DropBlot: single-cell western blotting of chemically fixed cancer cells

Abstract

To further realize proteomics of archived tissues for translational research, we introduce a hybrid microfluidic platform for high-specificity, high-sensitivity protein detection from individual chemically fixed cells. To streamline processing-to-analysis workflows and minimize signal loss, DropBlot serially integrates sample preparation using droplet-based antigen retrieval from single fixed cells with unified analysis-on-a-chip comprising microwell-based antigen extraction followed by chip-based single-cell western blotting. A water-in-oil droplet formulation proves robust to the harsh chemical (SDS, 6M urea) and thermal conditions (98°C, 1-2 hr.) required for sufficient antigen retrieval, and the electromechanical conditions required for electrotransfer of retrieved antigen from microwell-encapsulated droplets to single-cell electrophoresis. Protein-target retrieval was demonstrated for unfixed, paraformaldehyde-(PFA), and methanol-fixed cells. We observed higher protein electrophoresis separation resolution from PFA-fixed cells with sufficient immunoreactivity confirmed for key targets (HER2, GAPDH, EpCAM, Vimentin) from both fixation chemistries. Multiple forms of EpCAM and Vimentin were detected, a hallmark strength of western-blot analysis. DropBlot of PFA-fixed human-derived breast tumor specimens (n = 5) showed antigen retrieval from cells archived frozen for 6 yrs. DropBlot could provide a precision integrated workflow for single-cell resolution protein-biomarker mining of precious biospecimen repositories.

Electromigration of Charged Analytes Through Immiscible Fluids in Multiphasic Electrophoresis

“…We then compared the results to a scenario with zero oil layer thickness (Figure 2A). Using the method reported by Liu and Herr 21 , we calculated and plotted the dispersion in the gel phase along the x-axis and y-axis for each oil layer thickness and noted a progressive reduction in x-axis variance with decreasing oil layer thickness (y-axis variance is negligible compared to x-axis variance) (Figure 2B). To further explore the underlying dispersion mechanism, we applied Probstein’s method, as illustrated in Figure 2C (L: 1 mm).…”

“…Building on these advancements, recently, our lab has developed a hybrid microfluidic system termed “DropBlot”, which combines water-in-oil droplet-based single-cell sample preparation with microfluidic and western blotting technologies 21 . This platform aims to facilitate the understanding of putative protein biomarkers using archived cell and tissue specimens.…”

Ten “Cheat Codes” for Measuring Oxidative Stress in Humans

“…For example, and with reference to cheat code 5, a promising recent approach leveraged an enzymatic conjugation strategy to identify and quantify novel F2-isoprostanes in human urine (Milne et al, 2024). Novel analytical approaches, such as the drop blot for single cell western analysis (Hughes et al, 2014;Liu and Herr, 2023), and artificial intelligence breakthroughs allowing for the production of designer proteins, notably antibodies (Hie et al, 2023;Madani et al, 2023), provide fertile ground for further advances. For example, a designer enzyme to selectively reduce sulfenic acids over other chemotypes.…”