Electrokinetic Preconcentration and Label-Free Electrical Detection of Sars-Cov-2 RNA at a Packed Bed of Bioconjugated Microspheres

Abstract

In diagnostics, point-of-care (POC) devices, such as lateral flow assays, play an important role due to their ease of use, practical application, and fast response. However, one of the major limitations of POC devices is low sensitivity. To address this limitation, electrokinetic techniques have been utilized to enrich the target analyte prior to detection. In one such technique, ion concentration polarization (ICP), an ion depleted zone (IDZ) is formed near an ion permselective membrane or electrode following the application of an external electric field. The low conductivity of the IDZ supports the formation of an extended electric field gradient. Enrichment arises from focusing of a charged analyte along this electric field gradient in the presence of opposing fluid flow. ICP focusing is a reliable technique for the enrichment and mobility-based separation of charged species, such as DNA, RNA, or proteins. However, ICP focusing is negatively impacted by electroconvective vortices that drive unwanted mixing.We utilize an out-of-plane faradaic ion concentration polarization (fICP) technique that employs a 3D porous electrode and an adjacent packed bed of insulating microbeads to improve the stability of the enriched plug. In this work, we exhibit the detection of SARS-CoV-2 RNA, extracted from lysed virus, at subpicomolar concentrations in this microfluidic device. Here, the insulating beads are bioconjugated with an uncharged peptide nucleic acid (PNA) oligoprobe that hybridizes with the target RNA. The resulting increase in negative charge on the bead surface leads to a shift in a current-voltage curve between the 3D electrode and a driving electrode at the microchannel inlet. This shift in conductivity serves as a non-optical sensing mechanism for detection of the hybridized SARS-CoV-2 RNA. Most notably, this enrichment and sensing scheme utilizes off-the-shelf streptavidin-modified beads, making it highly versatile, while the reagent-free approach and low driving voltage render it amenable to POC implementation.This work is supported by a grant from the Roy J. Carver Charitable Trust.