Luminita Fritea

Andrew James Gross

Karine Gorgy

Rachel K O'reilly

Alan Le Goff

Serge Cosnier

Alan Le Goff

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de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A bifunctional triblock polynorbornene/carbon nanotube buckypaper bioelectrode for low-potential/high-current thionine-mediated glucose oxidation by FAD-GDH

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is a glucose-oxidizing enzyme that has been proposed as a true alternative to the widely-investigated glucose oxidase for the catalytic oxidation of glucose in glucose biosensors and glucose biofuel cells. 1,2 FAD-GDH is not inhibited by O2, nor does it produce H2O2 from the concomitant reduction of O2. This has the important advantage that FAD-GDH can efficiently operate in the presence of O2, which is of tremendous importance for glucose/O2 fuel cells. 2,3 Furthermore, the presence of H2O2 is known to have deactivating effects on O2-reducing cathodes. 2 However, for the wiring of FAD-GDH at electrodes, a redox partner or a redox mediator is required in most cases. Different redox mediators have been studied so far for the wiring of FAD-GDH such as organic quinone derivatives, [4][5][6] and osmium, 1,7-9 ruthenium 10 and metallocene complexes 2 . These redox mediators have been mostly adsorbed either directly on electrodes, or covalentlyattached to a polymer backbone then immobilized on the electrode. Thionine, which features a phenothiazine core, has also been very recently envisioned as a low-potential redox mediator for FAD-GDH. 5,11 In many of these examples, nanomaterials have been studied for the immobilization of the redox mediator and the wiring of FAD-GDH. In particular, multiwalled carbon nanotubes (MWCNTs) have proven to be a useful nanostructured platform for the co-immobilization of the enzyme and the redox mediator. [12][13][14][15][16] Furthermore, the recent use of buckypapers (BPs) as CNT-based electrode supports has permitted the easy processability of flexible bioelectrodes for wearable and implantable fuel cell systems. 17 In particular, we have recently developed the fabrication of freestanding buckypaper biocathodes based on functionalizedpolynorbornene/MWCNT matrices. 18,19 A multifunctional polynorbornene BP was used to bind 2-aminoanthraquinone and laccase from Trametes versicolor for bioelectrocatalytic oxygen reduction via the orientation of immobilized enzymes. 18 The multifunctional polynorbornene copolymers were composed of a pyrene-based building block, for the reinforcement of the MWCNT matrix, and an NHS "grafting block", for the binding of proteins. In this work, we have developed the use of this type of multifunctional polynorbornene-based BP for the coimmobilization of both FAD-GDH and a redox mediator, thionine, for the MET-based oxidation of glucose. This freestanding BP was compared with a commercial MWCNT BP (from NanoTechLabs Inc.) modified via a previously-developed pyrene-NHS functionalization approach used for GDH wiring at glassy carbon-CNT electrodes 20 . We developed and compared the fabrication processes for both types of MWCNT BPs towards the immobilization and wiring of FAD-GDH at flexible electrodes for low-potential mediated bioelectrocatalytic oxidation of glucose. First, the preparation of the homemade free-standing BP was made according to our previously-described procedure with slight modification. 18 In brief, a mixed suspension comprising 66 mg of MWCNTs and 16.5 mg of polynorbornene triblock Please do not adjust margins Please do not adjust margins copolymer (Py33-b-NHS66-b-Py33, 50 kg mol -1 ) was prepared in 66 mL of DMF, filtered through a PTFE membrane under high vacuum, then washed, dried, and cut into 6 mm disk electrodes. The weight ratio of 20% polymer to 80% CNTs for the dispersion was chosen to give an optimal compromise between conductivity and mechanical stability. 18 It is noted that the linear polymer comprises an ABA triblock sequence with an average of 50% of each of the pyrene and NHS ester groups. The polymer was characterised by IR, 1 HNMR and size exclusion chromatography (SEC) (Figure S1-S3). For preparation of the bioelectrode, both thionine and FAD-GDH were successively coimmobilized via coupling to the NHS groups (Figure 1). Thionine was immobilized in a first step in order to maximize its immobilization. The enzyme was then immobilized in a second step via unreacted NHS groups and physical adsorption. Control experiments showed that the chemical grafting initially of the enzyme then of the mediator gave electrodes that did not show catalysis. In order to study and compare a similar strategy, we explored the use of a commercial MWCNT BP, postfunctionalized with 1-pyrenebutyric acid NHS ester (pyreneNHS), followed by the immobilization of thionine and then FAD-GDH (Figure 1). Characterization of buckypapers was done using laser scanning microscopy, conductivity measurements, X-ray photoelectron spectroscopy (XPS) and electrochemistry. Figure 2a shows the impressive flexibility of the polynorbornene-based BP. This is in contrast to the more fragile and less bendable commercial BP. The average conductivity (and thickness) for the pyreneNHSand polynorbornene-based BPs are 1.8 S cm -1 (270 µm) and 5.8 S cm -1 (360 µm), respectively, based on four-point probe and Vernier caliper measurements at room temperature (average values obtained from 6 independent BP samples). The higher conductivity at the polynorbornene BP electrode, despite the insulating nature of the polymer, suggests improved interconnectivity between conductive CNTs in the homemade BP. The optical microscopy recorded at the visibly smoother sides of each BP revealed a more homogeneous surface structure at the polymer-based electrode (Figure 2b). PyreneNHS-based and polynorbornene-based BP electrodes were subsequently characterized by XPS after covalent immobilization of thionine (Figure S4 andS5). The XPS analysis confirms the presence of thionine at the surface of both types of BP, as indicated by the peaks at 164.2 and 165.4 eV, which are assigned to the S 2p1/2 and 2p3/2 binding energies, respectively. The element ratio N/S was calculated as 3.6 instead of 3 for pyreneNHS-based BP and 2.8 instead of 4 for polynorbornene-based BP. This indicates that pyreneNHS molecules are not fully functionalized by thionine in the pyreneNHS-based commercial BP while a relatively larger amount of non-specifically adsorbed thionine, entrapped in the copolymer, might account for the low N/S ratio observed for polynorbornene-based BP. The redox properties of pyreneNHS-modified and polynorbornene-modified BP electrodes, after immobilization of thionine, were studied without glucose in pH 7 0.1 mol L -1 PB at 25°C by cyclic voltammetry (CV, Figure 3). A single reversible system is observed at E1/2 = -0.14 V vs. Ag/AgCl, corresponding to the electroactivity of thionine. The electroactivity of thionine at neutral pH corresponds to a 2 electron/1 proton process leading to leucothionine. Oxidation and reduction peak currents are highly stable upon multiple CV scans at both types of BP (Figure S6). Furthermore, a linear dependence of oxidative and reductive peak currents towards the square root of the scan rate is observed. The peak-to-peak separation is also > 59 mV for scan rates in the range 2-100 mV s -1 . This underlines that electron transfer is diffusion-controlled, as is commonly observed in redox hydrogels employed for MET-Please do not adjust margins Please do not adjust margins based wiring of enzymes. [21][22][23][24][25] By integration of the charge under the thionine redox system at low scan rate, a surface concentration of 0.16 nmol cm -2 and 0.27 nmol cm -2 was estimated for thionine immobilized on pyreneNHS-based BP and polynorbornene-based BP, respectively. A higher amount of thionine at the surface of polynorbornene-based BP likely arises from the higher number of available NHS groups, facilitated by the flexible NHS linker (n = 7), at the surface of the polymer-coated CNTs, as compared to the pyreneNHS-modified CNTs. This behaviour can be expected owing to the supramolecular nature of pi-stacking of pyrene molecules onto CNT sidewalls, 20 which is governed by a Langmuir isotherm and leads to an incomplete coverage of CNT sidewalls. The bioelectrocatalytic properties of each of the functionalized BPs were studied in the presence of glucose (Figure 4). For both types of BPs, upon addition of 150 mmol L -1 glucose, an electrocatalytic irreversible wave is observed with an onset potential of -0.25 V vs. Ag/AgCl. This confirms that MET is achieved between immobilized thionine and FAD-GDH. It is noteworthy that it is a gain of almost 100 mV in terms of overpotential for the MET wiring of FAD-GDH compared to an organic redox mediator such as phenanthrolinequinone, and a gain of around 250 mV compared to an osmium-based or ruthenium-based redox mediator. 4,9,10 Maximum current densities of 3.03 mA cm -2 and 1.98 mA cm -2 were observed at 0.2 V for polynorbornene-based BP and pyreneNHS-based BP, respectively. Chronoamperometric measurements were performed at Ep = 0.2 V vs Ag/AgCl in the presence of increasing concentrations of glucose (Figure 5A). Both types of BP exhibit a typical Michaelis-Mentens dependence with a K M value of 25 mmol L -1 for both BPs (Figure 5B). Maximum current densities were obtained for homemade polynorbornene-based BPs with 3.7 mA cm -2 beyond 50 mmol L -1 glucose. A linear region is observed between 0 and 15 mmol L -1 glucose with sensitivity of 0.12 mA mol -1 L cm -2 (R 2 = 0.999) and 0.093 mA mol -1 L cm -2 (R 2 = 0.982) for polynorbornenebased BP and pyreneNHS-based BP, respectively, accompanied with a limit of detection of 600 µM (Figure S7). Furthermore, stability experiments examined by 30-min chronoamperometric measurements at 0.2 V vs Ag/AgCl show that polynorbornenebased BP bioelectrodes retain 68% of their electrocatalytic activity after 7 days (Figure S8A) while pyreneNHS-based BPs exhibit 20% of their initial activity after 7 days (Figure S8B). The enhanced catalytic stability observed for the polynorbornenebased electrode is attributed, at least in part, to improved enzyme stabilization via non-covalent enzyme entrapment in the polymer-CNT matrix. The stabilisation may be due to factors such as improved retention of the enzyme at the electrode and preservation of the enzyme's active conformation. The polymer-CNT matrix benefits from effective cross-linking, owing to the presence of multiple pyrene groups per polymer chain that can cross-link CNTs and thionine. In contrast, the BP electrode with pyrene-NHS cannot effectively cross-link. The cross-linked polynorbornene-CNT electrode is therefore expected to be more robust than the pyrene-NHS electrode. Furthermore, the higher number of available NHS groups at the surface of the polynorbornene BPs induces a higher number of thionine wired enzyme per surface unit as compared to the supramolecular pyrene-NHS modification, the later being a reversible phenomenon governed by Langmuir insotherm. 20 For comparison, FAD-GDH entrapped in osmium hydrogels at hierarchically structured porous electrodes exhibit maximum current densities of 100 mA cm -2 (but at a much higher potential of 0.5 V vs. Ag/AgCl, with onset potentials of 0.0 V, and only under hydrodynamic conditions) 9 . For comparison, thionine and FAD-GDH adsorbed on gold nanoparticle-modified electrodes exhibit a maximum current density of 1.6 mA cm -2 with an onset potential of -0.24 V 11 . We recently reported a powerful catalytic glucose oxidation but with limited stability at a homemade BP electrode with adsorbed FAD-GDH and phenanthrolinequinone This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins which exhibited a maximum current density of 5.4 mA cm -2 with onset potentials of -0.15 V 4 . The catalytic current of the phenanthroline quinone electrode was only around ca. 35% of its initial catalytic activity after 7 days, however, highlighting further beneficial performance of the polynorbornene-based thionine BP bioelectrode in terms of stability.

Conclusions

Here is described a performant biomaterial based on buckypapers for bioelectrocatalytic glucose oxidation via immobilization of both FAD-GDH enzyme and thionine redox mediator using an ABA triblock polynorbornene copolymer substituted with alternating pyrene and NHS groups. Compared to buckypaper prepared with the classical molecular linker, pyreneNHS, the buckypaper with the polynorbornene exhibited higher current densities of 3.7 mA cm -2 at a lower potential with increased stability, flexibility and conductivity. The interest of using triblock polynorbornene copolymers for bioanode construction is therefore highlighted for the first time towards the goal of wearable and implantable bioelectrodes for biofuel cell and biosensor applications

Figure 1 .

 1 Figure 1. Schematic representation of the functionalization of MWCNT-based BP with (A) pyrene-NHS and (B) polynorbornene-pyrene-NHS polymer followed by the sequential immobilization of thionine and FAD-GDH

Figure 2 .

 2 Figure 2. (A) Photograph of the flexible free-standing polynorbornene/MWCNT BP; (B) laser scanning microscopy image of the surface topography of (a) pyreneNHS BP and (b) polynorbornene BP

Figure 3 .

 3 Figure 3. CV scans for (A) pyreneNHS-based BP and (B) polynorbornene-based BP at different scan rates: 2, 5, 10, 20, 30, 50, 80 and 100 mV s -1 (0.1 M PB, pH 7, 25°C); (C) Evolution of oxidation and reduction peak current towards square root of scan rate for () pyreneNHS-based BP and () polynorbornene-based BP with immobilized thionine

Figure 4 .

 4 Figure 4. CV scans recorded before and after addition of 150 mmol L -1 glucose for (A) pyreneNHS-based BP and (B) polynorbornene-based BP with immobilized thionine and FAD-GDH (1 mV s -1 , 0.1 mol L -1 PB , pH 7, 25°C).

Figure 5 .

 5 Figure 5. (A) Chronoamperometric measurements performed at Ep = 0.2 V for (a) pyreneNHS-based BP and (b) polynorbornene-based BP with immobilized thionine and FAD-GDH after addition of successive amounts of glucose with stirring in 0.1 mol L -1 PB pH 7, 25 °C; (B) Evolution of the current density with the glucose concentration accompanied with a fitting curve using a classic Michaelis-Mentens dependence. Error bars correspond to one standard deviation from at least two electrodes.

Acknowledgments

This work was supported by the Labex ARCANE, CBH-EUR-GS (ANR-17-EURE-0003) and Campus France. The authors wish to acknowledge the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG). The authors are grateful both to Yannig Nedellec, for conductivity and thickness measurements, and to Valerie Flaud from ICGM Montpelier for XPS characterization.

Notes and references


Jules L Hammond

Andrew J Gross

Fabien Giroud

Christophe Travelet

Redouane Borsali

Serge Cosnier

Solubilized Enzymatic Fuel Cell (SEFC) for Quasi-Continuous Operation Exploiting Carbohydrate Block Copolymer Glyconanoparticle Mediators

Enzymatic biofuel cells are ecofriendly power sources that can deliver μW-mW outputs from renewable substrates, but their stability is a major issue owing to enzyme fragility. The vast majority of reported biofuel cells can only generate power continuously for relatively short periods of time. Here we report a novel "solubilized enzymatic fuel cell (SEFC)" concept for continuous long-term operation. Avoiding surface immobilization techniques allows biocatalytic activity to be easily restored or replenished. The biofuel cell exploits freely diffusing enzymes and β-cyclodextrin-coated glyconanoparticles with entrapped quinone and thiazoline redox mediators, for mediated glucose and oxygen conversion. The cell was designed with permselective membranes to enable substrate and proton diffusion while trapping the enzymes and glyconanoparticles in separate compartments. The SEFC exhibited a peak power loss of only 26.3% after 7 days of continuous charge-discharge cycling at 50 μA; thus, SEFCs may be envisaged to power lab-on-a-chip devices for periods of several weeks. 47 combined with surface modification chemistry, is considered to 48 be the best way to stabilize the enzyme. However, this method 49 eventually leads to a buildup of inactive enzyme at the surface, 50 making it extremely difficult for the catalytic activity to be 51 restored or replenished. 7 Enzyme immobilization and entrap-52 ment methods can also fix the enzymes in inefficient 53 orientations, restricting their degrees of freedom and reducing 54 their activity. Irreversible physical damage to the enzyme upon 55 binding or cross-linking to the electrode is also an issue. Redox 56 mediators are often immobilized together with the enzyme to 57 facilitate electron shuttling to and from the enzyme's active site 58 (mediated electron transfer (MET)). Covalent attachment of 59 the enzyme followed by entrapment of the mediator by cross-60 linking, 8 encapsulation in polypyrrole films, 9 or forming a 61 mixed compression with graphite particles 2 is typically used to 62 stabilize enzymes at electrodes. Such techniques are considered 63 to mitigate catalytic instability and minimize leaching 10 but at 64 the expense of reduced activity. High concentrations of

E nzymatic biofuel cells convert chemical energy into electrical energy by electroenzymatic reactions. 1 The use of enzymes provides several advantages compared to conventional noble metal catalysts, including very high specificity toward their substrates, high turnover and activity under mild conditions, and the ability to transform complex organic fuels. Enzymes are also biocompatible and environmentally benign, although intrinsically complex and fragile. Another major drawback of enzymes is their limited stability after immobilization on artificial electrode surfaces. Currently there is an urgent need to develop superior strategies to preserve, optimize, and enhance enzyme stability and activity at electrodes for bioelectrocatalysis under various conditions.

With state-of-the-art prototype biofuel cells already capable of powering small electronic devices such as pacemakers, lactate sensors, and wireless communication systems for short periods of time, their long-term operational stability remains a crucial parameter for their future success. [1][START_REF] Cinquin | A Glucose BioFuel Cell Implanted in Rats[END_REF][START_REF] Macvittie | Cyborg" Lobsters to a Pacemaker Powered by Implantable Biofuel Cells[END_REF][START_REF] Monsalve | A H 2 /O 2 Enzymatic Fuel Cell as a Sustainable Power for a Wireless Device[END_REF] One of the key factors determining the lifetime of a biofuel cell is the stability of the enzymes and mediators, presenting pivotal concern for their practical application. [START_REF] Moehlenbrock | Extended Lifetime Biofuel Cells[END_REF] Immobilization of the enzyme on the electrode, for example, using carbon nanostructures, [START_REF] Mazurenko | Impact of Substrate Diffusion and Enzyme Distribution in 3D-Porous Electrodes: A Combined Electrochemical and Modelling Study of a Thermostable H 2 /O 2 Enzymatic Fuel Cell[END_REF] often immobilized mediators are typically required to maximize power density, but this can be difficult to realize, expensive, and unsafe due to toxicity, caused by progressive leaching.

The aim of this work is to depart from the traditional approach of immobilizing the bioelectrocatalytic components of the fuel cell on the electrodes. Instead, we adopt here a strategy of utilizing enzymes and redox-mediating glyconanoparticles (GNPs) solubilized (i.e., nonimmobilized) in solution. Enzymes and mediators in solution can freely diffuse and rotate for dynamic orientation with each other for effective electron transfer and bioelectrocatalysis. The use of enzymes in solution can also avoid issues related to poor mass transfer [START_REF] Johnston | In Situ Measurement of Activity and Mass Transfer Effects in Enzyme Immobilized Electrodes[END_REF] and reproducibility due to complicated enzyme immobilization procedures. [START_REF] Zhao | Kinetic Limitations of a Bioelectrochemical Electrode Using Carbon Nanotube-Attached Glucose Oxidase for Biofuel Cells[END_REF] Importantly, Zhu and Zhang [START_REF] Zhu | Use of Nonimmobilized Enzymes and Mediators Achieved High Power Densities in Closed Biobatteries[END_REF] previously demonstrated that higher power densities can be obtained at a nonimmobilized bioanode of a closed biobattery when compared to an immobilized bioanode.

If both the enzymes and mediators are solubilized, as explored/reported herein, fresh biocatalysts can be introduced at high concentrations to restore or replenish performance by a simple exchange of the anode and cathode solutions. This will further prolong the biofuel cell lifetime and would address the major problem of conventional biofuel cells whereby the bioelectrodes with immobilized enzymes must be reconstructed when the enzyme becomes inactive. The concept of refueling the fuel solution of a biofuel cell has been previously demonstrated by Sony Corporation with an immobilized system. However, the cell voltage collapsed to 0.2 V after 40 fuel exchanges (200 min total operation). [START_REF] Fujita | A Repeatedly Refuelable Mediated Biofuel Cell Based on a Hierarchical Porous Carbon Electrode[END_REF] Taking into account that redox mediators and substrates such as glucose are similar in size, it was not possible to consider a solubilized bioelectrode system that was capable of retaining redox mediators while still permitting diffusion of glucose. In this context, the engineering of redox supramolecular entities exhibiting a similar size to proteins is a promising route in the design of bioelectrodes based on a sizeexclusion process. GNP synthesis [START_REF] Gross | Redox-Active Glyconanoparticles as Electron Shuttles for Mediated Electron Transfer with Bilirubin Oxidase in Solution[END_REF] not only allows hydrophobic and insoluble redox-active species to be solubilized but simultaneously enables size-dependent entrapment within a fuel cell compartment due to their large size via a size-exclusion membrane. Replacing classical redox mediators with redoxactive GNPs opens up the new possibility to separate the anolyte from the catholyte and avoid cross-reaction. Without size exclusion there would be an inevitable drop in open-circuit voltage (OCV), close to zero, due to a mixed potential between the bioanode and biocathode. Furthermore, the use of GNPs can allow the redox-active anolyte/catholyte or fuel to be specifically replenished or restored.

In summary, we develop here the use of two types of freely diffusing, solubilized redox glycocopolymer nanoparticles for the electrical wiring of enzymes both at the anode and cathode. Furthermore, we have engineered a new solubilized enzymatic fuel cell (SEFC) device that integrates permselective membranes, allowing us to exploit the size of the GNPs to prevent mediator leaching into the fuel stream or away from their respective anode or cathode compartments.

The biofuel cell can be envisaged as the combination of two half-cells that concomitantly undergo oxidation and reduction reactions at the anode and cathode, respectively. Electrons produced by oxidation at the anode flow through the external circuit, delivering power to the load, and are then transferred to the cathode where they participate in the reduction reaction. As it is desirable to utilize abundant and low-cost organic 128 energy sources, we base our system on glucose and oxygen as 129 the fuel and oxidant, respectively. These substrates have 130 attracted considerable attention for implantable and wearable S1). Importantly, the electroactivity of the 176 PS-βCD-PQ GNPs is approximately doubled (1:1.91) when 177 doubling the concentration of the mediator, as demonstrated 178 by the scan-rate dependence shown on cyclic voltammograms 179 (SI, Figures S4a,b and Table S2).

TEM images showed no change in the GNPs after 2 months or after addition of pH 7.0 McIlvaine buffer (data not shown). This is consistent with the apparent stability previously observed for our PS-βCD-P 2 ABTS GNPs. [START_REF] Gross | Redox-Active Glyconanoparticles as Electron Shuttles for Mediated Electron Transfer with Bilirubin Oxidase in Solution[END_REF] The electrocatalytic activity of the new PQ GNPs was then confirmed at glassy carbon electrodes (GCEs) with increasing concen-f3 trations of glucose in DI H 2 O (Figure 3). Furthermore, system performance was found to be limited by the enzyme mass (120 μg•mL -1 ) in the system (SI, Figure S5). At the higher bound of a typical blood glucose level (5 mM), the catalytic current was around 2.5 ± 0.2 μA.

f4

A new fuel cell (Figure 4) was developed specifically for our system and consists of three main parts. The central chamber (V = 1.5 mL) is connected to a peristaltic pump to provide a constant flow of fuel to the adjacent compartments (V = 1 mL) that contain the anode and cathode biocatalytic components. To prevent loss of the enzymes and mediators from their respective compartments, dialysis membranes with a cutoff value of 25 kDa are used as separators and sealed with a double O-ring system. This cutoff value translates to an approximate R min of 1.9 nm (D min = 3.9 nm), calculated using the following equation, [START_REF] Erickson | Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy[END_REF] R min = 0.066(Da) 1/3 , and the cutoff value is sufficiently smaller than the diameter of PQ (20.0 ± 7.0 and 43.9 ± 27.7 nm at 34 and 68 μM, respectively) and P 2 ABTS (43.3 nm ±18.8 nm) GNPs and the enzymes (BOx = 66-68 kDa and FAD-GDH = 95-135 kDa). Carbon nanotube buckypaper offers properties such as high specific surface area, mechanical flexibility, and high electrical conductivity. [START_REF] Gross | A High Power Buckypaper Biofuel Cell: Exploiting 1,10-Phenanthroline-5,6-Dione with FAD-Dependent Dehydrogenase for Catalytically-Powerful Glucose Oxidation[END_REF] To increase power output, electrodes with increased area (5 × 40 mm) were fabricated by attaching commercial carbon nanotube buckypaper to graphite foil current collectors using carbon paste. To limit evaporation and secure the electrodes in place, the compartments were sealed with tape and then the electrodes terminated with conductive adhesive copper tape. The electrocatalytic behavior of the bioelectrodes was verified for both half-cells (SI, Figures S6 andS7). Here we can compare the catalytic performance with our previous work on buckypaper electrodes [START_REF] Gross | A High Power Buckypaper Biofuel Cell: Exploiting 1,10-Phenanthroline-5,6-Dione with FAD-Dependent Dehydrogenase for Catalytically-Powerful Glucose Oxidation[END_REF] with immobilized enzyme and mediator bioelectrodes (see the SI for further details). Using a similar but not identical quinone mediator and FAD-GDH enzyme, we produced a catalytic output of 0.637 mA.cm -2 • mg -1 . In contrast, the solubilized system with PQ GNPs and FAD-GDH reported here achieved 2.05 mA•cm -2 •mg -1 . Likewise, for the biocathode, prepared with immobilized BOx, we achieved 1.77 mA•cm -2 •mg -1 . This compares to a catalytic output of 3.79 mA•cm -2 •mg -1 for the solubilized BOx A typical charge-discharge profile is shown in Figure 6a,b.

254

The charge and discharge times were then calculated at Figures S9-S11). This fuel cell provided relatively stable operational performance with discharge time falling from 20 to 15 s (-25%) and charge time increasing from 158 to 340 s (+115%). This demonstrates the ability to operate the biofuel cell at higher discharge currents over long periods of time while retaining a reasonable level of charge-discharge time stability.

The recirculating fuel solution was tested for the presence of any enzymes and mediators after completion of the operational stability tests and showed no evidence for catalysis (black lines), as shown by the control experiments (red/blue lines for f7 anode/cathode, respectively; Figure 7a,b). This confirms that the biocatalytic elements are retained in their respective chambers and do not leach into the fuel stream. This is a testament to the GNPs, which, owing to their markedly larger sizes compared to classical redox mediators, provide the ability to constrain them within the anode and cathode compartments while allowing diffusion of the substrates, glucose, and oxygen.

It is emphasized that redox mediators without nanoparticle encapsulation would not be retained owing to their smaller molecular size. A control experiment using classical redox mediators in place of the GNPs is described further below.

To check for the presence of adsorbed biocatalytic components, discharge profiles were performed following 7 days of operational cycling at 50 μA and after using different rinse techniques (SI, Figures S12 andS13). Although an OCV develops (after resting the cell for 2 h between each rinse), there is negligible power generated and no operational cycling can be performed, ruling out significant adsorption of the biocatalysts. The presence of only trace quantities of biocatalytic components is particularly attractive as the cell chambers can be effectively refreshed.

Finally, a control experiment without PS-βCD GNPs to encapsulate the mediators but instead with solubilized mediators was carried out. This control test confirmed that the addition of GNPs in the system helps to efficiently stabilize and improve performance. Without the GNPs, the performance was lower and rapidly degraded, with the charge times steadily increasing to the point that the cell failed to recharge after 20 h (SI, Figures S14 andS15). This was most likely

  131 applications, owing to their availability in the body in μM-mM 132 concentrations by human metabolism and consumption, but 133 are equally viable for portable applications. 1 134 At the anode, we perform catalytic oxidation of glucose with 135 fungal flavin adenine dinucleotide-dependent glucose dehy-136 drogenase (FAD-GDH) from Aspergillus sp. using 9,10-137 phenanthrenequinone (PQ) as the organic redox mediator. 138 PQ was recently shown to be an effective mediator in solution 139 for glucose oxidation with this enzyme compared to other 140 quinone mediators. 16 For the cathode, we reduce oxygen via a 141 four-electron process to water using the bilirubin oxidase 142 (BOx) enzyme from Myrothecium verrucaria, mediated by bis-143 pyrene-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 144 (P 2 ABTS). 15 In both cases, the mediator is encapsulated 145 within a GNP using a guest-host interaction via β-cyclodextrin 146 inclusion complexes. The two separate reactions occurring at 147 f1 the anode and cathode are illustrated in Figure 1. 148 The redox GNPs were prepared using an amphiphilic β-149 cyclodextrin (β-CD) polystyrene diblock copolymer based on 150 a click chemistry reaction. The β-CD GNPs are obtained by 151 self-assembly with controlled encapsulation of the mediator in 152 a high yield via the nanoprecipitation technique.17

  Further 153 details of the P 2 ABTS GNP preparation, characterization, and 154 use for catalysis within a cathodic half-cell have been published 155 previously by our group. 15 To complement the P 2 ABTS GNPs 156 and complete the aqueous biofuel cell, new GNPs with 157 entrapped PQ as a redox mediator were developed for the 158 anodic half-cell. We formed PS-βCD-PQ GNPs with 159 approximate PQ concentrations of 34.2 and 68.4 μM (1:1 160 and 1:2, PS-βCD:PQ, mass). These concentrations are much 161 higher than the maximum concentration achieved when 162 dissolving PQ in deionized (DI) H 2 O with ultrasonic agitation, 163 of around 5 μM [SI, Figure S1]. This demonstrates how 164 increased quantities of a hydrophobic mediator can be 165 incorporated in solution for catalysis via host-guest 166 encapsulation in our GNP system.

  167 f2 TEM imaging (Figure 2) of the PQ GNPs showed 168 nanoparticles with average diameters of 20.0 ± 7.0 and 43.9 169 ± 27.7 nm at 34.2 and 68.4 μM, respectively. This compares to 170 43.3 ± 18.8 nm for the PS-βCD-P 2 ABTS GNPs (SI, Figure 171 S2). Interestingly, increasing the concentration of the guest 172 altered the average GNP diameter, as shown in the size

Figure 1 .

 1 Figure 1. Scheme showing the reactions occurring at the anode (left) and cathode (right)

Figure 2 .

 2 Figure 2. TEM images and histograms showing the typical size distribution of PS-βCD-PQ GNPs.

Figure 3 .

 3 Figure 3. Cyclic voltammograms and glucose dose response for an anode half-cell using GCEs, 2.5 mL of 34.2 μM PS-βCD-PQ GNPs, and 0.3 mg of FAD-GDH in 25 μL of pH 7.0 McIlvaine buffer, v = 5 mV•s -1 .

A

  peristaltic pump operating at a flow rate of 80 μL•min -1 (mimicking typical blood vessel flow 20 ) was used to deliver a continuous stream of pH 7.0 McIlvaine buffer containing ambient O 2 and 5 mM glucose from a 50 mL reservoir. A volume of 800 μL of PQ GNPs with 120 μg of FAD-GDH and 800 μL of P 2 ABTS GNPs with 200 μg of BOx were injected into the anode and cathode compartments, respectively.

  233 system obtained in this work. We therefore clearly observe 234 better catalytic performances per amount of enzyme catalyst in 235 this work compared to state-of-the-art buckypaper anodes and 236 cathodes with immobilized enzymes.

  237 f5Polarization plots (Figure5) were performed for the biofuel 238 cell by increasing the discharge current and recording the cell 239 voltage after 30 s. A maximum power point of 116.2 ± 21.3 240 μW was observed at a discharge current of 350 μA. The 241 observed OCV of the fuel cell was 0.685 V ± 26 mV. 242 Operational stability of the fuel cell was tested by performing 243 constant charge-discharge cycling for extended periods. The 244 cell is discharged at a current of 50 μA until it reaches 0.25 V 245 and then is allowed to recover until the voltage reaches 0.65 V. 246 The glucose oxidation and oxygen reduction reactions are 247 continuous. The selected current and voltage values provide a 248 compromise between charge/discharge times, maximizing the 249 duty cycle of power generation while also preventing damage 250 to the enzyme. It is important to note that many previous 251 reports of biofuel cell stability operate the device periodically 252 for relatively short periods of time. 253 f6

Figure 4 .

 4 Figure 4. 3D annotated drawing (left) showing the fuel cell components and photograph (right) of the setup when connected.

Figure 5 .

 5 Figure 5. Cell performance (n = 3) showing voltage (black, left axis) and power (blue, right axis) as a function of current at a fuel flow rate of 80 μL•min -1 (pH 7.0 McIlvaine buffer with 5 mM glucose and ambient O 2 ), with 120 μg of FAD-GDH, 200 μg of BOx, and 800 μL of each GNP suspension.

Figure 6 .

 6 Figure 6. Typical charge-discharge cycles (a), zoomed period between 39 and 40 h (b), and plots of fuel cell discharge (blue, square marker) and charge (red, diamond marker) stability at 10 h intervals (c), all at a 50 μA discharge current and fuel flow rate of 80 μL•min -1 (pH 7.0 McIlvaine buffer with 5 mM glucose and ambient O 2 ) with 120 μg of FAD-GDH, 200 μg of BOx, and 800 μL of each GNP suspension.

Figure 7 .

 7 Figure 7. CVs showing response at GCEs for biofuel cell residues (black) with (a) +300 μM [Fe(CN) 6 ] 3-(red, dashed) and +120 μg of FAD-GDH (red), all in saturated Ar with an Ar blanket, 2 mV•s -1 , and (b) +300 μM ABTS (blue, dashed) and +200 μg of BOx (blue), all in saturated O 2 with an O 2 blanket, 2 mV•s -1 .

DOI: 10.1021/acsenergylett.8b01972 ACS Energy Lett. XXXX, XXX, XXX-XXX


Serge Cosnier
email: serge.cosnier@univ-grenoble-alpes.fr

Andrew J Gross

Fabien Giroud

Michael Holzinger

Anna Maria Oliveira Brett

Beyond the hype surrounding biofuel cells: What's the future of enzymatic fuel cells?

After a short comparison of biofuel cells based on enzymes 1 and microorganisms, several important developments and 2 applications of enzymatic fuel cells (EFCs) are discussed. This 3 discussion emphasizes how to evaluate the performance of 4 EFCs, and highlights the influence of temperature and how it 5 must be carefully considered for practical use of EFCs as 6 power sources. Some of the latest and most important 7 innovations in EFC design using buckypapers and redox 8 nanoparticles are briefly reviewed.

Introduction 15

The development of sustainable and renewable energy 16 sources with greater respect for the environment is a cru-17 cial challenge for the 21st century. Among various alterna-18 tive energy sources, fuel cells have generated great inter-19 est for the production of clean electrical energy. Fuel cells come the depletion of fossil fuels and enter the category of renewable energy resources such as hydroelectric, solar, and wind energy. The scope of applications for EFCs and MFCs is restricted, at least for now, to electricity generation for powering microelectronic systems such as actuators and sensors [3,4,5 Enzymatic and microbial biofuel cells differ vastly in terms of their volumetric size (cm 3 to m 3 ), power density output (μW to mW) and their targeted applications (smallto large-scale). Another important consideration lies in the cost of the biocatalyst, which ranges from low-cost bacteria or yeast cells, for MFCs, to the higher cost of purified wild-type and engineered enzymes for EFCs. MFCs have been successfully employed as long-term powergenerating systems, for example, for self-powered sensors placed in "hostile" environments or remote locations [9] . Although MFCs have successfully powered different generations of robots, this approach remains at the academic level because of limitations such as the large volume of MFCs required and their long amortization cost [8] . Nevertheless, MFCs constitute an original and ecofriendly system for producing electrical energy, e.g. from waste, and have become an attractive bioreactor concept for purification of wastewaters. EFCs on the other hand, because of their superior power density and compactness, are better suited to miniaturization and use as portable power sources for wearable and implantable electronics devices. EFCs appear to carry greater promise as batteryfree power solutions, but it is necessary to define if this is a simple scientific curiosity or if their potentialities really do have conceivable real-world applications. This opinion article attempts to shed light on some hitherto neglected facets of EFCs, to briefly revisit their possible applications, and to reveal new insights concerning future directions. It is emphasized that this review does not cover all of the exciting facets and developments in EFC research. Recent reviews are available for further information on important topics such as the evolution of bioinspired strategies for the electrical wiring of enzymes and the development of high surface area bioelectrodes [10,11] . Notable innovations on high surface area electrodes include the hierarchically porous MgO-templated carbon and carbon nanotube (CNT) yarn textile electrodes [12 with glucose EFCs, a major advantage of hydrogen EFCs lies in the size of the fuel. Hydrogen is markedly smaller than glucose or various redox mediators used for electrical enzyme wiring. The small size can thus be exploited, via careful electrode design with permselective membranes, to prevent the leakage of redox mediators whilst allowing H 2 to efficiently diffuse into the cell. Hydrogen EFCs have been successfully developed with gas-diffusion electrodes, like air-breathing cathodes or H 2 -breathing anodes, which make it possible to increase the availability of dissolved O 2 and H 2 in quiescent solutions. This is an essential development for portable EFCs used outside of the body. Owing to electrode nanostructuration, and improvements in enzyme orientation and electrical wiring, great improvements in power densities have been achieved, which can match, or even surpass, the performance of glucose EFCs. However, the use of hydrogen for EFCs confronts us with problems of H 2 storage and transport. This is in contrast to the use of glucose, which is a safe, abundant and easy-to-handle energy compound. 131 Hydrogen can be physically stored as either a gas or a liq-132 uid but generally requires high pressure and large volume 133 tanks. Practical high density hydrogen storage remains a 134 major challenge for EFCs dedicated to portable applica-135 tions.

136 How to define EFC power? 137 One of the problematic points for the comparison and 138 continuous optimization of EFC performance concerns 139 their power output and how it is defined. As already 140 pointed out by Katz and coworkers, the power density of 141 an EFC is an important parameter that must be associ-142 ated with the real power generated by the EFC in order 143 to determine which applications can actually be consid-144 ered [START_REF] Gamella | Biofuel cells -activation of micro-and macro-electronic devices[END_REF] . There are a multitude of electronic and wireless 145 electrical products that consume microwatt to milliwatt 146 powers during operation. Many electronic devices have 147 multiple power modes such as a "standby" mode, where 148 low power is consumed, and "active modes", for example, 149 for data transmission. High milliwatt powers, for example, 150 may therefore only be required for very short periods of 151 peak activity. Future advances in ultra-low power and low 152 voltage electronics in the next decade are expected which 153 will further valorise energy-harvesting devices with μW 154 to mW outputs. For instance, an ultra-low power 3.5 μW-155 consuming cell phone without a battery is under develop-156 ment [START_REF] Talla | Battery-Free Cellphone[END_REF] .

157

In the research field of biological fuel cells, power den-158 sities can be misleading. For example, microwatt power 159 densities obtained using microelectrodes seem extremely 160 attractive, but in reality, are almost useless when the actual 161 power output is on the order of nanowatts. Another issue 162 for bioelectrodes is that the power does not evolve propor-163 tionally to the surface area owing to the electrode mor-164 phology that plays, for example, on the continuous sup-165 ply and removal of reactants and products. The power is 166 highly dependent on electrode morphology, including the 167 structure and thickness of the adsorbed enzymes, and the 168 crucial mass transport and charge propagation processes 169 that occur inside and outside of the biocatalyst layer(s) 170 [START_REF] Rajendran | Mathematical modeling of nonlinear reaction-diffusion processes in enzymatic biofuel cells[END_REF] .

171 Regarding the electrode architecture, which ranges from 172 simple planar electrodes with monolayers of enzymes 173 to 3D-structured mesoporous carbon, CNT pellets, and 174 CNT yarns with embedded enzymes, the electroactive 175 surfaces and enzyme loadings can be vastly different 176 [12 • ,13] . Despite this, the power densities of biofuel cells 177 are conventionally calculated only with respect to the ge-178 ometric surface of the electrodes. So what about the fact 179 that most bioelectrodes are now based on 3D architec-180 tures with huge differences in electrode thickness and 181 surface area? Another difficulty in defining power also 182 lies sometimes in the design of EFCs that exploit anodes 183 and cathodes with different sizes. For example, a H 2 /O 2 184 EFC combines a 6 cm 2 bilirubin oxidase-modified cath-185 ode with a 1.2 cm 2 hydrogenase-modified anode to bal-186 ance the catalytic performance as a function of the H 2 -air 187 mixture used [18 stant physiological temperature of 37 °C. Nevertheless, this does not mean that relevant enzymes are unsuitable for operation at elevated temperatures. In fact, it appears that the power of EFCs can be increased by increasing the temperature, as demonstrated by Lojou and coworkers for H 2 /O 2 EFCs using thermostable enzymes such as hydrogenase from Aquifex aeolicus and bilirubin oxidase from Bacillus pumilus [17 • ,41] . High power densities have been obtained under these (relatively) mild conditions, making it possible to power a wireless transmission system [START_REF] Monsalve | A H 2 /O 2 enzymatic fuel cell as a sustainable power for a wireless device[END_REF] as illustrated in Figure 1 . However, to accurately evaluate the performance and practicality of such hydrogen EFCs, it is necessary to also consider the power required to maintain the EFC at 60 °C. Rather than the possibility for improved EFC power output, the main advantage of this approach lies in the use of thermostable enzymes that allow the EFC to withstand significant temperature variations (e.g. from 30 -80 °C) without denaturation.

As well as temperature considerations, the type of solution used in the biofuel cell is another critical but often overlooked factor in EFC research. Until now, relatively few studies have investigated EFC performance in matrices for practical applications such as biological fluids, environmental waters or wastewater. The vast majority of experiments have simply been performed in aqueous buffer solutions. Towards human body-integrated applications, recent studies have started to address bioelectrode and EFC performance directly in human serum, urine, saliva and blood [START_REF] Cadet | An enzymatic glucose/O2 biofuel cell operating in human blood[END_REF][START_REF] Kwon | Stability of carbon nanotube yarn biofuel cell in human body fluid[END_REF][START_REF] Milton | Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum[END_REF][START_REF] Conghaile | A fully enzymatic membrane-less glucose|oxygen fuel cell provides 0.275 mA cm -2 in 5 mM glucose operates in human physiological solutions and powers transmission of sensing data[END_REF][START_REF] Gobel | Operation of a carbon nanotube-based glucose/oxygen biofuel cell in human body liquids-performance factors and characteristics[END_REF] . These works have highlighted complications that reduce EFC power output and stability due to low substrate concentrations and interferences from ascorbic acid, chloride, uric acid and biophysical matter. Reported strategies to address interferences include the use of a dialysis bag as a protective membrane, a strategy first reported by our group in 2010 for an EFC implanted in a rat [START_REF] Cinquin | Beyond the hype surrounding biofuel cells: What's the future of enzymatic fuel cells?[END_REF] . to be effective bioelectrodes for EFCs and will certainly 326 have an important role to play in future implantable or 327 wearable EFCs. To improve the limited performance of 328 biocathodes, which commonly suffer from oxygen limi-329 tations, we note the cathode design from Atanassov and 330 his colleagues that combined a gas-diffusion layer with a 331 catalytic buckypaper such that the individual benefits of 332 both materials could be exploited [61] . Some examples of 333 buckypaper interfaces and their applications are sketched 334 in Figure 2 .

335

Perspectives on nanoparticle-based rechargeable EFCs 336

EFCs are faced with two major technological bottle-337 necks that currently block their development, namely 338 their short lifetime, and to a lesser degree, their weak 339 power output. The low stability of EFCs is largely re-340 lated to the deactivation of immobilized enzymes, the 341 intrinsic catalytic component, and this seems somewhat 342 ineluctable. In particular, enzymes have a limited stabil-343 ity that depends on the environment in which they are 344 used (temperature, pH, inhibitors...). Weak power out-345 put is related to factors including the sub-optimal fixa-346 tion of enzymes and redox mediators to electrodes, which 347 reduces markedly their mobility and flexibility, and in 348 some cases, fixes enzymes in poor orientations for elec-349 tron transfer. An attractive solution lies in the devel-350 opment of bioelectrodes based on confined solutions of 351 enzymes and supramolecular assemblies of redox media-352 tors or electron transfer-promoting molecules. The elec-353 trical wiring of enzymes in solution with electrodes form-354 ing a conductive chamber only permeable to the enzyme 355 12.

  At the very beginning, EFCs were developed to generate electrical energy mainly through the oxidation of saccharides at the anode coupled with the red]uction of oxygen at the cathode[4,14] . In addition to glucose, which is by far the most exploited fuel at the anode, other fuels Q2 that have been explored include fructose, pyruvate, hydrogen, lactate, methanol and ethanol [3,4,6] . Since glucose is an essential and relatively abundant source of energy in living organisms, special attention has been given to the development of implantable EFCs for powering implanted medical devices from physiological fluids [15] . In parallel, EFCs are considered for powering portable electronic devices such as mobile phones, sensors, digital music players, laptops, and GPS systems. A fascinating flagship application, proposed by many authors for more than a decade, is the use of EFCs as a lifetime power supply for cardiac pacemakers. However, despite all of the promises, there are many hurdles and roadblocks to overcome. It is now time to recognize that the pacemaker application is unrealistic. Besides sterilization and biocompatibility problems, the major issue with implanted EFCs is their operational stability that ranges from a few hours to one year compared to 5 -8 years for sealed batteries [16] . A recent and promising generation of biofuel cells concerns H 2 /O 2 EFCs [17 • ,18 •• ] . The adsorption, entrapment and chemical immobilization of NiFe and FeFe hydrogenases, and their "electrical wiring", is at the origin of bioanodes for electrocatalytic interconversion of protons and hydrogen [19-23] . Hydrogenases entrapped in redox polymers are particularly active for bioelectrocatalytic hydrogen oxidation [24 • ,25,26] . Recent hydrogenase electrodes exhibit characteristics that are competitive to those of Pt-based electrodes such as long-term stability, mW power densities, insensitivity to fuel impurities such as CO and sulfides, and tolerance to O 2 . EFCs with hydrogenase bioanodes thus show great promise as noble metal-free hydrogen fuel cells [27-31] . Compared

  194A crucial power definition for the future, which will be-195 come more relevant as EFCs emerge for practical device 196 powering, is the volumetric power density of the single 197 or stacked EFC in cm 3 , taking into account the space be-198 tween the anode(s) and the cathode(s) and the entire cell. 199 For a single EFC, the optimisation of distance between 200 the bioelectrodes, which also plays on the resistance, sub-201 strate supply, and by-product formation, will become a 202 key consideration for the power/size ratio. 203 Influence of temperature on the operation of EFCs 204 Besides the difficulty of designing EFCs whose enzy-205 matic reactions at the anode and the cathode must op-206 erate at the same pH, another major problem for EFCs 207 concerns the influence of temperature on the rate and sta-208 bility of enzyme-catalyzed reactions, and hence on EFC 209 performance. This key parameter has often been under-210 estimated or neglected, yet can drastically change the 211 perspectives of EFCs for different applications. Depend-212 ing on the origin of the enzyme, the temperature/activity 213 profile may show optima ranging from low temperatures 214 (3 -5 °C) to high temperatures (50 -80 °C). Regarding the 215 more common glucose/O 2 EFCs, cathodes are usually 216 based on immobilized bilirubin oxidase or laccase for the 217 reduction of oxygen. Laccase from Trametes versicolor ex-218 hibits its greatest stability at 30 °C and undergoes a ther-219 mal deactivation above 50 °C, which for example, leads to 220 a loss in activity of 53% and 90% after 6 and 48 h rs , respec-221 tively [36] . In contrast, bilirubin oxidase from Myrothecium 222 verrucaria is considered relatively stable in the range of 223 40 -60 °C but loses 40% of its activity when the temper-224 ature decreases from 40 to 25 °C [37] . At the anode, the 225 oxidation of glucose is mainly catalyzed by glucose oxi-226 dase. The latter has a maximum activity at 20 -35 °C but 227 retains only 65% of its activity at 45 °C, and exhibits total 228 thermal inactivation at 60 °C [38-40] . 229 The different thermal behavior of oxidoreductase en-230 zymes is a real problem for the performance and stability 231 of EFCs, especially considering that an EFC must em-232 ploy two very different enzymes at the anode and cathode. 233 The influence of temperature may call into question the 234 use of EFCs for powering portable electronics exposed 235 to certain environmental conditions with wide variations 236 in temperature. This problem actually becomes an attrac-237 tive point for implanted biofuel cells, of which one of the 238 main features is their exposure, by definition, to a con-239

  Carbon nanotubes are a widely used material in bioelectrode design [6 • ] . In the last 5 -7 years, free-standing thin films of CNTs, called buckypapers, have emerged for use in biofuel cells [49 • ] . Buckypapers are in fact at the origin of remarkable advances in the field of EFCs [50-53] , particularly concerning implanted biofuel cells [51 •• ,54

Figure 1

 1 Figure 1

  different shapes and sizes compared to electrodes based on glassy carbon. Until now, buckypapers have mainly been modified via π -stacking interactions with pyrene derivatives, which are used for covalent or non-covalent binding of enzymes and/or mediators, and have led to high performance bioelectrodes for in-vivo biofuel cells. In particular, Katz and coworkers have highlighted the practical advantages of buckypaper for implantation compared to traditional bulk electrodes (e.g. glassy carbon), such as their ideal microscale thickness and tunable dimensions [54 •• ,55] . Owing to attractive properties such as their flexibility, Minteer and coworkers developed a miniaturized buckypaper-based lactate/O 2 EFC embedded in a contact lens for operation in human tears [56] . Besides increasing the length and alignment of CNTs, another possibility to improve the strength and flexibility of buckypapers lies with the incorporation of an organic crosslinker. An elegant approach is to "molecularly" cross-link the CNTs of buckypaper with bis-pyrene-2,2azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (bispyrene-ABTS), which enhances the mechanical stability of the buckypaper and gives it redox properties for wiring multicopper enzymes [57,58] . Furthermore, Cosnier and coworkers reported a new approach for fabricating robust and flexible buckypaper bioelectrodes based on the intimate association of CNTs and linear polynorbornene polymers [59,60] . These polymers were functionalized with pyrene groups, for cross-linking, and activated esters, for covalent attachment of enzymes and/or molecules for electrical enzyme wiring. Besides improved flexibility and handleability, enzyme-modified buckypapers are proving

Figure 2

 2 Figure 2

Figure 3 403•

 3 Figure 3

502 22 .•

 22 Liu J, Wu W-J, Fang F, Zorin NA, Chen M, Qian D-J: 503 Immobilization of hydrogenase on carbon nanotube 504 polyelectrolytes as heterogeneous catalysts for 505 electrocatalytic interconversion of protons and hydrogen . J 506 Nanoparticle Res 2016, 18 :220. 507 23. Karyakin AA, Morozov SV, Karyakina EE, Varfolomeyev SD, 508 Zorin NA, Cosnier S: Hydrogen fuel electrode based on 509 bioelectrocatalysis by the enzyme hydrogenase . Electrochem 510 Commun 2002, 4 :417-420. Morozov SV, Vignais PM, Cournac L, Zorin NA, Karyakina EE, 512 Karyakin AA, Cosnier S: Bioelectrocatalytic hydrogen 513 production by hydrogenase electrodes . Int J Hydrog Energy 514 2002, 27 :1501-1505. 515 This is the first report of a redox polypyrrole-hydrogenase electrode. 516 This paper also demonstrates the production of hydrogen by mass 517 spectrometry and electrochemistry 518 25. Morozov SV, Voronin OG, Karyakina EE, Zorin NA, Cosnier S, 519 Karyakin AA: Tolerance to oxygen of hydrogen enzyme 520 electrodes . Electrochem Commun 2006, 8 :851-854. 521 26. Karyakin AA, Morozov SV, Voronin OG, Zorin NA, Karyakina EE, 522 Fateyev VN, Cosnier S: The limiting performance characteristics 523 in bioelectrocatalysis of hydrogenase enzymes . Angew Chem 524 Int Ed 2007, 46 :7244-7246. 525 27. Pandelia M-E, Fourmond V, Tron-Infossi P, Lojou E, Bertrand P, 526 Léger C, Giudici-Orticoni M-T, Lubitz W: Membrane-bound 527 hydrogenase I from the hyperthermophilic bacterium aquifex 528 aeolicus: enzyme activation, redox intermediates and oxygen 529 tolerance . J Am Chem Soc 2010, 132 :6991-7004. 530 28. Vincent KA, Cracknell JA, Lenz O, Zebger I, Friedrich B, 531 Armstrong FA: Electrocatalytic hydrogen oxidation by an 532 enzyme at high carbon monoxide or oxygen levels . Proc Natl 533 Acad Sci 2005, 102 :16951-16954. 534 29. Xu L, Armstrong FA: Optimizing the power of enzyme-based 535 membrane-less hydrogen fuel cells for hydrogen-rich H 2 -air 536 mixtures . Energy Environ Sci 2013, 6 :2166-2171.

537 30 .

 30 Plumeré N, Rüdiger O, Oughli AA, Williams R, Vivekananthan J, 538 Pöller S, Schuhmann W, Lubitz W: A redox hydrogel protects 539 hydrogenase from high-potential deactivation and oxygen 540 damage . Nat Chem , vol 6 2014.

  •• ,6 • ] and for use in environmental remediation and sanitation systems [7 • ,8] .

  •• ] . The raw power output must surely 188 now be reported in all cases? It now seems valuable to de-

	189
	190	fine the volumetric power of bioelectrodes in mW cm -3 ,
	191	especially if 3D electrodes with thicknesses ranging from
	192	micrometres and centimetres are being used. Indeed, we
	193	have recently reported the raw power together with the
		current density of an EFC in both cm 2 and cm 3 [35 • ] .

high glucose current on a hierarchically structured porous carbon electrode with "Wired" flavin adenine dinucleotide-dependent glucose dehydrogenase

	This paper demonstrates exceptionally high catalytic currents at
	templated mesoporous carbons using the FAD-GDH enzyme, and
	brings forward the important concept of pore size engineering for
	bioelectrocatalysis
	13. Kwon CH, Lee S-H, Choi Y-B, Lee JA, Kim SH, Kim H-H,
	Spinks GM, Wallace GG, Lima MD, Kozlov ME, Baughman RH,
	Kim SJ: Nat Commun 2014, 5 :3928.
	14. Mano N: A. de Poulpiquet, O2 reduction in enzymatic biofuel
	cells . Chem Rev 2017, 5 :2392-2468.
	15. Cosnier S, Goff AL, Holzinger M: Towards

•

Tsujimura S, Murata K, Akatsuka W: Exceptionally . J Am Chem Soc 2014, 136 :14432-14437.

glucose biofuel cells implanted in human body for powering artificial organs: review

  This review summarizes the latest advances in the field of H 2 /O 2 EFCs

	481
	482	in terms of hydrogenase immobilization, hydrogenase structure,
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. Electrochem Commun 2014, 38 :19-23. 16. Reuillard B, Abreu C, Lalaoui N, Le Goff A, Holzinger M, Ondel O, Buret F, Cosnier S: One-year stability for a glucose/oxygen biofuel cell combined with pH reactivation of the laccase/carbon nanotube biocathode . Bioelectrochemistry 2015, 106 :73-76. Current Opinion in Electrochemistry 2018, 000 :xxx-xxx www.sciencedirect.com Please cite this article as: Cosnier et al. , Beyond the hype surrounding biofuel cells: What's the future of enzymatic fuel cells? Current Opinion in Electrochemistry (2018), https://doi.org/10.1016/j.coelec.2018.06.006 fuel cells: from proof-of-concept to powerful devices . Sustain 479 Energy Fuels 2017, 1 :1475-1501. 480 498 Giudici-Orticoni MT, Lojou E: Electrochemistry,

AFM, and 499 PM-IRRA spectroscopy of immobilized hydrogenase: Role of a 500 hydrophobic helix in enzyme orientation for efficient H 2 501

oxidation . Angew Chem Int Ed 2012, 51 :953-956.

JID: COELEC[mNS; July 3, 2018;1:14 ]

Please cite this article as: Cosnier et al. , Beyond the hype surrounding biofuel cells: What's the future of enzymatic fuel cells? Current Opinion in Electrochemistry (2018), https://doi.org/10.1016/j.coelec.2018.06.006

Acknowledgement

398

The authors are grateful to the Agence Nationale de la Recherche for Q3

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