Zinc ion detection fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to a zinc ion detection fluorescent probe as well as a preparation method and application thereof, and particularly provides an indole compound N '-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridine-4-yl) methylene)-1H-indole-2-carbohydrazide which can be used as a zinc ion detection fluorescent probe and can be used for detecting zinc ions. High sensitivity and high selectivity are shown on Zn < 2 + > in a solution.

Description

[0001] The application belongs to the field of organic materials, and particularly relates to a zinc ion detection fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] The threat of heavy metal pollution to the natural environment and human health is increasingly attracting people's attention. Zinc, as an essential trace element for humans, can maintain a series of human activities, and plays an important role in a series of biological activities such as DNA and peptide synthesis, RNA transcription, cell apoptosis, metal enzyme regulation, etc. Zn 2+ Ion content in the organism is crucial, and excessive or insufficient intake of zinc ions can cause various diseases. For example: intake of less zinc ions can cause the organism to have low immunity, growth retardation, metabolic disorder, etc., and can cause prostate cancer, Parkinson's disease, diabetes. Excessive intake of zinc ions is toxic to important organs of the body, including the liver, kidneys, heart and nervous system. Therefore, it is of great significance to develop an effective method for identifying Zn 2+ in the environment and biological samples.

[0003] At present, the conventional detection of Zn 2+ has multiple analysis methods, such as atomic absorption spectrometry, inductively coupled plasma spectrometry and electrochemical technology. Compared with all these complex and expensive methods, fluorescent probes have the advantages of high sensitivity, simplicity and real-time detection. So far, many fluorescent probes for rapid detection of Zn 2+ have been reported. It is a great challenge to develop Zn 2+ selective fluorescent probes that can distinguish Zn 2+ from other closely related metal ions. SUMMARY

[0004] The present application provides a compound or a salt thereof, which is The compound as a fluorescent probe has high selectivity for zinc ions, low detection limit, fast response time, and is anti-interference, and can rapidly detect trace amounts of zinc ions in the environment system.

[0005] The present application also provides a method for preparing the aforementioned compound, which comprises the step of reacting 1H-indole-2-formylhydrazine with pyridoxal.

[0006] In some embodiments, the reaction solution is a protic solvent, such as ethanol.

[0007] In some embodiments, the reaction temperature is selected from 60-100℃, including 60℃, 70℃, 80℃, 90℃, 100℃ or any value between two numbers.

[0008] In some embodiments, the 1H-indole-2-carboxylic acid hydrazide is reacted with pyridoxal at 80°C.

[0009] In some embodiments, the method comprises the steps of dissolving pyridoxal hydrochloride and 1H-indole-2-carboxylic acid hydrazide in ethanol, and reacting at 80°C.

[0010]

[0011] In some embodiments, the method comprises the steps of dissolving pyridoxal hydrochloride and 1H-indole-2-carboxylic acid hydrazide in ethanol, and reacting at 80°C.

[0012]

[0013] In some embodiments, the method of the present application further comprises one or more steps of filtration, recrystallization, concentration, and drying.

[0014] In some embodiments, the metal ion is zinc ion.

[0015] In some embodiments, the method of detecting zinc ion in a solution comprises the steps of adding compound X6 into the solution to be tested, and detecting by a fluorescence spectrometer.

[0016] In some embodiments, the fluorescence spectrometer detects excitation wavelength less than or equal to 400 nm. In some embodiments, the fluorescence spectrometer detects emission wavelength less than or equal to 480 nm.

[0017] In some embodiments, the solution is selected from a methanol solution, or a solution containing methanol.

[0018] In the present application, the numerical values are measured values by instruments, and there is a certain degree of error. Generally speaking, plus or minus 10% is within a reasonable error range. Of course, the context in which the numerical value is used should be considered, for example, the particle size of the active ingredient, the numerical value after measurement error change is not more than plus or minus 10%, which can be plus or minus 9%, plus or minus 8%, plus or minus 7%, plus or minus 6%, plus or minus 5%, plus or minus 4%, plus or minus 3%, plus or minus 2%, or plus or minus 1%, preferably plus or minus 5%.

[0019] In the present application, the salt of the compound includes, for example, hydrochloride.

[0020] Advantages: Compared with the prior art, the present application has the following advantages:

[0021] The present application takes pyridoxal hydrochloride and 1H-indole-2-formylhydrazine as a fluorescent group, and prepares a zinc ion fluorescent detection probe with a novel structure taking pyridoxal hydrochloride and 1H-indole-2-formylhydrazine as a parent by a condensation reaction. 2+ Compared with the reported fluorescent probes based on rhodamine, chromone, benzothiazole and naphthaldehyde as a parent, the present probe takes pyridoxal hydrochloride and 1H-indole-2-formylhydrazine as a parent to prepare an electron-rich Schiff base, has rich N, O coordination sites, and is beneficial to complex with Zn 2+ ion. We synthesize a Schiff base (X6) taking pyridoxal hydrochloride and 1H-indole-2-formylhydrazine as a parent, which is used for high-sensitivity, high-selectivity, low-detection-limit, high-efficiency and dual-purpose Zn 2+ detection signal mechanism, and provides comparison results and is suitable for real water sample analysis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 UV absorption spectrum and color change diagram of the zinc ion fluorescent probe prepared in Example 1 in CH3OH:H2O:Hepes (v / v / v=9:1:0.1) solution for different concentrations of zinc ions;

[0023] Figure 2 Fluorescence spectrum diagram of the zinc ion fluorescent probe prepared in Example 1 in CH3OH:H2O:Hepes (v / v / v=9:1:0.1) solution for different metal ions;

[0024] Figure 3 Fluorescence corresponding intensity of the zinc ion fluorescent probe prepared in Example 1 in different solvents for zinc ions;

[0025] Figure 4 Fluorescence spectrum response diagram and color change diagram of the fluorescent probe prepared in Example 1 in CH3OH:H2O:Hepes (v / v / v=9:1:0.1) solution for different concentrations of zinc ions (Zn 2+ );

[0026] Figure 5 Fluorescence response diagram of the fluorescent probe prepared in Example 1 in CH3OH:H2O:Hepes (v / v / v=9:1:0.1) solution for different metal ions to interfere with the detection of Zn 2+ ;

[0027] Figure 6 Fluorescence spectrum response diagram and color change diagram of the fluorescent probe prepared in Example 1 in CH3OH:H2O:Hepes (v / v / v=9:1:0.1) solution for different concentrations of zinc ions (Zn 2+Job-plot curve of complexation ratio;

[0028] Figure 7 Response time plot of the fluorescent probe prepared in Example 1 for detection of zinc ion (Zn 2+ ) detection limit;

[0029] Figure 8 Response time plot of the fluorescent probe prepared in Example 1 for detection of zinc ion (Zn 2+ ) binding constant Ka plot;

[0030] Figure 9 Response time plot of the fluorescent probe prepared in Example 1 for detection of zinc ion (Zn 2+ ) detection limit;

[0031] Figure 10 Response time plot of the fluorescent probe prepared in Example 1 for detection of zinc ion (Zn 2+ ) detection limit;

[0032] Figure 11 Selectivity plot of the fluorescent probe prepared in Example 1 for detection of zinc ion after complexation with different anions;

[0033] Figure 12 Color change plot of the fluorescent test paper prepared in Example 1 for detection of zinc ion after complexation with different concentrations of zinc ion;

[0034] Figure 13 DFT calculation plot of the fluorescent probe prepared in Example 1 and the fluorescent probe after complexation with zinc ion.

[0035] Figure 14 Cytotoxicity plot of the fluorescent probe prepared in Example 1 for detection of zinc ion.

[0036] Figure 15 Cell experiment plot of the fluorescent probe prepared in Example 1 for detection of zinc ion

[0037] Figure 16 Mobile phone detection plot of the fluorescent probe prepared in Example 1 for detection of zinc ion. DETAILED DESCRIPTION

[0038] The present disclosure will be further described in conjunction with the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0039] The experimental methods in the examples of the present application, unless otherwise specified, are generally performed according to conventional conditions, or according to the conditions suggested by the manufacturer of the raw materials or commercial products. The reagents, unless otherwise specified, are commercially available conventional reagents.

[0040] The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR shift (δ) is given in units of 10-6 (ppm).

[0041] The NMR determination is performed using a Bruker AVANCE-400 nuclear magnetic instrument, and the determination solvent is deuterated dimethyl sulfoxide (d6-DMSO).

[0042] The MS determination is performed using a Waters Micromass Quattro micro API triple quadrupole mass spectrometer, scanning in positive / negative ion mode, and the mass scan range is 120-1300.

[0043] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 silica gel plate, and the thin layer chromatography (TLC) uses silica gel plate with a specification of 0.2 mm±0.03 mm, and the thin layer chromatography separation and purification product uses a specification of 0.4 mm-0.5 mm.

[0044] Example 1:

[0045]

[0046] Dissolve 1H-indole-2-carboxylic acid hydrazide (A) (0.087 g, 0.5 mmol) and pyridoxal hydrochloride (B) (0.101 g, 0.5 mmol) in 2 mL of ethanol, stir the reaction system at 80°C under reflux, after the reaction is completed, recrystallize to obtain 0.10 g of the target fluorescent probe X6 solid, the yield is 62%.

[0047] 1 H NMR (400 MHz, d6-DMSO): δ 13.06 (s, 1H), 12.01 (s, 1H), 8.91 (s, 1H), 8.2 (s, 1H), 7.74-7.72 (d, 1H), 7.50-7.46 (m, 2H), 7.45-7.26 (m, 1H), 7.13-7.09 (m, 1H), 4.77 (s, 1H), 3.17 (s, 3H), 2.59-2.50 (m, 2H).

[0048] 13C NMR (400 MHz, d6-DMSO): 158.16, 153.04, 144.13, 143.45, 137.80, 136.57, 129.06, 127.34, 125.21, 122.66, 120.87, 113.03, 106.10, 58.66, 15.65.

[0049] MS (ESI): m / z 325.1 [M+1] + .

[0050] Example 2

[0051] The zinc ion detection fluorescent probe X6 prepared in Example 1 was configured into a 1 mM probe stock solution with DMSO, and each metal ion was configured into a 3 mM metal ion stock solution with deionized water, 30 μL of the probe stock solution and 50 μL of the metal ion stock solution were added to 3 mL of a blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1), and detected with a fluorescence spectrometer and a UV spectrophotometer. It was found that the maximum excitation wavelength of the fluorescent probe was 400 nm, and the maximum emission wavelength was 480 nm. The specific test results are as follows:

[0052] Two cuvettes were taken, and 3 mL of a blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1) and 30 μL of a probe stock solution were added to each of the cuvettes, and different volumes of zinc ion stock solution were added to one of the cuvettes, and the other cuvette was not added with zinc ion stock solution, and UV spectrum test was carried out. As shown in Figure 1 , the fluorescent probe itself has a strong UV absorption at a wavelength of λ = 326 nm, and when zinc ions are added to the solution, the UV absorption peak gradually weakens; however, the fluorescent probe itself has almost no UV absorption at a wavelength of λ = 421 nm, and when zinc ions are added, the UV absorption peak gradually increases. The results show that the probe has high sensitivity to Zn 2+ , and the naked eye can see the color change due to the formation of a new complex between the probe and zinc ions.

[0053] As shown in Figure 2 , the zinc ion detection fluorescent probe has high selectivity to various common ions (Co 2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Al 3+ , Cu + , Mn 2+ , Zn 2+ , Mg 2+ , Ba 2+ , Pb 2+ , Fe3+ Sn 2+ K + Ca 2+ Sr 2+ Na + Ag + Cd 2+ The selective fluorescence response spectrum of the probe was observed. 30 μL of probe stock solution and 50 μL of various metal ion stock solutions were added to 3 mL of a buffer solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1, pH = 7.4). The results showed that only the addition of zinc ions significantly enhanced the fluorescence intensity at 490 nm. Other metals and zinc ions showed only slight enhancements in fluorescence intensity at 490 nm, which were negligible compared to the change in fluorescence intensity caused by zinc ions. That is, the fluorescent probe of this invention exhibits high selectivity for zinc ions, and a significant shift in fluorescence intensity occurs.

[0054] like Figure 3 The image shows the fluorescence intensity spectra of the zinc ion detection fluorescent probe for zinc ions in different solutions. 30 μL of probe stock solution and 50 μL of zinc ion stock solution were added to 3 mL of different solvents (Acetone, DMSO, CH3OH, CH3CN, CH3CH2OH, THF, H2O), respectively. The results showed that the zinc ion detection fluorescent probe exhibited the strongest fluorescence intensity for zinc ions in methanol solution.

[0055] like Figure 4 As shown, the fluorescent probe for zinc ion detection reacts to different concentrations of zinc ions (Zn). 2+ The fluorescence titration spectrum response of the probe was obtained. 30 μL of probe stock solution and 1–50 μL (1, 2, 3…10, 15, 20, 25, 30, 35, 40, 45, 50 μL) of zinc ion solution (3 mM zinc ion stock solution) were added to 3 mL of blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1). The fluorescent probe itself showed almost no fluorescence in solution, but the fluorescence at 490 nm increased continuously with increasing zinc ion concentration; that is, the fluorescence intensity increased with increasing zinc ion concentration, indicating that Zn... 2+ The ions complex with the probe, inhibiting the free rotation of C=N and hindering the transfer of single electrons, thus enhancing fluorescence, indicating that probe X6 is a fluorescence-enhanced probe.

[0056] like Figure 5 The figure shows a bar chart of fluorescence intensity after the zinc ion detection fluorescent probe reacts with zinc ions in the presence of different interfering metal ions. 30 μL of probe stock solution and 50 μL of any other metal ion (Co) were added to 3 mL of blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1).2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Al 3+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Pb 2+ , Fe 3+ , Sn 2+ , Cd 2+ , K + , Ca 2+ , Sr 2+ , Na + , Ag + ) stock solution, and finally 50 μL of Zn 2+ stock solution was added to the blank solution, and the fluorescence intensity was tested. The results showed that the presence of other metal ions, except for Cu + , Cu 2+ , Fe 3+ covalent metal ions, had no obvious interference with the recognition of zinc ions by the zinc ion fluorescent probe compound of the present application.

[0057] As shown in Figure 6 , the complex ratio of the probe and Zn 2+ was studied by the Job's plot method. A certain volume of probe stock solution (1 mM) and Zn 2+ stock solution (3 mM) was added to 3 mL of blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1) so that the total concentration of the zinc ion detection fluorescent probe and zinc ions was 50 μM. By changing the concentration ratio of the two (the molar ratio of the zinc ion detection fluorescent probe and zinc ions was 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, respectively), the difference between the fluorescence intensity at 490 nm and the self-fluorescence intensity of the zinc ion fluorescent probe compound at the concentration was plotted against the proportion of ions in the total concentration. It can be seen from this Figure 6 that the ordinate reaches the highest value when the proportion of zinc ions is 0.5, and it can be determined that the fluorescent probe compound mainly combines with zinc ions in the form of 1:1 to form a stable complex.

[0058] As shown in Figure 7 , the formula (LOD = 3S B1 / S, where S B1 is the standard deviation of the blank solution) was used to calculate the detection limit of X6 for Zn 2+ as 40.0 nm from the calibration chart and the fluorescence titration data.

[0059] AsFigure 8 As shown, based on the Benesi-Hildebrand formula and fluorescence titration data, probe X6 and Zn 2+ The complexation constant (Ka) is 6.464 × 10⁻⁶. 4 M -1 .

[0060] like Figure 9 As shown, 30 μL of probe stock solution and 50 μL of Zn were added to 3 mL of blank buffer CH3OH:H2O:Hepes (v / v / v = 9:1:0.1). 2+ In the stock solution, the fluorescence intensity of the probe rapidly increases to its peak and reaches a stable value within 1 minute. Furthermore, upon adding Zn... 2+ Within 10 minutes of the response, the fluorescence intensity of the probe remained unchanged, indicating that the probe is effective for Zn. 2+ The detection is stable enough.

[0061] like Figure 10 As shown, 30 μL of probe stock solution and 50 μL of Zn were added to 3 mL of blank buffer CH3OH:H2O:Hepes (v / v / v = 9:1:0.1), respectively. 2+ Stock solution was prepared to obtain probe X6 stock solution (10 μL) and [X6-Zn]. 2+ The stock solution was then prepared, and the probe stock solutions and [X6-Zn] were adjusted to different pH values ​​(from 2.0 to 12.0) using 1M HCl and 1M NaOH. 2+ Stock solution. Testing, probe X6 and X6-Zn 2+ The fluorescence response intensity of the complex varied within a variable pH range of 2.0 to 12.0. The probe X6 itself exhibited varying fluorescence intensity from pH 2 to 7, but remained constant from pH 7 to 12; however, X6-Zn... 2+ The complex exhibits significantly enhanced fluorescence intensity at 490 nm within a pH range of 4.0–12.0, reaching its maximum intensity at pH 7. X6-Zn 2+ No obvious fluorescence signal was observed under strongly acidic conditions (pH < 4.0), possibly because the probe complexation site was protonated, inhibiting its interaction with Zn. 2+ The complexation of X6-Zn. 2+ Under alkaline conditions (pH>8.0), the fluorescence signal was significantly reduced, possibly due to the formation of insoluble Zn(OH)₂,X₆-Zn. 2+ The concentration decreased. Therefore, X6 was used to detect Zn. 2+ The optimal pH range is 5-8, indicating that it can detect Zn in biological environments. 2+ The ability.

[0062] As shown in Figure 11 , the fluorescence spectrum response diagram of the fluorescent probe after complexing with zinc ions to detect different anions. Add 30 μL of probe stock solution and 50 μL of Zn 2+ stock solution to 3 mL of blank buffer CH3OH:H2O:Hepes (v / v / v = 9:1:0.1), and add 50 μL of different anion stock solution respectively to test the change of fluorescence intensity. The results show that the presence of other anions has no obvious interference with the recognition of zinc ions by the zinc ion fluorescent probe of the application.

[0063] As shown in Figure 12 , the test paper coated with X6 is prepared for rapid and quantitative detection of Zn 2+ ions. Initially, the X6 fluorescent test paper is prepared by immersing the filter paper in a CH3OH:H2O:Hepes (v / v / v = 9:1:0.1) stock solution containing the fluorescent probe X6 (10 μM), and then it is dried. Subsequently, the prepared fluorescent test paper is further soaked in different concentrations of Zn 2+ ion (0, 0.1 mM and 1.0 mM) standard solution for 30 minutes and naturally dried. Under the ultraviolet lamp, the fluorescent test paper containing different concentrations of X6-Zn 2+ loaded shows different color changes, indicating that the newly designed X6 can be used to conveniently and rapidly detect Zn 2+ in the environment, i.e., trace amounts of zinc ions in the environment can be quantitatively detected in a solid state.

[0064] As shown in Figure 13 , the optimal configuration diagram of the fluorescent probe X6 and the [X6-Zn 2+ ] complex and the energy level diagram of its corresponding frontier orbital, X6 has a higher HOMO energy level (-6.03 eV), which may lead to its better hole transport performance, and the low LUMO energy (-1.93 eV) should help to accept electrons. The LUMO-HOMO energy difference of the fluorescent probe X6 after combining with zinc ions is reduced, and the DFT calculation result further proves the enhanced stability of the [X6-Zn 2+ ] complex.

[0065] As shown in Figure 14 , the PK-15 cells are inoculated into a 96-well culture plate at a density of about 7000 cells per well and adhered at 37°C in a humidified air with 5% CO2 for 12 hours. Then, 100 μL of fresh medium with different X6 concentrations (0, 2, 4, 6, 8 and 10 μM) is added to each well and cultured for 24 hours. Finally, 10 μL of MTT reagent is added to each well and cultured for another 3 hours. As Figure 10The results show that the cell survival rate is greater than 90% when the concentration of X6 is in the range of 0-10 μM, which indicates that X6 has very low cytotoxicity and good biocompatibility. On this basis, we did cell experiments.

[0066] As shown in Figure 15 , PK-15 cells were incubated with X6 (10 μM) in growth medium at 37 °C for 30 min. After washing with sterilized PBS buffer for three times, the excess X6 on the cell surface was removed. Then, the X6-prepared PK-15 cells were further exposed to 10 μM Zn 2+ solution for another 30 min, and then washed with PBS to remove the excess Zn 2+ . Finally, all the prepared cell samples were detected by confocal fluorescence microscopy.

[0067] As shown in Figure 16 , the linear calibration curve of Zn 2+ concentration and color intensity based on RGB mode. To 3 mL of blank solution CH3OH:H2O:Hepes (v / v / v = 9:1:0.1), 30 μL of probe stock solution and 0, 5, 15, 25, 50 μL of zinc ion solution (3 mM) were added. The high-resolution photos of the solution containing probe X6 and different concentrations of Al 3+ under 365 nm ultraviolet light were taken, and the photos were converted into corresponding RGB data using the "Color Assistant" application on the mobile phone. The color intensity was calculated by the formula I = 0.3R + 0.59G + 0.11B, and the results showed that there was a good linear relationship between the color intensity and the concentration of zinc ions in the range of 0-50 μM.

[0068] The above experiments show that the fluorescent probe compound X6 exhibits high sensitivity and high selectivity for Zn 2+ in solution.

Claims

1. A compound or its salt, which is 2. A method for preparing the compound of claim 1 or a salt thereof, characterized in that, The method includes the step of reacting 1H-indole-2-formylhydrazide with pyridoxal.

3. The method according to claim 2, characterized in that, The reaction temperature is 60–100°C.

4. The method according to claim 2, characterized in that, The reaction solvent is selected from protic solvents, preferably ethanol.

5. The method according to claim 2, characterized in that, The method includes the following reaction steps:

6. The application of the compound of claim 1 or a salt thereof in the preparation of a fluorescent probe for detecting metal ions in solution, wherein the metal ion is preferably a zinc ion.

7. A method for detecting zinc ions in a solvent, characterized in that, The method includes the steps of adding compound X6 to the test solution and detecting it using a fluorescence spectrometer.

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