Immunojanus Particles for low-volume and isolation-free unlabeled characterization of small Extracellular Vesicle in biofluids: Characterization of disease type by surface marker profiling

Small extracellular vesicles (sEVs) are vital for cellular communication and serve as critical biomarker carriers for diseases such as cancer. However, quantifying and profiling sEV surface markers presents significant challenges due to the low concentration of specific sEV-bound proteins and interference by more abundant dispersed proteins. This paper presents Immunojanus Particles (IJPs), a new method that enables the direct detection of sEVs in less than an hour without isolation. The design of IJPs incorporates fluorescent and non- fluorescent halves, utilizing rotational Brownian motion to detect captured sEVs through the change in the blinking rate, without interference from the smaller dispersed proteins. We demonstrate a detection limit of 2E5 sEVs/mL with low sample volumes and the capability to characterize sEVs directly from plasma, serum, cell culture media, and urine. In a small pilot study involving 87 subjects, including individuals with colorectal cancer, pancreatic ductal adenocarcinoma, glioblastoma, Alzheimers disease, and healthy controls, our method accurately identified the type of disease with a high 0.90-0.99 AUC in a blind setting. Compared with an orthogonal ultracentrifugation plus surface plasmon resonance (UC+SPR) method that requires about 24 hours, the sensitivity and dynamic range of IJP are better by 2 logs.