Step-by-Step Assembled Enzyme–Polymer–Carbon Nanotubes for

Jun 10, 2019 - Protein-conjugated single-walled carbon nanotubes (SWCNTs) have received much attention for their diverse applications in molecular bio...
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Step-by-Step Assembled Enzyme−Polymer−Carbon Nanotubes for Solution-Processed Bioreactive Composites Hsiu-Pen Lin,†,⊥ Jun Akimoto,† Yaw-Kuen Li,*,⊥,§ Yoshihiro Ito,*,†,# and Masuki Kawamoto*,†,#,∥

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Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ⊥ Department of Applied Chemistry, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan § Center for Emergent Functional Matter Science, National Chiao Tung University, Hsinchu 30010, Taiwan # Nano Medical Engineering Laboratory, RIKEN Center for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ∥ Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan S Supporting Information *

ABSTRACT: Protein-conjugated single-walled carbon nanotubes (SWCNTs) have received much attention for their diverse applications in molecular biology. Intrinsically waterinsoluble SWCNTs avoid conjugation with proteins, which leads to limited availability of biomolecule−nanocarbon composites. Because protein functions are directly affected by assembled structures, the synthesis of heterogeneous composites with bioreactive responses is a great challenge. We demonstrate that step-by-step assembled enzyme/ polymer/SWCNTs are obtained by using noncovalentbonding methodologies in aqueous media. A multifunctional polymer containing aromatic, cationic, and redox-active units allows for a direct aqueous dispersion of SWCNTs through π interactions and a subsequent charge attraction to the enzyme, which yields the ternary composites. The resulting composites show bioreactive responses in enzyme-conjugated SWCNT networks. The solution-processed glucose oxidase (GOx)/polymer/SWCNT composite displays a high current density of 1420 μA cm−2 by enzymatic oxidation of glucose. Only 2.4 μg of GOx is shown to be necessary for the enzymatic reaction with a sensitivity of 72 μA mM−1 cm−2. This high sensitivity results from the assembled structure through noncovalent-bonding interactions. We demonstrate that the bioreactive composite allows energy conversion from a glucose-including beverage (cola) to electricity. Lactate oxidase-driven bioreactivity also takes place on the structurally organized composite. This step-by-step methodology would be beneficial for enzyme-assisted energy conversion nanocomposites. KEYWORDS: single-walled carbon nanotubes, self-assembly, multifunctional polymers, noncovalent modification, bioreactive composites

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as a “soft” dispersion approach.6−9 When dispersants, such as surfactants, polymers, and biomaterials, are attached to the surface of SWCNTs through hydrophobic or delocalized π interactions or both, the resulting dispersant−SWCNT composite exhibits homogeneous dispersion without deformation of the sp2-bonded carbon atoms. The dispersed solutions allow for solution-processed SWCNT composites. We have developed novel functional dispersants of fullerene-based nanoparticles for photoelectric conversion at air mass 1.5,10 as well as thermally cleavable polythiophenes for substrate-free thermoelectrics.11 Protein-conjugated SWCNTs have been developed for a wide range of biological applications including drug delivery,12 tissue engineering,13 imaging,14 biofuel cells,15 and sensors.16

INTRODUCTION Single-walled carbon nanotubes (SWCNTs) are one-dimensional nanocarbons with exceptional electrical conductivity, mechanical strength, and chemical durability.1,2 These characteristics have contributed to next-generation technologies, including lightweight and flexible energy-conversion devices.3,4 SWCNTs also exhibit high surface areas, which result from an extended hexagonal lattice of sp2-bonded carbon atoms. The adhesion of functional materials at the SWCNT surface yields SWCNT-based composites that show catalytic activities and the adsorption of gases.5 Despite the diverse applications of SWCNTs, they also have some drawbacks. Because the pristine SWCNTs exhibit poor dispersion abilities, precipitation occurs immediately in most organic solvents. The surface of SWCNTs is hydrophobic, which leads to unfavorable aggregation in aqueous media resulting in limited biological applications. Noncovalent bonding functionalization of SWCNTs has been categorized © 2019 American Chemical Society

Received: April 25, 2019 Accepted: June 10, 2019 Published: June 10, 2019 4323

DOI: 10.1021/acsanm.9b00769 ACS Appl. Nano Mater. 2019, 2, 4323−4332

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ACS Applied Nano Materials

Figure 1. (a) Chemical structure of the random copolymer PVImQOs. (b) Step-by-step processing of a GOx/PVImQOs/SWCNT composite in aqueous media.

mediator for efficient electron transfer from GOx to SWCNTs. We also expect that enzyme-conjugated SWCNT networks contribute to good electron transport to an electrode, which results in a highly efficient bioreactive system.

For example, glucose oxidase (GOx)−CNT hybrid biomaterials have been widely investigated for electrochemical sensing of glucose over the past decade.17,18 As mentioned above, homogeneous dispersion of SWCNTs is necessary for a heteroassembly of the biomolecule−nanocarbon composite. Furthermore, the intrinsic activity of the protein is also crucial for biologically reactive (bioreactive) responses after conjugation to SWCNTs. An assembled structure of a composite is considered as one of the most essential steps toward efficient responses.19 Nishizawa et al. reported that assembled enzyme− SWCNT composites show high-performance biocatalytic reactions for biofuel cells.20,21 Adsorption of GOx at the surface of aligned SWCNTs prepared by chemical vapor deposition yielded a self-regulating enzyme−SWCNT composite. The resulting composite exhibited effective electron transfer to an electrode through the aligned SWCNTs after enzymatic oxidation of glucose. Unfortunately, highly specific techniques through chemical vapor deposition are necessary for fabrication of the aligned SWCNTs.22 In this study, we report the first example of step-by-step assembled enzyme/polymer/SWCNTs using a polymer dispersant for solution-processed bioreactive composites. The multifunctional polymer dispersant of the random copolymer PVImQOs contains an imidazole group as an aromatic unit, an imidazolium group as a cationic unit, and an osmium (Os) complex as a redox-active unit (Figure 1a). For the step-bystep methodology, we expect the following interactions to achieve a GOx/PVImQOs/SWCNT composite: (i) the aromatic imidazole and cationic imidazolium units are attached to the SWCNT surface through delocalized π interactions, which leads to an aqueous dispersion of the PVImQOs/ SWCNT composite and (ii) direct assembly of GOx and PVImQOs on SWCNTs occurs by charge attraction between the positively charged imidazolium unit in the polymer and the negatively charged GOx (Figure 1b). The dispersed solution facilitates the solution-processed bioreactive composite. If GOx in the composite shows catalytic oxidation of glucose, a redoxactive unit of an Os complex in PVImQOs would act as a

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RESULTS AND DISCUSSION

Synthesis and Characterization of Multifunctional Polymers. Full synthesis details are provided in the Supporting Information. Radical polymerization of 1-vinylimidazole using 2,2′-azobis(isobutyronitrile) (AIBN; 1.1 mol % of the monomer) in toluene produced poly(1-vinylimidazole) (PVIm). From the 1H NMR spectra of PVIm in D2O (see Figure S12 in Supporting Information), the polymerization proceeded successfully, because the peak of the vinyl group of 1-vinylimidazole at 5.25 ppm diminished in Figure S11 (Supporting Information). The viscosity-average molecular weight (Mv) was estimated to be 40 000 using a viscometer. Partially quaternized PVIm (PVImQ) was synthesized using 2-(2-chloroethoxy)ethanol in ethanol at 70 °C for 24 h under nitrogen. 1H NMR spectra revealed that quaternization of PVIm occurred owing to the observance of a methylene group at 3.45−3.81 ppm in D2O (see Figure S13 in Supporting Information). The ratio of imidazole and imidazolium units in PVImQ was estimated to be 0.79:0.21 from the integration of peak a and peaks d−g. Further modification of PVImQ was achieved by the conjugation of osmium bis(2,2′-bipyridine)chloride (Os(bpy)2Cl2) to the imidazole group. Because the aromatic peaks of 2,2′-bipyridine (peak h) in Os(bpy)2Cl2 overlapped with those of the imidazole and imidazolium groups (peak c) (see Figure S14 in Supporting Information), the loading ratio of Os(bpy)2Cl2 in the polymer was carefully estimated (see Supporting Information). We proposed that PVImQOs had the following properties: (i) the Os complex in PVImQOs exhibited a specific absorption spectrum with the maximum wavelength, λmax, at 296 nm, which corresponded to the ligand-centered π−π* transition of the bipyridine group, and (ii) the conjugation of Os(bpy)2Cl2 took place only with the imidazole 4324

DOI: 10.1021/acsanm.9b00769 ACS Appl. Nano Mater. 2019, 2, 4323−4332

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ACS Applied Nano Materials group of PVImQ. From the Beer−Lambert law and 1H NMR spectra of PVImQ, the loading ratio of the Os complex in PVImQOs was estimated to be 0.05 (Figure 1a). Figure S1 (Supporting Information) shows photographs and absorption spectra of the polymers in water. λmax of PVIm was 208 nm, which was attributed to the aromatic imidazole unit (see Figure S1b(i) in Supporting Information).23 The absorption spectrum of PVImQ was similar to that of PVIm even after quaternization (see Figure S1b(ii) in Supporting Information). Because the optical band gap corresponding to the π−π* transition of the imidazolium unit in water (6.9 eV, 180 nm) is larger than that of the imidazole unit in water (6.0 eV, 207 nm),24 the imidazolium unit in PVImQ could not be detected with our experimental conditions. In contrast, the absorption spectrum of PVImQOs exhibited clear electronic structures between 250 and 600 nm, which resulted in a colored solution (see Figure S1a(iii),b(iii) in Supporting Information). The electronic transitions observed in the visible region were attributed to metal-to-ligand charge transfer (MLCT) in the Os complex.25 In comparison with the absorption spectrum of Os(bpy)2Cl2 in water (see Figure S1b(iv) in Supporting Information), the Os complex was successfully introduced into the polymer backbone. Aqueous Dispersion Behavior of SWCNTs Using Water-Soluble Polymers. We investigated the aqueous dispersion behavior of SWCNTs (the index of chirality (n,m), (6,5)) using water-soluble polymers (Figure 2). Raw SWCNTs (0.1 mg mL−1) in water showed precipitation, which resulted in no light absorption (Figure 2a(i),b(i)). Unfortunately, PVIm showed a poorer ability to disperse SWCNTs in water; after ultrasonication for 15 min, most of the mixtures

yielded a precipitation of SWCNTs in water, and the upper limit of the dispersed PVIm/SWCNTs was estimated to be 1.0:0.2 w/w. In contrast, aqueous-dispersed solutions of PVImQ/SWCNTs were successfully obtained after ultrasonication (Figure 2a(iii)). Absorption spectra exhibited characteristic electronic structures of (6,5)SWCNTs corresponding to the first (E11; 1023 nm) and second (E22; 588 nm) optical transitions (Figure 2b(iii)). 26 Changes in the absorption spectra of PVImQ/SWCNTs in water were consistent with the concentrations of SWCNTs (see Figure S2a in Supporting Information). To explore the dispersion ability of SWCNTs, the relationship between absorbance and SWCNT concentration was investigated using the Beer− Lambert law (see Figure S2b in Supporting Information). The Beer−Lambert law represents a linear relationship between the absorbance and concentration of a medium and is defined as A = εcl, where A, ε, c, and l are the absorbance, molar absorption coefficient, concentration, and optical path length, respectively.27 Linear relationships between A and c were observed until the PVImQ/SWCNT composite contained 1.2 wt % SWCNTs, with coefficients of determination (R2) > 0.981 for the E11 and E22 optical transitions. A change in the slope occurred when the concentration of SWCNTs exceeded 1.2 wt % (see Figure S2b in Supporting Information). This observation arose from the saturated dispersion of SWCNTs in water.10 We also observed that the imidazolium group is crucial for the dispersion of SWCNTs, because a cationic group not only provides a hydrophilic property but also facilitates cation−π interactions28 on the SWCNT surface, which results in efficient dispersion behavior. PVImQOs also exhibited an ability to disperse SWCNTs in water (Figure 2a(iv)). The dispersed solution of the PVImQOs/SWCNTs (1.0:0.2 w/w/w) composite remained unchanged even after 7 days (see Figure S3 in Supporting Information). From the concentration dependence of the absorption spectra (Figure 2c), a linear relationship between A and c was maintained until 0.8 wt % SWCNTs with R2 > 0.996 (Figure 2d). This observation reveals the saturating concentration owing to the aggregated PVImQOs/SWCNTs composite in water. The upper limit of the dispersed concentration for PVImQOs/SWCNTs was lower than that for PVImQ/SWCNTs. These results suggested that the conjugated Os complex in PVImQOs may exhibit less activity for an aqueous dispersion of SWCNTs. Step-by-Step Assembly of Enzyme/Polymer/SWCNT Composite. GOx/PVImQOs/SWCNTs ternary composite was successfully prepared by step-by-step assembly (Figure 3a). Ultrasonication of raw SWCNTs (0.1 mg mL−1) and PVImQOs (0.5 mg mL−1) in water yielded the PVImQOs/ SWCNT (1.0:0.2 w/w) composite. Following the addition of GOx (0.4 mg mL−1), a ternary composite of GOx/PVImQOs/ SWCNTs (0.8:1.0:0.2 w/w/w) in phosphate buffer (pH 7.4) was observed as a homogeneous dispersion. Because GOx possesses negative charges at pH 7.4,29 electrostatic attraction occurred for the positively charged PVImQOs on the surface of SWCNTs. SWCNT-based samples were prepared by drop-casting asprepared solutions on silicon (Si) substrates. The SEM images showed different surface morphologies (Figure 3b). Raw SWCNTs displayed lumpy surfaces owing to aggregation (Figure 3b(i)). In contrast, network structures of the PVImQOs/SWCNT (1.0:0.2 w/w) composite with a diameter of 10−30 nm were observed (Figure 3b(ii)). Because the

Figure 2. Photographs (a) and absorption spectra (b) of (i) raw SWCNTs, (ii) PVImQOs (0.5 mg mL−1), (iii) PVImQ/SWCNT (1.0:1.2 w/w) composite, and (iv) PVImQOs/SWCNT (1.0:0.8 w/ w) composite in water. (c) Absorption spectra of PVImQOs/ SWCNT composites at various concentrations of SWCNTs in water. (d) Absorbance at 588 nm (blue) and 1023 nm (red) as a function of the concentration of SWCNTs. 4325

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Figure 3. (a) Preparation of the dispersed solution of GOx/PVImQOs/SWCNTs. (b) SEM images of (i) raw SWCNTs, (ii) PVImQOs/SWCNT (1.0:0.2 w/w) composite, and (iii) GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite on Si substrates.

Figure 4. (a) Schematic illustration of enzymatic and redox reactions for the GOx/PVImQOs/SWCNT composite. (b) Cyclic voltammograms of composites in 20 mM phosphate buffer (pH 7.4). (c) Electrocatalytic responses of composites upon adding 10 mM glucose in 20 mM phosphate buffer (pH 7.4). (black) GOx/SWCNTs (0.8:0.2 w/w), (blue) GOx/PVImQOs (0.8:1.0 w/w), (green) GOx/PVImQ/SWCNTs (0.8:1.0:0.2 w/w/ w), (orange) a bilayer of GOx and PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w), and (red) GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composites. Scan rate: 5 mV s−1.

diameter of (6,5)SWCNTs is 0.757 nm,30 small bundles of PVImQOs-covered SWCNTs were proposed to lead to entangled structures in the network. The GOx/PVImQOs/ SWCNTs (0.8:1.0:0.2 w/w/w) composite resulted in relatively distorted network structures with a diameter of approximately 100 nm (Figure 3b(iii)). The change in the diameter of the ternary composite structures arose from noncovalent binding of GOx at the PVImQOs/SWCNTs surface. The resulting GOx/PVImQOs/SWCNTs composite gave rise to thicker fibrils in the SWCNT networks. Enzymatic Responses of Solution-Processed Enzyme/ Polymer/SWCNT Composites. To investigate the enzymatic activities of the GOx/PVImQOs/SWCNT composite, we examined the electrochemical properties. The enzymatic activities of GOx for glucose involve the following reactions (Figure 4a): (i) the enzymatic reaction of glucose yields

gluconolactone, (ii) electron transfer occurs from reduced GOx to a mediator of the Os complex, and (iii) the redox reaction from Os(II) to Os(III) through oxidation affords electron transfer from the SWCNTs to an electrode. A composite was obtained by drop-casting a dispersed solution in phosphate buffer on a gold working electrode (see Figure S4a in Supporting Information). To avoid elution of the composite in phosphate buffer (pH 7.4), a chitosan layer was deposited by drop-casting the 1.0wt% acetic acid solution (including 1.0wt% chitosan, 3 μL) for the post-treatment.31 Because chitosan is insoluble under near-neutral conditions, watersoluble GOx and PVImQOs can be protected by the chitosan layer. We also expected that the chemical structure of chitosan would be inert to electrical conductivity, which results in smooth electron transfer to the electrode through the SWCNT networks. 4326

DOI: 10.1021/acsanm.9b00769 ACS Appl. Nano Mater. 2019, 2, 4323−4332

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Figure 5. (a) Amperometric responses of GOx/PVImQOs/SWCNT composite upon adding 10 mM glucose in 20 mM phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl at various loading amounts of GOx. GOx/PVImQOs/SWCNTs (i) 0:1.0:0.2, (ii) 0.3:1.0:0.2, (iii) 0.5:1.0:0.2, (iv) 1.0:1.0:0.2, and (v) 0.8:1.0:0.2 w/w/w. (b) Amperometric responses of GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite upon adding 10 mM glucose in 20 mM phosphate buffer (pH 7.4) at various applied voltages versus Ag/AgCl.

transfer from GOx to the Os complex occurred only at the interface between GOx and the PVImQOs/SWCNT composite, a low current density of the bilayer was observed. These results suggested that the step-by-step methodology is essential for efficient electron transfer to the electrode through the enzymatic reaction. CV of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite without the chitosan layer in 20 mM phosphate buffer (pH 7.4) was examined as a control experiment (see Figure S6a in Supporting Information). We found that the current densities decreased with the increasing number of cycles; the current density at E01/2 for the fourth cycle was 8% lower than that for the first cycle (see Figure S6b in Supporting Information). Decrease in the current density arose from the elution of water-soluble GOx and PVImQOs in phosphate buffer. The chitosan layer is necessary for the stable electrochemical response. Amperometric Responses for Enzyme/Polymer/ SWCNT Composites. Next, we investigated the amperometric properties of the GOx/PVImQOs/SWCNTs composites driven by enzymatic oxidation of glucose. To understand the enzymatic responses, changes in the current densities at various loading amounts of GOx were explored on the working electrode (Figure 5a). After adding the glucose solution (10 mM), the current density immediately increased owing to the enzymatic reaction. The loading amount of GOx increased with increasing the current density because of the enzymatic electron transfer. The maximum current density reached 338 μA cm−2 with the GOx/PVImQOs/SWCNT composite (0.8:1.0:0.2 w/w/w). We found that the current density decreased with increasing the loading amount of GOx (Figure 5a(iv)). Because GOx shows an electrically insulating nature, excess enzyme in the composite may lead to the disturbance of electron transfer to the mediator PVImQOs.33 The loading amount of GOx in GOx/PVImQOs/SWCNTs (0.8:1.0:0.2 w/ w/w) was confirmed by the Bradford assay (see Figure S5 in Supporting Information).34 The calibration curve revealed that the concentration of the GOx solution was estimated to be 0.243 mg mL−1. Because 10 μL of the dispersed solution was used for the GOx/PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w) composite, the loading amount of GOx was 2.4 μg. Figure 5b shows amperometric response of the GOx/ PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite at

Figure 4b shows cyclic voltammograms (CVs) of composites in phosphate buffer. The GOx/SWCNT (0.8:0.2 w/w) composite displayed no redox responses because of no mediator PVImQOs. In contrast, the GOx/PVImQOs/ SWCNT (0.8:1.0:0.2 w/w/w) composite exhibited a redox couple of Os(II)/Os(III) at an oxidation−reduction potential (E0 1/2) of 0.25 V versus Ag/AgCl. The current density of GOx/PVImQOs/SWCNTs at E01/2 (7.19 μA cm−2) was 17times higher than that of GOx/PVImQOs (0.8:1.0 w/w) at E01/2 (0.43 μA cm−2). These results suggest that (6,5) SWCNTs are crucial for the conducting medium, to result in effective electron transfer to the electrode.32 A large change in the current density of the GOx/ PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite (272 μA cm−2) was achieved by adding a glucose solution (10 mM) (Figure 4c). Because the reduction peak diminished, catalytic oxidation of glucose occurred through the enzymatic reaction. We also found that the current density of the GOx/SWCNTs composite without PVImQOs was only 0.24 μA cm−2. The current density for the GOx/SWCNTs composite was approximately 1130 times lower than that for the GOx/ PVImQOs/SWCNT composite, even though we used the same loading ratios of SWCNTs showing the same surface area and electrical conductivities. These results indicated that the redox reaction of the Os complex in PVImQOs is crucial for the high current density. The GOx/PVImQOs composite without SWCNTs showed a small current density of 2.86 μA cm−2 owing to the lack of electron transfer to the electrode. Furthermore, the current density for the GOx/PVImQ/ SWCNT (0.8:1.0:0.2 w/w/w) composite without the Os complex was 1.19 μA cm−2. This result suggested that no mediator in the ternary composite resulted in inefficient electron transfer to the electrode. To clarify the step-by-step methodology, we investigated the electrochemical properties of a bilayer of GOx and the PVImQOs/SWCNT composite (0.8:1.0:0.2 w/w/w) on the electrode (orange in Figure 4b,c). The CV of the bilayer exhibited the redox couple of Os(II)/Os(III) at E01/2. Unfortunately, the current density for the bilayer (1.89 μA cm−2) was approximately four-times lower than that for the GOx/PVImQOs/SWCNT composite (7.19 μA cm−2) (Figure 4b). The current density of the bilayer reached 31 μA cm−2 after adding 10 mM glucose (Figure 4c). Because electron 4327

DOI: 10.1021/acsanm.9b00769 ACS Appl. Nano Mater. 2019, 2, 4323−4332

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Figure 6. (a) Time-dependent amperometric responses of GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite upon adding 0.5−80 mM glucose in 20 mM phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl on a working electrode. (b) Relations between current densities and concentrations of glucose (n = 3): (i) step-by-step assembly; (ii) simple mixing, respectively.

Figure 7. (a) Amperometric responses for GOx/PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w) composite by adding 10 mM glucose in 20 mM phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl at various storage times on the working electrode. (b) Current density at 0.5 V versus Ag/AgCl as a function of storage time. Current densities were monitored after 80 s in Figure 7a.

various applied voltages. A current density of 269 μA cm−2 at 0.5 V versus Ag/AgCl was obtained after 60 s. However, the current density (258 μA cm−2) slightly decreased when the applied voltage was 0.6 V versus Ag/AgCl. This indicated that the enzymatic oxidation of glucose exhibited a different steadystate value depending on the applied voltage. From CV of the GOx/PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w) composite, no degradation occurred at 0.5 V versus Ag/AgCl (Figure 4c). We concluded that 0.5 V versus Ag/AgCl was suitable as the monitoring voltage for the bioreactive responses. Figure 6a(i) shows the time-dependent amperometric responses of the step-by-step assembled GOx/PVImQOs/ SWCNT (0.8:1.0:0.2 w/w/w) composite by adding glucose in phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl on the working electrode. The enzymatic response displayed a high current density of 1420 μA cm−2 with 90% of the steady-state values within 5 s after addition of the glucose solution. Furthermore, the steady-state current densities remained almost unchanged, which indicated that diffusion restriction was negligible for the enzymatic oxidation of glucose. Linear relationships between current densities and concentrations of glucose were obtained in the range of 0.5−8.0 mM glucose with R2 > 0.996 (see Figure S7 in Supporting Information). The sensitivity reached 72 μA mM−1 cm−2 for the GOx/

PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite on the working electrode (Figure 6b(i)). Since the enzymatically generated electrons exhibited electron transfer from reduced GOx to the mediator of the Os complex directly, hydrogen peroxide could not be detected by the reduction of oxygen (Figure 4a(ii)). In Figure 3b(iii), the GOx/PVImQOs/SWCNT composite showed SWCNT networks covered with GOx. Owing to this assembled structure, gluconolactone has almost no chance to contact the SWCNTs. We also found that no obvious degradation occurred during the bioreactive responses even at a high concentration of glucose. From these results, we believe that the sidewalls of SWCNTs in the GOx/PVImQOs/SWCNT composite remain unchanged. Several groups have reported assembled SWCNT composites. Gao et al. prepared a GOx/polymer/SWCNT composite on a screen-printed carbon electrode using a layer-by-layer method.35 The layer-by-layer approach allows for facile deposition of an alternately charged Os polymer/SWCNT composite on the working electrode. The device displayed a sensitivity of 32 μA mM−1 cm−2. Unfortunately, they did not estimate the current densities of the layer-by-layer composites. A modified working electrode through a Au−thiol interaction exhibited an increase in the sensitivity.36 Negatively charged 11-mercaptoundecanoic acid on the gold working electrode 4328

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Figure 8. (a) Amperometric responses of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite upon adding 200 μL of (i) cola and (ii) diet cola in 20 mM phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl. (b) Amperometric responses of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite upon adding 200 μL of cola and diet cola repeatedly.

Figure 9. (a) Time-dependent amperometric responses of LOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite upon adding 0.25−4.0 mM lactic acid in 20 mM phosphate buffer (pH 7.4) at 0.5 V versus Ag/AgCl on a working electrode. (b) Relations between current densities and concentrations of lactic acid (n = 3).

the mixed composite (Figure 6b(ii)). Because a negatively charged enzyme is attached to the positively charged PVImQOs by charge attraction, the resulting electron shows good electron transfer from GOx to the Os complex in the polymer after the enzymatic oxidation of glucose. The directly attached polymer at the SWCNT surface resulted in effective electron transfer to the working electrode. To investigate the stabilities of our composites, the amperometric responses at various storage times were determined (Figure 7a). We found that the current densities of the GOx/PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w) composite after adding 10 mM of glucose did not change significantly even for a storage time of 8 h. This stability was confirmed by the relationship between the current density after injection and storage time. The relative standard deviation (RSD) of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite to 10 mM glucose was 4.88% (Figure 7b). We also investigated the amperometric responses of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composites with various numbers of active layers (see Figure S8 in Supporting Information). We expected that an increase in the number of active layers would show a higher sensitivity through the enzymatic reaction. The multilayer composites were prepared by drop-casting the dispersed GOx/PVImQOs/ SWCNT solution and the chitosan solution onto the working

formed the layer-by-layer assembly by adsorption of the Os polymer, which resulted in a sensitivity of 56 μA mM−1 cm−2 with a current density of 440 μA cm−2. Pang et al. reported that conjugated polyelectrolyte complexes of polythiophene and polyethynylphenylene derivatives show good sensitivity.37 Their assembled composite was prepared by mixing the polyelectrolyte, SWCNTs, and GOx in phosphate buffer. The sensitivity of the GOx/polyelectrolyte/SWCNTs composite (157 μA mM−1 cm−2) was twice as large as that of the GOx/ PVImQOs/SWCNTs (0.8:1.0:0.2 w/w/w) composite (72 μA mM−1 cm−2). However, the loading amount of GOx in their composite (50 μg) was over 20 times higher than that in our composite (2.4 μg). These results suggested that our step-bystep methodology would be advantageous for a highly efficient enzymatic reaction. To evaluate the effects of the step-by-step assembly, the amperometric response of a ternary composite prepared by simple mixing of GOx, PVImQOs, and SWCNTs (0.8:1.0:0.2 w/w/w) was examined as a control experiment (Figure 6a(ii)). We found that the maximum current density of the mixed composite (137 μA cm−2) was 10 times lower than that of the step-by-step assembled composite (1420 μA cm−2). More importantly, an 87% decrease in the sensitivity (9.3 μA mM−1 cm−2) indicated that the assembled structure is crucial for the high sensitivity even though SWCNTs are incorporated into 4329

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ACS Applied Nano Materials

sensitivity reached 72 μA mM−1 cm−2 for the GOx/ PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite within the linear range of the glucose concentration from 0.5 to 8.0 mM. We also found that the loading amount of GOx for our composite was only 2.4 μg; the step-by-step methodology allows direct assembly of PVImQOs and GOx by charge attraction at the surface of SWCNTs, which leads to an effective electron transfer from GOx to the working electrode. We demonstrated that energy conversion from food to electricity is achieved using the glucose-including cola. Our synergetic assembly using LOx is also applicable for the bioreactive composite driven by enzymatic oxidation of lactic acid. This step-by-step methodology will be beneficial for enzyme-assisted energy conversion nanocomposites.

electrode alternately (see Figure S4b in Supporting Information). Unfortunately, the sensitivity could not be improved; the sensitivities for the two-layer and four-layer composites were 18 and 21 μA mM−1 cm−2, respectively (see Figure S8b in Supporting Information). These results suggested that the multilayer composite showed less efficiency for electron transfer to the electrode. We also found that the response time for 90% of the steady-state value decreased with an increasing number of active layers; the response time for the single-layer, two-layer, and four-layer composites was 3, 5, and 11 s, respectively. Because the layered structure made an interface between each GOx/PVImQOs/SWCNT composite, the multilayer composite may result in slow electron transfer to the electrode. We propose that the assembled structure of the single-layer composite is advantageous for high sensitivity. Our step-by-step assembled composite allowed bioreactive responses to beverages (Figure 8). We selected beverages of cola (sugar content 0.113 g mL−1) and diet cola (sugar content 0 g mL−1). A rapid change in the current density of the GOx/ PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite (80 μΑ cm−2) was observed after adding 200 μL of cola (Figure 8a(i)). In sharp contrast, no bioreactive response occurred upon addition 200 μL of diet cola (Figure 8a(ii)). This clear difference indicated that the assembled composite showed the enzymatic reaction of glucose in the beverage. Furthermore, the bioreactive response was also displayed by adding cola and diet cola repeatedly, which suggested that our assembled composite responded not only to the glucose solution but also to the glucose-including beverage with food contaminants (Figure 8b). Our investigations demonstrate that the structurally organized composite would be applicable for enzyme-assisted energy conversion from food to electricity. Our enzyme/polymer/SWCNT composites could be driven by the enzymatic reaction of lactic acid (Figure 9). Because lactate oxidase (LOx), a flavin adenine dinucleotide (FAD)based enzyme, can oxidize lactic acid to pyruvate,38 electron transfer would occur from the active site of FAD in LOx to the Os complex in PVImQOs. A dispersed solution of the LOx/ PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite was obtained in phosphate buffer (pH 7.4) through the charge attraction of the polymer−enzyme interaction. The solutionprocessed LOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w/w) composite on the working electrode exhibited a sensitivity of 54 μA mM−1 cm−2 with a linear range of 0.25−1.0 mM (see Figures 9b and S9 in Supporting Information). Amperometric responses at various storage times revealed that the LOx/ PVImQOs/SWCNT composite exhibited a high stability with an RSD of 7.21% (see Figure S10 in Supporting Information).

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

PVImQOs/SWCNT Binary Composites. Raw SWCNTs ((6,5) chirality, 1.0 mg) were added to deionized water containing PVImQOs (0.5 g L−1, 10 mL). The mixture was sonicated for 15 min using a tip-type ultrasonic homogenizer (Branson Sonifer 250, Branson Ultrasonics, Danbury, CT, USA; power output 40 W) in an ice bath. The mixture after ultrasonication was centrifuged at 7500g for 30 min at 25 °C to remove any large bundles of SWCNTs. A homogeneous PVImQOs/SWCNT (1.0:0.2 w/w) composite was obtained from the supernatant. Binary composites with different SWCNT concentrations were also prepared at concentrations up to PVImQOs/SWCNTs = 1.0:2.0 w/w. GOx/PVImQOs/SWCNT Ternary Composites. GOx (5 mg mL−1) in phosphate buffer (0.8 μL, pH 7.4) was added to the dispersed solution of PVImQOs/SWCNTs (1.0:0.2 w/w) in phosphate buffer (including 0.09% chitosan in acetic acid, 11 μL) at 25 °C for 1 h. The resulting solution (total volume 11.8 μL) was kept at 4 °C overnight, which yielded a dispersed solution of the GOx/PVImQOs/SWCNT (0.8:1.0:0.2 w/w) composite. The loading ratios of GOx for the GOx/PVImQOs/SWCNT composites were varied from 0:1.0:0.2 w/w/w to 1.0:1.0:0.2 w/w/w. In a control experiment, samples were obtained by simply mixing GOx, PVImQOs, and SWCNTs (0.8:1.0:0.2 w/w/w, 0.8 μL) in phosphate buffer (including 0.09% chitosan in acetic acid, 11 μL) (pH 7.4), without dispersion treatments such as ultrasonication and centrifugation. Precipitation occurred immediately in phosphate buffer because of the large bundles of SWCNTs. Bilayer of GOx and PVImQOs/SWCNT Composite. The PVImQOs/SWCNT composite (1.0:0.2 w/w) was obtained by drop-casting the dispersed solution in phosphate buffer (including 0.09% chitosan in acetic acid, 10 μL) (pH 7.4) on the electrode. After drying under a reduced pressure for 1 h at 25 °C, GOx was prepared by drop-casting the solution in phosphate buffer (including 0.09% chitosan in acetic acid, 10 μL) (pH 7.4) on the composite, and then the resulting bilayer was dried under a reduced pressure for 1 h at 25 °C. Finally, the chitosan layer was deposited by drop-casting an aqueous solution (1.0 wt % chitosan in 1.0 wt % acetic acid, 3 μL), which yielded the bilayer of GOx and the PVImQOs/SWCNT composite (0.8:1.0:0.2 w/w/w).

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CONCLUSIONS We demonstrated structurally organized enzyme/polymer/ SWCNT ternary composites using step-by-step assembly in aqueous media. The multifunctional polymer PVImQOs yielded an aqueous dispersion of the PVImQOs/SWCNT composite through cation−π interactions. Following the addition of the enzyme GOx, the GOx/PVImQOs/SWCNT composite was achieved by charge attraction between the positively charged PVImQOs and the negatively charged GOx. The solution-processed ternary composites showed bioreactive properties driven by enzymatic glucose oxidation. The step-bystep assembled composites resulted in current densities as high as 1420 μA cm−2, which was a 10-fold increase over the response of composites prepared by a simple mixing. The high

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.9b00769. Further details of experimental procedures and characterization including synthetic procedures, absorption spectra, determination of the loading amount of GOx and bioreactive behavior of enzyme/polymer/SWCNT composites (PDF) 4330

DOI: 10.1021/acsanm.9b00769 ACS Appl. Nano Mater. 2019, 2, 4323−4332

Article

ACS Applied Nano Materials

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.-K.L.). *E-mail: [email protected] (Y.I.). *E-mail: [email protected] (M.K.). ORCID

Yoshihiro Ito: 0000-0002-1154-253X Masuki Kawamoto: 0000-0003-3101-4416 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the JSPS KAKENHI, Grant Number JP15K05639, for M.K. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This work was also supported by the Ministry of Science and Technology of Taiwan Grant Number MOST108-3017-F-009004 and MOST106-2113-M-009-004 for Y.-K.L. and the Center for Emergent Functional Matter Science of National Chiao Tung University from The Featured Areas Research Center Program within the framework of the Higher Education Support Project and the Scientist Cultivation Program for Y.K.L. from the Ministry of Education (MOE) of Taiwan. We thank Keisuke Tajima and Kyohei Nakano of the Emergent Functional Polymers Research Team, RIKEN Center for Emergent Matter Science (CEMS), for the ultraviolet− visible−near-infrared spectrophotometry results. We thank the RIKEN Brain Science Institute for the high-resolution mass spectrometry studies. We thank Daisuke Hashizume and Daishi Inoue of the Materials Characterization Support Team, RIKEN CEMS, for the SEM measurements and for their expertise. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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