Fabrication of Amine-Modified Magnetite-Electrochemically Reduced Graphene Oxide Nanocomposite Modified Glassy Carbon Electrode for Sensitive Dopamine Determination

Amine-modified magnetite (NH2–Fe3O4)/reduced graphene oxide nanocomposite modified glassy carbon electrodes (NH2–Fe3O4/RGO/GCEs) were developed for the sensitive detection of dopamine (DA). The NH2-Fe3O4/RGO/GCEs were fabricated using a drop-casting method followed by an electrochemical reduction process. The surface morphologies, microstructure and chemical compositions of the NH2–Fe3O4 nanoparticles (NPs), reduced graphene oxide (RGO) sheets and NH2–Fe3O4/RGO nanocomposites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-Ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy. The electrochemical behaviors of DA on the bare and modified GCEs were investigated in phosphate buffer solution (PBS) by cyclic voltammetry (CV). Compared with bare electrode and RGO/GCE, the oxidation peak current (ipa) on the NH2–Fe3O4/RGO/GCE increase significantly, owing to the synergistic effect between NH2–Fe3O4 NPs and RGO sheets. The oxidation peak currents (ipa) increase linearly with the concentrations of DA in the range of 1 × 10−8 mol/L – 1 × 10−7 mol/L, 1 × 10−7 mol/L – 1 × 10−6 mol/L and 1 × 10−6 mol/L – 1 × 10−5 mol/L. The detection limit is (4.0 ± 0.36) ×10−9 mol/L (S/N = 3). Moreover, the response peak currents of DA were hardly interfered with the coexistence of ascorbic acid (AA) and uric acid (UA). The proposed NH2–Fe3O4/RGO/GCE is successfully applied to the detection of dopamine hydrochloride injections with satisfactory results. Together with low cost, facile operation, good selectivity and high sensitivity, the NH2–Fe3O4/RGO/GCEs have tremendous prospects for the detection of DA in various real samples.


Introduction
Dopamine (DA) is one of the most important neurotransmitters, playing a key role in the regulation of the functions of the renal, hormonal, the central nervous and cardiovascular systems [1]. Abnormal levels of DA may cause serious neurological disorders such as Huntington's disease, Parkinson's disease and schizophrenia [1][2][3]. Generally, the basal DA concentration in the central nervous system is 10 −6 mol/L-10 −8 mol/L. Therefore, it is essential to develop a low cost, good selectivity and high sensitivity method for the detection of DA at the physiological level. Traditional techniques including high performance liquid chromatography [4,5], mass spectrometry [6], fluorescent spectrometry [7,8] and electrochemoluminescence [9,10] have been widely used for the detection of DA. Although these methods possess high precision and reliability, they often involve complicated and time-consuming experimental protocols with expensive instrumentations. Due to its low cost, facile operation, high selectivity and sensitivity, the electrochemical method has received growing attention in biosensors recently [11,12]. As is well known, bare glassy carbon electrodes (GCE) have always suffered from serious problems such as interference and electrode fouling, which can result in poor selectivity and reproducibility. To resolve these problems, various chemicals (including conducting polymers, metal or metal oxide nanomaterials and carbon-based nanomaterials) and modified electrodes have been developed for the sensitive detection of DA [13][14][15][16][17][18].
Magnetite nanoparticles (Fe 3 O 4 NPs) are widely used in the biomedical field due to their good biocompatibility, large surface area, excellent magnetic target properties and distinct catalytic activity [19][20][21][22][23]. The advantages of Fe 3 O 4 NPs are their low cost, ease of preparation and excellent water solubility [24]. More importantly, they have excellent electronic and magnetic properties due to electron transfer between Fe 3+ and Fe 2+ [25]. However, Fe 3 O 4 NPs have poor electrical conductivity and inclination of agglomeration, which will eventually cause poor sensing performances with a narrow linear range and limited detection limits [26]. The aforementioned problem can be effectively solved by compositing the Fe 3 O 4 NPs with graphene. Owing to its excellent electrical conductivity, large surface-to-volume ratio and rapid heterogeneous electron transfer rate, graphene has been considered one of the most promising materials for electrochemical sensors in recent years [27]. As a result, a Fe 3 O 4 /graphene composite combines the individual merits of each component, including large surface area, excellent electrical conductivity and distinct catalytic activity, which can be used to detect dopamine, hydrogen peroxide, guanosine and N-acetylcysteine [24,[28][29][30][31].
Amine-modified Fe 3 O 4 (NH 2 -Fe 3 O 4 ) can bind with graphene-type through hydrogen bonds between the amino-groups attached to NH 2 -Fe 3 O 4 and the available carboxylic groups of graphene-type. Consequently, the NH 2 -Fe 3 O 4 /graphene-type nanocomposite has good dispersibility and stability. Recently, the NH 2 -Fe 3 O 4 /graphene-type has been widely used for the removal of environmental pollutants such as heavy metal ions due to their strong adsorb ability and ease of separation from waste water [32][33][34]. In our previous report, the oxidation of DA is an adsorption-controlled electrochemical process [35]. Therefore, NH 2 -Fe 3 O 4 /graphene-type nanocomposites are expected to enhance the adsorption of DA on the electrode and improve the sensing performance. However, to our best knowledge, only a few papers have reported NH 2 -Fe 3 O 4 /graphene-type toward the sensitive detection of DA. Wu and coworkers prepared an NH 2 -Fe 3 O 4 NPs/graphene-type modified glassy carbon electrode (Fe 3 O 4 /Gr/GCE) toward DA detection, combining the advantages of NH 2 -Fe 3 O 4 NPs and chemically reduced graphene oxide. The proposed electrode displayed a linear range of 0.2-38 µM and a detection limit of 0.126 µM [24]. For the purpose of detecting DA at the physiological level (10 −6 mol/L -10 −8 mol/L), the dynamic range and detection limits still need to be further improved. Moreover, chemical reduction is not eco-friendly since it requires some poisonous reductants such as hydrazine and metal hydride [36]. In this case, the oxygen-containing groups have almost been removed from graphene due to the use of strong reductant, which is not favorable for the electrochemical oxidation of dopamine [37,38].
Inspired by Wu's work, we developed NH 2 -Fe 3 O 4 /RGO nanocomposites modified GCE (NH 2 -Fe 3 O 4 /RGO/GCE) for detecting DA at the physiological level. In contrast to Wu's work, the NH 2 -Fe 3 O 4 /RGO/GCE is prepared by way of drop-casting the NH 2 -Fe 3 O 4 /RGO dispersion on the bare GCE and subsequently undergoing a facile electrochemical reduction of GO process. In contrast, the electrochemical reduction method is green and eco-friendly due to the elimination of the reductants. Moreover, the electrochemical performance can be easily regulated by electrochemical reduction conditions (including reduction potential as well as time). Second-order derivative linear sweep voltammetry (SDLSV) shows higher sensitivity and selectivity as compared to the other electrochemical methods such as differential pulse voltammetry (DPV) and square wave voltammetry (SWV) [39]. Hence, SDLSV was employed to detect the DA samples. The morphologies, microstructure and chemical composition of NH 2 -Fe 3 O 4 NPs, RGO and NH 2 -Fe 3 O 4 /RGO were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy accordingly. Then the cyclic voltammetry (CV) behaviors of DA on the surface of bare and modified GCEs were also investigated. Moreover, the detection conditions including pH, scan rate, accumulation potential as well as time were further optimized. The sensing performances in terms of anti-interference, linear range and detection limit of DA on the NH 2 -Fe 3 O 4 /RGO/GCEs were evaluated systematically. Finally, the proposed NH 2 -Fe 3 O 4 /RGO/GCEs were used to detect DA in real samples.

Synthesis of NH 2 -Fe 3 O 4 NPs
Amine-modified Fe 3 O 4 NPs were synthesized by solvothermal method according to previous report [24]. In brief, 1.0 g of FeCl 3 ·6H 2 O was dissolved in 20 mL of EG. Then, 3 g of NaAc, 0.4 g NaOH and 10 mL of ED were added into the solution and vigorously stirred under ultrasound for 30 min. Afterwards the mixture was transferred to 30 mL Teflon-lined stainless-steel autoclave and autoclaved at 200 • C for 8 h. After cooling down to room temperature naturally, the black precipitates (NH 2 -Fe 3 O 4 NPs) were obtained with an external magnetic field and washed with DI water and ethyl alcohol alternately. Finally, the resulting products were dispersed in DI water to obtain 1 mg/mL NH 2 -Fe 3 O 4 NPs solution.

Synthesis of NH 2 -Fe 3 O 4 /GO Nanocomposites
Graphene oxide (GO) was prepared using the modified Hummers' method [40]. Typically, 0.5 g of graphite powder and 0.5 g of NaNO 3 were slowly added into 23 mL cooled concentrated H 2 SO 4 under vigorous mechanically stirring. Then, 3.0 g of KMnO 4 were gradually added into the mixture with continuous stirring under ice bath. Afterwards, the mixture was transferred to 35 • C water bath and stirred for 2 h. The reaction was terminated by adding 100 mL of DI water into the mixture. The mixture added into 20 mL of 30% H 2 O 2 aqueous solution in batches, which turned the color form dark golden yellow. The as-obtained suspension was filtered washed with 150 mL of hydrochloric acid (1:10) and 150 mL of DI water repeatedly and then vacuum-dried at 50 • C overnight to obtain graphite oxide. 100 mg of graphite oxide was dispersed in 100 mL DI water and exfoliated to GO by ultrasonication for 2 h. It was then centrifuged at 6000 rpm for 30 min to remove unexfoliated graphite oxide and excess graphite. Finally, 1 mL of NH 2 -Fe 3 O 4 NPs solution (1 mg/mL) was added into 20 mL of GO solution (1 mg/mL) under ultrasound exposure for 2 h and the NH 2 -Fe 3 O 4 /GO nanocomposite dispersion were obtained.

Fabrication of NH 2 -Fe 3 O 4 /RGO/ GCE
Prior to electrode modification, the GCE was polished to form mirror-like surface using α-Al 2 O 3 with different fine sizes (1.0 µm, 0.3 µm and 0.05 µm), then continuously ultrasonicated in ethyl alcohol and DI water (each for 1 min). The NH 2 -Fe 3 O 4 /GO/GCE were fabricated via drop-casting of 5 µL NH 2 -Fe 3 O 4 /GO dispersion on the surface of GCE and dried under infrared lamp radiation. The NH 2 -Fe 3 O 4 /GO/GCEs were finally obtained by electrochemical reduction of GO. Then NH 2 -Fe 3 O 4 /RGO/GCE was prepared by electrochemical reducing of GO in NH 2 -Fe 3 O 4 /GO/GCE at a suitable constant potential for a period in PBS (pH 6.5). The optimum reduction potential as well as time was also explored.

Characterization
The surface morphologies and microstructure of NH 2

Electrochemical Experiments
Cyclic voltammetry (CV) and second-order derivative linear sweep voltammetry (SDLSV) were performed with a standard three-electrode system. Bare or modified GCEs was used as working electrode, platinum wire electrode and saturated calomel electrode (SCE) was acted as auxiliary electrode and reference electrode, respectively. The electrochemical active areas of bare and modified GCEs were estimated using CV recorded in a freshly prepared [Fe(CN) 6 ] 3−/4− solution (5.0 × 10 −4 mol/L). The CV behavior of DA on NH 2 -Fe 3 O 4 /RGO/GCE was measured in a 10 mL electrochemical cell containing 5.0 × 10 −5 mol/L DA and 0.1 mol/L PBS. SDLSV was used to detect DA due to its high resolution and sensitivity. Both the CVs and SDLSVs were recorded at a scan rate of 100 mV/s, after a suitable accumulation period under stirring at 500 rpm and a 5 s rest. The potential scan ranges of both the CV and SDLSV were 0-1.0 V. Before electrochemical detection, pure N 2 was bubbled through the standard solution of DA to remove O 2 dissolved the solution.

Analysis of Real Samples
Dopamine hydrochloride injections were purchase from Aladdin Reagent Co. (Shanghai, China). 2 mL dopamine hydrochloride injections (containing 2 mg dopamine hydrochloride) were diluted to 100 mL with 0.1 M PBS (pH 3.5) to obtain DA diluents. Then DA diluent was further diluted with 0.1 M PBS (pH 3.5) to prepare DA samples of various concentration. Under the optimal detection conditions, the content of dopamine in the dopamine samples was detected using SDLSV by standard addition method.

Optimization of Electrochemical Reduction Conditions
The reduction potential as well as time is two crucial parameters for GO reduction. Firstly, the NH 2 -Fe 3 O 4 /RGO/GCEs were fabricated under various reduction potential for 300 s and then the oxidation peak currents (i pa ) of as-obtained electrodes were compared. As shown in Figure 1A, the maximal i pa is obtained when the reduction potential is at −1.5 V. The reduction degree of GO increases with the reduction potential shifts from −0.8 V to 1.5 V. Accordingly, the electrical conductivity of NH 2 -Fe 3 O 4 /RGO/GCE may increase due to the restoration of conductive carbon-conjugate structure [41]. However, the i pa decrease when the reduction potential shifts to more negative direction, probably because the oxygen-containing groups closely relating to dispersibility [42] and electrocatalytic active sites are almost removed. Furthermore, the reduction time was optimized with the electrochemical reduction of −1.5 V. The largest i pa is obtained when the reduction time is 120 s ( Figure 1B). There is no doubt that the reduction degree of GO increased over time. However, the i pa decreased with the reduction time beyond 120 s, since the oxygen-containing groups were almost removed due to excessive reduction of GO. Thus, the optimal reduction conditions are suggested as −1.5 V and 120 s.    Figure 3B). More interestingly, the NH 2 -Fe 3 O 4 NPs themselves have mesoporous structures on their surface ( Figure 3C). The average particle size is estimated to be ca. 50 nm. Moreover, the surface of NH 2 -Fe 3 O 4 NPs were successfully coated with the thin RGO nanosheets ( Figure 3D).

The Electrochemical Behavior of Modified Electrodes
The CV behaviors of DA (1.0 × 10 −5 mol/L) on the surface of the bare and modified GCEs are shown in Figure 5. A pair of broad and weak redox peaks is observed on the bare GCE, indicating the electrochemical performance is poor. In this case, the oxidation peak current (i pa ) of DA is 2.3 × 10 −6 A. A pair of well-defined redox peaks appears on the RGO/GCE and the i pa (2.2 × 10 −5 A) is approximately an order of magnitude higher than that on the bare GCE. The significant increase of i pa on the RGO/GCE is mainly due to good electrical conductivity and large electrochemical active area of RGO. Moreover, the π-π interactions between RGO and DA promote the charge transfer and DA adsorption process. When the NH 2 -Fe 3 O 4 /RGO/GCE acted as working electrode, the redox peaks are sharp and reversible and the i pa (2.9 × 10 −5 A) is nearly 13 times as compared to that on the bare GCE, due to the synergetic effect between mesoporous NH 2 -Fe 3 O 4 and RGO sheets. These results confirmed that NH 2 -Fe 3 O 4 /RGO with much higher electrocatalytic activity toward the oxidation of DA.

The Influence of pH Value
The electrochemical oxidation of DA is strong dependent upon the pH value, so it deserves further investigation the effect of pH on the oxidation peak current. Plot of oxidation peak currents (i pa ) versus pH is presented in Figure 6A. It is obvious that the i pa increases with the increase of pH and highest i pa is obtained with pH of 3.5. Afterwards, the i pa gradually decreases when the pH increases further from 3.5 to 5.0. Thus, the pH of 3.5 was chosen for the subsequent experiments. Furthermore, peak potential (E p ) shifted negatively with the increase of pH ( Figure 6B).The linear equation relating E p to pH is E p (mV) = −53.03 pH + 632.24 (R 2 = 0.989) and the slope is −53.03 mV/pH. According to Nernst equation, the slope (−53.03) suggests the oxidation of DA is an equal number of electrons and protons transferred electrochemical process [45].

The Influence of Scan Rate
The scan rate is an important parameter that influences the sensing performance of DA. The CVs of 1 × 10 −5 mol/L of DA on the NH 2 -Fe 3 O 4 /RGO/GCE recorded at various scan rates are presented in Figure 7A. Both i pa and i pc increased obviously with the increase of scan rates. Meanwhile, the background currents also increase with the increase of scan rates. In order to enhance signal to noise ratio (SNR) and reduce the background currents, 100 mV/s was selected for subsequent experiments. As shown in Figure 7B, the redox currents (i pa and i pc ) is well linear to scan rates (v) and the linear equations are expressed as: i pa = 0.0244v + 0.4023 (R 2 = 0.998) and i pc = −0.0271v + 0.3132 (R 2 = 0.996), respectively. These results suggest that the electrochemical oxidation of DA on the NH 2 -Fe 3 O 4 /RGO/GCE is an adsorption-controlled process [13]. Thus, accumulation step is adopted to increase the response peak currents for subsequent experiments. In addition, i pa shifts positively with the increase of scan rates (v) while i pc shifts to negative direction, demonstrating that the oxidation of DA is quasi-reversible process. As shown in Figure 7C, the redox peak potentials (E pa and E pc ) are linear to Napierian logarithm of scan rate (lnv). The linear equations are E pa = 0.0282 lnv + 0.449 (R 2 =0.999) and E pc = −0.0246 lnv + 0.232 (R 2 = 0.973), respectively. According to Lavrion equation [46]: where E 0 is formal potential (V), T is Kelvin temperature (K), α is charge transfer coefficient, n number of electron transfer, k 0 is heterogeneous electron transfer rate, F is Faraday constant (96,480 C·mol −1 ), R is ideal gas constant (8.314 J·mol −1 ·K −1 ). As for a quasi-reversible process, α is generally assumed to be 0.5. Then n is calculated to be around 2. So, the oxidation of DA is two electrons and two protons transferred quasi-reversible process. The electrochemical redox mechanism of DA is summarized in Figure 8.

Effect of Accumulation Potential and Time
Before the detection of DA, accumulation was carried out to increase the concentration of DA on the surface of NH 2 -Fe 3 O 4 /RGO/GCE. The effect of accumulation conditions (accumulation potential and time) on the oxidation peak currents (i pa ) was also investigated. The i pa of DA was measured after a 210 s accumulation step with various accumulation potentials. As shown in Figure 9A, the i pa of DA are highly dependent on the accumulation potential and the highest i pa appears when the accumulation potential is 0 V. Moreover, the effect of accumulation time on the i pa of DA was also investigated when the accumulation potentials were fixed to 0 V ( Figure 9B). The i pa increases gradually with the increase of accumulation time within the first 180 s. Afterwards the i pa is almost unaltered with the further increase of accumulation time, suggesting that the adsorption achieved saturation rapidly. Thus, 0 V and 210 s is chosen for further experiments.

Interference Studies
High selectivity is critical to the detection of real samples since DA often coexists with AA and UA in blood samples. In this section, the electrochemical responses of DA (2 × 10 −5 mol/L), AA (1 × 10 −5 mol/L) and UA (1 × 10 −5 mol/L) are investigated by SDLSV method. As shown in Figure 10, the response peaks of AA, DA and UA separate well each other and the peak potentials of AA, DA and UA are 216 mV, 444 mV and 620 mV, respectively. The peak potential differences (∆E p ) of AA-DA and DA-UA are 228 mV and 176 mV, respectively. Moreover, the response peak currents of DA were not interfered even in the excess of AA and UA. This result indicates that the synergistic effect between NH 2 -Fe 3 O 4 NPs and RGO improve the selectivity and anti-interference property significantly.

Analysis of Real Samples
The practicability of NH 2 -Fe 3 O 4 /RGO/GCE was validated using SDLSV under the optimal detection conditions. As listed in Table 2, satisfactory results are obtained on the NH 2 -Fe 3 O 4 /RGO/GCE.
The determination values of DA are well consistent with standard values with the RSD of −1.63~2.20% and the recovery rate is 97.1~103.9%. These results indicate that the NH 2 -Fe 3 O 4 /RGO/GCE have tremendous application prospects on the sensitive detection of DA in various real DA samples.

Conclusions
In summary, the NH 2 -Fe 3 O 4 /RGO/GCE are successfully prepared via drop-casting the NH 2 -Fe 3 O 4 /RGO dispersion on the bare GCE, followed by an electrochemical reduction of GO process. The optimal reduction potential is −1.5 V and reduction time is 120 s for electrochemical reduction of GO. The NH 2 -Fe 3 O 4 /RGO nanocomposites not only have the inherited advantages from the component materials but also improve properties due to synergetic effect between NH 2 -Fe 3 O 4 NPs and RGO. As a result, the electrochemical active area of NH 2 -Fe 3 O 4 /RGO/GCE increased greatly as compared to those of bare GCE and RGO/GCE. A pair of well-shaped redox peaks is observed on the NH 2 -Fe 3 O 4 NPs/RGO/GCE, suggesting the oxidation of DA is a quasi-reversible process. The electrochemical oxidation of DA on the NH 2 -Fe 3 O 4 /RGO/GCE is a two-electrons, two-protons transferred and adsorption-controlled process. The wide linear range (1 × 10 −8 -1 × 10 −7 mol/L, 1 × 10 −7 -1 × 10 −6 mol/L and 1 × 10 −6 -1 × 10 −5 mol/L) and low detection limit (4.0 × 10 −9 mol/L) are also obtained. Moreover, the response peak currents of DA were not interfered by the coexistence species such as AA and UA. Finally, the proposed NH 2 -Fe 3 O 4 /RGO/GCE are successfully applied to detect DA in real samples with satisfactory results.