Electrochemical logic sensing of protease activity patterns for stratifying breast cancer metastatic tendencies

Breast cancer metastasis is shaped by organ-specific microenvironments, but blood-based assays that report metastatic tendency through functional enzyme activity remain limited. Here, we developed a dual-channel electrochemical logic sensor to read protease activity patterns associated with different metastatic states. The sensing interface was assembled on gold electrodes using two PNA peptide chimeric duplexes. One duplex coupled cathepsin B and cathepsin K responsive segments to a methylene blue output, while the other coupled cathepsin B and cathepsin S responsive segments to a ferrocene output. Matched protease combinations cleaved the corresponding duplexes and released redox-labelled fragments from the electrode surface, producing separated current decreases and a parallel change in charge-transfer resistance. Stepwise impedance analysis, fluorescent substrate cleavage, differential pulse voltammetry and interference tests supported the construction of the logic interface and its protease-combination-dependent response. In clinical sample analysis, three electrochemical features, ΔI_MB, ΔI_Fc and R_ct, generated distinguishable patterns among non-metastatic, bone metastatic, brain metastatic and multiple metastatic breast cancer samples. A random forest model was used to integrate these features and provided stratification results in both the training set and an independent validation set. The work shows an electrochemical route for reading protease activity combinations instead of single markers, and suggests a practical format for functional stratification of breast cancer metastatic tendencies.