An Effective Electrochemical Platform for Chloramphenicol Detection Based on Carbon-Doped Boron Nitride Nanosheets

Currently, accurate quantification of antibiotics is a prerequisite for health care and environmental governance. The present work demonstrated a novel and effective electrochemical strategy for chloramphenicol (CAP) detection using carbon-doped hexagonal boron nitride (C-BN) as the sensing medium. The C-BN nanosheets were synthesized by a molten-salt method and fully characterized using various techniques. The electrochemical performances of C-BN nanosheets were studied using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results showed that the electrocatalytic activity of h-BN was significantly enhanced by carbon doping. Carbon doping can provide abundant active sites and improve electrical conductivity. Therefore, a C-BN-modified glassy carbon electrode (C-BN/GCE) was employed to determine CAP by differential pulse voltammetry (DPV). The sensor showed convincing analytical performance, such as a wide concentration range (0.1 µM–200 µM, 200 µM–700 µM) and low limit of detection (LOD, 0.035 µM). In addition, the proposed method had high selectivity and desired stability, and can be applied for CAP detection in actual samples. It is believed that defect-engineered h-BN nanomaterials possess a wide range of applications in electrochemical sensors.


Introduction
Chloramphenicol (CAP) is a nitrobenzene derivative and was first obtained from Streptomyces venezuelae in 1947 [1]. This compound is highly active against a variety of bacteria and thus has been widely employed as an antibiotic to treat various diseases [2,3]. However, the abuse of CAP has been a significant threat to environmental security and human health. For instance, CAP can easily accumulate to cause persistent environmental hazards due to its poor biodegradability [4,5]. Furthermore, these CAP residuals enter the human body through the food chain and result in serious diseases [6,7]. For this reason, the use of CAP in aquatic products and animal feeds has been prohibited in many countries [8]. Unfortunately, CAP residuals have still been detected in biological and environmental samples because of their low cost and high effectiveness [9][10][11]. Accordingly, it is imperative to develop an effective method for CAP detection.
Spectrophotometric and chromatographic methods have been developed for CAP detection [12][13][14][15][16][17]. Nevertheless, these methods are always time-consuming, expensive, and have cumbersome preprocessing [18]. Hence, most researchers have focused on electrochemical techniques due to their merits of simple preparation, fast response, good stability and in situ analysis [19,20]. Chloramphenicol is an electroactive substance and is able to produce an electrochemical signal when the redox reactions of CAP occur at the surface of an electrode. Generally, a bare GCE only gives rise to a poor electrochemical response of the samples. The UV-Vis diffuse reflectance spectroscopy (DRS) was recorded with a UV3600 spectrophotometer (Shimadzu Corporation, Japan).
A CHI 660E workstation (Shanghai China) was utilized for electrochemical tests, which were performed in conventional three-electrode installation at room temperature (25 ± 2 • C). The electrochemical installation consists of a saturated calomel electrode (SCE, reference electrode) and a platinum wire (counter electrode). Unmodified/modified GCEs were adopted as working electrodes. The CV and EIS measurements were carried out in a KCl solution (0.1 M) containing 1 mM [Fe(CN) 6 ] 3−/4− . The DPV plots were acquired in PBS solution. The scanning parameters were −0.8-−0.4 V for potential window, 50 mV for modulation amplitude, 4 mV for potential increment and 0.05 s for pulse width.

Synthesis of Carbon-Doped BN
Carbon-doped boron nitride (C-BN) was prepared modified from a previous report [35]. In general, cyanuramide aqueous solution is weakly basic, and H 3 BO 3 is weakly acidic. They can interact with each other through acid-base interactions to form C 3 N 6 H 6 ·2H 3 BO 3 [39]. To successfully introduce carbon sources, excessive cyanuramide was added to the reaction. In a typical procedure for preparing carbon-doped BN nanosheets, 3.0 g H 3 BO 3 was dissolved into 200 mL DI water and heated to 95 • C (water bath). After that, 5.0 g cyanuramide was added into the hot boric acid solution partially three times. The above mixture was vigorously stirred (95 • C) for 2 h and then subjected to the removal of water by vacuum rotary evaporation. The solid mixture was dried overnight at 80 • C and crushed to obtain a white precursor. Afterwards, 2 g precursor was well-mixed with 10 g NaCl/KCl (mass ratio of 45/55) eutectic matrix and transferred to a high-alumina crucible. The mixture was calcined at 900 • C for 3 h under a reducing atmosphere (carbon). To remove free carbon residues, the powders were washed with hot dilute nitric acid (0.2 M) and hot water several times. The final yellow powder was ground and dried and named C-BN.

Construction of the C-BN/GCE Sensor
The C-BN/GCE was obtained using a casting method. First, successive mirror polishing of the GCE was carried out on a suede using Al 2 O 3 slurries (300 and 50 nm) and cleaned with ethanol and deionized water. Next, the cleaned GCE was treated in 1.0 M H 2 SO 4 solution until a stable cyclic voltammetry curve was obtained. Meanwhile, C-BN aqueous suspensions (1 mg/mL) were obtained by dispersing C-BN nanosheets in deionized water under ultrasonication. Then, the suspension was spread on the surface of the pretreated GCE and dried naturally. Finally, the C-BN/GCE was carefully rinsed with deionized water to remove any unbound compounds. As a reference, an h-BN-modified GCE (h-BN/GCE) was constructed with the same method.

Sample Preparation
A CAP stock solution (10 mM) was freshly obtained by dissolving 0.323 g CAP in 100 mL deionized water and stored at 4 • C in the dark. The practicability of the proposed method was investigated by analyzing CAP in real samples (chloramphenicol eye drops and human serum), which were first diluted using 0.2 M PBS (pH 7.0). The final CAP concentrations were then examined by the C-BN/GCE sensor using a standard addition method.

Characterization
To identify the crystal structure of the products, powder XRD measurements were performed. Figure 1a displays the XRD patterns of C-BN and h-BN. As a reference, the standard diffraction data (JCPDS No. 34-0421) of hexagonal BN are also provided in Figure 1a. For both samples, all the diffraction peaks could be well indexed by standard diffraction data, and no additional diffraction peaks were found, indicating good phase purity. Different from h-BN, C-BN showed weak and broad diffraction peaks, suggesting that the hexagonal crystal structure was disturbed by carbon doping. A peak shift was also observed in the C-BN XRD pattern, which is attributed to the successful incorporation of carbon in the h-BN structure [39,40]. In the FTIR spectra (Figure 1b), both C-BN (red curve) and h-BN showed two strong absorption bands at 1389 and 805 cm −1 , which should be ascribed to the in-plane B-N stretching and out-of-plane B-N-B bending vibrations [41,42]. For the C-BN sample, some new IR absorption bands were visible. Several bands in the 1000-1210 cm −1 range can be assigned to C-N and C-B vibrations, and C = N vibrations contribute to the absorption band at 1662 cm −1 [40,42]. The results suggested that carbon atoms were successfully incorporated into the h-BN crystal. On the other hand, the existence of O-H and N-H was evidenced by absorption bands at 3428 cm −1 and 3182 cm −1 . These functional groups are beneficial for adsorbing target objects for further catalytic reactions.   Figure 2b) exhibited a more uniform morphology and smaller size, which would provide a more active surface for adsorbing target compounds. Figure S1 shows the EDS profile of C-BN, confirming the presence of B, N and C. The band structure of semiconductors plays an important role in their electrocatalytic activity. The DRS was measured to investigate the influence of carbon doping on the band structure. As shown in Figure 3a, there was a drastic absorption in the ultraviolet window for both samples, which corresponds to the bandgap energy of a semiconductor. Compared to that of h-BN, the absorption edge of C-BN obviously shifts to the low energy direction. In addition, a wide absorption band appeared in the whole visible range for the C-BN sample.
The results indicate that carbon doping has a significant impact on the band structure of h-BN and then modulates its photoelectronic properties. Figure 3b shows the PL spectra of the h-BN and C-BN samples under excitation at 230 nm. The C-BN shows multiple emission bands, which were not observed for h-BN. These emission peaks of C-BN were ascribed to carbon-linked defects, which modulated the behaviors of electrons [43].  Among all the electrodes, C-BN/GCE exhibited the sharpest and highest redox peaks. This reveals that the electron transfer rate of C-BN/GCE is faster than that of the others. In addition, the EASA of the electrode was calculated based on the dependence of the CV on the scan rate ( Figure S2a,c,e) using the Randles-Sevcik equation [44] I p = 2.69 × 10 5 ·(n 3/2 ) A·D 1/2 ·C·ν 1/2 (1) where I p is the peak current and ν is the scan rate (V/s). For 1 mM [Fe(CN) 6 ] 3−/4− (C) in 0.1 M KCl solution, n equals 1 and the diffusion coefficient (D) is 6.5 × 10 6 ·cm 2 /s. Accordingly, the EASA (A) of electrodes can be estimated based on the fitting results between I p and ν 1/2 ( Figure S2b,d,f). The EASA values of GCE, h-BN/GCE and C-BN/GCE were 0.19 cm 2 , 0.17 cm 2 , and 0.23 cm 2 , respectively. The good electron transfer ability and large EASA of C-BN/GCE should be ascribed to the defect structures induced by carbondoping, as they not only modulate the electronic structure of h-BN but also provide more active sites for electrochemical reactions. The EIS technique was utilized to study the interface nature of different electrodes, and the results are shown in Figure 4b. In the Nyquist plot, each curve consists of a semicircle part and a linear part. The semicircle diameter is proportional to the charge transfer resistance (R ct ) of an electrode, reflecting the electron transport ability at the electrode/electrolyte interface. As shown in Figure 4b, there are significant differences between the plots, revealing the distinct electrochemical behaviors of these electrodes. By fitting with the equivalent Randle circuit (inset of Figure 4b), the R ct values are 843 Ω, 3383 Ω and 275 Ω for the GCE, h-BN/GCE and C-BN/GCE, respectively. The R ct of C-BN/GCE was less than that of the other electrodes. This result is consistent with the above CV data, indicating the outstanding charge transferability of C-BN/GCE. Therefore, it is expected that carbon-doped BN has potential applications in electrochemical sensors.

Electrochemical Behaviors of CAP over C-BN/GCE
The electrochemical behaviors of CAP over different electrodes were first investigated by CV in 0.2 M PBS (pH = 7.0) with a scan rate of 100 mV/s. Figure 5a displays CV profiles obtained at the C-BN/GCE in the absence/presence of 100 µM CAP. No redox signals were discerned in the absence of CAP. In contrast, there was a pair of redox peaks in the presence of 100 µM CAP with an oxidation peak at −0.039 V and a reduction peak at −0.675 V (vs. SCE), respectively. The −0.675 V peak resulted from the nitro group reduction of CAP to hydroxylamine and the −0.039 V was ascribed to the oxidation of hydroxylamine to a nitroso group [45]. As depicted in Figure 5b, both the GCE and h-BN/GCE exhibited similar voltammetric signals toward CAP because they are electroactive. Interestingly, the redox signal produced by C-BN/GCE was remarkably larger than those obtained from bare GCE and h-BN/GCE. Its reduction peak current (88.9 µA) was approximately 2.6 times that of h-BN/GCE (34.1 µA) and 2.7 times that of GCE (32.9 µA). The favorable electrocatalytic effect of C-BN in the redox of CAP is attributed to its defect structure induced by carbon doping, which facilitates electron transfer and provides abundant active sites. Therefore, C-BN/GCE shows high sensitivity in the detection of CAP. As the reduction step of CAP at −0.68 V exhibited a better current response compared to the others, this peak was adopted for later experiments.   Figure 6a shows that the reduction peak current has a positive relation to the scan rate from 25 to 200 mV/s. A linear relationship between the reduction peak current and the square root of the scan rate (υ 1/2 ) was observed (Figure 6b). The regression equation was defined as I pc = 9.477 υ 1/2 − 6.788.

Effect of Scan Rate
The good linear relationship (R 2 = 0.998) revealed that electrochemical reduction of CAP over C-BN/GCE is a diffusion-controlled process [2].

Effect of pH
It is well known that the pH of the electrolytes plays an important role in an electrochemical reaction. Figure 7a presents the influence of pH on the CV response of C-BN/GCE toward CAP. The current response gradually increased with increasing pH from 5.0 to 7.0 and then significantly decreased with further increasing pH (Figure 7b, left). Meanwhile, the larger pH value makes the reduction peak potential shift to a more negative direction, implying that protons participated in the electrochemical reaction. As illustrated in Figure 7b (right), the peak potential showed a good linear relationship to pH with a regression equation of I pc = −0.0303 pH-0.468 (R 2 = 0.994). The slope value of −0.0303 V/pH is very close to the theoretical value of −0.059/2 V/pH. Therefore, the electroredox of CAP over C-BN/GCE is an irreversible process involving one electron and two protons, which is consistent with previous reports [45,46]. According to literature [47], the pH value has an impact on the structure of CAP and further on its adsorption on the electrode surface. On the other hand, increasing the pH of the alkaline electrolyte could inhibit the reduction reaction of CAP. As a result, the best current response was achieved at pH 7.0, and was adopted for the later studies. The possible electrochemical reaction mechanism of CAP on C-BN/GCE can be described by Figure S3

Detection of CAP Using the DPV Method
The DPV technique is more effective and sensitive than CV for the quantification of organic substances and thus was employed for CAP detection. Figure 8a shows a series of DPV curves of C-BN/GCE for different CAP concentrations. The current response monotonically increased with increasing CAP concentration from 0.1 µM to 700 µM. Figure 8b illustrates the linear calibration plots between the peak current and concentration. The regression equations can be described as I pc (µA) = 0.422 C (µM) + 5.02 (R 2 = 0.994) in the low concentration region (0.1-200 µM) and I pc (µA) = 0.160 C (µM) + 58.5 (R 2 = 0.989) in the high concentration region (200-700 µM). The limit of detection (LOD) was calculated using the equation LOD = 3σ/S according to the IUPAC definition [18], in which σ is the standard deviation of 5 blank measurements and S is the slope of the calibration plot. The proposed method shows a low LOD of 0.035 µM. The analytical performance of C-BN/GCE was compared with that of other electrodes reported previously. As summarized in Table 1, C-BN/GCE is a suitable platform for CAP detection. Moreover, the construction procedure of C-BN/GCE is facile, and all elements, including carbon, boron, and nitrogen, are earth-abundant.

Selectivity and Stability
The selectivity of the C-BN/GCE for the electrocatalytic redox of CAP was investigated to ensure its practical applications. The DPV measurements were conducted using C-BN/GCE to detect 50 µM CAP in the absence/presence of various interfering compounds (IC), such as 10-fold concentrations of Zn 2+ , Cu 2+ , Fe 3+ , NO 3 − , glucose, Cys, NO 2 − , DA, CTC, and AAP. Figure 9a displays the ratios of peak current before and after adding various interferents. All the investigated compounds had a negligible influence on the current response of CAP with a current deviation of less than 5.0%, indicating the good selectivity of the developed method. The satisfied selectivity of the proposed method should be ascribed to the π-π interaction and specific surface adsorption of CAP molecules by C-BN nanosheets. Five different C-BN/GCEs were fabricated independently through the same process and used for CAP detection under the same conditions. All the electrodes displayed similar peak current values with a relative standard deviation (RSD) of 2.76% (Figure 9b, left), confirming the favorable reproducibility of the proposed method. Meanwhile, ten cycle tests were performed on the same C-BN/GCE in the presence of 50 µM CAP. The RSD of the current responses was 2.44%. Therefore, the modified electrode possesses prominent repeatability. For long-term stability, the current response of the same C-BN/GCE toward 50 µM CAP was measured every week. After 4 weeks of storage at room temperature, the retention rate of the peak current was 89.6% (Figure 9b, right), demonstrating that the sensor was stable.

Real Sample Analysis
To assess the practical feasibility of the proposed method, C-BN/GCE was applied to detect CAP in real samples using a standard addition method. Real samples (human serum and eye drops) were first diluted using 0.2 M PBS (pH 7.0) and spiked with defined CAP content. The final CAP concentrations were tested by the DPV technique, and three parallel tests were conducted for every concentration. As listed in Table 2, C-BN/GCE showed satisfactory recoveries and small RSDs (less than 5%), confirming that the proposed method is reliable and applicable.

Conclusions
In conclusion, a novel and efficient CAP detection method was developed using C-BN nanosheets as sensing materials. The C-BN nanosheets were successfully synthesized via a molten-salt process and well characterized. Modulation of the band structure in defective h-BN was observed by carbon doping, which improved the electrochemical performance of C-BN/GCE, such as conductivity and EASA. As a result, C-BN/GCE showed enhanced electrocatalytic activity in the redox of CAP compared to that of h-BN. Under the optimized conditions, the proposed method exhibited a low LOD of 0.035 µM and a wide linear range from 0.1 µM to 700 µM in the detection of CAP. Moreover, the good anti-interference ability, high stability, and desired reproducibility of C-BN/GCE were experimentally authenticated. The good recovery results obtained in real sample analyses suggested the practical feasibility of the as-fabricated sensor for CAP detection. Hence, defect-engineered h-BN nanomaterials are potential candidates for electrochemical sensing applications.