The development of paper-based electroanalytical strips as powerful diagnostic tools has gained a lot of attention within the sensor community. In particular, the detection of nucleic acids in complex matrices represents a trending topic, especially when focused towards the development of emerging technologies, such as liquid biopsy. The DNA-based biosensors have been largely applied in this direction and, currently, there are two main approaches based on target/probe hybridization re-ported in literature, namely Signal ON and Signal OFF. In this technical note, the two approaches are evaluated in combina-tion with paper-based electrodes, using a single strand DNA relative to H1047R (A3140G) missense mutation in exon 20 in breast cancer as the model target. A detailed comparison among the analytical performances, detection protocol, and cost as-sociated with the two systems is provided, highlighting the advantages and drawbacks depending on the application. The present work is aimed to a wide audience, particularly for those in the field of point-of-care, and it is intended to provide the know-how to manage with the design and development stages, and to optimize the platform for the sensing of nucleic acids using a paper-based detection method.