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 (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.