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Design of Laccase-Metal Organic Frameworks based Bioelectrodes for Biocatalytic Oxygen Reduction Reaction Snehangshu Patra, Saad Sene, Christine Mousty, Christian Serre, Annie Chausse, Ludovic Legrand, and Nathalie Steunou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05289 • Publication Date (Web): 22 Jul 2016 Downloaded from http://pubs.acs.org on July 23, 2016

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ACS Applied Materials & Interfaces

Design of Laccase-Metal Organic Frameworks based Bioelectrodes for Biocatalytic Oxygen Reduction Reaction Snehangshu Patra,,,‡,# Saad Sene,‡ Christine Mousty, Christian Serre,‡ Annie Chaussé,, Ludovic Legrand, *,, Nathalie Steunou*,‡ 

CNRS UMR 8587 Bd François Mitterrand 91025 Evry, France



Université d’Evry, Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement

(LAMBE), Université Evry, Université Paris Saclay, Bd François Mitterrand 91025, Evry, France. ‡

Institut Lavoisier de Versailles, UMR CNRS 8180, Université de Versailles St Quentin en Yvelines,

Université Paris Saclay, 45 avenue des Etats-Unis 78035 Versailles Cedex. France. #Present address : Center for Excellence in Green Energy and Sensors Systems (CEGESS), Indian Institute of Engineering Science and Technology (IIEST), Shibpur, Howrah, 711103, West Bengal, India; 

Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, UMR-CNRS

6296, BP 10448 F-63000 Clermont-Ferrand, France. ABSTRACT Laccase in combination with 2,2’azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a mediator is a well-known bioelectrocatalyst for the 4-electron oxygen reduction reactions (ORR). The present work deals with the first exploitation of mesoporous iron (III) trimesate based metal organic frameworks (MOF) MIL-100(Fe) (MIL stands for Materials from Institut Lavoisier) as a new and efficient immobilization matrix of laccase for the building-up of biocathodes for ORR. First, the immobilization of ABTS in the pores of the MOF was studied by combining micro-Raman spectroscopy, X-ray powder diffraction (XRPD) and N2 porosimetry. The ABTS-MIL-100(Fe) based modified electrode presents excellent properties in terms of charge transfer kinetics and ionic conductivity as well as a very stable and reproducible electrochemical response, showing that MIL1 ACS Paragon Plus Environment


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100(Fe) provides a suitable and stabilizing microenvironment for electroactive ABTS molecules. In a second step, laccase was further immobilized on the MIL-100(Fe)-ABTS matrix. The Lac-ABTS-MIL100(Fe)-CIE bioelectrode presents a high electrocatalytic current density of oxygen reduction and a reproducible electrochemical response characterized by a high stability over a long period of time (3 weeks). These results constitute a significant advance in the field of laccase based bioelectrocatalysts for ORR. According to our work, it appears that the high catalytic efficiency of Lac-ABTS-MIL-100(Fe) for ORR may result from a synergy of chemical and catalytic properties of MIL-100(Fe) and laccase. Keywords: bioelectrocatalysis, oxygen reduction reaction, ABTS, laccase, MOFs, MIL-100(Fe).

 INTRODUCTION Enzymatic biofuel cells (EBFCs) is a sub-class of fuel cells, based on redox enzymes involved in electrocatalytic reactions.1,2 When considering power output, EBFC cannot compete with conventional fuel cells. However, in contrast to metal catalysts, the high specificity of enzymes towards their respective substrates is of great interest together with their ability to achieve high catalytic turnovers at mild conditions (20–40 °C within a reasonable pH range 5–8).

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Therefore, such electrochemical

generators are intended to operate in complex media such as physiological fluids or vegetal. As an energy-producing cell, these devices must be designed such that overpotentials due to kinetics, ohmic resistance, and mass transfer are minimized and current density (i. e. current per unit volume) is maximized.2 Recently, fascinating results have been obtained with glucose EBFCs implanted in mammals.4,5 Since glucose and O2 are both present in living organisms, in blood or extracellular fluids, these implanted EBFCs devices can generate electrical power from the oxidation of bioavailable glucose and reduction of oxygen.4,5 Oxygen-reducing cathode is one of the key elements of biofuel cells. However, the low-overpotential four-electron reduction of molecular oxygen (i. e. O2 + 4H+ + 4e- = 2H2O) at neutral pH is one of the most difficult electrocatalytic process in the case of small substrate activation.3 While most metal catalysts require high overpotentials, biocathodes based on laccases have 2 ACS Paragon Plus Environment

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demonstrated exceptional performances for oxygen reduction reaction at medium pH and are thus currently used in EBFC devices.

6,7,8,9,10,11,12,13

Laccases (polyphenoloxidases) are a group of blue

multicopper oxidases that simultaneously combine the oxidation of a broad range of analytes (polyphenols, aromatic compounds, diamines…) and the reduction of O2 to H2O.14 At pH 5, while the thermodynamic O2/H2O redox potential is set at 0.93 V vs NHE, laccases from Trametes versicolor (Tv) or Trametes hirsuta can achieve electrocatalytic reduction of O2 at medium pH (pH 4–7) with exceptional low overpotentials of 30–70 mV.3 However, for their practical use, EBFCs must have lifetimes ranging from months to years to justify implanted, highly distributed, or consumer portable applications and the stability of numerous EBFCs still remains an issue.2,3 In particular, the performance of laccase based bioelectrocatalytic systems is strongly dependent on the pH stability of the enzyme.4 It is also strongly dependent on the rate of the electron transfer between the redox sites of the immobilized enzyme and the electrode. This electron transfer can be achieved either by using redox mediators (mediated electron transfer, MET) or by a direct electron transfer (DET). Both mechanisms have their own advantages/disadvantages depending on the bioelectrode systems used. In MET, the close contact between the mediators and the redox centers of the enzyme allows the electrical wiring of the enzyme leading to a free flow of electrons from the enzyme to the electrode.6,7,15 In such a process, the enzyme does not need to orientate itself relative to the surface of the electrode since all redox centers of the enzyme are wired to the electrode through the mediators in a three-dimensional manner. The 2,2`-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is known to be a good redox mediator to play the role of electron shuttle between the redox center of laccase and the electrode.7 It has been reported that MET-based laccase bioelectrodes are able to produce high catalytic current densities of O 2 reduction.6,7,15, 16 However, this method requires the co-immobilization of redox mediators in the biomembrane for which the leaching is often evidenced for numerous bioelectrodes under operating conditions. In contrast, DET was mainly reported with conducting carbon based bioelectrodes and particularly high values of catalytic currents were measured for several bioelectrodes. However, DET 3 ACS Paragon Plus Environment


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requires a short distance (less than 15-20 Å) between the redox centers of the enzyme and the electrode surface and thus a peculiar favorable orientation of the enzyme with their most exposed redox centers facing the electrode surface.10,11,12 Such configuration is not straightforward in many cases. Metal Organic Frameworks (MOFs) are porous crystalline hybrid materials built up from a wide range of inorganic subunits (transition metals, 3p metals, lanthanides…) and organic polytopic linkers (carboxylates, imidazolates, phosphonates …). Most of them exhibit a very large and monodisperse porosity with tunable pore sizes and volumes (0.2-4 cm3.g-1; SBET300-6000 m2/g; pore diameter 360 Å) often exceeding those of traditional crystalline porous solids while one can easily introduce chemical functionalities (acid Lewis, Bronsted or redox sites, polar or apolar groups…) through direct synthesis or post-synthetic treatments. 17,18

Due to these outstanding features, MOFs have attracted

great attention for a wide range of applications in societal and economical key fields such as gas storage or separation,19 biomedicine,20 catalysis,21 sensors,22 among others. Recently, the use of such porous hybrid materials as host matrices for proteins23,24,25 was studied. In particular, the immobilization of enzymes in MOFs was explored as a strategy to enhance the performance of existing bioelectrocatalysts for which the efficient immobilization and stabilization of enzymes under wide operating conditions still remains an issue.26,27 It is thus strongly desired to develop novel, cost-effective biocompatible host matrices in which enzymes may retain their biocatalytic activity even in unnatural environment.28 In this context, MOFs may bring novel advantages due to their modular nature (i. e. tunable cavity size and functionality of the pore walls). Such properties make MOFs appealing host matrices able to create a stabilizing microenvironment for enzymes through specific host-guest interactions and confinement effect.23,24 The encapsulation of some microenzymes inside the pores of mesoporous MOFs was reported 29 and this approach was extended thereafter to larger enzymes (i. e. cytochrome C, myoglobin). 30 , 31 It was found that the encapsulation method is effective in preventing any leaching of enzyme and improving its stability in different solutions such as non-physiological media; however, this strategy is 4 ACS Paragon Plus Environment

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restricted to a limited number of enzymes. Therefore, it was also reported that the immobilization of enzymes in MOF matrices may also proceed successfully by adsorption,32,33 covalent grafting,34 or host-guest interactions between tag-functions on the protein and MOF cavities. 35 Recently, MOFs-enzyme composites were prepared by embedding enzymes during the mineralization process of MOFs. 36,37,38,39 Until recently, a few MOFs-enzymes bioreactors have been reported with an interesting biocatalytic activity and recyclability.24,31,34,35,40 In comparison, enzyme-MOFs based composites were rarely investigated for biosensing applications.38, 41 , 42 Biosensing based on electrical and electrochemical transduction schemes was minimally explored with MOFs due mainly to the insulating character of most of these materials. One example is the use of Cu-MOF-tyrosinase biosensors for the sensitive electrochemical detection of bisphenol A. 43 An alternative approach was proposed through the use of ZIFs (zeolitic imidazolate frameworks) for building-up glucose biosensors with interesting sensitivity and selectivity properties.44 The electroactivity of a few MOFs candidates such as Fe(III) based MOFs was demonstrated in the past few years and these hybrid materials were used for the colorimetric titration of glucose. 45 , 46 , 47 Recently, we have shown that glucose amperometric biosensors based on the biocompatible mesoporous iron(III) trimesate MIL-100(Fe) and glucose oxidase (GOx) supported on platinium nanoparticles present very interesting properties, namely a high sensitivity (71 mA.M-1.cm-2), good response time (10 U mg-1), Glutaraldehyde, Albumin (from bovine serum (BSA), 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich. Ultrapure O2 cylinder was purchased from Air Liquide. All other chemicals and reagents are of analytical grade and were prepared using milipore water. Synthesis of MIL-100(Fe) nanoparticles. Mesoporous iron(III) trimesate nanoparticles were synthesized with a significant yield following a previously published protocol based on the microwaveassisted hydrothermal process in water at a temperature of 130 °C and within 7 minutes.49 Activation of the solids consists in the dispersion of the as-synthesized MIL-100(Fe) nanoparticles into ethanol as previously reported.49 MIL-100(Fe) was further characterized by combining X-ray powder diffraction (XRPD) and FT-IR spectroscopy. The surface charge and diameter of MOFs nanoparticles were evaluated by ζ-potential and DLS. The composition and purity of MOFs nanoparticles were evaluated by thermogravimetric analyses (TGA) and FT-IR while their porosity measured by N2 adsorption at 77 K. Preparation of bioelectrodes. Bioelectrodes were prepared in two consecutive steps. First, ABTS was encapsulated in the MIL-100(Fe) matrix through an impregnation method. The MIL-100(Fe) nanoparticles (9 mg) were dispersed in 1 mL of double distilled water and sonicated 3 times for 5 min. Then, several ABTS encapsulation tests with various ABTS/MIL-100(Fe) weight ratios (1:6, 1:2 or 1:1) were performed through the dispersion of an amount of as received ABTS, into the orange MIL-100(Fe) dispersion under vigorous stirring overnight at room temperature. A visible change of color of the solution from orange to green was observed after ABTS addition. ABTS-MIL-100(Fe) mixtures were 6 ACS Paragon Plus Environment

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then centrifuged and washed 3 times with H2O to remove unanchored ABTS followed by drying at 50oC for about 3-4 hours. It is noteworthy that an excess of ABTS was observed in solution for weight ratios of 1:2 or 1:1. Therefore an ABTS/MIL-100(Fe) weight ratio of 1:6 was kept for further synthesis and characterization as well as laccase immobilization. Composite ABTS-MIL-100(Fe) carbon ink electrodes (CIE) were prepared by mixing ABTS-MIL100(Fe) (10 wt%) and carbon ink (Acheson, 90 wt %) along with few drops of acetone. The resulting slurry was painted with a brush on 20 mm diameter stainless steel 304L (SS) electrode disk followed by drying at 50oC for 45 minutes. The surface area (A) of the electrode is 3 cm-2. For the bioelectrode Lac-ABTS-MIL-100(Fe)-CIE preparation, laccase powder (7 wt %) was physically ground with ABTS-MIL-100(Fe) (3 wt %) and bovine serum albumin powder (BSA) (3 wt%) and the resulting composite was mixed with carbon ink ( 87 wt %). Electrodes without MOF were fabricated by grinding ABTS (1 wt %), laccase (7 wt %), BSA (3 wt%) followed by mixing with carbon ink (89 wt %). The final weight of the composite layer on the electrode is close to 20-22 mg. After drying, bioelectrodes were exposed to the saturated glutaraldehyde vapor for 1 hour in order to cross-link biomembranes. Bioelectrodes were finally soaked in buffer solution of pH=5.1 for 5 minutes for their further usage. Instrumentation and electrochemical characterizations. A EG & G PAR VersaSTAT 3 potentiostat equipped with the software Versa Studio was used. The Ag/AgCl reference electrode was home-made by electrolysis of Ag wire as previously reported.48 The counter electrode was a Nickel mesh. For chronoamperometry, a Metrohm 802 Stirrer rotator was used to control mass transport of oxygen while measuring the currents with respect to duration of the experiments. The temperature in the electrochemical cell controlled by a POLYSTAT 5hp thermostat (Bioblock Scientific) was equal to 37°C. For electrochemical impedance spectroscopy (EIS) measurements, a sinusoidal potential modulation with amplitude of 10 mV (peak to peak) was applied with a frequency from 10 mHz to 100 kHz. FT-IR spectra were recorded using a Bruker IFS28 FTIR spectrometer equipped with a reflection 7 ACS Paragon Plus Environment


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absorption tool (Grazeby Specac) that allowed us to analyze the film directly on the disc surface without scraping procedure. The background measurement was taken with a gold disc that had been mirror-like polished. Twenty scans were performed for background and samples. Dynamic light scattering (DLS) was carried out with a Malvern Zetasizer Nano-ZS ZEN 3600. Scanning electron microscopy (SEM) images were acquired on gold-coated samples using a JEOL JSM-7001F microscope. X-ray powder diffraction patterns (XRPD) were acquired with a Siemens D5000 diffractometer (θ−2θ) with Cu Kαaverage radiation (λ = 1.54059 Å). Surface area of MOF and composites was evaluated by N2 adsorption using BJH method in a BELSORP-mini II porosimeter at 77 K. Thermogravimetric analyses (TGA) were recorded on a Perkins Elmer SDA 6000 equipment. Solids were heated up to 600°C with a heating rate of 5 °C.min-1 in an oxygen atmosphere.

 RESULTS AND DISCUSSION: Description of the MIL-100 (Fe) host matrix. Nanoparticles of the mesoporous iron trimesate MIL-100(Fe)) were synthesized by microwave assisted hydrothermal synthesis, as previously reported.49 MIL-100(Fe) is an iron(III) polycarboxylate based MOFs whose mesoporous 3D cubic structure is built by the combination of trimers of iron octahedra and trimesate linkers, leading to hybrid supertetrahedra (ST). The subsequent assembly of these ST gives rise to a zeolitic framework of MTNtype (Mobil Thirty Nine) (Scheme 1)50 that present mesoporous cavities of free apertures of about 25 and 29 Å accessible through windows (5.6 Å and 8.6 Å). One of the most intriguing features of MIL100(Fe) is the presence of a large amount of iron(III/II) Lewis and/or redox acid active sites which are of a great interest for separation51 but also for the controlled release of polar drugs bearing complexing groups (-NH2, -COOH, -POOH…).52

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Scheme 1. (a) oxo-centered trimers of iron octahedra (b) combined with trimesate linker leading to the MIL-100(Fe) framework with a zeolitic topology of the MTN-type. (c) molecular structure of ABTS

According to N2 adsorption measurements at 77 K, the BET specific surface area of MIL-100(Fe) nanoparticles is close to 1350 m2.g-1. As determined previously by TEM,49 nanocrystals of MIL-100(Fe) present a well-faceted octahedral shape and their diameter is about 130 ± 30 nm. At pH 5.1, the surface of MIL-100(Fe) nanoparticles is negatively charged according to the ζ-potential value (-10 mV) Synthesis and physico-chemical characterization of ABTS-MIL-100(Fe) hybrid materials. Due to the nanoscale 3D porosity of MIL-100(Fe) and the presence of a suitable amphiphilic environment (Scheme 1), ABTS molecules with the concomitant presence of sulfonate polar groups and aromatic moieties are expected to be strongly encapsulated in the mesoporous cages of MIL-100(Fe), thereby avoiding any rapid leaching under operating conditions. The size of ABTS (6.4 x 6.4 x 17.4 Å) is also compatible with the one of the hexagonal windows of MIL-100(Fe) (8.6 Å) which would indicate that a priori, ABTS shall be loaded into the larger cages only. The ABTS-MIL-100(Fe) hybrid was prepared by adding an amount of ABTS powder to an orange MIL-100(Fe) suspension at variable ABTS/MIL100(Fe) ratios (see experimental part) followed by vigorous stirring overnight. Noteworthy, right after 9 ACS Paragon Plus Environment


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ABTS addition, the suspension turned green indicative of a redox reaction between ABTS and MIL100(Fe). Since ABTS+• is stable and blue-green radical cation, it may be suggested that ABTS molecules are oxidized by the Fe(III) centers of MIL-100(Fe).

Figure 1. Different steps of the synthesis route of ABTS-MIL-100(Fe).

For the 1:6 weight ratio, a green precipitate was isolated after centrifugation. and the colorless character of the supernatant solution obtained indicates that ABTS+• cations are not presumably present in solution, and thus strongly immobilized into the MIL-100(Fe) matrix. As mentioned in the experimental section, for higher ABTS/MIL-100(Fe) weight ratios, an excess of ABTS is detected in solution. The precipitates collected were washed repeatedly with water in order to remove the excess or loosely anchored ABTS. A green color solid was obtained after drying at 50 oC for about 3-4 hours (Figure 1). Due to the a priori redox reaction between ABTS and MIL-100(Fe), it was crucial to determine if the crystalline structure is preserved upon ABTS immobilization. ABTS-MIL-100(Fe) materials were thus characterized by combining X-ray powder diffraction (XRPD), nitrogen porosimetry and micro-Raman spectroscopy. The XRPD pattern of the ABTS-MIL-100(Fe) composite prepared with a ABTS/MIL100(Fe) = 1:6 wt % (i. e. ABTS-MIL-100(Fe) (1:6)) (Figure 2(a)) is fully consistent with that of the parental MIL-100(Fe) indicating that the crystalline structure of MIL-100(Fe) is preserved after ABTS immobilization. As expected from any change in the pore content of this mesoporous MOF, a change in the relative intensity ratio (R) between the Bragg peak at 2θ=3.4° and peak at 2θ=4.2° (R=0.73 to 0.37) is noticeable after encapsulation.52


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ABTS-MIL-100(Fe)

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Figure 2. (a) XRPD patterns (Cu1.5406 Å) and (b) N2-adsorption isotherm (77K, P0=1 atm.) of ABTS-MIL-100(Fe) (1:6) in comparison with pure MIL-100(Fe).

The N2 adsorption isotherm of ABTS-MIL-100(Fe) (1:6) is still characteristic of a mesoporous solid although the typical sub-step of MIL-100(Fe), associated with the presence of two types of mesoporous cages and microporous windows, is less visible compared with pure MIL-100(Fe) in agreement with a decrease in the free pore size/volume upon ABTS loading. These results are consistent with the absence of any alteration of the framework of MIL-100(Fe) after ABTS addition. The decrease of the specific surface area of ABTS-MIL-100(Fe) (1:6) is in agreement with the insertion of a significant amount of ABTS within the porosity of MIL-100(Fe) (SBET 1332 m2 g−1 (Vp ~1.32 cm3.g−1) vs. 1180 m2.g-1 (Vp ~0.71 cm3.g−1) for MIL-100(Fe) and ABTS-MIL-100(Fe) (1:6) respectively). When further increasing the amount of ABTS (1:1 ratio), one can observe a further decrease of the BET surface and pore volume as a result of a larger amount of ABTS encapsulated (i. e. see Figure S1 of SI). As discussed above, one expects here a direct coordination of the SO3- moieties of ABTS+• to the Lewis acid iron(III) sites of 11 ACS Paragon Plus Environment


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MIL-100(Fe) as previously reported for sulfonic acid functionalized mesoporous MOFs bearing coordinatively unsaturated sites (CUS) (i. e. MIL-101(Cr)). 53 The comparison of the micro-Raman spectrum of ABTS-MIL-100(Fe) (Figure S2 of SI) with that of ABTS and MIL-100(Fe) with characteristic vibration bands of both MIL-100(Fe) and ABTS components is in agreement with the efficient immobilization of ABTS in the MIL-100(Fe) host matrix whose crystalline structure is preserved. SEM images of ABTS-MIL-100(Fe) (1:6) (Figure S3 of SI) show a strong aggregation of MIL-100(Fe) nanoparticles. In comparison with pure MIL-100(Fe), no major modification of the morphology of MOFs nanoparticles is evidenced after ABTS encapsulation. The quantification of ABTS loading in ABTS-MIL-100(Fe) (1:6) was performed by thermogravimetric analysis leading to a value close to 9 wt % (see below and Figure S4 of SI). The TGA curves of ABTS-MIL-100(Fe) (1:6) and MIL-100(Fe) show a first weight loss below 80°C corresponding to the removal of free water molecules while the slight weight loss between 100 and 200°C may correspond to water molecules in stronger interactions with the MOF framework and/or ABTS molecules. It is noteworthy that the hydration rate of ABTS-MIL-100(Fe) (1:6) is higher than in pure MIL-100(Fe) in agreement with the high hydrophilic character of ABTS. The main weight loss between 300°C and 400°C is assigned to the degradation of the benzenetricarboxylate linker of MIL-100(Fe) and/or ABTS. According to the difference of weight loss (i. e. 51 wt% for ABTS-MIL-100(Fe) (1:6) vs. 59 wt% for MIL-100(Fe)) between 300 and 400°C, the ABTS content in ABTS-MIL-100(Fe) (1:6) is close to 95 wt% which is lower than the initial amount of ABTS (i. e. 17 wt%). To shed some light on the encapsulation of ABTS in the pores of MIL-100(Fe), it is known that ABTS displays a maximum calculated size of 17.4 Å in a planar conformation which is smaller than the one of the mesoporous cages (25 and 29 Å). As mentioned above, this is likely that most of the ABTS molecules are lying into the larger cages by considering the dimension of the pentagonal windows (~4.7 x 5.5 Å) of the smaller mesoporous cages with regard to the hydrodynamic radius of the ABTS molecule ( 6.7 Å). 54 This would also be in agreement with the presence of a significant residual 12 ACS Paragon Plus Environment

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porosity after ABTS encapsulation (i. e. SBET  1180 m2.g-1 and Vp ~0.71 cm3.g−1). If one considers the volume of the large cage (12750 Å3) and only one ABTS molecule per cage, the ABTS theoretical loading would be of 4.3 wt%. This means that the maximum ABTS loading would almost be reached in ABTS-MIL-100(Fe) (1:6). One cannot nevertheless exclude that more than one ABTS molecule per cage is present particularly with the (1:1) composite with a higher ABTS loading. As shown by its electrochemical characterization, the ABTS loading of ABTS-MIL-100(Fe) (1:6) is adequate to impart a high electron transfer kinetics between ABTS molecules and the electrode (see vide infra). Electrochemical characterization of ABTS-MIL-100(Fe) hybrid materials. In order to confirm that ABTS, once loaded in the pores of MIL-100(Fe), still acts as a redox mediator of laccase, it is of outmost importance to ascertain that the electroactivity of ABTS is preserved in the microenvironment of the MOF pores. Figure 3(a) displays representative cyclic voltammograms of ABTS-MIL-100(Fe) (1:6)-CIE modified SS electrode in 0.1 M acetate buffer of pH 5.1. A couple of peaks situated at mean peak potential (E½=Epa + Epc)/2) of ~0.26 V vs. Ag/AgCl (ΔEp = 0.30 V at 0.01 V.s−1) is observed in the potential window between -0.20 and 0.50 V and can be attributed to the one-electron reversible redox process of ABTS (see Figure 3(a), curve (ii) and Figure 3(b)). Interestingly, neither any additional set of CV peaks nor the appearance of a green color in the solution that could be the result of a significant leaching of ABTS+• were observed.55 For a potential range up to 1 V (Figure 3a, curve (iii)), an additional oxidation peak is observed which corresponds to the oxidation of ABTS+• radical cation to the dication ABTS 2+. The surface concentration in electroactive adsorbed ABTS ( mol.cm-2) can be calculated by integration of the peak current recorded at low scan rate ( ≤ 10 mV/s) (Fig. 3(a) curve (ii)) based on the equation (1):56 (1)



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where Ip is the peak current,  the surface coverage of the electroactive adsorbed species (mol. cm-2), A the electrode surface area (3 cm2), Q the quantity of charge (C) which is calculated from the peak area of the cyclic voltammogram,  the sweep rate and n the number of electrons transferred per electroactive species. Other symbols have usual meanings. From Eq. (1),  can be calculated according to the following equation: (2) According to the integration of the peak, the surface coverage  of ABTS is equal to 6×10-8 mol.cm-2 (33 g cm-2 of ABTS). This value indicates that 90±5% of the ABTS initially introduced is electrochemically accessible. Moreover, it is worth noting that only a very small current density is detected at ABTS-CIE modified SS electrode within this potential range (Figure 3a(i)), in contrast to modified electrodes based on ABTS-MIL-100(Fe). This result clearly points out the advantage of entrapping ABTS in the MIL-100(Fe) host matrix for an efficient electrochemical response. The oxidation peak potential value of Epa=0.40 V is close to that of earlier report on immobilization of ABTS in polypyrrole films (0.46 V)57 or onto carbon nanotubes (0.47 V)58 and in pure [ZnCr-ABTS] matrix (0.47 V).7 This CV study confirms that ABTS is indeed electroactive even after its incorporation within the frameworks of electronically insulating MIL-100(Fe). Moreover, long term stability of ABTS-MIL-100(Fe)-CIE modified SS electrode has been studied by comparing the change in voltammetric peak current of the modified electrode before and after potential cycling in acetate buffer of pH = 5.1. ABTS-MIL-100(Fe)-CIE modified SS electrode has shown an exceptional stability in cycling since stable potentiodynamic responses are observed as demonstrated by only insignificant differences between the 1st and 500th cyclic voltammograms. In addition, after soaking ABTS-MIL100(Fe)-CIE modified SS electrodes continuously for 5 days in 0.1 M acetate buffer of pH=5.1, a negligible amount of ABTS could be detected in the supernatant solution as indicated by UV-Vis


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(a)

ABTS

.+

ABTS

2+

800µ

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Current density (A.cm )

1m

600µ

ABTS

ABTS

.+

400µ 200µ

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ABTS/CIE MIL-100(Fe)-ABTS/CIE MIL-100(Fe)-ABTS/CIE upto 1 V

-600µ -0.2

600µ 400µ

-2


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0.2

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Potential (V vs. AgCl/Ag)

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1st cycle 2nd cycle 10th cycle 50 th cycle 100th cycle 400th cycle 500th cycle

0

-200µ -400µ -600µ -0.3 -0.2 -0.1

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0.4


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Figure 3. (a) Cyclic voltammograms of ABTS/CIE (i) and ABTS- MIL-100(Fe(1:6))/CIE (ii and iii) electrode at a sweep rate of 10 mV s-1 in 0.1 M acetate buffer of pH=5.1. (b) The scheme of redox transformation of ABTS to radical cation (ABTS+•) and dication (ABTS2+). (c) Cyclic voltammograms of ABTS-MIL-100(Fe)/CIE electrodes recorded for 500 cycles.

spectroscopy (see Figure S5(b) of SI). This is also a benefit from the very good chemical stability of MIL-100(Fe) in the acetate buffer at pH=5.148 which prevents from any release of the ABTS molecules. In contrast a significant amount of ABTS was released in solution after soaking ABTS-CIE modified SS 15 ACS Paragon Plus Environment


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electrode in similar conditions (Figure S5(a) of SI). These results clearly demonstrate that ABTS is significantly anchored in the MIL-100(Fe) host matrix. The effect of scan rate (ν) on the electrochemical behavior of ABTS-MIL-100(Fe)-CIE modified SS electrodes has been investigated (see Figure S6 (a)). One can observe that the cathodic peak potentials shift to more negative values while the corresponding anodic peak potentials to more positive values with increasing scan rate. The reversibility of the electrochemical process decreases when the scan rate increases. As shown in Figure S6(b), the peak currents are proportional to the square root of the scan rate which is consistent with a diffusioncontrolled redox process. As a consequence, the charge transfer kinetics is certainly limited by the slow electron hopping across the MIL-100(Fe) matrix. By considering that the diffusion process may proceed through the transport of counter ions inside the pores of MIL-100(Fe), the calculated apparent diffusion coefficient (D0) is close to 0.8±0.4×10-8 cm2 s-1 (see SI for the calculation of D0). This diffusion value lies between those commonly obtained for ABTS in aqueous solution (~3-4 10-6 cm2 s-1at 293 K),54,55 , in polymers (2.1 10-6 cm2 s-1at 293 K in polyazetidine prepolymer),54 and those reported for incorporated anions in clay films (~10-11- 10-12 cm2 s-1).55 This value of ionic diffusion may suggest a good penetration of aqueous electrolyte in the pores of MIL-100(Fe) which is probably favored by the 3D mesoporosity of this phase. This would also benefit from the presence of the dual porosity of MIL100(Fe) with the ABTS lying only in the large mesoporous cages while the electrolyte would diffuse easily within the arrays of smaller mesoporous cages. To possibly unravel the origin of such interesting electrochemical activity of ABTS-MIL100(Fe)(1:6)-CIE electrode, electrochemical impedance spectroscopy (EIS) measurements were performed at open circuit potential (OCP). A typical Faradaic impedance spectrum presented as a Nyquist plot was obtained (see Figure S7 of SI). The impedance data were fitted using the equivalent circuit, adapted from the model proposed by Conway et al59 for porous electroactive material/electrolyte interfaces with faradic impedance in parallel with a double layer capacitance. The Ret parameter which describes the electron transfer resistance was extracted from these EIS experiments. Both ABTS-MIL16 ACS Paragon Plus Environment

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100(Fe)-CIE and MIL-100(Fe)-CIE are characterized by Ret values equal to 139 Ω and 243 Ω, respectively. Even though these electronic transfer resistance values are higher than those reported for conducting carbon based bioelectrodes, it is noteworthy that they are about 103-104 lower than those of non-conductive ABTS-layered double hydroxides (i. e. Co2Al-ABTS and Zn2Al-ABTS). 60 Cyclic voltammetry together with EIS experiments clearly show that ABTS-MIL-100(Fe)-CIE presents excellent properties in terms of charge transfer kinetics and ionic conductivity. Moreover, this modified electrode presents an excellent, very stable and reproducible electrochemical response, showing that MIL-100(Fe) provides a suitable and stabilizing microenvironment for electroactive ABTS molecules. In the next step, ABTS-MIL-100(Fe) (1:6) has been used as a host matrix for laccase immobilization and electrochemical studies for ORR. Immobilization of laccase in ABTS-MIL-100(Fe) (1:6) and bioelectrocatalytic O2 reduction reaction (ORR). For the purpose of ORR studies, laccase (Tv) with a redox potential of +0.8 V vs. standard hydrogen electrodes (SHE) was immobilized on ABTS-MIL-100(Fe) (1:6) (i. e. Lac-ABTSMIL-100(Fe)) as described in the experimental section. Since laccase presents a molecular mass of about 60-70 kDa and a spherical shape with a diameter of 6 nm (6.5 x 5.5 x 4.5 nm),9,10 the encapsulation of this enzyme in the porosity of MIL-100(Fe) can be ruled out taking into account the diameter of the mesoporous cages (25-29 Å) and windows (5.5-8.6 Å). However, this protein can be adsorbed at the outer surface of MIL-100(Fe) nanoparticles. Since the point of zero charge of MIL100(Fe) is close to 4 and the isoelectric point of laccase (Tv) is about 3.5, 61 repulsive electrostatic interactions are expected between both components. Therefore, the optimization of the laccase adsorption was carried out by performing a co-reticulation with BSA and cross-linking of the biomembrane with glutaraldehyde. As previously reported for numerous laccase-based biomembranes, the efficient immobilization of laccase (Tv) on host matrices is difficult and requires this double reticulation step.7



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In the following section, we report the bioelectrocatalytic O2 reduction reaction in ABTS-MIL-100(Fe). Laccase belongs to the family of blue multicopper oxidases. The active site of this metalloprotein contains one mononuclear copper site (type-1(T1) center) which is situated near the substrate binding pocket and a trinuclear copper cluster (one T2 and two T3 copper centers) embedded in the protein structure.14,61 The electroenzymatic reduction of oxygen by laccase proceeds through a ABTS mediated 4-electron reduction mechanism, converting O2 completely to H2O.62 This is a well-documented process previously reported for laccase/ABTS systems in solution 63,64,65 or laccase/ABTS co-immobilized on electrode surfaces.3,7,14,16,57 It involves four steps: (i)

The first step is the reduction of the oxidation state of Cu2+ at the T1 site of laccase by ABTS to form the radical cation ABTS+. ABTS + Lac(Cu2+, T1) → ABTS+. + Lac(Cu+, T1)

(i)

The T1 copper center functions as the primary electron acceptor site and shuttles electron through an histidine-cysteine-histidine transfer pathway to the T2/T3 cluster: 2 Lac(Cu+, T1) + Lac(Cu2+, T2, T3) → 2 Lac(Cu2+, T1) + Lac(Cu+, T2, T3)

(ii)

Then, oxygen is reduced by the reduced state of the central Cu ions at its T 2 and T3 sites in laccase Lac(Cu+, T2, T3): Lac(Cu+, T2, T3) + ½ O2 + 2H+ →Lac(Cu2+, T2, T3) + H2O

(iii)

The last step in the catalytic cycle is the electrochemical reduction of the ABTS+. radical cation to regenerate ABTS: ABTS+. + e- → ABTS

Figure 4 shows the CV of Lac-ABTS-MIL-100(Fe)-CIE and Lac-ABTS-CIE modified SS electrodes in 0.1 M acetate buffer of pH=5.1. The CV of Lac-ABTS-MIL-100(Fe)-CIE under saturated argon (Figure 4(i)) is characterized by a pair of peaks centered at mean potential of 0.37 V (ΔEp = 300 mV) due to the redox process of ABTS as detailed previously. Under saturated ambient air, an increase in the cathodic peak current density at the expense of the anodic peak current density is observed, which 18 ACS Paragon Plus Environment

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implies the oxidation of ABTS by laccase. The regeneration of the enzyme is achieved by the concomitant four-electron reduction of oxygen to water. This is a typical electrocatalytic reduction process of oxygen by laccase which is mediated by ABTS. In this configuration, ABTS entrapped in the MIL-100(Fe) matrix ensures the electrical wiring of laccase. 150µ Lac-ABTS-MIL-100(Fe)-CIE under argon Lac-ABTS-MIL-100(Fe)-CIE under air Lac-ABTS-MIL-100(Fe)-CIE under O2

100µ

Lac-ABTS-CIE under O2

-2


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50µ 0 -50µ

(iv)

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-150µ

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-200µ -0.1

0.0

0.1

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0.5


0.6

0.7

Figure 4. Cyclic voltammograms of Lac-ABTS-MIL-100(Fe)-CIE modified bioelectrodes in 0.1 M acetate buffer (pH=5.1) electrolyte at a sweep rate of 10 mV s-1 under(i) argon, (ii) air and (iii) pure O2 flow, and (iv) Lac-ABTS -CIE under O2.

A further increase in ORR current density was observed in O2-saturated solution. It is noteworthy that the CV of Lac-ABTS-CIE electrode displays only negligible ORR response under similar conditions (Figure 4(iv)). In the framework of our experimental conditions (non stirred electrolyte, inadequate flow of oxygen etc.), we were not able to achieve the “true” electrocatalytic wave. However, as revealed by the ratio between cathodic and anodic peak current density which increases from ~1.0 to 2.7 when argon was replaced by O2 atmosphere, these results shows clearly the electrocatalytic reduction of oxygen by laccase. The electrochemical response of the Lac-ABTS-MIL-100(Fe)-CIE bioelectrode was optimized by varying physico-chemical parameters such as pH of the electrolyte, amount of BSA and laccase. Since ABTS oxidation is known to be reversible and pH independent, the influence of pH may mainly impact the laccase activity. By varying pH in the range between 3 and 7, the maximum electrochemical response was obtained at pH 5 (see Figure S8(a) of SI). This pH value is identical to that previously reported for laccase (Tv) immobilized in [Zn-Cr-ABTS] layered double hydroxide7 or graphenesupported carbon nanotubes13 and close to the pH of the optimal activity of fungal laccase (3

Design of Laccase–Metal Organic Framework ... - ACS Publications

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