A high signal-to-noise ratio fluorescent probe for detecting changes in gastric viscosity and its application

Abstract

This invention belongs to the technical field of organic small molecule fluorescent probes and provides a fluorescent probe for viscosity detection. In high-viscosity solutions, the probe exhibits strong fluorescence in the near-infrared region, with high sensitivity, a large Stokes shift, and a high signal-to-noise ratio. This probe can be used not only to detect viscosity changes within mitochondria in living cells but also to measure viscosity in situ gastritis, demonstrating significant advantages in biological analysis and detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule fluorescent probes, and in particular relates to a high signal-to-noise ratio fluorescent probe for detecting changes in gastric viscosity and an application thereof. Background Art

[0002] A suitable microenvironment is crucial for maintaining normal life activities in biological systems. Viscosity, as an important microenvironmental parameter, plays a vital role in many life activities, such as the transport and diffusion of substances, the interaction between biomolecules, and the transduction of chemical signals. Abnormal changes in viscosity in organisms are considered to be important contributing factors or detection indicators of many diseases and physiological dysfunctions, such as diabetes, inflammation, and cancer. Therefore, developing an effective method to visualize viscosity changes in organisms is of great significance for the study and diagnosis of viscosity-related diseases.

[0003] Conventional viscometers (such as rotational viscometers, falling ball viscometers, and capillary viscometers) can only measure the viscosity of macroscopic fluids and are not suitable for monitoring viscosity changes in vivo. In recent years, fluorescence imaging has become an important analytical tool for detecting pathophysiological microenvironments and active molecules due to its unique advantages, including simplicity, non-invasiveness, high spatial and temporal resolution, and sensitivity. In particular, near-infrared fluorescence imaging has attracted great interest in biological sciences due to its inherent properties, including deep tissue penetration, low optical damage, and large signal-to-noise ratio.

[0004] Gastritis is the most common digestive disease and one of the inflammatory responses of organisms, affecting millions of people worldwide. Previous reports have shown that inflammation is often accompanied by an increase in the viscosity of organisms. However, due to the lack of suitable tools, the relationship between gastritis and viscosity has never been confirmed. Therefore, there is an urgent need to develop a fluorescent probe that can detect viscosity changes in gastritis. However, most of the reported viscosity fluorescent probes have some disadvantages: (1) the emission wavelength is short, which cannot effectively avoid background interference and achieve deeper tissue penetration; (2) the Stokes shift is small, which will cause serious self-quenching during imaging; (3) the signal-to-noise ratio is low, which will reduce the sensitivity of the probe to imaging viscosity changes in vivo. To solve this problem, there is an urgent need to develop a near-infrared fluorescent probe with a longer emission wavelength, a larger Stokes shift, and a higher signal-to-noise ratio for in situ imaging of viscosity changes in gastritis. Summary of the Invention

[0005] In order to solve the problems of existing probes being subject to large background interference, self-quenching and low sensitivity, the present invention provides a fluorescent probe for detecting viscosity, which can be used not only for detecting living cells but also for detecting viscosity of in situ gastritis.

[0006] Another object of the present invention is to provide an application of the above fluorescent probe in detecting the viscosity of biological cells and tissues.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] A fluorescent probe for detecting viscosity, whose chemical structure is shown in formula (I):

[0009]

[0010] The preparation method of the fluorescent probe comprises the following steps:

[0011] (1) 6-bromo-2-naphthol, Na2S2O5 and dimethylamine were heated to react, cooled to room temperature, filtered for separation, dried and purified to obtain intermediate 1:

[0012] (2) Intermediate 1, triphenylphosphine, K2CO3 and palladium acetate were dissolved in N-methylpyrrolidone and stirred at room temperature under a nitrogen atmosphere. 4-vinylpyridine was then added dropwise. After heating for reaction, water was added to the reaction solution. The solution was separated by suction filtration, dried and purified to obtain Intermediate 2:

[0013] (3) Dissolve the intermediate 2 and 1,4-dichlorobenzyl in toluene, heat under reflux under nitrogen protection, and purify to obtain the product: Abbreviated as BDB-1.

[0014] The preparation reaction formula of the above probe is as follows:

[0015]

[0016] A use of the fluorescent probe in preparing a reagent for detecting the viscosity of a solution, cell, organ or organism.

[0017] The mechanism of the present invention is as follows:

[0018]

[0019] The fluorescent probe for detecting viscosity changes described in this invention emits a fluorescence signal that changes from weak to strong under viscous conditions and exhibits near-infrared molecular characteristics. This phenomenon demonstrates that the probe responds to viscosity and can be used for imaging deep tissues. This phenomenon provides a reliable theoretical foundation for bioimaging applications. It is expected that this invention will play an important role in viscosity detection.

[0020] The present invention has the following advantages:

[0021] In this study, a novel near-infrared fluorescent probe with a large Stokes shift and high signal-to-noise ratio was constructed using naphthalene as a fluorescent platform, N,N-dimethyl as an electron-donating group, and pyridinium salt as an electron-withdrawing group. This probe was used to image viscosity changes in gastritis in situ. In high-viscosity solutions, the probe exhibited strong fluorescence in the near-infrared region, demonstrating high sensitivity, a large Stokes shift, and a high signal-to-noise ratio. This probe can be used not only to monitor viscosity changes within mitochondria in living cells but also to monitor viscosity in situ gastritis, demonstrating significant advantages in biological analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Here is the NMR characterization of the probe: 1 H NMR spectra and 13 C NMR spectrum;

[0023] Figure 2 This is the absorption spectrum of the probe in methanol and glycerol; probe concentration: 10 μM;

[0024] Figure 3 This is the fluorescence image of the probe detecting solutions with different viscosities: the excitation wavelength is 490nm; the probe concentration is 10μM;

[0025] Figure 4 is the fluorescence spectrum of the probe in different solvents;

[0026] Figure 5 The probe is selective in phosphate buffer; the excitation wavelength is 490 nm; the probe concentration is 10 μM;

[0027] Figure 6 This is the fluorescence intensity graph of the probe at different pH values; the excitation wavelength is 490 nm; the probe concentration is 10 μM;

[0028] Figure 7 It is a test of the toxicity of different concentrations of probes to cells;

[0029] Figure 8 This is the localization map of the probe in the cell. Excitation wavelength: 488nm, emission band: 500-550nm and 650-750nm;

[0030] Figure 9 This is an imaging diagram of the probe detecting viscosity changes in cells; excitation wavelength: 488nm, emission band: 500-550nm and 650-750nm;

[0031] Figure 10 This is an imaging diagram of viscosity changes in gastritis detected by the probe; excitation wavelength: 500nm, emission wavelength: 710nm. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0033] Example 1 Synthesis of fluorescent probe

[0034] (1) Synthesis of Intermediate 1

[0035]

[0036] A mixture of 6-bromo-2-naphthol (3.346 g, 15 mmol), 40% aqueous dimethylamine solution (8.452 g, 75 mmol), Na2S2O5 (5.703 g, 30 mmol) and 20 mL of water was stirred in an autoclave at 165°C for 6 hours. After completion of the reaction, the mixture was cooled to room temperature, filtered, dried, and purified by column chromatography using dichloromethane-methanol (30:1) as the eluent. The solvent was removed from the effluent to obtain intermediate 1 with a yield of 44%.

[0037] (2) Synthesis of Intermediate 2

[0038]

[0039] Intermediate 1 (300 mg, 1.2 mmol), triphenylphosphine (95 mg, 0.36 mmol), K2CO3 (1.327 g, 9.6 mmol) and palladium acetate (18 mg, 0.08 mmol) were dissolved in 5 mL of N-methylpyrrolidone. After stirring at room temperature for 1 hour under nitrogen protection, 4-vinylpyridine (1 mL, 9.6 mmol) was added dropwise. The mixture was then reacted at 165°C under nitrogen protection for 48 hours. After completion of the reaction, an appropriate amount of water was added to the reaction solution, which was filtered and dried, and then purified by column chromatography using dichloromethane-methanol (20:1) as the eluent to obtain intermediate 2 with a yield of 67.8%.

[0040] (3) Synthesis of BDB-1

[0041]

[0042] Intermediate 2 (27 mg, 0.1 mmol) and 1,4-dichlorobenzyl (156 mg, 0.9 mmol) were dissolved in 3 mL of toluene and refluxed at 110°C under nitrogen for 48 hours. After completion of the reaction, the product was purified by thin-layer chromatography using dichloromethane-methanol (15:1) as the developing solvent to obtain the fluorescent probe BDB-1 with a yield of 57.9%.

[0043] Fluorescent probe BDB-1 1 H NMR spectra and 13 C NMR spectrum Figure 1(a) and (b) show:

[0044] 1 H NMR (400MHz, DMSO) δ9.05 (s, 2H), 8.23 (d, J = 5.8Hz, 2H), 8.13 (d, J = 16.1Hz, 1H), 8.01 (s, 1H), 7.86-7.6 8(m,3H),7.62-7.41(m,5H),7.27(d,J=9.2Hz,1H),6.98(s,1H),5.77(s,2H),4.79(s,2H),3.07(s,6H)

[0045] 13C NMR(101MHz,DMSO)δ154.19(s),150.07(s),144.53(s),142.74(s),139.13(s),136.47(s),135.33(s),130.96(s),130.64-130.46(m),130.13 (d, J=12.5Hz),129.45(s),128.87(s),127.24(s),125.93(s),124.46( s),124.04(s),121.17(s),116.95(s),105.88(s),61.90(s),45.99(s).

[0046] Example 2 Absorption spectra of fluorescent probes in solutions with different viscosities

[0047] Prepare a 4 mL dimethyl sulfoxide (DMSO) solution of the fluorescent probe BDB-1 prepared in Example 1 with a 1 mM stock solution. Take 20 μL of the probe stock solution and add 2 mL of methanol or glycerol, invert the solution to mix, and perform absorption spectrum measurement.

[0048] The results are as follows Figure 2 As shown: It can be seen from the figure that the maximum absorption peak of the probe in methanol solution is concentrated at 486nm; while in glycerol solution, the maximum absorption peak shifts from 486nm to 492nm; this indicates that in a viscosity environment, the rotation of the probe molecule is inhibited, resulting in increased conjugation.

[0049] Example 3 Fluorescence spectra of fluorescent probes in solutions with different viscosities

[0050] A 4 mL dimethyl sulfoxide (DMSO) solution of the fluorescent probe BDB-1 prepared in Example 1 was prepared with a 1 mM stock solution. The experimental solutions included 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% water-glycerol systems, with methanol as a control. 20 μL of the probe stock solution was added to 2 mL of water-glycerol systems at different ratios, and the solutions were mixed by inverting them. Fluorescence spectra were then measured (λ ex =490nm).

[0051] The results are as follows Figure 3 As shown in the figure: Under 490nm excitation, the probe BDB-1 shows almost no fluorescence emission in methanol solution. In contrast, the fluorescence intensity of BDB-1 at 706nm gradually increases with the increase of glycerol ratio, indicating that the probe can be used for viscosity detection.

[0052] Example 4 Fluorescence spectra of fluorescent probes in different solutions

[0053] Prepare a 4 mL dimethyl sulfoxide (DMSO) solution of the fluorescent probe BDB-1 prepared in Example 1 with a 1 mM stock solution. The experimental solution contains dimethylformamide (DMF), DMSO, phosphate buffered saline (PBS), acetone (BLE), methanol (MeOH), water, ethanol (EtOH), acetonitrile (ACN), and glycerol (Gly). Take 20 μL of the probe stock solution and add it to 2 mL of each solvent system. Invert the solution to mix thoroughly and perform fluorescence spectroscopy (λ ex =490nm).

[0054] The results are as follows Figure 4 As shown: The fluorescence signal of the probe is weak in other solvents, and the fluorescence intensity is significantly enhanced only in a viscosity environment, indicating that the probe can recognize viscosity without being affected by other solvents.

[0055] Example 5 Specificity of fluorescent probe

[0056] Prepare a 4 mL dimethyl sulfoxide (DMSO) solution of the fluorescent probe BDB-1 prepared in Example 1 with a stock solution concentration of 1 mM. Prepare 5 mL solutions of different compounds with a concentration of 40 mM for later use. Add 25 μL of the probe stock solution, 225 μL of DMSO, and the corresponding concentrations of various compound solutions to a 5 mL volumetric flask, dilute to volume with phosphate buffer, shake well, and perform fluorescence detection (λ ex =490nm,λ em =706 nm), and a histogram of fluorescence intensity and each ion was established.

[0057] The results are as follows Figure 5 As shown: 1-45 represent ClO -(200 μM), ONOO - (200μM),Na + (1mM),blank,H2O2(200μM),S 2- (500μM),F - (500μM),PO4 3- (500 μM), Cl - (500μM),HSO3 - (500μM), HS - (500μM), Ba 2+ (1mM),SO3 2- (500μM), N3 - (500μM), NO2 - (500 μM), Mg 2+ (1mM), SCN - (500μM), hemin (500μM), K + (1mM), NO3 - (500μM), di-tert-butyl peroxide (200μM), tert-butyl hydroperoxide (200μM), CH3COO - (500μM), arginine (5mM), cysteine (5mM), aspartic acid (5mM), glutathione (5mM), glutamine (5mM), adenosine triphosphate (1mM), glycine (5mM), glucose (1mM), ascorbic acid (1mM), Fe 3+ (1mM), serine (5mM), tryptophan (5mM), Zn 2+ (1mM),O2 - (200 μM), Cu 2+ (1mM),Fe 2+ (1 mM), trypsin (1 mg / mL), Ca 2+ (1mM), RNA (1mg / mL), chymotrypsin (1mg / mL), DNA (1mg / mL), glycerol. Figure 5 It can be found that other substances have almost no effect on the fluorescence of compound BDB-1, indicating that the probe can recognize viscosity without being affected by other molecules.

[0058] Example 6 Fluorescence intensity of fluorescent probe in solutions with different pH values

[0059] Prepare a 4 mL dimethyl sulfoxide (DMSO) solution of the fluorescent probe BDB-1 prepared in Example 1 with a 1 mM stock solution. The experiment included buffer solutions with pH values of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Take 20 μL of the probe stock solution and add it to 2 mL of different pH buffers or glycerol. Mix the solutions by inverting them and perform fluorescence spectroscopy (λ ex =490nm).

[0060] The results are as follows Figure 6 As shown: the fluorescence signal of the probe changes weakly at different pH levels, and the fluorescence intensity is significantly enhanced only in a viscosity environment, indicating that the probe can recognize viscosity without being affected by pH.

[0061] Example 7 Toxicity of fluorescent probe to cells

[0062] The density is 3×10 4 HeLa cells were seeded at a concentration of 100 μg / mL into a sterile 96-well plate and cultured in a CO2 incubator (37°C, 5% CO2). After cell attachment, the plates were incubated with different concentrations of BDB-1 probe (0-15 μM) for 12 hours. 10 μL of MTT (5 mg / mL) was then added to each well. After an additional 4 hours of incubation, the supernatant was aspirated, 100 μL of DMSO was added, the plates were shaken for approximately 10 minutes, and the absorbance was measured at 490 nm.

[0063] The results are as follows Figure 7 As shown: after incubation with different concentrations of probes (0-15 μM) for 12 hours, more than 80% of the cells can still survive, indicating that the probe has low cytotoxicity and can be used for testing in biological systems.

[0064] Example 8 Imaging Application of Fluorescent Probes in Living Cells

[0065] The density is 3×10 5 HeLa cells were seeded at a concentration of 100 μg / mL into a sterilized 35 mm culture dish and cultured in a CO2 incubator (37°C, 5% CO2). After the cells attached, the fluorescent probe BDB-1 (final concentration: 5 μM) and the commercial mitochondrial dye Mito-Tracker (1 μM) were added to the dish. The cells were cultured in the incubator for 0.5 hours, after which the cell culture medium was aspirated. The cells were rinsed three times with PBS buffer before fluorescence imaging (excitation wavelength: 488 nm, emission wavelengths: 500-550 nm and 650-750 nm).

[0066] The results are as follows Figure 8 As shown: The fluorescence overlap between the probe BDB-1 and the mitochondrial dye is high, indicating that the probe is mainly localized in the mitochondria.

[0067] Example 9 Imaging Application of Fluorescent Probes in Living Cells

[0068] The density is 3×10 5 HeLa cells were inoculated at a concentration of 1 μg / mL into a sterilized 35 mm culture dish and cultured in a CO2 incubator (37°C, 5% CO2). After the cells attached, monensin (final concentration of 10 μM) or nystatin (final concentration of 10 μM), which can stimulate changes in intracellular viscosity, was added to the dish and incubated in the incubator for 40 minutes. The fluorescent probe BDB-1 (final concentration of 5 μM) was then added and cultured in the incubator for a further 30 minutes. The cell culture medium was then aspirated, and the cells were rinsed three times with PBS buffer before fluorescence imaging (excitation wavelength: 488 nm, emission bands: 500-550 nm and 650-750 nm).

[0069] The results are as follows Figure 9 As shown: the fluorescence signal of cells stimulated by monensin or nystatin is significantly stronger than the fluorescence of the probe itself, indicating that the fluorescent probe BDB-1 can be used to detect changes in intracellular viscosity.

[0070] Example 10 Imaging Application of Fluorescent Probes in Organisms

[0071] Two groups of mice were prepared and fasted for 18 hours. The experimental group mice were then gavaged with 50 μL of 60% (v / v) ethanol, and an acute gastritis model was induced 4 hours later. The control group mice were orally administered with 50 μL of normal saline. Prior to in vivo imaging, each mouse was orally administered with 100 μL of a 500 μM solution of the fluorescent probe BDB-1 in normal saline. The mice were then anesthetized with 4% chloral hydrate solution and imaged using an in vivo imaging system (excitation at 500 nm, signal acquisition at 710 nm).

[0072] The results are as follows Figure 10 As shown: the fluorescence signal of the probe in gastritis mice is significantly stronger than that in normal mice, indicating that the viscosity of gastritis mice is higher than that of normal mice. The probe can be used to detect viscosity changes in mice.

Claims

1. A fluorescent probe for detecting viscosity, characterized in that: Its chemical structure is shown in formula (I):

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) 6-bromo-2-naphthol, Na2S2O5 and dimethylamine were heated and reacted, and then separated and purified to obtain intermediate 1: (2) Intermediate 1, triphenylphosphine, K2CO3 and palladium acetate were dissolved in N-methylpyrrolidone and stirred at room temperature under a nitrogen atmosphere. 4-vinylpyridine was then added dropwise. After heating for reaction, water was added to the reaction solution. The reaction solution was separated and purified to obtain Intermediate 2: (3) Dissolve the intermediate 2 and 1,4-dichlorobenzyl in toluene, heat under reflux under nitrogen protection, and purify to obtain the product:

3. Use of the fluorescent probe according to claim 1 in preparing a reagent for detecting the viscosity of a solution, cell, organ or organism.

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