Photometric determination of free cholesterol via cholesterol oxidase and carbon nanotube supported Prussian blue as a peroxidase mimic

The authors describe a photometric method for the determination of free cholesterol based on the oxidation of cholesterol by the catalytic action of the enzyme cholesterol oxidase. The hydrogen peroxide formed is used to oxidize the chromogenic compound 3,3′,5,5′-tetramethylbenzidine (TMB) by exploiting the peroxidase-like activity of carbon nanotube-supported Prussian blue (PB). This enzyme mimic was synthesized from a mixture of ferric chloride and hexacyanoferrate(III) in the presence of intrinsically reducing multi-walled carbon nanotubes. The composite can trigger the formation of a blue-green dye from TMB in the presence of H₂O₂. The findings were exploited to design a photometric (652 nm) assay that has a linear response in the 4 to 100 μM cholesterol concentration range and a 3 μM detection limit. The practicability of the method was verified by the successful analysis of free cholesterol in human blood samples.