布朗葡萄藻脂质含量的荧光光谱检测方法的改进

Improved fluorescence spectrometric determination of lipid content in Botryococcus braunii

摘要:

布朗葡萄藻是一种含脂量高、开发潜力大的能源微藻，但因其细胞呈集落态且胞壁较坚厚，应用传统的尼罗红荧光光谱法测定其脂质含量时，通常因灵敏度低、重复性差而难以获得可靠结果。本研究通过超声波分散集落、二甲基亚砜辅助尼罗红进入细胞、优化染色条件，有效地解决了上述问题。具体方法为：藻液先经频率为20 kHz、发射功率为100 W的超声波按1 s/1 s (开/关) 间歇处理20 s使细胞分散，而后依次加入与藻液等体积的15％二甲基亚砜溶液和3 μg/mL尼罗红溶液，充分混匀后于黑暗中40 ℃染色10 min，再于490 nm激发光下进行荧光光谱检测。上述改进方法与传统方法相比，检测灵敏度提高了196.6％，反映检测重复性的相对标准偏差 (RSD) 由10.91％降至1.84％。该方法为布朗葡萄藻育种与培养等研究提供了快速、灵敏的脂质含量检测技术。

Abstract:

Botryococcus braunii is a unique colonial green microalga and a great potential renewable resource of liquid fuel because of its ability to produce lipids. Due to the dense cell colonies and rigidly thick cell wall of B. braunii, the traditional Nile red method is usually of low sensitivity and bad repeatability and hard for the determination of lipid content in the cells. By dispersing the colony with ultrasonic, assisting permeation of Nile red across the cell wall with dimethyl sulfoxide and optimizing the staining conditions, we established an improved detection method. The details were as follows: after the colonial algal sample was treated by ultrasonic at 20 kHz for 20 s, 100 W transmitting power and with 1 s on/1 s off intermittent cycle, the equivoluminal 15% (V/V) dimethyl sulfoxide and 3 μg/mL Nile red were successively added and mixed evenly, then the staining system was incubated in dark at 40℃ for 10 min, and subsequently was measured by fluorescence spectroscopy detection with an excitation wavelength of 490 nm. Compared with the traditional method, the improved one not only had higher detection sensitivity which was increased by 196.6%, but also had obviously better detection repeatability whose characteristic parameter - relative standard deviation (RSD) was decreased from 10.91% to 1.84%. Therefore, the improved method could provide a rapid and sensitive detection of lipid content for B. braunii breeding and cultivation.