Embedding biocatalysts in a redox polymer enhances the performance of dye-sensitized photocathodes in bias-free photoelectrochemical water splitting.

Dye-sensitized photoelectrodes consisting of photosensitizers and molecular catalysts with tunable structures and adjustable energy levels are attractive for low-cost and eco-friendly solar-assisted synthesis of energy rich products. Despite these advantages, dye-sensitized NiO photocathodes suffer from severe electron-hole recombination and facile molecule detachment, limiting photocurrent and stability in photoelectrochemical water-splitting devices. In this work, we develop an efficient and robust biohybrid dye-sensitized NiO photocathode, in which the intermolecular charge transfer is enhanced by a redox polymer. Owing to efficient assisted electron transfer from the dye to the catalyst, the biohybrid NiO photocathode showed a satisfactory photocurrent of 141±17 µA·cm-2 at neutral pH at 0 V versus reversible hydrogen electrode and a stable continuous output within 5 h. This photocathode is capable of driving overall water splitting in combination with a bismuth vanadate photoanode, showing distinguished solar-to-hydrogen efficiency among all reported water-splitting devices based on dye-sensitized photocathodes. These findings demonstrate the opportunity of building green biohybrid systems for artificial synthesis of solar fuels.

Bioelectrocatalytic Cofactor Regeneration Coupled to CO2 Fixation in a Redox-Active Hydrogel for Stereoselective C-C Bond Formation.

The sustainable capture and conversion of carbon dioxide (CO2 ) is key to achieving a circular carbon economy. Bioelectrocatalysis, which aims at using renewable energies to power the highly specific, direct transformation of CO2 into value added products, holds promise to achieve this goal. However, the functional integration of CO2 -fixing enzymes onto electrode materials for the electrosynthesis of stereochemically complex molecules remains to be demonstrated. Here, we show the electricity-driven regio- and stereoselective incorporation of CO2 into crotonyl-CoA by an NADPH-dependent enzymatic reductive carboxylation. Co-immobilization of a ferredoxin NADP+ reductase and crotonyl-CoA carboxylase/reductase within a 2,2'-viologen-modified hydrogel enabled iterative NADPH recycling and stereoselective formation of (2S)-ethylmalonyl-CoA, a prospective intermediate towards multi-carbon products from CO2 , with 92±6 % faradaic efficiency and at a rate of 1.6±0.4 µmol cm-2 h-1 . This approach paves the way for realizing even more complex bioelectrocatalyic cascades in the future.

Reversible H2 Oxidation and Evolution by Hydrogenase Embedded in a Redox Polymer Film.

Efficient electrocatalytic energy conversion requires the devices to function reversibly, i.e. deliver a significant current at minimal overpotential. Redox-active films can effectively embed and stabilise molecular electrocatalysts, but mediated electron transfer through the film typically makes the catalytic response irreversible. Here, we describe a redox-active film for bidirectional (oxidation or reduction) and reversible hydrogen conversion, consisting of [FeFe] hydrogenase embedded in a low-potential, 2,2'-viologen modified hydrogel. When this catalytic film served as the anode material in a H2/O2 biofuel cell, an open circuit voltage of 1.16 V was obtained - a benchmark value near the thermodynamic limit. The same film also acted as a highly energy efficient cathode material for H2 evolution. We explained the catalytic properties using a kinetic model, which shows that reversibility can be achieved despite intermolecular electron transfer being slower than catalysis. This understanding of reversibility simplifies the design principles of highly efficient and stable bioelectrocatalytic films, advancing their implementation in energy conversion.

Spectroscopic Evidence for a Covalent Sigma Au-C Bond on Au Surfaces Using 13C Isotope Labeling.

The Au-C linkage has been demonstrated as a robust interface for coupling thin organic films on Au surfaces. However, the nature of the Au-C interaction remains elusive up to now. Surface-enhanced Raman spectroscopy was previously used to assign a band at 412 cm-1 as a covalent sigma Au-C bond for films generated by spontaneous reduction of the 4-nitrobenzenediazonium salt on Au nanoparticles. However, this assignment is disputed based on our isotopic shift study. We now provide direct evidence for covalent Au-C bonds on the surface of Au nanoparticles using 13C cross-polarization/magic angle spinning solid-state NMR spectroscopy combined with isotope substitution. A 13C NMR shift at 165 ppm was identified as an aromatic carbon linked to the gold surface, while the shift at 148 ppm was attributed to C-C junctions in the arylated organic film. This demonstration of the covalent sigma Au-C bond fills the gap in metal-C bonds for organic films on surfaces, and it has great practical and theoretical significance in understanding and designing a molecular junction based on the Au-C bond.

Artificial maturation of [FeFe] hydrogenase in a redox polymer film.

We demonstrate that the insertion of the dinuclear active site of [FeFe] hydrogenase into the apo-enzyme can occur when the enzyme is embedded in a film of redox polymer, under conditions of mediated electron transfer. The maturation can be monitored by electrochemistry, and is as fast as under conditions of direct electron transfer. This new approach further clears the way to the implementation of hydrogenases in large scale real life processes.

Reversible catalysis.

We describe as 'reversible' a bidirectional catalyst that allows a reaction to proceed at a significant rate in response to even a small departure from equilibrium, resulting in fast and energy-efficient chemical transformation. Examining the relation between reaction rate and thermodynamic driving force is the basis of electrochemical investigations of redox reactions, which can be catalysed by metallic surfaces and biological or synthetic molecular catalysts. This relation has also been discussed in the context of biological energy transduction, regarding the function of biological molecular machines that harness chemical reactions to do mechanical work. This Perspective describes mean-field kinetic modelling of these three types of systems - surface catalysts, molecular catalysts of redox reactions and molecular machines - with the goal of unifying concepts in these different fields. We emphasize that reversibility should be distinguished from other figures of merit, such as rate or directionality, before its design principles can be identified and used to engineer synthetic catalysts.

The electron as a probe to measure the thickness distributions of electroactive films.

Electron conducting films are ubiquitous in applications such as energy conversion, and their ability to fulfill their catalytic function can be greatly limited by inhomogeneities in their thickness or breaks within the film. Knowing the electroactive film thickness distribution would greatly facilitate optimization efforts, but techniques to measure this are lacking. Here, we present an electroanalytical method that provides the thickness distribution of the electrochemically accessible fraction of redox-active films in which the transfer of electrons is diffusional, i.e. by electron hopping. In this method, as the time scale of the experiment (the scan rate) is changed, the location of the diffusion layer boundary relative to the film roughness features is varied, allowing for the extraction of the film thickness distribution. In addition to being conveniently carried out in the solvated state, which is often the operational state of these conductive films, this approach is highly complementary to classical microscopy methods since it samples the entire modified electrode and is specific to the electroactive portions of the film. Therefore, this approach provides information on film morphology that is truly relevant for the catalytic processes being optimized, and thus can guide the optimization of catalyst integration in films towards macroscale cohesion and thickness homogeneity which are essential for optimal performances.

Reactivation of sulfide-protected [FeFe] hydrogenase in a redox-active hydrogel.

[FeFe] hydrogenases are highly active hydrogen conversion catalysts but are notoriously sensitive to oxidative damage. Redox hydrogels have been used for protecting hydrogenases from both high potential inactivation and oxygen inactivation under turnover conditions. However, [FeFe] hydrogenase containing redox hydrogels must be fabricated under strict anoxic conditions. Sulfide coordination at the active center of the [FeFe] hydrogenase from Desulfovibrio desulfuricans protects this enzyme from oxygen in an inactive state, which can be reactivated upon reduction. Here, we show that this oxygen-stable inactive form of the hydrogenase can be reactivated in a redox hydrogel enabling practical use of this highly O2 sensitive enzyme without the need for anoxic conditions.

Suppressing hydrogen peroxide generation to achieve oxygen-insensitivity of a [NiFe] hydrogenase in redox active films.

Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.

Complete Protection of O2-Sensitive Catalysts in Thin Films.

Energy conversion schemes involving dihydrogen hold great potential for meeting sustainable energy needs, but widespread implementation cannot proceed without solutions that mitigate the cost of rare metal catalysts and the O2 instability of biological and bioinspired replacements. Recently, thick films (>100 µm) of redox polymers were shown to prevent O2 catalyst damage but also resulted in unnecessary catalyst load and mass transport limitations. Here we apply novel homogeneous thin films (down to 3 µm) that provide protection from O2 while achieving highly efficient catalyst utilization. Our empirical data are explained by modeling, demonstrating that resistance to O2 inactivation can be obtained for nonlimiting periods of time when the optimal thickness for catalyst utilization and current generation is achieved, even when using highly fragile catalysts such as the enzyme hydrogenase. We show that different protection mechanisms operate depending on the matrix dimensions and the intrinsic catalyst properties and can be integrated together synergistically to achieve stable H2 oxidation currents in the presence of O2, potentially enabling a plethora of practical applications for bioinspired catalysts under harsh oxidative conditions.

Preventing the coffee-ring effect and aggregate sedimentation by in situ gelation of monodisperse materials.

Drop-casting and inkjet printing are virtually the most versatile and cost-effective methods for depositing active materials on surfaces. However, drawbacks associated with the coffee-ring effect, as well as uncontrolled aggregation of the coating materials, have impeded the use of these methods for applications requiring high control of film properties. We now report on a simple method based on covalent cross-linking of monodisperse materials that enables the formation of thin films with homogeneous thicknesses and macroscale cohesion. The coffee-ring effect is impeded by triggering gelation of the coating materials via a thioacetate-disulfide transition which counterbalances the capillary forces induced by evaporation. Aggregates are prevented by monodisperse building blocks that ensure that the resulting gel resists sedimentation until complete droplet drying. This combined strategy yields an unprecedented level of homogeneity in the resulting film thickness in the 100 nm to 10 µm range. Moreover, macroscale cohesion is preserved as evidenced by the long-range charge transfer within the matrix. We highlight the impact of this method with bioelectrocatalysts for H2 and NADPH oxidation. Peak catalytic performances are reached at about 10-fold lower catalyst loading compared to conventional approaches owing to the high control on film cohesion and thickness homogeneity, thus setting new benchmarks in catalyst utilization.

A gas breathing hydrogen/air biofuel cell comprising a redox polymer/hydrogenase-based bioanode.

Hydrogen is one of the most promising alternatives for fossil fuels. However, the power output of hydrogen/oxygen fuel cells is often restricted by mass transport limitations of the substrate. Here, we present a dual-gas breathing H2/air biofuel cell that overcomes these limitations. The cell is equipped with a hydrogen-oxidizing redox polymer/hydrogenase gas-breathing bioanode and an oxygen-reducing bilirubin oxidase gas-breathing biocathode (operated in a direct electron transfer regime). The bioanode consists of a two layer system with a redox polymer-based adhesion layer and an active, redox polymer/hydrogenase top layer. The redox polymers protect the biocatalyst from high potentials and oxygen damage. The bioanodes show remarkable current densities of up to 8 mA cm-2. A maximum power density of 3.6 mW cm-2 at 0.7 V and an open circuit voltage of up to 1.13 V were achieved in biofuel cell tests, representing outstanding values for a device that is based on a redox polymer-based hydrogenase bioanode.

Bioelectrocatalytic and electrochemical cascade for phosphate sensing with up to 6 electrons per analyte molecule.

Despite the availability of numerous electroanalytical methods for phosphate quantification, practical implementation in point-of-use sensing remains virtually nonexistent because of interferences from sample matrices or from atmospheric O2. In this work, phosphate determination is achieved by the purine nucleoside phosphorylase (PNP) catalyzed reaction of inosine and phosphate to produce hypoxanthine which is subsequently oxidized by xanthine oxidase (XOx), first to xanthine and then to uric acid. Both PNP and XOx are integrated in a redox active Os-complex modified polymer, which not only acts as supporting matrix for the bienzymatic system but also shuttles electrons from the hypoxanthine oxidation reaction to the electrode. The bienzymatic cascade in this second generation phosphate biosensor selectively delivers four electrons for each phosphate molecule present. We introduced an additional electrochemical process involving uric acid oxidation at the underlying electrode. This further enhances the anodic current (signal amplification) by two additional electrons per analyte molecule which mitigates the influence of electrochemical interferences from the sample matrix. Moreover, while the XOx catalyzed reaction is sensitive to O2, the uric acid production and therefore the delivery of electrons through the subsequent electrochemical process are independent of the presence of O2. Consequently, the electrochemical process counterbalances the O2 interferences, especially at low phosphate concentrations. Importantly, the electrochemical uric acid oxidation specifically reports on phosphate concentration since it originates from the product of the bienzymatic reactions. These advantageous properties make this bioelectrochemical-electrochemical cascade particularly promising for point-of-use phosphate measurements.

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Light-induced formation of partially reduced oxygen species limits the lifetime of photosystem 1-based biocathodes.

Interfacing photosynthetic proteins specifically photosystem 1 (PS1) with electrodes enables light-induced charge separation processes for powering semiartificial photobiodevices with, however, limited long-term stability. Here, we present the in-depth evaluation of a PS1/Os-complex-modified redox polymer-based biocathode by means of scanning photoelectrochemical microscopy. Focalized local illumination of the bioelectrode and concomitant collection of H2O2 at the closely positioned microelectrode provide evidence for the formation of partially reduced oxygen species under light conditions. Long-term evaluation of the photocathode at different O2 concentrations as well as after incorporating catalase and superoxide dismutase reveals the particularly challenging issue of avoiding the generation of reactive species. Moreover, the evaluation of films prepared with inactivated PS1 and free chlorophyll points out additional possible pathways for the generation of oxygen radicals. To avoid degradation of PS1 during illumination and hence to enhance the long-term stability, the operation of biophotocathodes under anaerobic conditions is indispensable.

Dual properties of a hydrogen oxidation Ni-catalyst entrapped within a polymer promote self-defense against oxygen.

The Ni(P2N2)2 catalysts are among the most efficient non-noble-metal based molecular catalysts for H2 cycling. However, these catalysts are O2 sensitive and lack long term stability under operating conditions. Here, we show that in a redox silent polymer matrix the catalyst is dispersed into two functionally different reaction layers. Close to the electrode surface is the "active" layer where the catalyst oxidizes H2 and exchanges electrons with the electrode generating a current. At the outer film boundary, insulation of the catalyst from the electrode forms a "protection" layer in which H2 is used by the catalyst to convert O2 to H2O, thereby providing the "active" layer with a barrier against O2. This simple but efficient polymer-based electrode design solves one of the biggest limitations of these otherwise very efficient catalysts enhancing its stability for catalytic H2 oxidation as well as O2 tolerance.

Interrogation of a PS1-Based Photocathode by Means of Scanning Photoelectrochemical Microscopy.

In the development of photosystem-based energy conversion devices, the in-depth understanding of electron transfer processes involved in photocurrent generation and possible charge recombination is essential as a basis for the development of photo-bioelectrochemical architectures with increased efficiency. The evaluation of a bio-photocathode based on photosystem 1 (PS1) integrated within a redox hydrogel by means of scanning photoelectrochemical microscopy (SPECM) is reported. The redox polymer acts as a conducting matrix for the transfer of electrons from the electrode surface to the photo-oxidized P700 centers within PS1, while methyl viologen is used as charge carrier for the collection of electrons at the reduced FB site of PS1. The analysis of the modified surfaces by SPECM enables the evaluation of electron-transfer processes by simultaneously monitoring photocurrent generation at the bio-photoelectrode and the associated generation of reduced charge carriers. The possibility to visualize charge recombination processes is illustrated by using two different electrode materials, namely Au and p-doped Si, exhibiting substantially different electron transfer kinetics for the reoxidation of the methyl viologen radical cation used as freely diffusing charge carrier. In the case of p-doped Si, a slower recombination kinetics allows visualization of methyl viologen radical cation concentration profiles from SPECM approach curves.