Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.

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