Observation of ice‑like two‑dimensional flakes on self‑assembled protein monolayer without nanoconfinement under ambient conditions

Interfacial water dictates protein stability, ligand recognition and biochemical reactivity, yet direct visualization of its structure under biologically relevant, non-confined conditions has remained elusive. A Tsinghua–Hangzhou Normal team led by Prof. Yuan Li and Prof. Jian-Qiang Zhong now reports the spontaneous formation of two-dimensional, ice-like water "flakes" on a cytochrome C (Cyt C) protein monolayer anchored to a self-assembled sulfonate monolayer (SAM). Remarkably, the crystalline water assembles at room temperature and ambient pressure without external nanoconfinement, offering a new model to decode protein–water interplay in real time.

Why These 2D Ice-Like Flakes Matter

• Ambient Stability: Uniform islands (0.7–3.3 nm thick) persist at 25 °C and 60 % RH, surviving AFM scans and only melting when locally heated by the cantilever laser, confirming solid-like order.
• Composition Fingerprint: Synchrotron nano-AFM-IR maps a sharp 3336 cm^-1 O–H stretch and Raman shows the 3390 cm^-1 ice signature inside each flake, while amide I/II bands verify co-embedded Cyt C.
• Reversible Phase Control: Raising humidity to 90 % expands the flakes; continuous scanning or gentle heating erases them, demonstrating true crystallization/melt cycles on demand.
• Stronger Water–Protein Bond: Temperature-programmed IR desorption reveals two-step water loss from Cyt C/SAM, with the high-temperature peak requiring 111 kJ mol^-1—almost twice the 61 kJ mol^-1 needed for desorption from bare SAM—proving that the protein surface locks water in an ice-like lattice.

Innovative Platform & Characterization

• Ultra-flat Auᵀˢ template (RMS < 0.5 nm) guarantees defect-controlled nucleation; sulfonate SAM provides charged, hydrophilic anchor for Cyt C physisorption.
• Blue-drive AFM eliminates laser heating artifacts, enabling direct height profiles and in-situ melt/growth movies.
• Nano-IR spectroscopy (20 nm spot) correlates local topography with chemical identity, while UHV-IRRAS quantifies desorption energetics via Polanyi–Wigner analysis.

Implications & Outlook

By coupling high-resolution morphology with site-specific vibrational data, the work delivers the first ambient-pressure model where protein-induced electrostatic fields compensate entropic penalties to nucleate 2D ice. The platform is extendable to antifreeze proteins, cryo-enzymes and hydration-controlled bioelectronics. Next steps include real-time mutagenesis to pinpoint residues responsible for ice templating and exploration of drug binding within the ice-like hydration shell. Watch for follow-up studies from the Tsinghua-Hangzhou collaborative team!