Isolation and Purification-Free Digital Single-Small Extracellular Vesicle Biosensing with Scalable Plasmonic Arrays.

Authors

Mohammad Sadman Mallick, Saswat Mohapatra, Abhay Kotnala, A B M Arafat Hossain, Wei-Chuan Shih

Published in

ACS sensors. Sep 23, 2026. Epub Sep 23, 2026.

Abstract

Recent advances in plasmonic biosensing and imaging have enabled label-free analysis of single biological nanoparticles. We previously developed PlAsmonic NanOapeRture lAbel-free iMAging (PANORAMA) for isolation and purification-free, digital counting and precise localization of antibody captured, tetraspanin-positive small extracellular vesicles (sEVs), with complementary fluorescence interrogation of surface and intravesicular biomarkers for quantitative molecular profiling. The fact that no isolation and purification is needed represents a crucial advantage because various specificity, efficiency, and time-consumption issues hinder quantitatively reproducible extraction of sEVs from biological fluids. PANORAMA achieves ultrahigh refractive-index sensitivity through arrayed gold nanodisks on invisible substrates (AGNIS) fabricated by nanosphere lithography (NSL). However, despite its simplicity and low cost, NSL is frequently constrained by poor large-area uniformity, which hinders scalable fabrication. Here, we introduce nanosphere settling lithography (NSSL) as an alternative to the gold-standard Langmuir-Blodgett trough (LBT) process, enabling highly uniform, large-area monolayers with reduced process stringency. AGNIS fabricated via NSSL exhibits high refractive-index sensitivity with low spatial variability across 60 mm × 24 mm substrates, sufficient for 60-well in standard 384-well plate format. The platform demonstrates exquisite sensitivity through PANORAMA digital counting and sizing of 25, 50, and 100 nm polystyrene beads, as well as single-vesicle characterization of antibody captured, tetraspanin-positive sEVs derived from H460 lung cancer cells. Combined PANORAMA and fluorescence imaging enables quantitative analysis of microRNA-21 (miR-21)-associated fluorescence among antibody captured sEV-like particles from liver cancer patient plasma, allowing label-free digital counting after selective affinity capture and simultaneous molecular profiling of the captured vesicle subset. Collectively, NSSL enables uniform, high-performance plasmonic biosensing over large areas, providing a scalable and economical pathway for high-throughput, digital analysis of antibody captured single sEVs and translational liquid biopsy applications.

PMID:
42776860
Bibliographic data and abstract were imported from PubMed on 24 Sep 2026.

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