Simultaneous, multiplex quantification of protease activities using a gold microelectrode array

Proteases are a large family of enzymes involved in many important biological processes. Quantitative detection of the activity profile of specific target proteases is in high demand for the diagnosis and monitoring of diseases such as cancers. This study demonstrates the fabrication and characterization of an individually addressable 3 x 3 Au microelectrode array for rapid, multiplex detection of cathepsin B activity based on a simple electrochemical method. The nine individual microelectrodes in the array show highly consistent cyclic voltammetric signals in Au surface cleaning experiments and detecting benchmark redox species in solution. The individual Au microelectrodes are further selectively functionalized with specific ferrocene-labeled peptide molecules which serve as the cognate substrates for the target proteases. Consistent proteolytic kinetics are measured by monitoring the decay of the AC voltammetry signal from the ferrocene label as the peptide molecules are cleaved by cathepsin B. Accurate activity of cathepsin B is derived with an improved fitting algorithm. Simultaneous detection of the proteolysis of cathepsin B on the microelectrode array functionalized with three different hexapeptides is demonstrated, showing the potential of this sensor platform for rapid detection of the activity profiles of multiple proteases in various diseases including many forms of cancer.