A Versatile and Ultrasensitive Electrochemical Sensing Platform for Detection of Chlorpromazine Based on Nitrogen-Doped Carbon Dots/Cuprous Oxide Composite

The excessive intake of chlorpromazine (CPZ) adversely affects human health profoundly, leading to a series of severe diseases such as hepatomegaly and dyskinesia. The rapid and precise detection of CPZ in real samples is of great significance for its effective surveillance. Herein, a versatile and sensitive electrochemical sensor was developed for the detection of antipsychotic drug CPZ based on a Nafion (Nf)-supported nitrogen-doped carbon dots/cuprous oxide (N-CDs/Cu2O) composite. The as-synthesized N-CDs/Cu2O composite was systematically characterized using various physicochemical techniques. The developed composite-based sensor displayed excellent performance towards CPZ determination in a dynamic linear range of 0.001–230 µM with the detection limit of 25 nM. Remarkably, the developed sensor displayed good performance in terms of sensitivity and selectivity. Furthermore, good anti-interference properties toward CPZ determination were attained despite the presence of highly concentrated interfering compounds. Therefore, this composite could be a notable potential modifier to enhance electrocatalytic activity onto the surface of the electrode. Finally, N-CDs/Cu2O/Nf-based sensor was effectively applied for quantification of CPZ in human urine and pharmaceutical formulation samples.


Introduction
As a psychosis medication and the first-generation antipsychotic drug used for the management of schizophrenia, chlorpromazine (CPZ) is used to control various mental disorders, hypoxia, psychomotor agitation, manic depression, paranoia, anxiety, and tension [1]. However, it is frequently used as a growth promotion agent for the livestock industry. The illegal overuse and wrongful administration of CPZ results in its chronic accumulation in the environment and human food, which could lead to severe health issues such as hematological disorders, central nervous system reactions, and hypotensive effects [2][3][4]. Therefore, a highly sensitive and selective tool for the emerging, rapid, and accurate sensing of CPZ is of great significance to the field of pharmacological research. orthophosphate and sodium phosphate dibasic were used to prepare buffer solutions. Deionized (DI) water was used throughout this work.
The electrochemical data were recorded using CHI 660E electrochemical workstation (CH Instruments, Shanghai, China). A conventional three electrodes (a glassy carbon electrode (GCE) or a modified GCE (working electrode), a platinum wire (axillary electrode) and an Ag|AgCl (3.0 M KCl) electrode (reference electrode)) electrochemical system was used. All the experiments were performed at room temperature only. The pH measurement of buffer solutions was carried out using pH meter (Shanghai Yueping Scientific Instrument Ltd., Shanghai, China). The diffraction patterns of as-synthesized materials were collected using X-ray diffractometer (XRD (Bruker ® AXS D8 adv, Bruker (Beijing) Scientific Technology Co., Ltd., Beijing, China)) and the chemical compositions were attained using Thermo-Fisher ® Microlab 350 X-ray photoelectron spectroscopy (XPS) system (Hillsboro, OR, USA). The surface morphological studies of as-synthesized materials were studied using Hitachi ® Field emission-scanning electron microscopy (FESEM, SU-70, Tokyo, Japan)) with energy-dispersive X-ray spectroscopy (EDS). The Fourier transform infrared spectrometer (Nicolet 6700) was employed to study FT-IR spectra. Transmission electron microscopic (TEM) analysis was carried out using field emission electron microscope (JEOL JEM-2100F, Tokyo, Japan).

Synthesis of N-CDs
In a typical synthesis of N-CDs, 1.0508 g of citric acid and 335 µL of ethylenediamine were dissolved in 10 mL of DI water and then transferred to 50 mL Teflon lined autoclave and heated at 200 • C for 5 h. After the autoclave was cooled to room temperature naturally, the obtained products were centrifuged at 3500 rpm to separate unreacted and large size particles. The outcome was freeze-dried for 72 h to get solid N-CDs.

Synthesis of N-CDs/Cu 2 O Composite
N-CDs/Cu 2 O composite was synthesized according to previous literature with minor modifications [30]. Typically, 500 mg of copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) and 10 mL of DI water containing 1 mg of N-CDs (solid) were dissolved in 40 mL of DI water under mechanical stirring. 10 mL of 1 M NaOH was slowly added to the above solution and followed by injecting 10 mL of 0.3 M glucose (acts as a reducing agent) after being heated to 60 • C. After aging at 60 • C for 3.5 h, the product was centrifuged and then washed 3 times with ethanol and DI water, respectively. Later, the product was dried at 65 • C and termed as N-CDs/Cu 2 O (Scheme 1). The similar procedure was used to synthesize pure Cu 2 O cubes without introducing N-CDs during the synthesis. Nanomaterials 2020, 10, x 3 of 15 dihydrogen orthophosphate and sodium phosphate dibasic were used to prepare buffer solutions. Deionized (DI) water was used throughout this work. The electrochemical data were recorded using CHI 660E electrochemical workstation (CH Instruments, Shanghai, China). A conventional three electrodes (a glassy carbon electrode (GCE) or a modified GCE (working electrode), a platinum wire (axillary electrode) and an Ag|AgCl (3.0 M KCl) electrode (reference electrode)) electrochemical system was used. All the experiments were performed at room temperature only. The pH measurement of buffer solutions was carried out using pH meter (Shanghai Yueping Scientific Instrument Ltd., Shanghai, China). The diffraction patterns of as-synthesized materials were collected using X-ray diffractometer (XRD (Bruker ® AXS D8 adv, Bruker (Beijing) Scientific Technology Co., Ltd., Beijing, China)) and the chemical compositions were attained using Thermo-Fisher ® Microlab 350 X-ray photoelectron spectroscopy (XPS) system (Hillsboro, OR, USA). The surface morphological studies of as-synthesized materials were studied using Hitachi ® Field emission-scanning electron microscopy (FESEM, SU-70, Tokyo, Japan)) with energy-dispersive X-ray spectroscopy (EDS). The Fourier transform infrared spectrometer (Nicolet 6700) was employed to study FT-IR spectra. Transmission electron microscopic (TEM) analysis was carried out using field emission electron microscope (JEOL JEM-2100F, Tokyo, Japan).

Synthesis of N-CDs
In a typical synthesis of N-CDs, 1.0508 g of citric acid and 335 µL of ethylenediamine were dissolved in 10 mL of DI water and then transferred to 50 mL Teflon lined autoclave and heated at 200 °C for 5 h. After the autoclave was cooled to room temperature naturally, the obtained products were centrifuged at 3500 rpm to separate unreacted and large size particles. The outcome was freeze-dried for 72 h to get solid N-CDs.

Synthesis of N-CDs/Cu2O Composite
N-CDs/Cu2O composite was synthesized according to previous literature with minor modifications [30]. Typically, 500 mg of copper (II) sulfate pentahydrate (CuSO4·5H2O) and 10 mL of DI water containing 1mg of N-CDs (solid) were dissolved in 40 mL of DI water under mechanical stirring. 10 mL of 1 M NaOH was slowly added to the above solution and followed by injecting 10 mL of 0.3 M glucose (acts as a reducing agent) after being heated to 60 °C. After aging at 60 °C for 3.5 h, the product was centrifuged and then washed 3 times with ethanol and DI water, respectively. Later, the product was dried at 65 °C and termed as N-CDs/Cu2O (Scheme 1). The similar procedure was used to synthesize pure Cu2O cubes without introducing N-CDs during the synthesis.

Preparation of N-CDs/Cu 2 O/Nf Composite Modified Electrode
First, 5 mg of as-synthesized N-CDs/Cu 2 O was dispersed in 5 mL of N-dimethylformamide by ultra-sonication for 20 min (solution A). Then, 20 µL of 5% Nf solution and 180 µL of ethanol were ultra-sonicated for 30 min (solution B). Next, an optimized mixture of solution A and B (1:32, v/v) were ultra-sonicated for 5 min to obtain a suspension. Afterwards, 5 µL of the suspension was dropped onto the surface of cleaned GCE and dried under the IR lamp. The resulted electrode was known as N-CDs/Cu 2 O/Nf/GCE. Similarly, 5 mg of N-CDs and 5 mg of Cu 2 O were used for preparing N-CDs/GCE and Cu 2 O/GCE, respectively.

Real Sample Preparation
The urine sample was collected from healthy personnel and centrifuged for 5 min at 5000 rpm to obtain supernatant solution. The obtained supernatant solution was diluted 10 times with 0.1 M phosphate buffer solution (PBS) (pH 7.0) and stored in refrigerator at 4 • C for study analytical application of proposed sensor in biological matrix.

Physicochemical Characterization
The morphological and structural features of as-synthesized Cu 2 O and N-CDs/Cu 2 O composite were studied using FESEM and TEM as shown in Figure 1. The pure Cu 2 O displayed a cube-like structure with an evidently smooth surface ( Figure 1A-C). With the introduction of N-CDs, N-CDs/Cu 2 O composite exhibited a sphere-like morphology in Figure 1D-F due to the coverage of CDs outside cubic Cu 2 O. The elemental mapping of N-CDs/Cu 2 O composite in Figure 1G was conducted to determine the homogeneous distribution of C, N, Cu, and O elements. The Cu and O signals were much higher than that of C and N elements, indicating the cuprous oxide is dominant in the composite materials. The structure of N-CDs/Cu 2 O composite can be obviously observed in the TEM image of Figure 1H [31]. The existence of carbon source for N-CDs was revealed by a broad peak at around 2θ = 24.73 • ( Figure 2B). However, no characteristic peak of carbon source from 23 • to 27 • was attained for N-CDs/Cu 2 O composite due to the presence of minor quantities, well dispersions and low crystallinity of N-CDs in N-CDs/Cu 2 O composite [30]. Moreover, no additional peaks were observed in the XRD pattern of both pure Cu 2 O and N-CDs/Cu 2 O composite and confirming the high purity of synthesized materials. The functionalities of N-CDs, Cu 2 O and N-CDs/Cu 2 O composite were further investigated by FT-IR spectra. As seen in Figure 2C [31]. The existence of carbon source for N-CDs was revealed by a broad peak at around 2θ = 24.73° ( Figure  2B). However, no characteristic peak of carbon source from 23° to 27° was attained for N-CDs/Cu2O composite due to the presence of minor quantities, well dispersions and low crystallinity of N-CDs in N-CDs/Cu2O composite [30]. Moreover, no additional peaks were observed in the XRD pattern of both pure Cu2O and N-CDs/Cu2O composite and confirming the high purity of synthesized materials. The functionalities of N-CDs, Cu2O and N-CDs/Cu2O composite were further investigated by FT-IR spectra. As seen in Figure 2C, the broad peaks at 3430 cm −1 and 3255 cm −1 represent O-H and N-H stretching vibrations, respectively. Moreover, bands of 1661 cm −1 (amide I) and 1554 cm −1 (amide II) are ascribed to the bending vibrations of the amide group [32]. The weak merged peaks at 2943 and 2879 cm −1 are related to the C-H bond stretching vibrations. All of them demonstrate the presence of N-CDs in the composite. A sharp and intense peak at 627 cm -1 in FT-IR spectra of N-CDs/Cu2O and Cu2O is primarily assigned to the characteristic stretching of Cu-O, which specifies the framework of Cu2O. Furthermore, the peak positions of N-CDs/Cu2O in FT-IR are almost similar to Cu2O, owing to small quantities of N-CDs in N-CDs/Cu2O composite.  Figure S1B), corresponding to its 2p3/2 and 2p1/2 spin-orbital, which is ascribed to Cu + of Cu2O [33]. The difference of less than 20 eV in binding energies (Cu 2p3/2 and Cu 2p1/2) confirming the existence of Cu2O in the prepared composite. The deconvoluted spectrum of C 1s of composite displayed the Gaussian peaks at 284.96, 285.7, 287.5 and 290 eV ( Figure S1C) are corresponding to C-C (sp 3 ), C-N (sp 3 ), C=O (sp 2 ) and O-C=O (sp 2 ) or C=N, respectively [34]. The presence of C-N bonding reveals nitrogen-doping in the CDs. The fitted two Gaussian peaks of O 1s at 531.72 and 534.0 eV ( Figure S1D) are ascribed to C=O and C-OH / C-O-C groups, respectively [35]. The peaks of N 1s at 399.5, 402.79 and 405.8 eV shown in Figure S1E specify the existence of nitrogen typically  Figure S1B), corresponding to its 2p 3/2 and 2p 1/2 spin-orbital, which is ascribed to Cu + of Cu 2 O [33]. The difference of less than 20 eV in binding energies (Cu 2p 3/2 and Cu 2p 1/2 ) confirming the existence of Cu 2 O in the prepared composite. The deconvoluted spectrum of C 1s of composite displayed the Gaussian peaks at 284.96, 285.7, 287.5 and 290 eV ( Figure S1C) are corresponding to C-C (sp 3 ), C-N (sp 3 ), C=O (sp 2 ) and O-C=O (sp 2 ) or C=N, respectively [34]. The presence of C-N bonding reveals nitrogen-doping in the CDs. The fitted two Gaussian peaks of O 1s at 531.72 and 534.0 eV ( Figure S1D) are ascribed to C=O and C-OH / C-O-C groups, respectively [35]. The peaks of N 1s at 399.5, 402.79 and 405.8 eV shown in Figure S1E specify the existence of nitrogen typically in the form of pyrrolic or pyridonic-N, graphitic-N and N-oxides [36]. Pyrrolic or pyridonic-N, graphitic-N, and N-oxides can generate different electronic environments for carbon atoms and further create electrochemically active sites. Therefore, based on deconvolution results of XPS analysis, it can be evidently confirmed that N-doping was perfectly taken place in CDs. Interestingly, the presence of pyridinic-N is additionally beneficial for enhancing the electrochemical sensing of organic compounds [37]. Moreover, graphitic-N can considerably increase the conductivity of N-CDs/Cu 2 O composite through additional electrons at nitrogen sites [38].

Electrochemical Characterization of N-CDs/Cu 2 O/Nf/GCE
The electrochemical response of bare and modified electrodes was studied using CV technique, in 0.1 M KCl containing 2.5 mM of [Fe(CN) 6 ] 3−/4− (equimolar) at a scan rate of 100mV s −1 . Figure 3A displays cyclic voltammetric response of GCE, N-CDs/GCE, Cu 2 O/GCE and N-CDs/Cu 2 O/Nf/GCE. As can be seen, typical redox peaks were attained with peak to peak separation (∆E p ) of 164 mV at bare GCE due to relatively poor reversibility of GCE in [Fe(CN) 6 ] 3−/4− . The N-CDs modified GCE showed a better performance with ∆E p of 134 mV with improved redox peak currents than GCE by the good electrical conductivity of N-CDs. Furthermore, Cu 2 O/GCE exhibited the ∆E p of 98 mV due to the good catalytic activity of Cu 2 O. A significant increase in the peak currents with decreased ∆E p (89 mV) was observed for N-CDs/Cu 2 O/Nf/GCE. Moreover, the intensities of peak currents at N-CDs/Cu 2 O/Nf/GCE were found to be 4.68 times higher than that of bare GCE. Obviously, remarkable enhancement in the peak currents, least ∆E p and perfect reversibility of redox system observed in cyclic voltammogram at this composite modified electrode signifying the excellent electrocatalytic enhancement. Moreover, the anodic peak current(i pa )/cathodic peak current(i pc ) was almost unity and can be treated as a significant factor to testify the stable electrochemical response for this redox system at the modified electrode. This has probably resulted from the synergistic effect caused by the high electrical conductivity of N-CDs and the good catalytic activity and high surface area of Cu 2 O.  where ip is the anodic peak current (A), Aeff is an effective surface area (cm 2 ), D is diffusion coefficient of potassium ferricyanide (7.60 × 10 −6 cm 2 s −1 ) [40], υ is the scan rate (V s −1 ), n is the number of electrons involved in the electrochemical reaction of potassium ferricyanide (n = 1) and C is the bulk concentration of the redox probe (mol. cm -3 ). By taking the slope of the plot of ipa vs ν 1/2 (Figure where i p is the anodic peak current (A), A eff is an effective surface area (cm 2 ), D is diffusion coefficient of potassium ferricyanide (7.60 × 10 −6 cm 2 s −1 ) [40], υ is the scan rate (V s −1 ), n is the number of electrons involved in the electrochemical reaction of potassium ferricyanide (n = 1) and C is the bulk concentration of the redox probe (mol. cm -3 ). By taking the slope of the plot of i pa vs ν 1/2 ( Figure 3B) into account, the effective surface area of N-CDs/Cu 2 O/Nf/GCE was found to be 0.1582 cm 2 which was higher than that of the effective surface area of N-CDs/GCE (00756 cm 2 ) and Cu 2 O/GCE (0.0945 cm 2 ). This evidence demonstrates that the N-CDs/Cu 2 O/Nf/GCE possesses a good electrochemical effective surface area.

Electrochemical Performance of N-CDs/Cu 2 O/Nf/GCE towards CPZ Oxidation
To estimate the influence of the amount of N-CDs/Cu 2 O/Nf on the surface of GCE, the electrochemical performance of N-CDs/Cu 2 O/Nf/GCE in 0.1 M PBS (pH 7.0) containing 1mM CPZ was recorded. Figure 4A displays the electrocatalytic oxidation peak current of CPZ is nonlinear over the increasing amount of N-CDs/Cu 2 O/Nf. Obviously, the highest peak current of CPZ was achieved when 5 µL of N-CDs/Cu 2 O/Nf was applied onto the surface of GCE. Thus, the optimized concentration was chosen for ultrasensitive detection of CPZ.
The electrochemical response of CPZ at modified and unmodified electrodes was verified using CV technique. Figure 4B shows cyclic voltammograms of 1 mM CPZ in 0.1 M PBS (pH 7.0) at GCE, N-CDs/GCE, Cu 2 O/GCE and N-CDs/Cu 2 O/Nf/GCE. Apparently, no characteristic peak was detected in 0.1 M PBS without CPZ in the given window of potential. The bare GCE exhibited an irreversible voltammogram with poor peak current for CPZ, while the N-CDs/GCE displayed higher electrochemical oxidation response due to the better conductive and stable nature of N-CDs [41]. A notable enlargement in the peak current of CPZ was observed at Cu 2 O/GCE when compared with GCE and N-CDs/GCE owing to the good electrons transfer property at the electrode surface that is endowed by the catalytic activity of Cu 2 O. Beyond that, N-CDs/Cu 2 O/Nf/GCE presented the superior peak current among all the electrodes with a shifting towards the less positive potential for electro-oxidation of CPZ, which is attributed to the high surface area of N-CDs/Cu 2 O on the surface of modified electrode.
The influence of scan rate on the oxidation peak current of CPZ was examined and Figure 4C displays the changes of anodic peak current with an increasing scan rate of 1 mM CPZ from 50 to 300 mV s −1 . The linear relationship between oxidation peak current and the square root of scan rate was evident in Figure 4D. Moreover, with the increasing of scan rate, an obvious shift of the anodic peak potentials towards the positive was emerged, indicating the limitation of charge transfer kinetics. The electrochemical observations revealed that the electro-oxidation of CPZ was a diffusion-controlled irreversible process at the surface of N-CDs/Cu 2 O/Nf/GCE. The electrochemical response of CPZ at modified and unmodified electrodes was verified using CV technique. Figure 4B shows cyclic voltammograms of 1 mM CPZ in 0.1 M PBS (pH 7.0) at GCE, N-CDs/GCE, Cu2O/GCE and N-CDs/Cu2O/Nf/GCE. Apparently, no characteristic peak was detected in 0.1 M PBS without CPZ in the given window of potential. The bare GCE exhibited an irreversible voltammogram with poor peak current for CPZ, while the N-CDs/GCE displayed higher electrochemical oxidation response due to the better conductive and stable nature of N-CDs [41]. A notable enlargement in the peak current of CPZ was observed at Cu2O/GCE when compared with GCE and N-CDs/GCE owing to the good electrons transfer property at the electrode surface that is endowed by the catalytic activity of Cu2O. Beyond that, N-CDs/Cu2O/Nf/GCE presented the superior peak current among all the electrodes with a shifting towards the less positive potential for electro-oxidation of CPZ, which is attributed to the high surface area of N-CDs/Cu2O on the surface of modified electrode.
The influence of scan rate on the oxidation peak current of CPZ was examined and Figure 4C displays the changes of anodic peak current with an increasing scan rate of 1 mM CPZ from 50 to 300 The electrochemical reactions of organic compounds are usually carried out with the involvement of protons which have a significant impact on speeding up the reactions. The pH of supporting electrolyte generally influences the electrochemical reaction of CPZ by shifting its oxidation potentials towards positive side due to the acid dissociation of CPZ. Hence, to optimize the pH of supporting electrolyte for the electrochemical oxidation of CPZ at the composite electrode, a cyclic voltammetric investigation was performed in 0.1 M PBS with the pH ranging from 4.5 to 9.0 ( Figure 4E). The negligible shift of CPZ peak potentials at various pH conditions indicates that the electro-oxidation of CPZ is a simple electron-transfer process rather than a proton transfer process. However, the anodic peak current of CPZ strongly depends on the pH of the supporting electrolyte. Figure 4F displays the relationship between the pH of analytes and their corresponding anodic peak currents for N-CDs/Cu 2 O/Nf composite electrode, in which the maximum peak current was attained at pH 7.0. Hence, the PBS with a pH of 7.0 was selected as an optimized condition throughout the following electrochemical study of CPZ at N-CDs/Cu 2 O/Nf composite-based sensor.
According to the results in Figure 4E, the redox peaks with reversible cyclic voltammogram can be observed from pH 4.5 to 6.5, whereas only oxidation peaks are seen from pH 7.0 to 9.0. A reversible redox system in acidic media is shown in Figure 5A. An oxidation peak was observed in the forward scan at 0.730 V and the counterpart reduction peak was attained at 0.571 V, which is due to the influence of the pH of supporting electrolyte on the peak potentials of redox peaks. The oxidation peak appeared in the forward scan is attributed to the formation of CPZ cation radical (CPZ ·+ ) through one-electron oxidation of CPZ, whereas the reduction peak in reverse scan relates to the reduction of CPZ ·+ by a slow disproportionation reaction (Scheme 2A) [42]. The CPZ cation radical is more stable in acidic media. However, Figure 5B displays an irreversible system in neutral and basic media. Obviously, only oxidation peak at 0.687 V in the forward scan was observed, which signifies that with the increasing of pH values, direct conversion of CPZ to CPZ 2·+ would take place and make the process unstable. It gets a conversion to the final product by taking oxygen from the water molecule by a chemical reaction without any electrochemical-reduction (Scheme 2B). oxidation potentials towards positive side due to the acid dissociation of CPZ. Hence, to optimize the pH of supporting electrolyte for the electrochemical oxidation of CPZ at the composite electrode, a cyclic voltammetric investigation was performed in 0.1 M PBS with the pH ranging from 4.5 to 9.0 ( Figure 4E). The negligible shift of CPZ peak potentials at various pH conditions indicates that the electro-oxidation of CPZ is a simple electron-transfer process rather than a proton transfer process. However, the anodic peak current of CPZ strongly depends on the pH of the supporting electrolyte. Figure 4F displays the relationship between the pH of analytes and their corresponding anodic peak currents for N-CDs/Cu2O/Nf composite electrode, in which the maximum peak current was attained at pH 7.0. Hence, the PBS with a pH of 7.0 was selected as an optimized condition throughout the following electrochemical study of CPZ at N-CDs/Cu2O/Nf composite-based sensor.
According to the results in Figure 4E, the redox peaks with reversible cyclic voltammogram can be observed from pH 4.5 to 6.5, whereas only oxidation peaks are seen from pH 7.0 to 9.0. A reversible redox system in acidic media is shown in Figure 5A. An oxidation peak was observed in the forward scan at 0.730 V and the counterpart reduction peak was attained at 0.571 V, which is due to the influence of the pH of supporting electrolyte on the peak potentials of redox peaks. The oxidation peak appeared in the forward scan is attributed to the formation of CPZ cation radical (CPZ ·+ ) through one-electron oxidation of CPZ, whereas the reduction peak in reverse scan relates to the reduction of CPZ ·+ by a slow disproportionation reaction (Scheme 2A) [42]. The CPZ cation radical is more stable in acidic media. However, Figure 5B displays an irreversible system in neutral and basic media. Obviously, only oxidation peak at 0.687 V in the forward scan was observed, which signifies that with the increasing of pH values, direct conversion of CPZ to CPZ 2·+ would take place and make the process unstable. It gets a conversion to the final product by taking oxygen from the water molecule by a chemical reaction without any electrochemical-reduction (Scheme 2B).

Analytical Performance
The analytical electrochemical performance of N-CDs/Cu2O/Nf/GCE was studied at various concentrations of CPZ under the optimal experimental conditions using differential pulse voltammetry (DPV) technique. The recorded voltammograms were presented in Figure 6A. It simplifies that there was no prominent peak of CPZ potential appeared in the absence of CPZ. The characteristic oxidation peaks of CPZ (≈ 0.65 V) in 0.1 M PBS (pH 7.0) increased with the increasing concentration of CPZ without affecting the oxidation potential in the range of 0.001 µM and 230 µM. The calibration plot of the CPZ concentration and its corresponding peak current was depicted in Figure 6B. The detection limit was found to be 25 nM (S/N = 3) and is comparable in a dynamic linear

Analytical Performance
The analytical electrochemical performance of N-CDs/Cu 2 O/Nf/GCE was studied at various concentrations of CPZ under the optimal experimental conditions using differential pulse voltammetry (DPV) technique. The recorded voltammograms were presented in Figure 6A. It simplifies that there was no prominent peak of CPZ potential appeared in the absence of CPZ. The characteristic oxidation peaks of CPZ (≈ 0.65 V) in 0.1 M PBS (pH 7.0) increased with the increasing concentration of CPZ without affecting the oxidation potential in the range of 0.001 µM and 230 µM. The calibration plot of the CPZ concentration and its corresponding peak current was depicted in Figure 6B. The detection limit was found to be 25 nM (S/N = 3) and is comparable in a dynamic linear range (0.001 to 230 µM), signifying the superior performance of the designed sensor. Additionally, the analytical merits of the present sensor are compared with previously reported sensing protocols of CPZ (Table 1)

Analytical Performance
The analytical electrochemical performance of N-CDs/Cu2O/Nf/GCE was studied at various concentrations of CPZ under the optimal experimental conditions using differential pulse voltammetry (DPV) technique. The recorded voltammograms were presented in Figure 6A. It simplifies that there was no prominent peak of CPZ potential appeared in the absence of CPZ. The characteristic oxidation peaks of CPZ (≈ 0.65 V) in 0.1 M PBS (pH 7.0) increased with the increasing concentration of CPZ without affecting the oxidation potential in the range of 0.001 µM and 230 µM. The calibration plot of the CPZ concentration and its corresponding peak current was depicted in Figure 6B. The detection limit was found to be 25 nM (S/N = 3) and is comparable in a dynamic linear range (0.001 to 230 µM), signifying the superior performance of the designed sensor. Additionally, the analytical merits of the present sensor are compared with previously reported sensing protocols of CPZ (Table 1) and enlightening the capability of the developed sensor towards the determination of CPZ. The outstanding analytical response of N-CDs/Cu2O/Nf modified electrode can be ascribed to the enhanced surface area of the composite onto the electrode which was produced by the incorporating of N-CDs into Cu2O.

Interference Study
It is well-known that the anti-interference is one of the most important parameters for electrochemical sensing of CPZ. Some organic compounds and inorganic ions may exist in pharmaceutical and biological samples in the quantification of CPZ. Figure 7 displays the comparison of the peak current of 200 µM CPZ in the presence of 100 folds of common interfering compounds at N-CDs/Cu 2 O/Nf/GCE. The negligible variations of the peak currents for various interfering substances reveal that the developed composite-based sensor has high reliability and good selectivity for ultrasensitive electrochemical sensing of CPZ.
It is well-known that the anti-interference is one of the most important parameters for electrochemical sensing of CPZ. Some organic compounds and inorganic ions may exist in pharmaceutical and biological samples in the quantification of CPZ. Figure 7 displays the comparison of the peak current of 200 µM CPZ in the presence of 100 folds of common interfering compounds at N-CDs/Cu2O/Nf/GCE. The negligible variations of the peak currents for various interfering substances reveal that the developed composite-based sensor has high reliability and good selectivity for ultrasensitive electrochemical sensing of CPZ.

Stability, Repeatability, and Reproducibility
Repeatability refers to the ability of a sensor to produce almost the same signals in consecutive electrochemical measurements. To verify the repeatability of the developed N-CDs/Cu2O/Nf composite-based sensor, 30 distinct cyclic voltammetric cycles were recorded with and without CPZ (20 µM) alternately. There was a little difference in anodic peak currents and the peak potentials of CPZ for these repeated tests. The relative standard deviation (RSD) for the measurements was calculated to be 1.53% demonstrating that the sensor has good repeatability. To investigate the reproducibility of the proposed sensor, three N-CDs/Cu2O/Nf composite-based sensors were

Stability, Repeatability, and Reproducibility
Repeatability refers to the ability of a sensor to produce almost the same signals in consecutive electrochemical measurements. To verify the repeatability of the developed N-CDs/Cu 2 O/Nf composite-based sensor, 30 distinct cyclic voltammetric cycles were recorded with and without CPZ (20 µM) alternately. There was a little difference in anodic peak currents and the peak potentials of CPZ for these repeated tests. The relative standard deviation (RSD) for the measurements was calculated to be 1.53% demonstrating that the sensor has good repeatability. To investigate the reproducibility of the proposed sensor, three N-CDs/Cu 2 O/Nf composite-based sensors were constructed under the same working conditions and RSD of 1.83% was presented for these sensors with 20 µM CPZ, confirming its good reproducibility. The long-term stability of N-CDs/Cu 2 O/Nf composite modified electrode was further determined by storing the electrode for four weeks at 4 • C. Remarkably, the anodic peak current of CPZ, after four weeks, maintained more than 97% of its initial signal, which signifies that the proposed sensor could be used for determination of pharmaceutical samples with acceptable operational stability.

Real Sample Analysis
To further appraise the reliability of the proposed sensor, examination of CPZ was carried out in pharmaceutical formulations ( Figure S2) and human urine sample ( Figure S3). Before the measurements, 0.1 mL of human urine sample was diluted with 9.0 mL of PBS (pH 7.0) to reduce the matrix effect for obtaining accurate results. The stock solution of tablet sample was prepared with the help of PBS (pH 7.0). Although spiking a known concentration of CPZ, the oxidation peak current of CPZ increased linearly without any peak potential shift. The content of CPZ in both the samples was determined at N-CDs/Cu 2 O/Nf composite modified electrode using the standard addition method. Each addition was measured three times using DPV technique under the optimal working conditions. The obtained results were tabulated in Table 2. The recovery profile reveals that the developed sensor is greatly reliable for determining CPZ in real sample analysis and confirming a promising analytical sensing platform for pharmaceutical drugs.

Conclusion
In this work, we have developed a versatile strategy for developing a sensing platform for CPZ based on nitrogen-doped carbon dots with cuprous oxide (N-CDs/Cu 2 O) composite. The as-synthesized composite was characterized using physicochemical and electrochemical techniques. With the support of Nafion (Nf), N-CDs/Cu 2 O composite was successfully employed as a sensing material for the detection of CPZ. The composite-based sensor showed excellent performance towards CPZ determination in a dynamic linear range of 0.001 -230 µM with a detection limit of 25 nM. The developed sensor displayed long-term stability, good anti-interfering property, repeatability, and reproducibility. Additionally, the potential applicability of N-CDs/Cu 2 O/Nf/GCE was successfully verified with satisfactory recoveries in pharmaceutical drug and human urine samples. The successful performance of N-CDs/Cu 2 O/Nf composite sensor was attributed to the synergetic effect of N-CDs and Cu 2 O, which made the effective electron-transfer ability at the surface of electrode. Therefore, the developed sensor can be potentially used for clinical applications.

Conflicts of Interest:
The authors declare no conflict of interest.