Small organic molecule fluorescent probe for lithium ion detection as well as synthesis method and application of small organic molecule fluorescent probe

Abstract

The invention belongs to the technical field of fluorescent probes and supramolecules, and particularly relates to an organic small molecule fluorescent probe for lithium ion detection as well as a synthesis method and application of the organic small molecule fluorescent probe. Aiming at the problems that an existing fluorescent probe for identifying Li < + > is easily influenced by pH and interfered by coexisting metal ions, and is poor in water solubility, low in sensitivity, poor in selectivity and the like, the crown ether fluorescent molecular probe for detecting the lithium ions is 4-aza-12-crown-4-ether-7-nitrobenzofuran; the compound is prepared from 4-chloro-7-nitrobenzofuran and aza 12-crown-4-ether in a substitution manner. The fluorescent probe has the advantages that 1) the structure is simple, the preparation is easy, and excitation and emission wavelengths are in a visible light region; 2) good water solubility is achieved; 3) the material has good selectivity on lithium ions; 4) the method has the advantages of high sensitivity, quick response and the like in lithium ion detection, and can be used for in-situ-real-time detection of lithium ions; and (5) the cell toxicity is low, and the biocompatibility is good.

Description

Technical Field

[0001] The present invention belongs to the technical fields of fluorescent probes and supramolecular, and particularly relates to an organic small molecule fluorescent probe for lithium ion detection, a synthesis method and an application thereof. Background Art

[0002] Lithium mainly exists in crustal rocks, salt lake brines and seawater, and is currently widely used in the manufacture of lithium ion power batteries, alloys, lubricating additives, glass products and clinical drugs. Lithium in nature will not have an impact on the ecological environment. However, with the widespread application of lithium batteries and the imperfect recycling of waste electronic products, to a large extent, lithium ion batteries will be discarded or disposed of at will, and the probability of lithium entering the soil and water bodies will increase greatly, which may cause the enrichment of lithium in the ecological environment and cause biological poisoning. In the medical field, lithium carbonate has been widely used as a drug for the treatment of bipolar disorder and is also used for the treatment of diseases related to dementia. However, a high concentration of lithium salt in the body will cause serious physiological poisoning. When the Li + concentration in human serum is higher than the normal range, it may damage the kidneys and nerves. When the Li + concentration in the body is relatively high, it may also affect the normal development of nerve cells, resulting in drowsiness, fatigue, etc., and may even lead to death in severe cases. Monitoring the reasonable dosage of patients taking drugs and studying the physiological toxicological mechanism of lithium is of great significance.

[0003] The advantage of azacrown ether is that the electron-donating ability of the nitrogen atom is stronger than that of oxygen, making the azacrown ether more active. It has good coordination ability with various metal ions and organic ions. N provides active sites for substituents and can be further functionalized, thus greatly improving the coordination ability and selectivity of the crown ether ring, and further broadening the application fields of azacrown ether. Summary of the Invention

[0004] Aiming at the problems of existing fluorescent probes for identifying Li + such as being easily affected by pH, interfered by coexisting metal ions, having poor water solubility, low sensitivity and poor selectivity, the present invention provides an organic small molecule fluorescent probe for lithium ion detection, a synthesis method and an application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an organic small molecule fluorescent probe for lithium ion detection, which is a crown ether-based fluorescent molecular probe, namely 4-aza-12-crown-4-ether-7-nitrobenzofuran, and its structural formula is as follows:

[0007]

[0008] The present invention also provides a method for synthesizing an organic small molecule fluorescent probe for lithium ion detection, comprising the following steps:

[0009]

[0010] Dissolve azacrown-12-ether and 4-chloro-7-nitrobenzofuran in acetonitrile, then add potassium carbonate. At room temperature, stir the mixture overnight, stop the reaction, filter to remove potassium carbonate, rotary evaporate to remove the solvent, subject the obtained oily substance to column chromatography separation, and elute through the column to obtain an orange-red solid, which is the target compound.

[0011] Furthermore, the molar ratio of azacrown-12-ether to 4-chloro-7-nitrobenzofuran is 1:1.

[0012] Furthermore, the silica gel for column chromatography is used as the stationary phase, and methanol and ethyl acetate are used as the eluents, and the volume ratio of methanol to ethyl acetate is 1:1.

[0013] The present invention also provides an application of an organic small molecule fluorescent probe for lithium ion detection, which is used for preparing a reagent for detecting lithium ions in an aqueous environment or a biological cell system.

[0014] Furthermore, the reagent for detecting lithium ions in an aqueous environment or a biological cell system includes fluorescence detection and cell imaging detection.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The fluorescent probe containing a crown ether group of the present invention can achieve the selective recognition function of Li + in an aqueous environment system, is not affected by other coexisting metal ions in the aqueous solution, and has strong anti-interference ability. The recognition response of this fluorescent molecular probe is very rapid and can complete the recognition of Li + in the water system environment within 2.5 seconds.

[0017] The fluorescent probe of the present invention has good cell permeability, good stability, good solubility and biocompatibility, and can perform microscopic imaging on the distribution of lithium ions in living cells.

[0018] The raw materials required for preparing the fluorescent molecular probe of the present invention are simple and easy to obtain, and the cost is low; the reaction conditions are mild, the product separation and purification process is simple, the yield is high, and the yield is as high as 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 1H NMR spectrum of the fluorescent molecular probe prepared in Example 1 of the present invention;

[0020] Figure 2 High-resolution mass spectrum of the fluorescent molecular probe prepared in Example 1 of the present invention;

[0021] Figure 3 High-resolution mass spectrum of the complex formed by the fluorescent molecular probe prepared in Example 1 of the present invention and lithium ions;

[0022] Figure 4 is the curve of the fluorescence intensity of the fluorescent molecular probe in Example 2 varying with the concentration of Li + ;

[0023] Figure 5 is the change of the fluorescence emission spectrum of the fluorescent molecular probe in Example 2 in the CH 3 CN / PBS buffer solution (v / v = 1 / 9, pH = 7.4) system after adding 30 μmol / L of Li + and 300 μmol / L of metal cations (Na + , K + , Mg 2+ , Ca 2+ , Al 3+ ), with the abscissa being the wavelength and the ordinate being the fluorescence intensity;

[0024] Figure 6 is the working curve for measuring Li + in Example 2;

[0025] Figure 7 is the response time for measuring the detection of Li + by the probe in Example 2;

[0026] Figure 8 is the fluorescence emission diagram of the sample measured in Example 2;

[0027] Figure 9 is the cell imaging diagram of the detection and imaging of lithium ions by the fluorescent molecular probe in Hela cells in Example 2. Detailed implementation manners

[0028] To further elaborate on the technical solution of the present invention, the present invention will be further described below through examples.

[0029] Example 1

[0030] An organic small molecule fluorescent probe for lithium ion detection, the structural formula of which is as follows:

[0031]

[0032] The synthesis method of this probe is carried out according to the following steps:

[0033] Dissolve 0.2110 g (1.2 mmol) of azacrown-12-ether and 0.2250 g (1.1 mmol) of 4-chloro-7-nitrobenzofuran (NBD-Cl) in 15 mL of acetonitrile, and then add 0.2760 g (2.0 mmol) of potassium carbonate. At room temperature, stir the mixture overnight, stop the reaction, filter off the potassium carbonate, remove the solvent by rotary evaporation, and subject the resulting oil to column chromatography. The stationary phase for column chromatography is silica gel, and the eluent is methanol:ethyl acetate (1:1, v / v). After passing through the column, an orange-red solid, i.e., the target compound (0.3474 g, yield 90.9%), is obtained.

[0034] The 1H NMR spectrum, high-resolution mass spectrum of the fluorescent molecular probe prepared in this example, and the high-resolution mass spectrum of the complex formed with lithium ions are as Figures 1 to 3 shown.

[0035] Example 2

[0036] 1. Prepare solutions

[0037] Prepare a PBS buffer solution with a concentration of 10 mM (pH = 7.4) and an acetonitrile solution of the lithium ion fluorescent probe with a concentration of 2 mM.

[0038] 2. Relationship between probe fluorescence intensity and Li + concentration

[0039] Add 1000 μL of PBS buffer solution with pH 7.4, 1000 μL of acetonitrile, and 30 μL of the acetonitrile solution of the lithium ion fluorescent probe to a fluorescence cuvette, and detect with a fluorescence spectrophotometer. As Li + (100 - 300 μM) is added, the fluorescence intensity at a wavelength of 550 nm in the emission spectrum gradually increases (as Figure 4 shown);

[0040] 3. Probe selectivity

[0041] In 7 fluorescence cuvettes, add 1000 μL of PBS buffer solution with pH 7.4, 1000 μL of acetonitrile, and 30 μL of the acetonitrile solution of the lithium ion fluorescent probe to each cuvette, and then add 100 μL (2×10 -3 M) of LiCl, and 100 μL (2×10 -2 M) of various metal ions: Li + , Na + , K + , Al 3+ , Ca 2+ , Mg 2+ respectively, and detect with a fluorescence spectrophotometer, and draw a bar chart of the fluorescence intensity at 550 nm corresponding to different metal ions (asFigure 5 shown), Li + makes the fluorescence intensity at 550 nm of the probe increase, and other metal ions basically do not cause changes in the fluorescence intensity of the reagent.

[0042] 4. Detection of Li by the probe + working curve

[0043] Add 1000 μL of PBS buffer solution with pH 7.4, 1000 μL of acetonitrile, and 30 μL of acetonitrile solution of the lithium ion fluorescence probe to another six fluorescence cuvettes. Then add LiCl solutions with volumes of 0, 100, 150, 200, 250, and 300 μM respectively, and measure the intensity corresponding to 550 nm on a fluorescence spectrophotometer. With the lithium ion concentration as the abscissa and the fluorescence intensity as the ordinate, the working curve for detecting lithium ions by the lithium ion fluorescence probe is obtained (as Figure 6 shown). The linear regression equation is: y = 2.63c + 119.11, where the unit of c is μM; according to the working curve and the detection limit formula (DL = 3σ / k) defined by IUPAC, the detection limit for detecting lithium ions by the lithium ion fluorescence probe is 0.25 μM, indicating that the probe has high sensitivity for the detection of Li + detection.

[0044] 5. Response time of the probe for detecting Li +

[0045] Add 1000 μL of PBS buffer solution with pH 7.4, 1000 μL of acetonitrile, and 30 μL of acetonitrile solution of the lithium ion fluorescence probe to a fluorescence cuvette. Then add 10-fold amount of LiCl and detect with a fluorescence spectrophotometer. As Figure 7 shown, when Li + is added, the fluorescence intensity increases to the maximum intensity after 2.5 s.

[0046] 6. Fluorescence intensity of the detection sample

[0047] Add 1000 μL of PBS buffer solution with pH 7.4, 1000 μL of acetonitrile, and 30 μL of acetonitrile solution of the lithium ion fluorescence probe to a clean fluorescence cuvette. Use a pipette to aspirate the lithium ion-containing sample solution to be measured and add it to this fluorescence cuvette, and then detect with a fluorescence spectrophotometer. The fluorescence intensity at 550 nm is measured to be 565 (as Figure 8 shown). Substitute the measured fluorescence intensity into the above linear regression equation, and the concentration c of lithium ions can be obtained as 2×10 -3 mol / L.

[0048] 7. Cellular imaging of Li detection by the probe in Hela cells +

[0049] ​​Hela cells stained with the nuclear dye Hoechst 33342 showed strong fluorescence in the blue channel and no fluorescence in the green channel after incubation with 30 μM of the fluorescent probe in the control group. For cells pretreated with different concentrations (20, 100, 150 μM) of LiCl and then incubated with 30 μM of the fluorescent probe, obvious green fluorescence could be observed in the green channel, and the fluorescence intensity in the cells increased correspondingly with the increase of the Li + concentration. As Figure 9 shown, it indicates that the fluorescent probe has good cell permeability, good stability, good solubility and biocompatibility, and can perform microscopic imaging on the distribution of lithium ions in living cells.

[0050] In summary, the fluorescent probe of the present invention has a simple structure and is easy to prepare. Both the excitation and emission wavelengths are in the visible light region (λ ex = 485 nm, λ em = 550 nm). The probe has good water solubility and can be dissolved in pure water. The probe has good selectivity for lithium ions, and other metal ions do not interfere with the detection. The probe has very good sensitivity, and has the advantages of high sensitivity (DL = 0.25 μM) and fast response (2.5 s) for detecting lithium ions, and can be used for in-situ and real-time detection of lithium ions. The probe has low cytotoxicity and good biocompatibility. Further, through fluorescence imaging studies, it was found that the probe can be used for the detection and imaging of lithium ions in Hela cells.

[0051] The above shows and describes the main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0052] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An organic small molecule fluorescent probe for lithium ion detection, characterized in that: The structural formula is as follows:

2. A method for synthesizing an organic small molecule fluorescent probe for lithium ion detection according to claim 1, characterized in that: The following steps are involved: Dissolve aza-12-crown-4 ether and 4-chloro-7-nitro-benzofuran in acetonitrile, then add potassium carbonate, stir the mixture overnight at room temperature, stop the reaction, filter out potassium carbonate, and remove the solvent by rotary evaporation. The resulting oil is separated by column chromatography to obtain an orange-red solid, i.e., the target compound.

3. The method for synthesizing an organic small molecule fluorescent probe for lithium ion detection according to claim 2, characterized in that: The molar ratio of the aza-12-crown-4 ether to 4-chloro-7-nitro-benzofuran is 1:

1.

4. The method for synthesizing an organic small molecule fluorescent probe for lithium ion detection according to claim 2, characterized in that: The column chromatography uses silica gel as the stationary phase, methanol and ethyl acetate as the eluents, and the volume ratio of methanol to ethyl acetate is 1:

1.

5. An application of the organic small molecule fluorescent probe for lithium ion detection according to claim 1, characterized in that: Used to prepare reagents for detecting lithium ions in water environments or biological cell systems.

6. The use of an organic small molecule fluorescent probe for lithium ion detection according to claim 5, characterized in that: The reagents for detecting lithium ions in a water environment or a biological cell system include fluorescence detection and cell imaging detection.

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