Hydrodynamic shearing of DNA in a polymeric microfluidic device

Nesterova, I.,Hupert, M.,Witek, M.,Soper, S. Royal Society of Chemistry 2012 Lab on a chip Vol.12 No.6

Simple replication methods for producing nanoslits in thermoplastics and the transport dynamics of double-stranded DNA through these slits

Chantiwas, Rattikan,Hupert, Mateusz L.,Pullagurla, Swathi R.,Balamurugan, Subramanian,Tamarit-Ló,pez, Jesú,s,Park, Sunggook,Datta, Proyag,Goettert, Jost,Cho, Yoon-Kyoung,Soper, Steven A. Royal Society of Chemistry 2010 Lab on a chip Vol.10 No.23

<P>Mixed-scale nano- and microfluidic networks were fabricated in thermoplastics using simple and robust methods that did not require the use of sophisticated equipment to produce the nanostructures. High-precision micromilling (HPMM) and photolithography were used to generate mixed-scale molding tools that were subsequently used for producing fluidic networks into thermoplastics such as poly(methyl methacrylate), PMMA, cyclic olefin copolymer, COC, and polycarbonate, PC. Nanoslit arrays were imprinted into the polymer using a nanoimprinting tool, which was composed of an optical mask with patterns that were 2–7 µm in width and a depth defined by the Cr layer (100 nm), which was deposited onto glass. The device also contained a microchannel network that was hot embossed into the polymer substrate using a metal molding tool prepared <I>via</I> HPMM. The mixed-scale device could also be used as a master to produce a polymer stamp, which was made from polydimethylsiloxane, PDMS, and used to generate the mixed-scale fluidic network in a single step. Thermal fusion bonding of the cover plate to the substrate at a temperature below their respective <I>T</I><SUB>g</SUB> was accomplished by oxygen plasma treatment of both the substrate and cover plate, which significantly reduced thermally induced structural deformation during assembly: ∼6% for PMMA and ∼9% for COC nanoslits. The electrokinetic transport properties of double-stranded DNA (dsDNA) through the polymeric nanoslits (PMMA and COC) were carried out. In these polymer devices, the dsDNA demonstrated a field-dependent electrophoretic mobility with intermittent transport dynamics. DNA mobilities were found to be 8.2 ± 0.7 × 10<SUP>−4</SUP> cm<SUP>2</SUP> V<SUP>−1</SUP> s<SUP>−1</SUP> and 7.6 ± 0.6 × 10<SUP>−4</SUP> cm<SUP>2</SUP> V<SUP>−1</SUP> s<SUP>−1</SUP> for PMMA and COC, respectively, at a field strength of 25 V cm<SUP>−1</SUP>. The extension factors for λ-DNA were 0.46 in PMMA and 0.53 in COC for the nanoslits (2–6% standard deviation).</P> <P>Graphic Abstract</P><P>Thermoplastic nanoslits were replicated from a simple molding tool and consisted of mixed-scale structures with successful DNA translocation through the slits demonstrated. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c0lc00096e'> </P>

UV activation of polymeric high aspect ratio microstructures: ramifications in antibody surface loading for circulating tumor cell selection

Jackson, J.,Witek, M.,Hupert, M.,Brady, C.,Pullagurla, S.,Kamande, J.,Aufforth, R.,Tignanelli, C.,Torphy, R.,Yeh, J. Royal Society of Chemistry 2014 Lab on a chip Vol.14 No.1

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of similar to 3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer's damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device's cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

Discrete geometry optimization for reducing flow non-uniformity, asymmetry, and parasitic minor loss pressure drops in Z-type configurations of fuel cells

Jackson, J.M.,Hupert, M.L.,Soper, S.A. Elsevier Sequoia 2014 Journal of Power Sources Vol.269 No.-

Parallel channel configurations, such as Z-type, used to distribute reagents in planar fuel cells provide lower overall pressure drop as compared to other channel designs. However, due to their inherent characteristics, flow maldistribution in parallel configurations is commonly observed and leads to starvation of reagents in middle channels. In addition, the Reynolds number dependent minor losses at branching tee junctions may cause asymmetric flow non-uniformity and reagent imbalance between the cathode and anode. Herein, we present a universal and simple optimization method to simultaneously reduce flow maldistribution, asymmetry, and parasitic pressure in Z-type parallel configurations of fuel cells or fuel cell stacks that has improved scalability relative to previous methods. A discrete model's governing equations were reduced to yield geometric ratios between headers. Increasing header widths to satisfy these ratios reduced flow maldistribution without modifying parallel channel geometry as validated by computation fluid dynamics (CFD) simulations. Furthermore, decreased Reynolds numbers throughout the headers reduced minor pressure drops and flow distribution asymmetry. We offer several methods to reduce the optimized geometry's footprint, including an adaptation of the discontinuous design.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

Battle, Katrina N.,Jackson, Joshua M.,Witek, Małgorzata A.,Hupert, Mateusz L.,Hunsucker, Sally A.,Armistead, Paul M.,Soper, Steven A. The Royal Society of Chemistry 2014 The Analyst Vol.139 No.6

<P>We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O<SUB>3</SUB> treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins <I>via</I> DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.</P> <P>Graphic Abstract</P><P>We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c3an02400h'> </P>

Parallel Affinity-Based Isolation of Leukocyte Subsets Using Microfluidics: Application for Stroke Diagnosis

Pullagurla, Swathi R.,Witek, Małgorzata A.,Jackson, Joshua M.,Lindell, Maria A. M.,Hupert, Mateusz L.,Nesterova, Irina V.,Baird, Alison E.,Soper, Steven A. American Chemical Society 2014 ANALYTICAL CHEMISTRY - Vol.86 No.8

<P/><P>We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as <I>S100A9</I>, <I>TCRB</I>, and <I>FPR1</I>, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated.</P>