Development of electrode array based biosensors for detecting the activity of cancer related proteases

Abstract

As an essential biomolecule involved in many physiological and metabolic processes, proteases are potential targets in cancer diagnosis and therapy. We have developed an electrochemical method for measuring the activity of proteases using two different working electrodes: vertically aligned carbon nanofiber nanoelectrode arrays (VACNF NEAs) and gold microelectrode arrays (MEAs). A conductive Cr layer coated on SiO₂-covered Si chip served as the substrate for the growth of VACNFs which were subsequently encapsulated in a SiO₂ matrix. After polishing and reactive ion etching, VACNF tips were exposed to form a NEA. Specifically designed ferrocene (Fc) labeled peptide substrates were covalently functionalized to the exposed NEA tips. The Fc moiety at the distal end of the peptide can be oxidized and measured with AC voltammetry (ACV). Compared with ordinary macro-electrodes, VACNF NEA has distinct properties due to its unique structure. The optimal ACV frequency is up to 1 kHz, enabling the kinetic measurement of proteolysis of surface-functionalized peptides by proteases. Different peptide substrates (tetrapeptide, hexapeptide, and octapeptide), designed and synthesized in Hua’s laboratory, have been used to functionalize the VACNF NEA. The detected proteolysis rate is highest for the hexapeptide, followed by the octapeptide and tetrapeptide. These results indicate the potential of using hexapeptide as a peptide probe for protease activity profiling of specific cancer-relevant proteases. In another study, we fabricated a gold MEA for multiplex protease activity profiling. The MEA contains nine individual microelectrodes, which have dimensions of 200 x 200 micrometers. The MEA shows highly consistent cyclic voltammetry signals in Au surface cleaning experiments and reliably detects benchmark redox species in solution, which indicates the identical surface area and electrochemical activity of the electrodes. The individual Au microelectrodes were selectively functionalized with three different Fc-labeled peptide substrates. The proteolytic kinetics were profiled by monitoring the Fc oxidation signal through AC voltammetry as the peptide substrates were cleaved by cathepsin B. Cathepsin B activity was derived with an improved fitting algorithm and the heterogeneous Michaelis-Menten model. The multiplex functionalization of an Au MEA for simultaneous detection of the proteolysis of cathepsin B on a single chip shows the potential of this sensor platform for a fast and multiple protease activities profiling in various diseases, including different types of cancer. The Au MEAs were further explored for detecting multiple proteases (such as cathepsin B and ADAM-17) in a common physiology-compatible buffer at pH = 7.4 for measuring human serum samples. This Au MEA shows a great potential toward multiplex detection.