Zinc ion fluorescent probe compound and preparation method and application thereof

Abstract

The application discloses a zinc ion fluorescent probe compound and a preparation method and application thereof, and relates to the technical field of fluorescent probes.The application forms a reversible fluorescent probe for detecting zinc ions by reasonably designing a probe molecular structure, taking 2-(phenylimino)methyl)phenol as a skeleton, and introducing coordination sites capable of being combined with zinc ions.From the perspective of Lewis acid-base theory, zinc ions are electron-deficient Lewis acids, are easy to be coordinated with heteroatoms such as N and O to form Lewis acid-base pairs, form a complex, and can realize accurate detection of zinc ions.Meanwhile, the complex fluorescent probe has reusability and high anti-interference property, the photophysical property of the probe can be recovered through a decomplexing agent after fluorescent recognition, the fluorescent detection can be realized again, the effect of reversible recognition is achieved, the use cost of the probe is reduced, and the pollution to the environment is reduced.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a zinc ion fluorescent probe compound, its preparation method, and its application. Background Technology

[0002] Zinc ions are among the most important metal ions in living organisms, participating in various physiological processes such as enzyme activity regulation, gene expression, cell signal transduction, and protein synthesis and folding. Under normal physiological conditions, zinc ion concentration remains stable, but abnormal changes are closely related to the development of various diseases, including neurodegenerative diseases, diabetes, cardiovascular diseases, and certain tumors. Therefore, developing highly sensitive and selective fluorescent probes for zinc ions is of great significance for studying the physiological functions and pathological mechanisms of zinc ions, as well as for the early diagnosis of related diseases.

[0003] In recent years, fluorescence sensing has experienced rapid development due to its excellent anti-interference ability, good biocompatibility, and fast response speed. Fluorescent probes, as an important detection tool, have been widely used for the detection of zinc ions. Fluorescent probes detect zinc ions by specifically interacting with them, causing significant changes in their fluorescence properties (such as fluorescence intensity and emission wavelength). This detection method offers advantages such as high sensitivity, high selectivity, ease of operation, and the ability to perform in-situ real-time detection, providing a powerful means for zinc ion detection.

[0004] Currently, fluorescence sensing has become an important method for zinc ion detection due to its advantages such as strong anti-interference ability, good biocompatibility, and fast response speed. Fluorescent probes exhibit excellent performance in zinc ion detection, with high sensitivity and high selectivity, and can specifically identify zinc ions without interference from other metal ions.

[0005] However, existing fluorescent probes still have certain shortcomings in terms of reversibility, detection sensitivity and selectivity, and there is an urgent need to develop zinc ion fluorescent probes with better performance. Summary of the Invention

[0006] The purpose of this invention is to provide a dual-Schiff base zinc ion fluorescent probe with high sensitivity and selectivity for zinc ions, good reversibility and biocompatibility, as well as its simple preparation method and wide application.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides a zinc ion fluorescent probe compound, chemically named 2-(diethylamino)-5-[{4-[4-[4-(diethylamino)-2-hydroxybenzylamino]phenylimino}methyl]phenol, abbreviated as AMEP, with the following structural formula:

[0009]

[0010] Secondly, the present invention provides a method for preparing the above-mentioned zinc ion fluorescent probe compound. Under conditions of 65°C, p-phenylenediamine and 4-(diethylamino)salicylaldehyde are added to methanol at a molar ratio of 1:1.5 and reacted for 24 h. The reaction progress is detected by TLC. After the reaction is complete, the mixture is filtered through a Buchner funnel to obtain an orange precipitate, which is then dried to obtain AMEP.

[0011] Thirdly, the present invention provides the application of the above-mentioned zinc ion fluorescent probe compound in the preparation of zinc ion detection reagents or test strips.

[0012] Furthermore, the zinc ions are zinc ions in the water sample or within cells.

[0013] Furthermore, in the zinc ion detection reagent, the optimal reaction solvent for AMEP is DMF, the optimal reaction time is 10s, and the optimal complexation ratio of zinc ions to AMEP is 2:1.

[0014] Compared with the prior art, the technical effects of the present invention are as follows:

[0015] This invention provides a zinc ion fluorescent probe compound. Through rational design of the probe molecule structure, using 2-(phenylimino)methylphenol as the backbone, coordination sites capable of complexing with zinc ions are introduced to form a reversible fluorescent probe for zinc ion detection. From the perspective of Lewis acid-base theory, zinc ions are electron-deficient Lewis acids, readily coordinating with heteroatoms such as N and O to form Lewis acid-base pairs, thus forming complexes and enabling precise detection of zinc ions. Simultaneously, the complexing fluorescent probe exhibits reusability and high anti-interference properties. After fluorescence recognition, the probe's photophysical properties can be restored using a decoupling agent, allowing for re-detection of fluorescence, achieving reversible recognition, reducing probe usage costs, and minimizing environmental pollution. Furthermore, from a practical application perspective, the reversible fluorescent probe can monitor the dynamic changes in zinc ion concentration in real time, providing more accurate information for studying the physiological functions and pathological mechanisms of zinc ions in biological systems. For example, in the dynamic monitoring of intracellular zinc ions, the reversible fluorescent probe can reflect changes in zinc ion concentration in real time, providing strong support for studying the role of zinc ions in cell signal transduction.

[0016] 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.

[0017] Figure 1 This diagram illustrates the synthesis of AMEP and its fluorescent recognition of zinc ions.

[0018] Figure 2 This is the hydrogen NMR spectrum of AMEP.

[0019] Figure 3 This is the carbon NMR spectrum of AMEP.

[0020] Figure 4 Fluorescence spectra of AMEP before and after the addition of zinc ions in different solvents.

[0021] Figure 5 The graph shows the fluorescence intensity changes at 525 nm for probe systems with different reaction times.

[0022] Figure 6 The fluorescence spectrum is the result of the reaction between the probe AMEP and zinc ions.

[0023] Figure 7 The curve showing the fluorescence intensity at 535 nm as a function of concentration is fitted.

[0024] Figure 8 Zn 2+ Titration with AMEP's Job's plot working curve.

[0025] Figure 9 The fluorescence spectra are those of AMEP (100 μM) and common metal ions (100 μM).

[0026] Figure 10 The bar chart shows the fluorescence intensity at 520 nm for the reaction of AMEP (100 μM) and common metal ions (100 μM).

[0027] Figure 11 For silicone plates for Zn 2+ Detection (under visible light).

[0028] Figure 12 For silicone plates for Zn 2+ Detection (under 365nm excitation light).

[0029] Figure 13 Test strips for different concentrations of Zn 2+ (Detection results under visible light, decreasing gradually from left to right)

[0030] Figure 14 Test strips for different concentrations of Zn 2+ Detection results (gradually decreasing from left to right) under 365nm excitation light.

[0031] Figure 15 Alternately add Zn to the probe2+ The fluorescence changes of triethylamine. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Preparation of AMEP

[0034] This invention provides a zinc ion fluorescent probe compound (AMEP), the synthesis process of which is as follows: Figure 1 As shown, it includes the following steps:

[0035] Step (1): At 65°C, p-phenylenediamine (1081.4 mg, 10 mmol), 4-(diethylamino)salicylaldehyde (2898.6 mg, 15 mmol), and 50 mL of methanol were added to a 100 mL flask and reacted for 24 h. The reaction progress was monitored by TLC until the reaction was complete.

[0036] Step (2): Filter the sample using a Buchner funnel to obtain an orange precipitate. Filter the precipitate to separate it, and dry the solid phase to obtain 3.1426 g of orange product, with a yield of 68.6%.

[0037] like Figure 2 and Figure 3 As shown, the proton NMR spectrum and carbon NMR spectrum confirm the structure of the probe. 1 H NMR (400MHz, CDCl3) δ15.54(s,2H),10.40(d,J=4.3Hz,2H),9.19(t,J=2.9Hz,4H),9.10(dd,J=8.7,5.1Hz,2H),8.18(dd,J=8. 6,2.7Hz,2H),8.07(d,J=2.6Hz,2H),7.23(d,J=5.7Hz,3H),5.31(q,J=6.4Hz,8H),3.46(s,5H),3.11(dt,J=7.8,4.1Hz,12H). 13 C NMR (101MHz, CDCl3) δ165.62,161.98,135.61,123.53,110.95,99.34,46.48,14.38.

[0038] Example 2 AMEP Solvent Screening

[0039] In solution systems, the solvent effect between solute and solvent molecules is a key factor influencing the system's properties. Its essence is the electrostatic interaction between solute and solvent molecules. This interaction includes different forms such as dipole-dipole, ion-dipole, and dispersion forces. The solvent effect can induce dissociation in the reaction medium, altering the pathway and kinetics of the chemical reaction and significantly impacting the reaction process. Particularly for fluorescent probe systems, when the same fluorescent probe is dissolved in solvents of different polarities, the different interactions between solvent and probe molecules lead to significant changes in the emission wavelength, intensity, and spectral shape of the fluorescence spectrum. This change provides important information for further research into the mechanism of probe-solvent interactions. Therefore, we investigated the effects of different solvents on AMEP and the fluorescence response after the addition of zinc ions.

[0040] 1. Preparation of AMEP solvent test solution:

[0041] (i) Zinc ion solution: Weigh 21.95 mg of zinc acetate, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of water to dilute to a 100 μM solution.

[0042] (ii) Probe solutions: Accurately weigh seven equal portions of 45.83 mg AMEP. For the first portion, add MeOH to a 10 mM stock solution and dilute 100 μL of the stock solution with 9.9 mL of MeOH to make a 100 μM solution. For the second portion, add THF to a 10 mM stock solution and dilute 100 μL of the stock solution with 9.9 mL of THF to make a 100 μM solution. For the third portion, add CH3CN to a 10 mM stock solution and dilute 100 μL of the stock solution with 9.9 mL of CH3CN to make a 100 μM solution. For the fourth portion, add DMSO to a 10 mM stock solution and dilute 100 μL of the stock solution with 9.9 mL of DMSO to make a 100 μM solution. For the fifth portion, add EtOH to a 10 mM stock solution and dilute 100 μL of the stock solution with 9.9 mL of EtOH to make a 100 μM solution. EtOH was diluted to a 100 μM solution; the sixth portion was diluted with DCM to a 10 mM stock solution, and 100 μL of the stock solution was diluted with 9.9 mL of DCM to a 100 μM solution; the seventh portion was diluted with DMF to a 10 mM stock solution, and 100 μL of the stock solution was diluted with 9.9 mL of DMF to a 100 μM solution.

[0043] 2. Fluorescence detection

[0044] Using a 100 μL pipette, 100 μM zinc acetate solution was mixed with 100 μM MeOH solution, 100 μM THF solution, 100 μM CH3CN solution, 100 μM DMSO solution, 100 μM EtOH solution, 100 μM DCM solution, and 100 μM DMF solution at a volume ratio of 2:1 in 2 mL centrifuge tubes. After reacting at room temperature for 30 min, fluorescence detection was performed.

[0045] The results are as follows Figure 4 As shown, AMEP exhibited fluorescence signals around 520 nm. Zn was added to THF, DCM, and EtOH solvents. 2+ Although the AMEP fluorescence intensity changed slightly before and after, the change was not significant. The fluorescence intensity increased significantly in MeOH, CH3CN, and DMSO solvents, with DMF showing the best increase. When the probe was in DMF solvent, the addition of zinc ions increased the fluorescence intensity to twenty-seven times its original value. This demonstrates that DMF achieves ideal detection results; therefore, DMF was chosen as the solvent for the next experimental step.

[0046] Example 3 Probe to Zn 2+ Response time research

[0047] Response time is one of the factors affecting the practical application of probes. Fluorescent probes with fast response times can more quickly capture the presence of zinc ions in water samples, improving detection sensitivity. Therefore, studying the changes in fluorescence intensity under different reaction times can optimize the measurement conditions of the entire system. To investigate the relationship between the probe and Zn... 2+ To determine the optimal reaction time, this invention mixes a zinc ion solution (100 μM) and a probe solution (100 μM) and reacts them at room temperature for different times to detect the fluorescence of the reaction solution.

[0048] 1. Preparation of fluorescent probe response time detection solution:

[0049] (i) Zinc ion solution: Weigh 21.95 mg of zinc acetate, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of water to dilute to a 100 μM solution.

[0050] (ii) Probe solution: Weigh 45.83 mg AMEP, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of DMF to dilute to a 100 μM solution.

[0051] 2. Fluorescence detection

[0052] The zinc ion solution and probe solution were mixed in a 2:1 ratio in a 2 mL centrifuge tube and the mixture was shaken at room temperature. Fluorescence was measured after 10 s, 30 s, 60 s, 90 s, and 120 s.

[0053] The results are as follows Figure 5 As shown, the fluorescence intensity reached its peak at 10s, therefore, this invention selected 10s as the optimal reaction time for the probe.

[0054] Example 4: Study on zinc ion concentration using a probe

[0055] To establish the relationship between zinc ion concentration and fluorescence intensity and obtain a feasible fluorescence curve, this invention, under the optimal conditions determined in the above experiments, mixes zinc ion solutions of different concentrations (0-400 μM) with a probe solution (100 μM), and then performs fluorescence detection on the reaction solution.

[0056] 1. Preparation of concentration titration test solution:

[0057] (i) Zinc ion solutions of different concentrations: Weigh 21.95 mg of zinc acetate, add DMF to make up to 10 mL in a volumetric flask, and prepare a 10 mM stock solution. Use a pipette to measure and prepare solutions from 0 μM to 400 μM.

[0058] (ii) Probe solution: Weigh 45.83 mg AMEP, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of DMF to dilute to a 100 μM solution.

[0059] 2. Fluorescence detection

[0060] The zinc ion solution and probe solution were mixed in a 2:1 ratio in a 2 mL centrifuge tube and allowed to react with shaking at room temperature. Fluorescence was measured after 10 seconds.

[0061] The results are as follows Figure 6 As shown, the fluorescence intensity initially increases with increasing zinc ion concentration, then tends to fluctuate regularly. Furthermore, the 0-250 μM zinc ion concentration curve in the figure exhibits a certain linear relationship. The fluorescence intensity reaches its highest value above 250 μM, and then shows a regular fluctuation trend. To establish a feasible fluorescence working curve, this invention further refines the concentration detection range from 0-250 μM.

[0062] Using a pipette, zinc ion solutions of different concentrations (0-400 μM) were mixed with probe solution (100 μM) in 2 mL centrifuge tubes, and fluorescence detection was performed after reacting at room temperature for 10 s.

[0063] The results are as follows Figure 7As shown, based on the obtained fluorescence spectral data, a good linear relationship was observed between the fluorescence intensity and the zinc ion concentration in the 0-250 μM range. Linear fitting was performed on the fluorescence intensity of zinc ions in the 0-30 μM range, yielding the correlation between the 535 nm fluorescence intensity and the Zn concentration. 2+ The concentration exhibits a linear growth relationship, R 2 =0.9966, indicating a good linear fit. Based on LOD = 3σ / S, the detection limit of the probe AMEP is 4.718 nM.

[0064] Example 5: Study on the complexation ratio of AMEP with zinc ions

[0065] Since this invention prepares a coordination-type fluorescent probe, it is necessary to investigate the complexation ratio between the metal ion and the probe. This invention uses the Job's plot method to determine the binding ratio of AMEP to zinc ions.

[0066] 1. AMEP Job's plot curve detection solution

[0067] (i) Probe solution: Weigh 45.83 mg AMEP, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of DMF to dilute to a 100 μM solution.

[0068] (ii) Zinc ion solution: Weigh 21.95 mg of zinc acetate, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, and take 100 μL of the stock solution and dilute it with 9.9 mL of water to make a 100 μM solution.

[0069] 2. Fluorescence detection

[0070] Zinc ion solution (100 μM) and probe solution (100 μM) were prepared into solutions in 2 mL centrifuge tubes at molar ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, respectively. After reacting at room temperature for 10 s, fluorescence was measured.

[0071] The results are as follows Figure 8 As shown, the optimal complexation ratio of AMEP to zinc ions is Zn 2+ When the ratio of / Probe = 2:1, the fluorescence intensity reaches its maximum value.

[0072] Example 6: Study on Selective Recognition of Probes

[0073] To further confirm AMEP's selective recognition function for zinc ions, this invention conducted an anti-interference experiment on AMEP against other common heavy metal ions.

[0074] 1. Preparation of AMEP metal ion selective detection solution

[0075] (i) Metal ion solution: Accurately weigh the corresponding masses of nickel sulfate hexahydrate (NiSO4·6H2O), praseodymium nitrate hexahydrate (Pr(NO3)3·6H2O), manganese sulfate monohydrate (MnSO4·H2O), neodymium nitrate hexahydrate (Nd(NO3)3·6H2O), yttrium nitrate hexahydrate (Y(NO3)3·6H2O), stannous chloride (SnCl2), aluminum acetate (Al(CH3COO)2), lead acetate (Pb(CH3COO)2), chromium trioxide (Cr2O3), and copper sulfate (CuSO4), etc., and dissolve and dilute with deionized water to prepare a 10 mL 10 mM solution as a metal ion stock solution. Dilute to 100 μM aqueous solutions of praseodymium ions, manganese ions, neodymium ions, nickel ions, yttrium ions, aluminum ions, cerium ions, tin ions, molybdenum ions, lead ions, chromium ions, aluminum ions, and copper ions.

[0076] (ii) Probe solution: Weigh 45.83 mg AMEP, add DMF to make up to 10 mL in a volumetric flask to prepare a 10 mM stock solution, take 100 μL of the stock solution and add 9.9 mL of DMF to dilute to a 100 μM solution.

[0077] 2. Fluorescence detection

[0078] Using a pipette, take 500 μL of probe solution and 500 μL of metal ion solution (100 μM) at a volume ratio of 1:1 into 2 mL centrifuge tubes. Incubate at room temperature for 30 min. At the same time, set up a control group (1 mL of zinc ion solution) in a 2 mL centrifuge tube. After incubating at room temperature for 30 min, perform fluorescence measurement.

[0079] The results are as follows Figure 9 and 10 As shown, AMEP did not exhibit a significant fluorescence response at 520 nm after reacting with praseodymium, manganese, neodymium, nickel, yttrium, tin, lead, chromium, aluminum, and copper ions. However, a significant fluorescence response appeared after adding zinc ion solution to the reaction solution, indicating that AMEP has good selectivity for these metal ions.

[0080] Fluorescence interference experiments showed that the presence of metal ions did not affect the probe's ability to recognize zinc ions, confirming AMEP's high sensitivity and excellent selectivity for zinc ions, thus creating favorable conditions for it to become an effective fluorescent sensor for detecting zinc ions.

[0081] Example 7: Application of probe for visual identification of zinc ions

[0082] Given that the probe compound of the present invention has good colorimetric effect, test strips made using the probe compound are made more convenient for detecting zinc ions in the environment.

[0083] AMEP probe test strip preparation method: Accurately weigh 45.83 mg AMEP, add DMF to a 100 mL volumetric flask, and prepare a 1 mM stock solution. Immerse the silica gel plate in the 1 mM probe solution, then remove and air dry. Use 1 mM Zn... 2+ The solution writes words on a silica gel plate. For example... Figure 11 , 12 As shown, the letters written on the silicone plate are clearly visible. This indicates that this probe can be used in the production of test strips, enabling convenient, accurate, and rapid Zn testing. 2+ The detection.

[0084] Cut filter paper into circles with a diameter of 2 cm. Immerse the circular filter paper in a 1 mM probe solution, then remove and air dry. Add zinc ions at concentrations of 100 mM, 10 mM, 2 mM, 1 mM, and 100 μM onto the test paper, respectively. Figure 13 , 14 As shown, the color of the test strip did not weaken under visible light and 365nm excitation light as the zinc ion concentration decreased, providing support for the future application of the test strip in the detection of low zinc ion concentrations.

[0085] Example 8: Study on the reversibility of the probe

[0086] When the probe is reversible, it can reduce costs and the possibility of environmental pollution, and enable dynamic monitoring of zinc ions, reflecting real-time changes in zinc ion concentration and state. Therefore, this invention studies the reversibility of zinc ions.

[0087] The detection of Zn by the probe AMEP was studied using triethylamine. 2+ The reversibility. The result is as follows: Figure 13 As shown, when zinc ion solution (100 μM) and triethylamine solution (100 μM) were alternately added to the probe solution (100 μM), the probe compound AMEP exhibited an "ON-OFF-ON" fluorescence change pattern, and the fluorescence efficiency loss was small after 4 cycles, indicating that the probe compound AMEP of the present invention is effective against Zn. 2+ The identification and detection have good reversibility.

[0088] In summary, to confirm whether the probe achieved the initially designed results, this experiment conducted a series of tests on the probe's fluorescence performance, determined that the optimal reaction solvent was DMF, the optimal reaction time was 10 s, and obtained a linear relationship (R0) between the probe and the zinc ion solution concentration. 2=0.9967), the optimal complexation ratio is 2:1 (n Zn :n AMEP The probe AMEP can be repeatedly cycled up to four times while maintaining good detection performance, demonstrating its excellent cyclic performance. Furthermore, it was verified that 10 heavy metal ions, such as praseodymium and copper, have no effect on the detection of zinc ions by the AMEP probe, thus confirming the high anti-interference advantage of this compound.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. The application of a zinc ion fluorescent probe compound in the preparation of zinc ion detection reagents or test strips, characterized in that, The chemical name of the zinc ion fluorescent probe compound is 2-(diethylamino)-5-[{4-[4-[4-(diethylamino)-2-hydroxybenzylamino]phenylimino}methyl]phenol, abbreviated as AMEP, and its structural formula is as follows: ； The zinc ion detection reagent or test strip is a reversible detection reagent or test strip. The specific detection method is as follows: using the zinc ion fluorescent probe compound AMEP as a fluorescent probe, the probe is restored to fluorescence performance by adding triethylamine, thereby realizing the reversible detection of zinc ions; the optimal reaction solvent for AMEP is DMF, the optimal reaction time is 10 s, and the optimal complexation ratio of zinc ions to AMEP is 2:

1.

2. The application according to claim 1, characterized in that, The zinc ions are zinc ions in the water sample or inside the cells.

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