Fluorescent probe for quantitatively detecting acetylcholin esterase in cells as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for quantitatively detecting the concentration of acetylcholin esterase in cells as well as a preparation method and application of the fluorescent probe, and belongs to the technical field of fluorescent molecular probe detection. The structural formula of the fluorescent probe for quantitatively detecting the concentration of the acetylcholin esterase in the cells is # imgabs0 #; the fluorescent probe for quantitatively detecting the concentration of acetylcholin esterase in cells is simple in preparation method and excellent in specificity, acetylcholin esterase can be accurately recognized and captured through ratio type fluorescence change, a unique product Cy565 with a determined structure is generated, quantitative determination is more accurate, and the concentration of acetylcholin esterase in cells can be quantitatively detected through fluorescence ratio change. The acetylcholin esterase is identified with high selectivity and obvious color change is accompanied; the fluorescent probe is applied to detection of living cells, has small harm to biological cells, can carry out fluorescence imaging without influencing normal physiological functions of the cells, and provides a powerful tool for revealing a complex physiological process in a life body.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence molecular probe detection, and particularly relates to a fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells, a preparation method thereof, and an application thereof. Background Art

[0002] Acetylcholinesterase is an enzyme that is ubiquitously present in organisms and is involved in many life activities such as metabolic regulation and signal transduction. The detection of the activity of acetylcholinesterase is of great significance for understanding the metabolic process in organisms, disease diagnosis, and drug development.

[0003] Existing traditional methods for detecting acetylcholinesterase have some limitations, such as low sensitivity, poor selectivity, and complex operation. Therefore, it is particularly important to develop new methods for detecting acetylcholinesterase. Fluorescence probes detect acetylcholinesterase in living cells with high sensitivity and specificity. Summary of the Invention

[0004] In view of the problems of low sensitivity, poor selectivity, and complex operation of traditional methods for detecting acetylcholinesterase, the present invention provides a fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells, a preparation method thereof, and an application thereof, which has high sensitivity, strong specificity, and is applied to the detection of living cells with less harm to biological cells.

[0005] The present invention is achieved through the following technical solutions: A fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells, the structural formula of which is shown as Cy667: 。

[0006] The present invention also discloses a preparation method of the above-mentioned fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells, comprising the following steps: (1) React IR-813 with 2-mercaptoethanol to obtain an intermediate product Cy625; (2) React the intermediate product Cy625 with acetyl chloride to obtain a fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells; The synthesis chemical formula is shown as follows: 。

[0007] Further, in step (1), IR-813 reacts with 2-mercaptoethanol in a dichloromethane solvent, and the molar ratio of IR-813 to 2-mercaptoethanol is 1:1 to 1:1.3.

[0008] Further, in step (1), the reaction temperature is 35 to 55 °C, and the reaction time is 2 to 4 h.

[0009] Furthermore, the crude product after the reaction in step (1) is purified by recrystallization with dichloromethane / petroleum ether solution.

[0010] Furthermore, the intermediate Cy625 in step (2) and acetyl chloride are dissolved and reacted in dichloromethane solvent, and the molar ratio of the intermediate Cy625 to acetyl chloride is 1:1 to 1:1.3.

[0011] Furthermore, the reaction condition in step (2) is to stir and react for 0.5 - 2.5 h under ice bath condition.

[0012] Furthermore, the crude product after the reaction in step (2) is purified by silica gel column chromatography to obtain a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells.

[0013] In the present invention, the application of the above-mentioned fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells in detecting the content of acetylcholinesterase in biological cells is also disclosed.

[0014] Beneficial effects 1) The fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells in the present invention uses a cyanine dye as a fluorescent group, and this molecule can emit bright and stable light in the near-infrared region, effectively reducing the possible interference between the fluorescence signal and other molecules or the background environment, thereby providing an efficient and accurate fluorescence imaging method; 2) The fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells in the present invention has excellent specificity, can accurately identify and capture acetylcholinesterase through ratio-type fluorescence changes, and generates a unique product Cy565 with a determined structure, making the quantitative determination more accurate; 3) The method for preparing the fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells in the present invention is simple and has extremely low impurity content, greatly reducing the interference of other factors; this probe molecule highly selectively recognizes acetylcholinesterase through fluorescence ratio changes and is accompanied by obvious color changes; 4) The fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells in the present invention is applied to the detection of living cells, has less harm to biological cells, and can perform fluorescence imaging without affecting the normal physiological functions of cells. This technology provides a powerful tool for revealing complex physiological processes in vivo. Brief description of the drawings

[0015] Figure 1 It is the mass spectrum of the fluorescent probe Cy667 for quantitatively detecting the concentration of acetylcholinesterase in cells; Figure 2 It is the 1H NMR spectrum of the fluorescent probe Cy667 for quantitatively detecting the concentration of acetylcholinesterase in cells; Figure 31H NMR spectrum of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells; Figure 4 Mechanism diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells to detect acetylcholinesterase; Figure 5 Ratio-type UV titration diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells to detect acetylcholinesterase. a is the UV absorption spectrum of the generated Cy565; b is the UV absorption spectrum of the consumed Cy667; Figure 6 Ratio-type fluorescence titration diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells to detect acetylcholinesterase. a is the fluorescence emission spectrum of the generated Cy565; b is the fluorescence emission spectrum of the consumed Cy667; Figure 7 Ratio-type linear diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells to detect acetylcholinesterase; Figure 8 Selectivity diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells for detecting acetylcholinesterase Figure 9 Confocal fluorescence imaging and fluorescence intensity diagram of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in cells for acetylcholinesterase in HepG2 cells. a is the confocal fluorescence imaging diagram; b is the fluorescence intensity diagram; Figure 10 Detection diagram of the cytotoxicity detection of the fluorescence probe Cy667 for quantitative detection of acetylcholinesterase concentration in HepG2 cells. Detailed implementation manners

[0016] The specific details of the present invention are further elaborated to fully understand the present invention. The terms used in the description of the present invention are only used to illustrate the advantages and characteristics of the present invention, not to limit the present invention. Unless otherwise defined, all professional and scientific terms used in the present invention have the same meaning as understood by those skilled in the technical field of the present invention. Now, the technical solutions of the present invention will be further described according to the specific implementation manners.

[0017] The HepG2 cells in the present invention are: Shanghai Fuheng Biotechnology Co., Ltd., human hepatoma cells, product number: FH0076; high performance liquid chromatography-mass spectrometry analysis is performed using a 1290 / 6545 UHPLC-Q-TOF mass spectrometry system (Agilent, USA), and high performance liquid chromatography separation is accomplished with the aid of an Agilent 1260 Infinity II; fluorescence imaging observation is carried out using an Olympus FV3000 laser confocal microscope; the separation and purification of the compound are achieved using a thin layer chromatography silica gel column, where the packing material is 300 - 400 mesh.

[0018] Example 1 Preparation of a fluorescence probe for quantitatively detecting the concentration of acetylcholinesterase in cells: (1) Add 0.1 mol of IR-813 and 0.12 mol of 2-mercaptoethanol to 20 mL of dichloromethane solution, and reflux and react at 35 - 55 °C for 2 - 4 h. After the reaction is completed, the crude product is recrystallized and purified with a dichloromethane / petroleum ether solution (V:V = 1:20) to obtain the intermediate product Cy625; (2) Dissolve 0.1 mol of the intermediate product Cy625 and 0.12 mol of formyl chloride in 30 mL of dichloromethane, and stir and react under an ice-water bath condition for 0.5 - 2.5 h. After the reaction is completed, the crude product is purified by silica gel column chromatography to obtain a fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells; The molecular formula of the above reaction is shown as follows: ; The mass spectrum of the fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells prepared in Example 1 is as Figure 1 shown, the 1H NMR spectrum is as Figure 2 shown, and the 13C NMR spectrum is as Figure 3 shown.

[0019] Example 2 The mechanism of the fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells to detect acetylcholinesterase is as Figure 4 shown. Cy667 undergoes a specific deacetylation reaction with acetylcholinesterase, and through enzymatic catalysis, a reactive oxygen anion intermediate is generated. This intermediate undergoes an intramolecular cyclization reaction to form an unstable transition state structure, and finally, a stable product Cy565 is generated through a 1,4-elimination reaction; Verify the ability of the fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells to detect different concentrations of acetylcholinesterase; (1) Prepare a 2 mM stock solution of Cy667, and the solvent is DMSO; (2) In 2 mL of ethanol, add the stock solution from step (1) to prepare a 10 μM solution, and then add acetylcholinesterase solutions at 0 U / mL, 4 U / mL, 8 U / mL, 12 U / mL, 16 U / mL, and 20 U / mL respectively. React for 1 h, and then perform the following detections after the reaction: a. Using the ultraviolet absorption wavelength as the abscissa and the absorbance as the ordinate, obtain the ultraviolet absorption spectrum of Cy667 detecting the generation of Cy565 by acetylcholinesterase. The results are as Figure 5 shown (a is the ultraviolet absorption spectrum of the generated Cy565; b is the ultraviolet absorption spectrum of the consumed Cy667). From Figure 5 a, it can be observed that the absorbance peak intensity of Cy565 at 560 nm increases significantly as the acetylcholinesterase concentration gradually increases from 0 U / mL to 20 U / mL; at the same time, Figure 5 b shows that the absorbance peak intensity of Cy667 at 821 nm shows a downward trend as the acetylcholinesterase concentration gradually increases from 0 U / mL to 20 U / mL; b. Using the emission wavelength as the abscissa and the fluorescence intensity as the ordinate, obtain the fluorescence emission spectrum of Cy667 detecting the generation of Cy565 by acetylcholinesterase. The results are as Figure 6 shown (a is the fluorescence emission spectrum of the generated Cy565; b is the fluorescence emission spectrum of the consumed Cy667); as Figure 6 shown in a, as the acetylcholinesterase concentration gradually increases from 0 U / mL to 20 U / mL, the fluorescence emission peak intensity of Cy565 at 636 nm shows a significant increasing trend; on the contrary, Figure 6 b shows that the fluorescence emission peak intensity of Cy667 at 831 nm shows an obvious downward trend as the acetylcholinesterase concentration increases (0 - 20 U / mL); c. Using the concentration of acetylcholinesterase as the abscissa and the corresponding fluorescence intensity as the ordinate, obtain the linear graph of the fluorescence intensity ratio (green light / red light) of Cy667 against the concentration of acetylcholinesterase Figure 7 , and it can be seen through Figure 7 that as the concentration of acetylcholinesterase increases, the fluorescence intensity of the product at 834 nm shows a linear relationship with the concentration of acetylcholinesterase. The detection limit of the molecular probe measured in the present invention is 0.046 U / mL.

[0020] Example 3 To verify the selective detection of acetylcholinesterase by the fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells, the following experiment was designed: Taking different targets as the abscissa (the targets include biological thiols and cationic solutions) and the change in green fluorescence intensity as the ordinate, a selectivity map of Cy667 was obtained, and the results are as Figure 8 shown, Figure 8 The corresponding relationship between the abscissa numbers and the targets is as follows: 1: BChE (butyrylcholinesterase), 2: AChE (acetylcholinesterase), 3: CES1 (carboxylesterase 1), 4: CES2 (carboxylesterase 2), 5: Na + (sodium ion), 6: K + (potassium ion), 7: Ca 2+ (calcium ion), 8: Fe 2+ (ferrous ion), 9: Fe 3+ (ferric ion), 10: H2O2 (hydrogen peroxide), 11: Arg (arginine), 12: Gly (glycine), 13: Glu (glutamic acid), 14: Cys (cysteine), 15: GSH (glutathione), 16: Tyr (tryptophan), 17: Lys (lysine), 18: Hcy (homocysteine). It can be seen through Figure 8 that the fluorescent probe (Cy667) has strong selectivity for acetylcholinesterase.

[0021] Example 4 The steps of fluorescence imaging of human hepatocellular carcinoma HepG2 cells with a fluorescent probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells include: (1) Prepare a 10 mM Donepezil solution, a 10 mM Rivastigmine solution, a 5 mM DMSO standard solution of Cy667, and a 5 mM DMSO standard stock solution of Cy565; (2) Cell culture: Resuscitate HepG2 cells and then place them in an incubator at 37 °C with a carbon dioxide concentration of 5%. The culture medium contains 89% DMEM, 10% fetal bovine serum, and 1% double antibody. After culturing for 24 h, well - growing cells can be obtained for later use; (3) Divide HepG2 cells into inoculation amounts of 2×10 7 ~9×10 7Four groups with a density of cells / mL were labeled as A, B, C, and D. After culturing for 24 h, only 2 μM of Cy667 was added to the HepG2 cells in group A and incubated for 30 min. For the HepG2 cells in group B, 10 μM of Donepezil was added first and cultured for 30 min, then 2 μM of Cy667 was added and incubated for 30 min. For the HepG2 cells in group C, 10 μM of Rivastigmine was added first and cultured for 30 min, then 2 μM of Cy667 was added and incubated for 30 min as well. Only 2 μM of Cy565 was added to the HepG2 cells in group D and incubated for 30 min.

[0022] Confocal laser fluorescence imaging was performed on HepG2 cells. For the red channel, the excitation wavelength was 594 nm and the emission wavelength was 650 - 750 nm. For the green channel, the excitation wavelength was 514 nm and the emission wavelength was 550 - 650 nm. Confocal images of the four groups of cells were obtained, namely Figure 9 a. The results showed that in the three groups of cells incubated with Cy667, the fluorescence intensity of the green channel in the untreated control group was relatively strong, indicating that a large amount of Cy565 was produced. Since the concentration of acetylcholinesterase in the cells was relatively high, the fluorescence intensity of the red channel was weak, indicating that Cy667 had basically completely reacted with the acetylcholinesterase in the cells. After adding the acetylcholinesterase inhibitors Donepezil solution and Rivastigmine solution, the activity of acetylcholinesterase was inhibited, the fluorescence intensity of the red channel was high, and the fluorescence intensity of the green channel became weak, indicating that Cy667 was not decomposed by acetylcholinesterase. For the cells incubated with Cy565, the green fluorescence was obvious and the red fluorescence was weak, which was consistent with the results of the Cy667 control group.

[0023] The fluorescence intensity of HepG2 cells was calculated using ImageJ, and the intensity maps of the fluorescence images of the four groups of cells were obtained, namely Figure 9 b. The results showed that the red fluorescence intensity in the Donepezil group and the Rivastigmine group was much stronger than that in the control group, indicating that Cy667 was not decomposed by acetylcholinesterase.

[0024] Example 5 Detect the effect of the fluorescence probe (Cy667) for quantitatively detecting the concentration of acetylcholinesterase in cells on cell viability. The specific steps are as follows: Add Cy667 with concentrations of 0 μmol / L, 2 μmol / L, 4 μmol / L, 6 μmol / L, 10 μmol / L, and 20 μmol / L to the cell culture medium, and culture in an incubator at a temperature of 37 °C and a carbon dioxide concentration of 5% for 24 h. Then add 20 μL of 5 mg / mL 4-methylthiazolyl tetrazolium MTT to the cell culture medium and culture for 4 h. Estimate the cell viability by the MTT colorimetric method. Use the relevant data of the cell group without adding Cy667 with a viability of 100% and the experimental groups with different concentrations of Cy667 to draw a relative bar chart. As Figure 10 shown, adding different concentrations of Cy667 to the cell culture medium will not have too much impact on cell survival, and the cell viability reaches 90% and above.

[0025] Although the present invention has been elaborated in detail through preferred embodiments, these embodiments are not intended to limit the scope of the present invention. Any person skilled in the art of chemistry, on the premise of fully understanding the technical concept of the present invention, can make reasonable modifications such as adjusting reaction conditions, using equivalent alternative materials, or optimizing parameters through conventional experimental means. As long as these technical deformations do not deviate from the core innovative elements stated in the claims, they should be regarded as falling within the protection scope of the present invention. It should be particularly stated that the legal protection scope of this patent is ultimately defined by the literal expression of the claims and the equivalent extension scope of its technical features.

Claims

1. A fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells, characterized in that: The structural formula is shown below for Cy667: 。 2. A method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 1, characterized in that: The following steps are involved: (1) IR-813 reacts with 2-mercaptoethanol to obtain the intermediate product Cy625; (2) The intermediate product Cy625 reacts with acetyl chloride to obtain a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells; The synthetic chemical formula is as follows: 。 3. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: In step (1), IR-813 and 2-mercaptoethanol are reacted in a dichloromethane solvent, and the molar ratio of IR-813 to 2-mercaptoethanol is 1:1 to 1:1.

3.

4. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: In step (1), the reaction temperature is 35-55°C and the reaction time is 2-4 h.

5. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: The crude product after the reaction in step (1) is purified by recrystallization from a dichloromethane / petroleum ether solution.

6. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: In step (2), the intermediate product Cy625 is dissolved and reacted with acetyl chloride in a dichloromethane solvent, and the molar ratio of the intermediate product Cy625 to acetyl chloride is 1:1 to 1:1.

3.

7. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: The reaction conditions in step (2) are to stir the reaction in an ice bath for 0.5 to 2.5 hours.

8. The method for preparing a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells according to claim 2, characterized in that: The crude product after the reaction in step (2) is purified by silica gel column chromatography to obtain a fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells.

9. Use of the fluorescent probe for quantitatively detecting the concentration of acetylcholinesterase in cells as claimed in claim 1 in detecting the content of acetylcholinesterase in biological cells.

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