Bifunctional fluorescent probes and methods for simultaneous detection of hydroxyl radicals and iron ions

Abstract

This invention discloses a bifunctional fluorescent probe and method for simultaneously detecting hydroxyl radicals and iron ions, belonging to the field of analytical and environmental monitoring. The bifunctional fluorescent probe is prepared by adding a ruthenium complex [Ru(bpy)2(DA-phen)] during the synthesis of MOF-808. 2+ A one-pot method was used to prepare ruthenium complex-encapsulated MOF material, which is the bifunctional fluorescent probe MOF-808@Ru for the simultaneous detection of hydroxyl radicals and iron ions. This invention uses a zirconium oxide cluster as the metal center and trimesic acid as the bridging ligand, simultaneously encapsulating [Ru(bpy)2(DA-phen)]. 2+ A MOF-808@Ru composite fluorescent probe with dual recognition units was prepared. This probe enables in-situ visualization of endogenous ·OH and Fe in biological systems. 3+ In addition, it can also sensitively detect ·OH and Fe in environmental water samples. 3+ Furthermore, by using fluorescence imaging to dynamically reflect changes in Fenton's reagent during simulated wastewater treatment, it has broad application prospects in the field of environmental monitoring.

Description

Technical Field

[0001] This invention relates to a hydroxyl radical (·OH) and an iron ion (Fe). 3+ The method for simultaneous detection specifically involves a fluorescent probe (MOF-808@Ru) based on a metal-organic framework material encapsulated with ruthenium complexes for the detection of ·OH and Fe. 3+ Simultaneous detection methods belong to the field of analysis and environmental monitoring. Background Technology

[0002] Hydroxyl radicals, an important type of reactive oxygen species (ROS), maintain the balance of the redox system in organisms under normal levels. However, excessive ·OH can cause oxidative damage to various biomolecules, including DNA, glycosyl compounds, lipids, and proteins, leading to lipid peroxidation and ultimately apoptosis. Fe(III), an important trace element in biological systems, is an indispensable cofactor in many enzymatic reactions, and its concentration may induce diseases such as Huntington, Parkinson, and Alzheimer's disease. It is well known that both Fe(III) and ·OH are products of the Fenton reaction, which plays a crucial role in life sciences and environmental pollution control. Therefore, simultaneous detection of both can not only lay the foundation for further elucidating the link between intracellular ·OH generation pathways and the Fenton reaction, but also provide a feasible monitoring strategy for the removal efficiency of recalcitrant organic matter in wastewater treatment.

[0003] To date, various in vitro or in vivo detection methods for Fe have been reported. 3+ Analytical methods for Fe or ·OH include electron spin resonance, colorimetry, high-performance liquid chromatography, chemiluminescence, and fluorescence methods. Among these methods, fluorescence has attracted much attention due to its high sensitivity, good selectivity, and the non-destructive, real-time, in-situ operation characteristics of fluorescence imaging technology. However, to date, most Fe... 3+ Or OH fluorescent probes still have many problems. First, due to the complex synthesis and purification steps, poor photostability and toxicity, these probes have certain limitations in practical applications. Second, most probes have a single recognition group and emission peak, which makes it impossible to detect different targets simultaneously. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies in the prior art and to provide a bifunctional fluorescent probe and method for simultaneously detecting hydroxyl radicals and iron ions.

[0005] The specific technical solution adopted in this invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing a bifunctional fluorescent probe that simultaneously detects hydroxyl radicals and iron ions, wherein a ruthenium complex [Ru(bpy)2(DA-phen)] is added during the synthesis of MOF-808. 2+ MOF material encapsulated with ruthenium complex was prepared using a one-pot method, which is the bifunctional fluorescent probe MOF-808@Ru that can simultaneously detect hydroxyl radicals and iron ions.

[0007] As a preferred embodiment, the one-pot method is as follows:

[0008] Dissolve 322.0 mg ZrOCl2·8H2O and 421.0 mg H3BTC in a mixed solution of 15 mL N,N-dimethylformamide and 15 mL formic acid by sonication, then add 78.0 mg [Ru(bpy)2(DA-phen)]. 2+ After being fully dissolved, the resulting transparent mixed solution was stirred continuously at 150°C for 6 hours. After naturally cooling to room temperature, it was washed by centrifugation with anhydrous ethanol. The collected pale yellow precipitate was vacuum dried at 60°C for 24 hours to obtain a bifunctional fluorescent probe that can simultaneously detect hydroxyl radicals and iron ions.

[0009] Furthermore, the centrifugal washing is performed at a speed of 6000 r / min, and the number of cycles is 3.

[0010] Preferably, the ruthenium complex [Ru(bpy)2(DA-phen)] is used. 2+ The preparation method is as follows:

[0011] 0.12 mmol and 25.0 mg of 5,6-diamino-1,10-o-phenanthroline and 0.12 mmol and 69 mg of Ru(bpy)₂Cl₂·2H₂O were dissolved in 10 mL of ethanol by sonication for 10 min. The resulting mixed solution was then refluxed at 80 °C for 12 h. After cooling to room temperature, 8 mL of saturated NH₄PF₆ solution was added to the resulting clear solution, and finally centrifuged to obtain [Ru(bpy)₂(DA-phen)]. 2+ Orange precipitate.

[0012] Furthermore, the centrifugation speed is 8000 r / min.

[0013] Secondly, the present invention provides a bifunctional fluorescent probe for the simultaneous detection of hydroxyl radicals and iron ions, obtained by any of the preparation methods described in the first aspect.

[0014] Thirdly, the present invention provides a method for simultaneously detecting hydroxyl radicals and iron ions based on the bifunctional fluorescent probe described in the second aspect, as follows:

[0015] A bifunctional fluorescent probe was added to a test solution containing hydroxyl radicals and iron ions. After adjusting the pH, the mixed solution was incubated. Subsequently, the fluorescence intensity of the mixed solution at emission wavelengths of 453 nm and 612 nm was measured under 365 nm excitation, with both excitation and emission slits fixed at 5 nm. Based on the fluorescence intensity at 453 nm emission wavelength, combined with the linear relationship between fluorescence enhancement efficiency and different concentrations of H2O2, the concentration of hydroxyl radicals in the test solution was obtained. Based on the fluorescence intensity at 612 nm emission wavelength, combined with the linear relationship between quenching efficiency and different concentrations of iron ion standard solution, the concentration of iron ions in the test solution was obtained, thus achieving simultaneous detection of hydroxyl radicals and iron ions.

[0016] Preferably, the mixed solution comprises 200.0 μL, 200 mM, pH 6.0 PBS phosphate buffer, 30.0 μL, 10.0 g / L MOF-808@Ru solution, 0.1-50 μM H2O2 standard solution, and / or 0.5-50 μM Fe 3+ Standard solution.

[0017] Preferably, the incubation method is: irradiation under a 320nm ultraviolet lamp for 20 minutes or incubation with shaking at 37°C for 20 minutes.

[0018] Preferably, the concentration of the bifunctional fluorescent probe in the mixed solution is 150.0 mg / L; the pH during measurement is 6.0; and the response time is 20 min.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] This invention uses a zirconium oxide cluster as the metal center and pyromellitic tricarboxylic acid as the bridging ligand, while simultaneously encapsulating [Ru(bpy)2(DA-phen)]. 2+ A MOF-808@Ru composite fluorescent probe with dual recognition units was prepared. Tristyric acid enables selective detection of ·OH, and the Ru complex can target Fe. 3+ For specific recognition, the fluorescence emission peaks of the two molecules are located at 453 nm and 612 nm, respectively. Their simultaneous responses do not interfere with each other, thus enabling their simultaneous detection. This probe can also visualize endogenous ·OH and Fe in situ within biological systems. 3+ In addition, it can sensitively detect ·OH and Fe in environmental water samples. 3+ Furthermore, by using fluorescence imaging to dynamically reflect changes in Fenton's reagent during simulated wastewater treatment, it has broad application prospects in the field of environmental monitoring. Attached Figure Description

[0021] Figure 1 For different concentrations of Fe3+ Effects of (A) and ·OH (B) on the fluorescence spectrum of MOF-808@Ru. MOF-808@Ru: 150.0 mg / L, PBS buffer: 20.0 mM (pH = 6.0), λ ex =365nm.

[0022] Figure 2 In the middle, (A) pH affects Fe 3+ (A) Effect of pH on quenching of MOF-808@Ru fluorescence system; (B) Effect of pH on ·OH-enhanced MOF-808@Ru fluorescence system; (C) Effect of response time on Fe 3+ The effect of the presence and absence of Fe on the fluorescence intensity of the MOF-808@Ru system; (D) The effect of response time on the fluorescence intensity of the MOF-808@Ru system with and without ·OH. F0 and F represent the fluorescence intensity of the MOF-808@Ru system with and without Fe. 3+ Fluorescence intensity of the MOF-808@Ru system at 612 nm and 453 nm in the absence and presence of ·OH. MOF-808@Ru: 150.0 mg / L, Fe 3+ :10.0μM, ·OH: 4.0μM, PBS buffer: 20.0mM (pH=6.0), λ ex =365nm.

[0023] Figure 3 (F0-F) / F0 and (F-F0) / F0 are respectively related to different concentrations of Fe. 3+ Linear relationship diagram between (A) and ·OH(B). F0 and F represent the linear relationship when Fe 3+ Fluorescence intensity at 612 nm and 453 nm of the MOF-808@Ru system in the absence and presence of ·OH. MOF-808@Ru: 150.0 mg / L, PBS buffer: 20.0 mM (pH = 6.0), λ ex =365nm.

[0024] Figure 4 MOF-808@Ru fluorescent probe for Fe 3+ The selectivity test, (A) cation, (B) anion, (C) active substance. F0 and F represent the different reactions when Fe... 3+ Fluorescence intensity of MOF-808@Ru at 612 nm in its absence and presence. MOF-808@Ru: 150.0 mg / L, Fe 3+ 10.0 μM, Cu 2+ : 10.0 μM, other substances concentration 100.0 μM, PBS buffer: 20.0 mM (pH = 6.0), λ ex =365nm.

[0025] Figure 5 This study describes the selectivity assay of the MOF-808@Ru fluorescent probe for ·OH. (A) Common intracellular ions, (B) Active substances. F0 and F represent the fluorescence intensity of MOF-808@Ru at 453 nm in the absence and presence of ·OH, respectively. MOF-808@Ru: 150.0 mg / L, ·OH: 4.0 μM, other substances: 100.0 μM, PBS buffer: 20.0 mM (pH = 6.0), λ ex =365nm. Detailed Implementation

[0026] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0027] This invention provides a method for preparing a bifunctional fluorescent probe (i.e., MOF-808@Ru composite fluorescent probe) that simultaneously detects hydroxyl radicals and iron ions. This method involves adding a ruthenium complex [Ru(bpy)2(DA-phen)] during the synthesis of MOF-808. 2+ A one-pot method was used to prepare MOF materials encapsulated with ruthenium complexes, thus obtaining a bifunctional fluorescent probe that can simultaneously detect hydroxyl radicals and iron ions.

[0028] In practical use, this method includes the following steps:

[0029] (1) Synthesis of ruthenium complex ([Ru(bpy)2(DA-phen)]) 2+ ):

[0030] 5,6-Diamino-1,10-o-phenanthroline (0.12 mmol, 25.0 mg) and Ru(bpy)₂Cl₂·2H₂O (0.12 mmol, 69 mg, i.e., cis-bis(2,2-dipyridyl)ruthenium(II) dichloride dihydrate) were dissolved in 10 mL of ethanol by sonication for 10 min. The resulting mixture was then transferred to a 50.0 mL flask and refluxed at 80 °C for 12 h. After cooling to room temperature, 8 mL of a saturated solution of NH₄PF₆ (i.e., ammonium hexafluorophosphate) was added to the resulting clear solution. Finally, the mixture was centrifuged at 8000 rpm to obtain [Ru(bpy)₂(DA-phen)]. 2+ Orange precipitate.

[0031] (2) Preparation of MOF-808@Ru composite fluorescent probe:

[0032] Dissolve 322.0 mg ZrOCl2·8H2O (zirconium oxychloride octahydrate) and 421.0 mg H3BTC (tristyric acid) in N,N-dimethylformamide / formic acid (15 mL / 15 mL) by sonication, then add 78.0 mg [Ru(bpy)2(DA-phen)]. 2+ After the solution was fully dissolved, a transparent mixed solution was obtained and stirred continuously at 150°C for 6 hours. After naturally cooling to room temperature, the solution was washed three times by centrifugation with anhydrous ethanol (6000 r / min). The collected pale yellow precipitate was vacuum dried in an oven at 60°C for 24 hours.

[0033] The bifunctional fluorescent probe prepared using the above method is effective against ·OH and Fe. 3+ The fluorescence detection method is as follows:

[0034] A bifunctional fluorescent probe was added to a test solution containing hydroxyl radicals and iron ions. After adjusting the pH, the mixed solution was incubated. Subsequently, the fluorescence intensity of the mixed solution at emission wavelengths of 453 nm and 612 nm was measured under 365 nm excitation, with both excitation and emission slits fixed at 5 nm. Based on the fluorescence intensity at 453 nm emission wavelength, combined with the linear relationship between fluorescence enhancement efficiency and different concentrations of H2O2, the concentration of hydroxyl radicals in the test solution was obtained. Based on the fluorescence intensity at 612 nm emission wavelength, combined with the linear relationship between quenching efficiency and different concentrations of iron ion standard solution, the concentration of iron ions in the test solution was obtained, thus achieving simultaneous detection of hydroxyl radicals and iron ions.

[0035] The methods and effects of the present invention will be specifically illustrated below through examples.

[0036] Example

[0037] This embodiment describes the preparation of a bifunctional fluorescent probe for the simultaneous detection of hydroxyl radicals and iron ions. The specific method is as follows:

[0038] (1) Synthesis of ruthenium complex ([Ru(bpy)2(DA-phen)]) 2+ ):

[0039] 5,6-Diamino-1,10-o-phenanthroline (0.12 mmol, 25.0 mg) and Ru(bpy)₂Cl₂·2H₂O (0.12 mmol, 69 mg) were dissolved by sonication in 10 mL of ethanol for 10 min. The resulting mixture was then transferred to a 50.0 mL flask and refluxed at 80 °C for 12 h. After cooling to room temperature, 8 mL of a saturated solution of NH₄PF₆ (ammonium hexafluorophosphate) was added to the resulting clear solution. Finally, the mixture was centrifuged at 8000 rpm to obtain [Ru(bpy)₂(DA-phen)]. 2+ Orange precipitate.

[0040] (2) Preparation of MOF-808@Ru composite fluorescent probe:

[0041] Dissolve 322.0 mg ZrOCl2·8H2O (zirconium oxychloride octahydrate) and 421.0 mg H3BTC (tristyric acid) in N,N-dimethylformamide / formic acid (15 mL / 15 mL) by sonication, then add 78.0 mg [Ru(bpy)2(DA-phen)]. 2+ After the solution was fully dissolved, a transparent mixed solution was obtained and stirred continuously at 150°C for 6 hours. After naturally cooling to room temperature, the solution was washed three times by centrifugation with anhydrous ethanol (6000 r / min). The collected pale yellow precipitate was vacuum dried in an oven at 60°C for 24 hours.

[0042] Based on the bifunctional fluorescent probe prepared above, 200.0 μL of PBS phosphate buffer (200 mM, pH = 6.0), 30.0 μL of MOF-808@Ru solution (10.0 g / L), and a certain concentration of H2O2 (0.1-50 μM) or Fe were added sequentially to a 2.0 mL colorimetric tube. 3+ (0.5-50 μM) standard solutions were prepared and then diluted to 2.0 mL with ultrapure water. The solutions were then irradiated under a 320 nm UV lamp for 20 min or incubated at 37 °C with shaking for 20 min. Finally, fluorescence spectra with emission peaks at 453 nm and 612 nm were recorded using 365 nm as the excitation wavelength, with the slit width set to 5 / 5 nm.

[0043] MOF-808@Ru composite fluorescent probe for ·OH and Fe 3+ fluorescence response such as Figure 1 As shown, the composite fluorescent probe has only one fluorescence peak at 612 nm. When Fe is added to this detection system... 3+ After ionization, Fe 3+ It can only quench the fluorescence peak at 612 nm, while the addition of ·OH generates a new fluorescence peak at 453 nm. The two do not interfere with each other, thus enabling the composite fluorescent probe to target ·OH and Fe. 3+ Simultaneous measurement was performed. Two excitation spectra were obtained by scanning at fixed emission peaks of 453 nm and 612 nm. These two excitation spectra highly overlapped, and the excitation wavelength of 365 nm was finally selected.

[0044] This embodiment optimizes the measurement conditions of the MOF-808@Ru composite fluorescent probe, specifically optimizing the probe's detection conditions, including pH and response time. The specific method is as follows:

[0045] pH has a significant impact on fluorescent systems containing acidic or basic groups. Different pH PBS buffer solutions (pH 3.5-10.0) were added to 150.0 mg / L MOF-808@Ru probe solutions, followed by the addition of 10.0 μM Fe. 3+ Alternatively, use 4.0 μM OH, react thoroughly for 20 min, and then measure the fluorescence intensity at 453 nm and 612 nm. Results are as follows: Figure 2 A and Figure 2 As shown in B, the probe is used to probe Fe under weakly acidic conditions. 3+ The probe exhibits good response to ·OH, which may be because the protonation of the probe functional groups under acidic conditions enhances its interaction with Fe. 3+ The coordination between them, and although H2O2 produces more ·OH under alkaline conditions, it easily forms dihydroxybenzoic acid and benzoquinone structures under the action of hydroxide ions, resulting in a decrease in fluorescence intensity at 453 nm. Therefore, the fluorescence enhancement efficiency of ·OH and Fe at pH=6.0 are affected. 3+ The quenching efficiency is relatively high, and considering practical applications such as wastewater treatment, acidic media are also conducive to the Fenton reaction. Therefore, the pH of the system was determined to be 6.0 as the condition for further analytical experiments.

[0046] Equilibrium time is another important factor in fluorescence sensing. Different concentrations of Fe were added to a 150.0 mg / L MOF-808@Ru probe solution. 3+ And ·OH, and then the fluorescence intensity at 453 nm and 612 nm at different time points was recorded. The results are as follows. Figure 2 As shown in C and 2D, the fluorescence intensity of the probe at 453 nm and 612 nm remained stable within 60 min, and the probe could achieve Fe oxidase activity within 10 min. 3+ The system achieves a complete response. However, for the detection of ·OH, since the generation of ·OH from UV-activated H₂O₂ is a continuous and dynamic process, the fluorescence intensity of the probe at 453 nm continuously increases with the increase of detection time. Ultimately, to enable the simultaneous analysis and detection of Fe... 3+ To maintain consistency in the reaction time with ·OH, 20 min was selected as the response time for subsequent experiments.

[0047] The results showed that the optimal probe concentration was 150.0 mg / L, and the optimized pH and response time were as follows: Figure 2 As shown. By Figure 2 It can be seen that pH 6.0 and response time 20 min are the optimal measurement conditions.

[0048] Under optimized experimental conditions, the probe was tested for its effects on OH· and Fe. 3+ The quantitative detection method is as follows:

[0049] Add 200.0 μL of PBS phosphate buffer (200 mM, pH = 6.0), 30.0 μL of MOF-808@Ru solution (10.0 g / L), and a certain concentration of H2O2 (0.1-50 μM) or Fe to a 2.0 mL colorimetric tube in sequence. 3+ (0.5-50 μM) standard solutions were prepared and then diluted to 2.0 mL with ultrapure water. The solutions were then irradiated under a 302 nm UV lamp for 20 min or incubated at 37 °C with shaking for 20 min. Finally, fluorescence spectra with emission peaks at 453 nm and 612 nm were recorded using 365 nm as the excitation wavelength, with the slit width set to 5 / 5 nm.

[0050] Experimental results are as follows Figure 3 As shown. In the range of 0.5–12.0 μM, the fluorescence quenching efficiency (F0–F) / F0 at 612 nm is related to Fe. 3+ There is a good linear relationship between the concentrations, and the linear equation is (F0-F) / F0=0.03218[Fe 3+ +0.04063(R) 2 =0.9932), Fe 3+ The detection limit (LOD, 3σ / k) was 0.35 μM (19.6 μg / L), which is far below the reference value (0.3 mg / L) for surface water specified in China's environmental quality standards. Therefore, this method can meet the requirements for Fe in surface water. 3+ The detection requirements; when the concentration of ·OH is in the range of 0.2-6.0 μM, it shows a good linear relationship with the fluorescence enhancement effect (F-F0) / F0, and the regression equation is (F-F0) / F0=1.1137[·OH]+0.3574(R 2 =0.9944), the detection limit (LOD, 3σ / k) is 54.0 nM. When the concentration of ·OH is in the range of 10.0-100.0 μM, it shows a good linear relationship with log[(F-F0) / F0)], and the regression equation is log[(F-F0) / F0)] = 0.01061[·OH] + 0.7684(R) 2 =0.9962), and the limit of detection (LOD, 3σ / k) was 5.6 μM.

[0051] To investigate the effect of the MOF-808@Ru composite fluorescent probe on ·OH and Fe 3+ The selectivity was tested with common metal ions, anions, and various reactive substances in redox equilibrium (AA, GSH, Cys, GLu, O2). 1 H2O2, NO·, ONOO - The specific method is as follows: ·OH),

[0052] Add 200.0 μL of PBS phosphate buffer (200 mM, pH = 6.0), 30.0 μL of MOF-808@Ru solution (10.0 g / L), and standard solutions of anions, cations, and various intracellular active substances of a certain concentration to a 2.0 mL colorimetric tube in sequence. 2 + [10.0 μM, other substances 100.0 μM], and finally diluted to 2.0 mL with ultrapure water. Irradiated under a 302 nm UV lamp for 20 min or incubated at 37 °C with shaking for 20 min. Finally, using 365 nm as the excitation wavelength, the fluorescence spectra with emission peaks at 453 nm and 612 nm were recorded, with the slit width set to 5 / 5 nm.

[0053] The results are as follows Figure 4 and Figure 5 As shown. By Figure 4 and Figure 5 As can be seen, these substances have no significant effect on the fluorescence intensity of the system. Therefore, the MOF-808@Ru composite fluorescent probe is effective against ·OH and Fe. 3+ It has good selectivity.

[0054] To verify the effectiveness of the MOF-808@Ru composite fluorescent probe against ·OH and Fe in real environmental water samples. 3+ To demonstrate the effectiveness of the testing application, two water samples were collected: tap water and lake water. After simple filtration, the water samples were analyzed for ·OH and Fe. 3+ Different concentrations of spiking were added, with the Fe in the unspiked water sample being... 3+ Comparative determinations were performed using atomic absorption spectrophotometry, and the results are shown in Table 1. Fe in the unspecified water sample... 3+ The results of this method and atomic absorption spectrophotometry were compared and found to be basically consistent. The recoveries of other spiked determinations were between 92.8% and 107.5%, which are satisfactory results.

[0055] Table 1. OH· and Fe in water samples 3+ Simultaneous detection

[0056]

[0057] ND - Not detected, RSD - Relative standard deviation, AAS - Atomic absorption spectrophotometry

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing a bifunctional fluorescent probe for simultaneously detecting hydroxyl radicals and iron ions, characterized in that, The ruthenium complex [Ru(bpy)2(DA-phen)] was added during the synthesis of MOF-808. 2+ The MOF material encapsulated with ruthenium complex was prepared by a one-pot method, which is the bifunctional fluorescent probe MOF-808@Ru that simultaneously detects hydroxyl radicals and iron ions; where DA-phen is 5,6-diamino-1,10-o-phenanthroline.

2. The method for preparing a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and iron ions according to claim 1, characterized in that, The one-pot method is described in detail below: Dissolve 322.0 mg ZrOCl2·8H2O and 421.0 mg H3BTC in a mixed solution of 15 mL N,N-dimethylformamide and 15 mL formic acid by sonication, then add 78.0 mg [Ru(bpy)2(DA-phen)]. 2+ After being fully dissolved, the resulting transparent mixed solution was stirred continuously at 150°C for 6 h. After naturally cooling to room temperature, it was washed by centrifugation with anhydrous ethanol. The collected pale yellow precipitate was vacuum dried at 60°C for 24 h to obtain a bifunctional fluorescent probe that can simultaneously detect hydroxyl radicals and iron ions.

3. The method for preparing a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and iron ions according to claim 2, characterized in that, The centrifugal washing was performed at a speed of 6000 r / min, and the number of cycles was 3.

4. The method for preparing a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and iron ions according to claim 1, characterized in that, The ruthenium complex [Ru(bpy)2(DA-phen)] 2+ The preparation method is as follows: 0.12 mmol (25.0 mg) of 5,6-diamino-1,10-o-phenanthroline and 0.12 mmol (69 mg) of Ru(bpy)₂Cl₂·₂H₂O were dissolved in 10 mL of ethanol by sonication for 10 min. The resulting mixture was then refluxed at 80°C for 12 h. After cooling to room temperature, 8 mL of saturated NH₄PF₆ solution was added to the resulting clear solution, and the mixture was centrifuged to obtain [Ru(bpy)₂(DA-phen)]. 2+ Orange precipitate.

5. The method for preparing a bifunctional fluorescent probe for simultaneous detection of hydroxyl radicals and iron ions according to claim 4, characterized in that, The centrifugal speed is 8000 r / min.

6. A bifunctional fluorescent probe for simultaneously detecting hydroxyl radicals and iron ions, obtained by the preparation method according to any one of claims 1 to 5.

7. A method for simultaneously detecting hydroxyl radicals and iron ions based on the bifunctional fluorescent probe of claim 6, characterized in that, Specifically as follows: A bifunctional fluorescent probe was added to a test solution containing hydroxyl radicals and iron ions. After adjusting the pH, the mixed solution was incubated. Subsequently, the fluorescence intensity of the mixed solution at emission wavelengths of 453 nm and 612 nm was measured under 365 nm excitation, with both excitation and emission slits fixed at 5 nm. Based on the fluorescence intensity at 453 nm emission wavelength, combined with the linear relationship between fluorescence enhancement efficiency and different concentrations of hydroxyl radicals, the concentration of hydroxyl radicals in the test solution was obtained. Based on the fluorescence intensity at 612 nm emission wavelength, combined with the linear relationship between quenching efficiency and different concentrations of iron ion standard solutions, the concentration of iron ions in the test solution was obtained, thus achieving simultaneous detection of hydroxyl radicals and iron ions.

8. The method for simultaneously detecting hydroxyl radicals and iron ions according to claim 7, characterized in that, The mixed solution includes 200.0 μL of 200 mM PBS phosphate buffer (pH 6.0), 30.0 μL of 10.0 g / L MOF-808@Ru solution, 0.1-50 μM H2O2 standard solution, and 0.5-50 μM Fe2+. 3+ Standard solution.

9. The method for simultaneously detecting hydroxyl radicals and iron ions according to claim 7, characterized in that, The incubation method is as follows: irradiate under a 320 nm ultraviolet lamp for 20 min or incubate with shaking at 37°C for 20 min.

10. The method for simultaneously detecting hydroxyl radicals and iron ions according to claim 7, characterized in that, The concentration of the bifunctional fluorescent probe in the mixed solution was 150.0 mg / L; the pH during the measurement was 6.0; and the response time was 20 min.

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