Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers

Single-cellwestern blotting (scWB) is a prevalent technique forhigh-resolution protein analysis on low-abundance cell samples. However,the extensive signal loss during repeated antibody stripping precludesmultiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cellresolution. The minimal size of aptamer probes allows rapid in-gelpenetration, diffusion, and elution. Meanwhile, the fluorophore-taggedaptamers, coordinated with complementary quenching strands, avoidthe massive signal loss conventionally caused by antibody strippingduring repeated staining. Such a strategy also facilitates multiplexprotein analysis with a limited number of fluorescent tags. We demonstratedFAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2,CA125) at single-cell resolution with lower signal loss and enhancedsignal-to-noise ratio compared to conventional antibody-based scWB.Being more time-saving (less than 25 min per cycle) and economical(1/1000 cost of conventional antibody probes), FAS-WB offers a highlyefficient platform for profiling multiplex proteins at single-cellresolution.

Automated summary

Single-cellwestern blotting (scWB) is a common method for high-resolution protein analysis on low-abundance cell samples. However, antibody stripping during repeated staining leads to signal loss, preventing multiplex protein detection. In this study, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. FAS-WB allows rapid penetration, diffusion, and elution of aptamer probes, while avoiding signal loss through the use of fluorophore-tagged aptamers coordinated with quenching strands.