Towards On-Chip integration of isothermal amplification for electrochemical DNA detection

Biochemical sensors are becoming increasingly important for the detection of biomarkers, as they can be used to realise small, easy to use handheld devices suitable for point-of-care (POC). Biomarkers are a variety of molecules, e.g. salts, metabolites, proteins, antibodies or nucleic acids, which indicate a person's state of health. The detection of these biomarkers plays a decisive role in medicine, particularly in the early detection or follow-up treatment of certain diseases. The presented thesis focuses on detecting PIK3CA point mutations as a biomarker specific for a hormone receptor positive, HER2 negative breast cancer type. Early phase detection of PIK3CA point mutations enables fast start of anticancer therapy and therefore a good survival rate of the patients. To improve the PIK3CA point mutation detection, small, cost-effective, and easy to use point of care (POC) systems are beneficial. To better realize For the realisation a POC system, isothermal recombinase polymerase amplification (RPA) with subsequent electrochemical detection of the amplified target DNA sequences on a screen-printed sensor is being investigated. In particular, the master's thesis is investigating whether RPA reagents can be embedded in a hydrogel reservoir so that they can later be integrated into a microfluidic system. To optimise the RPA and the electrochemical detection in the presence of the hydrogel reservoir material the following investigations were performed: First, the electrochemical mutation detection in the presence of the hydrogel matrix was studied. The hydrogel monomers were found to affect DNA hybridisation but not the electrochemical enzymatic detection. Secondly, to optimise RPA in the presence of hydrogel, the hydrogel washing steps (to reduce the monomers that interfere with DNA hybridisation), the drying procedure of the hydrogel, and the MgOAc concentration in the RPA buffer were investigated. Thirdly, the preparation of a dried RPA hydrogel reservoir was investigated, and the drying process of the hydrogel and the RPA reagents was optimised for this purpose. In the final and fourth step, the electrochemical mutation detection using RPA loaded hydrogels were investigated. As a result, it was found that the RPA reagents could be successfully embedded in the hydrogel, because it was still possible to amplify and measure the target DNA. However, the amplified target DNA must be purified before it can be electrochemically detected. Despite the promising results, there are still some open questions regarding the improvement of the RPA performance, fabrication of hydrogel reservoirs and the amplified target DNA purification in a microfluidic. These questions will be addressed in future research work.