Probing metastability, relaxation dynamics and interfacial energetics in lipase unfolding by hysteresis-encoded...

Proteins transduce thermal stress into electrical signatures through coupled changes in hydration, interfacial polarization, and charge transport. Here, we introduce a dynamic non-faradaic electrical impedance spectroscopy (NFEIS) approach that resolves protein unfolding in real time and yields a quantitative impedance-hysteresis marker of metastability and irreversibility. Using a temperature-programmable coplanar microelectrode, we measure non-equilibrium impedance trajectories in real time during continuous 298 K to 373 K ramps (single-frequency), while isothermal equilibrium spectra at selected setpoints (253 K to 343 K) provide auxiliary constraints on permittivity and resistance evolution. Dynamic measurements reveal a reproducible hysteresis loop that encodes thermal-history-dependent damage, while dielectric relaxation time analysis extracts redistribution of relaxation times (DRT) and Arrhenius analysis quantifies dc conductivity (σ₀). Orthogonal assays (circular dichroism (CD), activity, ζ-potential, differential scanning calorimetry (DSC)) delineate three regimes: a native compact state (≤298 K) with high |Z| and low permittivity; a metastable window (298 K to 323 K) with early α-helical perturbation and high electrical sensitivity (slope d|Z|/dT = -23.75 Ω K^-1); and an irreversible denatured state (≥323 K) with elevated permittivity, reduced |Z|, and an enthalpic transition. Heating produces a persistent ∼30% conductance increase, whereas cooling drives enthalpy-coupled dielectric reconfiguration, together defining a bidirectional relaxation-energetic map. This work advances NFEIS from static state sensing to trajectory-based, physics-informed stability metrics that are rapid, label-free, and microliter-scale, with direct relevance to formulation screening and cold-chain quality assurance of protein based biologics.