A novel sandwich structured SiO2 gel/cytochrome c (Cyt c)/SiO2 gel was designed and constructed on
conductive boron-doped diamond (BDD) film substrate. A SiO2 gel membrane was first in situ deposited
on the pretreated positive charged H-terminated BDD electrode with a simple and artful surface vapor
sol–gel method. Cyt c was subsequently immobilized onto the SiO2 membranes by electrostatic attraction,
followed by another SiO2 gel layer in situ depositing on it. The SiO2 interlayer was conceived to play
an important role in the resultant sandwich structured SiO2/Cyt c/SiO2/BDD electrode as a selective
‘‘semi-open’’ medium, which guaranteed the immobilized Cyt c to maintain high stability and perform
good electrochemistry and biocatalysis responses. The bioactivity of Cyt c was well protected and the
immobilized biomolecule even didn’t denature at extremely high or low pH condition. More attractively,
Cyt c in the sandwich structured electrode could be further oxidized into highly reactive Cyt c p-cation by
two-step electrochemical oxidation, which could oxidize NO2 into NO3 in the solution. A sensitive determination
approach of nitrite was accordingly built up based on this biocatalytic oxidative interactionfor the first time and a possible mechanism of the interaction was herein proposed.