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Bioconjugation of nanozyme and natural enzyme to enable a one-step cascade reaction for the detection of metabolites

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Abstract

Nanozyme, with enzyme-mimicking activity and excellent stability, has attracted extensive attention. However, some inherent disadvantages, including poor dispersion, low selectivity, and insufficient peroxidase-like activity, still limit its further development. Therefore, an innovative bioconjugation of a nanozyme and natural enzyme was conducted. In the presence of graphene oxide (GO), histidine magnetic nanoparticles (H-Fe3O4) were first synthesized by a solvothermal method. The GO-supported H-Fe3O4 (GO@H-Fe3O4) exhibited superior dispersity and biocompatibility because GO was the carrier and possessed outstanding peroxidase-like activity because of the introduction of histidine. Furthermore, the mechanism of the peroxidase-like activity of GO@H-Fe3O4 was the generation of •OH. Uric acid oxidase (UAO) was selected as the model natural enzyme and covalently linked to GO@H-Fe3O4 with hydrophilic poly(ethylene glycol) as a linker. UAO could specifically catalyze the oxidation of uric acid (UA) to generate H2O2, and subsequently, the newly produced H2O2 oxidized the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue ox-TMB under the catalysis of GO@H-Fe3O4. Based on the above cascade reaction, the GO@H-Fe3O4-linked UAO (GHFU) and GO@H-Fe3O4-linked ChOx (GHFC) were used for the detection of UA in serum samples and cholesterol (CS) in milk, respectively. The method based on GHFU exhibited a wide detection range (5–800 μM) and a low detection limit (1.5 μM) for UA, and the method based on GHFC exhibited a wide detection range (4–400 μM) and a low detection limit (1.13 μM) for CS. These results demonstrated that the proposed strategy had great potential in the field of clinical detection and food safety.

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Grant No. 21605114) and Tianjin Natural Science Foundation (Grant No. 17JCQNJC13300).

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  1. Jin-Ye Lang and Jia-Meng Zhao contributed equally to this work.

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  1. Tianjin Key Laboratory On Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Building B for School of Pharmacy, Tianjin Medical University, 22 Qixiangtai Road, Heping District, Tianjin, 300072, China

    Jin-Ye Lang, Jia-Meng Zhao, Ming-Jin Ren, Xin-Yu Wang, Le-Ping Chen, Xin-Chi Zhang, Xian-Hua Wang & Lin-Yi Dong

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The study (bc2022278) was approved by the institutional ethics committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China), and carried out in according with the ethical guidelines of the Declaration of Helsinki for experiments involving humans. All healthy volunteers from our laboratory received a detailed description of the study and provided written informed consent to participate in the study before providing their serum samples.

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Lang, JY., Zhao, JM., Ren, MJ. et al. Bioconjugation of nanozyme and natural enzyme to enable a one-step cascade reaction for the detection of metabolites. Anal Bioanal Chem 415, 3385–3398 (2023). https://doi.org/10.1007/s00216-023-04720-9

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