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Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force ([Formula: see text]) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 µl/h and 78.7% at 80 µl/h, for 100 µm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).
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This paper reports the use of a recent composite material, noted hereafter i-PDMS, made of carbonyl iron microparticles mixed in a PolyDiMethylSiloxane (PDMS) matrix, for magnetophoretic functions such as capture and separation of magnetic species. We demonstrated that this composite which combine the advantages of both components, can locally generate high gradients of magnetic field when placed between two permanent magnets. After evaluating the magnetic susceptibility of the material as a function of the doping ratio, we investigated the molding resolution offered by i-PDMS to obtain microstructures of various sizes and shapes. Then, we implemented 500 µm i-PDMS microstructures in a microfluidic channel and studied the influence of flow rate on the deviation and trapping of superparamagnetic beads flowing at the neighborhood of the composite material. We characterized the attraction of the magnetic composite by measuring the distance from the i-PDMS microstructure, at which the beads are either deviated or captured. Finally, we demonstrated the interest of i-PDMS to perform magnetophoretic functions in microsystems for biological applications by performing capture of magnetically labeled cells.
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We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500-1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions' cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.
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This paper describes two configurations that integrate electrochemical detection into microfluidic devices. The first configuration is a low-cost approach based on the use of PCB technology. This device was applied to electrochemiluminescence detection. The second configuration was used to carry out amperometric quantification of electroactive species using a serial dilution microfluidic system.
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Eletroquímica/métodos , Microfluídica/instrumentação , Engenharia Biomédica/métodos , Calibragem , Eletroquímica/instrumentação , Eletrodos , Desenho de Equipamento , Peróxido de Hidrogênio/química , Luminescência , Técnicas Analíticas Microfluídicas/métodos , Potenciometria/métodos , Propriedades de SuperfícieRESUMO
Microfluidic devices were designed to perform on micromoles of biological macromolecules and viruses the search and the optimization of crystallization conditions by counter-diffusion, as well as the on-chip analysis of crystals by X-ray diffraction. Chips composed of microchannels were fabricated in poly-dimethylsiloxane (PDMS), poly-methyl-methacrylate (PMMA) and cyclo-olefin-copolymer (COC) by three distinct methods, namely replica casting, laser ablation and hot embossing. The geometry of the channels was chosen to ensure that crystallization occurs in a convection-free environment. The transparency of the materials is compatible with crystal growth monitoring by optical microscopy. The quality of the protein 3D structures derived from on-chip crystal analysis by X-ray diffraction using a synchrotron radiation was used to identify the most appropriate polymers. Altogether the results demonstrate that for a novel biomolecule, all steps from the initial search of crystallization conditions to X-ray diffraction data collection for 3D structure determination can be performed in a single chip.
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Cristalografia por Raios X/instrumentação , Substâncias Macromoleculares/química , Técnicas Analíticas Microfluídicas/instrumentação , Cristalização , Dimetilpolisiloxanos/química , Polimetil Metacrilato/químicaRESUMO
This paper describes a microfluidic device fabricated in poly(dimethylsiloxane) that was employed to perform amperometric quantifications using on-chip calibration curves and on-chip standard addition methods. This device integrated a network of Au electrodes within a microfluidic structure designed for automatic preparation of a series of solutions containing an electroactive molecule at a concentration linearly decreasing. This device was first characterized by fluorescence microscopy and then evaluated with a model electroactive molecule such as Fe(CN(6))(4-). Operating a quantification in this microfluidic parallel approach rather than in batch mode allows a reduced analysis time to be achieved. Moreover, the microfluidic approach is compatible with the on-chip calibration of sensors simultaneously to the analysis, therefore preventing problems due to sensor response deviation with time. When using the on-chip calibration and on-chip standard addition method, we reached concentration estimation better than 5%. We also demonstrated that compared to the calibration curve approach, the standard addition mode is less complex to operate. Indeed, in this case, it is not necessary to take into account flow rate discrepancies as in the calibration approach.
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An acrylate monolith has been synthesized into a cyclic olefin copolymer microdevice for reversed-phase electrochromatography purposes. Microchannels, designed by hot embossing, were filled up with an acrylate monolith to serve as a hydrophobic stationary phase. A lauryl acrylate monolith was formulated to suit the hydrophobic material, by implementing 100% organic porogenic solvent. This new composition was tested in capillary prior to its transfer into the microfluidic device. Surface functionalization of the cyclic olefin copolymer surface was applied using UV-grafting technique to improve the covalent attachment of this monolith to the plastic walls of the microfluidic chip. The on-chip performances of this monolith were evaluated in detail for the reversed-phase electrochromatographic separation of polycyclic aromatic hydrocarbons, with plate heights reaching down to 10 microm when working at optimal velocity.
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Acrilatos/química , Eletrocromatografia Capilar/instrumentação , Eletrocromatografia Capilar/métodos , Cicloparafinas/química , Técnicas Analíticas Microfluídicas/instrumentação , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Polímeros/química , Técnicas Analíticas Microfluídicas/métodos , Hidrocarbonetos Policíclicos Aromáticos/análise , Reprodutibilidade dos TestesRESUMO
We present an instrumental development to implement electrochemiluminescence (ECL) microanalysis using printed circuit board (PCB) technology. PCB gold macro-(10 mm2) and micro- (0.09 mm2) electrodes and two ECL microfluidic devices are designed, fabricated and tested via luminol ECL detection. Potential modulation is performed between 0.7 and 0 V vs. Ag/AgCl for luminol oxidation, thus giving rise to on/off ECL responses in the presence of hydrogen peroxide. Synchronous detection is adopted to allow weak ECL signal recovery at a very low signal-to-noise ratio (SNR). The detection limit obtained with the two ECL microfluidic devices is 50 nM and 100 nM H2O2 for macroelectrodes and microelectrodes, respectively.
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Medições Luminescentes/métodos , Técnicas Analíticas Microfluídicas , Eletroquímica , Eletrônica , Peróxido de Hidrogênio/química , Substâncias Luminescentes/química , Medições Luminescentes/instrumentação , Luminol/química , Microeletrodos , Prata/química , Compostos de Prata/químicaRESUMO
It is well known that sample introduction via electrokinetic mode leads to a bias in conventional CE, which is proportional to the difference of electrophoretic mobilities between species. In electrophoretic separation chips using the gated injection mode, flow distribution at the crossjunction, which is linked to the electric field strength distribution during the loading step, induces an additional contribution to species discrimination. This second-order bias has a similar effect on quantitation like usual electrokinetic bias: the higher the analyte's apparent mobility, the larger the amount injected into the separation channel. The present paper assesses by numerical simulations the influence of several parameters, namely the injected amount, the electric field distribution, and the analyte-apparent Peclet number on this second-order bias.
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Campos Eletromagnéticos , Eletroforese em Microchip/métodos , Modelos TeóricosRESUMO
This communication presents an instrumental development based on the printed circuit board (PCB) technology to integrate electrochemiluminescence (ECL) analysis in microfluidic systems. PCB gold macro- (10 mm2) and micro- (0.09 mm2) electrodes and two ECL microfluidic devices are designed, fabricated and tested via luminol ECL detection. Potential modulation is performed between 0.7 and 0 V vs. Ag/AgCl for luminol oxidation, thus giving rise to on/off ECL responses in the presence of hydrogen peroxide. Synchronous detection is adopted to allow weak ECL signal recovery at a very low signal-to-noise ratio (SNR). The detection limit obtained with the two ECL microfluidic devices is 50 nM and 100 nM H2O2 for macroelectrodes and microelectrodes, respectively.
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Computadores , Técnicas Analíticas Microfluídicas/instrumentação , Eletroquímica/instrumentação , Eletroquímica/métodos , Eletrodos , Ouro , Peróxido de Hidrogênio/análise , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Microeletrodos , Técnicas Analíticas Microfluídicas/métodos , Sensibilidade e Especificidade , PrataRESUMO
This work investigates the efficiency of a chaotic micromixer using thermal lens spectrometry. The outlet of the mixing device was connected to a thermal lens detection head integrating the probe beam optical fibers and the sample capillary. The chaotic micromixer consisted of a Y-shaped poly(dimethylsiloxane) (PDMS) microchip in which ribbed herringbone microstructures were etched on the floor of the main channel. Due to the solvent composition dependence of the thermal lens response, the photothermal method was shown to be highly sensitive to nonhomogeneous mixing compared to fluorescence detection. The apparatus was applied to the determination of Fe2+ with 1,10-phenanthroline using flow injection analysis; a limit of detection of 11 microg L(-1) of iron was obtained.
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Carvão Mineral , Compostos Férricos/química , Modelos Teóricos , Eletroquímica , Eletrodos , Oxirredução , TemperaturaRESUMO
This paper describes a microfluidic device, fabricated in poly(dimethylsiloxane), that is used for potentiometric titrations. This system generates step gradients of redox potentials in a series of microchannels. These potentials are probed by microelectrodes that are integrated into the chip; the measured potentials were used to produce a titration curve from which the end point of a reaction was measured.
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Dimetilpolisiloxanos/química , Microfluídica/métodos , Silicones/química , Eletrodos , Microfluídica/instrumentação , Platina/química , Potenciometria/métodosRESUMO
This report describes a method to pattern mammalian cells using self-assembled monolayers (SAMs), and then to use electrochemical desorption of these monolayers to release cells from their patterns. This method uses an oligo(ethyleneglycol)-terminated SAM to prevent,-and a methyl-terminated SAM to allow-adsorption of proteins and attachment of bovine capillary endothelial cells. Electrochemical removal of the oligo(ethyleneglycol)-terminated SAM allowed proteins to adsorb onto areas that had been previously inert and enabled cells to migrate into these areas. This straightforward technique is useful in bioassays for drug screening and for fundamental studies in cell biology.
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Eletroquímica/métodos , Endotélio Vascular/citologia , Compostos de Sulfidrila/química , Animais , Proteínas Sanguíneas/química , Bovinos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Cicloexanos , Cicloeximida/farmacologia , Endotélio Vascular/efeitos dos fármacos , Proteínas da Matriz Extracelular/química , Ácidos Graxos Insaturados/farmacologia , Ouro/química , Nocodazol/farmacologia , SesquiterpenosRESUMO
This paper describes the design and characterization of a small, membraneless redox fuel cell. The smallest channel dimensions of the cell were 2 mm x 50 mum or x 200 mum; the cell was fabricated in poly(dimethylsiloxane) using soft lithography. This all-vanadium fuel cell took advantage of laminar flow to obviate the need for a membrane to separate the solutions of oxidizing and reducing components.
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Eletroquímica/métodos , Vanádio/química , Carbono/química , Eletroquímica/instrumentação , Eletrodos , Fontes Geradoras de Energia , Ouro/química , OxirreduçãoRESUMO
This article presents a strategy for the fabrication of ordered microstructures using concepts of design inspired by molecular self-assembly and template-directed synthesis. The self-assembling components are 4-microm-thick hexagonal metal plates having sides 10 microm in length ("hexagons"), and each template consists of a 4-microm-thick circular metal plate surrounding a central cavity, the perimeter of which is complementary in shape to the external edges of a two-dimensional, close-packed array of hexagons. The hexagons and templates (collectively, "pieces") were fabricated via standard procedures and patterned into hydrophobic and hydrophilic regions using self-assembled monolayers (SAMs). Templated self-assembly occurs in water through capillary interactions between thin films of a nonpolar liquid adhesive coating the hydrophobic faces of the pieces. The hexagons tile the cavities enclosed by the templates, and the boundaries of the cavities determine the sizes and shapes of the assemblies. Curing the adhesive with ultraviolet light furnishes mechanically stable arrays having well-defined morphologies. By allowing control over the structures of the resulting aggregates, this work represents a step toward the development of practical methods for microfabrication based on self-assembly.
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Química Orgânica/métodos , Coloides/química , Moldes Genéticos , Química Orgânica/instrumentação , Substâncias MacromolecularesRESUMO
A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.
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Eletroforese em Gel Bidimensional/métodos , Proteínas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel Bidimensional/instrumentação , Escherichia coli/química , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Proteoma/isolamento & purificação , Serpinas/isolamento & purificaçãoRESUMO
A ceramic electrochemical reactor (CEM) devoted to electrosyntheses was developed. The CEM was constituted by the assembly of five structured ceramic layers. On one layer, platinum interdigitated band electrodes with a submillimetre interelectrode gap were screen-printed. The microreactor chamber was constituted by seven channels and its volume was less than 100 microL. After a sintering step at 850 degree C for 1 h, the CEM appeared as a solid and compact unit. The CEM was directly connected to the six port valve of a mass spectrometer allowing an on-line sampling and analysis of the reaction mixture. The methoxylation of the methyl-2-furoate was carried out and the effect of the residence time in the CEM was investigated thanks to mass spectrometry analyses.