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.

Circulating microRNAs are associated with variability in fasting blood glucose over 12-months and target pathways related to type 2 diabetes: A pilot study

Introduction MicroRNAs (miRs) may be important regulators of risk for type 2 diabetes (T2D). Circulating miRs may provide information about which individuals are at risk for T2D. The purpose of this study was to assess longitudinal associations between circulating miR expression and variability in fasting blood glucose (FBG) and to identify miR-targeted genes and biological pathways. Methods Variability in FBG was estimated using standard deviation from participants ( n = 20) in a previously completed yoga trial. Expression of 402 miRs was measured using hydrogel particle lithography. MirTarBase was used to identify mRNAs, and miRPathDB was used to identify pathways targeted by differentially expressed miRs. Results Six circulating miRs (miR-192, miR-197, miR-206, miR-424, miR-486, and miR-93) were associated with variability in FBG and targeted 143 genes and 23 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Six mRNAs ( AKT1, CCND1, ESR1, FASN, SMAD7, and VEGFA) were targeted by at least two miRs and four of those were located in miR-targeted KEGG pathways. Conclusions Circulating miRs are associated with variability in FBG in individuals at risk for T2D. Further studies are needed to determine whether miRs may be prodromal biomarkers that can identify which individuals are at greatest risk to progress to T2D and which biological pathways underlie this risk.

Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical labs. We present a lab-on-a-particle solution to digital counting of thousands of single enzymatic reactions. Hydrogel particles are used to bind enzymes and template the formation of droplets that compartmentalize reactions with simple pipetting steps. These hydrogel particles can be made at a high throughput, stored, and used during the assay to create ~500,000 compartments within 2 minutes. These particles can also be dried and rehydrated with sample, amplifying the sensitivity of the assay by driving affinity interactions on the hydrogel surface. We demonstrate digital counting of β-galactosidase enzyme at a femtomolar detection limit with a dynamic range of 3 orders of magnitude using standard benchtop equipment and experiment techniques. This approach can faciliate the development of digital ELISAs with reduced need for specialized microfluidic devices, instruments, or imaging systems.

Ionotropic Gelation Fronts in Sodium Carboxymethyl Cellulose for Hydrogel Particle Formation

Hydrogel microparticles (HMPs) find numerous practical applications, ranging from drug delivery to tissue engineering. Designing HMPs from the molecular to macroscopic scales is required to exploit their full potential as functional materials. Here, we explore the gelation of sodium carboxymethyl cellulose (NaCMC), a model anionic polyelectrolyte, with Fe3+ cations in water. Gelation front kinetics are first established using 1D microfluidic experiments, and effective diffusive coefficients are found to increase with Fe3+ concentration and decrease with NaCMC concentrations. We use Fourier Transform Infrared Spectroscopy (FTIR) to elucidate the Fe3+-NaCMC gelation mechanism and small angle neutron scattering (SANS) to spatio-temporally resolve the solution-to-network structure during front propagation. We find that the polyelectrolyte chain cross-section remains largely unperturbed by gelation and identify three hierarchical structural features at larger length scales. Equipped with the understanding of gelation mechanism and kinetics, using microfluidics, we illustrate the fabrication of range of HMP particles with prescribed morphologies.

Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

Counting of numerous compartmentalized enzymatic reactions underlies quantitative and high sensitivity immunodiagnostic assays. However, digital enzyme-linked immunosorbent assays (ELISA) require specialized instruments which have slowed adoption in research and clinical...

Poroelastic shape relaxation of hydrogel particles

We describe a straightforward way to determine the poroelastic diffusion coefficient of a hydrogel particle by first indenting it via swelling in a granular packing, and then monitoring how its indented shape relaxes after it is removed.

Viscoelasticity of non-colloidal hydrogel particle suspensions at the liquid–solid transition

The liquid–solid transition occurs across a viscoelastic–liquid regime for non-colloidal, polydisperse, frictional soft hydrogel particle suspensions converse to the discrete transition expected for these suspensions of large particles.