Novel Strategies for Micro-Contact Printing Based Protein-Protein Interaction Detection

Abstract

Originally developed for application in micro-electronics, micro-patterning has spread over most areas of science, including biology. Micro-patterning allows for the specific design of cell micro-environment and now covers a large number of cell biology applications, from stem cell culture and differentiation to printing of purified proteins or other biomolecules for in vitro assays. Recent efforts led to the development of much simpler, cheaper and more versatile methods. Nevertheless, there are still some technical difficulties and unresolved problems which need to be addressed.We have recently introduced a method that is capable of detecting and characterizing protein-protein interactions (PPIs) in a live cell context by combining Total Internal Reflection Fluorescence microscopy and micro-patterned surfaces. Recent experiments showed that different biological questions require different pattern sizes and variations in the microstructure, respectively. Here we report on the advantages of PDMS-alternatives for an improved and fast micro-contact printing process. Using those materials we could print proteins in various structures (including pillars, lines and grids) and sizes down to 100 nm. For characterization of pattern quality we used TIRF and super-resolution microscopy and focused on the validation of well-known protein-protein interactions including the one of EGF-, Insulin/IGF1- or beta-adrenergic receptors with intracellular binding partners. As TIRF microscopy requires homogenously adhered cells on the micro-patterned surface, we especially focused on the verification of cell adhesion efficiency when using different pattern shapes.An important issue which we are currently addressing is the production of a micro-structured and functionalized multiwell plate (96 and 384 well design). This development step will set a milestone concerning the throughput rates of the micro-patterning assay and increase the number of potential users interested in this methodology.