荧光探针法测定极地雪藻自由基含量的研究

Determination of Free Radicals in Snow Algae Chlamydomonas nivalis using Fluorescence Probe

摘要:

本文研究利用二氯荧光素双醋酸盐（DCFH-DA）作为荧光探针来测定极地雪藻Chlymydomonas nivalis中自由基的含量。DCFH-DA进入雪藻细胞后由还原型二氯荧光素（DCFH）迅速氧化成氧化型二氯荧光素DCF，其氧化过程的氧化量与自由基的含量成正比，通过荧光分光光度计可直接进行检测。研究表明最佳的测定条件为：荧光探针DCFH-DA用量为60 μmol/L，反应时间1~2.5 h，藻液细胞数为5106~20106 个/mL。荧光强度的测定条件为：激发波长：485 nm，发射波长：520 nm。在上述条件下能够稳定、快速地测定藻类细胞中自由基的含量。

Abstract:

The free radicals in snow algae Chlamydomonas nivalis were determined using 2’, 7’-dichlorodihydrofluorescin diacetate (DCFH-DA) as fluorescence probe. In the cell, DCFH-DA was hydrolyzed to 2’, 7’-dichlorodihydrofluorescin (DCFH), and then quickly oxidized to fluorescent compound 2’, 7’-dichlorofluorescin (DCF). The quantity of oxidation was in direct proportion to the content of free radicals. For the determination of free radicals by fluorescence spectrophotometer, the best dosage of DCFH-DA, cell counts and reaction time were 60μmol/L, 5×106~20×106 cell/mL and 1~2.5 h, respectively. And the best excitation and emission wavelength were 485 nm and 520 nm, respectively. Under those conditions, the free radicals in algae can be stably and rapidly determined.