Eco-Friendly Surface Functionalization of Fe ₃ O ₄ Nanoparticles with Cholinium Citrate-Based Deep Eutectic Solvents: A Sustainable Material To Improve the Stability and Efficiency of Enzymes

magnetic nanoparticles (MNPs) functionalized with deep eutectic solvents (DESs) based on cholinium citrate and ethylene glycol (EG). MNPs were synthesized via chemical coprecipitation and further coated using three different DESs formulations: mono-, di-, and tricholinium citrate-EG, referred to as MNPs-DES1, MNPs-DES2, and MNPs-DES3, respectively. Interactions between SB and MNPs-DESs were studied by employing a combination of spectroscopic techniques, thermodynamic, and kinetic analysis. Spectroscopic analyses revealed that MNPs functionalized with higher cholinium content (DES2 and DES3) showed stronger interactions and also induced structural instability and aggregation. On the other hand, MNPs-DES1 offered moderate binding while preserving the enzyme's conformation. Dynamic light scattering (DLS) and microscopic results further confirmed that DES1-modified MNPs led to smaller, more stable enzyme-MNPs complexes, reducing aggregation compared to MNPs or MNPs functionalized with DESs of higher alkyl chain length. Thermal fluorescence studies highlighted MNPs-DES1 as the most effective in enhancing SB's resistance to thermal denaturation, supported by favorable thermodynamic stability. Therefore, MNPs-DES1 is the best preserved material for SB structure and function, improving both thermal stability and activity across concentrations. These results demonstrate the importance of fine-tuning NPs surface properties and support the use of DES1-coated MNPs as promising candidates for enzyme stabilization in biotechnology, catalysis, and biomedical applications.