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A kind of two-photon lysosome pH fluorescent probe and its preparation method and application

A fluorescent probe, two-photon technology, applied in fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of inability to apply real-time high-resolution detection of living tissue, photodamage and photobleaching, and cell photodamage, etc. Achieving the effect of good cell membrane permeability, environmental protection and low cost

Inactive Publication Date: 2019-02-26
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] So far, most of the lysosomal pH fluorescent probes reported in the literature are single-photon fluorescent probes, which are usually limited to short-wavelength excitation (400-560nm), resulting in greater photodamage and photobleaching; they can also cause photodamage and autologous Luminescent, cannot be applied to real-time high-resolution detection of living tissue pH

Method used

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  • A kind of two-photon lysosome pH fluorescent probe and its preparation method and application
  • A kind of two-photon lysosome pH fluorescent probe and its preparation method and application
  • A kind of two-photon lysosome pH fluorescent probe and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The synthetic steps of probe of the present invention are as follows:

[0028]

[0029] (1) In a sealed tube, add 0.446g of 2-methylbenzimidazole, 0.237g of 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde and trimethyl Dissolve 2.28 ml of mole chlorosilane in N,N-dimethylformamide, and heat at 100°C for 24 hours.

[0030] (2) Add saturated Na to the reaction solution 2 CO 3 After adjusting the pH value of the solution to weak alkaline, stir for 1 hour, and use CH 2 Cl 2 Extracted 3 times, combined organic phase, anhydrous Na 2 SO 4 After drying, the crude product was obtained by distillation under reduced pressure.

[0031] (3) The crude product was separated by a silica gel column to obtain the pure product. 1 H-NMR (DMSO-d 6 ,400MHz)δ12.59(m,1H),8.47(d,J=1.2Hz,1H),8.24(d,J=7.6Hz,1H),7.83(d,J=16.4Hz,1H),7.77- 7.79(dd, J=1.2Hz, 1H), 7.64-7.69(dd, J=8.2Hz, 2H), 7.59(d, J=7.2Hz, 1H), 7.45-7.49(m, 2H), 7.22-7.26 (m,2H),7.13-7.20(m,2H),4.59(t,J=5.4Hz,2...

Embodiment 2

[0033] The probe in Example 1 was used to prepare a stock solution with a concentration of 1 mM in DMSO. In the experiment, the probe was diluted with water / DMSO (V / V=4 / 1) system to make the final concentration 30 μM. Regulate the pH value of this system with the HCl of 1mol / L, record the ultraviolet absorption spectrogram under different pH conditions ( figure 1 ). As the pH value decreased from 6.8 to 2.5, the absorption peak at 359nm decreased gradually, and the absorption peak at 396nm increased in turn, and there was an obvious isoelectric point at 380nm. figure 2 It shows that the color of the solution changes from colorless to yellow. The picture on the left is a colorless solution without adding a sample, and the picture on the right is a yellow solution with HCl added to a sample.

Embodiment 3

[0035] Similarly, the probe was diluted to 10 μM with water / DMSO (V / V=4 / 1) system for fluorescence spectrum titration, the excitation wavelength was fixed at 360 nm, and the fluorescence emission spectra under different pH conditions were recorded. like image 3 As shown, under the neutral condition of pH 6.8, the maximum fluorescence emission of the probe is located at 454nm. As the pH decreases, the fluorescence emission at 454nm decreases gradually, and a new fluorescence emission appears at 514nm and is significantly enhanced. exhibits remarkable ratiometric fluorescence emission properties (F 454nm / F 514nm ). At the same time, a clear isoelectric point appears at 500nm. Under the irradiation of ultraviolet light, the color of the solution changes from the blue of the left sample to the green of the right sample ( Figure 4 ). According to the fluorescence intensity ratio (F 454nm / F 514nm ) and the fitting curve of different pH values ​​to calculate pK a The valu...

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Abstract

The invention provides a two-photon lysosome pH fluorescence probe, a preparation method thereof and an application. The probe is a pH fluorescence probe based on an intramolecular charge transfer effect and structured by bridging benzimidazole with carbazole derivative. The preparation method includes steps of, dissolving benzimidazole and carbazole derivative in a few amount of N, N- dimethyl formamide; adding trimethylchlorosilane to catalyze and reacting it in a seal pipe; then adding saturated Na2CO3 solution and adjusting pH value to alkalescence; acquiring a pure product through vacuum concentration and silicagel column separation. The probe has pKa value approaching to pH value balance of lysosome. The probe has high sensitivity, good selectivity and big Stocks displacement to H+. After combining with H+, the probe has obvious color change, and can be identified by bared eyes. The probe takes the carbazole derivative as a two-photon fluorescence group; the two-photon fluorescence imaging technology is used for proving that the probe and the marketed green lysosome probe have good common polarization, and can be applied to detect pH value change in the HeLa cell lysosome.

Description

technical field [0001] The invention relates to a lysosome pH fluorescent probe, in particular to a benzimidazole two-photon lysosome pH fluorescent probe and a preparation method thereof, as well as the application of the probe in detecting intracellular lysosome pH. Background technique [0002] The pH value in the body is closely related to many important physiological processes of cells and organelles. Different cells and organelles have different pH balances. Lysosomes are typically acidic organelles with a pH range of 4.5-5.5, and abnormal lysosomal pH changes have been linked to a genetically inherited disorder called lysosomal storage disorders. Therefore, it is necessary to monitor the changes of acidic pH in lysosomes sensitively and accurately in real time, which is of great significance for studying the physiological and pathological processes of cells at the molecular level. [0003] So far, most of the lysosomal pH fluorescent probes reported in the literatur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D403/10C09K11/06G01N21/64
CPCC07D403/10C09K11/06C09K2211/1044G01N21/6486
Inventor 葛金印樊丽梁文婷张凯张雯佳双少敏黄文成董川
Owner SHANXI UNIV
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