Plasmon‐Enhanced Single Extracellular Vesicle Analysis for Cholangiocarcinoma Diagnosis

Abstract

Cholangiocarcinoma (CCA) is a fatal disease often detected late in unresectable stages. Currently, there are no effective diagnostic methods or biomarkers to detect CCA early with high confidence. Analysis of tumor-derived extracellular vesicles (tEVs) harvested from liquid biopsies can provide a new opportunity to achieve this goal. Here, an advanced nanoplasmonic sensing technology is reported, termed FLEX (fluorescence-amplified extracellular vesicle sensing technology), for sensitive and robust single EV analysis. In the FLEX assay, EVs are captured on a plasmonic gold nanowell surface and immunolabeled for cancer-associated biomarkers to identify tEVs. The underlying plasmonic gold nanowell structures then amplify EVs' fluorescence signals, an effective amplification process at the single EV level. The FLEX EV analysis revealed a wide heterogeneity of tEVs and their marker levels. FLEX also detected small tEVs not detected by conventional EV fluorescence imaging due to weak signals. Tumor markers (MUC1, EGFR, and EPCAM) are identified in CCA, and this marker combination is applied to detect tEVs in clinical bile samples. The FLEX assay detected CCA with an area under the curve of 0.93, significantly better than current clinical markers. The sensitive and accurate nanoplasmonic EV sensing technology can aid in early CCA diagnosis.

Rapid Evaluation of Lung Adenocarcinoma Progression by Detecting Plasma Extracellular Vesicles with Lateral Flow Immunoassays

“…The application scenarios should be explored and added markers on EVs should be developed in further study. Inspiringly, researchers attempted to develop various methods for ultra-sensitive detection of EVs − (Table S2). For instance, surface plasmon resonance (SPR) systems could construct a comprehensive EV atlas through single-EV analysis or improve the specificity of diagnosis through multiplex measurements …”

Fluorescence labeling of extracellular vesicles for diverse bio-applicationsin vitroandin vivo

“…This inherent property makes biofluid derived EVs promising biomarkers for disease diagnosis, prognosis, and treatment monitoring at various disease stages. [10][11][12] In comparison with floating biomarkers such as proteins, nucleic acids and metabolites, EVs have superior properties as a non-invasive liquid-biopsy candidate due to their non-invasive properties, abundant molecular information and capability of penetrating various biological barriers. 13,14 Recently, increasing research interest has been put on EV biomarker discovery and subsequent cancer diagnostics.…”

Precise Identification and Profiling of Surface Proteins of Ultra Rare Tumor Specific Extracellular Vesicle with Dynamic Quantitative Plasmonic Imaging

“…14−16 Third, not all tEVs contain cancerrelated molecules. 17 Existing EV isolation approaches, including ultracentrifugation, 18 polyethylene glycol (PEG)-based precipitation, 19 affinity capture, and size-exclusion chromatography 20 could not effectively separate tEV from clinical samples due to low purity and yield. Bulk EV analysis technologies, such as enzyme-linked immunosorbent assay (ELISA) 21−23 and magnetic bead-based capture and detection 24 remain disturbed by background signals arising from the nonspecific binding of EVs from normal cells and non-EV particles to the assay interface.…”

“…27,29,30 owing to the tiny size of EVs. 17 However, binding of nonspecific EVs, non-EV particles, or detection probes to the assay interface produces non-negligible background signals, although stringent washing or blocking may mitigate unwanted background noises to some extent. 22 Previously, single-molecule kinetic fingerprinting-based assays have been applied for single miRNA and protein analysis, improving the specificity.…”