Bioenzymatic Approach to Cholesterol Detection Using Cholesterol Oxidase-Immobilized Copper-Incorporated Zeolitic Imidazolate Framework-8.

A significant challenge in creating enzyme-based electrochemical biosensors is the immobilization of enzymes onto the electrode surface while retaining their activity. High surface area, tunable porosity, biocompatibility, and stability of metal-organic frameworks (MOFs) make them a suitable matrix for the immobilization of enzymes. Besides acting as an effective immobilization platform, MOFs exhibit interesting enzyme-mimicking capabilities as well. In this study, we demonstrate dual functionality of copper-incorporated zeolitic imidazolate framework-8 (Cu@ZIF-8) both as a peroxidase mimic and as an efficient immobilization matrix for the enzyme, cholesterol oxidase (ChOx), enabling the electrochemical sensing of cholesterol. ChOx enzyme is immobilized onto Cu@ZIF-8 using biomimetic mineralization strategy. Enhanced sensitivity and lowered Michaelis-Menten constant values are observed for enzyme-immobilized system compared to the nonimmobilized system, indicating the stronger substrate affinity of the developed sensing platform. This suggests that Cu@ZIF-8 acts as a scaffold for the enzyme immobilization and improves the enzyme stability and reaction kinetics. Moreover, this strategy also avoids the complications of using multiple enzymes for sensing purposes. This dual functionality of MOFs enhances the stability and selectivity of the biosensor while eliminating the use of multiple enzymes and thus paves the way for the fabrication of cost-effective and robust next-generation biosensors.