From Upconversion Nanoparticles to Proteins: Probing Hydration-Water Density Fluctuations by Luminescence Thermometry

ConspectusWater appears simple, yet its anomalous behavior reveals an unexpected structural complexity. A growing body of evidence indicates that many of water's anomalies arise from fluctuations between low-density (LD) and high-density (HD) local structural motifs, a form of polymorphism that is well established in the supercooled regime and increasingly supported at ambient conditions. Yet, how these structural motifs manifest within hydration layers, where water interacts with nanoparticles, proteins, and charged interfaces, remains far less understood. This interfacial water governs colloidal stability, biomolecular function, and chemical reactivity, but its microscopic organization is difficult to probe directly with conventional bulk techniques.In this Account, we describe how luminescence nanothermometry provides a powerful and versatile approach to accessing density fluctuations in the hydration layer. By monitoring temperature-dependent optical and Brownian observables of luminescent probes, structural reorganizations of the surrounding hydration layer can be inferred with nanoscale sensitivity. Over the past several years, our group has shown that lanthanide-doped upconversion nanoparticles (UCNPs) and fluorescent proteins, such as enhanced green fluorescent protein (EGFP), act as local reporters of hydration-water density fluctuations.A central observation emerging from these studies is the existence of a crossover temperature, <i>T</i>_c, at which hydration-water observables exhibit bilinear temperature dependencies. This <i>T</i>_c correlates with the depletion of LD motifs in the hydration shell and typically falls within the 315-330 K range, close to the minimum of water's isothermal compressibility. Importantly, <i>T</i>_c depends on the nature of the probe and its interaction with the surrounding water.By systematically varying nanoparticle size, pH, surface chemistry, and probe type, we show that previously contradictory trends in <i>T</i>_c can be unified by a single parameter: the effective surface charge density of the probe. When <i>T</i>_c is plotted against this quantity, data from UCNPs with different sizes and surface functionalizations, as well as from fluorescent proteins at different concentrations, collapse onto a master curve. This result demonstrates that interfacial electrostatics govern the stability of LD motifs in the hydration layer, providing a physically intuitive framework that links nanoscale charge distributions to local water structure.We further extend this framework by examining nuclear quantum effects through isotopic substitution. Using EGFP as a model biomolecular probe, we show that replacing H₂O with D₂O shifts <i>T</i>_c upward by ≈10 K and enhances protein thermal stability, consistent with stronger hydrogen bonding and the displacement of thermodynamic anomalies in heavy water. In contrast, several inorganic and molecular probes fail to resolve a comparable isotopic shift, highlighting that the detectability of LD/HD fluctuations might be probe-dependent. Control experiments in H₂¹⁸O confirm that hydrogen, rather than oxygen, dominates these quantum effects.Together, these results establish luminescent nanoprobes as sensitive reporters of hydration-water density fluctuations and reveal how interfacial charge, confinement, and quantum effects sculpt water structure at the nanoscale. Beyond resolving long-standing questions about water's anomalies, this approach opens new avenues for understanding protein stability, designing functional nanomaterials, and exploiting hydration-water density fluctuations in chemical and biological systems.