Application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection

Abstract

The application of a ratio fluorescent probe based on ligand charge transfer effect in phosphate ion detection belongs to the technical field of fluorescence detection, and the dual-emission ratio fluorescent probe comprises quantum dots stably emitting red light and metal organic framework material UiO-66-NH2 capable of emitting blue light and responding to phosphate. UiO-66-NH2 takes zirconium as a metal center and 2-amino terephthalic acid as an organic fluorescent ligand. Without the addition of phosphate, the ligand fluorescence is greatly weakened due to the existence of the ligand charge transfer effect. After the addition of phosphate, the phosphate is specifically combined with the Zr-O site in UiO-66-NH2, the ligand charge transfer effect is destroyed, and the ligand produces obvious fluorescence change. The red quantum dots with good distinguishability of ligand fluorescence emission are introduced to construct the dual-emission ratio fluorescent probe, which can effectively eliminate the influence of detection instruments, external environment and the like on the detection process, the obvious change from red to purple red and then to blue is caused by the addition of phosphate, and the fluorescence visual analysis of phosphate is realized.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to the application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection. Background Technology

[0002] Phosphate is an important inorganic anion, essential for living organisms. It is a crucial component for maintaining physiological pH and participates in physiological processes such as gene construction, energy storage, bone mineralization, and signal transduction. Phosphate levels are closely related to disease occurrence. Furthermore, phosphate plays a vital role in the ecological environment, serving as an essential nutrient for aquatic plant growth. However, excessive phosphate can lead to eutrophication, causing rapid growth of algae and other plankton, decreased oxygen levels in the water, water quality deterioration, and ultimately, the death of fish and other organisms. Therefore, the detection of phosphate ions is of great significance for ecological environment protection and human health monitoring.

[0003] Currently, commercially available kits for phosphate detection mainly use malachite green and molybdate as their main components. These kits form a blue-green complex with phosphate to indicate the phosphate content. However, colorimetric methods suffer from insufficient detection sensitivity. In contrast, fluorescence detection methods offer advantages such as ease of operation and high sensitivity, allowing them to play a greater role in phosphate detection.

[0004] Metal-organic frameworks (MOFs), a class of porous crystalline materials with periodic network structures formed by the self-assembly of metal centers and organic ligands through coordination bonds, can yield diverse structures and rich properties through the rational design of their central ions and ligands. UiO-66-NH2, a classic MOF composed of zirconium groups and 2-aminoterephthalic acid (BDC-NH2), possesses excellent chemical stability and abundant surface active sites. Furthermore, its ligands exhibit fluorescence in the free state, making UiO-66-NH2 a promising candidate for applications in biosensing. Gu et al. (Metal–organic frameworks with inherent recognition sites for selective phosphate sensing through their coordination-induced fluorescence enhancement effect. J. Mater. Chem. A, 2015, 3, 7445) prepared UiO-66-NH2 and used it for the fluorescence detection of phosphate ions. Inorganic phosphate ions specifically chelate with specific sites in UiO-66-NH2, achieving ligand fluorescence enhancement. However, this work mainly utilizes a single signal enhanced by ligand fluorescence, and changes in this single signal can easily affect the accuracy of the detection if they are interfered with by changes in the external environment. Summary of the Invention

[0005] To address the shortcomings of the aforementioned research methods, we introduced quantum dots into UiO-66-NH2 to achieve the application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection. The ratiometric fluorescent probe of this invention operates on the principle that phosphate ions disrupt the ligand charge transfer effect in UiO-66-NH2 without affecting the quantum dots. The stable fluorescence emission of the quantum dots is used to calibrate detection errors caused by environmental changes, enabling accurate phosphate ion detection. The ratiometric fluorescent probe provided by this invention is simple to synthesize, easy to use, and can achieve ratiometric fluorescence detection of phosphate ions in solution systems. Furthermore, a clear fluorescence color transition can be observed under a single excitation wavelength, facilitating the visual analysis of phosphate ions.

[0006] The present invention adopts the following technical solution:

[0007] An application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection includes the following steps:

[0008] (1) A metal-organic framework UiO-66-NH2 that specifically responds to phosphate was synthesized by a solvothermal method;

[0009] (2) Quantum dots were introduced to construct a ratiometric fluorescent probe based on ligand charge transfer effect to realize fluorescence detection and visualization analysis of phosphate ions.

[0010] Furthermore, the process of synthesizing the phosphate-specific metal-organic framework UiO-66-NH2 in step (1) using a solvothermal method is as follows:

[0011] Dissolve 0.0543 g of 2-aminoterephthalic acid in 5 mL of N,N-dimethylformamide and add 30 μL of triethylamine to obtain solution A. Dissolve 0.0668 g of zirconium oxychloride octahydrate in 5 mL of N,N-dimethylformamide to obtain solution B. Mix 1.38 mL of acetic acid with 5 mL of N,N-dimethylformamide to obtain solution C. Mix solutions A, B, and C, vortex, and add to a 10 mL glass bottle. Place the glass bottle in an 85℃ oven for 24 h and cool to room temperature to obtain a brownish-red turbid liquid. Centrifuge the reaction solution at 8000 rpm for 5 min, discard the supernatant, and collect the precipitate. Wash the precipitate three times each with 5 mL of N,N-dimethylformamide, 5 mL of methanol, and 5 mL of water. Disperse the product in 1 mL of water and store at room temperature for later use.

[0012] Furthermore, in step (2), quantum dots are introduced to construct a ratiometric fluorescent probe based on the ligand charge transfer effect, and the process of realizing the fluorescence detection and visualization analysis of phosphate ions is as follows:

[0013] A 20 nM quantum dot solution was prepared by diluting 8 μM quantum dots 400 times with water. This quantum dot solution was then mixed with the metal-organic framework material UiO-66-NH2 at a volume ratio of 1:6 to obtain a ratiometric fluorescent probe. 70 μL of this ratiometric fluorescent probe solution was taken, and phosphate solutions of different concentrations were added. Finally, ultrapure water was added to bring the reaction volume to 200 μL. After reacting at room temperature for 10 min, the emission intensities of the probe after adding the target were recorded at 430 nm and 620 nm, and the ratio F was calculated. 430 / F 620 Create a work curve.

[0014] Furthermore, the fluorescence visualization results of adding different concentrations of phosphate were observed under a 365 nm handheld ultraviolet lamp. The red fluorescence remained stable, while the blue fluorescence gradually appeared with the addition of phosphate, showing a visually observable color change from red to purplish-red and then to blue, providing a convenient method for the detection of phosphate ions in the ecological environment and living organisms.

[0015] The mechanism of this invention is as follows:

[0016] This invention achieves ratiometric fluorescence detection and visualization analysis of phosphate ions based on the different interaction mechanisms between phosphate ions and the metal-organic framework UiO-66-NH2 and quantum dots. The synthesized metal-organic framework UiO-66-NH2 uses 2-aminoterephthalic acid as a ligand, which emits blue fluorescence in its free state, with a maximum emission wavelength of 435 nm. When the ligand is inside the metal-organic framework, its fluorescence is greatly weakened due to the ligand charge transfer effect. The presence of phosphate ions disrupts this charge transfer effect, thus the ligand fluorescence gradually recovers with increasing phosphate concentration. The quantum dots used in this invention exhibit extremely high stability to phosphate ions; their luminescence properties do not change with phosphate concentration, and their maximum emission is at 620 nm, clearly distinguishable from the blue fluorescence of the ligand. When the two materials are mixed, the blue fluorescence gradually recovers with increasing phosphate ion concentration, while the red fluorescence remains unchanged. Ratiometric fluorescence detection of phosphate ions can be achieved by recording the emission intensity at these two wavelengths. Under 365 nm ultraviolet light, a clear fluorescence color change can be observed, which facilitates the visualization and analysis of phosphate ions.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. The application of the ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection provided by this invention has simple synthesis conditions, good fluorescence stability, and is easy to store.

[0019] 2. The ratiometric fluorescent probe provided by this invention utilizes the blue fluorescence generated by the response of UiO-66-NH2 to phosphate, and introduces stable luminescent red quantum dots that are unresponsive to phosphate, effectively eliminating the influence of detection instruments and the external environment on the detection process. Importantly, the addition of phosphate causes a significant color transition from red to purplish-red and then to blue, enabling the fluorescence visualization analysis of phosphate, which can promote the detection of phosphate and the development of phosphate-related physiological processes. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the detection principle used in this invention.

[0021] Figure 2 Basic fluorescence characterization of the ligand and the metal-organic framework UiO-66-NH2 is shown in Figure A, where A is the fluorescence emission spectrum of 2-aminoterephthalic acid; B is a photograph of 2-aminoterephthalic acid and the metal-organic framework UiO-66-NH2; and C is a fluorescence photograph of 2-aminoterephthalic acid and the metal-organic framework UiO-66-NH2 under 365 nm excitation.

[0022] Figure 3Potential changes before and after the addition of phosphate ions to the metal-organic framework UiO-66-NH2.

[0023] Figure 4 The images show the basic morphological and spectral characterization of quantum dots, where A, B, and C are transmission electron micrographs of quantum dots, particle size statistics of quantum dots, and fluorescence spectra of quantum dots, respectively.

[0024] Figure 5 The fluorescence spectra of a ratiometric fluorescent probe based on ligand charge transfer effect in the presence of different concentrations of phosphate are shown.

[0025] Figure 6 Fluorescence images of ratiometric fluorescent probes based on ligand charge transfer effects detecting different concentrations of phosphate. Detailed Implementation

[0026] To make the above features and advantages of the present invention clearer and easier to understand, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] Preparation of a ratiometric fluorescent probe based on ligand charge transfer effect

[0029] (1) Dissolve 0.0543 g of 2-aminoterephthalic acid in 5 mL of N,N-dimethylformamide and add 30 μL of triethylamine to obtain solution A. Dissolve 0.0668 g of zirconium oxychloride octahydrate in 5 mL of N,N-dimethylformamide to obtain solution B. Mix 1.38 mL of acetic acid with 5 mL of N,N-dimethylformamide to obtain solution C. Mix solutions A, B and C, vortex and add to a 10 mL glass bottle. Place the glass bottle in an 85℃ oven for 24 h and cool to room temperature to obtain a brownish-red turbid liquid. Centrifuge the reaction solution at 8000 rpm for 5 min, discard the supernatant and collect the precipitate. Wash the precipitate three times each with 5 mL of N,N-dimethylformamide, 5 mL of methanol and 5 mL of water. Disperse the product in 1 mL of water and store at room temperature for later use.

[0030] (2) Dilute 8 μM quantum dots with water 400 times to obtain a 20 nM quantum dot solution. Mix the quantum dot solution with the metal-organic framework material UiO-66-NH2 at a volume ratio of 1:6 to obtain a ratiometric fluorescent probe, which is then stored at room temperature.

[0031] Example 2

[0032] Ratio-fluorescent probes based on ligand charge transfer effects are used for fluorescence visualization analysis of phosphate ions.

[0033] Take 70 μL of ratiometric fluorescent probe solution, add phosphate solutions of different concentrations, and finally add ultrapure water to bring the reaction volume to 200 μL. After reacting at room temperature for 10 min, record the emission intensities of the probe at 430 nm and 620 nm after adding the target, and calculate the ratio F. 430 / F 620 Working curves were generated. Furthermore, the fluorescence visualization results of adding different concentrations of phosphate were observed under 365 nm handheld UV light.

[0034] Figure 1 According to the detection principle of this invention, when there is no phosphate in the reaction system, the ligands in the metal-organic framework UiO-66-NH2 show no fluorescence, while the quantum dots exhibit bright red fluorescence under 365 nm excitation. When phosphate is present, the phosphate disrupts the charge transfer effect of the ligands in the metal-organic framework UiO-66-NH2, and the ligand fluorescence is restored. At this time, the red fluorescence remains stable, exhibiting a mixed fluorescence of red and blue.

[0035] Figure 2 For basic fluorescence characterization of the ligand and the metal-organic framework UiO-66-NH2, the free ligand dissolved in N,N-dimethylformamide appears yellow and produces bright blue fluorescence under 365 nm UV light. Spectroscopic characterization results show that its maximum emission peak is located at 435 nm with obvious fluorescence. In contrast, the aqueous solution of the metal-organic framework UiO-66-NH2 is brick red and shows no fluorescence under 365 nm excitation, indicating that the presence of ligand charge transfer effect leads to the weakening of ligand fluorescence in the metal-organic framework.

[0036] Figure 3 The potential change of the metal-organic framework UiO-66-NH2 before and after the addition of phosphate is shown. Due to the presence of amino groups, UiO-66-NH2 is positively charged, but after the addition of phosphate, UiO-66-NH2 becomes negatively charged. As the concentration of phosphate increases, the potential of UiO-66-NH2 further decreases.

[0037] Figure 4 The basic morphology and spectroscopic characterization of the quantum dots show that the quantum dots are well dispersed in the solution and have uniform size. The particle size statistics show that the size of the quantum dots is 7 nm ± 2 nm, and they produce a strong fluorescence emission peak at 620 nm under 365 nm excitation.

[0038] Figure 5The detection of phosphate ions based on ratiometric fluorescent probes shows that as the concentration of phosphate ions increases, the fluorescence of the UiO-66-NH2 ligand is continuously restored due to the continuous disruption of the ligand charge transfer effect, and the fluorescence intensity at 435 nm continuously increases. However, since phosphate ions have no effect on quantum dots, the intensity of quantum dots at 620 nm remains basically unchanged.

[0039] Figure 6 For the visualization analysis of phosphate ions based on ratiometric fluorescent probes, it was shown that under the excitation of a 365 nm ultraviolet lamp, the fluorescence of the probe changes from red to purplish-red and then to blue as the concentration of phosphate ions increases.

Claims

1. An application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection, characterized in that: Includes the following steps: (1) The metal-organic framework UiO-66-NH2, which is specifically responsive to phosphate, was synthesized by a solvothermal method. The process is as follows: Dissolve 0.0543 g of 2-aminoterephthalic acid in 5 mL of N,N-dimethylformamide and add 30 μL of triethylamine to obtain solution A. Dissolve 0.0668 g of zirconium oxychloride octahydrate in 5 mL of N,N-dimethylformamide to obtain solution B. Mix 1.38 mL of acetic acid with 5 mL of N,N-dimethylformamide to obtain solution C. Mix solutions A, B and C, vortex and add to a 10 mL glass bottle. Place the glass bottle in an 85℃ oven and react for 24 h. After cooling to room temperature, a brownish-red turbid liquid is obtained. Centrifuge the reaction solution at 8000 rpm for 5 min, discard the supernatant, and take the precipitate. Wash the precipitate three times each with 5 mL of N,N-dimethylformamide, 5 mL of methanol and 5 mL of water. Disperse the product in 1 mL of water and store at room temperature for later use. (2) Quantum dots were introduced to construct a ratiometric fluorescent probe based on the ligand charge transfer effect, enabling fluorescence detection and visualization analysis of phosphate ions; the specific process is as follows: A quantum dot solution with a concentration of 8 μM was diluted 400 times with water to obtain a 20 nM quantum dot solution. The quantum dot solution was then mixed with the metal-organic framework material UiO-66-NH2 at a volume ratio of 1:6 to obtain a ratiometric fluorescent probe. The maximum emission wavelength of the quantum dots was 620 nm.

2. The application of a ratiometric fluorescent probe based on ligand charge transfer effect in phosphate ion detection according to claim 1, characterized in that, The detection process is as follows: Take 70 μL of the ratiometric fluorescent probe solution, add phosphate solutions of different concentrations, and finally add ultrapure water to bring the reaction system volume to 200 μL. After reacting at room temperature for 10 min, record the fluorescence emission intensity of the probe at 430 nm and 620 nm after adding the target, and calculate the ratio F. 430 / F 620 Working curves were constructed, and the fluorescence visualization results of adding different concentrations of phosphate were observed under a 365 nm handheld UV lamp. The red fluorescence remained stable, while the blue fluorescence gradually appeared with the addition of phosphate, showing a visually observable color change from red to purplish-red and then to blue.

Images