Deciphering the formation of biogenic nanoparticles and their protein corona: State-of-the-art and analytical challenges

Biogenic synthesis of nanoparticles (NPs) using biological organisms such as plants, fungi, and bacteria offers a sustainable, green alternative to conventional chemical synthesis. These green methods involve biological matrices mediating metal ion reduction to form NPs capped by complex biomolecular coronas. These protein coronas critically affect NP stability, biological identity, and functionality, including notable therapeutic potentials. Despite advancements, the dynamic and heterogeneous nature of in situ biogenic NP formation remains insufficiently understood, as most studies emphasize synthetic production using biological extracts combined with metal precursors. Moreover, characterization of biogenic NPs requires a suite of bioanalytical techniques, each offering diverse advantages but also inherent limitations, requiring a comprehensive understanding of their features to critically select the optimal combination of strategies to obtain a full descriptive picture of the studied NPs. This review critically discusses current biogenic synthesis methods alongside the complementary bioanalytical strategies employed to elucidate NP formation, corona composition, and their biological implications. By highlighting the strengths and weaknesses of available techniques, the review aims to identify key challenges and future directions necessary to advance the safe and effective development of green nanotechnologies.