A bifunctional fluorescent molecular probe, a preparation method thereof, and applications in detecting aluminum ions and / or zinc ions

Abstract

The present invention discloses a bifunctional fluorescent molecular probe, a preparation method thereof, and an application thereof in detecting aluminum ions and / or zinc ions, and relates to the technical field of aluminum ion and zinc ion detection. The bifunctional fluorescent molecular probe of the present invention is specifically prepared by reacting compound III with 2-furoyl hydrazide. Based on specific recognition, the bifunctional fluorescent molecular probe of the present invention can be used as a specific indicator for the presence of aluminum ions and zinc ions in living cells and zebrafish in vivo, and can provide real-time qualitative and quantitative information through fluorescence detection, with a color change, making it a highly specific indicator suitable for visual colorimetric detection; meanwhile, the probe has high reversibility and repeatability for the detection of aluminum ions and zinc ions, and can also be used as a sensor for detecting aluminum ions and zinc ions in living cells and zebrafish in vivo, and can be repeatedly detected without damaging its performance.

Description

Technical Field

[0001] The present invention relates to the technical field of aluminum ion and zinc ion detection, and particularly relates to a bifunctional fluorescent molecular probe, a preparation method thereof, and an application in detecting aluminum ions and zinc ions in living cells. Background Art

[0002] Aluminum ion is the most widely distributed form of aluminum element in nature, widely used in water bodies, soils, air, and most biological tissues, and widely applied in food packaging, water purification, pharmaceuticals, cosmetics, dye manufacturing, and cooking utensils. At the same time, aluminum is an essential trace element in organisms and plays an important role in their growth, development, and metabolism. However, when the concentration of Al 3+ exceeds a certain range, it will lead to the occurrence of many neurodegenerative and neurological diseases, including dementia, osteomalacia, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In addition, since Al 3+ increases the acidity of the soil, it has been determined that the concentration of aluminum is crucial for aquatic organisms and crop yields. The daily intake of aluminum ions that the human body can tolerate is approximately 3 - 10 milligrams per day. According to the World Health Organization, the maximum allowable content of Al 3+ in drinking water is 7.41 μM. Therefore, in order to prevent the accumulation of Al 3+ and its negative effects on human tissues, it is of great significance to develop an efficient and rapid aluminum identification and monitoring method.

[0003] Zinc is the second most common transition metal ion among micronutrients and plays a crucial role in many important biological processes in the human body, including gene transcription and expression, apoptosis, nerve signal transduction, immune system, and brain function. Therefore, a lack of Zn 2+ in the body can lead to various diseases, such as immune system damage, diarrhea, type D diabetes, and other diseases. In addition, too high Zn 2+ in the human body is easily prone to health problems, such as Parkinson's disease and Alzheimer's disease. According to the information provided by the World Health Organization, the maximum dosage of zinc ions in drinking water is 76 μM. Therefore, considering the ecological balance and physiological balance of zinc in nature and the human body, the development of a highly sensitive and selective Zn 2+ detection chemical sensor has become a research hotspot in the field of biochemistry.

[0004] Existing Al 3+ and Zn 2+Detection methods such as inductively coupled plasma spectrometry (ICP-MS), anodic stripping voltammetry, photochemical flame atomic absorption spectrometry, and electrochemical gel film extraction are applied in various fields and have the advantages of high sensitivity and fast response speed. However, these techniques also have limitations and disadvantages such as complex operation, weak sample stability, and inability to perform on-site real-time detection. Compared with instrumental analysis, fluorescence sensing detection has low cost, simple operation, no high requirements for operators, rapid response, and can be used for biological imaging. Therefore, the research on the fluorescence determination of aluminum ions and zinc ions is urgently needed. Summary of the Invention

[0005] The object of the present invention is to overcome the problems of complex synthesis steps, poor selectivity, and low sensitivity of the probes for detecting aluminum ions and zinc ions in the prior art, and to provide a bifunctional fluorescent molecular probe, a preparation method thereof, and an application thereof in detecting aluminum ions and zinc ions.

[0006] The present invention provides a bifunctional fluorescent detection agent for aluminum ions and zinc ions, which is simple, rapid, highly specific, and highly sensitive, and has broad application prospects, especially suitable for the field of in vivo imaging.

[0007] To achieve the above object, in the first aspect of the present invention, a bifunctional fluorescent molecular probe is provided, wherein the structural formula of the bifunctional fluorescent molecular probe is shown as Formula I below:

[0008]

[0009] In the second aspect of the present invention, a preparation method of the bifunctional fluorescent molecular probe described in the first aspect is provided. Specifically, compound III is reacted with 2-furoyl hydrazine to obtain the target product, and the reaction equation is shown as Formula II below:

[0010]

[0011] Further, in the above technical solution, the preparation method of the bifunctional fluorescent molecular probe includes the following steps: adding compound III to ultra-dry ethanol, then adding 2-furoyl hydrazine, and heating under reflux for reaction; after the reaction is completed, cooling the obtained product to room temperature, a large amount of precipitate is generated during the cooling process, filtering, pulping the obtained filter cake with absolute ethanol and then purifying and drying to obtain the bifunctional fluorescent molecular probe.

[0012] Specifically, in the above technical solution, the dosage of the ultra-dry ethanol may not be specifically limited as long as it can achieve the uniform dissolution of compound III. For example, in the preferred embodiment of the present invention, the dosage ratio of compound III to ultra-dry ethanol is 100 mg: 20 mL.

[0013] Preferably, in the above technical solution, the molar ratio of the compound III to 2-furoyl hydrazide is 1:1.1.

[0014] Preferably, in the above technical solution, the time of the heating reflux reaction is 4 - 8 h.

[0015] Furthermore, in the above technical solution, in the preferred embodiment of the present invention, the preparation method of the compound III is as follows, including the following steps:

[0016] Step 1: Prepare the compound I by using isophorone and malononitrile as reaction raw materials;

[0017] Step 2: React the compound I with p-hydroxybenzaldehyde to prepare the compound II;

[0018] Step 3: React the compound II with hexamethylenetetramine to prepare the said compound III.

[0019] Specifically, the synthesis reaction equation of the said compound III is as shown in Formula III below:

[0020]

[0021] Furthermore, in the above technical solution, the specific synthesis steps of the compound I in step S1 are as follows:

[0022] Dissolve isophorone in absolute ethanol, then successively add malononitrile and piperidine, and heat and stir for reflux reaction under a nitrogen atmosphere; after the reaction is completed, cool to room temperature, pour the obtained product into ice water, a large amount of precipitate is formed, filter by suction, and purify the obtained solid by column chromatography to obtain the said compound I.

[0023] Preferably, in the above technical solution, the molar ratio of the isophorone to the malononitrile is 5:6.

[0024] Preferably, the molar ratio of the isophorone to the piperidine is 50:3.

[0025] Preferably, the time of the reflux reaction is 6 h.

[0026] Furthermore, in the above technical solution, the specific synthesis steps of the compound II in step S2 are as follows:

[0027] Add p-hydroxybenzaldehyde to absolute ethanol, then successively add the compound I and piperidine, heat and reflux until the raw material spots disappear, stop the reaction; spin-dry the obtained product, and then purify it by column chromatography to obtain the said compound II.

[0028] Specifically, for the above technical solution, the dosage of absolute ethanol may not be specifically limited, as long as it can achieve uniform dissolution of p-hydroxybenzaldehyde. For example, in a preferred embodiment of the present invention, the dosage ratio of p-hydroxybenzaldehyde to absolute ethanol is 10 mmol: 40 mL.

[0029] Preferably, for the above technical solution, the molar ratio of Compound I to p-hydroxybenzaldehyde is 1:1.

[0030] Preferably, for the above technical solution, the molar ratio of Compound I to piperidine is 10:3.

[0031] Further, for the above technical solution, the specific synthesis steps of Compound III in Step S3 are as follows:

[0032] Dissolve Compound II in trifluoroacetic acid, then add hexamethylenetetramine, and heat under reflux; after the reaction is completed, cool the obtained product to room temperature, and then successively filter and purify by column chromatography to obtain the said Compound III.

[0033] Specifically, for the above technical solution, the dosage of trifluoroacetic acid may not be specifically limited, as long as it can achieve uniform dissolution of Compound II. For example, in a preferred embodiment of the present invention, the dosage ratio of Compound II to trifluoroacetic acid is 1.5 mmol: 10 mL.

[0034] Preferably, for the above technical solution, the molar ratio of Compound II to hexamethylenetetramine is 1:1.

[0035] The third aspect of the present invention provides the application of the bifunctional fluorescent molecular probe described in the first aspect or the bifunctional fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of aluminum ions and / or zinc ions, especially in the detection of aluminum ions and / or zinc ions in living cells.

[0036] The fourth aspect of the present invention provides a complex of a bifunctional fluorescent molecular probe with aluminum ions and / or zinc ions, wherein: the bifunctional fluorescent molecular probe is the bifunctional fluorescent molecular probe described in the first aspect or the bifunctional fluorescent molecular probe obtained by the preparation method described in the second aspect.

[0037] Further, for the above technical solution, the specific preparation method of the complex is as follows:

[0038] Mix the bifunctional fluorescent molecular probe with an equimolar amount of aluminum ions and / or zinc ions in a mixed solution of DMF and deionized water to obtain a complex of the bifunctional fluorescent molecular probe with aluminum ions and / or zinc ions.

[0039] In some embodiments of the present invention, the complex is used for sensing detection in living cells and zebrafish in vivo.

[0040] In some embodiments of the present invention, the sensing detection is selective sensing detection and competitive sensing detection.

[0041] In some embodiments of the present invention, the sensing detection is visual qualitative detection, ultraviolet detection, and fluorescence detection.

[0042] In some embodiments of the present invention, the sensing detection is quantitative detection under ultraviolet spectrum and fluorescence spectrum.

[0043] Through the above technical solutions, the beneficial technical effects achieved by the present invention are as follows:

[0044] (1) Based on specific recognition, the bifunctional fluorescent molecular probe of the present invention can be used as a specific indicator for the presence of aluminum ions and zinc ions in living cells and zebrafish in vivo. It can provide real-time qualitative and quantitative information through fluorescence detection, and undergo color changes, making it a highly specific indicator suitable for visual colorimetric detection. At the same time, the probe has high reversibility and repeatability for the detection of aluminum ions and zinc ions, and can also be used as a sensor for detecting aluminum ions and zinc ions in living cells and zebrafish in vivo, and can be repeatedly detected without damaging its performance.

[0045] (2) The synthesis of the bifunctional fluorescent molecular probe provided by the present invention only requires two steps or even one step, with easily available raw materials and simple operations.

[0046] (3) The present invention realizes the quantitative detection of aluminum ions and zinc ions by the different functions of the probe shown through ultraviolet and fluorescence spectra, with strong reversibility, multiple recycling capabilities, good selectivity, not easily affected by other ions, and low detection limits, and can be widely applied to detect aluminum ions and zinc ions existing in complex environments.

[0047] Aluminum ions: The ultraviolet detection limit is 4.00×10 -8 M, and the fluorescence detection limit is 1.42×10 -7 M;

[0048] Zinc ions: The ultraviolet detection limit is 3.32×10 -8 M, and the fluorescence detection limit is 1.27×10 -7 M. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0050] Figure 1 It is the high-resolution mass spectrum of the bifunctional fluorescent molecular probe H-4 synthesized in Example 1 of the present invention.

[0051] Figure 2 It is the nuclear magnetic resonance 1 1H NMR spectrum of the bifunctional fluorescent molecular probe H-4 synthesized in Example 1 of the present invention.

[0052] Figure 3 It is the nuclear magnetic resonance 13 13C NMR spectrum of the bifunctional fluorescent molecular probe H-4 synthesized in Example 1 of the present invention.

[0053] Figure 4 It is the fluorescence spectra of H-4, H-4+Al 3+ , H-4+Zn 2+ and other metal ions in Application Example 1 of the present invention.

[0054] Figure 5 It is the ultraviolet absorption spectra of H-4, H-4+Al 3+ and H-4+Zn 2+ in Application Example 1 of the present invention.

[0055] Figure 6 (A) It is the competitive detection graph of probe H-4 for Al 3+ and other cations in the DMF / water (5 / 5, v / v) system in Application Example 2 of the present invention; (B) It is the competitive detection graph of probe H-4 for Zn 2+ and other cations in the DMF / water (5 / 5, v / v) system.

[0056] Figure 7 (A) It is the fluorescence titration curve of H-4+Al 3+ in Application Example 3 of the present invention, (B) It is the fluorescence titration curve of H-4+Zn 2+ in Application Example 3 of the present invention.

[0057] Figure 8 (A) It is the ultraviolet titration curve of H-4+Al 3+ in Application Example 3 of the present invention, (B) It is the ultraviolet titration curve of H-4+Zn 2+ in Application Example 3 of the present invention.

[0058] Figure 9 (A) It is the reversibility detection graph of H-4+Al 3+ in Application Example 4 of the present invention, (B) It is the reversibility detection graph of H-4+Zn 2+ in Application Example 4 of the present invention.

[0059] Figure 10 It is probe H-4, H-4+Al in Application Example 5 of the present invention.3+ and H-4+Zn 2+ Effect diagram under sunlight.

[0060] Figure 11 For the probes H-4, H-4+Al in Application Example 5 of the present invention 3+ and H-4+Zn 2+ Effect diagram under irradiation of 365nm ultraviolet lamp.

[0061] Figure 12 For the imaging of the application of the probe H-4 for detecting Al 3+ and Zn 2+ in living cells in Application Example 6 of the present invention.

[0062] Figure 13 For the imaging of the application of the probe H-4 for detecting Al 3+ and Zn 2+ in zebrafish in Application Example 7 of the present invention. Detailed implementation mode

[0063] Existing fluorescent probes only quantitatively detect metal ions by fluorescence spectroscopy. The dual-functional fluorescent molecular probe of the present invention can combine ultraviolet ratio quantitative detection with fluorescence spectroscopy quantitative detection to provide a variety of different data, increasing the accuracy and reliability of the detection of metal ions, namely aluminum ions and zinc ions. The ultraviolet spectrum can provide higher contrast and sensitivity. The most intuitive phenomenon is that the dual-functional fluorescent molecular probe of the present invention has a chromogenic sensing function, which not only causes a change in the color of the solution under sunlight, but also produces a change in the fluorescence color under irradiation of a 365nm ultraviolet lamp. This dual observation mode makes it a chromogenic sensing fluorescent probe suitable for different lighting conditions.

[0064] The present invention will be further described in detail below through implementation cases. These implementation cases are implemented on the premise of the technology of the present invention, and the detailed implementation modes and specific operation processes are given to illustrate the creativity of the present invention. However, the protection scope of the present invention is not limited to the following implementation cases.

[0065] In the ranges disclosed in the present invention, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0066] The first aspect of the present invention provides a dual-functional fluorescent molecular probe, wherein the structural formula of the dual-functional fluorescent molecular probe is as follows:

[0067]

[0068] In the second aspect of the present invention, a method for preparing the bifunctional fluorescent molecular probe described in the first aspect is provided. Specifically, reacting compound Ⅲ with 2-furoyl hydrazine gives the target product, and the reaction equation is shown as follows:

[0069]

[0070] Compound Ⅲ used in the present invention is disclosed in the prior art. For example, compound Ⅲ is synthesized by the synthesis method disclosed in the application publication number CN117945994A. The method includes the following steps:

[0071] Step 1: Prepare compound Ⅰ using isophorone and malononitrile as reaction raw materials;

[0072] Step 2: React compound Ⅰ with p-hydroxybenzaldehyde to prepare compound Ⅱ;

[0073] Step 3: React compound Ⅱ with hexamethylenetetramine to prepare compound Ⅲ.

[0074] In some embodiments of the present invention, the progress of the reaction is monitored by TLC.

[0075] In the third aspect of the present invention, an application of the bifunctional fluorescent molecular probe described in the first aspect or the bifunctional fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of aluminum ions and zinc ions is provided.

[0076] In some embodiments of the present invention, the bifunctional fluorescent molecular probe is used for the sensing detection of aluminum ions and zinc ions in living cells and zebrafish in vivo.

[0077] In the fourth aspect of the present invention, a complex of the bifunctional fluorescent molecular probe described in the first aspect or the bifunctional fluorescent molecular probe obtained by the preparation method described in the second aspect with aluminum ions and / or zinc ions is provided.

[0078] In the present invention, the preparation method of the complex may include: mixing the bifunctional fluorescent molecular probe with equimolar amounts of aluminum ions and / or zinc ions in a mixed solution of DMF and deionized water to obtain a complex of the bifunctional fluorescent molecular probe with aluminum ions and / or zinc ions.

[0079] In some embodiments of the present invention, the sensing detection is selective sensing detection and competitive sensing detection.

[0080] In some embodiments of the present invention, the sensing detection is visual qualitative detection, ultraviolet light detection, fluorescence detection, and reversible detection.

[0081] In some embodiments of the present invention, the sensing detection is quantitative detection under ultraviolet spectrum and fluorescence spectrum.

[0082] In the following examples and comparative examples, those without specific conditions noted are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial channels.

[0083] The present invention will be described in detail below through examples.

[0084] Example 1

[0085] This example is used to illustrate the preparation of the target compound H-4

[0086] Step 1: Prepare compound I using isophorone and malononitrile as the main reaction raw materials; the specific method is as follows:

[0087] Dissolve 6.91 g of isophorone (50.1 mmol) in 100 mL of absolute ethanol, then sequentially add 3.84 g of malononitrile (60.1 mmol) and 0.3 mL of piperidine (3.0 mmol), heat and stir under reflux in a nitrogen atmosphere for 6 h, monitor the complete reaction of isophorone by TLC, stop the reaction, and let it stand and cool to room temperature; then pour the obtained product into 200 mL of ice water to form a large amount of grayish-white precipitate, and the solid obtained by suction filtration is separated and purified by column chromatography to obtain 7.23 g of white crystalline solid, namely compound I;

[0088] Step 2: React compound I with p-hydroxybenzaldehyde to prepare compound II; the specific method is as follows:

[0089] Add p-hydroxybenzaldehyde (10.2 mmol) to a round-bottom flask containing 40 mL of absolute ethanol, then sequentially add 10.5 mmol of compound I and 3.0 mmol of piperidine, heat and reflux the reaction until the disappearance of the raw material spots is monitored by TLC spotting, and stop the reaction; the product obtained after drying the reaction solution by rotary evaporation is separated and purified by column chromatography to obtain compound II;

[0090] Step 3: React compound II with hexamethylenetetramine to prepare compound III; the specific method is as follows:

[0091] Dissolve compound II (1.5 mmol) in 10 mL of trifluoroacetic acid, then add 1.5 mmol of hexamethylenetetramine, heat and reflux for 6 h, monitor the complete reaction by TLC, and stop the reaction; cool the obtained product to room temperature, then slowly pour it into 50 mL of ice water, and the product obtained by filtration is separated and purified by column chromatography to obtain compound III;

[0092] Step 4: React compound III with 2-furoylhydrazine to obtain the target product; the specific method is as follows:

[0093] 100 mg of Compound III was added to a round-bottom flask containing 20 mL of ultradry ethanol, and then 1.1 equivalents of 2-furoylhydrazide of Compound III was added. The mixture was heated under reflux for 6 h until the reaction was monitored by TLC to be complete; the reaction was stopped, and the resulting product was cooled to room temperature. A large amount of precipitate was formed during the cooling process. The precipitate was filtered, and the filter cake was slurried and purified with anhydrous ethanol to obtain the target product: a bifunctional molecular probe for identifying aluminum ions and zinc ions.

[0094] The target compound H-4 obtained in this example is a novel compound that has not been reported. After purification, it was characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR). The high-resolution mass spectrum of probe H-4 is as Figure 1 shown. The molecular formula of [M+H + + is C 25 H 22 N4O3. The calculated theoretical value is 427.1770, and the actual value found in the mass spectrum is 427.1768. The nuclear magnetic resonance 1 H NMR of probe H-4 is as Figure 2 shown. The nuclear magnetic resonance 13 C NMR of probe H-4 is as Figure 3 shown. 1 H NMR (400 MHz, DMSO-d6) (δ ppm) 12.24 (s, 1H), 11.60 (s, 1H), 8.64 (s, 1H), 7.98 (d, J = 0.8 Hz, 1H), 7.87 (s, 1H), 7.70 (dd, J = 8.6, 1.8 Hz, 1H), 7.34 (d, J = 2.7 Hz, 1H), 7.28 (s, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.83 (s, 1H), 6.73 (dd, J = 3.4, 1.7 Hz, 1H), 2.54 (s, 4H), 1.02 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) (δ ppm): 170.4, 159.6, 156.5, 150.0, 150.2, 138.2, 137.5, 131.2, 130.5, 128.4, 128.0, 127.3, 123.1, 122.3, 119.7, 117.8, 114.2, 113.4, 76.4, 43.2, 39.4, 32.1, 27.9.

[0095] In summary, the structural formula of the target compound H-4 synthesized in this example is as follows:

[0096] ​

[0097] Example 2

[0098] Dissolve the fluorescent molecular probe H-4 synthesized in Example 1 in DMF to prepare a DMF / deionized water (5 / 5, v / v) buffer system with a concentration of 10 μM, namely the probe H-4 solution.

[0099] Prepare metal cations and disodium ethylenediaminetetraacetate (EDTA) into an aqueous solution with a concentration of 0.01 mol / L.

[0100] The H-4 solution used in the following application examples is the probe H-4 solution prepared in Example 2.

[0101] The metal cation aqueous solution and disodium ethylenediaminetetraacetate aqueous solution used in the following application examples are both prepared in Example 2.

[0102] Application Example 1

[0103] Add 25 μL of different metal cations (Zn 2+ , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3 + , Cu 2+ , Fe 2+ , Fe 3+ , Hg 2+ , K + , Mg 2+ , Mn 2+ , Na + , Pb 2+ , Ni 2+ , Ag + and Al 3+ ) to 5 mL of the probe H-4 solution, and the fluorescence spectrum is as Figure 4 shown. It can be seen from Figure 4 that after adding Al 3+ , the fluorescence intensity increases significantly, while the addition of Zn 2+ results in a relatively large Stokes shift, and the addition of other metal ions does not cause obvious changes in fluorescence intensity.

[0104] Figure 5 are the ultraviolet spectra of the probe H-4 solution and the solution after adding Zn 2+ and Al 3+ . It can be seen that after adding Zn 2+ and Al 3+After that, the 426 nm ultraviolet absorption peak decreased and red-shifted to 480 nm and 450 nm respectively.

[0105] The above results indicate that Al 3+ can enhance the fluorescence of the probe H-4, and Zn 2+ can produce a large Stokes shift. Compared with other ions, it is unique. The probe H-4 can specifically recognize Al 3+ and Zn 2+ .

[0106] Application Example 2

[0107] Using the H-4 probe solution to detect Al 3+ for different metal cations Zn 2+ , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu 2+ , Fe 2+ , Fe 3+ , Hg 2+ , K + , Mg 2+ , Mn 2+ , Na + , Pb 2+ , Ni 2+ , Ag + competitiveness, the fluorescence intensities of the H-4 probe solution and after adding different metal cations to it were measured respectively, and the fluorescence intensity of the system after adding 25 μL of other metal cations to 5 mL of the H-4 probe solution first and then adding an equal amount of Al 3+ was measured. As shown in Figure 6 (A). It can be seen that the probe H-4 has good anti-interference ability for the recognition of Al 3+ . The presence of other metal ions in the system will not affect its recognition effect.

[0108] Using the H-4 probe solution to detect Zn 2+ for different metal cations Al 3+ , Ba 2+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu 2+ , Fe 2+ , Fe 3+ , Hg 2+ , K + , Mg 2+ , Mn 2+ , Na +, Pb 2+ , Ni 2+ , Ag + 's competitiveness, as shown in Figure 6 (B). It can be seen that except for Al 3+ , the probe H-4 also has good anti-interference ability for the recognition of Zn 2+ .

[0109] Application Example 3

[0110] Add aluminum ions or zinc ions with different concentrations to the H-4 probe solution and detect the changes in its fluorescence spectrum, as shown in Figure 7 .

[0111] Figure 7 (A) shows the relationship between the change in the fluorescence spectrum of the system and the concentration of Al 3+ . As the concentration of aluminum ions increases, the fluorescence intensity gradually increases, and the change in fluorescence intensity is obvious, proving that the probe H-4 can be used for the quantitative detection of Al 3+ , and the phenomenon is obvious and easy to distinguish.

[0112] Figure 7 (B) shows the relationship between the change in the fluorescence spectrum of the system and the concentration of Zn 2+ . As the concentration of zinc ions increases, the fluorescence gradually redshifts, and an obvious Stokes shift occurs, proving that the probe H-4 can be used for the quantitative detection of Zn 2+ , and the phenomenon is obvious and easy to distinguish.

[0113] Add aluminum ions or zinc ions with different concentrations to the H-4 probe solution and detect the changes in its ultraviolet spectrum, as shown in Figure 8 .

[0114] Figure 8 (A) shows the relationship between the change in the ultraviolet spectrum of the system and the concentration of Al 3+ . As the concentration of aluminum ions increases, the ultraviolet absorbance at 426 nm gradually weakens and redshifts to 450 nm, and the change in the ultraviolet spectrum is obvious, proving that the probe H-4 can be used for the quantitative detection of Al 3+ , and the phenomenon is obvious and easy to distinguish.

[0115] Figure 8 (B) shows the relationship between the change in the ultraviolet spectrum of the system and the concentration of Zn 2+ . As the concentration of zinc ions increases, the ultraviolet absorbance at 426 nm gradually weakens and redshifts to 480 nm, and the change in the ultraviolet spectrum is obvious, proving that the probe H-4 can be used for the quantitative detection of Zn 2+ , and the phenomenon is obvious and easy to distinguish.

[0116] Application Example 4

[0117] As shown Figure 9 (A), 5 μL of EDTA solution and Al were alternately added in equal amounts to the H-4 probe solution 3+ and then the reversible change in the fluorescence intensity at 630 nm was detected.

[0118] As shown Figure 9 (B), 5 μL of EDTA and Zn (concentration: 1×10 2+ mol / L) were alternately added in equal amounts to 5 mL of the H-4 probe solution, and then the reversible change in the fluorescence intensity at 650 nm was detected. -5 The results showed that the probe H-4 had good reversibility in the recognition of Zn

[0119] and Al 2+ and could be used repeatedly for many times. 3+

[0120] Application Example 5

[0121] Figure 10 The color change of the solution under sunlight after adding 25 μL of aluminum ions or zinc ions to 5 mL of the H-4 probe solution was as follows. It could be seen that after adding Al 3+ , the color change was small, from light yellow to dark yellow on the left side. After adding Zn 2+ , the color changed from light yellow to orange on the right side. The probe of the present invention could detect aluminum ions and zinc ions by visual colorimetry.

[0122] Figure 11 The color change of the solution under irradiation with a 365 nm ultraviolet lamp after adding 25 μL of aluminum ions or zinc ions to 5 mL of the H-4 probe solution was as follows. It could be seen that after adding Al 3+ , the color changed from yellow to orange, and after adding Zn 2+ , the color changed to orange-red on the right side. The probe of the present invention could also achieve visual qualitative detection of aluminum ions and zinc ions under ultraviolet irradiation.

[0123] Application Example 6

[0124] The fluorescence images of aluminum ions or zinc ions detected by the probe H-4 in Hela cells (purchased from Cybiogene (Shanghai) Biotechnology Co., Ltd.) were as shown Figure 12 .

[0125] As shown Figure 12 (A), the fluorescence image of Al detected by the probe H-4 in Hela cells: Hela cells were respectively combined with 0.01 mol / L Hepes (CAS: 7365-45-9, purchased from Aladdin Reagent (Shanghai) Co., Ltd.) buffer solution, H-4 (10 μM), H-4 (10 μM) + Al 3+ ​3+ (5 μM), H-4 (10 μM) + Al 3+ (10 μM) and the changes in their fluorescence imaging after cultivation. As the concentration of Al 3+ increases, the green fluorescence signal becomes stronger and stronger.

[0126] As Figure 12 (B) shows, the fluorescence images of probe H-4 detecting Zn in Hela cells: Hela cells were cultured with Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Zn 2+ (5 μM), H-4 (10 μM) + Zn 2+ (10 μM) respectively, and the changes in their fluorescence imaging after cultivation. As the concentration of Zn 2+ increases, the green fluorescence signal becomes weaker and weaker, and the red fluorescence signal becomes stronger and stronger. 2+ The results show that probe H-4 can be used to monitor and study the dynamic changes of metal ion levels such as aluminum ions and zinc ions in the cell environment in real time.

[0127] Application Example 7

[0128] The fluorescence images of probe H-4 detecting aluminum ions / zinc ions in zebrafish are as

[0129] shown. Figure 13 shown.

[0130] As Figure 13 (A) shows, the fluorescence images of probe H-4 detecting Al in zebrafish: Zebrafish were cultured with Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Al 3+ (5 μM), H-4 (10 μM) + Al 3+ (10 μM) respectively, and the changes in their fluorescence imaging after cultivation. As the concentration of Al 3+ increases, the green fluorescence signal becomes stronger and stronger. 3+ The fluorescence images of probe H-4 detecting Zn in zebrafish are as

[0131] As Figure 13 (B) shows: Zebrafish were cultured with Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Zn 2+ (5 μM), H-4 (10 μM) + Zn 2+ (10 μM) respectively, and the changes in their fluorescence imaging after cultivation. As the concentration of Zn 2+ increases, the fluorescence in the green channel becomes weaker and weaker, and the fluorescence in the red channel becomes stronger and stronger. 2+ The results show that probe H-4 can be used as an effective tool for real-time and differential detection of aluminum ions and zinc ions in animals.

[0132] The results show that probe H-4 can be used as an effective tool for real-time and differential detection of aluminum ions and zinc ions in animals.

[0133] The present invention designs and synthesizes a fluorescent molecular probe for specific recognition of both aluminum ions and zinc ions. Probe H-4 has a weak fluorescence intensity in the absence of aluminum ions and zinc ions, but shows a significant fluorescence enhancement at 538 nm after the addition of aluminum ions, and the addition of zinc ions causes a large red shift in fluorescence, with high selectivity and anti-interference ability. In addition, probe H-4 has a low detection limit for aluminum ions, with an ultraviolet detection limit of 4.00×10 -8 M and a fluorescence detection limit of 1.42×10 -7 M; probe H-4 has a low detection limit for zinc ions, with an ultraviolet detection limit of 3.32×10 -8 M and a fluorescence detection limit of 1.27×10 -7 M. It can effectively detect aluminum ions and zinc ions present in water environment, living cells and zebrafish in vivo with high sensitivity, which has certain guiding significance for production activities.

[0134] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Application of a bifunctional fluorescent molecular probe in sensing and detecting aluminum ions and zinc ions, characterized in that: The structural formula of the bifunctional fluorescent molecular probe is shown as Formula I below:

2. The application according to claim 1, characterized in that: The preparation method of the bifunctional fluorescent molecular probe is to react compound Ⅲ with 2-furoyl hydrazide to obtain the target product, and its reaction equation is shown as Formula II below:

3. The application according to claim 2, characterized in that: It includes the following steps: Add compound Ⅲ to ultradry ethanol, then add 2-furoyl hydrazide, and heat under reflux; after the reaction is completed, cool the obtained product to room temperature, filter, slurry the obtained filter cake with absolute ethanol and then purify and dry it to obtain the bifunctional fluorescent molecular probe.

4. The application according to claim 3, characterized in that: The molar ratio of compound Ⅲ to 2-furoyl hydrazide is 1:1.

1.

5. The application according to claim 4, wherein: The time of the heating reflux reaction is 4 - 8 h.

6. The application according to claim 2, characterized in that: The preparation method of compound Ⅲ is as follows, including the following steps: Step 1: Prepare compound Ⅰ using isophorone and malononitrile as reaction raw materials; Step 2: React compound Ⅰ with p-hydroxybenzaldehyde to prepare compound Ⅱ; Step 3: React compound Ⅱ with hexamethylenetetramine to prepare the said compound Ⅲ.

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