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Rising Mesopores to Realize Direct Electrochemistry

of Glucose Oxidase toward Highly Sensitive Detection
of Glucose
Taotao Liang, Long Zou, Xiaogang Guo, Xiaoqing Ma, Chenke Zhang, Zhuo Zou,
Yuhuan Zhang, Fangxin Hu, Zhisong Lu, Kanglai Tang,* and Chang Ming Li*

1. Introduction
Direct electrochemistry, a direct electron transfer process between
enzymes and electrode possesses, has important fundamental signifi­ Diabetes is a fatal disease requiring fre-
cance in bioelectrochemistry while offering very efficient electrocatalysis quently sensitive and reliable diagnosis
for enzyme-based sensors. Herein, the pore structure of bacterial cellu­ of glucose for medical treatment and
lose porous carbon nanofibers (BPCNFs) is tailored by controlled thermal healthcare for diabetic patients.[1] Most
commercial glucose sensors mainly rely
carbonization. It is discovered that rising mesopores can realize a fast
on glucose oxidase (GOx) for high sen-
direct electrochemistry of glucose oxidase (GOx) for highly sensitive sitivity and selectivity. Flavin adenine
detection of glucose, achieving a sensitivity of 123.28 µA mmol L−1 cm−2 dinucleotide-dependent glucose oxidase
and a detection limit of 0.023 µmol L−1. The enhancement mechanism for (FAD-GDH) enzymes have been reported
the mesopores is ascribed to the most adequate mesopores of BPCNF900, for sensitive glucose detections.[2] GOx
is a structurally rigid glycoprotein of
which offer size-matched “nests” to trap GOx for intimate contacts with
160 kDa with diameters over 6–10 nm,
the conductive carbon nanofiber enabling fast direct electrochemistry. In consisting of two identical polypeptide
addition, with the BPCNF900 sensing platform, the mechanisms for GOx- chains with a flavin adenine dinucleotide
direct-electrochemistry-catalyzed glucose oxidation and oxygen reduction (FAD/FADH2) redox center. Although
are systematically investigated to further clarify the confusions of glucose the structures of the active sites of FAD-
sensing in air and N2-saturated solutions. This work demonstrates fun­ GDH are similar to FAD-GOx and have
been reported to have fast direct electro-
damental insights for the direct electrochemistry enabled by rationally
chemistry, they cannot be used to replace
designing a pore structure matching the target proteins, thus possessing as the enzyme in commercial blood
universal significance in protein-based electrochemical devices while sugar biosensors due to their rarity and
offering a facile route to fabricate a highly sensitive glucose sensor for being difficult to produce.[3] The GOx
practical clinic diagnosis. catalyzes glucose oxidation occurs as
follows:[2]

T. Liang, Dr. L. Zou, Dr. X. Ma, Dr. Z. Zou, Dr. Y. Zhang, Prof. Z. Lu, Prof. X. Guo, Dr. X. Ma
Prof. C. M. Li College of Chemistry and Chemical Engineering
Institute of Clean Energy and Advanced Materials Yangtze Normal University
School of Materials and Energy Chongqing 408100, P. R. China
Southwest University C. Zhang, Prof. K. Tang
Chongqing 400715, P. R. China Chongqing Sports Medicine Center
E-mail: ecmli@swu.edu.cn Department of Orthopedic Surgery
T. Liang, Dr. L. Zou, Dr. X. Ma, Dr. Z. Zou, Dr. Y. Zhang, Prof. Z. Lu, Southwest Hospital
Prof. C. M. Li The Third Military Medical University
Chongqing Key Laboratory for Advanced Materials and Technologies Chongqing 40038, P. R. China
of Clean Energies E-mail: tangkanglai@hotmail.com
Chongqing 400715, P. R. China Dr. F. Hu, Prof. C. M. Li
Dr. L. Zou Institute of Materials Science and Devices
College of Life Science Suzhou University of Science and Technology
Jiangxi Normal University Suzhou 215011, P. R. China
Nanchang 330022, P. R. China Prof. C. M. Li
Institute of Advanced Cross-field Science & College of Life Science
The ORCID identification number(s) for the author(s) of this article
Qingdao University
can be found under https://doi.org/10.1002/adfm.201903026.
Qingdao 200671, P. R. China
DOI: 10.1002/adfm.201903026

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GOx − FAD + glucose → GOx − FADH2 + gluconic acid (1)
GOx − FADH2 → GOx − FAD + 2H+ + 2e − (2)
Equations (1) and (2) show that GOx-FAD is the catalyst
toward glucose oxidation. Nevertheless, direct electron transfer
(DET) between FAD and electrode is actually inhibited because
it is deeply buried in a poorly conductive glycoprotein shell.
The developed three generations including the Clark glucose
enzyme sensor,[4] the artificial mediator-based glucose sen-
sors,[5] and DET-involved highly sensitive sensors[6] are mainly
designed to solve the electron transfer problem, of which the
mediator-based ones require small diffusive redox species to
mediate the electron transfer between FAD and electrode and
fast DET-based sensor have the highest sensitivity and selec-
tivity due to their high energy conversion efficiency. DET pro-
cess has important scientific significance in bioelectrochemistry
and broad applications in biosensors and bioenergies. Various
advanced materials with unique nanostructures, including
nanoparticles,[7] nanotubes,[8] graphene,[9] and graphene oxide
(GO)[10] have been developed to realize the DET of GOx. Rivas
et al. have reported a DET process achieved by electrical wiring
GO.[11] Muguruma et al. immobilized glucose dehydroge-
nase-FAD (FAD-GDH) on single-walled carbon nanotubes
(SWCNTs) for a mediator-less DET between oxygen-insensitive
GDH-FAD and SWCNTs.[12] Liu et al. studied the activity of
CNTs for DET by immobilization of GOx on CNTs.[13] Wu et al.
developed a graphene-based biosensing platform to enable DET
of GOx.[14] The DET catalytic process is mostly ascribed to the
unique nanostructures achieving very close proximity between
the electrode surface and GOx-FAD for short distance allowing
quantum hoping or/and direct electron transfer.
It is known that the most electrodes including nanostruc-
tured ones in practical electrochemical devices are porous.[15]
Material pores are classified into three types: macropores
(>50 nm), mesopores (2–50 nm), and micropores (<2 nm).[16]
The high porosity of an electrode can offer high surface area,
to which the micropores make the most contribution, while
the macropores and the mesopores are mainly responsible for
mass transport and interconnections for high rate capacity.[17] A
porous glassy carbon electrode has been reported to enable DET
for glucose biosensor due to its well-defined redox behaviors.[18]
A porous TiO2 material with uniform pore-size to trap GOx for
the DET.[19] Although reports often ascribe the mechanism of
the direct electrochemistry to the electrode nanostructures for
close proximity between the electrode surface and enzyme,
there are lack of both experimental evidence and fundamental
explanation for the effect of pore structures on the direct elec-
trochemistry as well as how to rationally design an optimal pore
structure for fast DET leading to high-performance glucose
sensors, which is indeed highly demanded.
Carbon nanofibers (CNFs) have been investigated and used
in electrochemical biosensors.[20] The CNFs-formed network
can be tuned by their diameter for optimal biosensor perfor-
mance.[21] Palladium-modified CNFs biosensor enables simulta-
neous and highly sensitive detections of hydrogen peroxide and
glucose.[22] In particular, Vamvakaki et al. have demonstrated
that CNF is one of the best matrix to well trap or immobilize
enzymes for biosensing platform.[23] The glucose sensor made
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from nanofibers exhibits fast response and high sensitivity.[24]
Porous CNFs have also exhibited the DET of heme-proteins,
which is generally attributed to the microenvironment without
further scientific exploration.[25] In a short, porous CNFs could
be an excellent material for the use in superior biosensors due
to their high specific surface area, good conductivity, fast reac-
tivity, and strong immobilization ability, and thus can render a
good platform to further explore the DET fundamentals.
In this work, bacterial cellulose porous carbon nanofibers
(BPCNFs) prepared from microbial fermentation at high-
temperature under Argon atmosphere were used to construct
porous electrodes for immobilization of GOx as glucose sen-
sors. The pore structure of the BPCNFs can be delicately tai-
lored by thermal treatment and were further used to study the
effect of pore structure on the DET. Moreover, the electrochem-
ical analysis of the electron transfer reaction among optimal
BPCNFs electrodes modified with electroactive GOx has been
measured at a structurally well-defined interface. This work
provides a credible foundation and novel concept on how an
optimal pore structure can be designed to effectively improve
the adsorption or load of target molecules for fast DET process
in field of biosensing and bioelectrochemistry.
2. Results and Discussion
2.1. Characterizations of BPCNFs
BPCNFs were prepared by delicately controlled pyrolysis tem-
peratures and were examined by scanning electron microscope
(SEM) (Figure S1, Supporting Information) and field emission
scanning electron microscopy (FESEM) (Figure  1a1,b1,c1,d1).
SEM and FESEM images show that all obtained mono-
lithic BPCNFs after the pyrolysis treatment can maintain
the original 3D porous interconnected network structures
of the bacterial cellulose (BC) but are much rougher. Among
all samples, BPCNF900 shows the most uniform and porous
structure. In addition, with increased pyrolysis temperatures,
the BPCNFs turn to more nanofiberized with less aggrega-
tions (Figure 1a2,b2,c2,d2). Transmission electron microscopy
(TEM) images (Figure  2) display that the BPCNFs with a
diameter of 10–20 nm are highly interconnected with many
junctions to form networked mesopores. Obviously, BPCNF900
(Figure 2c,e,f) has the most uniform and smallest sizes with
more links than other BPCNFs (Figure 2a,b,d).
The pore structures obtained by different pyrolysis tempera-
tures were studied by Bruneian–Emmett–Teller (BET) and N2
adsorption–desorption isotherms measurement at 77 K. The
result in Figure  3a shows that all samples have quick rise of
the N2 isotherms at low-pressure range (P/P0  < 0.01) indi-
cating existence of certain amount of micropores. Nevertheless,
BPCNF900 and BPCNF1000 exhibit faster rise at 0.5–0.8 P/P0 than
that of BPCNF700 and BPCNF800 representing that the formers
possess more mesopores than the latters. This is in good agree-
ments with the morphologies shown in Figure 2. In addition,
the N2 adsorption–desorption isotherms at high relative pres-
sures (P/P0  > 0.9) can signal the formation of macropores:
the faster increase means formation of more macropores.
Apparently, BPCNF900 and BPCNF1000 display quicker rise at
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Figure 1.  FESEM images of BPCNFs prepared at different temperatures
of a1) low resolution, a2) high resolution) 700 °C,b1,b2) 800 °C, c1,c2)
900 °C, and d1,d2) 1000 °C.
(P/P0  > 0.9) than that of BPCNF700 and BPCNF800, denoting
formation of more macropores at higher pyrolysis tempera-
tures.[26] On the basis of Barrett–Joyner–Halenda (BJH) model
in Figure 3b, mesopore size distributions of BPCNFs are cal-
culated and displayed, which have a pore distribution over
2–50 nm with similar volume peaks around 2 nm, followed by
an almost flat tail covering mesopores region over 2–50 nm in
diameters. The BPCNF700, BPCNF800, and BPCNF900 exhibit
the coexistence of micropores and the mesopores with the pore
size ranging from 1.5–2 to 2–10 nm, respectively. Apparently,
for BPCNF800 and BPCNF900, the volumes of mesopores are
dominant, while for BPCNF700 mesopores are relatively less
than micropores. The specific surface area gradually grows
from 484.67 to 1015.08 m2 g−1 with increasing the pyrolysis
temperature from 700 to 900 °C (Table S1, Supporting Infor-
mation), which could be attributed to a more gas emission to
become more porous when increasing temperature during
the pyrolysis of BC. However, for BPCNF1000, the specific sur-
face area sharply drops to 921.61 m2 g−1. It is likely that the
temperature higher than 900 °C leads to the conversation of
more micropores and mesopores into larger mesopores or
macropores. The lack of mesopores (2–10 nm) may lead to
the less loading of GOx and the slow mass transport of elec-
trolyte ions.[27] As the pyrolytic temperature increases from
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Figure 2.  TEM images of a) BPCNF700, b) BPCNF800, d) BPCNF1000, and
c,e,f) BPCNF900.
700 to 900 °C, the mesopore density of the BPCNFs enhances
from 34.9% to 50%, but further rising of the temperature to
1000 °C causes a sharp reduction of mesopore density to 36.6%
(Table S1, Supporting Information). This also indicates the
mesopore damages. Desorption average pore width (DAPW)
of BPCNFs increases as the pyrolysis temperature rises
(Figure 3c). Interestingly, the DAPW of BPCNF900 is ≈17.40 nm
closing to the size of GOx (≈6–10 nm).[3] Compared with other
samples, it seems that BPCNF900 owns more reasonably sized
mesopores and thus is more suitable to absorb GOx for glucose
biosensors.
Figure S2 (Supporting Information) displays X-ray powder
diffraction (XRD) pattern for BPCNF900, which comprises
a broad peak and a weak peak representing the graphitic
stacking of (002) and the reflections of the overlapped (101)
face. The effect of high temperature treatment of BC on pore-
formation of biomass was further studied. BC as precursors
prepared with freezing dry were examined by Fourier trans-
form infrared (FT-IR) spectrometer (Figure 4a), showing com-
plex absorption bands of the biomass organic polysaccharide
structure of BC. However, FT-IR absorption bands of BPCNFs
(Figure 4b) are different from that of BC with less absorp-
tion peaks and also weaker intensity, which indicates that the
pyrolysis loses a large amount of functional groups. However,
the different BPCNFs produced by different pyrolysis tem-
peratures display similar absorption bands, clearly confirming
that the pyrolized products have almost the same chemical
structure. In more details, peaks at 3353 and 1060 cm−1 can
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Figure 3.  a) N2 adsorption–desorption isotherm of the BPCNFs. b) BJH
mesopore size distributions of BPCNFs at different pyrolytic tempera-
tures. c) Desorption average pore width (DAPW) of BPCNFs (BPCNF700,
BPCNF800, BPCNF900, and BPCNF1000).
be assigned to the typical acylamino (RCONH2 with absorp-
tion peak range of 3050–3500 cm−1) and carboxylic anhydride
((RCO)2O with absorption peak range of 1050–1170 cm−1)
adsorption bands, respectively. The two major character-
istic bands at 666 and 1591 cm−1, agree well with OH
(650–750 cm−1) and CO (1540–1640 cm−1) with adjoin CC
(1580–1620 cm−1) adsorption bands, respectively. It is worth
noting that the CO and CC stretching band of the sample
obtained by pyrolysis treatment has a slightly redshift. This
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Figure 4.  FT-IR spectra of a) BC and b) BPCNFs prepared by pyrolysis at
different temperatures.
could indicate that only a few of CO groups existing in BC
may be decomposed under high-temperature treatment into
H2O, CO2, and so on.[28]
2.2. Immobilization of GOx on BPCNFs and Its Direct
Electrochemistry
TEM was used to investigate the optimal pore structure of
BPCNFs for GOx immobilization (Figure  5). Results indicate
that mesopores mainly formed by the fiber-crossovers, which
lead to effective immobilization of GOx on the BPCNFs. The
BPCNF700 only exhibits a few mesopore spots and has the
lowest loading of GOx with ≈10 nm diameters (Figure 5a).
BPCNF800 and BPCNF1000 display better GOx immobilization,
in which more GOx molecules are well adsorbed at the sites of
fiber-crossovers (Figure 5b,d). In contrast, BPCNF900 (Figure 5c)
has the most abundant mesopores in sizes of 2–10 nm formed
thus resulting in much more immobilized GOx than other
BPCNFs. This gives strong evidence that the pore structure
can be delicately tuned by the pyrolysis temperature and the
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Figure 5.  The TEM images of GOx immobilized on BPCNFs a) BPCNF700/

GOx, b) BPCNF800/GOx, c) BPCNF900/Gox, and d) BPCNF1000/GOx.

mesopore sizes around 2–10 nm obtained at 900 °C could well

accommodate GOx immobilizations.
The effect of pore structure of BPCNFs on DET was inves-
tigated by cyclic voltammetry (CV) in 0.01 mol L−1 pH 7.0
nitrogen-saturated phosphate buffer solution (PBS) at 50 mV s−1
as shown in Figure  6a, of which the current was corrected by
background current. One can clearly see that the GOx/glassy
carbon electrode (GCE) has no redox current (Figure 6a: curve
a), indicating that the direct electrochemistry could not be
achieved on conventional bare electrode due to the redox centers
deeply buried in a poorly conductive glycoprotein shell.[29]
The BPCNF900 electrode without GOx immobilization
only exhibits typically squared capacitive CV curves caused
by double layer capacitance, indicating that it has a large sur-
face area but no redox reaction occurs (Figure 6a: curve b). Figure 6.  a) Cyclic voltammograms recorded at different modified GCE
BPCNFs/GCEs prepared from BPCNFs obtained at different in 0.01 mol L−1 N2-saturated PBS at 50 mV s−1 with curve a) GOx, curve,
pyrolysis temperatures have the similar CV curves (date not b) BPCNF900, curve, c) BPCNF700/GOx, curve, d) BPCNF800/GOx, curve
shown). Figure 6b shows that both mesopore ratio of BPCNFs e) BPCNF900/GOx, and curve f) BPCNF1000/GOx. b) The relationships of
both mesopore ratio (Vmes/Vtot%) of BPCNFs and the redox peak-current
(top Figure of Figure 6b) and the redox peak-current (bottom
of BPCNFs/GOx versus pyrolysis temperatures.
figure of Figure 6b) of BPCNFs/GOx are related closely with
the pyrolysis temperatures. Apparently, only the BPCNFs/
GOx/GCE electrodes show well-defined redox peaks (Figure 6a: shown in Figure  7a. In a pH range of 5.4–8.0, the peak cur-
curves b), c), e), and f), among which the BPCNF900/GOx one rent increases with the increased pH until pH of 7.0, at
(curve e) delivers the largest current and has the most nega- which the current reaches the highest, and then decreases
tive redox potential (−0.45 V vs Ag/AgCl), clearly indicating with the increased pH (Figure 7b). In addition, both anodic
that BPCNF900/GOx traps the highest GOx while possessing and cathodic peak potentials simultaneously move to more
the highest direct electron transfer activity by its most negative positive with increased pH (Figure 7a), indicating that the
redox potential. Importantly, the redox potential (−0.45 V) of direct electrochemistry of GOx involves hydrogen ion. The
BPCNF900/GOx is consistent with the reported value for redox redox peak currents of GOx are also dependent on pH, which
potential of FAD/FADH2, the confirmed redox pair of GOx, at increases with increased pH until the maximum peak cur-
pH 7.0.[19] This indisputably proves that a DET process of GOx rent is achieved at pH 7.0, and then decreases with further
indeed occurs at BPCNF900/GOx, and confirms that rising the increasing of pH (Figure 7b), possibly due to the decreased
mesopores can realize fast direct electrochemistry of glucose proton concentration and deteriorated bioactivity of the
oxidase. immobilized GOx. It has been reported that the direct elec-
Cyclic voltammograms of the constructed BPCNF900/GOx trochemistry of GOx on the electrode involves a two-proton
show a strong dependence of DET rates on solution pH as redox reaction as follows

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Figure 7.  a) The cyclic voltammograms of the BPCNF900/GOx/GCE in 0.01 mol L−1 PBS with different pH values at 50 mV s−1. b) Plot of peak current
versus pH value. Inset of (b): Plot of E0’ versus pH value. c) Cyclic voltammograms recorded at BPCNF900 film electrode in 0.01 mol L−1 PBS at different
scan rates (from 20 to 200 mV s−1, inside to outside). d) Plots of peak currents versus scan rate.

direct electron transfer process between GOx and the electrode

GOx − FAD + 2e − + 2H+ ↔ GOx − FADH2
It is fair to assume that the concentrations of GOx-FADH2
and GOx-FAD are constant during pH changes. The peak
potential of a reversible electrochemical reaction is[23]
2.3RT CGOx −FAD × (C + )2 
E = E0 + log  H
 (4)
nF  CGOx −FADH2 
2.3RT × 2
E = E0 − pH
nF
E 0 ′ = constant + 0.059 pH
where E, n, R, T, C, F, and E0’ are the peak potential, the
number of electron transfer involved, the gas constant, tem-
perature, the concentrations, Faraday constant, and peak poten-
tial, respectively. The slope of E0’ versus pH should theoretically
be 59.0 mV pH−1. The slope of the E0’ versus pH line plotted
from our experimental results was 54.0 mV pH−1, which is very
close to the theoretical one. Hence, it can be concluded that the
direct electrochemistry of GOx is a two-proton coupled two-
electron transfer process. The peak current of the immobilized
GOx is linearly proportional with the increase of the scan rate
as displayed in Figure 7c, clearly indicating a surface-controlled
electrochemical redox process,[30] thus further verifying the
(3)
surface since GOx is actually fixed on the electrode surface by
immobilization. As shown in Figure S3 (Supporting Informa-
tion), the cathodic peak potential of GOx is linearly proportional
to the natural logarithm of the scan rate (lnV). The calculated
electron-transfer coefficient (α) is 0.015 and the standard heter-
ogeneous transfer constant (Ks)[31] of GOx on BPCNF900/GOx/
GCE is 1.17 s−1, which is larger than that for GOx immobilized
SWNT (0.3 s−1) modified electrodes.[32] The great biomolecule
immobilization capacity is obviously caused by the adequate
mesoporous nanostructure of the synthesized BPCNF900.
(5)
(6) 2.3. Biosensing Applications of BPCNF900/GOx Electrode
The new mesoporous BPCNF900 with excellent negative redox
potential of GOx direct electrochemistry offers great electro-
chemical catalytic activity and strong anti-interference ability. To
further confirm whether the oxidation of glucose is carried out
under a direct electrochemistry process of GOx on BPCNF900/
GOx surface, CV measurements on the BPCNF900/GOx/GCE
were carried out in air- and N2-saturated 0.01 mol L−1 PBS as
well as N2- and air-saturated 0.01 mol L−1 PBS +2.5 mmol L−1
glucose, respectively (Figure  8a–c). The observed pair of well-
defined symmetric redox peaks in Figure 8a can be certainly
assigned to the direct electrochemistry of GOx. However,
although the anodic peak current has no change, the cathodic

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Figure 8.  Cyclic voltammograms of the mesoporous BPCNF900/GOx/GCE in different condition and the response to glucose: a) in air- and N2-saturated
PBS, b) BPCNF900/GOx/GCE in N2-saturated PBS and N2-saturated PBS+2.5 mmol L−1 glucose, and c) in air-saturated PBS and air-saturated PBS +
2.5 mmol L−1 glucose. d) The competing mechanism of both oxygen reduction and glucose oxidation on BPCNF900/GOx electrode involve the direct
electrochemistry of GOx in N2-saturated PBS + glucose (I) and air-saturated PBS + glucose (II).

peak current has great increase (Figure 8a), which is undoubt-
edly produced by the oxygen reduction. Interestingly, oxidation
and reduction current are generated in air- and N2-saturated
0.01 mol L−1 PBS +2.5 mmol L−1 glucose solutions, respec-
tively, which obviously corresponds to the glucose oxidation
and oxygen reduction. In addition, the current of oxygen reduc-
tion decreases when adding glucose in. Since the presence
of oxygen cannot be avoid in clinical diagnosis and the cur-
rent decrease is significant with the increase of glucose con-
centration, the oxygen reduction current is used to detect the
glucose concentration. It has been long argued that there is no
direct electrochemistry of GOx, in particular in the presence
of oxygen, which is considered to be an electron mediator for
the glucose oxidation.[33] Fundamentally, an electron mediator
in an electrochemical enzymatic reactions cannot contribute a
net current but the results in Figure 8a,c clearly indicate that
oxygen is actually reactant to produce a large current. In addi-
tion, Figure 8b further confirms that the glucose can directly
be oxidized on the BPCNF900/GOx surface without oxygen pres-
ence or any another electron mediator to produce anodic cur-
rent. The continuously decreased reduction current of oxygen
with the successive addition of glucose solution in air-saturated
PBS occurs only on the GOx-immobilized surface and thus
it can be reasonably explained by the competing adsorption
of glucose and oxygen molecules on the GOx redox centers
resulting in the decreased current of oxygen reduction when
adding glucose. The competing mechanism has been reported
by our earlier work.[2] Apparently, both oxygen reduction and
glucose oxidation on BPCNF900/GOx electrode involve the
direct electrochemistry of GOx, which is schematically shown
in Figure 8d.
Scheme  1 further demonstrates that the rich mesopores of
BPCNF900 in a range over 2–10 nm, which are about sizes of
GOx to not only offer “nests” to stably trap GOx, but also make
intimate contacts with the very conductive CNFs in close prox-
imity to enable direct electron transfer between GOx-FAD/GOx-
FADH2 and BPCNFs. This is why rising mesopores of electrode
materials can realize direct electrochemistry of GOx. To further
prove this mechanism while exploring its universe significance,
horseradish peroxidase (HRP, EC.1.11.1.7, 42 kDa) and cata-
lase (CAT, EC 1.11.1.6, 240 kDa) were selected as modification
enzymes on BPCNF900 to match or unmatch the mesopore
sizes. Results in Figure S4a (Supporting Information) show that
BPCNF900-GC only produces capacitive response (black line);
in contrast, the BPCNF900/HRP-GC in PBS displays a well-
defined pair of redox waves, unambiguously confirming that
HRP enables direct electrochemistry on BPCNF900 (red line).
Further, the BPCNF900-GC in 2 × 10−3  m H2O2 has no response
over the capacitive current (dotted black line), indicating there
is no reduction of H2O2 on the electrode; nevertheless, the
BPCNF900/HRP-GC responds to the added 2 × 10−3  m H2O2
for obviously increased reduction current (red dotted line),

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Scheme 1.  Schematic illustration of BPCNFs electrode materials realizing the direct electro- and the highest affinity toward the substrate.
chemistry of GOx.
evidencing that H2O2 reduction undergoes a DET process
(Figure S4a, Supporting Information). Figure S4b (Supporting
Information) exhibits that the cathodic current on BPCNF900/
HRP-GC increases linearly with the increased H2O2 concentra-
tion for a sensitivity of 45.39 µA mmol L−1 cm−2 (R2  = 0.991).
This result vividly indicates that BPCNF900/HRP-GC can also
enable DET process due to its size matching with the range of
mesopores of BPCNF900. Conversely, CAT cannot generate well
defined redox current on BPCNF (Figure S4c, Supporting Infor-
mation).It is very likely that CAT is too large to match with the
mesopores of BPCNF900 since its molecular mass is 1.6 times
[34]
larger than GOx. Those results indicate that the direct electro-
chemistry of enzymes on the mesopores-rich BPCNF900 is not
only for GOx but can be also extended to other enzymes with
the size lower than that of GOx.
Figure  9a shows a typical current-time plot for the
BPCNF900/GOx electrode measured at of −0.45 V versus
Ag/AgCl by successive injections of 2.5 µmol glucose into
the air-saturated and stirred 0.01 mol L−1 pH 7.0 PBS with
an applied potential of −0.45 V. It is observed that each suc-
cessive addition results in a decrease of the steady state
current with fast response time of 3.7 s (Figure 9b). The
calibration plot in Figure 9c obtained from Figure 9a shows
a well linear relationship, a typical behavior of an enzy-
matic kinetic reaction with a linear regression equation of
Y1( x =0.0004 −0.01×10−3 M)   = 1.319 + 7.852X (n  = 3, R2  = 0.995) and
Y2( x =0.01−3.8×10−3 M)   =  −0.0636  + 4.060X (n  = 3, R2  = 0.996), where ibility of the as-prepared sensors was investigated as shown
Y1 and Y2 are the peak current, X is glucose concentration,
n is the number of electrodes measured, and R2 is correla-
tion coefficient, demonstrating a sensitivity of 111.14 and
57.5  µA mmol L−1 cm−2 and a linear response range of
0.01  µmol L−1 to 3.8 mmol L−1. The apparent Michaelis–
Menten constant (KMapp) can predict the enzyme-substrate
kinetics, of which a smaller value indicates a higher enzymatic
activity and higher affinity toward the substrate. By using the
electrochemical version of the Lineweaver–Burk equation[35]
Adv. Funct. Mater. 2019, 1903026 1903026  (8 of 11)
www.afm-journal.de
1 kMapp 1 1
= + (7)
iss imax c imax
where iss is the steady-state current, imax
is the maximum current, and C is the
glucose concentration, a linear plot of cur-
rent−1 versus concentration−1 (Figure 9d) is
obtained to calculate KMapp of sample from
its intercept and slope. The calculated KMapp
for GOx/BPCNF900 is 0.61 mmol L−1, which
is much smaller than 8.5 mmol L−1 for the
GOx/SWCNT electrode,[36] 2.2 mmol L−1
for a GOx/p-NiO/n-Bi4Ti3O12 electrode,[37]
5.5 mmol L−1 for the GOx/CNF electrode,[38]
and 7.01 mmol L−1 for the GOx/Al2O3/PPy
electrode.[39] The results undoubtedly indi-
cate that the mesoporous GOx/BPCNF900
has the highest enzymatic activity to GOx
The current responses versus glucose con-
centrations measured by successive addi-
tions of 1 µmol glucose in N2-saturated PBS is linear with a
developed plateau part, which represents a typical enzymatic
kinetic nature. The inset of Figure 9e shows a typical current-
time plot for the BPCNF900/GOx electrode measured at of
−0.45 V versus Ag/AgCl by successive injections of 1 µmol
glucose into the N2-saturated and stirred 0.01 mol L−1 pH 7.0
PBS at an applied potential of −0.45 V. The calibration plot
in Figure 9e shows a good linear relationship with a linear
regression equation of Y1( x =0.0002−0.1×10−3 M)   =  −0.040  + 8.712X
(n  = 3, R2  = 0.982) and Y2( x =0.1−1.3×10−3 M)   =  −0.13  + 0.94X
(n  = 3, R2  = 0.995), demonstrating a sensitivity of 123.28 and
13.31 µA mmol L−1 cm−2, of which the sensitivity of the sensor
in N2-saturated solution is even higher than that in the air-
saturated solution. This further confirms the fast DET pro-
cess enabled by BPCNF900. Figure 9f reveals good selectivity
of the prepared biosensor for glucose in the presence of var-
ious interferences, in which there is no significant change of
the current response. The current response caused by addi-
tion of 0.5 mmol L−1 ascorbic acid (AA), dopamine (DA), uric
acid (UA), and NaNO3 (Na+, NO3−) are less than 1/7 of that
induced by 0.25 mmol L−1 glucose. The reported enzyme-
modified porous carbon nanomaterials-based glucose sensors
and their sensitivity, detection limit, and detection range, are
listed in Table  1, indicating that the mesoporous BPCNF900/
GOx sensor shows the highest sensitivity and very low detec-
tion limit. In addition, the storage stability and the reproduc-
in Figures S5 and S6 of the Supporting Information.[42] After
7 d of storage at 4 °C, current responses of the sensors in the
air- and N2-saturated PBS could be retained at 92% and 88% of
the original level, respectively, demonstrating the ideal storage
stability. The current responses of five independent sensors,
which were prepared with our approach, toward 2.5 mmol L−1
glucose were tested in the air-saturated PBS to evaluate the
reproducibility. The relative standard deviation (RSD) is only
≈3.3%, showing the excellent reproducibility of the sensors.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.afm-journal.de

Figure 9.  a) Typical current-time trace for succesive additions of 2.5 µmol glucose to stirred pH 7.0 air-saturated PBS with an applied potential of
−0.45 V. b) Response time of mesoporous BPCNF900/GOx/GCE sensor toward 20 µmol glucose in air-saturated PBS. c) The linear plot of calibra-
tion curve of steady-state current response of (a) versus glucose concentration. d) Lineweaver-Burk plots of current−1 versus concentration−1 from
(c). e) Typical current-time trace for succesive additions of 1 µmol glucose in N2-saturated PBS with an applied potential of −0.45 V. Inset of (e) is the
linear plot of calibration curve of steady-state current response versus glucose concentration. f) Effect of 0.5 mmol L−1 interfering species (AA, DA,
UA, and NaNO3) on the biosensor response for 0.25 mmol L−1 glucose in air-saturated PBS.

To verify the feasibility, the BPCNF900/GOx sensor was 3. Conclusions

used to detect the glucose concentrations in serum samples,
and the results summarized in Table S2 (Supporting Informa-
tion) reveal that RSD values and the recovery are acceptable,
indicating that the detected concentrations are in good agree-
ment with the results measured by the commercial blood
glucose test strip, and the variations of the detected concen-
tration for three times is less than 4% for our sensor, thus
demonstrating the good reproducibility of the BPCNF900/GOx
sensor.
In brief, the BPCNFs with highly mesoporous structure
were successfully prepared, and the mesopore structure and
mesopore density were delicately regulated by controlling the
pyrolysis temperatures. The results indicate BPCNF900 with
the most adequate mesopores offers size-matched “nests” to
trap GOx for intimate contacts with the conductive carbon
nanofiber enabling fast direct electrochemistry of GOx
without any electron mediator. The glucose sensor made by

Adv. Funct. Mater. 2019, 1903026 1903026  (9 of 11) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.afm-journal.de

Table 1.  Comparison for the sensor performance of the previous reports based on enzyme-modified porous carbon materials glucose sensor and this
work.

Electrode Sensitivity Detection limit Detection range DET/Not DET Reference

[µA mmol L−1 cm−2] [µmol L−1] [mmol L−1]
GOx/Pd-HCNF 13.0 3.0 0.06–6.0 Not DET Jia et al.[22]
GOx/1DHS TiO2 9.9 1.29 0.0025–0.015 DET Si et al.[40]
GOx/CNDs/GC 6.1 1.07 0–0.64 DET Wang et al.[38]
GOx/HPGC-PDA / 0.013 0.001–0.12 Not DET Fu et al.[41]
GOx/3D-KSC/NCNTs 29.4 1.93 0.0112–12 DET Song et al.[42]
GOx/BPCNF900 in Air PBS 57.5 5.22 0.01–3.8 Not DET This work
GOx/BPCNF900 in Air PBS 111.14 0.026 0.0004–0.01 Not DET This work
GOx/BPCNF900 in N2 PBS 13.31 2.25 0.10–1.30 DET This work
GOx/BPCNF900 in N2 PBS 123.28 0.023 0.0002–0.10 DET This work

the BPCNF900 shows a fast direct electrochemistry of glucose
oxidase to catalyze glucose oxidation for a highly sensitive and
selective sensors with a rapid response time, low detection
limit and small apparent Michaelis–Menten constant. With
the BPCNF900 sensing platform, the competing mechanisms
for GOx-direct electrochemistry catalyzed glucose oxidation
and oxygen reduction are investigated to further clarify the
confusions of glucose sensing in air and N2-saturated solu-
tions. This work holds a great promise for fabrication of a new
direct electrochemistry based glucose sensors, but also sheds
a scientific light on the roles of pore structures in electron
transfer paths.
4. Experimental Section
Preparation of Bacterial Cellulose-Derived Porous Carbon Nanofibers with
Different Pore Structures: BPCNFs were prepared according to the methods
reported by Zou et al.[43] and Yu et al.[44] In this work, the typical preparation
procedure was to first neutralize sliced BC (received as a gift from Ms. C.
Y. Zhong, Hainan Yeguo Foods Co., Ltd.) and then was frozen in liquid
nitrogen (−196  °C) followed by freeze-drying at a temperature of −80  °C
and a pressure of 0.021 mbar. The as-obtained BC was then pyrolized at
different temperatures in an argon flow to finally produce BPCNFs. The
pore structures of the BPCNFs were delicately tuned by different pyrolized
temperatures, which was started from room temperature to rise at
2  °C min−1 to 500 °C for 1 h and then at 5 °C min−1 to different higher
temperatures for 1 h to yield black BPCNFs for different pore structures.
Enzyme Electrode Construction: GCE with 3 mm diameter was
sequentially polished by 0.3, 0.05 µm aluminum powder slurry to a
mirror-like surface, followed by successively rinsing, sonicating with
deionized water, and drying in H2 flow at room temperature before use.
The enzyme immobilization on BPCNFs was achieved by dissolving
GOx (10 mg) (obtained from Sigma-Aldrich Co.) and as-prepared
BPCNFs (10 mg) in pH 7.0, 0.01 mol L−1 PBS (1 mL). The mixture
(10 mg mL−1) was shaking in an oscillator at 30 °C for 5 h and then
stored at 4 °C longtime to obtain bioconjugates suspension. As-prepared
bioconjugates suspension (5 µL) was deposited on the pretreated GCE
and dried at room temperature resulting in BPCNFs/GOx bioelectrodes.
Then the nanofiber was washed with buffer to remove nonadsorbed
enzymes and vacuum-dried. Nafion solution (5%) was dropped onto
the fiber containing the adsorbed GOx, to give the BPCNFs/GOx
bioelectrode ready for investigation. For comparison, Other enzyme
(CAT, HRP, and SOD) was modified on BPCNF900 following the same
procedure. All other chemicals were purchased from Sigma-Aldrich and
used as received.
Electrochemical Measurements: Electrochemical measurements were
performed using an electrochemical workstation (CHI660D), in which
a three-electrode system with the premodified GCE, a platinum wire and
a saturated Ag/AgCl electrode as the working electrode, the auxiliary
electrode and the reference electrode, respectively, was used. The PBS
was deoxygenated by bubbling highly pure N2 for at least 20 min and
a N2 atmosphere environment was kept in whole electrochemical
measurements. All chemicals are of analytic grade and used as received
without further purification. For all electrochemical measurements,
10 mmol L−1 PBS with pH 7.0 as electrolyte was used, and temperature
was at 25 °C. AA, DA, UA, and NaNO3 were selected as interfering species.
Serum Samples Preparation: Human serum samples were obtained
from Sigma-Aldrich Co. and the serum sample was separated from
impure deposits for removal by a centrifuge. The glucose concentration
of the human serum sample was determined using both commercial
blood glucose test strip and biosensor prepared with the BPCNF900
modified electrode. The testing samples were prepared by mixing
tenfold dilution human serum samples + PBS + glucose with different
concentrations by adding prepared concentrated glucose solution.
Material Characterization: The morphology and nanostructures were
characterized by SEM (JSM-6700F), FESEM (JEOL-7800F), and TEM
(JEM-2100F). The crystalline structure of BPCNFs was studied by an
XRD (Shimadzu-7000). Nitrogen adsorption/desorption experiments
were carried out at 77 K by BET surface area and pore size analyzer
(ASAP 2020). The structures of BC and BPCNFs were examined by FTIR
(Nicolet 6700).
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors would like to gratefully acknowledge the financial support
from National Program on Key Basic Research Project of China (973
Program under Contract No. 2013CB127804) and Institute for Clean
Energy & Advanced Materials (Southwest University, Chongqing, China).
Conflict of Interest
The authors declare no conflict of interest.

Adv. Funct. Mater. 2019, 1903026 1903026  (10 of 11) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Keywords
bioelectrochemistry, direct electrochemistry, glucose sensors, mesopores,
porous carbon nanofibers
Received: April 15, 2019
Revised: August 9, 2019
Published online:
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