Label-free and amplification-free viral RNA quantification from primate biofluids using a trapping-assisted optofluidic nanopore platform.

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.

Nano/microfluidic device for high-throughput passive trapping of nanoparticles.

We present a design and a fabrication method for devices designed for rapid collection of nanoparticles in a fluid. The design uses nanofluidic channels as a passive size-based barrier trap to isolate particles near a central point in the channel, which is also covered by a thin membrane. Particles that enter the collection region are trapped with 100% efficiency within a 6-12 µm radius from a central point. Flow rates for particle-free fluid range from 1.88 to 3.69 nl/s for the pressure and geometries tested. Particle trapping tests show that high trapped particle counts significantly impact flow rates. For suspensions as dilute as 30-300 aM (20-200 particles/µl), 8-80 particles are captured within 500 s.

Optofluidic Particle Manipulation: Optical Trapping in a Thin-Membrane Microchannel.

We demonstrate an optofluidic device which utilizes the optical scattering and gradient forces for particle trapping in microchannels featuring 300 nm thick membranes. On-chip waveguides are used to direct light into microfluidic trapping channels. Radiation pressure is used to push particles into a protrusion cavity, isolating the particles from liquid flow. Two different designs are presented: the first exclusively uses the optical scattering force for particle manipulation, and the second uses both scattering and gradient forces. Trapping performance is modeled for both cases. The first design, referred to as the orthogonal force design, is shown to have a 80% capture efficiency under typical operating conditions. The second design, referred to as the gradient force design, is shown to have 98% efficiency under the same conditions.

Optofluidic Particle Manipulation Platform with Nanomembrane.

We demonstrate a method for fabricating and utilizing an optofluidic particle manipulator on a silicon chip that features a 300 nm thick silicon dioxide membrane as part of a microfluidic channel. The fabrication method is based on etching silicon channels and converting the walls to silicon dioxide through thermal oxidation. Channels are encapsulated by a sacrificial polymer which fills the length of the fluid channel by way of spontaneous capillary action. The sacrificial material is then used as a mold for the formation of a nanoscale, solid-state, silicon dioxide membrane. The hollow channel is primarily used for fluid and particle transport but is capable of transmitting light over short distances and utilizes radiation pressure for particle trapping applications. The optofluidic platform features solid-core ridge waveguides which can direct light on and off of the silicon chip and intersect liquid channels. Optical loss values are characterized for liquid and solid-core structures and at interfaces. Estimates are provided for the optical power needed to trap particles of various sizes.

Solid-state membranes formed on natural menisci.

We present a method to create robust, nanoscale solid-state membranes using the natural shape of a liquid meniscus as a template. A narrow, open channel is etched into a silicon substrate and then a photoresist polymer is introduced into the channel through spontaneous capillary action. The natural concave meniscus formed by the polymer is then covered by a thin chemical vapor deposited membrane. The polymer is removed by sacrificial etching, leaving behind a suspended membrane. Membranes as large as 20 µm by 9 mm can be fabricated with a thickness as low as 50 nm.