A highly sensitive electrode modi fied with graphene, gold nanoparticles,and molecularly imprinted over-oxidized polypyrrole for electrochemical determination of dopamine Do Phuc Tuyena,
Trang 1A highly sensitive electrode modi fied with graphene, gold nanoparticles,
and molecularly imprinted over-oxidized polypyrrole for
electrochemical determination of dopamine
Do Phuc Tuyena, Do Phuc Quana,⁎ , Nguyen Hai Binhb, Van Chuc Nguyenb, Tran Dai Lamb,⁎⁎ , Le Trong Huyenc,
a
Research Centre for Environmental Technology and Sustainable Development, Hanoi University of Science, 334, NguyenTrai Road, Hanoi, Viet Nam
b
Institute of Materials Science, Vietnam Academy of Science and Technology, 18, Hoang Quoc Viet Road, Hanoi, Viet Nam
c
School of Chemical Engineering, Hanoi University of Science and Technology, 1, Dai Co Viet Road, Hanoi, Viet Nam
d National Institute of Hygiene and Epidemiology, 1, Yersin Street, Hanoi, Viet Nam
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 7 March 2014
Received in revised form 20 July 2014
Accepted 21 July 2014
Available online xxxx
Keywords:
Dopamine
Over-oxidized polypyrrole
Graphene
Gold nanoparticles
A glassy carbon electrode (GCE), modified with graphene (Gr) films, molecularly imprinted (MIP) over-oxidized polypyrrole (OPPy), and electrochemically de posited gold nanoparticles (AuNP) was used for rapid detection of dopamine (DA) The as-prepared electrode (AuNP/Gr/OPPy-MIP/GCE) was characterized by Raman spectroscopy and atomic force microscopy (AFM) Electrochemical determination of DA was conducted via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) The results showed that the modified electrodes exhibited high electrocatalytic activity towards the oxidation of DA in 0.1 M phosphate buffer solution (pH 6.9) Linear depen-dency of electrocatalytic oxidation current on DA concentrations was observed from 0.5μM to 8 μM and detection limit was estimated to be 0.1μM (S/N = 3) The developed electrode could serve as a viable platform for further studies on in vivo detection of DA
© 2014 Elsevier B.V All rights reserved
1 Introduction
Dopamine (DA) is a neurotransmitter which belongs to the family of
the catecholamine and phenethylamine Dopamine plays an important
role in the functions of the central nervous, cardiovascular, renal and
hormonal systems and Parkinson's disease[1] Several techniques
have been developed for DA determination[1,2], but their sensitivity
and selectivity are still not satisfactory Generally, the performances of
these methods are adversely affected by interferences of other active
biomolecules such as ascorbic acid (AA) and uric acid (UA) which
co-exist in biologicalfluids Recently, electrochemistry-based analyses
have been attempted for determination of DA Nevertheless, these
approaches are hindered by the similar electrochemical behaviors of
DA, AA and UA[3] For this reason, research challenges are to obtain
clear separation of the electrochemical signals of these three
com-pounds The concept of modified electrodes using different materials
is an exciting development in electroanalytical chemistry Combination
of various materials such as organic redox mediators, nanoparticles,
polymers, self-assembled monolayers and graphene have been
employed in the modification of electrodes for DA determination [4–11] Among these materials, graphene (Gr) has attracted consid-erable attention due to high electrical conductivity, surface-to-volume ratio, electron transfer rate and exceptional thermal stability[12] The unique and superior properties of Gr have been extensively exploited for the fabrication of electrochemical sensors and biosensors[13] Gold nanoparticle (AuNP) is another material which has been widely used in electrochemical analyses due to its excellent catalytic activity, mass transport and high effective surface area[14,15]
The technology of molecular/ionic imprinting in networked polymers has provided a new generation of recognition materials, capable of competing with biological specific receptors[16] Molec-ularly imprinted polymer (MIP) is formed by the copolymerization
of functional monomers and crosslinkers in the presence of a target molecule The extraction of molecular template creates a comple-mentary cavity with chemical affinity for the original molecule Mechanistically, this is similar to antigen–antibody or ligand–recep-tor interactions of key-lock recognition Polypyrrole (PPy) is one of the conducting polymers which has many attractive features as a molecular recognition system[17,18] PPy can be over-oxidized at high positive potential and when the doping ions are expelled, PPy lost its conductivity due to introduction of oxygen-containing groups into its backbone[19,20] Such over-oxidation may result
Journal of Molecular Liquids xxx (2014) xxx–xxx
⁎ Corresponding author Tel.: +84 4 38588152; fax: +84 438587964.
⁎⁎ Corresponding author Tel.: +84 4 37564129; fax: +84 438360705.
E-mail addresses: doquan@vnu.edu.vn (P.Q Do), lamtd@ims.vast.ac.vn (D.L Tran).
http://dx.doi.org/10.1016/j.molliq.2014.07.029
0167-7322/© 2014 Elsevier B.V All rights reserved.
Contents lists available atScienceDirect
Journal of Molecular Liquids
j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / m o l l i q
Trang 2in carbonyl and carboxylic groups being incorporated into the PPy
backbone which may attract electropositive groups (\NH3+) of DA
and repel anionic molecules like AA Consequently, it can be
expect-ed that OPPyfilm obtained by MIP technique has better cation
exchange and molecular sieve properties as compared to
conven-tional PPy This would enhance selectivity and sensitivity of the
ma-terial towards DA detection The decreased conductivity of OPPy
modified electrodes is to be compensated by the combination of
AuNP and Gr layers which possibly helps improve the sensitivity of
the electrochemical sensor for DA detection The objectives of this
study were a) to investigate a new design of the glassy carbon
elec-trode modified by graphene, gold nanoparticles, and particularly
molecularly imprinted overoxidized polypyrrole and b) to apply
the as-prepared electrode for electrochemical determination of
do-pamine using cyclic voltammetry (CV) and differential pulse
volt-ammetry (DPV)
2 Experimental
2.1 Chemicals and reagents
Pyrrole (Py) was purchased from Fluka (Switzerland) Ascorbic
acid (AA), dopamine hydrochloride (DA.HCl), uric acid (UA) and
other chemicals were purchased from Sigma (Germany) and used
without further purification All other reagents were of analytical
reagent grade All solutions were de-aerated with purified nitrogen
prior to use
2.2 Apparatus and measurement
Electropolymerization of PPy and electrochemical measurements
were performed using Autolab PGSTAT-30 Potentiostat/Galvanostat
with GPES software (EcoChemie, The Netherland) The three-electrode
system was employed in which reference electrode was based on Ag/
AgCl/saturated KCl; auxiliary electrode was Pt wire and working
elec-trodes were either bared GCE or modified GCE Electrochemical
detection for DA was performed in 0.1 M phosphate buffer solution
(PBS, pH 6.9) by using CV and DPV techniques The DPV parameters
were 50 mV pulse amplitude, a scan rate of 50 mV/s and the potential
range from−0.2 to 0.5 V All aqueous solutions were prepared with DI
water of 18 MΩ·cm resistivity Prior to the electropolymerization of
PPy-MIP, the surface of the GCE was polished with 15μm and 0.3 μm
alumina slurry and cleaned by sonication in DI water
2.3 Preparation of modified electrodes
2.3.1 Preparation of Gr/GCE
Graphenefilms (Gr) were prepared by CVD method at growth
tem-perature of 1000 °C in Ar environment The Grfilm was then transferred
to glassy carbon electrode by chemical etching (Fig 1) Briefly, a thin
layer of polymethyl methacrylate (PMMA) was pre-coated on top of
ob-tained Grfilms on Cu tapes Subsequently, the samples were annealed at
180 °C in air for 1 min The Gr/PMMAfilms were released from the Cu
tapes by chemical etching of the underlying Cu in Fe(NO3)3solution
Then, suspendedfilms were transferred to deionized water to
re-move the residuals of Cu etching stages Next, Gr/PMMAfilms
were transferred onto electrode surfaces Gr transfer process was
improved by using a second PMMA coating step after the Gr/
PMMA being placed on the substrate[21] Finally, the PMMAfilms
were dissolved by acetone and the samples were cleaned by rinsing
several times in deionized water The crystalline quality and
thick-ness of Gr layers were characterized by Raman spectroscopy and
atomic force microscopy, respectively
2.3.2 Preparation of AuNP/Gr/OPPy-MIP/GCE DA-imprinted PPy was electrochemically synthesized on Gr/GCE in aqueous solution consisting of 1 mM DA, 10 mM Py and 0.1 M KCl using cyclic voltammetry The polymerization was performed infive cy-cles with potential ranging from 0.0 to 0.80 V, versus Ag/AgCl (sat KCl) and scan rate of 10 mV/s The template molecules DA were removed by immersing the MIP-PPy in 95% ethanol and DI water for 60 min The over-oxidation of DA-imprinted PPyfilms was conducted using ten cy-cles of cyclic voltammetry from 0.0 to 1.0 V (vs Ag/AgCl), at a scan rate
of 10 mV/s in 0.5 M KOH Gold nanoparticles (AuNP) were electrochem-ically deposited on the OPPy surface using 10 potential scans from 0.0 to 1.1 V in 2.5 mM HAuCl4and 0.1 M KNO3solution at a scan rate of 50 mV/s The preparation of AuNP/Gr/OPPy-MIP/GCE electrodes, involving four main steps (except Gr transfer onto GCE) is shown inFig 2
3 Results and discussion 3.1 Characterization of modified electrodes The Grfilms were characterized with Raman spectroscopy, a powerful method to determine the crystalline quality of Gr layers (Fig 3)
It was evidenced that I2D/IGdepended on the number of Gr layers (monolayer: the ratio I2D/IG~ 2–3; bilayer: 1 b I2D/IGb 2; multilayer:
I2D/IGb 1)[12,13] In this study, the ratio I2D/IGobtained was 0.44, indi-cating that the Grfilm grown on the Cu tape had multilayered structure Raman spectra (632.8 nm excitation laser) of Gr transferred on the elec-trode exhibit three peaks at ~ 1333, ~ 1582 and ~2660 cm−1, in which the latter is assigned to 2D characteristic peak of Gr It is worth mention-ing that the intensity of the D-band at∼1333 cm−1, an important
Cu substrate
Graphene/Cu
PMMA/Graphene/Cu
Etching Cu
Removing PMMA
Electrochemical sensor
Fig 1 Schematic representation of Gr transfer process onto GCE.
Trang 3indicator of defects in the Gr, is quite low, demonstrating the successful
transfer of Gr onto the given electrode surface One of the main
drawbacks of the Gr transfer process is the incomplete contact between
Gr and substrate surface The unattached regions tend to break easily
when the PMMA coating is dissolved When an appropriate amount of
liquid PMMA was dropped on the cured PMMA layer, dissolution of
the pre-coated PMMA was expected to minimize formed cracks/defects
As a result, the contact of Gr with the electrode surface was improved
[12,13,20] AFM image shows that the thickness of the Grfilm is about 5–6 nm (Fig 4) which meant that the grown Grfilm consists of about 15 layers
DA-imprinted PPy was synthesized by electrochemical polymeriza-tion (Fig 5A) The affinity of template molecules DA and OPPy network were previously ascribed to interactions of hydrogen bonds[16,17,19] The over-oxidation of DA-imprinted PPyfilms is illustrated inFig 5B During the over-oxidation of the OPPyfilm, the formation of carbonyl and carboxylic groups could provide a better permselectivity for DA with the electrostatic force[19] The CVs describing deposition of AuNP on OPPy surface are given inFig 5C The cathodic peak at ~0.3 V exhibits the deposition of AuNP onto the electrode surface The voltammetric response of AuNP-modified electrode in 50 mM
K4Fe(CN)6and 0.1 M KCl solution presented the strongest reversible redox peak current of ferrocyanide/ferricyanide couple after 10 cycles
It suggests that AuNP electrodeposited onto the OPPy surface rendered OPPy better conductive On the other hand, compared with the Gr/GCE, the values ofΔEpdecreased significantly at the modified AuNP/Gr/ OPPy-MIP/GCE (from 155 to 84 mV) This downshifting demonstrated that the introduction of AuNP in AuNP/Gr/OPPy-MIP/GCE played an im-portant role in providing the conducting bridges for the electron trans-fer of trans-ferrocyanide/trans-ferricyanide couple, leading to enhanced electron transfer kinetics of AuNP/Gr/OPPy-MIP/GCE as compared to the Gr/GCE
3.2 Electrochemical detection of DA Cyclic voltammograms of AuNP/Gr/OPPy-MIP/GCE in 0.1 M PBS containing DA were subjected to various scan rates and were shown inFig 6A Both the values of anodic peak current (Ipa) and cathodic peak current (Ipc) exhibited linear relationship with scan rate over the range of 10–125 mV/s The linear regression equations,
Ipa(μA) = − 5.53 + 2.3v1/2(mV/s)1/2(R = 0.975) and Ipc(μA) = + 4.77− 1.7v1/2(mV/s)1/2(R = 0.981) were obtained (inset in Fig 6A) These results imply that the electrocatalytic reaction is dif-fusion controlled, which is ideal for quantitative analysis in practical applications
The typical DPV calibration curve at the AuNP/Gr/OPPy-MIP/GCE for DA detection in the concentration range from 0.54 to 8μM is shown inFig 6B The current response of the modified electrode showed a linear dependence on DA concentration with ipa(μA) =
− 0.16 + 1.76 × CDA(μM), (R = 0.990) It is well known that DA,
AA and UA can be oxidized at practically the same potential at bare electrodes, resulting in the peak overlapping as well as poor
Fig 3 Raman spectra of the synthesized Gr films.
Fig 2 Schematic fabrication process of AuNP/Gr/OPPy-MIP/GCE and its recognition for DA.
Trang 4response resolution in DA determination Therefore, in this study,
UA and AA were chosen as the interfering molecules to evaluate
the DA selectivity of the electrodes The CVs of 0.01 mM DA at the
Gr/OPPy-MIP/GCE and AuNP/Gr/OPPy-MIP/GCE in the presence of
1 mM AA and 50μM UA were studied (Fig 7A) There were only
electrochemical signals of DA and UA in the cyclic voltammograms
at 0.135 V and 0.281 V (vs Ag/AgCl), respectively The oxidation peaks of DA and UA were clearly separated from each other for about 146 mV, signifying that anions such as AA can be rejected by OPPy-MIPfilms The CV results indicated that OPPy-MIP was selective
-20 0 20 40 60 80 100
120
PPy(DA)
E / V vs Ag/AgCl
E / V vs Ag/AgCl
E / V vs Ag/AgCl
E / V vs Ag/AgCl
D C
Py(DA) oxidation
0 20 40 60 80 100 120 140
1st cycle
2nd - 10th cycles
-200
-100
0 100
200
300
400
500
Au3+
1st cycle
2nd cycle
3rd cycle
Au0
Au3+ Au0
-40 -20 0 20 40
60
Gr/GCE AuNP/Gr/OPPy-MIP/GCE
Fig 5 (A) Electrochemical preparation of DA-imprinted PPy by CVs in Py(DA) solution (B) Over-oxidation of DA-imprinted PPy by CVs in 0.5 M KOH solution (C) Electrochemical depo-sition of AuNP on OPPy film (D) CVs of 50 mM K 4 Fe(CN) 6 and 0.1 M KCl at the Gr/GCE (black curve), and AuNP/Gr/OPPy-MIP/GCE (red curve), respectively The scan rate is 50 mV/s.
Fig 4 The AFM image of the Gr films.
Trang 5for DA in the presence of AA and UA Furthermore, the current responses
of the AuNP/Gr/OPPy-MIP/GCE were much better than those of the Gr/
OPPy-MIP/GCE, which confirmed the role of AuNP in the overall
perfor-mance of the modified AuNP/Gr/OPPy-MIP/GCE electrodes DPV
re-sponses of electrochemical signals to different DA concentrations in
the presence of 1 mM AA and 50μM UA are demonstrated inFig 7B
While Ipaincreased linearly with varying concentration of DA, no
obvi-ous changes in the peak current of UA were observed Therefore, with
the modified electrode AuNP/Gr/OPPy-MIP/GCE, interferences of AA
and UA during DA determination were minimized
In order to examine the matrix effect in biologicalfluid samples
such as rabbit serum and urine, the standard addition method has
been used The rabbit serum and urine samples were diluted with
0.1 M PBS solution (ratio 1:1) and were analyzed for DA
determina-tion using AuNP/Gr/OPPy-MIP/GCE The obtained results,
summa-rized inTable 1, clearly indicated that DPV method with AuNP/Gr/
OPPy-MIP/GCE was reliable for determining DA in the real sample
matrix
The selectivity of developed electrode was also studied by
compar-ing the electrochemical responses of DA-imprinted PPy-MIP modified
electrodes with those of non-imprinted polymer (PPy-NIP) modi
fica-tion The current responses of the AuNP/Gr/OPPy-MIP/GCE were
con-siderably higher than those of the AuNP/Gr/OPPy-NIP/GCE Thus,
imprinting effect produced in the presence of DA helped impart
excel-lent selective recognition capacity of AuNP/Gr/OPPy-MIP/GCE towards
the template molecule (Fig 8)
4 Conclusion
In this study, DA-imprinted modified electrodes of AuNP/Gr/OPPy-MIP/GCE have been successfully produced and characterized The elec-trochemical behaviors of the above electrodes were fully investigated The prepared electrode showed a good recognition capacity for tem-plate molecule (DA) in the presence of other structurally similar mole-cules (AA, UA) A linear relationship between the DA concentration (from 0.5μM to 8 μM) and the current response was obtained with good reproducibility and a low detection limit of 10−7M
Acknowledgments This work was supported by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant no 104.07.108.09
-10 -8 -6 -4 -2 0 2 4 6
8
UA
E / V vs Ag/AgCl
AuNP/Gr/OPPy-MIP/GCE Gr/OPPy-MIP/GCE
DA
A
6 8 10
14 12
16
18
3.5 M DA
50 M UA
E / V vs Ag/AgCl
B
2.0 M DA
Fig 7 (A) CVs for 1 mM AA, 50μM UA and 10 μM DA, in 0.1 M PBS at AuNP/Gr/OPPy/GCE The scan rates is 50 mV/s (B) DPVs of mixture solution of different concentrations (2.0, 3.5 and 5.0 μM) of DA, 1 mM AA, 50 μM UA in 0.1 M PBS at AuNP/Gr/OPPy-MIP/GCE.
Table 1 Determination of DA in rabbit serum and urine samples (n = 5).
Sample DA added
(μM)
Found (μM)
R.S.D (%)
Recovery (%) Rabbit serum 3.0 2.818 4.2 93.9
5.0 4.492 4.8 89.8 Rabbit urine 3.0 3.114 1.9 103.8
5.0 4.881 2.1 97.6 Note: R.S.D: relative standard deviation
-20
-10
0
10
20
30
30 mV/s
50 mV/s
70 mV/s
90 mV/s
-10 0 10 20
v1/2 / (mV/s) 1/2
E /V vs Ag/AgCl
A
3
6
9
12
15
18
21
4.70 M
2.60 M
1.10 M
0 1 2 3 4 5 6 7 8 9 0
4 8 12 16
CDA / M
8,00 M
E / V vs Ag/AgCl
B
0.54 M
Fig 6 (A) Electrochemical response of 10 μM DA in 0.1 M PBS at AuNP/Gr/OPPy-MIP/GCE
with different scan rates (10, 30, 50, 70, 90 and 125 mV/s) Inset: I pa -v 1/2
and I pc -v 1/2
plots.
(B) DPVs of mixture solution of different concentration of DA in 0.1 M PBS at AuNP/Gr/
OPPy-MIP/GCE Inset: The linear regression curve of peak current vs DA concentration.
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0
2
4
6
8
10
AuNP/Gr/OPPy-NIP/GCE AuNP/Gr/OPPy-MIP/GCE
Electrodes
5 M DA
50 M UA
Fig 8 Comparison of the DPV peak currents for AuNP/Gr/OPPy-MIP/GCE and AuNP/Gr/
OPPy-NIP/GCE.