A fluorescent probe for detecting viscosity and its preparation method and application

Abstract

The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for detecting viscosity, and its preparation method and application. The fluorescent probe is JD-V, which is based on twisted intramolecular charge transfer. It uses a thiazole hemicyanine structure as a fluorophore and a carbon-carbon double bond as a specific response group. In low-viscosity solvents, the free rotation of the molecular single bond causes energy to be dissipated in a non-radiative form, resulting in a low fluorescence intensity. However, as the viscosity of the solvent increases, the molecular rotation is restricted, and energy is released more in the form of fluorescence, thereby significantly enhancing the fluorescence intensity. The preparation method of the fluorescent probe JD-V is simple, and the kit is easy to operate and cheap; it has high sensitivity, strong anti-interference ability, and a high multiple of viscosity fluorescence intensity increase; JD-V is applied to drug-induced cell viscosity changes and real-time monitoring, and can be used to detect viscosity changes caused by food thickeners, providing a tool for food detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for detecting viscosity, a preparation method thereof, and an application thereof. Background Art

[0002] Cell microenvironment viscosity refers to the fluid resistance properties of the environment surrounding cells, which has a significant impact on cellular physiological functions and behaviors. Changes in cell microenvironment viscosity are closely related to various physiological and pathological processes, such as cell division, cell migration, mechanotransduction, and intracellular trafficking. Furthermore, alterations in cell microenvironment viscosity are associated with the development and progression of diseases such as cancer and neurodegenerative diseases. Abnormal changes in cell microenvironment viscosity are closely linked to the development and progression of various diseases. In cancer, the viscosity of the tumor cell microenvironment often changes, affecting the invasion and metastasis of tumor cells. For example, increased viscosity of the matrix surrounding tumor cells may promote tumor cell invasion. Changes in cell microenvironment viscosity also play a significant role in neurodegenerative diseases. When the microenvironment changes, organelle function becomes abnormal, potentially leading to cancer, neurodegeneration, autoimmune diseases, myocardial damage, and other diseases. Viscosity, as a fundamental parameter of the cell microenvironment, is closely related to many life processes, such as metabolism and signal transduction. Therefore, quantifying the viscosity of living cells is of great significance.

[0003] Conventional viscosity measurement instruments are unable to monitor microenvironmental viscosity within living cells because they are designed for macroscopic fluids, such as falling ball viscometers, capillary viscometers, and rotational viscometers, and are not applicable to cellular-scale viscosity measurements. There is an urgent need to develop new technologies that can monitor viscosity within cellular microenvironments. Fluorescent probes have proven to be effective methods for monitoring microviscosity, allowing for real-time, in situ, and non-invasive detection of changes in viscosity within living cells. In recent years, the combination of fluorescent probes and bioimaging techniques has been widely applied to monitor the intracellular microenvironment and various small molecules, providing new insights into the development and progression of related diseases in vivo and opening up new approaches for early clinical diagnosis and treatment of tumor-related diseases. The ability of probes to detect viscosity is due to their rotatable bonds within the molecule. Solution viscosity affects the rotation of the probe bonds, leading to changes in the molecular structure and, consequently, the fluorescence of the probe. In low-viscosity solutions, the probe molecules can rotate freely, allowing the excited molecules to return to the ground state via nonradiative transitions. However, in high-viscosity systems, bond rotation is hindered, and nonradiative transition modes are restricted, resulting in enhanced fluorescence. Therefore, these freely rotatable bonds can be used to monitor changes in viscosity within solutions and living cells.

[0004] Among the probes reported for viscosity measurement, most fluorescent probes exhibit fluorescence only in the visible region, but this response is slow and susceptible to interference. Furthermore, most fluorescent probes exhibit only fluorescence without any noticeable color change, significantly limiting their application. Therefore, developing a fluorescent probe for viscosity monitoring with excellent selectivity, high sensitivity, ease of operation, strong anti-interference ability, visualization, and widespread applicability is of great significance for measuring viscosity changes at the cellular level and for detecting viscosity in food additives. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide a fluorescent probe for detecting viscosity, a preparation method thereof, and an application thereof. The probe is based on twisted intramolecular charge transfer (TICT), with a thiazole hemicyanine structure as a fluorophore and a carbon-carbon double bond as a specific response group. In low-viscosity solvents, the free rotation of the molecular single bond causes energy to be dissipated in a non-radiative form, resulting in a low fluorescence intensity. However, as the solvent viscosity increases, the molecular rotation is restricted, and more energy is released in the form of fluorescence, thereby significantly enhancing the fluorescence intensity.

[0006] The present invention provides a fluorescent probe for detecting viscosity, the structural formula of the fluorescent probe is:

[0007] .

[0008] The present invention also provides a method for preparing the above-mentioned fluorescent probe for detecting viscosity, comprising the following steps:

[0009] S1, 6-hydroxy-2-naphthaldehyde was dissolved in trifluoroacetic acid, followed by addition of hexamethylenetetramine, and the mixture was heated under reflux and then cooled to room temperature, H2SO4 was added, and reflux stirring was continued. After the reaction was completed, the mixture was cooled, and the mixture was successively extracted, dried and evaporated under reduced pressure to obtain a crude product, which was purified by a first column chromatography to obtain an intermediate product;

[0010] S2. Dissolve 3-ethyl-2-methylbenzothiazol-3-ium iodide and the intermediate product in anhydrous ethanol, then add piperidine dropwise, and stir to react at room temperature. After the reaction is completed, collect the filtrate by suction filtration and washing, and evaporate under reduced pressure to obtain a crude product, which is purified by a second column chromatography to obtain a fluorescent probe.

[0011] According to the preparation method provided by the present invention, the molar volume ratio of 6-hydroxy-2-naphthaldehyde, trifluoroacetic acid, hexamethylenetetramine and H2SO4 in S1 is 0.5-1.5 mmol: 5-15 mL: 1-3 mmol: 5-15 mL, and the concentration of H2SO4 is 33-98%.

[0012] According to the preparation method provided by the present invention, the temperature of the heating reflux reaction in S1 is 75° C., the time of the heating and stirring reaction is 2 to 4 h, and the time of the reflux stirring is 2 to 4 h.

[0013] According to the preparation method provided by the present invention, the extraction agent in S1 is ethyl acetate, the number of extractions is 3 to 5 times, the drying desiccant is anhydrous Na2SO4, and the first column chromatography purification uses ethyl acetate / n-hexane as the eluent, and the elution ratio is 2 to 3:5 to 7.

[0014] According to the preparation method provided by the present invention, the molar volume ratio of 3-ethyl-2-methylbenzothiazol-3-ium iodide, the intermediate product, anhydrous ethanol and piperidine in S2 is 0.75-1 mmol: 0.50-1.5 mmol: 4-8 mL: 0.05-0.15 mL.

[0015] According to the preparation method provided by the present invention, the stirring reaction time in S2 is 30 to 60 minutes, the washing detergent is anhydrous ethanol, and the second column chromatography purification uses dichloromethane / methanol as the eluent, and the elution ratio is 8 to 10:1 to 2.

[0016] The present invention also provides an application of the above-mentioned fluorescent probe for detecting viscosity, wherein the fluorescent probe is used to detect viscosity changes caused by food thickeners and monitor them in real time.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention provides a fluorescent probe for detecting viscosity, a preparation method, and an application thereof. The fluorescent probe is JD-V, which is based on twisted intramolecular charge transfer (TICT). It uses a thiazole hemicyanine structure as a fluorophore and a carbon-carbon double bond as a specific response group. In low-viscosity solvents, the free rotation of molecular single bonds causes energy to be dissipated in a non-radiative form, resulting in a low fluorescence intensity. However, as the solvent viscosity increases, molecular rotation is restricted, and more energy is released in the form of fluorescence, thereby significantly enhancing the fluorescence intensity.

[0019] The fluorescent probe JD-V is simple to prepare, and the kit is easy to operate and inexpensive. It has high sensitivity, strong anti-interference ability, and a high fluorescence intensity increase multiple with viscosity. JD-V is used to monitor drug-induced cell viscosity changes and real-time monitoring. It can be used to detect viscosity changes caused by food thickeners, providing a tool for food and environmental testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 is the H NMR spectrum of probe JD-V;

[0022] Figure 2 is the mass spectrum of probe JD-V;

[0023] Figure 3 The UV and fluorescence spectra of probe JD-V in methanol and probe JD-V in methanol: 1% glycerol: 99% thiophenol are shown;

[0024] Figure 4 The following are the UV absorption of probe JD-V in different solvents, the fluorescence histogram at 550 nm under 450 nm excitation, and the photos under fluorescent light and UV light in different solvents;

[0025] Figure 5 (a) Fluorescence response of probe JD-V to different viscosities (0.59-945 cP) in MeOH-Gly systems with different viscosity ratios; (b) Linear fit of JD-V to fluorescence intensity at 555 nm with different viscosities (0.59-945 cP); (c) UV absorption spectra of probe JD-V (25 μmol / L) at different viscosities (Gly ratio 0, 20, 40, 60, 99%); (d) Photos of the probe under fluorescent light and UV light at different viscosities (Gly ratio 0, 20, 40, 60, 80, 99%).

[0026] Figure 6 This is the anti-interference ability test chart of probe JD-V; the concentration of probe JD-V is 25 μM, and common cations (Al 3+ 、Cu 2+ 、Fe 3+ 、Mn 2+ Mg 2+ 、Cd 2+ , K + 、Na + Cr 3+ 、Ni 2+ , Ca 2+ NH4 + ), common anions (NO3 - 、Cl - 、HCO3 - Br - 、CH3COO- 、SO3 2- 、S2O3 2- 、HSO3 - ), common amino acids: cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH) and human serum albumin (HSA); the concentration of HSA was 10 mg / mL, and the concentrations of the other analytes were all 500 μmol / L;

[0027] Figure 7 (a) is the fluorescence intensity of probe JD-V in different concentrations of xanthan gum; (b) is the absorbance of probe JD-V in different concentrations of xanthan gum; (c) is a photograph of probe JD-V in different concentrations of xanthan gum under a fluorescent light; (d) is the fluorescence intensity of probe JD-V in different concentrations of pectin; (e) is the absorbance of probe JD-V in different concentrations of pectin; (f) is a photograph of probe JD-V in different concentrations of pectin under a fluorescent light;

[0028] Figure 8 The probe JD-V was used for fluorescence imaging of A549 cell viscosity induced and treated with cell drugs; the JD-V group was the control group (50 μmol / L probe incubated for 30 min); LPS (8 μg / mL, 3 h) induced viscosity changes in A549 cells, followed by incubation with JD-V (50 μmol / L, 30 min); APAP (500 μmol / L, 12 h) induced viscosity changes in A549 cells, followed by incubation with JD-V (50 μmol / L, 30 min); APAP+NAC: A549 cells were incubated with APAP (500 μmol / L, 12 h), NAC (1 mmol, 30 min), and JD-V (50 μmol / L, 30 min);

[0029] Figure 9 The JD-V probe was used for fluorescence imaging of A549 cells induced by different concentrations of LPS: A549 cells were incubated with 0, 2, 4, 6, 8, and 10 μg / mL LPS for 3 h, and then incubated with 50 μmol / L of the probe for 30 min;

[0030] Figure 10 It is a real-time monitoring of LPS-induced A549 cells by probe JD-V. DETAILED DESCRIPTION

[0031] Example 1

[0032] This embodiment provides a fluorescent probe for detecting viscosity and a preparation method thereof, comprising the following steps:

[0033] S1. In a 50 mL round-bottom flask, dissolve 1 mmol of 6-hydroxy-2-naphthaldehyde in 5 mL of trifluoroacetic acid (TFA), then add 2 mmol of hexamethylenetetramine and heat under reflux at 75 °C for 2 h.

[0034] The mixture was cooled to room temperature with cold water at a temperature of 0-4°C, and 7 mL of 33% H2SO4 was added. Reflux and stirring were continued for 2 h. Thin-layer chromatography (TLC) was used for real-time spot plate monitoring. After the reaction was completed, cold water was poured into the mixture, and the mixture was extracted three times with ethyl acetate. The organic layer was separated, dried over anhydrous Na2SO4, and evaporated under reduced pressure to remove the solvent to obtain a crude product. The crude product was purified by column chromatography using ethyl acetate / n-hexane as the eluent in a ratio of 3:7 to obtain a white solid intermediate JD-1.

[0035] ;

[0036] S2. In a 25 mL round-bottom flask, 0.75 mmol of 3-ethyl-2-methylbenzothiazol-3-ium iodide and 0.50 mmol of JD-1 were dissolved in 4 mL of anhydrous ethanol. 0.1 mL of piperidine was then added dropwise as a catalyst and stirred at room temperature for 30 min.

[0037] The reaction was monitored by TLC real-time spot plate. After completion of the reaction, the reaction was filtered, the filter residue was washed with anhydrous ethanol, the filtrate was collected, and the solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was purified by a second column chromatography using dichloromethane / methanol as eluent at an elution ratio of 10:1 to obtain the purple solid fluorescent probe JD-V.

[0038] .

[0039] The reaction equations for the steps provided in this embodiment are:

[0040] .

[0041] The structural characterization data of the fluorescent probe JD-V are as follows: Figure 1 As shown:

[0042] 1H NMR (600 MHz, Methylene Chloride: Methanol=2:1-d2), δ (ppm): 11.94(s, 1H), 10.77 (d, J = 13.6 Hz, 1H), 10.59 (d, J = 13.3 Hz, 1H), 10.08 (d, J= 8.8 Hz, 1H), 10.00 (s, 1H), 9.86 (dd, J = 25.08, 8.8 Hz, 2H), 9.68 (d, J =9.3 Hz, 1H), 9.60 (d, J = 12.2 Hz, 2H), 9.48 (d, J = 7.7 Hz, 1H), 8.81 (d, J= 9.2 Hz, 1H), 6.47 (q, J = 7.3 Hz, 2H), 3.55 (t, J = 7.5 Hz, 3H).

[0043] The mass spectrometry monitoring results of probe JD-V are as follows Figure 2 As shown;

[0044] The JD-V probe structure is shown above. Its principle is based on twisted intramolecular charge transfer (TICT), with a thiazole hemicyanine structure as the fluorophore and a carbon-carbon double bond as the specific response group. In low-viscosity solvents, the free rotation of the molecular single bonds results in non-radiative energy dissipation, resulting in low fluorescence intensity. However, as the solvent viscosity increases, molecular rotation becomes restricted, and more energy is released as fluorescence, resulting in a significant increase in fluorescence intensity.

[0045] Fluorescent probe JD-V and viscosity detection mechanism:

[0046] .

[0047] Example 2

[0048] This embodiment provides a fluorescent probe for detecting viscosity and a preparation method thereof, comprising the following steps:

[0049] S1. In a 50 mL round-bottom flask, dissolve 0.5 mmol of 6-hydroxy-2-naphthaldehyde in 10 mL of trifluoroacetic acid, then add 1 mmol of hexamethylenetetramine and heat under reflux at 75 °C for 3 h.

[0050] The mixture was cooled to room temperature, and 5 mL of 98% H2SO4 was added. Reflux stirring was continued for 3 h, and the mixture was monitored by TLC real-time spot plate. After the reaction was completed, cold water was poured into the mixture, and the mixture was extracted with ethyl acetate five times. The organic layer was separated, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using ethyl acetate / n-hexane as the eluent in a ratio of 2:5 to obtain a white solid intermediate product JD-1.

[0051] S2. In a 25 mL round-bottom flask, 0.9 mmol of 3-ethyl-2-methylbenzothiazol-3-ium iodide and 1 mmol of JD-1 were dissolved in 6 mL of anhydrous ethanol, followed by dropwise addition of 0.05 mL of piperidine as a catalyst, and stirred at room temperature for 45 min.

[0052] The reaction was monitored by TLC real-time spot plate. After the reaction was completed, the reaction was filtered, the filter residue was washed with anhydrous ethanol, the filtrate was collected, and the solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography for the second time using dichloromethane / methanol as eluent at an elution ratio of 8:2 to obtain the purple solid fluorescent probe JD-V.

[0053] Example 3

[0054] This embodiment provides a fluorescent probe for detecting viscosity and a preparation method thereof, comprising the following steps:

[0055] S1. In a 50 mL round-bottom flask, dissolve 1.5 mmol of 6-hydroxy-2-naphthaldehyde in 15 mL of trifluoroacetic acid, then add 3 mmol of hexamethylenetetramine and heat under reflux at 75 °C for 4 h.

[0056] The mixture was cooled to room temperature, and 15 mL of 98% H2SO4 was added. The mixture was refluxed and stirred for 4 h. The mixture was monitored by TLC real-time spot plate. After the reaction was completed, cold water was poured into the mixture, and the mixture was extracted with ethyl acetate five times. The organic layer was separated, dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using ethyl acetate / n-hexane as the eluent in a ratio of 2:6 to obtain a white solid intermediate product JD-1.

[0057] S2. In a 25 mL round-bottom flask, 1 mmol of 3-ethyl-2-methylbenzothiazol-3-ium iodide and 1.5 mmol of JD-1 were dissolved in 8 mL of anhydrous ethanol, followed by the dropwise addition of 0.15 mL of piperidine as a catalyst, and the mixture was stirred at room temperature for 60 min.

[0058] The reaction was monitored by TLC real-time spot plate. After completion of the reaction, the reaction was filtered, the filter residue was washed with anhydrous ethanol, the filtrate was collected, and the solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was purified by a second column chromatography using dichloromethane / methanol as eluent at an elution ratio of 9:1 to obtain the purple solid fluorescent probe JD-V.

[0059] Example 4

[0060] This embodiment provides a spectrum test of the probe JD-V prepared in Example 1.

[0061] The probe JD-V was prepared into a 1 mM stock solution using dimethyl sulfoxide, and mixed solutions of methanol and glycerol in different ratios were prepared.

[0062] A 25 μmol / L stock solution of the probe JD-V was added to a mixture of methanol (MeOH) and glycerol (Gly) in different proportions and shaken thoroughly. The solution was transferred to a micro-cuvette using an ultrasonic instrument to remove bubbles. The fluorescence spectrum and UV-visible absorption spectrum of the solution were measured.

[0063] The results of the test are as follows Figure 3 As shown in the figure, the probe JD-V solution appears purple in MeOH solution and has almost no fluorescence. The UV absorption intensity is strong at 565 nm and weak at 450 nm. When the MeOH:Gly ratio is 1:99, the solution appears light yellow and has strong fluorescence. The fluorescence intensity at 550 nm increases significantly, the UV absorption at 565 nm decreases significantly, and the UV absorption at 450 nm increases significantly, and the solution color changes from purple to light yellow.

[0064] Example 5

[0065] This example provides a study on the spectral performance of the probe JD-V prepared in Example 1 for viscosity.

[0066] In order to deeply evaluate the sensitivity of the fluorescent probe JD-V to viscosity and polarity, a variety of different solution systems were selected, including EtOH (ethanol), EtCN (acetonitrile), MeOH, PBS (pH=7.4), H2O, DMSO (dimethyl sulfoxide), EG (ethylene glycol), DCM (dichloromethane), DMF (N,N-dimethylformamide), EA (ethyl acrylate) and Gly. The fluorescence intensity at 520-570 nm was measured with an excitation wavelength of 430-470 nm, and its response ability was measured and analyzed.

[0067] The test results are as follows Figure 4 As shown in the UV-visible absorption spectrum, the absorption peak of JD-V in Gly solution is mainly concentrated at 450 nm, while in other solvents, its absorption peak is mainly distributed in the range of 550-580 nm; when excited at 450 nm, as shown in Figure 5 As shown, when the solvent was Gly, a stronger fluorescence signal was detected.

[0068] Example 6

[0069] This embodiment provides an anti-interference ability test of the probe JD-V prepared in Example 1.

[0070] 25 μM probe JD-V was mixed with common cations (Al 3+ 、Cu 2+ 、Fe 3+ 、Mn 2+ Mg 2+ 、Cd 2+ , K + 、Na + Cr 3 + 、Ni 2+ , Ca 2+ NH4 + ), common anions (NO3 - 、Cl - 、HCO3 - Br - 、CH3COO - 、SO3 2- 、S2O3 2- 、HSO3 2- ), common amino acids: cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), human serum albumin (HSA) and Gly.

[0071] The concentration of HSA was 10 mg / mL, and the concentrations of the other analytes were 500 μmol / L, and then the fluorescence emission spectra were measured.

[0072] The test results are as follows Figure 6 As shown, it can be seen that the fluorescence response of JD-V to high viscosity is significantly higher than that to other tested potential factors.

[0073] Example 7

[0074] This example provides a quantitative detection of viscosity using the probe JD-V prepared in Example 1.

[0075] First, a 25 μM probe solution was prepared and added with different proportions of methanol-glycerol solution (0.59 - 945 cP). The excitation wavelength was 430-470 nm, and the fluorescence intensity at 520-570 nm was measured and recorded as F. The viscosity was used as the horizontal axis and the fluorescence intensity value F was used as the vertical axis to draw a curve. The curve equation was y=81.66+4.86x, R 2=0.9950; then prepare a 25 μM probe solution and add it to the methanol-glycerol solution to be tested. Use 430-470 nm as the excitation wavelength and measure the fluorescence intensity at 520-570 nm, record it as F', and substitute it into the above curve equation to obtain the viscosity of the unknown solution.

[0076] Example 8

[0077] This embodiment provides a method for detecting the viscosity of a food thickener using the probe JD-V prepared in Example 1.

[0078] Deionized water was used to prepare stock solutions of xanthan gum and pectin at concentrations of 0, 1, 2, 3, 5, and 10 g / kg, respectively. 25 μM probe solution was added to the stock solutions of xanthan gum and pectin, and the bubbles were removed by ultrasonication. The fluorescence intensity at 520-570 nm was measured using an excitation wavelength of 430-470 nm. The test results are shown in Figure 2. Figure 7 As shown, the results showed that xanthan gum had a better thickening effect than pectin.

[0079] Example 9

[0080] This example provides an application of the probe JD-V prepared in Example 1 for cell imaging of viscosity.

[0081] A549 cells were plated in cell culture dishes and incubated in a confocal microplate reader at 37°C for 24 hours before imaging. Different drug inductions were used: the JD-V group was incubated with 50 μmol / L of the probe for 30 minutes; the LPS (lipopolysaccharide) group was incubated with 8 μg / mL of LPS for 3 hours, followed by incubation with 50 μmol / L of the probe for 30 minutes; the APAP (acetaminophen) group was incubated with 500 μmol / L of APAP for 12 hours, followed by incubation with 50 μmol / L of the probe for 30 minutes; and the APAP+NAC (acetylcysteine) group was incubated with 500 μmol / L of APAP for 12 hours, followed by incubation with 1 mmol / L of NAC for 30 minutes, followed by incubation with 50 μmol / L of the probe for 30 minutes.

[0082] LPS induction at different concentrations: Six dishes of cells were incubated with LPS (8 μg / mL) at concentrations of 0, 2, 4, 6, 8, and 10 μg / mL for 3 hours, followed by incubation with 50 μmol / L of the probe for 30 minutes. Real-time monitoring: Four dishes of cells were incubated with LPS for 0, 60, 120, and 180 minutes, followed by incubation with 50 μmol / L of the probe for 30 minutes.

[0083] All groups were rinsed with PBS buffer solution (pH 7.4) and subjected to fluorescence imaging using a confocal laser scanning microscope.

[0084] The excitation wavelength of JD-V is 488 nm, and the emission wavelength collection range is 500-630 nm.

[0085] The test results are as follows Figures 8-10 As shown, it can be seen that the probe JD-V can effectively monitor the changes in cell viscosity.

[0086] The fluorescent probe JD-V prepared by the present invention is based on twisted intramolecular charge transfer, with a thiazole hemicyanine structure as the fluorophore and a carbon-carbon double bond as the specific response group. In low-viscosity solvents, the free rotation of the molecular single bonds causes non-radiative energy dissipation, resulting in low fluorescence intensity. However, as the solvent viscosity increases, molecular rotation is restricted, and more energy is released as fluorescence, resulting in a significant increase in fluorescence intensity.

[0087] When the probe JD-V was excited at 450 nm, a strong fluorescence signal was detected when the solvent was Gly. The fluorescence response of JD-V to high viscosity was significantly higher than that to other potential factors tested. The curve equation was y=81.66+4.86x, R 2 =0.9950 can be used to quantitatively detect viscosity and the thickening effect of food thickeners. It can also effectively monitor changes in cell viscosity through fluorescence imaging.

[0088] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A fluorescent probe for detecting viscosity, characterized in that: The structural formula of the fluorescent probe is: 。 2. A method for preparing a fluorescent probe for detecting viscosity as claimed in claim 1, characterized in that: The following steps are involved: S1, 6-hydroxy-2-naphthaldehyde was dissolved in trifluoroacetic acid, followed by addition of hexamethylenetetramine, and the mixture was heated under reflux and then cooled to room temperature, H2SO4 was added, and reflux stirring was continued. After the reaction was completed, the mixture was cooled, and the mixture was successively extracted, dried and evaporated under reduced pressure to obtain a crude product, which was purified by a first column chromatography to obtain an intermediate product; S2. Dissolve 3-ethyl-2-methylbenzothiazol-3-ium iodide and the intermediate product in anhydrous ethanol, then add piperidine dropwise, and stir to react at room temperature. After the reaction is completed, collect the filtrate by suction filtration and washing, and evaporate under reduced pressure to obtain a crude product, which is purified by a second column chromatography to obtain a fluorescent probe.

3. The preparation method according to claim 2, characterized in that The molar volume ratio of 6-hydroxy-2-naphthaldehyde, trifluoroacetic acid, hexamethylenetetramine and H2SO4 in S1 is 0.5-1.5 mmol: 5-15 mL: 1-3 mmol: 5-15 mL, and the concentration of H2SO4 is 33-98%.

4. The preparation method according to claim 2, characterized in that The temperature of the heating reflux reaction in S1 is 75° C., the time of the heating reflux reaction is 2 to 4 h, and the time of the reflux stirring is 2 to 4 h.

5. The preparation method according to claim 2, characterized in that The extraction agent used in S1 is ethyl acetate, the number of extractions is 3 to 5 times, the drying agent is anhydrous Na2SO4, and the first column chromatography purification uses ethyl acetate / n-hexane as the eluent, and the elution ratio is 2 to 3:5 to 7.

6. The preparation method according to claim 2, characterized in that The molar volume ratio of 3-ethyl-2-methylbenzothiazol-3-ium iodide, the intermediate product, anhydrous ethanol and piperidine in S2 is 0.75-1 mmol: 0.50-1.5 mmol: 4-8 mL: 0.05-0.15 mL.

7. The preparation method according to claim 2, characterized in that The stirring reaction time in S2 is 30 to 60 minutes, the washing detergent is anhydrous ethanol, and the second column chromatography purification uses dichloromethane / methanol as the eluent, and the elution ratio is 8 to 10:1 to 2.

8. An application of the fluorescent probe for detecting viscosity as claimed in claim 1, characterized in that: The fluorescent probe is used to detect viscosity changes caused by food thickeners and monitor them in real time.

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