Sequential separation and profiling of extracellular vesicles using antibody-aptamer conjugates

Extracellular vesicles (EVs) are membrane-bound vesicles secreted by cells, exhibiting diverse compositions reflective of their cellular origin. With significant potential as biomarkers for liquid biopsies, EV research has led to various isolation techniques. However, a consensus on the optimal strategy remains elusive. Immunoprecipitation, selectively capturing EVs based on surface markers, is promising but hindered by cost, low yields, and potential damage during release. In this study, we propose an innovative Antibody-Aptamer Conjugate (AAC): a three-component separation reagent for the separation of EVs. Combining an EV-specific antibody, a streptavidin-binding aptamer, and a unique barcode DNA sequence, this conjugate serves dual roles, facilitating both EV separation and subsequent multiplexed analysis. We detail the development and validation of the AAC, demonstrating its efficacy in isolating intact EVs from complex samples. The unique barcode DNA sequence enables high-throughput analysis on a DNA microarray chip, addressing limitations of existing methodologies. This approach offers a valid and cost-effective alternative for selective EV isolation and analysis, with implications for diagnostic and therapeutic advancements in liquid biopsy applications. • Innovative EV separation and analysis using a three-component reagent. • Components include antibody, streptavidin-binding aptamer, and barcode DNA sequence. • Aim: to separate EV subpopulations and enable multiplex analysis on a DNA microarray chip. • Advantages: overcomes limitations of traditional immunoprecipitation, preserves EV integrity. • Potential impact: enhanced diagnostic accuracy in liquid biopsies.