A self-assembled peptide forms α-helical nanopores for ultrasensitive biomarker profiling.

α-Helical nanopores are attractive molecular sensors, yet their rational design and assembly remain challenging. Here we show that the single peptide pPorA, derived from porin PorACj, self-assembles into flexible α-helical nanopores, inserts in lipid membranes and exists in distinct small- and large-conductance states. By strategically incorporating unnatural amino acids, we engineered small- and large-diameter pores exhibiting single-channel conductances of 2.4 nS and 3.5 nS in 1 M KCl, respectively, while retaining a common octameric architecture. These nanopores enabled the detection of sugars, peptide enantiomers and intrinsically disordered disease proteins that form dynamic, heterogeneous assemblies. The large pores detected multiple α-synuclein (α-syn) variants, including a pathogenic Parkinson's disease-associated C-terminal deletion mutant with nanomolar affinity (KD ≈ 20 nM). Selective electrostatic trapping of the α-syn N-terminus enabled charge-resolved identification of individual α-syn species within heterogeneous mixtures. The nanopores further resolved time-dependent and inhibitor-modulated α-syn aggregation pathways from monomers to toxic oligomers and fibrils. The small pores detected humanin and superoxide dismutase peptides associated with apoptosis and amyotrophic lateral sclerosis, demonstrating tunable sensing through pore-size control. These conformationally programmable α-helical nanopores provide a versatile platform for ultrasensitive profiling of disease biomarkers.