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

Abstract

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.

Constrained Volume Micro- and Nanoparticle Collection Methods in Microfluidic Systems

“…Recent developments have been made in many particle collection methods in the last few years, including physical [ 12 , 47 , 53 ], optical [ 8 ], and electrical [ 104 ] mechanisms. Mechanisms like dielectrophoresis that have a long standing in microfluidic systems have the distinct advantage of well-established fabrication and use processes.…”

“…As particles flow through the channel, optical gradient and radiation forces guide and push them into a geometrically defined protrusion where the particles are collected, as depicted in Figure 7 . The particles are trapped near a thin membrane spanning the channel top; the membrane may serve as a platform for a sensor such as a nanopore [ 8 , 68 ].…”

“…A few groups have used particle collection methods to improve nanopore sensors in this way. Most of these were addressed previously in the trapping methods sections, and they included barrier-based and optical particle trapping [ 8 , 12 , 33 , 34 , 46 , 68 ].…”

“…Enrichment and immobilization of particles into highly constrained volumes has significance in enhancing the capability of tools designed to handle, evaluate, and use those particles. Many types of sensors have detectable limits that may be met or surpassed using dilute samples which have had their concentration enhanced [ 7 , 8 ]. Likewise, many particle processing methods benefit from having subject particles immobilized in small, pre-determined volumes, allowing for more precise control and observation of the particles being processed [ 9 , 10 ].…”