A Hydrogel-Interfaced Nanopore System for Sub-100 bp Nucleic Acid Sizing and Multiplexed Virus Detection

Introduction: : Nucleic acid (NA) sizing is a fundamental technique in diagnostics, next-generation sequencing (NGS), gene editing, and circulating DNA analysis. Conventional methods such as gel or capillary electrophoresis offer high resolution but are complex, time-consuming, and require labels. Solid-state nanopores provide a simple, label-free alternative for NA sizing but are limited by fast translocation rates, low signal-to-noise ratio (SNR), and poor resolution for short DNA fragments. While hydrogels interfaced with nanopores have shown potential for slowing DNA transport and enhancing resolution, current implementations face challenges in fabrication and integration. We developed a facile hydrogel-interfaced glass nanopore (HIGN) system by embedding 10 nm glass nanopores into a 7.5% PEG-DMA hydrogel for label-free sizing of short DNA fragments. Using 4 M LiCl as electrolyte, the HIGN system leverages ion-mediated charge neutralization and hydrogel-induced barrier to achieve more than 10-fold increase in DNA dwell time compared to bare-solution nanopores. The 7.5% HIGN demonstrated enhanced SNR, higher event frequency, and effective sizing resolution down to 50 bp. Spatial configuration analysis confirmed robust performance irrespective of hydrogel insertion location and depth. We further validated HIGN's capability for multiplexed detection of respiratory viruses, successfully distinguishing influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) amplicons from a combined RT-PCR product. Our results establish HIGN as a scalable and sensitive platform for sub-100 bp NA sizing with potential applications in improved multiplexed diagnostics.

Materials and

Methods: : Fig. 1a shows a schematic of HIGN system, developed by inserting glass nanopores into PEG-DMA hydrogel. The HIGN system was characterized using an Axopatch 200 B amplifier at 100 kHz sampling frequency for ionic current time (I-t) data recording. Nanopores were fabricated using glass capillaries, laser-pulled with a Sutter P-2000 (recipe: heat 650, filament 5, velocity 50, delay 140, pull 165) to produce sub-10 nm pores. Pore sizes were estimated from open pore conductance values through IV curve analysis as shown in Figure 1b. Three different %w/v (5, 7.5, and 10%) hydrogels were synthesized in 2 M LiCl, 10 mM Tris-EDTA (pH 8.0) by a chemical cross-linking method, imaged using scanning electron microscopy (SEM) and then used to characterize three respective HIGN systems. A controlled micro positioning system was used to maneuver the glass nanopore into and out of the hydrogels, whereas DNA samples were loaded in the nanopore itself at know concentrations. Following nanopore depth optimization in three different HIGN systems, DNA fragments with different lengths (50 – 400 bp) were characterized using 7.5% HIGN system to establish sizing resolution. To demonstrate the applicability of HIGN for multiplexed diagnostics, RT-PCR was performed using qScript XLT Master Mix (2×) in 20 µL reactions with IAV, SARS-CoV-2, or both primers, and 2 µL RNA per reaction. Thermal cycling was performed on Bio-Rad CFX96 with custom protocol. PCR products were analyzed using 5% agarose gel electrophoresis. The amplicons from single plex and multiplexed RT-PCR assays were then analyzed using 7.5% HIGN system.

Results, Conclusions, and Discussions:: The HIGN system effective slowed down 200 bp short dsDNA translocations (Figure. 1b) by more than 10× confirming the presence of entropic barriers offered by hydrogel mesh fibers at hydrogel nanopore interface. The entropic barrier significantly increased as demonstrated in Figure 1c as the % w/v or PEG-DMA polymer is increased from 5% to 10%. However, a 10% hydrogel was not included for further analysis because of frequent clogging. And a comparison of 5% and 7.5% HIGN systems (Figure 1d) suggested improved signal-to-noise ratio for a 7.5% hydrogel with insignificant improvement in dwell. Further investigation on the depth of nanopore insertion (30 – 120 μm) into the hydrogel suggested a negligible effect on the dwell time, blockage amplitudes and event frequencies of 200 bp dsDNA (Figure 2a,b).
To determine the sizing resolution of our HIGN system short dsDNA targets of 50, 100, 150, 200, 250, 300, and 400 bp length were tested individually by filling the nanopore with target solution and inserting in 7.5% w/v hydrogel at fixed insertion depth of ~ 60 μm for scatter plot, blockage amplitude analysis (Figure 1c,d). The normalized blockage amplitudes (ΔI/Io) were linearly related to the DNA length with high R2  0.99 (Figure 1e) suggests a sensitivity of ~0.0001 of ΔI/Io change per bp length which demonstrated the ability of our HIGN to resolve 50 bp with ΔI/Io of ~0.005.
To demonstrate the applicability of our HIGN system for multiplexed detection, we performed multiplexed RT-PCR assay for amplification of respiratory viruses IAV and SARS-CoV-2. The amplicons from the multiplexed reactions were separated by a 75 bp difference in 5% agarose gel electrophoresis (Figure 3a), confirmed by gel bands of respective single plex reactions. The high-resolution sizing capability of our HIGN platform also indicated two separate scatter populations along with distinguishable gaussian fittings of blockage amplitudes (Figure 3b). The blockage amplitude thresholding was performed as guided by the I-t traces and mean values of blockage amplitudes obtained from amplicons of single plex RT-PCR reactions (Figure 3c). These validations highlight the high potential of our HIGN system for label-free multiplexed molecular diagnostic applications.

Acknowledgements and/or References (Optional):: This work was partially supported by the National Science Foundation (1902503, 1912410, 2045169). Any opinions, findings, conclusions, or recommendations expressed in this work are those of the authors and do not necessarily reflect the views of the National Science Foundation.