Colorimetric determination of cholesterol based on the peroxidase-like activity of Cu-Salt-Fe nanozyme with multiple...

A strategy to enhance the peroxidase-like activity of the hemin composite is presented. The Cu-Salt-Fe with enhanced activity was synthesized by one-step heat treatment and applied to the colorimetric determination of free cholesterol in human serum. Phosphate can act in complexing Cu²⁺ to form a carrier Cu-Floc with a large specific surface area. The coordination effect of Cu-Floc with hemin was used to disperse and load hemin to form Cu-Floc-Hemin from which Cu-Salt-Fe was prepared. The Cu-Salt-Fe exhibits a synergistic catalytic effect of Cu-Salt, Fe²⁺, Fe³⁺, or Fe-Nx active sites in amplification of H₂O₂ oxidation. As expected, Cu-Salt-Fe triggered H₂O₂-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), resulting in H₂O₂-dependent absorbance changes at 652 nm. A cholesterol oxidase (ChOx)/Cu-Salt-Fe-TMB colorimetric sensing system exhibited excellent cholesterol determination performance in the range 5-1200 μM with a detection limit of 2.73 μM. Cholesterol recoveries from the three-level spiked serum ranged from 92.2 to 98.9% (RSD ≤ 5.4). This colorimetric sensing system not only provided a strategy for the determination of endogenous substances with H₂O₂ as an intermediate, but also provided a new design idea (carrier selection, activity enhancement method) for the development of other artificial enzymes with the same catalytic core.