Immobilisation of biomolecules onto organised molecular assemblies: Dissertation

This thesis describes immobilisation techniques for biomolecules on solid surfaces via an intermediate self-assembled or Langmuir-Blodgett (LB) film. Such films are advantageous, because the biological activity can be optimised by tailoring the layer composition. Factors like charge density, density of the linking group and composition of the monolayer matrix have particularly large effects on the activity. In the first part, oxidase enzymes were immobilised on self-assembled, conductive layers of bispyridiniumthiophene oligomers (thienoviologens) on gold substrates. The enzyme was bound by electrostatic interaction of the negatively charged enzyme with the positively charged pyridinium groups. The enzymatic activity of glucose oxidase was dependent on the surface density of the conductor and on the ionic strength during adsorption. In the second part of the thesis, Fab'-fragments were bound to LB-films of various linker lipids and the immobilisation efficiency (relative amount of binding sites) was optimised by variation of the constitution of the lipid LB-film. The films were deposited onto various substrates by vertical contact transfer of a preformed mixed Langmuir film of the linker lipid and a matrix lipid. The immobilisation efficiency was investigated with radioassay, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements, while also atomic force microscopy (AFM) was used to image the structure of the films at different stages of binding. The immobilisation efficiency of the LB-films appeared to be much higher as that of more conventional methods for antibody immobilisation. Between 20 and 70% of the binding sites could be preserved, depending on the type of linker and film matrix composition, while that of the conventional methods was less than 10%. Films with dipalmitoyl-phosphatidylcholine as the matrix lipid and a maleimide derivative of dipalmitoylphosphatidyl-ethanolamine showed the highest sensitivity (and lowest detection limit) in QCM measurements.