Enhancing Extracellular Vesicle Detection via Cotargeting Tetraspanin Biomarkers

Extracellular vesicles (EVs) are emerging as key diagnostic biomarkers due to their widespread presence in body fluids and the proteins on their surfaces, which reflect the identity and condition of their parent cells. Research has focused on detecting EVs with biosensors that target individual transmembrane proteins (TMPs) like tetraspanins. However, due to TMP heterogeneity and the formation of tetraspanin-enriched microdomains (TEMs), cotargeting multiple TMPs is a promising strategy for enhancing EV detection. In this work, we introduce a dual-antibody surface functionalization approach using surface plasmon resonance (SPR) biosensors to cotarget tetraspanins on EVs derived from mouse macrophages. The expression of EV tetraspanin markers followed the trend of CD9 > CD63 > CD81, which was consistent with the EV detection targeting their nontetraspanin partners, exhibiting LFA-1 > ICAM-1 > VCAM-1, and suggesting a differential role of tetraspanins with their associated TMPs. Cotargeting EV tetraspanins