This contribution summarizes some of our efforts in designing, fabricating, and characterizing interfacial binding matrices that allow for a sensitive detection of hybridization reactions between surface-attached catcher probe strands and oligonucleotide targets or PCR amplicons from solution. The multilayer architectures that we employ for the in-situ and real-time detection of the association (hybridization) reaction as well as for the dissociation upon rinsing are based on self-assembly strategies using the well-established biotin-streptavindin conjugates. For the optical characterization of the hybridization processes we use two novel surface plasmon optical techniques, i.e., surface plasmon diffraction for label-free detection, and surface plasmon fluorescence spectroscopy with its unmatched sensitivity for monitoring interfacial binding reactions with LOD values in the sub-femtomolar concentration range. In addition, we employ electrochemical methods as complementary techniques to quantify surface reactions. In a first series of experiments we analyze the details of the hybridization between catcher oligonucleotides (typically 15mers with 15 thymines as spacers) and a variety of single stranded targets differing, in particular, in length and the degree of nucleotide mismatch to the catcher sequence. For the detection of PCR amplicons typically 125 -200 bases long we developed a strategy that employs thermal treatment of the samples in order to separate (melt) the double strands followed by quenching the solution at low temperature to a low ionic strength buffer. This way, we prevent rehybridization in solution but rather allow for efficient association of the single sense strands to the sensor surface. This works best for peptide nucleic acids (PNAs) as catcher strands which interact with the DNA independently of the ionic strength.