A fluorescent probe for detecting tyrosinase and its preparation method and application
A technology of fluorescent probe and tyrosinase, which is applied in the field of fluorescent probe for detecting tyrosinase and its preparation, and achieves the effects of easy popularization and application, detection wavelength and good biocompatibility
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Embodiment 1
[0058]Embodiment 1, the preparation of the fluorescent probe (formula I) that tyrosinase detects
[0059] according to figure 1 The chemical reaction scheme shown in the preparation was carried out.
[0060] Step 1): Dissolve 0.78 mmol of resorcinol in acetonitrile, add 0.78 mmol of potassium carbonate, and continue stirring for 15 minutes under the protection of Ar gas, then add 0.31 mmol of IR-780, and continue the reaction at 60°C for 5 hours. After filtration, the filtrate was evaporated to dryness under reduced pressure to obtain the crude product. Separation and purification by column chromatography yielded a hemicyanine as a purple solid.
[0061] Step 2): Dissolve 0.5 mmol of hemicyanine in anhydrous acetonitrile, then add 1 mmol of sodium hydride, stir for 15 minutes under the protection of Ar gas, and then add 3-hydroxybenzyl bromide. After reacting at 60°C for 12 hours, evaporate to dryness under reduced pressure to obtain the crude product. Separated by chromat...
Embodiment 2
[0065] Embodiment 2, the preparation of tyrosinase detection kit
[0066] Weigh 1.29 mg of the compound shown in Formula I obtained in Example 1 as a fluorescent probe, dissolve it in 10 mL of solvent dimethyl sulfoxide, and make a fluorescent probe stock solution (also known as mother solution, 0.2 mM), to obtain tyrosinase Detection kit.
[0067] The kit of the present invention detects tyrosinase specifically according to the following steps:
[0068] 1) Make a standard curve:
[0069] Taking 670nm as the excitation wavelength, measure the concentration of tyrosinase respectively 2mL of tyrosinase standard reaction solution of 0, 10, 20, 40, 60 and 80U / mL, and the fluorescence intensity at the emission wavelength of 708nm, record for F;
[0070] And measure the fluorescence intensity of the reagent blank at an emission wavelength of 708nm, denoted as F 0 ;
[0071] Taking the concentration C of tyrosinase as the abscissa, the change value of fluorescence intensity ΔF=F...
Embodiment 3
[0087] Embodiment 3, the application of tyrosinase detection kit
[0088] 1. Detection of tyrosinase in cells
[0089] 1) At 37°C and 5% CO 2 Under these conditions, the cells were cultured with DMEM medium containing 10% (v / v) fetal calf or calf serum, 100 U / mL penicillin, and 100 μg / mL streptomycin (the cultured cells were human cervical cancer cell HeLa and mouse melanin tumor cell B16 cells). Cells were washed with DMEM before use.
[0090] 2) Add 1mL DMEM to the cells, then add fluorescent probe (5μM) and incubate for 2h, then perform confocal fluorescence imaging, the excitation wavelength is 635nm, and the collection wavelength range is 650-750nm.
[0091] Such as Figure 6 Shown are the confocal imaging images of the kit of the present invention used in human cervical cancer cell HeLa (low expression of tyrosinase) and mouse melanoma cell B16 (high expression of tyrosinase).
[0092] Such as Figure 7 Shown is the confocal imaging image of B16 cells previously in...
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