A fluorescent probe for detecting pH value, its preparation method and special detection kit
A ph value and reducing agent technology, applied in the fields of biological analysis and chemical analysis, can solve the problems of short analysis wavelength and inability to measure ratio, and achieve the effect of fast color reaction.
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Embodiment 1
[0082] Embodiment 1, the preparation of fluorescent probe Lyso-pH
[0083] Step a): Dissolve 276 mg 2,4-dihydroxybenzaldehyde and 174 mg morpholine in 30 mL 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.forC 11 h 15 NO 3 , 208.0969; found, 208....
Embodiment 2
[0087] Spectral properties of the compound shown in embodiment 2, formula III
[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 i...
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 (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) for 5min, wash with PBS three times, Perform confocal fluorescence imaging for 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 RedDND-99 of American Probe Company for cell imaging was obtained by Figure 5 It...
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