Abstract
<jats:p>Rapid, absolute counting of objects in a flowing fluid is central to cell analysis but is challenging for sub-micron structures because of the large dimensions of reproducible flow streams. Here, we report a fluorescence-based absolute single-particle flow instrument (SPFlow) constructed from etched silicon microchannels for rapid quantification of nanoscale macromolecules. Multi-channel coincidence detection was used to address dark-noise limits of megahertz-rate optical sensors, resulting in near-zero background events over 10-minute runs. A near-absolute standard curve (slope = 0.95) was obtained with a 0.61 fM limit of detection for 0.5 µL sample volumes in 10-minute detection time using fluorescent nanoparticles as small as ~25 nm that were undetectable by nanoparticle tracking analysis (NTA) and flow cytometry. SP-Flow was applied for digital microRNA quantification after targets were enzymatically extended as DNA nanoflowers, which could be colorimetrically multiplexed by microRNA sequence with a 0.67 fM limit of detection. The instrument was further applied to quantify and multiparametrically characterize extracellular vesicles of ~100 nm size isolated from adipose tissue. These microchannels and sensing formats may expand the options for rapid flow-based assays to quantify biomolecules smaller than 100 nm.</jats:p>