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Herein, we report a nanochannel sensing platform for sequential ion detection, constructed via a protein-phase-transition-induced in situ interface engineering strategy. Bovine serum albumin (BSA) is converted into phase-transited BSA (PTB) through tris(2-carboxyethyl)phosphine (TCEP)-mediated disulfide bond cleavage, forming a uniform PTB layer within conical nanochannels. The abundant thiol (\u2013SH) groups on PTB enable ultrasensitive Hg2+ detection via strong Hg\u2013S interactions, achieving an exceptionally low detection limit of 2.26 \u00d7 10\u201314 M. Building on this robust template which is essential for uniform gold deposition, chloroauric acid (HAuCl4) undergoes in situ reduction to generate a continuous gold nanolayer on the nanochannel inner surface, which is subsequently functionalized with glutathione (GSH), producing PTB\u2013Au\u2013GSH-modified nanochannels for second-step Ce3+ detection with a detection limit of 1.4 \u00d7 10\u201312 M. This hierarchical stepwise design provides tailored regulation of interfacial charge and nanoconfined transport, enabling detection of emerging contaminants such as Ce3+ with limited intra-lanthanide selectivity. The resulting system combines tunable multilevel gating, enhanced signal amplification, and robust interfacial stability, offering a generalizable and extensible platform for ultrasensitive detection of both conventional and emerging metal ions via sequential interface reconstruction on a single nanochannel membrane without substrate replacement. This work provides new insights into biomimetic ion transport regulation and paves the way for advanced biosensing, micro\/nanofluidic devices, and environmental monitoring technologies.<\/jats:p>","DOI":"10.1021\/acsami.6c14356","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T10:02:15Z","timestamp":1790330535000},"page":"54252-54262","source":"Crossref","is-referenced-by-count":0,"title":["Protein Phase Transition Engineering of Nanochannels: A Stepwise Functionalization Strategy for Sequential Ultrasensitive Metal Ion Detection"],"prefix":"10.1021","volume":"18","author":[{"given":"Jincan","family":"Yang","sequence":"first","affiliation":[{"name":"School of Chemistry and Chemical Engineering, Northwestern Polytechnical University , , No. 127, Youyi Road (West) , , ,","place":["Xi'an, Shaanxi, P. R. 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