Europium-doped carbon quantum dot fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a europium-doped carbon quantum dot fluorescent probe, a preparation method thereof, a tetracycline detection method and a cell imaging method, and belongs to the technical field of fluorescent probes. According to the preparation method, pineapple peel is taken as a carbon source, atomic-scale uniform doping of Eu < 3 + > is realized through a one-step hydrothermal method, ion leakage caused by physical adsorption is avoided, the stability of the probe is improved, and tedious synthesis steps are avoided. The europium-doped carbon quantum dot fluorescent probe has the advantages of high stability, high sensitivity and strong anti-interference performance, and can synchronously realize environmental water sample and cell imaging. The europium-doped carbon quantum dot fluorescent probe shows bimodal fluorescence at 440 nm and 618 nm, the tetracycline detection method is based on a self-calibration mechanism of F618 / F440 ratio signals of the europium-doped carbon quantum dot fluorescent probe, environmental interference is eliminated, and the detection sensitivity is improved. The problems that an existing tetracycline detection material is poor in stability, low in sensitivity and difficult to be compatible with living body imaging are solved.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, specifically to a europium-doped carbon quantum dot fluorescent probe, its preparation method, and its application. Background Technology

[0002] Tetracycline (TC), a broad-spectrum antibiotic, is widely used in livestock farming, aquaculture, and clinical medicine due to its low cost, strong antibacterial efficacy, high bioavailability, and relatively few toxic side effects. However, due to its long-term overuse and lack of regulation, TC residues in the environment and food have posed a serious threat to ecological security, food health, and public health.

[0003] Currently, the detection of tetracycline mainly relies on liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), chemiluminescence, and electrochemical analysis. While these methods offer high detection accuracy, they generally suffer from drawbacks such as demanding equipment requirements, complex detection procedures, long processing times, and reliance on specialized operators, limiting their application in rapid on-site screening and routine monitoring.

[0004] In recent years, fluorescence sensing-based detection technologies have become an important development direction in the field of tetracycline detection due to their advantages such as rapid response (minute-level), high sensitivity (detectable at trace concentrations), small sample volume (micro-saturation), simple operation, good selectivity, and low cost. However, existing fluorescent probes still have significant limitations. For example, Chinese patent CN115960607B discloses a method based on copper nanoclusters and attached Eu... 3+ While these probes are suitable for detection, their synthesis is complex, and europium ions are primarily loaded through physical adsorption, which can easily lead to ion leakage and signal drift, resulting in poor detection stability and repeatability. Furthermore, fully chemically synthesized materials are highly toxic and have poor biocompatibility, making them difficult to apply to in vivo or intracellular detection. More importantly, these probes have a single function and cannot simultaneously achieve highly sensitive environmental trace detection and live cell imaging, hindering their application in cross-scale correlation studies between the environment and biology.

[0005] Therefore, developing a fluorescent probe that is easy to synthesize, highly stable, biocompatible, and capable of both environmental detection and cell imaging has significant research value and application prospects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a europium-doped carbon quantum dot fluorescent probe, its preparation method, and its application. The europium-doped carbon quantum dot fluorescent probe has high stability, high sensitivity, and strong anti-interference ability, and can simultaneously achieve environmental water sample and cell imaging. The preparation method is green, environmentally friendly, and has low toxicity.

[0007] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a method for preparing a europium-doped carbon quantum dot fluorescent probe, comprising the following steps: S1. After freeze-drying the pineapple peel, grind it to obtain freeze-dried pineapple peel powder; In step S1, freeze-drying the pineapple peel can gently remove moisture under low temperature conditions, while retaining the organic components and active functional groups in the pineapple peel to the greatest extent, providing a stable and uniform carbon precursor for subsequent carbonization. Grinding the glass peel into freeze-dried powder can increase the specific surface area of ​​the pineapple peel, making it easier to disperse in the subsequent hydrothermal reaction system and enhancing the contact efficiency with europium salts and nitrogen sources. S2. Dissolve Eu(NO3)3·6H2O, N,N'-diethylthiourea, and the pineapple peel freeze-dried powder from step S1 in water to obtain a mixed solution; the mass ratio of Eu(NO3)3·6H2O, N,N'-diethylthiourea, and pineapple peel freeze-dried powder is (3-7):(7-11):(4-8); the mixed solution is subjected to hydrothermal reaction at 160-220 °C to obtain a reaction product solution; In step S2, the carbon source provided by the pineapple peel freeze-dried powder forms carbon quantum dots under hydrothermal conditions. The surface of these carbon quantum dots is rich in hydroxyl (-OH), carboxyl (-COOH), carbonyl (C=O), and pectin groups. These functional groups can interact with Eu... 3+ Strong coordination occurs; N,N'-diethylthiourea, as a dopant, contains N and S atoms that can form coordination sites with carbon quantum dots, allowing Eu(NO3)3·6H2O to form coordination sites. 3+ Anchored to the carbon quantum dot framework through atomic-level coordination; a specific raw material mass ratio (3-7):(7-11):(4-8) synergistically works with a hydrothermal reaction at 160-220 ℃ to construct stable coordination sites for the N and S atoms provided by N,N'-diethylthiourea and the carbon source formed by pineapple peel carbon dots, thereby anchoring Eu into the carbon quantum dot framework. 3+ Firmly anchored inside and on the surface of carbon quantum dots, forming an atomic-level bonded structure; S3. The reaction product solution described in step S2 is centrifuged to obtain a supernatant; the supernatant is filtered and dialyzed to obtain a europium-doped carbon quantum fluorescent probe.

[0008] In step S3, insoluble large-particle carbides and unreacted solid residues generated during the reaction are first rapidly removed by centrifugation. Then, submicron-sized aggregates or colloidal particles are further retained by filtration to ensure that the resulting solution is a truly nanoscale, well-dispersed carbon quantum dot colloidal solution. Finally, free Eu is removed by dialysis. 3+ Ions, ensuring the acquisition of Eu 3+ Fluorescent probes stably bound to carbon quantum dots.

[0009] The method for preparing europium-doped carbon quantum dot fluorescent probes provided by this invention uses pineapple peel as the main carbon source, realizing the high-value utilization of biomass resources, reducing raw material costs, and exhibiting good environmental friendliness and sustainability. By carrying out a hydrothermal reaction of europium salt (Eu(NO3)3·6H2O), nitrogen source (N,N'-diethylthiourea), and pineapple peel carbon source together, the method achieves the high-value utilization of biomass resources, reduces raw material costs, and possesses good environmental friendliness and sustainability. 3+ Atomic-level uniform doping in the carbon quantum dot framework. Compared to traditional "free Eu" 3+ "Mixed" or "Physical Adsorption Eu" 3 + The process of atomic-level doping effectively avoids Eu. 3+ Leakage caused by solvent erosion and matrix interference during the detection process is prevented by addressing the root cause, ensuring the structural stability of the probe in aquatic environments and biological systems, thereby ensuring stable fluorescence signals. Furthermore, a one-step hydrothermal method is used to achieve Eu... 3+ Atomic-level uniform doping of the carbon quantum dot framework simplifies the process and improves preparation efficiency. The prepared europium-doped carbon quantum dots exhibit dual emission characteristics (typically around 440 nm and 618 nm), enabling ratiometric detection based on the fluorescence intensity ratio at the two wavelengths (F618 / F440). This mechanism effectively counteracts environmental interference, enhancing detection sensitivity and anti-interference capabilities. The pineapple peel carbon source, doped with N,N'-diethylthiourea, forms hydrophilic functional groups such as -OH and -NH2, resulting in probes with excellent solubility in water, allowing for uniform dispersion in water samples and achieving efficient detection without the need for surfactants. Utilizing a natural carbon source and achieving stable europium doping, the resulting probe exhibits low toxicity and good biocompatibility, enabling simultaneous detection of tetracycline in environmental water samples and intracellular imaging, achieving cross-scale analysis from environmental to biological systems.

[0010] Further, in step S2, the mass-to-volume ratios of Eu(NO3)3·6H2O, N,N'-diethylthiourea, and the pineapple peel freeze-dried powder to water are 0.15-0.35 g / mL, 0.35-0.55 g / mL, and 0.2-0.4 g / mL, respectively. By optimizing the mass-to-volume ratios of Eu(NO3)3·6H2O, N,N'-diethylthiourea, and the pineapple peel freeze-dried powder to water, while ensuring Eu... 3+ While effectively embedding into the carbon quantum dot lattice to form stable fluorescence centers, Eu(NO3)3·6H2O, N,N'-diethylthiourea, and pineapple peel lyophilized powder further enhance the crystallinity and dispersibility of europium-doped carbon quantum dot fluorescent probes through synergistic effects.

[0011] Furthermore, the hydrothermal reaction time in step S2 is 6-12 h. By optimizing the hydrothermal reaction time to 6-12 h, the Eu...3+ While gradually diffusing into and stably binding with the carbon quantum dot framework, it can also balance the lattice growth rate of the carbon quantum dots, reduce surface defect states, and help improve the fluorescence quantum yield of europium-doped carbon quantum dot fluorescent probes.

[0012] Secondly, this invention provides a europium-doped carbon quantum dot fluorescent probe, prepared by the above-described method for preparing europium-doped carbon quantum dot fluorescent probes. In the europium-doped carbon quantum dot fluorescent probe provided by this invention, Eu... 3+ Entering the carbon quantum dot framework, Eu 3+ Europium-doped carbon quantum dot fluorescent probes, which are atomically uniformly doped into carbon quantum dots, have the advantages of high stability, high sensitivity, and strong anti-interference ability.

[0013] Thirdly, the present invention provides a tetracycline detection method, comprising the following steps: M1. Dissolve the above europium-doped carbon quantum dot fluorescent probe in ultrapure water to obtain a solution with a mass-volume concentration of 8-25 μg / mL; M2. First, place the solution described in step M1 into a colorimetric tube, then add tetracycline to the colorimetric tube to obtain a mixed solution. After the mixed solution is allowed to stand and incubate, a europium-doped carbon quantum dot fluorescent probe-tetracycline composite system is obtained. M3. Perform fluorescence spectroscopy analysis on the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system described in step M2, and construct a ratiometric detection standard curve based on the dual emission fluorescence intensity ratio F618 / F440.

[0014] The tetracycline detection method provided by this invention uses the aforementioned europium-doped carbon quantum dot fluorescent probe, which has dual emission characteristics: the 440 nm blue fluorescence of the carbon quantum dot itself and the Europium-doped carbon quantum dot fluorescence. 3+ The 618 nm red fluorescence after chelation with TC is self-calibrated during detection by calculating the "F618 / F440 ratio": the influence of complex matrices (such as turbidity in water samples and background fluorescence in cells) on the 440 nm and 618 nm fluorescence is synchronous, and the ratio calculation can cancel out this non-specific interference, retaining only the specific signal changes caused by TC, significantly improving the accuracy and reproducibility of detection. Furthermore, the aforementioned europium-doped carbon quantum dot fluorescent probe can only specifically chelate with the β-diketone structure of TC to form a stable six-membered ring complex, while remaining unaffected by other antibiotics (such as amoxicillin) and metal ions (such as Ca) in the water sample. 2+ Mg 2+ It has no binding capacity to intracellular amino acids (such as Cys and Ala), thus effectively eliminating interference from other antibiotics, common metal ions, and organic matter.

[0015] Furthermore, the concentration of tetracycline in the mixed solution described in step M2 is 1-200 μM. Controlling the tetracycline concentration to 1-200 μM can cover the needs of practical application scenarios in environmental water samples, medical and biological samples.

[0016] Furthermore, the static incubation time in step M2 is ≥1 min. A static incubation time of ≥1 min allows for stronger coordination between tetracycline and the europium-doped carbon quantum dot fluorescent probe in the mixed solution. 3+ The characteristic fluorescence enhancement is beneficial to improving the fluorescence intensity ratio F618 / F440.

[0017] Furthermore, the pH of the solution described in step M2 is 5-10. Controlling the pH of the solution to 5-10 provides a more favorable Eu ratio. 3+ Coordination environment with Eu-CQDs, thereby enhancing Eu 3+ The characteristic fluorescence is improved by increasing the fluorescence intensity ratio F618 / F440.

[0018] Fourthly, the present invention provides a cell imaging method, comprising the following steps: N1. Dissolve the above europium-doped carbon quantum dot fluorescent probe in ultrapure water to obtain a solution with a mass-volume concentration of 8-25 μg / mL; N2. The cells are seeded into a cell culture plate for the first culture, and then cultured in a medium with a tetracycline concentration ≤250 μM for the second culture. After washing away the residual tetracycline on the surface, the cells are cultured in the solution described in step N1 for the third culture. After washing away the residual solution on the surface, the sample to be tested is obtained. N3. Perform laser confocal microscopy imaging on the sample to be tested described in step N2.

[0019] The cell imaging method provided by this invention first culturees cells with tetracycline, then cultures them with europium-doped carbon quantum dot fluorescent probes. Utilizing the synergistic effect between the europium-doped carbon quantum dot fluorescent probes and tetracycline, and by controlling the concentrations of both the europium-doped carbon quantum dot fluorescent probe solution and tetracycline, cell survival and sufficient fluorescence intensity are ensured, enabling laser confocal microscopy imaging of the cells. The imaging analysis based on the ratio changes of dual-channel fluorescence signals effectively counteracts interference from the complex intracellular microenvironment, local concentrations, and cell autofluorescence, making the imaging results more accurately reflect the actual concentration distribution of tetracycline. This cell imaging method can directly display the localization and relative concentration changes of tetracycline in the cytoplasm without damaging the cell structure, providing direct evidence for studying its cellular uptake, subcellular distribution, and metabolic processes. The europium-doped carbon quantum dot fluorescent probes used in this imaging method are derived from green synthesis and have minimal impact on the viability of various cell lines at their working concentrations, ensuring that the imaging process is performed under essentially normal physiological conditions, making the observation results more biologically significant.

[0020] Furthermore, the excitation wavelength of the laser confocal microscopy is 380 nm. The laser confocal microscopy acquires fluorescence images in the blue and red channels. The wavelength range of the blue channel is 425-475 nm, and the wavelength range of the red channel is 570-620 nm. When the excitation wavelength is 380 nm, the excitation efficiency is maximized. The wavelength range of the blue channel (425-475 nm) corresponds to the Eu-CQDs carbon nucleus emission peak at 434 nm, and the wavelength range of the red channel (570-620 nm) corresponds to the Eu-CQDs carbon nucleus emission peak at 434 nm. 3+ Characteristic peak at 618 nm.

[0021] The positive and progressive effects of this invention are as follows: This invention provides a method for preparing europium-doped carbon quantum dot fluorescent probes, using pineapple peel as a carbon source and achieving Eu doping via a one-step hydrothermal method. 3+ Atomic-level uniform doping avoids ion leakage caused by physical adsorption, improves probe stability, and avoids cumbersome synthesis steps. The europium-doped carbon quantum dot fluorescent probe prepared using the method provided in this invention exhibits bimodal fluorescence at 440 nm and 618 nm. The tetracycline detection method provided in this invention is based on the self-calibration mechanism of the F618 / F440 ratio signal of the europium-doped carbon quantum dot fluorescent probe, eliminating environmental interference and improving detection sensitivity. Furthermore, the europium-doped carbon quantum dot fluorescent probe combines environmental detection and cell imaging functions. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope image of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 2 This is a high-resolution transmission electron microscope image of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 3 This is a histogram showing the particle size distribution of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 4 The infrared spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 5 The XPS full spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 6 High-resolution XPS spectra of C 1s, N 1s, O 1s and Eu 3d in the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 7 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different static incubation times in Example 4; Figure 8 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different pH solution conditions in Example 5; Figure 9 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different static incubation temperatures in Example 6; Figure 10 The UV-Vis absorption spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1; Figure 11 The fluorescence spectra of the Eu-CQDs-TC composite system under different excitation wavelengths in Example 7 are shown. Figure 12 The fluorescence spectra of the Eu-CQDs-TC composite system with different tetracycline concentrations in Example 8 are shown. Figure 13 The graph shows the linear relationship between tetracycline concentration and F618 / F440 in the Eu-CQDs-TC complex system in Example 8. Figure 14 The graphs show the fluorescence lifetime of the Eu-CQDs-TC composite system in Example 1 and Example 4. Figure 15 The graph shows the F618 / F440 fluorescence intensity ratio data of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 for different antibiotics, ions and amino acids. Figure 16 Cell viability data for three cell types under europium-doped carbon quantum dot fluorescent probes prepared in Example 1 at different concentrations; Figure 17The images show the fluorescence response of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 to human liver cancer cells treated with different concentrations of tetracycline, as captured by confocal fiber microscopy. Figure 18 The images show confocal fiber microscope images of the fluorescence response of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 to human breast cancer cells treated with different concentrations of tetracycline. Detailed Implementation

[0023] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0024] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0026] The technical solution of the present invention will be further described below with reference to specific embodiments and comparative examples. All reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing. The instruments used in the embodiments are also commercially available.

[0027] Example 1 This embodiment provides a europium-doped carbon quantum dot fluorescent probe, which is prepared by the following method: S1. Chop the pineapple peel and put it into a freeze dryer. Freeze dry for 72 hours, then grind to obtain freeze-dried pineapple peel powder. S2. Dissolve 0.5 g of Eu(NO3)3·6H2O, 0.6 g of pineapple peel freeze-dried powder from step S1, and 0.9 g of N,N'-diethylthiourea in 20 mL of ultrapure water, and sonicate for 20 min to obtain a mixed solution. S3. Place the mixed solution from step S2 into a stainless steel reactor lined with polytetrafluoroethylene, and react at 180 °C for 8 h to obtain the reaction product solution. S4. After the reaction product solution in step S3 is allowed to cool naturally to room temperature, it is centrifuged at 10,000 rpm for 10 min to obtain a supernatant. The supernatant is filtered through a membrane with a pore size of 0.22 μm to remove the carbonized insoluble substances and obtain a filtrate. The filtrate is dialyzed through a 100 Da membrane for 48 h to obtain a yellow europium-doped carbon quantum dot fluorescent probe (Eu-CQDs) solution. Finally, the solution is lyophilized to obtain europium-doped carbon quantum dot fluorescent probe solid powder.

[0028] Example 2 This embodiment provides a europium-doped carbon quantum dot fluorescent probe, which is prepared by the following method: S1. Chop the pineapple peel and put it into a freeze dryer. Freeze dry for 72 hours, then grind to obtain freeze-dried pineapple peel powder. S2. Dissolve 0.5 g of Eu(NO3)3·6H2O, 0.5 g of pineapple peel freeze-dried powder from step S1, and 0.8 g of N,N'-diethylthiourea in 20 mL of ultrapure water, and sonicate for 20 min to obtain a mixed solution. S3. Place the mixed solution from step S2 into a stainless steel reactor lined with polytetrafluoroethylene, and react at 200 °C for 8 h to obtain the reaction product solution. S4. After the reaction product solution in step S3 is allowed to cool naturally to room temperature, it is centrifuged at 10,000 rpm for 10 min to obtain a supernatant. The supernatant is filtered through a membrane with a pore size of 0.22 μm to remove the carbonized insoluble substances and obtain a filtrate. The filtrate is dialyzed through a 100 Da membrane for 48 h to obtain a yellow europium-doped carbon quantum dot fluorescent probe (Eu-CQDs) solution. Finally, the solution is lyophilized to obtain europium-doped carbon quantum dot fluorescent probe solid powder.

[0029] Example 3 This embodiment provides a europium-doped carbon quantum dot fluorescent probe, which is prepared by the following method: S1. Chop the pineapple peel and put it into a freeze dryer. Freeze dry for 72 hours, then grind to obtain freeze-dried pineapple peel powder. S2. Dissolve 0.6 g of Eu(NO3)3·6H2O, 0.6 g of pineapple peel freeze-dried powder from step S1, and 0.9 g of N,N'-diethylthiourea in 20 mL of ultrapure water, and sonicate for 20 min to obtain a mixed solution. S3. Place the mixed solution from step S2 into a stainless steel reactor lined with polytetrafluoroethylene, and react at 220 °C for 8 h to obtain the reaction product solution. S4. After the reaction product solution in step S3 is allowed to cool naturally to room temperature, it is centrifuged at 10,000 rpm for 10 min to obtain a supernatant. The supernatant is filtered through a membrane with a pore size of 0.22 μm to remove the carbonized insoluble substances and obtain a filtrate. The filtrate is dialyzed through a 100 Da membrane for 48 h to obtain a yellow europium-doped carbon quantum dot fluorescent probe (Eu-CQDs) solution. Finally, the solution is lyophilized to obtain europium-doped carbon quantum dot fluorescent probe solid powder.

[0030] Example 4 This embodiment provides a method for detecting tetracycline, including the following steps: M1. Dissolve the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 in ultrapure water to obtain a solution with a mass-volume concentration of 15 μg / mL. M2. Take eight 5 mL colorimetric tubes. First, add 2.5 mL of the solution from step M1 to each of the eight colorimetric tubes. Then, add 2.5 mL of 25 μM tetracycline (TC) to each of the eight colorimetric tubes to obtain a mixed solution. Adjust the pH of the mixed solution to 7 using sodium hydroxide or hydrochloric acid. Incubate the eight colorimetric tubes at 25 °C for 1, 2, 3, 4, 5, 6, 7, and 8 min, respectively, to obtain the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system (Eu-CQDs-TC). M3. Perform fluorescence spectroscopy analysis on the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system in step M2. Tetracycline was detected using an excitation wavelength of 380 nm, and the emission spectrum was set in the range of 380-650 nm. The slits for both excitation and emission were set to 5 nm, and the scan rate was 1200 nm / min. Each sample was tested three times, and the detection results were analyzed. A ratiometric detection standard curve was constructed based on the dual emission fluorescence intensity ratio F618 / F440.

[0031] Example 5 This embodiment provides a method for detecting tetracycline, which differs from Embodiment 4 in that: M2. Take seven 5 mL colorimetric tubes. First, add seven 2.5 mL portions of the solution from step M1 to the seven colorimetric tubes respectively. Then, add seven 2.5 mL portions of 25 μM tetracycline (TC) to the seven colorimetric tubes respectively to obtain a mixed solution. Adjust the pH value of the mixed solution in the seven colorimetric tubes to 4, 5, 6, 7, 8, 9 and 10 respectively by sodium hydroxide or hydrochloric acid. Incubate at 25 ℃ for 4 min to obtain the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system (Eu-CQDs-TC).

[0032] Example 6 This embodiment provides a method for detecting tetracycline, which differs from Embodiment 4 in that: M2. Take five 5 mL colorimetric tubes. First, add five 2.5 mL portions of the solution from step M1 to the five colorimetric tubes respectively. Then, add five 2.5 mL portions of 25 μM tetracycline (TC) to the five colorimetric tubes respectively to obtain a mixed solution. Adjust the pH of the mixed solution to 7 using sodium hydroxide or hydrochloric acid. Incubate the mixed solutions in the five colorimetric tubes at 20, 25, 30, 35, and 40 °C for 4 min respectively to obtain the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system (Eu-CQDs-TC).

[0033] Example 7 This embodiment provides a method for detecting tetracycline, which differs from Embodiment 4 in that: M2. Take a 5 mL colorimetric tube, first add 2.5 mL of the solution from step M1 to the colorimetric tube, then add 2.5 mL of 25 μM tetracycline (TC) to the colorimetric tube to obtain a mixed solution; adjust the pH of the mixed solution to 7 using sodium hydroxide or hydrochloric acid, and incubate at 25 ℃ for 4 min to obtain the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system (Eu-CQDs-TC). M3. Fluorescence spectroscopy analysis was performed on the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system in step M2. Tetracycline was detected using excitation wavelengths of 360, 365, 370, 375, 380, 385, 390, 395 and 400 nm, respectively. The emission spectrum was set in the range of 380-650 nm. The slits for both excitation and emission were set to 5 nm. The scan rate was 1200 nm / min. Each sample was tested three times, and the detection results were analyzed. A ratiometric detection standard curve was constructed based on the dual emission fluorescence intensity ratio F618 / F440.

[0034] Example 8 This embodiment provides a method for detecting tetracycline, which differs from Embodiment 4 in that: M2. Take six 5 mL colorimetric tubes. First, add 2.5 mL of the solution from step M1 to each of the six colorimetric tubes. Then, add 2.5 mL of tetracycline (TC) at concentrations of 1, 25, 50, 100, 150, and 200 μM to each of the six colorimetric tubes to obtain mixed solutions. Adjust the pH of the mixed solutions to 7 using sodium hydroxide or hydrochloric acid. Incubate at 25 °C for 4 min to obtain the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system (Eu-CQDs-TC).

[0035] Example 9 This embodiment provides a cell imaging method, including the following steps: N1. Dissolve the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 in ultrapure water to obtain solutions with a mass-volume concentration of 25 μM. N2. Human liver cancer cells (HepG2) or human breast cancer cells (MCF-7) were seeded into six-well plates and cultured for 10 h. Then, human liver cancer cells (HepG2) or human breast cancer cells (MCF-7) were cultured in medium with tetracycline concentrations of 0, 10, 31.25, 62.5, 125, and 250 μM for 14 h. The cells were washed three times with phosphate-buffered saline (PBS) to remove residual tetracycline. The cells were then co-cultured with the solution from step N1 for 10 h. The cells were washed three times with PBS to remove residual solution, and the sample to be tested was obtained. N3. Perform laser confocal microscopy on the sample to be tested in step N2. The excitation wavelength of laser confocal microscopy is 380 nm. Laser confocal microscopy acquires fluorescence images of the blue channel and the red channel. The wavelength range of the blue channel is 425-475 nm and the wavelength range of the red channel is 570-620 nm. The combined channel images are captured after the fluorescence signal is acquired to avoid photobleaching.

[0036] The europium-doped carbon quantum dot fluorescent probes in the examples were characterized and tested, and the results are as follows: Figure 1 The image shown is a transmission electron microscope (TEM) image of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Figure 1 The overall distribution of Eu-CQDs can be seen. Eu-CQDs are relatively uniformly dispersed in the sample, and the particle size is approximately in the range of a few nanometers, indicating that the Eu-CQDs prepared in Example 1 have good monodispersity.

[0037] Figure 2The image shows a high-resolution transmission electron microscope image of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Clear lattice fringes can be observed in the image, with a lattice spacing of d = 0.209 nm and a corresponding crystal plane index C(100), indicating that Eu-CQDs have high crystallinity. Good crystallinity is beneficial to improving the optical performance and stability of Eu-CQDs.

[0038] Figure 3 This is a histogram showing the particle size distribution of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Figure 3 The results show that most Eu-CQDs have a particle size range of 2.5 to 3.5 nm, with an average particle size of about 3.54 nm. The narrow particle size distribution indicates that the Eu-CQDs prepared in Example 1 have good size control, which is very important for ensuring the consistency and repeatability of material properties.

[0039] comprehensive Figures 1-3 The data shows that the Eu-CQDs prepared in Example 1 have uniform size distribution, good monodispersity and high crystallinity. Eu-CQDs can be an ideal material for fluorescent probe drug delivery and bioimaging.

[0040] Figure 4 The infrared spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 is shown below. Figure 4 It can be known that 3425cm -1 The absorption peak at 1628 cm⁻¹ is the stretching vibration absorption peak of OH / NH. -1 The absorption peak at 1530 cm⁻¹ is the bending vibration absorption peak of OH / C=N. -1 The absorption peak at 1356 cm⁻¹ is the stretching vibration absorption peak of C=C. -1 The absorption peak for the in-plane bending vibration of CH is at 1187 cm⁻¹. -1 The absorption peak for the stretching vibration of CO is at 875 cm⁻¹. -1 The peak at this point is the out-of-plane bending vibration absorption peak of CH. This infrared spectroscopy result indicates that the surface of Eu-CQDs contains hydrophilic groups such as C=O, -OH, and -NH2. These hydrophilic groups give Eu-CQDs good water solubility and stability.

[0041] Figure 5 This is the XPS full spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Figure 5 Distinguished binding energy peaks at 284, 399, 531, and 1135 eV can be observed, belonging to C 1s, N 1s, O 1s, and Eu 3d, respectively. The measured mass percentages of C 1s, N 1s, O 1s, and Eu 3d are 65.68%, 24.81%, 9.41%, and 0.1%, respectively.

[0042] Figure 6 High-resolution XPS spectra of C 1s, N 1s, O 1s and Eu 3d in the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Figure 6 (a) is the high-resolution XPS spectrum of C 1s. Figure 6 (b) is the high-resolution XPS spectrum of O 1s. Figure 6 (c) is the high-resolution XPS spectrum of N 1s. Figure 6 (d) is the high-resolution XPS spectrum of Eu 3d. Figure 6 The high-resolution XPS spectrum of C 1s in (a) shows three peaks at 283.88, 285.33 and 286.92 eV, respectively, which are derived from the binding energies of CC, CO and C=O. Figure 6 (b) The high-resolution XPS spectrum of O 1s can be fitted with three peaks at 529.92, 531.24 and 532.61 eV, which actually originate from Eu-O, C=O and CO, respectively. Figure 6 In (c), the three peaks at 398.08, 399.18 and 400.52 eV in the high-resolution XPS spectrum of N 1s correspond to C=NC, N(C)3 and NH (d), respectively. Figure 6 The high-resolution XPS spectrum of Eu 3d in (d) shows that Eu has been successfully doped into Eu-CQDs.

[0043] comprehensive Figure 5 and Figure 6 The data shows that N-element doping and Eu-element doping were successfully achieved in Eu-CQDs. 3+ Coordination modification is employed, and the surface of Eu-CQDs contains oxygen / nitrogen functional groups such as hydroxyl (-OH), amino (-NH2), and carboxyl (-COOH), whose chemical structural characteristics are corroborated by FTIR spectral analysis results. The presence of hydrophilic components such as hydroxyl, amino, and carboxyl groups endows Eu-CQDs with excellent solubility, giving them a dual advantage in the detection of aquatic pollutants and bioimaging: on the one hand, the hydrophilic groups enhance the targeted capture ability of pollutants such as tetracycline through hydrogen bonding and electrostatic interactions, significantly improving detection sensitivity; on the other hand, the surface functional groups endow the material with good biocompatibility and cell membrane permeability, combined with Eu... 3+ Its fluorescence properties enable low-toxicity, high-contrast intracellular imaging, providing an integrated, multifunctional nanomaterial solution for environmental monitoring and bioimaging.

[0044] Figure 7 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different static incubation times in Example 4. Figure 7The fluorescence intensity ratio F618 / F440 showed an increasing trend within 1-3 minutes, indicating that the reaction between Eu-CQDs and TC proceeded gradually. This phenomenon may be attributed to the gradually strengthening coordination between TC molecules and Eu-CQDs, leading to Eu... 3+ The characteristic fluorescence gradually increases. When the reaction time reaches 3-4 min, the fluorescence intensity ratio tends to stabilize, indicating that the reaction between Eu-CQDs and TC is basically complete, forming a stable complex. During the 4-8 min period, the fluorescence intensity ratio remains essentially unchanged with slight fluctuations. This is likely because the fluorescence signal of the complex stabilizes after reaching equilibrium, and the experimental error is within an acceptable range. Therefore, 4 min can be determined as the optimal reaction time for this detection system, at which point the reaction between Eu-CQDs and TC has reached equilibrium, the fluorescence signal is stable, and it is suitable for quantitative detection.

[0045] Figure 8 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different pH solution conditions in Example 5. From... Figure 8 It can be seen that the fluorescence intensity ratio F618 / F440 first increases and then decreases with increasing pH, gradually increasing between pH 4 and 7. This may be due to Eu 3+ The coordination environment with Eu-CQDs is more favorable under neutral conditions, thereby enhancing the coordination of Eu-CQDs. 3+ The characteristic fluorescence of the sample was observed. As the pH increased further above 7, the F618 / F440 ratio gradually decreased. Therefore, the optimal pH was 7, at which point the F618 / F440 ratio reached its maximum, indicating that the chelation of Eu-CQDs with TC was most effective.

[0046] Figure 9 This is a graph showing the F618 / F440 fluorescence intensity ratio data of Eu-CQDs-TC under different static incubation temperatures in Example 6. Figure 9 It is evident that the Eu-CQDs probe system exhibits excellent temperature adaptability within the 20-40 ℃ range. The small fluctuation range of the fluorescence intensity ratio F618 / F440 indicates that the impact of temperature changes on the stability of the detection signal is negligible. This allows the detection system to maintain consistent results without the need for precise temperature control at room temperature, demonstrating its convenience for practical applications, especially suitable for rapid on-site detection in water bodies in the field.

[0047] Figure 10 The image shows the UV-Vis absorption spectrum of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1. Figure 10A significant absorption peak is observed at 380 nm, which originates from the n-π* electronic transitions of carboxyl / amino functional groups in the surface states of Eu-CQDs. This absorption characteristic indicates that selecting an excitation wavelength near 380 nm maximizes excitation efficiency, providing an ideal photophysical basis for subsequent fluorescence detection.

[0048] Figure 11 The images show the fluorescence spectra of the Eu-CQDs-TC composite system under different excitation wavelengths in Example 7. Figure 11 It is observed that within the excitation wavelength range of 360-400 nm, the emission peak of the Eu-CQDs-TC composite system redshifts from 440 nm to 485 nm, accompanied by a decrease in fluorescence intensity. This wavelength-dependent redshift phenomenon is closely related to the size polydispersity of Eu-CQDs and the distribution of surface defect states. Shorter wavelength excitation preferentially activates the bandgap emission of smaller CQDs, while longer wavelength excitation triggers radiative recombination dominated by larger particles or surface states. When the excitation wavelength matches the TC absorption band (λ... ex At 380 nm, the system exhibits Eu at 618 nm. 3+ The characteristic emission peak.

[0049] Figure 12 The fluorescence spectra of the Eu-CQDs-TC composite system with different tetracycline concentrations in Example 8 are shown. Figure 12 It can be seen that as TC concentration increases, Eu... 3+ The fluorescence intensity at 618 nm showed a linear increase, while the optimal emission of Eu-CQDs at 440 nm was significantly suppressed due to the internal filtration effect (IFE) and static quenching (SQE).

[0050] Figure 13 This is a linear relationship graph between tetracycline concentration and F618 / F440 in the Eu-CQDs-TC complex system in Example 8. Figure 13 It can be seen that the ratiometric detection standard curve constructed based on the dual emission fluorescence intensity ratio F618 / F440 shows excellent linearity in the range of 1-200 μM, with the linear equation F618 / F440=0.1248+0.00236CTC, R2=0.9902, and the limit of detection (LOD) reaching 15.1 nM (S / N=3).

[0051] Figure 14 The images show the fluorescence lifetime curves of the Eu-CQDs-TC composite systems in Example 1 and Example 4. Figure 14The fluorescence lifetime of the Eu-CQDs-TC composite system decreased slightly from 6.42 ns to 5.46 ns after the introduction of TC. This result indicates that the fluorescence quenching of Eu-CQDs by TC does not depend on dynamic collisions or energy transfer mechanisms, but is more likely achieved through a static quenching pathway, forming a ground-state complex with the functional groups on the surface of Eu-CQDs. This response mechanism is beneficial to the stability of the probe in complex environments, avoiding signal fluctuations caused by molecular collisions or energy dissipation, thereby improving detection reproducibility.

[0052] Figure 15 The image shows the F618 / F440 fluorescence intensity ratio data of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 for different antibiotics, ions, and amino acids (*p<0.05, **p<0.01, ***p<0.001). The concentrations of amoxicillin (AMX), the antibiotics ampicillin (AMP), enrofloxacin (ERFX), chlortetracycline (KM), cysteine ​​(Cys), alanine (Ala), leucine (Leu), and various ions used in the tests were all 37.5 μM. Figure 15 Monitoring the fluorescence intensity changes of Eu-CQDs at an excitation wavelength of 380 nm revealed that only TC significantly enhanced the F618 / F440 ratio fluorescence signal, while the influence of other interfering substances on the fluorescence signal was negligible. This result indicates that Eu-CQDs' recognition of TC relies on its unique coordination or energy transfer mechanism, rather than non-specific adsorption. Combined with its excellent resistance to matrix interference, the ratiometric signal of the Eu-CQDs probe can effectively overcome background fluorescence and turbidity interference in complex aquatic environments, making it highly suitable for rapid in-situ detection of trace TC in real water bodies.

[0053] The europium-doped carbon quantum dot fluorescent probe prepared in Example 1 demonstrated excellent reliability in detecting tetracycline in actual lake water samples. Spiked recovery experiments were conducted to simulate tetracycline pollution in real water bodies, and the results are shown in Table 1. As can be seen from Table 1, within the tetracycline concentration range of 5-200 μM, the probe achieved a recovery rate of 96.2%-103.8% for tetracycline in the lake water matrix, with a relative standard deviation (RSD) of less than 5%, confirming its excellent resistance to interference from complex matrices. The probe's unique dual-signal self-calibration mechanism effectively eliminates interference from coexisting humic acids, metal ions, and suspended particles in lake water, eliminating the need for complex pretreatment. Measurement errors were relatively large in the 0.1-1 μM tetracycline concentration range, mainly because the F-value was close to the intercept of the standard curve, significantly amplifying small fluctuations; however, the recovery rate was still below 110%. This indicates that the detection method has good accuracy and precision for detecting TC in lake water samples.

[0054] Table 1. Results of spiked recovery experiments simulating tetracycline pollution in real water bodies.

[0055] Figure 16 Cell viability data for three cell types under different concentrations of europium-doped carbon quantum dot fluorescent probes prepared in Example 1. Figure 16 It was found that when the Eu-CQDs concentration was between 0.04207 and 0.21033 g / L, the cell viability of mouse mononuclear macrophage leukemia cells (RAW264.7) was above 80%, and the cell viability gradually decreased with increasing concentration after 0.21033 g / L. Similarly, when the Eu-CQDs concentration was between 0.04207 and 0.3155 g / L, the cell viability of human breast cancer cells (MCF-7) was above 80%, and the cell viability gradually decreased with increasing concentration after 0.3155 g / L. Likewise, when the Eu-CQDs concentration was between 0.04207 and 0.1262 g / L, the cell viability of human liver cancer cells (HepG2) was above 80%, and the cell viability gradually decreased with increasing concentration after 0.1262 g / L. With increasing Eu-CQD concentration, the viability of all cell lines decreased, indicating that its toxicity is concentration-dependent. The MCF-7 cell line showed strong tolerance to Eu-CQDs, followed by the RAW264.7 cell line, while the HepG2 cell line was more sensitive, especially at higher concentrations where cell viability significantly decreased. This difference may be related to cell metabolic characteristics and the intracellular transport efficiency of nanoparticles. Overall, the prepared Eu-CQDs exhibit good biocompatibility and low toxicity.

[0056] Figure 17 The images show confocal fiber microscope images of the fluorescence response of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 to human liver cancer cells treated with different concentrations of tetracycline. Figure 18 The images show confocal fiber microscope images of the fluorescence response of the europium-doped carbon quantum dot fluorescent probe prepared in Example 1 to human breast cancer cells treated with different concentrations of tetracycline. Figure 17 and Figure 18 In the diagram, AF represents cells treated with tetracycline at concentrations of 0, 10, 31.25, 62.5, 125, and 250 μM, respectively; i represents the blue channel; ii represents the red channel; and iii represents the combined blue, red, and bright-field channels. HepG2 and MCF-7 cells were treated with different concentrations of TC and then labeled with Eu-CQDs. Fluorescence images of the blue channel (425–475 nm) and red channel (570–620 nm), as well as bright-field images, were acquired using laser confocal microscopy. The results showed that with increasing TC concentration, the fluorescence signal of the blue channel gradually weakened, while the fluorescence signal of the red channel gradually increased. This may be due to the interaction between TC and Eu-CQDs and Eu... 3+The interaction between the two leads to fluorescence quenching and enhancement effects. The results indicate that the Eu-CQD-based dual-channel fluorescence imaging method can effectively detect changes in intracellular TC concentration, and exhibits good biocompatibility and high sensitivity.

[0057] 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; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a europium-doped carbon quantum dot fluorescent probe, characterized in that, Includes the following steps: S1. After freeze-drying the pineapple peel, grind it to obtain freeze-dried pineapple peel powder; S2. Dissolve Eu(NO3)3·6H2O, N,N'-diethylthiourea, and the pineapple peel freeze-dried powder from step S1 in water to obtain a mixed solution; the mass ratio of Eu(NO3)3·6H2O, N,N'-diethylthiourea, and pineapple peel freeze-dried powder is (3-7):(7-11):(4-8); the mixed solution is subjected to hydrothermal reaction at 160-220 ℃ to obtain a reaction product solution; S3. The reaction product solution described in step S2 is centrifuged to obtain a supernatant; the supernatant is filtered and dialyzed to obtain a europium-doped carbon quantum fluorescent probe.

2. The method for preparing the europium-doped carbon quantum dot fluorescent probe according to claim 1, characterized in that, In step S2, the mass-to-volume ratios of Eu(NO3)3·6H2O, N,N'-diethylthiourea, and the pineapple peel freeze-dried powder to the water are 0.15-0.35 g / mL, 0.35-0.55 g / mL, and 0.2-0.4 g / mL, respectively.

3. The method for preparing the europium-doped carbon quantum dot fluorescent probe according to claim 1, characterized in that, The hydrothermal reaction time in step S2 is 6-12 h.

4. A europium-doped carbon quantum dot fluorescent probe, characterized in that, It is prepared by the method described in any one of claims 1-3 for preparing europium-doped carbon quantum dot fluorescent probes.

5. A method for tetracycline detection using a europium-doped carbon quantum dot fluorescent probe as described in claim 4, characterized in that, Includes the following steps: M1. Dissolve the europium-doped carbon quantum dot fluorescent probe in water to obtain a solution with a mass-volume concentration of 8-25 μg / mL; M2. First, place the solution described in step M1 into a colorimetric tube, then add tetracycline to the colorimetric tube to obtain a mixed solution. After the mixed solution is allowed to stand and incubate, a europium-doped carbon quantum dot fluorescent probe-tetracycline composite system is obtained. M3. Perform fluorescence spectroscopy analysis on the europium-doped carbon quantum dot fluorescent probe-tetracycline composite system described in step M2, and construct a ratiometric detection standard curve based on the dual emission fluorescence intensity ratio F618 / F440.

6. The tetracycline detection method according to claim 5, characterized in that, The concentration of tetracycline in the mixed solution in step M2 is 1-200 μM.

7. The tetracycline detection method according to claim 5, characterized in that, The static incubation time described in step M2 is ≥1 min.

8. The tetracycline detection method according to claim 5, characterized in that, The pH value of the solution described in step M2 is 5-10.

9. A cell imaging method using a europium-doped carbon quantum dot fluorescent probe as described in claim 4, characterized in that, Includes the following steps: N1. Dissolve the europium-doped carbon quantum dot fluorescent probe in water to obtain a solution with a mass-volume concentration of 8-25 μg / mL; N2. The cells are seeded into a cell culture plate for the first culture, and then cultured in a medium with a tetracycline concentration ≤250 μM for the second culture. After washing away the residual tetracycline on the surface, the cells are cultured in the solution described in step N1 for the third culture. After washing away the residual solution on the surface, the sample to be tested is obtained. N3. Perform laser confocal microscopy imaging on the sample to be tested described in step N2.

10. The cell imaging method according to claim 9, characterized in that, The excitation wavelength of the laser confocal microscopy is 380 nm. The laser confocal microscopy acquires fluorescence images of the blue channel and the red channel. The wavelength range of the blue channel is 425-475 nm, and the wavelength range of the red channel is 570-620 nm.

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