English

• 0.   Introduction

  Ornidazole (ONZ) is a 5-nitroimidazole antibiotic used to treat anaerobic bacterial infections^[1-2] and conditions such as amebic liver abscesses, duodenal ulcers, giardiasis, intestinal lambliasis, and trichomoniasis of the urogenital tract, as well as bacterial vaginosis^[3-4]. However, several studies have reported side effects associated with ONZ, including abdominal discomfort, vomiting, nausea, and diarrhea. Misuse can lead to adverse effects ranging from discomfort to severe conditions such as cancer^[5-7]. Therefore, it is crucial to monitor and prevent its overuse. Traditional methods for detecting and separating ONZ include capillary electrophoresis (CE)^[8-9], high-performance liquid chromatography (HPLC)^[10], liquid chromatography-tandem mass spectrometry^[11], spectrophotometry^[12-13], electrochemical methods^[14-15], euzymelinked immunosorbent assay (ELISA)^[16], and spectrofluorimetry^[17-19]. Luminescence methods have shown promise in detecting antibiotic residues in recent years due to their sensitivity and rapid detection capabilities. However, selecting the appropriate sensor material is critical. Various materials, including silver nanoparticles and coordination polymers, have been utilized in the luminescence detection of ONZ^[20].

  Metal nanomaterials have been increasingly employed in various fields due to their unique optoelectronic properties, excellent biocompatibility, and potential noncytotoxicity^[21-22]. Unlike metal nanoparticles, metal nanoclusters (MNCs) consist of a few to hundreds of metal atoms encapsulated by ligands, offering smaller dimensions and unique luminescence properties^[23-24]. The synthesis and application of MNCs have been extensively studied in recent decades^[25-27]. Despite their advantages, MNCs suffer from drawbacks such as poor stability, low quantum yields (QYs), and challenging preparation methods, which limit their applications. To address these issues, researchers have employed strategies such as metal doping, encapsulation in metal-organic frameworks, ligand exchange, and dual-ligand modification^[28-32]. Copper, with its high natural abundance and low cost compared to noble metals, has attracted significant attention for nanocluster applications^[33-34]. Numerous copper nanoclusters (CuNCs) have been synthesized and applied in luminescence analysis in recent years^[35-36]. The incorporation of dual ligands in CuNCs has been particularly explored to enhance fluorescence intensity and stability. This approach has garnered attention and development due to its potential advantages over traditional noble MNCs^[37-39].

  In this study, a stable bovine serum albumin (BSA) and glycine (Gly) dual-ligand-modified CuNCs (BSA-Gly CuNCs) were synthesized using a one-step chemical reduction method. The introduction of the electron-rich ligand Gly significantly improved the QYs (5.27%) and stability of BSA ligand-modified CuNCs (BSA CuNCs). Compared to BSA CuNCs, BSA-Gly CuNCs exhibited a markedly enhanced optical response to ONZ. Based on this property, a novel fluorescence method utilizing BSA-Gly CuNCs as a fluorescence probe was successfully developed for the sensitive and selective detection of ONZ. The synthesis and detection conditions were thoroughly investigated, and the determination mechanism was discussed in detail. Furthermore, the method was successfully applied to determine the ONZ content in both sodium chloride injections and pharmaceutical tablets (Fig. 1).

  Figure 1

  

  Figure 1.  Schematic illustration of BSA-Gly CuNCs for the detection of ONZ

  The green circular area represents the green fluorescence emitted by the CuNCs.

  下载: 全尺寸图片 幻灯片

  1.   Experimental

  1.1   Materials and instruments

  BSA and Gly were obtained from Aladdin (Shanghai, China). Disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), and sodium chloride (NaCl) were purchased from Xi′an Chemical Reagent Factory (Xi′an, China). Citric acid (CA) and copper sulfate pentahydrate (CuSO₄·5H₂O) were purchased from the Tianjin Beilian Fine Chemical Reagent Factory (Tianjin, China). All reagents were of analytical grade. Deionized water (18.25 MΩ·cm) was used in all experiments. Standard ONZ (99.9%) was obtained from Beijing Biotechnology Co., Ltd (Beijing, China). ONZ tablets (0.25 g per tablet, 221201) and ONZ in sodium chloride injection (0.5 g per 100.00 mL, B231219-1) were purchased from local pharmacies.

  A multiposition magnetic stirrer (IKA, Germany) and high-speed centrifuge (CENCE, China) were used for the synthesis of nanoclusters. Fluorescence measurements were performed using an LS 55 spectrofluorimeter (PerkinElmer, UK). FLS920 (Edinburgh Instruments, UK) fluorescence spectrophotometer was used for fluorescence lifetime measurement. UV-2550 (Shimadzu, Japan) was used for the UV-Vis spectrum. Fourier transform infrared spectra were obtained with an FTIR-440 spectrophotometer (Shimadzu, Japan). The morphological measurements were conducted on a JEM-F200 transmission electron microscope (TEM, JEOL, Japan) operated at an acceleration voltage of 200 kV. The analysis of valence states of elements was conducted using an X-ray photoelectron spectroscopy spectrometer (XPS, Thermo Fisher Scientific, K-Alpha, USA).

  1.2   Synthesis of BSA-Gly CuNCs

  BSA-Gly CuNCs were prepared with modifications based on the literature^[40]. In brief, 180 mg of BSA and 20 mg of Gly were dissolved in 10.00 mL of ultrapure water and the mixture was stirred for 30 min. Subsequently, 200 μL CuSO₄·5H₂O (50 mmol·L^-1) solution was added to the stirred solution. The pH value of the solution was then adjusted to 11 using 0.2 mol·L^-1 NaOH. The mixed solution was then stirred and reacted at 45 ℃ (water bath temperature) for 10 h. After the reaction, the final mixture became a clear and transparent dark blue solution. The product was then dialyzed and stored at 25 ℃ for further investigations. The synthesis of BSA CuNCs involved dissolving 180 mg of BSA in 10.00 mL of ultrapure water, followed by the same procedure as described for BSA-Gly CuNCs. The dialysis process was conducted using a dialysis bag with a molecular weight cutoff of 3 500 Da, which effectively allows the removal of small molecules while retaining larger entities.

  1.3   Fluorescence detection of ONZ

  1.00 mL of the BSA-Gly CuNCs dialysate was mixed with 10.00 mL of Na₂HPO₄-CA (pH=7.00) solution and the mixture was adjusted with ultra-pure water to a total volume of 20.00 mL to obtain BSA-Gly CuNCs solution. The solution was then stabilized, and 2.00 mL of the solution was taken and placed in the cuvette to measure the fluorescence intensity. The fluorescence intensity at 400 nm (with an excitation wavelength of 322 nm and a slit width of 5 nm) of the BSA-Gly CuNCs solution was measured and recorded as I₀. Then, 50 μL of ONZ solution was added to the BSA-Gly CuNCs solution, and the fluorescence intensity was measured and recorded as I. The fluorescent quenching efficiency (ΔI/I₀, ΔI=I₀-I) was used for the quantitative determination of ONZ.

  1.4   Measurement of the QY of CuNCs

  Quinine sulfate (Its QY was 55% at 310 nm excitation wavelength.) was used as the reference to calculate the fluorescence QY of BSA-Gly CuNCs. Quinine sulfate was dissolved in 0.1 mol·L^-1 of H₂SO₄ solution, and the absorbance of both quinine sulfate and BSA-Gly CuNCs was obtained by UV-Vis absorption spectrum (The absorbance of both solutions was less than 0.05.). Subsequently, the fluorescence-integrated area of the two solutions was obtained by fluorescence spectroscopy. The QY of BSA-Gly CuNCs was calculated using the following formula^[41]:

  $ Y_{\mathrm{CuNCs}}=Y_{\mathrm{ST}}\left(I_{\mathrm{CuNCs}} / I_{\mathrm{ST}}\right) \times\left(A_{\mathrm{ST}} / A_{\mathrm{CuNCs}}\right) \times 100 \% $

  where Y_CuNCs and Y_ST are the fluorescence QY of BSA-Gly CuNCs and quinine sulfate, respectively; I_CuNCs and I_ST are the integrated areas of fluorescence intensity of BSA-Gly CuNCs and quinine sulfate, respectively; A_CuNCs and A_ST are the absorbances of BSA-Gly CuNCs and quinine sulfate at the excitation wavelength of 310 nm, respectively.

  2.   Results and discussion

  2.1   Characterization of BSA-Gly CuNCs

  The as-prepared BSA-Gly CuNCs underwent comprehensive characterization using various analytical techniques, including UV-Vis absorption spectroscopy, fluorescence spectroscopy, TEM, FTIR spectroscopy, and fluorescence lifetime analysis.

  In Fig. 2a, the synthesized BSA-Gly CuNCs exhibited fluorescence with excitation and emission peaks at 322 and 400 nm, respectively. These peaks were consistent with those of BSA CuNCs but showed significantly enhanced fluorescence intensity. The QY of BSA-Gly CuNCs increased from 2.78% to 5.27% compared to BSA CuNCs. This enhancement may be attributed to the electron-rich groups of Gly, which stabilize CuNCs and intensify their fluorescence. Additionally, Gly molecules may bind with copper within the BSA matrix to restrict ligand rotation, further enhancing fluorescence intensity^[39].

  Figure 2

  

  Figure 2.  (a) Fluorescence spectra of BSA-Gly CuNCs and BSA CuNCs; (b) UV-Vis spectra of BSA-Gly CuNCs, BSA CuNCs, BSA, and Gly; (c) FTIR spectra of BSA, Gly, and BSA-Gly CuNCs; (d) TEM image of BSA-Gly CuNCs; (e) Fluorescence stability of BSA-Gly CuNCs and BSA CuNCs; (f) Salt tolerance of BSA-Gly CuNC

  Ex: excitation; Em: emission; Inset in d: particle size distribution histogram with Gaussian fit from TEM analysis.

  下载: 全尺寸图片 幻灯片

  Fig. 2b indicated no localized surface plasmon resonance (LSPR) absorption peak of Cu nanoparticles (CuNPs) at 500-600 nm, confirming the absence of larger CuNPs in the BSA-Gly CuNCs. This absence validates successful CuNCs synthesis. Moreover, an absorption peak at 285 nm suggests the presence of copper atoms in the synthesized nanoclusters^[42].

  In Fig. 2c, FTIR spectra of BSA-Gly CuNCs, BSA, and Gly revealed distinctive functional groups. BSA exhibited characteristic peaks at ca. 3 450 cm^-1 (C—OH stretching), ca. 1 650 cm^-1 (C=O stretch of protein amide ⅰ), and ca. 1 550 cm^-1 (C—N stretch coupled with N—H bending of amide ⅱ)^[43]. For Gly, the absorption peaks were mainly distributed in 2 500-3 300 cm^-1, 1 630-1 330 cm^-1, 1 100 cm^-1, 913 cm^-1, and 689 cm^-1, among which the wide absorption peaks at 2 500-3 300 cm^-1 are the asymmetric stretching vibration of NH₄⁺ and O—H, the sharp peak at 1 690 cm^-1 corresponds to C=O stretching, the 1 630 and 1 592 cm^-1 peaks are assigned to COO^- antisymmetric stretching (ν_as) and the 1 499 cm^-1 peak corresponds COO^- symmetric stretching (ν_s), showing characteristic splitting due to crystal packing effects, the 1 350 cm^-1 peak is identified as C—N stretching, while the 1 230 and 950 cm^-1 peaks originate from C—O stretching and NH₃ deformation modes, respectively. Upon conjugation with copper, shifts in these peaks were observed: the amide ⅰ band shifted to lower wavenumbers for BSA and higher wavenumbers for Gly, consistent with previous reports on BSA MNCs and glycine metal ion complexes^[44-45]. Meanwhile, the characteristic absorption peaks of amino acids at 2 125 cm^-1 disappeared in BSA-Gly CuNCs. These all showed that BSA and Gly were coordinated with copper.

  TEM images (Fig. 2d) indicated good dispersion and an average particle size of approximately 1.24 nm for BSA-Gly CuNCs. This result confirms the formation of small and well-dispersed nanoclusters. Fluorescence lifetime analysis and stability tests (Fig. 2e) demonstrated that BSA-Gly CuNCs exhibited improved stability compared to BSA CuNCs, indicating their potential for long-term applications. Fig. 2f illustrates the excellent salt tolerance of BSA-Gly CuNCs, further highlighting their robustness and suitability for applications.

  XPS was employed to analyze the elemental composition and states of the Cu in BSA CuNCs and BSA-Gly CuNCs, providing more information about their chemical states. As illustrated in Fig. 3, for BSA CuNCs, the XPS survey spectrum (Fig. 3a) showed the presence of C, N, O, and Cu. The Cu2p spectrum was deconvoluted into two peaks with binding energies of 932.6 and 952.5 eV (Fig. 3b), indicating the presence of Cu⁰2p_3/2 and Cu⁰2p_1/2^[46]. Since the difference in binding energy between Cu⁰ and Cu⁺ was only around 0.1 eV, the as-prepared CuNCs may constitute of Cu⁰ and Cu^{+ [47-48]}. Further analysis by Auger electron spectroscopy indicates the presence of Cu⁰ in CuNCs. The kinetic energy of the Auger electron was calculated by subtracting the measured peak position (567.38 eV in Fig. 3c) from the X-ray source energy (1 486.60 eV), yielding a value of 919.22 eV, which matches the standard kinetic energy of metallic copper (Cu⁰)^[49]. The peak position of the Cu²⁺2p_3/2 was approximately 942 eV, but no peak appeared at around 942 eV in Fig. 3b, indicating the absence of Cu²⁺ on the material surface^[50]. As for BSA-Gly CuNCs, the XPS survey spectrum showed the typical elements C, N, O, and Cu (Fig. 3a). The Cu2p XPS peaks of CuNCs were located at 933.1 and 953.3 eV, corresponding to Cu⁰2p_1/2 and Cu⁰2p_3/2. Notably, a comparative analysis of the binding energy shifts between BSA CuNCs and BSA-Gly CuNCs (Fig. 3b) revealed ligand-dependent variations. This phenomenon likely originates from the differential coordination interactions of copper with the functional groups present in BSA versus the BSA-Gly hybrid ligands. The observed electronic structure modulation further confirms the successful synthesis of BSA-Gly CuNCs through ligand engineering^[51].

  Figure 3

  

  Figure 3.  (a) Survey and (b) Cu2p XPS spectra of BSA CuNCs and BSA-Gly CuNCs; (c) Auger electron spectrum of copper element

  Inset in a: atomic fractions (%) of elements identified by XPS analysis.

  下载: 全尺寸图片 幻灯片

  In conclusion, the characterization of BSA-Gly CuNCs through multiple analytical techniques confirms their enhanced fluorescence properties, stability, and specific interactions with copper, making them promising candidates for various biomedical and analytical applications.

  2.2   Fluorescence sensing behavior of BSA-Gly CuNCs

  It was observed that the addition of ONZ caused a decrease in the fluorescence of BSA-Gly CuNCs and BSA CuNCs, as depicted in Fig. 4a. Notably, ONZ exhibited higher quenching efficiency towards BSA-Gly CuNCs compared to BSA CuNCs. Furthermore, the fluorescence intensity of BSA-Gly CuNCs decreased within a specific concentration range of ONZ (Fig. 4b), indicating that BSA-Gly CuNCs can serve as a fluorescence probe for the quantitative detection of ONZ.

  Figure 4

  

  Figure 4.  (a) Fluorescence spectra of BSA-Gly CuNCs and BSA CuNCs with and without ONZ; (b) Sensing ability of BSA-Gly CuNCs to ONZ; (c) UV-Vis spectra of BSA-Gly CuNCs, BSA-Gly CuNCs+ONZ, ONZ, and fluorescence spectrum of BSA-Gly CuNCs; (d) Fluorescence decay curves of BSA-Gly CuNCs and BSA-Gly CuNCs+ONZ

  Inset in a: quenching efficiencies of BSA-Gly CuNCs and BSA CuNCs to ONZ under the same conditions; The concentrations from A to F of ONZ in b were 0, 0.277, 1.64, 5.53, 11.0, and 21.7 μmol·L^-1.

  下载: 全尺寸图片 幻灯片

  To investigate the mechanism underlying the fluorescence quenching of BSA-Gly CuNCs by ONZ, fluorescence spectra, UV-Vis absorption spectra, and fluorescence lifetime tests were conducted. As shown in Fig. 4c, the absorption peaks of BSA-Gly CuNCs did not exhibit significant changes upon the addition of ONZ, and no new absorption peaks were observed in the UV-Vis absorption spectrum. This suggests that ONZ and BSA-Gly CuNCs did not form non-radiative composite ground-state species^[52]. In Fig. 4c, the absorption spectrum of ONZ overlaps with the excitation spectrum of BSA-Gly CuNCs. Consequently, in a solution where both ONZ and BSA-Gly CuNCs were present, absorption competition occurred. This competition results in the absorption of excitation light by ONZ, thereby quenching the fluorescence of BSA-Gly CuNCs. Comparison of the fluorescence lifetime attenuation curves of BSA-Gly CuNCs before and after the addition of 50 μL ONZ (1 mmol·L^-1), as shown in Fig. 4d. The fluorescence lifetime parameters (Table 1) revealed a drastic reduction in the amplitude-weighted average lifetime (τ) from 0.32 to 0.020 ns upon addition of ONZ. Here, τ represents the average time for the excited-state population to decay, calculated by weighting each lifetime component (τ₁, τ₂, τ₃) with its corresponding amplitude (A₁, A₂, A₂, expressed as a percentage of the total emission signal). The dominant contribution of shorter lifetime components after ONZ binding indicates efficient fluorescence quenching through non-radiative energy dissipation.

  Table 1

  

  Table 1.  Fitting parameters for fluorescence decay curves

  下载: 导出CSV

  Sample	τ1/ns	A1/%	τ2/ns	A2/%	τ3/ns	A3/%	τ/ns
  BSA-Gly CuNCs	0.012	24.77	0.11	54.36	0.20	20.87	0.32
  BSA-Gly CuNCs+ONZ	0.007 0	71.89	0.017	28.11			0.020

  Such a decrease in fluorescence lifetime typically indicates dynamic quenching^[53]. Based on the above findings, it can be concluded that the quenching mechanism of ONZ toward BSA-Gly CuNCs involves absorption competition and dynamic quenching.

  2.3   Optimization of experimental conditions

  To enhance the response of BSA-Gly CuNCs to ONZ and improve the sensitivity of the determination, various determination conditions, such as pH, buffer type, buffer amount, temperature, and time, were thoroughly investigated and optimized.

  Initially, Britton-Robinson (BR) buffer solutions with pH ranging from 4.10 to 8.36 were prepared separately at room temperature. A 1.00 mL aliquot of BSA-Gly CuNCs was taken and diluted with BR buffers of different pH values in a series of 20.00 mL colorimetric tubes. Then, 2.00 mL of the diluted BSA-Gly CuNCs solutions were taken into the cuvette, and fluorescence intensities were measured immediately. Subsequently, 50 μL of ONZ (1 mmol·L^-1) were added respectively, and the fluorescence intensity of the solution was measured to calculate the fluorescence quenching efficiency. The results indicated that the highest quenching efficiency was achieved at pH=7.00 (Fig. 5a). Although the isoelectric points of BSA and Gly were 4.70 and 6.00^[54-55], respectively, the fluorescence intensity of the fluorescent probes was most sensitive around pH=7.00, while it was insensitive at pH ranges of 4.10-6.37 and 7.24-8.36.

  Figure 5

  

  Figure 5.  Optimizing experimental conditions for BSA-Gly CuNCs detecting ONZ: (a) pH value; (b) type of buffer solution; (c) volume of buffer solution; (d) temperature; (e) reaction time

  The bar chart from 1 to 7 was Barbitone sodium-HCl, H₂O, PBS, Na₂HPO₄-CA, Na₂HPO₄-NaH₂PO₄, Na₂HPO₄-KH₂PO₄, and KH₂PO₄-NaOH, respectively.

  下载: 全尺寸图片 幻灯片

  Then, BSA-Gly CuNCs were diluted with different types of buffer solutions (pH=7.00, ultrapure water, Barbitone sodium-HCl, PBS, Na₂HPO₄-CA, Na₂HPO₄-NaH₂PO₄, Na₂HPO₄-KH₂PO₄, KH₂PO₄-NaOH) at the same dilution ratio, and the fluorescence intensities were measured before and after the adding of ONZ. The quenching efficiency was determined using the same method, and it was concluded that the effect of Na₂HPO₄-CA was the best (Fig. 5b).

  Subsequently, the optimal dosage of the buffer solution was determined at room temperature. A mixture of 1.00 mL of BSA-Gly CuNCs and different volumes of Na₂HPO₄-CA buffer solution (pH=7.00) was diluted with ultrapure water to a total volume of 20.00 mL. The fluorescence intensity before and after the addition of ONZ (1 mmol·L^-1) was measured, and the fluorescence quenching efficiency was calculated. The results showed that the highest quenching efficiency was obtained when 10.00 mL of the buffer solution was added (Fig. 5c).

  Finally, the interaction temperature and time of ONZ with BSA-Gly CuNCs were optimized. BSA-Gly CuNCs were diluted with Na₂HPO₄-CA using the aforementioned method, and the quenching efficiency was subsequently measured following the addition of ONZ under systematically varied temperature conditions (25-60 ℃) and incubation time intervals (0-120 min). It was observed that the quenching efficiency was highest at room temperature (Fig. 5d). However, the efficiency decreased as the temperature increased, possibly due to the structural changes in BSA-Gly CuNCs caused by high temperatures^[56]. Moreover, the degree of fluorescence quenching remained almost unchanged with increased incubation time (Fig. 5e), indicating a fast ONZ detection process.

  2.4   Sensing performance of BSA-Gly CuNCs to ONZ

  The sensitivity of the method was tested under optimized conditions in a Na₂HPO₄-CA buffer solution (pH=7) at room temperature, with a 1∶10 volume ratio of BSA-Gly CuNCs to buffer solution, followed by immediate measurement. As shown in Fig. 6a, the fluorescence intensity of BSA-Gly CuNCs continuously decreased with increasing ONZ concentration. Within the range of 0.28 to 52.60 μmol·L^-1, the quenching efficiency exhibited a linear correlation with ONZ concentration (Fig. 6b). The linear regression equations were ΔI/I₀=7 411.21c+0.149 19 (R²=0.996 0) over the range of 0.28-10.99 μmol·L^-1 and ΔI/I₀=19 474.56c+0.007 31 (R²=0.998 7) at 10.99-52.60 μmol·L^-1, with a detection limit (LOD) of 0.069 μmol·L^-1 in Fig. 6b. The LOD was calculated by the formula LOD=3σ/k, where σ is the standard deviation of the blank and k is the slope of the calibration curve. Table 2 summarizes the reported methods for ONZ detection along with their corresponding LOD.

  Figure 6

  

  Figure 6.  (a) Fluorescence spectra of BSA-Gly CuNCs response to concentration of ONZ; (b) Linear relationship between ΔI/I₀ and concentration of ONZ; (c) Selectivity test results of BSA-Gly CuNCs for detection of ONZ

  The concentrations of ONZ were from 0, 2.33, 7.32, 12.50, 16.70, 23.65, 31.87, 37.36, 41.45, 48.76, to 52.60 μmol·L^-1.

  下载: 全尺寸图片 幻灯片

  Table 2

  

  Table 2.  Comparison of different analytic methods for the detection of ONZ

  下载: 导出CSV

  Method	Linear concentration range/(μmol·L-1)	LOD/(μmol·L-1)	Ref.
  CE	4.56-2 374.42	3.20	[59]
  Linear sweep voltammetry (LSV)	55.66-419.31	4.47	[60]
  Reversed-phase HPLC	0-228.31	4.68	[40]
  HPLC	2.83-228.31	0.91	[42]
  Zn-MOF	n.d.*	0.016	[61]
  Fluorescence	0.28-52.60	0.069	This work

  Additionally, the selectivity of the method was investigated. Under optimal testing conditions, the selectivity was evaluated by adding 50 μL each of 0.001 mol·L^-1 ONZ and 0.01 mol·L^-1 potential interferents (including ions, pharmaceutical auxiliaries, and pharmaceutical molecules) to the detection system, followed by fluorescence intensity measurement. The results shown in Fig. 6c demonstrated that BSA-Gly CuNCs had no noteworthy response to K⁺, Pb²⁺, Co²⁺, NO₃^-, S₂O₈^2-, starch, glucose (Glu), lincomycin hydrochloride (LHC), benzylpenicillin sodium (BPS), roxithromycin (ROX), and chloramphenicol (CAM). At the same time, it was found that molecules similar in structure to ONZ, such as metronidazole (MNZ) and dimetridazole (DMZ), can also produce fluorescence quenching. Since ONZ, MNZ, and DMZ belong to the class of nitroimidazole antibiotics and only one of these medications is used at a time^[57-58], their interference can be ignored in the actual determination. Furthermore, the measured response for a mixture of 0.001 mol·L^-1 ONZ and 0.01 mol·L^-1 of all the above interferents other than MNZ and DMZ was similar to that of ONZ alone. These results indicated that BSA-Gly CuNCs had good selectivity for ONZ.

  2.5   Sample analysis

  The accuracy and potential applications of the proposed method were validated through the determination of ONZ in real samples. ONZ tablets and ONZ in sodium chloride injection were chosen as samples for analysis, and their ONZ content was measured. A spiked recovery test was conducted by adding ONZ at different concentrations (10.70, 21.16, 36.46 μmol·L^-1). The results, detailed in Table 3, showed that the recoveries of the samples ranged from 95.6% to 103.5%, with a relative standard deviation (RSD) between 3.8% and 8.7%. These findings indicate that the method is well-suited for analyzing ONZ in authentic samples.

  Table 3

  

  Table 3.  ONZ detection in real samples

  下载: 导出CSV

  Sample	Specified mass	Measured mass/g	Spiked concentration/(μmol·L-1)	Found/(μmol·L-1)	Recovery/%	RSD (n=3)/%
  Tablet	0.25 g per tablet	0.24	10.70	10.23	95.6	5.4
  			21.16	20.78	98.2	7.5
  			36.46	37.74	103.5	3.8
  Injection	0.50 g per 100.00 mL	0.49	10.70	10.42	97.4	4.2
  			21.16	20.93	98.9	8.7
  			36.46	37.48	102.8	7.4

  3.   Conclusions

  In summary, dual-ligand-modified BSA-Gly CuNCs were successfully synthesized using a simple one-pot method. The introduction of Gly effectively enhanced the fluorescence intensity, stability, and sensing ability of the BSA CuNCs. Utilizing the BSA-Gly CuNCs, a sensitive and selective fluorescence method for detecting ONZ was developed. This method was applied to determine ONZ in real samples and can potentially provide a new approach for the simple and rapid determination of ONZ, as well as find applications in other assays. Additionally, it offers a new methodology for the design and preparation of MNCs.

  
  
• 1. [1]

     DU R B, BA K, YANG Y X, ZHAO Y Y, LIN Y P. Efficacy of ornidazole for pericoronitis: A meta-analysis and systematic review[J]. Arch. Med. Sci., 2024, 20(1):  189-195. doi: 10.5114/aoms/171907

  2. [2]

     LI S C, CAO M, ZHOU Y, SHU C, WANG Y. Ornidazole transfer into colostrum and assessment of exposure risk for breastfeeding infant: A population pharmacokinetic analysis. pharmaceutics[J]. Pharmaceutics, 2023, 15(11):  2524. doi: 10.3390/pharmaceutics15112524

  3. [3]

     HARIKA S C, KUMAR Y S, RAO Y M, SRIRAM P, SHANKAR U. Design and evaluation of sustained release of ornidazole by dental inserts[J]. Curr. Drug Metab., 2021, 22(7):  572-580. doi: 10.2174/1389200222666210222152940

  4. [4]

     GÓMEZ-MUÑOZ M T, GÓMEZ-MOLINERO M Á, GONZÁLEZ F, AZAMI-CONESA I, BAILÉN M, GARCÍA PIQUERAS M. Avian oropharyngeal trichomonosis: Treatment, failures and alternatives, a systematic review[J]. Microorganisms, 2022, 10(11):  2297. doi: 10.3390/microorganisms10112297

  5. [5]

     ETTADILI F E, AZRIOUIL M, MATROUF M, TAHIRI ALAOUI O, LAGHRIB F, FARAHI A. Materials framework based biosensors for the detection of ornidazole and metronidazol antibiotics in environment and foodstuffs[J]. Inorg. Chem. Commun., 2022, 140:  109416. doi: 10.1016/j.inoche.2022.109416

  6. [6]

     MANCIU F S, GUERRERO J, PENCE B C, MARTINEZ LOPEZ L V. Assessment of drug activities against giardia using hyperspectral Raman microscopy[J]. Pathogens, 2024, 13(5):  358. doi: 10.3390/pathogens13050358

  7. [7]

     MEMIŞ A C, ALCAN S Y, TEMIZ S G, BAŞAR F, ARSLAN K. Managing unpredictable challenge of a liver injury in ornidazole use: A case report[J]. Med. Rep., 2024, 6:  100097.

  8. [8]

     ZHANG L Y, ZHANG Z J, WU K. In vivo and real time determination of ornidazole and tinidazole and pharmacokinetic study by capillary electrophoresis with microdialysis[J]. J. Pharm. Biomed. Anal., 2006, 41(4):  1453-1457. doi: 10.1016/j.jpba.2006.03.016

  9. [9]

     SEE K L, ELBASHIR A A, SAAD B, ALI A S M, ABOUL-ENEIN H Y. Simultaneous determination of ofloxacin and ornidazole in pharmaceutical preparations by capillary zone electrophoresis[J]. Biomed. Chromatogr., 2009, 23(12):  1283-1290. doi: 10.1002/bmc.1251

  10. [10]

      BABU N R D. Development and validation of stability indicating RP-HPLC method for quantitative estimation of ornidazole and its impurities in ornidazole injection[J]. Res. J. Pharm. Technol., 2022, 25(2):  82-88.

  11. [11]

      AGRAWAL G P, MAHESHWARI R K, MISHRA P. Validation of ultra performance liquid chromatography-tandem mass spectrometry coupled with electrospray ionization method for quantitative determination of ornidazole in solid dispersion[J]. Curr. Pharm. Anal., 2020, 16(5):  487-493. doi: 10.2174/1573412914666181024145937

  12. [12]

      KELANI K M, GAD A G, FAYEZ Y M, MAHMOUD A M, ABDEL-RAOOF A M. Three developed spectrophotometric methods for determination of a mixture of ofloxacin and ornidazole; Application of greenness assessment tools[J]. BMC Chem., 2023, 17(1):  16. doi: 10.1186/s13065-023-00932-3

  13. [13]

      KUMAR VASHISTHA V, BALA R, VSR PULLABHOTLA R. Derivatizing agents for spectrophotometric and spectrofluorimetric determination of pharmaceuticals: A review[J]. J. Taibah Univ. Sci., 2023, 17(1):  2206363. doi: 10.1080/16583655.2023.2206363

  14. [14]

      EL HAYAOUI W, TAJAT N, RADAA C, BOUGDOUR N, ZOUBIR J, IDELAHCEN A. In situ preparation of eggshell@Ag nanocomposite electrode for highly sensitive detection of antibiotic drug ornidazole in water sample[J]. Nanotechnol. Environ. Eng., 2022, 7(3):  635-646. doi: 10.1007/s41204-022-00272-y

  15. [15]

      WANG H X, BO X J, ZHOU M, GUO L P. DUT-67 and tubular polypyrrole formed a cross-linked network for electrochemical detection of nitrofurazone and ornidazole[J]. Anal. Chim. Acta, 2020, 1109:  1-8. doi: 10.1016/j.aca.2020.03.002

  16. [16]

      ZHAO L, LI J Y, LI Y, WANG T G, JIN X L, WANG K, RAHMAN E, XING Y, JI B P, ZHOU F. Preparation of monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for ornidazole detection[J]. Food Chem., 2017, 229:  439-444. doi: 10.1016/j.foodchem.2017.02.100

  17. [17]

      RAWAT A, KANZARIYA D B, LAMA P, PAL T K. A Zn²⁺ coordination polymer as a dual sensor for ppb level detection of antibiotics and organo-toxins in a green solvent[J]. Spectroc. Acta Pt. A‒Molec. Biomolec. Spectr., 2023, 295:  122579. doi: 10.1016/j.saa.2023.122579

  18. [18]

      MAGDY G, ABOELKASSIM E, EL-DOMANY R A, BELAL F. Green synthesis, characterization, and antimicrobial applications of silver nanoparticles as fluorescent nanoprobes for the spectrofluori-metric determination of ornidazole and miconazole[J]. Sci. Rep., 2022, 12(1):  21395.

  19. [19]

      SONG X M, HOU X F, DANG M X, ZHAO Q X, LIU S, MA Z H, REN Y X. Design and preparation of a multi-responsive Cd-based fluorescent coordination polymer for smart sensing of nitrobenzene and ornidazole[J]. Spectroc. Acta Pt. A ‒ Molec. Biomolec. Spectr., 2024, 320:  124656.

  20. [20]

      BAO H L, LIU Y H, LI H, QI W X, SUN K Y. Luminescence of carbon quantum dots and their application in biochemistry[J]. Heliyon, 2023, 9(10):  e20317.

  21. [21]

      LEONG C Y, WAHAB R A, LEE S L, PONNUSAMY V K, CHEN Y H. Current perspectives of metal-based nanomaterials as photocatalytic antimicrobial agents and their therapeutic modes of action: A review[J]. Environ. Res., 2023, 227:  115578.

  22. [22]

      PEI G X, ZHANG L L, SUN X Y. Recent advances of bimetallic nanoclusters with atomic precision for catalytic applications[J]. Coord. Chem. Rev., 2024, 506:  215692.

  23. [23]

      KAWAWAKI T, EBINA A, HOSOKAWA Y, OZAKI S, SUZUKI D, HOSSAIN S. Thiolate-protected metal nanoclusters: Recent development in synthesis, understanding of reaction, and application in energy and environmental field[J]. Small, 2021, 17(27):  2005328.

  24. [24]

      MATUS M F, HÄKKINEN H. Understanding ligand-protected noble metal nanoclusters at work[J]. Nat. Rev. Mater., 2023, 8(6):  372-389.

  25. [25]

      BURRATTI L, CIOTTA E, BOLLI E, KACIULIS S, CASALBONI M, DE MATTEIS F, GARZON-MANJON A, SCHEU C, PIZZOFERRATO R, PROSPOSITO P. Fluorescence enhancement induced by the interaction of silver nanoclusters with lead ions in water[J]. Colloid Surf. A-Physicochem. Eng. Asp., 2019, 579:  123634.

  26. [26]

      XUE R, GENG X, LIANG F, LIU Y, YANG W, HUANG Z. Natural plant compounds in synthesis and luminescence modulation of metal nanoclusters: Toward sustainable nanoprobes for sensing and bioimaging[J]. Mater. Today Adv., 2022, 16:  100279. doi: 10.1016/j.mtadv.2022.100279

  27. [27]

      ZOU X J, KANG X, ZHU M Z. Recent developments in the investigation of driving forces for transforming coinage metal nanoclusters[J]. Chem. Soc. Rev., 2023, 52(17):  5892-5967. doi: 10.1039/D2CS00876A

  28. [28]

      YANG J, YANG F, ZHANG C S, HE X B, JIN R C. Metal nanoclusters as biomaterials for bioapplications: Atomic precision as the next goal[J]. ACS Mater. Lett., 2022, 4(7):  1279-1296. doi: 10.1021/acsmaterialslett.2c00237

  29. [29]

      NIE Y M, TAO X L, ZHANG H W, CHAI Y Q, YUAN R. Self-assembly of gold nanoclusters into a metal-organic framework with efficient electrochemiluminescence and their application for sensitive detection of rutin[J]. Anal. Chem., 2021, 93(7):  3445-3451. doi: 10.1021/acs.analchem.0c04682

  30. [30]

      XIE B, DING B S, MAO P, WANG Y, LIU Y N, CHEN M R, ZHOU C J, WEN H M, XIA S J, HAN M, PALMER R E, WANG G G, HU J. Metal nanocluster-metal organic framework-polymer hybrid nanomaterials for improved hydrogen detection[J]. Small, 2022, 18(23):  2200634. doi: 10.1002/smll.202200634

  31. [31]

      XU J, LI J M, ZHONG W C, WANG M Y, SUKHUROROKOV G, LI S. The density of surface ligands regulates the luminescence of thiolated gold nanoclusters and their metal ion response[J]. Chin. Chem. Lett., 2021, 32(8):  2390-2394. doi: 10.1016/j.cclet.2021.02.037

  32. [32]

      JIANG M Z, XU X Y, LIU S, LIU L L, WANG X M, JIANG H. Enhancement of nanozyme activity by second ligand modification on glutathione protected gold nanoclusters for regulation of intracellular oxidative stress[J]. Inorg. Chem. Commun., 2024, 161:  112115. doi: 10.1016/j.inoche.2024.112115

  33. [33]

      ZHAO P, XU L J, LI B H, ZHAO Y F, ZHAO Y S, LU Y, CAO M H, LI G Q, WENG T C, WANG H, ZHENG Y J. Non-equilibrium assembly of atomically-precise copper nanoclusters[J]. Adv. Mater., 2024, 36(28):  2311818. doi: 10.1002/adma.202311818

  34. [34]

      ZHANG L L, WONG W Y. Atomically precise copper nanoclusters as ultrasmall molecular aggregates: Appealing compositions, structures, properties, and applications[J]. Aggregate, 2023, 4(1):  e266. doi: 10.1002/agt2.266

  35. [35]

      OUYANG X Y, WANG M F, GUO L J, CUI C J, LIU T, REN Y G, ZHAO Y, GUO X N, XIE G, LI J, FAN C H, WANG L H. DNA nanoribbon-templated self-assembly of ultrasmall fluorescent copper nanoclusters with enhanced luminescence[J]. Angew. Chem.‒Int. Edit., 2020, 59(29):  11836-11844. doi: 10.1002/anie.202003905

  36. [36]

      BARNWAL N H, NANDI N, SARKAR P, SAHU K. White light emission from Zn²⁺ and DMSO-induced copper nanocluster assembly[J]. Chem.‒Asian J., 2024, 9(9):  e202400633.

  37. [37]

      HUANG X, ZHAO H N, QIU W, WANG J, GUO L H, LIN Z Y, PAN W, WU Y, QIU B. A fluorescence signal amplification strategy for modification-free ratiometric determination of tyrosinase in situ based on the use of dual-templated copper nanoclusters[J]. Microchim. Acta, 2020, 240(4):  240.

  38. [38]

      PU S, XIA C Y, WU L, XU K L. CuNCs modified with dual-ligand to achieve fluorescence visualization detection of Tin (Ⅳ)[J]. Microchem. J., 2022, 183(20):  108086.

  39. [39]

      FAN Y, YU W H, LIAO Y W, JIANG X H, WANG Z H, CHENG Z J. Ratiometric detection of doxycycline in pharmaceutical based on dual ligands-enhanced copper nanoclusters[J]. Spectroc. Acta Pt. A‒Molec. Biomolec. Spectr., 2022, 267:  120509. doi: 10.1016/j.saa.2021.120509

  40. [40]

      BİLKAY M, KARA H. Fluorometric determination of ornidazole by using BSA coated copper nanoclusters as a novel turn off sensor[J]. Turk. J. Chem., 2022, 46(2):  475-486. doi: 10.55730/1300-0527.3321

  41. [41]

      NAWARA K, WALUK J. Improved method of fluorescence quantum yield determination[J]. Anal. Chem., 2017, 89(17):  8650-8655. doi: 10.1021/acs.analchem.7b02013

  42. [42]

      KALIA A, KAUR G. Biosynthesis of nanoparticles using mushrooms[M]//SINGH B, LALLAWMSANGA, PASSARI A. Biology of Macrofungi. Fungal Biology. [S. l.]: Springer, Cham, 2018.

  43. [43]

      BARTH A. Infrared spectroscopy of proteins[J]. Biochim. Biophys. Acta, 2007, 1767(9):  1073-1101. doi: 10.1016/j.bbabio.2007.06.004

  44. [44]

      DING C F, XU Y J, ZHAO Y N, ZHONG H, LUO X L. Fabrication of BSA@AuNC-based nanostructures for cell fluoresce imaging and target drug delivery[J]. ACS Appl. Mater. Interfaces, 2018, 10(10):  8947-8954. doi: 10.1021/acsami.7b18493

  45. [45]

      LE GUÉVEL X, HÖTZER B, JUNG G, HOLLEMEYER K, TROUILLET V, SCHNEIDER M. Formation of fluorescent metal (Au, Ag) nanoclusters capped in bovine serum albumin followed by fluorescence and spectroscopy[J]. J. Phys. Chem. C, 2011, 115(22):  10955-10963. doi: 10.1021/jp111820b

  46. [46]

      WANG L, MIAO H, ZHONG D, YANG X M. Synthesis of dopamine-mediated Cu nanoclusters for sensing and fluorescent coding[J]. Anal. Methods, 2016, 8(1):  40-44. doi: 10.1039/C5AY02494C

  47. [47]

      VILAR-VIDAL N, BLANCO M C, LOPEZ-QUINTELA M A. Electrochemical synthesis of very stable photoluminescent copper cluster[J]. J. Phys. Chem. B, 2010, 114(38):  15924-15930.

  48. [48]

      HAN A, YANG Y, ZHANG Q, TU Q, FANG G, LIU J, WANG S, LI R. Electrochemistry and electrochemiluminescence of copper metal cluster[J]. J. Electroanal. Chem., 2017, 795:  116-122. doi: 10.1016/j.jelechem.2017.04.058

  49. [49]

      杨斌, 杨防祖, 黄令, 许书楷, 姚光华, 周绍民. 2, 2-联吡啶在化学镀铜中的作用研究[J]. 电化学, 2007,13,(4): 425-430. YANG B, YANG F Z, HUANG L, XU S K, YAO G H, ZHOU S M. Study on the role of 2, 2-bipyridine in chemical copper plating[J]. Electrochemistry, 2007, 13(4):  425-430.

  50. [50]

      ZHAO M, CHEN A Y, HUANG D, ZHUO Y, CHAI Y Q, YUAN R. Cu nanoclusters: Novel electrochemiluminescence emitters for bioanalysis[J]. Anal. Chem., 2016, 88:  11527-11532. doi: 10.1021/acs.analchem.6b02770

  51. [51]

      BALOGH L, VALLUZZI R, LAVERDURE K S, GIDO S P, HAGNAUER G L, TOMALIA D A. Poly (amidoamine) dendrimer-templated nanocomposites. 1. Synthesis of zerovalent copper nanoclusters[J]. J. Am. Chem. Soc., 1998, 120(29):  7355-7356. doi: 10.1021/ja980861w

  52. [52]

      PAN Y, WEI X L. A novel FRET immunosensor for rapid and sensitive detection of dicofol based on bimetallic nanoclusters[J]. Anal. Chim. Acta, 2022, 1224:  340235. doi: 10.1016/j.aca.2022.340235

  53. [53]

      MATOSSI F. Theory of dynamic quenching of photoconductivity and luminescence[J]. J. Electrochem. Soc., 1956, 103(12):  662-667. doi: 10.1149/1.2430187

  54. [54]

      XU Y L, SHERWOOD J, QIN Y, CROWLEY D, BONIZZONI M, BAO Y. The role of protein characteristics in the formation and fluorescence of Au nanoclusters[J]. Nanoscale, 2014, 6(3):  1515-1524. doi: 10.1039/C3NR06040C

  55. [55]

      ANDREWS B, TORRIE B H, POWELL B M. Intermolecular potentials for alpha-glycine from Raman and infrared scattering measurements[J]. Biophys. J., 1983, 41(3):  293-298. doi: 10.1016/S0006-3495(83)84441-5

  56. [56]

      TIAN L, LI Y F, REN T T, TONG Y L, YANG B S, LI Y Q. Novel bimetallic gold-silver nanoclusters with "Synergy"-enhanced fluorescence for cyanide sensing, cell imaging and temperature sensing[J]. Talanta, 2017, 170:  530-539. doi: 10.1016/j.talanta.2017.03.107

  57. [57]

      THULKAR J, KRIPLANI A, AGARWAL N. A comparative study of oral single dose of metronidazole, tinidazole, secnidazole and ornidazole in bacterial vaginosis[J]. Indian J. Pharmacol., 2012, 44(2):  243-245. doi: 10.4103/0253-7613.93859

  58. [58]

      KURT Ö, GIRGINKARDESLER N, BALCIOGLU I C, ÖZBILGIN A, OK Ü Z. A comparison of metronidazole and single-dose ornidazole for the treatment of dientamoebiasis[J]. Clin. Microb. Infect., 2008, 14(6):  601-604. doi: 10.1111/j.1469-0691.2008.02002.x

  59. [59]

      LEI M Y, WANG X H, ZHANG T J, SHI Y, WEN J H, ZHANG Q F. Homochiral Eu³⁺@MOF composite for the enantioselective detection and separation of (R/S)-ornidazole[J]. Inorg. Chem., 2022, 61(18):  6764-6772. doi: 10.1021/acs.inorgchem.1c03695

  60. [60]

      CARTER D C, HO J X. Structure of serum albumin[J]. Adv. Protein Chem., 1994, 45:  153-203.

  61. [61]

      陈小莉, 刘露, 商璐, 蔡苗, 崔华莉, 杨华, 王记江. 一种高灵敏、多响应的Zn-MOF荧光传感器对Fe³⁺、2, 4, 6-三硝基苯酚和奥硝唑的检测[J]. 无机化学学报, 2022,38,(4): 735-744. CHEN X L, LIU L, SHANG L, CAI M, CUI H L, YANG H, WANG J J. A highly sensitive and multi-responsive Zn-MOF fluorescent sensor for detection of Fe³⁺, 2, 4, 6-trinitrophenol, and ornidazole[J]. Chinese J. Inorg. Chem., 2022, 38(4):  735-744.

• 

  Figure 1  Schematic illustration of BSA-Gly CuNCs for the detection of ONZ

  The green circular area represents the green fluorescence emitted by the CuNCs.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  (a) Fluorescence spectra of BSA-Gly CuNCs and BSA CuNCs; (b) UV-Vis spectra of BSA-Gly CuNCs, BSA CuNCs, BSA, and Gly; (c) FTIR spectra of BSA, Gly, and BSA-Gly CuNCs; (d) TEM image of BSA-Gly CuNCs; (e) Fluorescence stability of BSA-Gly CuNCs and BSA CuNCs; (f) Salt tolerance of BSA-Gly CuNC

  Ex: excitation; Em: emission; Inset in d: particle size distribution histogram with Gaussian fit from TEM analysis.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  (a) Survey and (b) Cu2p XPS spectra of BSA CuNCs and BSA-Gly CuNCs; (c) Auger electron spectrum of copper element

  Inset in a: atomic fractions (%) of elements identified by XPS analysis.

  下载: 全尺寸图片 幻灯片
  

  Figure 4  (a) Fluorescence spectra of BSA-Gly CuNCs and BSA CuNCs with and without ONZ; (b) Sensing ability of BSA-Gly CuNCs to ONZ; (c) UV-Vis spectra of BSA-Gly CuNCs, BSA-Gly CuNCs+ONZ, ONZ, and fluorescence spectrum of BSA-Gly CuNCs; (d) Fluorescence decay curves of BSA-Gly CuNCs and BSA-Gly CuNCs+ONZ

  Inset in a: quenching efficiencies of BSA-Gly CuNCs and BSA CuNCs to ONZ under the same conditions; The concentrations from A to F of ONZ in b were 0, 0.277, 1.64, 5.53, 11.0, and 21.7 μmol·L^-1.

  下载: 全尺寸图片 幻灯片
  

  Figure 5  Optimizing experimental conditions for BSA-Gly CuNCs detecting ONZ: (a) pH value; (b) type of buffer solution; (c) volume of buffer solution; (d) temperature; (e) reaction time

  The bar chart from 1 to 7 was Barbitone sodium-HCl, H₂O, PBS, Na₂HPO₄-CA, Na₂HPO₄-NaH₂PO₄, Na₂HPO₄-KH₂PO₄, and KH₂PO₄-NaOH, respectively.

  下载: 全尺寸图片 幻灯片
  

  Figure 6  (a) Fluorescence spectra of BSA-Gly CuNCs response to concentration of ONZ; (b) Linear relationship between ΔI/I₀ and concentration of ONZ; (c) Selectivity test results of BSA-Gly CuNCs for detection of ONZ

  The concentrations of ONZ were from 0, 2.33, 7.32, 12.50, 16.70, 23.65, 31.87, 37.36, 41.45, 48.76, to 52.60 μmol·L^-1.

  下载: 全尺寸图片 幻灯片

  Table 1.  Fitting parameters for fluorescence decay curves

  Sample	τ1/ns	A1/%	τ2/ns	A2/%	τ3/ns	A3/%	τ/ns
  BSA-Gly CuNCs	0.012	24.77	0.11	54.36	0.20	20.87	0.32
  BSA-Gly CuNCs+ONZ	0.007 0	71.89	0.017	28.11			0.020

  下载: 导出CSV

  Table 2.  Comparison of different analytic methods for the detection of ONZ

  Method	Linear concentration range/(μmol·L-1)	LOD/(μmol·L-1)	Ref.
  CE	4.56-2 374.42	3.20	[59]
  Linear sweep voltammetry (LSV)	55.66-419.31	4.47	[60]
  Reversed-phase HPLC	0-228.31	4.68	[40]
  HPLC	2.83-228.31	0.91	[42]
  Zn-MOF	n.d.*	0.016	[61]
  Fluorescence	0.28-52.60	0.069	This work

  下载: 导出CSV

  Table 3.  ONZ detection in real samples

  Sample	Specified mass	Measured mass/g	Spiked concentration/(μmol·L-1)	Found/(μmol·L-1)	Recovery/%	RSD (n=3)/%
  Tablet	0.25 g per tablet	0.24	10.70	10.23	95.6	5.4
  			21.16	20.78	98.2	7.5
  			36.46	37.74	103.5	3.8
  Injection	0.50 g per 100.00 mL	0.49	10.70	10.42	97.4	4.2
  			21.16	20.93	98.9	8.7
  			36.46	37.48	102.8	7.4

  下载: 导出CSV
•