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
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  • 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.
[0

Method used

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  • 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

Example Embodiment

[0082] Example 1. Preparation of fluorescent probe Lyso-pH

[0083] Step a): Dissolve 276mg 2,4-dihydroxybenzaldehyde and 174mg morpholine in 30mL CH 3 In OH, slowly add 91mg NaBH under ice bath 4 After stirring at 25°C for 1 hour, quench with water, 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 resulting crude product by column chromatography (Silica gel G, 200~300 mesh), CH 2 Cl 2 / MeOH (v / v, 20:1) was used as an eluent for purification to obtain 67 mg of intermediate 4-(morpholinomethyl)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; found,208.0975.

[0085] Step b): Combin...

Example Embodiment

[0087] Example 2. Spectral properties of the compound represented by formula II

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

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

Example Embodiment

[0092] Example 3. Labeling of lysosomes and determination of lysosomal pH

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

[0094] 2) Add PBS (pH 7.4) 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 , Perform confocal fluorescence imaging and 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 For the lysosome co-localization map of the fluorescent probe Lyso-pH prepared in Example 1 and LysoTracker Red DND-99 of American Probe Company for cell imaging, Figure 5 It can be seen that the co-local...

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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

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Application Information

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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
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