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/cm² in a biofuel cell device.

Figure 1

Fabrication of a polydopamine-based biosensor. TeGDH or BOD is mixed with their respective redox mediator and polydopamine and then deposited on the GCE-MWCNT surface. Both biosensors can be combined to form a biofuel cell.

Figure 2

Cyclic voltammetry (CV) of a TeGDH-based biosensor. (a) CV measurement of a DCPIP-based sensor with (orange) and without (black) addition of 40 mM glucose. The inset represents a calibration curve at 0.1 V versus Ag/AgCl based on CV measurements with varying glucose levels. (b) CV measurement of a DCNQ-based sensor with (beige) and without (black) addition of 40 mM glucose. The inset represents a calibration curve at 0 V versus Ag/AgCl based on CV measurements with varying glucose levels.

Figure 3

Chronoamperometry (CA) of a DCNQ-based glucose biosensor for 1 day in 10 mM glucose at 0 V versus Ag/AgCl. After 14 h, another dose of 10 mM glucose was mixed into the solution. The solution was homogenized via pipetting during a brief pause in the measurement.

Figure 4

Interference assay for the DCNQ-based biosensor. (a) CA of the interference assay at 0 V versus Ag/AgCl. Analytes were added in the following order: UA—6.8 μg/mL uric acid, g—2.5 mM glucose, Asc—100 μM ascorbic acid, Aca—1.1 mM acetaminophen, and 2g—5 mM glucose. The solution was homogenized via pipetting during a brief pause in the measurement. (b) A plot of average current versus glucose concentration. The current values were taken from three separate CA measurements.

Figure 5

Power output of the TeGDH enzymatic biofuel cell. (a) Linear sweep voltammetry (LSV) describing the power output with 40 mM glucose under atmospheric conditions. (b) An LSV curve describing the power output with 40 mM glucose and enriched O₂. (c) A CV curve of the TeGDH bioanode and the BOD biocathode under 40 mM glucose and enriched O₂, respectively.