Ice-Confinement Overcomes the Diffusion-Reaction Limitation in Surface Assays

Diffusion-reaction coupling at solid-liquid interfaces has long constrained the performance of interfacial molecular assays, particularly surface-based DNA assays. Herein, we report an ice-confinement strategy that reprograms the mass transport at the solid-liquid interface. Through in situ electrochemistry and finite-element modeling, we reveal that directional freezing concentrates DNA oligonucleotides within a thin interfacial liquid layer, effectively shifting the reaction from diffusion-controlled to surface-confined regime. Moreover, with rationally designed probes, we demonstrate that ice confinement lowers interfacial energy barriers and can kinetically trap overequilibrium binding states, improving access to sterically hindered sites and structured targets. Furthermore, integrating PEGylated passivation expands the effective electrical double layer and, together with freezing, affords ultrasensitive miRNA detection to 100 aM within 30 min while suppressing amplification leakage and preserving labile RNA. Importantly, our strategy applies to different substrates (gold and glass) and multimodal signal outputs (fluorescence, Raman spectroscopy, and electrochemistry). Overall, our work establishes a kinetic modulation strategy via phase-transition-enabled physical confinement, with significant implications for biosensing, surface engineering, and molecular diagnostics.