Quantification of Protease Activity Using a Multiplex Microelectrode Array Sensor

Abstract

Proteases are a class of enzymes responsible for selectively breaking down proteins and thus they are involved in many biological processes ranging from apoptosis to hormone regulation. Overexpression of proteases is often indicative of several health conditions, such as muscle atrophy, bone loss and cancer progression. Consequently, the measurement of protease activity can serve as a biomarker for diagnosis as well as the efficacy of treatment regimens. Developing rapid low-cost techniques that can quantitatively detect multiple proteases is essential for these applications. Here we show the electrochemical characterization of a microfabricated sensor consisting of nine individually addressable microelectrodes. Subsequently, we demonstrate the application of this microelectrode array for the simultaneous, multiplex detection and quantification of protease activity. To accomplish this, each electrode is functionalized with a specific ferrocene labelled hexapeptide substrate and the electrochemical signal is monitored by AC voltammetry. In the presence of the target protease, the AC voltammetry signal decays as the target protease cleaves the peptide substrate, causing the ferrocene tags to leave the electrode surface. The proteolytic activity is determined by fitting the decay in signal to an exponential curve in accordance with the Michaelis-Menten model of heterogeneous kinetics. Our results show that each electrode of the MEA can be selectively modified with little or no contamination from adjacent electrodes. Additionally, we demonstrate that three different peptide substrates can be simultaneously used to quantify the protease activity of a sample. This microelectrode array sensor will be used to develop a portable instrument to track muscular atrophy progression during long duration space habitation in microgravity and to evaluate potential physiological and pharmaceutical countermeasures.