sEV CAPTURE FROM PLASMA
Normal Flow Filtration:
Small extracellular vesicle experiments were performed using purified human plasma (Equitech-Bio, Inc., Kerrville, TX). NFF experiments were performed using NPN chips with 50 nm thick freestanding membranes, with an average pore diameter of 50 nm and a porosity of 15% in a SepCon™ centrifuge cup (SiMPore Inc., Rochester, NY). A 500 μL sample of undiluted plasma was spun at 1500 x g through the membrane and the chip was extracted from the device. The chip was allowed to dry and was then imaged by scanning electron microscopy as described below.
Tangential Flow for Analyte Capture:
Nanoporous silicon nitride microfluidic devices were fabricated as described above. The NPN chip used had a 50 nm thick freestanding membrane with a 50 nm average pore diameter and a 15% porosity. 1 mL of plasma was passed tangentially to the membrane surface at a rate of 10 μL/min using a syringe pump (Chemyx Fusion 200 Syringe Pump, Chemyx Inc., Stafford, TX), while fluid was actively pulled through the membrane at a rate of 2 μL/min. After processing the full 1 mL volume, the device was unclamped and the chip extracted. Captured sEVs were labeled for CD63 (Abcam, Cambridge, MA) and imaged via scanning electron microscopy as outlined below.
Capture and Release
Flow experiments were performed using two Chemyx Fusion 200 syringe pumps (Chemyx Inc., Stafford, TX). Micron scale experiments with 10 μm polystyrene green fluorescent particles (Thermo Scientific, USA) were conducted on 8 μm track-etch membranes. The capturing step was performed using a sample supply flow rate of 90 μL/min and an ultrafiltration/pulling rate of 10 μL/min. Captured particles were released by a reversed flow of 10 μL/min through the membrane.
These experiments were conducted using 100 nm polystyrene green fluorescent particles (Thermo Scientific, USA) on PCTE or NPN membranes with 80 nm median pore size. Nanoparticles were captured by a supply flow rate of 5 μL/min and the ultrafiltration/pulling flow rate of 2 μL/min. The input channel was then cleaned by rinsing buffer to wash away the floating particles under the same flow condition as the capturing step. Finally, captured particles were released by a reversed flow of 2 μL/min through the membranes.