Selective capture and digital counting of intact HIV pseudovirus using designer DNA nets, tethered motion, and photonic resonator interferometric scattering microscopy.

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.