Facile Fabrication of CuO Nanoparticles Embedded in N-Doped Carbon Nanostructure for Electrochemical Sensing of Dopamine

In the present study, a highly selective and sensitive electrochemical sensing platform for the detection of dopamine was developed with CuO nanoparticles embedded in N-doped carbon nanostructure (CuO@NDC). The successfully fabricated nanostructures were characterized by standard instrumentation techniques. The fabricated CuO@NDC nanostructures were used for the development of dopamine electrochemical sensor. The reaction mechanism of a dopamine on the electrode surface is a three-electron three-proton process. The proposed sensor's performance was shown to be superior to several recently reported investigations. Under optimized conditions, the linear equation for detecting dopamine by differential pulse voltammetry is Ipa (μA) = 0.07701 c (μM) − 0.1232 (R2 = 0.996), and the linear range is 5-75 μM. The limit of detection (LOD) and sensitivity were calculated as 0.868 μM and 421.1 μA/μM, respectively. The sensor has simple preparation, low cost, high sensitivity, good stability, and good reproducibility.


Introduction
Dopamine (DA) is a common hormone in the family of catechol ethylamine which works as a neurotransmitter. It affects the renal, nervous, and cardiovascular systems as well as endocrine system of mammals [1]. DA controls certain physiological conditions, such as movement, learning, memory, behavior, mood, and mental cognition [2,3]. DA excites the heart and increases the blood flow. When dopamine (DA) levels are low, people lose the ability to control muscles and continue to tremble. In severe cases, this leads to Parkinson's, Alzheimer's, schizophrenia, and other symptoms [2,4]. e neurological disorders, depression, autism, congenital cardiopathy, hypertension, bronchial asthma, and septic shock are treated using DA clinically [5]. Due to such important uses and function of DA in human body, it is necessary to detect DA concentration very accurately. Among various methods of detection, electrochemical sensing is a proficient method which detects the biological molecules in very low detection range with high sensitivity and good selectivity [6,7]. Nanoparticles play an important role in electrochemical detection and are widely used in electrode fabrication for chemical sensors and biosensors [8][9][10][11]. Apart from electrochemical sensing applications, nanostructured materials are extensively used in various applications, such as heat transfer, photocatalysis, hydrogen production, solar cell, biomedical equipment, and therapeutics [12][13][14][15][16][17][18][19][20]. Various electro catalysts have performed efficiently in electrochemical-based sensors and contributing in this technique since decades [21,22].
In the catalytic analysis of small biological molecules, the sensitivity is improved to a certain extent and the detection limit is lowered. However, the expensive precious metal price limits the further popularization of these research results. Reducing costs and getting to the goal quickly and efficiently without reducing catalytic performance is one of the current problems facing by analysts. Compared to noble metals (such as Pt and Au), Cu is preferred because of its advantages such as great elasticity, good toughness, high wear resistance, affordable price, and good electrical conductivity. On this basis, CuO nanoparticles were embedded into the nitrogen-doped carbon matrix, and as a new nanocomposite material CuO@NDC was synthesized. Previous research found that adsorption of pollutants over the surface of the catalysts promotes the effectiveness. e doped-carbon materials can help for the surface adsorption and enhance the charge transfer process. In addition to this, nitrogen-doped carbon materials can participate in higher conductivity and improve electron transport, resulting in a potential electrode material for electrochemical sensing applications [23,24]. As a consequence, the produced CuO@ NDC nanocomposite, which combines the benefits of high conductivity N-doped carbon and unusual properties of CuO, may significantly improve electrochemical sensing performance [24].
When CuO nanoparticles are dispersed into N-doped carbon matrix, they show higher catalytic activity due to the synergy between the two. is material is used to modify the glassy carbon electrode and the DA content using differential pulse voltammetry.

Preparation of NDC and CuO@NDC.
e method was used to fabricate the samples with certain modifications as reported in the literature [23,25]. e polymeric metal complex was used to fabricate the nanocomposite. 1.20 g urea and 4.00 mL glutaraldehyde in a basic medium (at pH [8][9] were used to make the polymer-metal complex. 1.87 g copper(II) nitrate was added to the polymer matrix and heated at 60°C for 6 hours to get the polymer copper(II) complex. e hydrothermal method was adopted to fabricate the hydrochar, a 100 ml autoclave was filled with 3.00 gm of polymer Cu (II) complex, and 20 ml of distilled water was added and sealed properly, and the autoclave was heated to 250°C for 24 hours. After that, the autoclave was cooled and the resulting hydrochar was filtered, washed, and dried before being used for further heat treatment to produce the graphite carbon matrix. e resultant hydrochar was heated to 800°C at a rate of 5°C/min under argon flow and held at that temperature for 1 hour. Before use, the CuO@NDC was vacuum dried for 24 hours. NDC was produced without Cu(II) source using a similar method [23,25].

Characterization.
e powder X-ray diffraction (XRD) pattern of the as-synthesized nanocomposites powder was recorded using a Rigaku Ultima IV diffractometer (Cu K radiation). e FT-IR spectra were recorded in transmission mode on a Bruker Tensor spectrometer using KBr pellets with a scan range of 400-4000 cm −1 . Raman spectra were recorded on Aramis LabRam spectrometer. e Brunauer-Emmett-Teller (BET) technique of N 2 adsorption isotherms at 77.35 K; the Quantachrome Autosorb-1-MP analyzer was utilized to measure the aperture characteristics and specific surface area of the nanocomposite. Field emission scanning electron microscopy was used to study the shape and structure of manufactured materials (FESEM, JEOL, JSM-7600F). e samples were imaged and studied using a transmission electron microscope (TEM/HRTEM, JEOL, JEM-2100F) operating at 120 kV. e X-ray photoelectron spectroscopy (XPS) experiment was carried out with the help of ESCALAB250 equipment outfitted with an Al K X-ray source.

Fabrication of CuO@NDC Electrode.
e cyclic voltammetric (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were carried out at room temperature utilizing a CHI660C electrochemical workstation equipped with a three-electrode setup. e glassy carbon electrode (GCE) having 3 mm diameter was prepared by polishing alumina slurry and washed off with ethanol and distilled water and dried at room temperature. ereafter, 10 μL of the nanocomposite suspension made using ethanol Nafion solution (0.5 mg/mL) was drop cast over the surface of GCE and dried at room temperature. e cyclic voltammetry analysis was performed at several scanning rates ranging from 10 to 200 mV/s between 0.0 and + 0.8 V using 0.1 M NaOH solution as the background electrolyte. Individual CV tests were performed by adding 0.1 mL of newly produced aqueous solution of the DA analyte to 10 mL of 0.1 M NaOH medium. EIS measurements were carried out in solution of 5 mmol/L [Fe(CN) 6] 4− /[Fe(CN) 6] 3− in 0.1 mol/L KCl at a bias potential of 0.55 V using an AC voltage with a 5 mV amplitude in the frequency range of 0.1 Hz to 1 MHz. At low concentrations, interfering species such as ascorbic acid, glucose, uric acid, glutamic acid, and sodium chloride were also investigated.  [26]. ese featured peaks obtained at the specified two theta values suggest the successful formation of CuO@NDC nanostructures. e crystal size calculated using Scherrer's equation was obtained ∼21.5 nm for the highest peaks observed. e FTIR spectra of NDC and CuO@NDC are shown in Figure 1

Results and Discussion
e bands appeared at 626 cm −1 and 434 cm −1 ascribed to the vibration of Cu-O functional group [27]. e stretching vibrations of O-H functional group suggested at the ∼3400 cm −1 which is appeared as a broad band in the spectrum of both NDC and CuO@NDC. e strong band that appeared at 1625 cm −1 is due to the overlapping of bending vibrations of N-H and stretching vibrations of C � O in amide band [27]. e band that appeared in NDC at 1594 cm −1 is disappeared or shifted to 1625 cm −1 in nanocomposite due to heat treatment decompose-NHCO group [27]. e bands that appeared at 1424 cm −1 and 1147 cm −1 suggest the deformation vibration of C � N and C-N bonds, respectively, which indicate the successful synthesis of nitrogen doping in the carbon matrix [27]. e slight shifting of bands in the CuO@NDC nanocomposite confirms the successful decoration of CuO in NDC.
To evaluate the structure of the nanocomposites, the effective measurement of nanocomposite was employed in the form of Raman spectroscopy and the results are presented in Figure 1(c). Two intense peaks were observed, which are attributed to D and G bands, respectively. e D band appeared at 1350 cm −1 and G band appeared at 1594 cm −1 corresponding to the sp 2 -bonded carbon atoms which indicate a disordered graphitic structure and sp 3 carbon atom, respectively [28]. ere are total 3 modes (Ag +2 Bg) that are Raman active in CuO nanoparticles [29][30][31][32]. e other strong peak observed at 284 cm −1 is A 1 g Raman active optical-phonon Ag mode of monoclinic (C 2 /c) CuO NPs. e other significant peaks were observed at 332 cm −1 and 614 cm −1 which are known as B 1 g and B 2 g modes, respectively [33]. e porosity, surface area, and other properties of CuO@NDC nanocomposite were obtained by N 2 sorption isotherm at 77 K as presented in Figure 1(  found to be following the mesoporous sample. Brunauer-Emmett-Teller surface area was calculated for the nanocomposite and was obtained SBET � 140.32 m 2 /g. e high surface area can be attributed to the dense network of the CuO-decorated NDC matrix. Furthermore, the total pore volume of the CuO@NDC NC was found to be 0.424 cm 3 /g. e large surface area, pore volume, and hierarchical pore structure were very beneficial to the exposure of active sites and rapid transportation of gas molecules to catalytic sites [34,35]. Figure 2(a) represents the SEM image of the CuO@NDC, which states about the morphology and surface topology of the nanocomposite. e image shows that the CuO nanoparticles are embedded in the nitrogen-doped carbon matrix. e network of carbon matrix is so dense, and the CuO nanoparticles are decorated uniformly on the nitrogendoped carbon nanostructures, which indicate the successful formation of CuO@NDC. e TEM images are shown in Figure 2(b), which shows that the dark images of sphericalshaped CuO nanoparticles are embedded in a nitrogendoped carbon network. e size of CuO nanoparticles was found to be ∼15 nm. e HRTEM (Figure 2(c)) shows the lattice spacing of CuO, and it was found to be 0.25 nm, which is corresponding to the (002) crystallographic plane of CuO [26]. e XPS analysis is presented in Figure 3 and shows the survey scan, C, N, O, and Cu elements. e peaks for C, N, O, and Cu were obtained at ∼284.8 eV, 400.06 eV, 531.5 eV, and 934.6 eV, respectively, which are presented in Figure 3(a), which indicates the successful decoration of CuO@NDC. Further, deconvolution of the C 1s, N 1s, O 1s, and Cu 2p spectra is carried out to understand the atombinding states in the CuO@NDC nanocomposite. Four components of C 1s exist in CuO@NDC, corresponding to the graphite-like sp 2 C--C at 284.7 eV, 285.7 eV, 287.2 eV, and 289.07 eV, which are presented in Figure 3(b). Similarly, Figure 3(c) represents the three components of N 1s which can be found to exist at 399.5, 400.5, and 402.07 that are ascribed-NH 2 /pyridine, pyrrolic N/or graphitic N, and pyridine-N-oxide, respectively [36]. e deconvolution of 2p peaks of Cu can be done into 934.6 eV, for Cu 2p 3/2 and for Cu 2p 1/2 at 954.4 eV, while their satellite peaks can be observed at 943.4 eV and 962.4 eV, as shown in Figure 3(d). e high-resolution spectrum of O 1s can be seen in Figure 3(e) where three peaks centered at 529.4 eV, 531.2 eV, and 532.7 eV are assigned to the O 1s spectrum, respectively.

Electrochemical Sensing Performance.
e dopamine (DA) sensing performance was carried out at different modified electrodes as shown in Figure 4(a). e cyclic voltammetry (CV) (10 μM of DA) results revealed that in the case of glassy carbon electrode (GCE), the very small redox peaks were observed, while in the case of nanocomposite the intensity of these peaks was increased and the CuO@NDC redox peaks show higher current density. e sensing of the DA was characterized at different scanning rates and was carried out in the range of 25-200 mV/s. It was noticed that as the scanning rate was increased, the current density of the redox peak was increased linearly. e linear equations of redox peak current and sweep rate are I pa = 0.00908 v + 0.6259 (R 2 = 0.9679) and I pc = −0.0176 v − 0.8764 (R 2 = 0.9633). e results reveal that the current density of the redox peak increased linearly as the scanning rate was increased [37,38]. e linear equations of the logarithm of scan rate and redox peak potential were determined using the following Laviron Equation [39] as shown in Figure 4(f ).
where E pa and E pc represent the oxidation peak potential and the reduction peak potential, respectively, and v is the scan rate, e number of electron transferred coefficient (n) was obtained from Equation as below: where α is the charge transfer coefficient, n is the number of electrons transferred, T is the Kelvin temperature (273.16 K), F is the Faraday constant (96480 C/mol), and R is the molar gas constant (8.314 J/(mol/K)). e slope of the plot of E pa against log v was used for obtaining the value of n. e values of n and α were obtained as 3.17 and 0.0136, respectively. is value of n (∼3) means that three electrons were transferred during the redox process [38].  Bioinorganic Chemistry and Applications e differential pulse voltammetry (DPV) was also utilized for the detection of DA at different concentrations, and the DPV response results are illustrated in Figure 4(d).
e experimental data on varying concentration of DA was make fitted with linear, represented in Figure 4(e) and natural logarithm of scan rate versus potential has shown in Figure 4(f ). e results reveal that as the DA concentration was increased, the current density of the oxidation peak was also increased. It shows a good linear relationship in the range of 5∼75 μM. e linear equation is I pa (μA) � 0.07701 c (μM) − 0.1232 (R 2 � 0.996), and the detection limit was found to be 0.868 μM.
An interference experiment during the detection of DA (25 μM) was carried out in the presence of glucose, ascorbic acid (AA), glutamic acid, NaCl, and uric acid (UA) (Figure 5(a)). e results revealed that in the presence of glutamic acid (GA), ascorbic acid (AA), and uric acid (UA), no change in peak potential and current density was observed, suggesting that the fabricated electrode, CuO@NDC, has the excellent anti-interference ability and is expected to be used in real sample measurement. Although detection of DA in real sample is still challenging owing to its lower range in body fluids, moreover DA is found with many interfering species such as UA, AA, and other analytes. e coexistence of other analytes makes it difficult to detect, and therefore, an accurate, rapid, high sensitivity, and high selectivity sensors need to be developed.
Fayemi et al. [40] have analyzed the electrochemical detection of DA using polyaniline (PANI)-metal oxide (MO)-based nanocomposites (where MO � NiO, ZnO, and Fe 3 O 4 ). e best sensitivity and limit of detection were found about 0.153 × 10 −7 M. Due to the best detection limit, it is suggested that this sensor can be applied for real sample measurement. In another study, Minta et al. [41] have proposed the A ternary polyaniline/Fe 2 O 3 -SnO 2 /reduced graphene oxide (PFSG) sensor for the detection of DA in the presence of other interference. e proposed sensor provides the simultaneous detection of DA and UA and the OD was measured as 0.15 μM and 6.4 μM, respectively. e authors have suggested the possibility of detecting DA in presence of other analytes due to good results. Due to nearby range of detection of DA, the current study also suggest to investigate the real sample analysis with modification in the sensor design and other parameters. e stability and reproducibility measurement of CuO@ NDC nanocomposite during DA detection were measured every 5 days, and the parallel experiments were carried out each time. As shown in Figure 5(b), the results showed that the response remains around ∼96.6% after 25 days. ese outcomes showed that the produced nanocomposite has excellent stability due to the synergistic effect between CuO nanoparticles and the N-doped carbon.

Electrocatalytic Responses Using EIS Analysis
e semicircle diameter at high frequency can be used to calculate the electron-transfer resistance at the electrode surface, whereas the slope of the tail line at low frequency can be used to calculate the semi-infinite diffusion of species to the modified electrode [42]. As shown in Figure 6, the plot of NDC/GCE has a smaller semicircle diameter than CuO-NDC/GCE, indicating that CuO-NDC/ GCE has a lower electron transfer resistance. Furthermore, the plot of CuO-NDC/GCE in the low frequency tends to be more vertical to the real axis than CuO-NDC/GCE in the high frequency, indicating a higher ion diffusion rate due to the nitrogen-doped carbon nanostructures with good surface conductivity. An equivalent circuit (inset) was fitted with the EIS data ( Figure 6). EIS data was fitted with ZSimpWin 3.20 d program and an equivalent circuit (inset) was drawn ( Figure 6). Rct and Cdl represent charge transfer resistance and double-layer capacitance, respectively, and Zw is the vector sum of resistance and capacitive reactance, as shown in the circuitry. e experimental values obtained from the impedance data were combined with the EIS fitting data obtained from the equivalent circuits. When compared to NDC, the Rct fitting values for CuO@NDC nanocomposite decrease. e charge-transfer resistance is significantly reduced by embedding NDC into CuO nanoparticles, as evidenced by the nanocomposite's small Rct value, which is much lower than that of bare NDC. e Cdl values tend to trend in the opposite direction as Rct values [43,44]. e high electron transfer efficiency is indicated by the low Rct and high Cdl values, which further supports the hypothesis.

Conclusion
In this study, novel nanocomposites (CuO@NDC) were fabricated using the hydrothermal method and used for the electrochemical detection of DA. e results revealed that the DA concentration has a good linear relationship with its peak current, and the LOD and sensitivity were found to be 0.868 μM (S/N � 3) and 421.1 μA/μM, respectively. Strong anti-interference ability, UA, and AA do not interfere with the measurement for the detection of DA.
e electrode stability is found good and checked for the modified electrode after storing for several days and retained up to 98.6% on 10 th day and maintained up to 96% even after 25 days.

Data Availability
e data used to support the findings of the study are available upon request.  Bioinorganic Chemistry and Applications