Enzyme Assisted Nanolithography

Abstract

In this work a method is developed to chemically immobilize proteins on the apex of the tip of an atomic force microscope (AFM) in order to chemically or physically modify surfaces with high spatial resolution. The feasibility of this method is demonstrated exemplarily with the enzyme alka­line phosphatase and the substrate 5-­bromo­-4-­chloro­indoxyl­phosphate (BCIP) and the cofactor nitro­blue­tetrazol (NBT). The product of the enzymatic reaction precipitates and it is possible to deposit it locally by bringing the functionalized tip in the vicinity of a suitable surface. The deposition of single spots and continuous contours with typical dimensions of 10 nm in height and 150-­170 nm laterally is demonstrated. Estimation of the number of immobilized enzymes on the apex of the AFM tip suggest a number of approximately 40 enzymes. This novel method was dubbed Enzyme-­assisted Nanolithography. The universality of this technique gives it a "tool box" character, which allows the employment of many different proteins. The second part of this work deals with the attempt to regulate the activity of immobilized enzymes by adjusting the pH value in their vicinity by the application of electric surface potentials. So far it was not possible to demonstrate the feasibility of this concept. A theoretical analysis will be given and suggestions for improved experiments are made. The third part describes the development of a self-­built AFM. The instrument features small physical dimensions which make it suitable for fast imaging of samples. The software is based entirely on Open Source components, thus allowing easy modification and adaption of the instrument to new requirements.