Peptide substrate optimization toward multiplex protease activity profiling under neutral pH conditions using Au microelectrode arrays.

Cathepsin B (CB), a lysosomal cysteine protease, is associated with cancer and other diseases. Sensitive multiplex detection of CB and other extracellular proteases at neutral pH compatible with human serum is essential to advance biosensing platforms for disease diagnosis. Here, we present a strategy to enhance protease activity profiling based on a 3×3 gold (Au) microelectrode array (MEA). Ferrocene-tagged peptide substrates are functionalized on Au MEA. The proteolytic cleavage induces an exponential decay of the electrochemical signal, with the inverse of the decay time constant (1/τ) reflecting the proteolytic activity following a heterogeneous Michaelis-Menten model. The activity of CB is studied with Au MEA at pH = 5.0, 6.3, and 7.3, showing the highest value at pH 5.0, which decreased at pH 7.3. The limit of detection (LOD) of the active CB concentration was quantitatively determined at pH 5.0 and 7.3. Fluorogenic assays and molecular docking are used to identify the amino acid sites that are sensitive to pH value in CB detection, which reveal that incorporation of a non-proteinogenic norleucine (Nle) at the P3 position substantially restores the CB activity at neutral pH. Based on this strategy, the hexapeptide substrate Nle-Leu-Gly-Phe-Val-Ala shows an enhanced and highly specific CB activity on Au MEA at pH 7.3, with the LOD down to 0.43 ± 0.08 nM. Distinct activity profiles for CB, MMP-9, and ADAM-17 are obtained with four peptide substrates on Au MEA in the same neutral buffer. It lays the groundwork for developing multiplex electrochemical biosensors toward early disease diagnosis.