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Precipitated alkaline phosphatase fluorescence probe, and synthetic method and applications thereof

A fluorescent probe and synthesis method technology, applied in the field of fluorescent probes, can solve the problems of poor fluorescence imaging effect and easy diffusion of fluorescent probe signals, and achieve the effects of fast response, good dyeing effect, and simple synthesis method

Active Publication Date: 2017-11-21
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of precipitated alkaline phosphatase fluorescent probe and its synthesis method and application, to solve the in situ detection of endogenous alkaline phosphatase activity in living cells, and the easy diffusion of fluorescent probe signal in cell imaging And technical problems such as poor fluorescence imaging effect

Method used

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  • Precipitated alkaline phosphatase fluorescence probe, and synthetic method and applications thereof
  • Precipitated alkaline phosphatase fluorescence probe, and synthetic method and applications thereof
  • Precipitated alkaline phosphatase fluorescence probe, and synthetic method and applications thereof

Examples

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Effect test

Embodiment 1

[0036] Example 1 Synthesis of Precipitated Alkaline Phosphatase Fluorescent Probe HTPQA

[0037] (1) 4-Hydroxy-tetraphenylethylene (3.482g) and urotropine (2.102g) were dissolved in glacial acetic acid (50mL), heated under reflux for 3h, dichloromethane and petroleum ether were eluents (two The volume ratio of methyl chloride and petroleum ether is 1:5) to obtain a light yellow intermediate (1.316g, 35%) through column separation;

[0038] (2) Reflux and stir the light yellow intermediate (0.3761g), 2-amino-5-chlorobenzamide (0.2050g) and p-toluenesulfonic acid (10mg) in absolute ethanol for 5h, add dichlorodi Cyanobenzoquinone (0.5g), suction filtration obtains yellow solid, washes solid with ethanol, dries to obtain pure yellow compound (0.43g, 81.5%), i.e. novel solid-state fluorescent dye i.e. 6-chloro-2-( 2-Hydroxy-5-(1,2,2-triphenylethenyl)phenyl)-4(3hydro)-quinazolinone, named HTPQ;

[0039] Mass Spectrometry and NMR Characterization of HTPQ:

[0040] 1 HNMR(DMSO-d6...

Embodiment 2

[0048] Embodiment 2HTPQA probe is to the detection of alkaline phosphatase in test tube

[0049] The HTPQA probe was made into 1 mM DMSO stock solution and stored at -20°C. The detection system is TBS buffer solution (10 mM, pH 8.0, containing 5% DMSO). The reaction system of HTPQA probe and alkaline phosphatase was shaken at 37° C. for 30 minutes, and then its fluorescence emission spectrum was measured. The excitation wavelength of the fluorescence instrument is set to 410 nm, and the emission wavelength receiving range is 500-700 nm. The result is as figure 2 shown, from figure 2 It can be seen that the HTPQA probe has a good response to alkaline phosphatase.

Embodiment 3

[0050] The reaction kinetic investigation of embodiment 3HTPQA probe and alkaline phosphatase

[0051] The reaction system is TBS buffer solution (10 mM, pH 8.0, containing 5% DMSO). The fluorescence emission spectra of the HTPQA probe and alkaline phosphatase with different activities were measured every 2 minutes at 37 °C until the fluorescence intensity no longer changed. Then take the intensity of the maximum emission peak as the ordinate, and the reaction time as the abscissa to make a graph, as image 3 The reaction kinetic curve shown, from image 3 It can be seen that the HTPQA probe responds rapidly to phosphatases. The excitation wavelength of the fluorescence instrument is set to 410 nm, and the emission wavelength receiving range is 500-700 nm.

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Abstract

The invention discloses a precipitated alkaline phosphatase fluorescence probe, and a synthetic method and applications thereof. The synthetic method of the fluorescence probe comprises the following steps: 1, synthesizing a novel solid fluorescence dye (HTPQ); and 2, designing the dye to form the alkaline phosphatase probe (HTPQA). The applications comprise the response performance of the fluorescence probe to alkaline phosphatase in a buffer solution and the ability of fluorescence probe in in-situ detection of cell endogenous alkaline phosphatase and fluorescence signal stabilization indiffusion. The precipitated solid fluorescence dye is synthesized for the first time, so problems of no in-situ detection and easy signal diffusion of traditional fluorescence probes are solved; the fluorescence probe has solid fluorescence properties, can effectively reduce background fluorescence signals, and has improved sensitivity; and the fluorescence probe has the advantages of very good living cell dyeing effect, short dyeing time, high dyeing efficiency, and realization of in-situ detection of the cell endogenous alkaline phosphatase.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and relates to a precipitation-type alkaline phosphatase fluorescent probe and its synthesis method and application. Background technique [0002] Small-molecule fluorescent probes can effectively monitor and image events related to cell life activities. In the past few decades, small-molecule fluorescent probes have been widely used in the detection of biological targets. However, most of the existing fluorescent probes are designed based on cytoplasmic-soluble dyes, such as coumarin, rhodamine, and fluorescein. These fluorescent probes, if not localized to specific organelles, generally cannot provide in situ information on biological events because fluorescent signaling molecules diffuse rapidly away from the target reaction site. Fluorescent signaling molecules can then diffuse out of the cell, reducing the fluorescent signal, resulting in low signal-to-noise ratio and low sensiti...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 张晓兵刘红文谭蔚泓
Owner HUNAN UNIV
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