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

## TL;DR

This study develops a highly sensitive test using nanoparticles to detect urinary extracellular vesicles, which could help diagnose bladder cancer.

## Contribution

A novel upconverting nanoparticle-based lateral flow immunoassay for rapid and sensitive detection of CD63-positive urinary extracellular vesicles.

## Key findings

- The UCNP-LFIA detected CD63-positive EVs with a limit of detection of 4 × 10⁷ EVs/mL.
- Bladder cancer patients had a 2.3-fold increase in CD63-positive EVs compared to benign conditions.
- CD63-positive EV concentrations in bladder cancer were 16-fold higher than in healthy controls.

## Abstract

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) (n = 62), benign prostatic hyperplasia (BPH) (n = 50) and healthy (n = 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 (p = 0.007), and a 16‐fold increase compared to healthy controls (p = 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.