Surfactant-free microemulsion as a fluid scaffold for the thermal stabilization of lysozyme

Electrostatic forces supported by hydrogen-bonding (H-bonding) interactions in the presence of surfactants generally stabilize microemulsions. So, contrary to this common wisdom, developing a surfactant-free microemulsion (SFME) that is predominantly stabilized by weak but large number of H-bonding interactions would be remarkable. Herein, the formulation and characterization of an SFME comprising a hydrophobic ionic liquid (IL) and a deep eutectic solvent (DES) exhibiting high thermal stability are reported. The constituents of DES, namely, ethylene glycol (EG) and choline chloride (ChCl), served as polar and amphiphile components, respectively, and an IL, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, worked as a hydrophobic entity to form the SFME. The formation mechanism and high-temperature thermal stability of the SFME are discussed in terms of relative changes in the thickness of interfacial films, predominantly stabilizing the polar and non-polar pseudo-domains via alteration in H-bonding interactions, which is supported by computational studies. The sufficiently low interfacial energy in the SFME is exploited to thermally stabilize lysozyme (LYZ) in SFME, resulting in remarkable thermal stability of up to 150 °C, which is higher than that observed in buffer, as revealed by comparative enzyme activity at room temperature after heating. This study not only adds to the existing knowledge about the formation and stability of SFMEs but is also expected to prompt other researchers to design relatively greener IL or deep eutectic solvent (DES)-based SFMEs for various biological and other applications.