With increasing knowledge about the diverse roles of exosomes in the biological process, much attention has been paid to develop analytical methods for detection and quantification of exosomes. Immunoassays based on the recognition of exosomal protein markers by antibodies were widely used. However, considering that exosomal protein composition varies with the cell type, the protein markers should be carefully selected for a sensitive and selective analysis of target exosomes.
Researchers from Tohoku University have developed a new class of exosome-binding fluorogenic probes based on membrane curvature (MC) sensing of amphipathic helical (AH) peptides for exosome analysis without the need to use protein markers on the exosomal membranes. The C-terminal region of apolipoprotein A-I labeled with Nile red (ApoC-NR) exhibited a significant fluorescence enhancement upon selective binding to the highly curved membranes of synthetic vesicles. Circular dichroism (CD) measurements involving 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-2-dioleoyl-sn-glycerol (DOG) vesicles suggested that ApoC-NR recognizes the lipid packing defects in the surface of highly curved membranes via the hydrophobic insertion of the α-helix structure of the ApoC unit. ApoC-NR exhibited a stronger binding affinity for exosome-sized vesicles and a higher MC selectivity compared to all other previously reported peptide probes. ApoC-NR can be used in a simple and rapid “mix and read” analysis of various kinds of exosomes derived from different cell types (limit of detection: –105 particles/μL) without being influenced by the variation in the expression of the surface proteins of the exosomes, which stands in sharp contrast to immunoassays.
Schematic illustration of the exosome analysis with the
AH peptide-based fluorescent probe ApoC-NR
A helical wheel representation of ApoC sequence is also shown, wherein the hydrophobic and the polar residues are orange and blue, respectively.