Label-Free Profiling of Immunoglobulin Isotypes Using Solid-State Nanopores.

Accurate profiling of antibody isotypes is essential to monitor immune responses, diagnose disease, and guide therapy, yet existing immunoassays remain limited in capturing their conformational dynamics with molecular precision. Nanopore sensing offers a label-free route to such resolution, but most implementations rely on fragmentation, probes, or functionalization, leaving intact isotypes inaccessible. Here, a nanopore-based assay that directly discriminates full-length IgG, monomeric IgA, and IgM under non-denaturing conditions, resolving intrinsic molecular signatures in real time is presented. In a lithium chloride electrolyte, glass nanopipettes operate in an electroosmotic-flow-dominated regime where flow-driven transport and nanoscale confinement produce distinct ionic fingerprints for each isotype. Affinity-based signal modulation with protein G, which selectively binds the IgG Fc domain, further introduces a tunable contrast mechanism that sharpens IgG-IgA separability while preserving native structure and binding functionality. Quantitative analysis demonstrates robust separation (p < 0.0001) in binary and ternary mixtures with low misclassification. Evaluation in diluted human serum with a physiologically representative IgG:IgA:IgM ratio (7:2:1) shows that isotype-specific fingerprints remain distinguishable in a serum-derived background. The EOF-governed assay establishes a scalable framework for direct, multiplexed immunoprofiling and extends label-free nanopore analysis beyond antibodies toward broader proteomic targets.