Preparation of functionalized star polymer nanoparticles by RAFT polymerization and their application in positionally assembled enzymes for cascade reactions

A well-defined functional star polymer nanoparticle was prepared using an “arm first” approach via RAFT polymerization. In this work, N-adamantylacrylamide and N-succinimidyl acrylate were copolymerized with N-isopropylacrylamide sucessfully. The obtained diblock copolymer regarded as an “arm” endowed the star polymer with multiple conjugate functions. By adjusting the amount of crosslinking agent, the size and polydispersity of the star polymer were controlled. With the star polymer as the scaffold, a sequential multi-enzyme system was constructed, where HRP was placed in the inner layer through covalent conjugation, and β-cyclodextrin-modified GOx was assembled in the outer layer through host–guest recognition. More interestingly, the activity of the enzyme modified by β-cyclodextrin was improved. In addition to the enhanced thermal stability, this multienzyme system also exhibited a reduced Km value (from 2.18 mM to 0.39 mM) and an excellent substrate affinity. The specificity constant (Kcat/Km) for GOx in the star polymer@HRP@GOx system was 1.7-fold higher than that of the free HRP with free GOx system. This strategy will promote the development of biocatalysis reactions and biosensors as a promising method.