Target-induced proximity ligation triggers polymerase chain reaction for subset tracing of small extracellular vesicles.

The considerable abundance and remarkable stability of sEVs provide substantial benefits for diagnosing Alzheimer's disease. Therefore, precise tracking subtypes of small extracellular vesicles (sEVs) is crucial for screening novel diagnostic biomarkers and developing therapeutic technologies. We propose a three-target recognition-mediated proximity ligation assay for the precise identification of sEV subtypes utilizing three specifically designed probes: one for the exosomal surface protein CD63 recognition, one for fixing the biolipid layer, and the third for the identification of distinctive protein associated with a specific subtype of sEVs (L1CAM positive sEVs). The developed sEVs subtype tracing approach integrates proximity ligation of the three probes to specifically bind to surface biomarkers and polymerase chain reaction (PCR) for signal amplification, enabling "AND" logic analysis of three essential components on sEVs. This method can be utilized for both sEVs quantification and subtype tracing. The proposed approach demonstrated a low limit of detection for neuronal sEVs at 2.5 particles/μL, according to this design. In addition, we utilized this technique to measure plasma sEV levels in individuals with Alzheimer's disease and examined its early diagnostic effectiveness. The approach can assess the concentration ratios of neuronal sEVs and cancer-derived sEVs, highlighting its potential for clinical applications. In addition, the approach enables precise tracing and identification of sEVs subtypes, hence facilitating extensive applications in biological science, biomedical engineering, and personalized medicine.