A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae
A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae
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DOI：
10.1016/j.mimet.2009.01.001
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发表时间：
2009-04-01
影响因子：
2.2
通讯作者：
Hu, Qiang
中科院分区：
文献类型：
--
作者：
Chen, Wei;Zhang, Chengwu;Hu, Qiang
分离高中性脂质含量的微藻是基于微藻的生物燃料生产取得商业成功的关键。尼罗红荧光法已成功地应用于某些微藻中的脂质测定，但在许多其他微藻中，特别是那些具有厚的刚性细胞壁的微藻中并不成功，这些细胞壁阻止了荧光染料的渗透。传统的“一次一个样品”的方法也很耗时。在这项研究中，溶剂二甲基亚砜（DMSO）被引入到微藻样品作为染色载体在高温下。使用96孔板在荧光分光光度计上测定和定量细胞中性脂质，激发波长为530 nm，发射波长为575 nm。一个优化的程序产生了较高的相关系数（R-2 = 0.998）与脂质标准品三油酸甘油酯和重复测量的重复。应用改进的方法对几种绿色藻进行了测定，在2 μ g/mL和20 μ g/mL两个浓度水平上的重复性和重现性的相对标准误差分别为8.5%、3.9%和8.6%、4.5%。此外，改进的尼罗红染色法的检测限和定量限分别为0.8 μ g/mL和2.0 μ g/mL的中性脂质标准三油酸甘油酯。改进的方法和传统的重量测定方法提供了类似的结果，对重复样品。基于96孔板的尼罗红方法可用作高通量技术，用于快速筛选更广谱的天然存在的和遗传修饰的藻类菌株和突变体，用于高中性脂质/油生产。(C)2009年由Elsevier B. V.出版
Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.