Mediatorless Direct Electron Transfer between Flavin Adenine

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Mediatorless Direct Electron Transfer between Flavin Adenine DinucleotideDependent Glucose Dehydrogenase and Single-Walled Carbon Nanotubes Hitoshi Muguruma, Hisanori Iwasa, Hiroki Hidaka, Atsunori Hiratsuka, and Hirotaka Uzawa ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b02470 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016

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Mediatorless Direct Electron Transfer between Flavin Adenine Dinucleotide-Dependent Glucose Dehydrogenase and Single-Walled Carbon Nanotubes Hitoshi Muguruma,*,† Hisanori Iwasa,‡ Hiroki Hidaka,† Atsunori Hiratsuka,‡ Hirotaka Uzawa,‡ †

Graduate School of Engineering and Science, Shibaura Institute of Technology, 3-7-5 Toyosu,

Koto, Tokyo 135-8548, Japan. ‡

Nanomaterials Research Institute, National Institute of Advanced Industrial Science and

Technology (AIST), Tsukuba Central 5-41, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

ABSTRACT The flavoenzymes flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and oxidase (FAD-GOx) do not undergo direct electron transfer (DET) at conventional electrodes, because the FAD cofactor is buried deeply (~1.4 nm) below the protein surface. We present a mediatorless DET between oxygen-insensitive 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 due to 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 multi-walled 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 mediatorless 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 FADGDH molecule is 4.5×5.6×7.8 nm, which is larger than the 1.2-nm diameter of an individual

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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 FADGDH molecules and debundled SWCNTs. We demonstrate that DET can be explained by quantum tunneling theory. Electrochemical experiments with various FAD-GDHs suggest (i) DET with debundling SWCNT can be applied to any type of FAD-GDH, (ii) the electrode with various kinds 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.

KEYWORDS: flavin adenine dinucleotide-dependent glucose dehydrogenase, direct electron transfer, single-walled carbon nanotube, debundling, biosensor

1. INTRODUCTION Electrochemical biosensors with biospecific enzyme reactions have become an active research area because they can help diabetes mellitus patients to monitor their daily sugar levels1 and are also used in environmental studies, agriculture, and the food industries. Glucose oxidase (GOx) is the most common enzyme used as a biological component in such applications.2 Direct electron transfer (DET) between enzyme and electrode (electron collector) is the main issue because it strongly affects the performance of the resulting devices. A cofactor of the widely used GOx is flavin adenine dinucleotide (FAD), which is tightly and deeply embedded within a structurally rigid glycoprotein shell,3 thereby making DET difficult. The classical approach to achieve DET is the utilization of electron transfer mediators such as ferrocene,4,5 osmium complexes,6 hydroquinone,7 and phenothiazine.8 Small and mobile mediators ferry electrons from FAD, which originates from enzyme-catalytic reaction, to the electron collector. We define mediated electron transfer (MET) as


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small and mobile mediators having the specific redox potential. However, some practical limitations in MET exist, such as the complicated procedure for the immobilization of mediators, and leaching of mediators, which reduces the sensitivity and causes poisoning problems. Therefore, a mediatorless approach to DET (i.e, non MET) has increased in popularity over the last few decades. One of the most promising strategies for mediatorless DET is based on nanocarbon materials9-11 such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), and graphenes. The different approaches to achieve mediatorless DET are that wiring or plugging with reconstruction of apo-GOx were conducted to achieve distances close to that required for DET.12-15 Another feasible approach was the combination of protein engineering and binding nanoparticles.16,17 However, the reported DET procedures still involve some problems: the elimination of oxygen or non-DET active GOx is required to achieve maximum performance (large turnover rate),12-17 and many cumbersome steps are unavoidable. Therefore, the motivation for this study is to develop DET with a simple and versatile strategy. GOx has substantial drawbacks in that its enzyme-catalytic reaction depends strongly on the concentration of dissolved oxygen. The enzymatic reaction of glucose dehydrogenase (GDH) is independent of the presence of oxygen. Unlike GOx, there are many varieties of GDH for the application of electrochemical biosensors. Nicotinamide adenine dinucleotide (NAD)-dependent GDH18-22 functions based on the redox reaction of a factor between the oxidized (NAD+) and reduced (NADH) forms. However, the drawbacks are (i) that exogenous addition is required, because the NAD cofactor is not bound to the enzyme, and (ii) high oxidation potential and the irreversible reaction of NADH are mitigating factors. Pyrroloquinoline quinone (PQQ)-dependent GDH has also been reported,23-28 although the water-soluble PQQ-GDH lacks selectivity, it is used to monitor blood sugar levels for the purpose of acute control (i.e., insulin injection) with type I diabetes. From a fundamental perspective, there is a variety of structures around the glucose binding site;29 therefore, the DET strategy is dependent on the type of PQQ-GDH. FAD-GDHs are potential


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candidates for many applications.30-41 Recently, the structure of FAD-GDH was reported,42 and it was revealed that the structures of the active sites (vicinity of FAD) in FAD-GOx3 and FAD-GDH42 were similar. Thus, the difficulty of DET in FAD-GDH is similar to that in FAD-GOx; however, the strategy for FAD-GOx can be directly applied to FAD-GDH. For example, glucose biosensors and biofuel cells based on MET of FAD-GDH with electron transfer mediators such as osmium complexes30,31,34,35 and phenothiazine,37 which have already been used in FAD-GOx, have been reported. In contrast, it has not been done on the mediatorless DET of FAD-GDH. We consider that there is a possibility of mediatorless DET between SWCNTs and FADGDH, firstly because Guiseppi-Elie et al.43 reported DET between SWCNTs and FAD-GOx, and secondly because oxygen-insensitive FAD-GDH provides evidence of DET without oxygen elimination. This is the first report of mediatorless DET between FAD-GDH and SWCNTs.

2. EXPERIMENTAL 2.1. Materials and Reagents. Distilled water, potassium dihydrogenphosphate, disodium hydrogenphosphate, D-glucose, L-ascorbic acid, uric acid, acetonitrile, and sodium cholate (SC) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Aspergillus niger FAD-GOx (EC 1.1.3.4, type VII-S) and NAD+ (oxidized form) was procured from Sigma (St. Louis, MO). Bacillus species NAD-GDH (EC 1.1.1.47), bovine serum albumin (BSA), and MWCNTs (diameter, 10–30 nm; average length, 1 µm) were obtained from Wako (Osaka, Japan). SWCNTs (1.2–1.7 nm diameter, 0.1–4 µm long, PureTubes powder) was purchased from Nanointegris (Boisbrin, Canada). Aspergillus oryzae FAD-GDH (EC 1.1.5.9, AoGDH) was produced by TOYOBO Co., LTD. (Osaka, Japan). Thermophilic filamentous fungus Thermoascus crustaceus FAD-GDH (TcGDH) was recently discovered by the authors’ group, and recombinant TcGDH was expressed by Escherichia coli (EcTcGDH) and Pichia pastoris (PpTcGDH).44 T. crustaceus FAD-GDH was also expressed by Saccharomyces cerevisiae (ScTcGDH). S. cerevisiae INVSc1 strain and pYES2 expression vector were purchased from Thermo Fisher Scientific (Waltham, USA). In both P. pastoris and S.


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cerevisiae expression systems, the α-factor secretion signal was fused to FAD-GDH, and FADGDH was secreted into the culture media. FAD-GDH was purified using methods reported elsewhere.44 The enzymes used in this research are summarized in Table S1 (Supporting Information) and the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results are shown in Fig. S1 (Supporting Information). The enzymes expressed by E. coli, P. pastoris, and S. cerevisiae have different glycoforms. 2.2. Electrode Preparation. The DET electrode has a sandwich-like structure and was fabricated by the layer-by-layer process shown in Fig. 1. The device was formed on sputtered Au. The width of the opening for the working electrode was 9 mm2. A plasma generator (Ulvac, VEP1000) was used to deposit a 2-nm-thick acetonitrile plasma-polymerized film (PPF) layer onto the Au layer. A SWCNT-SC solution was prepared with 0.15% SWCNT (w/v) and 2.0% SC in water (Fig. 1A). A SWCNT solution without SC was prepared with 0.1% SWCNTs dispersed in a 1:1 mixture of ethanol and water (Fig. 1B). A MWCNT-SC solution was prepared with 0.1% MWCNTs and 1.0% SC in water (Fig. 1C). Then, 3–5 µL aliquots of the CNT solutions were dropped onto the PPF surface and dried. The CNT-adsorbed surface was subsequently treated with acetonitrile plasma using the following parameters: power, 100 W; flow rate, 15 mL min-1; pressure, 0.6 Pa; exposure time, 30 s (thickness PpTcGDH > EcTcGDH (Fig. S1, Supporting Information). Removal of the glycan chain by treatment with glycosidase is one of the DET strategies with FAD-GDH30,31,37 and FADGOx.33,55,56 Expression by prokaryote E. coli, which has no ability of post translational modification, is equivalent to removal of the glycosylated layer. The molecular mass of GDH without a glycan chain is 58 kDa, which is almost the same as EcTcGDH. The apparent masses of glycan chains in PpTcGDH and ScTcGDH are 55–180% and 72–330% as much as peptide chains, respectively. The glycan chains are mainly composed of oligosaccharide chains of mannose and Nacetylglucosamine.57 Those oligosaccharide chains mainly connect residues of asparagine (N58, N129, N178, N187, N239, N256, N320, N341, N363, N387, N511) at the surface of GDH. The difference in the amount of glycan chains between PpTcGDH and ScTcGDH is due to the tendency of S. cerevisiae-expressed proteins to be hyper-glycosylated.57 The electrode fabrication process was the same as the AoGDH electrode. CVs of PPF/TcGDH/SWCNT-SC/PPF/Au are shown in Fig. 2A. Glucose concentration vs. current plots


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with the CV data are shown in Fig. S8 (Supporting Information). Currents were observed for all of the PPF/TcGDH/SWCNT-SC/PPF/Au electrodes. The large GCDC due to an increase in the polarized potential increased steeply and the current increase began at around +0.1 V without the appearance of the FAD/FADH2 redox peak at –0.45 V (Fig. 2A). The CV profiles and onset potentials (+0.1 V) for all of the PPF/TcGDH/SWCNT-SC/PPF/Au electrodes were similar to those for the PPF/AoGDH/SWCNT-SC/PPF/Au electrode (Fig. 1A’), which indicates that this procedure for mediatorless DET can be applied to any type of FAD-GDH. Figure 2B shows a comparison of the various enzyme electrodes (PPF/enzyme/SWCNT-SC/PPF/Au). The response current of the FAD-GDH electrodes (except ScTcGDH) is much larger than that of FAD-GOx. The response current of AoGDH and PpTcGDH electrodes is much larger than that of NAD-GDH electrodes. This is probably attributed to the difference nature of enzyme (e.g., catalytic ability) not to the amount of glycan chains. This indicates that FAD-GDH is the most suitable enzyme for applications of bioelectronics devices.

Figure 2

The much larger response of the glycoprotein PpTcGDH electrode than that of the deglycosylated protein EcTcGDH is unexpected because the common strategy for DET is deglycosylation to shorten the distance between the electrode and the cofactor FAD.25,30,34,55,56 A possible explanation is that one role of the glycan chains is stabilization of the tertiary structure of the protein. The deglycoprotein EcTcGDH is more fragile than the glycoprotein PpTcGDH. Thus, EcTcGDH positioned closely to a SWCNT may unfold and lose its activity. On the other hand, the response of the ScTcGDH electrode is much smaller than that of the PpTcGDH electrode. One explanation is that the number of DET-enzyme complexes in ScTcGDH electrode is less than that in the PpTcGDH electrode because the thicker glycan shell in ScTcGDH prevents SWCNT positioning close to FAD. The origin is the number of DET-enzyme complexes, and is not the distance between


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GDH and SWCNT. This is supported by the similar onset potentials (+0.1 V). If the distance was different, then the potential would be different (discussed in Section 3.3). It is concluded that FADGDH expressed by P. pastoris has so suitable amount of glycan chains for positioning close to FAD without unfolding. The recently reported FAD-cellobiose dehydrogenase58 is a structurally related enzyme to FAD-GDH, which suggests that this procedure can be applied to FAD-dehydrogenase enzymes for the construction of mediatorless DET. 3.3. Study of DET between FAD-GDH and SWCNTs. We have demonstrated that DET occurred for the combination of FAD-GDH and debundled SWCNTs, but did not occur for combination with bundled SWCNTs or debundled MWCNTs. In this section, the details of DET between FAD-GDH and SWCNTs are discussed. The recently reported structure of FAD-GDH is from Aspergillus flavus,42 of which the amino acid sequence perfectly corresponds to AoGDH. The entire sequence of TcGDH is different from that of A. flavus FAD-GDH (55.1% sequence identity); however, the partial sequence at a glucose binding site (vicinity of FAD) correspond perfectly to A. flavus FAD-GDH and AoGDH (HHYERNHHLW) in Table S2 (Supporting Information). Threedimensional structure modeling of TcGDH was performed based on the structure of A. flavus FADGDH using the SWISS-MODEL server,59 and the results are shown in Fig. S9 (Supporting Information). The structure and size of TcGDH were almost the same as those of A. flavus FADGDH with a global model quality estimation score of 0.76, which suggests that the modeled structure has high reliability. The calculated root mean square deviation (RMSD) for the distances between aligned Cα atom pairs of TcGDH and A. flavus FAD-GDH indicated only a few, short regions with high pairwise RMSD. Therefore, we can discuss DET between SWCNT and FADGDH based on the protein structural data of A. flavus FAD-GDH. The problem is how the individual SWCNT positions with respect to a single FAD-GDH molecule. The speculative scheme of piercing a balloon is not appropriate because the probability that FAD-GDH meets the edge of a 1-µm-long SWCNT is low. The feasible speculative scheme60 is that the side wall of the SWCNT is plugged into the groove of FAD-GDH, as shown in Figs. 3A


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and 3B, because the SWCNT axis is embedded on the surface of the electrode and FAD-GDH has a groove that a 1.2-nm-diameter individual SWCNT can enter into. The individual SWCNT can thus be close to 1.0 nm in distance from FAD positioned at the bottom of the groove (Figs. 3C and 3D). The 1.0-nm distance is sufficient for electron tunneling.61 Alternatively, a MWCNT with a diameter of greater than 10 nm, which is larger than the GDH molecule (near-plane structure), cannot be plugged into the groove (Fig. 3C). The bundled SWCNTs cannot be plugged into the groove either, because two bundled SWCNTs cannot pass through the gate of the groove with the size of an individual SWCNT. Furthermore, it is probable that the presence of glycan chains maintain the catalytic activity of FAD-GDH when the complex of SWCNT and FAD-GDH is formed. We think that the existence of driving force lets SWCNTs meet with specific groove position with an appropriate direction effectively. The driving force for the interaction of GDH and debundled SWCNT is attributed to π-π interaction between an individual SWCNT and the aromatic side chains of amino acids such as tyrosine, tryptophan, and phenylalanine.62 In fact, there are several amino acid residues with benzene rings (Y75, F79, Y221, F503, Y522) at the groove of the FAD-GDH molecule structure in Fig. S10 (Supporting Information). However, we do not think that those residues are related to DET firstly because there were no redox peaks due to benzene rings in CV data and secondly because π-electrons at benzene ring are isolated at the peptide chain in the enzyme.

Figure 3 Figure 4

TEM and AFM observations were performed to obtain evidence that the side wall of the SWCNT was plugged into the groove of FAD-GDH, as illustrated in Fig. 3. Figure 4 shows both TEM and AFM images with the 5-nm-wide wire-like structure and the 8–10 nm globular structure assigned as an individual SWCNT and a single GDH molecule, respectively. The sizes of observed GDH molecules were larger than the diameter of an individual SWCNT; therefore, the speculative


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scheme of an individual SWCNT plugged into the groove of a GDH molecule (Fig. 3) is reasonable for stable attachment. The observed sizes of individual SWCNTs (5 nm) were larger than their real sizes (1.2 nm) in both images. The reason for the TEM image (Fig. 4A) is attributed to staining of uranyl acetate. In Fig. S11 (Supporting Information), the size of the non-modified SWCNT observed using TEM corresponds to the diameter of an individual SWCNT. In the AFM image (Fig. 4B), the AFM tip used (~10 nm size) was out of the resolution limit for the object in the horizontal direction. On the other hand, the height from the cross-sectional profile in Fig. S12 (Supporting Information) corresponds to the diameter of an SWCNT. We investigate the same size protein, bovine serum albumin (BSA), as a control study in Fig. S13 (Supporting Information). The BSA molecule was not observed on debundled SWCNT. Therefore, we think that the Fig. 4 proves the adsorption of FAD-GDH onto SWCNTs by our proposed scheme. It is concluded that (i) the SWCNT bundles are completely dispersed into individual SWCNTs by the surfactant (no small bundled SWCNTs), and (ii) an individual SWCNT is plugged into the groove of GDH. We propose a DET model (Fig. 5A) according to quantum tunneling theory derived from the Schrödinger equation:63 −1

 2πV 2 sinh 2 h −1 [2m (V − η )]1 / 2 d  T = 1 +  , 4η (V − η )  

(5)

where T is the tunneling probability, h is the Planck constant, V is the barrier height for electron tunneling DET, η is the overpotential, and d is the distance between the SWCNT and FAD. η is a controllable parameter in electrochemical measurements. T is related to the electron transfer rate; therefore, the observed current I, can be represented with Eq. (5) as: I = I0T(η),

(6)

where I0 is the exchange current, which is determined by various conditions of the electrode, such as the activity of the enzyme and the electric double layer. Linear sweep voltammetry (LSV) with the addition of 48 mM glucose is adopted to obtain the η-I data shown in Fig. 5B. The redox potential of FAD (–0.45 V) is defined as η = 0 V. The barrier height for electron tunneling DET is


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the protein shell, so V is determined to 1.4 V.61 The data calculated using Eqs. 5–6 almost correspond to the LSV data, which suggests that the DET quantum tunneling model is feasible. d is strongly correlated with the onset potential in the I-η characteristics Xiao et al.12 reported that the length of a wiring spacer of reconstructed GOx affected η (onset potential). In the present case, a value of d = 0.95 nm produces results consistent with the experimental observations. The onset potential for all of the FAD-GDHs was +0.1 V, which suggests that the SWCNT is positioned at a distance of around 1 nm from FAD. This is supported by the similar structures and sizes of AoGDH and TcGDH. The tunneling effect is the reason why they do not observe DET at an onset potential near to that of the FAD within GDH. To the contrast, MET can facilitate at lower onset potential (ca. -0.2 V)31 because low molecular mass mediator can move close to FAD and get electron at the specific potential.

Figure 5

The onset potential in the present experiment is similar to that of the FAD-GOx wiring electrode aligned with a CNT.13 The onset potential of other DET electrodes, such as PQQ-wiring,15 rotaxane-wiring,14 and GOx-Au nanoparticles,16 were –0.1, –0.2, and –0.4 V, respectively. The calculation shows that the difference in the onset potential is well represented by d than by I0 (see red and green dashed lines in Fig. 5B). From these results and reports, it is reasonable that the decrease in the onset potential is attributed to a shorter d for DET. It is concluded that mediatorless DET is completed and the mechanism for mediatorless DET can be explained by quantum tunneling theory. 3.4. Sensor performance. The sensor performance of the PPF/AoGDH/SWCNTSC/PPF/Au electrode is demonstrated in this section. Amperometric measurements at fixed potential are widely used to evaluate and analyze the performance of PPF/AoGDH/SWCNTSC/PPF/Au as a glucose biosensor. Figure 6A shows the steady-state amperometric response of the


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PPF/AoGDH/SWCNT-SC/PPF/Au electrode at +0.6 V vs. Ag/AgCl. The potential at +0.6 V is available for time-based measurements with a fixed potential. The sequential increase in the glucose concentration at regular intervals is observed, of which the range of glucose concentrations can cover the physiological range. The small background current compared to the glucose response is a significant characteristic in the present results that indicates there is no need to perform background subtraction for the glucose measurement. Because of the artifact and drift at higher glucose concentration, it can be seen as if the current response didn’t reach a stable step during sequential glucose addition. We confirmed reaching the stable steps. The response time was less than 7 s. The detection characteristics at very low concentrations (2.5–30 µM) were excellent (Fig. 6B) because AoGDH has higher enzymatic activity than other enzymes such as FAD-GOx and NAD-GDH. The detection limit was 0.83 µM when the signal-to-noise (S/N) ratio was 3. The effect of interferent compounds such as ascorbic acid and uric acid on the sensing characteristics (Fig. 6C) was negligible for use with samples in the physiological range. Figures 6D and 6E show the current vs. glucose concentration based on the data from Figs. 6A and 6B. The sensitivity determined from the slopes obtained with the PPF/AoGDH/SWCNTSC/PPF/Au electrode was 110 µA mM-1 cm-2 (r = 0.98 in the linear range of 0.05–2.5 mM). The sensitivity can also be evaluated at much lower concentrations (2.5–35 µM, Fig. 6E). The wide dynamic range from lower concentrations is a specific characteristic of FAD-GDH biosensors. The sensitivity to physiological glucose levels was 7.1 µA mM-1 cm-2 (r = 0.98 in the linear range of 5.2–26 mM). Saturation from linearity is observed at higher (>10 mM) glucose concentrations, which represents a typical characteristic of the Michaelis-Menten model. This is a reactioncontrolled step to which the Michaelis-Menten analysis can be applied. Figure 6F shows a Lineweaver-Burk plot (double reciprocal plot), from which the apparent Michaelis-Menten activity (KMapp) can be calculated as an indication of the enzyme-substrate kinetics for the biosensor: 1 K Mapp 1 1 = + , I I max C I max


(7)

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where I is the steady-state current, Imax is the maximum current under stationary substrate conditions, KMapp is the apparent Michaelis constant, and C is the glucose concentration. Imax and KMapp were obtained from extrapolation of the plot. KMapp for immobilized AoGDH was estimated to be 4.4 mM, which is smaller than that for AoGDH in solution (29 mM). It is well-known that immobilized enzymes have increased affinity toward the substrate. The large Imax (0.67 mA cm-2) indicates a highly effective electronic contact. Finally, it was confirmed that the electrochemical response of the devices that retained a current response due to continuous polarization at +0.6 V in the presence of 9.6 mM glucose was greater than 90% of the initial current after 24 h, as shown in Fig. S14 (Supporting Information).

Figure 6 Table 1

Table 1 shows a comparison of the characteristics and performance of the fabricated FADGDH biosensors are compared with those for other FAD-GDH biosensors. Since the biosensors with FAD-GDH have appeared recently, the comparative data is for the first time. It is confirmed that the present sensor based on mediatorless DET exhibited better performance compared with the other FAD-GDH biosensors based on MET in most statements. The fact that the polarized potential at mediatorless DET sensing (+0.6V) is larger than that at MET sensing is disadvantage. Nevertheless, the present DET biosensor is not affected by interferants in the physiological range (Fig. 6C). Furthermore, we confirmed that there is a possibility of performing at lower potential (+0.2V) based on CV data (Fig. S15, Supporting Information). This is attributed to the large glucose response current with FAD-GDH/SWCNT electrode. However, MET involves some disadvantages such as the complicated procedure and harmful chemicals. Therefore, our procedure of mediatorless DET between debundled SWCNT and FAD-GDH can overcome MET sensing.

4. CONCLUSION


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An actual example of mediatorless DET between glycosylated FAD-GDH and debundled SWCNTs is presented. The enzyme electrode is formed by a simple layer-by-layer process in which a droplet of SWCNT-surfactant solution is placed onto a current collector and allowed to evaporate, followed by a droplet of enzyme solution onto the SWCNT layer. A GCDC was observed for the electrode with a combination of FAD-GDH and debundled SWCNTs, whereas no such currents were observed with bundled SWCNTs or debundled MWCNTs. Therefore, clear evidence of mediatorless DET between a glycosylated FAD-GDH and debundled SWCNTs was presented for the first time. The three-dimensional globular FAD-GDH molecule is 4.5×5.6×7.8 nm, which is larger than the 1.2-nm diameter of an individual SWCNT and is smaller than the 10-nm diameter of an individual MWCNT and bundled SWCNTs. Only debundled SWCNTs can be plugged into the groove of FAD-GDH to a distance within 1.0 nm from FAD while maintaining catalytic activity. TEM and AFM observations support the configuration of the FAD-GDH and debundled SWCNT complex. Electrochemical measurements showed that the current output due to an increase of the polarized potential increases steeply (+0.1 V of onset) without the appearance of a FAD redox peak at –0.45 V, which indicates that DET can be explained by a quantum tunneling model. Electrochemical measurements with various FAD-GDHs suggest (i) mediatorless DET between any type of FAD-GDH and debundled SWCNTs is possible, (ii) FAD-GDH electrodes exhibit superior properties, i.e., the response of the FAD-GDH electrodes is much larger than that of FAD-GOx and NAD-GDH electrodes, and (iii) glycan chains present on FAD-GDH prevent denaturation when the SWCNT is close to FAD. An optimized glucose biosensor had a high sensitivity of 110 µA mM-1 cm-2, a broad linear dynamic range of 0.05–3.2 mM, selectivity toward an interferent, a fast response time of 7 s, and a low detection limit of 0.83 µM. The advantage of the proposed method is its simple requirements that it can be extended and combined for the versatile utilization of biological devices and systems without practical limitations.

ASSOCISTED CONTENT


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxx/acscatal. SDS-PAGE of GDH, amino acid sequences of reaction center of GDH, optimization of surfactant, glucose concentration vs. current plot of CV data in Figs. 1 and 2, background current in CV, DET of other SWCNT, qualitative estimation of enzyme structure, various FAD-GDH data, sequence at glucose binding site, TEM image of debundled SWCNTs, AFM height profile of GDH and debundled SWCNT complex, representation of aromatic side chains of amino side in AoGDH interacting with SWCNT (PDF), stability.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax: +81-3-5859-8201; Notes The authors declare no competing financial interest.


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Figure captions Figure 1. Evidence of DET between FAD-GDH and SWCNT. (A–C) Optical and AFM images of CNT layer on AN PPF. Solution for drop-cast formation of CNT solution: (A) SWCNT-SC; (B) solo SWCNT (control); (C) MWCNT-SC (control). Scale bars in the optical microscopy images correspond to 250 µm. Horizontal scales of AFM images are all 1×1 µm. Vertical scales are (A) 20 nm, (B) 1000 nm, and (C) 100 nm. Surface roughness values (Rrms) are (A) 1.6 nm and (C) 10.5 nm. (A’-C’) AFM image and CV profiles of the resulting AoGDH electrodes. Horizontal scales of AFM images are all 1×1 µm. Vertical scales are (A,B) 20 nm and (C) 100 nm. Rrms are (A) 1.2 nm, (B) 1.1 nm, and (C) 7.5 nm. Sweep rate: 50 mV s-1. Glucose concentrations: 0 (black) and 48 mM (blue). pH 7.4 phosphate buffer solution. (A”–C”) CV profiles of the resulting GOx electrodes as a control. The solid and dashed lines represent the presence and the absence of oxygen, respectively. The conditions are the same as A’–C’. Figure 2. (A) CV profiles for PPF/GDH/SWCNT-SC/PPF/Au electrodes with AoGDH and T. crustaceus FAD-GDHs expressed with respect to the various host. Glucose concentrations: 0 and 48 mM. Sweep rate: 50 mV s-1. pH 7.4 phosphate buffer solution. The dashed line represents the background current. (B) Comparison of PPF/enzyme/SWCNT-SC/PPF/Au electrodes for response to 48 mM glucose at +0.6 V. Abbreviations for enzymes are given in the Experimental section. Figure 3. Speculative scheme of DET between FAD-GDH and SWCNT. The diameter of a SWCNT represents 1.2 nm. (A) Vertical and (B) side views with respect to the direction of the SWCNT-axis. (C) Enlargement of the area indicated by the green box in (A). (D) Enlargement of the area indicated by the green box in (C). Protein data was obtained from PDB file 4YNT,42 and the surface model was drawn with PyMOL.60 Figure 4. (A) TEM and (B) AFM images of FAD-GDH attachment to an individual SWCNT. (A) The SWCNT surfactant solution (20 µg/mL) was dropped onto a TEM grid and dried. AoGDH solution (0.2 µM) was then dropped onto the TEM grid and dried. Subsequently, uranyl acetate solution was dropped onto the TEM grid and dried. Magnification: 62000×. (B) The SWCNT


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surfactant solution (0.15 mg/mL) was dropped on a silicon substrate modified with acetonitrile PPF. AoGDH solution (0.5 mg/mL) was subsequently dropped onto the acetonitrile PPF and dried. Horizontal scale: 200×200 nm. Vertical scale: 5 nm. Figure 5. (A) DET model with quantum tunneling. (B) Linear wave voltammetry for PPF/AoGDH/SWCNT-SC/PPF/Au and PPF/PpTcGDH/SWCNT-SC/PPF/Au electrodes in the presence of 48 mM glucose. Sweep rate: 5 mV s-1. pH 7.4 phosphate buffer solution. V = 1.4 V. a is an arbitrary constant. Figure 6. (A) Time-current response for sequential glucose (G) addition at concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.39, 0.49, 0.59, 0.82, 1.1, 1.3, 1.5, 1.9, 2.4, 2.8, 3.2, 3.9, 4.6, 5.2, 5.8, 7.7, 9.5, 13, 17, 19, 23, 26, 33, 38, 44, and 48 mM with the PPF/AoGDH/SWCNT-SC/PPF/Au electrode. (B) Timecurrent response for sequential glucose (G) addition at very low concentrations. (C) The effect of an interferent species (0.1 mM ascorbic acid (AA), 0.1 mM and 0.1 mM uric acid (UA)) on the response at the PPF/AoGDH/SWCNT-SC/PPF/Au electrode. The concentrations of glucose (G) were sequentially 2.5 and 4.9 mM. The polarization potential was +0.6 V vs. Ag/AgCl with an electrolyte of pH 7.4 20 mM phosphate buffer solution. (D) Calibration plot for glucose response using the data in A and B. The sensitivities of the electrodes were 110 µA mM-1 cm-2 (r = 0.98 in the linear range of 0.05–3.2 mM) and 7.1 µA mM-1 cm-2 (r = 0.98 in the linear range of 5.2–26 mM), respectively. (E) Calibration plot in the very low concentration range. (F) A Lineweaver-Burk plot (Imax = 0.64 mA cm-2, KMapp = 3.1 mM, r=0.99) using the data presented in D. Table 1. Comparison of the Performance Parameters of Glucose Biosensors based on FAD-GDH.


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-20 -20 0.0 0.4 0.8 -0.4 0.0 0.4 0.8 -0.4 0.0 0.4 0.8 Potential (V) Potential (V) Potential (V) Figure 1. Evidence of DET between FAD-GDH and SWCNT. (A–C) Optical and AFM images of CNT layer on AN PPF. Solution for drop-cast formation of CNT solution: (A) SWCNT-SC; (B) solo SWCNT (control); (C) MWCNT-SC (control). Scale bars in the optical microscopy images correspond to 250 mm. Horizontal scales of AFM images are all 1×1 mm. Vertical scales are (A) 20 nm, (B) 1000 nm, and (C) 100 nm. Surface roughness values (Rrms) are (A) 1.6 nm and (C) 10.5 nm. (A’-C’) AFM image and CV profiles of the resulting AoGDH electrodes. Horizontal scales of AFM images are all 1×1 mm. Vertical scales are (A,B) 20 nm and (C) 100 nm. Rrms are (A) 1.2 nm, (B) 1.1 nm, and (C) 7.5 nm. Sweep rate: 50 mV s-1. Glucose concentrations: 0 (black) and 48 (blue) mM. pH 7.4 phosphate buffer solution. (A”–C”) CV profiles of the resulting GOx electrodes as a control. The solid and dashed lines represent the presence and the absence of oxygen, Paragon respectively. The conditions are the ACS same as A’–C’. Plus Environment -0.4

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0.0 0.4 0.8 Potential (V) Figure 2. (A) CV profiles for PPF/GDH/SWCNTSC/PPF/Au electrodes with AoGDH and T. crustaceus FADGDHs expressed with respect to the various host. Glucose concentrations: 0 and 48 mM. Sweep rate: 50 mV s-1. pH 7.4 phosphate buffer solution. The dashed line represents the background current. (B) Comparison of PPF/enzyme/SWCNT-SC/PPF/Au electrodes for response to 48 mM glucose at +0.6 V. Dashed line represents background current. Abbreviations for enzymes are given in the Experimental section.


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L423

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Graphene Figure 3. Speculative scheme of DET between FAD-GDH and SWCNT. The diameter of a SWCNT represents 1.2 nm. (A) Vertical and (B) side views with respect to the direction of the SWCNT-axis. (C) Enlargement of the area indicated by the green box in (A). (D) Enlargement of the area indicated by the green box in (C). Protein data was obtained from PDB file 4YNT,42 and the surface model was drawn with PyMOL.60


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Figure 4. (A) TEM and (B) AFM images of FADGDH attachment to an individual SWCNT. (A) The SWCNT surfactant solution (20 mg/mL) was dropped onto a TEM grid and dried. AoGDH solution (0.2 mM) was then dropped onto the TEM grid and dried. Subsequently, uranyl acetate solution was dropped onto the TEM grid and dried. Magnification: 62000×. (B) The SWCNT surfactant solution (0.15 mg/mL) was dropped on a silicon substrate modified with acetonitrile PPF. AoGDH solution (0.5 mg/mL) was subsequently dropped onto the acetonitrile PPF and dried. Horizontal scale: 200×200 nm. Vertical scale: 5 nm.


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Figure 5. (A) DET model with quantum tunneling. (B) Linear wave voltammetry for PPF/AoGDH/SWCNT-SC/PPF/Au and PPF/PpTcGDH/SWCNT-SC/PPF/Au electrodes in the presence of 48 mM glucose. Sweep rate: 5 mV s-1. pH 7.4 phosphate buffer solution. V = 1.4 V. a is an arbitrary constant.


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Figure 6. (A) Time-current response for sequential glucose (G) addition at concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.39, 0.49, 0.59, 0.82, 1.1, 1.3, 1.5, 1.9, 2.4, 2.8, 3.2, 3.9, 4.6, 5.2, 5.8, 7.7, 9.5, 13, 17, 19, 23, 26, 33, 38, 44, and 48 mM with the PPF/AoGDH/SWCNT-SC/PPF/Au electrode. (B) Time-current response for sequential glucose (G) addition at very low concentrations. (C) The effect of an interferent species (0.1 mM ascorbic acid (AA), 0.1 mM and 0.1 mM uric acid (UA)) on the response at the PPF/AoGDH/SWCNT-SC/PPF/Au electrode. The concentrations of glucose (G) were sequentially 2.5 and 4.9 mM. The polarization potential was +0.6 V vs. Ag/AgCl with an electrolyte of pH 7.4 20 mM phosphate buffer solution. (D) Calibration plot for glucose response using the data in A and B. The sensitivities of the electrodes were 110 mA mM-1 cm-2 (r = 0.98 in the linear range of 0.05–3.2 mM) and 7.1 mA mM-1 cm-2 (r = 0.98 in the linear range of 5.2–26 mM), respectively. (E) Calibration plot in the very low concentration range. (F) A LineweaverBurk plot (Imax = 0.64 mA cm-2, KMapp = 3.1 mM, r=0.99) using the data presented in D.


ACS Catalysis

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Table 1. Comparison of the Performance Parameters of Glucose Biosensors based on FAD-GDH Sensitivity Linear Detection Applied Response K app M Origin of Principle (mAmM-1 range limit potential time (mM) Ref. FAD-GDH (mM) (mM) (V) (s) cm-2) 0.05-3.2 0.83 4.4 This work DET 7 SWCNT/SC 0.6 A. oryzae 110 N/A-30 10 65 MET 200 (33) Fe(CN)630.5 A. terreus 5.3 0.005-18 3.4 14 N/A (35) Osmium polymer G. cingulata MET 0.175 30 0.5-12 N/A 2.6 MET N/A (37) Phenothiazine 0.1 N/A 42 0.07-0.62 4.2 3.1 MET 60 (38) Phenazinea 0.05 A. oryzae 70 aPhenazine methyl sulfate Functional material for electrode


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ACS Catalysis

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Table of Contents

Direct electron transfer between FAD-GDH and SWCNT Glucose 20 nm

e-

Surfactant

SWCNT 200 SWCNT

150 100 50 0

(mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(V) -0.4 0.0 0.4 0.8


Mediatorless Direct Electron Transfer between Flavin Adenine

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