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Beta-galactosidase (β-Gal) is an important enzyme utilized to determine cell senescence and understand disease pathology, among being utilized for several biosensing applications. This key biomarke...

The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens ( Hi CDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5–6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the Hi CDH film was studied. It was found that the presence of charged pyridinium groups was needed to successfully establish direct electron contact between the enzyme and electrode. SAMs formed from hydrophilic charged thiols achieved nearly two times higher current densities compared to hydrophobic charged thiols. Additionally, the results also indicated proportionality between Hi CDH catalytic constant and water content of the enzyme film. Enzyme films on charged pyridine thiols had smaller variations in water content and viscoelastic properties than films adsorbed on the more hydrophobic thiols. This work highlights several perspectives on the underlying factors affecting performance of immobilized Hi CDH.

Carbon paste paper electrodes (CPPEs) were fabricated by coating a regular paper strip with carbon paste made from graphite powder and mineral oil, followed by coating with polyaniline. The CPPEs were evaluated as anodes in bioelectrochemical cells (BECs) using Shewanella oneidensis MR-1 as bacteria that donate electrons through extracellular electron transfer. The CPPE was compared to a carbon felt electrode (CFE) modified with polyaniline under the same conditions. The BEC using the CPPE anode produces current continuously for at least 4 days without the need for additional fuel (lactate). Twenty-four hours after inoculation, the BEC using the CPPE anode generates a current density more than two times greater than that of the cell using the CFE, with a competitive maximum value of 2.2 A m–2. The simple fabrication, ease of modification, and low cost of the CPPE make it a promising new bioelectrode material for microbial fuel cells.

Direct electron transfer (DET) between cellobiose dehydrogenase from Humicola insolens ascomycete (HiCDH) and gold nanoparticles (AuNPs) was achieved by modifying AuNPs with a novel, positively charged thiol N-(6-mercapto)hexylpyridinium (MHP). The DET enabled the use of the HiCDH enzyme as an anodic biocatalyst in the design of a mediatorless carbohydrate/oxygen enzymatic fuel cell (EFC). A biocathode of the EFC was based on bilirubin oxidase from Myrothecium verrucaria (MvBOx) directly immobilised on the surface of AuNPs. The following parameters of the EFC based on Au/AuNP/MHP/HiCDH bioanode and Au/AuNP/MvBOx biocathode were obtained in quiescent air saturated PBS, pH 7.4, containing: (i) 5 mM glucose-open-circuit voltage (OCV) of 0.65 ± 0.011 V and the maximal power density of 4.77 ± 1.34 μW cm−2 at operating voltage of 0.50 V; or (ii) 10 mM lactose-OCV of 0.67 ± 0.006 V and the maximal power density of 8.64 ± 1.91 μW cm−2 at operating voltage of 0.50 V. The half-life operation times of the EFC were estimated to be at least 13 and 44 h in air saturated PBS containing 5 mM glucose and 10 mM lactose, respectively. Among advantages of HiCDH/MvBOx FCs are (i) simplified construction, (ii) relatively high power output with glucose as biofuel, and (iii) the absence of the inhibition of the HiCDH based bioanode by lactose, when compared with the best previously reported CDH based bioanode.

Bioelectrocatalytic reduction of oxygen to water at electrodes modified with gold nanoparticles and a new laccase from Trichaptum abietinum (TaLc) was studied. The bioelectrocatalytic current was found to be much higher at TaLc modified electrodes than at similarly prepared electrodes modified with a broadly used laccase from Trametes hirsuta (ThLc). To explain this difference the bioelectrocatalysis was described in terms of kinetic rate constants based on simultaneous cyclic voltammetry and quartz crystal microbalance measurements. From analysis of the rate constants both laccases appeared to possess similar rates (k0) of direct electron transfer. However, the enzyme turnover (kcat) was about three-fold higher for gold nanoparticle bound TaLc than for ThLc, calculated using surface concentration of enzyme established by QCM-D. Near reversible potential-induced reorientation of adsorbed proteins was observed by surface enhanced Raman spectroscopy.

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 μW cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode.

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