Single-Tube Capture, Concentration, and Genomic Extraction of a Human Norovirus Surrogate Using Magnetic Ionic Liquids

Abstract

Capture and concentration of viruses from contaminated foods are crucial for sensitive detection. Magnetic ionic liquids (MILs) are ideal capture reagents for portable applications since they require minimal equipment and no cold storage. However, a similarly portable genomic extraction method is also needed for use with portable nucleic-acid-based detection methods. Since MILs effectively bind both intact virus and viral RNA, they can potentially be used as a binding substrate for both viral capture and genomic extraction. To evaluate this, a protocol was developed for single-tube capture, concentration, and genomic extraction based on a commercial method for magnetic bead-based RNA extraction that utilized MILs as the binding substrate. Two of the MIL formulations showed a recovery efficiency comparable to that of commercial magnetic beads and were capable of target enrichment when lysis, wash, and elution buffers from a commercial kit were used. These commercial buffers were proprietary and optimized for use with magnetic silica beads; therefore, next, various combinations of known lysis, wash, and elution buffers were tested with both magnetic silica beads and MILs until RNA recovery was comparable to that achieved with the commercial reagents. Though further research is needed, this work represents the first reported instance of single-tube capture, concentration, and genomic extraction of a microbial target using MILs as a universal substrate capable of mediating all three actions and a step toward fully portable microbial detection.

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MIL chemical structures. (A) Chemical structures for transition metal-based MILs where X = Co (II), Mn (II), or Ni (II). (B) Chemical structures for rare earth metal-based MILs where Y = Dy (III). Reprinted with permission from Stoufer et al. Copyright 2025 Springer-Nature.

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MIL-based capture and recovery protocol. Basic schematic for MIL-based viral capture and recovery, created with BioRender.com.

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Complete MIL-based sample preparation scheme. This method would combine (A) viral capture and concentration, (B) capsid lysis, (C) wash, and (D) elution into a single tube. Created with Biorender.com.

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MIL-based viral capture and recovery. Separation of (A) MNV and (B) TV from PBS suspension was performed using various MIL formulations. The vertical axis indicates the recovered copy number as quantified by RT-qPCR after nucleic acid extraction. Percentages for capture and recovery for each condition are indicated above bars.

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MIL-based viral capture and recovery with Tween. Separation of (A) MNV and (B) TV from PBS suspension supplemented with 0.05% Tween-20 was performed using various MIL formulations. The vertical axis indicates the recovered copy number as quantified by RT-qPCR after nucleic acid extraction. Percentages for capture and recovery for each condition are indicated above bars.

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Influence of dispersion time on capture efficiency. Percentage of TV captured from suspension by different MIL formulations after vortex dispersion of MIL droplets for different time points.

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Influence of dispersion time on elution efficiency. Percentage of TV recovered from a suspension by different MIL formulations after vortex dispersion of MIL droplets for different time points, with a 60 s dispersion for the initial target capture.

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MIL-based viral RNA extraction initial testing. Capture efficiency of TV from aqueous suspension using MILs and recovery of viral RNA after extraction using either (A) a magnetic silica bead-based commercial extraction kit (Zymo Quick DNA/RNA Viral MagBead Kit) or (B) the MILs themselves in the same vial that was used for target capture. All reagents used were from the commercial kit and all incubation times were consistent with the provided instructions. The vertical axis indicates the recovered copy number as quantified by RT-qPCR after nucleic acid extraction. Percentages for capture and recovery for each condition are indicated above bars.

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Magnetic silica bead-based RNA extraction protocol development. The buffer condition deemed most favorable for each step is outlined in black. (A) Lysis buffers consisted of 4 M GuSCN in varying concentrations of pH 8.0 Tris buffer. (B) Wash buffers consisted of 5 M GuHCl and 50% v/v isopropanol in varying concentrations of pH 8.0 Tris buffer. 75% ethanol was used as a secondary wash buffer in all trials. (C) Elution buffers consisted of either pure water (recommended by the kit) or varying concentrations of pH 8.0 Tris buffer. (D) Lysis, wash, and elution buffers found to give the highest viral RNA recovery were used for genomic extraction following MIL-based target capture and concentration, using either magnetic silica beads or MILs as the binding substrate.

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MIL-based RNA extraction protocol development. The buffer condition deemed most favorable for each step is outlined in black. (A) Lysis buffers consisted of either the kit buffer, a minimal GuSCN buffer (4 M GuSCN in 50 mM Tris buffer, pH 8.0), or the GuSCN + buffer (4 M GuSCN, 5 mM EDTA, 25 mM NaCl, and 0.02% SDS in 50 mM Tris, pH 8.0). (B) Wash buffers consisted of either the kit buffers, a GuHCl-based buffer system (5 M GuHCl, 50% isopropanol in 50 mM Tris, pH 8.0) followed by 75% ethanol, or 100% ethanol followed by 75% ethanol. (C) Elution buffers consisted of either pure water (recommended by the kit), 100 mM Tris buffer (pH 8.0), or LBT. (D) Lysis, wash, and elution buffers found to give the highest viral RNA recovery were used for genomic extraction following MIL-based target capture and concentration, using either magnetic silica beads or MILs as the binding substrate.

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Residual buffer removal via air drying vs two-phase wash. Different methods were tested to remove residual wash buffer from samples prior to elution. These included 10 min air drying (+air dry), pipet removal following gravitational separation (−air dry, two phase), and two-phase wash with 1-undecanol (+two phase).