arXiv subjects

Zhengfu Huang

Publications and source records attributed to Zhengfu Huang.

2 recordsLinked to original sources

arXiv2026

Gel-Confined Rolling-Circle Amplification Enables Sensitive Single-Cell Proteoform Analysis

Protein abundance alone does not capture the molecular diversity generated by protein processing and post-translational modification, yet most single-cell protein assays do not resolve these proteoform states. Here, we develop RCAmp-scWB, a post-separation amplification strategy that performs rolling-circle amplification directly within the polyacrylamide gel after single-cell protein electrophoresis. Reaction-transport modeling identifies a balance between reagent access and confinement of the template-associated amplification product that supports signal amplification while retaining the electrophoretically encoded spatial readout. Using purified protein standards, RCAmp-scWB produced a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting. Across six human cancer cell lines, RCAmp-scWB quantified protein abundance and resolved distinct Vimentin and PD-L1 proteoform states whose relative abundance and single-cell distributions were not captured by total protein measurements alone. Extension to individual small extracellular vesicles further revealed source-dependent shifts in Vimentin proteoform composition despite comparatively similar abundance of one major Vimentin species. By separating electrophoretic molecular discrimination from signal amplification, RCAmp-scWB enables proteoform-resolved analysis of heterogeneous cells and small extracellular vesicles.

q-bio.QM↗

arXiv2026

Stiff-FCS: Single-Cell Stiffness Profiling With Integrated Molecular and Functional Analysis

Cell stiffness is a key determinant of how cells deform, migrate, and adapt to mechanically restrictive environments, yet existing single-cell stiffness assays remain difficult to combine with molecular analysis and downstream functional studies. To address these limitations, we introduce a microfluidic platform, stiffness-based ferrohydrodynamic cell sorting (Stiff-FCS), designed for high-throughput quantification of single-cell stiffness, on-chip molecular analysis, and post-assay cell recovery. Stiff-FCS combines ferrofluid-driven actuation with graded confinement channels to control cell movement, induce deformation, and spatially separate cells based on stiffness. An inverse computational model converts cell position and morphology into quantitative Young's modulus values. We demonstrate stiffness profiling of hundreds to thousands of cells per chip within minutes, same-cell fluorescence-based protein analysis, and recovery of stiffness-defined cells for downstream assays. Across diverse human and mouse cell lines, Lamin A/C showed the most consistent association with stiffness, whereas softer cells exhibited greater migratory capacity than stiffer cells. In a series of human head and neck cancer cell models, Stiff-FCS further resolved a stiff, less migratory subpopulation enriched in a higher-molecular-weight Vimentin state, offering a workflow for linking single-cell stiffness to molecular heterogeneity and cell behavior.

q-bio.CB↗