Microcontact printed diaphorase monolayer on glass characterized by atomic force microscopy and scanning electrochemical microscopy

Micropatterns of diaphorase (Dp) were fabricated on glass substrates by the microcontact printing (μCP) method and characterized with atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM). AFM images of the printed samples revealed that the mean height of the Dp patterns was 3-5 nm, indicating the formation of a monolayer pattern. The Dp molecules on the surface organized themselves into two-dimensional arrays. We used two kinds of inking solutions: Dp-phosphate buffer solution (PBS) (pH 7.0) and Dp-PBS (pH 7.0) with glutaraldehyde (GA, 1% v/v) as a cross-linking reagent. Although the AFM imaging showed high-quality Dp monolayer patterns in both cases, SECM measurements indicated that the enzymatic activity of Dp was almost lost when Dp-PBS with GA was used as the inking solution, whereas clear enzymatic activity was found when Dp-PBS was used.