Unbiased, Cell-free Profiling of Single Influenza Genomes at High-throughput.

The segmented structure of the Influenza A virus (IAV) genome facilitates reassortment, segment exchange during co-infection. When divergent strains mix across human, agricultural, and wildlife reservoirs novel strains are generated, which has been the source of pandemics. Due to the limited throughput and infection-based assays, IAV reassortment studies has been limited to permissive reassortment. We have developed DE-flowSVP to achieve extremely high throughput, direct profiling of as many as 10 5 IAV particles in a single-day experiment and enabled quantitative profiling of reassortment propensity between divergent strains for the first time. By profiling reassortants between two naturally circulating low-pathogenicity avian IAVs, we confirmed that molecular incompatibility yields strong preference toward within-strain mixing. Surprisingly, we revealed that two-to-three particle aggregation contributed primarily to genome mixing (75-99%), suggesting that aggregation mediated by sialic acid binding by viral surface proteins provides a secondary pathway to genome mixing while avoiding the co-packaging fitness cost. We showed that genome mixing is sensitively dependent on co-infection timing, relative segment abundances, and viral surface-protein background. DE-flowSVP enables large-scale survey of reassortment potential among the broad diversity of IAV strains informing pandemic strain emergence.

Multiplexed electrical detection of whole viruses from plasma in a microfluidic platform.

The advancement of point-of-care diagnostics is crucial to improving patient outcomes, especially in areas with low access to hospitals or specialized laboratories. In particular, rapid, sensitive, and multiplexed detection of disease biomarkers has great potential to achieve accurate diagnosis and inform high quality care for patients. Our Coulter counting and immunocapture based detection system has previously shown its broad applicability in the detection of cells, proteins, and nucleic acids. This paper expands the capability of the platform by demonstrating multiplexed detection of whole-virus particles using electrically distinguishable hydrogel beads by demonstrating the capability of our platform to achieve simultaneous detection at clinically relevant concentrations of hepatitis A virus (>2 × 103 IU mL-1) and human parvovirus B19 virus like particles (>106 IU mL-1) from plasma samples. The expanded versatility of the differential electrical counting platform allows for more robust and diverse testing capabilities.

Electrochemical point-of-care devices for the diagnosis of sepsis.

Sepsis is a life-threatening dysfunction of organ systems caused by a dysregulated immune system because of an infectious process. It remains one of the leading causes of hospital mortality and of hospital readmissions in the United States. Mortality from sepsis increases with each hour of delayed treatment, therefore, diagnostic devices that can reduce the time from the onset of a patient's infection to the delivery of appropriate therapy are urgently needed. Likewise, tools that are capable of high-frequency testing of clinically relevant biomarkers are required to study disease progression. Electrochemical biosensors offer important advantages such as high sensitivity, fast response, miniaturization, and low cost that can be adapted to clinical needs. In this review paper, we discuss the current state, limitations, and future directions of electrochemical-based point-of-care detection platforms that contribute to the diagnosis and monitoring of sepsis.

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis.

Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent drop contents from mixing. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer DE drop maker compatible with simple surface treatment using a plasma cleaner. The device allows for the robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets eliminate content mixing and maintain compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.

Rapid, multiplexed detection of biomolecules using electrically distinct hydrogel beads.

Rapid, low-cost, and multiplexed biomolecule detection is an important goal in the development of effective molecular diagnostics. Our recent work has demonstrated a microfluidic biochip device that can electrically quantitate a protein target with high sensitivity. This platform detects and quantifies a target analyte by counting and capturing micron-sized beads in response to an immunoassay on the bead surface. Existing microparticles limit the technique to the detection of a single protein target and lack the magnetic properties required for separation of the microparticles for direct measurements from whole blood. Here, we report new precisely engineered microparticles that achieve electrical multiplexing and adapt this platform for low-cost and label-free multiplexed electrical detection of biomolecules. Droplet microfluidic synthesis yielded highly-monodisperse populations of magnetic hydrogel beads (MHBs) with the necessary properties for multiplexing the electrical Coulter counting on chip. Each bead population was designed to contain a different amount of the hydrogel material, resulting in a unique electrical impedance signature during Coulter counting, thereby enabling unique identification of each bead. These monodisperse bead populations span a narrow range of sizes ensuring that all can be captured sensitively and selectively under simultaneously flow. Incorporating these newly synthesized beads, we demonstrate versatile and multiplexed biomolecule detection of proteins or DNA targets. This development of multiplexed beads for the electrical detection of biomolecules, provides a critical advancement towards multiplexing the Coulter counting approach and the development of a low cost point-of-care diagnostic sensor.

Modeling Oil Shale Pyrolysis: High-Temperature Unimolecular Decomposition Pathways for Thiophene.

The thermal decomposition mechanism of thiophene has been investigated both experimentally and theoretically. Thermal decomposition experiments were done using a 1 mm × 3 cm pulsed silicon carbide microtubular reactor, C4H4S + Δ â†’ Products. Unlike previous studies these experiments were able to identify the initial thiophene decomposition products. Thiophene was entrained in either Ar, Ne, or He carrier gas, passed through a heated (300-1700 K) SiC microtubular reactor (roughly ≤100 µs residence time), and exited into a vacuum chamber. The resultant molecular beam was probed by photoionization mass spectroscopy and IR spectroscopy. The pyrolysis mechanisms of thiophene were also investigated with the CBS-QB3 method using UB3LYP/6-311++G(2d,p) optimized geometries. In particular, these electronic structure methods were used to explore pathways for the formation of elemental sulfur as well as for the formation of H2S and 1,3-butadiyne. Thiophene was found to undergo unimolecular decomposition by five pathways: C4H4S → (1) S═C═CH2 + HCCH, (2) CS + HCCCH3, (3) HCS + HCCCH2, (4) H2S + HCC-CCH, and (5) S + HCC-CH═CH2. The experimental and theoretical findings are in excellent agreement.

Thermal Decomposition Mechanism for Ethanethiol.

The thermal decomposition of ethanethiol was studied using a 1 mm × 2 cm pulsed silicon carbide microtubular reactor, CH3CH2SH + Δ â†’ Products. Unlike previous studies these experiments were able to identify the initial ethanethiol decomposition products. Ethanethiol was entrained in either an Ar or a He carrier gas, passed through a heated (300-1700 K) SiC microtubular reactor (roughly ≤100 µs residence time) and exited into a vacuum chamber. Within one reactor diameter the gas cools to less than 50 K rotationally, and all reactions cease. The resultant molecular beam was probed by photoionization mass spectroscopy and IR spectroscopy. Ethanethiol was found to undergo unimolecular decomposition by three pathways: CH3CH2SH → (1) CH3CH2 + SH, (2) CH3 + H2C═S, and (3) H2C═CH2 + H2S. The experimental findings are in good agreement with electronic structure calculations.