Abstract: We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 [small mu ]m h-1 healing rate).
Abstract: Doppler Optical Coherence Tomography (DOCT) is a biomedical imaging technique that allows simultaneous structural imaging and flow monitoring inside biological tissues and materials with spatial resolution in the micrometer scale. It has recently been applied to the characterization of microfluidic systems. Structural and flow imaging of novel microfluidics platforms for cytotoxicologic applications were obtained with a real-time, Near Infrared Spectral Domain DOCT system. Characteristics such as flow homogeneity in the chamber, which is one of the most important parameters for cell culture, are investigated. OCT and DOCT images were used to monitor flow inside a specific platform that is based on microchannel division for a better flow homogeneity. In particular, the evolution of flow profile at the transition between the microchannel structure and the chamber is studied.
Abstract: Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.
Abstract: This article reports on recent electrical and optical techniques for investigating cellular signaling reactions in artificial and native membranes immobilized on solid supports. The first part describes the formation of planar artificial lipid bilayers on gold electrodes, which reveal giga-ohm electrical resistance and the insertion and characterization of ionotropic receptors therein. These membranes are suited to record a few or even single ion channels by impedance spectroscopy. Such tethered membranes on planar arrays of microelectrodes offer mechanically robust, long-lasting measuring devices to probe the influence of different chemistries on biologically important ionotropic receptors and therefore will have a future impact to probe the function of channel proteins in basic science and in biosensor applications. In a second part, we present complementary approaches to form inside-out native membrane sheets that are immobilized on micrometer-sized beads or across submicrometer-sized holes machined in a planar support. Because the native membrane sheets are plasma membranes detached from live cells, these approaches offer a unique possibility to investigate cellular signaling processes, such as those mediated by ionotropic or G protein-coupled receptors, with original composition of lipids and proteins.
Abstract: A complexing gel integrated microelectrode (CGIME) for direct measurements of free metal ion concentrations in natural waters has been developed. It is prepared by the successive deposition of microlayers of a chelating resin, an antifouling agarose gel and Hg on a 100-interconnected Ir-based microelectrode array. The trace metals of interest are in a first step accumulated on the chelating resin in proportion to their free ion concentration in solution, then released in acidic solution and detected simultaneously by using square wave anodic stripping voltammetry (SWASV). The reliability of this sensor for the simultaneous measurement of copper, lead and cadmium has been studied by a series of replicate laboratory tests. The proportionality between the voltammetric peak current intensity and the free metal ion concentrations in solution has been demonstrated by using malonate as a model ligand. Finally, the CGIME sensor was applied to the Cu and Pb free concentration measurement in sea water samples and the results compared to the free metal ion concentrations measured using hollow fiber based permeation liquid membrane (HF-PLM) coupled to inductively coupled plasma mass spectrophotometer (ICP-MS). Comparable concentration values were found for both metals with both techniques allowing to validate the CGIME measurements in complex media
Abstract: In this study, we present the development and the characterization of a generic platform for cell culture able to monitor extracellular ionic activities (K+, NH4+) for real-time monitoring of cell-based responses, such as necrosis, apoptosis, or differentiation. The platform for cell culture is equipped with an array of 16 silicon nitride micropipet-based ion-selective microelectrodes with a diameter of either 2 or 6 microm. This array is located at the bottom of a 200-microm-wide and 350-microm-deep microwell where the cells are cultured. The characterization of the ion-selective microelectrode arrays in different standard and physiological solutions is presented. Near-Nernstian slopes were obtained for potassium- (58.6 +/- 0.8 mV/pK, n = 15) and ammonium-selective microelectrodes (59.4 +/- 3.9 mV/pNH4, n = 13). The calibration curves were highly reproducible and showed an average drift of 4.4 +/- 2.3 mV/h (n = 10). Long-term behavior and response after immersion in physiological solutions are also presented. The lifetime of the sensors was found to be extremely long with a high recovery rate.
Abstract: In this contribution we present the development of three microfabricated devices for the study of neuronal and cellular networks. Together, these devices form an attractive toolbox, which is useful to stimulate and record signals of both electrical and chemical nature. One approach consist of microelectrode arrays for the study of neuronal networks, and allow for the electrical stimulation of individual cells in the network, while the other electrodes of the array record the electrical activity of the remaining cells of the network. We also present the use of micropipettes that can measure the extra- and intracellular concentrations of ions in cells cultures. A third approach exploits the laminar flows in a microfluidic device, to deliver minute amounts of drug to some cells in a cellular network. These three illustrations show that microfabricated platforms are appealing analytical tools in the context of cell biology.
Abstract: The development of a high-density active microelectrode array for in vitro electrophysiology is reported. Based on the Active Pixel Sensor (APS) concept, the array integrates 4096 gold microelectrodes (electrode separation 20 microm) on a surface of 2.5 mmx2.5 mm as well as a high-speed random addressing logic allowing the sequential selection of the measuring pixels. Following the electrical characterization in a phosphate solution, the functional evaluation has been carried out by recording the spontaneous electrical activity of neonatal rat cardiomyocytes. Signals with amplitudes from 130 microVp-p to 300 microVp-p could be recorded from different pixels. The results demonstrate the suitability of the APS concept for developing a new generation of high-resolution extracellular recording devices for in vitro electrophysiology.
Abstract: In this paper, the design and the characterization of batch fabricated SixNy micropipette arrays with diameters ranging from 6 µm down to 250 nm are described. The process used to fabricate the micromachined pipettes includes a deep reactive ion etching step, followed by the deposition of two successive layers, a thermal oxide layer and a low stress, low pressure chemical vapor deposited silicon nitride layer, respectively. The diameter of the micropipettes could be modulated simply by choosing the thicknesses of the oxide sacrificial layer and of the nitride walls of the micropipettes. The reactive ion etching of the micropipette top layer in deep cavities and in confined and deconfined configurations is discussed. The mechanical resistance of the micropipette array was qualitatively tested and it was demonstrated that a force of 0.25 mN/micropipette could be applied without rupture of the micropipettes.
Abstract: The design and the fabrication of an ion-selective electrode array platform aimed at in vitro intracellular recording are presented. The platform is composed of two parts: (i) a glass chip with microchannels whose bottom part is patterned with a platinum layer and (ii) a silicon chip in which an array of 50Â [mu]m long silicon nitride micropipettes with an inner diameter of 5Â [mu]m are structured. The 24 micropipettes are individually filled with a Ca2+ selective membrane, based on the neutral ionophore ETH 129. Preliminary tests in Ca2+ buffered solutions have shown a linear range from 10-6 to 0.1Â M Ca2+ with a sensitivity of 27Â mV/decade.
Abstract: Scanning electrochemical microscopy (SECM) was used to characterize addressable microelectrode arrays patterned with 25 regularly spaced (100 μm center to center) 10 μm diameter platinum microdisks. Both the feedback mode and the substrate generation/tip collection (SG/TC) mode were used where only the ultramicroelectrode (UME) tip current was recorded. A range of UME tip diameters (1, 2, 5, 10, and 25 μm) was used to image the surface of the arrays. The microdisks on the array were connected in parallel, kept electrically independent, or a predetermined combination of the two. The microelectrode array was also biased or unbiased. Combined, the SECM feedback and SG/TC modes gave valuable information regarding the conductivity, connectivity, and reactivity of the microdisks on the array. SECM was also found to be a unique method for screening the integrity of the insulator coating of the underlying tracks serving as connections to each microdisk in the array. Steady-state cyclic voltammetry and scanning electron microscopy were used to provide additional information regarding the nature of the microdisk surfaces on the array and the stability of the currents generated.
Abstract: The development of a voltammetric microsystem for monitoring and speciation studies of trace elements in natural waters is presented. This system was designed to provide an efficient coupling of the square-wave anodic stripping voltammetry (SWASV) detection with the permeation liquid membrane (PLM) technique. It consists of a voltammetric microcell, based on a gel-integrated Ir(Hg) microdisk array, a PLM and two micromachined channels for the sample and strip solutions respectively. The analytical performance of the microsystem was first assessed for Pb(II) and Cd(II) and then the simultaneous accumulation and detection of Cd(II), Pb(II) and Cu(II) from a synthetic sample solution was performed. The estimated detection limit of the free metal ion is 2 pM for Pb(II) and 75 pM for Cd(II) using 5 min as deposition time.
Abstract: A new micro total analysis system (μTAS) designed for volumetric nanotitrations, integrating two electroosmotically driven nanopumps and a sensor unit, is presented. The unique feature of the integrated electroosmotic pumps is their ability to pump widely different solutions, independent of intrinsic characteristics such as pH or ionic strength, with the exception of viscosity. Typical flow rates achieved are in the range of 2â65 nl/s, depending on the number of microchannels connected in parallel and on the applied voltage. The pulsation-free flows developed in two nanopumps push out the solutions contained in two reservoirs. The solutions are fed into a three-dimensional mixer, where the chemical reaction takes place. The potential difference as a function of the sample concentration is detected by means of a pseudo-reference electrode located in one channel and an indicator electrode placed downstream from the mixer in the sensor unit.
Abstract: This paper describes theoretically the behavior of partial electroosmotic and hydrodynamic flow in complex capillary systems. The equivalent hydraulic resistance and the equivalent electrical resistance of microfluidic systems are considered as the relevant parameters. The predicted flow rates and back-pressure characteristics agreed well with experimental results, as obtained with a novel electroosmotic pump consisting of up to six microfabricated microchannels connected in parallel.
The theory permits the tailored design and optimization of new electroosmotic pumps with specific features, such as defined flow rate range, back-pressure range and desired range of the applied voltage.
Abstract: Coulometric nanotitrations were realized in a microchannel system using a continuous-flow titration technique with a triangle current-time profile. Redox and acid-base titrations were carried out on Fe(II) and nitric acid samples, respectively, with the same nanotitrator device. A linear relation between the concentration and the coulometric current transferred to the solution was found. The advantages of this universally applicable nanotitrator are fast response, low sample volume, high sensitivity, and high reproducibility as well as the convenience of handling an automated analyzer of the flow-through type.
Abstract: Recent contributions to the design, development, and fabrication of microtechnological devices for chemical analysis are summarized. The discussion includes microdisk-electrode arrays for voltammetric analysis of trace metals, and micro total-analysis systems for coulometric nanotitrations of different analytes.
Abstract: This paper presents a numerical simulation of the flow field on a one-dimensional pneumatic actuator. Unlike conventional actuators, this model uses dynamic pressure instead of friction to drive a slider. The objective of this simulation is to find the detail of the flow field under the slider as well as the influence of its levitation on the horizontal transportation. Secondary vortices to be formed under the slider may cause an instability of the slider movement. To further assure a stable transportation of the slider, absence of secondary vortices in the gap is desirable, which can be achieved by narrowing the gap width. However, a too narrow gap might cause a significant increase of flow impedance and thus sacrifice the horizontal transportation. Here, two cases with gap width of 100 and 50 μm were investigated. With a gap width of 50 μm, there was no secondary vortex formed; however, the horizontal transportation was greatly sacrificed. In contrast, with a gap width of 100 μm, several secondary vortices of a size one to two times the gap width were formed. However, the horizontal driving force was about eight times larger than that in the case of a gap width of 50 μm
Abstract: This paper reports and fully characterizes a coulometric nanotitrator and its application for four different types of titrations: precipitation, complex-formation, redox and acidâbase nanotitrations. This simple device, composed of Pt or Ag planar electrodes located in a microchannel, gives very reproducible results and yields a linear relationship between the analyte concentration and the equivalence time for concentrations up to 10â2 M of analyte.
Abstract: This paper presents a one-dimensional (1-D) pneumatic actuator fabricated by combining several micromachining technologies such as microelectrodischarge machining (micro-EDM) as well as isotropic and anisotropic wet etching. Unlike the existing pneumatic actuators, which usually convey the object by means of friction, this device employs the dynamic pressure of inclined driving jets in order to enhance the horizontal transportation performance. Typical slider speeds of up to 5 cm/s can be obtained. Comparisons between different types of sliders are presented. By an appropriate patterning of the slider bottom surface, the speed could be increased by 50%-60%. Similarly, a maximum tangential force of 20 μN (equivalent shear stress: 2.2 μN/mm2) was obtained using this dynamic pressure concept. The latter is about two times larger than that of a slider with a smooth surface
