Mediatorless Direct Electron Transfer between Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase and Single-Walled Carbon Nanotubes

Abstract

The flavoenzymes flavin adenine dinucleotidedependent glucose dehydrogenase (FAD-GDH) and oxidase (FAD-GOx) do not undergo direct electron transfer (DET) at conventional electrodes, because the flavin adenine dinucleotide (FAD) cofactor is buried deeply (∼1.4 nm) below the protein surface. We present a mediator-less DET between oxygeninsensitive FAD-GDH and single-walled carbon nanotubes (SWCNTs). A glucose-concentration-dependent current (GCDC) is observed at the electrode with the combination of glycosylated FAD-GDH and debundled SWCNTs; the GCDC, because of an increase in the polarized potential during potential sweep voltammetry, increases steeply (+0.1 V of onset, 1.2 mA cm −2 at +0.6 V 48 mM glucose) without the appearance of the FAD redox peak at −0.45 V. In the control experiment, the GCDC is not observed at the counterpart with either bundled SWCNTs or debundled multiwalled carbon nanotubes (MWCNTs). In the control experiment, the GCDC is observed at an analogous electrode based on oxygen-sensitive FAD-GOx with all CNT types (bundled SWCNTs, debundled SWCNTs, and debundled MWCNTs) in the presence of oxygen because oxygen acts as a natural and mobile mediator. Therefore, observation of the GCDC at the electrode with oxygen-insensitive FAD-GDH and debundled SWCNTs provides evidence of mediator-less DET, even though oxygen is present. Details of the DET are discussed with respect to the recently reported crystallographic model of FAD-GDH. The three-dimensional globular FAD-GDH molecule is 4.5 nm × 5.6 nm × 7.8 nm, which is larger than the 1.2 nm diameter of an individual SWCNT and smaller than the 10 nm diameter of an individual MWCNT and the 1 μm size of a SWCNT bundle. Only individual SWCNTs can be plugged into the groove of FAD-GDH, which is close to and within 1.0 nm of FAD, while maintaining their catalytic activity. Images obtained using transmission electron and atomic force microscopies support the stated configuration of FAD-GDH molecules and debundled SWCNTs. We demonstrate that DET can be explained by quantum tunneling theory. Electrochemical experiments with various FAD-GDHs suggest that (i) DET with debundling SWCNT can be applied to any type of FAD-GDH, (ii) the electrode with various types of FAD-GDH implements superior functions (compared to an analogous electrode with FAD-GOx and nicotineamide adenine dinucleotide-GDH), and (iii) glycan chains present on FAD-GDH prevent denaturation when the SWCNT is close to FAD.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase

“…As a consequence, at least η = +0.30 V of overpotential is required for DET. 1 The CV profile of a novel fungal FAD-GDH/CNT electrode is similar to that with the conventionally used FAD-GDH counterpart, suggesting that the novel fungal FAD-GDH has a similar structure to the conventionally used FAD-GDH, and so the DET model in Fig. 1(B) is valid.…”

“…Oxygen-insensitive flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) has been given much attention recently and there has been an increasing numbers of related reports on biosensors [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] and biofuel cells. 13,14,[16][17][18] Recently, the structure of FAD-GDH from Aspergillus flavus was unveiled and it was found that an FAD cofactor is buried deeply (∼1.4 nm) below the protein surface.…”

Tackling the Challenges of Enzymatic (Bio)Fuel Cells

“…Examination of values of P max greater than 1 mW cm -2 ( Table 1) indicates that the majority of high-power-density EFCs are based on carbon nanomaterials (CNMs)-based electrodes. CNMs including buckypaper 46,117,303 , carbon felt 74 , carbon cloth, carbon black 16 , CNTs 62,72,[303][304][305][306][307][308][309][310][311][312][313] , carbon fiber 65,273,[314][315][316][317][318] , graphene 73,93,255,319 , porous carbon 11,63,66,320,321 , carbon nanodots 322 , and their combinations thereof 15,35,185,[323][324][325][326][327] have been widely used for the preparation of bioelectrodes. Although allowing significantly improved current densities, two main challenges of using CNMs based high-surface-area electrodes need to be addressed.…”

Nanocatalysts Containing Direct Electron Transfer-Capable Oxidoreductases: Recent Advances and Applications

“…This resulted in the biocathode exhibiting good performance-the system was able to operate in electrolyte solutions at a broad pH range and in the presence of high fluoride concentrations. Bioanodes with DET-capable oxidoreductases were also developed, for example, cellobiose dehydrogenase [93,94], fructose dehydrogenase [95], glucose dehydrogenase [96][97][98], and alcohol dehydrogenases [99,100]. Our group has also been working in this field-recently, we have demonstrated a DET bioanode system with glucose dehydrogenase from Ewingella americana, immobilized using gold nanoparticle and polyaniline nanocomposite, which exhibited exceptional performance towards glucose oxidation and operated in whole human blood [101].…”

“…One of the most viable and simple approaches to designing an enzyme-based catalytic material is a random immobilization of two different DET-capable oxidoreductases on a conductive carbon nano/micro particle, as carbon is protein-compatible [98]. Carbon nanomaterials, for example, Figure 2.…”