Protein Phase Transition Engineering of Nanochannels: A Stepwise Functionalization Strategy for Sequential Ultrasensitive Metal Ion Detection

Water-soluble heavy metal ions pose persistent threats to ecosystems and human health, necessitating highly sensitive and selective detection strategies. 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 (-SH) groups on PTB enable ultrasensitive Hg2+ detection via strong Hg-S interactions, achieving an exceptionally low detection limit of 2.26 × 10-14 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-Au-GSH-modified nanochannels for second-step Ce3+ detection with a detection limit of 1.4 × 10-12 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.