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Preparation method of a two-photon fluorescent pH probe and its application in cell imaging

A two-photon fluorescence and cell technology, which is applied in the field of preparation of two-photon fluorescence pH probes, to achieve the effects of improved clarity, high detection sensitivity, and good cell permeability

Active Publication Date: 2020-12-04
ZUNYI NORMAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the research process of the dicyano-stilbene two-photon fluorescent dye, the researchers of the present invention focused on the application of the compound probes code-named D1-1 and D3-1 in solvent polarity determination, micro-viscosity determination, environmental Research on temperature measurement and solid hardness detection (see invention patent application CN200710010784.6). The detection of intracellular sugar chain antigen activity detection and temperature sensing detection and other aspects have been studied and reported, but the D1 compound containing long flexible groups based on dicyano-stilbene is used for intracellular pH detection and The invention of imaging has not been reported

Method used

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  • Preparation method of a two-photon fluorescent pH probe and its application in cell imaging
  • Preparation method of a two-photon fluorescent pH probe and its application in cell imaging
  • Preparation method of a two-photon fluorescent pH probe and its application in cell imaging

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Effect test

Embodiment 1

[0041] Embodiment 1 Molecular structure and synthetic route

[0042] A two-photon fluorescent labeling probe D1 has the following molecular structure:

[0043]

[0044] The synthetic route is:

[0045]

[0046] Wherein, intermediates 2, 3, 4, 5, and 6 were all synthesized according to the following documents:

[0047] [1] H. Huang, Q. He, H. Lin, F. Bai, Z. Sun and Q. Li, Polym. Adv. Technol., 2004, 15 (): 84—88

[0048] [2] Huang C., Fan J., Peng X., Lin Z., Guo B., Ren A., Cui J., Sun S., J. Photochem. Photobio. A: Chem., 2008, 199 (2) –3):144—149

Embodiment 2

[0049] The preparation of embodiment 2 intermediate 7,8 and target compound D1

[0050] Preparation method: (1) Synthesize intermediates 2, 3, 4, 5 and 6 according to conventional methods;

[0051] (2) Synthesis of Intermediate 7: Accurately weigh carbazole (5g, 30mmol) and potassium hydroxide (3g, 53.6mmol) into a 100mL dry single-necked flask filled with 30mL DMSO, and accurately measure 4mL with a pipette 1-Bromooctane is placed in a constant pressure dropping funnel, and a constant pressure dropping funnel is installed on the flask; the flask is placed in an oil bath at 80-85°C, and 1-bromooctane is dripped under stirring, and reacted for 3 hours. After cooling, add 100mL of distilled water and mix; after standing still, a light yellow solid precipitates out, filter and dry; recrystallize with absolute ethanol to obtain white needle-like crystals, the yield is 68% (5.7g), and the melting point is 68-70°C;

[0052] 1 H NMR (CDCl 3 ,400MHz)δ:8.074(d,J=7.6Hz,2H),7.428(t,J ...

Embodiment 3

[0057] The performance research of embodiment 3 probe D1

[0058] The photophysical properties of probe D1 are shown in Table 1 below.

[0059] Table 1 Photophysical properties of D1

[0060]

[0061] Note: a) Maximum single-photon absorption wavelength (nm); b) Maximum single-photon emission wavelength (nm); c) Fluorescence quantum yield; d) Maximum two-photon excitation wavelength (nm); e) Stokes shift ( nm); f) Maximum two-photon absorption cross section (GM).

[0062] As in Table 1, attached Figure 1-2 As shown, D1 has a rather large two-photon absorption cross-section (δ), at CH 2 Cl 2 with H 2 The δ in O is as high as 3000 and 1540GM, respectively.

[0063] combined with image 3 It can be seen that the fluorescence intensity of probe D1 increases as the pH of the solution increases, and when the pH of the solution is close to 9, the fluorescence intensity hardly increases.

[0064] according to Figure 5 It can be seen that the Sigmoidal fitting curve of th...

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Abstract

The invention belongs to the technical field of fine chemical engineering, and particularly relates to a preparation method of a two-photon fluorescence pH probe and application in cell imaging thereof. The probe is prepared by taking dicyan-biphenyl-ethylene as a matrix, and comprises carbazolyl, di-cyano and n-octyl. Because the molecular structure of the probe has a longer flexible chain, the lipophicity is stronger, the probe can penetrate a cell wall (or cell membrane) into a cell to stain the cell for detecting the pH in the cell; the molecular structure of the probe belongs to D-pi-A type molecules, the two-photon absorption cross section (delta) is improved, so that the probe has higher imaging definition and resolution; in addition, the probe has a large Stokes shift in the marking process, thereby greatly improving the imaging accuracy and precision; the fluorescence quantum yield (phi) of the probe in a solvent is higher, the two-photon emission cross section is larger, so that the higher the brightness of the imaging is, the clearer the image is, and the higher the resolution is.

Description

technical field [0001] The invention relates to the field of fine chemical industry, in particular to a preparation method of a two-photon fluorescent pH probe and its application in cell imaging. Background technique [0002] Most activities within cells are pH-sensitive, including cell volume regulation, vesicle trafficking, cell polarization, fiber contraction, and scaffold establishment. Changes in intracellular pH affect intracellular signaling molecules such as Ca 2+ , cAMP activity, thus affecting the transduction of intracellular signals. The methods for measuring intracellular pH mainly include weak acid and weak base distribution method, nuclear magnetic resonance method, microelectrode method, and immunofluorescence probe method which is widely used at present. [0003] After continuous development, fluorescent probe technology is becoming more mature. There are many kinds of pH fluorescent dyes, and each dye has a different working pH range. The widely used one...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D209/86C09K11/06G01N21/64
CPCC07D209/86C09K11/06C09K2211/1007C09K2211/1029G01N21/6402G01N21/6458G01N21/6486
Inventor 黄池宝
Owner ZUNYI NORMAL COLLEGE
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