The Determination of Dopamine in Presence of Serotonin on Dopamine-Functionalized Electrochemically Prepared Graphene Biosensor

Reported herein is the synthesis of electrochemically prepared graphene with dopamine (DA) functionalization and its application for the electrochemical detection of DA in presence of serotonin (5-HT). Graphene oxide-DA (GO-DA) resulted from the condensation reaction of GO and DA. It was characterized via several instrumental methods. The electrochemical reduction of GO-DA (ERGO- DA) has been employed for the electrochemical detection of DA without and with 5-HT. The electrochemical detection of DA has been veriﬁed through cyclic voltammetry, differential pulse voltammetry, and amperometric techniques in a 0.1 M phosphate buffer saline (PBS, pH 7.4). The inﬂuence of the accumulation time and pH of the PBS at ERGO-DA modiﬁed electrode for DA oxidation has been investigated. The interference has also been performed using interfering substances such as 5-HT, glucose, ascorbic acid, H 2 O 2 , and uric acid. The detection limit and linear range of DA have been calculated as 0.04 μ M and 0.5–100 μ M, respectively, (where s/n = 3) at the ERGO-DA modiﬁed electrode. The real sample analysis has done without any difﬁculty and this biosensor can be used up to 10 days.

Graphene oxide (GO) can be electrochemically reduced by applying a more negative potential, which can reduce the oxygen functional groups present on its surface. [1][2][3] The electrochemically reduced GO (ERGO) sheets were found to be more conductive than those prepared through chemical reduction in electrocatalysis of dopamine (DA), and was found as the highly pure by using electrochemical approach while this method is green, fast, and the result could be contamination free from the reduced material. 1 Due to its high electrical conductivity, large surface area, long-term stability and unique mechanical flexibility, 4-6 graphene has already been used in various fields, especially in sensors, 2,7,8 capacitor 9 and electrochemical catalysis. [10][11][12][13] DA, the most important among the classes of catecholamines, plays an important role in the brain system. It is responsible for the functions of the central nervous, renal, hormonal, and cardiovascular systems. 14 The serotonin (5-HT) is also an important bimolecular in physiological systems, playing a vital role in the regulation of mood, sleep, and appetite. In the past, dopamine was also employed as a reducing agent through the oxidation of catechol groups to the quinone form. 15 Recently, Kaminska et al. successfully used dopamine derivatives to reduce and noncovalently functionalize GO, 16 implying that DA or its derivatives are good reducing reagents for GO. It is, however, the GO contains epoxy and carboxyl functional groups along with the other oxygenated groups 17,18 which providing a number of chemically active sites for addition with amine group of DA via a condensation reaction. 13 This approach cannot only prevent the agglomeration of graphene due to the bigger space between two sheets for DA molecules 19 but also can further improve the electrocatalytic activity due to surface functionalization. 17 Thus, it is promising to utilize DA to reduce GO, and then to incorporate it via condensation reaction, because of the advantages of DA addition including the non-toxic property, restraining agglomeration, and covalent functionalization of DA-GO. [20][21][22][23] Chemically modified electrodes based electrochemical analysis has been proven as a sensitive and selective method for the determination of DA. 24,25 For example, DA has been detected using carbon nanotube (CNT) modified electrodes, 26 graphene-modified electrodes, 27 DA self-polymerization, 28 enzyme electrodes, 29 and nitrogen-doped porous carbon nanopolyhedra. 30 To improve the oxidation current of DA, metal-decorated GO used modified electrodes, Au rod and nanoparticles (NPs), 31-34 reduced GO-CNT, 35 PdNPs-modified GO, 36 and various metal (Co, SnO 2 , Pt) NPs. [37][38][39] Also, research interest has focused on the development of electrodes for simultaneous electrochemical detection of DA and z E-mail: swjeon3380@naver.com 5-HT in the biological system. It is, however, there are few reports in the literature about the simultaneous detection of DA and 5-HT by use of electrochemical methods. [40][41][42] So far, for the first time we are reporting on DA functionalized ERGO for the determination of DA in presence of 5-HT.
In this paper, the synthesized GO-DA is characterized via Fourier transform-infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). ERGO-DA has been employed for the electrochemical detection of DA in the presence of 5-HT. The potential and current of DA oxidation have been verified via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometric techniques in a 0.1 M phosphate buffer saline (PBS, pH 7.4). The influence of the accumulation time, pH of PBS and interference has also been investigated at ERGO-DA electrode for DA oxidation.

Experimental
Materials and chemicals.-The graphite powder (∼325 mesh, 99.999%), 5-HT, DA, glucose, ascorbic acid (AA), H 2 O 2 , and uric acid (UA) were from Aldrich. The N,N-dimethylformamide (DMF) and ammonia solution were purchased from Daejung Chemicals & Metals. The toluene, chloroform, and hexane were purchased from OCI Co., Ltd. All the other reagents were of analytical grade and were used without further purification. A PBS was prepared with 0.1 M NaH 2 PO 4 , and its pH was adjusted with 0.1 M NaOH and 0.1 M H 3 PO 4 . Doubly distilled water was used in the preparation of the aqueous electrolyte solutions.
Characterization.-The FT-IR spectrum was observed at room temperature, on a Nicolet 6700 double-beam infrared (IR) spectrometer (Thermo Fisher Scientific, USA). The UV-Vis spectrum was observed with Sinco 3100. XPS was performed using a VG Multilab 2000 spectrometer (ThermoVG Scientific, Southend on Sea, Essex, UK) in an ultra-high vacuum chamber. Survey scan data were collected at the pass energy of 50 eV. The surface morphologies of the materials were measured via finite element scanning electron microscopy (FE-SEM), with a MIRA 3 LMU (TESCAN, Czech Republic). A three-electrode assembled cell was employed, consisting of a modified GCE (3.0 mm in diameter) as the working electrode, and a platinum wire electrode was used as an auxiliary electrode. An Ag/AgCl (3 M NaCl) electrode supplied by BAS (Model MF-2052) was used as the reference electrode. All the potentials were reported with respect to the Ag/AgCl electrode at room temperature, under an argon atmosphere. Electrochemical impedance spectroscopy (EIS) was performed with a Versa State 3, manufactured by Metek, USA. The electrochemical technique, including CV, was performed using a CHI electrochemical workstation (CH Instruments, Inc., USA) in a grounded Faraday cage. The pH measurements were conducted using a pH glass electrode with a JENCO meter.
Synthesis of GO-DA.-GO was obtained by oxidizing graphite using the improved Hummers method. 38,43 Briefly, a mixture of concentrated H 2 SO 4 :H 3 PO 4 (360:40 mL) was added to a mixture of graphite:KMnO 4 (3:18 g) at 50 • C, and resulting mixture was stirred for 12 h. The reaction was cooled to room temperature and was transferred onto ice with 30% H 2 O 2 (3 mL). The obtained solution was centrifuged and then filtered. The solid material was then washed with water and 30% HCl, and finally washed with 200 mL ethanol. The procedure of the synthesis of GO-DA is shown in Scheme 1. In this synthesis procedure, GO (30.0 mg) was loaded into a 100 mL three-neck flask equipped with a magnetic stirring bar, and 15.0 mL DMF was then added to create a homogenous suspension. Then DA (3.5 mM), AA (10 mM), and H 2 O 2 (7.0 mM) were added, and the mixture was stirred at room temperature for 24 h. The product was then sonicated, centrifuged, and washed several times with methanol to remove the unreacted DA. Finally, the resulting product was dried at 60 • C under vacuum conditions for 24 h.
Fabrication of ERGO-DA/GCE.-The GCE surfaces were polished with alumina paste and were then washed with distilled water and rinsed with methanol. The GO-DA suspension in H 2 O (1 mg/mL) was sonicated in a water bath for 1 h, and then 5.0 μL of it was used to coat the GCE surfaces. And the electrode dried room temperature. The GO-DA-coated GCE was electrochemically reduced through 30 successive cyclic voltammograms in an electrochemical cell containing a 0.05 M PBS (pH 5) over a potential window of 0 to -1.5 V, at 50 mV s -1 scan rate. 1 The resulting ERGO-DA/GCE was washed with distilled water before and after each experiment. All the experiments were carried out in an argon atmosphere at room temperature.

Results and Discussion
Characterization of GO-DA.-The GO-DA reacted with the carboxyl groups of GO and the amino group of DA through a condensation reaction. To understand the covalent grafting DA into GO surface the FT-IR has been employed first in Fig. 1A. Fig. 1A shows the FT-IR spectra of GO, DA, and GO-DA. The most characteristic feature of the FT-IR spectrum of GO is the absorption bands corresponding to the C=C sp 2 carbon stretching at 1610 cm -1 and the C=O carbonyl group stretching at 1720 cm −1 . The 1038, 1235 and 1415 cm -1 band corresponds to the C-O, epoxy and C-OH. 17,44 The DA spectrum also have those peaks such as, C=C at 1610 cm -1 , C-OH at 1415 cm -1 , N-H stretching at 3333 cm -1 and C-N at 1235 cm -1 . 45 At the GO-DA spectrum, however, the peaks at 1235 and 3333 cm -1 caused by the C-N and N-H stretching vibration, respectively, indicate that the amine group in DA is bonded to the graphene surface during the reaction process. The peak intensity of C-OH and C-O at 1038 and 1415 cm -1 , respectively, were increased due to addition of DA. Moreover, the peak intensity of C=O (1720 cm −1 ) GO-DA compared to GO was significantly decreased, that implying the partial reduction of GO via covalent functionalization.
The UV-Vis absorption spectra of GO, DA, and GO-DA in ethanol are shown in Fig. 1B. The UV-Vis absorption of GO display an absorption peak centered at 228 nm, corresponding to the π-π * transitions of the aromatic C-C bonds. 46 DA exhibits a typical absorption band at 202 and 288 nm. 47 When DA was introduced into GO, the peak at 202 nm shifted to 222 nm with higher intensity, which is a characteristic peak of GO-DA. 20 These red shifts can be attributed to the smaller amount of energy required for the n-π * and π-π * transitions due to the covalent linkage between DA and GO. The GO nanosheets were partially reduced, and the graphitic structure within the GO was partially restored. 17,48 The XPS spectra of ERGO and ERGO-DA are shown in Fig. 2. The C1s, O1s, and N1s peaks were located at around 285, 583, and 400 eV, respectively, 45,49 on ERGO-DA while N1s peak was not detected on ERGO ( Fig. 2A). High-resolution C1s XPS spectra were used to characterize the removal of the oxygen groups and the formation of chemical bonds on the surfaces of ERGO and ERGO-DA (Fig. 2B). As for ERGO, two different peaks centered at C-C/ C=C in graphite carbon (286 eV) and C-O/C=O in the epoxide group (287.3 eV) were observed, respectively. 49 The C 1s XPS spectra of the ERGO-DA peaks were centered at C-C/C=C (286 eV) and C-O/C=O/C-N (287.3 eV) respectively. Moreover, the intensity of a characteristic peak at 287.3 eV was significantly higher compared to ERGO in ERGO-DA, suggesting the C-N bond was influenced in here while DA was grafted onto GO. The N1s spectrum exhibited one peak at about 400 eV, corresponding to the C-N bonds for the ERGO-DA in Fig. 2C. A few signs of the C-N bonds in ERGO-DA can be seen because DA was a chemical bonded with GO.
The surface morphologies of ERGO and ERGO-DA are shown in Fig. 3. All the SEM images were observed on a GCE plate. The 5 μL of    50 The reaction impedance of the electrodes was determined as follows (Table I): bare ERGO-DA < GO-DA < Bare GCE. Also, the double layer capacity of the electrodes were determined as follows: bare ERGO-DA (9.53 × 10 −6 F) < GO-DA (2.40 × 10 −5 F) < Bare GCE (1.14 × 10 −2 F). It was concluded that the electrical conductivity of ERGO-DA was enhanced due to the effect of the electrochemical reduction.
The effect of the accumulation time of ERGO-DA/GCE in 0.1 M PBS containing 100 μM DA at a 100 mV s -1 scan rate was investigated (Fig. 4). The peak current of DA detection increased quickly at 2-150s (Fig. 4A); but after 150s the peak current did not significantly increase with the further increase of accumulation time due to proper dispersion in cell solution and surface saturation. It reached 97% of the maximum at 150s, indicating that the adsorption on the ERGO-DA/GCE surface was nearly completed after 150s (Fig. 4B). The DA sensitivity at lower concentrations was improved by increasing the accumulation time. Therefore, the 150s accumulation time was chosen further experiments.
The pH of the supporting electrolyte has a significant influence on the DA electrooxidation because it affects both in the peak current and the potential. Fig. 5 shows the CVs of 100 μM DA at different pH (4.2 to 9.1) (Fig. 5A) and illustrates the dependency of the DA peak currents and oxidation potentials at the pH of PBS. The DA peak currents increased with increasing pH, peaking at pH 7.4, and they decreased with the further increasing of pH in 7.4-9.1 range (Fig. 5B). The potentials of DA shifted negatively with increasing pH due to the participation of the protons in the electrode reaction (Fig. 5C). The DA potentials were proportional to the pH, following the linear regression equation E (mV) = 706.6-60.0 pH (R = 0.97). The potential shifted in the negative direction with increasing pH at the slope of -60 mV per pH unit, which is very close to the anticipated Nernstian dependence of -59 mV per pH unit. 51,52 Electrochemical application of ERGO-DA/GCE.-The kinetics of electrode reaction was investigated by evaluating the effect of scan rate on the redox peak current in Fig. 6. The scan rate of cyclic voltammetry exhibits a profound effect on the redox peak current of 100 μM DA in 0.1 M PBS at ERGO-DA/GCE. For the scan rates in the range of 10-300 mV s −1 , relationship is established between the redox peak current and the scan rate, indicating the surface controlled mechanism is significant at the low scan rate (Fig. 6A). The ERGO-DA/GCE was also investigated in 0.1 M PBS without DA at various scan rates but any identical redox peaks was not found, also indicating the DA which grafter into the ERGO surface has no any extra influence into the DA detection. (Fig 2A inset). The linear relationship is founded between the redox peak current and the scan rate, suggesting the diffusion controlled behavior is predominated at the high scan rate (Fig. 6B). 53 Such a variation on the reaction mechanism from the surface controlled to diffusion controlled at high sweeping rates demonstrates that the SPGNE possesses a faster electron transfer kinetics that could follow higher scan rates.      the oxidation peak currents increased with the increasing of both DA and 5-HT (Table II). The oxidation current of DA at in presence of the 5-HT was also proportional to the concentration, following the linear regression equation i p (μA) = 0.119×[C DA ] (μM) + 0.422. The plot also shows good linearity, with a 0.9906 correlation coefficient (Fig. 8C). Therefore, a wide linear range, lower detection limit, and good selectivity toward the electrocatalytic oxidation of DA and 5HT may observed by the excellent performance of proposed electrode, ERGO-DA/GCE, in 0.1 M PBS at pH 7.4. Fig. 9 illustrates the amperometric response of ERGO-DA/GCE to the subsequent addition of DA in the 0.1 M PBS (pH 7.4) at an applied potential of 250 mV. A steep increase in current responses was obtained after each addition of DA solution, and a steadystate current was achieved within very short time, indicating that the ERGO-DA catalyst exhibits very sensitive and rapid response to DA (Fig. 9A). This might be due to the fact that ERGO-DA is highly electro conductive, which provides a low resistance pathway and promotes the electron transfer and reduces the response time (as found in EIS). Fig. 9B depicts the calibration curve for the electrochemical responses of the ERGO-DA electrode to DA. The response to DA exhibits a good linear range from 10 to 500 μM with a correlation coefficient of 0.994. One of the major challenges in DA detection is to eliminate the electrochemical response generated by some easily oxidizable endogenous interfering compounds. The effect of common interfering electroactive substances such as glucose, AA, 5-HT, H 2 O 2 , and UA was investigated (Fig. 10). The response to the sequential addition of 20 μM DA, 5-HT, glucose, AA, H 2 O 2, UA, and DA in 0.1 M PBS at pH 7.4 was measured at an applied potential of 250 mV. The interfering species glucose, AA, H 2 O 2 , and UA did not show interferences with DA detection (signal change below 5%), but the 20 μM 5-HT interfered in a minimum level (signal change ∼10%) due to probably inappropriate applied potential. The interferences showed an effect on the DA response, demonstrating the high selectivity of the DA among all analytes.
Six human serum samples from healthy volunteers (that had been collected with the help of a local hospital) were used to determine the DA with the modified electrode. To fit into the linear range and reduce the matrix effect, the serum samples were diluted 200 times with 0.1 M PBS (pH 7.4) before analyses, without further addition of reagent. A standard addition method was employed under the previously described optimal experimental conditions. An oxidation potential was observed at 310 mV when the serum sample was added in  the PBS. The peak intensity quantificationally increased after the addition of standard DA solution, which conclusively indicates that the oxidation peak increase was caused by DA addition in the serum sample. The recoveries determined by spiking the serum samples with the amount of standard DA solution was found to be in the range of 96.5% to 102.8%, indicating the modified electrode can be applied to the determination of DA in human serum samples with satisfactory results.
Stability and reproducibility.-The stability of ERGO-DA/GCE for the detection of 100 μM DA was also investigated through amperometric responses for 1000s (Fig. 11A). After 1000s, the initial responses of DA were retained (changed responses: 15.78%, 200 mV; 9.33%, 250 mV; and 1.18%, 300 mV), respectively. Also the robustness of this electrode was investigated by CVs experiment (Fig. 11B). After 12 days only 7.7% oxidation current was reduced from the first day. Therefore, the lifetime of this electrode could be determined as 10 days for better response. The excellent long-period stability and reproducibility of the composite modified electrode make it attractive for the fabrication of electrochemical sensors. The biosensor electrode exhibited good reproducibility in the detection of DA with a relative standard deviation (R.S.D.) of ca. 4.68% found over 5 repeated measurements of 100 μM DA.

Conclusions
The GO-DA was successfully prepared through the condensation reaction between GO and DA. The electrochemically reduced GO-DA exhibited remarkable electrochemical detection of DA detection even in presence of 5-HT while ERGO-DA also weekly sensitive to the 5-HT in pH 7.4 PBS. The ERGO-DA modified electrode showed exhibited long term stability with 10 days life time, selectivity and