A fluorescent probe NHO for aluminum ion detection and its preparation method and application

Abstract

The present invention discloses a fluorescent probe (NHO) for aluminum ion detection, its preparation method, and applications. The probe exhibits strong selectivity for aluminum ions, a fast response time, significant fluorescence enhancement visible to the naked eye after complexation, and a low detection limit. Its structure is shown in Formula I below. The Schiff base-type fluorescent probe (NHO) is prepared by a condensation reaction using amino acid derivatives and 4-(diethylamino) salicylaldehyde as fluorescent groups. The preparation method is simple, the raw materials are readily available, and the resulting product is a solid powder that is easy to store.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe NHO for detecting aluminum ions, a preparation method thereof, and an application thereof. Background Art

[0002] Aluminum (Al) is a common nonferrous metal with good ductility, wear resistance, corrosion resistance, castability, and good room temperature mechanical properties. It can be made into alloys with various metals. It is widely distributed in the earth's crust and natural water bodies, and is also widely used in food additives, water treatment, packaging materials, medicines, cookware, etc. 3+ Excessive aluminum exposure not only causes distortion of enzymes, neurotransmitters, and nerve fibers in the central nervous system, but can also cause severe damage to organs such as bones, kidneys, and liver, leading to various diseases such as Parkinson's disease. Therefore, developing a rapid, simple, and quantitative method for measuring aluminum content is of great value.

[0003] Amino acids have multiple nitrogen and oxygen atoms, making them easy to complex with hard-base aluminum ions. Compared to other organic ligands, they are not only inexpensive and nontoxic, but also simple to synthesize. Furthermore, linking amino acids to aromatic compounds with conjugated systems can easily form organic ligands with semi-rigid backbones. This method can effectively modify the flexibility of amino acids, thereby expanding their application in the construction of metal-organic complexes.

[0004] Atomic absorption spectrometry (AAS), disulfide hydrazone colorimetry, inductively coupled plasma emission spectrometry, inductively coupled plasma mass probe and electrochemical methods are currently the most effective methods for aluminum ion detection. However, these methods require expensive instruments, rigorous experimental conditions, complex sample pretreatment and relatively long measurement time. In comparison, fluorescent probes designed based on organic small molecules have the advantages of high selectivity, high sensitivity, short detection time, simple operation, low detection cost, low toxicity and visualization, and have attracted much attention. Therefore, it is necessary to design and develop Al ion detectors with high sensitivity, high selectivity and low detection limit. 3+ Fluorescent probes have important practical value. Based on the fluorescence enhancement phenomenon, a smartphone application is used to output the RGB values ​​of the captured image for the colored solution. The smartphone-based ratiometric sensor can use the R / B value in the sample to give the linear range and detection limit for Al 3+ Visible fluorescence quantification. Through blue-red dual-emission fluorescence, the smartphone-based fluorescent probe provides on-site, rapid and sensitive detection of Al in complex samples. 3+ Provides an economical, portable and reliable method. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a fluorescent probe NHO for aluminum ion detection. The fluorescent probe NHO has excellent selectivity for aluminum ions, a low detection limit, a short response time, and a color change visible to the naked eye. It can quickly detect trace amounts of aluminum ions in environmental systems on site.

[0006] The present invention also provides a preparation method and application of the aluminum ion detection fluorescent probe NHO.

[0007] Technical Solution: To achieve the above-mentioned purpose of the invention, the present invention provides a fluorescent probe NHO for aluminum ion detection. The aluminum ion detection fluorescent probe NHO uses an amino acid derivative and 4-diethylamino salicylaldehyde as fluorescent groups and is prepared by a one-step Schiff base condensation. Its structure is shown in Formula I below:

[0008]

[0009] The method for preparing the aluminum ion detection fluorescent probe NHO of the present invention comprises the following steps:

[0010] Hippuric acid and benzaldehyde are reacted in the presence of acetic anhydride and potassium acetate to obtain the intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one; then, 4-benzylidene-2-phenyl-2-oxazolidin-5-one is used as a raw material and hydrazine hydrate is reacted to obtain the final target raw material (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide through a nucleophilic ring-opening reaction; the target raw material (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide and the compound 4-diethylaminosalicylaldehyde are reacted to obtain the fluorescent probe NHO (N-((Z)-3-(2-(Z)-4-(diethylamino)-2-hydroxybenzylidene)hydrazino)-3-oxo-1-phenylprop-1-en-2-yl)benzamide) through a condensation reaction.

[0011] The reaction route for preparing the aluminum ion detection fluorescent probe NHO is as follows:

[0012]

[0013] In the reaction route, the intermediate is (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (A); 4-diethylaminosalicylaldehyde (B); and N-((Z)-3-(2-(Z)-4-(diethylamino)-2-hydroxybenzylidene)hydrazino)-3-oxo-1-phenylprop-1-en-2-yl)benzamide (NHO), which is the fluorescent probe molecule for detecting aluminum ions described in the present invention.

[0014] The hippuric acid, benzaldehyde, acetic anhydride and potassium acetate are mixed and then heated to melt. After the reaction is complete, the reaction material is cooled to room temperature, and the solid is washed with saturated sodium carbonate. The crude product is extracted with ethyl acetate, separated, dried, and the solvent is removed by distillation under reduced pressure. Then, the intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one is obtained by column chromatography.

[0015] The intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one and hydrazine hydrate are dissolved in an organic solvent, methanol, and then heated under reflux with stirring. After the reaction is complete, the reaction solution is cooled to room temperature, the organic solvent is removed by distillation under reduced pressure, and the target raw material (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide is obtained by column chromatography.

[0016] The (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide and 4-diethylaminosalicylaldehyde are dissolved in an organic solvent, ethanol, and the mixture is refluxed with stirring. After the reaction is completed, the reaction material is cooled to room temperature, the solvent is removed by distillation under reduced pressure, and the crude product is purified by recrystallization to obtain a fluorescent probe NHO.

[0017] The invention provides an application of the aluminum ion detection fluorescent probe NHO in detecting trace aluminum ions in a solution.

[0018] Furthermore, the aluminum ion detection fluorescent probe NHO is prepared into fluorescent test paper for use in detecting trace aluminum ions in a solution.

[0019] The aluminum ion detection fluorescent probe NHO of the present invention is used in detecting trace aluminum ions in cells.

[0020] The present invention relates to the application of the aluminum ion detection fluorescent probe NHO in combination with a smartphone in real-time, on-site aluminum ion detection.

[0021] Furthermore, the aluminum ion detection fluorescent probe NHO can be combined with a smartphone to quickly detect the aluminum ion content in drugs.

[0022] Preferably, the preparation process of NHO comprises:

[0023] (1) Preparation of (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (A)

[0024] Hippuric acid, benzaldehyde, acetic anhydride and potassium acetate are mixed and then heated to melt. After the reaction is complete, the reaction mass is cooled to room temperature and the solid is washed with saturated sodium carbonate. The crude product is extracted with ethyl acetate, separated and dried, and the solvent is removed by distillation under reduced pressure. The intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one is then obtained by column chromatography (ethyl acetate: petroleum ether = 1:10).

[0025] The intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one and hydrazine hydrate were dissolved in methanol and heated under reflux with stirring for 4 hours. The reaction solution was cooled to room temperature and the organic solvent was removed by distillation under reduced pressure. Column chromatography (methanol:dichloromethane = 1:15) gave the yellow target material (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (A).

[0026] (2) Preparation of fluorescence-enhanced aluminum ion fluorescent probe NHO

[0027] (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (A) and 4-diethylaminosalicylaldehyde (B) were dissolved in ethanol solvent, and the reaction system was refluxed and stirred at 80°C. After the reaction was completed, the reaction system was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was purified by recrystallization to obtain the fluorescent probe molecule NHO.

[0028] The present invention designs a novel Schiff base fluorescent probe NHO based on a salicylaldehyde derivative. In the parent structure of the aluminum ion detection fluorescent probe, 4-diethylamino salicylaldehyde is a good fluorophore with excellent stability and multiple modification sites, and the diethylamino group can serve as an electron donor group. Hippuric acid derivatives have many advantages as fluorescent groups, and have good biocompatibility and low biotoxicity. At the same time, compared with the probes based on purine structure in the prior art, this probe has a short synthesis route, high yield, good solubility, low raw material cost, and is sensitive to Al. 3+ With unique sensitivity (not interfered by other ions), low detection limit, colorimetric fluorescence dual channel, it can successfully prepare fluorescent test paper to detect trace Al in aqueous solution. 3+ .

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] The present invention uses a one-step method to prepare a colorimetric fluorescent dual-channel fluorescent probe NHO by condensing 4-diethylamino salicylaldehyde and (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide. It has specific recognition for aluminum ions, short response time, high sensitivity, and visible color change. At the same time, combined with the RGB software auxiliary function of smart phones, it can be used to detect trace amounts of Al in the environment. 3+ The NHO probe provides a simple, convenient, and low-cost measurement tool with a detection limit as low as 24.8 nM, effectively enabling real-time, on-site aluminum ion detection. Importantly, the probe is simple to prepare, resulting in a solid powder that is easy to store and exhibits excellent stability. Furthermore, the probe exhibits low toxicity and strong cell-penetrating ability, effectively enabling the detection of trace aluminum ions in HeLa cells. In summary, the advantages of NHO's high-performance aluminum ion detection provide a foundation for the subsequent development of a portable, simple aluminum ion detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The aluminum ion fluorescent probe prepared in Example 1 was used to detect different concentrations of aluminum ions (Al 3+ )'s UV absorption spectrum and color change diagram ( Figure 1 The left side of the illustration is colorless, and the right side is yellow-green);

[0032] Figure 2 The selective fluorescence spectra of the aluminum ion fluorescent probe prepared in Example 1 to different metal ions in a (MeOH:Hepes buffer) (v / v=9:1) solution;

[0033] Figure 3 The fluorescent probe prepared in Example 1 was tested for different concentrations of aluminum ions (Al 3+ )'s fluorescence spectrum response diagram and color change diagram;

[0034] Figure 4 Graph showing the fluorescence response of the fluorescent probe prepared in Example 1 to selective interference detection of different metal ions in a (MeOH:Hepes buffer) (v / v=9:1) solution;

[0035] Figure 5 The fluorescent probe prepared in Example 1 was mixed with (MeOH:Hepes buffer) (v / v=9:1) and aluminum ions (Al 3+ ) Job-plot curve of complexation ratio;

[0036] Figure 6This is a response time diagram of the fluorescent probe prepared in Example 1 when detecting aluminum ions;

[0037] Figure 7 The fluorescent probe prepared in Example 1 was mixed with (MeOH:Hepes buffer) (v / v=9:1) and aluminum ions (Al 3+ ) Fluorescence response graph at different pH values ​​(2 to 12);

[0038] Figure 8 The fluorescent probe prepared in Example 1 and the optimal configuration diagram and frontier orbital energy level diagram after the fluorescent probe is complexed with aluminum ions;

[0039] Figure 9 The color change diagram of the fluorescent test paper prepared with the fluorescent probe prepared in Example 1 when testing different concentrations of aluminum ions;

[0040] Figure 10 This is a diagram showing the cytotoxicity test results of the fluorescent probe prepared in Example 1;

[0041] Figure 11 The fluorescent probe prepared in Example 1 is used to detect trace aluminum ions in active HeLa cells;

[0042] Figure 12 The fluorescent probe prepared in Example 1 is based on the Al2O3 RGB mode. 3+ Linear calibration curve plot of concentration versus color intensity;

[0043] Figure 13 The nuclear magnetic resonance of the fluorescent probe prepared in Example 1 1 H NMR spectrum;

[0044] Figure 14 The nuclear magnetic resonance of the fluorescent probe prepared in Example 1 13 C NMR spectrum;

[0045] Figure 15 is the mass spectrum MS spectrum of the fluorescent probe prepared in Example 1;

[0046] Figure 16 This is the infrared IR spectrum of the fluorescent probe prepared in Example 1. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and examples.

[0048] Unless otherwise specified, the experimental methods used in this invention are conventional methods. The materials and reagents used in the experiments, unless otherwise specified, can be obtained from commercial sources. All the following reagents used in the examples were commercially available of analytical or chemical purity.

[0049] The various metal ion solutions in the embodiments are prepared by adding deionized water to chloride chemical reagents with a purity of more than 99%, such as anhydrous aluminum chloride, anhydrous ferric chloride, zinc chloride, etc.

[0050] Example 1

[0051] Specific synthetic route of the fluorescent probe NHO based on salicylaldehyde derivatives:

[0052] (1) Preparation of intermediates

[0053] In a round-bottom flask, hippuric acid (0.89 g, 5 mmol), benzaldehyde (0.53 g, 5 mmol), potassium acetate (0.49 g, 5 mmol) and acetic anhydride (1.53 g, 15 mmol) were added respectively, heated to 120 ° C to melt, and after the reaction was complete, the reaction mass was cooled to room temperature, centrifuged and then the excess acetic acid was neutralized with saturated sodium carbonate. The reaction system was extracted with ethyl acetate, separated, and the organic phase was dried. The solvent was distilled off under reduced pressure, and then the yellow solid 4-benzylidene-2-phenyl-2-oxazolidin-5-one (1.25 g, 50%) was obtained by column chromatography (ethyl acetate: petroleum ether = 1:10).

[0054] The structural formula of the intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one obtained is:

[0055]

[0056] (2) Preparation of intermediate (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide

[0057] To a reaction flask containing 10 ml of methanol, the intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one (0.49 g, 2 mmol) and hydrazine hydrate (0.15 g, 3 mmol) were added. After stirring to dissolve, the reaction system was heated to 65°C and stirred at reflux for 4 hours. After cooling the reaction solution to room temperature, the organic solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (methanol:dichloromethane = 1:15) to obtain (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (0.52 g, 92%) as a yellow solid.

[0058] The structural formula of the obtained intermediate (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide is:

[0059]

[0060] (6) Preparation of aluminum ion fluorescent probe NHO based on salicylaldehyde derivatives

[0061] In a round-bottom flask containing 5 ml of ethanol, (Z)-N-(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide (A) (0.281 g, 1 mmol) and 4-diethylaminosalicylaldehyde (B) (0.193 g, 1 mmol) were added. After stirring to dissolve, the reaction system was heated to 80°C and refluxed for 4 hours. TLC was used to monitor the reaction. After completion of the reaction, the reaction system was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was purified by recrystallization (EtOH:H2O = 1:10) to obtain a yellow solid fluorescent probe molecule NHO (0.434 g, 95%).

[0062] The obtained fluorescent probe compound has the structural formula:

[0063]

[0064] Characterization data of this fluorescent probe 1 H NMR (400MHz, DMSO-d6) δ11.62(s,1H),11.47(s,1H),10.08(s,1H),8.35(s,1H),8.00(d,J=7.5Hz,2H),7.58(d,J=7.4Hz,3H),7.51(d,J=7.5Hz ,2H),7.39–7.26(m,3H),7.15(s,1H),7.12(d,J=8.8Hz,1H),6.22(d,J=8.8Hz,1H),6.08(s,1H),3.31(q,J=7.2Hz,4H),1.06(t,J=6.9Hz,6H); 13 C NMR(101MHz,DMSO-d6)δ166.09,161.42,159.85,150.20,149.91,134.29,133.52,131.98,131.73,1 29.61,128.99,128.72,128.53,128.06,106.60,104.59,103.72,97.60,96.01,43.91,12.67.ESI-MS m / z:[M+H]+calcd for C 27 H 28 N4O3456.5,found455.9.IR(KBr cm -1 ):3431,3234,1633,1593,1352.

[0065] The hydrogen spectrum of the fluorescent probe prepared in Example 1 ( 1 H NMR), carbon spectrum ( 13C NMR), mass spectra MS and infrared spectra are shown in Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown, it shows that the fluorescent probe of the present invention was successfully prepared.

[0066] Example 2

[0067] The aluminum ion detection fluorescent probe NHO prepared in Example 1 was configured with DMSO to a 1 mM probe stock solution, and each metal ion was configured with deionized water to a 3 mM metal ion stock solution. 30 μL of the probe stock solution and 50 μL of the metal ion stock solution were added to 3 mL of a blank solution (MeOH:Hepes buffer) (v / v=9:1) and detected using a fluorescence spectrometer and an ultraviolet spectrophotometer. The test showed that the maximum excitation wavelength of the fluorescent probe was 418 nm, and the maximum emission wavelength was 537 nm.

[0068] Take two cuvettes, add 3mL of mixed solution (MeOH:Hepes buffer) (v / v=9:1) containing probe stock solution (30μL) to each cuvette, and add 50μL of aluminum ion stock solution to one of the cuvettes for UV spectrum test. Figure 1 As shown in the figure, the fluorescent probe itself has almost no ultraviolet absorption at wavelength λ = 410nm. Once aluminum ions are added, the ultraviolet absorption peak gradually increases; moreover, the addition of aluminum ions changes the color of the probe solution from colorless to yellow-green. The results show that the probe has a strong affinity for Al 3+ Has excellent sensitivity and selectivity.

[0069] like Figure 2 Figure 2 shows the selective fluorescence spectra of the aluminum ion detection fluorescent probe for various common metal ions. 30 μL of the probe stock solution and 50 μL of the metal ion stock solution (3 mM) were added to 3 mL of a blank solution (MeOH:Hepes buffer) (v / v = 9:1). The experimental results show that only with the addition of aluminum ions does the fluorescence intensity at 537 nm increase significantly. Furthermore, the fluorescence intensity is significantly superior to that observed with the addition of other metal ions, demonstrating the excellent selectivity of the fluorescent probe for aluminum ions.

[0070] like Figure 3 As shown in the figure, the aluminum ion detection fluorescent probe reacts with different concentrations of aluminum ions (Al 3+) is a fluorescence spectrum response diagram. 30 μL of the probe stock solution and 0-100 μL of aluminum ion solution (3 mM aluminum ion stock solution) were added to 3 mL of a blank solution (MeOH:Hepes buffer) (v / v=9:1). The fluorescent probe itself has almost no fluorescence in the solution, but as the aluminum ion concentration increases, the fluorescence at 537 nm also increases with the increase in aluminum ion concentration, that is, the fluorescence intensity increases with the increase in aluminum ion concentration, and is accompanied by an obvious color change. This indicates that the probe NHO prepared in Example 1 of the present invention inhibits the rotation of the C=N double bond after complexing with aluminum ions, thereby inhibiting electron transfer, indicating that the probe NHO is a fluorescence-enhancing probe.

[0071] like Figure 4 As shown in the figure, the fluorescence intensity bar graph of the aluminum ion detection fluorescent probe after reacting with aluminum ions in the presence of different interfering metal ions. 30 μL of the probe stock solution and 50 μL of any other metal ion (Cu + ,Co 2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Pd 2+ , Sn 2+ , Fe 2+ , K + , Ca 2+ , Sr 2+ , Na + , Ag + and Cd 2+ ) stock solution, and finally add 50 μL of Al 3+ The results showed that other metal ions had no significant interference on the recognition of aluminum ions by the aluminum ion fluorescent probe of the present invention, indicating that the probe prepared by the present invention has good specificity.

[0072] like Figure 5 As shown in the figure, the interaction between the probe and Al was studied by Job's plot method. 3+ To determine the binding rate, a certain volume of probe stock solution (1 mM) and Al 3+The stock solution (3mM) was prepared so that the total concentration of the aluminum ion detection fluorescent probe and aluminum ions was 50μM. By changing the concentration ratio of the two (the molar ratio of the aluminum ion detection fluorescent probe and aluminum ions was (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1), the difference between the fluorescence intensity at 537nm and the combined fluorescence intensity of the aluminum ion fluorescent probe at this concentration was obtained, and the ratio of ions to the total concentration was plotted. Figure 5 It can be seen that when the proportion of aluminum ions is 0.7, the vertical axis reaches the highest value, which can be determined that the fluorescent probe and aluminum ions form a stable complex [NHO-Al 3+ ].

[0073] like Figure 6 As shown, 30 μL of probe stock solution and 50 μL of Al were added to 3 mL of blank buffer (MeOH:Hepes buffer) (v / v=9:1). 3+ The fluorescence intensity of the probe rapidly increased to the maximum and reached a stable value within 2 minutes. In addition, the fluorescence intensity of the probe remained basically unchanged in the following 30 minutes, which shows that the probe prepared by the present invention is effective for Al 3+ The detection is stable and very fast.

[0074] like Figure 7 As shown, 1 M HCl and 1 M NaOH were used to adjust the probe NHO stock solution (10 μL) and [NHO-Al 3+ ] stock solution (10 μL) in MeOH H2O solution (1 mL), where [NHO-Al 3+ The stock solution was prepared by dissolving the probe and aluminum chloride in double distilled water to the concentration of 10uM and 50uM respectively, so that it had different pH values. Then, the fluorescence intensity of the two systems was tested in different pH ranges. The probe NHO itself had almost no fluorescence intensity in the pH range of 2 to 12, but [NHO-Al 3+ ] The fluorescence (537nm) intensity of the system is significantly enhanced in the pH range of 3.0-7.0, and reaches the maximum value at pH=7. 3+ ] Under strong conditions (pH < 3.0), the fluorescence signal is relatively weak. The possible reason is that the complexing point of the fluorescent probe NHO is protonated and is not easy to complex with aluminum ions. 3+ ] Under alkaline conditions (pH>9.0), the fluorescence signal gradually decreased, which may be due to the formation of Al(OH)3 precipitation, which reduced the [NHO-Al 3+ ] concentration. Therefore, the most suitable pH range for NHO is 4-7. NHO has the ability to detect Al in biological environments. 3+ability.

[0075] like Figure 8 As shown, the fluorescent probe and [NHO-Al 3+ ]The optimal configuration diagram of the complex and its corresponding energy level diagram. The DFT calculation results further prove that the probe NHO and aluminum ion are a four-coordinate complex formed by -C=N, -OH and C=O.

[0076] like Figure 9 As shown, the filter paper was immersed in a MeOH:Hepes buffer (v / v=9:1) stock solution containing the fluorescent probe (1mM) for half an hour, and then the test strip was removed and air-dried to obtain a dry test strip containing the probe. The test strip was immersed in 0mM, 0.1mM, and 1mM aluminum ion concentration solutions for 30 minutes, then dried. The fluorescent test paper prepared with the fluorescent probe NHO solution and its color change when testing different concentrations of aluminum ions were measured under ultraviolet light, indicating that the color of the probe NHO changes with the aluminum ion concentration and can be used to quantitatively detect trace aluminum ions in the environment in a solid state.

[0077] like Figure 10 As shown, HeLa cells were seeded into 96-well culture plates at a density of approximately 7,000 cells per well and allowed to adhere for 12 hours at 37°C in a humidified atmosphere of 5% CO2. Then, 100 μL of fresh culture medium with different NHO concentrations (0, 2, 4, 6, 8, and 10 μM) was added to each well and cultured for 24 hours. Finally, 10 μL of MTT reagent was added to each well and cultured for another 3 hours. Figure 10 The results showed that when the NHO concentration ranged from 0 to 10 μM, the cell survival rate was greater than 95%, indicating that NHO had very low cytotoxicity and good biocompatibility. Based on this, relevant cell experiments were carried out.

[0078] like Figure 11 As shown, HeLa cells were seeded into 96-well plates at a density of approximately 7,000 cells. The HeLa cells were incubated with NHO (10 μM) in growth medium at 37°C for 30 minutes. Excess NHO on the cell surface was then removed after washing three times with sterile PBS buffer. The NHO-prepared HeLa cells were then further exposed to 10 μM Al 3+ The solution was left for another 30 min and then washed with PBS to remove excess Al 3+ Finally, all the cell samples were tested using confocal fluorescence microscopy. The cells treated with NHO alone did not change color under 488nM blue light. However, the cells treated with both NHO and Al showed no color change. 3+The treated cells showed blue color under 488 nM blue light irradiation, which proved that the probe of the present invention can effectively detect aluminum ions.

[0079] like Figure 12 As shown, Al based on RGB mode 3+ Linear calibration curve of concentration and color intensity. 30 μL of probe stock solution and 0, 5, 15, 25, and 50 μL of aluminum ion solution (3 mM aluminum ion stock solution) were added to 3 mL of blank solution (MeOH:Hepes buffer) (v / v=9:1). The color intensity of the probe NHO and different concentrations of Al were captured by a smartphone. 3+ A high-resolution photo of the solution under 365nm UV light was taken and converted into corresponding RGB data using the "Color Assist" application on the mobile phone. The color intensity was calculated using the formula I = 0.3R + 0.59G + 0.11B. The results showed that in the range of 0-50μM, the color intensity was consistent with that of Al 3+ There is a good linear relationship between the concentrations of the probes of the present invention. 3+ The detection limit of the solubility tester can reach 24.8nM, which can be used in real time for the detection of aluminum ions in real samples in environmental systems.

[0080] The above experiments show that the fluorescent probe NHO with salicylaldehyde is prepared by condensation reaction using hippuric acid derivatives and 4-diethylamino salicylaldehyde as fluorescent groups. 3+ Shows high sensitivity and high selectivity.

Claims

1. A fluorescent probe NHO for detecting aluminum ions, characterized in that The structure of the aluminum ion detection fluorescent probe NHO is shown in the following formula I: 。 2. A method for preparing the aluminum ion detection fluorescent probe NHO according to claim 1, characterized in that: The steps include: The intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one is obtained by condensation reaction of hippuric acid and benzaldehyde in the presence of acetic anhydride and potassium acetate; then the target raw material ( Z )- N -(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide; with the target raw material ( Z )- N The fluorescent probe NHO was obtained by condensation reaction of -(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide and compound 4-diethylaminosalicylaldehyde.

3. The preparation method according to claim 2, characterized in that The reaction scheme for preparing the aluminum ion detection fluorescent probe NHO is as follows: 。 4. The preparation method according to claim 2, characterized in that The hippuric acid, benzaldehyde, acetic anhydride and potassium acetate are heated and melted, and after the reaction is complete, the solid is cooled to room temperature, and then washed with saturated sodium carbonate, extracted with ethyl acetate, separated, dried, and after removing the solvent, the intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one is obtained by column chromatography.

5. The preparation method according to claim 2, characterized in that The intermediate 4-benzylidene-2-phenyl-2-oxazolidin-5-one was dissolved in an organic solvent, methanol, and then hydrazine hydrate was added, heated under reflux and stirred. After the reaction was complete, the reaction solution was cooled to room temperature, and the organic solvent was removed by vacuum distillation. The final target raw material ( Z )- N -(3-hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide.

6. The preparation method according to claim 2, characterized in that The Z )- N -(3-Hydrazino-3-oxo-1-phenylprop-1-en-2-yl)benzamide and 4-diethylaminosalicylaldehyde were dissolved in an organic solvent, ethanol, and the mixture was refluxed with stirring. After the reaction was completed, the reaction material was cooled to room temperature, the solvent was removed by distillation under reduced pressure, and the crude product was purified by recrystallization to obtain the fluorescent probe NHO.

7. Use of the aluminum ion detection fluorescent probe NHO according to claim 1 in preparing a reagent for detecting trace aluminum ions in a solution.

8. Use of the aluminum ion detection fluorescent probe NHO according to claim 1 in preparing a reagent for detecting trace aluminum ions in cells.

9. Use of the aluminum ion detection fluorescent probe NHO according to claim 1 in combination with a smartphone in the preparation of a tool for real-time, on-site aluminum ion detection.

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