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When designing
DNA
biosensors, the immobilization of specific DNA probes is one of the most essential parts. Unfortunately, many of the existing strategies (e.g., adsorption, complexation, and entrapment) can only be used on standard microscope slides, while for more tailored surfaces alternative strategies are still required. In the case of gold surfaces, the self-assembly of mixed DNA/alkanethiol films is a very common approach to directly couple single-stranded DNA probes. The quality of these mixed films greatly depends on different parameters including the sensitivity and the detection limit. We have shown a positive relation between the length of the used carbon spacer and the general performance of the DNA biosensor. In this chapter an extended protocol for the controlled immobilization and subsequent hybridization of DNA is described.