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 HRP1–MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRPx–MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRPx–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/HRPx–MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction viaH2O2 production. This model bienzyme–MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.
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