Fluorescent probe for detecting aluminum ions in pure water and cells and preparation method thereof

Abstract

The invention discloses a fluorescent probe for detecting aluminum ions in pure water and cells and a preparation method of the fluorescent probe, and belongs to the technical field of fluorescent probes, the structural formula of the fluorescent probe is # imgabs0 #, the fluorescent probe for detecting aluminum ions in pure water and cells and the preparation method of the fluorescent probe are adopted, and a synthesized probe molecule L has good AIE characteristics and can be used for detecting aluminum ions in pure water and cells. The probe molecule L can rapidly identify and detect aluminum ions in a mixed solution of DMF and water, has high selectivity and good anti-interference performance, successfully realizes conversion of detection of Al < 3 + > from a semi-aqueous phase to a pure water phase under the assistance of SDS, can specifically identify Al < 3 + > in pure water, and can be used for detection of tap water samples and Al < 3 + > in cells.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly to a fluorescent probe for detecting aluminum ions in pure water and cells and a preparation method thereof. Background Art

[0002] In recent years, the recognition of metal ions has gradually become an important branch of supramolecular chemistry, and the specific recognition and detection of metal ions are involved in various fields of production and life. Aluminum is the most abundant metal element on the earth. In daily life, as a common metal, aluminum is widely used by people and occupies an important position in both industry and agriculture. However, excessive aluminum ions will not only cause environmental pollution, but also harm human health and plant growth. Among them, the hygienic standard for domestic drinking water stipulates that the maximum allowable content of aluminum is 0.20 mg / L. Excessive intake of aluminum will damage the brain, heart, liver function and immune function of the human body, and induce Alzheimer's disease; after being absorbed by the digestive system, aluminum is toxic to the activity of osteoblasts and inhibits the synthesis of bone matrix, resulting in an increase in urinary calcium excretion and a decrease in calcium in the human body; aluminum binds to transferrin, causing interference in the distribution and metabolism of iron in plasma, resulting in non-iron-deficiency anemia. In addition, excessive aluminum intake can also cause various diseases such as renal failure and reduced gastric juice secretion.

[0003] The detection methods of metal ions have experienced a development process from traditional analytical methods to instrumental analytical methods, and from single detection means to the combination of multiple technologies. Currently, the commonly used analytical detection methods include complexometric titration, spectrophotometry, chemiluminescence analysis, electrochemistry analysis, chromatography, atomic spectrometry, and mass spectrometry, etc. Among them, complexometric titration and spectrophotometry are simple to operate and the equipment is economical, but the error is relatively large. Chemiluminescence analysis and electrochemistry analysis have high sensitivity, a wide linear range, and poor selectivity. Chromatography technology is relatively advanced and the conventional indicators are perfect, but the instrument maintenance cost is high and the detection time is long. Compared with the above several analytical methods, spectral analysis methods are rich in variety and excellent in various performances, and are commonly used for the determination of trace and ultra-trace metal elements.

[0004] Currently, the commonly used spectral analysis methods can be further divided into atomic spectrometry and molecular spectrometry. Among them, molecular spectrometry includes ultraviolet-visible spectrophotometry, infrared spectrometry, molecular fluorescence spectrometry, and molecular phosphorescence spectrometry, etc. Fluorescence spectrometry has been widely developed and utilized due to its excellent sensitivity and fast response speed, and has gradually become an important detection method in the fields of analytical chemistry, medicine, biochemistry, environmental chemistry, etc. Fluorescent probes can interact with specific target analytes, causing changes in fluorescence signals to achieve the purpose of detecting specific target analytes. Therefore, they have received extensive attention from the scientific community in recent years. Fluorescent probes can be applied to various fields such as environmental protection and intracellular fluorescence imaging.

[0005] Most fluorescent probes can only be applied in organic phases or semi-aqueous phases due to the aggregation-caused quenching (ACQ) effect. To overcome this situation, in recent years, people have begun to attempt to synthesize new fluorescent probes with AIE characteristics and apply them to the detection of ions in pure water. However, most of these probes are fluorescent probes designed based on tetraphenylethylene derivatives. These molecules not only have complex synthesis routes but also relatively expensive raw materials, which greatly increase the requirements and difficulties for production technology and processes during actual application, and also increase the production cost.

[0006] Schiff base compounds have a simpler synthesis route, more readily available reaction materials, and better application prospects, so they have received increasing attention. However, like most organic small molecule fluorescent probes, due to solubility problems, hydrolysis, and the existence of the ACQ effect, such probes cannot be applied in pure water phases. There are even fewer fluorescent probes based on Schiff base structures that can detect Al 3+ in water. It can be said that this work is somewhat difficult and is also an opportunity and challenge in the scientific research field. Summary of the Invention

[0007] The object of the present invention is to provide a fluorescent probe for detecting aluminum ions in pure water and cells and its preparation method. The synthesized probe molecule L has good AIE characteristics. The probe molecule L can quickly recognize and detect aluminum ions in a mixed solution of DMF and water, and has high selectivity and good anti-interference ability. With the assistance of SDS, the detection of Al 3+ has been successfully realized from the semi-aqueous phase to the pure water phase, and can specifically recognize Al 3+ in pure water, and can also be used for the detection of Al 3+ in cells.

[0008] To achieve the above object, the present invention provides a fluorescent probe for detecting aluminum ions in pure water and cells. The structural formula of the fluorescent probe is:

[0009]

[0010] The structural characterization is:

[0011] 1HNMR (500 MHz, DMSO) δ (ppm) 13.48 (s, 1H), 9.06 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.86 (dd, J = 8.6, 2.5 Hz, 1H), 7.38 (s, 4H), 7.11 (d, J = 8.6 Hz, 1H), 2.98–2.91 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13 CNMR (500 MHz, DMSO) δ (ppm) 162.93, 161.38, 148.19, 145.97, 144.16, 133.39, 132.08, 131.56, 129.48, 127.86, 127.19, 121.79, 120.13, 117.99, 109.79, 33.56, 24.34。

[0012] The present invention provides a preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells, comprising the following steps:

[0013] Step 1: Take cyanobiphenol, hexamine, and trifluoroacetic acid in a round-bottom flask, heat and reflux in an oil bath, then perform multiple extractions with dichloromethane and hydrochloric acid. After extraction and layering, wash with water two to four times, and then wash with saturated brine;

[0014] Step 2: Dry the organic layer with sodium sulfate, filter, perform rotary evaporation, and dry. Then add 4-isopropylaniline and ethanol as solvents, reflux in an oil bath, and then perform rotary evaporation, recrystallization, and suction filtration;

[0015] Step 3: Perform structural characterization on the product.

[0016] Preferably, the dosages of the raw materials used in Step 1 are: 0.2 - 1 g of cyanobiphenol, 1 - 4 g of hexamine, and 30 - 50 mL of trichloroacetic acid.

[0017] Preferably, the temperature of the oil bath in Step 1 is 75 - 90 °C, and the reflux time is 4 - 8 hours.

[0018] Preferably, the dosage of dichloromethane in Step 1 is 100 - 200 mL, the dosage of hydrochloric acid is 80 - 150 mL, and the concentration is 1 mol / L.

[0019] Preferably, the dosages of the solvents used in Step 2 are: 0.1 - 0.4 g of 4-isopropylaniline and 80 - 150 mL of ethanol.

[0020] Preferably, the temperature of the oil bath in Step 2 is 70 - 95 °C, and the reflux time is 6 - 10 hours.

[0021] Therefore, the present invention adopts the above-mentioned fluorescent probe for detecting aluminum ions in pure water and cells and its preparation method, and has the following beneficial effects:

[0022] (1) The synthesized fluorescent probe molecule L has good AIE properties and can emit orange-yellow fluorescence in the aggregated state.

[0023] (2) The fluorescent probe molecule L can rapidly recognize and detect aluminum ions in a mixed solution of DMF and water, with high selectivity and good anti-interference ability. Its ultraviolet and fluorescence spectra have obvious changes. At the same time, along with the spectral changes, under the irradiation of an ultraviolet lamp, the fluorescence color of its solution changes from orange-yellow to green, and this phenomenon can achieve the effect of naked-eye recognition.

[0024] (3) With the assistance of SDS, the detection of aluminum ions can be successfully realized from the semi-aqueous phase to the pure water phase, and aluminum ions can be specifically recognized in pure water.

[0025] (4) By means of mass spectrometry, nuclear magnetic resonance, fluorescence spectroscopy, etc., the mechanism of the probe molecule's recognition of aluminum ions was explored. The research shows that the probe molecule L first undergoes a 2:1 chelation reaction with Al 3+ and then the C=N double bond in its molecular structure breaks, generating the reactant molecule 1 with AIE effect, thereby releasing the green fluorescence of the AIE of molecule 1.

[0026] (5) It can be used for the detection of Al 3+ in cells.

[0027] Next, through the attached drawings and examples, the technical solutions of the present invention will be further described in detail. Description of the Drawings

[0028] Figure 1 is the ultraviolet absorption spectrum and fluorescence spectrum of the probe molecule L in the embodiment of the fluorescent probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention in a mixed solvent of different ratios of THF / water;

[0029] Figure 2 is the fluorescence spectrum of molecule 1 in the mixed system of different ratios of THF / water in the embodiment of the fluorescent probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention;

[0030] Figure 3 is the fluorescence spectrum of the probe molecule L in the mixed solution of DMF / water added with 10-fold amounts of different metal ions in the embodiment of the fluorescent probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention and the fluorescence intensity diagram of the probe molecule L at 506 nm;

[0031] Figure 4 Figure of fluorescence spectra of probe molecule L in a mixed solution of SDS and water with 10-fold amounts of different metal ions added, and fluorescence intensity plot of probe molecule L at 506 nm, which are examples of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention;

[0032] Figure 5 Figure of fluorescence emission spectra of probe molecule L (50 μM) of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention in DMF / water (2:8, v:v) solvent with gradual addition of Al 3+ (0 - 2 equiv);

[0033] Figure 6 Figure of fluorescence emission spectra of probe molecule L (50 μM) of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention in DMF / water solvent with gradual addition of Al 3+ (0 - 2 equiv) and the linear relationship between the ratio of (I0 - I) / I0 at 552 nm and the concentration of Al 3+ ;

[0034] Figure 7 Figure of Job’s plot of probe molecule L of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention in DMF / water mixed solvent (f w = 80%);

[0035] Figure 8 Mass spectra of probe molecule L of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention in organic solvent methanol, and mass spectra of probe molecule L in organic solvent methanol with 2-fold amount of Al 3+ added;

[0036] Figure 9 1H NMR spectra of probe molecule L, probe molecule L + Al 3+ and probe molecule 1 of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention;

[0037] Figure 10 Mechanism diagram of the reaction between probe molecule L and Al 3+ of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention;

[0038] Figure 11 Effect of different concentrations of probe molecule L on the proliferation of HeLa cells studied by MTT assay of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method of the present invention;

[0039] Figure 12 It is the fluorescence imaging of aluminum ions in HeLa cells by the probe molecule L of an embodiment of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method according to the present invention;

[0040] Figure 13 It is the 1H NMR spectrum of the probe molecule L of an embodiment of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method according to the present invention;

[0041] Figure 14 It is the 13C NMR spectrum of the probe molecule L of an embodiment of a fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method according to the present invention. Detailed implementation manners

[0042] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0043] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings as understood by those of ordinary skill in the field to which the present invention belongs.

[0044] Example 1

[0045] As Figure 1 shown in Figure 2 the present invention provides a fluorescence probe for detecting aluminum ions in pure water and cells, and the structural formula of the fluorescence probe is:

[0046]

[0047] The structural characterization is:

[0048] 1 1H NMR (500 MHz, DMSO) δ (ppm) 13.48 (s, 1H), 9.06 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.86 (dd, J = 8.6, 2.5 Hz, 1H), 7.38 (s, 4H), 7.11 (d, J = 8.6 Hz, 1H), 2.98–2.91 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13 13C NMR (500 MHz, DMSO) δ (ppm) 162.93, 161.38, 148.19, 145.97, 144.16, 133.39, 132.08, 131.56, 129.48, 127.86, 127.19, 121.79, 120.13, 117.99, 109.79, 33.56, 24.34.

[0049] The present invention also provides a preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells, comprising the following steps:

[0050] Step 1: Take 0.5 g of cyanobiphenyl, 2.15 g of hexamethylenetetramine and 40 mL of trifluoroacetic acid in a round-bottom flask, heat and reflux in an oil bath at 79 °C for 5 hours, then extract with 130 mL of dichloromethane and 100 mL of 1 mol / L hydrochloric acid for multiple times. After extraction and stratification, wash with water two to four times, and then wash with saturated brine;

[0051] Step 2: Dry the organic layer with sodium sulfate, then add 0.21 g of 4-isopropylaniline and 100 mL of ethanol as a solvent, reflux in an oil bath at 88 °C for 8.5 hours, and then perform rotary evaporation, recrystallization and suction filtration;

[0052] Step 3: Characterize the structure of the product.

[0053] The spectral test conditions and methods of the prepared fluorescent probe include:

[0054] 1. Detection of AIE properties: Prepare samples of the probe molecule L with different ratios of THF / water solvents, and make the final concentration of the probe molecule L in the whole system 50 μM. Test the luminescence properties of this molecule by a fluorescence spectrometer and an ultraviolet-visible absorption spectrometer.

[0055] The probe molecule L is easily soluble in organic solvents such as THF and insoluble in water. Therefore, by dispersing the probe molecule L in a mixed system of different ratios of THF / water, the aggregation-induced fluorescence enhancement performance of the probe molecule L, that is, the AIE performance, is studied. Figure 1 In (a), the ultraviolet absorption spectra of the probe molecule L in mixed solutions of different ratios of THF / water are shown. Figure 1 In (b), the fluorescence spectra of the probe molecule L in mixed solutions of different ratios of THF / water are shown.

[0056] Figure 1 As can be observed in (a), when the proportion of water in the mixed solution is greater than 70%, the absorption peak of the system increases and an obvious tailing phenomenon appears in the long-wave region, indicating that the probe molecule L aggregates at this time. Compared with the absorption spectrum in the dilute THF solution, the ultraviolet absorption peak of the aggregated state shows a slight red shift phenomenon.

[0057] Figure 1(b) shows that when the molecule is in pure THF, the probe molecule L has basically no fluorescence, but when the water content exceeds 70%, the fluorescence intensity of the probe molecule L at 546nm suddenly increases, and as the water content increases, the peak continues to increase until the fluorescence reaches the strongest when the water content reaches 95%. Compared with the fluorescence intensity of the probe molecule L in pure THF, the fluorescence intensity of the probe molecule L in a mixed solution with a water content of 95% increases by 11 times, because when the water content is small, the CC single bond and CN single bond on the phenyl group of the probe molecule L can rotate freely, resulting in non-radiative decay and weak fluorescence emitted by the molecule. However, in the aggregated state, due to the formation of intramolecular hydrogen bonds (N...HO-), the rigid structure of the molecule increases, thereby limiting the internal rotation of the molecule, and ultimately enhancing the fluorescence of the system. This result shows that the probe molecule L is likely to have good AIE properties.

[0058] Some Schiff base derivatives emit strong fluorescence in water, and people sometimes mistakenly believe that this is due to the AIE phenomenon caused by the aggregation of these compounds in water. However, in fact, it is because these compounds will hydrolyze into corresponding aldehydes and amines in water, and the aldehydes have AIE properties, so they will emit fluorescence in water. In other words, the fluorescence of these salicylaldehyde Schiff base compounds in water is not due to their own luminescent properties, but a phenomenon caused by the aldehyde compounds with AIE produced after their hydrolysis.

[0059] In order to further investigate whether the probe molecule L really has AIE properties, whether it will hydrolyze in water, and whether the light it emits in water is caused by its own properties or is related to molecule 1, molecule 1 was dissolved in mixed solutions of THF / water in different proportions, and its luminescence properties were studied.

[0060] The structural formula of molecule 1 is:

[0061]

[0062] Figure 2 (a) is the fluorescence spectrum of the probe molecule 1 in a THF / water mixture system with a ratio of 0-70%, Figure 2 (b) is the fluorescence spectrum of molecule 1 in a THF / water mixture system with a ratio of 70-99%. Figure 2As shown, molecule 1 itself hardly emits fluorescence in pure THF. However, when the volume of water is between 30 - 80%, molecule 1 emits strong green fluorescence at 506 nm. By comparison, it is found that the fluorescence at this point is completely different from the orange-yellow fluorescence emitted by the product probe molecule L at 546 nm. Thus, it can be seen that the fluorescence emitted by probe molecule L has nothing to do with probe molecule 1 and should be caused by the aggregation-induced emission (AIE) property of the molecule itself. This further indicates that compound L has good AIE properties and is relatively stable in water without hydrolysis occurring.

[0063] 2. Ion selective detection: (1) Dissolve probe molecule L in DMF organic solvent, and then separately add chloride salts of seventeen common metal ions, namely Fe 3+ , Mn 2+ , Cu 2+ , Co 2+ , K + , Ba 2+ , Li + , Na + , Al 3+ , Ni + , Ca 2+ , Cr 2+ , Cd 2+ , Pb 2+ , Hg 2+ , Mg 2+ and Zn 2+ to the solution. Keep the final concentration of the molecule at 50 μM, the concentration of the ions at 500 μM, and the volume ratio of DMF to water at 2:8. Test the fluorescence emission spectrum and ultraviolet absorption spectrum of the solution in the presence of different metal ions using a fluorescence spectrometer and an ultraviolet-visible absorption spectrometer. (2) Dissolve probe molecule L in acetonitrile with a stock solution concentration of 5 mM. Add 10 mM SDS solution, and then separately add various metal ions in method (1). Keep the final concentration of the molecule at 50 μM and the concentration of the ions at 500 μM, and test its fluorescence emission spectrum and ultraviolet absorption spectrum.

[0064] 3. Anti-interference ability detection: (1) Add different ions (final concentration 500 μM) dropwise to a DMF / water (2:8, v:v) mixed solution containing 50 μM of probe molecule L and 500 μM of aluminum ions, and detect the changes in the fluorescence emission spectrum and ultraviolet absorption spectrum of the mixed solution containing various interfering ions. (2) Add different ions with the same concentration dropwise to a mixed solution of 50 μM of probe molecule L and 500 μM of aluminum ions in SDS. The concentration of SDS is 10 mM, and detect the fluorescence emission spectrum and ultraviolet absorption spectrum of the solution. By comparing the co-mixed ions with Al 3+By examining the effect on the fluorescence emission spectrum of this mixed solution, the interference of competing ions on the recognition of probe molecule L with Al can be investigated. 3+ Recognition interference.

[0065] Figure 3 (a) in Figure 6 is the fluorescence spectrum of probe molecule L in a mixed solution of DMF / water (f w = 80%, λ ex = 310 nm) after adding 10-fold amounts of different metal ions. Figure 3 (b) in Figure 6 is the fluorescence intensity graph of probe molecule L at 506 nm (black: different metal ions added to probe molecule L, white: Al 3+ and different metal ions added to probe molecule L, f w = 80%, λ ex = 310 nm).

[0066] As Figure 3 shown in (a) of Figure 6, the molecule itself emits strong fluorescence in a dilute solution of DMF / water due to the AIE effect, with a peak at 552 nm. However, when 10-fold amounts of aluminum ions are added, the intensity of this peak decreases, and a new emission peak appears at a wavelength of 506 nm. The addition of other cations does not cause such changes in the solution.

[0067] Along with this spectral change, the change in its fluorescence color can be easily observed by the naked eye. That is, after adding aluminum ions to the solution of probe molecule L, the fluorescence of the solution changes from orange-yellow to green, achieving the effect of naked-eye recognition for aluminum ions. This indicates that the molecule has good selectivity for aluminum ions.

[0068] Under the same conditions, 10-fold amounts of different types of metal cations are respectively added to the solution containing probe molecule L and aluminum ions. The fluorescence spectra are as Figure 3 shown in (b) of Figure 6. Except for Fe 3+ , other ions do not interfere with the detection of aluminum ions by probe molecule L.

[0069] (2) Probe molecule L is dissolved in acetonitrile with a mother liquor concentration of 5 mM. A 10 mM SDS solution is added, and then various metal ions in method (1) are respectively added dropwise, keeping the final concentration of the molecule at 50 μM and the concentration of the ions at 500 μM, and its fluorescence emission spectrum and ultraviolet absorption spectrum are tested.

[0070] To achieve the detection and recognition of aluminum ions in pure water, the surfactant SDS is introduced into the detection system to assist probe molecule L. By the same method, the selectivity and anti-interference ability of probe molecule L for the detection of aluminum ions in pure water are investigated. The results are as Figure 4 shown.Figure 4 (a) in it is the fluorescence spectrogram of adding 10-fold amounts of different metal ions to the probe molecule L in a mixed solution of DMF / water (f w = 80%, λ ex = 310 nm). Figure 4 (b) in it is the fluorescence intensity diagram of the probe molecule L at 506 nm (black: different metal ions added to the probe molecule L, white: Al added to the probe molecule L 3+ and different metal ions, f w = 80%, λ ex = 310 nm). From the results, we found that it is basically the same as the experimental phenomenon in the mixed solvent of DMF / water, that is, the probe molecule L still has good selectivity and anti-interference ability for the recognition of aluminum ions in the pure water phase.

[0071] 4. Al 3+ Titration experiment and determination of detection limit: In a mixed solution of DMF / water (2:8, v:v) containing 50 μM of the probe molecule L, a certain concentration of aluminum ion solution was gradually added, and its linear response range and response sensitivity were investigated by detecting the changes in its corresponding fluorescence spectrum and ultraviolet-visible absorption spectrum. The detection limit was calculated by the formula DL = 3σ / K, where σ is the standard deviation of the blank sample and K is the slope of the calibration line. The detection of aluminum ions can achieve quantitative analysis within a certain range (0 - 65 μM), and its detection limit is 0.049 μM.

[0072] As Figure 5 shown, the probe molecule L (50 μM) was dissolved in a mixed solvent of DMF / water (2:8, v:v). When the concentration of Al 3+ was in the range of 0 - 100 μM, with the addition of Al 3+ , the fluorescence intensity of the probe molecule L solution at 506 nm gradually increased, while the peak at 552 nm gradually decreased. When the addition amount of Al 3+ reached 70 μM, the fluorescence of the solution was the strongest. Subsequently, when the concentration of Al 3+ exceeded 70 μM, the fluorescence of the solution gradually decreased. Along with the change of the fluorescence spectrum, under the irradiation of a hand-held ultraviolet lamp, the color of the solution could be observed with the naked eye to gradually change from orange-yellow to green.

[0073] As Figure 6 shown, within a relatively wide range of Al 3+ concentration (0 - 65 μM), the (I0 - I) / I0 of the probe molecule L showed a good linear relationship with the concentration of aluminum ions. This indicates that the probe molecule L can achieve quantitative detection of Al 3+ within a relatively large range. In addition, according to the calculation, its detection of Al 3+The detection limit is 4.9×10 -8 M, which is significantly lower than the World Health Organization standard (7.41 μM), the acceptable level of aluminum ions in drinking water (1.85 μM), and the previously reported Al 3+ probe. The above research results indicate that the probe molecule L has high sensitivity for the detection of Al 3+ , indicating that this molecule can meet the needs of more detection conditions.

[0074] 5. Job’s plot curve drawing: The probe molecule L and Al 3+ were added to the mixed solution of DMF / water (1:9, v:v) in different ratios, keeping the total concentration of the two at 20 μM. The intensities of the fluorescence spectrum and the ultraviolet spectrum changed with the ratio. By plotting a graph, the ratio of the probe molecule L to Al 3+ was determined.

[0075] As Figure 7 shown, the binding stoichiometry between the probe molecule L and Al 3+ was studied through the Job’s plot curve. It can be seen from the figure that the coordination ratio between the probe molecule L and aluminum ions is 2:1. To further confirm the existence of this complex, mass spectrometry was used to study it. Figure 8 In (a) is the mass spectrum of the probe molecule L in the organic solvent methanol. Figure 8 In (b) is the mass spectrum of the probe molecule L in the organic solvent methanol after adding 2-fold amount of Al 3+ . However, no peak of the complex of the probe molecule L and Al Figure 8 was found in (b), but the peaks of molecule 1 and isopropylamine were found. After the probe molecule L binds to Al 3+ , other reactions may have occurred. 3 + As

[0076] shown, after adding an excess of Al Figure 9 (2-fold amount) to the probe molecule L, the proton peak (H 3+ ) of the imine (-HC=N-) at 9.06 ppm weakened, and the proton peak (H a ) of the hydroxyl group (-OH) at 13.48 ppm disappeared. At the same time, a proton peak (H b ) of the aldehyde group (-CHO) in the probe molecule 1 appeared at 10.32 ppm. In addition, two peaks at 1.24 ppm and 2.94 ppm (H c’ ), H f , H e) also belong to the methyl group (-CH3) and the methine group (-CH) in the structure of isopropyl aniline respectively. This indicates that aluminum ions do initiate the hydrolysis of the probe molecule L, causing the carbon-nitrogen double bond of L to break, generating molecule 1 and 4-isopropyl aniline. Therefore, the reaction mechanism between the probe molecule L and aluminum ions can be divided into two steps: the probe molecule L coordinates with aluminum ions in a 2:1 ratio, and then a decomposition reaction occurs, generating the probe molecule 1 and 4-isopropyl aniline, as Figure 10 shown.

[0077] To improve the portability of the probe molecule L for detecting aluminum ions, the fluorescent probe was made into a test paper, and the performance of the test paper for detecting aluminum ions was studied. First, the test paper loaded with the probe molecule L was immersed in SDS solutions containing different concentrations of aluminum ions respectively. After 10 minutes, it was irradiated under a 365 nm hand-held ultraviolet lamp, and it was found that the filter paper containing aluminum ions showed green fluorescence, and as the concentration of aluminum ions increased, the green fluorescence on the filter paper gradually enhanced. Therefore, by using this simple and low-cost material, the "naked-eye" detection of the aluminum ion content in aqueous solutions can be achieved conveniently and quickly.

[0078] Taking the tap water in the laboratory as the research sample, different concentrations of aluminum ions were added to it, and its fluorescence spectrum was monitored. The obtained data was brought into the previously measured aluminum ion titration curve, and it was found that the measured results were basically consistent with the actual concentration added to the tap water sample, the recovery rate was also good, and the standard deviation was very small. This experimental result shows that the probe molecule L has good practical application value in the detection of aluminum ions.

[0079] Table 1 Determination of aluminum ion content in tap water by the probe molecule L

[0080]

[0081] Note: λ ex = 310 nm, DMF / water = 2:8.

[0082] After that, the probe molecule L was applied to imaging, and the cytotoxicity experiment of the probe molecule L was studied. As Figure 11 shown, even when the concentration of the probe molecule L reaches 100 μM, the cell viability is still more than 90%. From the results, it can be seen that the probe molecule L has good biocompatibility and does not produce obvious toxicity to living cells in a long time, and this result also lays a foundation for the subsequent cell imaging experiment.

[0083] As Figure 12 shown, through fluorescence microscopy observation, it was found that the probe molecule L itself shows red fluorescence in HeLa cells. But in Al 3+It turns into green fluorescence when present. The above research results indicate that the probe molecule L can be applied to the field of intracellular Al 3+ monitoring.

[0084] Therefore, the present invention adopts the above-mentioned fluorescence probe for detecting aluminum ions in pure water and cells and its preparation method. The synthesized probe molecule L has good AIE properties. The probe molecule L can rapidly recognize and detect aluminum ions in a mixed solution of DMF and water, and has high selectivity and good anti-interference ability. With the assistance of SDS, the detection of Al 3+ has been successfully realized from the semi-aqueous phase to the pure water phase, and can specifically recognize Al 3+ in pure water, and can be used for the detection of tap water samples and intracellular Al 3+ detection.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A fluorescent probe for detecting aluminum ions in pure water and cells, characterized in that: The structural formula of the fluorescent probe is as follows: The structural characterization is as follows: 1 HNMR (500 MHz, DMSO) δ (ppm) 13.48 (s, 1H), 9.06 (s, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.86 (dd, J = 8.6, 2.5 Hz, 1H), 7.38 (s, 4H), 7.11 (d, J = 8.6 Hz, 1H), 2.98–2.91 (m, 1H), 1.23 (d, J = 6.9 Hz, 6H). 13 CNMR (500 MHz, DMSO) δ (ppm) 162.93, 161.38, 148.19, 145.97, 144.16, 133.39, 132.08, 131.56, 129.48, 127.86, 127.19, 121.79, 120.13, 117.99, 109.79, 33.56, 24.34。 2. A preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells as described in claim 1, characterized in that: It includes the following steps: Step 1: Take cyanobiphenylol, hexamine, and trifluoroacetic acid in a round-bottom flask, heat and reflux in an oil bath, then extract multiple times with dichloromethane and hydrochloric acid. After extraction and layering, wash with water two to four times, and then wash with saturated brine; Step 2: Dry the organic layer with sodium sulfate, filter, rotary evaporate, and dry. Then add 4-isopropylaniline and ethanol as solvents, reflux in an oil bath, and then perform rotary evaporation, recrystallization, and suction filtration; Step 3: Conduct structural characterization on the product.

3. The preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells according to claim 2, characterized in that: The dosages of the raw materials used in Step 1 are as follows: 0.2 - 1 g of cyanobiphenylol, 1 - 4 g of hexamine, and 30 - 50 mL of trichloroacetic acid.

4. The preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells according to claim 2, characterized in that: The temperature of the oil bath in Step 1 is 75 - 90 °C, and the reflux time is 4 - 8 hours.

5. The preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells according to claim 2, characterized in that: The dosage of dichloromethane in Step 1 is 100 - 200 mL, the dosage of hydrochloric acid is 80 - 150 mL, and the concentration is 1 mol / L.

6. The preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells according to claim 2, characterized in that: The dosages of the solvents used in Step 2 are as follows: 0.1 - 0.4 g of 4-isopropylaniline and 80 - 150 mL of ethanol.

7. The preparation method of a fluorescent probe for detecting aluminum ions in pure water and cells according to claim 2, characterized in that: The temperature of the oil bath in Step 2 is 70 - 95 °C, and the reflux time is 6 - 10 hours.

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