Difunctional fluorescent molecular probe, preparation method thereof and application of difunctional fluorescent molecular probe in detection of aluminum ions and / or zinc ions

Abstract

The invention discloses a bifunctional fluorescent molecular probe, a preparation method thereof and application of the bifunctional fluorescent molecular probe in detection of aluminum ions and / or zinc ions, and relates to the technical field of detection of the aluminum ions and the zinc ions. The bifunctional fluorescent molecular probe disclosed by the invention is specifically prepared by reacting a compound III with 2-furoyl hydrazine. Based on specific recognition, the bifunctional fluorescent molecular probe disclosed by the invention can be used as a specific indicator for existence of aluminum ions and zinc ions in living cells and zebra fish bodies, real-time qualitative and quantitative information can be provided through fluorescence detection, color change occurs, and the detection sensitivity is high. Therefore, the indicator becomes a highly specific indicator suitable for visual colorimetry detection; meanwhile, the probe has high reversible repeatability on the detection of the aluminum ions and the zinc ions, can be used as a sensor for detecting the aluminum ions and the zinc ions in living cells and zebra fish bodies, and can perform repeated detection without damaging the performance of the probe.

Description

Technical Field

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

[0002] Aluminum ions are the most widely distributed form of aluminum in nature. They are widely used in water, soil, air and most biological tissues, and are widely used in food packaging, water purification, medicines, 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 Al 3+ When the concentration of Al exceeds a certain range, it will lead to many neurodegenerative and neurological diseases, including dementia, osteomalacia, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. 3+ Aluminum concentrations have been identified as being critical for aquatic life and crop yields due to increased soil acidity. The tolerable daily intake of aluminum ions by humans is approximately 3-10 mg per day. According to the World Health Organization, Al 3+ The maximum allowable content of Al in drinking water is 7.41 μM. 3+ Considering the accumulation of aluminum and its negative effects on human tissues, it is of great significance to develop an efficient and rapid method for aluminum identification and monitoring.

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

[0004] Existing Al 3+ and Zn 2+There are many detection methods such as inductively coupled plasma spectrometry (ICP-MS), anodic stripping voltammetry, photochemical flame atomic absorption spectrometry and electrochemical gel electromembrane extraction. These methods are applied in various fields and have the advantages of high sensitivity and fast response speed. However, these technologies 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 is low-cost, easy to operate, has no high requirements on operators, responds quickly, and can be used for biological imaging. Therefore, research on the fluorescence determination of aluminum ions and zinc ions is urgently needed. Summary of the invention

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

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

[0007] In order to achieve the above object, the first aspect of the present invention provides a bifunctional fluorescent molecular probe, wherein the structural formula of the bifunctional fluorescent molecular probe is shown in the following formula 1:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the bifunctional fluorescent molecular probe described in the first aspect, specifically, reacting compound III with 2-furoylhydrazide to obtain the target product, and the reaction equation is shown in the following formula 2:

[0010]

[0011] Furthermore, in the above technical scheme, the preparation method of the bifunctional fluorescent molecular probe comprises the following steps: adding compound III to ultra-dry ethanol, then adding 2-furoylhydrazide, heating and reflux 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, and purifying the obtained filter cake after pulping with anhydrous ethanol, and drying to obtain the bifunctional fluorescent molecular probe.

[0012] 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.

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

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

[0015] 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:

[0016] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0017] Step 2, reacting compound I with p-hydroxybenzaldehyde to prepare compound II;

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

[0019] Specifically, the synthesis reaction equation of the above-mentioned compound III is shown in the following formula 3:

[0020]

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

[0022] 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.

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

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

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

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

[0027] 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.

[0028] 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.

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

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

[0031] Furthermore, in the above technical scheme, the specific synthesis steps of compound III in step S3 are as follows:

[0032] 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 filtered and purified by column chromatography to obtain the compound III.

[0033] 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.

[0034] Preferably, in 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 use 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 and 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 and 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] Furthermore, in the above technical solution, the specific preparation method of the complex is as follows:

[0038] The bifunctional fluorescent molecular probe is mixed 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 and the 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.

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

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

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

[0043] Through the above technical solution, 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, and 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 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, and can perform repeated detections 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, the raw materials are easily available, and the operation is simple.

[0046] (3) The present invention realizes the quantitative detection of aluminum ions and zinc ions by the different functions of the probe displayed by ultraviolet and fluorescence spectra. It has strong reversibility, can be recycled many times, has good selectivity, is not easily affected by other ions, has a low detection limit, and can be widely used to detect aluminum ions and zinc ions in complex environments.

[0047] Aluminum ion: UV detection limit is 4.00×10 -8 M, the fluorescence detection limit is 1.42×10 -7 M;

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

[0049] 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.

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

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

[0052] Figure 3 The nuclear magnetic resonance of the bifunctional fluorescent molecular probe H-4 synthesized in Example 1 of the present invention is 13 CNMR image.

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

[0054] Figure 5 H-4 and H-4+Al in Application Example 1 of the present invention 3+ and H-4+Zn 2+ Ultraviolet absorption spectrum of .

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

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

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

[0058] Fig. 9 (A) is H-4+Al in Application Example 4 of the present invention 3+ Reversibility detection diagram, (B) is H-4+Zn 2+ Reversibility detection diagram.

[0059] Fig.10 The probes H-4 and H-4+Al in Application Example 5 of the present invention are3+ and H-4+Zn 2+ The effect in daylight.

[0060] Fig.11 The probes H-4 and H-4+Al in Application Example 5 of the present invention are 3+ and H-4+Zn 2+ The effect diagram under 365nm ultraviolet light.

[0061] Fig.12 The present invention uses the probe H-4 in Example 6 to detect Al in living cells. 3+ and Zn 2+ Application imaging diagram.

[0062] Fig.13 The present invention uses the probe H-4 in Example 7 to detect Al in zebrafish 3+ and Zn 2+ Application imaging diagram. DETAILED DESCRIPTION

[0063] Existing fluorescent probes only use fluorescence spectra to quantitatively detect metal ions. The bifunctional fluorescent molecular probe of the present invention can provide a variety of different data by combining ultraviolet ratio quantitative detection with fluorescence spectrum quantitative detection, increasing the accuracy and reliability 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 bifunctional 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 365nm ultraviolet light. This dual observation mode makes it a chromogenic sensing fluorescent probe suitable for different lighting conditions.

[0064] The present invention is further described in detail below through implementation cases. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following implementation cases.

[0065] 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.

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

[0067]

[0068] The second aspect of the present invention provides a method for preparing the bifunctional fluorescent molecular probe described in the first aspect, specifically, reacting compound III with 2-furoylhydrazide to obtain the target product, and the reaction equation is shown below:

[0069]

[0070] 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:

[0071] Step 1, using isophorone and malononitrile as reaction raw materials to prepare compound I;

[0072] Step 2, reacting 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] The third aspect of the present invention provides the use 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.

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

[0077] The fourth aspect of the present invention provides 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 and aluminum ions and / or zinc ions.

[0078] In the present invention, the preparation method of the complex may include: mixing 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 and aluminum ions and / or zinc ions.

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

[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 spectroscopy and fluorescence spectroscopy.

[0082] 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.

[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: using isophorone and malononitrile as main reaction raw materials to prepare compound I; the specific method is as follows:

[0087] 6.91 g of isophorone (50.1 mmol) was dissolved in 100 mL of anhydrous ethanol, and then 3.84 g of malononitrile (60.1 mmol) and 0.3 mL of piperidine (3.0 mmol) were added in sequence. The mixture was heated, stirred and refluxed for 6 h under a nitrogen atmosphere. The isophorone was completely reacted as monitored by TLC, and the reaction was stopped. The mixture was allowed to stand and cool to room temperature. The obtained product was then poured into 200 mL of ice water to generate a large amount of off-white precipitate. The solid obtained by suction filtration was separated and purified by column chromatography to obtain 7.23 g of a white crystalline solid, i.e., compound I.

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

[0089] p-Hydroxybenzaldehyde (10.2 mmol) was 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 were added in sequence, and the mixture was heated under reflux for reaction until the raw material spot disappeared as monitored by TLC spot plate, and the reaction was stopped; the reaction solution was spin-dried and the obtained product was separated and purified by column chromatography to obtain compound II;

[0090] Step 3, reacting compound II with hexamethylenetetramine to prepare compound III; the specific method is:

[0091] Compound II (1.5 mmol) was dissolved in 10 mL of trifluoroacetic acid, and then 1.5 mmol of hexamethylenetetramine was added, and the mixture was heated under reflux for 6 h. The reaction was stopped after TLC monitoring. The obtained product was cooled to room temperature, and then slowly poured into 50 mL of ice water. The product obtained by filtration was separated and purified by column chromatography to obtain compound III.

[0092] Step 4: react compound III with 2-furancarboxylic acid hydrazide to obtain the target product; the specific method is:

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

[0094] The target compound H-4 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-4 is shown in Figure 1 As shown, [M+H + ] + The molecular formula is C 25 H 22 N4O3, the theoretical value is 427.1770, and the actual value found in the mass spectrum is 427.1768. 1 H NMR Figure 2 As shown, the NMR of probe H-4 13 C NMR Figure 3 shown. 1 H NMR(400MHz, DMSO-d6)(δppm)12.24(s,1H),11.60(s,1H),8.64(s,1H),7.98(d,J=0.8Hz,1H),7.87(s,1H),7.70(dd,J=8.6,1.8Hz, 1H), 7.34 (d, J = 2.7Hz, 1H), 7.28 (s, 2H), 6.98 (d, J = 8.6Hz, 1H), 6.83 (s, 1H), 6.73 (dd, J = 3.4, 1.7Hz, 1H), 2.54 (s, 4H), 1.02 (s, 6H). 13 C NMR(101MHz,DMSO-d6)(δppm):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] The fluorescent molecular probe H-4 synthesized in Example 1 was dissolved 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] Metal cations and disodium ethylenediaminetetraacetate (EDTA) are prepared into a 0.01 mol / L aqueous solution.

[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 ethylenediaminetetraacetic acid aqueous solution used in the following application examples are all prepared according to Example 2.

[0102] Application Example 1

[0103] To 5 mL of probe H-4 solution, 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+ ), fluorescence spectra such as Figure 4 As shown. Figure 4 It can be seen that adding Al 3+ After that, the fluorescence intensity increased significantly, while Zn 2+ The addition of ions resulted in a large Snox shift, while the addition of other metal ions did not cause obvious changes in fluorescence intensity.

[0104] Figure 5 The probe H-4 solution and Zn 2+ and Al 3+ UV spectrum after addition of Zn 2+ and Al 3+After that, the 426nm ultraviolet absorption peak decreased and red-shifted to 480nm and 450nm respectively.

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

[0106] Application Example 2

[0107] Detection of Al using H-4 probe solution 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 + The fluorescence intensity of the H-4 probe solution and the different metal cations added thereto were measured, and the fluorescence intensity of the H-4 probe solution was measured by adding 25 μL of other metal cations to 5 mL of the H-4 probe solution and then adding an equal amount of Al 3+ The fluorescence intensity of the system is Figure 6 As shown in (A). It can be seen that probe H-4 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.

[0108] Detection of Zn using H-4 probe solution 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 + competitiveness, such as Figure 6 (B) As shown. It can be seen that except Al 3+ In addition, probe H-4 has a strong affinity for Zn 2+ It also has good anti-interference ability.

[0109] Application Example 3

[0110] Different concentrations of aluminum ions or zinc ions were added to the H-4 probe solution, and the changes in its fluorescence spectrum were detected, such as Figure 7 shown.

[0111] Figure 7 (A) shows the fluorescence spectrum change of the system and Al 3+ The relationship between the concentration of aluminum ions and the fluorescence intensity is shown in Table 1. 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-4 can be used for Al 3+ The quantitative detection is clear and easy to identify.

[0112] Figure 7 (B) shows the fluorescence spectrum change of the system with Zn 2+ With the increase of zinc ion concentration, the fluorescence gradually red-shifted and an obvious Snokes shift occurred, which proved that probe H-4 can be used for Zn 2+ The quantitative detection is clear and easy to identify.

[0113] Different concentrations of aluminum ions or zinc ions were added to the H-4 probe solution, and the changes in its UV spectrum were detected, such as Figure 8 shown.

[0114] Figure 8 (A) shows the change of the UV spectrum of the system and Al 3+ With the increase of aluminum ion concentration, the 426nm ultraviolet absorbance gradually weakened and red-shifted to 450nm. The changes in the ultraviolet spectrum were obvious, proving that probe H-4 can be used for Al 3+ The quantitative detection is clear and easy to identify.

[0115] Figure 8 (B) shows the change of the UV spectrum of the system and Zn 2+ With the increase of zinc ion concentration, the 426nm ultraviolet absorbance gradually weakened and red-shifted to 480nm. The changes in the ultraviolet spectrum were obvious, which proved that probe H-4 can be used for Zn 2+ The quantitative detection is clear and easy to identify.

[0116] Application Example 4

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

[0118] like Fig. 9 As shown in (B), 5 μL of EDTA and Zn were added alternately to 5 mL of H-4 probe solution. 2+ (Concentration is 1×10 -5 mol / L) and then detect the reversible change of its fluorescence intensity at 650nm.

[0119] The results showed that probe H-4 had a strong affinity for Zn 2+ and Al 3+ The recognition has good reversibility and can be used repeatedly.

[0120] Application Example 5

[0121] Fig.10 The color change of the solution under sunlight after adding 25 μL aluminum ions or zinc ions to 5 mL H-4 probe solution. 3+ The color changes slightly from light yellow to dark yellow on the left. Adding Zn 2+ The color changes from light yellow to orange on the right. The probe of the present invention can detect aluminum ions and zinc ions by visual colorimetry.

[0122] Fig.11 After adding 25 μL aluminum ions or zinc ions to 5 mL H-4 probe solution, the solution color changes under 365 nm ultraviolet light. 3+ The color changes from yellow to orange after adding Zn 2+ The color then changes to orange-red on the right. The probe of the present invention can also achieve visual qualitative detection of aluminum ions and zinc ions under ultraviolet irradiation.

[0123] Application Example 6

[0124] The fluorescence image of aluminum ions or zinc ions in Hela cells (purchased from Synbio (Shanghai) Biotechnology Co., Ltd.) detected by probe H-4 is shown in Figure 2. Fig.12 shown.

[0125] like Fig.12 As shown in (A), probe H-4 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-4 (10 μM), H-4 (10 μM) + Al3+ (5μM), H-4(10μM)+Al 3+ (10 μM) after incubation. 3+ As the concentration increases, the green fluorescence signal becomes stronger and stronger.

[0126] like Fig.12 As shown in (B), probe H-4 detects Zn in Hela cells 2+ Fluorescence images of Hela cells incubated with Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Zn 2+ (5μM), H-4(10μM)+Zn 2+ (10 μM) after incubation. 2+ As the concentration increases, the green fluorescence signal becomes weaker and the red fluorescence signal becomes stronger.

[0127] The results showed 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 cellular environment in real time.

[0128] Application Example 7

[0129] Fluorescence images of probe H-4 detecting aluminum ions / zinc ions in zebrafish, such as Fig.13 shown.

[0130] like Fig.13 (A) shows that probe H-4 detects Al in zebrafish 3+ Fluorescence images of zebrafish: Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Al 3+ (5μM), H-4(10μM)+Al 3+ (10 μM) after incubation. 3+ As the concentration increases, the green fluorescence signal becomes stronger and stronger.

[0131] like Fig.13 (B) Probe H-4 detects Zn in zebrafish 2+ Fluorescence images of zebrafish incubated with Hepes buffer solution, H-4 (10 μM), H-4 (10 μM) + Zn 2+ (5μM), H-4(10μM)+Zn 2+ (10 μM) after incubation. 2+ As the concentration increases, the fluorescence of the green channel becomes weaker and weaker, while the fluorescence of the red channel becomes stronger and stronger.

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

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

[0134] 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 dual-function fluorescent molecular probe, characterized in that: The structural formula of the bifunctional fluorescent molecular probe is shown in Formula 1 below:

2. A method for preparing the bifunctional fluorescent molecular probe according to claim 1, characterized in that: The target product is obtained by reacting compound III with 2-furancarboxylic acid hydrazide. The reaction equation is shown in the following formula 2:

3. The preparation method according to claim 2, characterized in that: The method comprises the following steps: adding compound III to ultra-dry ethanol, then adding 2-furoylhydrazide, heating and refluxing for reaction; after the reaction is completed, cooling the obtained product to room temperature, filtering, and purifying and drying the obtained filter cake after pulping with anhydrous ethanol to obtain the bifunctional fluorescent molecular probe.

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

1.

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

6. The preparation method according to claim 1, 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 bifunctional fluorescent molecular probe according to claim 1 or the bifunctional fluorescent molecular probe prepared by the preparation method according to any one of claims 2 to 6 in sensing and detecting aluminum ions and / or zinc ions.

8. A complex of a bifunctional fluorescent molecular probe and aluminum ions and / or zinc ions, characterized in that: The bifunctional fluorescent molecular probe is the bifunctional fluorescent molecular probe described in claim 1 or the bifunctional fluorescent molecular probe prepared by the preparation method described in any one of claims 2-6.

9. The method for preparing the complex of the bifunctional fluorescent molecular probe and aluminum ion and / or zinc ion according to claim 8, characterized in that: The method steps are as follows: The bifunctional fluorescent molecular probe is mixed 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 and the aluminum ions and / or zinc ions.

10. Use of the complex of the bifunctional fluorescent molecular probe and aluminum ions and / or zinc ions as claimed in claim 8 or the complex of the bifunctional fluorescent molecular probe and aluminum ions and / or zinc ions prepared by the method according to claim 9 in in vivo sensing detection of living cells and zebrafish.

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