Biofunctionalised surfaces for molecular sensing: Dissertation

In many application fields, like in biosensors, the sensing biomolecules are immobilized on solid surfaces to enable measuring of very small concentrations of molecules to be analysed. Such application fields are, for example, diagnostics, detection of abused drugs, environmental monitoring of toxins and tissue engineering. This thesis studies the immobilization of biomolecules (antibodies and Fab'-fragments, avidins and oligonucleotide sequences) on gold surfaces in biosensors. In order to achieve high nanomolar sensitivity even in difficult sample matrices, the effect of the sensing molecule immobilization type and concentration within these biomolecular surfaces were studied in detail. The suitability of these surfaces for neuronal stem cell attachment was also one of the topics. Real-time label-free detection was performed with surface plasmon resonance (SPR). The molecular surfaces in this study were constructed of biomolecules and repellent molecules, which formed self-assembled monolayers on gold. The molecules were immobilized on surfaces via reactive thiol- or disulphide groups. On surfaces assembled of proteins, the non-specific binding was minimized by hydrophilic polymer molecules and on surfaces assembled of oligonucleotides by means of lipoate molecules embedded on the surface in between the biomolecules, respectively. With these highly sensitive biomolecular surfaces, a nanomolar detection of small sized molecules such as the 3,4-methylenedioxymethamphetamine (MDMA) drug was achieved. MDMA was analysed from a difficult sample matrix of diluted saliva. Improved orientation of surface immobilized Fab'-fragments leading to a higher sensitivity was shown with surfaces constructed of cys-tagged avidins: Fab'-fragments immobilized via thiol-biotinylation to a surface constructed of cys-tagged avidins bound almost ten times the amount of antigen when compared to a conventional surface constructed of non-oriented wild-type avidins. Polymer molecules embedded in between the biomolecules efficiently reduced non-specific binding. Selective neuronal cell attachment was also shown with polymer and neuronal-specific antibody molecules physisorbed on cell culture plates. Only the differentiated neuronal cells attached to surfaces physisorbed with neuronal-specific antibodies, while the non-differentiated neurospheres did not. Selective surfaces were also developed for oligonucleotide sequences. Lipoate-based molecules efficiently reduced the non-specific binding of proteins and non-complementary DNA. A nanomolar detection range was achieved for single-stranded, breast cancer-specific polymerase chain reaction (PCR) products. First, the shorter single-stranded PCR-products were analysed and a nanomolar detection range was achieved in buffer. In the following study, the DNA-surfaces were analysed in the presence of diluted serum. Even in diluted serum matrix, nanomolar concentrations of longer single- stranded sequences could be analysed due to the efficient blocking of non-specific binding of serum proteins. It was found that sensitive detection surfaces for biomolecular recognition can be achieved, when optimal function of the biomolecules is ensured by immobilizing the molecules on surfaces in an oriented manner towards the analyte. Efficient reduction of non-specific binding is also important in reaching highly sensitive label-free detection. The surfaces were also found to be effective in selective neuronal stem cell attachment.