Immobilizing redox enzymes at mesoporous and nanostructured electrodes

Three key challenges are stimulating intensive research in the development of productive direct electron transfer mode enzyme electrodes: proper enzyme orientation, high enzyme loading, and full retention of enzyme activity. In this review, we summarize some significant advances that have been reported in the last years on the design of mesoporous and nanostructured electrodes as enzyme scaffolds and of innovative methodologies for wiring enzymes to electrodes. Particular attention is given to investigations on physical factors that determine a favorable enzyme immobilization, to provide rational guidelines for the design of productive enzymatic electrodes. Finally, some emerging trends focused on the spatial organization of either single enzymes or enzyme cascades are also briefly addressed.     

Magnetic control of chemical transformations: application for programmed electrocatalysis and surface patterning

Lateral translocation of electrocatalyst-modified magnetic particles was achieved upon application of an external magnetic field. Programmed electrocatalytic reactions at different electrodes or different areas of an electrode were performed. The spatially controlled electrocatalytic reactions were exemplified with NADH electrocatalytic oxidation and with bioelectrocatalytic electrode patterning. The method will be particularly useful for programmed electrochemical reactions at interdigitated electrodes.     

Direct Bioelectrocatalysis by NADP‐Reducing Hydrogenase from Pyrococcus furiosus

The direct bioelectrocatalysis by an NAD(P)‐reducing hydrogenase is reported for the first time. In contrast to previous attempts to involve similar enzymes in bioelectrocatalysis [1–4], which were in fact unsuccessful, in our report an effective electrocatalysis by Pyrococcus furiosus hydrogenase is convincingly shown by (i) achievement of the hydrogen equilibrium potential and (ii) a high current of hydrogen oxidation (0.3 mA cm^?2 at 100 mV overpotential and at 75 °C). The latter is just a few times lower compared to enzyme electrodes based on NAD(P)‐independent hydrogenases.     

辣根过氧化物酶／聚邻苯二胺膜电极的制备与性能研究

辣根过氧化物酶（ＨＲＰ）／聚邻苯二胺（ＰＰＤ）膜电极由ｐＨ７．０磷酸盐缓冲溶液介质中邻苯二胺在玻碳电极上的电聚合而制得。讨论了ＨＲＰ电化学固定化的过程。所得酶电极呈现生物催化活性，可以没有电子传递体存在的情况下催化Ｈ２Ｏ２还原。该反应发生在聚邻苯二胺氧化还原的电位区，聚合物参与了酶的电子转移过程。分析了旋转ＨＲＰ／ＰＰＤ电极上酶反应的动力学，讨论了动力学常数的影响因素。     

Direct electrochemistry of recombinant tobacco peroxidase on gold

Direct electron transfer (DET) reactions of recombinant tobacco peroxidase (rTOP), namely direct electroreduction of Compound I/Compound II and heme Fe^3+/2+ conversion, were studied on gold electrodes. rTOP of wild type, non-glycosylated, was produced using an Escherichia coli expression system. At pH 5.0, the redox potential for direct electrochemical transformation of the Fe^3+/2+ of the peroxidase heme was −143 mV vs. AgAgCl, and 0.26 ± 0.07 pmol of the adsorbed rTOP were in DET contact with the gold electrode. The total amount of the adsorbed rTOP estimated from QCM data was 53 ± 5 pmol/cm² or 1.67 pmol when referred to the surface area of the electrodes used for electrochemical measurements. Of 1.67 pmol of adsorbed rTOP, only 0.76 pmol were catalytically active. DET between Au and the enzyme was also studied in the reaction of the bioelectrocatalytic reduction of H₂O₂ by cyclic voltammetry and amperometric detection of H₂O₂ at +50 mV with rTOP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Maximal bioelectrocatalytic current response of the rTOP-modified gold electrodes to H₂O₂ was observed at pH 5.0 and stemmed from its bioelectrocatalytic reduction based on DET between Au and the active site of rTOP. Kinetic analysis of the DET reactions gave 52% of the adsorbed rTOP molecules active in DET reactions (0.4 pmol of adsorbed catalytically active rTOP, correspondingly), which correlated well with the non-catalytic-voltammetry data. DET was characterised by a heterogeneous ET rate constant of 13.2 s⁻¹, if one takes into account the QCM data, and 19.6 s⁻¹, if the amount of rTOP estimated from the data on DET transformation of Fe^3+/2+ couple of rTOP is considered. The sensitivity for H₂O₂ obtained for the rTOP-modified Au electrodes was 0.7 ± 0.1 A M⁻¹ cm⁻². These are the first ever-reported data on DET reactions of anionic plant peroxidases on bare gold electrodes.     

Direct evidence of electron flow via the heme c group for the direct electron transfer reaction of fructose dehydrogenase using a silver nanoparticle-modified electrode

The direct electron transfer reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. on alkanethiol-modified silver nanoparticles (AgNPs) was examined using cyclic voltammetry and surface-enhanced resonance Raman scattering (SERRS). Using cyclic voltammetry, catalytic oxidation currents (based on the direct electron transfer reaction of FDH) were observed from a potential of approximately −100 mV (vs. Ag/AgCl, 3 M NaCl) in the presence of d-fructose, without a mediator. A comparison of the SERRS spectra and the resonance Raman spectra of FDH in solution indicated that the heme c site retained its six-coordinated low-spin heme after immobilization. Moreover, SERRS also demonstrated that the heme c of the adsorbed FDH was the electron transfer site within the enzyme.     

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase

Massive efforts are invested in developing innovative CO₂-sequestration strategies to counter climate change and transform CO₂ into higher-value products. CO₂-capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO₂, it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO₂-reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO₂ reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO₂-fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.     

酶直接电化学与第三代生物传感器

本文详细地评述并展望了酶直接电化学与第三代生物传感器这个领域已取得的成果，主要内容涉及生物电催化的三个发展阶段，实现酶与电极之间的直接电子转移方法和相应机理、以及第三代酶传感器的研制。     

Enhancement of Bioelectrocatalytic Processes by the Rotation of Mediator‐Functionalized Magnetic Particles on Electrode Surfaces: Comparison with a Rotating Disk Electrode

The rotation of redox‐functionalized magnetic particles (MPs) by means of an external magnet is a common practice for enhancing bioelectrocatalytic processes and for the amplification of biosensing events. The current densities generated by rotating redox‐functionalized MPs in two bioelectrocatalytic systems are compared to the current densities generated by rotating disc electrodes (RDE) functionalized with similar redox functionalities. The bioelectrocatalytic systems consist of pyrroloquinoline quinone (PQQ)‐functionalized MPs that oxidize NADH, and ferrocene‐functionalized MPs that mediate the bioelectrocatalyzed oxidation of glucose in the presence of glucose oxidase. The results reveal that only ca. 1% of the area of the redox‐functionalized MPs are electrically contacted with the electrode. Also, the current densities generated by the rotating MPs at high rotation speeds are lower than theoretically expected, presumably due to lose of electrical contact between the MPs and the electrode, and incoherent rotation of the particles on the electrode, due to insufficient magnetization. The comparison of the current densities in the bioelectrocatalytic systems in the presence of the rotating redox‐functionalized MPs to the analogous RDE systems allows us to elucidate the kinetics of electron transfer at the redox‐active MPs.     

TTF‐TCNQ Functionalized Ormosil Based Electrocatalytic Biosensor: A comparative study on Bioelectrocatalysis

A novel electrocatalytic biosensor for glucose is reported that incorporate encapsulation of tetrathifulvalene‐tetracyanoquinodimethane (TTF‐TCNQ) functionalized organically modified sol gel glass (ormosil). The new ormosil is made using palladium‐linked glycidoxypropyltrimethoxysilane precursor, trimethoxysilane, HCl and TTF‐TCNQ powder at 25 °C. The ormosil is converted into fine powder and incorporated within graphite paste electrode along with glucose oxidase. The bioelectrochemistry of GOD and TTF‐TCNQ functionalized ormosil is examined based on cyclic voltammetry and amperometric measurements. A large electrocatalytic current to the order of 8000 μA/cm² is recorded on the addition of 300 mM glucose. Typical responses of new biosensor are reported. The sensitivity of glucose analysis is found relatively much better as compared to earlier reported glucose biosensors. The role of palladium and TTF‐TCNQ introduction within ormosil and its advantages on bioelectrocatalysis are discussed.