Direct identification of amino acid-derived hormones with single-atom resolution using an engineered aerolysin nanopore

The amino acid-derived hormones (AAHs), which are relatively small hydrophobic molecules derived from the amino acid tyrosine and tryptophan, play critical roles in the regulation of organism development and metabolism process. Direct identification of AAHs and their metabolites is highly desirable but very challenging because they simultaneously possess the features of small volume, hydrophobicity and highly structural similarity. Nanopore, capable of sensing the analytes in a single-molecule manner, may be engineered to identity AAHs. The key aporia is the ion current instability caused by the introduction of hydrophobic amino acids inside the nanopore confinement that is essential to improve the spatial and temporal resolution for small hydrophobic molecules sensing. Herein, we design a mutant aerolysin that precisely introduces a hydrophobic chemical environment in the critical sensing region with a highly controlled hydrogen bonds network to balance the ion follow stability and hydrophobic interactions. With this hydrophobic enhanced aerolysin nanopore, direct identification of AAHs was achieved, including thyroid hormone, pineal hormone and adrenal medullary hormone. Furthermore, three thyroid hormones with single iodine atom difference (T4, T3, and T2), were observed to be clearly discriminated in their mixture. Our engineered nanopore can even distinguish isomers of T3 and rT3 that vary only slightly in structure. Assisted with a custom clustering machine learning algorithm, the thyroid hormones T4, T3, and T2 in their mixture could be quantified, which suggests a simple and rare approach to measure the AAHs in clinical samples.