Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Fluorescent probe for detecting pH value and preparation method thereof, and special detection kit

A ph value and carbon number technology, applied in the fields of biological analysis and chemical analysis, can solve the problems of inability to ratio measurement and short analysis wavelength, and achieve the effect of good biocompatibility and cell membrane permeability, and fast color reaction speed.

Active Publication Date: 2014-10-08
INST OF CHEM CHINESE ACAD OF SCI
View PDF3 Cites 28 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These probes have drawbacks such as short analytical wavelengths, or inability to perform ratiometric determinations
However, a near-infrared probe capable of labeling lysosomes and ratiometrically measuring lysosomal pH has not yet been reported.
[0003] Hemicyanine dyes have good stability and are widely used in biological analysis; however, ratiometric near-infrared fluorescent probes based on hemicyanine dyes that can be selectively labeled and detect lysosomal pH have not been reported yet.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescent probe for detecting pH value and preparation method thereof, and special detection kit
  • Fluorescent probe for detecting pH value and preparation method thereof, and special detection kit
  • Fluorescent probe for detecting pH value and preparation method thereof, and special detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the preparation of fluorescent probe Lyso-pH

[0083] Step a): Dissolve 276 mg of 2,4-dihydroxybenzaldehyde and 174 mg of morpholine in 30 mL of CH 3 OH, slowly add 91mg NaBH under ice bath 4 , after stirring at 25°C for 1 h, add water to quench, adjust the pH of the solution to 6 with 2M hydrochloric acid, extract with ethyl acetate, collect the organic phase, dry and concentrate with anhydrous sodium sulfate, and separate the obtained crude product by column chromatography (silica gel G, 200-300 mesh), CH 2 Cl 2 / MeOH (v / v, 20:1) was used as the eluent to obtain 67 mg of intermediate 4-(morpholinemethyl)phenyl 1,3-diol (yield: 16%). The product is a white solid.

[0084] 1 H NMR (400MHz, CDCl 3 ): δ6.83(d, J=8.0Hz, 1H), 6.34(d, J=2.4Hz, 1H), 6.29(dd, J=2.4Hz, 1H), 3.75(s, 4H), 3.64(s ,2H),2.56(s,4H); 13 C NMR (400MHz, CDCl3): δ158.5, 157.1, 129.7, 112.8, 106.7, 103.5, 66.9, 61.4, 52.8. HR-ESI-MS (m / z): [M]calcd.for C 11 h 15 NO 3 , 208.0969; f...

Embodiment 2

[0087] Spectral properties of the compound shown in embodiment 2, formula II

[0088] Weigh 6.3mg of Lyso-pH, dissolve it in 10mL DMSO, and prepare the mother solution (1mM) to obtain the Lyso-pH kit. Add 50 μL of this mother solution dropwise into 0.2M phosphate buffered saline solution with different pH values, and dilute to 5 mL with the corresponding phosphate buffered saline solution. Measure its fluorescence emission spectrum. Deexcite with 635nm during fluorescence emission spectrum measurement, the intensity ratio of emission peak is 1 670 / I 708 or I 708 / I 670 ; The slit width for excitation and emission is 10 nm, and the voltage is 700 V.

[0089] figure 2 It is the color response diagram of the Lyso-pH kit to aqueous solutions of different pH values, by figure 2 It can be seen that with the increase of the pH value, the naked eye can observe that the solution gradually changes from blue to green, which proves that the kit of the present invention has an in...

Embodiment 3

[0092] Embodiment 3, the labeling of lysosome and the mensuration of lysosome pH value

[0093] 1) At 37°C and 5% (v / v) CO 2 Under these conditions, HeLa cells were cultured with DMEM medium containing 10% (v / v) FBS (fetal bovine serum), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were washed with PBS buffer before use.

[0094]2) Add PBS (pH7.4) to HeLa cells, then add LysoTracker Red DND-99 (50nM) and incubate for 5min, wash with PBS three times, add Lyso-pH (50nM) and incubate for 5min, wash with PBS three times , for confocal fluorescence imaging of colocalization. Among them, the excitation wavelength of LysoTracker Red DND-99 is 559nm, and the collection band is 570-650nm; the excitation wavelength of Lyso-pH is 635nm, and the collection band is 650nm-750nm.

[0095] Figure 5 The lysosome co-localization map of the fluorescent probe Lyso-pH prepared in Example 1 and the LysoTracker Red DND-99 of American Probe Company for cell imaging was obtained by Fi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a fluorescent probe for detecting pH value and a preparation method thereof, and a special detection kit. The structure of the fluorescent probe for detecting pH value is disclosed as Formula I. The invention also discloses a preparation method of the compound disclosed as Formula I, which comprises the following steps: (1) carrying out reduction reaction on dihydroxybenzaldehyde disclosed as Formula IV and morpholine to obtain substituted morpholinyl methoxyphenyl diol disclosed as Formula V; and (2) in an inert atmosphere, carrying out substitution decomposition rearrangement reaction on the substituted morpholinyl methoxyphenyl diol disclosed as Formula V and tricarbocyanine dye disclosed as Formula VI in the presence of alkali to obtain the compound disclosed as Formula I. The kit for detecting pH value comprises the compound disclosed as Formula I and a solvent. The fluorescent probe and special detection kit thereof have favorable responsivity for pH value of solutions, can implement marking of lysosome and detection of pH value of the cytolysosome, is simple to operate, has the advantages of low cost, high speed, high sensitivity and the like, and can easily implement popularization and application.

Description

technical field [0001] The invention relates to a fluorescent probe for detecting pH value, a preparation method thereof and a special detection kit, belonging to the fields of chemical analysis and biological analysis. Background technique [0002] Lysosomes (lysosomes) are organelles in eukaryotic cells, with a pH value of about 3.8-5.0. They are single-membrane-coated sac-like structures with a diameter of about 0.025-0.8 microns, and contain various hydrolytic enzymes. The basic function of lysosomes is to digest and remove substances such as biological macromolecules, and play an important role in maintaining normal metabolic activities of cells and defending against microbial infection. Therefore, it is of great significance to detect the pH value of lysosome and its change with environmental factors. At present, the probes with the function of labeling lysosomes on the market include: LysoTracker Red DND-99 (Abs: 577nm; Em: 590nm) of American Probe Company, LysoTrack...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07D405/06C09K11/06C09B23/10G01N21/64
CPCC07D405/06C09B23/105C09K11/06C09K2211/1029C09K2211/1033C09K2211/1088G01N21/64
Inventor 马会民万琼琼
Owner INST OF CHEM CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com