Microchip-based electrokinetic biosensor: microfabrication and application in membrane protein profiling of extracellular vesicles
Detection of analyte using streaming current has been previously explored using both experimental and theoretical approaches. However, little has been done to develop a viable microchip which can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrate the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by their characterization and optimization for highly sensitive detection of small extracellular vesicles (sEVs). We show that the sensitivity of the devices, estimated using a common protein-ligand pair and sEVs significantly outperforms previous reports using the same principle. Two versions of the microchips, denoted as enclosed and open-top microchip, were developed and compared aiming to discern the importance of high-pressure measurement vs easier and better surface preparation capacity. A custom-built chip-manifold allowing easy interfacing with standard microfluidic connections was also developed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the open-top configuration offers several practical benefits including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compare two common surface functionalization strategies and show that the proposed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for sEV detection. At the optimum condition, we demonstrate that the microchip can detect sEVs reaching a LoD of 104 sEV/mL, which is among the lowest in the reported microchip-based methods.