A fluorescence-enhanced aluminum ion detection fluorescent probe NMA and its preparation method and application

Abstract

The present invention discloses a fluorescence-enhanced aluminum ion detection fluorescent probe NMA and its preparation method and application. The aluminum ion detection fluorescent probe NMA uses chromone-3-carbaldehyde and benzohydrazide as fluorescent groups, and prepares a Schiff base-type fluorescent probe NMA through a simple condensation reaction. Its structure is shown in the following formula I. The synthesis steps of the aluminum ion detection fluorescent probe NMA of the present invention are simple, the raw materials are easily available, and it has strong selectivity for aluminum ions, a fast response time, obvious fluorescence enhancement after complexation, and a low detection limit. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a fluorescence-enhanced aluminum ion detection fluorescent probe NMA, a preparation method thereof, and an application thereof. Background Art

[0002] Aluminum is the most abundant metallic element in the earth's crust and plays an important role in human life and industrial and agricultural production. In daily life, with the widespread use of aluminum cookware, aluminum-containing leavening agents, baking powder, and water purifying agents, people inevitably absorb aluminum and its compounds. However, Al is a non-essential element for the human body, and abnormal Al 3+ levels can endanger human health and cause some neurological diseases such as Alzheimer's disease. At the same time, the accumulation of high concentrations of Al 3+ in the soil can also inhibit the growth of animals and plants.\ Considering the widespread use of aluminum ions and their harm to human health and the environment, it is imperative to develop efficient Al 3+ probes. [[ID=1st]]

[0003] Currently, many traditional analytical techniques, such as ion chromatography (IC), atomic absorption spectrometry (AAS), and inductively coupled plasma mass spectrometry (ICP-MS), are the most commonly used analytical techniques for determining Al 3+ and other metal ions. Although these techniques can usually provide accurate results, for the determination process, expensive large-scale instruments, professional technicians, and complex sample processing procedures are required. Fluorescent molecular probes designed based on organic small molecules as the matrix have the advantages of simple operation, high sensitivity, strong selectivity, short response time, etc. in the detection of metal ions, and have become a research hotspot in the fields of environment and life sciences.

[0004] Schiff base derivatives mainly refer to a class of organic compounds containing imine or methylimine characteristic groups (-RC=N-), usually formed by the condensation of amines and active carbonyl compounds. Schiff base-type fluorescent probes contain the C=N functional group. The lone pair of electrons on the nitrogen atom in the C=N group not only provides a complexation site for metal ion complexation, but also once complexed, the rotation of C=N is inhibited, and the fluorescence will be significantly enhanced, enabling visual detection. It is a good matrix for the design of organic small molecule fluorescent probes. Al 3+ is a strong Lewis acid and tends to complex with hard atoms N or O. Schiff base-type fluorescent probes have rich N,O complexation sites, which can provide a hard base environment for Al 3+ provide a hard base environment. Summary of the Invention

[0005] Objective of the Invention: Aiming at the problems existing in the prior art, the present invention provides a fluorescence-enhanced aluminum ion detection fluorescent probe NMA. This fluorescent probe NMA has high selectivity for aluminum ions, a low detection limit, and a short response time, and can quickly and real-time detect trace amounts of aluminum ions in the environmental system on-site.

[0006] The present invention also provides a preparation method, theoretical calculation, and multiple applications of the aluminum ion detection fluorescent probe NMA.

[0007] Technical Solution: To achieve the above-mentioned objective of the invention, the present invention provides a fluorescence-enhanced aluminum ion detection fluorescent probe NMA. The aluminum ion detection fluorescent probe NMA uses chromone-3-carbaldehyde and benzohydrazide as fluorescent groups, and its structure is shown in the following formula I:

[0008]

[0009] The preparation method of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA of the present invention includes the following steps:

[0010] (1) Using benzoic acid and methanol as raw materials, reacting to obtain methyl benzoate; then reacting with hydrazine hydrate to obtain benzohydrazide;

[0011] (2) Using benzohydrazide and chromone-3-carbaldehyde as raw materials, obtaining the fluorescence-enhanced aluminum ion detection fluorescent probe NMA through a Schiff base condensation reaction.

[0012] Among them, the reaction route for the preparation of the aluminum ion detection fluorescent probe NMA is as follows:

[0013]

[0014] In the said reaction route: the intermediate is chromone-3-carbaldehyde (A); benzohydrazide (B); (E)-N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide (NMA) is the fluorescent probe molecule for detecting aluminum ions of the present invention.

[0015] Among them, in step (1), benzoic acid is dissolved in the organic solvent methanol, then concentrated sulfuric acid is added dropwise as a catalyst, heated under reflux and stirred. After the reaction is complete, the reaction solution is cooled to room temperature, the pH is adjusted to neutral, extracted with ethyl acetate, separated, dried, and after removing the solvent, methyl benzoate is obtained through column chromatography.

[0016] Among them, in step (1), the intermediate methyl benzoate is dissolved in the organic solvent methanol, then hydrazine hydrate is added, heated under reflux and stirred. After the reaction is complete, the reaction solution is cooled to room temperature, the organic solvent is removed by vacuum distillation, extracted, separated, dried, and after removing the solvent, benzohydrazide is obtained through column chromatography.

[0017] Among them, in step (2), chromone-3-carbaldehyde and benzoyl hydrazine are dissolved in the organic solvent methanol, the mixed material is refluxed and stirred, 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 the fluorescent probe NMA.

[0018] Application of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA of the present invention in the detection of aluminum ions.

[0019] Among them, the fluorescence-enhanced aluminum ion detection fluorescent probe NMA complexes with aluminum ions through a single electron transfer and C=N double bond free rotation reaction mechanism, and the fluorescence is significantly enhanced after complexation in the application of aluminum ion detection.

[0020] Application of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA of the present invention in the real-time and rapid on-site detection of trace aluminum ions in solutions and cells.

[0021] Application of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA of the present invention prepared into a fluorescent test paper to qualitatively and quantitatively detect aluminum ions in a solution in a solid state.

[0022] Preferably, the preparation process of NMA includes:

[0023] (1) Preparation of benzoyl hydrazine (B)

[0024] Benzoic acid is dissolved in the organic solvent methanol, then a small amount of concentrated sulfuric acid is added dropwise as a catalyst, heated under reflux and stirred, the reaction progress is detected by TLC, after the reaction is complete, the reaction solution is cooled to room temperature, the pH of the reaction system is adjusted to neutral with diluted sodium hydroxide aqueous solution, extracted with ethyl acetate, separated, dried, and the solvent is removed to obtain the crude product methyl benzoate. Then the crude product methyl benzoate is dissolved in the organic solvent methanol, then hydrazine hydrate is added, heated under reflux and stirred, after the reaction is complete, the reaction solution is cooled to room temperature, the organic solvent is removed by distillation under reduced pressure, extracted, separated, dried, and the solvent is removed, and the final target raw material benzoyl hydrazine (B) is obtained by column chromatography (ethyl acetate: petroleum ether = 1:3).

[0025] (2) Preparation of the aluminum ion fluorescent probe NMA based on chromone-3-carbaldehyde as the parent

[0026] Chromone-3-carbaldehyde (A) and benzoyl hydrazine (B) are dissolved in the organic solvent methanol, the mixed material is refluxed and stirred, after the reaction is completed, the reaction material is cooled to room temperature, the solvent is removed by distillation under reduced pressure, the crude product is purified by recrystallization, filtered, and dried to obtain the white solid fluorescent probe NMA.

[0027] Preferably, the synthesis route is shown in the following formula:

[0028]

[0029] The present invention designs a novel Schiff base fluorescent probe NMA based on chromone-3-carbaldehyde. Organic small molecule fluorescent probes have become a research hotspot for the qualitative and quantitative detection of metal ions in environmental and life sciences due to their simple synthesis steps, convenient operation, low cost, low cytotoxicity, strong tissue penetration, and no sample damage. Chromone-3-carbaldehyde contains aldehyde group, carbonyl group and C=C double bond in its molecule, and is prone to various chemical reactions such as addition and substitution, and can be used to synthesize a variety of bioactive compounds, such as anti-cancer, anti-inflammatory, antioxidant, anti-hypertensive, antiviral drugs, and has received extensive attention from scientific researchers. Benzoylhydrazide derivatives have a rigid planar structure and a large π-bond conjugated system, and at the same time have the advantages of low cytotoxicity and modifiable structure, and are good chromophores. Compared with the existing probes (WYW) based on purine as the parent and the probes (NPP, NBP, NIQ) based on 1,8-naphthalimide as the parent, this probe has a short preparation route, high yield, low raw material cost, and has unique sensitivity and low detection limit for Al 3+ Moreover, the synthesis raw materials are simpler, the solubility is better, which is more suitable for the comprehensive experiments of undergraduates and meets the development of multiple disciplines. Secondly, due to its low toxicity and broad pH application range, the fluorescence enhancement type fluorescent probe NMA has been successfully used to detect trace aluminum ions in living HeLa cells.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0031] The present invention prepares a fluorescent probe NMA with chromone-3-carbaldehyde as the parent by one-step condensation of chromone-3-carbaldehyde and benzoylhydrazide. The synthesized molecule is simple, has good solubility, and has little environmental pollution. At the same time, the prepared NMA has specific recognition for aluminum ions, a wide pH application range, high sensitivity, a low detection limit (30 nM), and a fast response time. At the same time, the prepared NMA can quickly qualitatively analyze aluminum ions in actual water samples through obvious color changes. More importantly, the prepared NMA has low cytotoxicity and strong cell penetration ability, and has been successfully used to detect trace aluminum ions in living HeLa cells; through DFT theoretical calculation, the complexation mode of the prepared NMA with Al 3+ is obtained. Description of the Drawings

[0032] Figure 1 The ultraviolet absorption spectra and color change diagrams ( 3+ ) of the aluminum ion fluorescent probe NMA prepared in Example 1 in a (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different concentrations of aluminum ions (Al Figure 1 The left side in the illustration is colorless and the right side is light yellow);

[0033] Figure 2 Selective fluorescence spectra of the aluminum ion fluorescent probe NMA prepared in Example 1 in a (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different metal ions;

[0034] Figure 3 Fluorescence spectral response and color change diagrams of the fluorescent probe NMA prepared in Example 1 in a (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different concentrations of aluminum ions (Al 3+ ), changing from colorless to bright green;

[0035] Figure 4 Fluorescent response diagrams for the detection of selective interference of different metal ions by the fluorescent probe NMA prepared in Example 1 in a (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution;

[0036] Figure 5 Job-plot curve of the complexation ratio of the fluorescent probe NMA prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) with aluminum ions (Al 3+ );

[0037] Figure 6 Time response diagram of the fluorescent probe NMA prepared in Example 1 for detecting aluminum ions;

[0038] Figure 7 Fluorescent response diagrams of the fluorescent probe NMA prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) with aluminum ions (Al 3+ ) in the range of different pH values (2 to 12);

[0039] Figure 8 Optimal configuration diagrams of the fluorescent probe prepared in Example 1 and the fluorescent probe NMA after complexation with aluminum ions;

[0040] Figure 9 Color change diagrams of the fluorescent test paper prepared with the fluorescent probe NMA prepared in Example 1 for testing different concentrations of aluminum ions;

[0041] Figure 10 Toxicity test diagram of the fluorescent probe NMA prepared in Example 1 in biological cells;

[0042] Figure 11 Cell imaging diagram of the fluorescent probe NMA prepared in Example 1 in biological cells;

[0043] Figure 121H NMR spectrum of the fluorescent probe NMA prepared in Example 1 1 1H NMR spectrum;

[0044] Figure 13 13C NMR spectrum of the fluorescent probe prepared in Example 1 13 13C NMR spectrum;

[0045] Figure 14 Ms spectrum of the fluorescent probe prepared in Example 1

[0046] Figure 15 Infrared IR spectrum of the fluorescent probe prepared in Example 1 Detailed implementation mode

[0047] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0048] The experimental methods used in the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the experiments can be obtained from commercial channels unless otherwise specified. All the following reagents selected in the examples are commercially available analytical pure or chemical pure.

[0049] Among them, the various metal ion solutions in the examples are prepared by adding deionized water to chloride chemical reagents with a purity of more than 99% such as anhydrous zinc chloride, anhydrous iron chloride, anhydrous aluminum chloride, etc. or nitrate chemical reagents such as Pb(NO3)2.

[0050] Example 1

[0051] Specific synthesis route of the fluorescent probe NMA based on chromone-3-carbaldehyde as the parent body:

[0052] (1) Preparation of intermediate benzohydrazide (B)

[0053] Dissolve benzoic acid (1.22 g, 10 mmol) in methanol solvent (50 mL), stir and dissolve at room temperature, add commercially available concentrated sulfuric acid (98%) (1 mL) as a catalyst, and then heat the reaction system to 70 °C and stir under reflux for 12 hours. Cool the reaction system to room temperature, remove the organic solvent by vacuum distillation to obtain the crude product methyl benzoate. Add hydrazine hydrate (0.1 g, 20 mmol) and organic solvent methanol (30 mL) to the round-bottom flask containing the crude product, and continue to heat to 70 °C and stir under reflux for 3 hours. After the reaction is completed, cool the reaction system to room temperature, remove the solvent by vacuum distillation, and obtain the target raw material benzohydrazide (1.09 g, 80%) by column chromatography (ethyl acetate: petroleum ether = 1:3).

[0054] The structural formula of the obtained intermediate B is:

[0055]

[0056] (3) Preparation of the aluminum ion fluorescent probe NMA based on chromone-3-carbaldehyde as the parent

[0057] In a 50 mL round-bottom flask containing 20 mL of the organic solvent methanol, chromone-3-carbaldehyde (A) (0.174 g, 1 mmol) and benzoyl hydrazide (B) (0.136 g, 1 mmol) were added respectively. After stirring and dissolving, the reaction system was heated to 80 °C and refluxed with stirring for 3 hours. The reaction was monitored by TLC. 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 (methanol) to obtain the white solid fluorescent probe molecule NMA (0.248 g, 85%).

[0058] The structural formula of the obtained fluorescent probe NMA is as follows:

[0059]

[0060] Characterization data of this fluorescent probe 1 H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.84 - 8.83 (m, 1H), 8.65 - 8.64 (m, 1H), 8.14 - 8.11 (m, 1H), 7.95 - 7.92 (m, 2H), 7.86 (t, J = 8.1 Hz, 1H), 7.74 - 7.71 (m, 1H), 7.62 - 7.51 (m, 4H); 13 C NMR (100 MHz, DMSO-d6) δ 175.61, 163.50, 156.32, 155.06, 140.87, 135.23, 133.70, 132.40, 129.04, 128.20, 126.63, 125.75, 123.86, 119.28, 118.89. ESI-MS m / z: [M+H] + calc d for C 17 H 12 N2O3 292.08, found 292.1; IR (KBr cm -1 ): 3281, 3036, 1678, 1617, 1524, 1459, 767.

[0061] The hydrogen spectrum ( 1 H NMR), carbon spectrum ( 13 C NMR), mass spectrum MS, and infrared spectrum IR of the fluorescent probe prepared in Example 1 are as shown in Figure 12 , Figure 13 , Figure 14 , Figure 15 respectively, indicating that the fluorescent probe of the present invention was successfully prepared.

[0062] Example 2

[0063] The aluminum ion detection fluorescent probe NMA prepared in Example 1 was configured into a 1 mM probe stock solution with DMSO, and each metal ion was configured into a 3 mM metal ion stock solution with deionized water. 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:H2O:Hepes) (v / v / v = 9:1:0.1), and detection was carried out using a fluorescence spectrometer and an ultraviolet spectrophotometer. It was tested that the maximum excitation wavelength of the fluorescent probe was 418 nm and the maximum emission wavelength was 510 nm. The specific test results are as follows:

[0064] Take two cuvettes, add 3 mL of the blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) and 30 μL of the probe stock solution to each respectively. Add 50 μL of the aluminum ion stock solution to one of the cuvettes, and do not add the aluminum ion stock solution to the other cuvette, and perform ultraviolet spectrum testing. As Figure 1 shown, the fluorescent probe itself has almost no ultraviolet absorption at a wavelength of λ = 418 nm. 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. The results show that the probe has high sensitivity and selectivity for Al 3+ . The visible color change may be due to the formation of a new complex between the probe and Al 3+ .

[0065] As Figure 2 shown, the selective fluorescence spectrogram 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 were added to 3 mL of the blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1). The experimental results show that only when aluminum ions are added, the fluorescence intensity at 510 nm of the fluorescence spectrum significantly increases. Moreover, the fluorescence intensity is significantly better than that when other metal ions are added, indicating that the fluorescent probe of the present invention has good selectivity for aluminum ions.

[0066] As Figure 3 shown, the aluminum ion detection fluorescent probe for different concentrations of aluminum ions (Al 3+Fluorescence spectral response diagram of (). Add 30 μL of the probe stock solution and 0 - 150 μL (0, 5, 10... 50, 60... 150 μL, 3 mM) of aluminum ion stock solution to 3 mL of blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1). This fluorescent probe has almost no fluorescence in the solution itself, but as the concentration of aluminum ions increases, the fluorescence at 510 nm also continuously increases with the increase of aluminum ion concentration, that is, the fluorescence intensity increases with the increase of aluminum ion concentration, and is accompanied by an obvious color change. It shows that after the probe NMA prepared in Example 1 of the present invention complexes with aluminum ions, the rotation of the C=N double bond is inhibited, thereby inhibiting electron transfer, indicating that the probe NMA is a fluorescence-enhanced probe.

[0067] As Figure 4 shown, the bar chart of fluorescence intensity after the aluminum ion detection fluorescent probe reacts with aluminum ions in the presence of different interfering metal ions. Add 30 μL of the probe stock solution and 50 μL of any one of the other metal ions (Pb 2+ , Co 2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Zn 2+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Sn 2+ , Fe 3+ , K + , Sr 2+ , Ca 2+ , Sr 2+ , Na + , Ag + and Cd 2+ ) stock solution, and finally add 50 μL of Al 3+ stock solution to the blank solution, and test its fluorescence intensity. The results show that other metal ions have no obvious 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 specificity.

[0068] As Figure 5 shown, the binding mode of the probe to Al 3+ was studied by the Job's plot method. Add a certain volume of the probe stock solution (1 mM) and Al 3+ to 3 mL of blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1)Stock solution (3 mM) was used to make the total concentration of the aluminum ion detection fluorescent probe and aluminum ions 50 μM. By changing the concentration ratio of the two (the molar ratio of the aluminum ion detection fluorescent probe to aluminum ions was 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 in turn), the difference between the fluorescence intensity at 510 nm and the self-fluorescence intensity of the aluminum ion fluorescent probe at this concentration was obtained, and a graph was plotted with the proportion of the ion in the total concentration. From Figure 5 It can be seen that when the proportion of aluminum ions is 0.5, the ordinate reaches the highest value, and it can be determined that the fluorescent probe and aluminum ions form a stable complex [NMA-Al 3+ in a 1:1 ratio.

[0069] As Figure 6 shown, 30 μL of the probe stock solution and 50 μL of the Al 3+ stock solution were added to 3 mL of blank buffer (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1). The fluorescence intensity of the probe gradually increased to the highest and reached a stable value within 10 min. In addition, the fluorescence intensity of the probe remained basically unchanged within the subsequent 60 min, indicating that the probe is stable enough for the detection of Al 3+ and has a fast response time.

[0070] As Figure 7 shown, 1M HCl and 1M NaOH were used to adjust the MeOH / H2O buffer solution (1 mL) containing the probe NMA stock solution (10 μL) and the [NMA-Al 3+ stock solution (10 μL) respectively. The [NMA-Al 3+ stock solution was prepared by dissolving the probe and aluminum chloride in secondary distilled water to concentrations of 10 uM and 50 uM respectively, so that it had different pH change values. Then, the fluorescence intensities of these two systems were tested in different pH value ranges. The probe NMA itself had almost no fluorescence intensity in the pH range from 2 to 12. However, the fluorescence (510 nm) intensity of the [NMA-Al 3+ complex system increased significantly in the pH range of 2.0 - 7.0, and the fluorescence intensity reached the maximum at pH = 7. The [NMA-Al 3+ complex system had a relatively weak fluorescence signal under strong acidic conditions (pH < 3.0). The possible reason is that the complexation site of the fluorescent probe NMA was protonated and it was not easy to complex with aluminum ions. The [NMA-Al 3+ complex system had a weakened fluorescence signal under alkaline conditions (pH > 8.0). Maybe under alkaline conditions, Al 3+Combined with hydroxide ions to form Al(OH)3, reducing the concentration of aluminum ions in the system, resulting in weakened or even disappeared fluorescence. Therefore, the most suitable pH range for NMA is 5.0 - 8.0, indicating that NMA has the ability to detect Al 3+ in the biological environment.

[0071] As Figure 8 shown, the optimal configuration diagrams of the fluorescent probe and the [NMA - Al 3+ complex and their corresponding orbital energy level diagrams. The DFT calculation results further prove that the probe NMA forms a complex with aluminum ions through -C=N, C=O, and C=O.

[0072] As Figure 9 shown, the filter paper was immersed in a stock solution of MeOH:H2O:Hepes (v / v / v = 9:1:0.1) containing the fluorescent probe NMA (1 mM) for half an hour, then the test strip was taken out and dried in the air to obtain a dried test strip containing the probe. The test strip was immersed in solutions with aluminum ion concentrations of 0 mM, 0.1 mM, and 3.0 mM for 30 minutes, then air-dried. Under ultraviolet light, the fluorescence test paper prepared with the fluorescent probe NMA solution and its color changes when testing different concentrations of aluminum ions were measured, indicating that the color of the probe NMA changes with the change of aluminum ion concentration, and it can quantitatively detect trace aluminum ions in the environment in a solid state.

[0073] As Figure 10 shown, the cytotoxicity study of the aluminum ion detection fluorescent probe NMA on HeLa cells was carried out by MTT experiment at different concentrations (0 - 10 μM). In a 96-well plate, first, a solution containing 10 μM of aluminum ions (100 μL / well) was inoculated, then a cell suspension containing 10 μL of NMA probe with concentrations of 0 - 15 μM (0, 2, 4, 6, 8, 15 μM) was inoculated into the wells, and finally, the culture plate was pre-cultured in an incubator (under the conditions of 37 °C and 5% CO2). Secondly, 10 μL of MTT solution was injected into each well, and the culture plate was incubated in the incubator for another 2 h, and the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay reader. The results of HeLa cell viability showed that after 24 hours, the survival rate of HeLa cells exceeded 90%. The results of this MTT experiment indicate that the aluminum ion probe of the present invention has low cytotoxicity in the experimental environment, indicating that the probe has potential application value in detecting aluminum ions in living cells.

[0074] As Figure 11 shown, the imaging diagrams of the fluorescent probe in HeLa cells before and after binding to aluminum ions. 1×10 5HeLa cells were seeded into 35-mm glass-bottom tissue culture dishes. When the cell density reached 60%, the DMEM medium was replaced with serum-free medium containing 8 μM aluminum chloride and incubated at 37 °C for 30 minutes. Then the cells were washed three times with PBS. Subsequently, fresh DMEM medium supplemented with NMA (8 μM) was added and the incubation was continued for 30 minutes. Then the obtained cells were washed three times with PBS. Then, cells cultured in DMEM medium containing NMA without aluminum chloride treatment (incubated for 30 min) were washed three times with PBS as a control. The results of laser confocal microscopy imaging experiments (excitation wavelength 418 nm) showed that a significant increase in the blue fluorescence of the cells could be observed in the cells treated with the NMA probe and Al 3+ , probably due to the formation of the NMA-Al 3+ complex. Therefore, the results of the cell imaging experiments indicated that the probe NMA could penetrate the cell membrane and could be effectively used for the imaging of trace Al 3+ in living cells, further demonstrating that the aluminum ion fluorescent probe NMA of the present invention could be applied to biological experiments.

[0075] The above experiments showed that a fluorescent probe NMA with chromone-3-carbaldehyde as the parent was prepared by a condensation reaction using chromone-3-carbaldehyde and benzoylhydrazine as fluorescent groups, which showed high sensitivity and high selectivity to Al 3+ in solution, and had low cytotoxicity and could be used for the detection of trace Al 3+ in living cells.

Claims

1. Use of a fluorescence-enhanced aluminum ion detection fluorescent probe NMA in the preparation of a reagent for detecting aluminum ions; the aluminum ion detection fluorescent probe NMA uses chromone-3-carbaldehyde and benzoylhydrazine as fluorescent groups, and its structure is shown in the following formula I: 。 2. Use of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA according to claim 1 in the preparation of a reagent for detecting trace aluminum ions in solutions and cells in real time and rapidly on-site.

3. Use of the fluorescence-enhanced aluminum ion detection fluorescent probe NMA according to claim 1 in the preparation of a fluorescent test paper for detecting aluminum ions in solutions in a solid state, qualitatively or quantitatively.

Images