Rolling circle amplification for plasmonic biosensors

The detection of specific nucleic acids holds great potential to investigate the presence of various genes which are associated with genetic and infectious diseases. Particularly for the diagnosis of infectious diseases, there is a great need for medical devices to obtain fast, accurate and reliable information of such sequence-specific nucleic acids. This thesis deals with the detection of antibiotic resistance genes, by employing a ligation-based padlock probe concept on an optical biosensor. Circularized padlock probes were enzymatically amplified by rolling-circle amplification (RCA) at room temperature. The combination of surface plasmon resonance (SPR) with surface-plasmon fluorescence spectroscopy (SPFS) is used to detect minute amounts of the fluorophore labeled amplification products. As a first step, the assay concept, including padlock circularization and subsequent isothermal nucleic acid amplification through RCA is demonstrated in the liquid phase. The generation of RCA product was visualized by standard agarose gel electrophoresis. Secondly, the assay was implemented on a plasmonic biosensor. SPR and SPFS detection principle allowed to monitor and confirm the surface-initiated growth of single-stranded DNA strands. The reaction was investigated towards the specificity of fluorophore labeling of the RCA product as well as hybridization of the padlock to the sensor surface. The time-dependent growth of densely packed DNA brushes was monitored in-situ by optical waveguide spectroscopy (OWS). The length of the surface-bound DNA and the extension rate of φ29-DNA polymerase was determined. Additionally, a concept to control the growth of the long DNA strands to the sensor surface is shown and a possible route for the rapid detection of antibiotic resistance genes in a multiplexed format is proposed.