Monolayer-fluorescence counting for ultrasensitive detection of tumour cell-derived extracellular vesicles using a step-wedge microfluidic platform

Extracellular vesicles have emerged as significant noninvasive tumour biomarkers, and ultrasensitive detection methods are important for early cancer diagnosis. Here, a monolayer-fluorescence counting strategy was developed for the ultrasensitive detection of extracellular vesicles using a microfluidic platform. Extracellular vesicles were specially captured and separated from complex matrix by immune magnetic microbeads (IMBs). With an enhancing acoustic wave microfluidic channel, the magnetic-extracellular vesicles immune complex was adequately mixed and reacted with antibody-modified fluorescence microbeads (IFBs). Using a newly designed step-wedge microfluidic structure, micrometer-size particles were trapped in large numbers with monolayer in the step zone, and the fluidic resistance was reduced through the wedge structure. This detection signal of the fluorescence-labelled immune sandwich complex could be read by counting, avoiding background interference and achieving an ultrasensitive detection with a detection limit of 850 particles/mL. Moreover, this method showed strong reliability and specificity in clinic samples. In addition, a miniaturized detection device was developed based on this microfluidic platform, improving this detection automation.