The present invention belongs to the field of organic small molecule fluorescent probes, and in particular to a preparation method and application of a viscosity fluorescent probe.
Cell viscosity plays an extremely important role in the transport of various molecules in vivo, and it also affects the diffusion of biological processes, such as cell apoptosis and the transport of subcellular organelles in blood. It turns out that abnormal cell viscosity may cause a variety of diseases, such as arteriosclerosis, Alzheimer's disease, diabetes, etc. Given that changes in cell viscosity are associated with many diseases, the development of cell viscosity-responsive sensors and the development of biological research and medical diagnostic strategies have attracted great attention.
In recent years, instruments for measuring liquid viscosity have emerged in an endless stream, such as falling ball viscometers, rotational viscometers, piston viscometers, etc. However, the use of these viscometers is complicated and has shortcomings such as long use time, and thus cannot be used to detect viscosity changes at the cellular level. With the development of fluorescence imaging technology, it is possible to implement visualization by converting biological information into detectable signals. Compared with traditional viscometers, the fluorescence imaging technology has advantages such as short time, easy operation, high sensitivity, strong specificity, cell imaging, and direct imaging with confocal microscope, and it has become one of the most powerful tools for biological testing. Interestingly, there are many viscosity fluorescent probes reported so far, but there are almost no viscosity fluorescent probes based on 1,3,5 triazine dyes, and pH-resistant viscosity probes are also rare.
An object of the present invention is to provide a viscosity fluorescent probe TzAr-Nap-OH based on 1,3,5 triazine dye. The probe shows strong sensitivity to viscosity, has little effect on fluorescence intensity over a wide pH range, has good cytocompatibility, and responds well to viscosity.
The present invention adopts the following technical solution to synthesize an organic small molecule fluorescent probe that may detect viscosity.
The molecular structural formula of the viscosity fluorescent probe synthesized in the present invention is as follows:
The synthesis route of the probe TzAr-Nap-OH is as follows:
2,4-diphenyl-6-methyl-1,3,5-triazine (0.2 mmol), p-hydroxynaphtaldehyde (0.2 mmol), potassium hydroxide (1.0 mmol), and methanol (1.0 mL) were added in sequence into a 10 mL reaction tube and stirred at 100° C. for 24 hours. The solution was then cooled to room temperature and extracted three times with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered with suction. The resulting filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane:ethyl acetate=80:1), to obtain a yellow-green product TzAr-Nap-OH (15.15 mg, 48%).
Application of the viscosity fluorescent probe of the present invention: the fluorescent probe may be applied to detect cell viscosity of complex organisms; and fluorescence spectrum testing and cell imaging were performed on the probe.
Beneficial effects: in view of the problems currently posed for viscosity fluorescent probes, the present invention provides a viscosity fluorescent probe TzAr-Nap-OH based on 1,3,5 triazine dye. The fluorescent probe shows strong sensitivity to viscosity, has little effect on fluorescence intensity over a wide pH range, has good cytocompatibility, and responds well to viscosity. In cell imaging experiments, the fluorescent probe has low cytotoxicity and may accurately locate cells, which provides a feasible tool for studying the important role of cell viscosity in complex physiological processes of organisms. The fluorescent probe may be applied to detect cell viscosity of complex organisms, and the synthesis steps of the fluorescent probe are simple, with a wide range of raw material sources. Therefore, the present invention relates to a cell viscosity fluorescent probe with high sensitivity, low interference and low toxicity, which has broad application prospects in the field of biomolecular detection.
In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, a brief description will be given below with reference to the need to be used in the embodiments. It is obvious that the drawings in the following description are only some embodiments of the present invention, and it would have been obvious for a person skilled in the art to obtain other drawings according to these drawings without involving any inventive effort.
In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the protection scope of the present invention.
2,4-diphenyl-6-methyl-1,3,5-triazine (0.2 mmol), p-hydroxynaphtaldehyde (0.2 mmol), potassium hydroxide (1.0 mmol) and methanol (1.0 mL) were added in sequence into a 10 mL reaction tube and stirred at 100° C. for 24 hours. The solution was then cooled to room temperature and extracted three times with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and filtered with suction. The resulting filtrate was concentrated under reduced pressure and purified by column chromatography (dichloromethane:ethyl acetate=80:1), to obtain a yellow-green product TzAr-Nap-OH (15.15 mg, 48%) 1H NMR (600 MHZ, DMSO-d6): δ 10.07 (s, 1H), 8.71-8.69 (m, 4H), 8.57 (d, J=15.8 Hz, 1H), 8.25 (s, 1H), 7.99-7.96 (m, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.78 (d, J=8.6 Hz, 1H), 7.73-7.69 (m, 2H), 7.68-7.64 (m, 4H), 7.40 (d, J=15.8 Hz, 1H), 7.21-7.15 (m, 2H); 13C NMR (150 MHz, DMSO-d6): δ 172.3, 171.0, 167.4, 157.3, 143.2, 136.2, 135.9, 133.3, 132.2, 132.0, 130.9, 130.8, 130.0, 129.4, 129.0, 128.0, 127.4, 124.9, 124.8. 119.8, 109.5;
The 1H NMR spectrum of the probe is shown in
The molecular formula of the probe TzAr-Nap-OH is: C27H19N3O.
The fluorescent probe TzAr-Nap-OH was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol/L stock solution. 20 μL probes were added dropwise in different solvents, with a final volume of 2 mL, and UV absorption spectra of the probes were measured, as shown in
20 μL of the fluorescent probe stock solution prepared in Example 2 were taken, and added dropwise to different solvents, resulting in a final volume of 2 mL. The fluorescence emission spectra of the probe is measured (λex=360 nm), as shown in
The spectral response of the probe to solution viscosity was measured, as shown in
20 μL of the fluorescent probe stock solution were taken and added dropwise into a PBS/THF (4/1) system, resulting in a final volume of 2 mL. The effects of different pH of the solution on the probe were measured, as shown in
20 μL of the fluorescent probe stock solution were taken and added dropwise into the PBS/THF (4/1) system, into which 10 μM of other interfering substance solutions were added dropwise, resulint in a final volume is 2 mL. The effects of the interfering substances on TzAr-Nap were measured, as shown in
Cytotoxicity experiments were performed on TzAr-Nap-OH, as shown in
Cell imaging of TzAr-Nap-OH response to viscosity was shown in
Although the present invention has been described in detail with reference to the above embodiments, it is only a part but not all the embodiments of the present invention, and other embodiments may be obtained without inventive step according to the embodiments, which all fall within the scope of the present invention.
Number | Date | Country | Kind |
---|---|---|---|
2023102073203 | Mar 2023 | CN | national |