Deciphering the Kinetics of Spontaneous Generation of H₂O₂ in Individual Water Microdroplets

Spontaneous generation of H₂O₂ in sub-10 μm-sized water microdroplets has received increasing interest since its first discovery in 2019. On the other hand, due to the short lifetime of these microdroplets (rapid evaporation) and lack of suitable tools to real-time monitor the generation of H₂O₂ in individual microdroplets, such a seemingly thermodynamically unfavorable process has also raised vigorous debates on the origin of H₂O₂ and the underlying mechanism. Herein, we prepared water microdroplets with a long lifetime (>1 h) by virtue of microwell confinement and dynamically monitored the spontaneous generation of H₂O₂ in individual microdroplets via time-lapsed fluorescence imaging. It was unveiled that H₂O₂ was continuously generated in the as-prepared water microdroplets and an apparent equilibrium concentration of ∼3 μM of H₂O₂ in the presence of a H₂O₂-consuming reaction can be obtained. Through engineering the geometry of these microdroplets, we further revealed that the generation rates of H₂O₂ in individual microdroplets were positively proportional to their surface-to-volume ratios. This also allowed us to extract a maximal H₂O₂ generation rate of 7.7 nmol m^-2 min^-1 in the presence of a H₂O₂-consuming reaction and derive the corresponding probability of spontaneous conversion of interfacial H₂O into H₂O₂ for the first time, that is, ∼1 of 65,000 water molecules in 1 s. These findings delivered strong evidence that the spontaneous generation of H₂O₂ indeed occurs at the surface of microdroplets and provided us with an important starting point to further enhance the yield of H₂O₂ in water microdroplets for future applications.