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>

Surface charge, electroosmotic flow and DNA extension in chemically modified thermoplastic nanoslits and nanochannels

Uba, Franklin I.,Pullagurla, Swathi R.,Sirasunthorn, Nichanun,Wu, Jiahao,Park, Sunggook,Chantiwas, Rattikan,Cho, Yoon-Kyoung,Shin, Heungjoo,Soper, Steven A. The Royal Society of Chemistry 2015 The Analyst Vol.140 No.1

<P>Thermoplastics have become attractive alternatives to glass/quartz for microfluidics, but the realization of thermoplastic nanofluidic devices has been slow in spite of the rather simple fabrication techniques that can be used to produce these devices. This slow transition has in part been attributed to insufficient understanding of surface charge effects on the transport properties of single molecules through thermoplastic nanochannels. We report the surface modification of thermoplastic nanochannels and an assessment of the associated surface charge density, zeta potential and electroosmotic flow (EOF). Mixed-scale fluidic networks were fabricated in poly(methylmethacrylate), PMMA. Oxygen plasma was used to generate surface-confined carboxylic acids with devices assembled using low temperature fusion bonding. Amination of the carboxylated surfaces using ethylenediamine (EDA) was accomplished <I>via</I> EDC coupling. XPS and ATR-FTIR revealed the presence of carboxyl and amine groups on the appropriately prepared surfaces. A modified conductance equation for nanochannels was developed to determine their surface conductance and was found to be in good agreement with our experimental results. The measured surface charge density and zeta potential of these devices were lower than glass nanofluidic devices and dependent on the surface modification adopted, as well as the size of the channel. This property, coupled to an apparent increase in fluid viscosity due to nanoconfinement, contributed to the suppression of the EOF in PMMA nanofluidic devices by an order of magnitude compared to the micro-scale devices. Carboxylated PMMA nanochannels were efficient for the transport and elongation of λ-DNA while these same DNA molecules were unable to translocate through aminated nanochannels.</P> <P>Graphic Abstract</P><P>We report the surface modification of thermoplastic nanochannels and the evaluation of the surface charge density, zeta potential and electroosmotic flow (EOF). <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c4an01439a'> </P>

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.

Characterization of activated cyclic olefin copolymer: effects of ethylene/norbornene content on the physiochemical properties

O'Neil, Colleen E.,Taylor, Scott,Ratnayake, Kumuditha,Pullagurla, Swathi,Singh, Varshni,Soper, Steven A. The Royal Society of Chemistry 2016 The Analyst Vol.141 No.24

<P>The ethylene/norbornene content within cyclic olefin copolymer (COC) is well known to affect the chemical and physical properties of the copolymer, such as the glass transition temperature (<I>T</I>g) and transparency. However, no work has been reported evaluating the effects of the ethylene/norbornene content on the surface properties of COC following UV/O3 or O2 plasma activation. Activation with either O2 plasma or UV/O3 is often used to assist in thermal assembly of fluidic devices, increasing the wettability of the surfaces, or generating functional scaffolds for the attachment of biological elements. Thus, we investigated differences in the physiochemical surface properties of various ethylene/norbornene compositions of COC following activation using analytical techniques such as water contact angle (WCA), ATR-FTIR, XPS, TOF-SIMS, UV-VIS, AFM and a colorimetric assay utilizing Toluidine Blue O (TBO). Results showed that increased norbornene content led to the generation of more oxygen containing functionalities such as alcohols, ketones, aldehydes and carboxyl groups when activated with either UV/O3 or O2 plasma. Specifically, COC with ∼60% norbornene content showed a significantly higher -COOH functional group density when compared to COC with a 50% norbornene content and COC with a 35% norbornene content following UV/O3 or O2 plasma activation. Furthermore, COC with large norbornene contents showed a smaller average RMS roughness (0.65 nm) when compared to COC containing low norbornene contents (0.95 nm) following activation making this substrate especially suited for nanofluidic applications, which require smooth surfaces to minimize effects arising from dielectrophoretic trapping or non-specific adsorption. Although all COC substrates showed >90% transparency at wavelengths >475 nm, COC possessing high norbornene contents showed significantly less transparency at wavelengths below 475 nm following activation, making optical detection in this region difficult. Our data showed distinct physiochemical differences in activated COC that was dependent upon the ethylene/norbornene content of the thermoplastic and thus, careful selection of the particular COC grade must be considered for micro- and nanofluidics.</P>