Development of a suitable bioreceptor for C-reactive protein: Aptamer selection, validation & sensor development

Not many research areas are as evolving as biosensor research. Started in the fifties, it is now expanded to a technology-driven, multidisciplinary field with large impact on society and hence commercial interest. A myriad of applications exists for a device that very specifically and sensitively measures the presence and concentration of a target molecule of choice, e.g. in environmental monitoring (contaminants, pesticides), food analysis (pathogens, antibiotics) and especially in healthcare. Diagnostics of disease specific markers is one of the main but not the only application in the field of medicine. High-end technologies deliver biosensor devices that measure the interaction of bioreceptors or biomolecules of choice with other analytes in very high detail. These technologies provide information on affinity and moreover, on the rate of interaction. Therefore, therapeutic applications as biological activity evaluation of drugs, protein-DNA or protein-protein interactions and compound screening for new drug development have increased the performance of this research field even more. Biosensors are true examples of the multidisciplinary approach: a biological target binding element (molecular biology) on a binding signal transducing material (physics and chemistry) and the resulting biochemical or biophysical signal that is translated to a quantifiable value (engineering). The research presented in this dissertation is focused on the biological receptor element of the biosensor and on one type in particular: aptamers or selected synthetic DNA that binds very specifically to a target of choice. This dissertation starts at the onset of the development of a new biosensing application, the selection procedure. Suitable aptamers are selected that bind Creactive protein (CRP), an acute-phase protein and inflammation marker. By performing a selected and optimized SELEX procedure (Systematic Evolution of Ligands by Exponential Enrichment) nanomolar affinity aptamer candidates are selected and pitfalls of the SELEX selection procedure are addressed. By increasing the number of negative selection steps, reducing the number of amplification cycles and addition of non-specific single-stranded DNA (ssDNA), frequently encountered problems as non-specific binding and introduction of amplification bias are untangled. The research presented shows it is of utter importance to carefully monitor and test the applied selection conditions (target immobilization procedure, buffer compositions and applied SELEX design), preferably by different methods. An interesting tool for monitoring the selection progress is explored in this dissertation. By performing remelting curve analysis (rMCA) on the selection pools of three different SELEX set-ups, the method is proven to be very useful for monitoring SELEX conditions and dynamics in the DNA selection pool. By applying the real-time DNA amplification analysis as suggested here, the SELEX procedure and conditions are tailored in more detail, monitored and made more efficient. Detailed binding analysis of the selected aptamers is performed on different platforms, ranging from standard immunosorbent-like techniques to high-end instrumentations such as surface plasmon resonance (SPR) analysis. This has shown high affinity (nanomolar) of the aptamers for CRP when applied as they were selected: unmodified with reporter molecules and in solution over the target. This proofs aptamer functionality but also the high demands aptamers have for the proper binding conditions. This limits the versatility of the selected bioreceptors and the possible biosensor designs using this receptor to label-free homogeneous assays. The experiments performed on different read-out systems show that it is possible to optimize the aptamer functionality after the SELEX procedure with detailed binding condition optimizations on highly informative read-out systems as SPR. More ideally, aptamer versatility and flexible functionality is obtained during the selection procedure, by changing the selection conditions or by modifying a priori part of the selection library. The selected aptamers for CRP and another well validated aptamer sequence for thrombin are also used in this dissertation to test the applicability of aptamers in a recently developed read-out platform. This new platform measures the change in heat-transfer resistance over a solid-liquid interface when the binding event takes place on that interface. The experiments performed here show that the binding of the target or the selected aptamers does not result in a measurable insulation signal change in the prototype set-up. Nevertheless, indications from the work with DNA hybridization and denaturation show that target induced strand displacement is an interesting option to get this read-out system operational for aptamer applications.

Publication year:2014

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