Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

A microfluidic array with cellular valving for single cell co-culture

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (～99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (～70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.     

Cell sorting by deterministic cell rolling

This communication presents the concept of "deterministic cell rolling", which leverages transient cell-surface molecular interactions that mediate cell rolling to sort cells with high purity and efficiency in a single step.     

Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems

A one-step immunomagnetic separation technique was performed on a microfluidic platform for the isolation of specific cells from blood samples. The cell isolation and purification studies targeted T cells, as a model for low abundance cells (about 1:10,000 cells), with more dilute cells as the ultimate goal. T cells were successfully separated on-chip from human blood and from reconstituted blood samples. Quantitative polymerase chain reaction analysis of the captured cells was used to characterize the efficiency of T cell capture in a variety of flow path designs. Employing many (4-8), 50 microm deep narrow channels, with the same overall cross section as a single, 3 mm wide channel, was much more effective in structuring dense enough magnetic bead beds to trap cells in a flowing stream. The use of 8-multiple bifurcated flow paths increased capture efficiencies from approximately 20 up to 37%, when compared to a straight 8-way split design, indicating the value of ensuring uniform flow distribution into each channel in a flow manifold for effective cell capture. Sample flow rates of up to 3 microL min(-1) were evaluated in these capture beds.     

A novel approach to the uniform distribution of liquid in multi-channel (electrochemical) flow-through cells

Four-channel flow-through electrochemical cell working in thin-layer regime was designed, fabricated and characterized experimentally and in computational fluid dynamics (CFD) simulations. The new principle of operation allows reproducible splitting of a stream of liquid into multiple flow channels. Systems comprising of 2-, 3-, 4- and 8-channels were tested. The proper function of the cell is given by the ratio of the cross-sections of the fluidic element collecting chamber and the particular flow paths among which the liquid is distributed. Suitable flow rates providing uniform liquid distribution were evaluated and the results were compared to CFD modeling. The flow-through cells designed according to the proposed principle can be simply incorporated in automated routine analysis as only one inlet and one common outlet are required.     

Microfluidic depletion of endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions

Interactions between ligands and cell surface receptors can be exploited to design adhesion-based microfluidic cell separation systems. When ligands are immobilized on the microfluidic channel surfaces, the resulting cell capture devices offer the typical advantages of small sample volumes and low cost associated with microfluidic systems, with the added benefit of not requiring complex fabrication schemes or extensive operational infrastructure. Cell-ligand interactions can range from highly specific to highly non-specific. This paper describes the design of an adhesion-based microfluidic separation system that takes advantage of both types of interactions. A 3-stage system of microfluidic devices coated with the tetrapeptides arg-glu-asp-val (REDV), val-ala-pro-gly (VAPG), and arg-gly-asp-ser (RGDS) is utilized to deplete a heterogeneous suspension containing endothelial cells, smooth muscle cells, and fibroblasts. The ligand-coated channels together with a large surface area allow effective depletion of all three cell types in a stagewise manner.     

DNA linearization through confinement in nanofluidic channels

Stretching DNA has emerged as a vital process for studying the physical and biological properties of these molecules. Over the past decade, there has been increasing research interest in utilizing nanoscale fluidic channels to confine and stretch single DNA molecules. Nanofabricated systems for linearizing DNA have revealed new and important insights into the conformation changes of DNA molecules. They also have emerged as innovative techniques for efficiently separating DNA molecules based on size and for physically mapping genetic information along the genome. This review describes physical theories of DNA linearization, current DNA stretching techniques based on nanofabricated channels, and breakthroughs resulting from the use of nanofluidic channels for DNA linearization.     

Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

Microfabricated porous glass channels for electrokinetic separation devices

Electrically insulated porous SiO2 channels for electrokinetic separation devices were fabricated based on a mask-less etching process for creation of high aspect ratio needles in silicon. The silicon needles are converted to SiO2 by oxidation and integrated within the interior of a fluidic channel network. The channels are about 5 microm high with a pore size of 0.5+/-0.2 microm. An electrophoretic separation of a mixture of fluorescein and 5-carboxyfluorescein using epi-fluorescence detection was performed to verify proper electrokinetic transport in the porous channels. The plate height was about 170,000 m-1 for a field strength of 170 V cm-1. In the near future, it is intended to extend the fabrication scheme to include an array of porous pillars for capillary electrochromatography experiments.     

Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes

We present a new lab-on-a-chip system for electrophysiological measurements on Xenopus oocytes. Xenopus oocytes are widely used host cells in the field of pharmacological studies and drug development. We developed a novel non-invasive technique using immobilized non-devitellinized cells that replaces the traditional "two-electrode voltage-clamp" (TEVC) method. In particular, rapid fluidic exchange was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimetre-sized cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of human epithelial sodium channels (ENaC). Our results show that the response time of this ion channel to a specific reactant is about an order of magnitude faster than could be estimated with the traditional TEVC technique.     

Cell adhesion and locomotion on microwell-structured glass substrates

The purpose of this study was to investigate the effect of microstructured material surface on cell adhesion and locomotion in real-time. ArF excimer laser direct-writing ablation was used to fabricate microwell patterns with precise control of size and spacing on glass. The influence of the ablation process parameters (laser fluence, pulse number and repetition rate) on the micromachining quality (depth, width, aspect ratio and edge effects) of the microwells was established. Human fibroblast cells, as an example of anchorage-dependent cells, were seeded onto the microstructured glass substrate and time-lapse microscopy was used to study cell adhesion and locomotion. The interaction with microstructured materials resulted in fibroblast cell repulsion and the cells exhibited a higher locomotion speed (75.77±3.36 μm/h) on the structures in comparison with plane glass control (54.01±15.53 μm/h). Further studies are needed to firmly establish the potential of microstructuring, for example, in elongating the life spans of implantable devices.     

Effect of surface nanotopography on immunoaffinity cell capture in microfluidic devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.     

Covalent immobilization of p-selectin enhances cell rolling

Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.     

Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays

We present a soft lithographic method to fabricate multiphenotype cell arrays by capturing cells within an array of reversibly sealed microfluidic channels. The technique uses reversible sealing of elastomeric polydimethylsiloxane (PDMS) molds on surfaces to sequentially deliver various fluids or cells onto specific locations on a substrate. Microwells on the substrate were used to capture and immobilize cells within low shear stress regions inside channels. By using an array of channels it was possible to deposit multiple cell types, such as hepatocytes, fibroblasts, and embryonic stem cells, on the substrates. Upon formation of the cell arrays on the substrate, the PDMS mold could be removed, generating a multiphenotype array of cells. In addition, the orthogonal alignment and subsequent attachment of a secondary array of channels on the patterned substrates could be used to deliver fluids to the patterned cells. The ability to position many cell types on particular regions within a two dimensional substrate could potentially lead to improved high-throughput methods applicable to drug screening and tissue engineering.     

Rapid Analysis of Cell–Nanoparticle Interactions using Single‐Cell Raman Trapping Microscopy

Iron oxide nanoparticles have been used in preclinical studies to label stem cells for non‐invasive tracking and homing. The search continues for novel particle candidates that are suitable for clinical applications. Since standard analyses to investigate cell–particle interactions and safety are labor‐intensive, an efficient procedure is required to guide future particle development and to exclude adverse health effects. The application of combined Raman trapping microscopy with fluidic chips is reported for the analysis of single cells labeled with different types of aminated iron oxide particles. Multivariate data analysis revealed Raman signal differences that could be clearly assigned to cell–particle interactions and cytotoxicity, respectively. A validation dataset verified that more than 95 % of the spectra were correctly classified. Thus, our approach enables rapid discrimination of non‐hazardous from cytotoxic nanoparticles as a prerequisite for safe clinical applications.     

Vascular mimetics based on microfluidics for imaging the leukocyte--endothelial inflammatory response

We describe the development, validation, and application of a novel PDMS-based microfluidic device for imaging leukocyte interaction with a biological substrate at defined shear force employing a parallel plate geometry that optimizes experimental throughput while decreasing reagent consumption. The device is vacuum bonded above a standard 6-well tissue culture plate that accommodates a monolayer of endothelial cells, thereby providing a channel to directly observe the kinetics of leukocyte adhesion under defined shear flow. Computational fluid dynamics (CFD) was applied to model the shear stress and the trajectory of leukocytes within the flow channels at a micron length scale. In order to test this model, neutrophil capture, rolling, and deceleration to arrest as a function of time and position was imaged in the transparent channels. Neutrophil recruitment to the substrate proved to be highly sensitive to disturbances in flow streamlines, which enhanced the rate of neutrophil-surface collisions at the entrance to the channels. Downstream from these disturbances, the relationship between receptor mediated deceleration of rolling neutrophils and dose response of stimulation by the chemokine IL-8 was found to provide a functional readout of integrin activation. This microfluidic technique allows detailed kinetic studies of cell adhesion and reveals neutrophil activation within seconds to chemotactic molecules at concentrations in the picoMolar range.     

Cell handling using microstructured membranes

Gentle and precise handling of cell suspensions is essential for scientific research and clinical diagnostic applications. Although different techniques for cell analysis at the micro-scale have been proposed, many still require that preliminary sample preparation steps be performed off the chip. Here we present a microstructured membrane as a new microfluidic design concept, enabling the implementation of common sample preparation procedures for suspensions of eukaryotic cells in lab-on-a-chip devices. We demonstrate the novel capabilities for sample preparation procedures by the implementation of metered sampling of nanoliter volumes of whole blood, concentration increase up to three orders of magnitude of sparse cell suspension, and circumferentially uniform, sequential exposure of cells to reagents. We implemented these functions by using microstructured membranes that are pneumatically actuated and allowed to reversibly decouple the flow of fluids and the displacement of eukaryotic cells in suspensions. Furthermore, by integrating multiple structures on the same membrane, complex sequential procedures are possible using a limited number of control steps.     

Nanoporous micro-element arrays for particle interception in microfluidic cell separation

The ability to control cell-surface interactions in order to achieve binding of specific cell types is a major challenge for microfluidic immunoaffinity cell capture systems. In the majority of existing systems, the functionalized capture surface is constructed of solid materials, where flow stagnation at the solid-liquid interface is detrimental to the convection of cells to the surface. We study the use of ultra-high porosity (99%) nanoporous micro-posts in microfluidic channels for enhancing interception efficiency of particles in flow. We show using both modelling and experiment that nanoporous posts improve particle interception compared to solid posts through two distinct mechanisms: the increase of direct interception, and the reduction of near-surface hydrodynamic resistance. We provide initial validation that the improvement of interception efficiency also results in an increase in capture efficiency when comparing nanoporous vertically aligned carbon nanotube (VACNT) post arrays with solid PDMS post arrays of the same geometry. Using both bacteria (～1 μm) and cancer cell lines (～15 μm) as model systems, we found capture efficiency increases by 6-fold and 4-fold respectively. The combined model and experimental platform presents a new generation of nanoporous microfluidic devices for cell isolation.     

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

A novel single-step fabrication technique to create heterogeneous poly(ethylene glycol) hydrogel microstructures containing multiple phenotypes of mammalian cells

In this study, a novel method for the one-step fabrication of stacked hydrogel microstructures using a microfluidic mold is presented. The fabrication of these structures takes advantage of the laminar flow regime in microfluidic devices, limiting the mixing of polymer precursor solutions. To create multilayered hydrogel structures, microfluidic devices were rotated 90 degrees from the traditional xy axes and sealed with a cover slip. Two discreet fluidic regions form in the channels, resulting in the multilayered hydrogel upon UV polymerization. Multilayered patterned poly(ethylene glycol) hydrogel arrays (60 mum tall, 250 mum wide) containing fluorescent dyes, fluorescein isothiocyanate, and tetramethylrhodamine isothiocyanate were created for imaging purposes. Additionally, this method was used to generate hydrogel layers containing murine fibroblasts and macrophages. The cell adhesion promoter, RGD, was added to hydrogel precursor solution to enhance fibroblast cell spreading within the hydrogel matrix in one layer, but not the other. We were able to successfully generate patterns of hydrogels containing multiple phenotypes by using this technique.