15 research outputs found
Microfluidics: reframing biological enquiry
The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science
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Performance implications of chemical mobilization after microchannel IEF.
Chemical mobilization following IEF enables single-point detection of an ideally stationary equilibrium electrophoresis mode. Despite prior studies exploring optimization of chemical mobilization conditions and recent insight from numerical simulations, understanding of both chemical mobilization mechanisms and the implications of mobilization on IEF analytical performance remains limited. In this study, we utilize full-field imaging of microchannel IEF to assess the performance of a range of canonical chemical mobilization schemes. We empirically demonstrate and characterize key areas where limited understanding of performance implications exists, including: the effects of mobilization solution pH and ion concentration, differences between ionic and zwitterionic mobilization, and diffusion as a source of zone broadening. We utilize Simul5 simulations to gain insight into the sources of the measured performance differences. Measurements of the location, linearity, and slope of the IEF pH gradient (via fluorescent pH markers imaged before and during mobilization) as well as mobilization-associated broadening of focused analytes were performed to quantify performance and determine the dominant sources of variability. Our results suggest that nonuniform broadening of the pH gradient and changes in the pH gradient linearity stem from conductivity nonuniformities in the separation channel and not diffusion-associated band broadening during mobilization
Recommended from our members
Performance implications of chemical mobilization after microchannel IEF.
Chemical mobilization following IEF enables single-point detection of an ideally stationary equilibrium electrophoresis mode. Despite prior studies exploring optimization of chemical mobilization conditions and recent insight from numerical simulations, understanding of both chemical mobilization mechanisms and the implications of mobilization on IEF analytical performance remains limited. In this study, we utilize full-field imaging of microchannel IEF to assess the performance of a range of canonical chemical mobilization schemes. We empirically demonstrate and characterize key areas where limited understanding of performance implications exists, including: the effects of mobilization solution pH and ion concentration, differences between ionic and zwitterionic mobilization, and diffusion as a source of zone broadening. We utilize Simul5 simulations to gain insight into the sources of the measured performance differences. Measurements of the location, linearity, and slope of the IEF pH gradient (via fluorescent pH markers imaged before and during mobilization) as well as mobilization-associated broadening of focused analytes were performed to quantify performance and determine the dominant sources of variability. Our results suggest that nonuniform broadening of the pH gradient and changes in the pH gradient linearity stem from conductivity nonuniformities in the separation channel and not diffusion-associated band broadening during mobilization
Detection of Isoforms Differing by a Single Charge Unit in Individual Cells
To measure protein isoforms in individual mammalian cells we report single-cell resolution isoelectric focusing (scIEF) and high-selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control transport and handling of each 17 pL cell lysate during analysis. scIEF resolves protein isoforms with resolution down to single-charge unit differences, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells
Microchamber Integration Unifies Distinct Separation Modes for Two-Dimensional Electrophoresis
By combining isoelectric focusing
(IEF) with subsequent gel electrophoresis,
two-dimensional electrophoresis (2DE) affords more specific characterization
of proteins than each constituent unit separation. In a new approach
to integrating the two assay dimensions in a microscope slide-sized
glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide
(PA) gel elements housed in a millimeter-scale, 20-Ī¼m-deep chamber.
The microchamber minimizes information loss inherent to channel network
architectures commonly used for microfluidic 2DE. To define the IEF
axis along a ālaneā at the top of the chamber, we used
free solution carrier ampholytes and immobilized acrylamido buffers
in the PA gels. This approach yielded high-resolution (0.1 pH unit)
and rapid (<20 min) IEF. Next, protein transfer to the second dimension
was accomplished by chemical mobilization perpendicular to the IEF
axis. Mobilization drove focused proteins off the IEF lane and into
a region for protein gel electrophoresis. Using fluorescently labeled
proteins, we observed transfer-induced band broadening factors ā¼7.5-fold
lower than those observed in microchannel networks. Both native polyacrylamide
gel electrophoresis (PAGE) and pore-limit electrophoresis (PLE) were
studied as the second assay dimension and completed in <15 min.
PLE yields protein molecular mass information without the need for
ionic surfactant or reducing agents, simplifying device design and
operation. Microchamber-based 2DE unifies two independent separation
dimensions in a single device with minimal transfer-associated information
losses. Peak capacities for the total assay ranged from 256 to 35
with <1 h assay duration. The rapid microchamber 2DE assay has
the potential to bridge an existing gap in targeted proteomics for
protein biomarker validation and systems biology that may complement
recent innovation in mass spectrometry
Systematic characterization of degas-driven flow for poly(dimethylsiloxane) microfluidic devices
Degas-driven flow is a novel phenomenon used to propel fluids in poly(dimethylsiloxane) (PDMS)-based microfluidic devices without requiring any external power. This method takes advantage of the inherently high porosity and air solubility of PDMS by removing air molecules from the bulk PDMS before initiating the flow. The dynamics of degas-driven flow are dependent on the channel and device geometries and are highly sensitive to temporal parameters. These dependencies have not been fully characterized, hindering broad use of degas-driven flow as a microfluidic pumping mechanism. Here, we characterize, for the first time, the effect of various parameters on the dynamics of degas-driven flow, including channel geometry, PDMS thickness, PDMS exposure area, vacuum degassing time, and idle time at atmospheric pressure before loading. We investigate the effect of these parameters on flow velocity as well as channel fill time for the degas-driven flow process. Using our devices, we achieved reproducible flow with a standard deviation of less than 8% for flow velocity, as well as maximum flow rates of up to 3 nLās and mean flow rates of approximately 1ā1.5 nLās. Parameters such as channel surface area and PDMS chip exposure area were found to have negligible impact on degas-driven flow dynamics, whereas channel cross-sectional area, degas time, PDMS thickness, and idle time were found to have a larger impact. In addition, we develop a physical model that can predict mean flow velocities within 6% of experimental values and can be used as a tool for future design of PDMS-based microfluidic devices that utilize degas-driven flow
Nonfouling, Encoded Hydrogel Microparticles for Multiplex MicroRNA Profiling Directly from Formalin-Fixed, Paraffin-Embedded Tissue
MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.NIH-NIBIB (Grant 5R21EB024101-02
Spatially resolved and multiplexed MicroRNA quantification from tissue using nanoliter well arrays
Ā© 2020, The Author(s). Spatially resolved gene expression patterns are emerging as a key component of medical studies, including companion diagnostics, but technologies for quantification and multiplexing are limited. We present a method to perform spatially resolved and multiplexed microRNA (miRNA) measurements from formalin-fixed, paraffin-embedded (FFPE) tissue. Using nanoliter well arrays to pixelate the tissue section and photopatterned hydrogels to quantify miRNA, we identified differentially expressed miRNAs in tumors from a genetically engineered mouse model for non-small cell lung cancer (K-rasLSL-G12D/+; p53fl/fl). This technology could be used to quantify heterogeneities in tissue samples and lead to informed, biomarker-based diagnostics