Development of an impedimetric sensor based on carbon dots and chitosan nanocomposite modified electrode for Cu(II) detection in water

The fast and sensitive detection of copper ions would be essential for water monitoring. Herein, we report a novel development of an impedimetric sensor based on carbon dots/chitosan nanocomposite. Carbon dots (CDs) were synthesized by simple heating of an acidic aqueous solution of glucose. The prepared CDs were characterized by TEM, FTIR, XRD, UV-visible, and PL. These measurements revealed that the CDs possess a mean size of 3.2 nm, a graphitic structure with carboxyl and hydroxyl groups on the surface, and shows particular optical properties. A glassy carbon electrode (GCE) was modified with a carbon dot/chitosan nanocomposite and was characterized by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The CDs-CHITO/GCE electrode exhibits a large surface area, good conductivity film, and charge transfer at film/electrolyte interface. The proposed impedimetric sensor exhibits a linear response to Cu(II) ranging from 10−9 M to 10−5 M with a detection limit about 5 × 10−10 M. In addition, the sensor shows a good selectivity toward Cu(II) ions, which is less than 5%. Therefore, the as-developed impedimetric sensor exhibits good reproducibility, stability, selectivity, and a low limit of detection, which augur well for its application in water safety control processes.


Introduction
Nowadays, the determination of heavy metal ions is highly significant for human health and environmental safety [1,2]. One of the heavy metal ions, copper is the third most abundant metal ion that plays vital roles in various human beings' biological functions such as blood formation, connective tissue developments, transcriptional events, and the functioning of several enzymes [3][4][5][6]. However, excess of copper ions can cause gastrointestinal disturbance, neurodegenerative diseases, and kidney and liver damages [7,8]. The World Health Organization (WHO) decided that the permissible limit of Cu 2+ ions in drinking water at 30 nM [9, 10]. Thus, it is important to determine the low concentration of copper in the environment.
Several methods for the determination of copper ions have been proposed such as atomic absorption spectroscopy, chromatography spectrofluorimetry, and spectrophotometry [11][12][13][14]. However, these methods are characterized by the highest cost, the consuming time, and the control feasibility. Therefore, to avoid these drawbacks, electrochemical sensor has attracted great attention, for the detection of copper ion, because of their intrinsic advantages such as simple operation, high sensitivity, low-cost, rapidity, high sensitivity, and selectivity [15].
One of the most important nanostructured carbons, CDs have received major attention and appear as a new alternative due to their excellent physicochemical properties, high conductivity, environmental friendliness, high biocompatibility, high surface area, simple synthetic routes, and surface functionalization [49][50][51]. In this context, the nanocomposite can be prepared by dispersion CDs in CHITO to enhance the electrochemical properties for chemical sensing applications.
In this work, we have developed impedimetric sensors based on carbon dots (CDs) and chitosan (CHITO) nanocomposite modified glassy carbon electrode (CDs-CHITO/GCE) for copper ion detection. Carbon dots were synthesized from glucose by simple heating and were characterized by transmission electron microscope (TEM), X-ray diffraction (XRD), FTIR spectroscopy, UV-Vis absorption, and photoluminescence spectroscopy. The nanocomposite was deposited onto the GCE electrode by drop-casting. The morphological and electrochemical properties of nanocomposite were investigated by scanning electronic microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The linear range, limit detection, reproducibility, reproducibility, and selectivity of the prepared sensors were discussed. The CDs-CHITO/GCE sensor was applied to determine copper ions in real water samples.

Synthesis of carbon dots (CDs) from glucose
Three grams of glucose powder and 1 ml of H 2 SO 4 were dissolved in 10 ml of DI water. In addition, the solution was stirred at a temperature of 100°C (Fig. S1). A yellow-brownish color was obtained indicating the formation of CDs. Then, the solution was neutralized using NaOH, centrifuged at 14,000 rpm, filtered, and dried at a temperature of 80°C for 6 h.

Preparation of modified glassy carbon electrode (GCE)
The surface of the bare GCE electrode was polished with powder of alumina of 0.05 and 0.3 μm. Then, the electrode was washed with ethanol and distilled water successively in an ultrasonic bath and dried at room temperature before used.
In the proposed chemical sensor, we have used the chitosan to facilitate the immobilization of carbon dots on GCE electrode and improve the sensor properties. The CHITO-CD nanocomposite solutions were prepared as follows: 0.5 mL of 1.0% CHITO solution was added to 1.5 mL of CD solution with ultrasonication.
Then, 8 μL of the nanocomposite solution was dropped on the surface of the electrode, and it was dried at 60°C for 30 min.

Instrumentation
The synthesized CDs were selected for further study via transmission electron microscope (JEOL JEM 2010), X-ray diffractometer (Bruker AXS D8 Advance), Fourier transform infrared spectroscopy (FTIR) (Perkin Elmer, Spectrum Two), UV-Vis spectrophotometry (Specord 210 Plus), and spectrophotometer (HORIBA Jobin Yvon). The CHITO-CDs nanocomposite film was characterized by scanning electron microscopy (Jeol JSM-5400 emission) and impedance analyzer (EC-LAB BIOLOGIC). The electrochemical impedance spectroscopy (EIS) measurements were carried out in the 0.01-100-kHz frequency ranges with sinusoidal excitation signal amplitude of 10 mV. The electrochemical cell is formed by two components: an electrolyte (ammonium acetate, 0.1 M, pH = 7) and three electrodes (working electrode (GCE), counter electrode (platinum), reference electrode (Ag/AgCl)). The EIS measurements were conducted on three electrodes. The experimental data were fitted with the ZView software.

Characterization of carbon dots
To further explore the morphology, structure, chemical functionalities, and optical of CDs, we have used TEM, XRD, FTIR, UV-Vis absorption, and photoluminescence spectroscopies.
The TEM image of the prepared CDs is shown in Fig. 1a. It is observed that the CDs have a spherical shape with a narrow size distribution. In addition, the CD size is distributed in the range of 1.5-6 nm (3.2 nm mean size of CDs).
The XRD pattern of CDs is exhibited in Fig. 1b. The spectrum showed a broad peak at 2θ = 22.35°, corresponding to (002) hkl plane of the graphitic structure [52,53]. Figure 1c shows the FTIR spectrum of the CDs. It is obvious that the peaks at 3319 cm −1 , 1727 cm −1 , 1642 cm −1 , and 1092 are assigned to O-H, C=O, C=C and C-O, respectively [54,55]. The results reveal that carboxyl and hydroxyl groups exist on the CDs surface.
As shown in Fig. 1d, The UV-Vis absorption -spectrum of CDs exhibited two absorption band located at 280 and 375 nm, which are ascribed to π-π* and n-π* transitions of C=C Fig. 1 Characterization of CDs: a TEM images of CDs, b XRD pattern, c FTIR spectra of CDs, d UV-visible spectra, e PL spectra of CDs excited at 360 nm and C=O bonds, respectively. From this spectrum, the CD energies band gap was calculated using Eq. 1 [56]: where λ edge is the onset value of the most intense absorption bands. The optical band gap (Eg) was estimated to be 3.52 eV. Figure 1e presents the PL spectrum of the CDs excited at 360 nm. It noted that the PL emission peak of CDs located at 470 nm is the blue light luminescent spectral region [57].
Additionally, the PL spectrum shows a wide PL band with the full width at half maximum (FWHM) of 135 nm. This is related to the effect distribution of the different sizes of the CDs. To inform the origin of luminescent of CDs, the PL spectrum is fitted by two Gaussian bands (Fig. 1e). The first band (E m1 ) at 440 nm is attributed to radiative recombination of the clusters of carbon dots, and the second band (E m2 ) at 515 nm is assigned to the surface states of CDs [58].

Characterization of the modified GCE
Surface morphology of the modified GCE electrodes with CHITO and CHITO-CD nanocomposite films is investigated by SEM. The bare GCE electrode displays a flat and smooth surface (Fig. 2a). Further, the CHITO surface layer is rough and uniformly distributed on GCE electrode surface (Fig. 2b). After the addition of CDs into CHITO (Fig. 2c), the surface of CHITO-CD nanocomposite film is rough with a few apparent pores. Thus, the incorporation of CDs provided a large active surface area for CHITO-CDs/GCE electrode.
For sensor measurements, the potential polarization has a vast influence on impedimetric sensor response. It leads to an improved sensitivity for the recognition of the target  analyte [59]. Figure 3 shows the impedance spectra of the CHITO/GCE electrode at different potentials (0, −0.2, −0.4, and −0.6 V vs. Ag/AgCl). Under bias voltages, it is very clear that the Warburg straight line is reduced and the diameter of the semicircle is decreased. It is noticed that the best diameter of the half-circle was found at −0.6 V vs. Ag/AgCl, which can be allowed to observe the ion kinetic at the film/electrolyte interface [60]. Hence, the potential polarization is selected at −0.6 V along with the following work.
Nyquist diagrams of the bare and modified GCE electrodes are presented in Fig. 4. It is noted that the diameters of the semicircle increased after modification of GCE electrode. This is attributed to the immobilization of the membrane. However, with the addition of CDs onto CHITO, it is observed that there is a decreasing of the total impedance. This is due to the variation of the electric properties of the film with the incorporation of CDs.
The impedance spectra of functionalized GCE electrodes were fitted using the equivalent circuit model illustrated in Fig. 5. The given model is a combination of three parts. The first component is a series resistance of the electrolyte solution (R s ). The second component at higher frequencies, which is attributed to the electrode/film interface, is formed by a film resistance (R f ) and a film capacitance (CPE f ). The last component at the low frequency, which is attributed to the film/electrolyte interface, is consisted of a charge transfer resistance (R itc ) and a double-layer capacitance (CPE dl ). The reached values from the fitting model are given in Table 1.
To provide information on the electrical properties of the CHITO-CDs/GCE and CHITO/GCE structures, the values of R f and R itc are studied. As it has been seen in the Table 1, the CHITO film R f value is 37 ± 1.1 kΩ. Compared to R f CHITO film, the R f value of the CHITO-CDs film has been decreased to 13.5 ± 0.8 kΩ. This is indicated that CDs improve the electrical conductivity.
Moreover, the R itc parameter of the CHITO modified electrode is around 99 ± 2 kΩ. For the CHITO-CD modified electrode, the R itc value has been decreased to 79 ± 1 kΩ. This is indicated that there is a charge transfer enhancement.
Hence, additional CDs improve the conductivity of the film, increase the surface area, and facilitate charge transfer at the film/electrolyte interface. This clearly proves that the CHITO-CDs/GCE structures can be used as a promising candidates for impedimetric sensor application.

Detection of copper ions
The electrochemical response of copper at the CHITO/GCE and CHITO-CDs/GCE is examined using EIS in various concentrations of Cu 2+ ions.
The Nyquist plot of the two structures after the addition of concentration of Cu 2+ ion (from 10 −9 to 10 −5 M) is shown in Fig. 6a and b. It is mentioned that the total impedance decreased slightly for CHITO modified electrode with the increases of copper concentrations, whereas the diameter of the impedance spectra of the CHITO-CD modified electrode decreased clearly by the addition of the Cu 2+ concentrations. This indicates that the nanocomposite film has more sensitivity versus Cu(II) ions comparing to CHITO film. Thus, the   The response of CHITO-CDs modified electrode to Cu 2+ ions is due to a complexation process as shown in Fig. 7. Indeed, there is preferential coordination between Cu 2+ ions and chemical chitosan groups such as amide and hydroxyl ones. Moreover, the addition of CDs increases the surface area and the number of active sites facilitating consequently the interaction between the CHITO-CDs modified electrode surface and copper ions.
It is for this reason that further experiments were carried using CHITO-CDs/GCE structure to detect copper ions. The equivalent electrical circuit described above has been used to fit the experimental impedance data related to the copper ion detection. The values of electrical parameters are illustrated in Table 2.
It is observed in Table 2 that the value of R f remains relatively constant by the addition of Cu 2+ ions. This can be indicated that the copper ion interacts only on the CHITO-CDs modified electrode surface. However, the value of R it decreases when increasing the copper ion concentration, which lead to an enhancement of the charge transfer at the film/ electrolyte interface. This can be attributed to the charge redistribution and/or conductivity changes in the given  interface. Therefore, the charge transfer resistance is presented as the main parameter influencing on the response of the sensors to copper ions. Indeed, the calibration curve of the sensor for copper detection is determined by plotting the variation of R it (Fig. 8). The analytical performance of impedimetric copper sensors based on CHITO-CDs/GCE structure was compared with other chemical sensors from the literature. As seen in Table 3, the proposed impedimetric sensor shows an easy and simple preparation process, a broad linear range, and a lowest limit of detection (LOD).

Repeatability and interference
The repeatability of the impedimetric sensor at CHITO-CDs/ GCE was examined by three measurements of the response at 10 −6 M of copper in the electrolyte (pH 7.0). The relative standard deviation (RSD) was found to be around 3.93%. This shows that the proposed sensors has a good repeatability.
The selectivity of the proposed impedimetric sensor toward Cu 2+ ions was examined in the presence of interfering ions at concentrations 100-fold higher than that of Cu(II) ions. The interfering ions including Hg 2+ , Cd 2+ , Pb 2+ , Ni 2+ , Zn 2+ , Na + , Ca + , and K + was tested. For example, a typical Nyquist diagram of Cu 2+ solution in the absence and in the presence of Pb 2+ is shown in Fig. 9. Figure S2 presents the relative signal change ( in the presence of these interfering ions, where R it0 and R it are the resistances of charge transfer corresponding to the sensor response toward Cu 2+ ions in the absence and presence of the interfering ions, respectively. It is obvious that the relative signal change is less than 5%. The results indicated that CHITO-CDs/GCE structure exhibited a good selectivity to Cu 2+ in the presence of other ions.

Conclusion
A facile and a low-cost electrochemical impedimetric sensor for copper ion detection has been developed with a sustainable approach based on carbon dots/chitosan nano-biocomposites. Electrochemical impedance spectroscopy has been shown to be a very sensitive tool for analytical copper ion detection. The as-developed impedimetric sensor has shown a good selectivity and reproducibility with a high sensitivity and a very low limit of detection. The as-prepared highly sensitive Cu sensor provides a promising cost-effective nano-platform for water monitoring applications.