Electrochemical urine biopsy with a peptide/peptide nucleic acid tandem probe for Cathepsin B analysis

The integration of in vivo detection and liquid biopsy through engineered artificial biomarkers has garnered significant attention. These biomarkers overcome limitations of traditional liquid biopsy, such as low abundance and rapid clearance of endogenous biomarkers, by enabling quantifiable detection of disease-specific targets in body fluids. Herein, we report a spherical peptide nucleic acid (PNA)-based artificial biomarker system (Gold-Peptide-PNA, GPP) for ultrasensitive analysis of tumor-associated Cathepsin B (CB). The GPP system comprises AuNP-loaded tandem peptide-PNA probes, where the peptide serves as a CB-responsive substrate and linker, while the enzymatically stable PNA acts as a shielded reporter. Upon CB-mediated cleavage in tumor tissues, PNA probes are released into circulation, excreted via urine, and detected using an Nb. Bbv CI-assisted DNA walker based electrochemical sensor. The sensor leverages competitive binding between PNA and a locked DNA walker to trigger methylene blue (MB) signal amplification. In vitro and in vivo studies demonstrated that GPP achieves a detection limit of 17.5 pg/ mL for CB, exhibits robust stability under enzymatic and physiological challenges, and enables tumor-specific fluorescence/electrochemical signal readouts within 2–3 h of post-injection. This work pioneers a multi-functional, enzyme-responsive nanoplatform for non-invasive tumor diagnosis, combining the programmability of PNA with the sensitivity of electrochemical sensing.