Hybridization of surface-tethered oligonucleotide brushes

Abstract

Part 1. Introduction In this chapter the chemical structures of DNA and oligonucleotides as building materials for subsequent use in biosensor application are described. The general concepts of modern DNA-based biosensors are presented and the commonly used strategies of immobilization of biomolecules on surfaces are briefly depicted. This chapter also introduces the scope of the thesis and the motivation of the research and chosen methodological strategy. Part 2. Preparation and Characterization of Substrates for Oligonucleotide Immobilization Stable, predictable and irreversible immobilization of oligonucleotides on a surface is a crucial step in the development of DNA-based biosensors. Unfortunately, physical chemistry characterizations at each stage of the sample preparation are still missing. In this chapter the experimental approaches for covalent immobilization of oligonucleotides onto silicon surfaces are described. The existing methods of surface silanization and glutaraldehyde coupling were optimized for preparation of homogeneous layers covalently bound to silicon surfaces. The obtained results showed that the oligonucleotides bind to glutaraldehyde-modified substrates with preservation of their functional properties. Part 3. Optimal Hybridization Efficiency upon Immobilization of Oligonucleotide Double Helices For the first time, the covalent immobilization of oligonucleotides double helices on surfaces prior to sequential denaturation and rehybridization was proven to lead to optimal hybridization efficiency. Two indirect methods were used for monitoring these processes in situ: the quartz crystal microbalance with dissipation monitoring (QCM-D) and the wavelength interrogated optical sensor (WIOS, Bright Reader®). Both techniques led to the result that with this immobilization approach one could reach nearly 100% hybridization efficiency. Moreover, having applied the existing theory of polymer adsorption on surfaces to the surface tethering of nucleotide sequences, we demonstrated that for single stranded sequences the coil conformation prevails over the stretched one. Part 4. A Microcontact Printing Approach to the Immobilization of Oligonucleotide Brushes The microcontact printing of oligonucleotide double helices on surfaces enables their surface tethering into brushes in patterned structures, which further can be evidenced by efficient and selective sequential dehybridization and rehybridization. This concept was proved via exchange of the fluorescently labeled complementary sequence of the surface tethered nucleotide sequence. Hybridization with complementary sequences labeled with fluorescent tags emitting at different wavelength enabled to assess the sequential de-hybridization and re-hybridization cycle via fluorescence microscopy. Part 5. General Conclusions and Outlook The obtained results are summarized. Further investigations and potential applications are suggested.