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Highly sensitive and simultaneous detection of dopamine and uric acid at graphene nanoplatelet-modified fluorine-doped tin oxide electrode in the presence of ascorbic acid
Accepted Manuscript Highly sensitive and simultaneous detection of dopamine and uric acid at graphene nanoplatelet-modified fluorine-doped tin oxide electrode in the presence of ascorbic acid
Md. Mahbubur Rahman, Nasrin Siraj Lopa, Myung Jong Ju, JaeJoon Lee PII: DOI: Reference:
S1572-6657(17)30216-3 doi: 10.1016/j.jelechem.2017.03.038 JEAC 3203
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
28 September 2016 20 March 2017 21 March 2017
Please cite this article as: Md. Mahbubur Rahman, Nasrin Siraj Lopa, Myung Jong Ju, Jae-Joon Lee , Highly sensitive and simultaneous detection of dopamine and uric acid at graphene nanoplatelet-modified fluorine-doped tin oxide electrode in the presence of ascorbic acid. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jeac(2017), doi: 10.1016/j.jelechem.2017.03.038
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ACCEPTED MANUSCRIPT Highly Sensitive and Simultaneous Detection of Dopamine and Uric Acid at Graphene Nanoplatelet-Modified Fluorine-Doped Tin Oxide Electrode in the Presence of Ascorbic Acid
Nanotechnology Research Center and Department of Applied Life Science, College of Biomedical and Health
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Md. Mahbubur Rahmana, †, Nasrin Siraj Lopab, †, Myung Jong Juc, and Jae-Joon Leeb,*
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Department of Energy & Materials Engineering, Dongguk University, Seoul, 100-715, Korea
Ulsan National Institute of Science and Technology (UNIST), School of Energy and Chemical Engineering/Centre
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Science, Konkuk University, Chungju 380-701, Korea
†
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for Dimension-Controllable Organic Frameworks, 50, UNIST, Ulsan 689-798, Korea
Both authors contributed equally to this work
*Author to whom correspondence should be addressed: E-Mail: [email protected] (J.J. Lee); Tel.: +82-2-2260-4979
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ACCEPTED MANUSCRIPT Abstract We developed a graphene nanoplatelet-modified fluorine-doped tin oxide electrode (GNP/FTO) for the simultaneous detection of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA) and investigated the interaction mechanisms of DA, UA, and AA with GNPs
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considering their charging states at different pH values. Owing to the unique structure and
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properties originating from the oxygen and nitrogen functional groups at the edges, GNPs
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showed high electrocatalytic activity for the electrochemical oxidations of AA, DA, and UA with peak-to-peak potential separations (ΔEP) between AA-DA and DA-UA of ca. 0.23 and 0.17 V,
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respectively. These values are sufficiently high to allow the simultaneous detection of DA and
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UA without interference from AA. The highly sensitive and stable GNP/FTO sensor showed sensitivities of ca. 0.150.004 and 0.140.007 µA/µM, respectively, with detection limits of ca.
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0.220.009 and 0.280.009 µM, respectively, for DA and UA. The sensor could detect DA and
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UA concentrations in human serum samples with excellent recoveries.
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Keywords: Graphene nanoplatelet; fluorine-doped tin oxide; electrospray; dopamine; uric acid.
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ACCEPTED MANUSCRIPT 1. Introduction Dopamine (3,4-dihydroxyphenethylamine, DA) is one of the most essential catecholamine neurotransmitters in the mammalian central nervous system. It is produced in several areas of the brain and transmits signals between neurons to corroborate smooth muscle movement [1,2]. The
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normal level of DA in biological systems is 10 nM–1 µM and unbalanced DA concentration is
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strongly responsible for brain dysfunction and Parkinson’s disease [3,4]. Uric acid (UA), which
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is another neurotransmitter amine, normally coexists with DA in biological fluids in the concentration range of 207–444 µM [5]. Unbalanced UA concentration is responsible for several
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diseases such as kidney damage, gout, diabetes mellitus, and various malignant diseases. In
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addition, heart disease and related fatalities are responsible for elevated UA levels [6,7]. Hence, methodologies for the simultaneous detection of DA and UA have attracted significant interest
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from chemists. Ascorbic acid (AA) is considered the primary interference in the detection of DA
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and UA. The concentration of AA in the intracellular fluids of the central nervous system is in the range of 1–2 mM, which is several orders of magnitude higher than the concentrations of DA
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and UA [8]. Owing to the very low concentrations of DA and UA as well as the existence of AA
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in biological fluids, a highly sensitive and selective analytical method is necessary for the detection of DA and UA.
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Electrochemical detection methods are highly advantageous over other detection approaches including optical, colorimetric, and chromatographic methods from the points of view of selectivity, sensitivity, and simplicity [9,10]. The electrochemical oxidation potentials of AA, DA, and UA are very similar. Consequently, most of the conventional electrodes [e.g. glassy carbon electrode (GCE), Au, and Pt] cannot separate the oxidation signals of DA and UA from that of AA [11,12]. In addition, the product of DA oxidation catalyzes the oxidation of AA, 3
ACCEPTED MANUSCRIPT which contributes to the fouling of the electrode and decreases the selectivity and reproducibility of the detection method [13]. Thus, separating the electrochemical responses of DA and UA in the presence of high concentrations of AA with reasonably high sensitivity is one of the major goals in both electroanalytical and bioelectrochemical research. To achieve this goal, various
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nanomaterials including conducting polymers (CPs), graphenes (GPs), and nanocomposites have
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been examined as candidate materials for modifying the electrode surfaces to selectively
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distinguish between the oxidation signals of AA, DA, and UA [14-18].
Among these modifiers, GP, which is a one-atom-thick planar sheet of sp2-bonded carbon
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atoms, has attracted significant interest for the modification of conventional electrode surfaces to
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allow the sensing of different biological and clinically important compounds including DNA, cholesterol, and proteins [19-23]. This is owing to the excellent electronic properties of GP
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arising from the confinement of electrons in two-dimensions, in addition to its high surface area,
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and excellent chemical stability [19,22]. Additionally, GP has been utilized as a potential nanoscale building block material for the development of field-effect transistors, solar cells, and
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gas sensors [23-25]. Recently, various types of GPs including doped GPs, functionalized GPs,
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and GP nanocomposites have been utilized for the detection of DA and UA in the presence of AA [12,26]. Generally, most GP-based sensors can easily separate the oxidation signals of AA,
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DA, and UA, while others such as nitrogen-doped GPs, GP-oxides, and GP composites suppress the AA signal from those of DA and UA [26-28]. The signal separation and suppression properties along with the extent of sensitivity for AA, DA, and UA detection significantly depend on the chemical properties of the GP-based composites and the pH of the solutions used. This is attributed to the fact that DA (pKa1 = 8.87) and AA (pKa1 = 4.10)/UA (pKa1 = 5.7) are
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ACCEPTED MANUSCRIPT oppositely charged at pH 7.0 and the design of most of the GP-modified sensors relies on the electrostatic, H-bonding, and - stacking interactions among these compounds [29]. Recently, fluorine-doped tin oxide (FTO) has attracted significant interest over conventional electrode materials for the development of disposable sensors, owing to its wide electrochemical
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potential window, low cost, and high thermal stability [9]. In this report, we utilized graphene
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nanoplatelet (GNP)-modified FTO electrodes for the sensitive and simultaneous detection of DA
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and UA without interference from AA. By combining the advantageous features of GNPs with
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improved functionality (e.g. –COOH and N) at its edges, the GNP/FTO electrodes can easily separate the oxidation signals from AA, DA, and UA with different sensitivities at pH 7.0. The
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GNP/FTO sensors exhibit significantly high sensitivity towards detecting DA oxidation, owing to the electrostatic, H-bonding, -, and cation- interactions with the GNPs, while UA also
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shows reasonably high sensitivity, which is possibly due to the - and H-bonding interactions
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with the GNPs. Meanwhile, the sensitivity towards AA detection is significantly lower, owing to
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improved electrostatic repulsion with the negatively charged functional groups of GNPs. We also examined the interaction mechanisms of DA, UA, and AA with the GNPs at different pH values
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by considering their charging states and resonance structures. Various experimental parameters
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such as the amount of GNPs, pH, and scan rates were optimized to obtain high sensitivity and selectivity for the simultaneous detection of DA and UA.
2. Experimental section 2.1. Materials
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ACCEPTED MANUSCRIPT GNPs with particle diameter, thickness, and surface area < 2 m, few nanometers, and 500 m2/g, respectively, were purchased from XG-Science (C-500, USA). Around 5.5 and 1.5 % of oxygen and nitrogen functional groups were present at the edges of the GNPs, respectively. Double-distilled water obtained from a Milli-Q water purifying system (18 MΩ·cm) was used
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throughout the experiments. Dopamine hydrochloride, L-ascorbic acid, UA, disodium hydrogen
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phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), and 2-propanol were
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purchased from Sigma-Aldrich (St Louis, USA). Phosphate buffer solutions (PBS) of different
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pH were prepared from the stock solutions of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 and the pH of the PBS solutions were adjusted by adding HCl (0.1M) and NaOH (0.1M). All the experiments
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2.2. Instruments and measurements
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were carried out under ambient conditions.
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The electrochemical experiments were performed using a CHI 430A electrochemical workstation. A conventional three-electrode system was used, in which FTO (TEC-8, Pilkington,
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USA) or GNP/FTO electrode (exposed area ca. 0.32 cm2, defined by an O-ring), platinum wire,
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and Ag/AgCl (aq. saturated KCl) were used as working, counter, and reference electrodes, respectively. Differential pulse voltammograms (DPVs) were measured by scanning in an
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appropriate potential range with 100 mV/s pulse amplitude, 2 ms pulse width, and 1000 ms pulse period. The surface topography of the electrodes was investigated by atomic force microscopy (AFM, XE100, PSIA, Korea) in the non-contact mode.
2.3. Preparation of the GNP/FTO electrode
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ACCEPTED MANUSCRIPT GNPs (0.1 wt. %) were homogenously dispersed in a 2-propanol solution by using ultrasonication bath for 1 h. The dispersion was then deposited directly onto the FTO electrodes using an e-spray system (NanoNC, ESR200RD, Korea), as shown in Figure 1A. Briefly, the GNP solution was loaded in a plastic syringe equipped with a 30-gauge stainless steel-based
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hypodermic needle, which was connected to a high voltage power supply (ESN-HV30). A
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voltage of ca. 6.5 kV was applied between a metal orifice and the FTO substrate placed at a
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distance of ca. 0.7 cm. The GNPs were deposited onto the FTO electrode at a constant flow rate
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of 150 L/min with deposition times of ca. 1, 2, 3, 5, and 7 min; the as-prepared GNP/FTO
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electrodes were heat-treated at 80 ºC for 30 min under vacuum.
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3. Results and Discussions
3.1. AFM characterization and electrochemical behaviors of AA, DA, and UA at GNP/FTO
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electrodes.
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Figure 1B and 1C show the topographical AFM images of bare FTO and GNP/FTO electrodes (deposition time: 5 min). The images clearly showed the homogeneous distribution of
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GNPs on FTO and the enhancement of the geometric surface roughness. It was found that the
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actual surface area of the GNP/FTO electrode is varying from 27.90 to 140.70 m2 depends on the preparation condition, meanwhile the surface roughness is enhanced up to ca. 20 - 30% upon deposition of GNPs with respect to the measured geometric area of FTO. The effect of the amount of GNPs on the electrochemical responses of AA, DA, and UA was determined by varying the electrospraying time (Figure S1). The optimal deposition time for maximizing the electrocatalytic properties of the GNP films was 5 min and this deposition time was used for further experiments. Figure 2A and 2B show the cyclic voltammograms (CVs) and 7
ACCEPTED MANUSCRIPT DPV responses, respectively, for mixtures of AA, DA, and UA (1 mM each in PBS, pH 7.0) at bare FTO and GNP/FTO electrodes. Both the CV and DPV results clearly demonstrate that the bare FTO electrode exhibits a wide and low-sensitivity oxidation signal originating from DA. A peak at ca. 0.36 V was observed in the DPV response. This peak was confirmed in the CV
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results for the individual solutions of AA, DA, and UA in PBS (pH 7.0) (Figure S2A). In the
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case of the GNP/FTO electrode, the oxidation signals originating from AA, DA, and UA could
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be easily separated with peaks observed at ca. 0.10, 0.33, and 0.50 V, respectively. These peaks were further confirmed by the individual CV responses of AA, DA, and UA at the GNP/FTO
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electrode (Figure S2B). The oxidation peak-to-peak potential separations (ΔEpa) between AA-
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DA and DA-UA were ca. 0.23 and 0.17 V, respectively, which are sufficiently high to allow the simultaneous detection of DA and UA without interference from AA. The sensitivities towards
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DA and UA detection at the GNP/FTO sensor were significantly high, whereas the AA detection
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sensitivity was significantly low. The bare FTO and GNP/FTO electrodes did not show any redox peaks in PBS (pH 7.0) in the working potential range of AA, DA, and UA, which clearly
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confirmed the strong catalytic activity of GNPs towards the oxidation of AA, DA, and UA.
3.2.Effect of scan rate and pH
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The effect of scan rate on the redox behavior of DA and UA were studied on the GNP/FTO sensor. The oxidation peak currents (Ipa) for both DA and UA increased with increasing scan rate (Figure 3A) and were directly proportional to the square root of scan rate in the range of 10 to 500 mV/s with the same regression coefficient (R2) of ca. 0.99. This strongly suggests that the redox reactions of DA and UA at the GNP/FTO sensor are diffusion-controlled processes [14].
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ACCEPTED MANUSCRIPT Figure 3B shows CVs of solution mixtures containing DA and UA measured at the GNP/FTO sensor at different pH values ranging from 3.0 to 8.0. Owing to the different charging states of DA and UA at different pH values, the estimated formal potential [(Eº′= (Epa + Epc)/2)] of DA and UA shifted negatively with increase in pH. This clearly confirmed that protons
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participate in the electrochemical redox reactions and the oxidation peak currents reached
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maximum values at pH 7.0 and pH 3.0 for DA and UA, respectively. Considering that the physiological pH is ca. 7.0, the remaining detection measurements for DA and UA were carried
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out at pH 7.0. The slopes of the plots of Epa vs. pH for DA and UA were −510.001 and −470.001 mV/pH (Inset of Figure 3B), respectively, which are close to the theoretical value of
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−59 mV/pH predicted by the Nernst equation [30]. This clearly suggests that the same number of
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protons and electrons (two protons and electrons) are involved in the oxidation of DA and UA to
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dopaminequinone (DQ) and quinoid diimine (qdU), respectively, and reduction of DQ and qdU
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to DA and UA, respectively [31].
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3.3. Selective detection of DA and UA The selective detection of DA at the GNP/FTO sensor was examined using DPV method in
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the presence of AA (0.5 mM) and UA (100 µM) with varying concentrations of DA (1−100 µM) as shown in Figure 4A. The value of Ipa increased linearly with increase in the concentration of DA, while the peak currents and peak potentials of AA and UA remained nearly constant. The linear calibration plot of Ipa vs. [DA] shown in the inset in Figure 4A may be fit to the linear regression equation Ipa (µA) = (0.170.004) × [DA] (µM) + (4.480.12) (R2=0.99). This
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ACCEPTED MANUSCRIPT corresponds to a sensitivity and detection limit (S/N =3) of ca. 0.170.004 µA/µM and 0.180.003 µM, respectively. Figure 4B shows the DPV responses recorded at various concentrations of UA (1−100 µM) at the GNP/FTO sensor at constant concentrations of AA and DA (0.5 mM and 100 µM,
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respectively). The value of Ipa increased linearly with increase in the concentration of UA, while
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the peak currents and peak potentials of AA and DA remained almost constant. The inset in
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Figure 4B shows the calibration plot of Ipa vs. [UA], which corresponds to the following linear
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regression equation: Ipa (µA) = (0.170.008) × [UA] (µM) + (0.070.02) (R2=0.99). This corresponds to a sensitivity and detection limit (S/N=3) of ca. 0.170.008 µA/µM and
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0.230.005 µM, respectively.
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We also quantitatively determined the analytical performance of the bare FTO sensor for the
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detection of DA in the presence of AA and UA. Figure 4C shows the DPV responses of DA at various concentrations (100−1000 µM) in the presence of AA (0.5 mM) and UA (100 µM). The
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inset in Figure 4C shows a calibration plot of Ipa vs. [DA], which corresponds to a very low
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sensitivity of ca. 0.0220.08 µA/µM and high detection limit of ca. 68.180.09 µm.
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3.4. Simultaneous detection of DA and UA The excellent electrocatalytic activity of GNPs is also promising for the simultaneous detection of DA and UA in the presence of a high concentration of AA. Figure 4D shows that the Ipa of DA and UA at the GNP/FTO sensor increased with increasing concentrations of DA and UA at a constant AA concentration of 0.5 mM. The corresponding linear detection ranges for DA and UA were 30−100 µM and 10−100 µM, respectively, which correspond to the linear 10
ACCEPTED MANUSCRIPT regression equations Ipa (µA) = (0.150.004) × [DA] (µM) + (3.570.25) (R2 = 0.99) and Ipa (µA) = (0.140.007) × [UA] (µM) + (0.060.001)
(R2 = 0.98), respectively [inset in Figure
4D]. The detection limits for DA and UA were ca. 0.220.009 and 0.280.009 µM, whereas the sensitivities were ca. 0.150.004 and 0.140.007 µA/µM, respectively. These results
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demonstrate that individual or simultaneous detection of DA and UA at the GNP/FTO sensor can
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be achieved with high sensitivity. The low detection limits and wide dynamic ranges are
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attributed to the excellent electrocatalytic activity of GNPs towards the oxidation of DA and UA.
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By comparing with the results published so far in the literature (Table S1), it is clear that the analytical results obtained using the GNP/FTO sensor are better or comparable with the results
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reported for the simultaneous detection of DA and UA at different modified electrodes.
3.5. Stability, repeatability, reproducibility, and real sample analysis
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The stability of the GNP/FTO sensor, expressed in terms of relative standard deviation
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(RSD), was investigated by consecutive CV cycling in a solution mixture containing AA, DA, and UA (1 mM each). The RSD values were ca. 1.11, 1.25, and 1.57 % for AA, DA, and UA,
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respectively (Figure S3), clearly suggesting that the GNP/FTO sensor is highly stable.
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Furthermore, the peak current of AA, DA, and UA (1 mM each) at the GNP/FTO sensor remained within 95.7, 92.8, and 95.8%, respectively, of the initial values after storage for one week in PBS (pH 7.0) at 4C in a refrigerator. The GNP/FTO sensor exhibited excellent repeatability with the RSD of 1.52 and 1.48%, respectively, for DA and UA, obtained from the CV of five reparative measurements using single electrode. The sensor has shown high reproducibility with the RSD of 1.95 and 1.81 %, respectively, for DA and UA, for three independent measurement carried out in three different electrodes. Human serum sample was 11
ACCEPTED MANUSCRIPT selected as a real biological target for analysis by the standard addition method. All the serum samples were diluted 100 times with PBS (pH 7.0) before spiking with known concentrations of DA and UA. The DA and UA concentrations were quantified using external calibration plots (Figure 4D) and the results are summarized in Table 1 and Figure S4. The recoveries for DA and
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UA were in the range of ca. 99.66%–100.33% and 99.6%–100.6%, respectively, which clearly
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indicate the practicability, reliability, and excellent accuracy of the GNP/FTO sensor for the
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simultaneous detection of DA and UA.
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3.6. Proposed mechanism for the interaction of DA, UA, and AA at GNPs
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The interactions of DA, UA, and AA with different types of doped and/or functionalized GNPs can be categorized into two types: (i) aromatic - stacking interactions and (ii)
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electrostatic or chemical bonding interactions [27,32]. Figure 5A(a) shows a schematic
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illustration of the different types of interactions of DA with GNPs at pH 7.0. Both the - stacking interactions between the delocalized electrons of the benzene ring of DA and GNPs as
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well as the hydrogen bonding interactions between the –OH groups of DA and oxygen-
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containing functional groups (e.g. –COO-) and N of GNPs can occur to a significant extent
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[27,32]. These - stacking interactions induce facile electron transfer between the DA and GNPs, whereas the H-bonding interactions prompt facile oxidation of DA by weakening the O-H bond. This concurrently enhances the sensitivity towards DA oxidation [10,14]. On the other hand, at pH 7.0, DA exists in the protonated form (Figure 5B), therefore, cation- interactions between the ammonium cation (–NH3+) of DA and electrons of the benzene ring of GNPs can also occur [29,33]. Meanwhile, it is reported that the negative charges on the graphene oxide (GO) and carbon nanotubes (CNTs) significantly control the redox behaviors and charge transfer 12
ACCEPTED MANUSCRIPT kinetics of redox mediators (IrCl63-, [Fe(CN)6]4-, [Ru(NH3)6]3+ etc.) and it is highly favorable for the positively charged redox mediators due to the improved electrostatic attraction between the positively charged mediators and the negatively charged GO or CNT [34,35]. Thus, the positively charged DA at pH 7.0 may also experience electrostatic interactions between the –
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NH3+ cations of DA and –COO- of GNPs can [29,33]. The H-bonding, cation-, and electrostatic
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interactions between DA and GNPs strongly depend on the pH of the solution (i.e. the charging
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state of DA), which was verified by the DPV measurements for DA oxidation at different pH
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values (Figure 5C). It can be seen that the oxidation current originating from DA slightly decreased with decreasing pH from 7.0 to 3.0. This can be attributed to the fact that at low pH
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values, the oxidation of DA by releasing protons is difficult, owing to the effect of H+ ions. Meanwhile, at pH 9.0, the oxidation current of DA again slightly decreased compared to pH 7.0
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and 8.0, which might be due to the absence of cation- or electrostatic interactions, since at pH
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9.0 (pKa1 = 8.87), DA exists in the neutral form [29].
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Figure 5A(b) and 5A(c) present schematic illustrations of the interactions of UA and AA with GNPs, respectively. UA contains heterocyclic 5- and 6-membered aromatic rings with a low
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de-localized electron density, whereas AA contains only one heterocyclic 5-membered
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aromatic ring with an even lower de-localized electron density, which results in weaker - stacking interactions of AA with GNPs. At pH 7.0, UA and AA exist as urate (UA-) (>98%) and ascorbate (AA-) (> 99.99%), respectively, by releasing protons from the NH at position 9 (pKa1 = 5.7) and –OH at C4 (pKa1 = 4.10), respectively (Figure 5B) [36,37]. Therefore, high proportions of negatively charged UA- and AA- experience electrostatic repulsion with the negatively charged functional groups of GNPs as similarly observed in other reports between the 13
ACCEPTED MANUSCRIPT negatively charged redox mediators (IrCl63- and [Fe(CN)6]4-) and the negatively charged GO or CNTs [34,35]. Meanwhile, the negatively charged UA- and AA- stabilize, owing to the delocalization of the negative charge by resonance (Figure 5B) [38,39], which increases the net electron density in UA- compared to UA. On the other hand, the electron density of AA- is the
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same as that of AA. Thus, UA- might experience stronger - stacking interactions with the
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delocalized electrons of GNPs as well as H-bonding interactions between the NH of UA- at
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position 7 and the -COO- or N groups of GNPs. On the other hand, AA might experience H-
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bonding interactions between the –OH of AA- at C2 and the functional groups of GNPs or weaker - stacking interactions might occur between AA and the GNPs. Therefore, the signal
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sensitivity towards AA- is significantly lower than the sensitivity towards UA- at the GNP/FTO
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sensor. This result is consistent with many other published reports [14,26-28]. As the solution pH decreased from 7.0 to 3.0, the signal sensitivity towards the oxidations of UA and AA increased
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(Figure 5C). This is attributed to the fact that with decrease in the solution pH, the proportions of
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the neutral forms of UA and AA increase and their electron transfer kinetics are enhanced, owing to decreased electrostatic repulsion with the GNPs. Therefore, the H-bonding interactions with
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GNPs are more facile, resulting in the release of protons from UA and AA. On the other hand, at
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higher pH values (> 7.0), UA and AA are further ionized by releasing one additional proton from the NH group at position 7 (pKa2 = 10.3) and –OH at C2 (pKa2 = 11.8), respectively [36,37]. Therefore, at higher pH values, the fractions of neutral UA and AA eventually approach 0% and almost all the UA and AA exist as UA-/UA2- and AA-/AA2- [36,37], respectively. Consequently, their signals are eventually diminished, owing to enhanced electrostatic repulsion with the negative charge of GNPs. Thus, at pH 9.0 the oxidation signals originating from UA and AA are completely suppressed at the GNP/FTO sensor (Figure 5C). 14
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4. Conclusion A highly sensitive and simple electrochemical method for the simultaneous and quantitative detection of DA and UA without interference from high concentrations of AA was developed
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using a GNP-modified FTO electrode. The GNP/FTO sensor showed well-defined oxidation
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peaks originating from AA, DA, and UA. Excellent electrocatalytic activity of DA and UA at the GNP/FTO electrode allowed their simultaneous detection with sensitivities of 0.150.004 and
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0.140.007 µA/µM and detection limits of 0.220.009 and 0.280.009 µM, respectively. The sensors showed excellent stability and recoveries of DA and UA from human serum samples.
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The plausible interaction mechanisms of DA, UA, and AA with GNPs at various pH values and
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the variations in the signal sensitivities at the GNP/FTO electrode were discussed. Additionally,
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compared to other modifier-based sensors reported in the literature, the GNP/FTO sensor has a simpler and easier fabrication process with better or comparable sensitivities and detection limits,
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allowing the simultaneous detection of DA and UA. Moreover, the sensor proposed in this study may be fabricated at a low cost and may be easily used. Therefore, this methodology is
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promising for the large-scale production of disposable-type sensors.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research
Foundation
of
Korea
(NRF)
funded
by
the
Ministry
of
Education
(2015R1D1A1A01057380, NRF-2015M1A2A2054996, NRF-2016R1A2B2012061). This work was also supported by the Dongguk University Research Fund of 2016. 15
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Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at
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http://dx:doi.org/xx.xxxx/j.talanta.2016.xx.xxx.
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ACCEPTED MANUSCRIPT Figure Captions Figure 1: (A) Schematic illustration of the deposition of GNP thin films onto FTO substrates by the e-spray technique, (B) tapping-mode AFM images of bare FTO, and (C) GNP-modified FTO
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Figure 2: (A) CVs of the GNP/FTO electrode in PBS (pH 7.0) and the solution mixture
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composed of AA, DA, and UA (1 mM each) at bare FTO and GNP/FTO electrodes; scan rate: 100 mV/s. (B) DPVs of the GNP/FTO electrode in PBS (pH 7.0) and solution mixtures of AA,
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DA, and UA (1 mM each) at bare FTO and GNP/FTO electrodes.
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Figure 3: (A) CVs of solution mixtures composed of AA, DA, and UA (1 mM each) at the GNP/FTO electrode measured at different scan rates (; a → e: 10, 50, 100, 400, and 500 mV/s).
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Inset show plots of Ipa vs. 1/2 for DA and UA. (B) CVs of solution mixtures containing DA and
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Figure 4: DPV (after subtracted background signals) profiles of GNP/FTO sensors in PBS (pH 7.0) (A) containing fixed concentration of AA (0.5 mM) and UA (100 µM) with varying
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concentrations of DA (a → g: 1, 10, 20, 30, 40, 50, and 100 µM), (B) containing fixed concentration of AA (0.5 mM) and DA (100 µM), with varying concentrations of UA (a → g: 1, 10, 20, 30, 40, 75, and 100 µM), (C) DPV profiles of bare FTO electrode in PBS (pH 7.0) containing fixed concentrations of AA (0.5 mM) and UA (100 µM), and varying concentrations of DA (a → j: 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 µM), and (D) DPV profiles 21
ACCEPTED MANUSCRIPT of GNP/FTO electrodes in PBS (pH 7.0) containing 0.5 mM AA and varying concentrations of DA (a → h: 30, 40, 50, 60, 70, 80, 90, and 100 µM) and UA (a → h: 10, 20, 30, 40, 50, 60, 80, and 100 µM). Inset: plots of Ipa as functions of [DA] and [UA].
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Figure 5. (A) Schematic illustration of the interaction mechanisms of (a) DA, (b) UA, and (c)
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Table 1: Recovery of DA and UA from human serum samples DA in diluted Sample Used
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ACCEPTED MANUSCRIPT Highlights
FTO was modified with graphene nanoplatelets (GNPs) by the e-spray method.
Dopamine and uric acid were simultaneously detected with GNP/FTO without interference from ascorbic acid. Sensor allows low detection limits of 0.22 and 0.28 µM for dopamine and uric acid,
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Sensor shows good stability and recoveries from human serum samples.
The interaction mechanisms between dopamine, uric acid, and ascorbic acid with GNPs were
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Md. Mahbubur Rahman, Nasrin Siraj Lopa, Myung Jong Ju, JaeJoon Lee PII: DOI: Reference:
S1572-6657(17)30216-3 doi: 10.1016/j.jelechem.2017.03.038 JEAC 3203
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
28 September 2016 20 March 2017 21 March 2017
Please cite this article as: Md. Mahbubur Rahman, Nasrin Siraj Lopa, Myung Jong Ju, Jae-Joon Lee , Highly sensitive and simultaneous detection of dopamine and uric acid at graphene nanoplatelet-modified fluorine-doped tin oxide electrode in the presence of ascorbic acid. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jeac(2017), doi: 10.1016/j.jelechem.2017.03.038
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ACCEPTED MANUSCRIPT Highly Sensitive and Simultaneous Detection of Dopamine and Uric Acid at Graphene Nanoplatelet-Modified Fluorine-Doped Tin Oxide Electrode in the Presence of Ascorbic Acid
Nanotechnology Research Center and Department of Applied Life Science, College of Biomedical and Health
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Md. Mahbubur Rahmana, †, Nasrin Siraj Lopab, †, Myung Jong Juc, and Jae-Joon Leeb,*
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Department of Energy & Materials Engineering, Dongguk University, Seoul, 100-715, Korea
Ulsan National Institute of Science and Technology (UNIST), School of Energy and Chemical Engineering/Centre
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Science, Konkuk University, Chungju 380-701, Korea
†
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for Dimension-Controllable Organic Frameworks, 50, UNIST, Ulsan 689-798, Korea
Both authors contributed equally to this work
*Author to whom correspondence should be addressed: E-Mail: [email protected] (J.J. Lee); Tel.: +82-2-2260-4979
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ACCEPTED MANUSCRIPT Abstract We developed a graphene nanoplatelet-modified fluorine-doped tin oxide electrode (GNP/FTO) for the simultaneous detection of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA) and investigated the interaction mechanisms of DA, UA, and AA with GNPs
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considering their charging states at different pH values. Owing to the unique structure and
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properties originating from the oxygen and nitrogen functional groups at the edges, GNPs
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showed high electrocatalytic activity for the electrochemical oxidations of AA, DA, and UA with peak-to-peak potential separations (ΔEP) between AA-DA and DA-UA of ca. 0.23 and 0.17 V,
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respectively. These values are sufficiently high to allow the simultaneous detection of DA and
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UA without interference from AA. The highly sensitive and stable GNP/FTO sensor showed sensitivities of ca. 0.150.004 and 0.140.007 µA/µM, respectively, with detection limits of ca.
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0.220.009 and 0.280.009 µM, respectively, for DA and UA. The sensor could detect DA and
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UA concentrations in human serum samples with excellent recoveries.
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Keywords: Graphene nanoplatelet; fluorine-doped tin oxide; electrospray; dopamine; uric acid.
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ACCEPTED MANUSCRIPT 1. Introduction Dopamine (3,4-dihydroxyphenethylamine, DA) is one of the most essential catecholamine neurotransmitters in the mammalian central nervous system. It is produced in several areas of the brain and transmits signals between neurons to corroborate smooth muscle movement [1,2]. The
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normal level of DA in biological systems is 10 nM–1 µM and unbalanced DA concentration is
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strongly responsible for brain dysfunction and Parkinson’s disease [3,4]. Uric acid (UA), which
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is another neurotransmitter amine, normally coexists with DA in biological fluids in the concentration range of 207–444 µM [5]. Unbalanced UA concentration is responsible for several
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diseases such as kidney damage, gout, diabetes mellitus, and various malignant diseases. In
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addition, heart disease and related fatalities are responsible for elevated UA levels [6,7]. Hence, methodologies for the simultaneous detection of DA and UA have attracted significant interest
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from chemists. Ascorbic acid (AA) is considered the primary interference in the detection of DA
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and UA. The concentration of AA in the intracellular fluids of the central nervous system is in the range of 1–2 mM, which is several orders of magnitude higher than the concentrations of DA
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and UA [8]. Owing to the very low concentrations of DA and UA as well as the existence of AA
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in biological fluids, a highly sensitive and selective analytical method is necessary for the detection of DA and UA.
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Electrochemical detection methods are highly advantageous over other detection approaches including optical, colorimetric, and chromatographic methods from the points of view of selectivity, sensitivity, and simplicity [9,10]. The electrochemical oxidation potentials of AA, DA, and UA are very similar. Consequently, most of the conventional electrodes [e.g. glassy carbon electrode (GCE), Au, and Pt] cannot separate the oxidation signals of DA and UA from that of AA [11,12]. In addition, the product of DA oxidation catalyzes the oxidation of AA, 3
ACCEPTED MANUSCRIPT which contributes to the fouling of the electrode and decreases the selectivity and reproducibility of the detection method [13]. Thus, separating the electrochemical responses of DA and UA in the presence of high concentrations of AA with reasonably high sensitivity is one of the major goals in both electroanalytical and bioelectrochemical research. To achieve this goal, various
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nanomaterials including conducting polymers (CPs), graphenes (GPs), and nanocomposites have
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been examined as candidate materials for modifying the electrode surfaces to selectively
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distinguish between the oxidation signals of AA, DA, and UA [14-18].
Among these modifiers, GP, which is a one-atom-thick planar sheet of sp2-bonded carbon
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atoms, has attracted significant interest for the modification of conventional electrode surfaces to
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allow the sensing of different biological and clinically important compounds including DNA, cholesterol, and proteins [19-23]. This is owing to the excellent electronic properties of GP
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arising from the confinement of electrons in two-dimensions, in addition to its high surface area,
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and excellent chemical stability [19,22]. Additionally, GP has been utilized as a potential nanoscale building block material for the development of field-effect transistors, solar cells, and
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gas sensors [23-25]. Recently, various types of GPs including doped GPs, functionalized GPs,
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and GP nanocomposites have been utilized for the detection of DA and UA in the presence of AA [12,26]. Generally, most GP-based sensors can easily separate the oxidation signals of AA,
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DA, and UA, while others such as nitrogen-doped GPs, GP-oxides, and GP composites suppress the AA signal from those of DA and UA [26-28]. The signal separation and suppression properties along with the extent of sensitivity for AA, DA, and UA detection significantly depend on the chemical properties of the GP-based composites and the pH of the solutions used. This is attributed to the fact that DA (pKa1 = 8.87) and AA (pKa1 = 4.10)/UA (pKa1 = 5.7) are
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ACCEPTED MANUSCRIPT oppositely charged at pH 7.0 and the design of most of the GP-modified sensors relies on the electrostatic, H-bonding, and - stacking interactions among these compounds [29]. Recently, fluorine-doped tin oxide (FTO) has attracted significant interest over conventional electrode materials for the development of disposable sensors, owing to its wide electrochemical
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potential window, low cost, and high thermal stability [9]. In this report, we utilized graphene
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nanoplatelet (GNP)-modified FTO electrodes for the sensitive and simultaneous detection of DA
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and UA without interference from AA. By combining the advantageous features of GNPs with
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improved functionality (e.g. –COOH and N) at its edges, the GNP/FTO electrodes can easily separate the oxidation signals from AA, DA, and UA with different sensitivities at pH 7.0. The
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GNP/FTO sensors exhibit significantly high sensitivity towards detecting DA oxidation, owing to the electrostatic, H-bonding, -, and cation- interactions with the GNPs, while UA also
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shows reasonably high sensitivity, which is possibly due to the - and H-bonding interactions
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with the GNPs. Meanwhile, the sensitivity towards AA detection is significantly lower, owing to
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improved electrostatic repulsion with the negatively charged functional groups of GNPs. We also examined the interaction mechanisms of DA, UA, and AA with the GNPs at different pH values
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by considering their charging states and resonance structures. Various experimental parameters
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such as the amount of GNPs, pH, and scan rates were optimized to obtain high sensitivity and selectivity for the simultaneous detection of DA and UA.
2. Experimental section 2.1. Materials
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ACCEPTED MANUSCRIPT GNPs with particle diameter, thickness, and surface area < 2 m, few nanometers, and 500 m2/g, respectively, were purchased from XG-Science (C-500, USA). Around 5.5 and 1.5 % of oxygen and nitrogen functional groups were present at the edges of the GNPs, respectively. Double-distilled water obtained from a Milli-Q water purifying system (18 MΩ·cm) was used
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throughout the experiments. Dopamine hydrochloride, L-ascorbic acid, UA, disodium hydrogen
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phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), and 2-propanol were
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purchased from Sigma-Aldrich (St Louis, USA). Phosphate buffer solutions (PBS) of different
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pH were prepared from the stock solutions of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 and the pH of the PBS solutions were adjusted by adding HCl (0.1M) and NaOH (0.1M). All the experiments
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2.2. Instruments and measurements
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were carried out under ambient conditions.
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The electrochemical experiments were performed using a CHI 430A electrochemical workstation. A conventional three-electrode system was used, in which FTO (TEC-8, Pilkington,
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USA) or GNP/FTO electrode (exposed area ca. 0.32 cm2, defined by an O-ring), platinum wire,
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and Ag/AgCl (aq. saturated KCl) were used as working, counter, and reference electrodes, respectively. Differential pulse voltammograms (DPVs) were measured by scanning in an
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appropriate potential range with 100 mV/s pulse amplitude, 2 ms pulse width, and 1000 ms pulse period. The surface topography of the electrodes was investigated by atomic force microscopy (AFM, XE100, PSIA, Korea) in the non-contact mode.
2.3. Preparation of the GNP/FTO electrode
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hypodermic needle, which was connected to a high voltage power supply (ESN-HV30). A
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voltage of ca. 6.5 kV was applied between a metal orifice and the FTO substrate placed at a
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distance of ca. 0.7 cm. The GNPs were deposited onto the FTO electrode at a constant flow rate
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of 150 L/min with deposition times of ca. 1, 2, 3, 5, and 7 min; the as-prepared GNP/FTO
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electrodes were heat-treated at 80 ºC for 30 min under vacuum.
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3. Results and Discussions
3.1. AFM characterization and electrochemical behaviors of AA, DA, and UA at GNP/FTO
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electrodes.
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Figure 1B and 1C show the topographical AFM images of bare FTO and GNP/FTO electrodes (deposition time: 5 min). The images clearly showed the homogeneous distribution of
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GNPs on FTO and the enhancement of the geometric surface roughness. It was found that the
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actual surface area of the GNP/FTO electrode is varying from 27.90 to 140.70 m2 depends on the preparation condition, meanwhile the surface roughness is enhanced up to ca. 20 - 30% upon deposition of GNPs with respect to the measured geometric area of FTO. The effect of the amount of GNPs on the electrochemical responses of AA, DA, and UA was determined by varying the electrospraying time (Figure S1). The optimal deposition time for maximizing the electrocatalytic properties of the GNP films was 5 min and this deposition time was used for further experiments. Figure 2A and 2B show the cyclic voltammograms (CVs) and 7
ACCEPTED MANUSCRIPT DPV responses, respectively, for mixtures of AA, DA, and UA (1 mM each in PBS, pH 7.0) at bare FTO and GNP/FTO electrodes. Both the CV and DPV results clearly demonstrate that the bare FTO electrode exhibits a wide and low-sensitivity oxidation signal originating from DA. A peak at ca. 0.36 V was observed in the DPV response. This peak was confirmed in the CV
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results for the individual solutions of AA, DA, and UA in PBS (pH 7.0) (Figure S2A). In the
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case of the GNP/FTO electrode, the oxidation signals originating from AA, DA, and UA could
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be easily separated with peaks observed at ca. 0.10, 0.33, and 0.50 V, respectively. These peaks were further confirmed by the individual CV responses of AA, DA, and UA at the GNP/FTO
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electrode (Figure S2B). The oxidation peak-to-peak potential separations (ΔEpa) between AA-
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DA and DA-UA were ca. 0.23 and 0.17 V, respectively, which are sufficiently high to allow the simultaneous detection of DA and UA without interference from AA. The sensitivities towards
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DA and UA detection at the GNP/FTO sensor were significantly high, whereas the AA detection
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sensitivity was significantly low. The bare FTO and GNP/FTO electrodes did not show any redox peaks in PBS (pH 7.0) in the working potential range of AA, DA, and UA, which clearly
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confirmed the strong catalytic activity of GNPs towards the oxidation of AA, DA, and UA.
3.2.Effect of scan rate and pH
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The effect of scan rate on the redox behavior of DA and UA were studied on the GNP/FTO sensor. The oxidation peak currents (Ipa) for both DA and UA increased with increasing scan rate (Figure 3A) and were directly proportional to the square root of scan rate in the range of 10 to 500 mV/s with the same regression coefficient (R2) of ca. 0.99. This strongly suggests that the redox reactions of DA and UA at the GNP/FTO sensor are diffusion-controlled processes [14].
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ACCEPTED MANUSCRIPT Figure 3B shows CVs of solution mixtures containing DA and UA measured at the GNP/FTO sensor at different pH values ranging from 3.0 to 8.0. Owing to the different charging states of DA and UA at different pH values, the estimated formal potential [(Eº′= (Epa + Epc)/2)] of DA and UA shifted negatively with increase in pH. This clearly confirmed that protons
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participate in the electrochemical redox reactions and the oxidation peak currents reached
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maximum values at pH 7.0 and pH 3.0 for DA and UA, respectively. Considering that the physiological pH is ca. 7.0, the remaining detection measurements for DA and UA were carried
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out at pH 7.0. The slopes of the plots of Epa vs. pH for DA and UA were −510.001 and −470.001 mV/pH (Inset of Figure 3B), respectively, which are close to the theoretical value of
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−59 mV/pH predicted by the Nernst equation [30]. This clearly suggests that the same number of
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protons and electrons (two protons and electrons) are involved in the oxidation of DA and UA to
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dopaminequinone (DQ) and quinoid diimine (qdU), respectively, and reduction of DQ and qdU
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to DA and UA, respectively [31].
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3.3. Selective detection of DA and UA The selective detection of DA at the GNP/FTO sensor was examined using DPV method in
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the presence of AA (0.5 mM) and UA (100 µM) with varying concentrations of DA (1−100 µM) as shown in Figure 4A. The value of Ipa increased linearly with increase in the concentration of DA, while the peak currents and peak potentials of AA and UA remained nearly constant. The linear calibration plot of Ipa vs. [DA] shown in the inset in Figure 4A may be fit to the linear regression equation Ipa (µA) = (0.170.004) × [DA] (µM) + (4.480.12) (R2=0.99). This
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ACCEPTED MANUSCRIPT corresponds to a sensitivity and detection limit (S/N =3) of ca. 0.170.004 µA/µM and 0.180.003 µM, respectively. Figure 4B shows the DPV responses recorded at various concentrations of UA (1−100 µM) at the GNP/FTO sensor at constant concentrations of AA and DA (0.5 mM and 100 µM,
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respectively). The value of Ipa increased linearly with increase in the concentration of UA, while
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the peak currents and peak potentials of AA and DA remained almost constant. The inset in
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Figure 4B shows the calibration plot of Ipa vs. [UA], which corresponds to the following linear
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regression equation: Ipa (µA) = (0.170.008) × [UA] (µM) + (0.070.02) (R2=0.99). This corresponds to a sensitivity and detection limit (S/N=3) of ca. 0.170.008 µA/µM and
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0.230.005 µM, respectively.
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We also quantitatively determined the analytical performance of the bare FTO sensor for the
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detection of DA in the presence of AA and UA. Figure 4C shows the DPV responses of DA at various concentrations (100−1000 µM) in the presence of AA (0.5 mM) and UA (100 µM). The
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inset in Figure 4C shows a calibration plot of Ipa vs. [DA], which corresponds to a very low
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sensitivity of ca. 0.0220.08 µA/µM and high detection limit of ca. 68.180.09 µm.
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3.4. Simultaneous detection of DA and UA The excellent electrocatalytic activity of GNPs is also promising for the simultaneous detection of DA and UA in the presence of a high concentration of AA. Figure 4D shows that the Ipa of DA and UA at the GNP/FTO sensor increased with increasing concentrations of DA and UA at a constant AA concentration of 0.5 mM. The corresponding linear detection ranges for DA and UA were 30−100 µM and 10−100 µM, respectively, which correspond to the linear 10
ACCEPTED MANUSCRIPT regression equations Ipa (µA) = (0.150.004) × [DA] (µM) + (3.570.25) (R2 = 0.99) and Ipa (µA) = (0.140.007) × [UA] (µM) + (0.060.001)
(R2 = 0.98), respectively [inset in Figure
4D]. The detection limits for DA and UA were ca. 0.220.009 and 0.280.009 µM, whereas the sensitivities were ca. 0.150.004 and 0.140.007 µA/µM, respectively. These results
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demonstrate that individual or simultaneous detection of DA and UA at the GNP/FTO sensor can
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be achieved with high sensitivity. The low detection limits and wide dynamic ranges are
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attributed to the excellent electrocatalytic activity of GNPs towards the oxidation of DA and UA.
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By comparing with the results published so far in the literature (Table S1), it is clear that the analytical results obtained using the GNP/FTO sensor are better or comparable with the results
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reported for the simultaneous detection of DA and UA at different modified electrodes.
3.5. Stability, repeatability, reproducibility, and real sample analysis
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The stability of the GNP/FTO sensor, expressed in terms of relative standard deviation
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(RSD), was investigated by consecutive CV cycling in a solution mixture containing AA, DA, and UA (1 mM each). The RSD values were ca. 1.11, 1.25, and 1.57 % for AA, DA, and UA,
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respectively (Figure S3), clearly suggesting that the GNP/FTO sensor is highly stable.
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Furthermore, the peak current of AA, DA, and UA (1 mM each) at the GNP/FTO sensor remained within 95.7, 92.8, and 95.8%, respectively, of the initial values after storage for one week in PBS (pH 7.0) at 4C in a refrigerator. The GNP/FTO sensor exhibited excellent repeatability with the RSD of 1.52 and 1.48%, respectively, for DA and UA, obtained from the CV of five reparative measurements using single electrode. The sensor has shown high reproducibility with the RSD of 1.95 and 1.81 %, respectively, for DA and UA, for three independent measurement carried out in three different electrodes. Human serum sample was 11
ACCEPTED MANUSCRIPT selected as a real biological target for analysis by the standard addition method. All the serum samples were diluted 100 times with PBS (pH 7.0) before spiking with known concentrations of DA and UA. The DA and UA concentrations were quantified using external calibration plots (Figure 4D) and the results are summarized in Table 1 and Figure S4. The recoveries for DA and
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UA were in the range of ca. 99.66%–100.33% and 99.6%–100.6%, respectively, which clearly
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indicate the practicability, reliability, and excellent accuracy of the GNP/FTO sensor for the
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simultaneous detection of DA and UA.
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3.6. Proposed mechanism for the interaction of DA, UA, and AA at GNPs
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The interactions of DA, UA, and AA with different types of doped and/or functionalized GNPs can be categorized into two types: (i) aromatic - stacking interactions and (ii)
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electrostatic or chemical bonding interactions [27,32]. Figure 5A(a) shows a schematic
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illustration of the different types of interactions of DA with GNPs at pH 7.0. Both the - stacking interactions between the delocalized electrons of the benzene ring of DA and GNPs as
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well as the hydrogen bonding interactions between the –OH groups of DA and oxygen-
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containing functional groups (e.g. –COO-) and N of GNPs can occur to a significant extent
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[27,32]. These - stacking interactions induce facile electron transfer between the DA and GNPs, whereas the H-bonding interactions prompt facile oxidation of DA by weakening the O-H bond. This concurrently enhances the sensitivity towards DA oxidation [10,14]. On the other hand, at pH 7.0, DA exists in the protonated form (Figure 5B), therefore, cation- interactions between the ammonium cation (–NH3+) of DA and electrons of the benzene ring of GNPs can also occur [29,33]. Meanwhile, it is reported that the negative charges on the graphene oxide (GO) and carbon nanotubes (CNTs) significantly control the redox behaviors and charge transfer 12
ACCEPTED MANUSCRIPT kinetics of redox mediators (IrCl63-, [Fe(CN)6]4-, [Ru(NH3)6]3+ etc.) and it is highly favorable for the positively charged redox mediators due to the improved electrostatic attraction between the positively charged mediators and the negatively charged GO or CNT [34,35]. Thus, the positively charged DA at pH 7.0 may also experience electrostatic interactions between the –
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NH3+ cations of DA and –COO- of GNPs can [29,33]. The H-bonding, cation-, and electrostatic
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interactions between DA and GNPs strongly depend on the pH of the solution (i.e. the charging
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state of DA), which was verified by the DPV measurements for DA oxidation at different pH
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values (Figure 5C). It can be seen that the oxidation current originating from DA slightly decreased with decreasing pH from 7.0 to 3.0. This can be attributed to the fact that at low pH
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values, the oxidation of DA by releasing protons is difficult, owing to the effect of H+ ions. Meanwhile, at pH 9.0, the oxidation current of DA again slightly decreased compared to pH 7.0
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and 8.0, which might be due to the absence of cation- or electrostatic interactions, since at pH
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9.0 (pKa1 = 8.87), DA exists in the neutral form [29].
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Figure 5A(b) and 5A(c) present schematic illustrations of the interactions of UA and AA with GNPs, respectively. UA contains heterocyclic 5- and 6-membered aromatic rings with a low
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de-localized electron density, whereas AA contains only one heterocyclic 5-membered
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aromatic ring with an even lower de-localized electron density, which results in weaker - stacking interactions of AA with GNPs. At pH 7.0, UA and AA exist as urate (UA-) (>98%) and ascorbate (AA-) (> 99.99%), respectively, by releasing protons from the NH at position 9 (pKa1 = 5.7) and –OH at C4 (pKa1 = 4.10), respectively (Figure 5B) [36,37]. Therefore, high proportions of negatively charged UA- and AA- experience electrostatic repulsion with the negatively charged functional groups of GNPs as similarly observed in other reports between the 13
ACCEPTED MANUSCRIPT negatively charged redox mediators (IrCl63- and [Fe(CN)6]4-) and the negatively charged GO or CNTs [34,35]. Meanwhile, the negatively charged UA- and AA- stabilize, owing to the delocalization of the negative charge by resonance (Figure 5B) [38,39], which increases the net electron density in UA- compared to UA. On the other hand, the electron density of AA- is the
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same as that of AA. Thus, UA- might experience stronger - stacking interactions with the
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delocalized electrons of GNPs as well as H-bonding interactions between the NH of UA- at
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position 7 and the -COO- or N groups of GNPs. On the other hand, AA might experience H-
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bonding interactions between the –OH of AA- at C2 and the functional groups of GNPs or weaker - stacking interactions might occur between AA and the GNPs. Therefore, the signal
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sensitivity towards AA- is significantly lower than the sensitivity towards UA- at the GNP/FTO
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sensor. This result is consistent with many other published reports [14,26-28]. As the solution pH decreased from 7.0 to 3.0, the signal sensitivity towards the oxidations of UA and AA increased
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(Figure 5C). This is attributed to the fact that with decrease in the solution pH, the proportions of
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the neutral forms of UA and AA increase and their electron transfer kinetics are enhanced, owing to decreased electrostatic repulsion with the GNPs. Therefore, the H-bonding interactions with
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GNPs are more facile, resulting in the release of protons from UA and AA. On the other hand, at
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higher pH values (> 7.0), UA and AA are further ionized by releasing one additional proton from the NH group at position 7 (pKa2 = 10.3) and –OH at C2 (pKa2 = 11.8), respectively [36,37]. Therefore, at higher pH values, the fractions of neutral UA and AA eventually approach 0% and almost all the UA and AA exist as UA-/UA2- and AA-/AA2- [36,37], respectively. Consequently, their signals are eventually diminished, owing to enhanced electrostatic repulsion with the negative charge of GNPs. Thus, at pH 9.0 the oxidation signals originating from UA and AA are completely suppressed at the GNP/FTO sensor (Figure 5C). 14
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4. Conclusion A highly sensitive and simple electrochemical method for the simultaneous and quantitative detection of DA and UA without interference from high concentrations of AA was developed
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using a GNP-modified FTO electrode. The GNP/FTO sensor showed well-defined oxidation
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peaks originating from AA, DA, and UA. Excellent electrocatalytic activity of DA and UA at the GNP/FTO electrode allowed their simultaneous detection with sensitivities of 0.150.004 and
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0.140.007 µA/µM and detection limits of 0.220.009 and 0.280.009 µM, respectively. The sensors showed excellent stability and recoveries of DA and UA from human serum samples.
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The plausible interaction mechanisms of DA, UA, and AA with GNPs at various pH values and
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the variations in the signal sensitivities at the GNP/FTO electrode were discussed. Additionally,
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compared to other modifier-based sensors reported in the literature, the GNP/FTO sensor has a simpler and easier fabrication process with better or comparable sensitivities and detection limits,
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allowing the simultaneous detection of DA and UA. Moreover, the sensor proposed in this study may be fabricated at a low cost and may be easily used. Therefore, this methodology is
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promising for the large-scale production of disposable-type sensors.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research
Foundation
of
Korea
(NRF)
funded
by
the
Ministry
of
Education
(2015R1D1A1A01057380, NRF-2015M1A2A2054996, NRF-2016R1A2B2012061). This work was also supported by the Dongguk University Research Fund of 2016. 15
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Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at
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http://dx:doi.org/xx.xxxx/j.talanta.2016.xx.xxx.
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ACCEPTED MANUSCRIPT Figure Captions Figure 1: (A) Schematic illustration of the deposition of GNP thin films onto FTO substrates by the e-spray technique, (B) tapping-mode AFM images of bare FTO, and (C) GNP-modified FTO
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electrode.
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Figure 2: (A) CVs of the GNP/FTO electrode in PBS (pH 7.0) and the solution mixture
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composed of AA, DA, and UA (1 mM each) at bare FTO and GNP/FTO electrodes; scan rate: 100 mV/s. (B) DPVs of the GNP/FTO electrode in PBS (pH 7.0) and solution mixtures of AA,
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DA, and UA (1 mM each) at bare FTO and GNP/FTO electrodes.
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Figure 3: (A) CVs of solution mixtures composed of AA, DA, and UA (1 mM each) at the GNP/FTO electrode measured at different scan rates (; a → e: 10, 50, 100, 400, and 500 mV/s).
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Inset show plots of Ipa vs. 1/2 for DA and UA. (B) CVs of solution mixtures containing DA and
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UA (1 mM each) at GNP/FTO electrodes at different pH values (a → e: 3, 5, 6, 7, and 8); scan
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rate 100 mV/s. Inset show plots of Epa vs. pH.
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Figure 4: DPV (after subtracted background signals) profiles of GNP/FTO sensors in PBS (pH 7.0) (A) containing fixed concentration of AA (0.5 mM) and UA (100 µM) with varying
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concentrations of DA (a → g: 1, 10, 20, 30, 40, 50, and 100 µM), (B) containing fixed concentration of AA (0.5 mM) and DA (100 µM), with varying concentrations of UA (a → g: 1, 10, 20, 30, 40, 75, and 100 µM), (C) DPV profiles of bare FTO electrode in PBS (pH 7.0) containing fixed concentrations of AA (0.5 mM) and UA (100 µM), and varying concentrations of DA (a → j: 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 µM), and (D) DPV profiles 21
ACCEPTED MANUSCRIPT of GNP/FTO electrodes in PBS (pH 7.0) containing 0.5 mM AA and varying concentrations of DA (a → h: 30, 40, 50, 60, 70, 80, 90, and 100 µM) and UA (a → h: 10, 20, 30, 40, 50, 60, 80, and 100 µM). Inset: plots of Ipa as functions of [DA] and [UA].
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Figure 5. (A) Schematic illustration of the interaction mechanisms of (a) DA, (b) UA, and (c)
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AA with GNPs at pH 7.0, (B) chemical structures of DA, UA, and AA at pH 7.0, and (C) DPV
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responses for the oxidation of the solution mixture containing DA, UA, and AA (100 µM each)
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at the GNP/FTO electrode at different pH values.
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0.2
CR
GNPs/FTO in PBS Bare FTO in Mixture GNPs/FTO in Mixture
0.4
0.6
DA
(B)
80
UA
M
PT
20
AA
ED
I / A
60 40
GNPs/FTO in PBS Bare FTO in Mixture GNPs/FTO in Mixture
AN
100
US
E (V vs. Ag/AgCl)
0.8
DA
0
0.2
0.4
E (V vs. Ag/AgCl)
AC
CE
0.0
Figure 2: J.-J. Lee et al.
24
0.6
ACCEPTED MANUSCRIPT
400
e
(A)
300
a
200
0
70
-200
50 40
IP
Ipa / A
-100
30 20
-300 -0.2
0.0
0.2
5 10 15 20 Scan rate 1/2 / (mV/s) 1/2
0.4
0.6
400
a
e
AN
(B)
200
M
-200
PT
-300 0.0
ED
0
DA UA
0.6 0.5 0.4 0.3 3
0.4
4
0.6
E (V vs. Ag/AgCl)
AC
CE
0.2
0.7
Epa
I / A
100
-100
0.8
US
E (V vs. Ag/AgCl)
300
T
DA UA
60
CR
I / A
100
Figure 3: J.-J. Lee et al.
25
5
6 pH
0.8
7
8
1.0
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
Figure 4: J.-J. Lee et al.
26
ACCEPTED MANUSCRIPT (A)
(a)
(b)
-NH3+
(c) -
Weak -
H-bond
O electrostatic
O-
COO-
O-
COO-
COO-
O-
H-bond
H-bond
O
O
H-bond H-bond
HO
(C)
HO
NH2
HO
NH3
DA+
DA
O H N
NH
O N H O NH
O
At pH 7.0
NH
O N H
N H
N H
O
N
UA-
O
UA
NH
O
OH HO HO
O
O
OH
O
OH O
O
OH
pH 5.0
0.2
0.4
E (V vs. Ag/AgCl)
AA-
CE
PT
AA
pH 7.0
pH 3.0
0.0
HO
HO O
O
O HO
O HO At pH 7.0
N
ED
N H
pH 8.0
M
O H N
T
20 A
O
O H N
pH 9.0
AN
H N
N
dAA
US
At pH 7.0
HO
IP
qdU DQ
(B)
AA
UA
DA
CR
H-bond
AC
Figure 5: J.-J. Lee et al.
27
0.6
ACCEPTED MANUSCRIPT
Table 1: Recovery of DA and UA from human serum samples DA in diluted Sample Used
Added (M)
Found (M)
Recovery (%)
0
30
30.1
100.5
0
30
30.0
0
30
IP
101.5
Added (M)
Found (M)
Recovery (%)
0
30
30.2
100.6
0
30
30.1
100.3
0
30
29.9
99.6
29.9
US
UA in diluted samples (M)
AC
CE
PT
ED
M
Urine samples
AN
Sample Used
99
CR
Urine samples
T
samples (M)
28
ACCEPTED MANUSCRIPT Graphical Abstract
UA DA
qdU
DQ
T
dAA
e-
e-
AN
US
e-
CR
IP
AA
GNPs/FTO
AC
CE
PT
ED
M
AA = ascorbic acid ; dAA = dehydroascorbic acid; DA = dopamine; DQ = dopamine-o-quinone; UA = Uric acid; qdU = quinoid diimine
29
ACCEPTED MANUSCRIPT Highlights
FTO was modified with graphene nanoplatelets (GNPs) by the e-spray method.
Dopamine and uric acid were simultaneously detected with GNP/FTO without interference from ascorbic acid. Sensor allows low detection limits of 0.22 and 0.28 µM for dopamine and uric acid,
T
IP
respectively.
Sensor shows good stability and recoveries from human serum samples.
The interaction mechanisms between dopamine, uric acid, and ascorbic acid with GNPs were
US
CR
AC
CE
PT
ED
M
AN
discussed and verified.
30