An electrochemical biosensing platform based on 1-formylpyrene functionalized reduced graphene oxide for sensitive determination of phenol

Article abstract of DOI:10.1039/c5ra27563f

A highly efficient enzyme-based screen printed electrode (SPE) was developed based on the covalent immobilization of tyrosinase (Tyr) on 1-formylpyrene (1-FP) functionalized reduced graphene oxide (rGO). Here, the bifunctional molecule 1-FP was assembled onto rGO sheets. Subsequently, a Tyr molecule was immobilized on the 1-FP forming a biocompatible nanocomposite, which was further coated onto the working electrode surface of the SPE. The performance of as-prepared biosensor was investigated by the detection of phenol in the presence of molecular oxygen. Wide linear range, low detection limit and high sensitivity were obtained with this biosensor due to the good conductivity of rGO as well as the high bioactivity of Tyr well retained by the 1-FP/rGO platform. Finally, the proposed biosensor was successfully employed for the detection of phenol in real water samples with satisfactory results. These findings suggest that this novel biosensor could offer great potential for rapid, cost-effective and on-field analysis of phenolic compounds.

Full text of DOI:10.1039/c5ra27563f

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PAPER
An electrochemical biosensing platform based on
1-formylpyrene functionalized reduced graphene
oxide for sensitive determination of phenol†
Cite this: RSC Adv., 2016, 6, 25427
*
Zulin Hua, Qin Qin, Xue Bai, Xin Huang and Qi Zhang
A highly efficient enzyme-based screen printed electrode (SPE) was developed based on the covalent
immobilization of tyrosinase (Tyr) on 1-formylpyrene (1-FP) functionalized reduced graphene oxide
(rGO). Here, the bifunctional molecule 1-FP was assembled onto rGO sheets. Subsequently, a Tyr
molecule was immobilized on the 1-FP forming a biocompatible nanocomposite, which was further
coated onto the working electrode surface of the SPE. The performance of as-prepared biosensor was
investigated by the detection of phenol in the presence of molecular oxygen. Wide linear range, low
detection limit and high sensitivity were obtained with this biosensor due to the good conductivity of
rGO as well as the high bioactivity of Tyr well retained by the 1-FP/rGO platform. Finally, the proposed
biosensor was successfully employed for the detection of phenol in real water samples with satisfactory
results. These findings suggest that this novel biosensor could offer great potential for rapid, cost-
effective and on-field analysis of phenolic compounds.
Received 23rd December 2015
Accepted 1st March 2016
DOI: 10.1039/c5ra27563f
www.rsc.org/advances
Tyrosinase (Tyr) is
a well-known multicopper-oxidase
1. Introduction
enzyme that could catalyze the oxidation of various phenolic
compounds with the concomitant reduction of oxygen.12,13
Thus, biosensors based on Tyr have been studied extensively for
phenol determination. The main problem in constructing such
an enzyme based biosensor is the effective immobilization of
enzyme molecules onto electrode surface.14–16 Various methods
have been reported for the immobilization of enzyme on suit-
able matrix, such as adsorption,17,18 covalent binding,19,20
entrapment21,22 and encapsulation.23,24 Among these methods,
covalent attachments to electrochemical materials have been
frequently used for enzyme immobilization due to the enzyme
stability as well as the high immobilizing efficiency. In most
studies of covalent immobilization, the key point is to introduce
functional groups onto the electrochemical materials which
could further interact with enzyme (usually the amino groups).
For example, Pang et al.25 used 1-aminopyrene to functionalize
carbon nanotubes (CNTs) via p–p interaction, then the func-
tionalized CNTs was linked to one aldehyde group of glutaral-
dehyde, leaving the other aldehyde group of glutaraldehyde
exible to interact with laccase. Wei et al.26 applied 1-pyr-
enebutanoic acid, succinimidyl ester (PASE) as the noncovalent
functionalization reagent adsorbing onto the graphene oxide
(GO) via p–p interaction, then Tyr was covalently bind with
PASE-functionalized GO by forming amide bond. However,
some of these procedures are relatively complicated, or require
the use of organic solvents, which would lead to relatively poor
stability and bioactivity of enzyme. Thus, searching for a simple
and reliable scheme to immobilize enzyme is of considerable
interest.
Phenol and its derivatives are widespread contaminants whose
sources are both natural and industrial. Phenol is massively
used in the production of plastics, fertilizer, paint, rubber,
adhesives, paper and soap.1,2 Due to its high toxicity and low
degradation, excess concentrations of phenol in the environ-
ment would cause environmental problems as well as human
disease.3 Therefore, sensitive detection of phenol is of great
importance for the protection of public health and the
environment.
Various approaches, such as high performance liquid chro-
matography (HPLC),4 UV-Vis spectrophotometry,5 ame ioni-
zation detection,6 etc. have been developed for the detection of
phenol. In fact, these methods are associated with certain
disadvantages, such as high cost, special instrumentation,
complicated sample pretreatment, unstable detection results,
and unsuitable on-site monitoring.7,8 For this reason, immense
efforts have been focused on the development of simple and
effective analytical methods for fast determination of phenol.
Amperometric biosensors based on enzymes have been proven
to be promising for this purpose due to their easy operation, fast
response and feasibility of miniaturization.9–11
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lake of
Ministry of Education, College of Environment, Hohai University, Nanjing 210098,
China. E-mail: baixue@hhu.edu.cn; Tel: +86 15951940543
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra27563f
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Screen printed electrodes (SPEs), for their especially low cost, 1-FP was identied by Fourier transform infrared spectroscopy
fast response and large-scale production, have been widely used (FTIR). The Tyr-1-FP/rGO composite was fully characterized by
in sensors.27,28 Regardless of these advantages, signal conduc- X-ray photoelectron spectroscopy (XPS) and electrochemical
tivity remains a weak point of SPE electrodes. To solve this impedance spectroscopy (EIS). The amperometric response of
problem, various materials such as carbon nanomaterials,29,30 the prepared enzyme electrode in the detection of phenol was
metal nanoparticles31,32 and polymers33,34 have been used to investigated. The results showed that the developed biosensor
modify SPEs. Graphene,35,36 a two-dimensional carbon atom exhibited good stability and high sensitivity for the detection of
monolayer, exhibits good mechanical strength, large specic phenol.
surface area, and excellent electronic properties, making it
a promising electrode modier. Besides, due to its unique
nanostructure of six-membered rings, various p-conjugative
structured materials, such as benzenes,37 pyrenes38 and heter-
2. Experimental
2.1. Reagents and materials
oaromatic compounds39 have been used to interact with gra-
phene through p–p stacking interaction, providing spacer
chains terminated with exible functional groups onto gra-
phene surface. Though graphene possesses those outstanding
characteristics, it suffers from very limited water-dispersing
ability,40,41 which would exert hurdles for the fabrication of
the enzyme-based electrodes. Thus, reduced graphene oxide
(rGO) is a much more attractive material for enzyme immobi-
lization due to its enhanced dispersion ability as well as good
electrical properties.42
1-Formylpyrene (1-FP) is a bifunctional molecule with a pyr-
enyl group and a aldehyde group. The pyrenyl group of 1-FP,
being highly aromatic in nature, can interact strongly with
graphene sheets via p–p stacking. By functionalization of gra-
phene with 1-FP, lots of aldehyde groups can be introduced
uniformly on the graphene surface and can be further used to
immobilize enzymes to construct biosensors. However, to the
best of our knowledge, there was no studies on the immobili-
zation of enzymes using 1-FP functionalized graphene by far.
In this paper, we applied 1-FP to functionalize rGO for Tyr
immobilization (Scheme 1). The obtained Tyr-1-FP/rGO bio-
composite was then coated on the SPE surface through a drop
casting method. The non-covalent functionalization of rGO with
Tyr (EC 1.14.18.1 from mushroom) and phenol were purchased
from Sigma-Aldrich (USA). 1-FP was bought from Aladdin
(Shanghai, China). GO was obtained from Nanjing XFNano
Materials Tech Co., Ltd. A 0.1 M phosphate buffer solution (PBS)
made from Na2HPO4 and NaH2PO4 was employed as the sup-
porting electrolyte. Absolute ethanol was used as rGO disper-
sant agent. Na2HPO4, NaH2PO4 and absolute ethanol were all
obtained from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China). Deionized water obtained with a Milli-Q
System (Billerica, MA, USA) was used for preparing all solu-
tions. All reagents were of analytical grade and were used as
received without further purication.
2.2. Apparatus and electrodes
All the electrochemical experiments were carried out using
a CHI 660E electrochemical workstation (Shanghai Chenhua
Instrument Co. Ltd, China). A three-electrode system, consist-
ing of the modied SPE (diameter, 3 mm) as the working elec-
trode, a platinum wire as the counter electrode, and a saturated
Ag/AgCl electrode as the reference electrode, was employed. The
FT-IR spectra of rGO, 1-FP and 1-FP/rGO were collected between
4000 and 500 cmꢀ1 (Mid infrared region) on a Nicolet Nexus
Avater 370 FTIR spectrophotometer (Nicolet, USA), with 256
Scheme 1 Schematic representation of assembly for Tyr-1-FP/rGO/SPE electrode.
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scans at a resolution of 4 cmꢀ1. XPS was obtained with an AXIS-
Ultra instrument from Kratos Analytical using monochromatic
Al Ka radiation (225 W, 15 mA, 15 kV) and low-energy electron
ooding for charge compensation. The EIS measurements were
performed in a 0.1 M KCl solution containing 5 mM K3[Fe(CN)6]
and K4[Fe(CN)6], the results were plotted in the form of Nyquist
plots with a frequency range from 0.01 Hz to 100k Hz.
2.3. Fabrication of Tyr-1-FP/rGO/SPE
Scheme 1 illustrates the schematic of the preparation of the Tyr-
1-FP/rGO/SPE biosensor. First of all, rGO was obtained by the
hydroiodic acid reduction of GO.43 10 mg rGO and 80 mg 1-FP
were added into 100 mL absolute ethanol and mixed under
ultrasonication for 2 h. Aer standing overnight at room
temperature, the mixture was centrifuged and washed with
ethanol for several times, and the resulted deposit was dried
Fig. 1 FTIR spectra of rGO (a), 1-FP (b) and 1-FP/rGO (c).
ꢁ
under 70 C to obtain 1-FP/rGO composites.
In the procedure of enzyme immobilization, 1-FP/rGO was
rst dispersed in 0.1 M pH 7.0 PBS with the aid of ultra-
sonication to give a 2 mg mLꢀ1 stable 1-FP/rGO suspension.
Aer that, 2 mg mLꢀ1 Tyr solution (dissolved in pH 7.0 PBS) was
mixed with 2 mg mLꢀ1 1-FP/rGO suspension (1 : 1, v/v) and
shaken for over 30 min to obtain a homogeneous 1-FP-Tyr/rGO
suspension.
For fabrication of the Tyr-1-FP/rGO/SPE electrode, 10 mL of
the Tyr-1-FP/rGO suspension was dropped onto the working
electrode surface of SPE and the modied electrode was allowed
to dry at 4 ꢁC overnight. For comparison, rGO/SPE and 1-FP/
rGO/SPE were fabricated with the similar procedures. All of
the modied electrodes were stored at 4 ꢁC in a refrigerator
before use.
of rGO and the 1-FP–rGO composites provide a promising
matrix for enzyme immobilization.
3.1.2. XPS of Tyr-1-FP/rGO. XPS measurement was con-
ducted here to conrm the preparation of Tyr-1-FP/rGO. As
expressed in Fig. 2, the existence of N 1s is denitely attributed
to Tyr, demonstrating that Tyr had been successfully linked to 1-
FP/rGO. Moreover, the –C]N at 399.8 eV is most likely due to
the condensation reaction between Tyr and 1-FP, which further
conrms the successful immobilization of Tyr.
Additionally, other XPS peaks of Tyr-1-FP/rGO were also
demonstrated in Fig. S1 (ESI†). The C 1s spectrum of the Tyr-1-
FP/rGO reveals that it consists of two main components origi-
nating from the C–O (ꢂ286.1 eV) and C]C/C–C (ꢂ284.6 eV)
groups and three minor components from C–H (ꢂ283.9 eV),
C]N (ꢂ287.0 eV) and O–C]O (ꢂ288.1 eV) groups. For
comparison purpose, the C 1s spectrum of the 1-FP/rGO was
presented in Fig. S2 (ESI†). As shown, the C]O bond exists
mainly due to aldehyde groups that was distributed uniformly
on the rGO surface. While aer interact with enzyme, the C]N
peak was observed instead of C]O, indicating the successful
condensation reaction between aldehyde groups of 1-FP/rGO
and amino groups of Tyr. Moreover, the Cu spectrum of the
Tyr-1-FP/rGO further conrms the successful immobilization of
enzyme although the Cu peaks aren't very high-resolution
3. Results and discussion
3.1. Physicochemical characterization
3.1.1. FT-IR of 1-FP functionalized rGO. In order to conrm
the successfully fabrication of 1-FP functionalized rGO. The
structures of the rGO, 1-FP and 1-FP/rGO lms were investigated
using FTIR (Fig. 1). In rGO (Fig. 1a), we observe a strong and
broad absorption at 3417 cmꢀ1 due to O–H stretching vibration.
Besides, the FTIR spectrum of rGO showed prominent absorp-
tion bands at 3417 cmꢀ1 O–H stretching vibration 1625 cmꢀ1
(for C]C stretching vibrations), 1075 cmꢀ1 (for C–O stretching
vibrations) and 2924, 2865 cmꢀ1 (for C–H stretching vibration).
Fig. 1b displays the spectrum of 1-FP, it can be seen that 1-FP
spectrum is consisted of prominent bands of aldehyde group
(2820, 2720 and 1703 cmꢀ1) and aromatic nucleus (1600, 1580,
1500, 1450 cmꢀ1). A series of peaks at 880, 840, 709 and 650
cmꢀ1 in the spectrum are attributed to the C–H out of plane
bending vibrations, and the peak at 3033 cmꢀ1 is ascribed to the
aromatic ring C–H stretching vibration. As shown in Fig. 1c, the
spectrum of 1-FP/rGO is nearly the same as that obtained from
the pure 1-FP, conrming the successful functionalization of
rGO with 1-FP via the p–p interaction between the pyrenyl
groups of 1-FP and the six-membered rings of rGO. Thus, the
Fig. 2 XPS spectrum of the Tyr-1-FP/rGO: high resolution scan for
aldehyde groups should be distributed uniformly on the surface N 1s.
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(Fig. S1D in ESI†), which maybe because the copper sites of electron transfer. The results of EIS analysis also support that
redox center in biomolecules is usually embedded deeply into the Tyr-1-FP/rGO had been successfully fabricated and modied
the large three dimensional structures of enzyme molecules.
3.1.3. Characterization of Tyr-1-FP/rGO/SPE. EIS was
applied to monitor the whole procedure in preparing Tyr-1-FP/
rGO modied electrodes, which could provide useful informa-
tion between each step and oen be used to study the surface
features of modied electrodes.44 A typical impedance spectrum
consists of a semicircle portion observed at a high frequency
range corresponding to the electron-transfer-limited process
and a linear portion at low frequencies representing the
diffusion-limited process. The electron transfer resistance (Rct)
at the electrode surface can be quantied using the diameter of
the semicircle. Fig. 3 presents the representative impedance
spectra of bare SPE, rGO/SPE, 1-FP/rGO/SPE and Tyr-1-FP/rGO/
SPE in 0.1 M KCl solution containing 5 mM [Fe(CN)6]3ꢀ/4ꢀ. To
understand clearly the electrical properties of the electrodes/
solution interfaces, the Randle's equivalent circuit (inset of
Fig. 3) was chosen to t the obtained impedance data.45 In the
Randle's circuit, Rct, W, Cdl and Rsol represent the charge
transfer resistance, the diffusion impedance, the interfacial
capacity and the electrolyte resistance, respectively. It was
assumed that Rct and W were both in parallel to Cdl. This
parallel combination of Rct and Cdl gives rise to a semicircle in
the complex plane plot of Z00 against Z0, and the semicircle
diameter equals the charge transfer resistance (Rct). This resis-
tance exhibits the electron transfer kinetics of the redox probe
at electrode interface. As shown in Fig. 3, a well-dened semi-
circle curve was observed with each modied electrode. The Rct
values of rGO/SPE (Fig. 3b), 1-FP/rGO/SPE (Fig. 3c) and Tyr-1-FP/
rGO/SPE (Fig. 3d) were much lower than that of bare the SPE
electrode (Fig. 3a), indicating that the introduction of rGO can
greatly improve the conductivity of electrode. Aer 1-FP stacked
onto rGO, a bigger semicircle at high frequencies was obtained,
indicating that the electron transfer of the redox probe was
obstructed. Moreover, an obvious increase in the interfacial
resistance was observed when Tyr was immobilized in 1-FP/
onto the SPE surface.
3.2. Optimization of preparation parameters
To optimize the mass ratio between 1-FP and rGO (R1-FP/rGO), 1-
FP/rGO composites with ve kinds of mass ratio (1/32, 1/16, 1/8,
1/4 and 1/2) were prepared in this paper. The electrochemical
performance of Tyr-1-FP/rGO/SPE electrodes with different
R1-FP/rGO values have been investigated by CV in O2-saturated
PBS buffer solution with 100 mM phenol, and the histograms
about the value of reduction peak current (Ipc) against R1-FP/rGO
are presented in Fig. 4A. As shown in Fig. 4A, signicant
increases of Ipc were observed from R1-FP/rGO ¼ 1/32 to 1/8, while
Ipc at R1-FP/rGO ¼ 1/8 is nearly as high as that at R1-FP/rGO ¼ 1/4
and 1/2. These results indicate that 1-FP is favorable for the
immobilization of Tyr onto rGO, and the six-membered rings of
rGO might have been stacked completely by 1-FP in the case of
R1-FP/rGO ¼ 1/8. Therefore, we choose 1/8 as the optimum value
of R1-FP/rGO in the experiments carried out in this paper.
To obtain the optimum amount of added enzyme, 1 mL of
various concentrations of Tyr was added into 1 mL of 2 mg
mLꢀ1 1-FP/rGO suspension, which was subsequently shaken for
over 30 min to obtain 1-FP-Tyr/rGO suspension. As shown in
Fig. 4B, the current response of phenol increased with
increasing concentration of Tyr and reached a maximum at 2
mg mLꢀ1, thereaer the current response decreased signi-
cantly. This could be presumed that the covalent bonding of Tyr
with 1-FP/rGO was saturated at 2 mg mLꢀ1, higher amount of
added enzyme leads to excess Tyr exists in the electrolyte solu-
tion. These free Tyr molecules would increase the hydropho-
bicity of the electrode surface and make the electron transfer
more difficult. Therefore, 2 mg mLꢀ1 Tyr was chosen for all
subsequent experiments.
3.3. Cyclic voltammetric behaviors of phenol
rGO, the increase of Rct might have been caused by the Fig. 5 displays the typical CVs of phenol in O2-saturated 0.1 M
hindrance of the macromolecular structure of Tyr to the PBS (pH 7.0) at SPE, rGO/SPE, 1-FP–rGO/SPE and Tyr-1-FP/rGO/
SPE. As demonstrated, there was no obvious redox peak in the
potential range of ꢀ0.6–0.6 V on the modied electrodes
without Tyr (Fig. 5a–c). However, a distinct reduction peak
ꢀ0.08 V was observed with Tyr-1-FP/rGO/SPE (Fig. 5d), which
was due to the reduction of o-quinone produced from the
enzymatic reaction on the electrode surface. Besides, the high
currents at the very negative potential is most possibly due to
oxygen reduction at the electrode surface.46
The electrocatalytic activity of Tyr-1-FP/rGO/SPE electrode
towards phenol is examined by CV analysis. Fig. 6A compares
the CVs of the Tyr-1-FP/rGO/SPE in the N2-saturated (curve a),
air-saturated (curve b) and O2-saturated (curve c) PBS buffer
solution with 50 mM phenol. It can be seen that no obvious
peaks was observed in the solution saturated with N2, while
Fig. 3 AC impedance spectrum of (a) bare SPE, (b) rGO/SPE, (c) 1-FP/
rGO/SPE and (d) Tyr-1-FP/rGO/SPE in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]
(1 : 1) containing 0.1 M KCl solution. The frequency range was from
a prominent cathodic wave at ꢀ0.08 V appeared in both air-
saturated and O2-saturated phenol solution, indicating
a typical electrocatalytic oxidation of phenols by Tyr occurs,
when oxygen accepts the electron that phenols lost at Tyr.
0.01 Hz to 100k Hz. The amplitude potential was 5 mV. Inset, the
Randle's equivalent circuit for EIS analysis.
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Fig. 4 Effect of the mass ratio of 1-FP to rGO (A) and Tyr concentration (B) on the reduction response of Tyr-1-FP/rGO/SPE electrode in O2-
saturated PBS (pH 7.0) with 100 mM phenol.
The effect of pH value on the current response of the
biosensor to 100 mM phenol was investigated with the pH range
from 5 to 8 and the results were shown in Fig. 6B. It can be seen
that the response current increased with the increase of pH
from 5.0 up to 7.0, and then it decreased at higher pH. The
maximum reduction peak was obtained at pH 7.0, which was in
good accordance with those reported by others.47–49 The
decrease in the response current at pH values greater than 7.0
may be due to the involvement of protons in the reduction
reaction of o-quinone, and at low pH the increase in ampero-
metric response with an increasing pH is attributed to the
increase of the enzyme activity.49,50 Therefore, in order to ach-
Fig. 5 CVs of (a) SPE, (b) rGO/SPE, (c) 1-FP/rGO/SPE and (d) Tyr-1-FP/
ieve high sensitivity, pH 7.0 was chosen for the determination of
phenols. In addition, with the increase of pH, the cathodic peak
potential shied negatively, and the linear relationship of the
rGO/SPE in O2-saturated 0.1 M PBS (pH 7.0) containing 50 mM phenol
at the scan rate of 100 mV sꢀ1
.
Fig. 6 (A) CVs of Tyr-1-FP/rGO/SPE in the (a–c) N2-, air- and O2-saturated PBS buffer solution (pH 7.0) containing 50 mM phenol at the scan rate
of 50 mV sꢀ1; (B) effect of pH value on the response of 100 mM phenol; (C) CVs of 50 mM phenol at different scan rates: 10, 30, 50, 70, 100, 130,
150, 170 and 200 mV sꢀ1, inset is plot of the peak current vs. scan rate; (D) the relationship of peak potential (Ep) vs. the logarithm of scan rate
(log v).
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reduction potential (Epc) as a function of pH was expressed as
Epc ¼ ꢀ0.0582pH + 0.3285 (r ¼ 0.9917, Epc in V). This slope value
of ꢀ58.2 mV pHꢀ1 is close to the theoretical value of ꢀ59 mV
pHꢀ1, indicating an equal number of protons and electrons
participating in the electrochemical process.
Fig. 6C shows the CVs of 50 mM phenol in PBS (pH 7.0) at the
Tyr-1-FP/rGO/SPE electrode with various scan rates ranging
from 10 mV sꢀ1 to 200 mV sꢀ1. It was found that the cathodic
peak current of phenol increased linearly with increasing scan
rate (inset of Fig. 6C), suggesting that the reduction of phenol at
Tyr-1-FP/rGO/SPE electrode is an irreversible surface-controlled
process. The relationship between the reduction peak potential
(Epc) and the logarithm of scan rate (log v) for the electro-
catalysis of phenol on the fabricated Tyr-1-FP/rGO/SPE is also
shown in Fig. 6D. As can be seen, log v is proportional to Epc
within the range of 10–200 mV sꢀ1, and the equation can be
expressed as: Epc ¼ ꢀ0.0709 log v ꢀ 0.2166 (r ¼ 0.9956, Epc in V,
v in mV sꢀ1). As for an adsorption-controlled and totally irre-
versible electrode process, the slope of the linear regression
equations is equal to ꢀ2.303RT/anF. Here, R is the thermody-
namic gas constant (8.314 J Kꢀ1 molꢀ1), F is the Faraday
constant (96 500 C molꢀ1), T represents temperature (298 K), n
is the electron transfer number and a is the charge transfer
coefficient. According to Laviron equation,45 an is calculated to
be 0.84. Generally, a is assumed to be 0.5 in totally irreversible
electrode process, so n is around 2. Because the number of
electrons and protons involved in the reduction process was
equal in this work, the electrochemical reduction of phenol at
the electrode is a two-electron and two-proton process. There-
fore, the electro-reduction mechanism of phenol at Tyr-1-FP/
rGO/SPE can be expressed as Scheme 2.
Fig. 7 DPV curves obtained at Tyr-1-FP/rGO/SPE in 0.1 M PBS (pH 7.0)
containing different concentrations of phenol: 0.5, 1, 5, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 125 and 150 mM (from bottom to top). Inset,
plot of current response vs. the concentration of phenol.
regression equation: I (mA) ¼ ꢀ0.1131CDA (mM) ꢀ 3.4312, R2 ¼
0.9975). Detection limit of this sensor was calculated to be 0.17
mM (S/N ¼ 3). This excellent performance of the proposed
electrode could be ascribed to the high electrocatalytic activity
of Tyr immobilized onto 1-FP/rGO surface. Table 1 compares
the detection of phenol by Tyr-1-FP/rGO/SPE with other elec-
trochemical sensors. It is evident that the Tyr-1-FP/rGO/SPE
biosensor exhibited superiority over some earlier reported
sensors towards sensitive determination of phenol. This excel-
lent performance of the proposed biosensor could be ascribed
to efficient immobilization of Tyr to 1-FP/rGO and outstanding
conductivity of rGO as well as the high bioactivity of Tyr retained
by 1-FP/rGO.
3.5. Reproducibility and stability of Tyr-1-FP/rGO/SPE
3.4. Electrochemical determination of phenol
The repeatability of the same Tyr-1-FP/rGO/SPE described above
Fig. 7 displays DPVs of phenol with different concentrations was evaluated by the detection of 20 mM phenol. A relative
from 0.5 to 150 mM obtained at the Tyr-1-FP/rGO/SPE biosensor. standard deviation (R.S.D.) value of 2.14% was obtained for ten
As can be seen, the cathodic peak current increases linearly with successive determinations, indicating that this electrode
increasing phenol concentration (inset of Fig. 7; linear possessed good repeatability. The fabrication reproducibility
Scheme 2 Schematic representation of the biosensor and its different parts, also the scheme depicts enzymatic reaction between Tyr and
phenol with electrochemical reduction of the quinone formed at the biosensor surface. Tyr-oxi: oxidized form of Tyr-and Tyr-red: reduced form
of Tyr.
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Table 1 Comparison of different sensors for the detection of DA
Sensor
Linear range (mM)
Detection limit (mM)
Sensitivity
References
PPO/PTS-PPY/GCPa
3.3–220.3
1.5–25
1–250
20–80
1–150
—
—
0.6
4.43
0.2
5
17.1 mA mMꢀ1
51
52
53
54
55
56
MWCNT/DTDAB/Tyr-NCPEb
Tyr/MgFe2O4–SiO2/CPEc
Layered PANI sheets electroded
Tyr-ZnO-BDNDe
2900 mA mMꢀ1
54.2 mA mMꢀ1
1485.3 mA mMꢀ1 cmꢀ2
287.1 mA mMꢀ1 cmꢀ2
155 mA mMꢀ1
Ni/Al LDH on Ptf
Tyr-1-FP/rGO/SPE
5–650
0.5–150
0.17
113.1 mA mmꢀ1, 1615.7 mA
mMꢀ1 cmꢀ2
This work
a
b
Tyrosinase/p-toluene sulfonate ion-doped polypyrrole/glassy carbon plate. Multiwall carbon nanotube/dimethylditetradecylammonium
c
bromide/tyrosinase-carbon paste electrode. Tyrosinase modied core–shell magnetic nanoparticles supported at a carbon paste electrode.
d
e
Layered polyaniline nanosheets electrode. Tyrosinase-biofunctional ZnO nanorod microarrays on the boron-doped nanocrystalline diamond
f
substrates. Ni/Al layered double hydroxide thin lms deposited on Pt electrodes.
was evaluated with ve electrodes prepared under the same indicating that the proposed Tyr-1-FP/rGO/SPE could be used
conditions. The R.S.D. was calculated to be 3.82%, which for the detection of phenol in real samples.
demonstrated the reliability of the fabrication procedure.
The long-term stability of the Tyr-1-FP/rGO/SPE electrode
4. Conclusions
was evaluated by measuring its response to 20 mM phenol for 2
ꢁ
months. When not in use, the electrode was stored at 4 C. Its
current response was tested every 3 days during the rst month.
Beyond this period, the experiment was carried out once per 10
days. The electrode remained about 85% of its initial response
aer 1 month, and decreased gradually to about 62% aer 2
months. Such a favorable stability could be attributed to the
effective covalent immobilization method.
In summary, we reported the rst use of 1-FP as a bifunctional
molecule in rGO functionalization and enzyme immobilization.
Using this method, we successfully fabricated Tyr-1-FP/rGO/SPE
biosensor. The biosensor provides a suitable microenvironment
for retaining bioactivity of Tyr due to the exible aldehyde chain
of 1-FP. The resulted biosensor exhibited good analytical
performance to phenol in terms of wide linear range, low
detection limit, high sensitivity and good stability. Practical
application in the determination of phenol in real waters was
also succeeded with satisfying results indicated that the
biosensor we prepared had a great potential for practical
application.
3.6. Analytical applications
To verify the actual feasibility of the proposed sensor, Tyr-1-FP/
rGO/SPE was applied to determine phenol added to natural
waters (Table 2). The percentage of the recovery values was
calculated by comparing the concentration obtained from the
proposed electrode with that obtained from HPLC. It was found
that the recoveries were in the range of 97.54% to 102.22%,
Acknowledgements
The authors gratefully acknowledge the support provided by
special fund of Specialized Research Fund for the Doctoral
Program of Higher Education (20130094120009), National
Natural Science Foundation of China (Grant No. 51308183,
51179052), Natural Science Foundation of Jiangsu Province of
China (Grant No. BK20130828), the Fundamental Research
Funds for the Central Universities (2013B32214, 2014B07414,
2015B36414), Jiangsu Province Ordinary University Graduate
Student Scientic Research Innovation Project (KYLX15_0475)
and A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions.
Table 2 Determination of added phenol in natural watersa
Water
sample
Added
(mM)
Found by
proposed sensor (mM)
Found by
HPLC (mM)
Recovery
(%)
A
B
C
D
10
50
100
10
50
100
10
50
100
10
50
100
9.92
49.81
98.73
10.01
50.87
100.11
9.79
51.09
99.89
10.03
48.98
98.97
10.17
50.14
100.28
10.14
49.92
101.23
10.02
49.98
98.25
10.24
49.62
99.12
97.54
99.34
98.45
98.72
101.90
98.89
97.70
102.22
101.67
97.95
98.71
99.85
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