Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76–81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

Fluorogenic probes are valuable tools for rapid detection and bioimaging of target molecules without requiring bound/free separation. Although antibody‐based probes are widely used, their large size, reliance on secondary labeling, and limited accessibility to some intracellular regions can restrict their utility. In contrast, fluorogenic peptide aptamers—compact, chemically synthesizable, and self‐reporting—offer a promising alternative. Here, we developed a method to prepare a fluorophore‐conjugated peptide library using cDNA display and applied it to the selection of fluorogenic peptides targeting heat shock protein 90α (Hsp90α), a clinically relevant biomarker. As the solvatochromic reporter, we used an environmentally sensitive fluorophore, 4‐N, N‐dimethylamino‐1,8‐naphthalimide (4‐DMN). One selected peptide, Peptide2, exhibited fluorescence enhancement in the presence of Hsp90α while remaining largely unresponsive to the homologous Hsp70. In fixed cells, Peptide2 produced intracellular fluorescence patterns that partially overlapped with those obtained using an anti‐Hsp90α antibody, supporting its ability to detect intracellular Hsp90α‐related signals. Peptide2 and the antibody also showed partially distinct staining distributions, suggesting differences in epitope accessibility and/or intracellular accessibility. These results establish a proof of concept for generating target‐responsive, self‐reporting peptide probes using bioorthogonal cDNA display.