On-chip antibody immobilization for on-demand and rapid immunoassay on a microfluidic chip

Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified.     

A microfluidic membrane device to mimic critical components of the vascular microenvironment

Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.     

Microfluidic wet spinning of chitosan-alginate microfibers and encapsulation of HepG2 cells in fibers

The successful encapsulation of human hepatocellular carcinoma (HepG2) cells would greatly assist a broad range of applications in tissue engineering. Due to the harsh conditions during standard chitosan fiber fabrication processes, encapsulation of HepG2 cells in chitosan fibers has been challenging. Here, we describe the successful wet-spinning of chitosan-alginate fibers using a coaxial flow microfluidic chip. We determined the optimal mixing conditions for generating chitosan-alginate fibers, including a 1:5 ratio of 2% (w∕w) water-soluble chitosan (WSC) solution to 2% (w∕w) alginate solution. Ratio including higher than 2% (w∕w) WSC solution increased aggregation throughout the mixture. By suspending cells in the WSC-alginate solution, we successfully fabricated HepG2 cell-laden fibers. The encapsulated HepG2 cells in the chitosan-alginate fibers were more viable than cells encapsulated in pure alginate fibers, suggesting that cross-linked chitosan provides a better environment for HepG2 cells than alginate alone. In addition, we found that the adhesion of HepG2 cells on the chitosan-alginate fiber is much better than that on the alginate fibers.     

DNA separation by cholesterol-bearing pullulan nanogels

We present an application of a novel DNA separation matrix, cholesterol-bearing pullulan (CHP) nanogels, for microchip electrophoresis. The solution of the CHP showed a unique phase transition around 30 mg∕ml and formed gel phase over this critical concentration. This gel phase consists of the weak hydrophobic interactions between the cholesterols could be easily deformed by external forces, and thus, loading process of the CHP nanogels into microchannels became easier. The high concentration of the CHP nanogels provided excellent resolutions especially for small DNA fragments from 100 to 1500 bp. The separation mechanism was discussed based on Ogston and Reptation models which had developed in gels or polymer solutions. The result of a single molecule imaging gave us an insight of the separation mechanism and the nanogel structures as well.     

Electrochemical biosensors for on-chip detection of oxidative stress from immune cells

Seamless integration of biological components with electrochemical sensors is critical in the development of microdevices for cell analysis. The present paper describes the integration miniature Au electrodes next to immune cells (macrophages) in order to detect cell-secreted hydrogen peroxide (H(2)O(2)). Photopatterning of poly(ethylene glycol) (PEG) hydrogels was used to both immobilize horseradish peroxidase molecules onto electrodes and to define regions for cell attachment in the vicinity of sensing electrodes. Electrodes micropatterned in such a manner were enclosed inside poly(dimethylsiloxane) fluid conduits and incubated with macrophages. The cells attached onto the exposed glass regions in the vicinity of the electrodes and nowhere else on the non-fouling PEG hydrogel surface. A microfluidic device was converted into an electrochemical cell by placing flow-through Ag∕AgCl reference and Pt wire counter electrodes at the outlet and inlet, respectively. This microdevice with integrated H(2)O(2)-sensing electrodes had sensitivity of 27 μA∕cm(2) mM with a limit of detection of 2 μM. Importantly, this microdevice allowed controllable seeding of macrophages next to electrodes, activation of these cells and on-chip monitoring of H(2)O(2) release in real time. In the future, this biosensor platform may be utilized for monitoring of macrophage responses to pathogens or for the study of inflammatory signaling in micropatterned cell cultures.     

Long-range and superfast trapping of DNA molecules in an ac electrokinetic funnel

In this work we report a microfluidic platform capable of trapping and concentrating a trace amount of DNA molecules efficiently. Our strategy invokes nonlinear electro-osmotic flow induced by charge polarization under high-frequency ac fields. With the asymmetric quadrupole electrode design, a unique converging flow structure can be created for generating focusing effects on DNA molecules. This focusing in turn transforms into a robust funnel that can collect DNA molecules distantly from the bulk and pack them into a compact cone with the aid of short-range dipole-induced self-attraction and dielectrophoresis. Our results reveal that not only can DNA molecules be concentrated within just a few seconds, but also they can be focused into threads of 1 mm in length, demonstrating the superfast and long-range trapping capability of this funnel. In addition, pico M DNA solutions can be concentrated with several decades of enhancement without any continuous feeding. Alternating concentration and release of DNA molecules is also illustrated, which has potentials in concentrating and transporting biomolecules in a continuous fashion using microdevices.     

Use of a virtual wall valve in polydimethylsiloxane microfluidic devices for bioanalytical applications

A simple method for micromanipulation of liquids and∕or small groups of cells is presented in this study. Microfabricated sieving structures composed of PDMS (polydimethylsiloxane) were used to segregate aqueous solutions. This microfluidic valving scheme was an application of Cassie-Baxter wetting and was termed "virtual walls" as a nonsolid barrier exists at an air∕water interface. The manipulation of the virtual-air-wall valve was accomplished by controlling the strength of surface-tension and hydrostatic-pressure forces. Virtual walls with a range of feature sizes were designed and characterized by monitoring air and water displacement in response to hydrostatic pressure. Thresholds for the virtual-air-wall valves to be turned on or off were quantified. The walls could also be formed or dissipated by the focused microbeam of a pulsed laser. As an illustration of the virtual wall utility, a series of microfluidic applications were demonstrated. First, the capability of virtual walls to temporarily segregate liquids was integrated into a device utilized to establish a chemical gradient. In a second application, the arraying of nonadherent cells within individual aqueous cavities created by the virtual walls was demonstrated. Individual cells were also released from the cavities on demand using a focused microbeam. The virtual walls were simple and easy-to-fabricate without the requirement for surface treatment or precision alignment, and should find usage in bioanalytical applications.     

High-throughput size-based rare cell enrichment using microscale vortices

Cell isolation in designated regions or from heterogeneous samples is often required for many microfluidic cell-based assays. However, current techniques have either limited throughput or are incapable of viable off-chip collection. We present an innovative approach, allowing high-throughput and label-free cell isolation and enrichment from heterogeneous solution using cell size as a biomarker. The approach utilizes the irreversible migration of particles into microscale vortices, developed in parallel expansion-contraction trapping reservoirs, as the cell isolation mechanism. We empirically determined the critical particle∕cell diameter D(crt) and the operational flow rate above which trapping of cells∕particles in microvortices is initiated. Using this approach we successfully separated larger cancer cells spiked in blood from the smaller blood cells with processing rates as high as 7.5×10(6) cells∕s. Viable long-term culture was established using cells collected off-chip, suggesting that the proposed technique would be useful for clinical and research applications in which in vitro culture is often desired. The presented technology improves on current technology by enriching cells based on size without clogging mechanical filters, employing only a simple single-layered microfluidic device and processing cell solutions at the ml∕min scale.     

Observation of nonspherical particle behaviors for continuous shape-based separation using hydrodynamic filtration

Selection of particles or cells of specific shapes from a complex mixture is an essential procedure for various biological and industrial applications, including synchronization of the cell cycle, classification of environmental bacteria, and elimination of aggregates from synthesized particles. Here, we investigate the separation behaviors of nonspherical and spherical particles∕cells in the hydrodynamic filtration (HDF) scheme, which was previously developed for continuous size-dependent particle∕cell separation. Nonspherical particle models were prepared by coating the hemisphere of spherical polymer particles with a thin Au layer and by bonding the Janus particles to form twins and triplets resembling dividing and aggregating cells, respectively. High-speed imaging revealed a difference in the separation behaviors of spherical and nonspherical particles at a branch point; nonspherical particles showed rotation behavior and did not enter the branch channel even when their minor axis was smaller than the virtual width of the flow region entering the branch channel, w(1). The confocal-laser high-speed particle intensity velocimetry system visualized the flow profile inside the HDF microchannel, demonstrating that the steep flow-velocity distribution at the branch point is the main factor causing the rotation behavior of nonspherical particles. As applications, we successfully separated spherical and nonspherical particles with various major∕minor lengths and also demonstrated the selection of budding∕single cells from a yeast cell mixture. We therefore conclude that the HDF scheme can be used for continuous shape-based particle∕cell separation.     

Simple surface modification of poly(dimethylsiloxane) for DNA hybridization

Here, we present a simple chemical modification of poly(dimethylsiloxane) (PDMS) by curing a mixture of 2 wt% undecylenic acid (UDA) in PDMS prepolymer on a gold-coated glass slide. This gold slide had been previously pretreated with a self-assembled hydrophilic monolayer of 3-mercaptopropionic acid (MPA). During curing of the UDA∕PDMS prepolymer, the hydrophilic UDA carboxyl moieties diffuses toward the hydrophilic MPA carboxyl moieties on the gold surface. This diffusion of the UDA within the PDMS prepolymer to the surface is a direct result of surface energy minimization. Once completely cured, the PDMS is peeled off the gold substrate, thereby exposing the interfacial carboxyl groups. These groups are then available for subsequent attachment of 5(')-amino terminated DNA oligonucleotides via amide linkages. Our results show that the covalently tethered oligonucleotides can successfully capture fluorescein-labeled complementary oligonucleotides via hybridization, which are visualized using fluorescence microscopy.     

Optofluidic planar reactors for photocatalytic water treatment using solar energy

Optofluidics may hold the key to greater success of photocatalytic water treatment. This is evidenced by our findings in this paper that the planar microfluidic reactor can overcome the limitations of mass transfer and photon transfer in the previous photocatalytic reactors and improve the photoreaction efficiency by more than 100 times. The microreactor has a planar chamber (5 cm×1.8 cm×100 μm) enclosed by two TiO(2)-coated glass slides as the top cover and bottom substrate and a microstructured UV-cured NOA81 layer as the sealant and flow input∕output. In experiment, the microreactor achieves 30% degradation of 3 ml 3×10(-5)M methylene blue within 5 min and shows a reaction rate constant two orders higher than the bulk reactor. Under optimized conditions, a reaction rate of 8% s(-1) is achieved under solar irradiation. The average apparent quantum efficiency is found to be only 0.25%, but the effective apparent quantum efficiency reaches as high as 25%. Optofluidic reactors inherit the merits of microfluidics, such as large surface∕volume ratio, easy flow control, and rapid fabrication and offer a promising prospect for large-volume photocatalytic water treatment.     

Transport of particles and microorganisms in microfluidic channels using rectified ac electro-osmotic flow

A new method is demonstrated to transport particles, cells, and other microorganisms using rectified ac electro-osmotic flows in open microchannels. The rectified flow is obtained by synchronous zeta potential modulation with the driving potential in the microchannel. Experiments were conducted to transport both neutral, charged particles, and microorganisms of various sizes. A maximum speed of 50 μm∕s was obtained for 8 μm polystyrene beads, without any electrolysis, using a symmetrical square waveform driving electric field of 5 V∕mm at 10 Hz and a 360 V gate potential with its polarity synchronized with the driving potential (phase lag=0°).     

DNA methylation profiling in nanochannels

We report the profiling of the 5-methyl cytosine distribution within single genomic-sized DNA molecules at a gene-relevant resolution. This method linearizes and stretches DNA molecules by confinement to channels with a dimension of about 250×200 nm(2). The methylation state is detected using fluorescently labeled methyl-CpG binding domain proteins (MBD), with high signal contrast and low background. DNA barcodes consisting of methylated and non-methylated segments are generated, with both short and long concatemers demonstrating spatially resolved MBD binding. The resolution of the technique is better than 10 kbp, and single-molecule read-lengths exceeding 140 kbp have been achieved.     

Polyelectrolyte multilayer surface functionalization of poly(dimethylsiloxane) (PDMS) for reduction of yeast cell adhesion in microfluidic devices

Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.     

Stakeholder interaction within research consortia on emerging technologies: Learning how and what?

One of the challenges for public-private R&D collaborations in emerging scientific fields is to actively include the demand side. Insight in how to facilitate learning between stakeholders is, however, lacking. In this paper we present an approach to facilitate and analyse learning processes in multi-stakeholder interactions within public-private research consortia working on new science and technologies. The learning processes that took place during dialogue meetings within the framework of the Dutch Ecogenomics Consortium were analysed, including a reflection on the actual effects. The results show that a carefully structured dialogue method facilitates learning between researchers, users and policy-related participants, and that this learning to some extent is anchored within the Ecogenomics Consortium. At the same time, the results point to the challenges of translating learning into action.     

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 nL∕s and mean flow rates of approximately 1-1.5 nL∕s. 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.     

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.     

A self-contained polymeric cartridge for automated biological sample preparation

Sample preparation is one of the most crucial processes for nucleic acids based disease diagnosis. Several steps are required for nucleic acids extraction, impurity washes, and DNA/RNA elution. Careful sample preparation is vital to the obtaining of reliable diagnosis, especially with low copies of pathogens and cells. This paper describes a low-cost, disposable lab cartridge for automatic sample preparation, which is capable of handling flexible sample volumes of 10 μl to 1 ml. This plastic cartridge contains all the necessary reagents for pathogen and cell lysis, DNA/RNA extraction, impurity washes, DNA/RNA elution and waste processing in a completely sealed cartridge. The entire sample preparation processes are automatically conducted within the cartridge on a desktop unit using a pneumatic fluid manipulation approach. Reagents transportation is achieved with a combination of push and pull forces (with compressed air and vacuum, respectively), which are connected to the pneumatic inlets at the bottom of the cartridge. These pneumatic forces are regulated by pinch valve manifold and two pneumatic syringe pumps within the desktop unit. The performance of this pneumatic reagent delivery method was examined. We have demonstrated the capability of the on-cartridge RNA extraction and cancer-specific gene amplification from 10 copies of MCF-7 breast cancer cells. The on-cartridge DNA recovery efficiency was 54-63%, which was comparable to or better than the conventional manual approach using silica spin column. The lab cartridge would be suitable for integration with lab-chip real-time polymerase chain reaction devices in providing a portable system for decentralized disease diagnosis.     

Emergence of creativity in IS development teams: A socio-technical systems perspective

Despite the crucial role of creativity in the dynamic and intricate practice of Information Systems Development (ISD), our understanding of its emergence in ISD teams is limited. Prior research has focused on isolated interventions into the people, structure, task, and technology components of ISD practices. However, the synergistic interactions between these components are often overlooked, resulting in interventions that may be ineffective or counterproductive. Drawing on Leavitt’s Socio-Technical Systems (STS) theory, this paper explores creativity as a synergistic outcome of interactions within and between the people, structure, task, and technology components. Our qualitative study at a multinational banking software provider reveals that creativity emerged from within-component interactions when innovators and facilitators in ISD teams interact empathetically during idea generation and evaluation, visualize ideas using technologies for representation and collaboration, and align their creative styles with the organization's formal and informal structures. Additionally, we found that interactions between the STS components in ISD team practices created stimulating environments for creativity to emerge. Our study advances an STS perspective on the emergence of creativity in ISD teams, emphasizing synergistic interactions between people, structure, task, and technology rather than isolated interventions. Practically, our findings provide insights into how IS professionals can nurture creativity by fostering empathetic leadership, aligning creative styles with organizational structures, and balancing tensions between control and drift.     

Biochip∕laser cell deposition system to assess polarized axonal growth from single neurons and neuron∕glia pairs in microchannels with novel asymmetrical geometries

Axon path-finding plays an important role in normal and pathogenic brain development as well as in neurological regenerative medicine. In both scenarios, axonal growth is influenced by the microenvironment including the soluble molecules and contact-mediated signaling from guiding cells and cellular matrix. Microfluidic devices are a powerful tool for creating a microenvironment at the single cell level. In this paper, an asymmetrical-channel-based biochip, which can be later incorporated into microfluidic devices for neuronal network study, was developed to investigate geometric as well as supporting cell control of polarized axonal growth in forming a defined neuronal circuitry. A laser cell deposition system was used to place single cells, including neuron-glia pairs, into specific microwells of the device, enabling axonal growth without the influence of cytophilic∕phobic surface patterns. Phase microscopy showed that a novel "snag" channel structure influenced axonal growth in the intended direction 4:1 over the opposite direction. In heterotypic experiments, glial cell influence over the axonal growth path was observed with time-lapse microscopy. Thus, it is shown that single cell and heterotypic neuronal path-finding models can be developed in laser patterned biochips.