尼罗红荧光优化法快速检测微拟球藻细胞内油脂含量

Modified rapid determination of lipid content in Nannochloropsis by Nile Red fluorescence staining

摘要:

【目的】利用尼罗红荧光染色法快速检测微拟球藻细胞内的油脂含量。【方法】系统性地调整激发波长与发射波长，确定最佳二甲基亚砜(DMSO)浓度、尼罗红终浓度、染色时间和细胞密度的范围，比较重量法和Triolein标准品的油脂含量分别与荧光强度之间的关系。【结果】分析得出了尼罗红荧光强度与微拟球藻细胞油脂含量的关系，确定优化后染色条件为：二甲基亚砜浓度为5%，尼罗红终浓度为1 mg/L，染色时间为6 min，且细胞密度为(0.5?3.0)×106 cells/mL的范围内，激发波长和发射波长分别为515 nm和570 nm。重量法和Triolein标准品的油脂含量分别与尼罗红染色荧光强度之间的关联性，表明尼罗红荧光染色方法可以用来快速准确地检测细胞内的油脂含量，且油脂含量与荧光强度之间正相关，相关系数R2为0.997 3。尼罗红染色优化后油脂的检测下限达到2 μg，大大减少了测定油脂含量所需细胞量。【结论】针对不同种属系统性地确认了荧光激发波长和发射波长，并优化验证得到了最佳尼罗红荧光染色条件，可以快速准确地检测微量微拟球藻细胞内的油脂含量，便于大规模筛选高产油突变藻株。

Abstract:

[Objective] This study was aimed to investigate the optimal Nile Red fluorescence staining method to quickly determine the quantification of total lipid in Nannochloropsis. [Methods] We systematically adjusted the excitation wavelength, emission wavelength and optimized the dimethyl sulfoxide (DMSO) concentration, Nile Red concentration, staining time and cell density range. Meanwhile, we evaluated the relationship between the florescence intensity of Nile Red staining and the lipid contents of gravimetric method and Triolein standard method, respectively. [Results] The relationship between the Nile Red fluorescence intensity and the lipid content of green algae Nannochloropsis was obtained. The results showed that the optimal dyeing conditions are as follows: DSMO concentration was 5%, the final concentration of Nile Red was 1 mg/L, staining time was 6 min and the cell number ranged from 0.5×106 to 3.0×106 cells/mL, under the excitation and emission wavelengths was 515 nm and 570 nm. The correlation between the lipid content from gravimetric method and Triolein standard indicated that the Nile Red fluorescence staining method can be used to rapidly and exactly detect the lipid content in Nannochloropsis. The lipid content was positively correlated with the fluorescence intensity, and the correlation coefficient R2 was 0.997 3. The detection limit of lipid content reached 2 μg after the optimization of Nile Red staining, which greatly reducing the amount of cells required for the determination of lipid content. [Conclusion] These results indicated that the Nile Red fluorescence staining method can be used to determine the lipid content rapidly and exactly in the Nannochloropsis with a microdose cell, and be applied in screening Nannochloropsis mutants with higher lipids content in large scale, conveniently.