Research Article Cite This: ACS Catal. 2018, 8, 10279−10289
pubs.acs.org/acscatalysis
Enhanced Direct Electron Transfer of Fructose Dehydrogenase Rationally Immobilized on a 2‑Aminoanthracene Diazonium Cation Grafted Single-Walled Carbon Nanotube Based Electrode Paolo Bollella,*,† Yuya Hibino,‡ Kenji Kano,‡ Lo Gorton,§ and Riccarda Antiochia† †
Department of Chemistry and Drug Technologies, Sapienza University of Rome P.le Aldo Moro 5, 00185 Rome, Italy Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan § Department of Analytical Chemistry/Biochemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden ACS Catal. 2018.8:10279-10289. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/27/19. For personal use only.
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S Supporting Information *
ABSTRACT: In this paper, an efficient direct electron transfer (DET) reaction was achieved between fructose dehydrogenase (FDH) and a glassy-carbon electrode (GCE) upon which anthracene-modified single-walled carbon nanotubes were deposited. The SWCNTs were activated in situ with a diazonium salt synthesized through the reaction of 2-aminoanthracene with NaNO2 in acidic media (0.5 M HCl) for 5 min at 0 °C. After the in situ reaction, the 2-aminoanthracene diazonium salt was electrodeposited by running cyclic voltammograms from +1000 to −1000 mV. The anthracene-SWCNT-modified GCE was further incubated in an FDH solution, allowing enzyme adsorption. Cyclic voltammograms of the FDH-modified electrode revealed two couples of redox waves possibly ascribed to the heme c1 and heme c3 of the cytochrome domain. In the presence of 10 mM fructose two catalytic waves could clearly be seen and were correlated with two heme cs (heme c1 and c2), with a maximum current density of 485 ± 21 μA cm−2 at 0.4 V at a sweep rate of 10 mV s−1. In contrast, for the plain SWCNT-modified GCE only one catalytic wave and one couple of redox waves were observed. Adsorbing FDH directly onto a GCE showed no nonturnover electrochemistry of FDH, and in the presence of fructose only a slight catalytic effect could be seen. These differences can be explained by considering the hydrophobic pocket close to heme c1, heme c2, and heme c3 of the cytochrome domain at which the anthracenyl aromatic structure could interact through π−π interactions with the aromatic side chains of the amino acids present in the hydrophobic pocket of FDH. KEYWORDS: fructose dehydrogenase (FDH), 2-aminoanthracene (2-ANT), diazonium coupling, hydrophobic pocket, single-walled carbon nanotubes (SWCNTs)
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diameter in the nanometer range.51 Their peculiar properties (e.g., high conductance, tensile strength, etc.) have attracted great attention for the development of electronic devices.52 Moreover, several methods have been developed for their synthesis and functionalization.53 However, the nanotube functionalization will affect the charge delocalization, offering different possibilities for protein/molecule tethering.54,55 (e.g., SWCNTs can be functionalized by using EDC-NHS covalent coupling, glutaraldehyde or other cross-linkers, chemical modification with aryl diazonium salts, etc.).56−58 The electrochemical reductive adsorption of aryl diazonium salts onto carbon nanotubes has been widely used for the development of new electrode platforms for biosensors and EFCs.59−62 The aryl diazonium salt electroreduction can be performed in either acetonitrile or aqueous acid solutions
INTRODUCTION Recently, the direct electron transfer (DET) mechanism of several redox proteins has been widely investigated in order not only to gain fundamental bioelectrochemical knowledge but also to develop DET-based biosensors and enzymatic fuel cells (EFCs).1−8 DET reactions have been observed for smaller electron transfer redox proteins9 such as cytochrome c,10 azurin,11 and ferredoxin, as well as for a whole range of the larger, more complex redox enzymes of great relevance in recent studies of biosensors and biofuel cells: e.g., hydrogenases,12−14 cellobiose dehydrogenase,15−17 multicopper oxidases,18−20 peroxidases,21−23 fructose dehydrogenase (FDH),24−28 alcohol PQQ dehydrogenase,29−33 sulfite oxidase,34,35 etc. Nevertheless, several strategies to improve DET have been developed, such as deglycosylation36−41 or bioengineering of redox enzymes42,43 and electrode nanostructuration44,45 by using both carbon and metal nanomaterials (e.g., carbon nanotubes, graphene, metal nanoparticles, highly porous gold, etc.).46−50 Among the carbon nanomaterials, single-walled carbon nanotubes (SWCNTs) exhibit a cylindrical shepe with a © 2018 American Chemical Society
Received: July 12, 2018 Revised: September 26, 2018 Published: September 27, 2018 10279
DOI: 10.1021/acscatal.8b02729 ACS Catal. 2018, 8, 10279−10289
Research Article
ACS Catalysis
Figure 1. Enzymatic sequence of the subunit II (CYTFDH) and interaction with the anthracenyl moieties onto the electrode. Red and green highlights denote cysteine and methionine, respectively, and residues that coordinate the heme cs and hydrophobic motif are underlined.
(pH
