A "off-on" near-infrared lead ion detection fluorescent probe YSQ and a preparation method and application thereof

Abstract

This invention discloses an "off-on" type near-infrared fluorescent probe YSQ for lead ion detection, its preparation method, and its applications. This near-infrared fluorescent probe YSQ exhibits strong selectivity for lead ions, a fast response time, significant fluorescence enhancement after complexation, and a low detection limit. Its structure is shown in Formula I below. This invention uses dicyanoisophorone and isoquinoline hydrazide as fluorescent groups to prepare the Schiff base-type fluorescent probe YSQ through a simple condensation reaction. The preparation method of this "off-on" type near-infrared fluorescent probe is simple, the raw materials are readily available, and the obtained product is a solid powder that is easy to store.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to a 'off-on' near-infrared lead ion detection fluorescent probe YSQ, a preparation method and application thereof. BACKGROUND

[0002] Lead is one of the most widely used heavy metals and is also a major heavy metal pollutant in the environment. With the increasing use of gasoline, storage batteries, metal products, chemical coatings and other products and the discharge of industrial and agricultural waste, lead has widely entered people's living environment in various ways and is showing an increasing trend. Not only does lead directly affect the growth, yield and quality of crops, but also indirectly causes serious harm to human health through the food chain. Lead pollution has attracted widespread social attention. At present, although the detection technology for Pb 2+ has made great progress, there are still a series of problems. Traditional lead ion detection technologies include electrochemical methods, atomic absorption methods, ion chromatography methods, catalytic wave polarography methods and the like, and have problems such as complicated operation steps, high detection limit, long response time, not obvious visual changes and short absorption wavelength which is not conducive to cell imaging. In recent years, fluorescent chemical sensor detection methods have developed rapidly, especially near-infrared fluorescent probes, and the visual fluorescence changes can realize in vivo imaging, bringing convenience to the medical field.

[0003] At present, the design alternatives of fluorescent probes mainly include purine rings, coumarin, fluorescein and porphyrin and the like. Most of the Pb 2+ detection fluorescent probes designed and developed by researchers are of the quenching type, and the background signal of the fluorescent quenching type probe detection is large, the sensitivity is low and it is not suitable for biological sample detection. SUMMARY

[0004] The application provides a 'off-on' lead ion detection fluorescent probe YSQ, which has high selectivity for lead ions, a low detection limit and a short response time, and can rapidly and real-time detect trace lead ions in an environmental system.

[0005] The application also provides a preparation method and application of the lead ion detection fluorescent probe YSQ.

[0006] Technical scheme: In order to achieve the above application purposes, the 'off-on' near-infrared lead ion detection fluorescent probe YSQ provided by the application is prepared by using dicyanoisophorone and isoquinoline hydrazine as fluorescent groups, and the structure is shown in the following formula I:

[0007]

[0008] The preparation method of the "on-off" type near-infrared lead ion detection fluorescent probe YSQ comprises the following steps:

[0009] 2-(3,5,5-trihydroxycyclohexane-2-enyl) malonitrile is obtained by reacting isophorone and malonitrile; then (E)-2-(3-(4-hydroxyphenylstyryl)-5,5-dimethyl-2-cyclohexenyl-1-ylidene) malonitrile is prepared by reacting with p-hydroxybenzaldehyde; and urotropine and trifluoroacetic acid are added to prepare dicyanisophorone aldehyde.

[0010] Isoquinoline hydrazine is obtained by esterification and amidation of isoquinoline carboxylic acid; and the "on-off" type near-infrared lead ion detection fluorescent probe YSQ is obtained by Schiff base condensation reaction of isoquinoline hydrazine and dicyanisophorone aldehyde.

[0011] The reaction route for preparing the lead ion detection fluorescent probe YSQ is shown as follows:

[0012]

[0013] In the reaction route: the intermediate is dicyanisophorone aldehyde (A); isoquinoline hydrazine (B); (N'-((E)-5-((E)-2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-2-hydroxybenzylidene) isoquinoline-1-carbonyl hydrazine (YSQ) is the lead ion detection fluorescent probe molecule.

[0014] In step (1), isophorone and malonitrile are dissolved in methanol under N2 protection, piperidine, glacial acetic acid and acetic anhydride are added, the reaction is completed, the reaction liquid is cooled to room temperature, then poured into ice water, filtered, and the obtained solid is recrystallized with ethanol to obtain the intermediate 2-(3,5,5-trihydroxycyclohexane-2-enyl) malonitrile.

[0015] In step (1), the intermediate 2-(3,5,5-trihydroxycyclohexane-2-enyl) malonitrile is dissolved in ethanol solution, p-hydroxybenzaldehyde and piperidine are added, and reflux stirring reaction is carried out; after the reaction is completed, the organic solvent is removed under reduced pressure, and the intermediate (E)-2-(3-(4-hydroxyphenylstyryl)-5,5-dimethyl-2-cyclohexenyl-1-ylidene) malonitrile is obtained by column chromatography.

[0016] In step (1), the intermediate (E)-2-(3-(4-hydroxyphenylstyryl)-5,5-dimethyl-2-cyclohexenyl-1-ylidene) malonitrile is dissolved in trifluoroacetic acid, urotropine is added, and reflux stirring is carried out; after the reaction is completed, it is added to ice water, extracted, dried, and column chromatography is carried out to obtain the final product dicyanisophorone aldehyde.

[0017] In step (2), the isoquinoline carboxylic acid is dissolved in an organic solvent methanol, concentrated sulfuric acid is added, and the mixture is stirred under heating and reflux, and after the reaction is completed, the reaction liquid is cooled to room temperature, the pH is adjusted to neutral, and then extraction, separation, drying, and removal of the solvent are performed, and the isoquinoline carboxylic acid methyl ester is obtained by column chromatography.

[0018] In step (2), the isophorone aldehyde and isoquinoline hydrazide are dissolved in an organic solvent methanol, the mixture is stirred under heating and reflux, and after the reaction is completed, the reaction liquid is cooled to room temperature, and the solvent is removed by distillation under reduced pressure, and the crude product is purified by recrystallization to obtain the fluorescent probe YSQ.

[0019] The lead ion detection fluorescent probe YSQ according to the present application has the following mechanism of action. 2+ Theoretical calculation of the mechanism of action.

[0020] The application of the lead ion detection fluorescent probe YSQ according to the present application in detecting lead ions.

[0021] The application of the lead ion detection fluorescent probe YSQ according to the present application in real-time, rapid, and on-site detection of trace lead ions in a solution and cells.

[0022] Preferably, the preparation process of the YSQ comprises:

[0023] (1) Preparation of dicyanoisophorone aldehyde (A)

[0024] Under N2 protection, isophorone and malononitrile are dissolved in methanol, piperidine is added, and a small amount of glacial acetic acid and acetic anhydride is added, and after the reaction is completed, the reaction liquid is cooled to room temperature and poured into ice water, and then filtration is performed, and the obtained solid is recrystallized with ethanol to obtain the intermediate 2-(3,5,5-trihydroxycyclohexane-2-enylidene) malononitrile.

[0025] The intermediate 2-(3,5,5-trihydroxycyclohexane-2-enylidene) malononitrile is dissolved in an ethanol solution, p-hydroxybenzaldehyde and a small amount of piperidine are added, and the mixture is stirred under reflux, and after the reaction is completed, the organic solvent is removed under reduced pressure, and the intermediate (E)-2-(3-(4-hydroxyphenyl)-5,5-dimethyl-2-cyclohexenyl-1-ylidene) malononitrile is obtained by column chromatography (ethyl acetate: petroleum ether = 5:1).

[0026] The intermediate (E)-2-(3-(4-hydroxyphenethyl)-5,5-dimethyl-2-cyclohexenyl-1- ylidene)propanedinitrile is dissolved in trifluoroacetic acid, urotropine is added, and the mixture is stirred under reflux at 100°C. After the reaction is complete, the reaction mixture is added to ice water, extracted with dichloromethane, and dried. The final product, dicyanisatyl aldehyde (A), is obtained by column chromatography (ethyl acetate: petroleum ether = 5:1).

[0027] (2) Preparation of isoquinoline hydrazide (B)

[0028] The isoquinoline carboxylic acid is dissolved in an organic solvent, methanol, and concentrated sulfuric acid is added. The mixture is stirred under reflux with heating. After the reaction is complete, the reaction mixture is cooled to room temperature, and the pH is adjusted to neutral with sodium hydroxide. The mixture is extracted with ethyl acetate, separated, dried, and the solvent is removed to obtain the crude isoquinoline carboxylic acid methyl ester. The crude isoquinoline carboxylic acid methyl ester is dissolved in an organic solvent, methanol, and hydrazine hydrate is added. The mixture is stirred under reflux with heating. After the reaction is complete, the reaction mixture is cooled to room temperature, and the organic solvent is removed under reduced pressure. The mixture is extracted, separated, dried, and the solvent is removed. The final target material, isoquinoline hydrazide (B), is obtained by column chromatography (ethyl acetate: petroleum ether = 1:3).

[0029] (3) Preparation of a lead ion fluorescent probe based on dicyanisatyl as a parent compound

[0030] The dicyanisatyl aldehyde and the isoquinoline hydrazide are dissolved in an organic solvent, methanol, and the mixture is stirred under reflux. After the reaction is complete, the reaction mixture is cooled to room temperature, and the solvent is removed under reduced pressure. The crude product is purified by recrystallization to obtain the fluorescent probe YSQ.

[0031] A preferred reaction scheme for the preparation of the lead ion detection fluorescent probe YSQ is shown below.

[0032]

[0033] The dicyanoisophorone used in the probe of the present application is a good fluorescent parent, which has the characteristics of large Stokes shift, near-infrared emission, easy synthesis and the like. The isoquinoline derivative has many advantages as a fluorescent group, and has good biocompatibility and low biological toxicity. Therefore, the present application designs and develops a novel structure of Schiff base type fluorescent probe YSQ based on dicyanoisophorone as a parent. The probe molecule has a near-infrared emission wavelength (> 600 nm), and has the advantages of low cytotoxicity, good compatibility with ecological environment and biology, strong tissue penetration, small cell tissue damage and the like, and has a large Stokes shift, which effectively avoids the large spectral overlap between the absorption spectrum and the emission spectrum, and improves the proportion of fluorescence radiation in the energy dissipation in the fluorescence emission. In addition, compared with the probe with a purine structure as a parent in the prior art, the synthesis route is short, the yield is high, the raw material cost is low, and the probe has high selectivity and sensitivity to Pb 2+ , and can successfully prepare fluorescent test paper to detect trace Pb 2+ in the environment.

[0034] At the same time, the present application also verifies the complexation mechanism of the Pb 2+ detection fluorescent probe YSQ and Pb 2+ by density functional theory (DFT) calculation. Through the optimal configuration theory calculation, the probe molecule (YSQ) is combined with Pb 2+ through the oxygen atom in the phenolic hydroxyl group, the nitrogen atom on the enamine, and the nitrogen atom on the isoquinoline ring, and the bond lengths are After complexation, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of YSQ-Pb 2+ have an orbital energy level difference of 1.9116 eV, which is much lower than the orbital energy level difference (2.8934 eV) of the probe molecule YSQ itself, indicating that the YSQ-Pb 2+ complex is more stable. From a theoretical basis, it is shown that the probe molecule YSQ has strong selectivity and sensitivity to Pb 2+ .

[0035] The present application designs a near-infrared fluorescent probe based on dicyanoisophorone as a parent to detect lead ions. Compared with the traditional lead ion detection technology, the "on-off" type near-infrared lead ion fluorescent probe detection method of the present application is more convenient for detecting lead ions, realizes low cost and high efficiency, and can realize real-time, rapid and on-site detection, effectively solving the problems of large background signal, low sensitivity and unsuitability for biological sample detection of the existing fluorescence quenching type Pb 2+ detection fluorescent probe.

[0036] Advantages: Compared with the prior art, the present application has the following advantages:

[0037] The present application prepares a fluorescent probe with dicyanoisophorone as the parent by condensation of dicyanoisophorone aldehyde and isoquinoline hydrazine by one-step method. The fluorescent probe has specific recognition for lead ions, short response time, high sensitivity, and high selectivity. The probe has low detection limit (18 nM) for Pb 2+ The probe has high sensitivity, high selectivity, and low detection limit (18 nM), and more importantly, the preparation method of the probe is simple, the obtained product is a solid powder, easy to store, and has good stability, and can realize real-time, rapid and on-site detection of trace lead ions in solution and cells. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The ultraviolet absorption spectrum and color change graph of the lead ion fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different concentrations of lead ions (Pb 2+ ) are shown in the following figure: Figure 1 The left side of the figure is colorless, and the right side is purple red;

[0039] Figure 2 The selective fluorescence spectrum graph of the lead ion fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different metal ions is shown in the following figure:

[0040] Figure 3 The fluorescence spectrum response graph and color change graph of the fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different concentrations of lead ions (Pb 2+ ) are shown in the following figure:

[0041] Figure 4 The fluorescence response graph of the fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different metal ions for selective interference detection is shown in the following figure:

[0042] Figure 5 The Job-plot curve of the fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) and lead ions (Pb 2+ ) complexation ratio is shown in the following figure:

[0043] Figure 6 The response time graph of the fluorescent probe prepared in Example 1 for detecting lead ions is shown in the following figure:

[0044] Figure 7 The fluorescence spectrum response graph and color change graph of the fluorescent probe prepared in Example 1 in (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) solution for different concentrations of lead ions (Pb 2+Fluorescence response graph of the fluorescent probe prepared in Example 1 in different pH value (2 to 12) range;

[0045] Figure 8 Optimal configuration graph of the fluorescent probe prepared in Example 1 and the fluorescent probe after complexing with lead ions;

[0046] Figure 9 Toxicity test graph of the fluorescent probe prepared in Example 1 in biological cells;

[0047] Figure 10 Cell imaging graph of the fluorescent probe prepared in Example 1 in biological cells;

[0048] Figure 11 Color change graph of the fluorescent test paper prepared by the fluorescent probe prepared in Example 1 in testing different concentrations of lead ions;

[0049] Figure 12 Mass spectrum of the fluorescent probe prepared in Example 1 1 H NMR spectrum;

[0050] Figure 13 Mass spectrum of the fluorescent probe prepared in Example 1 13 C NMR spectrum;

[0051] Figure 14 Ms spectrum of the fluorescent probe prepared in Example 1

[0052] Figure 15 Infrared IR spectrum of the fluorescent probe prepared in Example 1. DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with the accompanying drawings and examples.

[0054] The experimental methods used in the present application are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the experiments can be obtained from commercial channels unless otherwise specified. All the reagents selected in the examples are commercially available analytical pure or chemical pure.

[0055] Among them, the various ion solutions in the examples are prepared by adding deionized water to chemical reagents such as anhydrous zinc chloride, anhydrous ferric chloride, anhydrous aluminum chloride, etc. with a purity of more than 99% or nitrate chemical reagents such as Pb (NO3) 2.

[0056] Specific synthesis route of the fluorescent probe YSQ based on dicyanoisophorone as the parent:

[0057] (1) Preparation of intermediate dicyanoisophorone aldehyde (A)

[0058] Isophorone (4.3 g, 31.13 mmol) and malononitrile (1.87 g, 28.3 mmol) were dissolved in methanol (15 mL) under N2protection, piperidine (0.5 mL) was added, and glacial acetic acid (0.2 mL) and acetic anhydride (0.2 mL) were added. After the reaction was complete, the reaction solution was cooled to room temperature and poured into ice water, filtered, and the resulting solid was recrystallized from ethanol to obtain the intermediate 2-(3,5,5-trihydroxycyclohexane-2-enylidene)malononitrile (5 g, 86%).

[0059] The structure of the obtained intermediate 2-(3,5,5-trihydroxycyclohexane-2- enylidene)malononitrile is as follows:

[0060]

[0061] The intermediate 2-(3,5,5-trihydroxycyclohexane-2-enylidene)malononitrile (4.65 g, 25 mmol) was dissolved in ethanol (20 mL), p-hydroxybenzaldehyde (3.35 g, 27.5 mmol) and piperidine (0.5 mL) were added, and the reaction was stirred at 70°C for 2 hours. After the reaction was complete, the organic solvent was removed under reduced pressure, and the intermediate (E)-2-(3-(4-hydroxyphenyl)-5,5-dimethyl-2-cyclohexenyl-1-ylidene)malononitrile (3.62 g, 50%) was obtained by column chromatography (ethyl acetate: petroleum ether = 5:1).

[0062] The structure of the obtained intermediate (E)-2-(3-(4-hydroxyphenyl)-5,5-dimethyl-2- cyclohexenyl-1-ylidene)malononitrile is as follows:

[0063]

[0064] The intermediate (E)-2-(3-(4-hydroxyphenyl)-5,5-dimethyl-2-cyclohexenyl-1- ylidene)malononitrile (2.9 g, 10 mmol) was dissolved in trifluoroacetic acid (30 mL), and urotropine (1.68 g, 12 mmol) was added, and the reaction was stirred at 100°C for 2 hours. After the reaction was complete, the product was added to ice water, extracted with dichloromethane, and dried. The intermediate product isophorone aldehyde (A) (2.23 g, 55%) was obtained by column chromatography (ethyl acetate: petroleum ether = 5:1).

[0065] The structure of the obtained intermediate isophorone aldehyde is as follows:

[0066]

[0067] (2) Preparation of the intermediate isoquinoline hydrazide (B)

[0068] Isoquinoline-1-carboxylic acid (1.73 g, 10 mmol) was dissolved in methanol solvent (50 mL) and stirred until the solution was clear, then commercially available concentrated sulfuric acid (98%) (1 mL) was added dropwise as a catalyst, and then the reaction system was warmed to 70°C and stirred at reflux for 12 hours. The reaction system was cooled to room temperature, and the organic solvent was removed by distillation under reduced pressure to obtain the crude product methyl isoquinoline carboxylate. Hydrazine hydrate (0.1 g, 20 mmol) and organic solvent methanol (30 mL) were added to the round-bottom flask containing the crude product, and the temperature was continued to be warmed to 70°C and stirred at reflux for 3 hours. After the reaction was completed, the reaction system was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was obtained by column chromatography (ethyl acetate: petroleum ether = 1:3) to obtain the target raw material isoquinoline hydrazide (1.45 g, 78%).

[0069] The structure of the obtained intermediate isoquinoline hydrazide is:

[0070]

[0071] (3) Preparation of lead ion fluorescent probe YSQ based on dicyanoisoforone as the parent

[0072] In a 50 mL round-bottom flask containing 20 mL of organic solvent methanol, dicyanoisophorone aldehyde (A) (0.318 g, 1 mmol) and isoquinoline hydrazide (B) (0.187 g, 1 mmol) were added respectively, and after stirring and dissolving, the reaction system was warmed to 80°C and stirred at reflux for 3 hours. TLC tracking reaction detection, after the reaction was completed, the reaction system was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The crude product was purified by recrystallization (MeOH: PE = 1:20) to obtain the red solid fluorescent probe molecule YSQ (0.414 g, 85%).

[0073] The structure of the obtained fluorescent probe compound is:

[0074]

[0075] Characterization data of the fluorescent probe 1 H NMR (400 MHz, DMSO-d6) δ 12.65 (s, 1H), 11.76 (s, 1H), 8.92 (d, J = 0.84 Hz, 1H), 8.71 (s, 1H), 8.61 (d, J = 0.84 Hz, 1H), 8.09 - 8.05 (m, 2H), 7.84 - 7.81 (m, 2H), 7.75 (t, J = 0.84 Hz, 1H), 7.67 (d, J = 0.84 Hz, 1H), 7.25 (s, 2H), 6.96 (d, J = 0.84 Hz, 1H), 6.78 (s, 1H), 2.54 (s, 2H), 2.48 (s, 2H), 0.96 (s, 6H).13 C NMR (100 MHz, DMSO-d6) δ 169.25, 160.22, 154.07, 145.92, 137.67, 136.07, 130.91, 130.31, 129.62, 127.51, 127.39, 127.26, 125.69, 122.90, 118.33, 118.12, 113.65, 112.90, 78.01, 42.97, 39.21, 32.03, 28.05. ESI-MS m / z: [M+K] + calcd for C 30 H 25 N5O2K 526.1640, found 526.1662; IR (KBr cm -1 ): 2956, 2218, 1687, 1551, 1521, 1495, 1341.

[0076] The hydrogen spectrum (1H NMR), carbon spectrum (13C NMR), mass spectrum MS and infrared IR of the fluorescent probe prepared in Example 1 are shown in 1 13 Figure 12 Figure 13 Figure 14 Figure 15 The fluorescent probe of the present application is successfully prepared.

[0077] Example 2

[0078] The lead ion detection fluorescent probe YSQ prepared in Example 1 is configured into a 1 mM probe stock solution with DMSO, and each metal ion is 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 are added to 3 mL of a blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1), and detection is performed with a fluorescence spectrometer and a UV spectrophotometer, and it is found that the maximum excitation wavelength of the fluorescent probe is 490 nm, and the maximum emission wavelength is 678 nm, and the specific test results are as follows:

[0079] Two cuvettes are taken, 3 mL of a blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) and 30 μL of a probe stock solution are added to each cuvette, 50 μL of a lead ion stock solution is added to one of the cuvettes, and the other cuvette is not added with an aluminum ion stock solution, and UV spectrum test is performed. As shown in Figure 1 The fluorescent probe itself has almost no UV absorption at a wavelength λ = 490 nm, and once the lead ion is added, the UV absorption peak gradually increases; and the addition of the lead ion changes the color of the probe solution from colorless to purple red. The results show that the probe is sensitive to Pb​​​​​2+ High sensitivity and selectivity. The naked eye can see the color change may be due to the formation of a new complex between the probe and lead ions.

[0080] As shown in Figure 2 , the fluorescence spectrum of the lead ion detection fluorescent probe for various common metal ions. The experimental results show that only when the lead ion is added, the fluorescence intensity of the fluorescence spectrum at 678 nm is obviously enhanced. Moreover, the fluorescence intensity is significantly better than that when other metal ions are added, indicating that the fluorescence probe of the application has good selectivity for lead ions.

[0081] As shown in Figure 3 , the fluorescence spectrum response diagram of the lead ion detection fluorescent probe for different concentrations of lead ions (Pb 2+ ). To 3mL of blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) add 30μL of probe stock solution and 0-50μL (0, 1, 2, 3…10, 15…50μL) of lead ion solution (3mM of lead ion stock solution), the fluorescence probe itself has almost no fluorescence in the solution, but with the increase of lead ion concentration, the fluorescence at 678nm is enhanced with the increase of lead ion concentration, that is, the fluorescence intensity increases with the increase of lead ion concentration, and accompanied by obvious color change. It is shown that the probe YSQ prepared in example 1 of the application is complexed with lead ions, which inhibits the rotation of C=N double bond, thereby inhibiting the electron transfer, thereby indicating that the probe YSQ is a fluorescence enhancement type probe.

[0082] As shown in Figure 4 , the fluorescence intensity column chart of the lead ion detection fluorescent probe after reacting with lead ions in the presence of different interfering metal ions. To 3mL of blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) add 30μL of probe stock solution and 50μL of any other metal ion (Al 3+ , Co 2+ , Pd 2+ , Ni 2+ , Cu 2+ , Cr 3+ , Cu + , Mn 2+ , Mg 2+ , Ba 2+ , Pd 2+ , Sn 2+ , Fe 2+ , K + , Sr2+ Ca 2+ Sr 2+ Na + Ag + and Cd 2+ ) Stock solution, and finally add 50 μL of Pb to the blank solution. 2+ The stock solution was tested for fluorescence intensity. The results showed that, except for diamagnetic cobalt, nickel, aluminum, copper, chromium, iron, and tin ions, other metal ions did not significantly interfere with the recognition of lead ions by the lead ion fluorescent probe of this invention, indicating that the probe prepared in this invention has good specificity.

[0083] like Figure 5 As shown, the probe and Pb were studied using the Job's plot method. 2+ To determine the binding rate, a certain volume of probe stock solution (1 mM) and Pb were added to 3 mL of blank solution (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1). 2+ A stock solution (3 mM) was used to bring the total concentration of the lead ion detection fluorescent probe and lead ions to 50 μM. By changing the concentration ratio of the two (the molar ratio of lead ion detection fluorescent probe to lead ions was 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1), the difference between the fluorescence intensity at 678 nm and the fluorescence intensity of the lead ion fluorescent probe combined with itself at that concentration was obtained and plotted against the proportion of ions in the total concentration. This... Figure 5 It can be seen that the ordinate reaches its highest value when the proportion of lead ions is 0.5, which confirms that the fluorescent probe forms a stable complex [YSQ-Pb] with lead ions in a 1:1 ratio. 2+ ].

[0084] like Figure 6 As shown, 30 μL of probe stock solution and 50 μL of Pb were added to 3 mL of blank buffer (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1). 2+ In the stock solution, the fluorescence intensity of the probe rapidly increased to its peak and stabilized within 2 minutes. Furthermore, the fluorescence intensity remained essentially constant over the following 30 minutes, indicating that the probe is effective against Pb. 2+ The detection is stable enough.

[0085] like Figure 7 As shown, the solutions containing probe YSQ (10 μL) and [YSQ-Pb] were adjusted with 1 M HCl and 1 M NaOH, respectively. 2+ A 10 μL stock solution of MeOH / H2O buffer (MeOH:H2O:Hepes) (v / v / v = 9:1:0.1) (1 mL), containing [YSQ-Pb 2+The stock solution was prepared by dissolving the probe and lead chloride in double distilled water to get the concentration of 10 uM and 50 uM respectively, so that they have different pH change values. Then, the fluorescence intensity of the two systems in different pH value ranges was tested. The probe YSQ itself has almost no fluorescence intensity in the pH range from 2 to 7, but the fluorescence of the probe YSQ itself is obviously enhanced in the range of 7-12. This is because the deprotonation of the hydroxyl group under alkaline conditions inhibits the original ESIPT effect, on the other hand, the oxygen atom is negatively charged, which enables the ICT effect to open, and the fluorescence is enhanced. However, the [YSQ-Pb 2+ ] system has a significant increase in fluorescence (678 nm) intensity in the pH range of 7.0-10.0, and the fluorescence intensity reaches a maximum at pH = 10. [YSQ-Pb 2+ ] Under acidic conditions (pH < 7.0), the fluorescence signal is relatively weak, and the possible reason is that the complexing point of the fluorescent probe YSQ is protonated and is not easy to complex with lead ions. [YSQ-Pb 2+ ] Under neutral or alkaline conditions (pH > 7.0), the fluorescence signal becomes strong, and the possible reason is that the deprotonation of the hydroxyl group under alkaline conditions inhibits the original ESIPT effect, on the other hand, the oxygen atom is negatively charged, which enables the ICT effect to open, and the fluorescence is enhanced. Therefore, the optimal pH range of YSQ is 7.0-10.0, and YSQ has the ability to detect Pb 2+ in a biological environment.

[0086] As Figure 8 shown, the optimal configuration diagram of the fluorescent probe and the [YSQ-Pb 2+ ] complex and its corresponding orbital energy level diagram, the DFT calculation result further proves that the probe YSQ and lead ions form a four-coordinated complex through -C=N, -OH and C=O, and the probe YSQ is more stable after forming a complex with Pb 2+ .

[0087] As Figure 9The HeLa cell toxicity of the fluorescent probe was investigated by MTT assay at different concentrations (0-10 μM) of lead ions. In 96-well plates, 10 μM of lead ion solution (100 μL / well) was first inoculated, and then cell suspension containing 10 μL of 0-10 μM (0, 2, 4, 6, 8, 10 μM) of YSQ probe was inoculated into the well plate, and finally the culture plate was placed in the incubator for pre-culture (at 37°C, 5% CO2). Second, 10 μL of MTT solution was injected into each well, and the culture plate was incubated in the incubator for 2 h, and the absorbance at 450 nm was measured by a microplate reader. The HeLa cell viability results showed that the survival rate of HeLa cells was more than 90% after 24 hours. The MTT experiment results showed that the lead ion probe of the present application had low cytotoxicity in the experimental environment, indicating that the probe had potential application value in detecting lead ions in living cells.

[0088] As shown in Figure 10 , the imaging diagram of the fluorescent probe before and after binding with lead ions in HeLa cells. 1×10 5 HeLa cells were inoculated into 35 mm glass bottom tissue culture dishes, and when the cell density reached 60%, the DMEM culture medium was replaced with serum-free medium containing 8 μM lead nitrate and incubated at 37°C for 30 minutes, and the cells were washed with PBS for 3 times. Then, new DMEM medium containing YSQ (probe final concentration 8 μM) was added, and the incubation was continued for 30 minutes, and then the obtained cells were washed with PBS for 3 times. Then, the cells cultured with YSQ-containing DMEM medium without lead nitrate treatment (incubated for 30 min) were washed with PBS for 3 times as a control. The results of the laser confocal microscope imaging experiment (excitation wavelength 488 nm) showed that the blue fluorescence of the cells could be observed significantly increased after adding YSQ probe and Pb 2+ , which may be due to the formation of YSQ-Pb 2+ complex. Therefore, the results of the cell imaging experiment showed that the probe YSQ was cell membrane permeable and could be effectively used for imaging of trace Pb 2+ in living cells, further indicating that the lead ion fluorescent probe YSQ of the present application could be applied to biological experiments.

[0089] As shown in Figure 11As shown, the filter paper was immersed in MeOH:H2O:Hepes (v / v / v = 9:1:0.1) stock solution containing fluorescent probe (1 mM) for half an hour, then the test strip was taken out and dried in air to obtain the dried test strip containing probe. The test strip was immersed in 0 mM, 0.1 mM, 3.0 mM lead ion concentration solution respectively, after soaking for 30 minutes, air-drying, under the ultraviolet lamp, the fluorescence test paper prepared with fluorescent probe YSQ solution and its color change when testing different concentrations of lead ions were determined, which showed that the color of probe YSQ changed with the change of lead ion concentration, and trace lead ions in the environment could be quantitatively detected in solid state.

[0090] The above experiment shows that the fluorescent probe YSQ with dicyanisophorone as the parent is prepared by condensation reaction with dicyanoisophorone aldehyde and isoquinoline hydrazine as the fluorescent group, and the probe shows high sensitivity and high selectivity to Pb 2+ It also successfully detects trace metal lead ions in living Hela cells due to its structural stability, low toxicity and strong cell penetration ability; the preparation method of the fluorescent probe is simple, the raw materials are easy to obtain, the obtained product is a solid powder, easy to store, and has high application development prospect.

Claims

1. A "off-on" near-infrared lead ion detection fluorescent probe YSQ, characterized in that, The lead ion detection fluorescent probe YSQ takes dicyaniso-phorone and isoquinoline hydrazide as fluorescent groups, and its structure is shown in the following formula I.

2. A method for preparing the "off-on" near-infrared lead ion detection fluorescent probe YSQ according to claim 1, characterized in that, The method comprises the following steps: (1) reacting iso-phorone and malononitrile to obtain 2-(3, 5, 5-trihydroxylcyclohexane-2-alkyl) malononitrile, then reacting with p-hydroxybenzaldehyde to obtain (E)-2-(3-(4-hydroxyphenylstyryl)-5, 5-dimethyl-2-cyclohexenyl-1-alkyl) malononitrile, then adding urotropine and trifluoroacetic acid to obtain dicyaniso-phorone aldehyde (A); (2) obtaining isoquinoline hydrazide through esterification and amidation reactions of isoquinoline carboxylic acid, and obtaining the fluorescent probe YSQ by Schiff base condensation reaction of isoquinoline hydrazide (B) and dicyaniso-phorone aldehyde; The reaction route is shown in the following formula:

3. The preparation method according to claim 2, characterized in that, In step (1), iso-phorone and malononitrile are dissolved in methanol under N2 protection, piperidine, glacial acetic acid and acetic anhydride are added, the reaction is completed, the reaction solution is cooled to room temperature, then poured into ice water, filtered, and the obtained solid is recrystallized with ethanol to obtain the intermediate 2-(3, 5, 5-trihydroxylcyclohexane-2-alkyl) malononitrile.

4. The production method according to claim 2, characterized by, In step (1), the intermediate 2-(3, 5, 5-trihydroxylcyclohexane-2-alkyl) malononitrile is dissolved in ethanol solution, p-hydroxybenzaldehyde and piperidine are added, and reflux stirring is performed until the reaction is completed, then the organic solvent is removed under reduced pressure, and the intermediate (E)-2-(3-(4-hydroxyphenylstyryl)-5, 5-dimethyl-2-cyclohexenyl-1-alkyl) malononitrile is obtained by column chromatography.

5. The preparation method according to claim 2, characterized in that, In step (1), the intermediate (E)-2-(3-(4-hydroxyphenylstyryl)-5, 5-dimethyl-2-cyclohexenyl-1-alkyl) malononitrile is dissolved in trifluoroacetic acid, urotropine is added, and reflux stirring is performed until the reaction is completed, then the reaction mixture is added to ice water, extracted, dried, and dicyaniso-phorone aldehyde is obtained by column chromatography.

6. The method of claim 2, wherein, In step (2), isoquinoline carboxylic acid is dissolved in an organic solvent methanol, concentrated sulfuric acid is added, and reflux stirring is performed until the reaction is completed, then the reaction solution is cooled to room temperature, the pH is adjusted to neutral, extracted, separated, dried, and isoquinoline carboxylic acid methyl ester is obtained by column chromatography after removing the solvent; the intermediate isoquinoline carboxylic acid methyl ester is dissolved in an organic solvent methanol, hydrazine hydrate is added, and reflux stirring is performed until the reaction is completed, then the reaction solution is cooled to room temperature, the organic solvent is removed by distillation under reduced pressure, extracted, separated, dried, and isoquinoline hydrazide is obtained by column chromatography after removing the solvent.

7. The preparation method according to claim 2, characterized in that, In step (2), dicyaniso-phorone aldehyde and isoquinoline hydrazide are dissolved in an organic solvent methanol, the mixture is reflux stirred until the reaction is completed, then the reaction mixture is cooled to room temperature, the solvent is removed by distillation under reduced pressure, the crude product is purified by recrystallization to obtain the fluorescent probe YSQ.

8. Application of the lead ion detection fluorescent probe YSQ in the preparation of a reagent for detecting lead ions.

9. Application of the lead ion detection fluorescent probe YSQ in the preparation of a reagent for detecting trace lead ions in a solution and cells on site.

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