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We present a transparent and flexible self-charging biosupercapacitor based on an optimised mediator- and membrane-free enzymatic glucose/oxygen biofuel cell. Indium tin oxide (ITO) nanoparticles were spray-coated on transparent conducting ITO supports resulting in a flocculent, porous and nanostructured electrode surface. By this, high capacitive currents caused by an increased electrochemical double layer as well as enhanced catalytic currents due to a higher number of immobilised enzyme molecules were obtained. After a chemical pre-treatment with a silane derivative, bilirubin oxidase from Myrothecium verrucaria was immobilized onto the ITO nanostructured electrode surface under formation of a biocathode, while bioanodes were obtained by either immobilisation of cellobiose dehydrogenase from Corynascus thermophilus or soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus. The latter showed a lower apparent KM value for glucose conversion and higher catalytic currents at µM glucose concentrations. Applying the optimised device as a biosupercapacitor in a discontinuous charge/discharge mode led to a generated power output of 0.030mW/cm2 at 50µM glucose, simulating the glucose concentration in human tears. This represents an enhancement by a factor of 350 compared to the power density obtained from the continuously operating biofuel cell with a maximum power output of 0.086µW/cm2 under the same conditions. After 17h of charging/discharging cycles a remarkable current enhancement was still measured. The entire device was transferred to flexible materials and applied for powering a flexible display showing its potential applicability as an intermittent power source in smart contact lenses.

The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens ( Hi CDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5–6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the Hi CDH film was studied. It was found that the presence of charged pyridinium groups was needed to successfully establish direct electron contact between the enzyme and electrode. SAMs formed from hydrophilic charged thiols achieved nearly two times higher current densities compared to hydrophobic charged thiols. Additionally, the results also indicated proportionality between Hi CDH catalytic constant and water content of the enzyme film. Enzyme films on charged pyridine thiols had smaller variations in water content and viscoelastic properties than films adsorbed on the more hydrophobic thiols. This work highlights several perspectives on the underlying factors affecting performance of immobilized Hi CDH.

Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence and in the presence of substrates, on graphite electrodes and thiolic self-assembled monolayer (SAM)-modified Au electrodes. Seven different thiols with different spacer lengths were used, containing -COOH, -OH, and -NH2 end groups. The length and head functionality of the thiol govern the efficiency of the DET reaction and indicate different DET properties of CtCDH and HiCDH (Less)

We report on a hybrid bioelectrochemical system that integrates an energy converting part, viz. a glucose/oxygen enzymatic fuel cell, with a charge-storing component, in which the redox features of the immobilized redox protein cytochrome c (cyt c) were utilized. Bilirubin oxidase and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) were employed as the biocatalysts for dioxygen reduction and glucose oxidation, respectively. A bi-protein PQQ-GDH/cyt c signal chain was created that facilitates electron transfer between the enzyme and the electrode surface. The assembled supercapacitor/biofuel cell hybrid biodevice displays a 15 times higher power density tested in the pulse mode compared to the performance achieved from the continuously operating regime (4.5 and 0.3 μW cm−2, respectively) with an 80% residual activity after 50 charge/discharge pulses. This can be considered as a notable step forward in the field of glucose/oxygen membrane-free, biocompatible hybrid power sources.

A combination of thylakoid membranes (TMs) as photobiocatalysts with high-surface-area electroactive materials could hold great potential for sustainable “green” solar energy conversion. We have studied the orientated immobilization of TMs on high-surface-area graphene electrodes, which were fabricated by electroreduction of graphene oxide and simultaneous electrodeposition with further aminoaryl functionalization. We have achieved the highest performance to date under direct electron transfer conditions through a biocompatible “wiring” of TMs to graphene sheets. The photobiocurrent density generated by the optimized mediator-free TM-based bioanodes yielded up to 5.24 ± 0.50 μA cm–2. The photobioelectrochemical cell integrating the photobioanode in combination with an oxygen reducing enzymatic biocathode delivered a maximum power output of 1.79 ± 0.19 μW cm–2. Our approach ensures a simplified cell design, a greater load of photosynthetic units, a minimized overpotential loss, and an enhanced overall performance., The authors thank the following agencies for financial support: the European Commission (”Bioenergy” FP7-PEOPLE-2013-ITN-607793), the Ørsted-COFUND Postdoc fellowship at DTU (Agreement No. 2014-5908), and the Swedish Research Council (project 2014-5908), and Independent Research Fund Denmark-Nature Sciences (DFF-FNU, Project No. DFF-7014-00302).

We present a fuel-independent self-charging biosupercapacitor comprising an oxygen reducing enzymatic biocathode and an opposing bioelectrode, in which the supercapacitive properties of immobilised protein were utilised. Our findings disclose a novel hybrid type of bioelectrochemical systems, which can potentially be employed as an autonomous power supplier under substrate-deficient conditions.

Gold disk electrodes modified with gold nanoparticles have been used as a scaffold for the covalent immobilization of bilirubin oxidase. The nanostructured bioelectrodes were tested as mediator-less biosensors for oxygen in a buffer that mimics the content and the composition of human physiological fluids. Chronoamperometry measurements showed a detection limit towards oxygen of 6 ± 1 μM with a linear range of 6-300 μM, i.e. exceeding usual physiological ranges of oxygen in human tissues and fluids. The biosensor presented is the first ever-reported oxygen amperometric biosensor based on direct electron transfer of bilirubin oxidase., This work was funded by the FP7 project “3D-Nanobiodevice” (NMP4-SL-2009-229255). We thank Novozymes A/S for the preparation of Myrothecium verrucaria BOx. M.P. acknowledges the 2009 Ramon y Cajal program from the Spanish MINECO.

A microscale membrane-less biofuel cell, capable of generating electrical energy from human lachrymal liquid, was developed by utilizing the ascorbate and oxygen naturally present in tears as fuel and oxidant. The biodevice is based on three-dimensional nanostructured gold electrodes covered with abiotic (conductive organic complex) and biological (redox enzyme) materials functioning as efficient anodic and cathodic catalysts, respectively. Three-dimensional nanostructured electrodes were fabricated by modifying 100 μm gold wires with 17 nm gold nanoparticles, which were further modified with tetrathiafulvalene-tetracyanoquinodimethane conducting complex to create the anode and with Myrothecium verrucaria bilirubin oxidase to create the biocathode. When operated in human tears, the biodevice exhibited the following characteristics: an open circuit voltage of 0.54 V, a maximal power density of 3.1 μW cm(-2) at 0.25 V and 0.72 μW cm(-2) at 0.4 V, with a stable current density output of over 0.55 μA cm(-2) at 0.4 V for 6 h of continuous operation. These findings support our proposition that an ascorbate/oxygen biofuel cell could be a suitable power source for glucose-sensing contact lenses to be used for continuous health monitoring by diabetes patients.

After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Two sugar oxidizing enzymes, namely, pyranose dehydrogenase from Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DH(CDH)) were tested during the pre-screening. The enzymes were mixed, "wired" and entrapped in a low-potential Os-complex-modified redox-polymer hydrogel immobilized on graphite. This anode was used in combination with a cathode based on bilirubin oxidase from Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. The optimized biofuel cell with the AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 μW cm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.

Integrating photosynthetic cell components with nanostructured materials can facilitate the conversion of solar energy into electric power for creating sustainable carbon-neutral energy sources. With the aim at exploring efficient photoinduced biocatalytic energy conversion systems, we have used an amidated carbon nanotube (aCNT) networked matrix to integrate thylakoid membranes (TMs) for construction of a direct electron transfer-driven biosolar cell. We have evaluated the resulting photobioelectrochemical cells systematically. Compared to the carboxylated CNT (cCNT)-TMs system, the aCNT-TMs system enabled a 1.5-fold enhancement in photocurrent density. This system offers more advantages including a reduced charge-transfer resistance, a lower open-circuit potential, and an improved cell stability. More remarkably, the average power density of the optimized cells was 250 times higher than that of reported analogue systems. Our results suggest the significance of physical and electronic interactions between the photosynthetic components and the support nanomaterials and may offer new clues for designing improved biosolar cells.

We detail a mediator- and membrane-free enzymatic glucose/oxygen biofuel cell based on transparent and nanostructured conducting supports. Chemically modified indium tin oxide nanoparticle modified electrodes were used to substantially increase the active surface area without significantly compromising transparency. Two different procedures for surface nanostructuring were employed, viz. spray-coating and drop-coating. The spray-coated biodevice showed superior characteristics as compared to the drop-coated enzymatic fuel cell, as a result of the higher nanostructured surface area as confirmed by electrochemical characterisation, as well as scanning electron and atomic force microscopy. Subsequent chemical modification with silanes, followed by the immobilisation of either cellobiose dehydrogenase from Corynascus thermophiles or bilirubin oxidase from Myrothecium verrucaria, were performed to obtain the bioanodes and biocathodes, respectively. The optimised biodevice exhibited an OCV of 0.67 V and power output of up to 1.4 µW/cm2 at an operating voltage of 0.35 V. This is considered a significant step forward in the field of glucose/oxygen membrane- and mediator-free, transparent enzymatic fuel cells., This work was supported in part by the European Commission through the Marie-Curie Project “Bioenergy” (PITN-GA-2013-607793), by the Swedish Research Council (621-2013-6006), and by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft (DFG).

Trabajo presentado en el 6th workshop Early Stage Researchers in Nanoscience, celebrado en Madrid el 22 y 23 de junio de 2016., Graphene-based electrodes have been used for several applications, such as biosensing or as support for further immobilization of enzymes for the preparation of biocathodes and/ or bioanodes. In order to overcome the low reproducibility of the current fabrication methods of graphene-based electrodes, in the present work the electrodeposition of graphene oxide and its simultaneous electrochemical reduction on the electrode surface is proposed as a reproducible method for the fabrication of stable and high surface area electrodes. The electrodeposition process was optimized in terms of pH and conductivity of the solution and the obtained graphene electrodes were characterized by X-ray photoelectron spectroscopy, Fourirer transform infrared spectroscopy, scanning electron microscopy and electrochemical methods (cyclic voltammetry and impendanc e spectroscopy). The characterization results indicate that electrochemical reduction of GO yielded a highly porous conductive surface that has still oxygen based functional groups. These functional groups have been used to immobilize two distint biocatalysts: laccase and bilirubin oxidase. The enzymatic electrodes were tested as direct electron transfer based biocathodes and catalytic currents as high as 1 mA/cm2 were obtained.

Threonine aldolases are versatile pyridoxal 5' phosphate (PLP) dependent enzymes key to glycine serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give ß hydroxy a amino acids they are also attractive as biotechnological catalysts for the diastereoselective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts ß hydroxy a amino acids provided in the medium to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized l threonine aldolase from Caulobacter crescentus CB15 (Cc LTA) illustrate the power of this system. The codons for four active site residues His91 Asp95 Glu96 and Asp176 were simultaneously randomized and active variants selected. The results show that only His91 which p stacks against the PLP cofactor and probably serves as the catalytic base in the carbon carbon bond cleavage step is absolutely required for aldolase activity. In contrast Asp176 one of the most conserved residues in this enzyme superfamily can be replaced conservatively by glutamate albeit with a >5000 fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential they appear to modulate substrate binding and His91 activity respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases. © 2012 Elsevier Ltd. All rights reserved.