Ravi Kumar

Dissertation, Westfälische Wilhelms-Universität (WWU) (2016) 

The thesis deals with enhanced multi-color patterning by Polymer Pen Lithography (PPL) and the use of the generated arrays for biological and bio-medical applications. For this, the multiplex capabilities of PPL were substantially enhanced in regard to number of ink components and array pattern fidelity. To enhance stability by covalent bound arrays, two ink-substrate combinations (Azide-alkyne modified glass and oligonucleotideaminoreactive glass) are demonstrated. The introduction of a simple ink coating strategy, redesign of the stamp with different sections, an offset correction method and precise stamp positioning during lithography, expands the ability of PPL to multi-color patterning into the cm² area. Complex features with dots and lines, all in true multiplexed and interdigitated patterns are demonstrated. By fabricating pen array with four and five sections, the multi-color patterning of up to five different ink compounds can be achieved.

The profiling of allergic responses is a powerful tool in biomedical research and in judging therapeutic outcome in patients suffering from allergy. Novel insights into the signaling cascades and easier readouts can be achieved by shifting activation studies of bulk immune cells to the single cell level on patterned surfaces. The functionality of dinitrophenol (DNP) as a hapten in the induction of allergic reactions has allowed to study the activation process of single mast cells seeded on patterned surfaces following treatment with allergen specific Immunoglobulin E (IgE) antibodies. Here, a click-chemistry approach in combination with PPL is applied to pattern DNP-azide on alkyne-terminated surfaces to generate arrays of allergen. The large area functionalization offered by PPL allows an easy incorporation of such arrays into microfluidic chips. In such a setup, easy handling of cell suspension, incubation process and read-out by fluorescence microscopy would allow immune cell activation screening to be easily adapted for diagnostics and biomedical research.

In-vitro study of gene expression is generally dependent on conventional DNA microchips, however, these are expensive to fabricate. PPL together with DNA-directed Immobilization (DDI) provides a low cost immobilized DNA array, which can be easily read-out at a microscope. Introduction of the oligonucleotides for PPL generated arrays offers a virtual unlimited inventory of orthogonal binding tags for later hybridization and self-assembly of proteins. For a proof of concept, multi-color DNA oligonucleotides arrays are utilized to monitor cell-protein interaction at the extracellular membrane of MCF7 cells. These arrays can be useful in future for in-vitro study in genetics, expression profiling, diagnostics and drug development.

PPL can also be used to prepare arrays with size gradients over a large area. This is used to study different ink-transport mechanism depending on the contact pressure on the pens in case of phospholipid deposition on cleaned glass. When switching to more hydrophilic silicon oxide surfaces the techniques provides lateral size gradient arrays with constant height features of 7 nm (2 bilayer with a wetting layer). Also, a new strategy to prepare density gradients of bioactive-lipids features is applied. In conclusion, these arrays can be useful to study the interfacial energy of a lipid or in experiments on lipid membrane organization for the field of biotechnology or in detection and purification applications for biomaterials.