The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements—enzymes and electrodes—is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.

Lytic polysaccharide monooxygenase (LPMO) supports biomass hydrolysis by increasing saccharification efficiency and rate. Recent studies demonstrate that H₂O₂ rather than O₂ is the cosubstrate of the LPMO-catalyzed depolymerization of polysaccharides. Some studies have questioned the physiological relevance of the H₂O₂-based mechanism for plant cell wall degradation. This study reports the localized and time-resolved determination of LPMO activity on poplar wood cell walls by measuring the H₂O₂ concentration in their vicinity with a piezo-controlled H₂O₂ microsensor. The investigated Neurospora crassa LPMO binds to the inner cell wall layer and consumes enzymatically generated H₂O₂. The results point towards a high catalytic efficiency of LPMO at a low H₂O₂ concentration that auxiliary oxidoreductases in fungal secretomes can easily generate. Measurements with a glucose microbiosensor additionally demonstrate that LPMO promotes cellobiohydrolase activity on wood cell walls and plays a synergistic role in the fungal extracellular catabolism and in industrial biomass degradation.

A self-assembly hybrid of gold nanoparticles on graphene modified electrodes for low-potential NADH detection has been achieved. We used the natural polymer chitosan (Chit) to assist the stabilization of graphene in aqueous solution, and immobilize the electronegative Au nanoparticles (NPs) through electrostatic attraction. The synergy of Au NPs with graphene for catalytic oxidation of NADH made the overpotential ca. 220 mV less positive than that on the bare electrode, and remarkably increased the oxidation current. The amperometric sensors based on such modified electrodes for detection of NADH exhibited a good linearity from 1.5 to 320 μM, and showed high sensitivity with a low detection limit of 1.2 μM (S/N = 3). It could also exclude common interfering electroactive compounds like ascorbic acid and possessed good reproducibility and operational stability. Such eminent performance of the Au–RGO/Chit film together with the ability of graphene to significantly enhance the electron transfer between enzymes and the electrode suggested its promise for constructing novel graphene based dehydrogenase biosensors.

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H₂O₂ is a more efficient cosubstrate for the enzyme than O₂, which could greatly affect the utilization of LPMOs in industrial settings. Results: We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H₂O₂ as a cosubstrate. The measurements were also followed by continuous electrochemical H₂O₂ detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H₂O₂ and for the reduction of LPMO’s active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions: We found for all systems that the supply of H₂O₂ limited LPMO’s cellulose depolymerization activity, which supports the function of H₂O₂ as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

Described herein are Cu/Cu ₂ O nanohybrids prepared via potential oscillation, and their high performance for nonenzymatic glucose detection applications.