High-Throughput Single-Cell Extracellular Vesicle Secretion Analysis on a Desktop Scanner without Cell Counting

Abstract

Single-cell EV (extracellular vesicle) secretion analysis is emerging for a better understanding of non-genetic cellular heterogeneity regulating human health and diseases through intercellular mediators. However, the requirements of expensive and bulky instrumentations hinder its widespread use. Herein, by combining gold nanoparticle-enhanced silver staining and the Poisson distribution, we reported the use of a home-use scanner to realize high-throughput single-cell EV secretion analysis without cell counting. We applied the platform to analyze the secretions of different EV phenotypes with the human oral squamous cell carcinoma cell line and primary cells from patients, which generated single-cell results comparable with those of the immunofluorescence approach. Notably, we also realized the quantification of the number of EVs secreted from every single cell using their respective titration curves obtained from population samples, making it possible to directly compare different EV phonotypes in regard to their secretion number, secretion rate, and so forth. The technology introduced here is simple, easy to operate, and of low cost, which make it a potential, easily accessible, and affordable tool for widespread use in both basic and clinical research.

Recent Progress of Exosome Isolation and Peptide Recognition-Guided Strategies for Exosome Research

“…In addition, the isolation and analysis of single cell-derived exosomes is still a scientific problem. Zhu et al designed a microwell array chips to trap a single cell, and antibody-coated glass slide could capture exosomes secreted from the single cell ( Zhu F. et al, 2021 ). The number of exosomes secreted from every single cell was quantified by combining gold nanoparticle-enhanced silver staining, which reflecting the ability of individual cells to secrete exosomes.…”

Optical Sensing Strategies for Probing Single-Cell Secretion

“…However, these fluorescent methods mainly rely on expensive or unwieldy instruments, which hinder the practical application. On the bases of gold nanoparticle-enhanced silver staining and the Poisson distribution statistics, a home-use scanner platform was developed . After seeding single cells in microchamber arrays, gold nanoparticle-labeled antibodies were used to execute the sandwich immunoassay, which enabled further generation of a plasmonic silver coating with intense light scattering and reflection (Figure A) .…”

“…On the bases of gold nanoparticle-enhanced silver staining and the Poisson distribution statistics, a home-use scanner platform was developed . After seeding single cells in microchamber arrays, gold nanoparticle-labeled antibodies were used to execute the sandwich immunoassay, which enabled further generation of a plasmonic silver coating with intense light scattering and reflection (Figure A) . The method was used to analyze the different phenotypes and the number of extracellular vesicles secreted from the single cell, and has the characteristics of high-throughput and no cell counting …”

“…102 After seeding single cells in microchamber arrays, gold nanoparticle-labeled antibodies were used to execute the sandwich immunoassay, which enabled further generation of a plasmonic silver coating with intense light scattering and reflection (Figure 4A). 102 The method was used to analyze the different phenotypes and the number of extracellular vesicles secreted from the single cell, and has the characteristics of high-throughput and no cell counting. 102 Extracellular vesicles encapsulate a variety of substances such as microRNAs (miRNA) and proteins which play a pivotal role in the disease progression and have received wide attention as next-generation biomarkers.…”

“…102 The method was used to analyze the different phenotypes and the number of extracellular vesicles secreted from the single cell, and has the characteristics of high-throughput and no cell counting. 102 Extracellular vesicles encapsulate a variety of substances such as microRNAs (miRNA) and proteins which play a pivotal role in the disease progression and have received wide attention as next-generation biomarkers. 103 However, the low abundance and protection of lipid bilayer make it challenging to sense these substances.…”