A ruthenium (II) complex fluorescent probe based on Schiff base bridging ligand and its preparation method and application

Abstract

The present invention discloses a ruthenium(II) complex fluorescent probe based on a Schiff base-bridged ligand, its preparation method and application, belonging to the technical field of organic chemistry. The ruthenium(II) complex fluorescent probe is [Ru(bipy)2(FPPM)] 2+ , and its structural formula is #imgabs0#. 1-(Pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazin-2-yl)imine and dichlorobis(2,2'-bipyridine)ruthenium(II) dihydrate are added to an organic solvent for reflux reaction to construct the ruthenium(II) complex [Ru(bipy)2(FPPM)] 2+ . The probe prepared by the present invention has the advantages of visible light excitation, large Stokes shift, high selectivity, high sensitivity, low cytotoxicity, etc., and can realize fluorescence imaging of trace Zn in cells 2+ .

Description

Technical Field

[0001] The present invention relates to the technical field of organic chemistry, and in particular to a ruthenium (II) complex fluorescent probe constructed based on a Schiff base bridging ligand, and a preparation method and application thereof. Background Art

[0002] Zn 2+ Ion is the second most abundant transition metal ion in the human body. 2+ The total content of ions is about 2-3g, part of which is Zn 2+ The ions are bound to enzymes and proteins and exist in a fixed form, with some concentrations in the micromolar range as free Zn 2+ It exists in tissue cells in the form of ions and is an extremely important physiological metal ion. Studies have found that Zn in the human body 2 + Disorders in the metabolism of Zn ions will lead to diseases such as diabetes, Alzheimer's disease and Parkinson's disease. 2+ The detection of Zn ions is extremely important. Traditional detection methods, such as atomic absorption spectroscopy, ion chromatography, and voltammetry, are not suitable for the detection of Zn in biological systems due to their high cost, long time consumption, and difficult operation. 2+ ion detection. Compared with the traditional Zn 2+ Detection method, fluorescent probe method has low cost, good repeatability, high selectivity and sensitivity, and can achieve Zn in biological samples 2+ The advantages of real-time non-invasive monitoring of ions have gradually become the focus of current research.

[0003] Existing Zn 2+ The probes mainly use organic molecules such as rhodamine, coumarin, fluorescein and bipyridine as fluorophores, which have disadvantages such as short excitation wavelength and small Stökes shift. On the one hand, the use of ultraviolet light with a shorter wavelength can cause irreversible damage to biological samples. On the other hand, the small Stökes shift can also cause serious crosstalk between the excitation spectrum and the emission spectrum, reducing the sensitivity and accuracy of the detection.

[0004] Ruthenium exhibits excellent physical and chemical properties due to its unique six-coordination ability and diverse valence states. This complex has a wide range of application value in many fields due to its excellent thermodynamic stability, photochemical and photophysical properties, excited state reactivity and long-lasting luminescence performance. In chemiluminescence research, ruthenium complexes are core materials. They participate in electron transfer and nonlinear optical processes, providing possibilities for the development of new optoelectronic devices. In molecular photoswitches and molecular recognition technologies, the sensitive responsiveness of ruthenium complexes makes them ideal recognition probes. However, the current use of ruthenium complexes as fluorescent probes to detect Zn 2+ There are few reports on Zn2+ -enhanced Ru (II) photoluminescence directed by double-clamp structural ligand for selective Zn 2+ sensing and live-cell imaging (Longlong Li, Sensors and Actuators: B. Chemical 371 (2022) 132513) discloses a method for detecting Zn 2+ Although the ruthenium (II) complex fluorescent probe has high sensitivity, it is also highly cytotoxic and can only be used at low concentrations. 2+ When the probe concentration is increased, it is more conducive for the probe to penetrate the cell membrane and enter the cell by free diffusion. 2+ If the cytotoxicity is high, it will kill the cells, making it difficult to achieve Zn in living cells 2+ If the concentration of the complex is reduced, the accuracy of the detection will be affected. Therefore, a ruthenium (II) complex fluorescent probe with low cytotoxicity is needed to detect Zn 2+ , and then further developed into corresponding biological detection reagents for physiological and pathological research. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a ruthenium (II) complex fluorescent probe based on Schiff base bridging ligand, and its preparation method and application. The present invention obtains 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine bridging ligand by Schiff base reaction, and then coordinates with di(2,2'-bipyridine) dichloride ruthenium (II) dihydrate to prepare a ruthenium (II) complex fluorescent probe. The probe has the advantages of visible light excitation, large Stökes shift, high selectivity, high sensitivity, low cytotoxicity, etc., and can realize the detection of trace Zn in cells. 2+ Fluorescence imaging.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a ruthenium (II) complex fluorescent probe constructed based on a Schiff base bridging ligand is provided, wherein the ruthenium (II) complex fluorescent probe is formed by using an imine compound containing a fluorine group, a pyridine group and a pyrazine group as a bridging ligand coordinated with a ruthenium (II) compound; the number of fluorine groups in the imine compound is ≥1.

[0008] Preferably, the fluorine-containing group in the imine compound is a fluorine-containing phenyl group; the number of fluorine atoms in the fluorine-containing phenyl group is 5; and the ruthenium (II)-containing compound is bis(2,2'-bipyridine) dichlororuthenium (II) dihydrate.

[0009] Preferably, the imine compound is 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine, and its structural formula is

[0010] .

[0011] Preferably, the ruthenium (II) complex fluorescent probe is [Ru (bipy) 2 (FPPM)] 2+ , whose structural formula is

[0012] .

[0013] The second aspect of the present invention provides a method for preparing a ruthenium (II) complex fluorescent probe, the preparation method comprising:

[0014] 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine is dissolved in an organic solvent to obtain an imine solution, di(2,2'-bipyridyl)ruthenium(II) dichloride dihydrate is dissolved in an organic solvent to obtain a ruthenium(II) solution, the ruthenium(II) solution is added dropwise to the imine solution under stirring, the mixture is refluxed under an inert atmosphere, and a red precipitate is obtained by extraction after cooling, namely [Ru(bipy) 2 (FPPM)] 2+ .

[0015] Preferably, the 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine is prepared by the following method:

[0016] Pentafluorobenzaldehyde was dissolved in ethanol, and 2-amino-5-(2-pyridyl)pyrazine was added in several portions under an inert atmosphere for reflux reaction. After the reaction was completed, the mixture was filtered, concentrated and purified in sequence to obtain a light yellow solid, namely 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine.

[0017] Preferably, the molar ratio of pentafluorobenzaldehyde to 2-amino-5-(2-pyridyl)pyrazine is 7.65:11.5; 2-amino-5-(2-pyridyl)pyrazine is added in four times, each time with an interval of 6 hours; the reflux reaction temperature is 78-85°C, and the reflux reaction time is 24 hours.

[0018] Preferably, the molar ratio of 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine and (2,2'-bipyridine)dichlororuthenium(II) dihydrate is 2.86:3.71; the volume ratio of the imine solution and the ruthenium(II) solution is 2:1; the organic solvent is anhydrous methanol; the temperature of the reflux reaction is 64-70°C, and the reflux reaction time is 6 h.

[0019] The third aspect of the present invention provides a ruthenium (II) complex fluorescent probe for preparing Zn 2+ Detection of product applications.

[0020] Preferably, when the concentration of the ruthenium (II) complex fluorescent probe is 50 µmol / L, the cell survival rate is 90%; when the concentration of the ruthenium (II) complex fluorescent probe is 200 µmol / L, the cell survival rate is above 75%.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention synthesizes a novel bridging ligand 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine (FPPM) and then reacts it with [Ru(bipy) 2 ] 2+ A novel method for detecting Zn 2+ Ru(II) complex fluorescent probe [Ru(bipy) 2 (FPPM)] 2+ , which has the ability to accurately identify Zn 2+ The lowest excited state is at the MLCT energy level, and the fluorescence released when the electron returns from the excited state to the ground state has excellent optical properties such as large Stökes shift and good stability.

[0023] (2) The fluorescent probe provided by the present invention is a new type of ruthenium (II) complex fluorescent probe. It uses visible light excitation and has not only a large Stökes shift but also high selectivity and sensitivity, as well as cell membrane permeability and low toxicity, thus achieving a "robust" and viable detection of trace amounts of Zn in cells. 2+ The detection imaging can be further developed into corresponding biological detection reagents for physiological and pathological research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :(a)[Ru(bipy) 2 (FPPM)] 2+ UV absorption spectrum of [Ru(bipy) 2 (FPPM)] 2+ Fluorescence emission spectra in different solvents;

[0025] Figure 2 :(a)[Ru(bipy) 2 (FPPM)] 2+ Fluorescence emission spectra of selective recognition of metal ions; (b) Ru(bipy) 2 (FPPM)] 2+ Fluorescence emission histogram for selective recognition of metal ions;

[0026] Figure 3 :(a)[Ru(bipy) 2 (FPPM)] 2+ Fluorescence emission spectra of selective recognition of anions; (b) [Ru(bipy) 2 (FPPM)] 2+ MTT toxicity test bar graph;

[0027] Figure 4 :(a)[Ru(bipy) 2 (FPPM)] 2+ and Zn 2+ Fluorescence emission spectra at different molar ratios; (b) [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ Job'plot graph;

[0028] Figure 5 :(a)With the increase of Zn 2+ With the increasing concentration, [Ru(bipy) 2 (FPPM)] 2+ (2×10 -5 mol / L) fluorescence emission spectrum; (b) [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ Detection limit diagram of fluorescence spectrometry;

[0029] Figure 6 :Confocal fluorescence images of HepG2 cells; (a) cells were treated with 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ Dark field images of cells incubated for 12 h; (b) cells incubated with 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ Bright field images after 12 h of incubation; (c) cells were treated with 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+Confocal fluorescence images of cells incubated for 12 h; (d) cells were first treated with 25 μmol / L Zn 2+ After 12 h of incubation, 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ Dark field image after 12 h of incubation; (e) cells were first treated with 25 μmol / L Zn 2+ After 12 h of incubation, 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ Bright field image after 12 h of incubation; (f) cells were first treated with 25 μmol / L Zn 2+ After 12 h of incubation, 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ Confocal fluorescence images after 12 h of incubation;

[0030] Figure 7 :[Ru(bipy) 2 (FPPM)] 2+ The hydrogen spectrum of

[0031] Figure 8 :[Ru(bipy) 2 (FPPM)] 2+ Fluorine spectrum of

[0032] Fig. 9 : Hydrogen spectrum of FPPM;

[0033] Fig.10 : Carbon spectrum of FPPM;

[0034] Fig.11 : Fluorine spectrum of FPPM. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0036] As introduced in the background technology section, ruthenium complexes are currently used as fluorescent probes to detect Zn 2+ There are few reports on Zn ions, and the existing technology for detecting Zn 2+ The ruthenium (II) complex fluorescent probe has high cytotoxicity and can only be used at low concentrations.

[0037] Based on this, the purpose of the present invention is to provide a ruthenium (II) complex fluorescent probe based on Schiff base bridging ligand, and its preparation method and application. The present invention uses Schiff base reaction to connect 2-amino-5-(2-pyridyl) pyrazine and pentafluorobenzaldehyde with an imine bond to obtain a bridging ligand 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine (FPPM), and then reacts with [Ru(bipy) 2 ] 2+ A novel method for detecting Zn 2+ Ru(II) complex fluorescent probe [Ru(bipy) 2 (FPPM)] 2+ The nitrogen atom on the aromatic ring of FPPM was used as a coordinating atom to [Ru (bipy) 2 ] 2+ The ruthenium (II) complex [Ru (bipy) 2 (FPPM)] 2+ . [Ru (bipy) 2 (FPPM)] 2+ The uncoordinated nitrogen atom on the aromatic ring of FPPM and the nitrogen atom in the imine group are used to bind Zn 2+ Coordination, thus specifically recognizing Zn 2+ In the ruthenium(II) complex [Ru(bipy) 2 (FPPM)] 2+ In the bridging ligand FPPM, the electron-withdrawing fluorine atom can reduce the triplet energy and shift the emission wavelength of the Ru(II) complex toward the long wavelength direction. That is, the presence of FPPM can reduce the [Ru (bipy) 2 (FPPM)] 2+ The fluorescence intensity of Zn 2+ Before coordination, the fluorescence intensity is low, and the addition of Zn 2+ The difference in fluorescence intensity before and after coordination shows obvious fluorescence enhancement emission, thus improving the detection of Zn 2+ Compared with the fluorescent detection probes in the prior art, this fluorescent probe has the advantages of visible light excitation (λ=450 nm), large Stökes shift (170 nm), high selectivity, and high sensitivity. 2+ Detection, and low cytotoxicity, can achieve Zn 2+ Fluorescence imaging can be further developed into corresponding biological detection reagents for physiological and pathological research, which has very important practical significance and application value.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0039] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0040] Example 1 Preparation of 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazin-2-yl)imine (FPPM)

[0041] Before the reaction, the three-necked flask was evacuated and replaced with nitrogen three times. Then, under the protection of nitrogen, pentafluorobenzaldehyde (1.5 g, 7.65 mmol) and anhydrous magnesium sulfate (1.10 g, 9.18 mmol) were dissolved in 10 ml of ethanol. The inert gas was continuously bubbled for 1 hour. 2-amino-5-(2-pyridyl) pyrazine (1.97 g, 11.50 mmol) was added four times, each time with an interval of 6 hours, and refluxed at 80°C for 24 hours. After the reaction mixture was cooled to room temperature, the anhydrous magnesium sulfate was filtered, the reaction solution was concentrated, and then purified by silica gel column chromatography to obtain a light yellow solid product, namely FPPM, with a yield of 2.05 g (76.5%). 1 H NMR (400 MHz, Chloroform-d): δ 8.47 (d, J = 2.8 Hz, 2H), 8.22 (s, 1H), 8.02 (s, 1H), 7.64 –7.55 (m, 2H), 7.11 (m, 1H).

[0042] 13 C NMR (100 MHz, DMSO-d6) δ 156.45, 155.27, 154.78, 153.52, 149.64,149.45, 140.59, 140.52, 139.68, 138.67, 137.64, 137.48, 132.80, 131.48,123.45, 122.89.

[0043] 19 F NMR (376 MHz, DMSO-d6) δ -142.63 (dd, J = 71.8, 21.9 Hz, 2F), -153.85 (d, J = 64.0 Hz, 1F), -161.66 – -161.84 (m, 2F).

[0044] The hydrogen, carbon and fluorine spectra of FPPM are shown in Fig. 9 , 10 、11.

[0045] Example 2: Probe [Ru(bipy) 2(FPPM)] 2+ Synthesis

[0046] 1-(Pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine (FPPM) (1.0 g, 2.86 mmol) was dissolved in 10 ml of anhydrous methanol and nitrogen was bubbled for 30 min. Di(2,2'-bipyridyl)dichlororuthenium(II) dihydrate (1.93 g, 3.71 mmol) was dissolved in 5 ml of anhydrous methanol saturated with nitrogen and added dropwise to the FPPM reaction solution under constant stirring for 2 h. Under inert gas protection, the mixture was refluxed at 65 °C for 6 h, cooled to room temperature, and 20 ml of methyl tert-butyl ether was added to obtain a red precipitate, which was [Ru (bipy) 2 (FPPM)] 2+ , yield: 0.71 g (32.6%). 1 H NMR (400 MHz, DMSO-d6)δ 9.43 (s, 1H), 8.89-8.80 (m, 3H), 8.74 (t, J = 6.7 Hz, 1H), 8.60 (d, J = 8.3Hz, 1H), 8.25-8.03 (m, 5H), 7.78-7.48 (m, 6H), 7.35 (t, J = 6.5 Hz, 2H), 7.21(s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 6.67 (s, 1H). 19 F NMR (376 MHz, DMSO-d6) δ-142.68 (dd, J = 23.8, 7.5 Hz, 2F), -154.72 (t, J = 22.3 Hz, 1F), -162.12 (td, J = 23.3, 7.5 Hz, 2F).

[0047] [Ru (bipy) 2 (FPPM)] 2+ The hydrogen and fluorine spectra of Figure 7 and Figure 8 .

[0048] Example 3: Probe [Ru(bipy) 2 (FPPM)] 2+ Optical properties

[0049] The [Ru(bipy) 2 (FPPM)] 2+ The UV-visible absorption spectrum of the probe in solid state, such as Figure 1As shown in (a), the strong absorption band at 270-380 nm mainly comes from [Ru(bipy) 2 ] 2+ The auxiliary ligand 2,2-bipyridine and the bridging ligand 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazin-2-yl)imine (FPPM) with a π-π*-centered L bipy C and L FPPM C transition; the metal-to-ligand charge transfer transition (MLCT) characteristic absorption generated by Ru(II) coordination appears at 400-550 nm, originating from mixed singlet and triplet dπ(Ru)-π*(bipy) and dπ(Ru)-π*(FPPM) transitions. The appearance of MLCT in the absorption spectrum is the main reason for the Ru(II) complex [Ru (bipy) 2 (FPPM)] 2+ It has the basis of emission properties such as visible light excitation and large Stökes shift.

[0050] Probe [Ru(bipy) 2 (FPPM)] 2+ Dissolve in dichloromethane (CH 2 Cl 2 ) ethyl acetate (EA), methanol (MeOH), acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and PBS buffer solution (pH=7.4), the test concentration is: (2×10 -5 mol / L). The fluorescence emission spectrum of the probe was measured at room temperature.

[0051] The fluorescence spectra of the probe in different solvents are shown in Figure 2. Figure 1 As shown in (b), when the excitation wavelength is 450 nm, due to [Ru(bipy) 2 (FPPM)] 2+ The charge transfer (MLCT) from the inner metal (Ru(II)) to the ligand (bipy, FPPM) occurs, and a broad emission band appears at 580-750nm, with the maximum emission wavelength appearing at around 620nm. 2 (FPPM)] 2+ Dissolved in CH 2 Cl 2 , EA, MeOH, CH 3 CN, DMF, DMSO and PBS (pH = 7.4) and other solvents (C = 2 × 10 -5 mol / L), with the increase of solvent polarity, the maximum emission wavelength showed a slight blue shift, and the fluorescence emission intensity followed the CH 2 Cl 2<EA < MeOH < CH 3 CN < PBS(pH = 7.4) < DMF < DMSO increases gradually in this order.

[0052] Example 4: Probe [Ru(bipy) 2 (FPPM)] 2+ for Zn 2+ selectivity

[0053] As Figure 2 shown, in the PBS(pH = 7.4) solution of the probe [Ru(bipy) 2 (FPPM)] 2+ (2×10 -5 mol / L), equimolar amounts of Na + , K + , Ca 2+ , Mg 2+ , Ni 2+ , Cu 2+ , Co 2+ , Mn 2+ , Al 3+ and Fe 3+ are added, the MLCT emission peak intensity of [Ru(bipy) 2 (FPPM)] 2+ between 580 - 750 nm is quenched to varying degrees. 3 The blank in (a) is the PBS(pH = 7.4) solution of [Ru(bipy) Figure 2 (FPPM)] 2 (2×10 2+ mol / L). When Cd -5 2+ , Pb 2+ and Cr 3+ ions are added, the emission peak increases slightly. Only when equimolar Zn 2+ is added, the MLCT emission of [Ru((bipy) 2 (FPPM)] 2+ has a significantly increased fluorescence intensity compared to the blank solution, indicating that the MLCT energy level of the probe can be used as a signal source to detect Zn 2+ ions.

[0054] Figure 3 To test whether [Ru(bipy) 2 (FPPM)] 2+ has a similar selectivity for anions, as Figure 3 shown in (a), 10 equiv. of the anions F - , I - , Cl - , Br- 、SO 4 2- , CO 3 2- , C 2 O 4 2- , HCO 3 - 、NO 3 - , AC - 、NO 2 - , S 2- and S 2 O 3 2- Add to Ru(bipy) 2 (FPPM)] 2+ Blank solution (prepared with PBS buffer solution at pH = 7.4, concentration of 2 × 10 - 5 mol / L), the fluorescence intensity was quenched to varying degrees, indicating that [Ru(bipy) 2 (FPPM)] 2+ Not selective for anions. [Ru(bipy) 2 (FPPM)] 2+ Only in Zn 2+ In the presence of Zn, the fluorescence emission was significantly enhanced between 580 and 750 nm, indicating that the probe was sensitive to Zn 2+ Has good selectivity.

[0055] Example 5: Probe [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ Job's plot determination

[0056] Probe [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ The quantitative recognition relationship was determined by Job's plot. Under the condition of total concentration of 10 μmol / L, by changing [Ru(bipy) 2 (FPPM)] 2+ and Zn 2+ The concentration ratio of [Ru(bipy) 2 (FPPM)] 2+ With Zn 2+ Quantitative identification relationship. Figure 4 As shown, [Ru(bipy) 2 (FPPM)] 2+ and Zn2+ When the molar ratio in the solution is about 6.6:3.3, [Ru(bipy) 2 (FPPM)] 2+ The luminescence intensity reaches the maximum value, indicating that [Ru(bipy) 2 (FPPM)] 2+ With Zn 2+ A 2:1 complex is formed.

[0057] Example 6: Probe [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ Determination of the test line

[0058] like Figure 5 As shown, with the Zn 2+ With the increasing concentration, [Ru(bipy) 2 (FPPM)] 2+ (2×10 -5 mol / L) fluorescence is continuously enhanced, and in the range of 0-10 μmol, [Ru(bipy) 2 (FPPM)] 2+ With Zn 2+ The ion concentration showed a good linear correlation (R 2 =0.99201), and the 3δ (LOD=3δ / k) method was used to calculate [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ The detection limit (LOD) was 5.21×10 -8 mol / L, indicating that [Ru(bipy) 2 (FPPM)] 2+ Zn 2+ Identify the ability to detect with high sensitivity and quantification.

[0059] Example 7: Probe [Ru(bipy) 2 (FPPM)] 2+ Cell viability assay on HepG2 cells

[0060] HepG2 cells in the logarithmic growth phase were taken and the cell concentration was detected using a cell counting plate. The concentration of HepG2 cells was controlled at 5000-50000 / μL according to 1×10 4 The cells were inoculated into 96-well plates in an atmosphere containing 5% CO 2After one day of cultivation in a 37°C constant temperature incubator, 50, 100, 150, and 200 µmol / L of the probe were added to the well plate, respectively. Then, 20 µl of 5 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to the well plate. The culture was continued for 24 hours, and the absorbance of each well was read on a microplate reader for calculation.

[0061] Cell survival rate (%) = (OD value of experimental group / mean OD value of control group) × 100%; control group: no addition of [Ru(bipy) 2 (FPPM)] 2+ Probe; Experimental group: different concentrations of [Ru(bipy) 2 (FPPM)] 2+ Probe.

[0062] according to Figure 3 (b) Cytotoxicity test results show that HepG2 cells were exposed to 50 µmol / L [Ru(bipy) 2 (FPPM)] 2+ After incubation in the medium for 24 h, the cell survival rate was about 90%. 2 (FPPM)] 2+ When the concentration increased to 200 µmol / L, the cell survival rate remained above 75%. 2 (FPPM)] 2+ No obvious toxicity to cells.

[0063] Example 8: Probe [Ru(bipy) 2 (FPPM)] 2+ Effect of Zn on HepG2 cells 2+ Detection

[0064] HepG2 cells were immersed in 4% paraformaldehyde solution for half an hour, and then treated with 0.5% TritonX-100 solution for 2-3 minutes at room temperature to fix the cells. The control group was treated with 50 µmol / L [Ru(bipy) 2 (FPPM)] 2+ Probe staining HepG2 cells; the experimental group was first incubated with 25 μmol / L zinc chloride aqueous solution for 12 h, and then treated with 50 μmol / L [Ru(bipy) 2 (FPPM)] 2+ The probe was used to stain HepG2 cells; the two groups were incubated in a constant temperature cell culture incubator for 12 h and then observed under a confocal microscope. Figure 6As shown in the figure, the probe can well penetrate the cell membrane of living HepG2 cells and the Zn 2+ The complexation caused the cells to show bright red fluorescence, proving that [Ru(bipy) 2 (FPPM)] 2+ Excellent cell membrane permeability and detection of Zn 2+ Fluorescence imaging capability.

[0065] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A ruthenium (II) complex fluorescent probe based on a Schiff base bridging ligand, characterized in that: The ruthenium (II) complex fluorescent probe is formed by using an imine compound containing a fluorine group, a pyridine group and a pyrazine group as a bridging ligand coordinated with a ruthenium (II) compound; The imine compound is 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine, and its structural formula is ； The ruthenium (II)-containing compound is bis(2,2'-bipyridine) dichloride ruthenium (II) dihydrate; The ruthenium (II) complex fluorescent probe is [Ru (bipy) 2 (FPPM)] 2+ , whose structural formula is 。 2. The method for preparing the ruthenium (II) complex fluorescent probe according to claim 1, characterized in that: The preparation method is: 1-(Pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine is dissolved in an organic solvent to obtain an imine solution, di(2,2'-bipyridyl)ruthenium(II) dichloride dihydrate is dissolved in an organic solvent to obtain a ruthenium(II) solution, the ruthenium(II) solution is added dropwise to the imine solution under stirring, the mixture is refluxed under an inert atmosphere, and a red precipitate is obtained by extraction after cooling, namely [Ru(bipy)2 (FPPM)] 2+ .

3. The preparation method according to claim 2, characterized in that: The 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine is prepared by the following method: Pentafluorobenzaldehyde was dissolved in ethanol, and 2-amino-5-(2-pyridyl)pyrazine was added in several portions under an inert atmosphere for reflux reaction. After the reaction was completed, the mixture was filtered, concentrated and purified in sequence to obtain a light yellow solid, namely 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine.

4. The preparation method according to claim 3, characterized in that: The molar ratio of pentafluorobenzaldehyde to 2-amino-5-(2-pyridyl)pyrazine is 7.65:11.5; 2-amino-5-(2-pyridyl)pyrazine is added in four times, each time with an interval of 6 hours; the temperature of the reflux reaction is 78-85°C, and the reflux reaction time is 24 hours.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the 1-(pentafluorophenyl)-N-(5-(pyridin-2-yl)pyrazine-2-yl)imine and (2,2'-bipyridine)dichlororuthenium(II) dihydrate is 2.86:3.71; the volume ratio of the imine solution and the ruthenium(II) solution is 2:1; the organic solvent is anhydrous methanol; the temperature of the reflux reaction is 64-70°C, and the reflux reaction time is 6 hours.

6. The ruthenium (II) complex fluorescent probe according to claim 1 is used in the preparation of Zn 2+ Detection of product applications.

7. The use according to claim 6, characterized in that: When the concentration of the ruthenium (II) complex fluorescent probe is 50 μmol / L, the cell survival rate is 90%; when the concentration of the ruthenium (II) complex fluorescent probe is 200 μmol / L, the cell survival rate is above 75%.

Images