A fluorescent probe CPP for detecting aluminum ions, and its preparation method and application

Abstract

This invention discloses a CPP fluorescent probe for detecting aluminum ions, its preparation method, and its application. The CPP exhibits strong selectivity for aluminum ions, exhibits significant fluorescence enhancement under ultraviolet light after complexation, and exhibits a low detection limit. Its structure is shown in (I) (#imgabs0#). The Schiff base-type CPP fluorescent probe is prepared via a condensation reaction using chromone-3-formaldehyde and 6-hydroxypyridinium hydrazide as fluorescent groups. The preparation method is simple, the raw materials are readily available and inexpensive, and the resulting product is a solid powder that is easy to store.

Description

Technical Field

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

[0002] Aluminum is the most abundant metallic element in the Earth's crust, comprising approximately 8% of the Earth's crust, second only to oxygen and silicon. It is widely used in everyday life, such as in food packaging, medicine, and tableware. However, excessive aluminum intake by animals, plants, and humans can cause certain harmful effects, including Alzheimer's disease and amyotrophic lateral sclerosis. The average daily aluminum intake is reported to be approximately 3.0 to 10.0 mg, and the average weekly aluminum intake is approximately 7 mg / kg. Widely used aluminum-containing food additives (such as potassium aluminum sulfate, ammonium aluminum sulfate, and sodium aluminosilicate) are the primary source of aluminum in the daily diet. Strengthened testing and supervision of these foods are necessary to ensure public health. Compared to aluminum-containing food additives, aluminum-containing medications are more likely to cause excessive aluminum levels and pose a greater risk to human health. Common aluminum-containing medications include antiperspirants and antacids, and their use must be strictly restricted and gradually phased out. In short, we are often exposed to aluminum in our daily lives, and aluminum intake is closely related to human health. To be vigilant about aluminum pollution and strengthen the supervision of aluminum-containing foods and drugs, it is extremely necessary to develop methods that can quickly and conveniently detect aluminum ions. Therefore, developing analytical methods to detect and control concentrations of aluminum in the environment is of great practical significance.

[0003] Fluorescent probes have been shown to be very useful methods for detecting metal ions in vivo and in vitro due to their simple synthesis methods and high selectivity. In terms of metal ion detection, probe technology is non-destructive in organisms, has instantaneous response and a wide range of dye materials, which has great advantages over traditional methods. In summary, the design and development of a new Schiff base fluorescent sensor with rich N- and O- 3+ It is of great significance. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a fluorescent probe CPP for detecting aluminum ions. The fluorescent probe CPP has high selectivity for aluminum ions and a low detection limit, and can quickly detect trace amounts of aluminum ions in the environment to be tested.

[0005] The present invention also provides a preparation method and application of the above-mentioned fluorescent probe CPP for detecting aluminum ions.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A fluorescent probe CPP for detecting aluminum ions, wherein the fluorescent probe CPP for detecting aluminum ions uses chromone-3-carboxaldehyde and 6-hydroxypyridinium hydrazide as fluorescent groups, and the specific structure is shown in (I):

[0008] The preparation method of the fluorescent probe CPP for detecting aluminum ions is as follows:

[0009]

[0010] In the formula, the intermediate is 6-hydroxypyridine hydrazide (A); chromone-3-carboxaldehyde (B); and (E)-6-oxo-N'-((4-oxo-4H-chromium-3-yl)methylene)-1,6-dihydropyridine-2-carbohydrazide (CPP) is the fluorescent probe molecule for detecting aluminum ions described in the present invention.

[0011] The specific steps include:

[0012] Step 1: dissolving 6-hydroxypyridine-2-carboxylic acid in an organic solvent, methanol, and then adding concentrated sulfuric acid. The mixture is refluxed with stirring to carry out an esterification reaction. After the reaction is complete, the organic solvent is removed, and then column chromatography is performed to obtain the intermediate 2-hydroxypyridine acid methyl ester.

[0013] Step 2: dissolving methyl 2-hydroxypicolinate in methanol, an organic solvent, adding hydrazine hydrate, heating under reflux and stirring to carry out a substitution reaction. After the reaction is completed, the mixture is naturally cooled to room temperature, and the organic solvent is removed by distillation under reduced pressure. The liquid is extracted and dried. After removing the solvent, 6-hydroxypicolinohydrazide is obtained by column chromatography;

[0014] Step 3: dissolving 6-hydroxypicolinohydrazide and chromone-3-carboxaldehyde in an organic solvent, methanol, and refluxing the mixture with stirring. After the condensation reaction is completed, the mixture is naturally cooled to room temperature, the solvent is removed by distillation under reduced pressure, and the crude product is recrystallized and purified to obtain the fluorescent probe CPP.

[0015] The application of the fluorescent probe CPP for detecting aluminum ions in detecting trace aluminum ions in wastewater.

[0016] As an improvement, the detection pH of the fluorescent probe CPP is 5-7, and the lower detection limit is 271 nm.

[0017] The above-mentioned fluorescent probe CPP for detecting aluminum ions is used to detect trace amounts of aluminum ions in active HeLa cells.

[0018] Beneficial effects:

[0019] Compared with the existing technology, the present invention provides a fluorescent probe CPP for detecting aluminum ions, and its preparation method and application. It uses a one-step method to prepare a Schiff base fluorescent probe CPP with a new structure and chromone-3-carboxaldehyde as the parent compound through the condensation of chromone-3-carboxaldehyde and 6-hydroxypyridinium hydrazide. Specific advantages are as follows:

[0020] 1. The preparation method of the present invention is simple, the raw materials are low-cost, and the obtained product is a solid powder that is easy to store and has good stability. 6-Hydroxypicolinohydrazide derivatives have many advantages as fluorescent groups, including good biocompatibility and low biotoxicity.

[0021] 2. In the parent structure of the fluorescent probe of the present invention, chromone-3-carboxaldehyde has a rigid planar structure and a large π-bond conjugated system, is an excellent chromophore with good biocompatibility, strong cell penetration and low biotoxicity, and has been successfully used to detect trace amounts of aluminum ions in active HeLa cells.

[0022] 3. Compared with the existing probes based on purine structure, the synthesis route is short, the yield is high, the raw material cost is low, and it has specific recognition of aluminum ions, high sensitivity, and is sensitive to Al in solution. 3+ It shows short response time, high sensitivity, high selectivity and low detection limit (271nm), and can be successfully used to prepare fluorescent test paper to detect trace Al in aqueous solution. 3+ . BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The aluminum ion fluorescent probe prepared in Example 1 was used to detect different concentrations of aluminum ions (Al 3+ )’s ultraviolet absorption spectrum and color change diagram;

[0024] Figure 2 Graph showing the selective fluorescence spectra of the aluminum ion fluorescent probe prepared in Example 1 for different metal ions in a (MeOH:H2O:Hepes) (v / v / v=9:1:0.1) solution;

[0025] Figure 3 The fluorescent probe prepared in Example 1 was tested for the effect of different concentrations of aluminum ions (Al 3+ )'s fluorescence spectrum response diagram and color change diagram;

[0026] 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:H2O:Hepes) (v / v / v=9:1:0.1) solution;

[0027] Figure 5 The fluorescent probe prepared in Example 1 was mixed with (MeOH:H2O:Hepes) (v / v / v=9:1:0.1) and aluminum ions (Al 3+ ) Job-plot curve of complexation ratio;

[0028] Figure 6 This is a response time diagram of the fluorescent probe prepared in Example 1 when detecting aluminum ions;

[0029] Figure 7 The fluorescent probe prepared in Example 1 was mixed with (MeOH:H2O:Hepes) (v / v / v=9:1:0.1) and aluminum ions (Al 3+ ) Fluorescence response diagram at different pH values ​​(2-12);

[0030] Figure 8 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;

[0031] Figure 9 The mass spectrum of the fluorescent probe prepared in Example 1 1 H NMR spectrum;

[0032] Figure 10 is the mass spectrum MS spectrum of the fluorescent probe prepared in Example 1;

[0033] Figure 11 This is the mass IR spectrum of the fluorescent probe prepared in Example 1. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and examples.

[0035] 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.

[0036] 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 zinc chloride, anhydrous ferric chloride, potassium chloride, etc.

[0037] Example 1

[0038] Preparation of intermediate 6-hydroxypyridinoylhydrazide

[0039]

[0040] 6-Hydroxypyridine-2-carboxylic acid (0.556 g, 4 mmol) was dissolved in methanol solvent (20 mL). Under stirring, 98% concentrated sulfuric acid (1 mL) was added dropwise as a catalyst. The reaction system was heated to 80°C and refluxed with stirring for 4 hours. After the reaction material was cooled to room temperature, the solvent was distilled off under reduced pressure. The crude product was extracted with dichloromethane, separated, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product, methyl 2-hydroxypyridine (0.565 g, 92%).

[0041] The crude product, methyl 2-hydroxypicolinate (0.46 g, 3 mmol) and hydrazine hydrate (0.75 g, 15 mmol), were dissolved in methanol (10 mL) and the mixture was heated under reflux at 80°C with stirring for 2 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. Column chromatography (ethyl acetate:petroleum ether = 1:4) afforded 0.36 g of 6-hydroxypicolinohydrazide with a yield of 85%.

[0042] (2) Preparation of aluminum ion fluorescent probe CPP based on chromone-3-carboxaldehyde

[0043]

[0044] In a 50ml round-bottom flask containing 20ml of the organic solvent methanol, 6-hydroxypicolinohydrazide (A) (0.153g, 1mmol) and chromone-3-carboxaldehyde (B) (0.174g, 1mmol) were added separately. After stirring to dissolve, the reaction system was heated to 80°C and refluxed under N2 protection for 3 hours. TLC was used to track 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 (DMSO:H2O=1:20) to obtain a yellow solid fluorescent probe CPP (0.26g, 85%). 1 H NMR (400MHz, DMSO-d6) δ11.96 (s, 1H), 8.85 (s, 1H), 8.64 (s, 1H), 8.14 (d, J = 0.84Hz, 1H), 7.78 (t, J = 0.84Hz, 1H), 7.75 (t, J=0.84Hz, 2H), 7.56 (t, J=0.84Hz, 1H), 7.31 (t, J=0.84Hz, 1H), 6.8 (d, J=0.84Hz, 1H); ESI-MSm / z: [M+H] + calcd for C 16 H 11 N3O4309.1, found 310.0.IR(KBr cm -1 ):3283,3062,1652,1521,1445,1371.

[0045] Among them, the hydrogen spectrum of the yellow solid fluorescent probe CPP of the present invention ( 1 H NMR), mass spectra MS and infrared spectra are shown in Figure 9 , Figure 10 , Figure 11 .

[0046] Example 2

[0047] The fluorescent probe CPP for detecting aluminum ions prepared in Example 1 was prepared into a 1 mM probe stock solution with DMSO, and each metal ion was prepared into a 3 mM metal ion stock solution with deionized water. 30 μL of the fluorescent probe CPP stock solution and 50 μL of the metal ion stock solution were added to 3 mL of a blank solution (MeOH:H2O:Hepes) (v / v / v=9:1:0.1), and the solution was detected using a fluorescence spectrometer and an ultraviolet spectrophotometer. The test results showed that the maximum excitation wavelength of the fluorescent probe CPP was 410 nm, and the maximum emission wavelength was 516 nm. The specific test results are as follows:

[0048] Take two cuvettes and add 3 mL of a mixed solution (MeOH:H2O:Hepes) (v / v / v=9:1:0.1) containing the fluorescent probe CPP stock solution (30 μL) to each cuvette. Add 50 μL of aluminum ion stock solution to one of the cuvettes and perform UV spectroscopy testing.

[0049] like Figure 1 As shown in the figure, the fluorescent probe CPP itself has almost no ultraviolet absorption at wavelength λ = 400nm. 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 green, indicating that the fluorescent probe CPP has a strong affinity for Al 3+ It has high sensitivity and selectivity, among which, Figure 1 The solution in the left test tube in the upper right corner is colorless, and the solution in the right test tube is light green.

[0050] like Figure 2 Figure 2 shows the selective fluorescence spectra of the fluorescent probe CPP for detecting aluminum ions for various common metal ions. Adding 30 μL of the fluorescent probe CPP stock solution and 50 μL of the metal ion stock solution to 3 mL of a blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) reveals that only with the addition of aluminum ions does the fluorescence intensity at 500 nm increase significantly. Furthermore, the fluorescence intensity is significantly higher than when other metal ions are added, demonstrating the excellent selectivity of the fluorescent probe for aluminum ions.

[0051] like Figure 3 As shown in the figure, the fluorescent probe CPP for detecting aluminum ions reacts with different concentrations of aluminum ions (Al3+ ) is a fluorescence spectrum response diagram. 30 μL of the fluorescent probe CPP stock solution and 0 to 150 μL (0, 10, 20 ... 150 μL) of aluminum ion solution (3 mM aluminum ion stock solution) were added to 3 mL of a blank solution (MeOH:H2O:Hepes) (v / v / v=9:1:0.1). The fluorescent probe CPP itself has almost no fluorescence in the solution, but as the aluminum ion concentration increases, the fluorescence at 500 nm also increases with the increase in aluminum ion concentration. That is, the fluorescence intensity CPP increases with the increase in aluminum ion concentration, and is accompanied by a significant color change. This indicates that the fluorescent probe CPP prepared in Example 1 of the present invention, after complexing with aluminum ions, inhibits the rotation of the C=N double bond, thereby inhibiting electron transfer, indicating that the fluorescent probe CPP is a fluorescence-enhancing probe.

[0052] like Figure 4 As shown in the figure, the fluorescence intensity bar graph of the fluorescent probe CPP for detecting aluminum ions 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 (Zn 2+ ,Co 2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Pd 2+ , Sn 2+ , Fe 2+ , K + , Sr 2+ , Ca 2+ , Sr 2+ , Na + , Ag + and Cd 2+ ) stock solution, and finally add 50 μL of Al 3+ The results showed that, except for diamagnetic cobalt, nickel, and copper ions, other metal ions had no significant interference in 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.

[0053] 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) is prepared so that the total concentration of the aluminum ion detection fluorescent probe and aluminum ions is 50μM. By changing the concentration ratio of the two (the molar ratio of the aluminum ion detection fluorescent probe and aluminum ions is 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 500nm and the fluorescence intensity of the aluminum ion fluorescent probe at this concentration is obtained, and the ratio of ions to the total concentration is 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 with a ratio of 1:2 [CPP-Al 3+ ].

[0054] 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:H2O:Hepes) (v / v / v=9:1:0.1). 3+ The fluorescence intensity of the probe rapidly increased to the maximum value and reached a stable value within 5 minutes. In addition, the fluorescence intensity of the probe remained basically unchanged in the following 30 minutes, which shows that the probe has a good fluorescence effect on Al 3+ The detection is stable enough.

[0055] like Figure 7 As shown, 1 M HCl and 1 M NaOH were used to adjust the CPP stock solution (10 μL) and [CPP-Al 3+ ] stock solution (10 μL) in MeOH / H2O solution (1 mL), where [CPP-Al 3+ The stock solution was prepared by dissolving the probe and aluminum chloride in double distilled water to a 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 fluorescent probe CPP itself had almost no fluorescence intensity in the pH range of 2 to 12, but [CPP-Al 3+ The fluorescence intensity (500nm) of the system was significantly enhanced in the pH range of 4.0-7.0, and reached the maximum value at pH=7. 3+ ] Under strong acidic conditions (pH < 4.0), the fluorescence signal is relatively weak. The possible reason is that the complexing point of the fluorescent probe CPP is protonated and is not easy to complex with aluminum ions. 3+] Under strong alkaline conditions (pH>7.0), the fluorescence signal gradually decreased, which may be due to the formation of Al(OH)3 precipitation, which reduced the [CPP–Al 3+ ] concentration. Therefore, the most suitable pH range for CPP is 5-7. CPP has the ability to detect Al in biological environments. 3+ ability.

[0056] like Figure 8 As shown, filter paper was immersed in a stock solution of MeOH:H2O:Hepes (v / v / v = 9:1:0.1) containing a fluorescent probe (1 mM) 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 then immersed in solutions with 0 mM, 0.1 mM, and 1 mM aluminum ion concentrations for 30 minutes, then air-dried. The fluorescent test paper prepared with the fluorescent probe CPP solution and its color change when testing different aluminum ion concentrations were measured under ultraviolet light. This shows that the color of the probe CPP changes with the aluminum ion concentration and can quantitatively detect trace aluminum ions in the environment in a solid state.

[0057] Table 1 shows the Al content in real water samples obtained in Example 1. 3+ The recovery value

[0058]

[0059] As shown in Table 1, lake water (the lake water selected in this embodiment was taken from Haiyun Lake, Jiangsu University of Science and Technology) and mineral water (the mineral water selected in this embodiment was Yibao mineral water) were used instead of double distilled water to prepare Al3+ stock solutions of different concentrations. Then, the fluorescent probe CPP for detecting aluminum ions was tested on 4 μL and 6 μL of different concentrations of aluminum ions (Al 3+ ) fluorescence titration spectral response curve. 3+ The recovery values ​​(74.7%–96.3%) indicate that this newly designed probe CPP can be used as a quantitative probe for the detection of trace Al in real samples. 3+ sensor.

[0060] In summary, the present invention uses 6-hydroxypyridinium hydrazide and chromone-3-carboxaldehyde as fluorescent groups to prepare a fluorescent probe CPP with chromone-pyridinium hydrazide as the parent through condensation reaction. The probe is sensitive to Al in solution. 3+ Shows high sensitivity and high selectivity.

Claims

1. Application of a fluorescent probe CPP for detecting aluminum ions in detecting trace aluminum ions in wastewater, characterized in that: The aluminum ion fluorescent probe CPP uses chromone-3-carboxaldehyde and 6-hydroxypyridinium hydrazide as fluorescent groups, and its specific structure is shown in (I): (I) .

2. The use according to claim 1, characterized in that When the fluorescent probe CPP for detecting aluminum ions is used in detecting trace aluminum ions in wastewater, the detection pH of the fluorescent probe CPP is 5-7, and the lower detection limit of the fluorescent probe CPP is 271 nm.

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