| Summary: | In the era of liquid biopsies, the reliable and cost-effective detection and screening of cancer biomarkers has become of fundamental importance, thus paving the way for the advancement of research in the field of point-of-care testing and the development of new methodologies and technologies. Indeed, the latter ones can help designing advanced diagnostic tools that can offer portability, ease of use with affordable production and operating costs. In this respect, impedance-based biosensing platforms might represent an attractive alternative. In this work, we describe a proof-of-concept study aimed at designing portable impedimetric biosensors for the monitoring of human urokinase-type plasminogen activator (h-uPA) cancer biomarker by employing small synthetic receptors. Aberrant levels of h-uPA were correlated with different types of cancers. Herein, we report the use of two bicyclic peptides (P<sub>2</sub> and P<sub>3</sub>) which have been engineered to bind h-uPA with high affinity and exquisite specificity. The synthetic receptors were immobilized via biotin-streptavidin chemistry on the surface of commercial screen-printed electrodes. The impedimetric changes in the electrode/solution interface upon incubation of spiked h-uPA samples in the presence of a redox probe were followed via electrochemical impedance spectroscopy. The P<sub>3</sub>-based impedimetric assay showed the best outcomes in terms of dynamic range and linearity (0.01–1 μg mL<sup>−1</sup>) and sensitivity (LOD = 9 ng mL<sup>−1</sup>). To fully assess the performances of P<sub>3</sub> over P<sub>2</sub>, and to compare the label-free architecture vs. labelled architecture, a voltammetric assay was also developed.
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