Total membrane lipid assay (MLA): simple and practical quantification of exosomes based on efficient membrane-specific dyes unaffected by proteins†,Materials Chemistry Frontiers

当前位置： X-MOL 学术 › Mater. Chem. Front. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Total membrane lipid assay (MLA): simple and practical quantification of exosomes based on efficient membrane-specific dyes unaffected by proteins†
Materials Chemistry Frontiers ( IF 6.0 ) Pub Date : 2018-09-19 00:00:00 , DOI: 10.1039/c8qm00300a
Ning Li _{1,

2,

3,

4} , Zhenlong Huang _{1,

2,

3,

4} , Zhiwei Ye _{1,

2,

3,

4} , Xinfu Zhang _{1,

2,

3,

4} , Lingcheng Chen _{1,

2,

3,

4} , Yi Xiao _{1,

2,

3,

4}

Affiliation

There has been a rapidly rising interest directed toward exosomes owing to their potential application in diagnosis and prognosis of major diseases. However, forthright and low-cost but accurate quantification of isolated or purified exosomes remains a technical challenge. Herein, we have developed a practical fluorimetry method to ratiometrically detect exosomes. Different from the conventional BCA total protein assay to quantify exosomes, we choose the exosome membrane lipids as the analyte and propose a new exosome quantification method called Total Membrane Lipid Assay (MLA). Our idea is based on two fluorescent dyes of completely different nature. The first one (Dye A) should be a membrane-specific dye that is non-fluorescent in aqueous buffer solutions but emits strong fluorescence once inserted into the membrane phospholipid bimolecular layer. The second one (Dye B) should be a highly water-soluble dye that has no affinity to membranes of exosomes. Therefore, the former is in charge of sensing exosomes and the latter acts as the internal reference insensitive to exosomes. The ratio of Dye A's fluorescence intensity to Dye B's (R_A/B) can be used as the quantitative basis. We have screened several candidate dyes, and among them we have obtained two that meet our requirements for Dye A and Dye B, respectively. Then, a linear relationship between exosomes’ membrane lipid and R_A/B has been established, with the detection limit as low as 0.342 ng μL⁻¹ (MLA total membrane lipid content). Therefore, the analytical sensitivity of this ratiometric assay is recommendable. Finally, we confirm the practical applicability of this method by quantification of exosomes isolated from different real samples, including culture media of HeLa, MCF-7, MCF-10A cells and human serum.

中文翻译：

总膜脂质测定（MLA）：基于不受蛋白质影响的有效膜特异性染料，对外泌体进行简单，实用的定量分析†

由于外来体在重大疾病的诊断和预后中的潜在应用，因此人们对外来体的兴趣迅速上升。然而，分离的或纯化的外泌体的直接和低成本但准确的定量仍然是技术挑战。在这里，我们已经开发了一种实用的荧光检测方法来按比例检测外泌体。与传统的BCA总蛋白测定法定量外泌体不同，我们选择外泌体膜脂质作为分析物，并提出了一种新的外泌体量化方法，称为总膜脂质测定法（MLA）。我们的想法基于两种性质完全不同的荧光染料。第一个（染料A）应该是一种膜特异性染料，在水性缓冲溶液中不发荧光，但是一旦插入膜磷脂双分子层中就会发出强烈的荧光。第二种染料（染料B）应该是高度水溶性的染料，对外泌体的膜没有亲和力。因此，前者负责感测外泌体，后者充当对外泌体不敏感的内部参考。之比染料A的荧光强度，以染料B的（- [R _{A / B}）可以用作定量基础。我们已经筛选了几种候选染料，其中我们已经获得了两种满足我们对染料A和染料B要求的染料。，分别。然后，外来体膜脂质和之间的线性关系- [R _{A / B}已经建立，与检测限为0.342纳克μL ^-1（MLA总膜脂含量）。因此，这种比例测定的分析灵敏度是可取的。最后，我们通过量化从不同的真实样品（包括HeLa，MCF-7，MCF-10A细胞和人血清）的分离出的外泌体来证实该方法的实用性。

更新日期：2018-09-19

点击分享查看原文

点击收藏

阅读更多本刊最新论文