Utilizing Two Electrokinetic Techniques on a Single Device for Detection of Extracellular Vesicle‐Associated Protease Activity

Protease activity is an emerging biomarker for cancer detection as activity levels are often increased in tumor tissue compared to healthy tissue. Of particular interest is the activity of proteases carried by extracellular vesicle (EV) nanoparticles which are oversecreted by tumors into circulation. Current methods to analyze the activity of proteases bound to EVs require complex multi-instrument sample processing to separate EVs from plasma to quantify protease activity. This makes EV-based protease activity detection a challenge for diagnostic or point-of-care applications. Here, a method is reported that manipulates EV nanoparticles and charged molecular byproducts from protease activity using two different electrokinetic phenomena generated by a single electrode microarray within a microfluidic channel. Dielectrophoresis is first generated to recover EVs carrying active trypsin-like proteases from human plasma followed by electrophoresis for subsequent analysis of peptide cleavage products indicating protease activity. This method demonstrates signal amplification through protease catalytic activity in combination with concentrating mechanisms of dielectrophoresis and electrophoresis. Using this approach, a significant difference in protease activity is observed between patients with pancreatic cancer and benign cysts. This demonstrates dual-electrokinetic chip-based technology as a useful tool to manipulate different sized and charged analytes in a single device enabling future clinical translation of EV-based protease diagnostics.