Introduction: Small extracellular vesicles (sEVs) are integral to liquid biopsy and essential in physiological and pathological processes, including immune response [1, 2]. PD-L1 protein is a vital immune checkpoint, and sEV-bound PD-L1 has been detected in multiple cancer types and is a relevant biomarker for monitoring cancer progression and immunotherapy response [3, 4]. Here, for the first time, we developed a quantitative lateral flow assay (LFA) using highly doped upconversion nanoparticles (UCNPs) for ultrasensitive detection of PD-L1-positive sEVs.
Methods: sEVs were isolated from three human mesothelioma cell lines using ultracentrifugation. The NaYF4:40%Yb,4%Er@NaYF4 core-shell nanocrystals were synthesised, surface modified with copolymers, and bioconjugated with CD63 and PD-L1 antibodies. UCNP probes were used in the LFA to detect target analytes in samples using a strip reader integrated with a 980nm laser.
Results: The limit of detection (LoDs) for CD63 and PD-L1 proteins in the UCNP-LFA were 58 pg/ml and 10 pg/ml, respectively. The UCNP-LFA detected CD63 and PD-L1 positive sEVs at concentrations as low as 1.03 × 103 sEVs/µl and 2.15 × 103 sEVs/µl, respectively. The UCNP-LFA detected sEV markers from ~1000 lower sEV concentrations than traditional ELISA. It was also ~100 times more sensitive than other colorimetric LFA studies [5, 6].
Conclusions: PD-L1 expression on sEVs excellently reflects the tumour environment in cancer patients and has significant diagnostic potential [7]. The technology represents a push towards simple and sensitive sEV biomarker detection that facilitates non-invasive and dynamic analyses of early-stage cancer biomarkers, particularly in clinical settings.