Dual-Loading of Fe3O4 and Pd Nanoparticles on g-C3N4 Nanosheets Toward a Magnetic Nanoplatform with Enhanced Peroxidase-like Activity for Loading Various Enzymes for Visual Detection of Small Molecules

Enzyme mimics now play a significant role in biochemistry. Especially, peroxidase mimics have been widely used for developing colorimetric sensors of blood glucose. The peroxidase mimics previously reported could not be recycled for reusing and may generate scattering to cause unwanted optical interference when it was used for fabricating colorimetric sensors. We herein prepared a broad-applicable and reusable magnetic enzyme-loading nanoplatform with enhanced peroxidase-like activity by simultaneously loading Fe₃O₄ nanoparticles (Fe₃O₄NPs) and palladium nanoparticles (PdNPs) on graphitic carbon nitride (g-C₃N₄) nanosheets (Fe₃O₄NPs/PdNPs/g-C₃N₄). The prepared Fe₃O₄NPs/PdNPs/g-C₃N₄ possesses stable and enhanced peroxidase-like activity and good enzyme-loading capacity and can be used to load various natural enzymes to form highly-efficient and stable double-active nanozyme for fabricating colorimetric sensors for the visual detection of small molecules. Especially, the magnetic feature facilitates the magnetic separation of Fe₃O₄NPs/PdNPs/g-C₃N₄ from sample solution, which is in favor of recycling and eliminating the optical interference caused by nanozyme in colorimetric sensors. The prepared Fe₃O₄NPs/PdNPs/g-C₃N₄ has been successfully used to load glucose oxidase (GOx) and cholesterol oxidase (Chox) to form magnetic peroxidase-GOx and peroxidase-Chox double-active nanozymes, which can be used to fabricate colorimetric methods for the detection of glucose and cholesterol, respectively, with a visual detection limit of 15 μM and a spectrometry detection limit of 1.0 μM. With the developed glucose and cholesterol detection methods, we have successfully detected glucose and cholesterol in serum with a recovery of 98-104% and a RSD (n = 5) < 5%. With high peroxidase-like activity, good stability, reusable features, and broad applicability of loading enzyme, the developed magnetic Fe₃O₄NPs/PdNPs/g-C₃N₄ provided a promising approach for fabricating cost-effective, sensitive, and simple colorimetric sensors for the visual detection of various small molecules.