Bi-locus high-sensitivity pH fluorescent probe and synthesis and application thereof
A fluorescent probe and double-site technology, applied in the field of high-sensitivity pH fluorescent probe, high-sensitivity pH fluorescent probe and its synthesis, and double-site, can solve the problems of interference of excitation light intensity of response signal and influence of detection results, etc. , to achieve the effect of simple synthesis method and high yield
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[0032] Example 1 Synthesis of fluorescent probe
[0033] (1) Synthesis of compound 1
[0034] Using ethanol as a solvent, add 2,4-dihydroxybenzaldehyde (1.38g, 10 mmol) and Michler’s acid (1.44 g, 10mmol), add a catalytic amount of pyrrolidine, heat and reflux for 24 hours; after cooling to room temperature Pour into water, extract with dichloromethane, dry, and distill to obtain a crude product, which can be separated and purified to obtain 7-hydroxy-2 carboxycoumarin; purified by column chromatography to obtain 1.21g of pure product, which is milky white crystals, with a yield of 61% . Its proton nuclear magnetic resonance spectrum such as figure 1 Shown. 1 H NMR (400 MHz, DMSO- d 6 )δ 8.46 (s, 1H), 7.64 (d, J = 8.6 Hz, 1H), 6.80 (dd, J = 8.6, 2.2 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H);
[0035] (2) Synthesis of compound 2
[0036] Using ethanol as the solvent, add rhodamine b (4.79 g, 10 mmol) and ethylenediamine (6 mL, 100 mmol) into the flask, heat and reflux for 24 hours; cool t...
Example Embodiment
[0039] Example 2 Spectral measurement of the response of fluorescent probe CR-pH to pH value
[0040] The fluorescent probe CR-pH in Example 1 was weighed and prepared with dimethyl sulfoxide (DMSO) to prepare a 5 mM mother liquor.
[0041] Take 10 μL of the probe mother solution and add it to a 5 mL volumetric flask, add buffer solutions of different pH values to constant volume, shake up and perform a spectrum test. The excitation wavelengths are 405 nm and 561 nm. The fluorescence wavelength is the abscissa and the fluorescence intensity is For the ordinate Figure 5 a, b. Figure 5 c is the change of fluorescence intensity at 405nm and 455nm with pH; and the change of fluorescence intensity at 561nm and 588nm with pH. Figure 5 d is the ratio of the fluorescence intensity at 588nm to the fluorescence intensity at 455nm as a function of pH. It can be seen from the figure that the intensity of red light and blue light and the ratio of red and blue light change drastically with ...
Example Embodiment
[0042] Example 3 Cell imaging study of fluorescent probe CR-pH
[0043] (1) Fluorescence imaging
[0044] Set the density to 3 × 10 5 Cells / mL HeLa cells were seeded into a sterilized 35 mm imaging petri dish, 2 Incubator (temperature 37 ℃, 5% CO 2 ) Cultivate for more than 12 hours to make the cells adhere to the wall. Dilute the mother liquor described in Example 2 to 1 mM, and add the diluent to the dead cell culture dish to make the final concentration all 5 μM. Continue to incubate for 0.5 h under the same conditions, then aspirate the cell culture fluid, wash the cells with PBS buffer three times, use 405 nm and 561 nm as excitation wavelengths, and use the blue and red channels for imaging.
[0045] (2) Study the changes of intracellular pH during autophagy
[0046] Set the density to 3 × 10 5 Cells / mL HeLa cells were seeded into a sterilized 35 mm imaging petri dish, 2 Incubator (temperature 37 ℃, 5% CO 2 ) Cultivate for more than 12 hours to make the cells adhere to the wall...
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