Cholesterol oxidase functionalization of a polymerized crystalline colloidal array

A cholesterol-responsive polymerized crystalline colloidal array (PCCA) is fabricated by covalently attaching the amine groups of cholesterol oxidase (ChOx) to an epoxide-functionalized PCCA. Approximately 0.6 mM of ChOx is immobilized on a PCCA, with the bound enzyme retaining about 85% of its initial activity, compared to the unbound enzyme. Kinetic studies reveal that the stability and activity of the free and bound enzyme are significantly improved upon binding to the PCCA, with the bound enzyme retaining 62% of its initial activity after 20 days, compared to no residual activity for the free enzyme. Furthermore, the bound enzyme exhibits a better tolerance to pH and temperature changes, as well as to repeated use. Upon exposure to 5.0 mM cholesterol, we observe diffraction peak wavelength shifts of as much as 63 nm, demonstrating potential application as an optical cholesterol sensor.