Biomolecule micropatterns for quantification of protein-protein-interactions in living cells

Roland Hager

Research output: Types of ThesesDoctoral Thesis


Micropatterned surfaces can be used as unique and powerful biointerfaces to study cellular processes within the plasma membrane as well as in the cytosol. Several different methods for the generation of these functional substrates have been established within the last decades. Among those, a widespread and frequently used method for the fabrication of micropatterns is microcontact printing (µCP). This method, and an alternative to this approach (photolithography) are highlighted in this thesis. Furthermore, glass as a traditional substrate as well as polymer-based foils were tested for biomolecule patterning. In addition, two different possibilities for the functionalization of polymeric surfaces were described: functional chemical groups can be introduced either via plasma activation followed by epoxysilane-coating, or with a commercially available polymer metal ion coating (AnteoBindTM Biosensor reagent).

We demonstrated the suitability of our different surface modification approaches for subcellular micropatterning experiments in living cells: on the one hand, analysis of surface receptor mediated protein-protein-interactions (PPIs) and on the other hand, a method for immunopatterning of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with micropatterned antibody arrays are presented. Aim of this thesis was to improve existing micropatterning techniques and to investigate alternative polymeric materials that can be used for the fabrication of micropatterned surfaces. Furthermore, experimental throughput with these biosensors in combination with 384-well plates was increased. The results were published in four different scientific journals.

Fabrication, Characterization and Application of Biomolecule Micropatterns on Cyclic Olefin Polymer (COP) Surfaces with Adjustable Contrast. In this work we report on a technique using a photolithographic approach for the fabrication of biomolecule micropatterns on cyclic olefin polymer (COP) foils. Here we describe how to generate these substrates, and how to manipulate the biomolecule density on the micropatterned surface. By variation of plasma activation parameters (plasma energy and treatment time) the deposition of proteins on the polymeric material can be influenced. The validation of receptor interactions in living cells was tested with immobilized epidermal growth factor (EGF) and Jurkat cells expressing the EGF-receptor. Finally, the implementation of this setup to analyze PPIs in living cells via total internal fluorescence (TIRF) microscopy was successfully demonstrated.

A Simplified and Robust Activation Procedure of Glass Surfaces for Printing Proteins and Subcellular Micropatterning Experiments. In this study a simplified activation procedure for untreated glass surfaces without the need for physical and chemical substrate modification was described: a commercially available polymer metal ion coating (AnteoBindTM Biosensor reagent) that allows the strong attachment of biomolecules via avidity binding. This is a very fast and straightforward approach for the fabrication of micropatterned glass substrates via µCP that can be easily implemented in daily lab routine, and it can be adapted to polymer substrates as well. We characterized the optimum working concentrations of the solution and analyzed the protein binding capacity. Besides, the applicability of this approach for the evaluation of PPIs in living cells was demonstrated. With this method we realized a substantial simplification of the µCP process and proved the biocompatibility of the surface with micropatterning experiments in living cells and their characterization via TIRF microscopy.

Microcontact printing of biomolecules on various polymeric substrates: limitations and applicability for fluorescence microscopy and subcellular micropatterning assays. Here we describe the characterization of various polymer materials with respect to their optical parameters and their applicability for TIRF microscopy. Therefore, eleven different polymer foils were analyzed to evaluate their performance regarding plasma activation, surface functionalization, protein micropatterning, fluorescence microscopy and their applicability in live-cell assays. The introduction of functional groups on the polymer surface was achieved via plasma activation followed by epoxysilane-coating. Biomolecule micropatterns were finally produced via µCP. Three polymer substrates were identified to be a promising alternative to glass substrates: cyclic olefin copolymer (COC), cyclic olefin polymer (COP) and polymethylmethacrylate (PMMA).

Subcellular Dynamic Immunopatterning of Cytosolic Protein Complexes on Microstructured Polymer Substrates. A technique for dynamic immunopatterning of cytosolic protein complexes was developed. Importantly, a 384-well plate-based platform on micropatterned COP substrates was realized to increase experimental throughput and to overcome the drawbacks of glass surfaces. For this purpose, we used micropatterned substrates in combination with artificial transmembrane bait constructs. Protein arrays inside living cells were realized with this method and used to characterize the growth factor receptor-bound protein 2 (Grb2) mediated signaling pathways downstream of the epidermal growth factor receptor (EGFR). With this approach Src homology 2 (SH2) and Src homology 3 (SH3) protein domain inhibitors were studied in a live cell environment. With the presented method several cytosolic proteins of interest that are placed near the plasma membrane can be analyzed in a live cell environment.

Overall, this thesis describes two different methods for the fabrication of micropatterns on glass and polymer substrates as well as the analyzation of suitable polymer materials for micropatterning and subsequent fluorescence microscopy. Furthermore, applicability and relevance in live cell-based protein-protein-interaction characterization is highlighted.
Translated title of the contributionMikrostrukturierte Oberflächen zur Quantifizierung von Protein-Protein-Wechselwirkungen in lebenden Zellen
Original languageEnglish
QualificationDr. techn.
Awarding Institution
  • Johannes Kepler University Linz
  • Romanin, Christoph, Supervisor, External person
  • Weghuber, Julian, Supervisor
  • Lanzerstorfer, Peter, Supervisor
Award date30 Jul 2022
Place of PublicationLinz
Publication statusPublished - 2022


  • Biomolecule micropatterns
  • protein-pro
  • TIRF


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