High‐Sensitivity Detection of Urinary Extracellular Vesicles With Upconverting Nanoparticle‐Based Lateral Flow Immunoassay

Urinary extracellular vesicles (uEVs) are well-known to express tetraspanin family membrane proteins abundantly on their surface. In this study, we aimed to develop an upconverting nanoparticle (UCNP)-based lateral flow immunoassay (UCNP-LFIA) designed for the rapid and high-sensitivity detection of CD63-positive uEVs for direct urinalysis. The assay utilizes UCNPs reporter to detect low concentrations of EVs. Minimally processed uEV samples from bladder cancer (BlCa) (<i>n</i> = 62), benign prostatic hyperplasia (BPH) (<i>n</i> = 50) and healthy (<i>n</i> = 30) individuals were tested in sandwich UCNP-LFIA format, capturing uEVs with the same anti-CD63 antibody conjugated to UCNP and immobilized on the test zone. After 80 min, the strips were read with an upconversion luminescence reader device. This UCNP-LFIA measured CD63-positive EVs with high sensitivity, exhibiting a limit of detection (LoD) of 4 × 10⁷ EVs/mL. The concentration of CD63-positive EVs in BlCa patients showed a 2.3-fold increase compared to benign conditions (<i>p</i> = 0.007), and a 16-fold increase compared to healthy controls (<i>p</i> = 0.00001). The results demonstrate the potential of UCNP-LFIA platform for sensitive and quantitative detection of uEVs, highlighting its promise as a tool for EV detection at point-of-care diagnostics.