Dimethylene-Cyclopropanide Units as Building Blocks for Fluorescence Dyes.

Many organic dyes are fluorescent in solution. In the solid state, however, quenching processes often dominate, hampering material science applications such as light filters, light-emitting devices, or coding tags. We show that the dimethylene-cyclopropanide scaffold can be used to form two structurally different types of chromophores, which feature fluorescence quantum yields up to 0.65 in dimethyl sulfoxide and 0.53 in solids. The increased fluorescence in the solid state for compounds bearing malonate substituents instead of dicyanomethid ones is rationalized by the induced twist between the planes of the cyclopropanide core and a pyridine ligand.

A Potent Auto-Umpolung Ligand for Conjugative Radical Stabilization.

Carbenes with conjugatively connected redox system act as "auto-umpolung" ligands. Due to their electronic flexibility, they should also be particularly suitable to stabilize open-shell species. Herein, the first neutral radical of such sort is described in form of a dialkylamino-substituted bis(dicyanomethylene)cyclopropanide. Despite the absence of steric shielding, the radical is stable for an extended amount of time and was consequently characterized in solution via EPR measurements. These data and accompanying X-ray structural analyses indicate that the radical species is in equilibrium with aggregates (formed via π-stacking) and dimers (obtained via σ-bond formation between methylene carbons).

Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis.

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2'-bipyridine)2Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by ß-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

A Tandem Solar Biofuel Cell: Harnessing Energy from Light and Biofuels.

We report on a photobioelectrochemical fuel cell consisting of a glucose-oxidase-modified BiFeO3 photobiocathode and a quantum-dot-sensitized inverse opal TiO2 photobioanode linked to FAD glucose dehydrogenase via a redox polymer. Both photobioelectrodes are driven by enzymatic glucose conversion. Whereas the photobioanode can collect electrons from sugar oxidation at rather low potential, the photobiocathode shows reduction currents at rather high potential. The electrodes can be arranged in a sandwich-like manner due to the semi-transparent nature of BiFeO3 , which also guarantees a simultaneous excitation of the photobioanode when illuminated via the cathode side. This tandem cell can generate electricity under illumination and in the presence of glucose and provides an exceptionally high OCV of about 1 V. The developed semi-artificial system has significant implications for the integration of biocatalysts in photoactive entities for bioenergetic purposes, and it opens up a new path toward generation of electricity from sunlight and (bio)fuels.

Insight into Electron Transfer from a Redox Polymer to a Photoactive Protein.

Biohybrid photoelectrochemical systems in photovoltaic or biosensor applications have gained considerable attention in recent years. While the photoactive proteins engaged in such systems usually maintain an internal charge separation quantum yield of nearly 100%, the subsequent steps of electron and hole transfer beyond the protein often limit the overall system efficiency and their kinetics remain largely uncharacterized. To reveal the dynamics of one of such charge-transfer reactions, we report on the reduction of Rhodobacter sphaeroides reaction centers (RCs) by Os-complex-modified redox polymers (P-Os) characterized using transient absorption spectroscopy. RCs and P-Os were mixed in buffered solution in different molar ratios in the presence of a water-soluble quinone as an electron acceptor. Electron transfer from P-Os to the photoexcited RCs could be described by a three-exponential function, the fastest lifetime of which was on the order of a few microseconds, which is a few orders of magnitude faster than the internal charge recombination of RCs with fully separated charge. This was similar to the lifetime for the reduction of RCs by their natural electron donor, cytochrome c2. The rate of electron donation increased with increasing ratio of polymer to protein concentrations. It is proposed that P-Os and RCs engage in electrostatic interactions to form complexes, the sizes of which depend on the polymer-to-protein ratio. Our findings throw light on the processes within hydrogel-based biophotovoltaic devices and will inform the future design of materials optimally suited for this application.

Scalable Fabrication of Biophotoelectrodes by Means of Automated Airbrush Spray-Coating.

The fabrication and electrochemical evaluation of transparent photoelectrodes consisting of Photosystem I (PSI) or Photosystem II (PSII) is described, which are embedded and electrically wired by a redox polymer. The fabrication process is performed by an automated airbrush-type spray coating system, which ensures controlled and scalable electrode preparation. As proof of concept, electrodes with a surface area of up to 25 cm2 were prepared. The macro-porous structure of the indium tin oxide electrodes allows a high loading of the photoactive protein complexes leading to enhanced photocurrents, which are essential for potentially technologically relevant solar-powered devices. In addition, we show that unpurified crude PSII extracts, which can be provided in comparatively high yields for electrode modification, are suitable for photoelectrode fabrication with comparable photocurrent densities.

Reassessing the rationale behind herbicide biosensors: The case of a photosystem II/redox polymer-based bioelectrode.

Interfacing photosynthetic protein complexes with electrodes is frequently used for the identification of electron transfer mechanisms and the fabrication of biosensors. Binding of herbicide compounds to the terminal plastoquinone QB at photosystem II (PSII) causes disruption of electron flow that is associated with a diminished performance of the associated biodevice. Thus, the principle of electron transport inhibition at PSII can be used for herbicide detection and has inspired the fabrication of several biosensors for this purpose. However, the biosensor performance may reveal a more complex behavior than generally expected. As we present here for a photobioelectrode constituted by PSII embedded in a redox polymer matrix, the effect caused by inhibitors does not only impact the electron transfer from PSII but also the properties of the polymer film used for immobilization and electrical wiring of the protein complexes. Incorporation of phenolic inhibitors into the polymer film surprisingly translates into enhanced photocurrents and, in particular cases, in a higher stability of the overall electrode architecture. The achieved results stress the importance to evaluate first the possible influence of analytes of interest on the biosensor architecture as a whole and provide important insights for consideration in future design of bioelectrochemical devices.

Electroenzymatic Nitrogen Fixation Using a MoFe Protein System Immobilized in an Organic Redox Polymer.

We report an organic redox-polymer-based electroenzymatic nitrogen fixation system using a metal-free redox polymer, namely neutral-red-modified poly(glycidyl methacrylate-co-methylmethacrylate-co-poly(ethyleneglycol)methacrylate) with a low redox potential of -0.58 V vs. SCE. The stable and efficient electric wiring of nitrogenase within the redox polymer matrix enables mediated bioelectrocatalysis of N3- , NO2- and N2 to NH3 catalyzed by the MoFe protein via the polymer-bound redox moieties distributed in the polymer matrix in the absence of the Fe protein. Bulk bioelectrosynthetic experiments produced 209±30 nmol NH3 nmol MoFe-1 h-1 from N2 reduction. 15 N2 labeling experiments and NMR analysis were performed to confirm biosynthetic N2 reduction to NH3 .

Redox-Polymer-Based High-Current-Density Gas-Diffusion H2 -Oxidation Bioanode Using [FeFe] Hydrogenase from Desulfovibrio desulfuricans in a Membrane-free Biofuel Cell.

The incorporation of highly active but also highly sensitive catalysts (e.g. the [FeFe] hydrogenase from Desulfovibrio desulfuricans) in biofuel cells is still one of the major challenges in sustainable energy conversion. We report the fabrication of a dual-gas diffusion electrode H2 /O2 biofuel cell equipped with a [FeFe] hydrogenase/redox polymer-based high-current-density H2 -oxidation bioanode. The bioanodes show benchmark current densities of around 14 mA cm-2 and the corresponding fuel cell tests exhibit a benchmark for a hydrogenase/redox polymer-based biofuel cell with outstanding power densities of 5.4 mW cm-2 at 0.7 V cell voltage. Furthermore, the highly sensitive [FeFe] hydrogenase is protected against oxygen damage by the redox polymer and can function under 5 % O2 .

Redox-Polymer-Wired [NiFeSe] Hydrogenase Variants with Enhanced O2 Stability for Triple-Protected High-Current-Density H2 -Oxidation Bioanodes.

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2 -oxidation catalysts in H2 /O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low-potential viologen-modified redox polymers and evaluated with respect to H2 -oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) µA cm-2 for G491A and (476±172) µA cm-2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high-potential inactivation, establishing a triple protection for the bioanode. The use of gas-diffusion bioanodes provided current densities for H2 -oxidation of up to 6.3 mA cm-2 . Combination of the gas-diffusion bioanode with a bilirubin oxidase-based gas-diffusion O2 -reducing biocathode in a membrane-free biofuel cell under anode-limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm-2 at 0.7 V and an open-circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F.

Drug Release from Polymer Thin Films and Gel Pellets: Insights from Programmed Microplate Electroanalysis.

Robotic electroanalysis in 24-well microplates was used to determine Paracetamol (PCT) release from thin films of chitosan and two pH-sensitive synthetic polymers as well as blends of the polymers with each other and with agarose. Square-wave voltammograms were recorded automatically in a potential window of 0.35 V-0.85 V vs. Ag/AgCl/0.1 M KCl and their evaluation revealed time-dependent PCT release into acidic and basic media. Comparison of the release profiles showed that pure chitosan layers released PCT quickly in a single-phase process while liberation from synthetic polymer thin films was slower with a sigmoidal shape at pH 1.2 and pH 8.0 with a maximum release of PCT after approximately 150 and 140 min, respectively. The release profile from thicker agarose films was between those of the thin films. Agarose blended with chitosan or synthetic polymers formed films with biphasic release behavior. Chitosan linearized the initial section of the release profile in chitosan/polymer blends. The automated procedure for release testing offers the advantage of low-cost, labor-effective and error-free data acquisition. The procedure has been validated as a useful microplate assay option for release profile testing.

Light-controlled imaging of biocatalytic reactions via scanning photoelectrochemical microscopy for multiplexed sensing.

A light-controlled multiplexing platform has been developed on the basis of a quantum dot-sensitized inverse opal TiO2 electrode with integrated biocatalytic reactions. Spatially resolved illumination enables multiplexed sensing and imaging of enzymatic oxidation reactions at relatively negative applied potentials.

Polymer/enzyme-modified HF-etched carbon nanoelectrodes for single-cell analysis.

Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)2Cl]+ (bpy = 2,2'-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.

Glutamate detection at the cellular level by means of polymer/enzyme multilayer modified carbon nanoelectrodes.

Carbon nanoelectrodes in the sub-micron range were modified with an enzyme cascade immobilized in a spatially separated polymer double layer system for the detection of glutamate at the cellular level. The enzyme cascade consists of glutamate oxidase (GlutOx) that was immobilized in a hydrophilic redox silent polymer on top of a horseradish peroxidase (HRP)/redox polymer layer. In the presence of O2, glutamate was oxidized under concomitant reduction of O2 to H2O2 at GlutOx. H2O2 is further reduced to water by means of HRP and electrons are shuttled via the redox polymer matrix that wires the HRP to the electrode surface, hence delivering a current response proportional to the glutamate concentration. The nanometer-sized sensors could be successfully used to measure glutamate release from primary mouse astrocytes in 10 mM HEPES buffer.

High spatial resolution electrochemical biosensing using reflected light microscopy.

If the analyte does not only change the electrochemical but also the optical properties of the electrode/solution interface, the spatial resolution of an electrochemical sensor can be substantially enhanced by combining the electrochemical sensor with optical microscopy. In order to demonstrate this, electrochemical biosensors for the detection of hydrogen peroxide and glucose were developed by drop casting enzyme and redox polymer mixtures onto planar, optically transparent electrodes. These biosensors generate current signals proportional to the analyte concentration via a reaction sequence which ultimately changes the oxidation state of the redox polymer. Images of the interface of these biosensors were acquired using bright field reflected light microscopy (BFRLM). Analysis showed that the intensity of these images is higher when the redox polymer is oxidized than when it is reduced. It also revealed that the time needed for the redox polymer to change oxidation state can be assayed optically and is dependent on the concentration of the analyte. By combining the biosensor for hydrogen peroxide detection with BFRLM, it was possible to determine hydrogen peroxide in concentrations as low as 12.5 µM with a spatial resolution of 12 µm × 12 µm, without the need for the fabrication of microelectrodes of these dimensions.

Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase.

We report an amperometric biosensor for the urinary disease biomarker para-hydroxyphenylacetate ( p-HPA) in which the allosteric reductase component of a bacterial hydroxylase, C1-hpah, is electrically wired to glassy carbon electrodes through incorporation into a low-potential Os-complex modified redox polymer. The proposed biosensing strategy depends on allosteric modulation of C1-hpah by the binding of the enzyme activator and analyte p-HPA, stimulating oxidation of the cofactor NADH. The pronounced concentration-dependence of allosteric C1-hpah modulation in the presence of a constant concentration of NADH allowed sensitive quantification of the target, p-HPA. The specific design of the immobilizing redox polymer with suitably low working potential allowed biosensor operation without the risk of co-oxidation of potentially interfering substances, such as uric acid or ascorbic acid. Optimized sensors were successfully applied for p-HPA determination in artificial urine, with good recovery rates and reproducibility and sub-micromolar detection limits. The proposed application of the allosteric enzyme C1-hpah for p-HPA trace electroanalysis is the first successful example of simple amperometric redox enzyme/redox polymer biosensing in which the analyte acts as an effector, modulating the activity of an immobilized biocatalyst. A general advantage of the concept of allosterically modulated biosensing is its ability to broaden the range of approachable analytes, through the move from substrate to effector detection.

Extended Operational Lifetime of a Photosystem-Based Bioelectrode.

The development of bioelectrochemical assemblies for sustainable energy transformation constitutes an increasingly important field of research. Significant progress has been made in the development of semiartificial devices for conversion of light into electrical energy by integration of photosynthetic biomolecules on electrodes. However, sufficient long-term stability of such biophotoelectrodes has been compromised by reactive species generated under aerobic operation. Therefore, meeting the requirements of practical applications still remains unsolved. We present the operation of a photosystem I-based photocathode using an electron acceptor that enables photocurrent generation under anaerobic conditions as the basis for a biodevice with substantially improved stability. A continuous operation lifetime considerably superior to previous reports and at higher light intensities is paving the way toward the potential application of semiartificial energy conversion devices.

A Z-Scheme-Inspired Photobioelectrochemical H2 O/O2 Cell with a 1†V Open-Circuit Voltage Combining Photosystem†II and PbS Quantum Dots.

A biohybrid photobioanode mimicking the Z-scheme has been developed by functional integration of photosystem II (PSII) and PbS quantum dots (QDs) within an inverse opal TiO2 architecture giving rise to a rather negative water oxidation potential of about -0.55 V vs. Ag/AgCl, 1 m KCl at neutral pH. The electrical linkage between both light-sensitive entities has been established through an Os-complex-modified redox polymer (POs ), which allows the formation of a multi-step electron-transfer chain under illumination starting with the photo-activated water oxidation at PSII followed by an electron transfer from PSII through POs to the photo-excited QDs and finally to the TiO2 electrode. The photobioanode was coupled to a novel, transparent, inverse-opal ATO cathode modified with an O2 -reducing bilirubin oxidase for the construction of a H2 O/O2 photobioelectrochemical cell reaching a high open-circuit voltage of about 1 V under illumination.

Photoreduction of CO2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-artificial Z-Scheme Architecture.

Solar-driven coupling of water oxidation with CO2 reduction sustains life on our planet and is of high priority in contemporary energy research. Here, we report a photoelectrochemical tandem device that performs photocatalytic reduction of CO2 to formate. We employ a semi-artificial design, which wires a W-dependent formate dehydrogenase (FDH) cathode to a photoanode containing the photosynthetic water oxidation enzyme, Photosystem II, via a synthetic dye with complementary light absorption. From a biological perspective, the system achieves a metabolically inaccessible pathway of light-driven CO2 fixation to formate. From a synthetic point of view, it represents a proof-of-principle system utilizing precious-metal-free catalysts for selective CO2-to-formate conversion using water as an electron donor. This hybrid platform demonstrates the translatability and versatility of coupling abiotic and biotic components to create challenging models for solar fuel and chemical synthesis.