Selective capture and digital counting of intact HIV pseudovirus using designer DNA nets, tethered motion, and...

Rapid and quantitatively accurate detection of HIV (human immunodeficiency virus) viral load using a simple workflow, automated instrumentation, and real-time data processing with easily interpretable output is required for an approach to become practical for point-of-care environments. We recently demonstrated a form of interferometric scattering microscopy called Photonic Resonator Interferometric Scattering Microscopy (PRISM) that amplifies the contrast of surface-attached nano-objects via a photonic crystal (PC) surface. Recently, our team also developed net-shaped DNA nanostructures called "Designer DNA Nets" (DDN) that organize multivalent aptamer arrays to precisely match the pattern of proteins on the outer surface of intact virions to provide high-affinity and selective binding. In this work, we demonstrate the combination of DDNs and PRISM for detection of HIV by digital counting of captured viruses. We compare multivalent DDN-based viral capture to monomeric aptamer and nanobody capture, in which the captured virions are tethered to the PC surface by a DNA linker. We observe that tethered virions are not fully stationary and that their localized dynamic movement provides a route for label-free digital-resolution detection with a signal-to-noise ratio of 50, while disregarding the presence of image features not related to specific virus capture. We obtain a detection limit of 10⁴ virions/ml with a single-step, room temperature 30-min assay and excellent selectivity for non-detection of a nonspecific virus and the presence of a high concentration of extracellular vesicles. This study highlights PRISM's utility as a means for versatile detection of immobilized particles as part of an assay for affinity molecule evaluation.