Deep Eutectic Solvent- and Ionic Liquid-Based Surfactant-Free Microemulsion Stabilized by H-Bonded Clusters: Interfacial Stabilization and Superactivity of Cytochrome-c.

The need to develop novel and sustainable media without using conventional surfactants has driven the development of surfactant-free microemulsions (SFMEs). Deep eutectic solvents (DESs) and ionic liquids (ILs) are great alternatives to conventional organic solvents, using which SFMEs can be prepared. Herein, an SFME has been prepared using a DES based on the natural components L-arginine and lactic acid as the polar phase; 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonylimide), a hydrophobic ionic liquid (HIL), as the nonpolar phase; and ethanol as an amphiphile. The SFME has been duly characterized to establish its structural organization at the molecular level by employing various techniques such as conductivity measurements, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), Fourier-transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Investigations on the internal structure of the SFME reveal that ethanol does not form an effective barrier layer, instead, it forms hydrogen-bonded (H-bonded) solvent domains interacting mainly with DES nanoclusters, followed by the imidazolium cation at the HIL polar-nonpolar interface. The presence of such nanoinhomogeneity at the solvent interfaces imparts an associative nature to microemulsion droplets, which has never been reported before. The prepared SFME has been employed as a nanoreactor to explore the catalytic activity of cytochrome-c (Cyt-c). Variation in the phase composition of the SFME markedly affects the enzyme activity, wherein the DES-in-HIL phase of the SFME offers an optimal environment for ∼6.5 folds enhancement in enzyme activity as compared to that observed in buffer. The favorable cation-π and H-bonding interactions of arginine and lactic acid at the DES-ethanol interface, along with ethanol clusters, stabilize the structure of Cyt-c. This study provides new insights into the structural and functional aspects of SFMEs, which will encourage the development of new SFME systems for a broad spectrum of biocatalytic applications, along with the utilization of nanoinhomogeneity for constructing hierarchically self-assembled materials.