Development of an electrochemical assay for detection of breast cancer-associated PIK3CA point mutations using noble metal-based screen-printed sensors

Cancer patients nowadays benefit from the ongoing transition of anticancer therapy towards a personalized treatment strategy guided by molecular diagnostics. However, currently available diagnostic methods for analysis of tumor-associated point mutations, which play a central role in cancer diagnostics as predictive or prognostic biomarkers, are limited to laboratory-based detection techniques. These have the major disadvantages of being only available in specialized clinical laboratories and generally being very time-consuming and expensive. Hence, there is an unmet need for novel diagnostic techniques that can be used for routine mutation analysis at the point of care, allowing for fast, reliable, and more economic cancer diagnostics that are also suitable for resource-limited settings. Point-of-care (POC) diagnostic approaches for detecting point mutations in circulating tumor DNA have the potential to complement the steady progress in anticancer therapies, improve disease outcomes, and reduce patient’s burden by enabling early cancer diagnosis and guiding treatment decisions. However, further effort is required to reach the final goal of establishing a POC testing system for the analysis of tumor-associated mutations in circulating tumor DNA that can be applied in clinical practice.

Within the scope of this doctoral thesis, a contribution to this ambitious goal is to develop a highly sensitive and specific POC-compatible method for multiplex analysis of three breast cancer-associated point mutations in the PIK3CA gene (H1047R, E545K, and E542K) as one of the most frequently mutated and clinically relevant genes in breast cancer. The developed method relies on mutation-specific electrochemical detection with an enzyme-linked assay on multi-channel screen-printed sensors combined with prior isothermal recombinase polymerase amplification. For this purpose, first a mutation-specific hybridization-based assay was developed with the help of microarray technology on a gold-coated microarray platform, allowing for thorough assay optimization. The optimized assay protocol could then be successfully transferred to screen-printed gold sensors, thus allowing for realizing a mutation-specific enzyme-linked electrochemical assay. Subsequent investigations on the influence of the electrode material and morphology on sensing performance and hybridization specificity revealed that the highly porous surface of sintered gold nanoparticle-modified electrodes offers particularly favorable properties for mutation-specific DNA detection and should be regarded as the electrode material of choice for such applications. Finally, by combining the electrochemical assay with prior isothermal recombinase polymerase amplification, it was possible to significantly increase the sensitivity of the overall assay. This could be achieved by labelling of the target DNA with biotin during RPA and the generation of single-stranded target DNA either directly via asymmetric RPA or by subsequent enzymatic digestion. Additionally, a wild-type blocking strategy was developed to increase mutation-specificity during probe-target hybridization.