Random and Positional Immobilization of Multi-enzyme Systems.

In recent years, three key techniques including random co-immobilization, positional co-immobilization, and compartmentalization for multi-enzyme immobilization were extensively considered. Herein, we investigate random co-immobilization and positional co-immobilization techniques for multi-enzyme systems in detail. We describe randomly co-immobilized glucose oxidase (GOx) and horseradish peroxidase (HRP) on reduced graphene oxide (rGO) as the most used methods. Materials and methods are presented in terms of preparation of GO and rGO as well as enzyme immobilization procedure. Moreover, the principles of positional co-immobilization have been reviewed, and the relevant methods based on microfluidic systems and DNA structure considering HRP and GOx enzymes have been individually studied. It is believed that the benefits of using the methods associated with random and specifically positional immobilized multi-enzyme systems include not only enhanced cascade enzymatic activity via manipulated surface such as microfluidic systems (including porous materials) and DNA structure but also improved enzyme stability and ease of recovery for recycle.