Application of Lateral and Distance Spacers in an Oligonucleotide Based Immobilization System for Bioactive Molecules...

Immobilization of bioactive molecules (BAMs) on a nanometer scale is of great interest for functionalization of implant and scaffold surfaces in current biomaterials research. A system for immobilization of one or more compounds is described, which is based on nanomechanical fixation of single-stranded nucleic acids into an anodic titanium oxide layer and their subsequent hybridization with BAMs conjugated to the respective complementary strands. This paper focuses on further development and in depth understanding of the immobilization system, as some of the major findings established for common sensor applications for immobilization of single-stranded DNA onto gold surfaces cannot be transferred to the TiO2 surface. The first approach concerning the influence of the internal spacer sequence revealed the best performance for a polyadenine based sequence out of four homologous spacer sequences (A30, T30, C30, and G30). This overall best performance of the A30 spacer is attributed to an increased contour length by nucleotide staggering, which resulted in the best protection of the hybridization sequence from unfavorable interactions with the surface or damaging attacks by reactive oxygen species. The second approach comprises the implementation of a lateral spacer, also based on a homologous sequence of A30. Simultaneous as well as sequential adsorption of anchor strands and spacer strands were performed, and it could be shown that a preadsorption with high density of the spacer was most effective to increase hybridization efficiency.