Direct exosome quantification via bivalent-cholesterol-labeled DNA anchor for signal amplification

Exosomes, as an important subpopulation of extracellular vesicles (EVs), play an important role in intercellular communications in various important pathophysiological processes, especially cancer-related. However, reliable and convenient quantitative methods for their determination are still technically challenging. In this study, researchers at the East China University of Science and Technology developed an efficient and direct method by combining immunoaffinity and lipid membrane surface modification into a single platform for specific isolation and accurate quantification of exosomes. Exosomes are specifically captured by immunomagnetic beads, and then a bivalent-cholesterol (B-Chol)- labeled DNA anchor with high affinity is spontaneously inserted into the exosome membrane. The rationally designed sticky end of the anchor acts as the initiator for the subsequent horseradish peroxidase (HRP)-linked hybridization chain reaction (HCR) for signal amplification. Detection is based on the color change of HRP-catalyzed H2O2-mediated oxidation of 3,3′,5,5′- tetramethyl benzidine (TMB), which can be conveniently observed by the naked eye and monitored by UV-vis spectrometry. This proposed method enables sensitive detection of 2200 exosomes per microliter with a relative standard deviation of <5.6%, with 100-fold higher sensitivity compared to conventional ELISA. The researchers believe that their assay has considerable potential as a routine bioassay (cost-efficient, reliable, and easy to operate) for the accurate quantification of exosomes in clinical samples.

Schematic illustration of the proposed method for exosome quantification by a combination of immunoaffinity separation and cholesterol-based signal amplification.