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. 2020 Dec 8;5(50):32844–32851. doi: 10.1021/acsomega.0c05521

Constructive Optimization of a Multienzymatic Film Based on a Cascade Reaction for Electrochemical Biosensors

Kai Sasaki †,‡, Hiroyuki Furusawa ‡,§,∥,*, Kuniaki Nagamine †,∥, Shizuo Tokito †,∥,*
PMCID: PMC7758940  PMID: 33376922

Abstract

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The application of a multienzyme cascade reaction in electrochemical biosensors has the advantage of expanding the target substrates in addition to selectivity combining multiple enzymes on an electrode. However, the multienzyme system has the drawback of inefficient substance conversion because of the time-consuming passing of intermediates between the enzymes and/or diffusional loss of the intermediates. In this study, the optimal construction of a multienzymatic film in an ammonia detection sensor was investigated using a cascade reaction of l-glutamate oxidase and l-glutamate dehydrogenase as a model sensor. Three enzymatic films were prepared: (1) a mixed film designed to have a short diffusional distance between closely located enzymes, (2) a normal-sequential layered film arranged for the correct reaction pathway, and (3) a reverse-sequential layered film as a negative control. This was followed by comparison of the conversion efficiency of ammonia to hydrogen peroxide using time-dependent potentiometric measurements of a Prussian blue electrode determining the hydrogen peroxide amount. The results indicate that the conversion efficiency of the normal-sequential layered film was the highest among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction showed that compared to the mixed film (34%), a higher conversion efficiency of 92% was obtained in the first enzymatic reaction step. These findings will promote the use of multienzymatic cascade reaction systems not only in biosensors and bioreactors but also in various industrial fields.

Introduction

With the development of information and communication technology in recent years, the demand for sensors that quantify the surrounding environment as chemical information is increasing.1 Especially in the fields of agricultural automation and sustainable environmental monitoring, it is important to detect specific chemical substances.2 Enzymes are good candidates for receptors that capture specific chemicals on a sensor because of advantages such as excellent substrate selectivity and reaction specificity. In fact, enzymes were applied in fine and pharmaceutical chemistry,3,4 food manufacturing,5 and biological engineering (e.g., bioreactors, biosensors, and biofuel cells).6 Previously, an organic field-effect transistor (OFET)-based sensor was developed that detects a specific chemical substance in combination with an enzyme.7,8 Because an OFET-based sensor functions as an electrical potentiometric device, the chemical substance is detected as an electrochemical signal, which can be easily combined with an information transmission circuit.9 In the case of using a Prussian blue (PB)-carbon electrode as the sensing chip, various substance-specific oxidases can be applied as an efficient receptor because hydrogen peroxide as a byproduct of the oxidase reaction can oxidize Fe2+ ions in PB to Fe3+ ions, resulting in a positive potential increase of the PB-carbon electrode.7,10 Thus, if an oxidase that uses the target molecule as a substrate is available, a specific substance sensor can be built that combines a potentiometric device and a PB-carbon electrode on which the oxidase is immobilized. However, when the use of the enzyme-PB-device system is extended to various other fields, the available type of oxidases is limited. One approach to solve this drawback is to use a cascade reaction that combines multiple enzymes.

Multienzyme reaction systems using the cascade reaction were applied to bioreactors11,12 and biosensors13−15 to produce the desired compounds from target molecules in the systems. To develop a sensor for ammonia detection that monitors eutrophication in the environment,16 the multienzyme reaction system was applied because an enzyme that converts ammonia directly to hydrogen peroxide, such as ammonia oxidase, was not available.17 To build an electrochemical ammonia biosensor, a cascade reaction of l-glutamate oxidase (LGOX)18 and l-glutamate dehydrogenase (LGDH)19 was chosen, in which ammonia acts as an essential substrate (Figure 1A).20 Briefly, the multienzyme reaction system for ammonia detection includes three steps as follows: (1) an enzymatic amination reaction for the production of l-glutamate (L-glu) from α-ketoglutaric acid (α-KA) and NH3 induced by LGDH oxidizing the nicotinamide adenine dinucleotide phosphate reduced form (NADPH), (2) an oxidative deamination reaction to regenerate NH3 and α-KA from L-glu caused by LGOX, resulting in the production of hydrogen peroxide consuming oxygen, and (3) an oxidative reaction converting Fe2+ ions to Fe3+ ions in a PB-carbon electrode with reduction of hydrogen peroxide to two hydroxide ions. The ammonia detection was achieved measuring the accumulated electric potential changes in the PB-carbon electrode to which LGOX and LGDH were immobilized as a thin film mixed with chitosan as a film matrix.17 However, a multienzymatic cascade reaction system has disadvantages such as slow reaction rates21,22 and low yields11 because of the time-consuming transport of and/or diffusional loss of the intermediates during the transport between the reaction points. To resolve this issue, the enzymes were located more closely to shorten the diffusional distance.23,24 According to this approach, the enzymes were randomly or regularly immobilized on a porous silicate support25 coated with inorganic13 or other support materials.14,17,26−29 However, the diffusional distance and diffusive resistance affect each other because of the high-density immobilization in a solid of enzymatic materials.30 Therefore, the immobilization strategy needs further studies, and the optimization is still under debate.

Figure 1.

Figure 1

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(A) Illustration of a multienzyme reaction system for ammonia sensing on a Prussian blue (PB)-carbon electrode by using a cascade reaction of l-glutamate dehydrogenase (LGDH) and l-glutamate oxidase (LGOX), which produce hydrogen peroxide (H2O2) triggered by ammonia addition via the cycling reaction between α-ketoglutaric acid (α-KA) and l-glutamate (L-glu). (B) Illustration of the setup for potentiometric measurements with a working electrode (WE) and a reference electrode (RE) in response to the addition of H2O2, L-glu, and NH3 using a PB-carbon electrode covered by enzymatic (a) mixed film, (b) normal-sequential layered film, and (c) reverse-sequential layered film.

Our developed sensor, which is based on a LGOX–LGDH-reaction system and located on a PB-carbon electrode with a potentiometric device, has the advantage of a quantitative response as a function of the concentration of the target molecule and its intermediates.17 In this study, the relationship between the enzymatic film structure, such as homogeneously mixed or layered films, and the sensor responsiveness for the target or intermediate molecule was investigated. The strategy was to quantify each step of the cascade reaction. As shown in Figure 1B, three enzymatic films were prepared: (a) a mixed film in which the active sites of LGDH and LGOX were homogeneously arranged and closely located to each other to achieve a short diffusional distance, (b) a normal-sequential layered film assembled with a multilayered film of LGDH and LGOX stacked on a PB-carbon electrode to provide an enzymatic cascade reaction, and (c) a reverse-sequential layered film composed of a multilayered film structure in reversed order. To demonstrate the effect of the enzymatic film structures on ammonia detection, the electric potential change (ΔE) of each enzymatic film electrode in response to the ammonia addition was measured and the detection rate was evaluated. Furthermore, to investigate the conversion efficiency of the enzymatic film structures on the intermediate reaction in the detection process, the detection rates of the (a) mixed film and (b) normal-sequential layered film electrode against H2O2 and L-glu as intermediates were compared.

Results and Discussion

Potentiometric Ammonia Detection on the Enzymatic Film Electrode

First, the responsiveness of a PB-carbon electrode, covered by a homogeneous enzyme-mixed film (mixed film, Figure 1B(a)), to the addition of ammonia solution (final concentration: 50 μM) was confirmed as reported previously.17 A response curve of the electric potential change (ΔE) was observed similar to that previously reported17 (Figure 2, curve b). This increasing positive potential change indicates that the mixed film electrode detects ammonia in PB through the accumulation of positive Fe3+ ion charges according to the designated cascade reaction mechanism (Figure 1A) because the curve was not observed in the absence of NADPH, which is a cofactor of LGDH.17

Figure 2.

Figure 2

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Typical electric potential changes over time (ΔE, vs Ag/AgCl) in response to ammonia addition (final concentration: 50 μM) using the electrode covered by (a) normal-sequential layered film, (b) mixed film, and (c) reverse-sequential layered film. The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl, 2 mM NADPH, and 5 mM α-KA.

Next, an ammonia solution was added to an electrode with a normal-sequential layered film (Figure 1B(b)) that was placed in a measurement beaker. As shown in Figure 2, a larger ΔE was observed using the normal-sequential layered film on the electrode (curve a) compared to the mixed film (curve b). In contrast, the response of the electrode with a reverse-sequential layered film to the addition of ammonia solution was clearly less than that of the other films (curve c), despite the same film components and quantity. Thus, the ammonia responsiveness of each electrode was influenced by the structure of the enzymatic film on the PB-carbon electrode. These results indicate that the structural design of the enzymatic film could improve the detection of a target molecule, which should enhance the diffusion process of substrates and intermediates or the enzyme reaction. Because the placement of the enzyme films was highly effective (curves a and c), the main improvement might be related to the diffusion process.

Dependence of the Initial Response Rate on Ammonia Concentration

Plotting the initial rates of the ΔE response curve using the homogeneous mixed film against the ammonia concentration over time resulted in a saturation curve according to the Michaelis–Menten equation. The linear relationship was obtained in the low concentration range (0–100 μM) in a previous study.17 The slope of the linear relationship between the target molecule concentration and the detection signal of a device provides the limit of detection (LOD) according to eq 1

graphic file with name ao0c05521_m001.jpg 1

where SD is the standard deviation for 0 μM ammonia.31 According to eq 1, higher slope values result in lower LOD values, meaning a better sensor performance. Therefore, the slope values can be used to evaluate the performance of a sensor.

The ΔE responses of each enzymatic film electrode as a function of ammonia concentrations (0–100 μM) were observed, and the initial rate values of ΔE, [Δ(ΔE)i/Δt], were plotted against the ammonia concentration (Figure S1). The calculated slope values of these linear plots are summarized in Table 1. The slope value of the normal-sequential layered film was 5.4 ± 0.14 μV s–1 μM–1. This value was 1.8-fold higher than that of the mixed film (3.0 ± 0.21 μV s–1 μM–1), indicating that the LOD was improved 1.8 times. Thus, the normal-sequential layered film is compared to the mixed film, an effective enzymatic structure to improve the ammonia detection.

Table 1. Summary of Slope Values of the Linear Relationship between Substrate Concentrations and Initial Rate Values of ΔE, [Δ(ΔE)i/Δt], Measured by the Enzymatic Film-Attached Electrodesg.

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a

Each electrode is illustrated along with an enzyme layer construction, in which LGOX and LGDH are indicated in green and yellow, respectively.

b

The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl in the range of 0–25 μM H2O2.

c

The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl in the range of 0–100 μM L-glu.

d

The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl, 2 mM NADPH, and 5 mM α-KA in the range of 0–100 μM NH3.

e

The values in parentheses indicate the relative slope values calculated based on the slope value using a bare electrode for H2O2 detection.

f

LOD was calculated using eq 1 with SD = 0.19 (mV s–1).

g

The experimental errors were obtained from linear regression analysis.

In contrast, the slope value of the reverse-sequential layered film (1.0 ± 0.10 μV s–1 μM–1) was 3.0-fold lower than that of the mixed film. In case of the reverse-sequential layered film, the reduced ammonia responsiveness was related to the decrease of ammonia receptors (LGDH) at the film–solution interface. The ratio for the normal-sequential layered, mixed, and reverse-sequential layered film is estimated to be 2:1:0, respectively. The other causes were related to an increase in the average migration distance of the target substrate (NH3) and the intermediate substances (L-glu and H2O2) because of the back and forth motion at the electrode surface in the film, which resulted in the loss of intermediates because of diffusion to the bulk.

It was initially expected that the mixed film structure was advantageous for the substance conversion efficiency because the LGOX and LGDH were close to each other at a molecular level. However, the results indicate that the normal-sequential layered film structure is more effective as a sensor film. To clarify the reason for the better efficiency, additional experiments were performed to investigate the responsiveness of the sensors to intermediates (H2O2 and L-glu).

Conversion Efficiency for Intermediates in the Mixed Film

Typical response curves of ΔE to the addition of H2O2, L-glu, and NH3 solutions using the mixed film electrode (final concentration: 5 μM) are shown in Figure 3A. As a control experiment, we obtained a ΔE curve in response to the addition of H2O2 solution for a bare PB-carbon electrode that was not covered by any film. In theory, the response rate of the bare PB-carbon electrode to H2O2 molecules should be the fastest. Furthermore, the initial rates of ΔE, [Δ(ΔE)i/Δt], were plotted against changing concentrations (0–100 μM) of each substrate (Figure 3B). The slope values of these linear plots were calculated for quantitative comparison and are summarized in Table 1. As expected, the existence of the film (Figure 3A, curve b) reduced the apparent reactivity of PB with H2O2 in comparison with the bare electrode (curve a). The slope of the straight line (Figure 3B, line b) for the mixed film electrode in response to H2O2 was reduced approximately 0.14-fold compared to that of the bare PB-carbon electrode (line a). This decrease due to the film attachment was independent of the film thickness because the half-thickness film of only LGOX reduced the apparent reactivity of PB with H2O2 approximately 0.12-fold (see Table 1). It should be emphasized that the responsiveness with H2O2 of the PB covered by the mixed film was still proportional to the substrate concentration (Figure 3B, line b). This means that the concentration in the film is correlated with the concentration in the bulk. Thus, the decrease in apparent reactivity of PB with H2O2 at the surface of the electrode could be explained by a decrease in the collision frequency of H2O2 because of the small mass transport in the nonstirred film as compared to the well-stirred bulk. Moreover, the slope of the straight line for the L-glu responsiveness (line c) was 0.59-fold lower than that of H2O2 (line b). This indicates that the PB-carbon electrode reacted with H2O2 at an apparent concentration 0.59-fold higher than that of L-glu. Thus, the apparent LGOX conversion efficiency from L-glu to H2O2, including the loss of intermediates owing to diffusion, should be 0.59-fold. Similarly, the slope of the NH3 responsiveness (line d) was 0.34-fold lower than that of L-glu (line c). Thus, the apparent LGDH conversion efficiency from NH3 to L-glu should be 0.34-fold. These decreases in the slope depend on the increase in the number of reaction steps in the film (Figure 1A). Thus, the efficiency improvement of each reaction step in an enzymatic cascade reaction contributes to the detection efficiency in the sensing process.

Figure 3.

Figure 3

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(A) Typical electric potential changes (ΔE, vs Ag/AgCl) in response to the addition of (a,b) H2O2, (c) L-glu, and (d) NH3 (final concentration: 5 μM) using the (a) bare electrode and the (b)–(d) mixed film electrode. The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl for the H2O2 and L-glu or 200 mM KCl, 2 mM NADPH, and 5 mM α-KA for the NH3 detection. (B) Linear plots for the initial rate values of ΔE measured by the (a) bare electrode and the (b)–(d) mixed film electrode as a function of substrate concentrations: (a,b) H2O2 (0–25 or 100 μM), (c) L-glu (5–100 μM), and (d) NH3 (5–100 μM). The line (d) was refined by remeasurements from our previous report.17 The error bars indicate the standard deviation (n = 3).

Advantage of Normal-Sequential Layered Film in Ammonia Detection

To investigate the mechanism for improving the ammonia conversion efficiency to hydrogen peroxide in a normal-sequential layered film, the conversion efficiency of the intermediates in each step was evaluated similar to the mixed film. Figure 4A shows ΔE as function of substrate addition (final concentration: 5 μM) using a normal-sequential layered film electrode. Then, the initial rate values of ΔE were plotted against various concentrations (0−100 μM) (Figure 4B). The calculated slopes of these linear plots are summarized in Table 1. The decrease of the slope for the normal-sequential layered film in response to H2O2 (Figure 4B, line b) was similar to that of the mixed film (0.15- and 0.14-fold, respectively) compared to the bare PB-carbon electrode (line a). The decrease in responsiveness due to film attachment did not depend on the type of film. The slope for the L-glu responsiveness on the normal-sequential layered film electrode (Figure 4B, line c) was lower compared to that of the mixed film electrode (Figure 3B, line c). The reason for the higher decrease was the negative effect of the LGDH film cover, which limited the exposure to the bulk solution of LGOX. In fact, this negative effect reduced the slope value for the L-glu responsiveness from 7.9 (only LGOX Film) to 5.9 (normal-sequential layered film), which is approximately 0.75-fold (Table 1).

Figure 4.

Figure 4

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(A) Typical electric potential changes (ΔE, vs Ag/AgCl) as function of (a,b) H2O2, (c) L-glu, and (d) NH3 addition (final concentration: 5 μM), using the (a) bare and the (b)–(d) normal-sequential layered film electrode. The measurements were carried out at 25 °C in a 100 mM phosphate buffer solution (pH 8.0) containing 200 mM KCl for the H2O2 and L-glu detection, or 200 mM KCl, 2 mM NADPH, and 5 mM α-KA for the NH3 detection. (B) Linear plots for the initial rate values of ΔE measured by the (a) bare and (b)–(d) normal-sequential layered film electrode as a function of substrate concentrations: (a,b) H2O2 (0–25 or 100 μM), (c) L-glu (5–100 μM), and (d) NH3 (5–100 μM). The error bars indicate the standard deviation (n = 3). The curve a and line a are the same as those in Figure 3 for ease of comparison.

It was observed that the slope value for NH3 responsiveness (Figure 4B, line d) was not significantly different from that for L-glu (Figure 4B, line c) despite the increase in the number of reaction steps. This suggests that the ammonia conversion efficiency to L-glu in a normal-sequential layered film could be almost 100%. The conversion efficiency was 92% derived from the slope values of 5.4 (for NH3) and 5.9 (for L-glu) (Table 1).

The reasons are suggested to be the following: (1) exposure of ammonia receptors (LGDH) at a film–solution interface should be maximum, (2) L-glu conversion in the LGDH film was maintained because of a suppressed diffusion in the film, and (3) L-glu converted in the LGDH film is fluxed more in the direction of the LGOX film on the electrode than that of the mixed film because a concentration gradient is formed resulting from the immediate consumption of L-glu by LGOX.23

Effect of Three Layers of LGOX-Mixed Layer-LGDH on Ammonia Detection

As a more improved design of the film construction, a three-layered film was constructed, which has an enzyme-mixed layer between the LGOX and LGDH layer because the film possesses the features of (1) maximum exposure of ammonia receptors (LGDH) at the film–solution interface, (2) close proximity of LGOX and LGDH at the molecular level in the enzyme-mixed layer, and (3) the introduction effect of an L-glu concentration gradient resulting from the consumption of L-glu by the LGOX layer (Figure S2). However, the slope value for the ammonia responsiveness on the three-layered film electrode was 0.67-fold smaller than that on the mixed film electrode (lines a and b). The feature of (3) could not be demonstrated because of the presence of a moderate enzyme-mixed layer. Thus, an interface between two enzyme layers in a cascade reaction is required to achieve superior responsiveness of the biosensors. In other words, the results suggest that the technique used in this study to immobilize an enzyme on a sensor electrode as a layer could form the sequential layered enzyme film. Further research regarding the mechanism will require computational simulation of substrate/intermediate diffusion in the film.

Conclusions

The optimization of enzymatic film structures, which includes two enzymes that cause a cascade reaction on an electrode for a biosensor, was investigated based on a potentiometric measurement. To develop a sensor for NH3 detection, we demonstrated three enzymatic films: (1) a mixed LGDH–LGOX film to create adjacent active sites to obtain a short intermediate diffusion distance, (2) a normal-sequential layered film having a stacked LGDH–LGOX layer along the cascade reaction pathway, and (3) a reverse-sequential layered film electrode having the reversed order of a normal-sequential layered film. The ammonia responsiveness of the normal-sequential layered film on a PB-carbon electrode was the most effective among the three enzymatic films. The quantitative evaluation of the intermediate conversion efficiency of the cascade reaction using the enzymatic films on the electrode, similar to the ammonia detection, showed that the normal-sequential layered film structure provided a higher conversion efficiency (over 90%) in the first enzymatic reaction step than the mixed film (34%). This fact encourages the use of multienzymatic cascade reaction systems because the application of the film structure can avoid the fundamental problems of the systems, such as the low responsiveness due to the slow diffusion and the loss of reaction intermediates. One limitation of our enzyme-based biosensors is the need to externally and continuously supply NADPH consumed in the reaction. To overcome this, we are focusing on combining the biosensor with an NADPH regeneration system by using an electrochemical mediator.32−34 In addition, by advancing the enzyme immobilization technology research, it is expected that the multienzyme system will be applied not only in the biosensor field but also in the wider industry.

Experimental Section

Materials

l-glutamate oxidase (LGOX, recombinant, and E.C. 1.4.3.11, manufactured from YAMASA Corporation, Tokyo, Japan) and l-glutamate dehydrogenase (LGDH, from yeast, E.C. 1.4.1.4, manufactured from Oriental Yeast Co., Ltd., Tokyo, Japan), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), l-glutamic acid sodium salt monohydrate (L-glu), and hydrogen peroxide solution (30%) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Ethylene glycol diglycidyl ether (EGDE) and α-ketoglutaric acid (α-KA) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Chitosan was acquired from Junsei Chemical (Tokyo, Japan). Aqueous ammonia solution (14.8 M) was procured from Kanto Chemical (Tokyo, Japan). The Prussian blue (PB)-carbon electrode (electrode diameter: 6 mm) manufactured from Gwent Electronic Materials (London, UK) was provided by DIC Corporation (Tokyo, Japan). Other reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). Type 1 ultrapure water (MilliQ water, Merck Ltd., Tokyo, Japan) was used in all experiments.

Preparation of Enzymatic Films Using LGDH and LGOX on Electrodes

An aqueous solution of LGDH (1.25 U/μL) was prepared and then 2 μL was aliquoted into a microtube and stored at −80 °C until use. An aqueous solution of LGOX (0.25 U/μL) was also aliquoted in the same manner and stored. After dissolution of chitosan in 50 mM HCl solution to obtain a 1% solution, the pH was adjusted to 5.4 with a small amount of 1 M NaOH solution. A chitosan solution diluted to 0.1% was used in this experiment. A 1 v/v % solution of EGDE in 100 mM phosphate buffer (pH 8.0) was prepared just before use.

To form a mixed enzymatic film on a PB-carbon electrode (Figure 1B(a)), a 14 μL polyion-complex solution including 10 μL of 0.1% chitosan solution, 2 μL of LGOX (0.25 U/μL), and 2 μL of LGDH (1.25 U/μL) was drop-cast on the entire PB-carbon electrode. Then, the electrode was incubated in a dry oven at 30 °C for 20 min. Next, 10 μL of a 1 v/v % EGDE solution was drop-cast onto the electrode, followed by incubation at 30 °C for 20 min to cross-link the amino groups of chitosan and/or the enzymes (Figure 5A). The amounts of the enzymes, chitosan, and EGDE were same as those reported in a previous study and were optimized for maximal biosensor response.17 Regarding an effect of the cross-linking reaction on the enzyme, it has been confirmed that the enzyme is not deactivated even if the film preparation operation is divided into two times. This drop-cast procedure was applied to prepare a normal-sequential (Figure 1B(d)) and reverse-sequential layered film (Figure 1B(c)), drop-casting 7 μL of the polyion-complex solution containing only LGDH or LGOX and then 5 μL of 1 v/v % EGDE solution on the PB electrodes, resulting in the same amounts of LGDH, LGOX, chitosan, and EDGE on each electrode (Figure 5B). A SEM image of the PB-carbon electrode covered with the normal-sequential layered film together with that of a bare PB-carbon electrode is shown in Figure S3. The SEM images showed that the enzyme film smoothly covers the electrodes. The film thickness is estimated to be the amount required to smoothen the roughness of the carbon electrode, which seems to be a few micrometers.

Figure 5.

Figure 5

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Schematic illustration of preparation procedure for (A) homogeneously mixed and (B) layered enzymatic films on a PB-carbon electrode.

Potentiometric Measurements to Evaluate Enzymatic Films

The PB-carbon electrode with the attached enzymatic film was connected to a source measurement unit (Keithley model 2450 SourceMeter SMU, Tektronix Inc., USA) as a working electrode together with an Ag/AgCl reference electrode (RE-1B, BAS Inc., Tokyo, Japan) (Figure 1B). Potentiometric measurements were carried out using a mode for potential difference in 5 mL of 100 mM phosphate buffer solution (pH 8.0), which contained 200 mM KCl for the H2O2 and L-glu, or 200 mM KCl, 2 mM NADPH, and 5 mM α-KA for the NH3 measurement. Data were acquired every 0.083 s. The measurement solution was kept in a beaker at 25 °C and stirred using a magnetic stirrer bar at 1000 rpm. Before the potentiometric measurements, each film electrode was shunted to the Ag/AgCl electrode for 30 s to reduce Fe3+ in PB (redox potential: 206 mV vs Ag/AgCl). Then, the electric potential of the electrode was monitored until the potential stabilized at approximately 150 mV. After the pretreatment, the electric potential changes in response to the addition of each substrate solution (H2O2, L-glu, or NH3) were measured.

Acknowledgments

The authors acknowledge Tomoko Okamoto from the DIC Corporation for providing the PB electrode.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c05521.

  • Additional linear plot charts and scanning electron microscopy images of electrode surfaces (PDF)

Author Contributions

All authors have given approval to the final version of the manuscript.

This work was partially supported by JSPS KAKENHI grant number JP19K05181 and grant-in-aid for JSPS Fellows grant number JP20J12942.

The authors declare no competing financial interest.

Supplementary Material

ao0c05521_si_001.pdf (321.3KB, pdf)

References

  1. Wen F.; He T.; Lui H.; Chen H.-Y.; Zhang T.; Lee C. Advances in Chemical Sensing Technology for Enabling the Next-generation Self-sustainable Integrated Wearable System in the IoT Era. Nano Energy 2020, 78, 105155. 10.1016/j.nanoen.2020.105155. [DOI] [Google Scholar]; in press
  2. Mercante L. A.; Scagion V. P.; Migliorini F. L.; Mattoso L. H. C.; Correa D. S. Electrospinning-Based (Bio)Sensors for Food and Agricultural Applications: A Review. Trends Anal. Chem. 2017, 91, 91–103. 10.1016/j.trac.2017.04.004. [DOI] [Google Scholar]
  3. Reetz M. T. Biocatalysis in Organic Chemistry and Biotechnology: Past, Present, and Future. J. Am. Chem. Soc. 2013, 135, 12480–12496. 10.1021/ja405051f. [DOI] [PubMed] [Google Scholar]
  4. Bruggink A.; Roos E. C.; de Vroom E. Penicillin Acylase in the Industrial Production of ß-Lactam Antibiotics. Org. Process Res. Dev. 1998, 2, 128–133. 10.1021/op9700643. [DOI] [Google Scholar]
  5. Akoh C. C.; Chang S.-W.; Lee G.-C.; Shaw J.-F. Biocatalysis for the Production of Industrial Products and Functional Foods from Rice and Other Agricultural Produce. J. Agric. Food Chem. 2008, 56, 10445–10451. 10.1021/jf801928e. [DOI] [PubMed] [Google Scholar]
  6. Khan A. A.; Alzohairy M. A. Recent Advances and Applications of Immobilized Enzyme Technologies: A Review. Res. J. Biol. Sci. 2010, 5, 565–575. 10.3923/rjbsci.2010.565.575. [DOI] [Google Scholar]
  7. Furusawa H.; Ichimura Y.; Nagamine K.; Shiwaku R.; Matsui H.; Tokito S. Detection of 1,5-anhydroglucitol as a Biomarker for Diabetes Using an Organic Field-Effect Transistor-Based Biosensor. Technologies 2018, 6, 77. 10.3390/technologies6030077. [DOI] [Google Scholar]
  8. Tsuchiya K.; Furusawa H.; Nomura A.; Matsui H.; Nihei M.; Tokito S. Formaldehyde Detection by a Combination of Formaldehyde Dehydrogenase and Chitosan on a Sensor Based on an Organic Field-Effect Transistor. Technologies 2019, 7, 48. 10.3390/technologies7030048. [DOI] [Google Scholar]
  9. Shiwaku R.; Matsui H.; Nagamine K.; Uematsu M.; Mano T.; Maruyama Y.; Nomura A.; Tsuchiya K.; Hayasaka K.; Takeda Y.; Fukuda T.; Kumaki D.; Tokito S. A Printed Organic Circuit System for Wearable Amperometric Electrochemical Sensors. Sci. Rep. 2018, 8, 6368. 10.1038/s41598-018-24744-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Nagamine K.; Mano T.; Shiwaku R.; Furusawa H.; Matsui H.; Kumaki D.; Tokito S. An L-lactate Biosensor Based on Printed Organic Inverter Circuitry and with a Tunable Detection Limit. Sensor. Mater. 2019, 31, 1205–1213. 10.18494/sam.2019.2174. [DOI] [Google Scholar]
  11. Lopez-Gallego F.; Batencor L.; Hidalgo A.; Mateo C.; Fernandez-Lafuente R.; Guisan J. M. One-Pot Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid in the Absence of Hydrogen Peroxide. Adv. Synth. Catal. 2005, 347, 1804–1810. 10.1002/adsc.200505099. [DOI] [Google Scholar]
  12. Chi Y.; Scroggins S. T.; Fréchet J. M. J. One-Pot Multi-Component Asymmetric Cascade Reactions Catalyzed by Soluble Star Polymers with Highly Branched Non-Interpenetrating Catalytic Cores. J. Am. Chem. Soc. 2008, 130, 6322–6323. 10.1021/ja8013456. [DOI] [PubMed] [Google Scholar]
  13. Boujakhrout A.; Sánchez E.; Díez P.; Sánchez A.; Martínez-Ruiz P.; Parrado C.; Pingarrón J. M.; Villalonga R. Single-Walled Carbon Nanotubes/Au–Mesoporous Silica Janus Nanoparticles as Building Blocks for the Preparation of a Bienzyme Biosensor. ChemElectroChem 2015, 2, 1735–1741. 10.1002/celc.201500244. [DOI] [Google Scholar]
  14. Ma L.; Yue Z.; Huo G.; Zhang S.; Zhu B.; Zhang S.; Huang W. 3D Hydrogen Titanate Nanotubes on Ti Foil: A Carrier for Enzymatic Glucose Biosensor. Sensors 2020, 20, 1024. 10.3390/s20041024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jasti L. S.; Dola S. R.; Fadnavis N. W.; Addepally U.; Daniels S.; Ponrathnam S. Co-Immobilized Glucose Oxidase and ß-Galactosidase on Bovine Serum Albumin Coated Allyl Glycidyl Ether (AGE)–Ethylene Glycol Dimethacrylate (EGDM) Copolymer as a Biosensor for Lactose determination in Milk. Enzym. Microb. Technol. 2014, 64–65, 67–73. 10.1016/j.enzmictec.2014.07.005. [DOI] [PubMed] [Google Scholar]
  16. Timmer B.; Olthuis W.; Berg A. V. D. Ammonia sensors and their applications—a review. Sens. Actuators, B 2005, 107, 666–677. 10.1016/j.snb.2004.11.054. [DOI] [Google Scholar]
  17. Sasaki K.; Furusawa H.; Nagamine K.; Tokito S. Charge-accumulative Potentiometric Measurements for Ammonia Detection Using an Enzymatic Cascade Reaction on a Prussian Blue Electrode. Chem. Lett. 2018, 47, 1412–1415. 10.1246/cl.180692. [DOI] [Google Scholar]
  18. Arima J.; Tamura T.; Kusakabe H.; Ashiuchi M.; Yagi T.; Tanaka H.; Inagaki K. Recombinant Expression, Biochemical Characterization and Stabilization through Proteolysis of an L-Glutamate Oxidase from Streptomyces sp. X-119-6. J. Biochem. 2003, 134, 805–812. 10.1093/jb/mvg206. [DOI] [PubMed] [Google Scholar]
  19. Coulton J. W.; Kapoor M. Purification and Some Properties of the Glutamate Dehydrogenase of Salmonella Typhimurium. Can. J. Microbiol. 1973, 19, 427–438. 10.1139/m73-071. [DOI] [PubMed] [Google Scholar]
  20. Kayamori Y.; Katayama Y. An Enzymatic Cycling Method for the Determination of Ammonia. Bunseki Kagaku 1989, 38, 188–192. 10.2116/bunsekikagaku.38.4_188. [DOI] [Google Scholar]
  21. Ljungcrantz P.; Carlsson H.; Mansson M. O.; Buckel P.; Mosbach K.; Buelow L. Construction of an Artificial Bifunctional Enzyme, ß-Galactosidase/Galactose Dehydrogenase, Exhibiting Efficient Galactose Channeling. Biochemistry 1989, 28, 8786–8792. 10.1021/bi00448a016. [DOI] [PubMed] [Google Scholar]
  22. Zhang Y.-H. P. Production of Biocommodities and Bioelectricity by Cell-Free Synthetic Enzymatic Pathway Biotransformations: Challenges and Opportunities. Biotechnol. Bioeng. 2010, 105, 663–677. 10.1016/j.copbio.2010.05.005. [DOI] [PubMed] [Google Scholar]
  23. Spivey H. O.; Ovádi J. Substrate Channeling. Methods 1999, 19, 306–321. 10.1006/meth.1999.0858. [DOI] [PubMed] [Google Scholar]
  24. Geck M. K.; Kirsch J. F. A Novel, Definitive Test for Substrate Channeling Illustrated with the Aspartate Aminotransferase/Malate Dehydrogenase System†. Biochemistry 1999, 38, 8032–8037. 10.1021/bi983029c. [DOI] [PubMed] [Google Scholar]
  25. Mukai C.; Gao L.; Nelson J. L.; Lata J. P.; Cohen R.; Wu L.; Hinchman M. M.; Bergkvist M.; Sherwood R. W.; Zhang S.; Travis A. J. Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate. Angew. Chem., Int. Ed. 2017, 56, 235–238. 10.1002/anie.201609495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zhou L.; Tang W.; Jiang Y.; Ma L.; He Y.; Gao J. Magnetic Combined Cross-Linked Enzyme Aggregates of Horseradish Peroxidase and Glucose Oxidase: an Efficient Biocatalyst for Dye Decolourization. RSC Adv. 2016, 6, 90061–90068. 10.1039/c6ra12009a. [DOI] [Google Scholar]
  27. Elani Y.; Law R. V.; Ces O. Vesicle-Based Artificial Cells as Chemical Microreactors with Spatially Segregated Reaction Pathways. Nat. Commun. 2014, 5, 5305. 10.1038/ncomms6305. [DOI] [PubMed] [Google Scholar]
  28. Vriezema D. M.; Garcia P. M. L.; Oltra N. S.; Hatzakis N. S.; Kuiper S. M.; Nolte R. J. M.; Rowan A. E.; van Hest J. C. M. Positional Assembly of Enzymes in Polymersome Nanoreactors for Cascade Reactions. Angew. Chem., Int. Ed. 2007, 46, 7378–7382. 10.1002/anie.200701125. [DOI] [PubMed] [Google Scholar]
  29. Ghéczy N.; Sasaki K.; Yoshimoto M.; Pour-Esmaeil S.; Kröger M.; Stano P.; Walde P. A Two-Enzyme Cascade Reaction Consisting of Two Reaction Pathways. Studies in Bulk Solution for Understanding the Performance of a Flow-Through Device with Immobilized Enzymes. RSC Adv. 2020, 10, 18655–18676. 10.1039/d0ra01204a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Idan O.; Hess H. Origins of Activity Enhancement in Enzyme Cascades on Scaffolds. ACS Nano 2013, 7, 8658–8665. 10.1021/nn402823k. [DOI] [PubMed] [Google Scholar]
  31. Currie L. A. Nomenclature in Evaluation of Analytical Methods, Including Detect ion and Quantification CapabiIities (IUPAC Recommendations 1995). Pure Appl. Chem. 1995, 67, 1699–1723. 10.1351/pac199567101699. [DOI] [Google Scholar]
  32. Wang X.; Saba T.; Yiu H. H. P.; Howe R. F.; Anderson J. A.; Shi J. Cofactor NAD(P)H Regeneration Inspired by Heterogeneous Pathways. Chem 2017, 2, 621–654. 10.1016/j.chempr.2017.04.009. [DOI] [Google Scholar]
  33. Vuorilehto K.; Lütz S.; Wandrey C. Indirect Electrochemical Reduction of Nicotinamide Coenzymes. Bioelectrochemistry 2004, 65, 1–7. 10.1016/j.bioelechem.2004.05.006. [DOI] [PubMed] [Google Scholar]
  34. Hildebrand F.; Lütz S. Stable Electroenzymatic Processes by Catalyst Separation. Chem. —Eur J. 2009, 15, 4998–5001. 10.1002/chem.200900219. [DOI] [PubMed] [Google Scholar]

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