In Vitro Evaluation of Miniaturized Amperometric Enzyme Sensor Based on the Direct Electron Transfer Principle for Continuous Glucose Monitoring

Background: The bacterial derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (FADGDH) is the most promising enzyme for the third-generation principle-based enzyme sensor for continuous glucose monitoring (CGM). Due to the ability of the enzyme to transfer electrons directly to the electrode, recognized as direct electron transfer (DET)-type FADGDH, although no investigation has been reported about DET-type FADGDH employed on a miniaturized integrated electrode. Methods: The miniaturized integrated electrode was formed by sputtering gold (Au) onto a flexible film with 0.1 mm in thickness and divided into 3 parts. After an insulation layer was laminated, 3 openings for a working electrode, a counter electrode and a reference electrode were formed by dry etching. A reagent mix containing 1.2 × 10−4 Unit of DET-type FADGDH and carbon particles was deposited. The long-term stability of sensor was evaluated by continuous operation, and its performance was also evaluated in the presence of acetaminophen and the change in oxygen partial pressure (pO2) level. Results: The amperometric response of the sensor showed a linear response to glucose concentration up to 500 mg/dL without significant change of the response over an 11-day continuous measurement. Moreover, the effect of acetaminophen and pO2 on the response were negligible. Conclusions: These results indicate the superb potential of the DET-type FADGDH-based sensor with the combination of a miniaturized integrated electrode. Thus, the described miniaturized DET-type glucose sensor for CGM will be a promising tool for effective glycemic control. This will be further investigated using an in vivo study.

Cost Effective Synthesis of Graphene Nanomaterials for Non-Enzymatic Electrochemical Sensors for Glucose: A Comprehensive Review

The high conductivity of graphene material (or its derivatives) and its very large surface area enhance the direct electron transfer, improving non-enzymatic electrochemical sensors sensitivity and its other characteristics. The offered large pores facilitate analyte transport enabling glucose detection even at very low concentration values. In the current review paper we classified the enzymeless graphene-based glucose electrocatalysts’ synthesis methods that have been followed into the last few years into four main categories: (i) direct growth of graphene (or oxides) on metallic substrates, (ii) in-situ growth of metallic nanoparticles into graphene (or oxides) matrix, (iii) laser-induced graphene electrodes and (iv) polymer functionalized graphene (or oxides) electrodes. The increment of the specific surface area and the high degree reduction of the electrode internal resistance were recognized as their common targets. Analyzing glucose electrooxidation mechanism over Cu- Co- and Ni-(oxide)/graphene (or derivative) electrocatalysts, we deduced that glucose electrochemical sensing properties, such as sensitivity, detection limit and linear detection limit, totally depend on the route of the mass and charge transport between metal(II)/metal(III); and so both (specific area and internal resistance) should have the optimum values.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Syntrophic propionate-oxidizing bacteria in methanogenic systems

The mutual nutritional cooperation underpinning syntrophic propionate degradation provides a scant amount of energy for the microorganisms involved, so propionate degradation often acts as a bottleneck in methanogenic systems. Understanding the ecology, physiology, and metabolic capacities of syntrophic propionate-oxidizing bacteria is of interest in both engineered and natural ecosystems, as it offers prospects to guide further development of technologies for biogas production and biomass-derived chemicals, and is important in forecasting contributions by biogenic methane emissions to climate change. Syntrophic propionate-oxidizing bacteria are distributed across different phyla. They can exhibit broad metabolic capabilities in addition to syntrophy (e.g. fermentative, sulfidogenic, and acetogenic metabolism) and demonstrate variations in interplay with cooperating partners, indicating nuances in their syntrophic lifestyle. In this review, we discuss distinctions in gene repertoire and organization for the methylmalonyl-CoA pathway, hydrogenases and formate dehydrogenases, and emerging facets of (formate/hydrogen/direct) electron transfer mechanisms. We also use information from cultivations, thermodynamic calculations, and omic analyses as the basis for identifying environmental conditions governing propionate oxidation in various ecosystems. Overall, this review improves basic and applied understanding of syntrophic propionate-oxidizing bacteria and highlights knowledge gaps, hopefully encouraging future research and engineering on propionate metabolism in biotechnological processes.

Superior Non-Invasive Glucose Sensor Using Bimetallic CuNi Nanospecies Coated Mesoporous Carbon

The assessment of blood glucose levels is necessary for the diagnosis and management of diabetes. The accurate quantification of serum or plasma glucose relies on enzymatic and nonenzymatic methods utilizing electrochemical biosensors. Current research efforts are focused on enhancing the non-invasive detection of glucose in sweat with accuracy, high sensitivity, and stability. In this work, nanostructured mesoporous carbon coupled with glucose oxidase (GOx) increased the direct electron transfer to the electrode surface. A mixed alloy of CuNi nanoparticle-coated mesoporous carbon (CuNi-MC) was synthesized using a hydrothermal process followed by annealing at 700 °C under the flow of argon gas. The prepared catalyst’s crystal structure and morphology were explored using X-ray diffraction and high-resolution transmission electron microscopy. The electrocatalytic activity of the as-prepared catalyst was investigated using cyclic voltammetry (CV) and amperometry. The findings show an excellent response time of 4 s and linear range detection from 0.005 to 0.45 mM with a high electrode sensitivity of 11.7 ± 0.061 mA mM cm−2 in a selective medium.

Utilizing Spent Batteries to Fabricate Ni/ZnO-MnO2 Electrodes for Electrochemical Ammonia Oxidation

In this work, a novel Ni/ZnO-MnO2 electrode was fabricated by utilizing spent zinc-manganese batteries and then was applied to the electrochemical treatment of ammonia-containing wastewater. The obtained Ni/ZnO-MnO2 electrode was characterized by scanning electron microscopy, X-ray diffraction, and linear scanning voltammetry, suggesting that the fabricated electrode had a flower-like structure and showed high oxygen evolution potential and electrochemical activity. The electrochemical performance of the ZnO-MnO2 electrode in regard to ammonia removal and product selectivity was then investigated with different operating factors (i.e., electrolyte concentration, initial pH value, current density, and Cl− concentration), and the results indicated that the ammonia removal efficiency could reach 100% with a N2 selectivity of 91.8% under optimal conditions. Additionally, the mechanism of ammonia oxidation was proposed by cyclic voltammetry tests and active radical measurements, showing that ammonia was mainly oxidized via direct electron transfer, hydroxyl radicals, and active chlorine. Finally, the ZnO-MnO2 electrode was equipped for the treatment of actual pharmaceutical wastewater, results for which showed that ammonia could be completely removed with a current efficiency of 26.2% and an energy consumption of 52.7 kWh/kg N. Thus, the ZnO-MnO2 electrode prepared by recycling spent batteries is a promising anode for wastewater treatment.

Bioconversion of Carbon Monoxide to Formate Using Artificially Designed Carbon Monoxide:Formate Oxidoreductase in Hyperthermophilic Archaea

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between noninteracting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 348 ± 34 μmol/mg/min and 90.2 ± 20.4 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.