Magnetic multi-enzyme nanosystems have been prepared via co-precipitation of enzymes and metalorganic framework HKUST-1 precursors in the presence of magnetic Fe 3 O 4 nanoparticles. The spatial co-localization of two enzymes was achieved using a layer-by-layer positional assembly strategy.Glucose oxidase (GOx) and horseradish peroxidase (HRP) were used as the model enzymes for cascade biocatalysis. By controlling the spatial positions of enzymes, three bienzyme nanosystems GOx@HRP@HKUST-1@Fe 3 O 4 , GOx-HRP@HKUST-1@Fe 3 O 4 and HRP@GOx@HKUST-1@Fe 3 O 4 were prepared in which GOx and HRP containing layers were in close proximity, either encapsulated in the HKUST-1 inner layer, or immobilized on the HKUST-1 outer shell, or randomly distributed in the two MOF layers. Their properties were characterized by transmission electron microscopy, energydispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermal gravimetric analysis, and zeta potential measurements. The highest activity was observed at pH ¼ 6 and a temperature of 20 C. Thanks to the favorable positioning of enzymes, the GOx@HRP@HKUST-
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