Electrokinetic focusing of SARS-CoV-2 spike protein via ion concentration polarization in a paper-based lateral flow assay.

The COVID-19 pandemic highlighted the importance of designing sensitive and selective point-of-care (POC) diagnostic sensors for early and rapid detection of infection. Paper-based lateral flow assays (LFAs) are easy to use, inexpensive, and rapid, but they lack sensitivity. Preconcentration techniques can improve the sensitivity of LFAs by increasing the local concentration of the analyte before detection. Here, ion concentration polarization (ICP) is used to focus the analyte, SARS-CoV-2 Spike protein (S-protein), directly over a test line composed of angiotensin converting enzyme 2 (ACE2) capture probes. ICP is the enrichment and depletion of electrolyte ions at opposing ends of an ion-selective membrane under a voltage bias. The ion depleted zone (IDZ) establishes a steep gradient in electric field strength along its boundary. Enrichment of charged species (such as a biomolecule analyte) occurs at an axial location along this electric field gradient in the presence of a fluid flow that counteracts migration of those species - a phenomenon called ICP focusing. In this paper, running buffer composition and pretreatment solutions for ICP focusing in a paper-based LFA are evaluated, and the method of voltage application for ICP-enrichment is optimized. With a power consumption of 1.8 mW, S-protein is concentrated by a factor of 21-fold, leading to a 2.9-fold increase in the signal from the LFA compared to a LFA without ICP-enrichment. The described ICP-enhanced LFA is significant because the preconcentration strategy is amenable to POC applications and can be applied to existing LFAs for improvement in sensitivity.

Label-Free, Non-Optical Readout of Bead-Based Immunoassays with and without Electrokinetic Preconcentration.

In this article, we report a microfluidic bead-based lateral flow immunoassay (LFIA) with a novel sensing mechanism for label-free, non-optical detection of protein binding. This device comprises two packed beds of microbeads: first, bioconjugated microbeads that serve as a test line, and second, a three-dimensional (3D) electrode for sensing. As the protein target binds the bioconjugated microbeads, a shift in ionic conductivity across the bioconjugated beads is produced and can be directly measured at the surface of the 3D electrode by obtaining current-voltage curves before and after incubation of the analyte. We use a model antigen, rabbit IgG, for quantitative evaluation of this sensor, obtaining a limit of detection (LOD) of 50 nM for the LFIA. We demonstrate that this device can be used to measure binding kinetics, exhibiting a rapid (<3 min) increase in the signal after the introduction of the analyte and an exponential decay in the signal after replacing the sample with buffer only. To improve the LOD of our system, we implement an electrokinetic preconcentration technique, faradaic ion concentration polarization (fICP), to increase the local concentration of antigen available during binding as well as the time the antigen interacts with the test line. Our results indicate that this enrichment-enhanced assay (fICP-LFIA) has an LOD of 370 pM, an 135-fold improvement over the LFIA and a 7-fold improvement in sensitivity. We anticipate that this device can be readily adapted for point-of-care diagnostics and translated to any desired protein target by simply modifying the biorecognition agent on these off-the-shelf microbeads.

Quantification of capture efficiency, purity, and single-cell isolation in the recovery of circulating melanoma cells from peripheral blood by dielectrophoresis.

This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.