Hydrophobic Confinement Enables Direct Identification of Thyroid Hormones with Single-Atom Resolution

Abstract The direct detection of small hydrophobic biomolecules remains a major challenge for biological nanopores. This is due to the weak interactions provided by the hydrophilic lumens, which result in ultrafast translocation and insufficient molecular recognition. Here, to reconcile the hydrophilicity for ionic flow and hydrophobic interactions for molecule recognition, we engineer a hydrophobic confinement stabilized by an interconnected hydrogen-bond network. By introducing tyrosine and tryptophan into the primary sensing region, a localized hydrophobic environment is established while maintaining nanopore structural stability and low current noise. Molecular dynamics simulations demonstrated the formation of a localized hydrophobic environment stabilized by an interconnected hydrogen-bond network, enabling the direct identification of thyroid hormones, aromatic amino acid-derived hydrophobic biomolecules containing iodinated aromatic structures, through enhanced molecular interactions. We show that the hydrophobic confinement could discriminate single iodine atom differences within diverse thyroid hormones, of which individual events can be automatically classified using an unsupervised clustering algorithm without prior model training. Furthermore, the isobaric triiodothyronine and reverse triiodothyronine could be directly resolved in a mixture. This work establishes hydrophobic confinement engineering as an effective strategy for extending biological nanopores to the direct single-molecule measurement of clinically relevant hydrophobic biomolecules.