Lithographic Patterning of Nanoscale Arrays of the Oxidase Enzyme CotA: Effects on Activity and Stability

Abstract

This study compares the enzymatic activity of nanoscale arrays bearing the oxidase CotA that are produced by two lithographic methods: polymer pen lithography (PPL), a scanning probe lithography for small‐area fabrication (≈1 cm2); and stepper photolithography, a large‐scale method (>300 cm2) used in the microelectronics sector. In both cases, arrays of 600 nm gold features are produced and functionalized with CotA through a bioconjugation method that gives uniform protein orientation. The enzyme activity of these arrays is then quantified over 100 days. The enzyme arrays produced by photolithography give higher oxidation activities immediately after fabrication but degrade more rapidly when compared to those produced by PPL. This result is due to the poorer passivation on the bulk surface of photolithographically produced arrays, resulting in a greater amount of non‐specifically adsorbed enzymes. However, once the results are adjusted to account for the differences in passivation and surface area, it is found that the enzymes immobilized on the gold features give essentially the same activity regardless of the lithographic method used. Thus, these results suggest PPL is a suitable method for prototyping biodevices prior to scale‐up, provided that due consideration is given to the design of the fabricated features.

Versatile Biosynthetic Approach to Regioselective Enzyme Patterning for Direct Bioelectrocatalysis Application

“…Spatial localization of a redox enzyme on an electrode is of crucial importance in the prospective development of an enzymatic electrochemical platform that can be applied to green and sustainable electrical or biochemical energy harvesting systems. [ 1–5 ] In nature, redox enzymes are organized in the membrane matrix with controlled spacing and positions to accomplish enzymatic catalysis with high catalytic efficiency. [ 6,7 ] Specifically, such a spatial organization of enzymes enables the efficient delivery of metabolites among enzyme active sites while preventing the steric hindrance of a reaction site by adjacent proteins.…”