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

Abstract

Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-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 highly efficient platform for profiling multiplex proteins at single-cell resolution.

Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays

“…Several studies have compared aptamers to antibodies to detect proteins in immuno-assays. Aptamers have replaced antibodies in histochemical staining of both fresh-frozen and paraffin-embedded tissue sections 11 – 14 , flow-cytometry 14 – 17 , dot-blot 18 , 19 , western blot 20 , 21 , ELISA 22 , 23 and many other antibody-dependent assays. All the above studies are about the application of aptamers in comparison antibodies to a single assay.…”