Zinc ion fluorescent probe based on 8-aminoquinoline as well as preparation method and application of zinc ion fluorescent probe

Abstract

The invention discloses a zinc ion fluorescent probe based on 8-aminoquinoline as well as a preparation method and application of the zinc ion fluorescent probe based on 8-aminoquinoline, and the structural formula of the zinc ion fluorescent probe based on 8-aminoquinoline is shown in the specification. The fluorescent probe APYQ capable of recognizing Zn < 2 + > with high selectivity is designed and synthesized by taking 8-aminoquinoline as a parent, introducing acetamido at a site 2 and reacting with salicylaldehyde at a site 8, is not interfered by other metal ions, and is simple in synthesis step and easy in purification process. The fluorescent probe has excellent detection limit (1.87 nM) and response time (5s), and the two indexes have the best comprehensive performance compared with other Zn < 2 + > probes in recent 5 years. The fluorescent probe can be applied to'naked eye 'detection of Zn < 2 + > on an ethanol / HEPES buffer solution and test paper, can also be applied to detection of fluorescence imaging of Zn < 2 + > in Hela cells, and has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule fluorescent probes, and specifically relates to a zinc ion fluorescent probe based on 8-aminoquinoline and a preparation method and application thereof. Background Art

[0002] Zinc is the second most abundant transition metal element in the human body, and its optimal concentration in the human body is essential for cell replication, nucleic acid metabolism, tissue repair, growth and development. Zinc ion deficiency in the human body can lead to serious neurological diseases such as Alzheimer's disease. Similarly, excessive zinc ions can cause Parkinson's disease. In addition, excessive zinc ions in the environment can lead to heavy metal ion pollution, and the World Health Organization (WHO) stipulates that the concentration of zinc ions in drinking water should not exceed 76μM. Therefore, it is meaningful to develop rapid and highly sensitive zinc ion detection methods in the environmental and biological fields.

[0003] At present, traditional zinc ion detection technologies mainly include atomic absorption spectroscopy (AAS), electrochemical analysis, spectrophotometry, etc. However, the application of these technologies is limited by factors such as high equipment cost, long detection time, low sensitivity and complex operation process. Compared with these traditional technologies, fluorescence spectroscopy has attracted much attention due to its portability, simple operation, fast response speed and real-time in-situ detection.

[0004] In recent years, quinoline derivatives have been reported as fluorescent molecular probes, especially 8-aminoquinoline derivatives, which are often used to identify zinc ions. However, these reported zinc ion probes still have a lot of room for improvement and enhancement. On the one hand, cadmium ions have similar electronic configurations to zinc ions, which poses a problem for the highly selective identification of zinc ions. On the other hand, the detection limits of these probes (10 -5 ~10 -7 mol·L -1 ) is high, which limits its application range. Therefore, how to develop a fluorescent probe with high selectivity, high sensitivity and rapid detection of zinc ions has become a challenging topic. Summary of the invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention aims to provide a zinc ion fluorescent probe based on 8-aminoquinoline and its preparation method and application. The fluorescent probe compound of the present invention is for Zn 2+ It has high selectivity, high sensitivity (1.87nM) and rapid detection (5s), and can identify and detect Zn with the naked eye. 2+ , can be applied to Hela cell fluorescence imaging and has good application prospects.

[0006] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0007] A zinc ion fluorescent probe based on 8-aminoquinoline, the structural formula of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe, comprising the following steps:

[0010]

[0011] (1) reacting 2-acetamido-8-nitroquinoline with a reducing agent to obtain an intermediate AAQ;

[0012] (2) reacting the intermediate AAQ and salicylaldehyde under acidic conditions to obtain the zinc ion fluorescent probe APYQ.

[0013] As a specific implementation scheme, in step (1), the reducing agent is selected from iron; the solvent of the reaction is selected from acetic acid; the reaction is carried out under the protection of an inert gas at 60 to 70° C. for 1.5 to 2.5 hours; and the molar ratio of the 2-acetamido-8-nitroquinoline to the reducing agent is 1:4 to 1:6.

[0014] As a specific embodiment, in step (1), the post-treatment method of the reaction includes: filtering, washing, removing the solvent, extracting with ethyl acetate, drying, removing the solvent, purifying the crude product by silica gel column, and separating with ethyl acetate and petroleum ether = 1:2 to 1:3 (v / v) as eluent to obtain the intermediate AAQ.

[0015] As a specific implementation scheme, in step (2), the acid in the acidic condition is selected from acetic acid; the solvent of the reaction is selected from anhydrous ethanol; the reaction is carried out under the protection of an inert gas at 80-90° C. for 2.5-3.5 hours; the molar ratio of the intermediate AAQ to acetic acid is 1:1-1:3, and the molar ratio of the intermediate AAQ to salicylaldehyde is 1:1-1:1.2.

[0016] As a specific implementation scheme, in step (2), the post-treatment method of the reaction includes: filtering the precipitate, washing with anhydrous ethanol, and drying to obtain the zinc ion fluorescent probe APYQ.

[0017] As a more preferred embodiment, the method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe comprises the following steps:

[0018] A method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline is carried out according to the following steps:

[0019] (1) Preparation of AAQ: 2-acetamido-8-nitroquinoline was dissolved in acetic acid, iron powder was slowly added, and the mixture was stirred at 65° C. for 2 h under nitrogen protection. The mixture was filtered and washed while hot, and the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column, and separated using ethyl acetate and petroleum ether in a ratio of 1:2 to 1:3 (v / v) as eluent to obtain the intermediate AAQ.

[0020] (2) Preparation of APYQ: salicylaldehyde was dissolved in anhydrous ethanol, and then acetic acid was added as a catalyst. Under nitrogen protection, the mixture was refluxed at 85°C for 10 min, and the intermediate AAQ was added. The mixture was refluxed at 85°C for 3 h. The precipitate was filtered, washed three times with anhydrous ethanol, and dried to obtain the fluorescent probe APYQ.

[0021] Its synthetic route is as follows:

[0022]

[0023] The present invention also provides application of the zinc ion fluorescent probe based on 8-aminoquinoline in zinc ion detection.

[0024] Finally, the present invention provides a method for detecting zinc ions, which comprises using the zinc ion fluorescent probe based on 8-aminoquinoline to detect zinc ions in a solution.

[0025] As a specific implementation, the method includes detecting zinc ions in an ethanol / HEPES buffer solution; preferably, the pH of the ethanol / HEPES buffer solution is 7.4±0.5, wherein the volume ratio of ethanol to HEPES buffer solution is (7-9):(3-1).

[0026] As a specific implementation scheme, under the action of zinc ions, the solution containing the zinc ion fluorescent probe changes from colorless to yellow under visible light and from blue to green under ultraviolet light.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] (1) The present invention uses 2-acetamido-8-nitroquinoline as a raw material to synthesize a fluorescent probe, and the synthesis steps are simple and the product separation and purification process is easy.

[0029] (2) Fluorescent probe APYQ for Zn 2+ Highly selective and unaffected by Na + , K + , Li + , Ag + , Ba 2+ , Ca 2+ , Cd 2+ ,Co2+ , Fe 2+ , Cu 2+ ,Mg 2+ , Ni 2+ , Pb 2+ , Hg 2+ , Mn 2+ , Al 3+ ,Cr 3+ , Fe 3+ Interference from metal ions.

[0030] (3) In the range of 0 to 10 μM, Zn 2+ There is a good linear relationship between concentration and probe fluorescence intensity. The detection limit of the probe is as low as 1.87nM, and the response time is as low as 5s. Considering the detection limit and response time, the fluorescent probe APYQ has the best performance compared with the probes reported in the past five years.

[0031] (4) The fluorescent probe APYQ can identify and detect Zn with the naked eye 2+ , which can be actually applied to ethanol / HEPES buffer solution and probe-pretreated test paper, showing obvious color change. In biological applications, adding Zn 2+ The color change of cell imaging before and after is obvious, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The intermediate AAQ of the present invention 1 H NMR (400 MHz, DMSO-d6) spectrum;

[0033] Figure 2 The intermediate AAQ of the present invention 13 C NMR (100 MHz, DMSO-d6) spectrum;

[0034] Figure 3 The mass spectrum of the intermediate AAQ of the present invention;

[0035] Figure 4 The fluorescent probe APYQ of the present invention 1 H NMR (400 MHz, DMSO-d6) spectrum;

[0036] Figure 5 The fluorescent probe APYQ of the present invention 13 C NMR (100 MHz, DMSO-d6) spectrum;

[0037] Figure 6 The mass spectrum of the fluorescent probe APYQ of the present invention;

[0038] Figure 7The ultraviolet spectra of the fluorescent probe APYQ of the present invention with different metal ions added;

[0039] Figure 8 The fluorescence spectra of the fluorescent probe APYQ of the present invention when different metal ions are added;

[0040] Fig. 9 The fluorescent probe APYQ of the present invention is added with different concentrations of Zn 2+ UV spectrum of

[0041] Fig.10 The fluorescent probe APYQ of the present invention is added with different concentrations of Zn 2+ Fluorescence spectrum of

[0042] Fig.11 The fluorescence intensity of the fluorescent probe APYQ at 535 nm is similar to that of Zn 2+ Linear relationship graph of concentration;

[0043] Fig.12 The fluorescent probe APYQ of the present invention detects Zn 2+ Response time graph of

[0044] Table 1 The fluorescent probe APYQ of the present invention and other Zn 2+ Probe structural formula, detection limit and response time;

[0045] Fig.13 The fluorescent probe APYQ of the present invention and other Zn 2+ Probe detection of Zn 2+ Comparison chart of detection limit and response time performance;

[0046] Fig.14 The color change diagram of the fluorescent probe APYQ of the present invention when different metal ions are added under natural light;

[0047] Fig.15 The fluorescent probe APYQ of the present invention is added with different concentrations of Zn under natural light. 2+ Color change diagram of

[0048] Fig.16 The color change diagram of the fluorescent probe APYQ of the present invention when different metal ions are added under ultraviolet light;

[0049] Fig.17 The fluorescent probe APYQ of the present invention was added with different concentrations of Zn under ultraviolet light. 2+ Color change diagram of

[0050] Fig.18 Selectivity and sensitivity color change diagram of the test paper pretreated with the fluorescent probe APYQ of the present invention under natural light;

[0051] Fig.19Selectivity and sensitivity color change diagram of the test paper pretreated with the fluorescent probe APYQ of the present invention under ultraviolet light;

[0052] Fig. 20 The fluorescent probe APYQ of the present invention detects Zn in Hela cells 2+ Cell imaging diagram. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline comprises the following steps:

[0056] (1) 0.4 g of 2-acetamido-8-nitroquinoline was added to a 100 mL round-bottom flask and dissolved in 50 mL of acetic acid. 0.72 g of iron powder was slowly added. Under nitrogen protection, the round-bottom flask was placed in an oil bath and stirred at 65° C. for 2 h. After hot filtration and washing, the solvent was removed by distillation under reduced pressure. The mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column and separated using ethyl acetate and petroleum ether = 1:3 (v / v) as eluent to obtain 0.31 g of white powder intermediate AAQ with a yield of 89.1%;

[0057] (2) In a 50 mL round-bottom flask, 0.15 g of salicylaldehyde was dissolved in 15 mL of anhydrous ethanol, and then 150 μL of acetic acid was added as a catalyst. Under nitrogen protection, the round-bottom flask was placed in an oil bath and refluxed at 85°C for 10 min. 0.2 g of the intermediate AAQ was added and refluxed at 85°C for 3 h. The precipitate was filtered and washed three times with anhydrous ethanol. After drying, 0.22 g of orange-red powder fluorescent probe APYQ was obtained with a yield of 73.8%.

[0058] Example 2

[0059] The fluorescence probe APYQ obtained in Example 1 is effective for Zn 2+ The selectivity includes the following steps:

[0060] Prepare the fluorescent probe APYQ mother solution with a concentration of 1mM with anhydrous ethanol, and prepare various metal ion aqueous solutions with a concentration of 10mM with distilled water. Take 30μL of the fluorescent probe mother solution in the cuvette each time, add 30μL of various metal ion aqueous solutions in turn, and then take ethanol / HEPES (v / v=8 / 2, HEPES=10mM, pH=7.4) buffer solution to dilute the solution to 3mL, mix well, and use it for UV and fluorescence testing, such as Figure 7 and Figure 8 As shown. Figure 7 Only when Zn is added 2+ and Cd 2+ The UV spectrum changes, and Zn 2+ The changes are more obvious; Figure 8 Only when Zn is added 2+ The fluorescence spectrum showed a significant enhancement, while the addition of other ions did not cause obvious fluorescence changes. 2+ Shows excellent selective recognition ability.

[0061] Example 3

[0062] The fluorescence probe APYQ obtained in Example 1 is effective for Zn 2+ The concentration titration experiment includes the following steps:

[0063] Each time, 30 μL of 1 mM fluorescent probe stock solution was taken into the cuvette, and different concentrations of Zn 2+ , then dilute the solution to 3 mL with ethanol / HEPES (v / v=8 / 2, HEPES=10 mM, pH=7.4) buffer solution, mix well, and use for UV and fluorescence testing, such as Fig. 9 and Fig.10 As shown. 2+ With the increase of concentration, the UV peak at 434nm and the fluorescence peak at 535nm gradually increased; Zn 2+ There is a good linear relationship between the concentration and the peak intensity of fluorescence emission. Fig.11 As shown. The linear equation is: y = 80.0671x + 79.3784, the linear correlation coefficient R 2 = 0.99695, the probe pair Zn 2+ The detection limit of the probe was 1.87 nM, indicating that the probe was sensitive to Zn 2+ Has high sensitivity.

[0064] Example 4

[0065] The fluorescence probe APYQ obtained in Example 1 is effective for Zn 2+ The response time includes the following steps:

[0066] Take 30 μL of 1 mM fluorescent probe stock solution in a cuvette, add 30 μL of 10 mM Zn 2+ , then dilute the solution to 3 mL with ethanol / HEPES (v / v=8 / 2, HEPES=10 mM, pH=7.4) buffer solution for fluorescence testing. Fig.12 Add Zn 2+ After that, the fluorescence intensity reached the highest within 5 s, indicating that the probe can quickly respond to Zn 2+ .

[0067] Example 5

[0068] The fluorescent probe APYQ obtained in Example 1 was mixed with other Zn 2+ Probe detection of Zn 2+ The detection limit and response time performance comparison includes the following steps:

[0069] The Zn detection data in the past 5 years are listed. 2+ The probes with excellent performance are shown in Table 1. The detection limit and response time of probe APYQ are compared with them. Fig.13 As shown, it can be seen that the probe APYQ has the best comprehensive performance in terms of high sensitivity and fast response capability.

[0070] Table 1 Fluorescent probe APYQ and other Zn 2+ Comparison of probes

[0071]

[0072]

[0073]

[0074] Example 6

[0075] The fluorescent probe APYQ obtained in Example 1 can identify Zn with naked eyes in solution 2+ The experiment includes the following steps:

[0076] Each time, 10 μM fluorescent probe mother solution was taken into the bacterial culture bottle, and 100 μM aqueous solutions of various metal ions were added in sequence, and then ethanol / HEPES (v / v=8 / 2, HEPES=10mM, pH=7.4) buffer solution was taken to dilute the solution to 3 mL, and mixed evenly. Fig.14 and Fig.15 As shown. Fig.14 In visible light, only when Zn is added 2+ The solution color changes from colorless to bright yellow; Fig.15 In the ultraviolet light, Zn was added 2+ The solution color changed from blue to bright green. Further tests were performed by adding different concentrations of Zn2+ ,like Fig.16 and Fig.17 As shown in the figure, the color changes gradually under visible light and ultraviolet light, indicating that Zn can be identified with the naked eye. 2+ .

[0077] Example 7

[0078] The fluorescent probe APYQ obtained in Example 1 can identify Zn with naked eyes in the test paper 2+ The experiment includes the following steps:

[0079] Prepare a 1mM fluorescent probe APYQ stock solution with anhydrous ethanol, soak the test paper in the probe stock solution for one day, and then dry the test paper for subsequent testing. Add 1mM aqueous solutions of various metal ions on the test paper and add different concentrations of Zn 2+ On the test paper, dry, take a comparison photo, such as Fig.18 and Fig.19 As shown. Fig.18 Under visible light, only when Zn is added 2+ The color changes from colorless to yellow, and the color changes gradually under the corresponding sensitivity; Fig.19 Under medium ultraviolet light, only when Zn is added 2+ The color changes from blue to green, and the color changes gradually under the corresponding sensitivity, indicating that the probe can be used for "naked eye" identification and quantitative detection of Zn 2+ .

[0080] Example 8

[0081] Example 1 The fluorescent probe APYQ obtained detects Zn in cells 2+ The fluorescence imaging experiment includes the following steps:

[0082] Hela cells were injected onto a confocal dish and placed in a constant temperature incubator containing 5% carbon dioxide at 37°C for 24 hours to allow the cells to adhere. The adherent cells were stained with the probe APYQ (10 μM) for 30 minutes, washed three times with phosphate buffered saline (PBS, pH = 7.4) to remove excess dye, and then photographed using a confocal microscope. Zn was then added 2+ (10 μM) and cultured for 30 min, and photographs were taken, e.g. Fig. 20 As shown. It can be seen that adding Zn 2+ After that, there is obvious green fluorescence, which shows that the probe APYQ has good cell permeability and has good application prospects in the biological field.

[0083] The implementation modes of the present invention have been described in detail above in conjunction with specific embodiments, but the present invention is not limited to the above-mentioned implementation modes, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A zinc ion fluorescent probe based on 8-aminoquinoline, the structural formula of which is shown below:

2. The method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) reacting 2-acetamido-8-nitroquinoline with a reducing agent to obtain an intermediate AAQ; (2) reacting the intermediate AAQ and salicylaldehyde under acidic conditions to obtain the zinc ion fluorescent probe APYQ.

3. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, characterized in that: In step (1), the reducing agent is selected from iron; the solvent of the reaction is selected from acetic acid; the reaction is carried out under the protection of an inert gas at 60 to 70° C. for 1.5 to 2.5 hours; and the molar ratio of the 2-acetamido-8-nitroquinoline to the reducing agent is 1:4 to 1:

6.

4. The method for preparing a zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, characterized in that: In step (1), the post-treatment method of the reaction includes: filtering, washing, removing the solvent, extracting with ethyl acetate, drying, removing the solvent, purifying the crude product by silica gel column, and separating with ethyl acetate and petroleum ether = 1:2 to 1:3 (v / v) as eluent to obtain the intermediate AAQ.

5. The method for preparing the zinc ion fluorescent probe based on 8-aminoquinoline according to claim 2, characterized in that, In step (2), the acid in the acidic condition is selected from acetic acid; the solvent of the reaction is selected from anhydrous ethanol; the reaction is carried out under the protection of an inert gas at 80-90° C. for 2.5-3.5 hours; the molar ratio of the intermediate AAQ to acetic acid is 1:1-1:3, and the molar ratio of the intermediate AAQ to salicylaldehyde is 1:1-1:1.

2.

6. The method for preparing the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 2, characterized in that: In step (2), the post-treatment method of the reaction includes: filtering the precipitate, washing with anhydrous ethanol, and drying to obtain the zinc ion fluorescent probe APYQ.

7. Use of the 8-aminoquinoline-based zinc ion fluorescent probe according to claim 1 in zinc ion detection.

8. A method for detecting zinc ions, characterized in that, The method comprises using the 8-aminoquinoline-based zinc ion fluorescent probe described in claim 1 to detect zinc ions in a solution.

9. The detection method of zinc ions according to claim 8, wherein The method comprises detecting zinc ions in an ethanol / HEPES buffer solution; preferably, the pH of the ethanol / HEPES buffer solution is 7.4±0.5, wherein the volume ratio of ethanol to HEPES buffer solution is (7-9):(3-1).

10. The method for detecting zinc ions according to claim 8, wherein Under the action of zinc ions, the solution containing the zinc ion fluorescent probe changes from colorless to yellow under visible light and from blue to green under ultraviolet light.

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