CN113234039B

CN113234039B - Hydrogen polysulfide fluorescent probe and preparation method and application thereof

Info

Publication number: CN113234039B
Application number: CN202110320109.3A
Authority: CN
Inventors: 薛运生; 吴春丽; 张玲; 张君瑜; 刘云萍; 郑友广
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2022-04-29
Anticipated expiration: 2041-03-25
Also published as: CN113234039A

Abstract

The invention relates to a hydrogen sulfide polysulfide fluorescent probe and a preparation method and application thereof, wherein the fluorescent probe AIE-PS2 has the advantages of larger Stokes shift (254nM), good selectivity, high sensitivity, low detection limit (9nM), high fluorescence response multiple (75 times) and good biocompatibility. In PBS buffer, fluorescence intensity with Na₂S₄The concentration of (0-20 mu M) has good linear relation, which indicates that the probe is suitable for quantitative detection of the hydrogen sulfide in organisms; the probe AIE-PS2 can image endogenous and exogenous poly-hydrogen sulfide at a cellular level, and further realizes fluorescent imaging of the poly-hydrogen sulfide of the MCF-7 cell by using AIE-PS2, so that the probe AIE-PS2 is an effective tool for visualizing and quantitatively detecting the poly-hydrogen sulfide. The structural formula of the fluorescent probe AIE-PS2 is shown in the specification.

Description

Hydrogen polysulfide fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the field of chemistry and analysis detection, and particularly relates to a hydrogen sulfide polysulfide fluorescent probe based on dual mechanisms of aggregation-induced luminescence and intramolecular proton transfer, and a preparation method and application thereof.

Background

Hydrogen sulfide (Hydrogen sulfide, H)₂S) is an important gas signal molecule, physiological concentration of H₂S has protective effect on cells, and participates in processes such as nerve regulation, vascular tone regulation, oxidative stress, inflammatory reaction, mitochondrial energy supply and fibrosis. Poly (hydrogen sulfide)s，H₂S_nN is not less than 2) is H₂The oxidation product of S belongs to sulfanylthio compounds (sulfane sulfur). In the living system H₂S and H₂S_nCoexists to regulate the intracellular redox homeostasis, and both exist in a tandem relationship in terms of bioactivity and signaling pathways (cross-talk). H₂S_nThe sulfur in the intermediate has a zero oxidation state and is easily reacted with cysteine residues of proteins to form protein persulfides, and the process is called S-thiolation modification (also called S-persulfurization or S-oversulfurization) and participates in various biological functions. Recent studies have shown that in H₂In the relevant physiological process of S, the actual signal transduction molecule may be (at least in part) H₂S_n. The poly hydrogen sulfide plays an important physiological function in organisms, regulates the activities of receptors, enzymes and ion channels, participates in redox signal conduction, and has the functions of cytoprotection, anti-inflammation, anticancer, antioxidation and the like.

The fluorescent probe has the advantages of rapidness, sensitivity and suitability for noninvasive visual detection, and is widely applied to the fields of biosensing, optical materials, biological detection, identification and the like. However, organic fluorescent dyes tend to aggregate in aqueous media, can only be used in trace amounts (usually at nM levels), are susceptible to photobleaching, increase in fluorescence background, and exhibit a random staining phenomenon, decrease in their luminous power, and produce aggregation-induced quenching (ACQ).

Aggregation-induced emission (AIE) fluorophores are aggregated and emitted in an aqueous medium, so that the Aggregation-induced quenching phenomenon of common fluorophores can be effectively avoided, the stability is good, the fluorescence background interference is low, and the photobleaching resistance is realized. Therefore, the fluorescent probe based on the AIE has better application value and development prospect.

Many fluorescent probes for detecting poly-hydrogen sulfide have been developed at present, but in practical application, some problems are faced, such as the dye is easy to aggregate under high concentration to quench fluorescence, easy to be photobleached, poor in biocompatibility, incapable of being used in large amount, etc., and further development of fluorescent probes with good selectivity and high sensitivity, which are suitable for biological imaging, is still needed.

Disclosure of Invention

The invention aims to provide a hydrogen sulfide polysulfide fluorescent probe based on the prior art, which can quantitatively detect the hydrogen sulfide in organisms and has the advantages of low detection limit (9nM), high fluorescence response multiple (75 times), high sensitivity, good selectivity and larger Stokes shift (254 nM). In PBS buffer, fluorescence intensity with Na₂S₄The concentration of (0-20 mu M) has good linear relation, which indicates that the probe is suitable for quantitative detection of the hydrogen sulfide in organisms; and further realizes the fluorescence imaging of the MCF-7 cell poly hydrogen sulfide by using AIE-PS 2.

The invention also aims to provide a preparation method of the poly hydrogen sulfide fluorescent probe.

The invention also aims to provide the application of the poly hydrogen sulfide fluorescent probe in the detection of poly hydrogen sulfide.

The technical scheme of the invention is as follows:

a poly hydrogen sulfide fluorescent probe, the structural formula of which is as follows:

the design idea of the poly hydrogen sulfide fluorescent probe AIE-PS2 provided by the invention is as follows: (1) fluorescent mother nucleus: an AIE fluorophore with a large Stokes shift (TPE-HBT) is used as a fluorescent mother nucleus. A small stokes shift not only causes fluorescence quenching, but also excitation light and scattered light seriously affect the accuracy of detection. At present, the Stokes shift of the reported poly hydrogen sulfide fluorescent probe is maximum 215 nm. The Stokes displacement of the probe AIE-PS2 can reach 254nm, so that the problems of fluorescence quenching and sensitivity reduction caused by small Stokes displacement are effectively solved; (2) recognition group: 2-fluoro-5-nitrobenzoate is taken as a poly hydrogen sulfide recognition group. This is because 2-fluoro-5-nitrobenzoate, having nucleophilic and electrophilic properties, is effective with H₂S_nThe reaction occurs, while other biological mercaptan can only perform one nucleophilic substitution reaction and can not perform intramolecular cyclization reactionTo release the fluorescent parent nucleus, thus, 2-fluoro-5-nitrobenzoate para-H₂S_nHas better selectivity; (3) the TPE-HBT and the 2-fluoro-5-nitrobenzoic acid are connected through an ester bond to obtain a probe AIE-PS 2.

Fluorescent probe AIE-PS2 recognizes H₂S_nThe principle of (1): due to H₂S_nHas strong nucleophilicity and electrophilicity, therefore, H₂S_nWill form a persulfate intermediate with probe AIE-PS 2. Then, the naked sulfydryl attacks carbonyl carbon atoms, and nucleophilic reaction is carried out to carry out intramolecular cyclization, so that TPE-HBT is released. Due to the hindered rotation of the bond, the TPE-HBT aggregates in the hydrophilic phase and protons are transferred from oxygen (proton donor) to nitrogen (proton acceptor) via hydrogen bonds, and the process of ESIPT occurs, resulting in strong fluorescence.

To verify the reaction principle of the probe AIE-PS2 with hydrogen polysulfide: AIE-PS2 was incubated with disodium tetrasulfide in PBS buffer at 37 ℃ for 100 min. The results showed that reaction of AIE-PS2 with disodium tetrasulfide produced a yellow phosphor¹H NMR, HRMS confirmed that the yellow fluorescent substance was TPE-HBT. The reaction principle of the probe AIE-PS2 and the poly hydrogen sulfide is shown as follows:

the hydrogen polysulfide fluorescent probe AIE-PS2 provided by the invention can act on fluorescent dye synergistically based on dual mechanisms of AIE and ESIPT. The AIE properties can facilitate ESIPT emission by forming aggregates, favoring ESIPT induction, resulting in larger stokes shifts, particularly in highly polar or hydrogen-bonded donor solvents. The ESIPT property can promote intramolecular motion confinement by forming intramolecular hydrogen bonds, enhancing AIE emission. Compared with a fluorescent probe based on a single AIE or ESIPT mechanism, the hydrogen sulfide-containing fluorescent probe AIE-PS2 provided by the invention has more remarkable advantages: (1) hydrophilic modification is avoided. A commonly used hydrophilic strategy is to introduce ionic structures into the probe to enhance its water solubility, but the charge of the ionic dye structure at high concentrations may affect the membrane potential, perturbing the intracellular physiological environment. The fluorescent probe based on the dual mechanism of AIE and ESIPT reduces the fussy synthesis steps on one hand, avoids the interference to the normal physiological environment of cells on the other hand, and is safer; (2) with a large stokes shift. Fluorescence quenching can be caused by small Stokes shift, and light scattering is increased, so that quantitative detection is influenced; (3) there was no self-quenching at high concentrations. The AIE fluorophore overcomes ACQ phenomenon, has higher light stability, and is beneficial to biological imaging.

The synthetic route of the fluorescent probe AIE-PS2 for identifying the poly-hydrogen sulfide is as follows:

a fluorescent probe AIE-PS2 for identifying poly-hydrogen sulfide comprises the following steps:

the first step is as follows: under the condition of trifluoroacetic acid, carrying out reflux reaction on a compound TPE-OH and hexamethylenetetramine to prepare a compound TPE-CHO;

the second step is that: at H₂O₂And in the presence of HCl, carrying out chemical reaction on a compound TPE-CHO and the compound 1 to prepare a compound TPE-HBT;

the third step: and (3) carrying out chemical reaction on the compound TPE-HBT and the compound 2 in the presence of triethylamine to prepare a fluorescent probe AIE-PS 2.

In a preferable scheme, in the first step, the molar ratio of the compound TPE-OH to the compound hexamethylenetetramine is 1: 5-15, and under the condition of not influencing the effect of the invention, the molar ratio is further preferably 1: 10.

In a more preferable scheme, the mass-to-volume ratio of the compound TPE-OH to the trifluoroacetic acid is 1: 50-70 g/mL, for example, the mass-to-volume ratio of the compound TPE-OH to the trifluoroacetic acid is 1:60 g/mL.

Further, the reaction time is 2-10 h, preferably 3-6 h, for example 4 h.

For the invention, in the second step, the compound TPE-CHO and the compound 1 are used for reaction to prepare the compound TPE-HBT, and in a preferable scheme, the molar ratio of the compound TPE-CHO to the compound 1 is 1: 2.5-4.5, and is further preferably 1: 3.5. Wherein, the compound 1 is 2-aminobenzenethiol.

Further, during the reaction, in H₂O₂And the amount of HCl, can be adjusted as desired.

In a preferred embodiment, the reaction time is 20 to 40 ℃, and the temperature is commonly understood as room temperature.

Further, the reaction time is 2-10 h, preferably 3-6 h, for example 4 h.

In the third step, a compound TPE-HBT and a compound 2 are subjected to chemical reaction in the presence of triethylamine to prepare a fluorescent probe AIE-PS2, wherein in a preferable scheme, the molar ratio of the compound TPE-HBT to the compound 2 is 1: 1.5-4.5, and more preferably 1: 2.5.

Wherein the compound 2 is 2-fluoro-5-nitrobenzoyl chloride, and can be prepared by acylation reaction of 2-fluoro-5-nitrobenzoic acid and thionyl chloride.

Further, the molar ratio of the compound TPE-HBT to triethylamine is 1: 20-30, and under the condition that the effect of the invention is not affected, the molar ratio is more preferably 1: 24.4.

Further, in the third step, when the compound TPE-HBT and the compound 2 are used as raw materials to prepare the fluorescent probe AIE-PS2, the reaction temperature is low, for example, the reaction is performed in an ice bath.

Further, the reaction time is 8-24 h, preferably 10-18 h, for example 12 h.

The fluorescent probe AIE-PS2 prepared by the invention can be used for quantitatively detecting the poly hydrogen sulfide, and is particularly used for detecting the poly hydrogen sulfide at a cellular level.

By adopting the technical scheme of the invention, the advantages are as follows:

the fluorescent probe AIE-PS2 for identifying the poly-hydrogen sulfide provided by the invention has the advantages of larger Stokes shift (254nM), good selectivity, high sensitivity, low detection limit (9nM), high fluorescence response multiple (75 times) and good biocompatibility.

In PBS buffer, fluorescence intensity with Na₂S₄(020 μ M) concentration, indicating that the probe is suitable for quantitative detection of hydrogen sulfide in organisms; the probe AIE-PS2 can image endogenous and exogenous poly-hydrogen sulfide at a cellular level, and further realizes fluorescent imaging of the poly-hydrogen sulfide of the MCF-7 cell by using AIE-PS 2.

The fluorescent probe AIE-PS2 provided by the invention is an effective tool for visually and quantitatively detecting poly hydrogen sulfide, namely H₂S_nA visual and noninvasive detection method is provided in the physiological and pathological mechanism and the signal transduction path in the organism, and has important significance for revealing the physiological and pathological mechanism of the hydrogen sulfide in the human body.

Drawings

FIG. 1 is a schematic representation of the compound TPE-CHO¹H NMR spectrum;

FIG. 2 shows a compound TPE-HBT¹H NMR spectrum;

FIG. 3 shows a compound TPE-HBT¹³A C NMR spectrum;

FIG. 4 shows a fluorescent probe AIE-PS2¹H NMR spectrum;

FIG. 5 is an HRMS profile, HRMS (ESI), of fluorescent probe AIE-PS2⁺):(M+H)⁺calcd for C₄₀H₂₆FN₂O₄S,649.1597；found,649.1593；

FIG. 6 shows fluorescent probes AIE-PS2 and Na₂S₄High resolution Mass Spectrometry ((M + H) after reaction)⁺calculated for C₃₃H₂₄NOS,482.1579；found 482.1572)；

FIG. 7 is a graph showing fluorescence spectra and Dynamic Light Scattering (DLS) of TPE-HBT at different water contents in the DMSO/water mixed solution; wherein, A in FIG. 7 is the fluorescence spectrum of TPE-HBT (10 μ M) under different proportions of water content; at 600nm, the water contents represented by the curves from bottom to top are 30%, 40%, 0%, 10%, 20%, 50%, 60%, 70%, 80%, 90% and 99% in sequence; wherein, B in FIG. 7 is a DLS particle size diagram of TPE-HBT (10 μ M) under the condition of DMSO/water ratio of 1: 99;

FIG. 8 is an electron micrograph of a fluorescent matrix TPE-HBT in solvent; wherein, A in FIG. 8 is an electron microscope image of the fluorescent mother nucleus TPE-HBT in acetone; FIG. 8B is a partial enlarged view of FIG. 8A; in FIG. 8, C is an electron microscope image of the fluorescent mother-core TPE-HBT in a mixed solution of acetone and water-1/9; d in FIG. 8 is a partial enlarged view corresponding to C in FIG. 8;

FIG. 9 shows the fluorescence spectrum and UV spectrum of fluorescent probe AIE-PS2 after reaction with hydrogen polysulfide; wherein A in FIG. 9 is TPE-HBT, AIE-PS2 and Na₂S₄Fluorescence spectrum of + AIE-PS2 in PBS buffer (20mM, pH 7.4, 1% DMSO); in FIG. 9, B is TPE-HBT, AIE-PS2 and Na₂S₄Absorption spectrum of + AIE-PS2 in PBS buffer (20mM, pH 7.4, 1% DMSO);

FIG. 10 shows the AIE-PS2 probe (5. mu.M) with different Na concentrations₂S₄(0-100. mu.M) change in fluorescence response intensity after 100min incubation in PBS buffer (20mM, pH 7.4, 1% DMSO), where A in FIG. 10 is compared to different concentrations of Na₂S₄(0-100. mu.M) change in the fluorescence intensity of AIE-PS2 (5. mu.M) at 589nm after incubation; in FIG. 10, B is the fluorescence intensity in PBS buffer and Na₂S₄Linear relationship between concentrations (0-25 μ M);

FIG. 11 shows probes AIE-PS2 and Na₂S₄Fluorescence spectrum of reaction time, wherein A in FIG. 11 is AIE-PS2 (5. mu.M) and Na₂S₄(25 μ M) fluorescence spectra of 0,20,40,60,80,100,120,140 and 200min incubations in PBS buffer (20mM, pH 7.4, 1% DMSO) at 37 ℃ respectively; in FIG. 11B is AIE-PS2(5 μ M) in PBS buffer (20mM, pH 7.4, 1% DMSO) with Na₂S₄(25. mu.M) fluorescence responses at 37 ℃ for 0,20,40,60,80,100,120,140 and 200min, respectively;

FIG. 12 shows the selectivity of probe AIE-PS2 for poly (hydrogen sulfide), wherein A in FIG. 12 is AIE-PS2 (5. mu.M) and Na₂S₄(25. mu.M) and RSS (Na)₂S₂ 25μM、Na₂S₂100μM、1mM Cys、1mM GSH、1mM CysSSCys、100μM Hcy、10mM GSH、1mM GSSG、500μM S₈、500μM Na₂S₂、500μM Na₂SO₃、500μM Na₂SO₄、1mM Cys-polysulfide、100μM CH₃SSSCH₃) In PBS buffer (20mM, pH 7.4, 1% DMSO) at 37 deg.CIncubating the fluorescence spectrum for 100 min; in FIG. 12, B is AIE-PS2 (5. mu.M) and Na₂S₄(25μM)、Na₂S₂(25μM)、Na₂S₂(100. mu.M) and RSS incubations for 100min, each group representing the response of AIE-PS2 to RSS at 589nm and RSS to 25. mu.M Na, respectively₂S₄The response of the mixture of (a); 1.blank + Na₂S₄(25. mu.M), 2. blank + Na₂S₂(25. mu.M), 3. blank + Na₂S₂(100μM)、4.Na₂S(25μM)+Na₂S₄(25μM)、5.Cys(1mM)+Na₂S₄(25μM)、6.GSH(1mM)+Na₂S₄(25μM)、7.CysSSCys(1mM)+Na₂S₄(25μM)、8.Hcy(100μM)+Na₂S₄(25μM)、9.GSH(10mM)+Na₂S₄(25μM)、10.GSSG(1mM)+Na₂S₄(25μM)、11.S₈(500μM)+Na₂S₄(25μM)、12.Na₂S₂O₃(500μM)+Na₂S₄(25μM)、13.Na₂SO₃(500μM)+Na₂S₄(25μM)、14.Na₂SO₄(500μM)+Na₂S₄(25μM)、15.Cys-polysulfide(1mM)+Na₂S₄(25μM)、16.CH₃SSSCH₃(100μM)+Na₂S₄(25. mu.M); in the numbers 1 to 16 of B in FIG. 12, the left-hand bar graphs represent no Na₂S₄The left bar chart represents the graph containing Na₂S₄；

FIG. 13 shows the selectivity of probe AIE-PS2 for poly (hydrogen sulfide), wherein A in FIG. 13 is AIE-PS2 (5. mu.M) and Na₂S₄(25μM)、Na₂S₂(25. mu.M) and ion (Na)⁺、K⁺、Cu²⁺、Mg²⁺、Zn²⁺、Ca²⁺、Fe³⁺、Fe²⁺、CO₃ ^2-、HCO₃ ^-、Cl^-、Br^-、I^-、HPO₄ ^2-、H₂PO₄ ^-1mM) fluorescence pattern incubated in PBS buffer (20mM, pH 7.4, 1% DMSO) at 37 ℃ for 100 min; in FIG. 13, B is AIE-PS2 (5. muM) and Na₂S₄(25μM)，Na₂S₂(25. mu.M) and the fluorescence response of the ion incubation for 100min, each group representing the response of AIE-PS2 with ions at 589nm and ions with 25. mu.M Na, respectively₂S₄The sound of the mixture of (a); b:1 blank + Na₂S₄(25μM)、2.blank+Na₂S₂(25μM)、3.HCO₃ ^-+Na₂S₄、4.CO₃ ^2-+Na₂S₄、5.Fe²⁺+Na₂S₄、6.Fe³⁺+Na₂S₄、7.Zn²⁺+Na₂S₄、8.Mg²⁺+Na₂S₄、9.Ca²⁺+Na₂S₄、10.Cu²⁺+Na₂S₄、11.K⁺+Na₂S₄、12.Na⁺+Na₂S₄、13.Br^-+Na₂S₄、14.I^-+Na₂S₄、15.HPO₄ ^2-+Na₂S₄、16.H₂PO₄ ^-+Na₂S₄、17.Cl^-+Na₂S₄(ii) a In the numbers 1 to 17 of B in FIG. 13, the left-hand bar graphs represent no Na₂S₄The left bar chart represents the graph containing Na₂S₄；

FIG. 14 shows the selectivity of probe AIE-PS2 for poly (hydrogen sulfide), wherein A in FIG. 14 is AIE-PS2 (5. mu.M) and Na₂S₄(25μM)、Na₂S₂(25. mu.M) and active oxygen (O)₂ ^-、¹O₂、·OH、t-BuOOH、OCl^-、H₂O₂100 μ M), active nitrogen (NO, NO)₃ ^-、ONOO^-、NO₂ ^-100 μ M) in PBS buffer (20mM, pH 7.4, 1% DMSO) at 37 ℃ for 100 min; in FIG. 14, B is AIE-PS2 (5. mu.M) and Na₂S₄(25μM)，Na₂S₂(25. mu.M) and the fluorescence response of activated oxygen and activated nitrogen incubation for 100min, each group representing the response of AIE-PS2 to activated oxygen and activated nitrogen at 589nm and activated oxygen, activated nitrogen and 25. mu.M, respectivelyM Na₂S₄The response of the mixture of (a); 1.blank + Na₂S₄(25. mu.M), 2. blank + Na₂S₂(25μM)、3.ONOO^-+Na₂S₄、4.NO₂ ^-+Na₂S₄、5.O₂ ^-+Na₂S₄、6.¹O₂+Na₂S₄、7.·OH+Na₂S₄、8.t-BuOOH+Na₂S₄、9.OCl^-+Na₂S₄、10.H₂O₂+Na₂S₄、11.NO+Na₂S₄、12.NO₃ ^-+Na₂S₄(ii) a In the numbers 1 to 12 of B in FIG. 14, the left-hand bar graphs represent no Na₂S₄The left bar chart represents the graph containing Na₂S₄；

FIG. 15 shows the effect of the probe AIE-PS2 on cell viability, where A in FIG. 15 is the viability of MCF-7 cells incubated with AIE-PS2(0,5,10,15,20,30,40, 50. mu.M) for 24h cells; FIG. 15, panel B shows the survival rate of MCF-7 cells incubated with AIE-PS2 (10. mu.M) for various periods of time (0,6,12,18,24 h);

FIG. 16 is fluorescence imaging of poly-hydrogen sulfide detected by probe AIE-PS2, wherein A in FIG. 16 is incubation of MCF-7 cells with AIE-PS2(10 μ M) at 37 ℃ for 30 min; FIG. 16, B is cells pretreated with N-methylmaleimide (NMM, 1mM) for 1h, then incubated with AIE-PS2 (10. mu.M) for 30 min; FIG. 16, C is incubation of cells with AIE-PS2 (10. mu.M) for 30min, followed by Na₂S₄(10. mu.M) incubation for 100 min; FIG. 16, D is incubation of cells with AIE-PS2 (10. mu.M) for 30min, followed by Na₂S₄(20. mu.M) incubation for 100 min; a ', B', C 'and D' in E in FIG. 16 are the mean fluorescence intensities of the cells in A, B, C and D in FIG. 16, respectively;

a, B, C and D in FIG. 17 are bright field diagrams of cells corresponding to A, B, C and (D) in FIG. 16;

FIG. 18 is fluorescence imaging of endogenous poly-hydrogen sulfide detection by probe AIE-PS2, wherein in FIG. 18A is induced MCF-7 cells by LPS (1. mu.g/mL) for 16h, and then incubated with AIE-PS2 (10. mu.M) for 30 min; FIG. 18B is a 30min pre-incubation of cells with DL-propargylglycine (PAG, 200. mu.M), followed by a further 16h incubation with LPS (1. mu.g/mL) at 37 ℃ followed by a 30min incubation of cells with AIE-PS2 (10. mu.M); a 'and B' in C in FIG. 18 are the mean fluorescence intensities of the cells in A, B in FIG. 18, respectively;

a and B in FIG. 19 are the corresponding cell brightfield diagrams of A and B in FIG. 18.

Detailed Description

The hydrogen sulfide polysulfide fluorescent probe of the present invention is further illustrated by the following examples in conjunction with the attached drawings, but these examples do not limit the present invention in any way.

First, implement method

1. Materials and instruments

1.1 reagents

The other reagents are all domestic analytical purifiers

1.2 instruments

1.3 preparation of the solution

Preparing a probe solution: AIE-PS2(6.47mg, 0.01mmol) was dissolved in dimethyl sulfoxide (10mL) to prepare a 1mM probe solution.

Na₂S₄Preparing a stock solution: nitrogen was passed through 10mL of deionized water for 15 min. Under the condition of nitrogen, adding Na₂S₄(17.42mg, 0.1mmol) was dissolved in the above deionized water to prepare Na with a concentration of 10mM₂S₄Stock solution, adding Na according to experiment requirement₂S₄The stock solution is diluted to 1.0 mM-100. mu.M for use.

Na₂S₂Preparation of stock solutions (asH₂S₂According to the preparation method in the literature: inorganic Chemistry,2003,42,12, 3712-: nitrogen was passed through 10mL of deionized water for 15 min. Under the condition of nitrogen, adding Na₂S₂(11.01mg, 0.1mmol) was dissolved in the above deionized water to prepare Na with a concentration of 10mM₂S₂Stock solution, adding Na according to experiment requirement₂S₂The stock solution is diluted to 1.0 mM-100. mu.M for use.

Na₂Preparation of S stock solution (as H)₂Source of S): 5mg EDTA was dissolved in 10mL deionized water and purged with nitrogen for 15 min. Under the condition of nitrogen, adding Na₂S·9H₂O (24mg, 0.1mmol) was dissolved in the above solution to prepare 10mM Na₂S stock solution, adding Na according to experiment requirements₂S stock solution is diluted to 1.0 mM-100. mu.M solution for standby.

Preparing an L-cysteine (L-Cys) stock solution: cys (12.10mg, 0.1mmol) was dissolved in deionized water (10mL) to prepare a stock solution with a concentration of 10.0mM, and the stock solution was diluted to a solution of 1.0mM and 100. mu.M for use.

Preparation of homocysteine (Hcy) stock solution: hcy (13.5mg, 0.1mmol) was dissolved in deionized water (10mL) to prepare a stock solution with a concentration of 10.0mM, and the stock solution was diluted to a solution of 1.0mM and 100. mu.M for use.

Preparation of Glutathione (GSH) stock solution: GSH (30.7mg, 0.1mmol) was dissolved in deionized water (10mL) to prepare a stock solution with a concentration of 10.0mM, and the stock solution was diluted to 1.0mM and 10mM for further use.

Other analytes (Cys-polysulfide, GSSG, Na)₂S₂O₃，CysSSCys，CH₃SSSCH₃，S₈，Na₂S₂O₃，NaHSO₃，Na₂SO₃，Na₂SO₄) The preparation method of the stock solution is the same as the above.

All the preparation solutions are prepared and used.

1.4 cells

Species and strains: human breast cancer cell MCF-7. The source is as follows: cell bank of Chinese academy of sciences.

Second, example

2.1 preparation of Probe AIE-PS2

Preparation of TPE-CHO: adding a compound TPE-OH (0.5g, 1.37mmol) into a solution obtained by mixing hexamethylenetetramine (1.92g, 13.7mmol) and trifluoroacetic acid (30mL), stirring and refluxing for reaction for 4h, quenching the obtained reaction solution with distilled water (30mL) after the reaction is finished, and using CH (CH)₂Cl₂(3X 20mL) was extracted. The combined organic phases were successively saturated with Na₂CO₃(20mL), water (20mL) and brine (20 mL). With anhydrous Na₂SO₄After drying, concentration under reduced pressure was carried out to dryness. The residue was purified by column chromatography (PE: EA, 25:1v/v) to give a pale yellow solid, i.e., the compound TPE-CHO, with a yield of 72.6%. TLC (silica, PE: EA, 5:1 v/v): r_f0.5. The relevant spectrum is shown in FIG. 1.

Preparation of TPE-HBT: the compound TPE-CHO (20mg, 0.054mmol) and 2-aminobenzenethiol (

compound

1, 20. mu.L, 0.187mmol) were dissolved in 20mL of methanol, and 30% H was added₂O₂(20. mu.L) and 37% HCl (20. mu.L) were reacted at room temperature for 4 hours, and after the reaction was completed, the resulting reaction solution was quenched with distilled water (10mL), extracted with ethyl acetate, and extracted with anhydrous Na₂SO₄After drying, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give TPE-HBT as a pale yellow solid in 63.2% yield. TLC (silica, PE: EA, 60:1, v/v): r_f0.6. The relevant spectra are shown in FIGS. 2 and 3.

Preparation of 2-fluoro-5-nitrobenzoyl chloride: 2-fluoro-5-nitrobenzoic acid (204mg, 1.1024mmol) was added to 15mL of thionyl chloride and reacted at 80 ℃ under reflux for 8h, after the reaction was completed, the resulting reaction solution was concentrated under reduced pressure to give 2-fluoro-5-nitrobenzoyl chloride (compound 2) as a pale yellow solid with a yield of 60%. TLC (silica, DCM: MeOH, 20:1, v/v): r_f＝0.6。

Preparation of Probe AIE-PS 2: 2-fluoro-5-nitrobenzoyl chloride (compound 2, 20g, 0.1mmol) was dissolved in dichloromethane and added dropwise to a solution of TPE-HBT (20mg, 0.041mmol) and triethylamine (139.137. mu.L, 1.0mmol) in anhydrous dichloromethane and reacted overnight for 12h in an ice bath. After completion of the reaction, the resulting reaction mixture was concentrated under reduced pressure to remove methylene chloride, and the residue was washed with water (30mL), extracted with methylene chloride (3X 20mL) and concentrated under reduced pressure to give a crude product. The crude product was recrystallized from ethyl acetate (20mL) to give AIE-PS2 as pale yellow crystals in 66.1% yield. TLC (silica, PE: EA, 10:1, v/v): r_f0.5. The relevant spectra are shown in FIGS. 4 and 5.

2.2 mechanism of recognition of Poly-Hydrogen sulfide by Probe AIE-PS2

Probe AIE-PS2(64.8mg, 0.1mmol) was dissolved in DMSO (15mL), and Na dissolved in it was added₂S₄(312.10mg, 1.0mmol) of phosphate buffer (30mL, 20.0mM, pH 7.4) was stirred at 37 ℃ for 100 min. After completion of the reaction, the obtained reaction solution was extracted with ethyl acetate (3X 10mL), concentrated under reduced pressure, and the obtained product was isolated by extraction, and the reaction product was confirmed by HRMS, whereby it was confirmed that the probes AIE-PS2 and H were present₂S_nThe reaction principle of (1).

2.3 scanning Electron microscopy analysis

TPE-HBT (6.48mg, 0.01mmol) is dissolved in acetone (10mL) to prepare a mother solution with the concentration of 0.1mM, and the mother solution is respectively diluted with acetone and water to obtain a pure acetone solution with the concentration of TPE-HBT (0.01 mM) and an acetone-water mixed solution (the water content is 90%) with the concentration of TPE-HBT (0.01 mM). And (3) dropping a drop of the gold-plating solution on a silicon chip, naturally drying the silicon chip, spraying gold, and performing analytical test by using a Tenoe-VS field emission sequence scanning electron microscope.

2.4 measurement of fluorescence Spectroscopy

Probe AIE-PS2 was dissolved in DMSO, disodium tetrasulfide or disodium disulfide (as Na)₂S₄Or Na₂S₂As H₂S_nSource) at a concentration corresponding to the concentration of the substance to be detected (e.g., Na)₂S₄25mM) was mixed with AIE-PS2 and diluted with 20.0mM phosphate buffer (pH 7.4, 20mM) so that the test solution contained 99% of the aqueous phase and 1% of the organic phase. After incubation at 37 ℃, the test solution was added to a quartz cuvette and the fluorescence intensity was measured. The test solution without analyte was used as a blank. Each set of data was assayed at least in triplicate and the results are expressed as mean + -SD. Fluorescence spectrophotometer test conditions: the excitation wavelength is 357nm, the excitation slit width is 5nm, the emission slit width is 5nm, the scanning speed is 1200nm/min, and the emission spectrum range is 490-720 nm. The voltage of the photomultiplier was set to 800V.

2.5 determination of detection Limit

The fluorescence emission spectra of the probes were repeatedly measured 10 times, and the standard deviation of the fluorescence intensities thereof was calculated. The probe AIE-PS2 was then incubated with a range of Na concentrations₂S₄And (5) incubating to obtain a linear equation of the concentration and the fluorescence intensity. The calculation formula of the detection limit of the probe is as follows: 3 sigma/k. k represents the fluorescence intensity and Na₂S₄The slope of the concentration linear equation, σ, represents the standard deviation of the blank.

2.6 measurement of ultraviolet Spectrum

The ultraviolet spectrum was measured using a UV-2401PC UV-visible spectrophotometer. Adding the probe into a quartz cuvette, adding 20.0mM phosphate buffer solution to dilute the probe, and adding Na₂S₄After incubation, the absorption spectrum was measured.

2.7 determination of the cytotoxicity of the Probe AIE-PS2

The cytotoxicity of the probe AIE-PS2 was determined by MTT method. MCF-7 cells were seeded at a density of 50,000 cells/well in 96-well plates, charged with 5% CO₂Culturing in an incubator until the cells enter logarithmic phase, and removingThe culture solution was removed and the cells were incubated with different concentrations of AIE-PS2 for 24 h. Cells without AIE-PS2 added were used as controls. After 24h, 20. mu.L of MTT dye (3- [4, 5-dimethylthiazol-2-yl) was added to each well]2, 5-diphenyltetrazolium bromide, 5mg/mL in PBS buffer) and incubation at 37 ℃ was continued for 4 h. The remaining MTT solution was then removed and 150 μ L DMSO was added to each well to dissolve formazan crystals. Shaking the shaking table for 10min, and measuring the absorbance at 570nm by using a microplate reader. Each sample was replicated three times and the entire experiment was replicated three times.

2.8 cell culture and cell level Poly Hydrogen sulfide imaging Studies

MCF-7 cells (from cell banks of Chinese academy of sciences) were inoculated into cell culture medium (DMEM containing 10% calf serum, penicillin/streptomycin (100. mu.g/mL) at 37 ℃ with 5% CO₂Culturing in an incubator. When the growth reaches logarithmic phase, the cells are digested by pancreatin and inoculated in a special confocal dish. Control group: probe AIE-PS2 (10. mu.M, 5. mu.L DMSO) was incubated with MCF-7 for 30 min. Exogenous H₂S_nAn imaging group: probe AIE-PS2 (10. mu.M, 5. mu.L DMSO) was incubated with MCF-7 cells for 30min, followed by addition of Na₂S₄(10. mu.M, 2. mu.L physiological saline), (20. mu.M, 4. mu.L physiological saline) was incubated for 100 min. Before imaging, the cells were washed three times gently with PBS buffer. Endogenous H₂S_nAn imaging group: MCF-7 cells were incubated with lipopolysaccharide (LPS, 1. mu.g/mL, 10. mu.L physiological saline) for 16h and then with probe AIE-PS2 (10. mu.M, 5. mu.L DMSO) for 30 min. Before imaging, the cells were washed three times gently with PBS buffer. Inhibitor group: MCF-7 cells were incubated with NMM (1mM, 10. mu.L physiological saline) for 1h and then with probe AIE-PS2 (10. mu.M, 5. mu.L DMSO) for 30 min. Before imaging, the cells were washed three times gently with PBS buffer. MCF-7 cells were incubated with PAG (200. mu.M, 10. mu.L saline) for 30min, then LPS (1. mu.g/mL, 10. mu.L saline) for 16h, and finally with probe AIE-PS2 (10. mu.M, 5. mu.L DMSO) for 30 min. Before imaging, the cells were washed three times gently with PBS buffer. The photographs were taken with a Zeiss confocal laser microscope (63X oil lens). Excitation wavelength 410nm, emission wavelength 590 nm. Analysis was performed using Zeiss software (ZEN). All data are expressed as mean ± SD (n ═ 3).

2.9 data processing

Statistical analysis was performed using SPSS software. Data are expressed as means ± standard deviation (Mean ± SD), and comparisons between groups were performed using a one-way ANOVA (one-way ANOVA) completely randomized design. P < 0.05 indicates that the difference is statistically significant.

Third, effect verification

1.1 AIE Performance of TPE-HBT

The compound TPE-HBT has good solubility in DMSO and good solubility in H₂O is poorly soluble, and therefore, its AIE properties are in DMSO-H₂And (4) characterization in a mixed solution of O. In DMSO solution, TPE-HBT is well dispersed, and the solution is transparent and clear. The volume fraction of DMSO is 60-99 percent of DMSO-H₂In O mixed solution, intense yellow fluorescence was observed with a maximum emission wavelength of 589nm (A in FIG. 7). Dynamic Light Scattering (DLS) experiments can also demonstrate that TPE-HBT has AIE properties. The TPE-HBT has good solubility, so no particles exist in a pure solvent; aggregation of nanoparticles (average particle size 100nm) was seen in the 99:1 volume fraction water/DMSO mixed solution (B in fig. 7), indicating that TPE-HBT formed nanoaggregates. Similarly, in the results of the scanning electron microscope, the TPE-HBT particles in the pure acetone solution were small (A, B in FIG. 8), and a large amount of aggregation of the particles was observed at a water content of 90% (C, D in FIG. 8), indicating that the TPE-HBT had AIE properties.

DMSO/H in different volume ratios₂The fluorescence change of TPE-HBT in O solution is mainly caused by the combined action of EISPT process and AIE. DMSO/H in TPE-HBT₂In the O mixed solution, a significant change in fluorescence spectrum was observed as the proportion of water increased from 0% to 90%. At lower water content (0-40%) at 350nm excitation, ketogenic emission is generally more prone due to strong intramolecular hydrogen bonding and other factors (e.g., intramolecular motion-limited, hydrophobic environment). The TPE-HBT showed dual emission peaks, with short wavelength emission at 453nm and long wavelength emission at 550 nm. As the proportion of water increased from 50% to 90%, TPE-HBT formed an aggregated state, with the short wavelength emission peak completely disappeared, while the long wavelength emission increased significantly and a significant red shift occurred from 550nm to 589nm (A in FIG. 7). LaunchingThe red-shift of the peak may be the result of enhanced charge transfer due to aggregation. Due to the interaction between ESIPT and AIE processes, the ketogenic emission (longer wavelength) at 589nm increases dramatically, and TPE-HBT exhibits strong fluorescence emission and large Stokes shift in the aggregated state.

1.2 fluorescent probes AIE-PS2 and Na₂S₄Fluorescence and ultraviolet spectra of reactions

First, we detected the probes AIE-PS2 and Na₂S₄The change in the fluorescence spectrum and the UV spectrum after the reaction. As shown in FIG. 9, the probe AIE-PS2 itself was not fluorescent, and was Na₂S₄(100. mu.M) gave a bright yellow fluorescence with a maximum emission wavelength of 589 nm. This is consistent with the fluorescence spectrum of the fluorescent parent TPE-HBT. The probe AIE-PS2 has the maximum ultraviolet absorption peak at 262nm, and Na₂S₄After the reaction is finished, the maximum ultraviolet absorption peak is at 335nm and is consistent with the ultraviolet absorption spectrum of the TPE-HBT.

1.3 fluorescent probes AIE-PS2 and Na₂S₄Linear relationship of reaction and detection limit

Mixing AIE-PS2 with different concentrations of Na₂S₄(0-100. mu.M) incubation, and examining the applicability of the probe AIE-PS2 to quantitative detection of poly-hydrogen sulfide in biological samples. As shown in FIG. 10, the probe AIE-PS2 was used without adding Na₂S₄Almost no fluorescence before incubation; when the probe AIE-PS2 is mixed with Na with different concentrations₂S₄(0-20. mu.M) after incubation, the fluorescence intensity at 589nm is dependent on Na₂S₄Increasing concentration and increasing concentration (75 times), Na₂S₄Shows a good linear relationship with the fluorescence intensity in the range of 0-20 mu M, and the detection limit of the probe AIE-PS2 for detecting the poly hydrogen sulfide in the PBS buffer solution is 9 nM. When AIE-PS2 is mixed with Na₂S₄When the reaction ratio of (1) to (4) is 1:4, the fluorescence intensity reaches a peak value and the reaction tends to be saturated. The results show that the probe AIE-PS2 has good detection sensitivity and is suitable for quantitatively detecting the endogenous level of the hydrogen polysulfide of the complex organism. Probe AIE-PS2 and Na₂S₄The relevant spectrum after the reaction is shown in FIG. 6.

1.4 fluorescent Probe AIE-PS2 andNa₂S₄reaction time of

As shown in FIG. 11, the probe AIE-PS2 was mixed with Na₂S₄(25. mu.M) incubation for different periods of time, the fluorescence intensity was found to peak around 100min, indicating that about 100min AIE-PS2 was found to overlap with Na₂S₄(25. mu.M) the reaction was complete. The reaction time is prolonged to 200min, the fluorescence intensity is not weakened, and the fluorescence intensity is relatively stable.

1.5 fluorescent Probe AIE-PS2 detection of Na₂S₄Selectivity of (2)

To test the selectivity of the probe AIE-PS2, we combined the probe with active sulfur (RSS, including Na)₂S，Cys，GSH，CysSSCys，Hcy，GSSG，S₈，S₂O₃ ^2-，SO₃ ^2-，SO₄ ^2-，Cys-polysulfide，CH₃SSSCH₃) Reactive oxygen species (ROS, including O)₂ ^-,¹O₂,·OH,t-BuOOH,OCl^-,H₂O₂) Active nitrogen (RNS, including NO, NO)₃ ^-,ONOO^-,NO₂ ^-) And ions (Na)⁺,K⁺,Cu²⁺,Mg²⁺,Zn²⁺,Ca²⁺,Fe³⁺,Fe²⁺,CO₃ ^2-,HCO₃ ^-,Cl^-,Br^-,I^-,HPO₄ ^2-,H₂PO₄ ^-) After incubation the fluorescence intensity was measured. As shown in FIGS. 12-14, the probe was reacted with Na₂S₄、Na₂S₂Incubation produces a strong fluorescent response but little response with other substances. In addition, whether the presence of active sulfur affects the probe AIE-PS2 with Na was demonstrated by competitive experiments₂S₄In the absence of Na during incubation₂S₂Or Na₂S₄And the probes have no fluorescence response, and the experiment shows that the probe AIE-PS2 can selectively recognize Na₂S₂Or Na₂S₄Without interference from other substances.

2 cell level polyhydrosulfide fluorescence imaging

2.1 cytotoxicity assay

Before cytofluorescence imaging, a cytotoxicity test on the probe AIE-PS2 was first performed. The cytotoxicity of the probe AIE-PS2 was detected by MTT method using human breast cancer MCF-7 cells as the detection cell line. As shown in FIG. 15, the probe AIE-PS2 (0. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M) was incubated with MCF-7 cells for 24 hours, and as a result, the cell survival rate was still over 90%. The cell survival rate of the AIE-PS2(10 mu M) and MCF-7 is still over 90 percent after 6,12,18 and 24 hours of incubation. Thus, it was demonstrated that AIE-PS2 had little effect on MCF-7 cell survival and low toxicity.

2.2 cellular fluorescence imaging of exogenous Poly-Hydrogen sulfide

The probe AIE-PS2 has good sensitivity and selectivity under in vitro test conditions, and then whether the probe can detect the hydrogen sulfide in cells and carry out fluorescence imaging is continuously researched. MCF-7 cells are inoculated in a standard confocal culture dish, and weak red fluorescence can be seen after a probe AIE-PS2 is added and incubated for 30min (A in figure 16); the cells were given N-methylmaleimide (NMM, H) 1H in advance₂S_nScavenger) was incubated with probe AIE-PS2, and a significant decrease in intracellular fluorescence intensity was observed (B in fig. 16). This indicates that the red fluorescence in A in FIG. 16 is probably caused by physiological concentrations of endogenous poly-hydrogen sulfide, and the probe AIE-PS2 can detect endogenous H under physiological conditions in cells₂S_nAnd has high detection sensitivity. After incubating the cells with the probe for 30min in advance, Na was administered at different concentrations₂S₄(10. mu.M, 20. mu.M), it was found that bright red fluorescence appeared in the cells (C in FIG. 16 and D in FIG. 16). As is clear from FIG. 17, the cell morphology was good. In conclusion, the probe AIE-PS2 can detect the physiological concentration of endogenous H₂S_nAnd varying levels of exogenous H₂S_n。

2.3 cellular fluorescence imaging of endogenous PolyHydrogen sulfide

It was examined whether the probe AIE-PS2 could be used for fluorescence imaging of endogenous polyhydrosulfide in living cells. Cystathionine-gamma-lyase (CSE) is an endogenous polyhydrosulfide synthase, while Lipopolysaccharide (LPS) induces CSE mRNA expressionThereby promoting endogenous H₂S_nIs generated. Therefore, the present invention adopts the method of incubating LPS and cells to induce the production of endogenous poly-hydrogen sulfide. MCF-7 cells were treated with LPS and incubated for 16h before probe incubation, and MCF-7 cells showed bright red fluorescence as seen in A of FIG. 18. Cells were incubated with DL-propargylglycine (PAG, CSE enzyme inhibitor) for 30min, with LPS for 16h, and with the probe, with a significant decrease in fluorescence intensity, with only very weak fluorescence present (B in fig. 18). As is clear from FIG. 19, the cell morphology was good. The above experiment shows that the probe AIE-PS2 can carry out cell endogenous H₂S_nAnd (4) fluorescence imaging.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A poly hydrogen sulfide fluorescent probe, the structural formula of which is as follows:

2. a method for preparing a hydrogen sulfide polysulfide fluorescent probe of claim 1, characterized in that it comprises the following steps:

3. the method for preparing a hydrogen sulfide fluorescent probe according to claim 2, comprising the steps of:

4. The method for preparing the hydrogen sulfide fluorescent probe as claimed in claim 3, wherein in the first step, the molar ratio of the compound TPE-OH to the compound hexamethylenetetramine is 1: 5-15.

5. The method for preparing a poly-hydrogen sulfide fluorescent probe as claimed in claim 4, wherein in the first step, the molar ratio of the compound TPE-OH to the compound hexamethylenetetramine is 1: 10.

6. The method for preparing the hydrogen sulfide fluorescent probe as claimed in claim 3, wherein in the first step, the mass-to-volume ratio of the compound TPE-OH to the trifluoroacetic acid is 1: 50-70 g/mL.

7. The method for preparing the poly-hydrogen sulfide fluorescent probe as claimed in claim 6, wherein in the first step, the mass-to-volume ratio of the compound TPE-OH and the trifluoroacetic acid is 1:60 g/mL.

8. The method for preparing a hydrogen sulfide fluorescent probe as claimed in claim 3, wherein in the second step, the molar ratio of the compound TPE-CHO to the compound 1 is 1: 2.5-4.5.

9. The method for preparing a poly-hydrogen sulfide fluorescent probe as claimed in claim 8, wherein the molar ratio of the compound TPE-CHO and the compound 1 in the second step is 1: 3.5.

10. The method for preparing a hydrogen sulfide fluorescent probe according to claim 3, wherein in the third step, the molar ratio of the compound TPE-HBT to the compound 2 is 1: 1.5-4.5.

11. The method for preparing a hydrogen sulfide fluorescent probe according to claim 10, wherein in the third step, the molar ratio of the compound TPE-HBT to the compound 2 is 1: 2.5.

12. The method for preparing the hydrogen sulfide fluorescent probe as claimed in claim 3, wherein in the third step, the molar ratio of the compound TPE-HBT to the triethylamine is 1: 20-30.

13. The method for preparing a hydrogen sulfide fluorescent probe as claimed in claim 12, wherein the molar ratio of the compounds TPE-HBT and triethylamine in the third step is 1: 24.4.

14. Use of the hydrogen sulfide polysulfide fluorescent probe of claim 1 as a reagent for detecting hydrogen sulfide.

15. Use according to claim 14, characterized in that: the application of the poly-hydrogen sulfide fluorescent probe in preparing a reagent for detecting poly-hydrogen sulfide in a cell level.