A fluorescent probe, its preparation method and application
By preparing a near-infrared fluorescent probe containing lipophilic and electronegative sulfonic acid groups, the shortcomings of existing cell membrane probes are overcome, enabling rapid anchoring, long-term imaging, and viscosity detection, making it suitable for cell membrane viscosity detection and targeted applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INST OF CHEM CHINESE ACAD OF SCI
- Filing Date
- 2022-10-24
- Publication Date
- 2026-06-30
AI Technical Summary
Existing cell membrane probes have shortcomings such as slow staining speed, cumbersome washing before imaging, easy diffusion into cells, short wavelength or need for toxic solvents, which make it impossible to achieve long-term cell membrane imaging and viscosity detection.
A near-infrared fluorescent probe containing lipophilic and electronegative sulfonic acid groups was developed. It was prepared by condensation reaction and can be anchored to the cell membrane to detect viscosity and respond specifically in the near-infrared wavelength region. It avoids diffusion and is unaffected by the polarity and pH of the medium.
It enables rapid anchoring and long-term imaging of cell membranes without the need for washing. The probe responds to viscosity changes at near-infrared wavelengths, has good water solubility, is suitable for mass production and storage, and is applicable to cell membrane viscosity detection and targeted applications.
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Figure CN117924144B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a viscosity-sensitive fluorescent probe anchored to the cell membrane, its preparation method, and its application, belonging to the field of biochemical analysis technology. Background Technology
[0002] The cell membrane, the outermost and most important organelle, is approximately 5–8 nm thick. Its basic framework is a phospholipid bilayer mainly composed of phospholipids, cholesterol, and proteins. It serves as the first and crucial barrier protecting cells from external environmental disturbances, undertaking various physiological functions such as substance transport and signal transduction. Cell membrane viscosity controls the fluidity of the phospholipid bilayer, playing a vital role in maintaining intracellular homeostasis and normal function. Abnormal cell membrane viscosity can even lead to various serious diseases such as atherosclerosis, sclerosis, diabetes, and Alzheimer's disease.
[0003] Currently, the main cell membrane probes on the market include: lipophilic Di series probes such as DiO, DiD, and DIR, and CellMask series probes such as CellMask Green and CellMask Orange. However, these probes generally suffer from drawbacks such as slow staining speed, cumbersome washing procedures before imaging, and easy diffusion into cells, making long-term cell membrane imaging studies impossible. Furthermore, most cell membrane fluorescent probes have relatively short wavelengths or require the addition of dimethyl sulfoxide (DMSO), which is toxic to organisms. Therefore, developing viscosity-sensitive near-infrared fluorescent probes with good cell membrane anchoring capabilities is of great significance.
[0004] Therefore, this invention is proposed. Summary of the Invention
[0005] The purpose of this invention is to provide a viscosity-sensitive fluorescent probe capable of cell membrane anchoring, its preparation method, and its application.
[0006] Therefore, in a first aspect, the present invention provides a fluorescent probe, the structure of which is shown in Formula I.
[0007]
[0008] In Equation I, n = 0, 3, or 7.
[0009] Furthermore, the structure of the fluorescent probe is shown in Formula II.
[0010]
[0011] Secondly, the present invention provides a method for preparing the fluorescent probe, comprising: reacting compounds of formulas III and IV in an organic solvent via a condensation reaction to obtain the fluorescent probe of formula I.
[0012]
[0013] In Equation IV, the definition of n is the same as that in the fluorescent probe.
[0014] Furthermore, the compound shown in Formula IV can specifically be the compound shown in Formula V, and the fluorescent probe shown in Formula II can be prepared from it.
[0015]
[0016] Furthermore, the organic solvent may be at least one of acetic anhydride, ethanol, and toluene, preferably ethanol, or a mixture of ethanol with acetic anhydride or toluene, etc.
[0017] Furthermore, the reaction temperature of the condensation reaction can be 20 to 100°C, including but not limited to 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, etc., with 90°C being preferred; the reaction time of the condensation reaction can be 1 to 5 hours, including but not limited to 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, etc., with 4 hours being preferred.
[0018] Thirdly, the present invention provides the application of the fluorescent probe in viscosity detection.
[0019] Specifically, viscosity is determined by detecting the fluorescence response intensity of the fluorescent probe at a wavelength of 710 nm.
[0020] Fourthly, the present invention provides the application of the fluorescent probe in specifically targeting cell membranes.
[0021] Specifically, the cell membrane targeting ability of the fluorescent probe is demonstrated by detecting the colocalization effect of the fluorescence imaging patterns of the fluorescent probe and the commercial cell membrane probe.
[0022] Fifthly, the present invention provides a study on the ability of the fluorescent probe to anchor on the cell membrane.
[0023] Specifically, the cell membrane anchoring ability of the fluorescent probe is determined by detecting confocal fluorescence imaging at different time points.
[0024] In a sixth aspect, the present invention provides the application of the fluorescent probe in the detection of cell membrane viscosity.
[0025] Specifically, cell membrane viscosity is determined by detecting the fluorescence intensity of the fluorescent probe in the fluorescence collection band of 650-750 nm.
[0026] In summary, compared with the prior art, the fluorescent probe provided by the present invention has the following advantages:
[0027] 1) It can be anchored on the cell membrane to measure viscosity;
[0028] 2) The probe contains a structure with good lipophilic groups, which facilitates the diffusion of the probe into the cell membrane;
[0029] 3) The probe contains two electronegative sulfonic acid groups, which can generate electrostatic repulsion with the negative potential of the cell membrane, thus preventing the probe from diffusing into the cell;
[0030] 4) The cell membrane can be imaged immediately after the probe is incubated with the cell, without the need for washing.
[0031] 5) The probe can remain anchored on the cell membrane for an extended period of time;
[0032] 6) The probe can specifically respond to viscosity in the near-infrared wavelength region (710nm), and its fluorescence can be enhanced as the viscosity of the medium increases, while being unaffected by the polarity and pH value of the medium.
[0033] 7) It has good water solubility, which avoids the introduction of organic solvents that are toxic to organisms during use;
[0034] 8) The preparation method is simple, it can be mass-produced, and it is easy to store.
[0035] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description
[0036] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiments below. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:
[0037] Figure 1 The fluorescence response diagram of the fluorescent probe shown in Equation II to different viscosity values is shown.
[0038] Figure 2 The graph shows the linear relationship between the logarithm of the fluorescence response intensity of the fluorescent probe at a wavelength of 710 nm and the logarithm of the viscosity value, as shown in Equation II.
[0039] Figure 3 This is a co-localization fluorescence imaging diagram of the fluorescent probe shown in Formula II and the cell membrane dye DIO in AML12 cells;
[0040] Figure 4The image shows a time-dependent fluorescence imaging of the fluorescent probe shown in Formula II in AML12 cells.
[0041] Figure 5 This is a reversible fluorescence imaging diagram of the cell membrane viscosity of AML12 cells by the fluorescent probe shown in Formula II at alternating temperatures. Detailed Implementation
[0042] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.
[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.
[0044] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.
[0045] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0046] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.
[0047] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).
[0048] In the description of the embodiments of this application, unless otherwise expressly specified and limited, technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. For those skilled in the art, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.
[0049] Example 1: Preparation of the fluorescent probe shown in Formula II
[0050] This embodiment provides a method for preparing the fluorescent probe shown in Formula II, and its synthetic route is as follows:
[0051]
[0052] The specific steps are as follows:
[0053] The compound shown in Formula I1I (1 mmol, 557 mg) and the compound shown in Formula V (1 mmol, 233 mg) were dissolved in 20 mL of anhydrous ethanol and stirred at 90 °C for 4 h. After cooling, the reaction solution was evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (silica gel G, 200-300 mesh) using CH2Cl2 / MeOH (5:1, v / v) as eluent to obtain the final product (650 mg, yield 84%), which was a dark blue solid and was the fluorescent probe shown in Formula II. The prepared fluorescent probe was used in Examples 2-5.
[0054] 1 H NMR (700MHz, 298K, CD3OD): δ7.89-7.87(m,2H),7.78-7.62(m,2H),7.60-7.58(m, 3H),7.41-7.27(m,2H),7.26-7.09(m,3H),6.90-6.88(d,2H,J=14Hz),4.42-4.31( m,4H),3.56-3.54(t,4H,J=14Hz),2.88-2.86(m,4H),2.39(s,3H),2.08(s,3H),1. 98-1.86(m,8H),1.69-1.65(m,4H),1.45-1.40(m,4H),1.00-0.98(t,6H,J=14Hz). 13C NMR (175MHz, 298K, CD3OD): δ162.47,157.57,154.20,150.26,145.65,138.23,1 37.66,128.55,126.73,124.49,124.36,123.68,123.51,123.17,122.56,121.0 3,120.32,118.71,115.80,113.12,111.33,110.70,50.89,50.56,43.59,29.43 ,29.27,28.25,28.06,22.81,22.15,19.74,12.84,11.63,11.43.HR-ESI-MS:m / z calcd.for MYN-BS(C 43 H 54 N3O6S2 - [M] - ),772.3460; found,772.3453.
[0055] Example 2: The viscosity of methanol-glycerol mixtures with different volume ratios was determined using the fluorescent probe shown in Formula II.
[0056] A series of methanol-glycerol mixtures with different volume ratios were added to test tubes, followed by an appropriate volume of probe aqueous solution (1 mM) to achieve a final probe concentration of 5 μM. The solutions were placed on a shaker and continuously shaken at a constant speed for 1 hour to ensure thorough mixing, then allowed to stand for 30 minutes to eliminate air bubbles. The fluorescence intensity of the solution was measured using a fluorescence spectrophotometer with an excitation wavelength of 670 nm. A graph was plotted with the logarithm of the fluorescence intensity at 710 nm as the ordinate and the logarithm of the solution viscosity as the abscissa to obtain the relationship between fluorescence intensity and viscosity.
[0057] Figure 1 The fluorescence response diagram of the fluorescent probe shown in Equation II to different viscosity values is obtained from... Figure 1 It can be seen that the fluorescence at a wavelength of 710 nm gradually increases with the increase of viscosity. Figure 2 To obtain the relationship between fluorescence intensity and viscosity, a graph is plotted with the logarithm of fluorescence intensity at 710 nm as the ordinate and the logarithm of solution viscosity as the abscissa. Figure 2 It can be seen that the logarithm of fluorescence intensity and the logarithm of solution viscosity exhibit a good linear relationship, which is consistent with... equation.
[0058] Example 3: Co-localization experiment of the fluorescent probe shown in Formula II and the cell membrane dye DIO
[0059] 1) In a glass-bottomed culture dish for laser confocal fluorescence microscopy, at 37°C and under a 5% (v / v) CO2 atmosphere, AML12 cells (purchased from Jiangsu Kaiji Biotechnology Co., Ltd.) were cultured for 24 hours in a special culture medium for AML12 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., product catalog number CM-0602) to allow the cells to fully adhere to the bottom of the culture dish.
[0060] 2) After the cell culture in step 1) is completed, remove the culture medium from the culture dish, and then add 1 mL of phenol red-free DMEM culture medium (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., product catalog number PM150233A) containing 10 μM MDIO (commercially purchased from Thermo Fisher Scientific, product catalog number D275), and continue to incubate for 20 min.
[0061] 3) After the culture in step 2) is complete, aspirate the culture medium from the culture dish, wash the cells with 20mM phosphate buffer (pH 7.4), and finally add 1mL of phenol red-free DMEM culture medium containing 10μM of the probe shown in Formula II. Immediately place the culture dish on a confocal fluorescence microscope for fluorescence imaging. Green channel (DIO), excitation source is a 488nm laser, fluorescence signal collection range is 500-550nm; red channel (probe shown in Formula II), excitation source is a 635nm laser, fluorescence signal collection range is 650-750nm.
[0062] Figure 3 The image shows the co-localization fluorescence imaging of the fluorescent probe shown in Formula II and the cell membrane dye DIO in AML12 cells. In this image, a is the image of the probe shown in Formula II in the red channel; b is the image of DIO in the green channel; c is a superimposed image of a and b; and d is a scatter plot of the intensity of the two channels. Figure 3 It can be seen that the fluorescent dye shown in Formula II and the commercially available cell membrane fluorescent dye DIO can achieve good co-localization in AML-12 cells, indicating that the fluorescent dye prepared in this invention has excellent cell membrane targeting.
[0063] Example 4: Long-term fluorescence imaging of the fluorescent probe shown in Formula II on the cell membrane.
[0064] 1) The steps are the same as step 1) of Example 3.
[0065] 2) After cell culture in step 1) is complete, aspirate the culture medium from the culture dish, then add 1 mL of phenol red-free DMEM culture medium containing 10 μM of the probe shown in Formula II. Place the culture dish on a confocal fluorescence microscope for fluorescence imaging at different times. The excitation source is a 635 nm laser, and the fluorescence signal collection range is 650-750 nm.
[0066] Figure 4 This is a time-dependent fluorescence imaging map of the fluorescent probe shown in Formula II in AML12 cells, by... Figure 4 It can be seen that even when the probe is co-incubated with the cell for 1 hour, the outline of the cell membrane can still be clearly imaged, indicating that the fluorescent dye of the present invention has excellent cell membrane anchoring ability and can be used for long-term fluorescence imaging of the cell membrane.
[0067] Example 5: Fluorescence imaging of cell membrane viscosity detected by the fluorescent probe shown in Formula II under two different temperature alternations.
[0068] 1) The steps are the same as step 1) of Example 3.
[0069] 2) After the cell culture in step 1) is completed, remove the culture medium from the culture dish, then add 1 mL of phenol red-free DMEM culture medium containing 10 μM probe, and continue to culture at 37°C for 15 min.
[0070] 3) After the cultivation in step 2) is completed, place the culture dish directly on the confocal fluorescence microscope for the first fluorescence imaging.
[0071] 4) After step 3) is completed, transfer the cells to 41°C and continue culturing for 15 min.
[0072] 5) After the cultivation in step 4) is completed, place the culture dish directly on the confocal fluorescence microscope for a second fluorescence imaging.
[0073] 6) After completing step 5), transfer the cells back to 37°C and culture for 15 minutes, then proceed with steps 3), 4), and 5 in sequence.
[0074] 7) Finally, repeat step 6) once. The excitation source for the imaging channel is a 635nm laser, and the fluorescence signal collection range is 650-750nm. Changes in signal intensity represent changes in cell membrane viscosity.
[0075] Figure 5 The image shows a reversible fluorescence imaging pattern of the fluorescent probe shown in Formula II for the cell membrane viscosity of AML12 cells at alternating temperatures. Figure 5 It can be seen that the fluorescence signal intensity at 37℃ is higher than that at 41℃, and the fluorescence intensity shows a reversible change under the alternating temperature of these two conditions. This indicates that the viscosity of the cell membrane is reduced to a certain extent under heating conditions compared to physiological conditions, and this degree of change can be monitored by the probe shown in Formula II.
[0076] Finally, it should be noted that the above embodiments only illustrate the preparation and application of the fluorescent probe shown in Formula II. The preparation methods and testing conditions of other fluorescent probes are not listed one by one because they have similar structures and properties.
[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any way. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.
Claims
1. A fluorescent probe, characterized by, The structural formula is shown in Equation I below: (Formula I), In Equation I, n = 0, 3, or 7.
2. The fluorescent probe according to claim 1, wherein Its structural formula is as shown in Formula II. (Formula II).
3. The method for preparing the fluorescent probe according to claim 1, characterized in that, The probe shown in Formula I is obtained by condensation reaction of the compounds shown in Formula I11 and Formula IV in an organic solvent; (Formula IⅠⅠ)(Formula ⅠV) In Equation IV, n = 0, 3, or 7.
4. The preparation method according to claim 3, characterized in that, The compounds shown in Formula IV are specifically the compounds shown in Formula V. (Form V).
5. The preparation method according to claim 3 or 4, characterized in that, In the above preparation method, the organic solvent is at least one of acetic anhydride, ethanol, and toluene.
6. The preparation method according to claim 3 or 4, characterized in that, The organic solvent is ethanol.
7. The preparation method according to claim 3 or 4, characterized in that, The condensation reaction temperature is 20~100℃, and the reaction time is 1~5h.
8. The use of the fluorescent probe of claim 1 or 2 for non-disease diagnostic and therapeutic purposes in targeting cell membranes.
9. The application of the fluorescent probe of claim 1 or 2 in the detection of viscosity in various systems for non-disease diagnostic and therapeutic purposes.
10. The application of the fluorescent probe according to claim 1 or 2 for non-disease diagnosis and treatment purposes in fluorescent imaging for selective labeling of cell membranes and detection of cell membrane viscosity.