Rapid high-selectivity viscosity fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to a rapid and high-selectivity viscosity fluorescent probe as well as a preparation method and application thereof, and relates to the field of fluorescent probes. Specifically, the probe disclosed by the invention can be used as a viscosity probe for detecting and measuring viscosity change in a biological model and a beverage. The probe can realize at least one of the following technical effects: the rapid and high-specificity viscosity response can be realized, and the quantitative analysis of the viscosity can be realized; the viscosity change in a biological system and a beverage can be detected; the viscosity change in the liver injury process can be tracked; beverage viscosity change caused by the thickening agent can be monitored; the synthesis is simple, the property is stable, and long-term storage and use can be realized.

Description

Technical Field

[0001] The invention relates to the field of fluorescent probes, and in particular to a rapid and highly selective viscosity fluorescent probe and a preparation method and application thereof. Background Art

[0002] Viscosity plays a vital role in biological systems, profoundly affecting intracellular and intercellular molecular transmission, signal transduction, and interactions between biomacromolecules. For example, in the nucleus and cytoplasm, changes in viscosity are closely related to a variety of physiological processes, such as DNA replication and protein synthesis. In addition, in the beverage industry, viscosity is not only an important parameter that determines the taste and quality of the product, but also an indispensable key factor in achieving stability and consistency in product quality control during the production process.

[0003] Although existing technical means, such as atomic force microscopy and rheology, can provide high-precision viscosity data, these methods usually rely on complex equipment and operations, and are time-consuming, so they are not suitable for real-time monitoring and online applications. Traditional viscosity measurement methods, such as rotational viscometers, can meet actual needs to a certain extent, but they still face many problems in the production process, such as cumbersome operations, lengthy measurement times, and restrictions on sample conditions.

[0004] In this context, fluorescent probe technology has emerged with its unique potential for real-time monitoring and online applications, bringing new hope to the field of viscosity measurement. Fluorescent probes can obtain viscosity information in a quick and convenient way without the need for complex equipment. Their high sensitivity and specific response to viscosity changes make them ideal for detecting small viscosity changes in complex biological systems and beverage environments. Fluorescent probes usually consist of two parts: fluorophores and molecular rotors. As a sensor of viscosity changes, fluorophores can feedback viscosity information through fluorescence changes; while molecular rotors rotate when viscosity changes, thereby changing the excited state of the fluorophore, which in turn causes changes in fluorescence intensity or a significant extension of fluorescence lifetime.

[0005] In summary, the development of a fluorescent probe that can monitor viscosity changes in biological systems and beverages in real time is of great significance, and is particularly critical and urgent for promoting the development of viscosity detection technology in biomedical research and beverage industry. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a rapid and highly selective viscosity fluorescent probe and its preparation method and application. The probe can be used to track viscosity changes in biological systems and beverages, and has the advantages of simple synthesis, good selectivity, and the ability to quickly detect viscosity.

[0007] Specifically, the present invention provides a fluorescent probe for detecting viscosity, the chemical structure of which is shown in Formula I: .

[0008] The present invention provides a method for preparing a fluorescent probe of formula I, comprising the following steps: The fluorescent probe is prepared by reacting 1-(2-hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium compound with 9-formyl julolidine, and the reaction formula is as follows:

[0009] In some specific embodiments of the present invention, the preparation method of the fluorescent probe of formula I is as follows: 1-(2-hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium compound and 9-aldehyde julolidine are dissolved in 10 mL of anhydrous ethanol, and 100 μL of piperidine is added, and the mixture is heated to reflux at 80°C in a closed environment. After the reaction is completed, the solvent is removed by rotary evaporation to obtain a crude product; and the mixture is further separated by a chromatographic column using a mixed system of dichloromethane and methanol as an eluent to obtain a pure compound of formula I.

[0010] In some specific embodiments of the present invention, the molar ratio of 1-(2-hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium compound to 9-formyl julolidine is 1:1.5.

[0011] In some specific embodiments of the present invention, the reflux reaction time is 12 hours.

[0012] The present invention also provides a fluorescent probe composition for detecting viscosity, which comprises a fluorescent probe of formula (I).

[0013] In some specific embodiments of the present invention, the fluorescent probe composition further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

[0014] The present invention also provides a method for detecting viscosity in a sample or measuring a change in viscosity in a sample, comprising: a) contacting the fluorescent probe of formula I with a sample to form a fluorescent compound; b) Determine the fluorescence properties of fluorescent compounds.

[0015] In some specific embodiments of the present invention, the sample is a chemical sample, a biological sample or a food sample, or an aqueous environment sample.

[0016] In some specific embodiments of the present invention, the sample is a biological sample including water sample, beverage, animal cell or tissue.

[0017] The present invention also provides the use of the fluorescent probe of formula I in detecting changes in cell viscosity or determining cell viscosity values.

[0018] The present invention also provides the use of the fluorescent probe of formula I in cell fluorescence imaging.

[0019] The present invention also provides the use of the fluorescent probe of formula I in liver injury imaging.

[0020] The present invention also provides the use of the fluorescent probe of formula I in fluorescent imaging of mouse liver tissue.

[0021] The present invention also provides the use of the fluorescent probe of formula I in detecting viscosity changes in beverages or determining the viscosity value of beverages.

[0022] The present invention also provides a kit for detecting viscosity changes in beverages or measuring the viscosity value of beverages, which comprises the fluorescent probe of formula I.

[0023] Compared with the prior art, the present invention has the following significant advantages and effects: (1) Quick response The fluorescent probe of the present invention can quickly respond to viscosity, which is beneficial to the rapid and real-time detection of viscosity.

[0024] (2) Good selectivity The fluorescent probe of the present invention can selectively react specifically with viscosity to form a fluorescent compound. Compared with other common metal ions and other substances in the body, including but not limited to potassium ions, sodium ions, calcium ions, copper ions, zinc ions, ferrous ions, iron ions, nitrite ions, nitrate ions, carbonate ions, sulfate ions, bicarbonate ions, bisulfite ions, bisulfate ions, acetate ions, sulfide ions, bromide ions, iodide ions, chloride ions, cysteine, glutathione, sodium ascorbate, hypochlorous acid, hydrogen peroxide, hydroxyl radicals, singlet oxygen, etc., the fluorescent probe of the present invention can eliminate the interference of most substances and show excellent selectivity.

[0025] (3) Application in the detection of viscosity during liver damage The fluorescent probe of the present invention can be used as a molecular-level viscosity detection tool to detect viscosity changes during liver damage. This application prospect makes the present invention have great potential in the field of biology and is expected to become an efficient and accurate viscosity detection tool to improve the early diagnosis rate and treatment effect of liver diseases.

[0026] (4) Applied to the detection of viscosity in beverages The fluorescent probe of the present invention can be used as a molecular-level viscosity detection tool to accurately capture and reflect the viscosity changes in beverages. This application prospect makes the present invention have great potential in the field of food industry and is expected to become an efficient and accurate viscosity detection tool.

[0027] (5) Simple synthesis The fluorescent probe of the present invention is simple to synthesize and is conducive to commercial and industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that may be involved. It should be emphasized that the drawings described below are only some embodiments of the present invention, and for ordinary technicians working in this field, other embodiments can be obtained based on these drawings without creative work.

[0029] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the synthetic fluorescent probe of Example 1; Figure 2 This is the carbon nuclear magnetic resonance spectrum of the synthetic fluorescent probe of Example 1; Figure 3 is the mass spectrum of the synthetic fluorescent probe of Example 1; Figure 4 is the absorption spectrum of the fluorescent probe in low viscosity (PBS) and high viscosity (glycerol) environments; Figure 5 It is the fluorescence spectra of the fluorescent probe in low viscosity (PBS) and high viscosity (glycerol) environments; Figure 6 is the fluorescence spectrum of the fluorescent probe in different viscosities; Figure 7 is the log value of the fluorescence intensity of the fluorescent probe at 620 nm and the viscosity level ( η ) Linear relationship of log value; Figure 8 is the stability of the fluorescent probe and its response time to viscosity; Fig. 9 is the effect of different ion analytes (glutathione and cysteine ​​concentrations are 1 mM, and the others are 100 µM) on the fluorescence intensity of the probe. The bar graph represents the fluorescence intensity of the probe at 620 nm in the presence of different analytes; Fig.10 is the effect of different pH environments on fluorescent probes; Fig.11 This is a cell imaging image of the fluorescent probe detecting drug-induced viscosity changes in L-02 cells; Fig.12It is a statistical graph of the fluorescence intensity of the fluorescent probe in L-02 cells to detect drug-induced viscosity changes; Fig.13 The images and fluorescence intensity statistics of the fluorescent probe used to detect changes in liver tissue viscosity in liver tissue sections of mice with drug-induced liver injury and alcohol-induced liver injury, including (a) normal mouse liver tissue sections, (b) APAP-treated mouse liver tissue sections, (c) APAP and NAC-treated mouse liver tissue sections, (d) EtOH-treated mouse liver tissue sections, (e) EtOH and silymarin-treated mouse liver tissue sections, (f) relative intensity of Figure ae, scale bar: 100 μm; Fig.14 It is a fluorescent probe that detects viscosity changes caused by thickeners in beverages; Fig.15 It is the linear relationship between the fluorescence intensity value of the fluorescent probe at 620 nm and the mass concentration value of the thickener when the fluorescent probe is used to detect viscosity changes in beverages. DETAILED DESCRIPTION

[0030] In conjunction with the drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be clear that the described embodiments only represent a part of the implementation methods of the present invention and do not limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without performing creative work belong to the protection scope of the present invention.

[0031] Example 1: Synthesis of Fluorescent Probe of Formula I The synthetic route is as follows:

[0032] The specific steps are as follows: 1-(2-Hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium (204 mg, 1.0 mmol) and 9-formyl julolidine (301 mg, 1.5 mmol) were dissolved in 10 mL of anhydrous ethanol, and 100 μL of piperidine was added. The mixture was heated under reflux at 80 °C for 12 hours. After the reaction, the solvent was removed by rotary evaporation to obtain a crude product. The product was further separated by a chromatographic column, and a mixed system of dichloromethane and methanol (volume ratio of 20:1) was used as the eluent to obtain 445 mg of the pure compound of formula I with a yield of 73%.

[0033] The hydrogen spectrum, carbon spectrum and mass spectrum of the compound of formula I synthesized by this method are shown in the attached Figure 1 , Attachment Figure 2 , Attachment Figure 3 shown.

[0034] Example 2: Testing the changes in absorption and fluorescence spectra of fluorescent probes in low-viscosity and high-viscosity environments

[0035] Use 1 mL of DMSO to fully dissolve the probe to prepare a 10 mM probe stock solution. Prepare two low-viscosity test systems (1×PBS buffer solution, pH=7.4) and high-viscosity test systems (glycerol, purity 99%) with a probe concentration of 10 μM, respectively, and the volume of each test system is 3 mL. After shaking, remove the bubbles by ultrasound, and use an absorption spectrometer to measure the changes in its absorption spectrum. Next, prepare two low-viscosity test systems (1xPBS buffer solution, pH=7.4) and high-viscosity test systems (glycerol, purity 99%) with a probe concentration of 5 μM, and the volume of each test system is 4 mL. After shaking, remove the bubbles by ultrasound, and use a fluorescence spectrometer to measure the changes in its fluorescence spectrum. The measurement temperature of the fluorescence spectrum is 25 ℃, and the probe used is the one prepared in Example 1.

[0036] Depend on Figure 4 It can be clearly seen that in a high viscosity environment, the absorption peak of the absorption spectrum of the fluorescent probe at 590 nm is significantly enhanced.

[0037] Depend on Figure 5 It can be clearly seen that in a high viscosity environment, the emission peak of the fluorescence spectrum of the fluorescent probe at 620 nm is significantly enhanced.

[0038] Example 3: Testing the fluorescence spectrum changes of fluorescent probes in different viscosities Prepare a test system of glycerol / PBS mixed solution of different proportions (99%-0%), and the volume of each mixed solution is 4mL. Then, add 5 μM probe to the test system of different proportions. After mixing, remove bubbles by ultrasound, and use a fluorescence spectrometer to test the change of its fluorescence intensity. Each group of experiments was repeated three times, the measurement temperature of the fluorescence spectrum was 25 ℃, and the probe used was prepared in Example 1.

[0039] from Figure 6 It is clearly seen that with the increase of viscosity, the emission peak of the probe at 620 nm gradually increases. Figure 7 It can be seen that the log value of the probe fluorescence intensity is related to the viscosity of the mixed system ( η ) showed a good linear relationship, indicating that the fluorescent probe can be used for quantitative analysis of viscosity.

[0040] Example 4: Testing the stability of fluorescent probes and their response time to viscosity Two low-viscosity test systems (1×PBS buffer solution, pH=7.4) and high-viscosity test systems (glycerol, purity 80%) with a probe concentration of 5 μM were prepared, and the volume of each test system was 3 mL. After shaking, the bubbles were removed by ultrasound, and the change in fluorescence intensity was tested using a fluorescence spectrometer. Each group of experiments was repeated three times, and the measurement temperature of the fluorescence spectrum was 25 °C. The probe used was prepared in Example 1.

[0041] from Figure 8 It can be clearly seen that the probe has excellent stability in both low concentration systems and high viscosity systems, and changes in viscosity will immediately cause changes in fluorescence intensity, indicating that the fluorescent probe can be used for rapid analysis of viscosity.

[0042] Example 5: Testing the selectivity of fluorescent probes Prepare several low viscosity systems (1×PBS, pH=7.4) with a volume of 4 mL of test samples. Then add different analytes (the analytes are blank probe, potassium ion, sodium ion, calcium ion, copper ion, zinc ion, ferrous ion, iron ion, nitrite ion, nitrate ion, carbonate ion, sulfate ion, bicarbonate ion, bisulfite ion, bisulfate ion, acetate ion, sulfide ion, bromide ion, iodide ion, chloride ion, cysteine, glutathione, sodium ascorbate, hypochlorous acid, hydrogen peroxide, hydroxyl radical, singlet oxygen, glycerol; the analytes correspond to the attached Fig. 9 az and a´-b´ in the test system, where the concentrations of glutathione and cysteine ​​were 1 mM, and the concentrations of other analytes were 100 μM. Each group was added with a probe concentration of 5 μM, and after shaking evenly, the fluorescence intensity change was tested using a fluorescence spectrometer. Each group of experiments was repeated three times, and the measurement temperature of the fluorescence spectrum was 25 °C. The probe used was prepared in Example 1.

[0043] from Fig. 9 The results show that among these common bioactive substances, only viscosity significantly enhances the fluorescence intensity of the probe, while the effects of other analytes are almost negligible. This proves that the probe of the present invention has a high selectivity for viscosity and can be used as a specific identification and analysis tool for viscosity in biological systems and beverages.

[0044] Example 6: Testing the effect of different pH environments on fluorescent probes PBS test systems of different pH were prepared, with a volume of 4 mL in each group, and 5 μM probe was added to each group. After mixing, the bubbles were removed by ultrasound, and the fluorescence intensity was tested using a fluorescence spectrometer. Next, glycerol / PBS test systems of different pH (v / v, 4 / 1) were prepared, with a volume of 4 mL in each group, and 5 μM probe was added to each group. After mixing, the bubbles were removed by ultrasound, and the change in fluorescence intensity was tested using a fluorescence spectrometer. Each group of experiments was repeated three times, the measurement temperature of the fluorescence spectrum was 25 °C, and the probe used was prepared in Example 1. In the experiment, PBS buffer solutions of different pH were prepared by mixing 10 mM Na2HPO4 and NaH2PO4 in different volume ratios, and the accurate pH value was measured using a Mettler-Toledo FE-30 pH acidity meter.

[0045] from Fig.10 It is clearly visible that in a low viscosity environment, the probe exhibits high stability over a wide pH range. In a high viscosity environment, the fluorescence intensity of the probe increases significantly and is almost unaffected over a wide range (pH: 3-11).

[0046] Example 7: Detection of changes in cell viscosity caused by drug stimulation using fluorescent probes L-02 cells were divided into four groups. The control group L-02 cells were incubated with 10 μM probe for 30 minutes; the model groups were: L-02 cells were incubated with 10 mM acetaminophen for 12 hours, and then 10 μM probe was added for incubation for 30 minutes; L-02 cells were incubated with 10 μM dexamethasone for 2 hours, and then 10 μM probe was added for incubation for 30 minutes; L-02 cells were incubated with 10 μM nystatin for 4 hours, and then 10 μM probe was added for incubation for 30 minutes. Both groups were washed 3 times with PBS. The cells were imaged under a confocal microscope. The cells were cultured in an incubator containing 5% CO2 at 37°C, the cell imaging environment temperature was 25°C, and the probe used was prepared in Example 1. The test results are shown in the attached figure. Fig.10 , 11 shown.

[0047] from Fig.11 , 12 It can be clearly seen that the model group showed a strong red fluorescence signal. In contrast, the control group showed a weaker red fluorescence signal. The results confirmed that the probe can effectively detect viscosity changes caused by drug stimulation.

[0048] Example 8: Fluorescent probe detection of viscosity in liver tissue sections of mice with alcoholic and drug-induced liver injury The experimental mice were randomly divided into five groups, namely the normal group (0.1 ml / 10 g normal saline for intragastric administration); the model group (mice were given 56° Red Star Erguotou at a dose of 0.12 mL / g); and the treatment group (mice were first given silymarin at a dose of 0.06 mg / g for intragastric administration, and 30 minutes later, 56° Red Star Erguotou alcohol was given at a dose of 0.12 mL / g for intragastric administration). All groups were treated for 14 consecutive days. Two hours after the last intragastric administration, the mice were anesthetized, and then blood and liver tissue samples were collected. APAP (acetaminophen group) mice were intraperitoneally injected with APAP (the dosage was calculated according to the mouse body weight, 300 mg kg -1 , causing drug-induced liver injury); APAP and NAC (N-acetylcysteine) groups were intragastrically administered with NAC (dose calculated according to mouse body weight 100 mg kg) before APAP injection. -1 ) and then injected with APAP. The normal group of mice was intraperitoneally injected with the same volume of normal saline. After 12 hours, the mice were anesthetized with isoflurane, and then blood and liver tissue samples were collected. The probe was incubated with each group of liver tissue sections at 37 ° C for 1 hour, washed with PBS three times, and fluorescence microscope images were collected. The environmental temperature of tissue fluorescence microscopy was 25 ° C, and the probe used was prepared in Example 1. The test results are shown in the attached Fig.13 shown.

[0049] like Fig.13 As shown, the liver tissue of the model group showed a strong red fluorescence signal, while the liver tissue of the normal group showed a relatively weak red fluorescence signal. Compared with the normal group, the red fluorescence signal of the APAP group showed a significant enhancement. The results showed that the liver viscosity of patients with alcoholic liver injury was significantly increased, and the liver viscosity was also increased during the APAP-induced injury process. The probe was able to successfully detect the changes in liver viscosity during liver injury.

[0050] Example 9: Fluorescence intensity change experiment of fluorescent probe to detect thickener in green tea beverage Different amounts of thickener were added to the green tea beverage, and then the probe was added. After shaking, the fluorescence intensity change was tested using a fluorescence spectrometer. Each group of experiments was repeated three times. The fluorescence spectrum was measured at 25°C. The probe used was prepared in Example 1. The test results are shown in the attached figure. Fig.14 and Fig.15 shown.

[0051] As attached Fig.14 and Fig.15 As shown in the figure, as the amount of thickener added increases, the fluorescence intensity increases accordingly. Moreover, there is a good linear relationship between the fluorescence intensity and the mass concentration of the thickener.

[0052] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting viscosity, characterized in that: The chemical structure is shown in Formula I: 。 2. A method for preparing the fluorescent probe according to claim 1, characterized in that: The method comprises the following steps: reacting 1-(2-hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium compound with 9-aldehyde julolidine to prepare a fluorescent probe, and the reaction formula is as follows: .

3. The preparation method according to claim 2, characterized in that: The steps include: 1-(2-hydroxy-ethyl)-2,3,3-trimethyl-3H-indolium compound and 9-formyl julolidine are dissolved in anhydrous ethanol, and piperidine is added, and the mixture is heated to reflux at 80°C in a closed environment for reaction; after the reaction is completed, the solvent is removed by rotary evaporation to obtain a crude product; and the crude product is further separated by a chromatographic column, using a mixed system of dichloromethane and methanol as an eluent to obtain a pure compound of formula I.

4. A fluorescent probe composition for detecting viscosity, characterized in that: The method comprises the fluorescent probe as claimed in claim 1.

5. A method for detecting viscosity in a sample or determining a change in viscosity in a sample, characterized in that: include: a) contacting the fluorescent probe according to claim 1 with a sample to form a fluorescent compound; b) determining the fluorescence property of the fluorescent compound.

6. The method according to claim 5, characterized in that The sample is a chemical sample, a biological sample, an aqueous environment sample or a food sample.

7. Use of the fluorescent probe as claimed in claim 1 in detecting changes in cell viscosity or determining cell viscosity values, wherein the use is for non-disease diagnosis and treatment purposes.

8. Use of the fluorescent probe as claimed in claim 1 in cell fluorescence imaging, wherein the use is for non-disease diagnosis and treatment purposes.

9. Use of the fluorescent probe as claimed in claim 1 in liver injury imaging, wherein the use is for non-disease diagnosis and treatment purposes.

10. Use of the fluorescent probe as claimed in claim 1 in detecting viscosity changes in beverages or determining the viscosity value of beverages.

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