Controlled synthesis of hierarchical ZSM-5 for catalytic fast pyrolysis of cellulose to aromatics

Article abstract of DOI:10.1039/C8TA08930B

Zeolite materials play a significant role throughout the oil refining and petrochemical industry. Microporous ZSM-5 has a high degree of crystallinity but low mass transfer, while hierarchical ZSM-5 shows a low degree of crystallinity as well as acidity. In this work, we first report the synthesis of ZSM-5 with a new morphological structure, which has nanocrystalline ZSM-5 particles on the surface of an intact ZSM-5 zeolite. This not only improved the mass transfer in microporosity but also overcame disadvantages of hierarchical ZSM-5 including low degrees of crystallinity and acidity. This new and very efficient structure with both intracrystal microporosity and intercrystal macroporosity, formed by secondary crystallization after the intact ZSM-5 zeolite was synthesized, was verified by transmission electronic microscopy, N₂ adsorption and desorption, and X-ray diffraction. The obtained ZSM-5 zeolite showed a uniform size (～200 nm), high crystallinity acidity, and a suitable hierarchical structure that exhibited excellent properties in the catalytic fast pyrolysis of cellulose to produce aromatics.

Full text of DOI:10.1039/C8TA08930B

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Construction of myoglobin–amphiphilic alginate
caprylamide–graphene composite modified
electrode for the direct electron transfer between
redox proteins and electrode and electrocatalysis
of myoglobin†
Cite this: RSC Adv., 2018, 8, 38003
ab
Xiuqiong Chen,ab Huiqiong Yan,
Wei Sun,b Guangying Chen,a Changjiang Yu,b
*
ab
Wen Fengb and Qiang Lin
*
To achieve the dispersion of the hydrophobic graphene (GR), the amphiphilic alginate caprylamide (ACA)
was synthesized to fabricate electroactive Nafion/Mb–ACA–GR/CILE for the accurate determination of
trichloroacetic acid (TCA). SEM observation, FT-IR and UV-Vis spectroscopic analysis indicated that ACA
could tightly immobilize Mb and GR on the electrode surface by constructing biointerfaces, which not
only provided Mb a suitable microenvironment to maintain its biological activity, but also shortened the
distances between the active centers of Mb with carbon ionic liquid electrode (CILE), thus promoting the
electron transfer rate. The electrochemical characterization of Nafion/Mb–ACA–GR/CILE showed that
the direct electron transfer of Mb was realized on the modified electrode, which was attributed to the
high electrical conductivity and excellent electrocatalytic activity of GR and good biocompatibility of
Received 26th August 2018
Accepted 31st October 2018
ACA. Moreover, Nafion/Mb–ACA–GR/CILE exhibited good electrocatalytic activity towards TCA with the
linear range from 2.5 to 47.3 mmol Lꢀ1 and lower KM
value of 8.3 mmol Lꢀ1. Moreover, the modified
app
DOI: 10.1039/c8ra07117a
electrode also revealed good stability, reproducibility and accurate detection of tap-water, exhibiting
great potential for the applications as the third-generation electrochemical biosensors.
rsc.li/rsc-advances
Myoglobin (Mb) is one of the ideal redox proteins because of
its known structure, commercial availability, and relatively low
1 Introduction
molecular weight, and its direct electron transfer is attributed to
the bioactive heme Fe(III)/Fe(II) redox couple.6 However, the
direct electron transfer between the redox protein and the
surface of bare electrodes is oen difficult to achieve, which is
attributed to the deeply embedded redox active center in the
structure of proteins, the unfavorable orientation of protein
molecules and the irreversible adsorption of denatured proteins
on the electrode surface.7–9 For these reasons, many materials
such as polymers, metal oxide nanoparticles, and graphene10–12
have been applied to modify the substrate electrode. This could
help to direct the proteins in favorable orientation towards the
electrode thus decreasing the distances between the active
centers of redox proteins and the substrate electrode as well as
accelerating the electron transfer rate.13 Among the above-
mentioned materials, graphene is one of the most perspective
functional materials for the fabrication of electrochemical
biosensors because of its high surface area, good mechanical
strength, high electrical conductivity and excellent electro-
Trichloroacetic acid (TCA), a type of organohalide pollutant, has
become one of the major environmental issues for its extensive
application in industry, agriculture and public healthcare.1
Since it also appears in drinking water as a result of chlorine
disinfection and has high solubility in water, TCA has been
considered to be harmful to aquatic life and humans.2 There-
fore, accurate determination and regular monitoring of TCA
concentration has received considerable attention in recent
years. Among the various methods for the detection of TCA,
electrochemical biosensing based on the direct electrochem-
istry between an electrode and the immobilized redox protein is
particularly promising owing to its high selectivity, high sensi-
tivity and relatively low cost.3–5
aKey Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education,
College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou
571158, China. E-mail: yanhqedu@163.com; linqianggroup@163.com; Tel: +86
0898 65884995; +86 0898 65889422
bKey Laboratory of Water Pollution Treatment & Resource Reuse of Hainan Province,
College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou
571158, Hainan, China
catalytic activity.14,15 Graphene (GR) is
a sheet of two-
dimensional (2D) single layer sp2 hybridized carbon atoms
packed into a dense honeycomb-like crystal structure.16,17 It has
attracted a tremendous amount of interest for the application in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra07117a
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 38003–38012 | 38003

View Article Online
RSC Advances
Paper
the electrochemistry and electrochemical sensing elds due to (CILE) to fabricate the novel modied electrode, abbreviated as
its extraordinary physicochemical and structural properties Naon/Mb–ACA–GR/CILE, whose preparation procedure is
since its discovery in 2004.18 Gan and Hu had reviewed the illustrated in Scheme 1. The secondary structure of Mb in the
potential applications for electrochemistry and electroanalysis ACA–GR lm was characterized by UV-Vis and FTIR spectros-
of GR.19 Wu et al.20 used chitosan-dispersed GR to immobilize copy. In addition, the morphology and the electrochemical
cytochrome C for the electrocatalytic determination of nitric performance of Naon/Mb–ACA–GR/CILE were investigated by
oxide. Sun et al.16 adopted graphene and titanium dioxide scanning electron microscopy (SEM) and cyclic voltammetry,
nanocomposite-modied carbon ionic liquid electrode for the respectively. The main objective of this study was to fabricate
electrocatalytic determination of TCA. Zhan et al.8 employed the biointerfaces by amphiphilic ACA to promote the electron
Co2Al layered double hydroxide–GR nanocomposite and chito- transfer rate between the redox protein and the underlying
san to immobilize hemoglobin on the CILE for the electro- electrode for the new biosensor material, which is capable of
catalytic determination of TCA. In addition, the electrochemical achieving the desired properties. To the best of our knowledge,
behavior of GR modied electrode towards other important the attempt to use biocompatible and amphiphilic alginate
electroactive substances had been also reported.21
derivatives to disperse GR and immobilize the redox protein for
Since the single GR sheets are prone to irreversible aggre- the construction of electrochemical biosensors has been rarely
gation due to the van der Waals forces and p–p restacking in reported.
aqueous medium, the dispersion of graphene is poor, which is
disadvantageous for the construction of various potentially
2 Experimental
2.1 Reagent and materials
useful graphene-based biosensors.22,23 In practical electro-
chemical applications, the common method to improve the
dispersion of GR is chemical functionalization. However, the
functionalization process would certainly destroy the extraor-
dinary structure of GR, which may result in the signicant
reduction of the electrical conductivity and other properties.24
Therefore, other methods without involving functionalization
of GR should be proposed to improve the dispersion of GR for
the construction of electrochemical biosensors.
Graphene (GR, >98.3%) was purchased from Sinocarbon Tech-
nology Co. Ltd., China. Myoglobin (Mb, MW ¼ 17.8 kDa) and
Naon (5% ethanol solution) were purchased from Sigma, USA.
Ionic liquid N-hexylpyridinium hexauorophosphate (HPPF6)
was purchased from Lanzhou Greenchem (ILS, LICP, CAS,
China). Graphite powder (particle size 30 mm) was purchased
from Shanghai Colloid Chem. Co., China. Liquid paraffin and
KCl were purchased from Aladdin Chemical Reagent Co., Ltd.,
Shanghai, China. Trichloroacetic acid (TCA) was purchased
from Tianjin Kemiou Chemical Ltd. Co., China. The chemicals
were of analytical grade and used without further purication.
0.1 mol Lꢀ1 pH 7.0 phosphate buffer solution (PBS) were used as
the supporting electrolyte.
It has been reported that aqueous surfactants can facilitate
direct electron transfer between the substrate electrode and the
redox protein of myoglobin.25,26 The interactions between the
surfactant and the protein promote the formation of dynamic
complexes that could inhibit irreversible adsorption of the
protein on the electrode surface and assist in favorably orient-
ing the protein to the hydrophobic electrode surface. This has
been regarded as the main reason for difficulty in achieving
direct electron transfer between redox proteins and elec-
trode.7,27 In addition, alginate, as an anionic natural polymer,
possesses unique biocompatible property that could provide
a desirable microenvironment for the immobilization of the
protein while retaining its structure and activity, thus leading to
the wide application in the construction of electrochemical
biosensors with enhanced analytical performance.28,29 As
a result of available hydroxyl and carboxyl groups on the back-
bone of alginate, chemical modication could be achieved with
the aid of 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide
hydrochloride (EDC$HCl).30 Moreover, modication of algi-
nate by introducing hydrophobic groups onto its hydrophilic
backbone could make it amphiphilic, similar to the surfactant,
which could enhance its affinity for the hydrophobic GR.31
Hence, we could utilize amphiphilic alginate derivative to
In addition, the alginate caprylamide (ACA) was prepared
based on our previous study,32 which was synthesized by the
amidation reaction through alginate coupling with octylamine
in the presence of EDC$HCl. The structure of the as-synthesized
ACA was conrmed by FT-IR and 1H NMR, which are shown in
the ESI in Fig. 1S and 2S,† respectively. FTIR (KBr pellet
method): 3445 cmꢀ1, hydroxyl O–H stretching; 2854 cmꢀ1
,
aliphatic C–H stretching, 1380 cmꢀ1, aliphatic –CH2– bending.
1H NMR (400 MHz, D2O) d 5.0–3.5 (m, alginate backbone), 3.0 (t,
–CH2–NH– of octyl), 1.4–0.8 (t, –(CH2)6– of octyl), 0.6 (s, –CH3 of
octyl). Elemental composition of ACA determined by elemental
analysis: C, 32.47; N, 1.26; H, 5.32; S, 0.38. According to the data
collected by elemental analysis, the substitution degree of ACA
was calculated as 0.25.
2.2 Apparatus
disperse GR, which could not only promote efficient electron A CHI 750B electrochemical workstation with the conventional
transfer between the electrode and the protein, but also retain three-electrode system composed of the modied electrode as
the protein bioactivity and the GR physicochemical property.
working electrode, the platinum wire electrode as auxiliary
In this study, the amphiphilic alginate caprylamide (ACA) electrode and the saturated calomel electrode (SCE) as reference
was prepared by amide linkage attachment of octylamine onto electrode were used for cyclic voltammetry measurements at
the carboxylate group of alginate. Then, the mixture of Mb, ACA ambient temperature. The colloidal interface activity of ACA was
and GR was cast on the surface of carbon ionic liquid electrode evaluated by uorescence spectroscopy and dynamic light
38004 | RSC Adv., 2018, 8, 38003–38012
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Scheme 1 Schematic of the fabrication of Nafion/Mb–ACA–GR/CILE.
scattering (DLS). Fluorescence measurement was performed on respectively performed on a Tensor 27 Fourier transform
a Hitachi F7000 uorescence spectrophotometer using pyrene infrared spectrometer (Bruker, Germany) and a Cary 50 probe
as a uorescence probe, which was an efficient way to detect spectrophotometer (Varian, Australia).
self-aggregation property of the formation of micelles by intra-
or intermolecular associations of amphiphilic polymers.31 The
2.3 Fabrication of the modied electrode
size and zeta potential of the polymeric micelles were deter-
mined by dynamic light scattering using a Malvern Nano-ZS90
A carbon ionic liquid electrode (CILE) was prepared using the
HPPF6 as the binder and the modier. In short, 3.2 g of graphite
powder, 1.6 g of HPPF6 and 1.0 mL of liquid paraffin were mixed
thoroughly in a mortar to form a homogeneous carbon paste.
Then, a certain amount of this carbon paste was lled into one
end of a glass tube, and a copper wire was inserted through the
opposite end to establish an electrical contact, thus construct-
ing the CILE.
ꢁ
Zetasizer (UK) at a scattering angle of 90ꢁ at 25 C by employ-
ing an (He-Ne) argon laser (l ¼ 633 nm). The morphology of the
composite materials on the electrode surface and the secondary
structure of Mb in the composite matrix were characterized by
SEM, FTIR and UV-Vis spectroscopy. The morphology observa-
tion was conducted on a JSM-7100F scanning electron micro-
scope (Electron, Japan). FT-IR and UV-Vis measurements were
Fig. 1 (A) Plots of pyrene fluorescence intensity I1/I3 vs. the concentration of (a) sodium alginate and (b) ACA solutions. (B) Hydrodynamic
diameter distribution and (C) zeta potential distribution of ACA self-aggregated micelle.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 38003–38012 | 38005

View Article Online
RSC Advances
Paper
Fig. 2 SEM images of (a) GR, (b) ACA, (c) ACA–GR and (d) Mb–ACA–GR composite.
Subsequently, the CILE was polished to obtain a mirror-like solution. This resultant electrode was abbreviated as Naon/Mb–
surface with a piece of weighing paper. Then, 8.0 mL of homoge- ACA–GR/CILE. For comparison, the modied electrodes, namely,
neously mixed suspension solution composed of 6 mg mLꢀ1 Mb, Naon/ACA–GR/CILE, Naon/Mb–ACA/CILE and Naon/Mb–GR/
1.0 mg mLꢀ1 GR and 1.6 mg mLꢀ1 ACA was cast on the surface of CILE were prepared by the similar procedure.
CILE directly and le to dry at 4 ꢁC overnight. Aer the surface of
CILE was dry, 5.0 mL of 0.5% Naon solution was spread evenly
onto the surface of the modied electrode to further consolidate
the composite lm. Naon with excellent lm-forming ability
could improve the stability of protein immobilized on the elec-
3 Results and discussion
3.1 Colloidal interface activity of ACA
Since pyrene is very sensitive to the environment and can be
encapsulated in the hydrophobic microdomains formed by
trode surface and prevent its leakage from electrode into the
Fig. 3 (A) FT-IR spectra and (B) UV-Vis absorption spectra (in PBS) of (a) Mb and (b) Mb–ACA–GR composite.
38006 | RSC Adv., 2018, 8, 38003–38012
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 4 (A) Cyclic voltammograms of (a) Nafion/ACA–GR/CILE, (b) Nafion/Mb–GR/CILE and (c) Nafion/Mb–ACA/CILE and (d) Nafion/Mb–ACA–
GR/CILE in pH 7.0 PBS with the scan rate as 100 mV sꢀ1. (B) Cyclic voltammograms of Nafion/Mb–ACA–GR/CILE in pH 7.0 PBS at different scan
rates (from a to r: 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mV sꢀ1, respectively). (C) Linear
relationship of the cathodic and anodic peak current (Ip) versus scan rate (v). (D) Linear relationship of the anodic and cathodic peak potential (Ep)
versus ln v.
alkyl chains, the amphiphilic property of ACA could be assessed hydrodynamic diameter distribution and zeta potential distri-
through uorescence measurements using pyrene as the uo- bution of ACA self-aggregated micelles are presented in Fig. 1B
rescent probe.33 There are ve electronic vibration peaks and C. In Fig. 1B, the ACA self-aggregated micelles exhibited
observed in the pyrene uorescence spectrum of ACA. The ratio a narrow size distribution involving two ranges, and their
value of the rst peak at 372 nm (I1) to the third peak at 383 nm average hydrodynamic diameter (dH) was 295 nm with the PDI
(I3) is related to the micro-environmental polarity surrounding of 0.28. These results indicated that the molecular exibility of
pyrene molecules,34 which decreases with the reduction of the ACA was enhanced by hydrophobic association.36 In addition,
micro-environmental polarity. When the hydrophobic octyl the ACA micelles showed a relatively high negative zeta poten-
groups drive ACA to form self-aggregated micelles, the ratio tial at about ꢀ44.2 mV as a result of the presence of the residual
value of I1/I3 signicantly decreases. The corresponding onset free-curling carboxyl groups, as shown in Fig. 1C. ACA micelles
concentration was the critical aggregation concentration (CAC) are generally considered to be stable in aqueous solution when
of ACA (Fig. 1A), which was the lowest concentration of self- the absolute value of zeta potential is higher than 30 mV, as the
aggregate formation by intramolecular or intermolecular asso- strong repulsion forces prevent the aggregation among the
ciation.35 As ACA was graed with a certain amount of hydro- micelles.37 The abovementioned results were ascribed to the
phobic octylamine (degree of substitution of 0.25), its molecular partial graing of hydrophobic octyl groups onto the main
self-aggregation ability in aqueous solution was much larger chains of the alginate molecule, which could destroy the orig-
than that of alginate. As shown in Fig. 1A, ACA revealed good inal intramolecular hydrogen bonding to enhance its molecular
amphipathy with a low CAC value of 0.51 g Lꢀ1, whereas that of exibility. Therefore, ACA displayed good colloidal interface
sodium alginate was 2.13 g Lꢀ1. These results indicated that the activity, which was benecial for the dispersion of GR sheets via
amidation reaction by octylamine could endow alginate with hydrophobic associations, thus facilitating direct electron
good amphiphilic functionality.
transfer between the substrate electrode and the redox protein
The colloidal property of ACA was investigated by dynamic of myoglobin.
light scattering using Malvern Nano-ZS90 Zetasizer. The
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 38003–38012 | 38007

View Article Online
RSC Advances
Paper
Fig. 5 Cyclic voltammograms of Nafion/ACA–GR/CILE in pH 7.0 PBS with 17.3 mmol Lꢀ1 TCA (curve a) and Nafion/Mb–ACA–GR/CILE in pH 7.0
PBS containing 2.5, 4.8, 7.3, 9.3, 13.3, 17.3, 21.3, 25.3, 31.3, 37.3, 43.3, 47.3, 59.3, 69.3, 79.3 mmol Lꢀ1 TCA (curves b–p), respectively, with the scan
rate of 100 mV sꢀ1. Inset: Linear relationship of catalytic reduction peak currents and the TCA concentration.
surface roughness (Fig. 2d), indicating that the GR and Mb were
successfully immobilized on the electrode surface. It was the
biointerfaces constructed by ACA that shortened the distances
between the active centers of Mb with the substrate electrode
and protected Mb from denaturation, thus facilitating electron
transfer rate and stability of Naon/Mb–ACA–GR/CILE.38
The biological activity of Mb in the Mb–ACA–GR composite
was evaluated by detecting the secondary structure of redox
proteins by conducting FT-IR and UV-Vis spectroscopy. FT-IR
spectra of Mb and Mb–ACA–GR composite are presented in
Fig. 3A. It is clearly observed that Mb displays characteristic
peaks at 1654.89 and 1541.91 cmꢀ1, which are respectively
ascribed to the amide I and II infrared absorbance. Aer Mb,
ACA and GR were mixed together; as a result, the amide I and II
peaks of Mb appeared at 1651.50 and 1538.10 cmꢀ1. In contrast,
the Mb–ACA–GR composite exhibited relatively weak charac-
teristic peaks of Mb because of the hydrogen bonding or
shielding effect stemmed from the ACA–GR composite.
3.2 Morphology and spectroscopic analysis of the composite
materials on the electrode surface
The morphology of the composite materials on the electrode
surface was examined by SEM, with the results shown in Fig. 2.
It can be observed that the single GR revealed representative
sheet-like shape with slightly scrolled edges.16 Due to the
hydrophobic property of GR, most GR nanosheets gathered
together to form a loose and laminar cluster structure, as shown
in Fig. 2a. This uffy structure was ineffective for electron
transfer and easily resulted in the disengagement of GR from
the electrode surface. In spite of the partial graing of hydro-
phobic octyl groups, the ACA could still generate a good lm on
the electrode surface (Fig. 2b). Owing to the hydrophobic
association and lm-forming property of ACA, GR could be well
dispersed and tightly adhered on the electrode surface, as
shown in Fig. 2c. Additionally, the Mb–ACA–GR composite
mainly exhibited the morphology of ACA with the increase in
Table 1 Electrochemical parameters of different modified electrodes with Mb as the model for the detection of TCA
Linear range
Detection limit
app
Modied electrode formulation
ks (sꢀ1
)
(mmol Lꢀ1
)
(mmol Lꢀ1
)
KM
4.7
(mmol Lꢀ1
)
References
Naon/Mb–Co3O4–Au/IL-CPE
CS/Mb/3D RGO–Au/CILE
Naon/Mb/NiO/GR/CILE
Naon/Mb–SA–Fe3O4–GR/CILE
Naon/Mb–SA–TiO2/CILE
Naon–BMIMPF6/Mb/CILE
Naon/Mb–ACA–GR/CILE
0.799
0.71
—
0.234
0.511
0.532
0.886
2.0–20.0
0.2–36.0
0.002–0.21
1.4–119.4
5.3–114.2
1.6–19.6
2.5–47.3
0.5
6
38
47
48
49
50
0.06
0.002
0.174
0.152
0.2
9.88
10.67
29.1
32.3
90.8
8.3
0.114
This work
38008 | RSC Adv., 2018, 8, 38003–38012
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Table 2 Determination of TCA in tap-water samples using the modified electrode and the standard GC-MS method
Electrochemical biosensor
GC-MS
Added
Found
Recovery
(%)
RSD
(%)
Found
Recovery
(%)
Samples
(mmol Lꢀ1
)
(mmol Lꢀ1
)
(mmol Lꢀ1
)
RSD (%)
1
2
3
4
5
10
15
20
5.2
10.1
14.8
19.9
104.0
101.0
98.7
3.9
1.6
2.3
1.8
5.3
10.3
14.6
19.7
106.0
103.0
97.3
5.7
3.1
2.7
2.1
99.5
98.5
However, their positions and shapes were similar to that of Mb. ACA/CILE displayed higher redox peak currents than Naon/
As the shapes and positions of the amide I and II infrared Mb–GR/CILE, indicating that ACA had more inuence on the
absorbance peaks of Mb can provide detailed information on electron transfer of Mb than GR.40 Moreover, the synergistic
the secondary structure of the polypeptide chain,6 the above effects of ACA and GR made Naon/Mb–ACA–GR/CILE present
results indicated that Mb could retain its native structure in the biggest redox peak currents among the modied electrodes.
Mb–ACA–GR composite during the fabrication of the modied This result could be explained by the fact that ACA with good
electrode.
colloidal interface activity could not only disperse GR sheets via
The probable denaturation or the conformational variations hydrophobic associations, but also tightly immobilize Mb and
of Mb could be also examined via the UV-Vis adsorption spec- GR on the electrode surface. Therefore, the biointerfaces con-
troscopy by testing the change in the shape and position of the structed by ACA during the fabrication of Naon/Mb–ACA–GR/
Soret absorption peaks attributed to the four iron heme groups CILE could protect Mb from denaturation and shorten the
of Mb.39 Fig. 3B shows the UV-Vis absorption spectra of Mb and distances between the active centers of Mb with CILE, thus
Mb–ACA–GR mixture in pH 7.0 PBS. It can be seen that Mb and accelerating the electron transfer rate.38 As shown in the cyclic
the mixture solution of Mb–ACA–GR reveal very similar Soret voltammogram of Naon/Mb–ACA–GR/CILE, the cathodic peak
peaks at the same position (408 nm). This result indicated that potential (Epc) and the anodic peak potential (Epa) respectively
Mb molecules retained its native structure in the mixture of appeared at ꢀ0.295 V and ꢀ0.208 V. Its formal peak potential
0
0
Mb–ACA–GR, and ACA revealed good biocompatibility towards (E ) was calculated as ꢀ0.252 V (vs. SCE), a typical value of the
maintaining the biological activity of Mb.
active center of Mb Fe(III)/Fe(II) redox couple,6 implying that
most of the Mb retained its original structure aer being
incorporated in ACA–GR matrix. Therefore, the direct electron
transfer of Mb was attributed to the heme Fe(III)/Fe(II) redox
couple in the Mb molecules, which could be realized on the
Naon/Mb–ACA–GR/CILE with fast electron transfer rate.
The effects of scan rate on the cyclic voltammograms of
Naon/Mb–ACA–GR/CILE are shown in Fig. 4B. With the
increase in scan rate from 150 to 1000 mV sꢀ1, a pair of
symmetric redox peaks with nearly equal redox peak currents
appeared because of the reduction and reoxidation of electro-
active Mb Fe(III)/Fe(II) redox couple upon cyclic scanning, and
the redox peak current increased linearly with scan rate, as
shown in Fig. 4C. The linear regression equations were calcu-
lated as Ipc (mA) ¼ 148.784v (V sꢀ1) + 8.539 (n ¼ 21, g ¼ 0.998)
and Ipa (mA) ¼ ꢀ104.508v (V sꢀ1) ꢀ 3.829 (n ¼ 21, g ¼ 0.997).
These results indicated that the electron transfer of Naon/Mb–
3.3 Direct electrochemistry of the modied electrode
The effects of Mb, ACA and GR on the electrochemical proper-
ties of the modied electrode were investigated by cyclic vol-
tammetry. Direct electrochemical tests of the modied
electrode were performed in a 10 mL electrochemical cell con-
taining 0.1 mol Lꢀ1 pH 7.0 PBS. Before the measurements, PBS
was deaerated with highly puried N2 for 30 min and then,
nitrogen atmosphere was maintained over the cell during the
experiments. Cyclic voltammograms of the different modied
electrodes in deaerated pH 7.0 PBS at a scan rate of 100 mV sꢀ1
are presented in Fig. 4A. It can be seen that there are no distinct
redox peaks in the cyclic voltammogram of Naon/ACA–GR/
CILE due to the absence of electroactive substances of Mb,
elucidating that the direct electron transfer was performed
between redox proteins and the electrode surface. Attributed to
the redox reaction of immobilized Mb, Naon/Mb–GR/CILE,
Naon/Mb–ACA/CILE and Naon/Mb–ACA–GR/CILE exhibited
a pair of symmetric redox peaks. GR with high electrical
conductivity and excellent electrocatalytic activity can be used
as a good electronic mediator of Mb, and is conducive towards
enhancing the electron transfer rate. However, Naon/Mb–GR/
CILE exhibited lower electrochemical response in comparison
with that of Naon/Mb–ACA/CILE and Naon/Mb–ACA–GR/
CILE owing to the uffy structure of GR. As a result of good
biocompatibility of ACA that could provide Mb suitable micro-
environment to maintain its biological activity, Naon/Mb–
ACA–GR/CILE was
a surface-controlled thin-layer electro-
chemical reaction.16 The surface concentration (G*) of electro-
active Mb on the surface of Naon/Mb–ACA–GR/CILE could be
calculated using Faraday's law (G* ¼ Q/nAF), where Q is the
integration charge of the reduction peak, n is the number of
electron transferred, A is the surface area of the electrode, and F
is the Faraday constant.41 The charge value (Q) was nearly
constant at different scan rates and the average value of G* was
obtained as 1.79 ꢂ 10ꢀ9 mol cmꢀ2, which was much larger than
that of monolayer coverage (2.0 ꢂ 10ꢀ11 mol cmꢀ2).42 These
results indicated that the ACA–GR composite lm contained
multilayers of Mb, which may result from the laminar cluster
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 38003–38012 | 38009

View Article Online
RSC Advances
Paper
structure of GR with the increased surface area. Additionally, of the enzyme–substrate kinetics, could be calculated by the
the redox peak potentials were also shied with the increase in Lineweaver–Burk equation:46
scan rate (Fig. 4D), and the redox peak potentials exhibited
app
1
1
KM
¼
þ
Imax ImaxC
linear relationship with natural logarithm of scan rate (ln v)
according to the equations Epc (V) ¼ ꢀ0.059 ln v (V sꢀ1) ꢀ 0.329
(n ¼ 13, g ¼ 0.987) and Epa (V) ¼ 0.039 ln v (V sꢀ1) ꢀ 0.294 (n ¼
13, g ¼ 0.968). Based on the Laviron's equation,43,44 the elec-
trochemical parameters of the electrode reaction, namely, the
values of the number of electrons transferred (n), the electron
transfer coefficient (a) and the apparent heterogeneous electron
transfer rate constant (ks) were calculated as 1.09, 0.398 and
0.886 sꢀ1, respectively. The high apparent heterogeneous elec-
tron transfer rate constant indicated a fast electron transfer rate
with high efficiency.
ISS
where ISS is the steady-state current, Imax is the maximum
current under saturated substrate condition, and C is the bulk
concentration of the substrate. Based on Lineweaver–Burk
equation, the KMapp value was calculated as 8.3 mmol Lꢀ1. The
electrochemical parameters of Naon/Mb–ACA–GR/CILE in
comparison with those of other reported Mb-modied elec-
trodes are summarized in Table 1.6,38,47–50 It can be seen that the
KMapp value of Naon/Mb–ACA–GR/CILE was lower than that of
the previous reports, indicating that the biointerfaces, con-
structed by ACA in Mb–ACA–GR composite, made Mb display
high affinity to TCA with good enzymatic activity. On the basis
of the abovementioned results, Naon/Mb–ACA–GR/CILE
exhibited good electrocatalytic ability towards the reduction of
TCA.
3.4 Electrocatalytic ability to TCA
Since TCA usually exists in drinking water as a result of chlorine
disinfection, it has been regarded as a very toxic water pollutant
to aquatic life and humans even at very low concentrations.
Thus, accurate determination and regular monitoring of TCA
concentration has received considerable attention in recent
years.1 The electrocatalytic activity of Naon/Mb–ACA–GR/CILE
to TCA was also investigated by cyclic voltammetry. The elec-
trocatalysis of TCA was performed in a 10 mL electrochemical
cell containing 0.1 mol Lꢀ1 deaerated pH 7.0 PBS with various
concentrations of TCA, with the results presented in Fig. 5. It
can be observed that the cyclic voltammogram of Naon/ACA–
GR/CILE in the presence of TCA (curve a) did not reveal a direct
reduction peak, indicating that the Mb played a major role in
the electrocatalysis of TCA. The redox proteins possessing good
electrocatalytic activity to TCA made Naon/Mb–ACA–GR/CILE
reveal the reduction peak in PBS in the presence of TCA.41
With the increase in TCA concentration, the reduction peak
current gradually improved, while the oxidation peak current
disappeared, which is a typical characteristic of the electro-
catalytic reaction. The electrocatalytic reaction may involve the
reduction of Mb Fe(III) to Mb Fe(II) and oxidation of Mb Fe(II) to
Mb Fe(III) in the presence of TCA by the direct electron transfer
on the electrode surface,38,41,45 which can be expressed with the
following equations (eqn (1) and (2)):
3.5 Real samples determination and interference
The capability of Naon/Mb–ACA–GR/CILE to determine TCA in
real samples was evaluated via a recovery study. The recovery
performance of Naon/Mb–ACA–GR/CILE was studied by
spiking TCA at different concentrations (5, 10, 15 and 20 mmol
Lꢀ1) in tap-water. At the same time, a standard GC-MS method
was used for comparison. As shown in Table 2, the recovery of
the electrochemical biosensor was in the range from 98.5% to
104% with small RSD value in the range from 1.6% to 3.9%,
which was desirably consistent with the GC-MS results. In
addition, the effect of some possible interfering substances,
which might exist in tap-water, was also evaluated. When the
concentrations of Na+, K+ Ca2+, Mg2+, Fe3+, Clꢀ, NO3ꢀ, SO4
2ꢀ
and PO43ꢀ in the sample were 100 times of TCA, no signicant
interference was found. Additionally, similar molecules, such as
dichloroacetic acid and monochloroacetic acid at 20 times of
the TCA concentration did not interfere with the catalytic
reduction peak, with the current changes below ꢃ5%. These
results indicated that the as-fabricated electrochemical
biosensor had excellent accuracy for the detection of tap-water.
3.6 Stability and reproducibility
MbFe(III) + e / MbFe(II)
(1)
The stability and reproducibility of Naon/Mb–ACA–GR/CILE
were characterized by measuring the cyclic voltammetric peak
currents. The Naon/Mb–ACA–GR/CILE could stay at nearly
constant current values upon the continuous cyclic voltam-
metric sweep for 120 cycles. When stored at 4 ꢁC for more than 2
2MbFe(II) + Cl3CCOOH + H+ / 2MbFe(III)
+ Cl2CHCOOH + Clꢀ
(2)
In addition, the catalytic reduction peak currents propor- weeks, the Naon/Mb–ACA–GR/CILE retained 97.7% of the
tionally increased with the increase in TCA concentration in the initial current response. Aer 1 month, the peak current
range from 2.5 to 47.3 mmol Lꢀ1. The linear regression equation response decreased about 8.4%. The good long-term stability
was tted as Iss (mA) ¼ 6.998C (mmol Lꢀ1) + 5.371 (n ¼ 12, g ¼ can be attributed to the good biocompatibility of ACA and lm-
0.995) and the detection limit was calculated as 0.114 mmol Lꢀ1 forming property of Naon that retained proteins' bioactivity
(3s). When the TCA concentration exceeded 47.3 mmol Lꢀ1, the and prevented their leakage. Additionally, the electrocatalytic
catalytic reduction peak currents began to level off and reached ability of Naon/Mb–ACA–GR/CILE towards TCA by cyclic vol-
a plateau, implying a Michaelis–Menten kinetic process. The tammetry was also tested to investigate its reproducibility. The
apparent Michaelis–Menten constant (KMapp), being indicative relative standard deviation (RSD) of 12 successive
38010 | RSC Adv., 2018, 8, 38003–38012
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
measurements for current determinations of 20 mmol Lꢀ1 TCA 10 K. Y. Hwa and B. Subramani, Biosens. Bioelectron., 2014, 62,
was 6.7%, which indicated acceptable reproducibility. On the 127–133.
basis of the abovementioned results, Naon/Mb–ACA–GR/CILE 11 D. Wang and L. Chen, Electrochim. Acta, 2009, 54, 4316–4320.
exhibited good stability and reproducibility.
12 Z. Wang, S. Liu, P. Wu and C. Cai, Anal. Chem., 2009, 81,
1638–1645.
13 F. A. Armstrong and G. S. Wilson, Electrochim. Acta, 2000, 45,
2623–2645.
4 Conclusion
¨
14 H. Chen, M. B. Muller, K. J. Gilmore, G. G. Wallace and D. Li,
In the present study, the biocompatible and amphiphilic ACA
was used to disperse GR and immobilize Mb for the fabrication
of electroactive Naon/Mb–ACA–GR/CILE. Due to the good
colloidal interface activity, ACA could tightly immobilize Mb
and GR on the electrode surface by constructing the bio-
interfaces, which not only provided Mb a suitable microenvi-
ronment to maintain its biological activity, but also shortened
the distances between the active centers of Mb with CILE,
promoting the electron transfer rate. As a result of the high
electrical conductivity and excellent electrocatalytic activity of
GR and good biocompatibility of ACA, the as-fabricated Naon/
Mb–ACA–GR/CILE exhibited a pair of symmetric redox peaks,
and exhibited good electrocatalytic activity towards TCA with
the linear range from 2.5 to 47.3 mmol Lꢀ1 and low KMapp value
of 8.3 mmol Lꢀ1. Furthermore, the modied electrode also
presented good stability, reproducibility and accurate detection
of tap-water, thus possessing great potential for applications in
the electroanalytical eld.
Adv. Mater., 2008, 20, 3557–3561.
15 Y. W. Zhu, S. Murali, W. W. Cai, X. S. Li, J. W. Suk, J. R. Potts
and R. S. Ruoff, Adv. Mater., 2010, 22, 3906–3924.
16 W. Sun, L. L. Cao, Y. Deng, S. X. Gong, F. Shi, G. N. Li and
Z. F. Sun, Anal. Chim. Acta, 2013, 781, 41–47.
17 A. K. Geim and K. S. Novoselov, Nat. Mater., 2007, 6, 183–191.
18 H. Liang, X. Miao and J. Gong, Electrochem. Commun., 2012,
20, 149–152.
19 T. Gan and S. S. Hu, Microchim. Acta, 2011, 175, 1–19.
20 J. F. Wu, M. Q. Xu and G. C. Zhao, Electrochem. Commun.,
2010, 12, 175–177.
21 M. Zhou, Y. M. Zhai and S. J. Dong, Anal. Chem., 2009, 81,
5603–5613.
22 Y. Zhou, Q. Bao, L. A. L. Tang, Y. Zhong and K. P. Loh, Chem.
Mater., 2009, 21, 2950–2956.
23 J. Shen, Y. Hu, C. Li, C. Qin and M. Ye, Small, 2009, 5, 82–85.
24 Y. C. Si and E. T. Samulski, Nano Lett., 2008, 8, 1679–1682.
25 A. K. Udit, K. D. Hagen, P. J. Goldman, A. Star, J. M. Gillan,
H. B. Gray and M. G. Hill, J. Am. Chem. Soc., 2006, 128,
10320–10325.
Conflicts of interest
26 W. Shan, H. Liu, J. Shi, L. Yang and N. Hu, Biophys. Chem.,
2008, 134, 101–109.
There are no conicts to declare.
27 A. K. Udit, M. G. Hill and H. B. Gray, Langmuir, 2006, 22,
10854–10857.
28 M. Cheong and I. Zhitomirsky, Colloids Surf., A, 2008, 328,
73–78.
29 H. Y. Zhao, W. Zheng, Z. X. Meng, H. M. Zhou, X. X. Xu, Z. Li
and Y. F. Zheng, Biosens. Bioelectron., 2009, 24, 2352–2357.
30 J. S. Yang, Y. J. Xie and W. He, Carbohydr. Polym., 2011, 84,
33–39.
31 H. Yan, X. Chen, J. Li, Y. Feng, Z. Shi, X. Wang and Q. Lin,
Carbohydr. Polym., 2016, 136, 757–763.
Acknowledgements
We gratefully thank the nancial support from the Natural
Science Foundation of Hainan Province (218QN233) and the
National Natural Science Foundation of China (21566009).
Notes and references
1 T. Zhan, X. Wang, Y. Zhang, Y. Song, X. Liu, J. Xu and
W. Hou, Sens. Actuators, B, 2015, 220, 1232–1240.
32 X. Chen, H. Yan, W. Sun, Y. Feng, J. Li, Q. Lin, Z. Shi and
X. Wang, Polym. Bull., 2015, 72, 3097–3117.
33 K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc.,
1977, 99, 2039–2044.
34 L. Q. Yang, B. F. Zhang, L. Q. Wen, Q. Y. Liang and
L. M. Zhang, Carbohydr. Polym., 2007, 68, 218–225.
35 J. S. Yang, Q. Q. Zhou and W. He, Carbohydr. Polym., 2013,
92, 223–227.
36 H. R. Nie, A. H. He, J. F. Zheng, S. S. Xu, J. X. Li and C. C. Han,
Biomacromolecules, 2008, 9, 1362–1365.
37 Y. An, M. Chen, Q. Xue and W. Liu, J. Colloid Interface Sci.,
2007, 311, 507–513.
ˆ
´
2 J. Dojlido, E. Zbiec and R. Swietlik, Water Res., 1999, 33,
3111–3118.
3 F. Ahami, F. Soltani-Felehgari, T. Madrakian and
H. Ghaedi, Biosens. Bioelectron., 2014, 51, 379–385.
4 J. Xu, C. H. Liu and Z. F. Wu, J. Solid State Electrochem., 2012,
16, 361–366.
5 L. Chen, H. Xie and J. Li, J. Solid State Electrochem., 2012, 16,
3323–3329.
6 X. F. Wang, Z. You, H. L. Sha, S. X. Gong, Q. J. Niu and
W. Sun, Microchim. Acta, 2014, 181, 767–774.
7 J. Rajbongshi, D. K. Das and S. Mazumdar, Electrochim. Acta,
2010, 55, 4174–4179.
8 T. Zhan, X. Wang, X. Li, Y. Song and W. Hou, Sens. Actuators,
B, 2016, 228, 101–108.
38 F. Shi, J. Xi, F. Hou, L. Han, G. Li, S. Gong, C. Chen and
W. Sun, Mater. Sci. Eng., C, 2016, 58, 450–457.
39 W. Sun, Y. Guo, Y. Lu, A. Hu, F. Shi, T. Li and Z. Sun,
Electrochim. Acta, 2013, 91, 130–136.
9 L. Fang, B. Liu, L. Liu, Y. Li, K. Huang and Q. Zhang, Sens.
Actuators, B, 2016, 222, 1096–1102.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 38003–38012 | 38011

View Article Online
RSC Advances
Paper
40 C. F. Ding, M. L. Zhang, F. Zhao and S. S. Zhang, Anal. 46 R. A. Kamin and G. S. Wilson, Anal. Chem., 1980, 52, 1198–
Biochem., 2008, 378, 32–37. 1205.
41 W. Sun, Y. Q. Guo, X. M. Ju, Y. Y. Zhang, X. Z. Wang and 47 N. Maleki, A. Safavi, F. Sedaghati and F. Tajabadi, Anal.
Z. F. Sun, Biosens. Bioelectron., 2013, 42, 207–213. Biochem., 2007, 369, 149–153.
42 S. F. Wang, T. Chen, Z. L. Zhang, X. C. Shen, Z. X. Lu, 48 X. Chen, H. Yan, Z. Shi, Y. Feng, J. Li, Q. Lin and X. Wang,
D. W. Pang and K. Y. Wong, Langmuir, 2005, 21, 9260–9266.
43 E. Laviron, J. Electroanal. Chem., 1974, 52, 355–393.
44 E. Laviron, J. Electroanal. Chem., 1979, 101, 19–28.
Polym. Bull., 2017, 74, 75–90.
49 H. Yan, X. Chen, Z. Shi, Y. Feng, J. Li, Q. Lin, X. Wang and
W. Sun, J. Solid State Electrochem., 2016, 20, 1783–1792.
45 T. Zhan, M. Xi, Y. Wang, W. Sun and W. Hou, J. Colloid 50 W. Sun, X. Q. Li and K. Jiao, Electroanalysis, 2009, 21, 959–
Interface Sci., 2010, 346, 188–193. 964.
38012 | RSC Adv., 2018, 8, 38003–38012
This journal is © The Royal Society of Chemistry 2018