Microchip-based electrokinetic biosensor: microfabrication and application in membrane protein profiling of extracellular vesicles
Abstract In this study, we demonstrate the fabrication, characterization, and application of a microchip-based electrokinetic biosensor which exploits streaming current for signal transduction. The sensor chips, fabricated using standard silicon fabrication techniques, are based on Si-glass microfluidics offering precision, robustness, and transparency. A custom-built chip-manifold allowing easy interfacing with standard microfluidic connections is also developed which shows leak-free integration of the microchips up to 6 bar of applied pressure. Within this range of pressure, the devices show linear and highly reproducible values for flow rates and streaming current with RMS noise below 20 pA. The microchips designed for multiplexed measurements were tested with the detection of free proteins (streptavidin) and also transmembrane proteins of small extracellular vesicles (sEVs) to demonstrate the capacity of the microchips to detect various types of bio-analytes. The limit of detection (LOD) for streptavidin was estimated to be 0.5 nM while for the transmembrane protein (CD9), the LOD was found to be 1.2×10 6 sEVs/mL. The sensitivity (LOD) of the devices was found to be about 4 times better in targeting CD9 transmembrane protein of H1975 extracellular vesicles when compared to commercial silica capillary which was used previously. The improvement in LOD is attributed to the higher surface quality of the sensor in terms of the density of surface charges which may be further exploited for even lower LOD. In addition, optical detection of fluorophore-tagged standard proteins was done through the optical window of the chip manifold and the transparent glass cap of the microchip.