A coumarin-based Schiff base fluorescent probe and its preparation method and application

Abstract

A coumarin-based Schiff base fluorescent probe has the structural formula shown in the following formula (I): #imgabs0# (I). The coumarin-based Schiff base fluorescent probe (NaChro sensor) of the present invention has various advantages, such as strong fluorescence, low detection limit, excellent selectivity, fast response time and reversibility, low cytotoxicity, dual-ion detection, high recovery rate in water sample detection, and can be used for fluorescence imaging of cells and animal tissues, for detecting and tracing Hg 2+ and Pb 2+ ions in environmental samples and biological systems. The development of such highly efficient chemical sensors is of great significance for fields such as environmental monitoring and water quality monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of metal ion detection, and particularly relates to a coumarin-based Schiff base fluorescent probe, a preparation method thereof and an application thereof. Background Art

[0002] Heavy metals have attracted much attention due to their harmful effects in various structural forms. Mercury (Hg 2+ ) and lead (Pb 2+ ) are highly toxic metal ions and are thus widely regulated. Mercury poisoning is usually caused by mining activities, ingestion of contaminated fish, and exposure of industrial workers. On the other hand, lead poisoning is usually associated with lead-based paints, gasoline, batteries, and pipes used in old buildings. Mercury enters water bodies at trace concentrations and is converted into methylmercury, which gradually accumulates in marine organisms over time. Eventually, humans ingest this mercury through the consumption of seafood. This gradual mercury poisoning usually leads to brain damage, kidney dysfunction, organ failure, and impaired immune function. Similarly, lead poses significant health risks as it affects various organs in the body, causing nerve damage, memory loss, kidney damage, and in severe cases, even muscle paralysis.

[0003] To effectively address the problem of heavy metal pollution, having reliable detection methods is crucial for biological and environmental systems. Various techniques have been developed and widely documented. However, in this particular study, our focus will be on fluorescent probes because they are simple, user-friendly, and there is a large body of literature on the subject. Recent literature has reported many fluorescent probes based on rhodamine, BODIPY, benzothiazole, carbazole, anthracene, coumarin, and naphthalimide for the detection of Hg 2+ and / or Pb 2+ . However, developing an efficient chemical sensor with the ability to detect two metal ions, a turn-on response, and enhanced selectivity for two toxic metal ions remains challenging. Specifically as follows:

[0004] 1. Selectivity and specificity: Simultaneously detecting and differentiating different metal ions in the same sample is a difficult problem to solve. This is because many metal ions are very similar in chemical properties, making it difficult to accurately and selectively detect them. The sensor needs to be designed according to the unique characteristics of the target metal ion to avoid the influence of interfering substances.

[0005] 2. Sensitivity: The sensor must have sufficient sensitivity to detect the target metal ion at low concentrations. Especially in environmental monitoring and water quality detection, the concentrations of toxic metal ions are usually very low, so high-sensitivity sensors are required for accurate detection.

[0006] 3. Response speed: An efficient sensor should have a fast response speed and be able to accurately detect the presence of target metal ions within a short period. For some toxic metal ions, a rapid response is crucial as they can pose serious threats to human health and the environment.

[0007] 4. Affinity and stability: The sensing layer of the sensor (usually a chemical fluorescent probe or similar substance) needs to have high selectivity and affinity to form a stable coordination complex with the target metal ion. Meanwhile, this coordination complex should remain stable under various conditions to ensure the reliability and regenerability of the sensor.

[0008] 5. Interfering substances: In real samples, there may be various interfering substances that can affect the accuracy and selectivity of the sensor. Therefore, the sensor needs to reduce the influence of these interfering substances through intelligent design and effective pretreatment steps.

[0009] 6. Repeatability and durability: An efficient chemical sensor must be able to operate stably for a long time and maintain its performance after multiple uses. The sensor should have good repeatability and durability to obtain reliable results in practical applications.

[0010] 7. Encapsulation and application: Advancing the sensor from the laboratory stage to practical applications requires solving the problems of encapsulation and integration. The sensor must be able to work under different conditions and adapt to the requirements of different scenarios.

[0011] Generally speaking, developing such an efficient chemical sensor requires comprehensive consideration of the above challenges and interdisciplinary research and cooperation, involving fields such as materials science, nanotechnology, and chemical analysis, to overcome these challenges and achieve practical applications. Summary of the Invention

[0012] Technical problems to be solved: Aiming at the problems existing in the prior art, the present invention provides a coumarin-based Schiff base fluorescent probe and its preparation method and application. The prepared coumarin-based Schiff base fluorescent probe (NaChro sensor), by reasonably designing the structure of the Schiff base compound and making full use of various detection mechanisms, has multiple advantages, such as strong fluorescence, low detection limit, excellent selectivity, fast response time and reversibility, low cytotoxicity, dual-ion detection, high recovery rate in water sample detection, and can be used for fluorescence imaging of cells and animal tissues. The development of such an efficient chemical sensor is of great significance for fields such as environmental monitoring and water quality monitoring.

[0013] Technical solution: A coumarin-based naphthol hydrazone Schiff base chemical sensor NaChro, the structural formula of which is shown in the following formula (I):

[0014]

[0015] The preparation method of a coumarin-based naphthol hydrazone Schiff base chemical sensor NaChro is as follows:

[0016] Step 1. Dissolve 2-hydroxy-1-naphthaldehyde in methanol, then add hydrazine hydrate, stir at room temperature for 20 h to form a yellow precipitate, then filter, wash and dry to obtain the intermediate compound 1

[0017] Step 2. Then dissolve compound 1 in a methanol solution, add 3-acetylcoumarin and AcOH, reflux under nitrogen for 20 h, and filter and wash the formed precipitate to obtain the yellow solid product NaChro

[0018] Preferably, in step 1, the ratio of 2-hydroxy-1-naphthaldehyde, methanol, and hydrazine hydrate is 5 mmol: 25 mL: 3 mL, and in step 2, the ratio of compound 1, methanol, 3-acetylcoumarin, and AcOH is 1.88 mmol: 25 mL: 1.88 mmol: 0.15 mL.

[0019] Application of the coumarin-based naphthol hydrazone Schiff base chemical sensor NaChro in detecting and tracing heavy metal ions in environmental samples and biological systems.

[0020] Preferably, the heavy metal ion is Pb 2+ and / or Hg 2+ .

[0021] Beneficial effects: The coumarin-based Schiff base fluorescent probe of the present invention solves the following technical problems:

[0022] 1. Selectivity and synergy: By incorporating specific heteroatoms, Schiff base compounds can act synergistically with the target analyte to improve selectivity. The NaCro sensor utilizes the characteristics of Schiff base compounds, especially the chelation reaction of characteristic imine bonds, to generate significant detection signals.

[0023] 2. Multiple detection mechanisms: The sensor involves multiple detection mechanisms, such as fluorescence resonance energy transfer (FRET), aggregation-induced emission (AIE), chelation-enhanced fluorescence / quenching (CHEF / CHEQ), excited-state intramolecular proton transfer (ESIPT), photoinduced electron transfer (PET), and intramolecular charge transfer (ICT). These mechanisms can be applied according to the different structures and active parts of Schiff base compounds.

[0024] 3. Reversibility and cost reduction: The sensor uses a reversible multi-analyte probe, which means that the sensor can be used multiple times, reducing costs and resource consumption.

[0025] 4. Strong fluorescence, selectivity, and response time: The NaCro sensor has strong fluorescence characteristics, high selectivity for Hg 2+ and Pb 2+ , and a relatively fast response time.

[0026] 5. Structural diversity: The NaCro sensor combines the structural units of naphthol hydrazone and 3-acetylcoumarin, which endows it with tunable emission wavelengths, high solubility, and diverse heteroatoms, further enhancing the sensor's functionality.

[0027] 6. Detection sensitivity: The NaCro sensor can detect Hg 2+ and Pb 2+ at very low limits in aqueous media, with limit of detection values of 8.3 and 10.5 nM respectively, showing high detection sensitivity.

[0028] In summary, the NaCro sensor described in the present invention integrates hydrogen bond donors and acceptors, and parts with lone pair electrons and C=N double bonds, enabling it to simultaneously undergo ESIPT, PET, and C=N isomerization. Its novelty lies in the ability to exhibit multiple response characteristics in a single system. Traditional chemical sensors usually involve the use of one or two processes. By integrating three processes into a single chemical sensor molecule, several advantages are achieved. First, it allows for the simultaneous detection of multiple analytes (Hg 2+ and Pb 2+ ), providing more comprehensive and sensitive sensing capabilities.

[0029] Secondly, the combined action of ESIPT, PET, and C=N isomerization generates a unique fluorescence response that can be easily distinguished from other compounds, thereby improving selectivity. This integrated system also provides a platform for studying complex photophysical interactions and photoinduced processes in a single molecular framework. In addition, the presence of ESIPT in the probe structure also results in a larger Stokes shift, ensuring no overlap between the absorption and emission spectra. Other advantages include reversibility in solution and color development reactions when used on test strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Fluorescence emission spectra of NaChro upon addition of Hg 2+ and Pb 2+ , excitation wavelength: 386 nm. In the figure, (a) is upon addition of Hg 2 + , (b) is upon addition of Pb 2+ ;

[0031] Figure 2 For Hg 2+ and Pb2+ Linear fitting curve of concentration and fluorescence emission intensity. In the figure, (a) is for Hg 2+ , (b) is for Pb 2+ ;

[0032] Figure 3 Images of filter paper strips coated with 20 μM NaChro before and after treatment with different concentrations of Hg 2+ and Pb 2+ .

[0033] Figure 4 After incubation with 10, 20, 50, and 100 μM NaChro respectively, the cytotoxicity was detected by the MTT method.

[0034] Figure 5 Confocal fluorescence images of HeLa cells. In the figure, (a - b) are control cells, (c - d) are cells stained with 20 μM NaChro for 30 minutes, and cells treated with (e - f) 0.4 μM Hg 2+ , (g - h) 0.8 μM Hg 2+ , (i - j) 0.4 μM Pb 2+ and (k - l) 0.8 μM Pb 2+ .

[0035] Figure 6 Fluorescence images of zebrafish. In the figure, (a - b) are control cells, (c - d) are zebrafish stained with 20 μM NaChro for 15 minutes, and zebrafish treated with (e - f) 0.4 μM Hg 2+ and (g - h) 0.8 μM Hg 2+ .

[0036] Figure 7 Absorption spectra of different concentrations of Hg 2+ and Pb 2+ added to acetonitrile with 20 μM NaChro. In the figure, (A) is for Hg 2+ , (B) is for Pb 2+ .

[0037] Figure 8 Graph of the binding constant K 2+ of NaChro - Hg 2+ and NaChro - Pb A using 20 μM NaChro in acetonitrile. In the figure, (A) is for Hg 2+ , (B) is for Pb 2+ .

[0038] Figure 9 In (A), NaChro, Hg 2+and other ions, and (B) NaChro, Pb 2+ Selectivity and interference diagrams for and other ions. The cations studied were Al 3+ , Zn 2+ , Fe 2+ , Ca 2+ , Na + , Fe 3+ , Mn 2+ , Cu 2+ , Ag + , Cr 6+ , Cr 3+ , K + , Mg 2+ , Ba 2+ , Ga 3+ , Ni 2+ and Cd 2+ each at 10 mM.

[0039] Figure 10 Emission responses of 20 μM NaChro solutions for (C) NaChro and Hg 2+ and (D) NaChro and Pb 2+ at different pH values. Excitation wavelength: 386 nm.

[0040] Figure 11 Response times of (A) NaChro with Hg 2+ and (B) NaChro with Pb 2+ in.

[0041] Figure 12 Reversibility studies of (C) NaChro and Hg 2+ and (D) NaChro and Pb 2+ in.

[0042] Figure 13 Job’s plot for (A) NaChro with [Hg 2+ and (B) NaChro with [Pb 2 + using a MeCN solution of 20 μM NaChro.

[0043] Figure 14 HR-MS spectrum of NaChro-Hg 2+

[0044] Figure 15 HR-MS spectrum of NaChro-Pb 2+

[0045] Figure 16 Response of (A) NaChro with Hg in​​2+ Binding mechanism. Add (B) 0, (C) 1, and (D) 1.5 μM Hg 2+ 1H NMR titration peaks after addition.

[0046] Figure 17 In (A), Na Chro binds to Pb 2+ Binding mechanism of the binding of (A) Na Chro to Pb. Add (B) 0, (C) 1, (D) 1.5 μM Pb 2+ 1H NMR titration peaks after addition. Detailed implementation manners

[0047] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] The sources of the raw materials used in the embodiments of the specification of the present invention are as follows:

[0049] All solvents and reagents, including 3-acetylcoumarin, 2-hydroxy-1-naphthaldehyde, hydrazine hydrate, acetonitrile (MeCN), methanol, glacial acetic acid (AcOH), are purchased from the same supplier (Aladdin, China). All cations used are from Accustandard. The double-distilled water used throughout the experiment is from a Milli-Q purification system (Millipore Corp., Bedford, MA, USA). Cell culture reagents such as penicillin, Dulbecco's phosphate-buffered saline (DPBS), DMEM medium, streptomycin, fetal bovine serum (FBS), and HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer are from Hyclone (USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity kit is purchased from Beyotime Institute of Biotechnology.

[0050] Example 1: Synthesis of the fluorescent probe compound NaChro of the present invention

[0051] The process is as follows:

[0052]

[0053] Step 1: Synthesis of intermediate compound 1:

[0054] In a 100 mL two-necked flask, add 0.86 g (5 mmol) of 2-hydroxy-1-naphthaldehyde and 3 mL of hydrazine hydrate (mass fraction 85%) dissolved in 25 mL of methanol, and stir the mixture at room temperature for 20 hours. Monitor the reaction using TLC. After the reaction is completed, filter, wash, and dry to obtain intermediate compound 1 It is a light yellow solid.

[0055] The intermediate compound 1 is 2-hydroxy-1-naphthaldehyde hydrazone, and finally 0.72 g was obtained with a yield of 77%.

[0056] Step 2: Synthesis of the probe NaChro sensor

[0057] In a 100 mL two-necked flask, 0.353 g (1.88 mmol) of compound 1 and 0.35 g (1.88 mmol) of 3-acetylcoumarin were added, dissolved in 25 mL of methanol solution, and then 0.15 mL of AcOH was added. The mixture was refluxed under nitrogen for 20 hours. The formed precipitate was filtered and washed to obtain a yellow solid product NaChro (0.503 g, yield 76%), which is the coumarin-based Schiff base fluorescent probe.

[0058] 1H nuclear magnetic resonance (1H NMR) confirmed the structure:

[0059] Compound 1: 1 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 8.77 (s, 1H), 7.95 (s, 1H), 7.71 (s, 2H), 7.56 - 7.13 (m, 3H), 5.52 (s, 2H) (Fig. S1). 13 13C NMR (100 MHz, DMSO-d6) δ 161.08, 156.04, 149.52, 143.69, 137.23, 132.48, 126.21, 118.88, 116.22, 116.08, 115.11 (Fig. S2).

[0060] NaChro: 1 1H NMR (00 MHz, DMSO-d6): δ 13.19 (s, 1H), 9.64 (s, 1H), 8.77 (s, 1H), 7.74 (s, 1H), 7.71 - 7.50 (m, 3H), 7.47 (d, J = 2.2 Hz, 1H), 7.45 - 7.36 (m, 3H), 7.34 (s, 1H), 3.88 (s, 3H) (Fig. S3). 13 13C NMR (100 MHz, DMSO-d6) δ 169.86, 152.13, 152.02, 148.12, 141.25, 131.11, 129.07, 128.38, 128.18, 128.08, 126.17, 124.25, 120.13, 111.73, 106.12, 104.21, 96.12, 95.27, 95.04, 28.82, 20.01, 12.05 (Fig. S4).

[0061] Mass spectrometry (MS) confirmation of the structure:

[0062] MS (m / z) calculated for C 22 H 16 N2O3 356.38, found [M+H] + 357.12 (Fig. S5).

[0063] Example 2 Fluorescence spectral titration experiment of the fluorescent probe NaChro and determination of the detection limit

[0064] Take 3 mL of a MeCN solution of NaChro with a concentration of 20 μM in a quartz cuvette, and add 5 μL of an aqueous solution of Hg 2+ and Pb 2+ each time, shake well, and measure the fluorescence spectrum of the solution after equilibrium (as Figure 1 shown). With the addition of Hg 2+ and Pb 2+ ions, the fluorescence intensity of the fluorescent probe NaChro under excitation at an excitation wavelength of 386 nm gradually increases. From the relationship curve between ion concentration and fluorescence intensity, it can be seen that the ion concentrations of Hg 2+ and Pb 2+ show a good linear relationship with the fluorescence intensity (as Figure 2 shown R 2 Hg = 0.9969, R 2 Pb = 0.9931), and the detection limits of the fluorescent probe NaChro for Hg 2+ and Pb 2+ ions are 0.014 μM and 0.091 μM, respectively.

[0065] Example 3 Use of the fluorescent probe NaChro for test paper detection

[0066] The test papers are soaked in a MeCN solution of NaChro with a concentration of 20 μM for about 30 seconds, and then dried. Then they are soaked in aqueous solutions of 0.4 and 0.8 μM Hg 2+ or Pb 2+ for 2 h. Figure 3 It shows that there are obvious color differences in the test papers soaked in different concentrations of ions.

[0067] Example 4 Use of the fluorescent probe NaChro for water sample detection

[0068] The fluorescent probe is added to tap water, pond water, and river water samples respectively. 3 mL of a 20 μM NaChro fluorescent probe is used to detect Hg 2+ and Pb 2+Recovery rates are shown in Tables 1 and 2 below:

[0069] Table 1 shows the recovery rates of Hg in water samples from different sources by NaChro 2+

[0070]

[0071] Table 2 shows the recovery rates of Pb in water samples from different sources by NaChro 2+

[0072]

[0073] As shown in Tables 1 and 2, the fluorescent probe NaChro has high recovery rates for Hg 2+ and Pb 2+ ions, which are 98.3% to 99.4% and 98.5% to 99.3% respectively.

[0074] Example 5 Analysis of cell viability of fluorescent probe NaChro

[0075] Probe solutions with different concentrations (0, 10, 20, 50, 100 μM) were added to culture dishes containing HeLa cells, and the cell states were observed after 24 h. The cell viability data are as Figure 4 shown. At concentrations of 0 - 20 μM, the survival rate is higher than 95%. When the concentration increases to 50 μM or higher, the cell survival rate decreases. When the NaChro concentration further increases to 100 μM, the cell viability is still above 50%. These results show good biocompatibility, which means that NaChro can be used for low-concentration cell imaging.

[0076] Example 6 Application of fluorescent probe NaChro fluorescence microscopy imaging in HeLa cells

[0077] Cells were seeded into the medium in a 24-well plate at a density of 2×104 cells per well. After 24 hours, the cells were incubated with 20 μM NaChro at 37 °C for about 30 minutes. The remaining probe was removed using PBS, and then the cells were further treated with Hg 2 + / Pb 2+ (0.4 and 0.8 μM) for about 30 minutes. Next, images were taken using a confocal laser scanning microscope. As Figure 5 shown, (a - b) are control cells, (c - d) are cells stained with 20 μM NaChro for 30 minutes, and cells treated with (e - f) 0.4 μM Hg 2+ , (g - h) 0.8 μM Hg 2+ , (i - j) 0.4 μM Pb 2+ ​​and (k-l) 0.8 μM Pb 2+ The treated stained cells. Figures a and b show the control cells, while figures c and d show the non-fluorescent images after incubation in 20 μM NaChro. The addition of 0.4 and 0.8 μM Hg 2+ resulted in an enhanced yellow-green fluorescence (figures f and h). Figures j and l show the enhanced cyan fluorescence after the addition of 0.4 and 0.8 μM Pb 2+ The color patterns at each stage are different, making NaChro an effective bioimaging tool.

[0078] Example 7 Fluorescent Probe NaChro for Zebrafish Imaging

[0079] Zebrafish at 3 - 7 days old were incubated with 20 μM NaChro for about 15 minutes. The remaining probe was removed using PBS, and then the zebrafish were further treated with Hg 2+ (0.4 and 0.8 μM) in PBS for about 10 minutes. Next, images were taken using a Zeiss AXIOSCOPE A1 microscope. As Figure 6 shown, (a - b) are control cells, (c - d) are zebrafish stained with 20 μM NaChro for 15 minutes, and (e - f) are stained zebrafish treated with 0.4 μM Hg 2+ and (g - h) 0.8 μM Hg 2+ The control cells in figures a and b show no emission, while figures c and d show the fluorescent images after incubation in 20 μM NaChro, with relatively weak emission at the head of the fish. The addition of 0.4 and 0.8 μM Hg 2+ enhanced the yellow-green fluorescence, and the entire zebrafish body showed strong fluorescence.

[0080] Example 8 Determination of Association Constant and Detection Limit

[0081] The detection limit (LOD) was determined using the absorbance values from the UV-visible titration experiment (as Figure 7 ). The plot of the average maximum absorbance against the concentration of Hg 2+ / Pb 2+ gave the slope value, and the following equation was used to obtain the LOD value.

[0082]

[0083] where σb is the standard deviation of the blank and m is the slope of the fitted line. The LOD values for Hg 2+ and Pb 2+ were calculated to be 0.014 and 0.091 μM, respectively.

[0084] To quantify the binding affinity (KA) of NaChro with ions, plot log[(F0 - F) / F] against log[M 2+ , where n represents the number of binding sites, and F0 and F are the fluorescence emission values of NaChro before and after the addition of Hg 2+ / Pb 2+ (see Figure 1 ).

[0085]

[0086] As Figure 8 shown, the binding constant values of Hg 2+ and Pb 2+ are estimated to be 6.09×10 5 and 4.91×10 4 M -1 respectively using Equation 2. This also indicates that the coordination of the probe with Hg 2+ is stronger than that with Pb 2+ .

[0087] Example 9 Fluorescent Probe NaChro Selectivity and Anti-Interference Ability

[0088] As Figure 9 , take 3 mL of the probe solution with a concentration of 20 μM in a quartz cuvette. First, add 5 μL of various cation solutions with a concentration of 10 mM, and then add 5 μL of Hg 2+ / Pb 2+ ion solutions respectively, shake well, and measure the fluorescence emission spectrum after waiting for 1 minute. When adding other ions except Hg 2+ and Pb 2+ , the fluorescence of the solution only increases slightly, but when adding Hg 2+ and Pb 2+ , the fluorescence of the solution increases significantly, indicating that the fluorescent probe NaChro has high selectivity and good anti-interference ability for the recognition of Hg 2+ and Pb 2+ ions.

[0089] Example 10 Influence of pH Value on Fluorescent Probe NaChro

[0090] As Figure 10 , when only the fluorescent probe NaChro is present, the fluorescence is quite weak, and the difference in emission intensity within different pH ranges is small. When adding Hg 2+When the pH is below 4, the emission is very low, which may be due to the protonation of imine nitrogen. However, as the pH increases, the emission intensity increases and reaches a peak at pH 7. As the condition turns alkaline, when the pH exceeds 9, the emission value drops rapidly. This should be the result of the deprotonation of the naphthol - OH group under strong alkaline conditions. The addition of Pb 2+ shows the same trend, where the protonation and deprotonation of the naphthol - OH group change the emission pattern.

[0091] Example 11 Response Time of Fluorescent Probe NaChro

[0092] As Figure 11 , the response time was determined by measuring the emission intensity values at different Hg 2+ and Pb 2+ concentrations. Real - time analysis was performed using a MeCN solution of 20 μM NaChro and measurements were made for 0.2, 0.4, and 0.6 μM Hg 2+ and Pb 2+ . Both graphs show that the fluorescence intensity of Hg 2+ and Pb 2+ rises sharply until reaching a stable level at 39 seconds and 58 seconds respectively.

[0093] Example 12 Reversibility Analysis of Fluorescent Probe NaChro

[0094] As Figure 12 , the reversibility of the NaChro complex was studied using the chelating agent EDTA. By adding a strong chelating agent, EDTA - Hg 2+ / Pb 2+ complexes were generated to reverse the initial probe - metal complexes. The experiments show that the fluorescence intensity of the probe switches back and forth between high and low levels, enabling reversible switching of fluorescence.

[0095] Example 13 Detection Mechanism of Fluorescent Probe

[0096] (1) Fluorescent Probe Binding Ratio

[0097] As Figure 13 , the absorbance values of Hg 2+ and Pb 2+ at different molar ratios were recorded to study the exact binding stoichiometry. Here, the total concentration was kept constant while changing the mole fractions of NaChro and Hg 2+ / Pb 2+ . Figure 13 (A) and 13(B) of the Job's plots show absorption maxima at mole fractions of 0.5 and 0.3, representing NaChro - Hg 2+ and NaChro - Pb 2+The binding ratios of the complexes are 1:1 and 2:1. In addition, the 1:1 and 2:1 stoichiometric ratios of NaChro with Hg 2+ and Pb 2+ were further confirmed by mass spectrometry ( Figure 14 and 15 ). The prominent peaks at m / z 556.82 and 921.19 reflect the NaChro-Hg 2+ and NaChro-Pb 2+ complexes, respectively.

[0098] (2) Sensing mechanism

[0099] As Figure 16 , the recognition mechanism of the dual-responsive NaChro towards Hg 2+ and Pb 2+ was determined by 1H NMR titration spectra in deuterated DMSO solvent. Figure 16 (A) shows how the inhibition of the ESIPT and PET processes enables the detection of analytes by enhancing the emission intensity. Figure 16 (B)-(D) show the changes in chemical shifts with increasing Hg 2+ concentration. Due to the simultaneous presence of OH and NH groups in the compound, rapid proton exchange occurs, and NMR usually shows a single signal at the average chemical shift. By alternating solvents and observing the disappearance and splitting patterns of the recognition peaks, the OH shift appears at 13.34 ppm. When 1 and 1.5 μM of Hg 2+ were added, this peak shrank, probably due to deprotonation starting to occur upon coordination with the metal. Conversely, when 1 and 1.5 μM of Hg 2+ were added, three hydrogen atoms initially appearing at 3.94 ppm shifted down to 4.65 ppm and 5.92 ppm. The NMR titration results of the probe towards Pb 2+ are shown in Figure 17 . For Pb 2+ , a downfield shift was also observed, but it was less obvious compared to that upon addition of Hg 2+ (( Figure 17 (B)-(D))). These proton shifts indicate the coordination of naphthol oxygen, imine nitrogen, and carbonyl oxygen with these two ions.

Claims

1. A coumarin-based Schiff base fluorescent probe, characterized in that, Its structural formula is shown in the following formula (I): （I）。 2. The preparation method of a coumarin-based Schiff base fluorescent probe according to claim 1, characterized in that, The steps are as follows: Step 1. Dissolve 2-hydroxy-1-naphthaldehyde in methanol, then add hydrazine hydrate, stir at room temperature for 20 h to form a yellow precipitate, then filter, wash and dry to obtain the intermediate compound 1 ; Step 2. Then dissolve Compound 1 in a methanol solution, add 3-acetylcoumarin and AcOH, reflux for 20 h under nitrogen, filter and wash the formed precipitate to obtain the yellow solid product NaChro .

3. The preparation method of a coumarin-based Schiff base fluorescent probe according to claim 2, characterized in that, In the first step, the ratio of 2-hydroxy-1-naphthaldehyde, methanol, and hydrazine hydrate is 5 mmol: 25 mL: 3 mL. In the second step, the ratio of compound 1, methanol, 3-acetylcoumarin, and AcOH is 1.88 mmol: 25 mL: 1.88 mmol: 0.15 mL.

4. Use of a coumarin-based Schiff base fluorescent probe according to claim 1 in detecting and tracking heavy metal ions in environmental samples and biological systems, characterized in that, The heavy metal ion is Pb 2+ and / or Hg 2+ .

Images