Difunctional fluorescent molecular probe for identifying aluminum ions and / or pH value as well as preparation method and application of difunctional fluorescent molecular probe

Abstract

The invention discloses a difunctional fluorescent molecular probe for identifying aluminum ions and / or pH value and a preparation method and application thereof. The Schiff base bifunctional fluorescent molecular probe can be prepared by taking dicyanoisophorone as a fluorescent group to react with 2-thiophenecarboxylic acid hydrazide. The probe provided by the invention can realize double-channel sensing detection of aluminum ions and / or pH through changes of ultraviolet and fluorescence spectrums and changes of solution color, and can perform real-time qualitative and quantitative visual colorimetric detection. The invention also relates to application of the molecular probe in sensing detection of aluminum ions and / or pH in living cells and living zebra fish. The fluorescent molecular probe disclosed by the invention has the advantages of easily available synthesis raw materials, good specificity, good selectivity on recognized ions, strong anti-interference capability on other competitive ions, quick response, low detection limit and the like. Quantitative detection of aluminum ions and response to different pH values are displayed through ultraviolet and fluorescence spectrum and color change.

Description

Technical Field

[0001] The invention relates to the technical field of aluminum ion and pH detection, and in particular to a colorimetric-fluorescence dual-channel molecular probe and a preparation method thereof, and an application in aluminum ion and / or pH sensing detection in living cells or living zebrafish. Background Art

[0002] Aluminum is the most abundant metallic element in the Earth's crust and one of the most important industrial elements. Aluminum is widely used in industrial applications, including food packaging materials, water treatment, and pharmaceutical preparations. However, aluminum is harmful to plants and animals. 3+ ) is soluble in soil and has been shown to be harmful to the growth of plant roots. Studies have shown that aluminum is carcinogenic and can promote tumorigenesis in mammary epithelial cells. According to the World Health Organization, the daily tolerable aluminum intake for the human body is 3.0-10.0 mg. Al 3+ It can enter organisms through a variety of pathways, including water cycles, food chains, and other mechanisms, thereby causing damage to organisms. In order to prevent the harm caused by excessive aluminum ions, the concentration of aluminum in water and organisms must be determined. Therefore, methods for detecting aluminum ions are urgently needed.

[0003] pH is one of the important parameters in the fields of medicine, scientific research, etc. The stability of pH is necessary for life activities such as metabolism, apoptosis, and homeostasis of the internal environment. The pH value inside the mitochondria is 8.0. Abnormal changes in body pH often lead to the development of various functional disorders and diseases. Therefore, pH plays an important role in the study of disease conditions and pathology. For example, pathological processes, such as cancer and Alzheimer's disease, are accompanied by changes in pH; the pH of healthy skin becomes acidic, while the pH of inflamed and infected skin does not show alkalinity. Therefore, monitoring pH is of great significance for detecting the onset of diseases and their treatment.

[0004] At present, the means of metal ion detection include inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS), electrochemical methods and voltammetry, and the means of pH detection include ion sensitive field effect transistor method (ISEFT) and amperometry. However, these methods require expensive and complex instruments and professional technicians in the detection process, and have the disadvantages of cumbersome analysis process, long detection and analysis time, and high analysis cost, which cannot meet the requirements of rapid on-site evaluation required by modern environmental monitoring. In recent years, the fluorescence detection and analysis method based on chemical sensors has attracted special attention due to its simple operation, high selectivity and sensitivity, low detection limit, fast analysis time, non-destructive to samples, and convenient visual qualitative identification.

[0005] However, although many molecular probes for identifying aluminum ions and detecting pH values ​​have been reported, these probes have problems such as cumbersome synthesis steps, low selectivity and sensitivity, which reduce the applicability of the probes. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present application provides a colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values, and a preparation method and application thereof. The probe of the present invention can realize dual-channel sensing detection of aluminum ions and / or pH values ​​through changes in fluorescence spectrum and color, and can perform real-time qualitative and quantitative visual colorimetric detection. In addition, the probe of the present invention has good specificity, strong anti-interference ability to other metal ions, and low detection limit. The detection limit of aluminum ions is as low as 5.08×10 -8 M.

[0007] In order to achieve the above objectives, in a first aspect, the present application provides a colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values, the structural formula of the probe is shown in Formula 1:

[0008]

[0009] Specifically, in the above technical solution, the colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values ​​is a Schiff base probe with dicyanoisophorone and 2-thiophenecarboxylic acid hydrazide as fluorescent groups.

[0010] In the second aspect, the present application provides a method for preparing the colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values ​​as described in the first aspect, which is obtained by reacting compound III with 2-thiophenecarboxylic acid hydrazide, and the synthesis reaction equation thereof is shown in Formula 2 below:

[0011]

[0012] Furthermore, in the above technical solution, the method for preparing the colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and detecting pH value specifically comprises the following steps:

[0013] Compound III is added to ultra-dry ethanol, followed by addition of 2-thiophenecarboxylic acid hydrazide, and the mixture is heated to reflux for reaction; after stopping the reaction, the mixture is cooled to room temperature, the obtained product is filtered, and the obtained filter cake is slurried and purified with anhydrous ethanol to obtain the target product.

[0014] Specifically, in the above technical solution, the amount of the super dry ethanol is not specifically limited, as long as the uniform dissolution of compound III can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of compound III to super dry ethanol is 100 mg: 20 mL.

[0015] Preferably, in the above technical solution, the molar ratio of compound III to 2-thiophenecarboxylic acid hydrazide is 1:1.1.

[0016] Preferably, in the above technical solution, the heating reflux reaction time is 4-8h.

[0017] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the preparation method of the compound III is as follows, comprising the following steps:

[0018] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0019] Step 2, reacting compound I with p-hydroxybenzaldehyde to prepare compound II;

[0020] Step 3: react compound II with hexamethylenetetramine to prepare the compound III.

[0021] Specifically, the synthesis reaction equation of the compound III is shown in Formula 3 below:

[0022]

[0023] Furthermore, in the above technical scheme, the specific synthesis steps of compound I in step S1 are as follows:

[0024] Dissolve isophorone in anhydrous ethanol, then add malononitrile and piperidine in sequence, and heat, stir and reflux under nitrogen atmosphere to react; after the reaction is completed, cool to room temperature, pour the obtained product into ice water to generate a large amount of precipitate, filter, and separate and purify the obtained solid by column chromatography to obtain the compound I.

[0025] Preferably, in the above technical solution, the molar ratio of isophorone to malononitrile is 5:6.

[0026] Preferably, the molar ratio of isophorone to piperidine is 50:3.

[0027] Preferably, the reflux reaction time is 6 h.

[0028] Furthermore, in the above technical scheme, the specific synthesis steps of compound II in step S2 are as follows:

[0029] Add p-hydroxybenzaldehyde to anhydrous ethanol, then add compound I and piperidine in sequence, heat and reflux until the raw material point disappears, and then stop the reaction; spin dry the obtained product, and then separate and purify it by column chromatography to obtain the compound II.

[0030] Specifically, in the above technical solution, the amount of the anhydrous ethanol is not specifically limited, as long as the uniform dissolution of p-hydroxybenzaldehyde can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of p-hydroxybenzaldehyde to anhydrous ethanol is 10mmol:40mL.

[0031] Preferably, in the above technical solution, the molar ratio of compound I to p-hydroxybenzaldehyde is 1:1.

[0032] Preferably, in the above technical solution, the molar ratio of compound I to piperidine is 10:3.

[0033] Furthermore, in the above technical scheme, the specific synthesis steps of compound III in step S3 are as follows:

[0034] Compound II is dissolved in trifluoroacetic acid, and then hexamethylenetetramine is added, and the mixture is heated under reflux for reaction. After the reaction is completed, the obtained product is cooled to room temperature, and then slowly poured into ice water, and the obtained product is filtered and purified by column chromatography to obtain the compound III.

[0035] Specifically, in the above technical solution, the amount of trifluoroacetic acid used is not specifically limited, as long as the uniform dissolution of compound II can be achieved. For example, in a preferred embodiment of the present invention, the amount ratio of compound II to trifluoroacetic acid is 1.5 mmol: 10 mL.

[0036] Preferably, in the above technical solution, the molar ratio of compound II to hexamethylenetetramine is 1:1.

[0037] In a third aspect, the present application also provides the above-mentioned colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions for sensing and detecting the aluminum ion content and / or pH value in a water environment system.

[0038] In a fourth aspect, the present application provides a complex of a colorimetric-fluorescent dual-channel molecular probe and an aluminum ion, wherein: the colorimetric-fluorescent dual-channel molecular probe is the colorimetric-fluorescent dual-channel molecular probe described in the first aspect or the colorimetric-fluorescent dual-channel molecular probe obtained by the preparation method described in the second aspect.

[0039] In the present invention, the preparation method of the complex may include: uniformly mixing the colorimetric-fluorescent dual-channel molecular probe and an equimolar amount of aluminum ions in a mixed solution of DMF and deionized water to obtain a complex of the colorimetric-fluorescent dual-channel molecular probe and aluminum ions.

[0040] Furthermore, in the above technical solution, the sensing detection includes fluorescence detection, ultraviolet ratio detection, visual qualitative detection or reversible detection.

[0041] The technical solution provided by this application has at least the following beneficial effects compared with the prior art:

[0042] (1) Based on specific recognition, the colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values ​​of the present invention can be used as a specific indicator for the presence of aluminum ions in different water environments, living cells and zebrafish. 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 highly reversible repeatability for the detection of lead ions and can also be used as a sensor for detecting lead ions in living cells and living zebrafish, and can perform repeated detections without compromising its performance.

[0043] (2) The colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values ​​provided by the present invention can be synthesized in only two steps or even in one step, the raw materials are easily available, and the operation is simple.

[0044] (3) The probe of the present invention has good specificity, strong anti-interference ability to other metal ions, and low detection limit. The detection limit of aluminum ions is 5.08×10 -8 M. Based on its specificity, the probe of the present invention can be used as a specific indicator for colorimetric-fluorescent dual-channel detection of the presence of aluminum ions in aqueous solution, and can perform real-time qualitative and quantitative visual colorimetric detection. More importantly, the present invention can achieve rapid, reversible and repeated detection of aluminum ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 This is a high-resolution mass spectrum of the colorimetric-fluorescent dual-channel molecular probe H-5 synthesized in Example 1 of the present invention.

[0047] Figure 2 The nuclear magnetic resonance of the colorimetric-fluorescent dual-channel molecular probe H-5 synthesized in Example 1 of the present invention is 1 HNMR spectrum.

[0048] Figure 3 The nuclear magnetic resonance of the colorimetric-fluorescent dual-channel molecular probe H-5 synthesized in Example 1 of the present invention is 13 CNMR image.

[0049] Figure 4 H-5+Al in Application Example 1 of the present invention 3+ and other metal ions fluorescence spectra.

[0050] Figure 5 H-5 and H-5+Al in Application Example 1 of the present invention 3+ UV absorption spectrum.

[0051] Figure 6 This is a competitive detection diagram of probe H-5 in Application Example 2 of the present invention for different metal cations in DMF / water (6:4, v / v, 0.01 M Hepes, pH=7.4) system.

[0052] Figure 7 In the application example 3 of the present invention: (A) H-5+Al 3+ UV titration curve of H-5+Al 3+ Fluorescence titration curve.

[0053] Figure 8 In Application Example 4 of the present invention: (A) is the UV spectrum of H-5 under different pH conditions, and (B) is the fluorescence spectrum of H-5 under different pH conditions.

[0054] Fig. 9 [H-5+Al 3+ ] reversibility detection diagram.

[0055] Fig.10 In the application example 6 of the present invention: (A) is probe H-5, H-5+Al 3+ Effect diagram under sunlight; (B) probe H-5 and H-5+Al 3+ The effect diagram under 365nm ultraviolet light.

[0056] Fig.11 In Application Example 7 of the present invention: (A) is a diagram showing the color change of the solution of probe H-5 under different pH conditions; (B) is a diagram showing the color change of the solution of probe H-5 under different pH conditions and under 365nm ultraviolet light.

[0057] Fig.12 In the application example 8 of the present invention: (A) probe H-5 detects Al in living cells 3+ (B) Probe H-5 detects Al in zebrafish 3+ Application imaging diagram.

[0058] Fig.13 In Application Example 9 of the present invention: (A) is an imaging diagram of the application of probe H-5 in living cells at different pH values; (B) is an imaging diagram of the application of probe H-5 in zebrafish at different pH values. DETAILED DESCRIPTION

[0059] The present application provides a bifunctional fluorescent molecular probe for identifying aluminum ions and / or pH values, and a preparation method and application thereof. The Schiff base bifunctional fluorescent molecular probe can be prepared by reacting dicyanoisophorone as a fluorescent group with 2-thiophenecarboxylic acid hydrazide. The probe of the present invention can realize dual-channel sensing detection of aluminum ions and / or pH through changes in ultraviolet and fluorescence spectra and changes in solution color, and can perform real-time qualitative and quantitative visual colorimetric detection. The present invention also relates to the application of the bifunctional fluorescent molecular probe for sensing and detecting aluminum ions and / or pH in living cells and living zebrafish. The fluorescent molecular probe of the present invention has the advantages of easy availability of synthetic raw materials, good specificity, good selectivity for identifying ions, strong anti-interference ability of other competing ions, rapid response, low detection limit, etc. The present invention displays quantitative detection of aluminum ions and response to different pH values ​​through ultraviolet and fluorescence spectra and color changes.

[0060] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed in this article.

[0061] In a first aspect, the present invention provides a colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values, wherein the structural formula of the probe is as shown in the formula:

[0062]

[0063] The second aspect of the present invention provides a method for preparing the colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values ​​as described in the first aspect, which is obtained by reacting compound III with 2-thiophenecarboxylic acid hydrazide, and the reaction equation is shown below:

[0064]

[0065] The compound III used in the present invention is disclosed in the prior art. For example, the compound III is synthesized by the synthesis method disclosed in the application publication number CN117945994A, and the method comprises the following steps:

[0066] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0067] Step 2, reacting compound I with p-hydroxybenzaldehyde to prepare compound II;

[0068] Step 3: react compound II with hexamethylenetetramine to prepare compound III.

[0069] In some embodiments of the present invention, the progress of the reaction is monitored by TLC.

[0070] The third aspect of the present invention provides the use of the colorimetric-fluorescent dual-channel molecular probe described in the first aspect or the colorimetric-fluorescent dual-channel molecular probe obtained by the preparation method described in the second aspect in aluminum ion and / or pH value sensing detection.

[0071] In some embodiments of the present invention, the colorimetric-fluorescent dual-channel molecular probe is used for sensing and detecting the aluminum ion content and / or pH value in a water environment system.

[0072] The fourth aspect of the present invention provides a complex of the colorimetric-fluorescent dual-channel molecular probe described in the first aspect or the colorimetric-fluorescent dual-channel molecular probe obtained by the preparation method described in the second aspect and aluminum ions.

[0073] In the present invention, the preparation method of the complex may include: mixing the colorimetric-fluorescent dual-channel molecular probe with an equimolar amount of aluminum ions in a mixed solution of DMF and deionized water to obtain a complex of the colorimetric-fluorescent dual-channel molecular probe and aluminum ions.

[0074] In some embodiments of the present invention, the sensing assay is a selective sensing assay or a competitive sensing assay.

[0075] In some embodiments of the present invention, the sensing detection is visual qualitative detection, ultraviolet light detection, fluorescence detection or reversible detection.

[0076] In some embodiments of the present invention, the sensing detection is quantitative detection under ultraviolet spectroscopy or fluorescence spectroscopy.

[0077] The present invention will be described in detail below through examples.

[0078] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0079] The present invention will be described in detail below through examples.

[0080] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0081] Example 1

[0082] This example is used to illustrate the preparation of the target compound H-5

[0083] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I; specifically, 6.91 g of isophorone (50.1 mmol) was dissolved in 100 mL of anhydrous ethanol, followed by sequentially adding 3.84 g of malononitrile (60.1 mmol) and 0.3 mL of piperidine (3.0 mmol), and heating and stirring the mixture under reflux for 6 h under a nitrogen atmosphere, and the isophorone was completely reacted by monitoring by TLC, and the reaction was stopped; the reaction was allowed to stand and cool to room temperature, and then the reaction solution was poured into 200 mL of ice water to generate a large amount of off-white precipitate, and the solid obtained by suction filtration was separated and purified by column chromatography to obtain 7.23 g of a white crystalline solid, namely compound I;

[0084] Step 2, compound I is reacted with p-hydroxybenzaldehyde to prepare compound II; specifically, p-hydroxybenzaldehyde (10.2 mmol) is added to a round-bottom flask containing 40 mL of anhydrous ethanol, and then 10.5 mmol of compound I and 3.0 mmol of piperidine are added in sequence, and the mixture is heated under reflux to react until the raw material spot disappears as monitored by the TLC spot plate, and the reaction is stopped; the reaction solution is spin-dried and the product obtained is separated and purified by column chromatography to obtain compound II;

[0085] Step 3, reacting compound II with hexamethylenetetramine to prepare compound III; specifically, dissolving compound II (1.5 mmol) in 10 mL of trifluoroacetic acid, then adding 1.5 mmol of hexamethylenetetramine, heating to reflux, monitoring the reaction completion by TLC, and stopping the reaction; cooling the reaction solution to room temperature and then slowly pouring it into 50 mL of ice water, filtering the obtained product, and separating and purifying it by column chromatography to obtain compound III;

[0086] Step 4, reacting compound III with 2-thiophenecarboxylic acid hydrazide to obtain the target product; the specific method is to add 100 mg of compound III into a round-bottom flask containing 20 mL of ultra-dry ethanol, and then add 1.1 times the equivalent of compound III 2-thiophenecarboxylic acid hydrazide, heating and reflux for 5 hours, until TLC monitors the completion of the reaction; stop the reaction, cool the reaction liquid to room temperature, a large amount of precipitate is generated, filter, and purify the filter cake with ethanol to obtain the target product: a bifunctional molecular probe that recognizes aluminum ions and zinc ions.

[0087] The target compound H-5 obtained in this example is a new compound that has not been reported before. After purification, high-resolution mass spectrometry (HRMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR) to characterize it. The high-resolution mass spectrum of probe H-5 is shown in Figure 1 As shown, the NMR of probe H-5 1 H NMR Figure 2 As shown, the NMR of probe H-5 13 C NMR Figure 3As shown. NMR characterization: 87.2%. 1 HNMR(500MHz,DMSO-d6)(Fig.S1)δ(ppm)12.21(s,1H),11.57(s,1H),8.62(s,1H),8.42(s,1H),8.08(d,J=24.6Hz,1 H),7.90(s,1H),7.71(s,2H),7.66(s,2H),7.17(d,J=7.1Hz,1H),6.98(s,1H),2.55(d,J=24.9Hz,4H),1.07(s,6H). 13 C NMR(101MHz,DMSO-d6)δ(ppm)174.33,160.31,155.82,151.22,150.60148.74,137.66,137.02,132.39,130.26,129.08,1 28.77,127.65,124.57,121.67,118.47,116.85,114.76,112.64,75.92,42.47,38.12,30.37,26.49,25.45HRMS(m / z)(TOF MS ES+):calcd for C 25 H 22 N4O2S:443.1542,[M+H + ] - found:443.1542.

[0088] In summary, the structural formula of the target compound H-5 synthesized in this example is as follows:

[0089]

[0090] Example 2

[0091] The fluorescent molecular probe H-5 synthesized in Example 1 was dissolved in DMF / H2O (5 / 5, v / v) to a concentration of 1×10 -5 mol / L buffer system to obtain H-5 probe solution.

[0092] Metal cations and disodium ethylenediaminetetraacetate (EDTA) are prepared into a 0.01 mol / L aqueous solution.

[0093] The concentrations of the H-5 probe solution, metal cation solution and EDTA solution used in the following application examples are all 0.01 mol / L, which are all configured in this example.

[0094] Study on the spectral modeling of a colorimetric-fluorescent dual-channel molecular probe for the recognition of aluminum ions and / or pH value

[0095] Application Example 1

[0096] To 5 mL of the H-5 probe solution prepared in Example 2, 25 μL of different metal ions (Fe 3+ 、Na + , Fe 2+ , Hg 2+ 、Ni 2+ , K + 、Cd 2+ Mg 2+ , Ca 2+ , Ba 2+ 、Ag + 、Co 2+ 、Zn 2+ Cr 3+ , Cu 2+ , Pb 2+ , Mn 2+ ), and then use a fluorescence spectrophotometer to detect the change in fluorescence intensity, thereby detecting its fluorescence selectivity for different metal ions. The test results are as follows: Figure 4 As shown. Figure 4 It can be seen that the H-5 probe itself has a weak fluorescence emission peak at 590nm under 430nm wavelength excitation. 3+ After that, the fluorescence spectrum shows that Al 3+ The addition of , the fluorescence emission peak of the probe solution red-shifted to 628nm. In summary, probe H-5 can be used for fluorescence-specific recognition of Al 3+， The fluorescence intensity was significantly enhanced; other metal ions (Fe 3+ 、Na + , Fe 2 + , Hg 2+ 、Ni 2+ , K + 、Cd 2+ Mg 2+ , Ca 2+ , Ba 2+ 、Ag + 、Co 2+ 、Zn 2+ Cr 3+ , Cu 2+ , Pb 2+ , Mn 2+ ) had almost no effect on the fluorescence intensity.

[0097] Figure 5 5 mL of H-5 probe solution prepared in Example 2 was added with 25 μL of Al 3+ UV spectrum after adding Al 3+After that, the 430nm ultraviolet absorption peak decreased and red-shifted to 453nm.

[0098] The above results show that after adding metal ions to the probe H-5 solution, Al 3+ Compared with other ions, probe H-5 is unique and can specifically recognize Al 3+ .

[0099] Application Example 2

[0100] The H-5 probe solution prepared in Example 2 was used to detect Al 3+ For other different metal cations (concentration of 5×10 -5 mol / L)Cu 2+ , K + , Fe 3+ , Fe 2+ 、Na + Cr 3+ , Ca 2+ 、Zn 2+ Mg 2+ , Mn 2+ , Ba 2+ , Hg 2+ , Pb 2+ 、Cd 2+ 、Co 2+ 、Ni 2+ 、Ag + The competitiveness of the probe solution was measured by measuring the fluorescence intensity of 5 mL of H-5 probe solution and 25 μL of different metal cations added thereto, as well as the fluorescence intensity of 5 mL of H-5 probe solution after adding 25 μL of other metal cations and then adding an equal amount of Al 3+ The fluorescence intensity of the system is Figure 6 As shown. It can be seen that probe H-5 has a strong affinity for Al 3+ The recognition has good anti-interference ability, and the presence of other metal ions in the system will not affect its recognition effect.

[0101] Application Example 3

[0102] The concentration of aluminum ions (from 0 to 5×10 -5 mol / L) was added to the H-5 probe solution prepared in Example 2, and the changes in its fluorescence and ultraviolet spectrum were detected. Figure 7 shown.

[0103] Figure 7 (A) shows the change of the UV spectrum of the system and Al 3+ With the increase of aluminum ion concentration, the ultraviolet absorbance at 340nm and 453nm gradually weakened, and the ultraviolet spectrum changed significantly, proving that probe H-5 can be used to detect Al3+ The quantitative detection is clear and easy to identify.

[0104] Figure 7 (B) shows the fluorescence spectrum change of the system and Al 3+ The relationship between the concentration and fluorescence intensity is that with the increase of aluminum ion concentration, the fluorescence intensity gradually increases, and the change of fluorescence intensity is obvious, which proves that the probe H-5 can be used for Al 3+ The quantitative detection is clear and easy to identify.

[0105] Application Example 4

[0106] The H-5 probe solution prepared in Example 2 was adjusted to different pH values ​​(pH=3-12), and its ultraviolet and fluorescence spectra were detected as the pH value changed. Figure 8 shown.

[0107] Figure 8 (A) shows the relationship between the UV spectrum change of the system and the pH value (pH = 3-12). In the solution (pH = 3-8), the UV spectrum peak of H-5 decreases with the increase of pH. When the solution pH increases from 8.5 to 12, the absorption band of H-5 red-shifts from 410nm to 530nm, and the UV spectrum peak of H-5 decreases with the increase of pH. It proves that the probe H-5 can be used for pH detection, and the phenomenon is obvious and easy to identify.

[0108] Figure 8 (B) shows the relationship between the fluorescence spectrum change of the system and the pH value (pH = 3-12). When the solution pH value = 3-8, the fluorescence spectrum peak of H-5 decreases with the increase of pH. As the pH of the solution increases from 8.5 to 12, the absorption band of H-5 increases from 590nm to 670nm, and the fluorescence spectrum peak of H-5 increases with the increase of pH, proving that the probe H-5 can be used to detect pH value, and the phenomenon is obvious and easy to identify.

[0109] Application Example 5

[0110] like Fig. 9 As shown in (A), 5 μL of ETA and Al were added alternately to 5 mL of the H-5 probe solution prepared in Example 2. 3+ The reversible change of fluorescence intensity at 628 nm was then detected. 3+ ] can be reversibly cycled for at least four cycles. H-5 has good reversibility in recognizing aluminum and can be used repeatedly.

[0111] like Fig. 9As shown in (B), the H-5 probe solution was alternately adjusted to pH = 7 and pH = 11, and the ratio of its fluorescence intensity at 670nm and 590nm was detected. The results show that the reversible change caused by pH can be cycled for at least four cycles, and H-5 has good reversibility for pH detection and can be used repeatedly.

[0112] Application Example 6

[0113] After adding 25 μL of aluminum ions to the H-5 probe solution prepared in Example 2, the color change was detected under sunlight. Fig.10 (A) shown. Fig.10 As shown in (A), after adding aluminum ions, the solution changes from yellow to orange. The probe H-5 of the present invention can detect aluminum ions by visual colorimetry. The fluorescence color change is observed under 365nm ultraviolet light. The detection results are shown in Fig.10 As shown in (B), after adding aluminum ions, the fluorescence color changes from orange-yellow fluorescence to bright orange-red fluorescence under 365nm irradiation under ultraviolet light. The probe of the present invention can also realize visual qualitative detection of aluminum ions under ultraviolet irradiation.

[0114] Application Example 7

[0115] The H-5 probe solution prepared in Example 2 was adjusted to different pH values ​​and its color change under sunlight was detected. Fig.11 (A) Fig.11 (A) It can be seen that the solution changes from yellow to orange and then to purple from left to right. The probe of the present invention can detect the pH value of the system by visual colorimetry.

[0116] The H-5 probe solution prepared in Example 2 was adjusted to different pH values ​​and placed under a UV lamp at 365 nm to observe the fluorescence color change. Fig.11 As shown in (B), from left to right, when placed under 365nm ultraviolet light, the fluorescence color changes significantly from fluorescence. The probe of the present invention can also achieve visual qualitative detection of pH value under ultraviolet light.

[0117] Application Example 8

[0118] Hela cells were cultured in DMEM medium containing penicillin (100 units / mL), streptomycin (100 μg / mL) and 10% fetal bovine serum at 37°C. Hela cells were prepared at a concentration of 5×104 cells / mL, seeded in a 24-well plate containing a coverslip, and allowed to adhere overnight at 37°C and 5% CO2. The probe was prepared in a 10 mM H-5 stock solution containing DMSO and diluted to a 10 μM concentration in the culture medium. Al was diluted with ultrapure water. 3+Prepare at 10 mM and dilute to 50 μM in culture medium.

[0119] The cells were transferred to the culture medium in a 24-well plate and Al 3+ ions (0 μM, 10 μM, 20 μM), incubate for 30 min, then add probe medium, mix gently, incubate for another 30 min, rinse twice with buffer, and place the coverslip containing cells on a slide for confocal microscopy. The medium at a specific pH is prepared with buffer.

[0120] The cells were aspirated into the culture medium in a 24-well plate, a culture medium with a specific pH (pH = 4.0, pH = 7.0, pH = 10.0) was added, and the cells were incubated for 30 min, and then the probe culture medium was added, mixed gently, and the incubation continued for 30 min. The cells were rinsed twice with buffer, and the coverslip containing the cells was placed on a slide for confocal microscopy.

[0121] Four-day-old wild-type zebrafish were cultured in embryo medium without tyrosinase inhibitor (PTU) at 28.5°C for 36 hours. The zebrafish were divided into three main groups, one of which was treated with liver buffer as a control. The second group of zebrafish were exposed to different concentrations of H-5 (10 μM), H-5+Al 3+ (10μM, 5μM) and H-5+Al 3+ (10μM, 10μM) solution for 30 minutes. Similarly, the third group of zebrafish was incubated in H-5 (10μM, pH=4.0), H-5 (10μM, pH=7.0), and H-5 (10μM, pH=10.0) for 30 minutes. Prior to imaging, zebrafish larvae were washed twice with HEPES buffer and then fixed in the imaging tray. Fluorescence imaging parameters were similar to those for cell imaging. The fluorescence excitation wavelength was 405nm, and the aggregation ranges were 550-600nm and 590-670nm.

[0122] Fluorescence images of aluminum ions detected by probe H-5 in Hela cells (purchased from Shanghai Biotechnology Co., Ltd.) and zebrafish, such as Fig.12 shown.

[0123] like Fig.12 As shown in (A), probe H-5 detects Al in Hela cells 3+ Fluorescence images of Hela cells: 0.01 mol / L Hepes (CAS: 7365-45-9, purchased from Aladdin Reagent (Shanghai) Co., Ltd.) buffer solution, H-5 (10 μM), H-5 (10 μM) + Al 3+ (5μM), H-5(10μM)+Al 3+(10 μM) after incubation. 3+ As the concentration increases, the fluorescence signal gradually increases.

[0124] like Fig.12 (B) Probe H-5 detects Al in living zebrafish 3+ Fluorescence images of zebrafish: Hepes buffer solution, H-5 (10 μM), H-5 (10 μM) + Al 3+ (5μM), H-5(10μM)+Al 3+ (10μM) after incubation. 3+ As the concentration increases, the fluorescence signal gradually increases.

[0125] Application Example 9

[0126] Fluorescence images of probe H-5 in Hela cells and zebrafish at different pH values, such as Fig.13 shown.

[0127] like Fig.13 (A) shows the fluorescence images of probe H-5 under different pH conditions: the changes in fluorescence imaging of Hela cells after incubation with Hepes buffer solution, H-5 (10 μM) solution at pH = 4.0, 7.0, and 10.0. As the pH increases, the green fluorescence signal becomes weaker and the red fluorescence signal becomes stronger.

[0128] like Fig.13 (B) shows the fluorescence image of probe H-5 in living zebrafish: the changes in fluorescence imaging of Hela cells after incubation with Hepes buffer solution, H-5 (10 μM) solution at pH = 4.0, 7.0, and 10.0. As the pH increases, the green fluorescence signal becomes weaker and the red fluorescence signal becomes stronger.

[0129] The results showed that the probe H-5 can be used to monitor and study the dynamic changes of aluminum ion concentration and / or pH value in real time in cellular environments and living organisms.

[0130] The results showed that the probe H-5 can be used to monitor and study the dynamic changes of metal aluminum ion levels in real time in cellular environments and living organisms.

[0131] The present invention designs and synthesizes a dual-function fluorescent molecular probe that specifically recognizes aluminum ions and / or pH values. The probe H-5 has enhanced fluorescence after adding aluminum ions, and has high selectivity and anti-interference. In addition, the detection limit of the probe H-5 can reach 2.54×10 -9M; When the solution pH (pH = 3-12) changes, the fluorescence and ultraviolet spectra of H-5 change accordingly. When the solution pH = 3-8, the fluorescence spectrum peak of H-5 decreases with the increase of pH. As the solution pH increases from 8.5 to 12, the absorption band of H-5 increases, and the fluorescence spectrum peak of H-5 increases with the increase of pH. In the solution (pH = 3-8), the ultraviolet spectrum peak of H-5 decreases with the increase of pH. When the solution pH increases from 8.5 to 12, the absorption band of H-5 red-shifts, and the ultraviolet spectrum peak of H-5 decreases with the increase of pH.

[0132] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A colorimetric-fluorescent dual-channel molecular probe for identifying aluminum ions and / or detecting pH values, characterized in that: The structural formula of the probe is shown in Formula 1:

2. The method for preparing the colorimetric-fluorescent dual-channel molecular probe according to claim 1, characterized in that: It is obtained by reacting compound III with 2-thiophenecarboxylic acid hydrazide, and its synthesis reaction equation is shown in the following formula 2:

3. The preparation method according to claim 2, characterized in that: The method specifically comprises the following steps: Compound III is added to ultra-dry ethanol, followed by addition of 2-thiophenecarboxylic acid hydrazide, and the mixture is heated to reflux for reaction; after stopping the reaction, the mixture is cooled to room temperature, the obtained product is filtered, and the obtained filter cake is slurried and purified with anhydrous ethanol to obtain the target product.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the compound III to 2-thiophenecarboxylic acid hydrazide is 1:1.

1.

5. The preparation method according to claim 3, characterized in that: The heating reflux reaction time is 4-8h.

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

7. Use of the colorimetric-fluorescent dual-channel molecular probe according to claim 1 or the colorimetric-fluorescent dual-channel molecular probe according to any one of claims 2 to 6 in sensing the aluminum ion content and / or pH value in a water environment system.

8. The use according to claim 7, characterized in that: The sensing detection includes fluorescence detection, ultraviolet ratio detection, visual qualitative detection or reversible detection.

9. A complex of a colorimetric-fluorescent dual-channel molecular probe and an aluminum ion, characterized in that: The colorimetric-fluorescent dual-channel molecular probe is the colorimetric-fluorescent dual-channel molecular probe according to claim 1 or the colorimetric-fluorescent dual-channel molecular probe according to any one of claims 2-6.

10. The method for preparing the complex of the colorimetric-fluorescent dual-channel molecular probe and aluminum ion according to claim 9, characterized in that: The colorimetric-fluorescent dual-channel molecular probe and an equimolar amount of aluminum ions are uniformly mixed in a mixed solution of DMF and deionized water to obtain a complex of the colorimetric-fluorescent dual-channel molecular probe and the aluminum ions.

Images