Bioconjugation of nanozyme and natural enzyme for ultrasensitive detection of cholesterol

When nanozymes are used in biological analysis, higher activity can improve the detection sensitivity, and better selectivity can eliminate other interference. To improve the specificity and sensitivity, we fabricated an innovative bioconjugated nanozyme with natural enzyme (BNNZ), in which natural ChOx was immobilized onto histidine-modified Fe3O4 (His-Fe3O4) with hydrophilic poly(ethylene glycol) (PEG) as a linker. ChOx could specifically catalyze the oxidation of cholesterol to generate H2O2 molecule, and then the newly formed H2O2 oxidized the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue ox-TMB by peroxidase-like His-Fe3O4. According to the above cascade reaction, the BNNZ-based colorimetric strategy was proposed for the detection of cholesterol. Wherein, natural enzymes specifically catalyzed substrates, which endowed BNNZ with excellent specificity for target molecules; meanwhile, the introduction of histidine on His-Fe3O4 effectively increased the peroxidase-like activity of BNNZ, which provided a guarantee for sensitivity. Furthermore, BNNZ after reaction could be rapidly separated by an external magnetic field without interfering with colorimetric quantitative detection. The proposed strategy exhibited excellent sensitivity with limit of detection of 0.446 μM and was successfully used for the detection of cholesterol in spiked human serum sample with recovery and relative standard deviation in the range of 97.9-103.5% and 2.5-4.0%, respectively. This work indicates that the bioconjugation of nanozyme and natural enzyme may be a universal strategy for synthesis of high-performance enzyme-nanozyme systems, and the new-type BNNZ will be widely used in biological detection and disease treatment.