Zeolitic-imidazolate framework (ZIF-8)-based immobilized multi-enzymes integrated with a colorimetric sensor for cholesterol assay

Co-immobilization strategies are available to improve the catalytic activity, stability and reusability of enzymes. It has been frequently applied in recent year in reactions involving multi-step catalysis to optimize the performance. In this study, a zeolitic-imidazolate framework 8 (ZIF-8)-based nanoreactor (Chox&HRP@ZIF-8) including cholesterol oxidase (Chox) and horseradish peroxidase (HRP) was developed successfully. The Chox&HRP@ZIF-8 was characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy, respectively. The as-prepared materials were used as a biocatalytic system to realize the cascade reaction initiated by Chox and HRP. Next, the investigation of the catalytic activity and stability of Chox&HRP@ZIF-8 was performed in extreme environments. The results showed that the stability of the prepared Chox&HRP@ZIF-8 was significantly improved compared with that of free enzymes. In addition, the assay method for cholesterol detection developed based on Chox&HRP@ZIF-8 as the biocatalytic system showed high selectivity and good interference resistance. It was successfully applied to the determination of cholesterol in mice serum. It is worth mentioning that the relative activity of co-localized Chox&HRP remained above 60% after 5 cycles for cholesterol detection, confirming the feasibility of the multi-enzyme immobilization strategy using ZIF-8 as a carrier in the cascade reaction.