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.

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.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

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.

Mechanism of protection of catalysts supported in redox hydrogel films.

The use of synthetic inorganic complexes as supported catalysts is a key route in energy production and in industrial synthesis. However, their intrinsic oxygen sensitivity is sometimes an issue. Some of us have recently demonstrated that hydrogenases, the fragile but very efficient biological catalysts of H2 oxidation, can be protected from O2 damage upon integration into a film of a specifically designed redox polymer. Catalytic oxidation of H2 produces electrons which reduce oxygen near the film/solution interface, thus providing a self-activated protection from oxygen [Plumeré et al., Nat Chem. 2014, 6, 822-827]. Here, we rationalize this protection mechanism by examining the time-dependent distribution of species in the hydrogenase/polymer film, using measured or estimated values of all relevant parameters and the numerical and analytical solutions of a realistic reaction-diffusion scheme. Our investigation sets the stage for optimizing the design of hydrogenase-polymer films, and for expanding this strategy to other fragile catalysts.

A redox hydrogel protects the O2 -sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage.

The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed.

Engineered electron-transfer chain in photosystem 1 based photocathodes outperforms electron-transfer rates in natural photosynthesis.

Photosystem 1 (PS1) triggers the most energetic light-induced charge-separation step in nature and the in vivo electron-transfer rates approach 50 e(-) s(-1) PS1(-1). Photoelectrochemical devices based on this building block have to date underperformed with respect to their semiconductor counterparts or to natural photosynthesis in terms of electron-transfer rates. We present a rational design of a redox hydrogel film to contact PS1 to an electrode for photocurrent generation. We exploit the pH-dependent properties of a poly(vinyl)imidazole Os(bispyridine)2Cl polymer to tune the redox hydrogel film for maximum electron-transfer rates under optimal conditions for PS1 activity. The PS1-containing redox hydrogel film displays electron-transfer rates of up to 335±14 e(-) s(-1) PS1(-1), which considerably exceeds the rates observed in natural photosynthesis or in other semiartificial systems. Under O2 supersaturation, photocurrents of 322±19 µA cm(-2) were achieved. The photocurrents are only limited by mass transport of the terminal electron acceptor (O2). This implies that even higher electron-transfer rates may be achieved with PS1-based systems in general.

Scanning droplet cell for chemoselective patterning through local electroactivation of protected quinone monolayers.

A reagentless strategy for template-free patterning of uniformly inert surfaces is suggested. A layer of p-hydroquinone (HQ) protected by the tert-butyldimethylsilyl (TBDMS) group is electrografted onto glassy carbon electrodes. Chemoselective activation is performed through electrochemically controlled cleavage of the TBDMS group, which yields the redox-active surface-confined quinone moieties. The latter are shown to undergo electrochemically induced Michael addition, which serves for subsequent functionalization of the electrode surface. Patterning of the TBDMS-quinone-modified surface is accomplished by using selective localized cleavage of the protecting group. State-of-the-art direct-mode scanning electrochemical microscopy (SECM) patterning fails to yield the anticipated interfacial reaction; however, the electrochemical scanning droplet cell (SDC) is capable of conducting the localized chemoselective reaction. In a small area, dictated by the dimensions of the droplet, electrochemically induced cleavage of the protecting group can be performed locally to give rise to arrays of active quinone spots. Upon deprotection, the redox signals, attributed to the hydroquinone/benzoquinone couple, provide the first direct evidence for chemoselective electrochemical patterning of sensitive functionalities. Subsequent SECM studies of the resulting modified areas demonstrate spatial control of the proposed patterning technique.

Redox hydrogels with adjusted redox potential for improved efficiency in Z-scheme inspired biophotovoltaic cells.

The improvement of Z-scheme inspired biophotovoltaics is achieved by fine tuning the properties of redox hydrogels applied as immobilization and electron conducting matrices for the photosystem-protein complexes. The formal potentials of the redox hydrogels are adjusted to the respective redox sites in the photosystems for optimized electron transfer without substantial voltage loss. The anode is based on photosystem 2 (PS2) integrated in a phenothiazine modified redox hydrogel with a formal potential of -1 mV vs. SHE, which is 59 mV more positive than the QB acceptor site in PS2. The cathode is based on photosystem 1 (PS1) contacted via an Os-complex based redox hydrogel with a formal potential of 395 mV vs. SHE, i.e. 28 mV more negative than the primary P700 electron acceptor of PS1. The potential difference between the two redox hydrogels is 396 mV. An open circuit voltage (VOC) of 372.5 ± 2.1 mV could be achieved for the biophotovoltaic cell. The maximum power output is 1.91 ± 0.56 µW cm(-2) and the conversion efficiency (η) is 4.5 × 10(-5), representing a 125-fold improvement in comparison to the previously proposed device exploiting the photosynthetic Z-scheme for electrical energy production.

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.

Interferences from oxygen reduction reactions in bioelectroanalytical measurements: the case study of nitrate and nitrite biosensors.

Bioelectroanalytical procedures based on cathodic processes are often subject to interference from dissolved oxygen. At the potentials applied for analyte detection, oxygen reduction may occur directly at the electrode or may be catalyzed by the electron mediators or the sensing enzyme of the biosensor. These processes affect the background current and may thus result in erroneous analyte quantification. In this review, current strategies to circumvent these oxygen interferences are presented and critically assessed with respect to their compatibility for on-site monitoring with amperometric biosensing devices operating at low potential. The main strategies consist in (1) use of oxygen scavenging systems to remove dissolved oxygen from the sample, (2) design of bioelectroanalytical approaches to shift the applied potential for analyte detection to more positive values, and (3) development of electrode materials to increase the overpotential for the oxygen reduction reaction. The latest developments in these approaches have recently led to the first biosensing devices based on reductases fully compatible with on-site monitoring requirements and this opens up possibilities for their widespread application.

Enzyme-catalyzed O2 removal system for electrochemical analysis under ambient air: application in an amperometric nitrate biosensor.

Electroanalytical procedures are often subjected to oxygen interferences. However, achieving anaerobic conditions in field analytical chemistry is difficult. In this work, novel enzymatic systems were designed to maintain oxygen-free solutions in open, small volume electrochemical cells and implemented under field conditions. The oxygen removal system consists of an oxidase enzyme, an oxidase-specific substrate, and catalase for dismutation of hydrogen peroxide generated in the enzyme catalyzed oxygen removal reaction. Using cyclic voltammetry, three oxidase enzyme/substrate combinations with catalase were analyzed: glucose oxidase with glucose, galactose oxidase with galactose, and pyranose 2-oxidase with glucose. Each system completely removed oxygen for 1 h or more in unstirred open vessels. Reagents, catalysts, reaction intermediates, and products involved in the oxygen reduction reaction were not detected electrochemically. To evaluate the oxygen removal systems in a field sensing device, a model nitrate biosensor based on recombinant eukaryotic nitrate reductase was implemented in commercial screen-printed electrochemical cells with 200 µL volumes. The products of the aldohexose oxidation catalyzed by glucose oxidase and galactose oxidase deactivate nitrate reductase and must be quenched for biosensor applications. For general application, the optimum catalyst is pyranose 2-oxidase since the oxidation product does not interfere with the biorecognition element.

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.

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 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.