Identification and Discrimination of Sulfated and Unsulfated Gut‐Hormone Biomarkers in Human Serum by Nanopore

ABSTRACT

Cholecystokinin‐8, an octapeptide, is a gut hormone mainly involved in the digestive process and neuromodulation. Over the last four decades, its biological functions have been elucidated and shown to be critically modulated by tyrosine sulfation. Recently, this octapeptide has been identified as a potential biomarker in several tumours. Regrettably, there are no clinical or analytical methods capable of distinguishing between the unsulfated (CCK‐8) and sulfated (SO ₃ ‐CCK‐8) forms in controlled media (e.g., saline) or complex biofluids (e.g., human serum). Here, we use an aerolysin nanopore inserted in a lipid‐polymer membrane to discriminate them with high specificity and sensitivity at the single‐molecule level in both controlled media and complex biofluids, even at low nanomolar concentrations. We found that sulfated and unsulfated CCK‐8 generate highly resolutive electrical signatures. Analysis of these signals shows discrimination based on three independent parameters. Using a robust hybrid lipid‐coblock membrane, we successfully performed measurements in 20% (v/v) human serum, thereby enabling the identification of peptides differing by a subtle post‐translational modification. These results enable reliable discrimination of these biomarkers for diagnostic evaluation.