Synthesis of Copper–nitrogen Codoped Carbon Quantum Dots Using Frangipani as a Carbon Source and Application of Metronidazole Determination

High-uorescence Cu/N codoped carbon quantum dots (Cu/NCQDs) were prepared by a one-step hydrothermal method using frangipani as the carbon source and copper acetate as the copper source. The Cu/NCQDs exhibited high-intensity, stable blue uorescence that is independent of the excitation wavelength. Since metronidazole can effectively quench the uorescence intensity of Cu/NCQDs, a metronidazole uorescence-detection method using Cu/NCQDs as the uorescence probe was developed, and the quenching mechanism was studied.The method has the advantages of simplicity, speed, and low cost. Besides,it has a wider linear range and detection limit. Further, the metronidazole content in actual samples was determined by this method, with good results.


Introduction
Metronidazole (2-methyl-5-nitro-1H-imidazol-1-ethanol, MNZ), a 5-nitroimidazole antibiotic, is mainly used to treat or prevent systemic or local infectious diseases caused by anaerobic bacteria and protozoa, such as lower respiratory, female genital tract, and digestive tract infections. In animal husbandry, metronidazole is often used as a feed or drug additive to promote the growth of pigs, cattle, and sheep and to inhibit the growth of parasites [1][2][3]. However, studies have shown that metronidazole has teratogenic effects on animal and plant cells, including carcinogenic and mutagenic effects. If used irrationally or abused in livestock and poultry feed, metronidazole residues may persist in animal tissue and could be transmitted through the food chain, thereby indirectly causing damage to the human body, such as epileptic neuropathy [4,5]. As is well known, the abuse of antibiotics, release of antibiotics into the environment, and development of drug-resistant genes in natural ecosystems are increasingly serious global threats. Therefore, the establishment of a rapid and effective metronidazole-detection method is of signi cant importance in ensuring food safety.
CQDs are novel carbon nanomaterials that exhibit photoluminescence (PL) and uorescence properties. However, pure CQDs, because of the few functional groups on their surfaces, exhibit a low quantum yield (QY), which limits their practical application [14]. Studies have shown that surface functionalization and chemical heteroatomic doping are effective methods for improving the uorescence properties of CQDs and expanding their application range [15]. In this regard, N, P, S, B, Cl, and other non-metallic heteroatoms have been extensively employed as dopants for the single doping or co-doping of CQDs. In particular, when CQDs are doped with N, whose atomic radius and valence electron structure are similar to those of C, the surface of the CQDs becomes positively charged, which signi cantly improves not only the interaction between the target analyte and CQDs, but also the QY of the CQDs. Resultantly, many studies on the heteroatom doping of CQDs have employed the N atom as the dopant [16][17][18]. However, compared with nonmetal impurity atoms, metal ions, particularly transition metal ions, have more volatile electronics and do not occupy orbits; further, they have larger atomic radii than non-metallic atoms [19].
Thus, transition metal atoms can change the surface charge density of CQDs, which is more advantageous [20]. In recent years, the research on the metal-atom doping of CQDs has developed rapidly. Metal atoms, including Cu, Fe, Zn, and Mn, have been extensively utilized. Among them, Cu is widely employed in the doping of CQDs because of its safety; additionally, it facilitates the interaction between aminocarboxyl and hydroxyl groups on the surfaces of CQDs [21]. Moreover, the Cu atoms doped on the surface of CQDs can form coordination complexes with most small molecules, i.e., chelation, which is essential for the detection of analytes in the environment. Therefore, high-uorescence Cu/N co-doped carbon quantum dots (Cu/NCQDs) were prepared by onestep hydrothermal method using fractionis as carbon source and copper acetate as copper source.
Cu/NCQDs showed strong blue light and excitation independent uorescence properties. Since metronidazole can effectively quench the uorescence intensity of Cu/NCQDs, a metronidazole uorescence-detection method using Cu/NCQDs as the uorescence probe was developed, and the quenching mechanism was studied. The metronidazole content in actual samples was detected by this method, and good results were obtained. All drugs were analytically pure, and distilled water was used in the experiment without further puri cation.

Preparation of Cu/NCQDs
The speci c preparation method of CQDs is as follows: 1.0 g of frangipani powder was dispersed in 40 mL of deionized water. Subsequently, 0.15 mL of ethylenediamine (20 mmol/L) was added as the nitrogen source. After ultrasonic treatment for 10 min, the mixed solution was transferred into a 50 mL high-pressure reaction kettle and placed in an electric thermostatic air blow-drying oven at the temperature of 200℃. Thereafter, the reaction kettle was cooled naturally to room temperature, and the reaction solution was pumped and ltered. The ltrate was centrifuged at 4000 rpm for 10 min, and the supernatant was ltered again using a water-soluble lter membrane (pore size: 0.22 µm) to obtain the puri ed uorescent Cu/NCQD solution. The prepared CQD solution was stored in four refrigerators for later use.
Using the same preparation process, except with the addition of copper acetate monohydrate, N-doped CQDs (NCQDs) were prepared as contrast materials.

Fluorescence detection of metronidazole
The Cu/NCQD standard metronidazole (MNZ) solutions (100 µL portions) of different concentrations were successively poured into a series of 10 mL-colorimetric tubes and diluted to scale using a citric acidsodium dihydrogen phosphate buffer (pH = 4; Conc.: 0.2 mol/L). After shaking, the metronidazole solution was stable for 3 min. The uorescence spectrum was recorded with a 1-cm quartz colorimeter, using an RF-5301 PC uorescence spectrophotometer. The uorescence intensities with and without the MNZ solution were denoted as F and F 0 , respectively. The excitation wavelength was set at 355 nm, and the excitation and emission slit widths were both 3 nm.

Pretreatment of honey samples
Metronidazole tablets (0.2 g each) and honey were purchased from Ali Health Pharmacy(Nanning,China). Each metronidazole tablet was ground into powder using an agate mortar, dissolved in distilled water, ultrasonicated for 15 min, and dissolved in a 100-mL volumetric bottle for subsequent analysis. The speci c method for pretreating the honey was obtained from the literature [11], as follows: 10 mg of the honey sample was dissolved in 1 mL of an acetone solution (5%). After 15 min of ultrasonic treatment, 1 mL of the sample solution was poured into a 10 mL-colorimetric tube, and 100 µL of the Cu/NCQD solution was added. Afterward, the solution was diluted to scale with the citric acid-sodium dihydrogen phosphate buffer (pH = 4).

Determination of the metronidazole working curve
Cu/NCQD (100 µL) and metronidazole solutions (100 µL) of different concentrations were added separately to a series of 10 mL-colorimetric tubes and diluted to scale with the citric acid-sodium dihydrogen phosphate buffer (pH = 4). Thereafter, the solution was shaken and left for 5 mins ; afterward, F 0 and F were recorded using the uorescence spectrometer, and the uorescence intensity without and with metronidazole was determined using the formula: F = F 0 − F, as shown in Figure 2.  Figure 3-A, the prepared Cu/NCQDs exhibited a nearly spherical morphology and good dispersion in water. Figure 3-B shows the particle-size distribution of the Cu/NCQDs. Statistically, the particle-size range of Cu/NCQDs is 1.90-3.70 nm, and the Gaussian tting curve shows that the average particle size is 2.58 nm.  [21].The absorption peaks slightly lower than 3000 cm −1 and 1384 cm −1 correspond to the stretching vibration and bending vibration of the alkyl C-H bond, respectively, and the peak near 1710 cm −1 is related to the stretching vibration of the -COOH group. The absorption peaks at 1000 and 1217 cm −1 correspond to the symmetric and asymmetric stretching vibrations of carbon and oxygen compounds (C-O-C), respectively [21]. The above results indicate the abundance of hydrophilic groups (-OH, NH 2 , and -COOH) on the surface of the Cu/NCQDs, which explains their good water solubility. Further, the hydrophilic groups facilitate the chelation of Cu/NCQDs with Cu 2+ , as well as promote the surface passivation process of the CQDs and Cu doping. Notably, the 870-1000 cm −1 range is denoted as the metal-sensitive region [33, 21,22]. Different from the NCQDs, the Cu/NCQDs exhibited partial absorption in the metal-sensitive region. This is related to the stretching vibration of the N-Cu-N bond and con rms the formation of a Cu coordination complex in the Cu/NCQDs. The FT-IR spectra showed that N and Cu were successfully doped on the surface or the Cu/NCQDs in the form of related groups.
X-ray photoelectron spectroscopy (XPS) was conducted to further con rm the structural composition of the Cu/NCQDs. In the XPS full-scan spectra of the Cu/NCQDs (Figure 3-D), four peaks are observed at binding energies of 284.82, 399.98, 531.32, and 932.66 eV, belonging to C 1s, N 1s, O 1s, and Cu 2p, respectively. The C 1s high-resolution spectrum (Fig. 4-A) shows four peaks at 284.55, 285.94, 287.11, and 288.10 eV, indicating the existence of carbon in four different states: C-C/C=C, C-N/C-O, C=O, and COO- [15], respectively. Figure 4-B shows the high-resolution spectrum of O 1s, and the peaks at 530.99 eV and 532.09 eV correspond to the C=O and C-OH/C-O-C bonds [23,34], respectively. The N 1s highresolution spectrum (Fig. 4-C) con rmed the presence of C-N-C (399.72 eV), N-C3 (400.59 eV), and N-H bonds (401.87 eV) [35,22]. The Cu 2p spectra (Fig. 4-D) show two peaks at 933.0 eV and 952.50 eV, attributable to Cu 2p 2/3 and Cu 2p 1/3, respectively, indicative of the N-Cu-N covalent bond in porphyrin-metal complexes. In conclusion, the XPS results are consistent with the FT-IR spectroscopy results, which proves that Cu and N were successfully codoped in the Cu/NCQD skeleton.

Study on uorescence properties of Cu/NCQDs
To explore the uorescence performance of the Cu/NCQDs, ultraviolet-visible (UV-vis) and uorescence spectroscopies were performed. Figure 5-A shows the UV-vis absorption spectra of the Cu/NCQDs, as well as their uorescence excitation and emission spectra. The maximum excitation and emission wavelengths of the Cu/NCQDs are approximately 355 nm and 443 nm, respectively. As illustrated in Figure S3, the Cu/NCQDs exhibit a pale-yellow color in sunlight. The bright blue uorescence under 360 nm UV light indicates that the Cu/NCQDs have unique optical properties. Noticeably, the uorescence intensity increases gradually as the excitation wavelength increases from 300 nm to 355 nm. Therefore, 355 nm was adopted as the best excitation wavelength in subsequent experiments. Notably, with varying the excitation wavelengths, the maximum emission wavelength of the Cu/NCQDs remained 443 nm without any noticeable red or blue shift. This result shows that the watersoluble Cu/NCQDs exhibit PL behavior regardless of the excitation wavelength, which is in contrast to most reported CQDs, whose PL behaviors depend on the excitation wavelength. This PL behavior of the Cu/NCQDs is attributed to its narrow particle-size distribution, as well as its highly uniform surface structure and surface-energy trapping ability [24,25]. Notably, the emission peak of the Cu/NCQDs at 443 nm is independent of the excitation wavelength, which reduces the uorescence interference from the CQDs themselves in the detection process of the target. This evidences the application potential of Cu/NCQDs in the analysis and detection of analytes.

Study of the factors in uencing the uorescence performance of Cu/NCQDs
A series of experiments was conducted to investigate the factors affecting the uorescence performance of the Cu/NCQDs. The experimental results are shown in Figure 6-A. In salt solutions of different concentrations, the uorescence intensity of the Cu/NCQDs remained almost unchanged, indicating that they are not affected by the solution ionic strength and have excellent salt tolerance. Therefore, Cu/NCQDs can be applied in biochemical analyses. Furthermore, as shown in Figure 6-B, the uorescence intensity of the Cu/NCQDs is relatively strong and stable in acidic environments. With the increase in the pH, the uorescence intensity decreases signi cantly. This phenomenon may be related to the Cu ions on the surface of the Cu/NCQDs, which inhibit hydrolysis in an acidic environment and promote hydrolysis in an alkaline environment. Thus, the content of Cu ions on the quantum dots decreases under alkaline conditions, which decreases the uorescence intensity. Moreover, several studies have proven the relationship between the uorescence intensity and doped Cu ions. In addition, the emission wavelength of the Cu/NCQDs does not vary with pH, indicating that the prepared Cu/NCQDs have no dependence on pH. To further measure the photobleaching resistance of the Cu/NCQDs, the same amount of the Cu/NCQD solution was irradiated under a 360 nm UV lamp for 0, 15, 30, 45, 60, 75, 90, 105, and 120 min, after which its uorescence emission spectrum was measured. As shown in Figure 6-C, the Cu/NCQDs dissolved as the duration of UV irradiation increased. The uorescence intensity of the solution barely changed, indicating the excellent photobleaching resistance of the prepared Cu/NCQDs, which is essential for their practical application.

Selective detection of metronidazole using Cu/NCQDs
To evaluate the speci city of this method for metronidazole detection using Cu/NCQDs, the common potential interference substances in metronidazole drugs were studied. The Cu/NCQD solution (100 µL), 2 mL of the citric acid-sodium dihydrogen phosphate buffer (pH = 4), and the metronidazole solution (100 µmol/L) were added into a series of 10 mL-colorimetric tubes. Thereafter, the interference substances (200 mmol/L): K + , Mg 2+ , NH 4 + , Ca 2+ , O 3 2− , glucose, ascorbic acid, and sucrose, as well as β-cyclodextrin and L-cysteine (5 mmol/L), were added. The uorescence intensity was recorded using a uorescence spectrometer. As shown in Figure 7, the uorescence intensities of the solutions with interfering substances (50 times and 200 times) and without interfering substances barely changed. The results validated the excellent speci city of the Cu/NCQD uorescence probe for metronidazole detection.

Interaction mechanism between the Cu/NCQDs and metronidazole
The interaction between the Cu/NCQDs and metronidazole is shown in Figure 8-A. As can be observed, metronidazole exerts a uorescence quenching effect on the Cu/NCQDs, leading to a decrease in their uorescence intensity. Therefore, the variation in the uorescence intensity can be utilized as an indicator of the change in the concentration of metronidazole. Based on this, an on-off uorescence-detection method using Cu/NCQDs as the uorescence probe was established for the determination of the metronidazole content. Figure 8-A shows that after the addition of metronidazole, the uorescence emission peak of the Cu/NCQDs did not shift from 443 nm, neither did a new emission peak appear, indicating that the quenching effect of metronidazole on the Cu/NCQDs is independent of the uorescence resonance energy transfer. To further understand the quenching mechanism of metronidazole on the Cu/NCQD uorescence, the UV-vis absorption spectrum of metronidazole was compared with the uorescence excitation spectrum of the Cu/NCQDs (Figure 8-B). In Figure 8-B, metronidazole displays two absorption bands at ~230 nm and 318 nm, while the maximum excitation wave peak in the Cu/NCQD spectrum is located at ~355 nm. Evidently, the absorption spectrum of metronidazole overlaps with the maximum excitation spectrum of the Cu/NCQDs. Therefore, it can be concluded that the quenching effect of metronidazole on the Cu/NCQD uorescence is related to the inner lter effect (IFE). In the detection system, metronidazole is the absorber, while the Cu/NCQDs are the uorophore. Metronidazole absorbs the excitation energy of Cu/NCQDs, leading to a decrease in the uorescence intensity of the Cu/NCQDs. To further determine the quenching mechanism of the system, MNZ and the mixed solution of MNZ and Cu/NCQDs were subjected to UV-vis spectroscopy, as shown in Figure 8-C. After adding the Cu/NCQDs, the characteristic absorption peaks of MNZ did not change, and the electron-hole pair recombination did not occur, indicating that the quenching mechanism was unrelated to the photoinduced electron transfer process. Therefore, in this detection system, the quenching effect of metronidazole on the Cu/NCQDs can be explained by the IFE [26,27].

Analysis and test of actual samples
The metronidazole-treated tablet solution (20 µL, 11.685 mmol/L) was poured into a 10 mL-colorimetric tube, and 100 µL of the Cu/NCQD solution was added. Thereafter, the solution was diluted to scale with the citric acid-sodium dihydrogen phosphate buffer (pH = 4), and its uorescence intensity was measured using a uorescence spectrophotometer. A portion of the honey-treatment solution was directly utilized for the uorescence intensity measurement, and the labeled recovery experiment was conducted simultaneously. The experimental results are listed in Table 1. The calculated MNZ content in the metronidazole tablets was 0.1972 g/tablet, which was not signi cantly different from the theoretical value (0.2 g/tablet). The recovery rates of the metronidazole tablets and honey samples were 96.8-103.7%, and the relative standard deviation was lower than 6%. The results showed the signi cant application potential of the uorescence probe in detecting MNZ in actual samples. Compared with other uorescence detection methods, the detection limit of this method is not the lowest, but the linear range is wider. The results are listed in Table 2. It is noteworthy that the uorescent probe of this method uses natural biomass as carbon source and adopts one-step hydrothermal method, which is simpler and more environmentally friendly.  [28] GQDsembedded SMIP b 0.2-15 0.15 [29] g-C 3 N 4 nanosheets Thermal polymerization 0.058-0.58 0.05 [30] CDs Solvent hot method Acid magenta6B H 2 O 2 0-58.4 1.50 [27] FCNs Hydrothermal method gardenia 0.8 -225 0.28 [31] CDs Solvent hot method H. cordata 3.3-240 0.12 [32] Cu/NCQDs Hydrothermal method frangipani 0-350 0.28 work

Conclusion
Blue uorescent Cu/NCQDs were successfully synthesized by a simple and green one-step hydrothermal method using frangipani as the carbon source. By introducing Cu and N diheteroatoms, the surface-energy trap of the Cu/NCQDs was effectively passivated by nitrogen-containing groups and Cucontaining groups (N-Cu-N), leading to a non-excitation dependent uorescence behavior. Further analysis of Cu/NCQDs showed that the high-uorescence intensity was stable and long-lasting, and the uorescence emission was not affected by pH.Therefore, a uorescence-detection method using Cu/NCQDs as an effective uorescence probe to determine the metronidazole content in the range of 0-350 mol/L was established. The R 2 value was 0.996, and the detection limit of the method was 0.28 mol/L. This method was applied to analyze actual samples of medicine tablets and honey, and the results validated its potential for practical application. This method is expected to be advanced into a uorescence kit for future practical analyses.  Effects of different potential interfering substances on the detection system of Cu/NCQDs-MNZ