Articles
Electropolymerization Conditions of Methylene Green Used as an Electron Transfer Mediator for Coenzyme-dependent Oxidoreductases
2025 Volume 93 Issue 2 Pages 027017
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2025 Volume 93 Issue 2 Pages 027017
Biosensors and biofuel cells are human-friendly and eco-friendly technologies that utilize enzymatic redox reactions. To operate in a solution flow environment, enzymes and electron mediators must be immobilized on the electrodes. The state of the electron mediator at an enzyme-immobilized electrode affects not only the electron transfer between the enzyme and the electrode but also the retention and orientation of the enzyme. Phenazine dyes, often used as electron mediators, can be easily immobilized on an electrode by electropolymerization. However, little is known about what kind of polymer films result from the conditions of electropolymerization. Here, we show that the electropolymerized film of methylene green (MG), a phenazine dye, changes from a uniformly distributed mesoscale structure to a nonuniformly dispersed state of spherical particles as polymerization progresses. Furthermore, we found that polyMG films with a uniform surface are suitable for enzyme-immobilized electrodes. More MG was deposited on the electrode under conditions of wide potential sweeps, slower scan rates, and more cycles of electrolysis. Uniform surfaces were observed in films with dissipation (ΔD), a measure of film softness as measured by EQCM-D, of less than approximately 7 × 10−6. These results indicate that the electrochemical polymerization conditions of MG can control the surface morphology of the polymerized film, as well as the properties of the enzyme-immobilized electrode. This finding could be applied to NAD-dependent and FAD-dependent glucose dehydrogenases, in which MG is known to function as a mediator, and could contribute to improving the current of biosensors and biofuel cells in which they are used.
Biosensors and biofuel cells1,2 are technologies that use enzyme-catalyzed redox reactions, which are catalysts derived from living organisms, and can operate in mild environments. Since these are human-friendly and eco-friendly technologies, many applications have been reported for wearable devices and implantable medical devices.3–6 Biofuel cells are a hot technology that fits the concept of green chemistry because they can generate electricity in an environmentally friendly way via the use of enzymes.7,8 The “EBFC reactor (Enzymatic BioFuel Cell reactor)” that we are studying is a technology that acts as both a bioreactor and a biofuel cell and can be used for green chemistry and biorefineries. Our final goal is to construct a series of flow systems that produce useful substances from biomass-derived polysaccharides through enzymatic hydrolysis and oxidation reactions while extracting electrical energy.9 The most important issue for our flow system is the immobilization of the enzyme on the electrode to allow for continuous enzyme–electrode electron transfer in flowing solutions. Without immobilization, the enzyme will flow and be lost, and electron transfer will fail. The main elements that need to be immobilized on the electrode are enzymes. However, the redox center of the enzyme is often embedded within the enzyme, making it difficult for the enzyme alone to directly exchange electrons with the electrode. Therefore, mediated electron transfer (MET), in which electrons are transferred via electron mediators, is often used. When making MET-type enzyme-immobilized electrodes that can be used in flow environments, it is preferable to immobilize the mediator on the electrode along with the enzyme to prevent flow and loss. For immobilization of the mediator to the electrode, redox active complex modified polymers, i.e. redox polymers, are often used.10–12 Redox polymers are those with redox sites in their structure. Although redox polymers themselves have a long history of research, their use in enzyme-immobilized electrodes began in the late 1980s when Degani and Heller used them for electron transfer between enzymes and electrodes in biosensor applications.10,13 Most redox polymers have a backbone, such as linear polyethyleneimine (LPEI), and redox pendants, such as osmium complexes and ferrocene.10 In redox polymers, electrons obtained at the redox center of the enzyme are transferred to the electrode by hopping between redox sites within the polymer. Although the electron transfer properties of these polymers can be optimized by designing the distance between redox sites and the length of the pendants, the synthesis steps are cumbersome and often involve more than 10 steps.14–17 For some organic mediators, direct immobilization onto electrodes by electropolymerization of itself is an effective method.18 Electropolymerization is a method of polymerization in which monomers are electrolytically oxidized or reduced by applying an electric potential to generate radicals. Although the method of immobilizing mediators by electrochemical polymerization is limited in the types of mediators that can be applied, it has the advantage of not requiring complicated synthesis to combine redox sites and backbones, i.e., the number of compounds required and the process can be reduced. A further advantage is that it can be used as-is for electrodes after electrochemical fabrication. Mediators known to be able to polymerize by electropolymerization include viologen and phenazine dyes (e.g., phenazine, phenoxazine, and phenothiazine).19,20 Polyphenazine have attracted attention in enzyme-immobilized electrode applications.21 Phenazine dyes have been found to act as mediators for dehydrogenases often used in biosensors and biofuel cells, and in our EBFC reactor study, methylene green catalyzed pyrroloquinoline-quinone (PQQ)-dependent electron transfer in the enzyme.9 Methylene green (MG), a phenazine dye, has been reported to act as a mediator not only for PQQ-dependent but also for nicotinamide adenine dinucleotide (NAD)-dependent and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases,22 making MG electropolymerized polymers versatile materials for enzyme electrodes.
In enzyme-immobilized electrodes, where the enzyme is immobilized on a mediator, the mediator layer not only provides an electrical connection between the enzyme and the electrode but also affects the stable immobilization and orientation control of the enzyme. Therefore, the number of redox sites, surface morphology, hydrophilicity, and interaction with enzymes on the mediator layer are important factors that affect the enzyme reaction efficiency and electron transfer efficiency.
In order to elucidate the electrochemical polymerization mechanism and polymer structure of phenazine dyes, electrochemical methods such as cyclic voltammetry (CV)19,23,24 have been combined with electron microscopy or electrochemical quartz crystal microbalance (EQCM) measurements. Kariyakin and colleagues, who have conducted many studies on the electropolymerization of phenazine dyes, used electrochemical techniques and infrared (IR) and ultraviolet-visible (UV-vis) spectroscopy to analyze the conformational changes and polymerization mechanisms of the molecules.23,25 Structural observations on scales larger than the molecular structure have been combined with scanning electron microscopy (SEM)26 or atomic force microscopy (AFM)27 measurements of the electropolymerized films. EQCM measurements provide information on mass change in addition to information from CV in real-time analysis of the progress of polymerization on the electrode.27,28 Hosu et al. used CV, EQCM, and SEM in combination to study the effect of the polymerization mechanism of methylene blue in deep eutectic solvents.29 SEM observations revealed that the surface morphology of the resulting electropolymerized films in deep eutectic solvents depends on the sweep rate.
In order to immobilize the enzyme on the polymerized film, morphological information on the polymerized film formed under each condition is important. By using EQCM with dissipation (EQCM-D) and AFM together and examining the range of potential application, sweep rate, and number of sweeps, we were able to clarify the growth process of MG electropolymerization films and the influence of each factor on the morphology. An EQCM-D can measure not only the frequency shift, which reflects the change in mass on the electrode but also dissipation, which is an indicator of film softness. In this study, the effects of potential application conditions on the amount of MG polymerized and the surface morphology of the polymer were investigated via EQCM-D and AFM techniques to determine the electrochemical polymerization conditions of MG films suitable for enzyme-immobilized electrodes. Furthermore, the currents derived from enzyme reactions were evaluated for enzyme-immobilized electrodes constructed using MG films obtained under various conditions as mediators.
Ammonia solution (28 %), hydrogen peroxide (30 %), dipotassium hydrogen phosphate, potassium dihydrogen phosphate and D(+)-glucose were purchased from Wako Pure Chemical Industries, Ltd. (Japan). MG was purchased from Kanto Chemical Co., Inc. (Japan). Carbon felt (CF) was purchased from Tsukuba Materials Information Laboratory, Ltd. (Japan). PQQ-GDH was purchased from Toyobo Co., Ltd. (Japan). For the experiments in this study, a 0.5 mol dm−3 potassium phosphate buffer (pH 7.0) was used. Milli-Q water was used for reagent preparation.
2.2 Electrochemical quartz crystal microbalance with dissipation monitoring (EQCM-D)EQCM-D measurements were performed on a Q-Sense instrument (Biolin Scientific, Sweden). An Au-coated QCM sensor (QCM sensor) with a Ti adhesion layer was used as the working electrode (QSX338, Biolin Scientific, Sweden). The QCM sensor was placed in the Q-Sense Electrochemistry module (Biolin Scientific, Sweden). A platinum plate was used as the counter electrode, and a Ag/AgCl (3 mol dm−3 KCl) electrode was used as the reference. The electrochemical measurement in parallel with the QCM measurement was performed with an ALS627E potentiostat (BAS Inc., Japan). In all the cases, the temperature setting was 25 °C, the flow rates were set to 50 µL min−1, and the presented frequency shifts (Δf) were related to the 3rd overtone.
2.3 Electropolymerization of MG onto QCM sensorsThe QCM sensor was cleaned before use as follows: the QCM sensor was immersed in a 5 : 1 : 1 solution of water, NH3 (28 %), and H2O2 (30 %) at 80 °C for 5 min and then rinsed with water. The sensor was then cleaned with UV-O3 treatment (10 min) via a UV/Ozone Pro Cleaner (Bio Force Nanosciences, USA) and rinsed with water. The cleaned sensor was placed in the EQCM-D equipment. The buffer solution was passed through the sensor for approximately 15 min to stabilize the frequency. Subsequently, 0.5 mmol dm−3 MG/buffer solution was allowed to flow for 5 min, and then a potential was applied to the QCM sensor under set conditions to carry out electropolymerization. Constant potential measurements were performed via chronoamperometry (CA), and potential sweep measurements were performed via CV. After the sensor was rinsed with a flow of water, it was removed from the EQCM-D equipment. The sensor was further washed with water to obtain a polyMG-modified sensor.
2.4 Atomic force microscopy (AFM)The surface of polyMG was observed via an SPM-9700HT scanning probe microscope (Shimadzu Corporation, Japan). All the AFM images were recorded in noncontact mode using silicon cantilevers with a resonance frequency of 70 kHz and a force constant of 1.7 N m−1 (Olympus Corporation, Japan). The calculation of the root-mean-square roughness (RMS) value and the cross-sectional measurement were performed via the surface roughness analysis mode and cross-sectional shape analysis mode of the SPM-9700 software, respectively.
2.5 Measurement of the glucose oxidation current on the PQQ-GDH immobilized electrodeThe measurements were performed via three electrodes: a PQQ-GDH-immobilized CF electrode as the working electrode, CF with only UV-O3 treatment as the counter electrode, and Ag/AgCl (3 mol dm−3 NaCl) as the reference electrode. In the EQCM-D experiment, a reference electrode with an internal solution of 3 mol dm−3 KCl (Dri-Ref-2SH, World Precision Instruments, USA) was used, but in this experiment, a reference electrode with an internal solution of 3 mol dm−3 NaCl (RE-1B, BAS Inc., Japan) was used.
CF with a diameter of 10 mm and a thickness of 5 mm was hydrophilized by UV-O3 cleaning (10 min) with a UV/Ozone Pro Cleaner (Bio Force Nanosciences, USA) and ultrasonic cleaning (5 min) in deionized water. The absorbed water in the CF was forced out. The CF was electropolymerized in 0.5 mmol dm−3 MG/buffer solution (10 mL) via continuous cyclic sweeps from −0.5 to +1.23 V vs. Ag/AgCl (3 mol dm−3 NaCl) using an HZ-7000 potentiostat (Hokutodenko, Japan). The CF was rinsed twice with water, and the absorbed water in the CF was forced out. A PQQ-GDH solution (1 mg/100 µL) was dropped on the polyMG-modified CF, which was allowed to stand for 30 min.
The cyclic voltammetry measurements were carried out between −0.3 and +0.3 V vs. Ag/AgCl (3 mol dm−3 NaCl) at a scan rate of 2 mV s−1. The reaction solution was a buffer solution (7 mL) containing 0.1 mol dm−3 glucose.
To understand the relationship between potential conditions and the progress of polymerization in the electropolymerization of MG, EQCM-D was used to measure the amount of MG deposited in real time while a potential was applied to the electrode. The electrode used was a QCM sensor with a gold-coated surface. Previous reports of electropolymerization include two methods of potential application: one is to apply a constant potential, and the other is to sweep the potential with CV. In recent studies, potentials have often been applied via the sweep method, which is based on the recognition that oxidative polymerization also requires a reduction step.26 However, to the best of our knowledge, it is not clear what is happening at each potential. Therefore, we first investigated how polymerization proceeds at constant potential and potential sweeps, respectively, in EQCM-D.
Electropolymerization is initiated by the generation of radicals by the application of a potential, and in the case of MG, radicals are known to be generated at approximately +1.0 V (vs. SCE).28 The constant potential condition was set at +1.0 V (vs. Ag/AgCl), and the CV condition was set between −0.5 and +1.23 V (vs. Ag/AgCl) at 50 mV s−1, referring to the report by Zhou et al.24 The initial potential was set at +0.6 V, and the potential was swept in the positive direction to +1.23 V, then swept in the negative direction to −0.5 V, and returned to the initial potential, forming one cycle. Figure 1 shows the frequency shift when MG is polymerized under each condition. In both methods, the decrease in the frequency shift (Δf) became greater as the potential application time increased. This suggests that the amount of deposition on the electrode increased as the polymerization progressed. However, a comparison of Δf after 40 min revealed a large difference: −65 Hz for a constant potential and −490 Hz for a potential sweep. When the potential was held at +0.8 V and +1.2 V, the Δf values were −24 Hz and −96 Hz, respectively, and even when the potential was increased, these values were smaller than those obtained by the sweep method (Fig. S1). The results showed that the amount of deposition depended on the sweep range and that when a constant potential was applied, the amount of deposition reached a plateau at an early stage. Polymerization during sweeps proceeded as Δf repeatedly increased and decreased, which was considered to be the cause of the greater amount of deposition than in the case of a constant potential.
Frequency shift (Δf) recorded during the electropolymerization of MG in different potential ranges: sweep from −0.5 to +1.23 V at 50 mV s−1 (solid line) and constant potential of +1.0 V (dashed line). The electropolymerization was carried out in 0.5 mol dm−3 phosphate buffer solution containing 0.5 mmol dm−3 MG on AuQCM sensors.
Figure 2 shows the CV and EQCM-D results for the first cycle swept at 50 mV s−1, arranged with the horizontal axis unified with time. When the potential was positively swept from +0.6 V, an oxidation peak of +1.0 V was observed at approximately 10 s. This peak is consistent with the aforementioned radical generation potential. After that potential, the Δf value begins to decrease, indicating that oxidative polymerization probably occurs in the range of +1.0 V to higher potentials. Experiments confirmed that the +0.5 V reduction peak appearing at 20 to 30 s is not from MG but from the gold oxide film (Fig. S2). The reduction peak at pH 7 and the behavior of the peak shifting to a higher potential when the pH is changed to the acidic side are consistent with previously reported results in the literature.30 In the +0.3 V to negative potential region from 30 s to 60 s, Δf decreased (30–55 s) but then increased again (55–65 s), eventually returning to almost the initial level. If polymerization was taking place, Δf would be expected to remain low, so adsorption (30–55 s) and desorption (55–65 s) would have occurred during this process. The phenomenon that dissipation (ΔD), a measure of film softness, increased as Δf decreased also suggests the formation of a soft adsorption layer rather than a rigid polymer layer. Thus, the polymerization of MG involves both oxidative polymerization and adsorption/desorption processes, and it was shown that the adsorption/desorption process also contributes to the promotion of polymerization. The reason why polymerization is promoted by the adsorption/desorption process is expected to be that the monomers remaining on the electrode surface after adsorption are polymerized in the next cycle or trapped inside the polymer. For positively charged molecules such as MG, the continuous application of a positive potential, at which oxidative polymerization occurs, may have caused electrical repulsion between the electrode and the MG monomer, making it difficult for polymerization to proceed. Conversely, the inclusion of a negative potential in the potential range may promote polymerization.
Cyclic voltammogram (above) and EQCM-D (below) measurements of the first cycle of MG electropolymerization. The potential range was −0.5 to +1.23 V, and the scan rate was 50 mV s−1.
Sweeping over a potential range that includes both oxidative polymerization processes and adsorption/desorption processes was found to increase the amount of MG deposited on the electrode. Hence, the sweep method using the CV technique was employed to apply the potential in the electrolytic polymerization. The effects of two parameters, the scan rate and number of sweeps, on polymerization were investigated. The values of Δf and ΔD for each scan rate from the EQCM-D measurements are shown in Fig. 3. At all sweep rates, Δf decreased with increasing number of sweeps. Cyclic voltammograms during polymerization (Fig. S3) revealed that with increasing cycles, the potential of the oxidation peak shifted from the monomer-derived peak at +0.2 V to a higher potential, becoming broader.24 The Δf values at the end of 70 cycles were −490, −990, and −2370 Hz for scan rates of 200, 50, and 25 mV s−1, respectively, with Δf decreasing with decreasing speed; this is thought to be because the slower rate increases the time available to apply the potential at which polymerization and adsorption occur in each cycle. In short, more cycles and slower scan rates resulted in more polymerization. Furthermore, the slopes of Δf and ΔD are not constant, suggesting that the polyMG film would be formed through several stages with different morphologies. There are two main tipping points in the Δf and ΔD results at a scan rate of 25 mV s−1. The first point is approximately 10 cycles, where the slopes of both Δf and ΔD increase (Figs. 3a and 3b), and the second point is approximately 40 cycles, where the slope of ΔD increases rapidly (Fig. 3b). The rapid increase in ΔD suggested that the surface conditions changed from rigid to soft and may provide a means of estimating the change in surface topography in real time during polymerization.
EQCM-D study of the electropolymerization of MG at different scan rates: 25, 50 and 200 mV s−1. (a) Frequency shift (Δf) and (b) dissipation shift (ΔD). The electropolymerization was carried out in 0.5 mol dm−3 phosphate buffer solution containing 0.5 mmol dm−3 MG on the AuQCM sensor.
AFM images of the polyMG films obtained under each electropolymerization condition are shown in Fig. 4. The surface morphology varied greatly depending on the polymerization conditions. Slower scan rates and more cycles were observed to increase the number and size of the spherical particles. At 25 mV s−1, after 70 cycles, spherical particles growing to nearly 1 µm were also observed. The root-mean-square (RMS) roughness of the polyMG film calculated for the AFM scanning area was 3 nm in 10 cycles and 74 nm in 70 cycles at 25 mV s−1 and 3 nm in 10 cycles and 7 nm in 70 cycles at 200 mV s−1. These results indicate that the more cycles and the greater the deposition (lower Δf) in the electropolymerization of MG, the more nonuniform the film tends to be (Fig. S4). The mechanism of film nonuniformity was investigated via AFM and EQCM-D as follows. According to the AFM images of the polyMG films prepared at 200 mV s−1 from cycles 10 to 70, a nucleus-like structure appears to develop on the uniform film at approximately 40 cycles, and at 70 cycles, the nucleus appears to grow (arrows in Fig. 4). ΔD is approximately 200 mV s−1 after 40 cycles (Fig. 3b inset), and the slope changes after 30 cycles. Similar slope changes were observed for the other scan rates, and at 25 mV s−1, a change point at approximately 15 cycles was observed. Before the slope change occurs, Δf decreases at a constant pace, and the change in ΔD is small, which is expected to indicate a uniform buildup of rigid films. After the slope change of ΔD and Δf, the slope change of Δf is particularly large, indicating that polymerization is progressing at a fast pace. In addition, the increase and decrease in Δf within one cycle gradually increase; this is thought to be due to the increased surface area caused by the growth of the spherical particles, which in turn increased the amount of monomer adsorption and desorption. Thus, polyMG films with various surface morphologies could be fabricated by controlling the polymerization conditions. Although there was some variation with the scanning speed, a uniform film was observed when ΔD was smaller than approximately 7 × 10−6, i.e., before the slope of ΔD began to change (25 mV s−1 for 10 cycles), and as ΔD increased, a nucleus-like structure began to appear (200 mV s−1 for 40 cycles). The results revealed that the degree of growth of the spherical particles can be inferred from ΔD.
AFM images of polyMG films formed at different scan rates and under different numbers of sweep cycles on the AuQCM sensor. Red arrows indicate what are thought to be nuclei that are generated during the spheroid growth process.
Figure 5 shows the 3D view and cross-sectional structural analysis of the polymerized films fabricated at 25 mV s−1 for 10 cycles and 25 mV s−1 for 70 cycles, as observed via AFM. Cross-sectional information revealed that the surface obtained at 10 cycles formed a mesoscale structure with a swordpile-shaped structure of approximately 5 nm in height, which was repeated at 40–50 nm intervals (Fig. 5a). In contrast, the surface obtained at 70 cycles had a macroscale structure with dispersed spherical particles 200 nm in height (Fig. 5b). These results showed that the surfaces of the uniform films formed mesoscale structures, whereas the nonuniform surfaces formed even larger macroscale structures. The 3D image in Fig. 5a shows highly sharp structures such as spikes in places. This is also observed at 200 mV s−1, as indicated by the arrows in Fig. 4 above, and is considered to be a nucleus in the preliminary stage of sphere formation, since the number of spheres increases as the number of cycles increases, i.e., polymerization proceeds, and the structure gradually changes to a larger structure (Fig. S5).
2D AFM images, cross-sectional analysis, and 3D AFM images of polyMG films electropolymerized at a) 25 mV s−1 for 10 cycles and b) 25 mV s−1 for 70 cycles on the AuQCM sensor.
We investigated how MG-polymerized films obtained under different electropolymerization conditions affect the properties of enzyme-immobilized electrodes. Pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) was immobilized on a carbon felt electrode coated with polyMG. The RMS calculated from the AFM images before and after enzyme adsorption on the polyMG film (25 mV s−1, 10 cycles) slightly decreased from 3.6 nm to 2.5 nm, which could be attributed to the adsorption of the enzyme on the surface (Fig. S6). CV measurements of the enzyme-immobilized electrode in 0.1 mol dm−3 glucose/buffer solution revealed that an increase in current from 0 V to higher potentials, indicating a current resulting from the enzyme reaction (Fig. 6). The CV measurement results of the enzyme-immobilized electrode under each polyMG film formation condition are shown in Fig. S7. Figure 7 shows the catalytic current values of the enzyme-immobilized electrode for each polymerization condition of the polyMG film. The value of the catalytic current was determined at +0.3 V, where the current almost reached a steady state (Fig. 6). The condition with the highest catalytic current was 25 mV s−1 for 10 cycles, where a uniform mesoscale structure was observed, and the catalytic current was 4.5 times greater than the lowest condition (25 mV s−1 for 70 cycles). Thus, the films with higher uniformity (smaller RMS) tended to have higher catalytic currents. The second highest catalytic current, 200 mV s−1 for 70 cycles, also gives a mesoscale structure with a swordpile-shaped structure repeated at intervals of approximately 60 nm, similar to the structure formed at 25 mV s−1 for 70 cycles (Fig. S8). In general, mesoscale structures are known to adsorb enzymes,31 and in an example of evaluating enzyme adsorption on carbon gels with mesoscale structures, it was shown that the 140 kDa enzyme does not adsorb in the 32 nm pore but does adsorb in the 44 nm and 60 nm pores.32 Since the PQQ-GDH used in this study is approximately 100 kDa, we believe that the mesoscale structure of approximately 50 nm observed in Fig. 5a may have contributed to enzyme retention. We speculate that the difference in the catalytic current is due not only to the surface topography but also to the amount of MG deposition (= Δf). Catalytic currents were compared for two groups of conditions (25, 50, and 200 mV s−1 for 10 cycles and 200 mV s−1 for 10, 40, and 70 cycles) that form highly uniform films with RMS values less than 10. In both groups, the higher the deposition (i.e., the larger the negative Δf), the higher the catalytic current value (Fig. S9). These results indicate that the condition for polyMG films suitable for enzyme-immobilized electrodes is high surface uniformity; moreover, a high amount of MG molecules in the film is desirable.
Cyclic voltammograms of polyMG (50 mV s−1 for 10 cycles) and PQQ-GDH-modified CF electrodes in 0.5 mol dm−3 phosphate buffer (pH 7.0) in the presence (solid line) and absence (dashed line) of 0.1 mol dm−3 glucose. The scan rate was 2 mV s−1.
Catalytic currents of the PQQ-GDH immobilized electrode in 0.5 mol dm−3 phosphate buffer solution containing 0.1 mol dm−3 glucose. The electrode was prepared by electropolymerization under various conditions (scan rates and sweep cycles) followed by PQQ-GDH adsorption. The catalytic current was the current value at +0.3 V of CV measured at 2 mV s−1.
We used EQCM-D and AFM techniques to observe the progress of the polymerization of MG and the surface morphology of the resulting polyMG under various electropolymerization conditions. When the potential was applied in sweeps, polymerization was accelerated more than when the potential was applied at a constant potential, and at negative potentials, the monomer adsorbed on the electrode and polymerization accelerated. The slower the scan rate and the greater the number of cycles, the greater the amount of polyMG deposited on the electrode. The surface structure of polyMG changed from a mesoscale structure of uniformly formed fine bumps at intervals of several tens of nm to nonuniform spherical particles as the polymerization progressed. Evaluation of the surface roughness using the RMS value calculated from the AFM images showed that the surface tended to become rougher with increasing Δf, and it was also observed that the roughness decreased with the adsorption of enzymes. The formation of spherical particles occurs around the area where the slope of ΔD obtained via the EQCM-D measurement increases, suggesting the possibility of inferring the surface structure by focusing on ΔD. According to the catalytic current evaluation of enzyme-immobilized electrodes prepared by adsorbing PQQ-GDH on polyMG, the catalytic current of electrodes with polyMG prepared under conditions of highly uniform film formation tended to be higher. A comparison of polyMG films with greater uniformity revealed that the greater the amount of MG deposited was, the greater the catalytic current was, indicating that both the surface structure and the amount of MG deposited affected the function of polyMG as a mediator.
In this study, we were able to add information on the morphology of the polymerized film to the previous knowledge on the polymerization mechanism of phenazine dyes. Furthermore, this finding can guide the selection of polymerization conditions to obtain the desired polyMG films. We believe that this direction can contribute to improving the performance of various biosensors and biofuel cells that use phenothiazine dyes as mediators.
This study was supported by JSPS KAKENHI (Grant Number JP 19K15615).
The data that support the findings of this study are openly available under the terms of the designated Creative Commons License in J-STAGE Data at https://doi.org/10.50892/data.electrochemistry.28252502.
Tomoe Nakagawa: Data curation (Lead), Formal analysis (Lead), Funding acquisition (Equal), Investigation (Lead), Methodology (Equal), Writing – original draft (Lead), Writing – review & editing (Equal)
Tomoko Gessei: Data curation (Supporting), Investigation (Supporting), Methodology (Equal), Writing – review & editing (Equal)
Akira Monkawa: Conceptualization (Supporting), Funding acquisition (Equal), Project administration (Equal), Resources (Equal), Writing – review & editing (Supporting)
Nobuhumi Nakamura: Conceptualization (Lead), Funding acquisition (Equal), Investigation (Equal), Methodology (Equal), Project administration (Lead), Resources (Lead), Supervision (Lead), Validation (Lead), Writing – original draft (Equal), Writing – review & editing (Lead)
The authors declare no conflict of interest in the manuscript.
Japan Society for the Promotion of Science: 19K15615
A part of this paper has been presented in the 91th ECSJ Meeting in 2024 (Presentation S5_3_10).
The content of this paper has been published as a preprint in SSRN. URL: http://dx.doi.org/10.2139/ssrn.4996366
T. Nakagawa: ECSJ Active Member
N. Nakamura: ECSJ Fellow
