Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme

We report the controlled orientation of bilirubin oxidases (BOD) from Myrothecium verrucaria on multiwalled carbon nanotubes (MWCNTs) functionalised by electrografting of 6 ‐ carboxynaphthalenediazonium and 4 ‐ (2 ‐ aminoethyl)benzenediazonium salts. On negatively charged naphthoate ‐ modified MWCNTs, a high ‐ potential (0.44 V vs. SCE) oxygen reduction electrocatalysis is observed, occurring via the T1 copper centre. On positively charged ammonium ‐ modified MWCNTs, a low ‐ potential (0.15 V) oxygen reduction electrocatalysis is observed, occurring through a partially oxidised state of the T2/T3 trinuclear copper cluster. Finally, chemically modified naphthoate MWCNTs exhibit high bioelectrocatalytic current densities of 3.9 mA cm−2 under air at gas ‐ diffusion electrode.

Caught on the tube: The controlled orientation of bilirubin oxidases from Myrothecium verrucaria (MvBOD) on multiwalled carbon nanotubes (MWCNTs) functionalised by electrografting of 6 ‐ carboxynaphthalenediazonium and 4 ‐ (2 ‐ aminoethyl)benzenediazonium salts is reported. Specific covalent functionalisation of carbon nanotubes (see figure) enables control over electron transfer and oxygen diffusion to the oxygen ‐ reducing copper enzyme.

bilirubin oxidases; diazonium; metalloenzymes; nanotubes; oxygen reduction

Enzymatic fuel cells (EFCs) are based on the immobilisation of redox enzymes on electrodes and their efficient electronic contact.[1] , [2] , [3] , [4] , [5] In addition to a high catalyst loading, high power EFCs require the maximisation of electron transfer (ET) rates in order to achieve low potential/high current bioelectrocatalysis. This is especially the case at the cathode side where oxygen must be reduced into water in a 4 H+/4 e− process. For this purpose, high potential multicopper oxidases (MCOs) have been widely studied at the surface of electrodes for the ability of their multicopper active site to reduce oxygen at low overpotentials, either by direct electron transfer (DET) or mediated electron transfer (MET).[6] , [7] While a redox mediator can be used to transfer electrons by MET to the T1 copper centre,[8] , [9] a direct electron transfer (DET) is also favourable since the T1 copper centre is located at the surface of the protein. DET has many advantages over MET since redox behaviour of the copper clusters can be observed and the enzyme can catalyse oxygen reduction at low overpotentials of only a hundred millivolts at pH 7 as compared to the O2/H2O redox couple.[6] , [7] Bilirubin oxidases (BOD) belong to MCO family, in which a mononuclear type 1 (T1) copper centre acts as an electron relay from the substrate to a trinuclear Type 2/Type 3 active site (TNC). Recent spectroscopic and crystallographic studies on MCOs have shown that electron transfers via the T1 copper centre is required to trigger the full reduction of the T3 copper centre in the resting oxidised (RO) state of the TNC.[10] , [11] A different state, called alternative resting (AR), has also been characterised and is composed of a single oxidised copper(II) in the TNC. This AR form is only reduced through low ‐ potential reduction without the participation of the T1 unit.[10] , [12] The first evidence of a DET at the TNC for an immobilised enzyme in direct contact with the electrode was reported by Shleev et al. for BOD from Trachyderma tsunodae on gold electrodes.[13] In light of novel spectroscopic and crystallographic studies, it has been proposed that the low ‐ potential redox system corresponding to the TNC[13] , [14] could be attributed to this low ‐ potential alternative form of the TNC, as it has been suggested for BOD from Magnaporthe oryzae and Myrothecium verrucaria (MvBOD) immobilised on graphite electrodes and studied by protein film voltammetry.[10] , [11] , [12]

In order to achieve an efficient DET, many recent studies have demonstrated the crucial role of the functionalisation of the electrode in order to favour a specific orientation of the enzyme towards the electronic relay located at the surface of the protein. Owing to a set of hydrophobic amino acids, laccase from Trametes versicolor was immobilised on electrodes modified with hydrophobic groups.[15] , [16] For MvBOD, Blanford et al. have shown that substrate ‐ like functionalities could also favour a specific orientation towards the substrate pocket of the enzyme.[17] Furthermore, the role of the charge of the electrode surface has been underlined by several studies, showing that negative charges at the surface of gold electrodes promote a DET to the T1 of MvBOD while neutral or positive charges switch the DET off.[18] , [19] We have also recently shown that orientation of MvBOD on functionalised carbon nanotubes (CNTs) was driven by a combination of negatively charged molecules having structural relation with bilirubin.[20] In this matter, CNTs represent a fantastic electrode material for studying DET of oriented enzymes. Owing to a library of functionalisation techniques, CNTs are an ideal conductive substrate for studying the influence of surface modification towards enzyme orientation. These functionalisation strategies have been widely developed for laccases. Noncovalent modifications have relied on π ‐ stacking interactions of pyrenes modified with specific groups for covalent coupling[21] , [22] , [23] , [24] and substrate ‐ like interactions.[22] , [23] , [25] Covalent coupling has also been employed using reactions such as diazonium chemistry,[26] amination[27] or amide coupling.[28] We have recently investigated the chemical grafting of aryl radicals to modify SWCNTs and MWCNTs by naphthoate groups.[29] Naphthoate groups have been shown to favour orientation of MvBOD when grafted on graphite electrodes.[30] We have recently shown that naphthoate groups have a detrimental effect on BOD from Bacillus pumilus which could arise from a dipolar moment unfavourably pointing out at the opposite of the T1 copper centre.[29] In this work, we report the use of these naphthoate ‐ modified CNTs towards MvBOD. We compare this chemical functionalisation of CNTs with the corresponding electrochemical grafting by using a newly synthesised 6 ‐ carboxynaphthalenediazonium tetrafluoroborate salt. In parallel, a comparison was also made with an ammonium derivative to underline the influence of the MWCNT surface charge on the orientation of the enzyme and the selective targeting of the electron entry point via the T1 copper centre or via the TNC. We finally investigate the impact of these CNT functionalisation strategies over the oxygen diffusion into the film for their application at air ‐ breathing oxygen ‐ reducing bioelectrodes.

The electrochemical functionalisation process is based on the electrografting of a MWCNT film deposited on a GC electrode. A MWCNT film was formed by drop ‐ coating 20 μL of a 2.5 mg mL−1N ‐ methyl ‐ 2 ‐ pyrrolidone (NMP) dispersion of MWCNTs. 2 ‐ Amino ‐ 4 ‐ ethylphenyldiazonium derivative was prepared according to the previously described procedure and the 6 ‐ carboxynaphthalenediazonium tetrafluoroborate salt was synthesised by the diazotation of 6 ‐ amino ‐ 2 ‐ naphtoic acid with NOBF4. The new derivative was characterised by 1H NMR and infrared spectroscopy.

Electrografting was performed on GC and MWCNT electrodes. Figure [NaN]  A displays cyclic voltammograms (CVs) performed in a 2 mmol L−1 solution of 6 ‐ carboxynaphthalenediazonium tetrafluoroborate. The CVs exhibit a typical reductive behaviour for an aryldiazonium salt. On the first scan, an irreversible cathodic peak observed at Epred=−[NaN]

On the contrary, on MWCNT electrodes, faradaic current intensities are almost 3000 ‐ times higher; and only a small current decrease after each scan is observed which is due to the high nanostructured surface of CNTs and their resistance to passivation by polyphenylene formation.[31] Furthermore, a higher Ep of +0.22 V confirms the higher reactivity of MWCNTs as compared to GC towards electroreduction of aryldiazonium. This behaviour was also observed for the electrografting of 2 ‐ amino ‐ 4 ‐ ethylbenzenediazonium tetrafluoroborate.[31] This ammonium derivative, which was electrografted according to a previously described procedure,[31] , [32] was chosen as a positive counterpart of the 6 ‐ carboxynaphthalenediazonium. A Epred of −0.85 and −0.02 V was obtained on GC and MWCNT electrode, respectively (Figure [NaN]  B). Comparison of the redox potential of the irreversible reduction of both diazonium derivatives indicates that 4 ‐ carboxylatonaphtyldiazonium salt is easier to reduce as compared to 2 ‐ amino ‐ 4 ‐ ethylphenyldiazonium, owing to the high electrophilic character of the carboxynaphthyl group. For clarity, electrodes modified with 6 ‐ carboxynaphthalenediazonium will be noted CN ‐ modified electrodes while electrodes modified 4 ‐ (2 ‐ aminoethyl)benzenediazonium will be noted AE ‐ modified electrodes.

A negatively and a positively charged redox probe, Ru(NH3)62+ and Fe(CN)63−, were used to investigate the permselectivity and the barrier effect of modified GC and MWCNT electrodes.

For CN ‐ modified GC electrodes, the CV from Figure [NaN]  A indicates a sluggish redox system for Fe(CN)63−, arising from the blocked diffusion of this anionic species on the negatively charged CN ‐ modified electrode. On the contrary, no barrier effect was observed for the Ru(NH3)62+ probe. The CN ‐ modified electrode thus exhibits a large barrier effect for Fe(CN)63−, as compared to Ru(NH3)62+. However, the AE ‐ modified GC electrode exhibits the opposite effect towards Ru(NH3)62+ and Fe(CN)63− (Figure [NaN]  A). These blocking effects of surfaces modified with charged groups towards cationic or anionic redox probes have already been demonstrated for self ‐ assembled monolayers[33] and diazonium species.[34] These experiments confirm the presence of positive charges on AE ‐ modified electrodes and negative charges on CN ‐ modified electrodes. In the case of AE ‐ or CN ‐ modified MWCNT electrodes (Figure [NaN]  B), a negligible barrier effect was observed for both redox probes even in the case of electrografting performed by multiple CV scans (up to 20). In addition to the fact that the high specific nanostructured surface of MWCNTs is hardly passivated by the electrografting of a polyphenylene layer, as compared to the planar GC electrode, this confirms the excellent electron ‐ transfer properties of MWCNT electrode despite the presence of positive or negative charges on the surface of MWCNT sidewalls.[NaN]

AE ‐ modified MWCNT (AE ‐ MWCNT) electrodes and CN ‐ modified MWCNT electrodes (CN ‐ MWCNT) were compared for the immobilisation and orientation of MvBOD. CVs under argon of the BOD ‐ modified MWCNT electrodes are displayed in Figure [NaN]  A.[NaN]

Two different redox systems were observed on AE ‐ MWCNT and CN ‐ MWCNT. On CN ‐ MWCNT electrodes, a redox system at E1/2=0.49 V (ΔEp=30 mV) was observed. This system can be unambiguously attributed to the T1 copper centre. Different studies corroborate the value of this redox system either using redox titration[35] or protein film voltammetry of MvBOD on different types of electrodes.[13] , [20] , [36] , [37] On the contrary, on AE ‐ MWCNT electrodes, the T1 redox system is no longer observed. Instead, a reversible redox system is observed at E1/2=0.12 V (ΔEp=30 mV). This redox system has been only scarcely observed and was attributed to the redox potential of the TNC (E1/2=0.39 V vs. NHE or 0.15 V vs. SCE) as reported by spectroelectrochemical studies of MvBOD and BOD from Trachyderma tsunodae.[13] , [35]

Figure [NaN]  B and C (curve b) shows the CV scan under O2 for both types of electrode. For both types of modified electrode, an irreversible electrocatalytic signal is observed corresponding to the electrocatalytic reduction of O2 by MvBOD. In a control experiment where no BOD was immobilised on MWCNTs, a negligible catalytic current is observed underlining the poor catalytic activity of CNTs towards oxygen reduction. For the CN ‐ MWCNT electrode, a quasi ‐ ideal electrocatalytic wave is observed at a half ‐ wave potential of 0.44 V and reaches a plateau current of 4.0(±0.4) mA cm−2. When 2,2’ ‐ azinobis(3 ‐ ethylbenzothiazoline ‐ 6 ‐ sulfonate) (ABTS) was added (curve c, Figure [NaN]  C), no change of the electrocatalytic signal was observed. This behaviour demonstrates that all immobilised MvBOD participate in the DET and that DET to the T1 copper centre is highly efficient and does not limit the electrocatalytic activity of MvBOD towards direct oxygen reduction via the T1 copper relay centre. This behaviour has also been observed for MvBOD immobilised on MWCNTs functionalised with substrate mimics of the enzyme, protoporphyrin IX and hemin.[20] This result underlines the crucial importance of carboxylate moieties to orientate BOD, positioning the T1 copper in the vicinity of the CNT sidewalls. This is corroborated by the well ‐ defined noncatalytic reversible redox system attributed to the T1 copper centre (Figure [NaN]  A, black curve).

For AE ‐ MWCNT electrode, only a negligible electrocatalytic wave is observed starting at around 0.4 V, which would correspond to residual MvBOD transferring electrons via the T1 copper centre. Instead, a low ‐ potential electrocatalytic wave is observed with a half ‐ wave potential of 0.19 V and a maximum current density of 1.7(±0.5) mA cm−2. The half ‐ wave potential of this low ‐ potential electrocatalytic wave is in good agreement with the reversible redox system observed under argon and is attributed to the electroactivity of the TNC. Thus, on AE ‐ MWCNT electrode, the main electrocatalytic wave shows a low ‐ potential oxygen reduction catalysis which does not involve the T1 copper centre as electron relay. Similar low potential catalytic waves have been observed in some studies of BOD immobilised at porous graphitic electrodes.[10] , [12] , [14] In these works, a deconvolution of the signal was difficult owing to the porosity of the electrode and the low amount of wired enzymes per surface unit. In our case, thanks to the excellent specific surface of modified MWCNTs and highly efficient DET properties, well ‐ shaped electrocatalytic waves could be seen and were unambiguously attributed to a specific and statistically favoured orientation of the enzyme, positioning the TNC at a close distance to the positively charged AE ‐ MWCNT sidewalls. Interestingly, the addition of ABTS allows the recovery of a high ‐ potential oxygen reduction at a half ‐ wave potential of 0.42 V and a plateau current of 2.1(±0.5) mA cm−2 (Figure [NaN]  C, dashed curve c), which is similar to the electrocatalytic behaviour involving the wiring of the T1 copper centre at CN ‐ MWCNT electrode from Figure [NaN]  B. Despite high capacitive currents arising from the MWCNT film capacitance, integration of the charge under the reversible peak systems observed under argon gives an estimation of the BOD surface coverage, that is, 5.8 pmol cm−2 for BOD connected via the TNC and 6.6 pmol cm−2 for BOD connected via the T1 centre. According to electrocatalytic current densities for both types of oriented BOD, catalytic rate constants of 0.7×103 and 1.7×103 s−1 were estimated for oxygen electrocatalysis performed by BOD oriented on AE ‐ MWCNT and CN ‐ MWCNT, respectively. For AE ‐ MWCNT and CN ‐ MWCNT, Tafel slopes of 120 and 100 mV per decade (Figure S1 in the Supporting Information) were, respectively, measured in the potential region where the electrocatalytic current is the steepest. These respective values confirm that the kinetics of the four ‐ proton/four ‐ electron oxygen reduction bioelectrocatalytic process is governed by monoelectronic electron ‐ transfer steps for both types of functionalised electrode.[16] , [38]

Recent studies by Solomon and co ‐ workers on MCOs have demonstrated that two resting forms coexist for MCOs.[10] , [11] The RO form has a TNC in which all copper centres are oxidised and can be fully reduced via ET from the T1 centre with a redox potential of approximately 0.70 V versus NHE (0.46 V vs. SCE). The AR form, where only one copper centre is oxidised, cannot be reduced by ET from the T1 centre and is activated at low potential of approximately 0.40 V versus NHE (≈0.16 V vs. SCE). We can therefore conclude that, depending on the nature of the diazonium grafted at the surface of CNTs, a specific orientation of MvBOD can be targeted. This orientation induces two different catalytic pathways, one involving the T1 copper centre and the full reduction of the RO form of the TNC, and the other, shortcutting the T1 copper centre and involving the reduction of a partially oxidised form (AR form) of the TNC (Figure [NaN] ). In the latter mechanism, the T1 can further be reached by addition of ABTS which allows the full reduction of the RO form of TNC and a high ‐ potential electrocatalysis. MvBOD immobilised on CN ‐ MWCNT gives access to a high potential oxygen reduction at the redox potential of the T1 copper (E1/2(T1)=0.49 V vs. SCE or 0.73 V vs. NHE), which is the electrochemical control centre of the catalysis. MvBOD immobilised on AE ‐ MWCNT gives access to a low ‐ potential oxygen reduction at the redox potential of the AR state of the TNC (E1/2(TNC)=0.12 V vs. SCE or 0.36 V vs. NHE). The TNC is the electrochemical control centre and the fully reduced form of the enzyme is only accessible through the one ‐ electron reduction of the AR form. This is in good agreement with the Tafel slopes indicating a rate ‐ limiting monoelectronic electron transfer for both orientations of the enzyme.[NaN]

Electrochemical grafting of 6 ‐ carboxynaphthalenediazonium on MWCNTs was then compared to chemical grafting. MWCNTs were chemically functionalised by 6 ‐ carboxynaphthalenediazonium using a homogenous chemical reaction. This reaction is based on the “Tour” reaction in which an aryldiazonium intermediate is formed in situ by treating the amine derivative with isopentyl nitrite.[39] Chemically modified CN ‐ MWCNTs were synthesised as previously described.[29] In the electrochemical process, the electrografting is performed on a MWCNT film previously deposited from a NMP dispersion of CNTs as already mentioned. In the chemical process, the chemically modified CN ‐ MWCNTs are directly deposited on the electrode. The chemical process allows access to well ‐ characterised and water ‐ soluble MWCNTs.[29] This type of CNTs is easy ‐ to ‐ handle and can be reproducibly deposited on various types of electrodes. This is the reason why a solution of these CN ‐ MWCNTs was directly deposited on GC and GDE electrodes for comparison of both chemical and electrochemical techniques towards the immobilisation of MvBOD and bioelectrocatalytic oxygen reduction. GDE electrodes, used in conventional proton ‐ exchange membrane fuel cells (PEMFC), create a three ‐ phase boundary allowing the catalyst to work without mass ‐ transport limitations and without oxygen aqueous solubility problems owing to the oxygen gas supply at the back of the GDE. A proper combination of controlled porosity and hydrophilic/hydrophobic properties is required for the operation of this type of electrode. In our case, the GDE was operated under passive air diffusion. No drying effect was observed at the GDE interface which would require air ‐ flow humidification.

Figure [NaN] displays CV obtained for GC electrode and GDE modified with electrochemically and chemically modified CN ‐ MWCNTs. On GC electrodes (Figure [NaN]  A), both types of electrode exhibit a high potential oxygen reduction electrocatalytic wave. On pristine MWCNTs, the electrocatalytic waveshape is indicative of a distribution of orientation of MvBOD and an associated dispersion of heteregenous ET rates[20] (Figure [NaN]  A, curve b). A maximum catalytic current of 1.1(±0.3) mA cm−2 is reached at 0 V. While electrocatalytic waveshape for electrochemically modified CN ‐ MWCNTs exhibits a kinetically limited Nernstian behaviour (Figure [NaN]  A, curve d), the electrocatalytic waveshape for chemically modified CN ‐ MWCNTs indicates that oxygen reduction electrocatalysis is not completely limited by kinetics but is also in part limited by oxygen mass transfer limitations (Figure [NaN]  A, curve c). This induces a lower oxygen reduction current reaching 2.1(±0.3) mA cm−2 at 0 V under O2. In contrast to the behaviour observed for modified MWCNT on GC electrodes, the chemically modified CN ‐ MWCNT GDE exhibits a superior performance as compared to electrochemically modified CN ‐ MWCNT GCE (Figure [NaN]  B). High maximum current densities of 3.9(±0.5) mA cm−2 at 0 V under air were measured (Figure [NaN]  B, curve c). It is worth noting that no rotating electrode or magnetic stirrer is needed. These results indicate that MWCNTs modified with the same naphthoate groups, which favours an orientation of BOD towards the T1 centre, exhibit different electrochemical behaviour towards oxygen reduction reaction, depending on the oxygen ‐ supplying electrochemical system.[NaN]

To investigate the particular behaviour of these different functionalised CN ‐ MWCNT films, we compared the surface morphology of the chemically modified CN ‐ MWCNT film with a pristine MWCNT film at the macroscale using laser scanning microscopy (LSM) and at the nanoscale using scanning electron microscopy (SEM) (Figure [NaN] ).[NaN]

This is an important parameter which has to be investigated in order to study oxygen depletion inside the film while maximising catalyst loadings. Several reports have underlined the crucial importance of oxygen mass transport limitations for oxygen ‐ reducing biocathodes.[40] , [41] , [42] , [43] The morphology of the pristine MWCNT surface exhibits a homogenous rough surface arising from the starting MWCNT NMP dispersion (Figure [NaN]  A and B). On the contrary, chemically modified CN ‐ MWCNT forms a uniform and dense film morphology (Figure [NaN]  D and E). This morphology arises from the excellent homogeneity of the soluble CN ‐ MWCNT in NMP. This high solubility leads to the deposition of a dense MWCNT film with negligible roughness. On SEM images, a denser and highly porous MWCNT network can be observed at the nanoscale for chemically modified CN ‐ MWCNTs as compared to pristine MWCNT, confirming the dense film deposition of soluble CN ‐ MWCNTs. The important difference in film morphology for both types of MWCNT electrode is likely responsible for difference in oxygen electrocatalysis. On GC electrode, the highly porous surface of electrochemically modified CN ‐ MWCNT provides an excellent macroporosity for improved oxygen mass transport into the film and high access to wired enzymes. On the contrary, the dense and homogenous network formed by chemically modified CN ‐ MWCNTs is responsible for mass transfer limitations in oxygen ‐ saturated solutions. On GDE, the high density of CN ‐ MWCNTs is no longer an issue for oxygen access. This allows the GDE ‐ modified with CN ‐ MWCNTs to have a combination of excellent properties: 1) favourable orientation of the enzyme via naphthoate groups, 2) high electroactive and nanostructured surface, and 3) excellent balance between hydrophilicity and hydrophobicity for their operation in air ‐ breathing systems, as it has been shown in other MCO ‐ based air ‐ breathing electrodes.[44] These properties allow this GDE bioelectrode to achieve high current densities of almost 4 mA cm−2 under air which is among the best performances reported for a bioelectrocatalytic oxygen reduction reaction under air.

In conclusion, we achieved the electrochemical grafting of MWCNTs for the specific orientation of MvBOD. Depending on the nature of the charged surface, two oxygen reduction catalytic pathways can occur. One catalytic pathway involves the T1 copper centre and gives access to a high current/high potential oxygen reduction reaction. The second catalytic pathway involves the AR intermediate of the TNC as the electrochemical control centre of the bioelectrocatalysis and a low ‐ potential oxygen reduction reaction. This work thus emphasis the possibility to target a specific redox centre in the protein through control over the orientation of the enzyme, thanks to the corresponding CNT functionalisation. Finally, we show that CNT functionalisation can also have an important impact on the morphology of the CNT film and on the oxygen diffusion. The highly efficient air ‐ breathing biocathode, achieved by control over the orientation of the enzyme and over the porosity of the electrode, show promising perspectives in the controlled design of EFC bioelectrodes at the molecular, nano ‐ and macroscales.

Multiwalled carbon nanotubes (MWCNT; 9.5 nm diameter, purity >95 %) were obtained from Nanocyl and Unidym Inc., respectively, and were used as received without any purification. All the reagents were purchased from Sigma – Aldrich and were used without further purification. Chemically modified naphthoic ‐ acid ‐ functionalised MWCNTs (CN ‐ MWCNTs)[29] and 4 ‐ (2 ‐ aminoethyl)benzenediazonium tetrafluoroborate[45] were synthesised according previously described procedures. All solvents were of analytical grade. Distilled water was passed through a Milli ‐ Q water purification system. NMR spectra were recorded on a Bruker AVANCE 400 operating at 400.0 MHz for 1H. Infrared spectra were recorded on a Bruker Optics ALPHA FT ‐ IR spectrometer equipped with a diamond ATR accessory.

The electrochemical characterisation of the bioelectrodes were carried out in a three ‐ electrode electrochemical cell using a Biologic potentiostat. A platinum wire was used as the counter electrode and all potentials were referred to SCE electrode. Unless otherwise specified, the experiments were conducted at RT in Mc Ilvaine buffer solution (pH 7) as the supporting electrolyte. All currents are normalised with the geometric surface of the electrodes.

The morphology of the electrodes was investigated by SEM using an ULTRA 55 FESEM based on the GEMENI FESEM column with beam booster (Nanotechnology Systems Division, Carl Zeiss NTS GmbH, Germany) and tungsten gun. Raman spectra were recorded using a Renishaw inVia spectrometer. The 3D and profile images were taken using a Keyence VK ‐ X200 laser microscope.

A solution of 412 mg (2.2 mmol) 6 ‐ amino ‐ 2 ‐ naphtoic acid in 10 mL of MeCN was cooled to −40 °C under a stream of argon and then treated with 0.3 mL (2.2 mmol) of HBF4/Et2O. Then, 257 mg (2.2 mmol) of NOBF4 was gradually added. After 30 min of stirring the reaction mixture was allowed to warm and 70 mL of cold ether was added. The resulting precipitate was filtered off and washed with cold ether. Yield: 540 mg (85 %).

1H NMR (400 MHz, [D6]DMSO): δ=8.86 (d, J=8.7 Hz, 1 H), 8.65 (d, J=1.8 Hz, 1 H), 8.41 (d, J=1.8 Hz, 1 H), 8.17 (d, J=8.7 Hz, 1 H), 8.04 (dd, J=8.7, 1.8 Hz, 2 H). IR (ATR): ν˜ =1040 (BF4), 1215 (CO), 1672 (CO), 2290 (N2+), 3091 cm−1 (=CH).

The working electrodes were non ‐ treated carbon cloth electrodes purchased from Paxitech. For MWCNT ‐ modified GDE, NMP dispersions of MWCNT were prepared by 30 min sonication of 2.5 mg nanotubes in 1 mL NMP until a homogenous black suspension was obtained. Then, 20 μL of the MWCNT solution was drop ‐ casted on a carbon cloth electrode and NMP was removed under vacuum leaving a homogenous MWCNT film. A MWCNT film with a thickness of 6 μm was measured by laser scanning microscopy.

For CN ‐ MWCNT ‐ modified GDE, 3 mg of f ‐ MWCNT was dispersed in 3 mL of NMP using an ultrasonic bath for 5 min, followed by centrifugation for 15 min at 3200 rpm (750 g). Then 20 μL of the CN ‐ MWCNT supernatant were drop ‐ casted on a carbon cloth electrode, and NMP was removed under vacuum leaving a homogenous CN ‐ MWCNT film. A MWCNT film with a thickness of 0.5 μm was measured by laser scanning microscopy.

Electrodes were modified with BOD by drop ‐ coating 20 μL of the enzyme solution (2.5 mg mL−1MvBOD in McIlvaine buffer pH 5) on the MWCNT ‐ modified GDE followed by drying, overnight, at 4 °C. The resulting electrodes were then washed with Mc Ilvaine buffer solution before the electrochemical characterisation to remove the non ‐ adsorbed enzyme. The electrodes were then sealed in a cylindrical plastic chamber with a gasket joint allowing the electrodes to be in contact with both the electrolyte and air with an active surface of 0.07 cm2.

We gratefully acknowledge funding from the Agence Nationale de la Recherche through the project CAROUCELL (ANR ‐ 13 ‐ BIOME ‐ 0003 ‐ 02). The authors wish also to acknowledge the support from the ICMG Chemistry Nanobio Platform, Grenoble (PCN ‐ ICMG) and from the LabEx ARCANE (ANR ‐ 11 ‐ LABX ‐ 0003 ‐ 01).

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re ‐ organized for online delivery, but are not copy ‐ edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Graph: Cyclic voltammetry (CV) of a solution of: A) 4 ‐ carboxylatonaphtyldiazonium tetrafluoroborate, and B) 4 ‐ (2 ‐ aminoethyl)benzenediazonium tetrafluoroborate (2 mmol L−1) in MeCN/TBAP 0.1 mol L−1 at a GC and a MWCNT electrode (5 scans, v=20 mV s−1).

Graph: Cyclic voltammetry (CV) of a solution of Ru(NH3)6Cl2 and Fe(CN)6K3 (3 mmol L−1) in Mc Ilvaine buffer pH 7: A) CN ‐ and AE ‐ modified GC, and B) MWCNT electrodes (v=20 mV s−1). GC and MWCNT electrodes were modified according to the conditions shown in Figure . Dashed curves correspond to unmodified GC or MWCNT electrodes.

Graph: A) CVs of the MvBOD ‐ functionalised (grey) AE ‐ MWCNT and (black) CN ‐ MWCNT electrodes under argon in Mc Ilvaine buffer pH 7. B) CVs of the MvBOD ‐ functionalised CN ‐ MWCNT: a) under argon, b) in stirred oxygen ‐ saturated Mc Ilvaine buffer pH 7, and c) in the presence of 0.3 mmol L−1 ABTS. C) CVs of the MvBOD ‐ functionalised AE ‐ MWCNT electrode: a) under argon, b) in stirred oxygen ‐ saturated Mc Ilvaine buffer pH 7, and c) in the presence of 0.3 mmol L−1 ABTS in Mc Ilvaine buffer pH 7 (v=10 mV s−1). CVs were reproduced using at least three electrodes.

Graph: Schematic representation of the ET pathway on CN ‐ MWCNT and AE ‐ MWCNT involving two different intermediates in the catalysis.

Graph: A) CV of the MvBOD ‐ functionalised GC/MWCNT electrode: a) under argon and CV of the MvBOD ‐ functionalised electrode in stirred oxygen ‐ saturated Mc Ilvaine buffer pH 7, for b) pristine MWCNT electrode, c) chemically modified CN ‐ MWCNT electrode, and d) electrochemically modified CN ‐ MWCNT electrode. B) CV of the MvBOD ‐ functionalised GDE/MWCNT electrode: a) under argon and CV of the MvBOD ‐ functionalised electrode under air, for b) pristine MWCNT electrode, c) chemically modified CN ‐ MWCNT electrode, and d) electrochemically modified CN ‐ MWCNT electrode in Mc Ilvaine buffer pH 7 (v=10 mV s−1). CVs were reproduced using at least three electrodes. Inset: schematic representation of the GDE.

Graph: A) LSM optical/laser image; scale bar: 100 µm. B) LSM 3D height difference profile, and C) SEM images of the surface of a pristine MWCNT film; scale bar: 200 nm, and D) LSM optical/laser image; scale bar: 100 µm. E) LSM 3D height difference profile, and F) SEM images of the surface of chemically modified CN ‐ MWCNT film; same scale as C).

Graph: Supplementary