Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using 'Catch and Display' on Ultrathin Nanoporous Silicon Nitride Membranes

Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'catch and display for liquid biopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced via pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.