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 × 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.

蛋白酶是涉及许多重要生物过程的一大类酶。对特定靶蛋白酶活性谱的定量检测对于癌症等疾病的诊断和监测有着很高的需求。本研究展示了基于简单电化学方法快速、多重检测组织蛋白酶 B 活性的可单独寻址的 3 × 3 Au 微电极阵列的制造和表征。阵列中的九个单独的微电极在金表面清洁实验和检测溶液中的基准氧化还原物质中显示出高度一致的循环伏安信号。单独的 Au 微电极进一步选择性地用特定的二茂铁标记的肽分子进行功能化，这些肽分子充当目标蛋白酶的同源底物。当肽分子被组织蛋白酶 B 切割时，通过监测来自二茂铁标记的交流伏安信号的衰减来测量一致的蛋白水解动力学。组织蛋白酶 B 的准确活性是通过改进的拟合算法得出的。证明了在用三种不同的六肽功能化的微电极阵列上同时检测组织蛋白酶 B 的蛋白水解，显示了该传感器平台在快速检测包括多种癌症在内的各种疾病中的多种蛋白酶的活性谱的潜力。