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-

The main challenges in multienzymatic cascade reactions for CO2 reduction are the low CO2 solubility in water, the adjustment of substrate channeling, and the regeneration of co-factor. In this study, metal-organic frameworks (MOFs) were prepared as adsorbents for the storage of CO2 and at the same time as solid supports for the sequential co-immobilization of multienzymes via a layer-by-layer self-assembly approach. Amine-functionalized MIL-101(Cr) was synthesized for the adsorption of CO2. Using amine-MIL-101(Cr) as the core, two HKUST-1 layers were then fabricated for the immobilization of three enzymes chosen for the reduction of CO2 to formate. Carbonic anhydrase was encapsulated in the inner HKUST-1 layer and hydrated the released CO2 to HCO3-. Bicarbonate ions then migrated directly to the outer HKUST-1 shell containing formate dehydrogenase and were converted to formate. Glutamate dehydrogenase on the outer MOF layer achieved the regeneration of co-factor. Compared with free enzymes in solution using the bubbled CO2 as substrate, the immobilized enzymes using stored CO2 as substrate exhibited 13.1-times higher of formate production due to the enhanced substrate concentration. The sequential immobilization of enzymes also facilitated the channeling of substrate and eventually enabled higher catalytic efficiency with a co-factor-based formate yield of 179.8%. The immobilized enzymes showed good operational stability and reusability with a cofactor cumulative formate yield of 1077.7% after 10 cycles of reusing.

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A new thermally switchable molecularly imprinted monolith for the selective capture and release of proteins has been designed. First, a generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith reacted with ethylenediamine followed by functionalization with 2-bromoisobutyryl bromide to introduce the initiator for atom transfer radical polymerization. Subsequently, a protein-imprinted poly(N-isopropylacrylamide) layer was grafted onto the surface of the monolithic matrix by atom transfer radical polymerization. Scanning electron microscopy and energy-dispersive X-ray spectroscopy of the cross-sections of imprinted monoliths confirmed the formation of dense poly(N-isopropylacrylamide) brushes on the pore surface. The imprinted monolith exhibited high specificity and selectivity toward its template protein myoglobin over competing proteins and a remarkably large maximum adsorption capacity of 1641 mg/g. Moreover, this "smart" imprinted monolith featured thermally responsive characteristics that enabled selective capture and easy release of proteins triggered only by change in temperature with water as the mobile phase and avoided use of stronger organic solvents or change in ionic strength and pH.

New monolithic materials comprising zeolitic imidazolate framework (ZIF-8) located on the pore surface of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith previously functionalized with N-(3-aminopropyl)-imidazole have been prepared via a layer-by-layer self-assembly strategy. These new ZIF-8@monolith hybrids are used as solid-phase carriers for enzyme immobilization. Their performance is demonstrated with immobilization of a model proteolytic enzyme trypsin. The best of the conjugates enable very efficient digestion of proteins that can be achieved in mere 43 s.