Nicolas Plumeré

A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage

Hydrogenases are natures efficient catalysts for both the generation of energy via oxidation of molecular hydrogen and the production of hydrogen via the reduction of protons. However, their O2 sensitivity and deactivation at high potential limit their applications in practical devices, such as fuel cells. Here, we show that the integration of an O2-sensitive hydrogenase into a specifically designed viologen-based redox polymer protects the enzyme from O2 damage and high-potential deactivation. Electron transfer between the polymer-bound viologen moieties controls the potential applied to the active site of the hydrogenase and thus insulates the enzyme from excessive oxidative stress. Under catalytic turnover, electrons provided from the hydrogen oxidation reaction induce viologen-catalysed O2 reduction at the polymer surface, thus providing self-activated protection from O2. The advantages of this tandem protection are demonstrated using a single-compartment biofuel cell based on an O2-sensitive hydrogenase and H2/O2 mixed feed under anode-limiting conditions.

Combination of A Photosystem 1-Based Photocathode and a Photosystem 2-Based Photoanode to a Z-Scheme Mimic for Biophotovoltaic Applications†

In photosynthesis, conversion of solar energy into chemical energy follows a Z-scheme, which involves two sequential photoinduced charge separation steps (Figure 1 A). First, upon water splitting at photosystem 2 (PS2), the excited electrons are transferred through an electron transport chain that generates a chemiosmotic potential, which provides the energy for ATP synthesis. Then, at photosystem 1 (PS1), upon light absorption and charge separation, the electrons are transferred via ferredoxin to ferredoxin–NADP+ oxidoreductase for the production of NADPH. Figure 1 Electron-transfer pathways in the Z-scheme of natural photosynthesis (A) and in the proposed coupled PS2/PS1 system (B): All of the potentials are given versus the standard hydrogen electrode (SHE) in volts (data for the natural Z-scheme according to ... The charge separation processes in the Z-scheme inspired the design of photosynthesis-like systems based on organic and inorganic photosensitizers to convert solar energy into chemical energy.1 Exploiting the yield in light collection of photosynthetic proteins may further increase the efficiency of solar to chemical energy conversion in semi-artificial devices. Various photobioelectrochemical half-cells based either on PS12–4 or PS25–7 were suggested. However, until now autonomous solar to chemical energy conversion could not be demonstrated. The electrons provided by PS2 are insufficiently energetic, and PS1-based systems require sacrificial electron donors4 or an externally applied potential2 to sustain solar to chemical energy conversion. These limitations may be overcome by the serial coupling of both light excitation steps of PS1 and PS2 in a semi-artificial photosynthesis device (Figure 1 B). In analogy to the natural Z-scheme, PS2 would extract electrons from water, which are then transferred to PS1. The charge separation at PS1 would provide electrons which are energetic enough for H2 evolution if a suitable catalyst such as a hydrogenase was efficiently coupled to the PS1 reaction,8 as has been proposed previously5, 9, 10 to mimic the last step in the Z-scheme (that is, NADPH synthesis). To achieve maximum yields in solar energy conversion, the energy from the charge separation at PS2, that is, the first step in the Z-scheme, needs to be recovered as well. Herein, we present the first experimental set-up to serially couple PS2 and PS1. We focus on the generation of electrical energy from the difference in potential between the acceptor side of PS2 and the donor side of PS1 (Figure 1 B), that is, that part which contributes to ATP synthesis in the Z-scheme of natural photosynthesis. Our cell is designed to enable the extension of the principle to simultaneous electrical and chemical energy generation (full Z-scheme mimic). We have previously shown that electrochemical half-cells based on either PS2 or PS1 can be constructed separately: The reducing site of PS2 was contacted to an electrode via an Os-complex modified hydrogel, resulting in high photocurrent densities with unprecedented stability.6 Similarly, the oxidizing site of PS1 was contacted (again by means of an Os-complex modified redox hydrogel) with an electrode, which resulted in the generation of high cathodic photocurrents.2 In both cases the same redox polymer was used. The combination of a PS1-based photocathode and a PS2-based photoanode (Figure 2) results in a photovoltaic cell that operates as a closed system without any sacrificial electron donors or acceptors: Under illumination, water is oxidized to oxygen in the anodic compartment by PS2, and oxygen is reduced in the cathodic compartment by PS1 via methyl viologen (MV); the latter is reduced by PS1 and then regenerated by oxygen reduction, leading finally to water.12 Figure 2 Representation of the proposed biophotovoltaic cell combining a PS2-based photoanode and a PS1-based photocathode. Upon absorption of photons, water molecules are split into electrons and protons. The electrons are transferred to the cathodic half-cell ... Notably, the harvest of electrical energy from two coupled light reactions analogous to the Z-scheme in nature (Figure 1) requires different redox potentials of the respective redox hydrogels that wire PS1 and PS2 to their electrodes. The electrical power output of such a photobiovoltaic cell will be determined by 1) the difference in the formal potential of the two redox polymers and 2) the photocurrent density. The electron-transfer communication between the photoanode and PS2 was achieved with an imidazole-coordinated bispyridyl osmium complex-based redox hydrogel (polymer Os1, formal potential: 395 mV vs SHE). At an applied potential of 500 mV vs. SHE, photocurrent densities of up to 45 μA cm−2 are obtained.6 At the photocathode, PS1 was immobilized via a pyridine-coordinated bispyridyl osmium complex-based redox hydrogel13 (polymer Os2) with a more positive formal redox potential of 505 mV vs SHE (for structures of polymer Os1 and Os2, see the Supporting Information). At an applied potential of 200 mV vs. SHE, cathodic photocurrent densities up to 3 μA cm−2 are obtained (see the Supporting Information). The two-compartment cell with the PS2/Os1-based photoanode and the PS1/Os2-based photocathode allows separate electrolyte and buffer optimization for each protein complex. As the potential of the two redox polymers is almost pH-independent, the difference in pH values between the two compartments should not significantly affect the open-circuit voltage. Electrical connection and illumination of both half-cells generate a steady-state photocurrent of about 1 μA cm−2, which disappears upon switching off the light (Figure 3, left). Switching off illumination exclusively on the anode side (PS2) results in a decrease of the photocurrent, which could be restored by switching the light on again (Figure 3, right). Figure 3 Photocurrent density of the proposed biophotovoltaic cell combining the PS2/Os1-based photoanode and the PS1/Os2-based photocathode as shown in Figure 2. The light status of the respective photoelectrode is indicated by O=light on and C=light ... To further confirm the contribution of the PS2/Os1-based photoanode to the overall photocurrent, dinoterb (2,4-dinitro-6-tert-butylphenol), a herbicide that blocks the QB site of the D1 subunit of PS2, was added to the photoanode compartment to deactivate PS2 while both half-cells were continuously illuminated. As shown in the Supporting Information, Figure S3, the photocurrent density substantially decreases upon addition of dinoterb. To determine the short-circuit current density (ISC), the open-circuit voltage (VOC), and the maximal cell power output (Pcell), both photoanode and photocathode were externally connected by a variable resistor. ISC is given by the intersection point of the linear fit of the current density over cell voltage plot and the y axis, and VOC is determined by the intersection point with the x axis (Figure 4). The following values were obtained: ISC=(2.0±0.7) μA cm−2, VOC=(90±20) mV (Figure 4, left), Pcell=(23±10) nW cm−2 (Figure 4, right). The fill factor (ff) is 0.128. The conversion efficiency η for the system, that is, the ratio of power output to power input, is 3.6×10−7, with the maximal power input (349 W m−2) resulting from the LEDs used. Figure 4 Determination of the short-circuit current density (ISC), the open-circuit voltage (VOC), and the maximal cell power density output (Pcell) for the biophotovoltaic cell, combining PS2/Os1-based photoanode and PS1/Os2-based photocathode via an external ... The determined VOC correlates with the difference in the formal potentials of the two redox hydrogels Os1 and Os2, while the maximum current density of the complete photovoltaic cell is limited by the PS1/Os2-based photocathode. Thus, the photocurrent density of the PS1/Os2 half-cell in combination with the relatively low potential difference between the two redox hydrogels limits at this stage the performance of the biophotovoltaic cell. However, the main goal was to proof the feasibility of connecting a PS2-based photoanode and a PS1-based photocathode in a Z-scheme-analogue setup. In future, polymer design for a better electron transfer to PS1 may enable an increased current density by up to one or two orders of magnitude.2 Additionally, an enhancement in power density can be achieved by higher PS1 and/or PS2 loadings using for example, nanostructured electrode surfaces.14, 15 Furthermore, the potential difference between the polymer-tethered redox species may be tuned and optimally adapted to PS1 or PS2. While the potential of the redox polymer Os2 at the photocathode matches well with PS1, the redox polymer used with PS2 could be about 400 mV more negative to match the potential of the acceptor site of PS2 (Figure 1). This would enable a significant increase in cell voltage and power density. In conclusion, we have shown the serial coupling of two independent processes of light capturing by PS2 and PS1 yielding a fully closed and autonomous biophotovoltaic cell. This is fundamentally different from previously reported biophotovoltaic devices,14, 16 as it provides the basis for the future use of this “biobattery” in combination with various catalysts: The very reactive electrons may be used for chemical energy conversion instead of reducing oxygen by methyl viologen. Notably, the separation of the oxygen evolving PS2 photoanode from the PS1 photocathode opens the possibility to couple PS1 with oxygen-sensitive biocatalysts such as nitrogenases, CO2 reducing enzymes of the Calvin cycle, or hydrogenases for the production of biohydrogen.10 In future, this principle will allow the full energetic exploitation of the photosynthetic Z-scheme in a single set-up.

Enzymatic Oxygen Scavenging for Photostability without pH Drop in Single-Molecule Experiments

Over the past years, bottom-up bionanotechnology has been developed as a promising tool for future technological applications. Many of these biomolecule-based assemblies are characterized using various single-molecule techniques that require strict anaerobic conditions. The most common oxygen scavengers for single-molecule experiments are glucose oxidase and catalase (GOC) or protocatechuate dioxygenase (PCD). One of the pitfalls of these systems, however, is the production of carboxylic acids. These acids can result in a significant pH drop over the course of experiments and must thus be compensated by an increased buffer strength. Here, we present pyranose oxidase and catalase (POC) as a novel enzymatic system to perform single-molecule experiments in pH-stable conditions at arbitrary buffer strength. We show that POC keeps the pH stable over hours, while GOC and PCD cause an increasing acidity of the buffer system. We further verify in single-molecule fluorescence experiments that POC performs as good as the common oxygen-scavenging systems, but offers long-term pH stability and more freedom in buffer conditions. This enhanced stability allows the observation of bionanotechnological assemblies in aqueous environments under well-defined conditions for an extended time.

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.

Rational wiring of photosystem II to hierarchical indium tin oxide electrodes using redox polymers

Photosystem II (PSII) is a multi-subunit enzyme responsible for solar-driven water oxidation to release O2 and highly reducing electrons during photosynthesis. The study of PSII in protein film photoelectrochemistry sheds light into its biological function and provides a blueprint for artificial water-splitting systems. However, the integration of macromolecules, such as PSII, into hybrid bio-electrodes is often plagued by poor electrical wiring between the protein guest and the material host. Here, we report a new benchmark PSII–electrode system that combines the efficient wiring afforded by redox-active polymers with the high loading provided by hierarchically-structured inverse opal indium tin oxide (IO-ITO) electrodes. Compared to flat electrodes, the hierarchical IO-ITO electrodes enabled up to an approximately 50-fold increase in the immobilisation of an Os complex-modified and a phenothiazine-modified polymer. When the Os complex-modified polymer is co-adsorbed with PSII on the hierarchical electrodes, photocurrent densities of up to ∼410 μA cm−2 at 0.5 V vs. SHE were observed in the absence of diffusional mediators, demonstrating a substantially improved wiring of PSII to the IO-ITO electrode with the redox polymer. The high photocurrent density allowed for the quantification of O2 evolution, and a Faradaic efficiency of 85 ± 9% was measured. As such, we have demonstrated a high performing and fully integrated host–guest system with excellent electronic wiring and loading capacity. This assembly strategy may form the basis of all-integrated electrode designs for a wide range of biological and synthetic catalysts.

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.

Stöber silica particles as basis for redox modifications: particle shape, size, polydispersity, and porosity.

The synthesis of Stöber silica particles as basis for redox modifications is optimized for desired properties, in particular diameter in a wide sub-micrometer range, spherical shape, monodispersity, the absence of porosity, and aggregation free isolability for characterization and later covalent modification. The materials are characterized by SEM, DLS, nitrogen sorption isotherms, helium as well as Gay-Lussac (water) pycnometry, and DRIFT spectroscopy. Particles with diameters between approximately 50 and 800 nm are obtained by varying the concentrations of the reagents and reactants, the type of solvent as well as the temperature. The use of high water concentrations and post-synthetic calcination at 600 °C results in silica particles that can be considered as nonporous with respect to the size of the active molecules to be immobilized. The effect of reaction temperature on size distribution is identified. Low polydispersity is achieved by performing the reaction in a temperature range in which a change in temperature has only a weak or no effect on the final particle diameter. Upon optimization of the sol-gel process, the shape of the particles is still spherical. The agreement between experimental and geometric data is within the expected precision of the characterization techniques.

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.

Coupling of an enzymatic biofuel cell to an electrochemical cell for self-powered glucose sensing with optical readout.

A miniaturized biofuel cell (BFC) is powering an electrolyser invoking a glucose concentration dependent formation of a dye which can be determined spectrophotometrically. This strategy enables instrument free analyte detection using the analyte-dependent BFC current for triggering an optical read-out system. A screen-printed electrode (SPE) was used for the immobilization of the enzymes glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) for the biocatalytic oxidation of glucose and reduction of molecular oxygen, respectively. The miniaturized BFC was switched-on using small sample volumes (ca. 60 μL) leading to an open-circuit voltage of 567 mV and a maximal power density of (6.8±0.6) μW cm(-2). The BFC power was proportional to the glucose concentration in a range from 0.1 to 1.0 mM (R(2)=0.991). In order to verify the potential instrument-free analyte detection the BFC was directly connected to an electrochemical cell comprised of an optically-transparent SPE modified with methylene green (MG). The reduction of the electrochromic reporter compound invoked by the voltage and current flow applied by the BFC let to MG discoloration, thus allowing the detection of glucose.