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Sensors and Materials, Vol. 35, No. 10 (2023) 4721–4730
MYU Tokyo
S & M 3425

Nanocarbon-assisted Carbon-nanotube-based Composite
Electrodes for Improved Laccase Bio-electrocatalysis
Masato Tominaga,1* Takuya Takatori,1 Makoto Togami,2 and Masayuki Tsushida3
1Graduate School of Science and Engineering, Saga University,

1 Honjomachi, Saga 840-8502, Japan

2Graduate School of Science and Technology, Kumamoto University,

2-39-1 Kurokami, Kumamoto 860-8555, Japan

3Faculty of Engineering, Kumamoto University,

2-39-1 Kurokami, Kumamoto 860-8555, Japan(Received March 27, 2023; accepted October 2, 2023)

Keywords: carbon nanotube, nanocarbon, laccase, electrocatalysis, oxygen reduction

Wearable devices have rapidly developed in recent years. The practical applications ofwearable devices utilize secondary batteries such as lithium-ion batteries. Biofuel cells are apromising next-generation power source for these devices because they can be operated under
mild conditions. Nanocarbons are essential materials owing to their excellent electricalconductivity and high surface area. However, nanocarbons easily aggregate, which results in anelectrochemical active surface area significantly smaller than their theoretical individual surface
area. Although carbon nanotubes (CNTs) have high electrical conductivity owing to their
network structure and large specific surface area, it is well known that CNTs form bundled
structures by aggregation. In this study, to completely exploit the properties of CNTs, a modified
electrode was fabricated using a nanocarbon composite material with CNTs as the base material.
Laccase bio-electrocatalysis tests were conducted, and the results clearly demonstrated thecomposite effect of CNTs with different nanocarbon morphologies, such as Ketjen black.

1.

Introduction

Wearable devices for health maintenance have attracted considerable attention in recent
years. Currently, it is possible to continuously measure blood oxygen concentration, obtain anelectrocardiogram, and determine body temperature using a smart watch. However, the powersources for these measurements are secondary batteries such as lithium-ion batteries. Biofuel
cells that use independently driven enzyme catalysis are expected to be the next-generation
power sources for these wearable devices.(1) A biofuel cell is particularly suitable for combining
with a wearable biosensor, which is a concept of enzymatic biofuel-cell-powered biosensors.
This concept was easily achieved by Wang’s group using screen-printing technology.(2)Currently, many wearable biosensor types have been reported, such as the glucose biosensor,(3,4)
glutathione sensor,(5) and diaper sensor.(6) Such enzymatic biofuel-cell-powered biosensor*Corresponding author: e-mail: masato@cc.saga-u.ac.jp

https://doi.org/10.18494/SAM4407

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

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Sensors and Materials, Vol. 35, No. 10 (2023)

systems can be made from an anode and a cathode. Multicopper enzymes (MCEs) such aslaccase, bilirubin oxidase, copper efflux oxidase, and ascorbate oxidase are powerful cathodes
in enzymatic biofuel cells owing to their ability to reduce oxygen at a high potential without amediator.(7–14) Moreover, the MCE-catalyzed reaction can be achieved under mild conditions.
The effective utilization of nanocarbons, which have excellent electrical conductivity and highsurface area, is crucial for the realization of wearable biofuel cells.(15–18) However, tailoring
nanocarbons for application in electrodes is problematic because nanocarbons tend to
agglomerate, and thus their theoretical surface area cannot be achieved. Carbon nanotubes(CNTs) are promising electrode materials for biofuel cells because of their high electricalconductivity, which originates from their network structure and large specific surface area.
(16,18–20) In contrast, it is very difficult to completely utilize the properties of CNTs to prepare
electrodes because CNTs are significantly agglomerated compared with other nanocarbons.
Therefore, in this study, we aim to completely exploit the properties of CNTs by fabricating
electrodes using composites composed of nanocarbons with different morphologies and CNTs
for application in laccase bio-electrocatalysis. The obtained results show that laccase bioelectrocatalysis clearly demonstrates the composite effect of CNTs with different nanocarbon
morphologies, such as Ketjen black. Additionally, surface modification, which optimizes themolecular orientation of laccase adsorbed on the electrode, is demonstrated to be effective fornanocarbon composites with different surface properties.

2.

Materials and Methods

2.1

Materials

Fungal laccase (Lac) from Trametes sp. (EC1.10.3.2) was obtained from Amano Enzyme(Nagoya, Japan) and purified as previously reported.(21,22) All other chemical reagents were ofanalytical grade and were used as received without further purification. Single-walled carbon
nanotubes (SWCNTs, diameter: 3–5 nm), OSAB (diameter: 10–30 nm), HS100 (diameter: 40–
100 nm), H25 (size: 10–30 µm, thickness: ca. 10 nm), Ketjen black EC600JD (KB, diameter:
20–50 nm), and carbon black (CB, diameter: 1–5 µm) were used as nanocarbon materials.
SG101 SWCNTs were provided by Zeon Co., Ltd. (Tokyo, Japan). OSAB and HS100 were
provided by Denka Black Co., Ltd. (Tokyo, Japan). Plate-shaped pyrolytic graphite H25 was
obtained from XG Science (Michigan, USA). KB and CB were purchased from Lion Specialty
Chemicals Co., Ltd. (Tokyo, Japan) and Sigma–Aldrich (St. Louis, MO, USA), respectively.
Panasonic graphite PGX 05 (Matsushita Electric Co., Japan) was used as the highly oriented
pyrolytic graphite (HOPG). A glassy carbon (GC) disk electrode (outer diameter: 6 mm, innerdiameter: 3 mm) was purchased from BAS Inc. (Tokyo, Japan). Sodium cholate (SC) was
obtained from Wako Pure Chemical Industries (Osaka, Japan). All solutions were prepared using
deionized water (resistivity > 18.2 M cm) obtained from a Milli-Q water purification system(Millipore, Billerica, MA, USA).

Sensors and Materials, Vol. 35, No. 10 (2023)

2.2

4723

Electrode preparation and enzyme modification

The GC electrode was used as the support material for the nanocarbon modification process,which was performed as follows: The GC electrode was polished using a 0.05 µm alumina
slurry, followed by sonication in ethanol and deionized water. A dispersed nanocarbon aqueous
solution (5 mL) was prepared by mixing 5 mg of SWCNTs, 5 mg of nanocarbon, and 15 mg ofSC, followed by dispersion for 10 min using probe-type ultrasonication (Branson 5520 sonicator,
Kanagawa, Japan). Subsequently, a 5 µL aliquot of the dispersed carbon solution, which isequivalent to 10 µg of total mixed carbon (5 µg of SWCNTs and 5 µg of nanocarbon), was cast
onto the GC disk electrode surface. The modified GC electrode was then dried under vacuum(~0.06 MPa). The nanocarbon-modified GC electrode was immersed in a 0.2 wt% SC aqueous
solution for 30 min prior to enzyme modification.(21,22) The electrode was gently rinsed byimmersion in a 0.1 M acetate buffer (pH 5.0) to remove excess SC on the electrode surface.
Subsequently, the electrode was immersed in a 5.0 µM Lac solution for 30 min and finally gently
rinsed by immersion in a 0.1 M acetate buffer.
2.3

Instrumentation

Cyclic voltammetry measurements were performed on an electrochemical analyzer (ALS,
Model 660A) using a conventional three-electrode cell with a Ag|AgCl|saturated KCl electrode(+199 mV vs a normal hydrogen electrode, NHE) as the reference electrode and a platinum wireas the counter electrode. In this study, all the reported potentials correspond to Ag|AgCl|saturated
KCl at 25 °C. A 0.1 M acetate buffer solution (pH 5.0) was purged with high-purity argon before
the measurements. During the electrochemical measurements, the buffer solution was vigorously
stirred using a magnetic stirrer (800 rpm) to obtain voltammograms with a steady-state
sigmoidal shape (plateau current). The saturated oxygen concentration was analyzed to be 1.24
mM using an oxygen meter (FireSting GO2, PyroScience GmbH, Aachen, Germany). The celltemperature was controlled at 25 ± 2 °C using a thermostated incubator.
Scanning electron microscopy (SEM) imaging was performed using a JSM-7600F microscope(JEOL, Japan). Transmission electron microscopy (TEM) was performed using a Tecnai F-20
field-emission microscope (Philips Electron Optics, The Netherlands).
Raman spectroscopy measurements were performed using a Horiba (Jobin Yvon) LabRAM
HR-800 instrument with 514 nm (2.41 eV) laser excitation. Wavenumber calibration was
performed using the emission of silica slides at 520 cm−1.
The Brunauer–Emmett–Teller (BET) specific surface areas of the samples were determinedfrom the N2 adsorption and desorption isotherms at 77 K using a Quantachrome NOVA2200e
instrument. Prior to the measurements, the samples were heated to 300 K for 24 h under vacuum
to clean the sample surface. The peak radius and mesopore volume were determined using theBarrett–Joyner–Halenda method.(23)4724

Sensors and Materials, Vol. 35, No. 10 (2023)

3.

Results and Discussion

3.1

SEM and Raman spectroscopic characterization

Figure 1 shows SEM images of the nanocarbon materials, which were categorized into three
types: (1) 1D nanocarbons with a diameter of 3–5 nm such as SG101 SWCNTs [Fig. 1(a)], (2)spherical nanocarbons with an aggregated secondary structure such as OSAB, HS100, KB, and
CB [Figs. 1(b)–1(e)], and (3) 2D nanocarbons with a thickness of approximately 10 nm (H25)[Fig. 1(f)]. Raman spectroscopy is also useful for characterizing sp2-hybridized structures incarbon materials and provides information on defects and crystalline structures.(24,25) Figure 2
shows the Raman spectra of the nanocarbons. All the nanocarbons exhibited characteristic
peaks at approximately 1340 and 1570 cm−1. The 1570 cm−1 peak was assigned to the G-band,
which is the doubly degenerate phonon Raman-active mode for sp2-hybridized carbon networks.
The D-band peak at 1340 cm−1 was attributable to the localization of regions where the lattice
structure is imperfect, mostly at the edges and defects of sp2-hybridized carbon structures.
SWCNTs [Fig. 2(a)] and H25 [Fig. 2(f)] exhibited a highly shared G-band peak and a weak
D-band peak, which were similar to those observed in the Raman spectrum of the basal plane of
HOPG [Fig. 2(g)]. In contrast, OSAB, KB, HS100, and CB [Figs. 2(b), 2(c), and 2(e)] showed ahigh D-band intensity, indicating that these nanocarbons are typical sp3-hybridized graphite-likestructures with numerous structural defects at their surfaces. The intensity ratios of the G-bandand D-band peaks are summarized in Table 1.

Fig. 1.

SEM images of (a) SWCNT, (b) OSAB, (c) KB, (d) HS100, (e) CB, and (f) H25.

Sensors and Materials, Vol. 35, No. 10 (2023)

t

4725

Intensity/arb. unit
�

e)

a)

A

t

Intensity/arb. unit��� '--------)\___

.._

b)

f)

�

g)

c)

d)

1000

2000
Raman shift/cm-1

3000

�--....-----------�
0Fig. 2.

1000

2000

Raman shift/cm-1

3000

Raman spectra of (a) SWCNT, (b) OSAB, (c) KB, (d) HS100, (e) CB, (f) H25, and (g) HOPG.

Table 1
Intensity ratio of G-band (IG) to D-band (ID).
Carbon
IG/ID
SWCNT
27
OSAB
0.6
KB
0.8
HS100
0.7
CB
0.8
H25
25
HOPG
124

3.2

Laccase bio-electrocatalysis

Figure 3 shows the catalytic oxygen reduction current based on the direct electron transfer
reaction of laccase and each nanocarbon-modified electrode surface. A catalytic oxygen
reduction current was observed from approximately 0.68 V, which is similar to the potential
reported previously.(21,22) This bio-electrocatalytic current strongly depended on the type ofnanocarbon mixed with the SWCNTs. Compared with the SWCNT-modified electrode, theelectrodes modified with the KB-SWCNT [Fig. 3(c)] and OSAB-SWCNT [Fig. 3(b)] composites
exhibited almost 2 and 1.5 times higher current values, respectively. On the basis of these results,
the Lac bio-electrocatalytic performance characteristics of the KB-SWCNT- and OSABSWCNT-modified electrodes were investigated. However, the KB-SWCNT and OSAB-SWCNT
composites easily peeled off the GC electrode surface and were observed in the buffer solution.
This indicates that a certain degree of SWCNT aggregation is required to fabricate a stable4726

Sensors and Materials, Vol. 35, No. 10 (2023)a)b):- +-- -- - -- - -. ----------.-.
!

c)+:::�: _--_-_-_-_-_-_-_·_-_-_-�_-_· -+·::::______________ ::�·::··---- ------.

�---:

200 MA,cm·2

!
!

0

0.2

0.4

0.6

0.8

EN vs AglAgCllsat'd KCl

200 µAcm· 2

0

0.2

0.4

0.6

0.8

0

0.2

EN vs AglAgCljsat'd KCl

d)200 µAcm·2e)+===-----------------<---�+.------------------------;

+

!

!

!

0

200 µAcm· 2

0.2

0.4

0.6

EN vs AglAgCltsat' d KCl

0.8--------------------<---/--

0200 µAcm·2

0.2

0.4

0.6

EN vs AgJAgClJsat' d KCl

0.4

0.6

0.8

EN vs AgJAgClJsat'd KCl

0.8

_.

.·-.. --.-----. ---. _ _. �·····--··-----·······-·;r0200 µAcm·2

0.2

0.4

0.6

0.8

EN vs AgjAgCltsat' d KCl

Fig. 3. Cyclic voltammograms of (a) SWCNT, (b) OSAB-SWCNT, (c) KB-SWCNT, (d) HS100-SWCNT, (e) CBSWCNT, and (f) H25-SWCNT modified with Lac in 0.1 M acetate buffer solution (pH 5.0) in the presence of O2.

modified electrode. Electrodes modified with CB-SWCNT [Fig. 3(e)] and HS100-SWCNT[Fig. 3(d)] composites exhibited almost the same catalytic current magnitude as the SWCNTmodified electrode. However, the catalytic current of the HS25-SWCNT-modified electrode[Fig. (3f)] was slightly lower than that of the SWCNT-modified electrode [Fig. 3(a)]. Upon
oxygen reduction, an onset potential of approximately 0.68 V was observed, which was
independent of the type of nanocarbon used in the composite. Similarly, the half-wave potential,
which indicates the potential at half the oxygen catalytic current, did not depend on the type ofnanocarbon used; the half-wave potential was determined to be 0.58–0.59 V. These findings
strongly indicate that the electron transfer kinetics between Lac and the electrode are similar forthe different nanocarbon-SWCNT-modified electrodes. Thus, it is reasonable to suggest that thedifference in catalytic oxygen reduction current between the different nanocarbons is attributable
to the number of Lac molecules adsorbed on the nanocarbon surface.

Sensors and Materials, Vol. 35, No. 10 (2023)

3.3

4727

Specific surface area dependence of catalytic current

Table 2 (left column) shows the BET surface area (SBET) values determined by N2 adsorption
and desorption at 77 K. Generally, the trend in the catalytic current magnitude is related to themeasured surface area. That is, the KB-SWCNT composite exhibited the highest catalytic
current intensity and the highest surface area among the different nanocarbon-SWCNT
composites used in this study. In contrast, the surface area of H25 was approximately threetimes higher than that of the SWCNTs, although the catalytic current intensities were similar.
The surface area was analyzed more carefully because it is dependent on the size of the adsorbed
molecules. The Lac molecule (approximately 7 nm in diameter) is significantly larger than theN2 molecule, indicating that adsorption is limited for the Lac molecule compared with the N2
molecule.(26) Table 2 (middle and right columns) shows the surface area (S10nm) results, which
exclude pore sizes smaller than 10 nm, and the S10nm /SBET ratio of S10nm relative to the total BET
surface area SBET. For the SWCNTs, the S10nm /SBET ratio was 13%, which is reasonable because
it is well known that CNTs, especially SWCNTs, easily form bundles.(27) The bundled SWCNTs
provide approximately 10% of the SBET for effective Lac adsorption. Additionally, the S10nm /SBET ratio (13%) of the H25-SWCNTs was similar to that of the SWCNTs. Figure 4 shows a plotof the catalytic current as a function of SBET for the nanocarbon-SWCNT composites. The
catalytic current increased linearly with S10nm, except for the H25-SWCNT composite. Thisbehavior is interesting because the nanocarbon surface conditions show large differences in thedefects and crystalline structures, as shown in Fig. 2. Generally, it is recognized that enzyme
electrocatalysis strongly depends on the electrode surface conditions, such as hydrophobicity/hydrophilicity and negative/positive charges. In this study, the sodium collate of a biosurfactant
was used as the surface modifier.(11,22) Therefore, it was concluded that, although the surface
was not optimal for the electrode reaction with Lac, surface modification may provide optimal
conditions for the Lac electrode reaction.
Figure 5 shows TEM images of the SWCNTs and KB-SWCNTs. The aggregated and bundled
SWCNT structures were observed only in the SWCNTs [Fig. 5(a)]. In contrast, in the KBSWCNTs [Fig. 5(b)], the aggregation and bundling of the SWCNTs were inhibited well by KB,
which clearly demonstrates why the KB-SWCNT composite exhibited the highest S10nm and thehighest catalytic current among the different nanocarbon-SWCNT composites used in this study.
Also, the OSAB-SWCNT composite was significantly effective for increasing the Lac bioelectrocatalytic performance and S10nm. These results suggest that, taking into account the fact

Table 2
Surface areas of nanocarbon–SWCNT composites.
CarbonSurface area (S BET) / m 2 g−1 Surface area (~10 nm)(S10nm) / m 2 g−1 S10nm / S %
SWCNT
4.8
0.64
13
OSAB-SWCNT
362158
KB-SWCNT
51
38
75
HS100-SWCNT
18
7.7
43
CB-SWCNT
7.8
5.5
71
H25-SWCNT
16
2.0
13

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Sensors and Materials, Vol. 35, No. 10 (2023)

Fig. 4.

Catalytic oxygen reduction current as a function of S10nm (specific surface area = ~10 nm).

Fig. 5.

TEM images of (a) SWCNTs and (b) KB-SWCNTs.

that the KB and OSAB sizes are less than those of the other nanocarbons, the inhibition effect on
the aggregation and bundling of SWCNTs would strongly depend on the size of the nanocarbons.

4.

Conclusions

The composite effect of nanocarbons with SWCNTs on Lac bio-electrocatalysis was clarified
using a sodium-collate-modified carbon electrode. In particular, KB was considered effective as
a composite nanocarbon with SWCNTs. KB inhibited SWCNT aggregation and bundling,
resulting in an amplified catalytic oxygen reduction current compared with the catalytic current
obtained using only SWCNTs. Surface modification with sodium collate provides suitableSensors and Materials, Vol. 35, No. 10 (2023)

4729

electrode surface conditions for Lac bio-electrocatalysis. Thus, although the surfaces of thenanocarbon crystal structures were not the same, the magnitude of Lac bio-electrocatalysis
strongly depended only on the specific surface area. The results of this research provide useful
information for design and adjustment methods in cathode development for enzymatic biofuelcell-powered biosensors.

Acknowledgments
The authors acknowledge Denka Co., Ltd. for kindly providing OSAB and HS-100. The
authors also thank Zeon Co., Ltd. for kindly providing SG101 SWCNTs.

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