Method for detecting enrofloxacin based on composite nanocarbon dot-gold cluster new material ratio type fluorescence and application

Abstract

The application discloses a method for detecting enrofloxacin based on composite nanometer carbon dot-gold cluster new material ratio type fluorescence and application, and belongs to the field of advanced non-ferrous metal nanometer new materials and new material detection technology. The composite nanometer carbon dot-gold cluster new material AuNCs@CDs can be prepared by mixing gold nanometer cluster seeds and ascorbic acid in a certain proportion and microwave heating; under the same excitation wavelength, the AuNCs@CDs has two emission peaks of 440 nm and 640 nm. Different concentrations of enrofloxacin are added into the AuNCs@CDs solution, the fluorescence intensity ratio I 440 / I 640 of the AuNCs@CDs is measured; and a standard curve is drawn according to the relationship between the enrofloxacin solution concentration and the fluorescence intensity ratio of the AuNCs@CDs solution, so that the enrofloxacin concentration can be detected. In addition, the method can be applied to the experimental recovery rate detection of enrofloxacin in actual samples. The method has high sensitivity and selectivity, is simple and rapid, does not need any pretreatment, and shows extremely broad application prospects in many fields such as biomedical detection, environmental monitoring, food safety analysis and the like.

Description

Technical Field

[0001] This invention belongs to the field of advanced non-ferrous metal nanomaterials and new material detection technology, specifically involving a method and application of ratiometric fluorescence detection of enrofloxacin based on composite nanocarbon dots-gold clusters. Background Technology

[0002] With the continuous development of society, the public's attention to food safety and nutritional balance is increasing. Along with the growing public demand for food, antibiotics are widely used to prevent and treat animal diseases and increase food production. As a third-generation fluoroquinolone broad-spectrum antibiotic, the extensive use of enrofloxacin has led to its residues entering the food chain and environment, posing a significant potential threat to ecosystems and human health (Microchim. Acta, 2021, 188: 194.).

[0003] The main methods for detecting enrofloxacin include electrochemical methods, ultra-high performance liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay (ELISA), molecular imprinting, and fluorescence immunoassay (Biosens. Bioelectron., 2017, 87: 203). These methods have good sensitivity and accuracy, but they still suffer from drawbacks such as long detection times, expensive equipment, and cumbersome operation, limiting their application in rapid, on-site detection of enrofloxacin residues (Spectrochim. Acta A, 2023, 301:122985). Therefore, developing a rapid, sensitive, simple, and reliable method for detecting enrofloxacin is of great significance.

[0004] Nanomaterials have wide applications in fluorescence sensing, but most fluorescent probes currently built with nanomaterials are based on a single change in fluorescence emission intensity (fluorescence enhancement or quenching). These probes are often susceptible to interference from background noise, instrumentation, matrix, or probe concentration, which can affect the accuracy and sensitivity of detection (NanoRes., 2023, 16: 1064). Ratiometric fluorescent probes can effectively overcome these problems, improving the sensitivity and reliability of analytical detection.

[0005] Gold nanoclusters (AuNCs) and carbon dots (CDs) are star nanomaterials in the field of fluorescence sensing, exhibiting significant advantages in constructing sensing methods. By cleverly integrating gold nanoclusters with restaurant exploration, and leveraging the respective strengths of both, a dual-emission ratio fluorescence sensing method can be constructed, demonstrating extremely broad application prospects in numerous fields such as biomedical detection, environmental monitoring, and food safety analysis. Summary of the Invention

[0006] One objective of this invention is to address the shortcomings of the aforementioned methods by providing a ratiometric fluorescence detection method for enrofloxacin based on a novel composite carbon nanodot-gold cluster material. This invention applies the preparation of a novel composite carbon nanodot-gold cluster material to the detection of enrofloxacin, constructing a simple method for detecting enrofloxacin based on this new material. This method is efficient, accurate, and rapid, enabling quantitative analysis and detection of trace amounts of enrofloxacin.

[0007] The method for detecting enrofloxacin provided by this invention includes the following steps:

[0008] (1) Gold nanocluster seeds were synthesized by water bath method using chloroauric acid HAuCl4 and glutathione GSH as raw materials;

[0009] (2) A novel composite nanocarbon dot-gold cluster material AuNCs@CDs was synthesized by microwave method using gold nanocluster seeds and ascorbic acid as raw materials;

[0010] (3) Weigh out AuNCs@CDs solid quantitatively to prepare AuNCs@CDs solution;

[0011] (4) Add enrofloxacin solution to AuNCs@CDs solution. After reaction, record the corresponding fluorescence emission spectrum and calculate the ratio I of the fluorescence intensity of the two emission peaks at 440 nm and 640 nm of AuNCs@CDs. 440 / I 640 The change value;

[0012] (5) Based on the ratio of enrofloxacin solution concentration to AuNCs@CDs solution fluorescence intensity I 440 / I 640 By plotting a standard curve based on the relationship between the levels of enrofloxacin and the concentration of enrofloxacin, it is possible to detect the concentration of enrofloxacin.

[0013] Specifically, the method for synthesizing gold nanocluster seeds in step (1) is as follows: 5.00 mL of 20 mM chloroauric acid solution is mixed with 1.50 mL of 100 mM glutathione solution, and heated and stirred in a water bath at 80°C for 2 h; after the reaction is completed, a gold nanocluster seed solution is obtained.

[0014] Specifically, the synthesis method of AuNCs@CDs in step (2) is as follows: 40 mg of ascorbic acid is added to 10.00 mL of the gold nanocluster seed solution in step (1), and microwaved on high for 8 minutes; after the reaction is completed, 10 mL of distilled water is added to resuspend the reactants; the resuspension is filtered and dialyzed for 24 hours with a dialysis bag with a molecular weight cutoff of 1000 Da to obtain a pure AuNCs@CDs solution; the pure AuNCs@CDs solution is subjected to vacuum freeze-drying to finally obtain AuNCs@CDs solid powder.

[0015] Specifically, the final concentration of AuNCs@CDs in step (3) is 0.025 mg / mL.

[0016] Specifically, the pH of the AuNCs@CDs solution described in step (3) is 6.

[0017] Specifically, the reaction time of enrofloxacin and AuNCs@CDs in step (4) is 2 minutes.

[0018] Specifically, the standard curve mentioned in step (5) is I. 440 / I 640 = 4.4071 * enrofloxacin concentration + 0.9836.

[0019] Specifically, the enrofloxacin concentration corresponding to the standard curve described in step (5) is 0 - 1 mg / L.

[0020] Specifically, the detection limit of the standard curve in step (5) corresponding to the enrofloxacin concentration is 0.00159 mg / L.

[0021] The second objective of this invention is the application of a ratiometric fluorescence detection method for enrofloxacin based on a novel composite nanocarbon dot-gold cluster material, specifically for detecting the enrofloxacin content in real samples. These real samples include, but are not limited to, any one of milk, chicken, human serum, and fish.

[0022] The technical effect of this invention is that it achieves the fluorescence intensity ratio I of the AuNCs@CDs dual emission peaks. 440 / I 640 A standard curve was established to correlate the concentration of enrofloxacin with its concentration, enabling the detection of enrofloxacin. The novel composite nanomaterial AuNCs@CDs used is an organic fusion of gold nanoclusters and carbon dots, requiring no further surface modification or passivation to detect enrofloxacin. This method can be used to detect the enrofloxacin content in actual samples such as milk, chicken, human serum, and fish. This invention offers high sensitivity and selectivity, is simple and rapid, requires no pretreatment, and shows great promise for applications in biomedical detection, environmental monitoring, food safety analysis, and many other fields. Attached Figure Description

[0023] Figure 1 The fluorescence emission spectrum of AuNCs@CDs in Example 1 is shown.

[0024] Figure 2 The ratio of fluorescence intensity of AuNCs@CDs solution to various interfering substances and water in Example 2 was determined by I. 440 / I 640 Influence on the bar chart.

[0025] Figure 3 The ratio of fluorescence intensity of AuNCs@CDs solution to various interfering substances (antibiotics) and water in Example 2 was determined by I. 440 / I 640 Influence on the bar chart.

[0026] Figure 4 The fluorescence spectra of AuNCs@CDs solution with different concentrations of enrofloxacin in Example 2 are shown.

[0027] Figure 5 In Example 2, the ratio of enrofloxacin solution concentration to AuNCs@CDs solution fluorescence intensity I... 440 / I 640 The standard curve is drawn based on the relationship between the two curves.

[0028] Figure 6 The fluorescence spectrum of sample 1 (AuNCs@CDs solution with added deionized water and milk) in Example 3 is shown.

[0029] Figure 7 The fluorescence spectrum of sample 2, which was obtained by adding deionized water and milk to the AuNCs@CDs solution in Example 3.

[0030] Figure 8 The fluorescence spectra of AuNCs@CDs solution with added deionized water and chicken sample 1 in Example 3 are shown.

[0031] Figure 9 The fluorescence spectra of AuNCs@CDs solution with added deionized water and chicken sample 2 in Example 3 are shown.

[0032] Figure 10 The fluorescence spectrum of AuNCs@CDs solution with added deionized water and human serum sample 1 in Example 3 is shown.

[0033] Figure 11 The fluorescence spectrum of AuNCs@CDs solution with added deionized water and human serum sample 2 in Example 3 is shown.

[0034] Figure 12 The fluorescence spectrum of AuNCs@CDs solution with added deionized water and fish meat sample 1 in Example 3 is shown.

[0035] Figure 13 The fluorescence spectra of AuNCs@CDs solution with added deionized water and fish meat sample 2 in Example 3 are shown. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but this is not intended to limit the present invention.

[0037] Example 1: Preparation of AuNCs@CDs solution.

[0038] Preparation of gold nanocluster seed solution: 5.00 mL of 20 mM chloroauric acid solution was mixed with 1.50 mL of 100 mM glutathione solution and heated and stirred in a water bath at 80 °C for 2 h; after the reaction was completed, the gold nanocluster seed solution was obtained.

[0039] Preparation of AuNCs@CDs: 40 mg of ascorbic acid was added to 10.00 mL of gold nanocluster seed solution and microwaved on high for 8 minutes; after the reaction was completed, 10 mL of distilled water was added to resuspend the reactants; the resuspended solution was filtered and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da to obtain a pure AuNCs@CDs solution; the AuNCs@CDs solution was then freeze-dried under vacuum to finally obtain AuNCs@CDs solid powder.

[0040] Preparation of AuNCs@CDs solution: AuNCs@CDs solid was quantitatively weighed to prepare AuNCs@CDs solution, achieving a final concentration of 0.025 mg / mL; the pH of the solution was adjusted to approximately 6. The fluorescence spectrum of AuNCs@CDs was measured using a fluorescence spectrophotometer. Figure 1 ).from Figure 1 As can be seen, AuNCs@CDs have two fluorescence emission peaks (excitation peak: 330 nm), and the emission peak positions are 440 nm and 640 nm, respectively.

[0041] Example 2: Plotting the standard curve for enrofloxacin detection using AuNCs@CDs.

[0042] Take 20 centrifuge tubes of 4 mL each, labeled 1-20. Add 1.98 mL of the AuNCs@CDs solution prepared as described above to each of the 20 centrifuge tubes. Then, add 20 μL of deionized water or 20 μL of 100 mg / L interfering substance to each centrifuge tube, i.e., K. + Ca 2+ Na + Mg 2+ Fe 2+ Zn 2+ Glucose, Urea, H2PO4 - HPO4 2- , Ampicillin, Amoxicillin, Erythromycin, Roxithromycin, Chloramphenicol, Tetracycline, Fleroxacin, Levofloxacin, Ofloxacin (where Zn 2+ Fe 2+(Ions were added together with an equal amount of sodium ethylenediaminetetraacetate). After reacting for 2 minutes, the solutions in each centrifuge tube were sequentially tested in a fluorescence spectrophotometer, and the fluorescence intensity ratio of AuNCs@CDs to I was recorded. 440 / I 640 Then, 20 μL of 20 mg / L enrofloxacin solution was added to each centrifuge tube. After reacting for 2 minutes, the solutions in each centrifuge tube were tested sequentially in a fluorescence spectrophotometer, and the fluorescence intensity ratio of AuNCs@CDs to I was recorded again. 440 / I 640 ,like Figure 2 and 3 As shown. From Figure 2 and 3 It can be seen that the fluorescence intensity ratio I of AuNCs@CDs 440 / I 640 The fact that it only responded to enrofloxacin indicates that AuNCs@CDs have high selectivity for enrofloxacin.

[0043] Take seven 4 mL centrifuge tubes, labeled 1-8. Add 1.98 mL of the AuNCs@CDs solution prepared as described above to each of the eight tubes. Then, add 20 μL of deionized water or 20 μL of enrofloxacin solutions with concentrations of 10 mg / L, 20 mg / L, 35 mg / L, 50 mg / L, 70 mg / L, and 100 mg / L, respectively, to each tube sequentially. After reacting for 2 minutes, test the solutions in each tube sequentially using a fluorescence spectrophotometer and record the AuNCs@CDs fluorescence intensity ratio I. 440 / I 640 ( Figure 4 Based on the ratio of enrofloxacin solution concentration to AuNCs@CDs solution fluorescence intensity I... 440 / I 640 Plotting the standard curve based on the relationship ( Figure 5 Enrofloxacin concentration can be detected based on the standard curve.

[0044] Example 3: Experimental recovery rate of enrofloxacin in actual samples detected by AuNCs@CDs.

[0045] Take nine 4 mL centrifuge tubes, labeled 1-9. Add 1.98 mL of the AuNCs@CDs solution prepared by the above method to each of the nine centrifuge tubes. Then, add 20 μL of deionized water or 20 μL of milk sample 1 (containing 20 mg / L enrofloxacin), 20 μL of milk sample 2 (containing 80 mg / L enrofloxacin), 20 μL of chicken sample 1 (containing 20 mg / L enrofloxacin), 20 μL of chicken sample 2 (containing 80 mg / L enrofloxacin), 20 μL of human serum sample 1 (containing 20 mg / L enrofloxacin), 20 μL of human serum sample 2 (containing 80 mg / L enrofloxacin), 20 μL of fish sample 1 (containing 20 mg / L enrofloxacin), and 20 μL of fish sample 2 (containing 80 mg / L enrofloxacin) to each centrifuge tube in sequence. After reacting for 2 minutes, the solutions in each centrifuge tube were tested sequentially in a fluorescence spectrophotometer, and the fluorescence intensity ratio I of AuNCs@CD was recorded. 440 / I 640 ( Figure 6 - 13). The recovery rate and relative standard deviation (RSD) of enrofloxacin in each sample were calculated based on the standard curve, and the results are shown in Table 1.

[0046]

[0047] Table 1 shows that for milk sample 1, the recovery rate and relative standard deviation (RSD) of enrofloxacin were 108.5% and 0.83%, respectively. For milk sample 2, the recovery rate and RSD were 109.0% and 0.27%, respectively. For chicken sample 1, the recovery rate and RSD were 100.0% and 0.50%, respectively. For chicken sample 2, the recovery rate and RSD were 92.3% and 1.45%, respectively. For human serum sample 1, the recovery rate and RSD were 106.0% and 0.38%, respectively. For human serum sample 2, the recovery rate and RSD were 109.6% and 0.26%, respectively. For fish sample 1, the recovery rate and RSD were 99.5% and 0.60%, respectively. For fish meat sample 2, the recovery rate and relative standard deviation (RSD) of enrofloxacin were 109.4% and 0.79%, respectively.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; any obvious adjustments and modifications made to the technical solutions of the present invention that are part of the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for ratiometric fluorescence detection of enrofloxacin based on a novel composite nanocarbon dot-gold cluster material, characterized in that... Includes the following steps: (1) Gold nanocluster seeds were synthesized by water bath method using chloroauric acid HAuCl4 and glutathione GSH as raw materials; (2) A novel composite nanocarbon dot-gold cluster material AuNCs@CDs was synthesized by microwave method using gold nanocluster seeds and ascorbic acid as raw materials; (3) Weigh out AuNCs@CDs solid quantitatively to prepare AuNCs@CDs solution; (4) Add enrofloxacin solution to AuNCs@CDs solution. After reaction, record the corresponding fluorescence emission spectrum and calculate the ratio I of the fluorescence intensity of the two emission peaks at 440 nm and 640 nm of AuNCs@CDs. 440 / I 640 The change value; (5) Based on the ratio of enrofloxacin solution concentration to AuNCs@CDs solution fluorescence intensity I 440 / I 640 By plotting a standard curve based on the relationship between the levels of enrofloxacin and the concentration of enrofloxacin, it is possible to detect the concentration of enrofloxacin.

2. The method for ratiometric fluorescence detection of enrofloxacin based on a novel composite nanocarbon dot-gold cluster material according to claim 1, characterized in that: The method for synthesizing gold nanocluster seeds in step (1) is as follows: 5.00 mL of 20 mM chloroauric acid solution is mixed with 1.50 mL of 100 mM glutathione solution, and the mixture is heated and stirred in a water bath at 80°C for 2 h. After the reaction is completed, a gold nanocluster seed solution is obtained.

3. The method for detecting enrofloxacin based on a ratiometric fluorescence method using a novel composite nano-carbon dot-gold cluster material according to claim 1, characterized in that: The synthesis method of AuNCs@CDs in step (2) is as follows: 40 mg of ascorbic acid is added to 10.00 mL of the gold nanocluster seed solution in step (1), and microwaved on high for 8 minutes; after the reaction is completed, 10 mL of distilled water is added to resuspend the reactants; the resuspension is filtered and dialyzed for 24 hours with a dialysis bag with a molecular weight cutoff of 1000 Da to obtain a pure AuNCs@CDs solution; the pure AuNCs@CDs solution is freeze-dried under vacuum to finally obtain AuNCs@CDs solid powder.

4. The method for ratiometric fluorescence detection of enrofloxacin based on a novel composite nanocarbon dot-gold cluster material according to claim 1, characterized in that: The final concentration of the AuNCs@CDs solution prepared in step (3) is 0.025 mg / mL.

5. The method for ratiometric fluorescence detection of enrofloxacin based on a novel composite nanocarbon dot-gold cluster material according to claim 1, characterized in that: The pH of the AuNCs@CDs solution prepared in step (3) is 6.

6. The method for detecting enrofloxacin based on a ratiometric fluorescence method using a novel composite nano-carbon dot-gold cluster material according to claim 1, characterized in that: The reaction time of enrofloxacin and AuNCs@CDs in step (4) is 2 minutes.

7. The method for ratiometric fluorescence detection of enrofloxacin based on a novel composite nanocarbon dot-gold cluster material according to claim 1, characterized in that: The standard curve mentioned in step (5) is I 440 / I 640 = 4.4071 * enrofloxacin concentration + 0.9836.

8. The method for detecting enrofloxacin based on ratiometric fluorescence of composite nano-carbon dots-gold clusters according to claim 1, characterized in that: The enrofloxacin concentration corresponding to the standard curve described in step (5) is 0 - 1 mg / L.

9. The method for detecting enrofloxacin based on ratiometric fluorescence of composite nano-carbon dots-gold clusters according to claim 1, characterized in that: The detection limit for enrofloxacin concentration is 0.00159 mg / L.

10. The method for detecting enrofloxacin based on a ratiometric fluorescence of a novel composite nanocarbon dot-gold cluster material according to any one of claims 1-9, characterized in that: This method is used to detect the experimental recovery rate of enrofloxacin in real samples.

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