Method for detecting tobramycin by using ratio fluorescent probe based on organic-inorganic hybrid copper cluster

Abstract

The invention discloses a method for detecting tobramycin based on an organic-inorganic hybrid copper cluster ratio fluorescent probe. A metal doped cluster CuCoNCs (at) PVP emitting blue fluorescence is synthesized, on the basis, a second fluorescent dye probe fluorescein sodium is introduced through physical and chemical interaction, an organic-inorganic hybrid copper cluster ratiometric fluorescent probe (CuCoNCs (at) PVP / FluNa) is constructed, the ratio F513 / F417 of the fluorescence intensity of the system at 513 nm to the fluorescence intensity of the system at 417 nm is used as an output signal, and the fluorescence intensity of the CuCoNCs (at) PVP and the fluorescence intensity of the CuCoNCs (at) PVP are detected. The precise detection on the tobramycin is realized.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry, and in particular relates to a method for detecting tobramycin using a ratiometric fluorescent probe based on an organic-inorganic hybrid copper cluster. Background Art

[0002] Tobramycin is an aminoglycoside antibiotic extracted from the fermentation broth of Streptomyces. Due to its good water solubility, stability, and broad-spectrum antimicrobial properties, tobramycin has been widely used to treat infections caused by aerobic Gram-negative and some Gram-positive bacteria. However, excessive and uncontrolled use of tobramycin can lead to adverse residues in food and the environment, causing serious and irreversible side effects on human health, such as nephrotoxicity, neuromuscular blockade, hypersensitivity reactions, and ototoxicity. Currently, tobramycin is primarily detected using antibiotic microbiological assays or high-performance liquid chromatography. However, microbiological assays often suffer from poor specificity, large errors, and cumbersome procedures, while chromatographic methods require complex pre-column derivatization procedures and expensive and bulky instrumentation. Both methods struggle to meet the requirements of rapid, on-site testing. Therefore, there is an urgent need to develop new analytical methods for the convenient, accurate, and sensitive detection of tobramycin in samples.

[0003] Fluorescence detection technology, characterized by high sensitivity, strong selectivity, and ease of operation, has become one of the most effective tools for monitoring antibiotic residues. In the design of fluorescent sensors, researchers typically rely on a variety of chemical reactions to achieve sensitive detection of non-fluorescent substances, including fluorescence derivatization, quenching, or sensitization. Furthermore, by constructing fluorescent hybrid systems with multiple emission signals and leveraging multi-signal cross-validation to obtain fluorescent biosensors with internal self-calibration capabilities, analytical accuracy and sensitivity can be further improved, while reducing complex matrix interference in actual samples. This approach has demonstrated excellent feasibility and practicality in the detection of tobramycin residues.

[0004] Copper nanoclusters (Cu NCs), as zero-dimensional nanomaterials surrounded by capping ligands, exhibit molecular-like properties distinct from those of bulk metals, such as HOMO-LUMO transitions and structure-dependent photoluminescence (PL) properties. Compared to organic small molecule fluorophores, Cu NCs often possess higher fluorescence stability and larger Stokes shifts; they are also biocompatible and inexpensive to prepare. Therefore, Cu NCs can be used as a fluorescent signal source for widespread applications in chemical sensing, bioimaging, and light-emitting devices. However, most synthesized Cu NCs suffer from weak fluorescence, low quantum yield, and susceptibility to oxidation, hindering their analytical applications. Furthermore, no organic-inorganic hybrid ratiometric fluorescent probes based on Cu NCs have been reported for the analysis of tobramycin. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for detecting tobramycin based on a ratiometric fluorescent probe of organic-inorganic hybrid copper clusters.

[0006] The technical solution adopted in the present invention is as follows:

[0007] The present invention first provides a fluorescent material based on organic-inorganic hybrid copper clusters. The fluorescent material is formed by coating metal M-doped copper nanoclusters and sodium fluorescein with polyvinylpyrrolidone, denoted as CuM NCs@PVP / FluNa. Under an excitation wavelength of 343 nm, it exhibits maximum fluorescence emission peaks at 417 nm and 513 nm, respectively.

[0008] The present invention further provides a method for preparing the above-mentioned fluorescent material based on organic-inorganic hybrid copper clusters, comprising the following steps:

[0009] An aqueous copper salt solution (Cu) was mixed with another doped metal salt solution (M), a polyvinyl pyrrolidone solution (PVP), an ascorbic acid solution (AA), and a fluorescein sodium solution (Flu Na). The pH was adjusted to 4.0 with sodium hydroxide. The mixed solution was transferred to a flask, placed in an oil bath at 75°C for 3 h, and naturally cooled to room temperature to obtain a crude product. The crude product was transferred to a regenerated cellulose dialysis bag and dialyzed with ultrapure water to obtain a clear yellow liquid, which was CuM NCs@PVP / FluNa.

[0010] The pH of the above reaction system will affect the effect of AA on Cu 2+ The PL intensity is the highest at pH 4.0, and deviations from the pH value will lead to a decrease in PL. At 75°C, the PL intensity reaches its maximum when the reaction time is 3 h, while there is no significant change in the PL intensity when the reaction time is increased. When the temperature is too low or too high, NCs may be insufficiently reduced or slightly aggregated.

[0011] The copper metal salt is a water-soluble chloride, nitrate, or sulfate. The metal ion of the other doping metal salt M is any one of cobalt, iron, zinc, zirconium, cerium, tin, manganese, and nickel, and the metal salt thereof is a water-soluble chloride, nitrate, or sulfate. Preferably, the doping metal is cobalt.

[0012] Preferably, in the mixed solution, the molar ratio of the total molar amount of copper ions and doping metal ions M to ascorbic acid (Cu+M):AA = 4:10, and the molar ratio of copper ions to doping metal ions Cu:M = 0.2~5:1, such as 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1; preferably, the Cu:M = 3:1.

[0013] Preferably, the PVP is PVP-K30, the ratio of the PVP to the total metal ions is 5 g:20 μmol, and the final concentration thereof in the mixed solution is 40-42 mg / mL.

[0014] Preferably, the molar ratio of fluorescein sodium to total metal ions is 1.2 to 12:20, preferably 3:20. The concentration of fluorescein sodium in the mixed solution is 100-1000 μM, more preferably 250 μM.

[0015] Preferably, the dialysis purification uses a regenerated cellulose membrane with a molecular weight cutoff of 3500-100000 Da, and the dialysis time is 24-48 h.

[0016] The present invention further uses the fluorescent material based on the organic-inorganic hybrid copper cluster as a ratiometric fluorescent probe to detect tobramycin.

[0017] Specifically, the method for detecting tobramycin is as follows: CuCoNCs@PVP / FluNa and tobramycin solution are mixed to prepare a tobramycin standard solution; under an excitation wavelength of 343 nm, the fluorescence emission spectrum of the standard solution in the emission wavelength range of 370-650 nm is measured, and the fluorescence intensity at 513 nm and the fluorescence intensity at 417 nm are recorded respectively. The ratio of the fluorescence intensity at 513 nm to the fluorescence intensity at 417 nm is used to calculate the fluorescence intensity of the system. 513 / F 417 The output signal is used as the vertical coordinate, and the concentration of tobramycin solution is used as the horizontal coordinate. The standard curve is drawn and the fitting equation is obtained; the fluorescence intensity ratio F of the sample solution is measured and calculated. 513 / F 417 , the concentration of tobramycin in the sample solution was calculated according to the fitting equation.

[0018] The reaction time of CuCoNCs@PVP / FluNa and tobramycin is 5-60 min; preferably, the reaction time is 25-60 min.

[0019] The technical principles and beneficial effects of the present invention are:

[0020] The present invention uses PVP-K30 as a ligand to synthesize a metal-doped copper cluster (CuM NCs@PVP), which achieves fluorescence emission from the ligand to the metal copper core at 417 nm. Furthermore, by utilizing the high molecular entanglement and protective properties of PVP-K30, the organic dye molecule sodium fluorescein is wrapped into the cluster nanostructure through hydrophilic and hydrophobic interactions, constructing a ratiometric fluorescent probe of organic-inorganic hybrid copper clusters (CuCo NCs@PVP / FluNa), which has the blue fluorescence emission (λ em = 417 nm) and the green fluorescence emission of the sodium fluorescein dye molecule (λ em = 513 nm). After adding tobramycin, F 417 Descending, F 513 Enhanced. According to the band structure test and density functional theory (DFT) calculation, the LUMO and HOMO of sodium fluorescein are located between the LUMO and HOMO of tobramycin. Tobramycin can act as an energy donor, and sodium fluorescein can act as an energy acceptor. The two can enhance the fluorescence of sodium fluorescein at 513 nm through energy transfer. The HOMO orbital of CuCo NCs@PVP is the lowest, and its LUMO is located between the LUMO and HOMO of tobramycin. When excited by light, tobramycin can act as an electron donor, and CuCo NCs@PVP can act as an electron acceptor. Through the acceptor-photoinduced electron transfer effect (a-PET), electrons are allowed to transfer from the HOMO orbital of tobramycin to the HOMO orbital of CuCo NCs@PVP, resulting in the fluorescence quenching of CuCo NCs@PVP at 417 nm. According to the ratio of the fluorescence intensity of the system at 513 nm to the fluorescence intensity at 417 nm, F 513 / F 417 The relationship between the concentration of tobramycin and the concentration of tobramycin was studied, and a ratiometric fluorescence sensor was developed. The ratiometric fluorescence detection method of tobramycin provided by the present invention is simple, rapid, low-cost, and highly sensitive, and can detect trace amounts of tobramycin in complex matrices. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the fluorescence detection mechanism of the ratiometric fluorescence sensor of the present invention;

[0022] Figure 2 This is a schematic diagram of the synthesis of metal-doped copper clusters;

[0023] Figure 3 TEM image (a) and particle size distribution (b) of CuCo NCs@PVP / FluNa;

[0024] Figure 4are the fluorescence excitation, fluorescence emission, and absorption images of CuCo NCs@PVP / FluNa;

[0025] Figure 5 These are photos of CuCo NCs@PVP / FluNa under sunlight and UV light;

[0026] Figure 6 is the effect of different doping metals on the fluorescence properties of CuM NCs;

[0027] Figure 7 The effect of different polymers on the fluorescence properties of CuM NCs

[0028] Figure 8 is the effect of the molar ratio of copper salt and cobalt salt on the fluorescence properties of CuCo NCs@PVP / FluNa;

[0029] Figure 9 is the effect of dialysis time on the fluorescence properties of CuCo NCs@PVP / FluNa;

[0030] Figure 10 is the effect of FluNa content on the fluorescence properties of CuCo NCs @PVP / FluNa;

[0031] Figure 11 This is an investigation of the reaction time of influencing factors by the method of the present invention;

[0032] Figure 12 The fluorescence emission spectra of tobramycin at different concentrations detected by the method of the present invention (a), the ratio fluorescence response linear graph (b), and the fluorescence response linear graphs at F417 and F513 nm (c, d);

[0033] Figure 13 It is the interference of common antibiotics on tobramycin detection. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Preparation Method of a Ratio Fluorescent Probe of Organic-Inorganic Hybrid Copper Clusters

[0036] Figure 2This is a schematic diagram of the one-pot synthesis of CuCo NCs@PVP / FluNa. The specific preparation method includes the following steps: 500 mg of PVP-K30 was weighed and dissolved in 8 mL of water. Then, 1.0 mL each of CuCl2·2H2O (0.01 M), CoCl2·6H2O (0.01 M), and AA solution (0.1 M) were added, followed by 0.9 mL of FluNa solution (final concentration of 250 μM). The pH of the mixed solution was adjusted to 4.0, and the mixture was reacted at 75°C for 3 hours. Finally, the mixture was dialyzed using a 3500 Da dialysis bag for 48 hours. The resulting yellow solution was the target product, CuCo NCs@PVP / FluNa.

[0037] Transmission electron microscopy characterization ( Figure 3 a and 3b) show that the prepared CuCo NCs@PVP / FluNa is spherical and evenly distributed with an average particle size of about 17 nm. UV-visible absorption and fluorescence spectra characterization ( Figure 4 ) showed that the fluorescence (Ex.) excitation and emission (Em.) spectra and ultraviolet absorption (Abs.) spectra of the prepared CuCo NCs@PVP / FluNa showed the characteristic absorption of CuCo NCs@PVP and FluNa at 274 nm and 454 nm; the maximum fluorescence emission peaks were at 417 nm and 513 nm, respectively, and the maximum fluorescence excitation peak was at 343 nm. Figure 5 The color image of the prepared CuCo NCs @PVP / FluNa solution shows that under sunlight (left), the CuCo NCs @PVP / FluNa appears light yellow, while under UV light (right), it appears green. These results indicate that the CuCo NCs @PVP / FluNa solution was successfully prepared.

[0038] Example 2 Effects of different doping metals on the fluorescence properties of CuM NCs

[0039] On the basis of synthesizing Cu NCs@PVP, another metal salt aqueous solution was introduced to synthesize CuM NCs. The other metal salt was one of the water-soluble chloride, nitrate, and sulfate salts of cobalt, iron, zinc, zirconium, cerium, tin, manganese, and nickel. The fluorescence intensity of the synthesized CuM NCs@PVP was measured, as shown in FIG. Figure 6 The fluorescence intensity is ranked from highest to lowest as Co>Ni>Zn>Mn>Fe>Al>Cu>Sn>Zr>Ce, with CuCo NCs@PVP showing the most significant fluorescence enhancement. This result indicates that cobalt doping can significantly improve the fluorescence properties of copper clusters. Cobalt was ultimately selected as another doping metal in the present invention.

[0040] Example 3 Effects of different polymers on the fluorescence properties of CuM NCs

[0041] Polyvinyl pyrrolidone (PVP-K 30) and polyethyleneimine (PEI) were selected as ligands to synthesize CuCoNCs, and the fluorescence intensity of the synthesized clusters was measured. Figure 7 It was shown that the fluorescence intensity of CuCo NCs@PVP was significantly higher than that of CuCoNCs@PEI, so PVP was selected as the best ligand in the synthesis of CuM NCs.

[0042] Example 4 Effect of different molar ratios of copper salt and cobalt salt on the fluorescence properties of CuCo NCs@PVP / FluNa

[0043] The total molar amount of metal was fixed at 20 μmol, and CuCo NCs@PVP / FluNa with different bimetallic molar ratios (1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1) were synthesized, and the fluorescence intensity changes of the synthesized clusters were measured. Figure 8 It is shown that with the change of Cu:Co molar ratio, F 417 , F 513 and F 513 / F 417 The fluorescence performance of CuCoNCs@PVP at 417 nm is significantly improved by adding Co at a certain concentration. When Cu:Co=3:1, F 417 and F 513 The fluorescence intensity at the point is the largest and F 513 / F 417 It is close to 1.0, so the present invention finally selects the molar ratio of Cu:Co = 3:1 as the optimal condition in the synthesis process of CuCo NCs @PVP / FluNa.

[0044] Example 5 Effect of dialysis time on the fluorescence properties of CuCo NCs@PVP / FluNa

[0045] Samples were taken after 24, 48, 54, 66, and 72 h of dialysis of CuCo NCs@PVP / FluNa. Figure 9 It showed that after 48 hours of dialysis, F 417 and F 513 The fluorescence intensity at the position remains basically unchanged, and F 513 / F 417 It is close to 1.0, so the present invention finally selects dialysis for 48 h as the optimal condition in the synthesis process of CuCo NCs @PVP / FluNa.

[0046] Example 6 Optimization of FluNa content in CuCo NCs@PVP / FluNa for detection of tobramycin

[0047] During the synthesis of CuCo NCs@PVP / FluNa, the final FluNa concentrations were set at 100, 250, 500, 750, and 1000 μM. A series of CuCo NCs@PVP / FluNa ratiometric fluorescent probes containing varying amounts of FluNa were prepared according to the method of Example 1. These probes were prepared into 1 μM aqueous solutions and incubated with a tobramycin solution at room temperature for 25 minutes to measure the fluorescence spectra. Figure 10 It shows that with the increase of FluNa synthesis concentration, F 417 Gradually decreases, and F 513 When the concentration of FluNa synthesis was 250 μM, F 513 / F 417 It is close to 1.0, so the present invention finally selects a FluNa synthesis concentration of 250 μM as the optimal condition in the synthesis process of CuCo NCs @PVP / FluNa.

[0048] Example 7 Investigation of the incubation time of CuCo NCs@PVP / FluNa and tobramycin

[0049] Blank group: 100 μL CuCo NCs@PVP / FluNa and 900 μL deionized water were added to the centrifuge tube in sequence, mixed thoroughly, and reacted for 0-60 min. The ratio of the fluorescence intensity at 417 nm and 513 nm in the system was recorded. 513 / F417 change.

[0050] Experimental group: 100 μL CuCo NCs@PVP / FluNa, 100 μL 1 μM tobramycin solution, and 800 μL deionized water were added to a centrifuge tube in sequence, mixed thoroughly, and reacted at room temperature for 0-60 min. The ratio of the fluorescence intensity at 417 nm and 513 nm in the system was recorded. 513 / F 417 change.

[0051] CuCo NCs@PVP / FluNa was reacted with tobramycin at room temperature for different times, i.e., 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min, and the changes in the fluorescence intensity of the system were detected. Figure 11 It shows that as the reaction time increases, F 513 / F 417The reaction time gradually increased and leveled off after 25 minutes. The present invention selected 25 minutes as the optimal reaction time for subsequent detection.

[0052] Example 8 Drawing of standard curve

[0053] Blank group: 100 μL CuCo NCs@PVP / FluNa and 900 μL deionized water were added to the centrifuge tube, mixed thoroughly, and reacted for 25 min. The ratio of the fluorescence intensity at 417 nm and 513 nm in the system was recorded. 513 / F417 change.

[0054] Experimental group: Tobramycin concentrations were set at 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, and 10 μM. 100 μL of CuCo NCs@PVP / FluNa, 100 μL of tobramycin solution at different concentrations, and 800 μL of deionized water were added to a centrifuge tube, mixed thoroughly, and reacted at room temperature for 25 minutes. The ratio of the fluorescence intensity at 417 nm to 513 nm in the system was recorded. 513 / F 417 change.

[0055] Under the optimal detection conditions, the detection performance of the ratiometric fluorescence sensor for tobramycin was investigated. Figure 12 a shows that with the increase of tobramycin concentration, F 417 Gradually decrease, F 513 When the concentration of tobramycin is 0.5–10 μM, the system is at F 417 The fluorescence intensity at F was linearly correlated with the tobramycin concentration. The fitted linear regression equation was Y=5.19213-0.029 lg [TOB / μM], with a linear correlation coefficient of 0.995 and an average RSD of 1.66% (S / N = 3). When the tobramycin concentration was 0.1–10 μM, the system 513 The fluorescence intensity at nm was linearly correlated with the tobramycin concentration. The fitted linear regression equation was Y=225540.61+38693.26 [TOB / μM], with a linear correlation coefficient of 0.997 and an average RSD of 1.86% (S / N = 3). Figure 12 b shows that as the concentration of tobramycin increases, F 513 / F 417 When the concentration of tobramycin was gradually increased from 0.1 to 10 μM, the F 513 and F 417 The ratio of the fluorescence intensity at 513 / F 417) was linearly correlated with tobramycin concentration, with the fitted linear regression equation being Y = 1.4418 + 0.27403 [TOB / μM], a linear correlation coefficient of 0.999, an average RSD of 1.48%, a linear range of 0.1 μM-10 μM, and a limit of detection of 0.025 μM (S / N = 3). Compared with either CuCoNCs@PVP or sodium fluorescein alone, CuCoNCs@PVP exhibits greater applicability and reliability.

[0056] Example 9 Interference Experiment of Common Antibiotics

[0057] Using the detection method provided in Example 8 of the present invention, the recording system F 513 / F 417 The interference of common antibiotics on tobramycin detection was investigated by measuring the concentration of gentamicin sulfate at 10 mg / mL, while the concentrations of the other antibiotics were 100 μM. These antibiotics included amoxicillin, ceftazidime, gentamicin sulfate, penicillin sodium, cephradine, cefaclor, cefotezole sodium, ceftriaxone sodium, streptomycin sulfate, paromomycin sulfate, chloramphenicol, and sulfacetamide solution. Figure 13 It shows that common antibiotics have little interference with the system. When and only when tobramycin is present, F 513 / F 417 The maximum value indicates that the fluorescence system has good selectivity for tobramycin and strong anti-interference ability.

[0058] Example 10 Detection and spiked recovery of tobramycin in eye drops and urine

[0059] Urine sample processing: Fresh urine samples were diluted 1000 times and stored in a refrigerator at -20°C.

[0060] Blank urine sample group: Dilute the urine sample 1000-fold with deionized water to form the blank urine sample group. Add 100 μL of blank urine sample and 100 μL of CuCo NCs@PVP / FluNa solution to a centrifuge tube and dilute to 1 mL with deionized water. The remaining steps were the same as in Example 8 of the present invention, and fluorescence signal detection was performed.

[0061] For spiked urine samples, add 100 μL of blank urine sample solution, 100 μL of tobramycin standard solution (0.5, 1, and 2 μM), and 100 μL of CuCo NCs@PVP / FluNa solution to a centrifuge tube, and dilute to 1 mL with deionized water. The remaining steps are the same as in Example 7, and fluorescence signal detection is performed.

[0062] Tobramycin eye drops treatment: Take 38.9 μL of 0.3% (5 mL, 15 mg) tobramycin eye drops, dilute to 5 mL with deionized water, centrifuge, take the supernatant as the test solution, and store in a refrigerator at -20 ℃.

[0063] For the blank eye drop sample group, add 100 μL of the test solution and 100 μL of the CuCo NCs@PVP / FluNa solution to a centrifuge tube, and dilute to 1 mL with deionized water. The remaining steps are the same as those in Example 7, and fluorescence signal detection is performed.

[0064] For the spiked eye drop sample group, add 100 μL of the test solution, 100 μL of tobramycin standard solution (0.5, 1, and 2 μM), and 100 μL of the CuCo NCs@PVP / FluNa solution to a centrifuge tube, and dilute to 1 mL with deionized water. The remaining steps are the same as in Example 8, and fluorescence signal detection is performed.

[0065] Substitute the measured fluorescence signal and absorbance into the standard curve to calculate the tobramycin content in different sample groups. 加标样本 – C 空白样本 ) / C 标准品 × 100%, and the recovery rates in urine and eye drop samples were calculated. The results are shown in Tables 1 and 2. Tobramycin was not detected in the blank urine sample. The recovery rates in urine were 93.0% to 104.0%, and the recovery rates in eye drop samples were 94.0% to 108.0%, with RSDs < 10%. These results demonstrate that the detection method provided by the present invention has good application potential in urine and eye drop samples.

[0066] Table 1. Determination of Tobramycin in Urine

[0067]

[0068] Table 2. Determination of tobramycin in eye drops

[0069]

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting tobramycin based on a ratiometric fluorescent probe of organic-inorganic hybrid copper clusters, characterized in that: The fluorescent probe material is a fluorescent material formed by coating metal-doped copper nanoclusters and sodium fluorescein with polyvinylpyrrolidone, denoted as CuM NCs @PVP / FluNa, where M represents the doped metal; The method for detecting tobramycin is as follows: CuM NCs@PVP / FluNa and tobramycin solution are mixed to prepare a tobramycin standard solution; under an excitation wavelength of 343 nm, the fluorescence emission spectrum of the standard solution in the emission wavelength range of 370-650 nm is measured, and the fluorescence intensity at 513 nm and the fluorescence intensity at 417 nm are recorded respectively. The ratio of the fluorescence intensity at 513 nm to the fluorescence intensity at 417 nm is used as the fluorescence intensity ratio F. 513 / F 417 The output signal is used as the vertical coordinate, and the concentration of tobramycin solution is used as the horizontal coordinate. The standard curve is drawn and the fitting equation is obtained. The CuM NCs@PVP / FluNa and the sample solution are mixed, and the fluorescence intensity ratio F of the sample solution is measured and calculated. 513 / F 417 , the concentration of tobramycin in the sample solution was calculated according to the fitting equation.

2. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 1, wherein: The preparation method of CuM NCs@PVP / FluNa comprises the following steps: uniformly mixing a copper salt aqueous solution with a doped metal salt aqueous solution, a polyvinyl pyrrolidone aqueous solution, an ascorbic acid aqueous solution, and a sodium fluorescein aqueous solution, and adjusting the pH to 4.0 to obtain a mixed solution; reacting the mixed solution at 75°C for 3 hours, and naturally cooling to room temperature to obtain a crude product; and transferring the crude product to a dialysis bag and purifying it by dialyzing with ultrapure water to obtain CuM NCs@PVP / FluNa.

3. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: The metal copper salt is one of water-soluble chloride, nitrate and sulfate, the doping metal is one of cobalt, iron, zinc, zirconium, cerium, tin, manganese and nickel, and the metal salt is one of water-soluble chloride, nitrate and sulfate.

4. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: In the mixed solution, the ratio of the total molar amount of the metal copper salt and the doping metal salt to the molar amount of ascorbic acid is 4:

10.

5. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: The molar ratio of the metal copper salt to the doping metal salt is 0.2-5:

1.

6. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: The ratio of the mass of the polyvinyl pyrrolidone to the total molar amount of the metal copper salt and the doped metal salt is 5 g:20 μmol.

7. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: The total molar ratio of the sodium fluorescein to the metallic copper salt and the doped metal salt is 1.2-12:

20.

8. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 2, wherein: Dialysis purification uses a regenerated cellulose membrane with a molecular weight cutoff of 3500-100000 Da, and the dialysis time is 24-48 h.

9. The method for detecting tobramycin using a ratiometric fluorescent probe based on organic-inorganic hybrid copper clusters according to claim 1, characterized in that: The reaction time of CuM NCs@PVP / FluNa mixed with tobramycin solution or sample solution was 5-60 min.

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