Reduced Graphene Oxide/Organic Dye Composites for Bioelectroconversion of Saccharides: Application for Detection of Saccharides and α-Amylase Assessments.

In this study, PQQ-dependent glucose dehydrogenase (PQQ-GDH) was immobilized onto reduced graphene oxide (rGO) modified with organic dyes from three different classes (acridine, arylmethane, and diazo); namely, neutral red (NR), malachite green (MG), and congo red (CR) formed three types of biosensors. All three rGO/organic dye composites were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. The impact of three rGO/organic dye modifications employed in bioelectrocatalytic systems on changes in enzyme activity and substrate selectivity was investigated. The highest sensitivity of 39 µA/cm2 was obtained for 1 mM of glucose when a rGO_MG/PQQ-GDH biosensor was used. A significant improvement in the electrochemical response of biosensors was attributed to the higher amount of pyrrolic nitrogen groups on the surface of the rGO/organic dye composites. Modifications of rGO by NR and MG not only improved the surfaces for efficient direct electron transfer (DET) but also influenced the enzyme selectivity through proper binding and orientation of the enzyme. The accuracy of the biosensor's action was confirmed by the spectrophotometric analysis. Perspectives for using the proposed bioelectrocatalytic systems operating on DET principles for total or single monosaccharide and/or disaccharide determination/bioconversion systems or for diagnoses have been presented through examples of bioconversion of D-glucose, D-xylose, and maltose.

Development of a Disposable Polyacrylamide Hydrogel-Based Semipermeable Membrane for Micro Ag/AgCl Reference Electrode.

Currently, Ag/AgCl-based reference electrodes are used in most electrochemical biosensors and other bioelectrochemical devices. However, standard reference electrodes are rather large and do not always fit within electrochemical cells designed for the determination of analytes in low-volume aliquots. Therefore, various designs and improvements in reference electrodes are critical for the future development of electrochemical biosensors and other bioelectrochemical devices. In this study, we explain a procedure to apply common laboratory polyacrylamide hydrogel in a semipermeable junction membrane between the Ag/AgCl reference electrode and the electrochemical cell. During this research, we have created disposable, easily scalable, and reproducible membranes suitable for the design of reference electrodes. Thus, we came up with castable semipermeable membranes for reference electrodes. Performed experiments highlighted the most suitable gel formation conditions to achieve optimal porosity. Here, Cl- ion diffusion through the designed polymeric junctions was evaluated. The designed reference electrode was also tested in a three-electrode flow system. The results show that home-built electrodes can compete with commercial products due to low reference electrode potential deviation (~3 mV), long shelf-life (up to six months), good stability, low cost, and disposability. The results show a high response rate, which makes in-house formed polyacrylamide gel junctions good membrane alternatives in the design of reference electrodes, especially for these applications where high-intensity dyes or toxic compounds are used and therefore disposable electrodes are required.

Biosensor prototype for rapid detection and quantification of DNase activity.

DNases are enzymes that cleave phosphodiesteric bonds of deoxyribonucleic acid molecules and are found everywhere in nature, especially in bodily fluids, i.e., saliva, blood, or sweat. Rapid and sensitive detection of DNase activity is highly important for quality control in the pharmaceutical and biotechnology industries. For clinical diagnostics, recent reports indicate that increased DNase activity could be related to various diseases, such as cancers. In this paper, we report a new bioelectronic device for the determination of nuclease activity in various fluids. The system consists of a sensor electrode, a custom design DNA target to maximize the DNase cleavage rate, a signal analysis algorithm, and supporting electronics. The developed sensor enables the determination of DNase activity in the range of 3.4 × 10-4 - 3.0 × 10-2 U mL-1 with a limit of detection of up to 3.4 × 10-4 U mL-1. The sensor was tested by measuring nuclease activity in real human saliva samples and found to demonstrate high accuracy and reproducibility compared to the industry standard DNaseAlert™ï¸. Finally, the entire detection system was implemented as a prototype device system utilizing single-use electrodes, custom-made cells, and electronics. The developed technology can improve nuclease quality control processes in the pharmaceutical/biotechnology industry and provide new insights into the importance of nucleases for medical applications.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

Consecutive Marcus Electron and Proton Transfer in Heme Peroxidase Compound II-Catalysed Oxidation Revealed by Arrhenius Plots.

Electron and proton transfer reactions in enzymes are enigmatic and have attracted a great deal of theoretical, experimental, and practical attention. The oxidoreductases provide model systems for testing theoretical predictions, applying experimental techniques to gain insight into catalytic mechanisms, and creating industrially important bio(electro)conversion processes. Most previous and ongoing research on enzymatic electron transfer has exploited a theoretically and practically sound but limited approach that uses a series of structurally similar ("homologous") substrates, measures reaction rate constants and Gibbs free energies of reactions, and analyses trends predicted by electron transfer theory. This approach, proposed half a century ago, is based on a hitherto unproved hypothesis that pre-exponential factors of rate constants are similar for homologous substrates. Here, we propose a novel approach to investigating electron and proton transfer catalysed by oxidoreductases. We demonstrate the validity of this new approach for elucidating the kinetics of oxidation of "non-homologous" substrates catalysed by compound II of Coprinopsis cinerea and Armoracia rusticana peroxidases. This study - using the Marcus theory - demonstrates that reactions are not only limited by electron transfer, but a proton is transferred after the electron transfer event and thus both events control the reaction rate of peroxidase-catalysed oxidation of substrates.