Utilization of FAD-Glucose Dehydrogenase from T. emersonii for Amperometric Biosensing and Biofuel Cell Devices

Abstract

Flavin-dependent glucose dehydrogenases (FAD-GDH) are oxygen-independent enzymes with high potential to be used as biocatalysts in glucose biosensing applications. Here, we present the construction of an amperometric biosensor and a biofuel cell device, which are based on a thermophilic variant of the enzyme originated from Talaromyces emersonii . The enzyme overexpression in Escherichia coli and its isolation and performance in terms of maximal bioelectrocatalytic currents were evaluated. We examined the biosensor’s bioelectrocatalytic activity in 2,6-dichlorophenolindophenol-, thionine-, and dichloro-naphthoquinone-mediated electron transfer configurations or in a direct electron transfer one. We showed a negligible interference effect and good stability for at least 20 h for the dichloro-naphthoquinone configuration. The constructed biosensor was also tested in interstitial fluid-like solutions to show high bioelectrocatalytic current responses. The bioanode was coupled with a bilirubin oxidase-based biocathode to generate 270 μW/cm 2 in a biofuel cell device.

An Oxygen-Insensitive Biosensor and a Biofuel Cell Device based on FMN L-lactate Dehydrogenase

“…Redox-active mediators with lower onset potentials such as dichloronaphthoquinone (DCNQ), anthraquinone sulfonate (AQS), methylene blue (MB), and thionine can theoretically allow measurement at lower potentials. Surprisingly, while DCNQ was an excellent redox mediator in our previous work [36,37], it failed to act as a redox mediator between ScLDH and the GCE/MWCNTs electrode. Thermodynamically, AQS and MB may act as redox mediators for the FMN cofactor positioned in the active site, (but not for the heme cofactor), yet both failed with ScLDH and the purified FMN catalytic subunit.…”

Enzymatic Galvanic Redox Potentiometry for In Vivo Biosensing

“…Compared with glucose oxidase and nicotinamide adenine dinucleotide-dependent glucose dehydrogenase or pyrroloquinoline quinone-dependent glucose dehydrogenase, FAD-GDH used here can effectively avoid the additional introduction of a cofactor and minimize the interference from O 2 , which improves the sensing accuracy and reliability. 43 According to the GRP sensing mechanism, the difference between the formal potentials of the sensing target and interfering species determines the sensing selectivity. 27 We used TH as the electron mediator in this study because it possesses a relatively low redox potential compared to those of neurochemicals commonly coexisting with glucose in the cerebral system, enabling large output signal and high selectivity over other redox species.…”

“…FAD-GDH is a dehydrogenase with tightly conjugated FAD as an enzyme cofactor (Figure S1). Compared with glucose oxidase and nicotinamide adenine dinucleotide-dependent glucose dehydrogenase or pyrroloquinoline quinone-dependent glucose dehydrogenase, FAD-GDH used here can effectively avoid the additional introduction of a cofactor and minimize the interference from O 2 , which improves the sensing accuracy and reliability . According to the GRP sensing mechanism, the difference between the formal potentials of the sensing target and interfering species determines the sensing selectivity .…”

Stimuli-Responsive DNA-Based Hydrogels on Surfaces for Switchable Bioelectrocatalysis and Controlled Release of Loads

“…A major effort in the area of bioelectrochemistry involves, however, the electrochemical activation of redox proteins, particularly, the activation of glucose oxidase, GOx, toward the oxidation of glucose, in the absence of oxygen, as shown in eq . While redox proteins, usually, lack direct electrochemical communication between their redox centers and electrode surfaces, the development of means to electrically communicate between the redox centers of proteins and the conductive support has attracted substantial research efforts. − Diverse approaches to “electrically wire” redox proteins and electrodes were reported, including the application of diffusional electron mediators, , the functionalization of the redox proteins with redox mediators, , the wiring of redox proteins by means of electron transporting nanomaterials, such as Au-nanoparticles , or carbon nanotubes, , the reconstitution of apo-proteins with cofactor-relay conjugates, , and the application of redox-modified soft polymer matrices loaded with the redox proteins. − The methods to electrically wire redox protein with electrode supports were broadly employed for the development of electrochemical biosensors, especially glucose sensors. , The electrical activation of glucose oxidase, as shown in eq , provides, however, a means to generate pH changes (acidification of the reaction medium) through the bioelectrocatalyzed oxidation of glucose. Accordingly, we argued that the bioelectrocatalyzed oxidation of glucose by the GOx-functionalized pH-responsive hydrogel could provide a means to control the stiffness of the hydrogel matrix and to stimulate the electrically-driven release of loads from the hydrogel.…”