Preparation method and application of dual-channel multifunctional multicolor fluorescent probe

Abstract

The invention discloses a preparation method and application of a dual-channel multifunctional multicolor fluorescent probe, and relates to the technical field of fluorescent sensing detection.The dual-channel multifunctional multicolor fluorescent probe is prepared through the following steps that pyromellitic acid and ZrCl4 solids are dispersed in deionized water, Zr-MOF is prepared through a hydrothermal method, pamoic acid and melamine are dispersed in deionized water, and the Zr-MOF is prepared through a hydrothermal method; the preparation method comprises the following steps: preparing green fluorescent carbon dots (CDs) by using a hydrothermal method, dispersing the CDs and Zr-MOF in deionized water according to a certain proportion, reacting, collecting a brown solid, washing, drying, dispersing the solid in ethanol, adjusting the pH to be alkaline, adding Eu (NO3) 3.6 H2O, reacting, filtering, and drying in vacuum to obtain a target probe. The probe can accurately distinguish Cu < 2 + > ions of tetracycline antibiotics (TCs) through two independent fluorescent channels, not only displays completely independent spectral response, but also has the advantages of self-calibration, high sensitivity, interference resistance, field visual detection and the like. The multifunctional fluorescent probe has important significance for improving detection efficiency, reducing cost and energy consumption, optimizing resource utilization and promoting green low-carbon economy.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence sensing detection technology, and particularly relates to a method that can simultaneously distinguish between tetracycline antibiotics and heavy metal copper ions (Cu). 2+ Preparation method and application of dual-channel multifunctional multicolor fluorescent probe. Background Technology

[0002] Tetracycline antibiotics (TCs) are a class of broad-spectrum antibiotics, mainly including doxycycline (DOX), chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC), widely used in clinical and livestock fields. However, drug residues caused by abuse enter ecosystems through multiple pathways, affecting the structure and function of soil microbial communities, disrupting the ecological balance of aquatic bodies, and causing potentially far-reaching damage to the ecological environment. Furthermore, drug residues in livestock products may bioaccumulate in the human body through the food chain, thereby inducing adverse health conditions such as drug resistance, gastrointestinal diseases, fatty liver disease, and pancreatitis. In addition, because different tetracycline antibiotics have significantly different side effects, accurate differentiation of TCs is crucial for accurate environmental monitoring and pollutant source attribution. Additionally, copper (Cu...) 2+ Cu, as an endocrine disruptor, has received widespread attention from scientists and the public due to its high toxicity. With the rapid increase in industrial wastewater and acidic mine wastewater, Cu... 2+ Pollution is now pervasive in all aspects of modern life. In summary, sensitive detection and accurate identification of TCs antibiotics and Cu... 2+ It is of great significance to environmental protection and human health.

[0003] Currently, Cu detection 2+ Methods for detecting Cu and TCs mainly include atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), and enzyme-linked immunosorbent assay (ELISA). However, these methods often require expensive and bulky instruments, specialized operators, and complex pretreatment procedures, making them unsuitable for on-site and rapid detection requirements. In contrast, fluorescence methods, as a mature visual signal transduction method, offer high operability and a simplified analytical workflow, demonstrating unique advantages in on-site detection. With the development of fluorescence sensing technology, especially the advancement of ratiometric fluorescence detection technology with self-calibration capabilities, the detection of Cu... 2+Significant progress has been made in fluorescent probes for TCs. However, most fluorescent probes are single-function systems, suffering from problems such as detecting only one substrate, high cost, and low efficiency, making it difficult to achieve highly sensitive and visual detection of multiple substrates coexisting in complex environments and to distinguish structurally similar substances. Therefore, the development of fluorescent probes that can be used for simultaneous, interference-free, self-calibrated, high-precision, high-sensitivity, and on-site visual detection of Cu for multiple pollutants is needed. 2+ The discovery of TC and multifunctional fluorescent probes that can accurately distinguish TC analogs is of great significance for improving detection efficiency, reducing costs and energy consumption, optimizing resource utilization, and promoting a green and low-carbon economy. Summary of the Invention

[0004] To overcome the above shortcomings, the purpose of this invention is to provide a method that can simultaneously distinguish between tetracycline antibiotics and heavy metal copper ions (Cu). 2+ Preparation method and application of dual-channel multifunctional multicolor fluorescent probe.

[0005] To achieve the above objectives, the present invention is implemented according to the following technical solution: A method for preparing a dual-channel, multi-functional, multicolor fluorescent probe includes the following steps: Preparation of S1 and Zr-MOF solid powder: A certain proportion of pyromellitic acid and ZrCl4 solid were dispersed in a certain volume of deionized water and stirred for several hours. The mixture was reacted at a certain temperature for a period of time, centrifuged, washed, and dried to obtain a white solid. S2. Preparation of green fluorescent carbon dots (CDs): A certain mass ratio of pamoic acid and melamine solids are dispersed in a certain amount of deionized water and stirred evenly. The mixture is heated at a certain temperature for a period of time, cooled to room temperature, and then centrifuged. The supernatant is placed in a dialysis bag for dialysis. The dialysis solution is freeze-dried to obtain brown carbon dot powder.

[0006] S3, Preparation of rare earth Eu 3+ A multifunctional fluorescent probe was obtained by ion-functionalizing Zr-MOF-based composite material encapsulating green carbon dots (CDs). CDs and Zr-MOF were mixed in deionized water at a specific ratio and stirred at a certain temperature for a period of time. The resulting brown solid product was collected by centrifugation, washed to remove residual impurities, dried, and then dispersed in a certain amount of ethanol solution. The pH was adjusted to alkaline using LiOH. A certain amount of Eu(NO3)3·6H2O was added to the solution, and the mixture was stirred at a certain temperature for a certain time. The solid was collected, washed, and vacuum dried to obtain the target product.

[0007] Preferably, the mass ratio of pyromellitic acid to ZrCl4 in S1 is 1.0~1.2, the reaction temperature is 110~130℃, and the reaction time is 12~36 hours; Preferably, the mass ratio of pamoic acid to melamine in S2 is 2-4, the temperature is 160-200℃, and the molecular weight cutoff of the dialysis bag is 500. Preferably, the mass ratio of CDs to Zr-MOF in S3 is 1:1, the reaction temperature is 60~80℃, the pH value is 7~9, and the mass ratio of added Eu(NO3)3·6H2O to Zr-MOF is 8~10. Preferably, the obtained green fluorescent carbon dots have a particle size range of 1-5 nanometers and an average particle size of 3.1 nanometers; Preferably, the obtained Zr-MOF and multifunctional probe are both cubic structures with a size of less than 30 μm.

[0008] This invention also provides a multifunctional fluorescent probe for detecting and accurately distinguishing TCs analogs and Cu. 2+ The specific steps for its application are as follows: S4. Detection and differentiation of Cu 2+ The method for detecting TCs was as follows: First, a certain amount of nanoprobe was dispersed in a certain volume of HCl-Tris buffer to obtain a standard detection solution. Then, TCs standard solutions of different concentrations were added to the above solution. Fluorescence intensity data at 520 nm and 616 nm were recorded at specific excitation wavelengths. 2+ The detection process is similar to that of TCs, except that the buffer solution is replaced with HEPES and the experimental data is recorded at a certain excitation wavelength.

[0009] Preferably, the concentration of the nanoprobe in S4 is 1~2 mg / mL, and the pH of both HCl-Tris and HEPES buffer is 7-9.

[0010] Preferably, the excitation wavelength range for detecting TCs in S4 is 350-390 nm, and the wavelength range for detecting Cu is... 2+ The excitation wavelength range is 260-300 nm.

[0011] Compared with existing technologies, the preparation method of the dual-channel multifunctional multicolor fluorescent probe of the present invention has the following advantages: 1. The dual-channel, multifunctional, multicolor fluorescent probe of this invention features a unique design, utilizing the abundant porous structure and ease of functionalization of MOF materials, combined with rare-earth Eu... 3+ Ions can achieve efficient energy transfer in a suitable ligand environment, thus producing strong red fluorescence. Combined with the excellent luminescence properties and low toxicity of carbon dots, a dual-channel proportional fluorescent probe with self-calibration function and excitation wavelength dependence was constructed.

[0012] 2. The dual-channel multifunctional multicolor fluorescent probe of the present invention responds to Cu 2+It has unique advantages in detecting and accurately distinguishing Cu from TCs, and can achieve Cu detection through two independent fluorescence channels. 2+ The precise differentiation from TCs not only demonstrates completely independent spectral responses but also exhibits high selectivity, high sensitivity, and anti-interference capabilities. Based on a competitive coordination substitution mechanism and intermolecular antenna effect, this nanoprobe shows excellent contrast between Cu and TCs at excitation wavelengths of 295 nm and 385 nm. 2+ The dual-channel cross-interference-free response was achieved with TCs, and Cu 2+ The detection limits (LODs) for this device and DOX are 10.58 nM and 2.71 nM, respectively, indicating extremely high sensitivity. The response time is rapid, at 3 minutes, meeting the needs of rapid on-site detection in practical applications.

[0013] 3. The dual-channel, multifunctional, multicolor fluorescent probe prepared in this invention can be fabricated into a smartphone-assisted wearable glove-based nanosensor, thereby improving the portability of rapid on-site detection. It has potential applications in the field of real-time monitoring of food and the environment. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a transmission electron microscope (TEM) image and particle size distribution diagram of green carbon dots in Example 1 of the present invention; Figure 2 This is a scanning electron microscope (SEM) image of the multifunctional fluorescent probe of Embodiment 1 of the present invention; Figure 3 The fluorescent probe of Example 1 of this invention responds to Cu 2+ Fluorescence spectra, linear relationships, and CIE plots of TCs; Figure 4 This is a comparison chart of the detection effects of the fluorescent probe of Example 1 of the present invention on different TCs; Figure 5 This invention relates to a portable glove-type fluorescence sensor based on a fluorescent probe, as described in Embodiment 1 of the present invention, for use in smartphone-assisted visual detection of Cu. 2+ Applications in TCs. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions herein are used to explain the present invention, but are not intended to limit the present invention.

[0017] Example 1 A method for preparing a dual-channel, multifunctional, multicolor fluorescent probe, the specific steps of which are as follows: A one-step hydrothermal method was used to prepare CDs: 30 mL of deionized water preheated to 60°C was added to 0.8 g of pamoic acid, and 26 mL of deionized water was added to 0.2 g of melamine. After the two mixtures were completely dispersed, they were mixed and stirred continuously for 1 h. The mixture was then transferred to a 100 mL polytetrafluoroethylene-lined reactor and heated at 200°C for 6 h. After cooling to room temperature, a dark brown mixed solution was obtained. After centrifugation, the supernatant was dialyzed in a dialysis bag containing an MWCO 500 for 24 h. The dialysate was freeze-dried to obtain brown CDs powder, the morphology and particle size distribution of which are shown in the figure. Figure 1 As shown.

[0018] Preparation of Zr-MOF: 0.087 g of pyromellitic acid and 0.087 g of ZrCl4 were dispersed in 40 mL of deionized water and stirred until homogeneous. The mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 120 °C for 24 hours. After the reaction, the solid was washed three times with water to remove unreacted impurities, yielding a white solid. This solid was then washed three times with ethanol and dried under vacuum to obtain Zr-MOF.

[0019] 40 mg of CDs and 40 mg of Zr-MOF powder were dispersed in 15 mL of distilled water. The resulting mixture was stirred at 60 °C for 16 hours. The brown solid product was then collected by centrifugation and washed alternately with deionized water and ethanol to remove residual impurities. After drying, the solid product was dispersed in 16 mL of ethanol solution, and the pH was adjusted to 7.0 with LiOH. Then, 0.36 g of Eu(NO3)3·6H2O was added to the above solution. After stirring at 60 °C for 12 h, the solid was collected and washed alternately with deionized water and ethanol to completely remove free CDs and Eu. 3+ Ions. Finally, the solid was dried in a drying oven at 60°C to obtain the target product.

[0020] Example 2 A one-step hydrothermal method was used to prepare CDs: 26 mL of deionized water preheated to 60°C was added to 0.6 g of pamoic acid, and simultaneously 26 mL of deionized water was added to 0.20 g of melamine. After the two mixtures were completely dispersed, they were mixed and stirred continuously for 1 h. The mixture was then transferred to a 100 mL polytetrafluoroethylene-lined reactor and heated at 190°C for 6 h. After cooling to room temperature, a dark brown mixed solution was obtained. After centrifugation, the supernatant was dialyzed in a dialysis bag containing an MWCO 500 for 12 h. The dialysate was freeze-dried to finally obtain brown CDs powder.

[0021] Preparation of Zr-MOF: 0.1 g of pyromellitic acid and 0.087 g of ZrCl4 were dispersed in 40 mL of deionized water and stirred until homogeneous. The mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 110 °C for 24 hours. After the reaction, the solid was washed three times with water to remove unreacted impurities, yielding a white solid. This solid was then washed three times with ethanol and dried under vacuum to obtain Zr-MOF.

[0022] 45 mg of CDs and 45 mg of Zr-MOF powder were dispersed in 18 mL of distilled water. The resulting mixture was stirred at 60 °C for 20 h. The brown solid product was then collected by centrifugation and washed alternately with deionized water and ethanol to remove residual impurities. After drying, the solid product was dispersed in 18 mL of ethanol solution, and the pH was adjusted to 8 with LiOH. Then, 0.45 g of Eu(NO3)3·6H2O was added to the above solution. After stirring at 70 °C for 12 h, the solid was collected and washed alternately with deionized water and ethanol to completely remove free CDs and Eu. 3+ Ions. Finally, the solid was dried in a drying oven at 60°C to obtain the target product, the morphology and size of which are as follows. Figure 2 As shown.

[0023] Example 3 A one-step hydrothermal method was used to prepare CDs: 28 mL of deionized water preheated to 60°C was added to 0.7 g of pamoic acid, and 26 mL of deionized water was added to 0.20 g of melamine. After the two mixtures were completely dispersed, they were mixed and stirred continuously for 1 h. The mixture was then transferred to a 100 mL polytetrafluoroethylene-lined reactor and heated at 160°C for 6 h. After cooling to room temperature, a dark brown mixed solution was obtained. The solution was centrifuged, and the supernatant was dialyzed in a dialysis bag containing an MWCO 500 for 12 h. The dialysate was freeze-dried to finally obtain brown CDs powder.

[0024] Preparation of Zr-MOF: 0.096 g of pyromellitic acid and 0.087 g of ZrCl4 were dispersed in 40 mL of deionized water and stirred until homogeneous. The mixture was transferred to a polytetrafluoroethylene-lined reactor and reacted at 130 °C for 36 hours. After the reaction, the solid was washed three times with water to remove unreacted impurities, yielding a white solid. This solid was then washed three times with ethanol and dried under vacuum to obtain Zr-MOF.

[0025] 35 mg of CDs and 35 mg of Zr-MOF powder were dispersed in 15 mL of distilled water. The resulting mixture was stirred at 60 °C for 20 h. The brown solid product was then collected by centrifugation and washed alternately with deionized water and ethanol to remove residual impurities. After drying, the solid product was dispersed in 15 mL of ethanol solution, and the pH was adjusted to 9 with LiOH. Then, 0.28 g of Eu(NO3)3·6H2O was added to the above solution. After stirring at 80 °C for 12 h, the solid was collected and washed alternately with deionized water and ethanol to completely remove free CDs and Eu. 3+ Ions. Finally, the solid was dried in a drying oven at 60°C to obtain the target product.

[0026] Example 4 Detection and differentiation of TCs and Cu 2+ Method: First, 1 mg of the nanoprobe was dispersed in 2 mL of HCl-Tris buffer solution (pH 7.0) to obtain a standard detection solution. Then, different concentrations of TCs standard solutions were added to the above solution. Fluorescence intensity data at 520 nm and 616 nm were recorded at an excitation wavelength of 385 nm. For Cu... 2+ The detection process is similar to that of TCs, except the buffer solution is replaced with HEPES (pH=9.0), and experimental data are recorded at an excitation wavelength of 295 nm. This fluorescent probe responds to and distinguishes Cu... 2+ Fluorescence spectra, linear relationships, CIE plots, differentiation diagrams, and device application diagrams of TCs are shown below. Figures 3-5 As shown;

[0027] Example 5 Detection and differentiation of TCs and Cu 2+ Method: First, 1.5 mg of the nanoprobe was dispersed in 2 mL of HCl-Tris buffer solution at pH = 8.0 to obtain a standard detection solution. Then, different concentrations of TCs standard solutions were added to the above solution. Fluorescence intensity data at 520 nm and 616 nm were recorded at an excitation wavelength of 385 nm. For Cu... 2+ The detection process is similar to that of TCs, except that the buffer solution is replaced with HEPES (pH = 8.0), and experimental data are recorded at an excitation wavelength of 295 nm.

[0028] Example 6 Detection and differentiation of TCs and Cu 2+Method: First, 2 mg of the nanoprobe was dispersed in 2 mL of HCl-Tris buffer solution at pH = 9.0 to obtain a standard detection solution. Then, standard solutions of different concentrations of TCs were added to the above solution. Fluorescence intensity data at 520 nm and 616 nm were recorded at an excitation wavelength of 385 nm. For Cu... 2+ The detection process is similar to that of TCs, except that the buffer solution is replaced with HEPES (pH = 7.0), and experimental data are recorded at an excitation wavelength of 295 nm.

[0029] like Figure 1 As shown, the obtained green carbon dots appear as uniformly dispersed spherical particles under an electron microscope.

[0030] Figure 2 This is a scanning electron microscope (SEM) image of the multifunctional fluorescent probe of Embodiment 2 of the present invention; Figure 3 The fluorescent probe of Example 4 of this invention responds to Cu 2+ Fluorescence spectra, linear relationships, and CIE plots of TCs; Figure 4 This is a comparison chart of the detection effects of the fluorescent probe of Example 3 of the present invention on different TCs; Figure 5 The portable glove-type fluorescence sensor based on a fluorescent probe, as described in Embodiment 3 of this invention, is used in smartphone-assisted visual detection of Cu. 2+ Applications in TCs.

[0031] This invention relates to a method for preparing and applying a dual-channel, multifunctional, multicolor fluorescent probe, which solves the problem that single-function probes cannot simultaneously detect multiple pollutants in complex environments. This method is applicable not only to Cu, but also to tetracycline antibiotics (TCs). 2 It exhibits a completely independent spectral response and possesses advantages such as self-calibration, high sensitivity, anti-interference, and on-site visual detection. This multifunctional fluorescent probe is of great significance for improving detection efficiency, reducing costs and energy consumption, optimizing resource utilization, and promoting a green and low-carbon economy.

[0032] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a dual-channel, multifunctional, multicolor fluorescent probe and its application, characterized in that, Green light CDs and Zr-MOF were dispersed in deionized water in a certain ratio. After the reaction, a brown solid was collected, washed, dried, and then dispersed in ethanol. The pH was adjusted to alkaline, and Eu(NO3)3·6H2O was added. After the reaction, the mixture was filtered and vacuum dried to obtain the target probe. This probe can achieve Cu2+ targeting of tetracycline antibiotics (TCs) through two independent fluorescence channels. 2+ Precise detection and differentiation of ions.

2. The method for preparing a dual-channel, multifunctional, multicolor fluorescent probe according to claim 1, characterized in that, The mass ratio of pyromellitic acid to ZrCl4 in the preparation of Zr-MOF is 1.0~1.2, the reaction temperature is 110~130℃, and the reaction time is 12~36 hours.

3. The method for preparing a dual-channel, multi-functional, multi-color fluorescent probe according to claim 1, characterized in that, When preparing green fluorescent CDs, the mass ratio of pamoic acid to melamine is 2-4, the temperature is 160-200℃, and the molecular weight cutoff of the dialysis bag is 500.

4. The method for preparing a dual-channel, multifunctional, multicolor fluorescent probe according to claim 1, characterized in that, The mass ratio of CDs to Zr-MOF is 1:1, the reaction temperature is 60~80℃, the pH value is 7~9, and the mass ratio of added Eu(NO3)3·6H2O to Zr-MOF is 8~10.

5. The application of the dual-channel multifunctional multicolor fluorescent probe according to claim 1, characterized in that, The concentration of the nanoprobe was 1–2 mg / mL, and the pH of both the HCl-Tris and HEPES buffers was 7–9.

6. The application of the dual-channel multifunctional multicolor fluorescent probe according to claim 1, characterized in that, The excitation wavelength range for detecting TCs is 350-390 nm, and the detection wavelength range for Cu is... 2+ The excitation wavelength range is 260-300 nm.

7. The method for preparing a dual-channel, multi-functional, multi-color fluorescent probe according to claim 1, characterized in that, The obtained green fluorescent carbon dots have a particle size range of 1-5 nm and an average particle size of 3.1 nm; the obtained Zr-MOF and multifunctional probe are both cubic structures with a size of less than 30 μm.

8. The fluorescent probe prepared according to any one of claims 1-7, characterized in that, The obtained probe can accurately distinguish tetracycline antibiotics in channels with excitation wavelengths of 350-390 nm, and can simultaneously specifically detect Cu in channels with excitation wavelengths of 260-300 nm. 2+ .

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