In this work, through the in situ growth of MnO2 nanosheets on the surface of terbium metal–organic frameworks (Tb–MOFs), MOF@MnO2 nanocomposites are prepared and the fluorescence of Tb–MOFs is quenched significantly by MnO2. Additionally, the hybrid nanoflowers are self-assembled by cholesterol oxidase (ChOx) and copper phosphate (Cu3(PO4)2·3H2O). Then a new strategy for cholesterol determination is developed based on MOF@MnO2 nanocomposites and hybrid nanoflowers. Cholesterol is oxidized under the catalysis of hybrid nanoflowers to yield H2O2, which further reduces MnO2 nanosheets into Mn2+. Hence, the fluorescence recovery of Tb–MOFs is positively correlated to the concentration of cholesterol in the range of 10 to 360 μM. The limit of detection (LOD) of cholesterol is 1.57 μM. On the other hand, the hierarchical and confined structure of ChOx–inorganic hybrid nanoflowers greatly improve the stability of the enzyme. The activity of hybrid nanoflowers remains at a high level for one week when stored at room temperature. Moreover, the hybrid nanoflowers can be collected by centrifugation and reused. The activity of hybrid nanoflowers can continue at a high level for five cycles of determination. Therefore, it can be concluded that the hybrid nanoflowers are more stable and more economic than free enzymes, and they show a similar sensitivity and specificity to cholesterol compared with free ChOx. Finally, this strategy has been further validated for the determination of cholesterol in serum samples with satisfactory recoveries.
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