Abstract
Nanomaterials, including carbon nanotubes, graphene oxide, metal-organic frameworks, metal nanoparticles, and porous carbon, play a crucial role as efficient carriers to enhance enzyme activity through substrate channeling while improving enzyme stability and reusability. However, there are significant debates surrounding aspects such as enzyme orientation, enzyme loading, retention of enzyme activity, and immobilization techniques. Consequently, these subjects have become the focus of intensive research in the realm of multi-enzyme cascade reactions. Researchers have undertaken the challenge of creating functional in vitro multi-enzyme systems, drawing inspiration from natural multi-enzyme processes within living organisms. Substantial progress has been achieved in designing multi-step reactions that harness the synthetic capabilities of various enzymes, particularly in applications such as biomarker detection (e.g., biosensors) and the development of biofuel cells. This review provides an overview of recent developments in concurrent and sequential approaches involving two or more enzymes in sequence. It delves into the intricacies of multi-enzyme cascade reactions conducted on nanostructured electrodes, addressing both the challenges encountered and the innovative solutions devised in this field.