Icaritin (ICT), a biologically active isoprenylated flavonoid, which has been shown to delay liver fibrosis, inhibit cancer cell proliferation and other therapeutic effects in the treatment of hepatocellular carcinoma. However, traditional methods for icaritin preparation suffer from significant drawbacks such as high environmental pollution, energy consumption, and low yield. In this study, snailase (SNA) was immobilized for the first time in hollow silica nanospheres (HSN) through facile and novel in situ immobilization and soft template removal method to construct highly active biocatalysts (SNA@HSN) where enzymes in the cavity exhibit “immobilized but not rigid state”. The resultant SNA@HSN showed excellent activity (78% activity recovery) and stability suffered from extreme pH, temperature, and organic solvents. Meanwhile, the reusability and storage stability of SNA@HSN were satisfactory. After 5 cycles, SNA@HSN still maintained 79% original activity. Especially, SNA@HSN exhibited highly efficient biotransformation of icariin (ICA) to ICT in deep eutectic solvents (DESs) system. ICT yield in choline chloride–propylene glycol (ChCl:P, 25% v/v) system was 111% higher than that of aqueous system, reached 374 mg/L. Thus, this study developed an efficient methodology for icaritin preparation through the integration of enzymatic catalysis and environmentally friendly catalytic medium.

Recent years, incorporation of multiple enzymes into matrix is considered as promising candidates for constructing highly sensitive glucose-responsive systems. However, the stochastic nature of traditional multi-enzyme immobilization techniques makes it challenging to precisely control the spatial arrangement of enzyme molecules. Here, a new hierarchical shell-shell (HSS) multi-enzyme structure was constructed using interfacial growth method and colloidal hydrated zinc sulphate to be used as a soft template (Cyt C@HSS@GOx). In this process, glutathione (GSH) hybrid ZIF-67 was used as the guest MOF and hollow ZIF-8 as the host MOF. The resultant Cyt C@HSS@GOx exhibited high activity by separating cytochrome C (Cyt C) and glucose oxidase (GOx) in two independent compartments. This method can accurately locate the spatial position of multiple enzymes, weaken enzyme-to-enzyme inhibition, and enhance the channeling effect of substrates, thus improving the catalytic performance of multiple enzymes. The resulting Cyt C@HSS@GOx showed 230% higher activity than free enzymes, and maintained good stability under extreme pH and temperature conditions. Furthermore, Cyt C@HSS@GOx demonstrated 71.25% retention of its initial activity following seven cycles, providing good reusability. Particularly, Cyt C@HSS@GOx has been demonstrated to possess a glucose detection limit as low as 0.38 μmol/L, with a reaction time of only three minutes. This strategy provides a rapid and accurate glucose detection method.