BOD is positively charged only below pH 4 and has a large negative charge at higher pHs (Figure 3d). Such difference should have a drastic influence on the orientation of those BODs on charged surfaces if the adsorption is controlled by electrostatic interactions. Taking into account the repulsion or attraction of two charges, the highest DET current for Mv BOD should be


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observed at negatively-charged surfaces, while Bp BOD should give the highest catalytic response on positively-charged surfaces at most pHs. This basic assumption will be discussed by following O2 catalytic reduction by the two immobilized BODs. O2 reduction with immobilized BODs The experiment was designed so that that the electroenzymatic response depends exclusively on the amount of enzyme molecules oriented favorably for the DET during the stage of adsorption. Eventhough the desorption and/or the reorientation of a part of the molecules may occur upon changing the pH, the influence of these processes should be negligible on the timescale of the experiment. Moreover, the ionic strength was kept the same in all experiments to eliminate its influence on the distance and force of electrostatic interactions. Once the DET signal was recorded, ABTS was added in the electrochemical cell to probe the MET process, and to access to the proportion of BOD for which the specific orientation does not allow a direct electrical connection. The MET current is probed at 0.0 V, i.e. at high overpotential where the heterogeneous electron transfer (DET) is rather fast. We can anticipate DET (if present) being much faster than MET in conditions where redox mediator concentration is low. Although it cannot be excluded that some mediated catalysis could displace direct catalysis, this would be expected only in the case of enzymes with an orientation that places the electronic relay far from the electrode, thus with low electron transfer rate. The resulting CVs obtained after modification of the electrodes with Mv BOD and Bp BOD are shown in Figure 4 and Figure 5 respectively. DET (black curves) and MET (red curves) voltammograms obtained in buffer solution pH 6 after immobilization at different pHs are plotted as a function of the type of CNTs, i.e. MWCNT-COOH (Figures 4a and 5a), MWCNT (Figures 4b and 5b) and MWCNT-PyrNH2 (Figures 4c and 5c).


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From these CVs, the DET currents for oxygen reduction at 0.0 V are extracted and plotted versus the pH at which the enzyme was adsorbed (Figure S5). The extent of MET is determined as the difference between the currents at 0.0 V in the presence and absence of redox mediator ABTS. This difference is further normalized to the DET current and the MET/DET ratio is plotted versus the pH of adsorption (Figure 6). MWCNT-COOH (“negatively” charged surface). According to the charge distribution, an electrostatic attraction should exist between MWCNT-COOH and the positively-charged T1 in the vicinity of Mv BOD at most pHs. Indeed, the electrode modified at pH 5-8 demonstrates high and steep cathodic current for oxygen reduction in the range 0.7 - 1 mA/cm2 (Figure 4a, black curves) that starts even to be limited by mass-transfer at lower potentials. Decrease of the DET current observed at more acidic pH can be explained by the relative Mv BOD instability in acidic conditions62 and by an unfavorable orientation of BOD on the electrode surface. At pH 3 and 4, the dipole moment is twice lower and does not point to the T1 center anymore which may prevent proper immobilization of the Mv BOD (Figure 4a, black curves). Upon addition of ABTS (Figures 4a, red curves), no current increase is observed in the all pH range indicating that all BODs are properly oriented, leading to a MET/DET ratio close to zero in the whole pH range (Figure 6a, red curve). A small decrease in current was observed occasionally and might be explained by a competitive adsorption of ABTS on the MWCNTCOOH resulting in a decrease of immobilized BOD quantity.63 The latter was further confirmed by the appearance and gradual growth of a pair of reversible peaks at the potential of ABTS while the enzymatic response was declining (data not shown). Contrary to Mv BOD, Bp BOD displays low DET responses on MWCNT-COOH, hardly distinguishable from the background in all the pH range (Figure 5a, black curves). This fact


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correlates well with the negative charge in the vicinity of T1 Cu above pH 4 that repels negatively-charged surface of CNTs, thus resulting in the orientation of the T1 center away from the surface. The DET current is not increased at pH 3 even though the residues around the T1 center have a slight positive charge. At this pH, the slight positive charge cannot compensate the orientation of the dipole moment that remains the same and keeps the T1 away from the electrode surface. However, upon addition of ABTS, MET currents are observed (Figure 5a, red curves). The MET/DET ratio is in the range 4-22 (Figure 6B, red curve) clearly showing the presence of a large quantity of Bp BOD not properly orientated to undergo DET on negatively charged surfaces. MWCNT (“neutral” surface charge). Mv BOD demonstrates increase of the DET current on MWCNT as the adsorption pH increases (Figure 4b, black curves, Figure S5). It is directly linked to a gradual appearance of negative charges on MWCNT that helps to orientate the positively-charged T1 center of Mv BOD towards the electrode surface. The change of orientation is seen even more clearly when considering the MET process (Figure 4b, red curves). Rather high MET/DET ratios (1.8-0.75) are observed at pH 3 and pH 4 indicating the presence of loosely orientated Mv BOD enzymes on the neutral CNT surface (Figure 6a, black curve). A low MET/DET value of ∼0.20 is observed at pHs 5-7.8 reinforcing the relation between a better orientation of enzyme molecules and the appearance of negative charge on the CNT-surface. Nevertheless, the orientation of Mv BOD on MWCNT at pHs 5-7.8 is worse than on the MWCNT-COOH for which the MET/DET ratio was close to zero. The explanation arises from the zeta-potential of MWCNT which is 5 times lower in these pH conditions than that for


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MWCNT-COOH. Weaker electrostatic interactions exist between Mv BOD and MWCNT compared to MWCNT-COOH inducing a higher distribution in orientations. In contrast to Mv BOD, Bp BOD presents a negatively-charged T1 environment. It is thus orientated in opposite way on the MWCNT surface at pH 6-7.8 resulting in weak DET currents (Figure 5b, Figure S5). At pH 3-5, when MWCNT lose their negative charge, the repulsion between residues around the T1 and the surface is decreasing. Doubling of the DET catalytic current is thus observed. The MET/DET ratio confirms this change in orientation. (Figure 6b, black curve). In fact, as soon as the electrode surface is gaining higher negative charges, more and more Bp BOD molecules are adsorbing in unfavorable orientation for DET (but still accessible for MET) resulting in the increase of the MET/DET ratio. MWCNT-PyrNH2 (“positively” charged surface). The same tendency of the DET current with pH is obtained when Mv BOD is adsorbed on MWCNT-PyrNH2 as in the case of MWCNT (Figure 4c). However, the CV shapes differ and will be discussed later in this manuscript. Due to similar zeta-potentials in the pH range 6-7.8, the DET current values are rather similar (Figure 4c and Figure S5). Further gradual decrease of the electrocatalytic current is observed upon decreasing the adsorption pH. It is in accordance with the appearance of positive charges on the MWCNT-PyrNH2 that repels positively-charged T1 center of Mv BOD (Figure 4c and Figure S5). Accordingly, the MET/DET ratio increases continually as the pH decreases (Figure 6a, blue curve). A slightly higher MET/DET ratio is observed on the MWCNT-PyrNH2 compared to MWCNT. It is particularly visible at pH 4-6 (Figure 6a, black and blue curves) and argues in favor of higher quantities of enzyme adsorbed in MET-orientation albeit similar zeta potential of MWCNT and MWCNT-PyrNH2 at these pHs. This is likely due to the presence of some areas with positive charge influencing enzyme orientation even if the total surface charge is negative.


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As expected from the charge of the T1 environment and the dipole moment, the highest DET currents are observed for Bp BOD on MWCNT-PyrNH2 (Figure 5c, Figure S5). A tricky balance between the charge of the CNT surface and the charge of the T1 environment occurs however. The highest DET current is observed at pH 5, where the T1 environment is still very negative but where CNTs have lost most of the negative charges due to the protonation of carboxylic groups. Therefore, the contribution of positively-charged Pyr-NH2 derivatives is increased. At higher pHs (or smaller pHs) the DET decreases because of electrostatic repulsions between the negatively (or positively) charged surfaces and negative (or positive) residues around the T1 (Figure S5). The MET/DET ratio increases with pH but remains below 1 in the pH range from 3 to 6 indicating rather good orientation of Bp BOD to undergo DET (Figure 6b, blue curve). The MET/DET ratio does not increase even at pH 3 where the repulsion of positive charges should exist. However, the direction of dipole moment remains the same at this pH and likely induces correct orientation of enzyme in spite of the repulsion. It should also be mentioned that immobilization support does not influence the thermal stability of the two enzymes. The activity of Mv BOD degraded rapidly above 35 °C whatever the CNT type, while Bp BOD, being a thermostable enzyme, remained stable and even more active at 50 °C (Figure S6). Modeling of the catalytic curve shape: access to the orientation parameter. The interfacial electron transfer constant k0 between the enzyme and the electrode surface depends, among other parameters, on the distance of the electron tunneling. Thus, the distribution in the enzyme orientations creates a dispersion of this constant that may lie in the range between kmin and kmax=k0. In case of a homogeneous distribution of enzyme orientations kmin=k0exp(-βd0), where βd0 is a parameter that represents the dispersion of possible orientations the enzyme molecules


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may adopt while maintaining electron exchange with the electrode. The βd0 parameter governs the shape of the voltammograms as modeled by Armstrong’s group.54,55,64 The model was however designed to account for the homogeneous distribution of distances between the enzymatic active center and the electrode surface, which may not be the case of some surfaces presenting regions with different properties. For example, MWCNT-PyrNH2 may present negatively-charged defects and positively-charged surface induced by adsorbed Pyr-NH2, imposing therefore opposite orientations of BOD and, as a consequence, non-homogeneous distributions of constants between kmin and kmax. This non-homogeneity should have an influence on the βd0 parameter by altering its actual meaning, but keeping the general tendency: narrower scattering of orientations with lower values of βd0. Typical fitted curves for the two BODs on the different CNTs are given in Figure 7 (All the fitted curves are shown in Figure S7). The parameters derived from the fitting of all enzymatic curves according to the equation (1) (see Methods) are given in Table 2. The resulting redox potentials of Cu T1 for Mv and Bp BODs show values of 0.5-0.51 and 0.39-0.40 V vs Ag/AgCl, respectively, in good agreement with previously reported values.41,65–67 Generally, the obtained dispersion parameter βd0 correlates well with the orientations predicted on the basis of the MET/DET ratios. Thus, the lowest βd0 (i.e. 5) - meaning the narrowest distribution of orientations for Mv BOD - is obtained at negatively-charged MWCNT-COOH in the range of pH from 5 to 7.8 (Table 2a). On the other hand, larger values (> 20) of βd0 are obtained on MWCNT-PyrNH2 in the range of pH from 3 to 7. This indicates wider dispersions of Mv BOD orientations on this kind of CNTs and statistically higher distances between T1 center and the electrode surface, in full agreement with electrostatic repulsion of two positively-charged surfaces. For the MWCNT bearing week negative charges, the value of βd0 gradually increases


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from 7 to 13 upon decreasing the pH due to the progressive charge loss upon deionization of groups on the surface (Table 2a). Similarly, the lowest orientation dispersion of Bp BOD (βd0 57) is obtained on the positively-charged MWCNT-PyrNH2 at pHs 4-5 and on MWCNT at pHs 34 (Table 2b). This confirms a better Bp BOD orientation on the positive or weekly-charged surfaces. One should notice also the changes in the parameter p which is 0.1 for Mv BOD on MWCNTCOOH and MWCNT, but one order of magnitude higher on MWCNT-PyrNH2 (i.e. 1.6). As , = -'0 /-+. in case of irreversible electrocatalysis and enzymatic turnover of Mv BOD is constant in all cases, the increase of the p value argues a proportional decrease of the maximal interfacial transfer rate constant kmax due to electrostatic repulsion that increases the minimal distance between T1 and the electrode surface. The lower kmax value together with elevated βd0 values contribute as well to a particular concave shape of cyclic voltammograms of Mv BOD on MWCNT-PyrNH2 that demonstrates typical sluggish electrocatalysis feature (Figure 4c). Correlation between the surface charge and MET/DET ratio. The MET/DET ratio seems to be more reliable than just the DET current in characterizing the orientation of enzyme molecules on charged surfaces.68,69 Being a relative value, the MET/DET ratio is free from errors introduced by a variation of adsorbed enzyme amount due to factors such as enzyme instability, slight differences in CNT electroactive surface area and changing adsorption capacities with pH. To demonstrate the relationship, the MET/DET ratio is plotted versus zeta-potential of different CNTs measured at the pHs of the adsorption (Figure 8). Non-linear correlations exist between these two parameters: positive for Mv BOD, i.e. better orientation on the negatively-charged surface (Figure 8a) and negative for Bp BOD, i.e. better orientation on positively-charged surface (Figure 8b). The origin of the non-linearity becomes clear if one recalls that MET/DET ratio is a


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reciprocal function of the amount of correctly orientated enzyme molecules. Moreover, this is the reason of large statistical errors appearing in MET/DET ratio when the DET current is very small in comparison with MET. In all cases, these results definitely confirm the importance of both the charge of the electrode surface and the charge of the residues around the T1 in order to ensure proper and efficient electrical connection of adsorbed enzymes. Our data tend also to indicate that Bp BOD adsorbs in the correct orientation for DET in case of weak total charge on the surface (cases of pH 3 and pH 4 on MWCNT). This suggests the occurrence of other types of interactions that may drive the orientation of molecules in the absence of strong electrostatic interactions. Apart from acidic residues, the environment of the T1 of Bp BOD contains 35% of nonpolar residues (against 29% for Mv BOD), and in particular 7 phenylalanines (only 2 for Mv BOD) (Figure S2). These residues might be responsible for the apparition of hydrophobic interactions with the non-charged surface of CNTs, helping therefore to orientate the Bp BOD molecule in a favorable position for DET. Furthermore, the presence of several phenylalanines may play a crucial role in the formation of π-π stacking interactions with the surface of CNTs, which dominate in absence of strong electrostatic interactions. This is also most probably the reason why high DET currents were previously obtained when adsorbing Bp BOD in a hydrophobic network composed of carbon nanofibers.21

Conclusions We demonstrate in this work that an in-depth understanding of the interactions between carbon nanotubes and enzymes are required to define the most efficient interface for enzymatic catalysis. O2 reduction by thermostable Bp BOD as a function of enzyme orientation on the surface of CNTs bearing different surface chemistry is compared to the well-known Mv BOD,


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and serves in the determination of the molecular basis of an efficient catalysis of O2 reduction. We propose a simple experimental set up that allows forcing enzyme adsorption into the optimal orientation by tuning the pH for enzyme adsorption. An original integrative approach is thus developed based on the analysis of the charges of the proteins and the CNTs as a function of pH, and a correlation with the electrochemical response both in the absence (DET) and presence of a redox mediator (MET). We clearly show that the 3D nanostructuration does not prevent orientation limitation. The drastic influence of the electrostatic interactions between the surface and enzyme molecules that drive a favorable orientation is instead underlined, although hydrophobic interactions and π-π stacking play a significant role in case of weekly charged surfaces. We connect the differences in amino acid composition and dipole moments of the two BODs with the optimal orientation of Mv BOD on the negatively-charged CNT-surface and of Bp BOD on the positively or weekly-charged surfaces. Modeling of the shapes of the electrocatalytic curves provides key information about the dispersion of enzyme orientations in correlation with the DET/MET ratio, confirming a more regular orientation in case of the T1 vicinity of BODs and the surface of CNTs of opposite charge. The use of enzymes as biocatalysts in bioreactors, biosensors or biofuel cells is attractive but still challenging especially because of poor long term stability. The methodology described in this work can be used for the pre-orientation of enzymes on the electrode prior to their covalent binding aiming to increase the stability of enzyme on the surface. Moreover, it can be applied in order to maximize the DET of other enzymes possessing appreciable dipole moment immobilized on various CNT-based electrodes. At least the molecular basis for efficient O2 reduction at high temperature by Bp BOD allows envisioning the design of a new high


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temperature H2/O2 enzymatic fuel cell with enlarged currents thanks to the entrapment of enzymes into porous carbon nanomaterials bearing the suitable positive functions.


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ASSOCIATED CONTENT Supporting Information. Cyclic voltammograms during enzyme adsorption; amino acids composition of two BODs; contact angle measurements; deconvolution of XPS C1s and O1s peaks; DET current at different pHs and CNTs; fitted voltammetric curves; Bp BOD response at different temperatures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Elisabeth Lojou, Aix Marseille Univ, CNRS, BIP, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France, [email protected] Author Contributions I.M

carried

out

the

electrochemistry

experiments,

zeta

potential

measurements,

electrochemistry modelling and analysis of the results. K.M. performed the electrochemistry experiments. J.R. performed the enzyme modeling. P.P. and C.L. performed the XPS measurements and analysis of the data. S.S. and R.B. helped in the zeta potential measurements. E.L is the initiator and director of the project and participated in all steps. N.M., A.L.G. and MT. G.-O. participated to the discussion of the results. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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ACKNOWLEDGEMENTS The authors thank the Région Aquitaine and ANR for financial support (RATIOCELLS-ANR12-BS08-0011-01 and CAROUCELL-ANR-13-BIOME-0003-02). This work was also supported by A*MIDEX Marseille (ANR-11-IDEX-0001-02). Part of this work was performed within the framework of the Labex AMADEus (ANR-10-LABX-0042-AMADEus) belonging to the program Initiative d’Excellence IdEx Bordeaux (ANR-10-IDEX-003-02). The authors are grateful to Dr Marianne Guiral and Dr Marianne Ilbert (BIP, Marseille, France), and to Iman Marhaba (CINAM, Marseille, France) for fruitful discussion.

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FIGURE CAPTIONS: Figure 1. XPS spectra of (black) MWCNT, (red) MWCNT-COOH and (blue) MWCNT-PyrNH2 deposited on the gold substrate Figure 2. Zeta potential measurements of (black) MWCNT, (red) MWCNT-COOH and (blue) MWCNT-PyrNH2 dispersions in 50 mM phosphate-citrate buffer at 25 °C Figure 3. Electrostatic properties of the two BODs. (a,b) Values (inset tables) and directions of the dipole moments of (a) Mv BOD and (b) Bp BOD calculated at different pHs. Figures were generated using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.) (c,d) Charge of (c) all enzyme molecule and (d) the T1 vicinity of (black) Mv BOD and (red) Bp BOD calculated at different pHs. Figure 4. Electrocatalytic response of Mv BOD. Cyclic voltammograms at pH 6 of (a) MWCNTCOOH, (b) MWCNT, (c) MWCNT-PyrNH2 with Mv BOD adsorbed at different pHs in the (black line) absence and (red line) presence of 10 µM ABTS. All measurements were made in 0.1 M PBS-Cit at 25°C and under O2 flow. v = 5 mV.s-1. Figure 5. Electrocatalytic response of Bp BOD. Cyclic voltammograms at pH 6 of (a) MWCNTCOOH, (b) MWCNT, (c) MWCNT-PyrNH2 with Bp BOD adsorbed at different pHs in the (black line) absence and (red line) presence of 10 µM ABTS. All measurements were made in 0.1 M PBS-Cit at 25°C and under O2 flow. v = 5 mV.s-1.


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Figure 6. The Ratio of MET vs. DET current densities measured at 0.0 V and pH 6 for (a) Mv BOD and (b) Bp BOD adsorbed at (red) MWCNT-COOH, (black) MWCNT and (blue) MWCNT-PyrNH2 as a function of adsorption pH. Figure 7. Electrocatalytic curves obtained at pH 6 after background subtraction (solid lines) and curves fitted to the equation (1) (unfilled circles) for (a) Mv BOD adsorbed at pH 6 and (b) Bp BOD adsorbed at pH 5 on (red) MWCNT-COOH, (black) MWCNT and (blue) MWCNTPyrNH2. Figure 8. Correlation between zeta-potential of CNTs and MET/DET ratio observed after adsorption of (a) Mv BOD and (b) Bp BOD. The dashed lines are given as guides to the eyes.


38

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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 1. XPS spectra of (black) MWCNT, (red) MWCNT-COOH and (blue) MWCNT-PyrNH2 deposited on the gold substrate


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Figure 2. Zeta potential measurements of (black) MWCNT, (red) MWCNT-COOH and (blue) MWCNT-PyrNH2 dispersions in 50 mM phosphate-citrate buffer at 25 °C


40

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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 3. Electrostatic properties of the two BODs. (a,b) Values (inset tables) and directions of the dipole moments of (a) Mv BOD and (b) Bp BOD calculated at different pHs. Figures were generated using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.) (c,d) Charge of (c) all enzyme molecule and (d) the T1 vicinity of (black) Mv BOD and (red) Bp BOD calculated at different pH.


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Figure 4. Electrocatalytic response of Mv BOD. Cyclic voltammograms at pH 6 of (a) MWCNTCOOH, (b) MWCNT, (c) MWCNT-PyrNH2 with Mv BOD adsorbed at different pHs in the (black line) absence and (red line) presence of 10 µM ABTS. All measurements were made in 0.1 M PBS-Cit at 25°C and under O2 flow. Scan rate 5 mV s-1.


42

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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 5. Electrocatalytic response of Bp BOD. Cyclic voltammograms at pH 6 of (a) MWCNTCOOH, (b) MWCNT, (c) MWCNT-PyrNH2 with Bp BOD adsorbed at different pHs in the (black line) absence and (red line) presence of 10 µM ABTS. All measurements were made in 0.1 M PBS-Cit at 25°C and under O2 flow. Scan rate 5 mV s-1.


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Figure 6. The Ratio of MET vs. DET current densities measured at 0.0 V and pH 6 for (a) Mv BOD and (b) Bp BOD adsorbed at (red) MWCNT-COOH, (black) MWCNT and (blue) MWCNT-PyrNH2 as a function of adsorption pH.


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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 7. Electrocatalytic curves obtained at pH 6 after background subtraction (solid lines) and curves fitted to the equation (1) (unfilled circles) for (a) Mv BOD adsorbed at pH 6 and (b) Bp BOD adsorbed at pH 5 on (red) MWCNT-COOH, (black) MWCNT and (blue) MWCNTPyrNH2.


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Figure 8. Correlation between zeta-potential of CNTs and MET/DET ratio observed after adsorption of (a) Mv BOD and (b) Bp BOD. The dashed lines are given as guides to the eyes.


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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


Elemental, Atomic % C 1s deconvolution, Atomic % 2

C 1s

O 1s

N 1s

94.9

5.0

0.1

58.1 26.8 6.2

3.6

0.2

MWCNT-COOH 83.5

14.8

1.7

47.4 15.0 8.9

7.5

MWCNT-PyrNH2 90.7

8.0

1.3

60.0

9.6 12.8 5.9

MWCNT

O 1s deconvolution, Atomic %

3

C sp C sp C-O C=O O=C-O H2O O-H C-O-C C=O C=O conjugated -

0.3

3.2

1.3

0.1

4.8

0.3 2.3

5.6

5.6

0.9

2.5

0.1 1.0

4.3

2.2

0.4

Table 1. Quantitative comparison of XPS spectra of different CNTs based on the deconvolution of C1s and O1s core levels (Figure S4).


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(a) MWCNT-COOH βd0

p

Jlim

MWCNT E0

βd0

p

MWCNT-PyrNH2 E0

Jlim

βd0

p

E0

Jlim

pH 3

7.3

-0.01

12.9

-0.01

22.1

-0.12

pH 4

7.6

-0.09

10.7

-0.04

22.4

-0.21

pH 5

5.1

pH 6

5.2

-0.78

8.5

-0.19

28.7

-0.48

pH 7

4.7

-0.88

7.7

-0.12

19.5

-0.52

pH 7.8

4.8

-0.61

7.1

-0.21

9.2

-0.20

0.16

-0.63

0.499

9.4

0.11

-0.09

23.3

0.509

1.63

-0.33

0.510

(b) MWCNT βd0

p

Jlim

MWCNT-PyrNH2 E0

βd0

p

Jlim

pH 3

5.1

-0.18

10.1

-0.34

pH 4

6.2

-0.15

6.5

-0.50

pH 5

26.0

pH 6

26.4

-0.15

9.6

-0.61

pH 7

38.4

-0.22

25.5

-1.20

pH 7.8

70.6

-0.18

29.7

-0.64

0.86

-0.45

0.388

7.0

0.37

-0.83

E0

0.404

Table 2. The parameters obtained from the fitting of enzymatic curves obtained at pH 6 for (a) Mv BOD and (b) Bp BOD adsorbed at different pH. n1 = 1, n2 = 4, Eeqm(O2/H2O) = 0.641 V, αc = 0.5


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TOC Graphic


49

How the Intricate Interactions between Carbon Nanotubes and Two

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