A fluorescent probe for detecting polyphenolic substances, a preparation method and a detection method

Abstract

This invention discloses a fluorescent probe for detecting polyphenols, its preparation method, and a detection method, as well as its application in distinguishing and / or quantifying polyphenols. The fluorescent probe is a complex formed by assembling rare-earth europium ions, an organic ligand containing a phenylboronic acid group, and a fluorescent signal molecule; the phenylboronic acid group is a specific recognition site for the catechol structure. This invention also relates to a method for distinguishing and / or quantifying specific polyphenols containing a catechol structure, utilizing the competitive substitution between this method and the fluorescent signal molecule to induce a fluorescence response, achieving high specificity distinction. Furthermore, a method for quantifying total phenol content is provided, achieving the detection of total phenol content by introducing copper ions and their competitive chelation effect. The probe and method of this invention possess both the dual functions of specific distinction of specific types of polyphenols and total phenol quantification. The detection process is simple, highly sensitive, and can be widely applied to the analysis and quality control of food, pharmaceuticals, and agricultural products.

Description

Technical Field

[0001] This application relates to the field of material detection and analysis technology. Specifically, it relates to a fluorescent probe for the qualitative and quantitative detection of polyphenolic substances, a preparation method, and a detection method, which is particularly suitable for the targeted identification of plant polyphenols containing catechol structures and the simultaneous determination of total phenol content. Background Technology

[0002] Plant polyphenols (such as tea polyphenols and proanthocyanidins) are core functional components related to the quality of agricultural product processing and the development of health foods. Accurate identification and content detection of polyphenols with different structural types are crucial prerequisites for regulating their activity and optimizing product characteristics.

[0003] Currently, the mainstream techniques for distinguishing and detecting polyphenols mainly include high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). These methods separate and quantify polyphenol components based on differences in the partition coefficients of different substances between the stationary and mobile phases or differences in the mass-to-charge ratio in the mass spectrometer. Their advantages include high selectivity and sensitivity. However, these methods rely on expensive chromatographic and mass spectrometric equipment, require specialized operators, and involve cumbersome sample pretreatment processes (such as extraction, purification, and gradient elution), resulting in long analysis times per session. This makes them unsuitable for the rapid screening and online monitoring of large numbers of samples in production practice.

[0004] Another widely studied technique is electrochemical sensing, which achieves electrochemical catalytic oxidation or selective recognition of polyphenols by modifying the electrode surface. It has a fast response speed and the instrument can be miniaturized. However, when distinguishing phenolic homologues with extremely similar structures, this method often faces the limitation of insufficient specificity due to overlapping oxidation potentials. Moreover, the long-term stability and reproducibility of the electrode are still bottlenecks in its application.

[0005] Therefore, there is an urgent need for a fluorescent probe, its preparation method, and its detection method for the qualitative differentiation and quantitative detection of polyphenols, in order to solve the problems existing in the prior art. Summary of the Invention

[0006] The purpose of this application is to address the aforementioned problems in the prior art by providing a fluorescent probe, preparation method, and detection method for polyphenol detection. By constructing a rare-earth fluorescent probe with a specific phenylboronic acid recognition site on its surface and loaded with fluorescent signal molecules, the binding of the target polyphenol to the recognition site induces competitive substitution of fluorescent molecules, or the competitive chelation reaction between polyphenols and metal ions induces characteristic changes in the fluorescent signal, thereby achieving specific differentiation and accurate quantitative detection of polyphenol structural types.

[0007] To achieve the above-mentioned objectives, this application adopts the following technical solution: a fluorescent probe, which is a complex formed by assembling rare earth europium ions, an organic ligand containing a phenylboronic acid group, and a fluorescent signal molecule; wherein, the phenylboronic acid group is a specific recognition site for the catechol structure, and the fluorescent signal molecule is used to generate a characteristic fluorescent response signal when it binds to or is replaced by the recognition site.

[0008] Furthermore, the organic ligand containing the phenylboronic acid group is 3,5-dicarboxyphenylboronic acid; the fluorescent signal molecule is 6,7-dihydroxycoumarin.

[0009] A method for preparing a fluorescent probe as described above includes the following steps:

[0010] (1) A soluble europium salt solution and an organic ligand solution containing phenylboronic acid groups are mixed in a solvent and subjected to a solvothermal reaction. After the reaction is completed, the rare earth organic framework precursor is obtained by solid-liquid separation, washing and drying. (2) Disperse the rare earth organic framework precursor in a solvent, mix it with the fluorescent signal molecule solution and incubate it to allow the fluorescent signal molecule to bind to the rare earth organic framework, thus obtaining the fluorescent probe.

[0011] Further, in step (1), the soluble europium salt is europium nitrate hexahydrate, the organic ligand containing the phenylboronic acid group is 3,5-dicarboxyphenylboronic acid, and the molar ratio of the two is 1:1; the solvent is a mixed solvent of N,N-dimethylformamide and water; and in step (2), the fluorescent signal molecule is 6,7-dihydroxycoumarin.

[0012] Further, in step (1), the volume ratio of N,N-dimethylformamide to water is 7:3, and the solvothermal reaction conditions are 115-125℃ for 10-14 hours; in step (2), the final concentration of 6,7-dihydroxycoumarin in the reaction system is 60.0-70.0 μM, and the incubation conditions are incubation at room temperature for at least 10 minutes.

[0013] An application of the above-described fluorescent probe in the qualitative differentiation and / or quantitative detection of samples containing polyphenols, wherein the polyphenols include types containing a catechol structure and types not containing a catechol structure.

[0014] A method for distinguishing and / or quantifying specific polyphenols in solution using the aforementioned fluorescent probe, wherein the polyphenols are polyphenols containing a catechol structure, the method comprising: The polyphenol solution to be tested was mixed with the fluorescent probe solution and reacted. After the reaction, the fluorescence signal intensity of the system was measured. When the test solution contains a polyphenol with a catechol structure, it specifically binds to the phenylboronic acid group of the probe through a borate bond, competitively displacing the fluorescent signal molecule, resulting in a characteristic change in the fluorescence signal intensity of the system. Polyphenols without a catechol structure do not cause this change. Based on the aforementioned changes in fluorescence signals, specific polyphenols can be qualitatively distinguished, or their content can be calculated by substituting the values ​​into a pre-established standard curve of fluorescence signal intensity versus specific polyphenol concentration.

[0015] Furthermore, the polyphenol containing the catechol structure is epigallocatechin gallate; the reaction time between the polyphenol solution to be tested and the fluorescent probe solution is 10 minutes.

[0016] A method for quantifying the total phenol content in a solution using the aforementioned fluorescent probe includes: First, the fluorescent probe solution is mixed with Cu 2+ Solution mixing, utilizing Cu 2+ The initial fluorescence is quenched by chelation of fluorescent signal molecules to obtain the baseline fluorescence signal; Then add the total phenol solution to be tested and react. After the reaction, measure the fluorescence signal intensity of the system. In this study, total phenols in the test solution act as competing ligands from Cu. 2+ The fluorescent signal molecules are captured and re-bound to the rare earth organic framework in the probe, resulting in the recovery of the fluorescent signal. The measured fluorescence signal intensity was substituted into the pre-established standard curve of fluorescence signal intensity versus total phenol concentration to calculate the total phenol content in the test solution.

[0017] Furthermore, Cu 2+ The concentration in the final reaction system was 50.0-70.0 μM; the total reaction time between the total phenol solution to be tested and the probe system was 10 minutes.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Excellent targeting and high specificity: The rare-earth fluorescent probe of this invention uses an organic ligand containing a phenylboronic acid group as a specific recognition site, which can competitively displace pre-loaded fluorescent signal molecules from polyphenols containing a catechol structure through specific binding. This synergistic design of the probe backbone and signal response unit significantly improves the recognition specificity of the target polyphenol, effectively solving the technical pain point of traditional methods in distinguishing polyphenols with highly similar structures.

[0019] 2. Dual Functions of Polyphenol Specific Differentiation and Precise Total Phenolic Quantification: This invention not only achieves selective identification and quantification of specific polyphenols through direct competitive substitution between the probe and polyphenols containing the catechol structure, but also introduces Cu...2+ The mediated competitive chelation mechanism induces the quenched fluorescent signal molecules to ionize and emit light again, enabling precise quantification of the total phenol content in the solution. This dual detection mode covers a wider range of detection scenarios.

[0020] 3. Simple operation and high quantitative accuracy: Compared with traditional chromatographic and mass spectrometric detection techniques, this invention is based on fluorescence spectrophotometry, which greatly simplifies the sample pretreatment process, has a fast detection speed and high efficiency, and shows an extremely stable linear response relationship between fluorescence signal intensity and specific polyphenol or total phenol concentration. It also shows good recovery rate and precision in the analysis of actual complex matrix samples. Attached Figure Description

[0021] Figure 1 A schematic diagram of the synthesis of the Eu / 3,5-DCBA fluorescent probe and the detection method for polyphenol differentiation and / or quantification based on fluorescence spectrophotometry; Figure 2 SEM, TEM and EDS characterization images of Eu-MOF and Eu / 3,5-DCBA; Figure 3 XPS high-resolution image of B1s for Eu-MOF; Figure 4 XPS high-resolution image of Eu-MOF at 1s; Figure 5 XPS high-resolution image of Eu 6 for Eu-MOF; Figure 6 XPS high-resolution image of Eu / 3,5-DCBA B 1s; Figure 7 XPS high-resolution image of Eu / 3,5-DCBA at 1s; Figure 8 XPS high-resolution image of Eu 6 for Eu / 3,5-DCBA; Figure 9 XPS full-spectrum images of Eu-MOF and Eu / 3,5-DCBA; Figure 10 Infrared spectra of Eu-MOF and Eu / 3,5-DCBA; Figure 11 The fluorescence response spectra of Eu / 3,5-DCBA to different concentrations of epigallocatechin gallate are shown. Figure 12 for Figure 11 The corresponding linear relationship graph; Figure 13 The fluorescence response spectra of Eu / 3,5-DCBA to different concentrations of epigallocatechin gallate in the total phenolic system are shown. Figure 14 for Figure 13 The corresponding linear relationship graph; Figure 15 Eu / 3,5-DCBA-Cu 2+ Fluorescence response spectra of total phenol solutions at different concentrations; Figure 16 This is the corresponding linear relationship diagram; Figure 17 The specific fluorescence response spectrum of the Eu / 3,5-DCBA fluorescent probe is shown. Figure 18 This is a reproducibility graph of the Eu / 3,5-DCBA fluorescent probe; Figure 19 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0024] This invention designs a rare-earth fluorescent probe assembled from rare-earth europium ions, an organic ligand containing a phenylboronic acid group, and a fluorescent signal molecule. This fluorescent probe utilizes the phenylboronic acid group as a specific recognition site for the catechol structure. When the recognition site binds to or undergoes a displacement reaction with the target polyphenol, the generated characteristic fluorescent response signal enables qualitative differentiation and quantitative detection of the polyphenol. Furthermore, by introducing a copper ion-mediated competitive chelation mechanism, the same probe can also be switched to a total phenol detection mode.

[0025] like Figure 1 As shown, Figure 1 Part A in the diagram is a schematic diagram of the synthesis of the Eu / 3,5-DCBA fluorescent probe, where the excitation wavelength is 275 nm, the slit width for both excitation and emission is 5 nm, and the scanning wavelength range is 350-650 nm. Figure 1 Part B in the diagram is a schematic diagram of a polyphenol differentiation method based on fluorescence spectrophotometry; Figure 1 Part C in the diagram is a schematic diagram of a method for quantifying the total amount of polyphenols based on fluorescence spectrophotometry.

[0026] like Figure 2 As shown, Figure 2 Parts A and D in the image are SEM images of Eu-MOF and Eu / 3,5-DCBA, respectively. Figure 2 Parts B and E in the image are TEM images of Eu-MOF and Eu / 3,5-DCBA, respectively. Figure 2 The C and F parts in the image are EDS images of Eu-MOF and Eu / 3,5-DCBA, respectively. The C and F parts each correspond to four sub-images, which correspond to the four key elements that make up the material: B (boron), C (carbon), O (oxygen), and Eu (eu).

[0027] Example 1: Preparation of rare earth fluorescent probe (Eu / 3,5-DCBA fluorescent probe) This embodiment provides a rare-earth fluorescent probe, which is a complex assembled from rare-earth europium ions, an organic ligand containing a phenylboronic acid group, and a fluorescent signal molecule. Specifically, in this embodiment, the organic ligand containing the phenylboronic acid group is selected from 3,5-dicarboxyphenylboronic acid, the fluorescent signal molecule is selected from 6,7-dihydroxycoumarin, and the rare-earth europium ions are derived from the soluble europium salt—europium nitrate hexahydrate. See also Figure 1 Part A and Figure 19 As shown, the preparation method of this embodiment includes the following steps: Step (1): Synthesis of rare earth organic framework precursors First, the polytetrafluoroethylene reactor was pre-treated by cleaning: the reactor was placed in a nitric acid solution with a volume ratio of nitric acid to water of 3:25, and treated in an oven at 160°C for 10 hours. Then it was washed with ultrapure water and dried for later use.

[0028] Weigh 0.1338 g of europium nitrate hexahydrate solid powder and 0.0630 g of 3,5-dicarboxyphenylboronic acid solid powder. Dissolve europium nitrate hexahydrate in 9 mL of ultrapure water and 3,5-dicarboxyphenylboronic acid in 21 mL of N,N-dimethylformamide (DMF), and dissolve them completely. Mix the two solutions to obtain a mixed solution with a molar ratio of europium nitrate hexahydrate to 3,5-dicarboxyphenylboronic acid of 1:1, and a volume ratio of N,N-dimethylformamide to water of 7:3. After stirring the mixed solution at room temperature for 2 hours, transfer it to a pretreated polytetrafluoroethylene reactor and carry out a solvothermal reaction at 120°C for 12 hours.

[0029] After the reaction was completed, the product was naturally cooled to room temperature and then separated into solid and liquid components by centrifugation. The resulting solid product was washed three times each with DMF and ethanol, and then placed in a vacuum drying oven and dried at 60°C for 12 hours to obtain a white powder product, which is the rare earth organic framework precursor Eu-MOF.

[0030] The morphology and structure of the rare-earth organic framework precursor prepared in this embodiment were characterized. See [link to documentation]. Figure 2 In part A of the image, the SEM image shows that Eu-MOF exhibits a regular morphology. See also... Figure 2 Part B of the image further confirms its size and morphological features using TEM images. See also Figure 2 In the C part of the structure, EDS elemental analysis confirmed that elements such as Eu, B, C, and O are uniformly distributed in the structure.

[0031] Step (2): Loading fluorescent signal molecules Weigh 0.03 mg of the prepared Eu-MOF and dissolve it in ultrapure water to obtain an Eu-MOF solution with a concentration of 0.0625 mg / mL. Simultaneously, weigh 0.67 mg of 6,7-dihydroxycoumarin and dissolve it in DMSO to prepare a 6,7-dihydroxycoumarin solution with a concentration of 125 μM.

[0032] The above Eu-MOF solution and 6,7-dihydroxycoumarin solution were mixed at a volume ratio of 1:1, so that the final concentration of 6,7-dihydroxycoumarin in the reaction system was 62.5 μM. The mixture was incubated at room temperature for 10 minutes to allow the fluorescent signal molecule 6,7-dihydroxycoumarin to be fully bound to the rare earth organic framework Eu-MOF through borate ester bonds or supramolecular interactions, thus obtaining the rare earth fluorescent probe of the present invention.

[0033] The prepared rare-earth fluorescent probes were characterized. See [link to documentation]. Figure 2 In part D of the image, SEM images show that after loading 6,7-dihydroxycoumarin, the probe retains its original morphology with slight surface roughening, indicating successful loading of the signal molecule. See also... Figure 2 The morphological feature in section E of the image was confirmed by TEM images. See also Figure 2 In the F portion of the spectrum, the proportions of C and O elements in the EDS elemental analysis are increased compared to Eu-MOF, further confirming the successful binding of 6,7-dihydroxycoumarin. See also... Figures 3 to 8 XPS high-resolution images, and Figure 9 XPS full spectrum images and Figure 10 The infrared spectra confirmed the successful coordination of europium ions with ligands and the binding of 6,7-dihydroxycoumarin with the rare earth organic framework.

[0034] Example 2: A method for distinguishing and / or quantifying specific polyphenols in solution This embodiment utilizes the rare-earth fluorescent probe prepared in Example 1 to provide a method for distinguishing and / or quantifying specific polyphenols in solution. The specific polyphenols are those containing a catechol structure. See also... Figure 1 Part B of the method states that the detection principle of this method is as follows: Polyphenols containing a catechol structure can specifically covalently bind to the phenylboronic acid group in the probe via a borate ester bond, competitively replacing the signal molecule 6,7-dihydroxycoumarin originally loaded on the rare earth organic framework, resulting in a characteristic change in the fluorescence signal intensity of the system; while polyphenols without a catechol structure cannot form a stable borate ester bond and therefore cannot trigger this competitive replacement reaction, thus not causing a change in fluorescence signal.

[0035] (a) Quantitative detection of specific polyphenols in a single system The rare-earth fluorescent probe solution prepared in Example 1 was placed in a fluorescence spectrophotometer. The excitation wavelength was set to 275 nm, and the excitation and emission slit widths were both 5 nm. The scanning wavelength range was 350-650 nm, and the initial fluorescence intensity at the characteristic emission wavelength was recorded as F0. A series of epigallocatechin gallate (EGCG, a typical polyphenol containing a catechol structure) standard solutions of different concentrations were added to the probe solution. After mixing and reacting at room temperature for 10 minutes, the fluorescence emission spectra were scanned.

[0036] See Figure 11 As the concentration of EGCG increases, the fluorescence intensity of the system exhibits a regular change. (See also...) Figure 12 A linear regression equation was obtained by plotting the logarithm of EGCG concentration on the x-axis and the corresponding change in fluorescence intensity on the y-axis. The equation was FL = 3.76 lgCEGCG + 0.66, with a linear correlation coefficient R² = 0.997, indicating a good linear relationship within this concentration range. Based on a signal-to-noise ratio of three times (3σ / S), the limit of detection (LOD) of this method was calculated to be 0.092 μM.

[0037] (II) Specific differentiation and quantification of specific polyphenols in mixed systems To verify the specific recognition ability of this method for catechol-type polyphenols, detection was performed against the background of interference from oxidized resveratrol (Ox-RES, a polyphenol without a catechol structure). A mixed standard solution of EGCG and oxidized resveratrol was added to the probe solution, with the final concentration of oxidized resveratrol fixed at 120 μM and the final concentration of EGCG varying in a gradient range of 0-60 μM. After reacting at room temperature for 10 minutes, the fluorescence emission spectrum was scanned.

[0038] See Figure 13 and Figure 14The obtained fluorescence response spectrum and linear relationship are highly consistent with the single EGCG system. The regression equation obtained after fitting is FL = 3.75 lgCEGCG + 0.73, with a linear correlation coefficient R² = 0.996 and a detection limit of 0.178 μM. Experimental results show that even against the background of high concentrations of non-catechol polyphenol interference, the fluorescent probe of this invention still exhibits excellent specificity and accurate quantification ability for specific polyphenols containing a catechol structure. Oxidized resveratrol, lacking a catechol structure, cannot trigger a competitive reaction and does not produce a fluorescence response signal, thus not interfering with the detection of EGCG.

[0039] Example 3: Method for quantifying total phenol content in solution This embodiment utilizes the rare-earth fluorescent probe prepared in Example 1 to provide a method based on copper ions (Cu). 2+ A method for the quantitative determination of total phenol content in solution mediated by [missing information]. See also [missing information]. Figure 1 In part C, the detection principle of this method lies in the introduction of Cu. 2+ First, it chelates with the 6,7-dihydroxycoumarin loaded in the probe, freeing the signal molecule from the rare-earth organic framework and quenching the initial fluorescence; upon addition of the total phenol solution to be tested, all polyphenol components in the solution can act as Cu. 2+ Competitive ligands from Cu 2+ Cu is extracted from the 6,7-dihydroxycoumarin chelate. 2+ This process causes the quenched 6,7-dihydroxycoumarin to become free again and re-bind with the rare earth organic framework, thereby restoring fluorescence. The degree of fluorescence recovery is directly proportional to the total phenol content in the test solution.

[0040] The specific steps are as follows: First, prepare a Cu solution with a concentration of 125 μM. 2+ The solution was mixed with the rare earth fluorescent probe solution prepared in Example 1 at a volume ratio of 1:1, so that Cu 2+ The concentration in the final reaction system was 62.5 μM (this concentration falls within the range of 50.0–70.0 μM). Incubation was performed at room temperature for 10 minutes, using Cu… 2+ The initial fluorescence was quenched by chelation with 6,7-dihydroxycoumarin, and the fluorescence intensity of the system at this point was measured as the baseline fluorescence signal F'0.

[0041] Then, to the above-mentioned Cu-containing 2+ A series of mixed standard solutions of EGCG and resveratrol oxide at different concentrations (EGCG to resveratrol concentration ratio of 1:1, total phenol concentration range of 0-80 μM) were added to the probe system. After reacting for 10 minutes at room temperature, the fluorescence emission spectrum was scanned.

[0042] See Figure 15 Fluorescence spectroscopy showed that the fluorescence intensity of the system gradually recovered and increased with increasing total phenol concentration. (See also...) Figure 16 A linear regression equation was obtained by plotting total phenol concentration on the x-axis and fluorescence intensity recovery value on the y-axis, resulting in FL = 0.025CTP + 0.243, with a linear correlation coefficient R0. 2 = 0.997, and the limit of detection (LOD) is 0.166 μM.

[0043] Example 4: Selectivity and Reproducibility of Rare Earth Fluorescent Probes To comprehensively evaluate the performance of the rare earth fluorescent probe of this invention, selectivity and reproducibility tests were conducted.

[0044] Selectivity testing: Under the same detection conditions, the fluorescence response of the rare-earth fluorescent probe to various potential interfering substances was examined. See also... Figure 17 The fluorescence responses of epigallocatechin gallate (EGCG), oxidized resveratrol (Ox-RES), gallic acid (GA), tannic acid (TA), ascorbic acid (AA), and glucose were tested. The results showed that only polyphenols containing a catechol group (EGCG, gallic acid, and tannic acid) elicited significant fluorescence response signals; substances without a catechol structure, such as oxidized resveratrol (resorcinol type), ascorbic acid, and glucose, showed fluorescence response signals that were essentially the same as the blank control, with no significant changes. This result confirms that the rare-earth fluorescent probe of this invention has a high specificity for recognizing catechol-type polyphenols.

[0045] Reproducibility test: To verify the reproducibility of the detection method, five parallel detections were performed on EGCG solutions of the same concentration using rare-earth fluorescent probes prepared in both the same and different batches. See [link to relevant documentation]. Figure 18 The relative standard deviation of the five parallel detection results is extremely small, indicating that the rare earth fluorescent probe and its detection method of the present invention have excellent intra-batch and inter-batch reproducibility.

[0046] Example 5: Application in Real Sample Analysis To evaluate the feasibility and accuracy of this method in actual sample analysis, commercially available green tea and white tea were used as actual samples to conduct a standard spike recovery experiment.

[0047] Sample pretreatment: Weigh 1.0 g each of green tea and white tea samples, add 100 mL of boiling water, steep for 10 minutes, filter, and collect the filtrate as the tea sample infusion solution. Dilute the tea sample infusion solution 100 times with ultrapure water to obtain the sample solution to be tested.

[0048] Spiked recovery experiment: Low, medium, and high concentrations of EGCG standard solution (for the catechol-type polyphenol specific detection channel) and a mixed standard solution of EGCG and oxidized resveratrol (concentration ratio 1:1, for the total phenol quantitative detection channel) were added to the test solutions of two tea samples, respectively. The final spiked concentrations of EGCG were 2.50 μM, 20.00 μM, and 50.00 μM, respectively; the final spiked concentrations of total phenols were 3.00 μM, 40.00 μM, and 80.00 μM, respectively. Detection was performed according to the methods of Examples 2 and 3. The target analytes in the samples were quantitatively analyzed using the established standard curves, and the spiked recovery rate was calculated.

[0049] The detection results are shown in Table 1. The spiked recoveries of both tea samples in the specific polyphenol detection channel ranged from 92.2% to 106.4%, with relative standard deviations (RSDs) below 4.2%. In the total phenol detection channel, the spiked recoveries ranged from 90.3% to 98.6%, with RSDs below 3.5%. These results fully demonstrate that the rare-earth fluorescent probe and corresponding dual detection mode constructed in this invention have good accuracy and precision for polyphenolic compounds in complex real-world sample matrices, and can meet the actual detection needs of polyphenolic functional components in agricultural products and food.

[0050] Table 1. Detection results of polyphenols in actual samples

[0051] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of the present invention should be covered within the scope of protection defined by the claims of the present invention.

[0052] It is understood that the term "a" should be understood as "at least one" or "one or more", that is, in one embodiment, the number of an element can be one, while in another embodiment, the number of the element can be multiple, and the term "a" should not be understood as a limitation on the number.

[0053] Although this document uses a significant amount of technical terminology, the possibility of using other terms is not excluded. These terms are used merely to facilitate the description and explanation of the nature of this application; interpreting them as any additional limitation would be contrary to the spirit of this application.

[0054] This application is not limited to the above-described preferred embodiments. Anyone can derive other products in various forms under the guidance of this application. However, regardless of any changes made to their shape or structure, any technical solution that is the same as or similar to that of this application falls within the protection scope of this application.

Claims

1. A fluorescent probe, characterized in that, The fluorescent probe is a complex formed by assembling rare earth europium ions, an organic ligand containing phenylboronic acid groups, and a fluorescent signal molecule. The phenylboronic acid group is a specific recognition site for the catechol structure, and the fluorescent signal molecule is used to generate a characteristic fluorescent response signal when it binds to or is replaced by the recognition site.

2. The fluorescent probe according to claim 1, characterized in that, The organic ligand containing the phenylboronic acid group is 3,5-dicarboxyphenylboronic acid; the fluorescent signal molecule is 6,7-dihydroxycoumarin.

3. A method for preparing a fluorescent probe as described in claim 1 or 2, characterized in that, Includes the following steps: (1) A soluble europium salt solution and an organic ligand solution containing phenylboronic acid groups are mixed in a solvent and subjected to a solvothermal reaction. After the reaction is completed, the rare earth organic framework precursor is obtained by solid-liquid separation, washing and drying. (2) The rare earth organic framework precursor is dispersed in a solvent, mixed with a fluorescent signal molecule solution and incubated to allow the fluorescent signal molecule to bind to the rare earth organic framework, thus obtaining the fluorescent probe.

4. The preparation method according to claim 3, characterized in that, The soluble europium salt in step (1) is europium nitrate hexahydrate, the organic ligand containing the phenylboronic acid group is 3,5-dicarboxyphenylboronic acid, and the molar ratio of the two is 1:1; the solvent is a mixed solvent of N,N-dimethylformamide and water; the fluorescent signal molecule in step (2) is 6,7-dihydroxycoumarin.

5. The preparation method according to claim 4, characterized in that, In step (1), the volume ratio of N,N-dimethylformamide to water is 7:3, and the solvothermal reaction conditions are 115-125℃ for 10-14 hours; in step (2), the final concentration of 6,7-dihydroxycoumarin in the reaction system is 60.0-70.0 μM, and the incubation conditions are incubation at room temperature for at least 10 minutes.

6. The use of the fluorescent probe according to claim 1 or 2 in the qualitative differentiation and / or quantitative detection of samples containing polyphenols, wherein, The polyphenols include types containing a catechol structure and types not containing a catechol structure.

7. A method for distinguishing and / or quantifying specific polyphenols in solution using the fluorescent probe according to claim 1 or 2, characterized in that, The polyphenol is a polyphenol containing a catechol structure, and the method includes: The polyphenol solution to be tested was mixed with the fluorescent probe solution and reacted. After the reaction, the fluorescence signal intensity of the system was measured. When the test solution contains a polyphenol with a catechol structure, it specifically binds to the phenylboronic acid group of the probe through a borate bond, competitively displacing the fluorescent signal molecule, resulting in a characteristic change in the fluorescence signal intensity of the system. Polyphenols without a catechol structure do not cause this change. Based on the aforementioned changes in fluorescence signals, the specific polyphenols can be qualitatively distinguished, or their content can be calculated by substituting the values ​​into a pre-established standard curve of fluorescence signal intensity versus the concentration of the specific polyphenol.

8. The method according to claim 7, characterized in that, The polyphenol containing the catechol structure is epigallocatechin gallate; the reaction time between the polyphenol solution to be tested and the fluorescent probe solution is 10 minutes.

9. A method for quantifying the total phenol content in a solution using the fluorescent probe according to claim 1 or 2, characterized in that, include: First, the fluorescent probe solution is mixed with Cu 2+ Solution mixing, utilizing Cu 2+ The initial fluorescence is quenched by chelation of fluorescent signal molecules to obtain the baseline fluorescence signal; Then add the total phenol solution to be tested and react. After the reaction, measure the fluorescence signal intensity of the system. In this study, total phenols in the test solution act as competing ligands from Cu. 2+ The fluorescent signal molecules are captured and re-bound to the rare earth organic framework in the probe, resulting in the recovery of the fluorescent signal. The measured fluorescence signal intensity was substituted into the pre-established standard curve of fluorescence signal intensity versus total phenol concentration to calculate the total phenol content in the test solution.

10. The method according to claim 9, characterized in that, The Cu 2+ The concentration in the final reaction system is 50.0-70.0 μM; the total reaction time between the total phenol solution to be tested and the probe system is 10 minutes.

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