Nanomolar Hydrogen Peroxide Detection Using Horseradish Peroxidase Covalently Linked to Undoped Nanocrystalline Diamond Surfaces

In this article, we report on the low-level detection of hydrogen peroxide, a key player in the redox signaling pathway and a toxic product in the cellular system, using a colorimetric solution assay. Amine-terminated undoped nanocrystalline diamond thin films were grown on glass using a linear-antenna microwave plasma CVD process. The diamond surface consists mainly of -NH2 termination. The aminated diamond surface was decorated with horseradish peroxidase (HRP) enzyme using carbodiimide coupling chemistry. The success of the HAP immobilization was confirmed by X-ray photoelectron spectroscopy (XPS). The enzymatic activity of immobilized HRP was determined with a colorimetric test based on the HRP-catalyzed oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sufonic acid (ABTS) in the presence of hydrogen peroxide. The surface coverage of active HRP was estimated to be Gamma = 7.3 x 10(13) molecules cm(-2). The use of the functionalized diamond surface as an optical sensor for the detection of hydrogen peroxide with a detection limit of 35 nM was demonstrated.