Nanopore Fingerprinting of Structurally Diverse Amino Acid-Conjugated Bile Acids

Bile acids are key signaling molecules implicated in various hepatobiliary and metabolic diseases. Synthesized via distinct pathways and undergoing diverse modifications, bile acids exhibit significant structural variation. The discovery of novel amino acid-conjugated bile acids (BA-AAs) has further expanded this structural diversity, posing substantial challenges for their precise identification. Here, an engineered aerolysin nanopore K238W was employed for detecting diverse BA-AAs, including cholic acid conjugates with various amino acids and those derived from structurally similar or isomeric bile acids. Using a high-ionic-strength electrolyte containing guanidinium chloride, the K238W nanopore produced distinctive blockade signal fingerprints specific to different BA-AAs. Subsequent machine-learning analysis allowed clear differentiation of diverse BA-AAs, as evidenced by 98% accuracy in identifying 20 amino acid-conjugated cholic acids. This approach offers a promising alternative for analyzing structurally similar or even isomeric bile acids, advancing their functional and clinical studies.