Nanotrap Particles Improve Nanopore Sequencing of SARS-CoV-2 and Other Respiratory Viruses

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40x improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher Apex system.

In Situ Synthesis of Magnetic Poly(DMAEAB-co-NIPAm)@Fe3O4 Composite Hydrogel for Removal of Dye from Water

Water pollution by toxic substances, such as dye molecules, remains a major environmental problem that needs to be solved. In the present work, the magnetic composite hydrogel based on the poly(2-(methacryloyloxy)-N-(2-hydroxyethyl)-N,N-dimethylethan-1-aminium bromide-co-N-isopropylacrylamide) copolymer with incorporated Fe3O4 particles ((poly(DMAEAB-co-NIPAm)@Fe3O4)) was prepared by an in situ synthesis technique for the efficient removal of dye molecules from water. The successfully synthesized magnetic hydrogel was characterized by FTIR, XRD, TGA, and TEM. The removal efficiency of the anionic dye bromophenol blue (BPB) and the cationic dye rhodamine B (RDM) by the prepared hydrogel adsorbents was evaluated. Various adsorption parameters, including the concentration of adsorbents and adsorption time, were also investigated. The results showed that the synthesized magnetic hydrogel had excellent BPB removal performance compared to the removal of RDM. The optimum adsorbent concentration for 0.5 mM BPB solution was approximately 0.5 g/L, and the removal efficiency was more than 99%. The kinetics data of BPB removal fitted well into the pseudo-2nd-order model, indicating that BPB dye adsorption involves chemical adsorption and physical adsorption. In addition, recycling studies were conducted to examine the reusability of the magnetic hydrogel for BPB removal for up to five cycles and the hydrogel could be reused without losing its high removal efficiency. The magnetic hydrogel poly(DMAEAB-co-NIPAm)@Fe3O4 with high removal efficiency, good selectivity, and reusability shows great potential for the removal of anionic dyes in wastewater treatment.

Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria

Background Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. Results CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. Conclusion Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods. Graphic Abstract

Magnetic Superporous Poly(2-hydroxyethyl methacrylate) Hydrogel Scaffolds for Bone Tissue Engineering

Magnetic maghemite (γ-Fe2O3) nanoparticles obtained by a coprecipitation of iron chlorides were dispersed in superporous poly(2-hydroxyethyl methacrylate) scaffolds containing continuous pores prepared by the polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate porogen. The scaffolds were thoroughly characterized by scanning electron microscopy (SEM), vibrating sample magnetometry, FTIR spectroscopy, and mechanical testing in terms of chemical composition, magnetization, and mechanical properties. While the SEM microscopy confirmed that the hydrogels contained communicating pores with a length of ≤2 mm and thickness of ≤400 μm, the SEM/EDX microanalysis documented the presence of γ-Fe2O3 nanoparticles in the polymer matrix. The saturation magnetization of the magnetic hydrogel reached 2.04 Am2/kg, which corresponded to 3.7 wt.% of maghemite in the scaffold; the shape of the hysteresis loop and coercivity parameters suggested the superparamagnetic nature of the hydrogel. The highest toughness and compressive modulus were observed with γ-Fe2O3-loaded PHEMA hydrogels. Finally, the cell seeding experiments with the human SAOS-2 cell line showed a rather mediocre cell colonization on the PHEMA-based hydrogel scaffolds; however, the incorporation of γ-Fe2O3 nanoparticles into the hydrogel improved the cell adhesion significantly. This could make this composite a promising material for bone tissue engineering.