Direct Electron Transfer-Type Oxidoreductases for Biomedical Applications.

Among the various types of enzyme-based biosensors, sensors utilizing enzymes capable of direct electron transfer (DET) are recognized as the most ideal. However, only a limited number of redox enzymes are capable of DET with electrodes, that is, dehydrogenases harboring a subunit or domain that functions specifically to accept electrons from the redox cofactor of the catalytic site and transfer the electrons to the external electron acceptor. Such subunits or domains act as built-in mediators for electron transfer between enzymes and electrodes; consequently, such enzymes enable direct electron transfer to electrodes and are designated as DET-type enzymes. DET-type enzymes fall into several categories, including redox cofactors of catalytic reactions, built-in mediators for DET with electrodes and by their protein hierarchic structures, DET-type oxidoreductases with oligomeric structures harboring electron transfer subunits, and monomeric DET-type oxidoreductases harboring electron transfer domains. In this review, we cover the science of DET-type oxidoreductases and their biomedical applications. First, we introduce the structural biology and current understanding of DET-type enzyme reactions. Next, we describe recent technological developments based on DET-type enzymes for biomedical applications, such as biosensors and biochemical energy harvesting for self-powered medical devices. Finally, after discussing how to further engineer and create DET-type enzymes, we address the future prospects for DET-type enzymes in biomedical engineering.

Exploiting CO2 laser to boost graphite inks electron transfer for fructose biosensing in biological fluids.

The possibility to print electronics by means of office tools has remarkedly increased the possibility to design affordable and robust point-of-care/need devices. However, conductive inks suffer from low electrochemical and rheological performances limiting their applicability in biosensors. Herein, a fast CO2 laser approach to activate printed carbon inks towards direct enzymatic bioelectrocatalysis (3rd generation) is proposed and exploited to build biosensors for D-fructose analysis in biological fluids. The CO2 laser treatment was compared with two lab-grade printed transducers fabricated with solvent (SB) and water (WB) based carbon inks. The use of the laser revealed significant morpho-chemical variations on the printed inks and was investigated towards enzymatic direct catalysis, using Fructose dehydrogenase (FDH) integrated into entirely lab-produced biosensors. The laser-driven activation of the inks unveils the inks' direct electron transfer (DET) ability between FDH and the electrode surface. Sub-micromolar limits of detection (SB-ink LOD = 0.47 µM; WB-ink LOD = 0.24 µM) and good linear ranges (SB-ink: 5-100 µM; WB-ink: 1-50 µM) were obtained, together with high selectivity due to use of the enzyme and the low applied overpotential (0.15 V vs. pseudo-Ag/AgCl). The laser-activated biosensors were successfully used for D-fructose determination in complex synthetic and real biological fluids (recoveries: 93-112%; RSD ≤8.0%, n = 3); in addition, the biosensor ability for continuous measurement (1.5h) was also demonstrated simulating physiological D-fructose fluctuations in cerebrospinal fluid.

Enhanced Electron Transfer Efficiency of Fructose Dehydrogenase onto Roll-to-Roll Thermal Stamped Laser-Patterned Reduced Graphene Oxide Films.

Herein, a strategy to stamp laser-produced reduced graphene oxide (rGO) onto flexible polymers using only office-grade tools, namely, roll-to-roll thermal stamping, is proposed, proving for the first time its effectiveness for direct bioelectrocatalysis. This straightforward, scalable, and low-cost approach allows us to overcome the limits of the integration of laser-induced rGO-films in bioanalytical devices. Laser-produced rGO has been thermally stamped (TS) onto different polymeric substrates (PET, PVC, and EVA) using a simple roll-laminator; the obtained TS-rGO films have been compared with the native rGO (untransferred) via morphochemical and electrochemical characterization. Particularly, the direct electron transfer (DET) reaction between fructose dehydrogenase (FDH) and TS-rGO transducers has been investigated, with respect to the influence of the amount of enzyme on the catalytic process. Remarkable differences have been observed among TS-rGO transducers; PET proved to be the elective substrate to support the transfer of the laser-induced rGO, allowing the preservation of the morphochemical features of the native material and returning a reduced capacitive current. Noteworthily, TS-rGOs ensure superior electrocatalysis using a very low amount of FDH units (15 mU). Eventually, TS-rGO-based third-generation complete enzymatic biosensors were fabricated via low-cost benchtop technologies. TS-rGOPET exhibited bioanalytical performances superior to the native rGO, allowing a sensitive (0.0289 µA cm-2 µM-1) and reproducible (RSD = 3%, n = 3) d-fructose determination at the nanomolar level (LOD = 0.2 µM). TS-rGO exploitability as a point-of-need device was proved via the monitoring of d-fructose during banana (Musa acuminata) postharvest ripening, returning accurate (recoveries 110-90%; relative error -13/+1%) and reproducible (RSD ≤ 7%; n = 3) data.

Directional propagation of action potential within a single cell and intercellular conduction within a cell aggregate using model cell systems.

The mechanism of directional propagation of action potential throughout a single cell was examined using a liquid-membrane model cell system. In the experiments on the liquid-membrane model cell system, liquid-membrane cells were constructed to mimic the function of K+ and voltage-gated Na+ channels, which play important roles in action potential propagation. These channel-mimicking cells were connected electrically, and a model cell system was composed of four parts within the one cell. When one voltage-gated Na+ channel-mimicking cell was connected to form the action potential and generated the inflow current at the one part, action potential occurred in the surrounding area due to the local circulating current and propagated to the other parts. The action potential propagation throughout the cell by a brief electrical stimulus (10 ms) was easier than that by a long electrical stimulus (2 s). The long electric stimulus thus caused hyperpolarized region within the cell. Moreover, the increase in resistance corresponding to the extracellular fluid weakened the action potential propagation. In the simulation experiments using the software LTspice, the characteristics of K+ and Na+ channel-mimicking cells were reproduced in the electrical circuit also. A model cell aggregate consisting of closely packed three model cells and the extracellular fluid was constructed in the electric circuit. When one cell fired, the electrical signal propagated to the neighboring cells through the intercellular and extracellular fluids. This result suggests that electrical propagation can occur between independent cells in closely packed tissues without chemical transmission or direct propagation across the gap junctions.

Structure and function relationship of formate dehydrogenases: an overview of recent progress.

Formate dehydrogenases (FDHs) catalyze the two-electron oxidation of formate to carbon dioxide. FDHs can be divided into several groups depending on their subunit composition and active-site metal ions. Metal-dependent (Mo- or W-containing) FDHs from prokaryotic organisms belong to the superfamily of molybdenum enzymes and are members of the dimethylsulfoxide reductase family. In this short review, recent progress in the structural analysis of FDHs together with their potential biotechnological applications are summarized.

Lab-made flexible third-generation fructose biosensors based on 0D-nanostructured transducers.

Herein, we report a scalable benchtop electrode fabrication method to produce highly sensitive and flexible third-generation fructose dehydrogenase amperometric biosensors based on water-dispersed 0D-nanomaterials. The electrochemical platform was fabricated via Stencil-Printing (StPE) and insulated via xurography. Carbon black (CB) and mesoporous carbon (MS) were employed as 0D-nanomaterials promoting an efficient direct electron transfer (DET) between fructose dehydrogenase (FDH) and the transducer. Both nanomaterials were prepared in water-phase via a sonochemical approach. The nano-StPE exhibited enhanced electrocatalytic currents compared to conventional commercial electrodes. The enzymatic sensors were exploited for the determination of D-fructose in model solutions and various food and biological samples. StPE-CB and StPE-MS integrated biosensors showed appreciable sensitivity (∼150 µA cm-2 mM-1) with µmolar limit of detection (0.35 and 0.16 µM, respectively) and extended linear range (2-500 and 1-250 µM, respectively); the selectivity of the biosensors, ensured by the low working overpotential (+0.15 V), has been also demonstrated. Good accuracy (recoveries between 95 and 116%) and reproducibility (RSD ≤8.6%) were achieved for food and urine samples. The proposed approach because of manufacturing versatility and the electro-catalytic features of the water-nanostructured 0D-NMs opens new paths for affordable and customizable FDH-based bioelectronics.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts.

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

Inhibition of direct-electron-transfer-type bioelectrocatalysis of bilirubin oxidase by silver ions.

In enzyme-based biosensors, Ag+ eluted from the reference electrode inhibits the enzyme activity. Herein, to suppress the inhibition of bilirubin oxidase (BOD) by Ag+, kinetic analysis was used to examine the effect of Ag+ on the activity of BOD. It was confirmed that the addition of Ag+ decreased the bioelectrocatalytic activity of BOD. Atomic absorption spectroscopy (AAS) suggested that Ag+ was attached to BOD. Moreover, the changes in the visible absorption spectra after Ag+ addition showed that Ag+ was bound to the type I Cu sites in BOD. During oxygen reduction by BOD, the direct-electron-transfer-type bioelectrocatalytic current decreased after Ag+ was added. The decay of the catalytic current was evaluated using kinetic analysis (assuming a pseudo-first-order reaction). Based on the analysis, the inhibition of BOD was suppressed when the Ag+ concentration was below 0.1 µM. Referring to the solubility product of AgCl, Cl- at a concentration of 1 mM suppressed the inhibition of the enzymatic activity by 95%.

Cyanide sensitivity in direct electron transfer-type bioelectrocatalysis by membrane-bound alcohol dehydrogenase from Gluconobacter oxydans.

An overexpression system of membrane-bound alcohol dehydrogenase (ADH) from Gluconobacter oxydans was constructed to examine its bioelectrocatalytic characteristics. The effects of cyanide (CN-) addition on the kinetics of direct electron transfer (DET)-type bioelectrocatalysis by ADH were analyzed. CN- enhanced the bioelectrocatalytic activity, while the catalytic activity in the solution remained unchanged, even in the presence of CN-. Electrochemical methods and electron spin resonance spectroscopy showed the detailed electron transfer pathway in the DET-type bioelectrocatalysis by ADH. Briefly, ADH is suggested to communicate with an electrode via a CN--insensitive and H+-sensitive heme c in DET. These characteristics of ADH with respect to CN- suggest the involvement of ADH in CN--insensitive respiration in G. oxydans.

Ion transport across bilayer lipid membranes in the presence of tetraphenylborate.

A pair of symmetrical cathodic and anodic peaks is observed in cyclic voltammograms for the ion transport across a bilayer lipid membrane (BLM) between two aqueous phases in the presence of tetraphenylborate (TPhB-). Although TPhB- serves as a carrier of a hydrophilic counter ion (Na+) under the steady-state condition, the reason for the appearance of symmetrical peaks has not been clearly explained until now. From the chronoamperometric analysis, it is turned out that the symmetrical peaks are attributed to the translocation of TPhB- between two adsorbed layers on the surface of the BLM.

Effects of N-linked glycans of bilirubin oxidase on direct electron transfer-type bioelectrocatalysis.

Bilirubin oxidase from Myrothecium verrucaria (mBOD) is a promising enzyme for catalyzing the four-electron reduction of dioxygen into water and realizes direct electron transfer (DET)-type bioelectrocatalysis. It has two N-linked glycans (N-glycans), and N472 and N482 are known as binding sites. Both binding sites located on opposite side of the type I (T1) Cu, which is the electrode-active site of BOD. We investigated the effect of N-glycans on DET-type bioelectrocatalysis by performing electrochemical measurements using electrodes with controlled surface charges. Two types of BODs with different N-glycans, mBOD and recombinant BOD overexpressed in Pichia pastoris (pBOD), and their deglycosylated forms (dg-mBOD and dg-pBOD) were used in this study. Kinetic analysis of the steady-state catalytic waves revealed that both size and composition of N-glycans affected the orientation of adsorbed BODs on the electrodes. Interestingly, the most favorable orientation was achieved with pBOD, which has the largest N-glycans. Furthermore, the effect of the orientation control by the N-glycans is cooperative with electrostatic interaction.

Quantification of leuco-indigo in indigo-dye-fermenting suspension by normal pulse voltammetry.

Quantification of leuco-indigo is most important for Aizome, Japanese indigo-dyeing; however, there has been no convenient quantitative method. This study demonstrated that normal pulse voltammetry under quiescent conditions can be used to detect leuco-indigo. As a result of quantification of leuco-indigo in the depth direction in fermenting suspensions, the steady-state concentrations of leuco-indigo showed sigmoidal profiles in the depth direction. The steady state is caused by competitive reactions of microbial reduction of indigo and autoxidation of leuco-indigo by O2 dissolved from the air interface of the suspension. In addition, we investigated the effects of stirring the suspension and adding some nutrients to the concentration profile. The weakened activity was partially recovered by the addition of ethanol and remarkably recovered by the addition of hipolypepton or glucose. Knowledge is essential for the proper management of indigo-dye-fermenting suspensions.

Multiple electron transfer pathways of tungsten-containing formate dehydrogenase in direct electron transfer-type bioelectrocatalysis.

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)-a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples-was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered.