Enhancement of Biosensors by Implementing Photoelectrochemical Processes.

Research on biosensors is growing in relevance, taking benefit from groundbreaking knowledge that allows for new biosensing strategies. Electrochemical biosensors can benefit from research on semiconducting materials for energy applications. This research seeks the optimization of the semiconductor-electrode interfaces including light-harvesting materials, among other improvements. Once that knowledge is acquired, it can be implemented with biological recognition elements, which are able to transfer a chemical signal to the photoelectrochemical system, yielding photo-biosensors. This has been a matter of research as it allows both a superior suppression of background electrochemical signals and the switching ON and OFF upon illumination. Effective electrode-semiconductor interfaces and their coupling with biorecognition units are reviewed in this work.

Metallacarboranes on the Road to Anticancer Therapies: Cellular Uptake, DNA Interaction, and Biological Evaluation of Cobaltabisdicarbollide [COSAN].

After uptake by U87 MG and A375 cancer cells, cobaltabisdicarbollide [COSAN]- distributes between membrane and nucleus and presents no relevant cytotoxicity against both cell lines even for long incubation times. The cytotoxicity of Na[COSAN] was also tested towards one normal cell line, the V79 fibroblasts, in order to ascertain the noncytotoxic profile of the compound. As the cell's nucleus contains DNA, the interaction between [COSAN]- and double-stranded calf thymus DNA (CT-dsDNA) has been investigated. There is a strong interaction between both molecules forming a nanohybrid CT-dsDNA-[COSAN] biomaterial, which was fully characterized. Moreover, Na[COSAN] shows characteristic redox peaks ascribed to the oxidation/reduction of Co3+/2+ at a formal potential of -1.444 V and it can be accumulated at a surface-immobilized DNA layer of glassy carbon electrodes. The equilibrium surface-binding constants (Kox /Kred ), which confirm that [COSAN]- interacts with DNA by an intercalative or electrostatic mode, depending on the ionic strength of the solution, were estimated. In addition, high binding affinity of Na[COSAN] to proteins was observed by 11 B{1 H} NMR and confirmed in vivo. Finally, biodistribution studies of [COSAN]- in normal mice were run. After administration, Na[COSAN] was distributed into many organs but mainly accumulated in the reticuloendothelial system (RES), including liver and spleen. After 1 h, the formation of aggregates by plasma protein interaction plays a role in the biodistribution profile; the aggregates accumulate mostly in the lungs. Na[COSAN], which displays low toxicity and high uptake by relevant cancer cells accumulating boron within the nucleus, could act as a suitable compound for further developments as boron neutron capture therapy (BNCT) agents.

Enzymatic Electrosynthesis of Alkanes by Bioelectrocatalytic Decarbonylation of Fatty Aldehydes.

An enzymatic electrosynthesis system was created by combining an aldehyde deformylating oxygenase (ADO) from cyanobacteria that catalyzes the decarbonylation of fatty aldehydes to alkanes and formic acid with an electrochemical interface. This system is able to produce a range of alkanes (octane to propane) from aldehydes and alcohols. The combination of this bioelectrochemical system with a hydrogenase bioanode yields a H2 /heptanal enzymatic fuel cell (EFC) able to simultaneously generate electrical energy with a maximum current density of 25 µA cm-2 at 0.6 V and produce hexane with a faradaic efficiency of 24 %.

Halides inhibition of multicopper oxidases studied by FTIR spectroelectrochemistry using azide as an active infrared probe.

An infrared spectroelectrochemical study of Trametes hirsuta laccase and Magnaporthe oryzae bilirubin oxidase has been performed using azide, an inhibitor of multicopper oxidases, as an active infrared probe incorporated into the T2/T3 copper cluster of the enzymes. The redox potential-controlled measurements indicate that N3- stretching IR bands of azide ion bound to the T2/T3 cluster are only detected for the oxidized enzymes, confirming that azide only binds to Cu2+. Moreover, the process of binding/dissociation of azide ion is shown to be reversible. The interaction of halide anions, which also inhibit multicopper oxidases, with the active site of the enzymes was studied by measuring the changes in the azide FTIR bands. Enzymes inhibited by azide respond differently upon addition of fluoride or chloride ions to the sample solution inhibited by azide. Fluoride ions compete with azide for binding at one of the T2/T3 Cu ions, whereas competition from chloride ions is much less evident.

Bioelectrochemical Haber-Bosch Process: An Ammonia-Producing H2 /N2 Fuel Cell.

Nitrogenases are the only enzymes known to reduce molecular nitrogen (N2 ) to ammonia (NH3 ). By using methyl viologen (N,N'-dimethyl-4,4'-bipyridinium) to shuttle electrons to nitrogenase, N2 reduction to NH3 can be mediated at an electrode surface. The coupling of this nitrogenase cathode with a bioanode that utilizes the enzyme hydrogenase to oxidize molecular hydrogen (H2 ) results in an enzymatic fuel cell (EFC) that is able to produce NH3 from H2 and N2 while simultaneously producing an electrical current. To demonstrate this, a charge of 60 mC was passed across H2 /N2 EFCs, which resulted in the formation of 286 nmol NH3  mg-1 MoFe protein, corresponding to a Faradaic efficiency of 26.4 %.

H2 -Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration.

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.

Bioelectrochemical oxidation of water.

The electrolysis of water provides a link between electrical energy and hydrogen, a high energy density fuel and a versatile energy carrier, but the process is very expensive. Indeed, the main challenge is to reduce energy consumption for large-scale applications using efficient renewable catalysts that can be produced at low cost. Here we present for the first time that laccase can catalyze electrooxidation of H2O to molecular oxygen. Native and laboratory-evolved laccases immobilized onto electrodes serve as bioelectrocatalytic systems with low overpotential and a high O2 evolution ratio against H2O2 production during H2O electrolysis. Our results open new research ground on H2O splitting, as they overcome serious practical limitations associated with artificial electrocatalysts currently used for O2 evolution.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.

Effect of Protection Polymer Coatings on the Performance of an Amperometric Galactose Biosensor in Human Plasma.

Galactose monitoring in individuals allows the prevention of harsh health conditions related to hereditary metabolic diseases like galactosemia. Current methods of galactose detection need development to obtain cheaper, more reliable, and more specific sensors. Enzyme-containing amperometric sensors based on galactose oxidase activity are a promising approach, which can be enhanced by means of their inclusion in a redox polymer coating. This strategy simultaneously allows the immobilization of the biocatalyst to the electroactive surface and hosts the electron shuttling units. An additional deposition of capping polymers prevents external interferences like ascorbic or uric acid as well as biofouling when measuring in physiological fuels. This work studies the protection effect of poly(2-methacryloyloxyethyl phosphorylcholine-co-glycidyl methacrylate (MPC) and polyvinylimidazole-polysulfostyrene (P(VI-SS)) when incorporated in the biosensor design for the detection of galactose in human plasma.

Single-Electron Charging of Thioctic Acid Monolayer-Protected Gold Clusters.

There is great interest in the use of Monolayer-Protected Gold Clusters (AuMPCs) as nanoscale capacitors in aqueous media for nanobiotechnological applications, such as bioelectrocatalysts, biofuel cells, and biosensors. However, AuMPCs exhibiting subattofarad double-layer capacitance at room temperature, and the resolution of single-electron charging, has been mainly obtained in an organic medium with nonfunctional capping ligands. We report here the synthesis of Thioctic Acid Monolayer-Protected Au Clusters (TA-AuMPCs) showing electrochemical single electron quantized capacitance charging in organic and aqueous solutions and when immobilized onto different self-assembled monolayer-modified gold electrodes. The presence of functional carboxylic groups opens a simple strategy for interfacing a nanoparticle assembly to biomolecules for their use as electron donors or acceptors in biological electron transfer reactions.

Implantable bioelectrochemistry: 70 years across "Cyborg" organisms and logically operated bioelectronics.

Professor Evgeny Katz (Department of Chemistry and Biomolecular Science, Clarkson University, USA) was born on 11th August 1952, and he turned 70 years old last summer. This special collection entitled Implanted Enzymatic Fuel Cells and Biosensors: Fundamentals to Applications is dedicated to Evgeny on this landmark occasion. This brief preface gives some personal insights into Evgeny's career beyond the scientific perspective.

Photobio-electrocatalytic production of H2 using fluorine-doped tin oxide (FTO) electrodes covered with a NiO-In2S3 p-n junction and NiFeSe hydrogenase.

Clean energy vectors are needed towards a fossil fuel-free society, diminishing both greenhouse effect and pollution. Electrochemical water splitting is a clean route to obtain green hydrogen, the cleanest fuel; although efficient electrocatalysts are required to avoid high overpotentials in this process. The combination of inorganic semiconductors with biocatalysts for photoelectrochemical H2 production is an alternative approach to overcome this challenge. N-type semiconductors can be coupled to a co-catalyst for H2 production in the presence of a sacrificial electron donor in solution, but the replacement of the latter with an electrode is a challenge. In this work we attach a NiFeSe-hydrogenase with high activity for H2 production with the n-type semiconductor indium sulfide, which upon visible irradiation is able to transfer its excited electrons to the enzyme. In order to enhance the transfer of the generated holes towards the electrode for their replenishment, we have explored the inclusion of a p-type material, NiO, to induce a p-n junction for H2 production in a photoelectrochemical biocatalytic system in absence of sacrificial reagents.

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor.

Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.

Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions.

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F1Fo-ATP synthase from Escherichia coli. Thus, H2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+.

Additively Manufactured 3D Micro-bioelectrodes for Enhanced Bioelectrocatalytic Operation.

The drive toward miniaturization of enzyme-based bioelectronics established a need for three-dimensional (3D) microstructured electrodes, which are difficult to implement using conventional manufacturing processes. Additive manufacturing coupled with electroless metal plating enables the production of 3D conductive microarchitectures with high surface area for potential applications in such devices. However, interfacial delamination between the metal layer and the polymer structure is a major reliability concern, which leads to device performance degradation and eventually device failure. This work demonstrates a method to produce a highly conductive and robust metal layer on a 3D printed polymer microstructure with strong adhesion by introducing an interfacial adhesion layer. Prior to 3D printing, multifunctional acrylate monomers with alkoxysilane (-Si-(OCH3)3) were synthesized via the thiol-Michael addition reaction between pentaerythritol tetraacrylate (PETA) and 3-mercaptopropyltrimethoxysilane (MPTMS) with a 1:1 stoichiometric ratio. Alkoxysilane functionality remains intact during photopolymerization in a projection micro-stereolithography (PµSLA) system and is utilized for the sol-gel reaction with MPTMS during postfunctionalization of the 3D printed microstructure to build an interfacial adhesion layer. This leads to the implementation of abundant thiol functional groups on the surface of the 3D printed microstructure, which can act as a strong binding site for gold during electroless plating to improve interfacial adhesion. The 3D conductive microelectrode prepared by this technique exhibited excellent conductivity of 2.2 × 107 S/m (53% of bulk gold) with strong adhesion between a gold layer and a polymer structure even after harsh sonication and an adhesion tape test. As a proof-of-concept, we examined the 3D gold diamond lattice microelectrode modified with glucose oxidase as a bioanode for a single enzymatic biofuel cell. The lattice-structured enzymatic electrode with high catalytic surface area was able to generate a current density of 2.5 µA/cm2 at 0.35 V, which is an about 10 times increase in current output compared to a cube-shaped microelectrode.

Gold nanoparticles as electronic bridges for laccase-based biocathodes.

Direct electron transfer (DET) reactions between redox enzymes and electrodes can be maximized by oriented immobilization of the enzyme molecules onto an electroactive surface modified with functionalized gold nanoparticles (AuNPs). Here, we present such strategy for obtaining a DET-based laccase (Lc) cathode for O(2) electroreduction at low overpotentials. The stable nanostructured enzymatic electrode is based on the step-by-step covalent attachment of AuNPs and Lc molecules to porous graphite electrodes using the diazonium salt reduction strategy. Oriented immobilization of the enzyme molecules on adequately functionalized AuNPs allows establishing very fast DET with the electrode via their Cu T1 site. The measured electrocatalytic waves of O(2) reduction can be deconvoluted into two contributions. The one at lower overpotentials corresponds to immobilized Lc molecules that are efficiently wired by the AuNPs with a heterogeneous electron transfer rate constant k(0) ≫ 400 s(-1).

Towards biochemical filters with a sigmoidal response to pH changes: buffered biocatalytic signal transduction.

We realize a biochemical filtering process by introducing a buffer in a biocatalytic signal-transduction logic system based on the function of an enzyme, esterase. The input, ethyl butyrate, is converted into butyric acid--the output signal, which in turn is measured by the drop in the pH value. The developed approach offers a versatile "network element" for increasing the complexity of biochemical information processing systems. Evaluation of an optimal regime for quality filtering is accomplished in the framework of a kinetic rate-equation model.

Bioelectrocatalytic Activity of W-Formate Dehydrogenase Covalently Immobilized on Functionalized Gold and Graphite Electrodes.

The decrease of greenhouse gases such as CO2 has become a key challenge for the human kind and the study of the electrocatalytic properties of CO2-reducing enzymes such as formate dehydrogenases is of importance for this goal. In this work, we study the covalent bonding of Desulfovibrio vulgaris Hildenborough FdhAB formate dehydrogenase to chemically modified gold and low-density graphite electrodes, using electrostatic interactions for favoring oriented immobilization of the enzyme. Electrochemical measurements show both bioelectrocatalytic oxidation of formate and reduction of CO2 by direct electron transfer (DET). Atomic force microscopy and quartz crystal microbalance characterization, as well as a comparison of direct and mediated electrocatalysis, suggest that a compact layer of formate dehydrogenase was anchored to the electrode surface with some crosslinked aggregates. Furthermore, the operational stability for CO2 electroreduction to formate by DET is shown with approximately 100% Faradaic yield.

Reversible "closing" of an electrode interface functionalized with a polymer brush by an electrochemical signal.

The poly(4-vinyl pyridine) (P4VP)-brush-modified indium tin oxide (ITO) electrode was used to switch reversibly the interfacial activity by the electrochemical signal. The application of an external potential (-0.85 V vs Ag|AgCl|KCl, 3M) that electrochemically reduced O(2) resulted in the concomitant consumption of hydrogen ions at the electrode interface, thus yielding a higher pH value and triggering the restructuring of the P4VP brush on the electrode surface. The initial swollen state of the protonated P4VP brush (pH 4.4) was permeable to the anionic [Fe(CN)(6)](4-) redox species, but the electrochemically produced local pH of 9.1 resulted in the deprotonation of the polymer brush. The produced hydrophobic shrunken state of the polymer brush was impermeable to the anionic redox species, thus fully inhibiting its redox process at the electrode surface. The interface's return to the electrochemically active state was achieved by disconnecting the applied potential, followed by stirring the electrolyte solution or by slow diffusional exchange of the electrode-adjacent thin layer with the bulk solution. The developed approach allowed the electrochemically triggered inhibition ("closing") of the electrode interface. The application of this approach to different interfacial systems will allow the use of various switchable electrodes that are useful for biosensors and biofuel cells with externally controlled activity. Further use of this concept was suggested for electrochemically controlled chemical actuators (e.g. operating as electroswitchable drug releasers).

Immobilization of Proteins on Gold Surfaces.

Gold has been a widely used support for protein immobilization in a nonspecific way through electrostatic and hydrophobic interactions. As no tools are available to predict the binding of proteins of biological interest to gold supports-for either nano, micro, or macroscopic sizes-smart, reliable, and reproducible protein immobilization protocols on gold are sought. This chapter will focus on a synthetic strategy which allows for the development of a multiplicity of architectures on gold that may be used for protein immobilization. Because of its simplicity, both from a conceptual and a practical point of view, the strategy demonstrated by this step-by-step synthesis of a functionally self-assembled monolayer (SAM) of thiols on gold is accessible to most laboratories working on enzyme technology, even those with limited organic synthesis facilities.