Nanoporous gold film encapsulating cytochrome c for the fabrication of a H₂O₂ biosensor

A layer-by-layer route to prepare nanoporous Au film materials on transparent ITO substrates is reported by alternatively assembling Au and Ag nanoparticles through 1,5-pentanedithiol as a cross-linker, followed by that Ag nanoparticles are dissolved at room temperature in HAuCl₄ solution. Electron transfer of cytochrome c (cyt. c) - an excellent model for investigation of biomolecules, is greatly facilitated at the nanoporous Au film with electron transfer rate constant (k_s) of 3.9 s^-1. Meanwhile, cyt. c adsorped onto the nanoporous Au film still maintain its enzymatic activity toward H₂O₂. On the basis of these experimental results, the cyt. c-nanoporous Au film is exploited to an amperometric biosensor for H₂O₂ with high selectivity, broad linear range, low detection limit, and long stability.