Issue 11, 2018

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

Abstract

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 (RA/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 RA/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.

Graphical abstract: Total membrane lipid assay (MLA): simple and practical quantification of exosomes based on efficient membrane-specific dyes unaffected by proteins

Supplementary files

Article information

Article type
Research Article
Submitted
18 जून 2018
Accepted
18 सितम्बर 2018
First published
19 सितम्बर 2018

Mater. Chem. Front., 2018,2, 2130-2139

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

N. Li, Z. Huang, Z. Ye, X. Zhang, L. Chen and Y. Xiao, Mater. Chem. Front., 2018, 2, 2130 DOI: 10.1039/C8QM00300A

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements