Convenient Colorimetric Detection of Cholesterol Using Multi-Enzyme Co-Incorporated Organic-Inorganic Hybrid Nanoflowers.

We have developed a microscale well-plate colorimetric assay for the multiplexed detection of cholesterol in clinical human blood samples. This system utilizes a novel multi-enzyme incorporated organic-inorganic hybrid nanoflower which entrap both cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) as the organic components with copper phosphate as the inorganic component to detect cholesterol levels in blood samples. The hybrid nanoflowers, synthesized via an extremely simple but rapid sonication-mediated method within 5 min at room temperature, enable an efficient one-pot two-enzyme cascade reaction. The ChOx in the nanoflowers catalyze the generation of H2O2 only in the presence of cholesterol in the sample. This subsequently activates the HRP co-entrapped in the nanoflowers, thereby leading to the conversion of the employed chromogenic substrate, 3,3',5,5'-tetramethylbenzidine (TMB), into a blue-colored product. This strategy can be used to detect target cholesterol concentrations as low as 8 μM, with a linear range from 10 to 70 μM, which is suitable to diagnose high levels of cholesterol (hypercholesterolemia) with excellent stability over three weeks at room temperature. The biosensor also exhibited an excellent selectivity to detect target cholesterol even in the presence of common interfering biomolecules in human blood and showed a high degree of precision when employing human blood serum samples. Therefore, this hybrid nanoflower-based assay can be used in clinical practice for the multiplexed and reliable quantification of cholesterol, and readily extended to other enzymes to prepare multi-step cascade enzymatic reactions for various biotechnological applications.