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1.
PLoS One ; 14(12): e0226571, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856234

RESUMO

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 µl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.


Assuntos
Eletroforese/instrumentação , Dispositivos Lab-On-A-Chip , MicroRNAs/isolamento & purificação , RNA Nucleolar Pequeno/isolamento & purificação , Fatores de Tempo
2.
Electrophoresis ; 40(22): 2988-2995, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31538669

RESUMO

We present a novel technique for continuous label-free separation of particles based on their dielectrophoretic crossover frequencies. Our technique relies on our unique microfluidic geometry which performs hydrodynamic focusing, generates a stagnation flow with two outlets, and simultaneously produces an isomotive dielectrophoretic field via wall-situated electrodes. To perform particle separation, we hydrodynamically focus particles onto stagnation streamlines and use isomotive dielectrophoretic force to nudge the particles off these streamlines and direct them into appropriate outlets. Focusing particles onto stagnation streamlines obviates the need for large forces to be applied to the particles and therefore increases system throughput. The use of isomotive (spatially uniform) dielectrophoretic force increases system reliability. To guide designers, we develop and describe a simple scaling model for the particle separation dynamics of our technique. The model predicts the range of particle sizes that can be separated as well as the processing rate that can be achieved as a function of system design parameters: channel size, flow rate, and applied potential. Finally, as a proof-of-principle, we use this technique to separate polystyrene bead and cell mixtures of the same diameters as well as mixtures of both particles with varying diameters.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Humanos , Células Jurkat , Tamanho da Partícula
3.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340481

RESUMO

We employed dielectrophoresis to a yeast cell suspension containing amyloid-beta proteins (Aß) in a microfluidic environment. The Aß was separated from the cells and characterized using the gradual dissolution of Aß as a function of the applied dielectrophoretic parameters. We established the gradual dissolution of Aß under specific dielectrophoretic parameters. Further, Aß in the fibril form at the tip of the electrode dissolved at high frequency. This was perhaps due to the conductivity of the suspending medium changing according to the frequency, which resulted in a higher temperature at the tips of the electrodes, and consequently in the breakdown of the hydrogen bonds. However, those shaped as spheroidal monomers experienced a delay in the Aß fibril transformation process. Yeast cells exposed to relatively low temperatures at the base of the electrode did not experience a positive or negative change in viability. The DEP microfluidic platform incorporating the integrated microtip electrode array was able to selectively manipulate the yeast cells and dissolve the Aß to a controlled extent. We demonstrate suitable dielectrophoretic parameters to induce such manipulation, which is highly relevant for Aß-related colloidal microfluidic research and could be applied to Alzheimer's research in the future.


Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Eletroforese/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Saccharomyces cerevisiae/química , Eletrodos , Eletroforese/instrumentação , Liofilização , Ligação de Hidrogênio , Cinética , Saccharomyces cerevisiae/citologia , Solubilidade , Temperatura
4.
Sensors (Basel) ; 19(14)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336874

RESUMO

We present a monolithic biosensor platform, based on carbon-nanotube field-effect transistors (CNTFETs), for the detection of the neurotransmitter glutamate. We used an array of 9'216 CNTFET devices with 96 integrated readout and amplification channels that was realized in complementary metal-oxide semiconductor technology (CMOS). The detection principle is based on amperometry, where electrochemically active hydrogen peroxide, a product of the enzymatic reaction of the target analyte and an enzyme that was covalently bonded to the CNTFET, modulated the conductance of the CNTFET-based sensors. We assessed the performance of the CNTs as enzymatic sensors by evaluating the minimal resolvable concentration change of glutamate in aqueous solutions. The minimal resolvable concentration change amounted to 10 µM of glutamate, which was one of the best values reported for CMOS-based systems so far.


Assuntos
Aminoácido Oxirredutases/química , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Ácido Glutâmico/análise , Nanotubos de Carbono/química , Calibragem , Eletrodos , Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Neurotransmissores/análise , Semicondutores , Sensibilidade e Especificidade , Soluções/química , Água/química
5.
Electrophoresis ; 40(20): 2718-2727, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31206722

RESUMO

Microelectrode arrays are used to sort single fluorescently labeled cells and particles as they flow through a microfluidic channel using dielectrophoresis. Negative dielectrophoresis is used to create a "Dielectrophoretic virtual channel" that runs along the center of the microfluidic channel. By switching the polarity of the electrodes, the virtual channel can be dynamically reconfigured to direct particles along a different path. This is demonstrated by sorting particles into two microfluidic outlets, controlled by an automated system that interprets video data from a color camera and makes complex sorting decisions based on color, intensity, size, and shape. This enables the rejection of particle aggregates and other impurities, and the system is optimized to isolate high purity populations from a heterogeneous sample. Green beads are isolated from an excess of red beads with 100% purity at a rate of up to 0.9 particles per second, in addition application to the sorting of osteosarcoma and human bone marrow cells is evidenced. The extension of Dielectrophoretic Virtual Channels to an arbitrary number of sorting outputs is examined, with design, simulation, and experimental verification of two alternate geometries presented and compared.


Assuntos
Separação Celular , Eletroforese , Processamento de Imagem Assistida por Computador/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Humanos , Tamanho da Partícula
6.
Electrophoresis ; 40(20): 2728-2735, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31219180

RESUMO

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA-MB-231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA-MB-231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA-MB-231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 Vpp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.


Assuntos
Neoplasias da Mama/patologia , Membrana Celular/fisiologia , Forma Celular/fisiologia , Eletroforese/instrumentação , Linhagem Celular Tumoral , Eletroforese/métodos , Feminino , Humanos , Microeletrodos
7.
Electrophoresis ; 40(18-19): 2541-2552, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31219183

RESUMO

Mathematical modeling is a fundamental component in the development of new microfluidics techniques and devices. Modeling allows for the rapid testing of new system configurations while saving resources. Microscale electrokinetic (EK) techniques have significantly benefited by the advances in modeling programs and software packages. However, EK phenomena are complex to model, as they dynamically affect system characteristics, including the physical properties of the particles and fluid within the system. Insulator-based dielectrophoresis (iDEP) is an EK technique that has received important attention during the last two decades. In particular, numerous research groups that study iDEP systems employ a combination of modeling and experimentation for developing new iDEP systems. An important fraction of these research groups has adopted the practice of employing "correction factors" to account for EK phenomena that cannot be accurately predicted in their models due to model complexity and limitations in computing resources. The present review article aims to provide the reader with an overview of the most common approaches in the use of correction factors for the modeling of iDEP systems.


Assuntos
Simulação por Computador , Eletroforese , Desenho de Equipamento , Eletricidade , Eletroforese/instrumentação , Eletroforese/métodos
8.
Biosensors (Basel) ; 9(2)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195725

RESUMO

Dielectrophoresis (DEP) is a nondestructive and noninvasive method which is favorable for point-of-care medical diagnostic tests. This technique exhibits prominent relevance in a wide range of medical applications wherein the miniaturized platform for manipulation (immobilization, separation or rotation), and detection of biological particles (cells or molecules) can be conducted. DEP can be performed using advanced planar technologies, such as complementary metal-oxide-semiconductor (CMOS) through interdigitated capacitive biosensors. The dielectrophoretically immobilization of micron and submicron size particles using interdigitated electrode (IDE) arrays is studied by finite element simulations. The CMOS compatible IDEs have been placed into the silicon microfluidic channel. A rigorous study of the DEP force actuation, the IDE's geometrical structure, and the fluid dynamics are crucial for enabling the complete platform for CMOS integrated microfluidics and detection of micron and submicron-sized particle ranges. The design of the IDEs is performed by robust finite element analyses to avoid time-consuming and costly fabrication processes. To analyze the preliminary microfluidic test vehicle, simulations were first performed with non-biological particles. To produce DEP force, an AC field in the range of 1 to 5 V (peak-to-peak) is applied to the IDE. The impact of the effective external and internal properties, such as actuating DEP frequency and voltage, fluid flow velocity, and IDE's geometrical parameters are investigated. The IDE based system will be used to immobilize and sense particles simultaneously while flowing through the microfluidic channel. The sensed particles will be detected using the capacitive sensing feature of the biosensor. The sensing and detecting of the particles are not in the scope of this paper and will be described in details elsewhere. However, to provide a complete overview of this system, the working principles of the sensor, the readout detection circuit, and the integration process of the silicon microfluidic channel are briefly discussed.


Assuntos
Técnicas Biossensoriais/instrumentação , Eletroforese/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Semicondutores , Animais , Eletrodos , Desenho de Equipamento , Análise de Elementos Finitos , Humanos , Dispositivos Lab-On-A-Chip , Metais/química , Óxidos/química , Tamanho da Partícula
10.
Electrophoresis ; 40(18-19): 2592-2600, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31127957

RESUMO

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem-like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen-based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells.


Assuntos
Eletroforese/métodos , Glioblastoma/patologia , Técnicas Analíticas Microfluídicas/métodos , Esferoides Celulares , Linhagem Celular Tumoral , Eletroforese/instrumentação , Desenho de Equipamento , Estudos de Viabilidade , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Esferoides Celulares/classificação , Esferoides Celulares/citologia , Células Tumorais Cultivadas
11.
Electrophoresis ; 40(10): 1446-1456, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30892709

RESUMO

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP-Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell-to-electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP-Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 µm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP-Raman devices to decrease the analysis time of bacteria.


Assuntos
Eletroforese/instrumentação , Mycobacterium/isolamento & purificação , Análise Espectral Raman/instrumentação , Condutividade Elétrica , Eletrodos , Eletroforese/métodos , Desenho de Equipamento , Humanos , Mycobacterium/química , Mycobacterium/classificação , Poliestirenos , Processamento de Sinais Assistido por Computador , Análise Espectral Raman/métodos
12.
Electrophoresis ; 40(10): 1408-1416, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30883810

RESUMO

Insulator-based dielectrophoresis (iDEP) is the electrokinetic migration of polarized particles when subjected to a non-uniform electric field generated by the inclusion of insulating structures between two remote electrodes. Electrode spacing is considerable in iDEP systems when compared to electrode-based DEP systems, therefore, iDEP systems require high voltages to achieve efficient particle manipulation. A consequence of this is the temperature increase within the channel due to Joule heating effects, which, in some cases, can be detrimental when manipulating biological samples. This work presents an experimental and modeling study on the increase in temperature inside iDEP devices. For this, we studied seven distinct channel designs that mainly differ from each other in their post array characteristics: post shape, post size and spacing between posts. Experimental results obtained using a custom-built copper Resistance Temperature Detector, based on resistance changes, show that the influence of the insulators produces a difference in temperature rise of approximately 4°C between the designs studied. Furthermore, a 3D COMSOL model is also introduced to evaluate heat generation and dissipation, which is in good agreement with the experiments. The model allowed relating the difference in average temperature for the geometries under study to the electric resistance posed by the post array in each design.


Assuntos
Eletroforese/instrumentação , Eletroforese/métodos , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Modelos Teóricos , Temperatura
13.
Lab Chip ; 19(8): 1427-1435, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30875418

RESUMO

Organic electronic ion pumps (OEIPs) have been used for delivery of biological signaling compounds, at high spatiotemporal resolution, to a variety of biological targets. The miniaturization of this technology provides several advantages, ranging from better spatiotemporal control of delivery to reduced invasiveness for implanted OEIPs. One route to miniaturization is to develop OEIPs based on glass capillary fibers that are filled with a polyelectrolyte (cation exchange membrane, CEM). These devices can be easily inserted and brought into close proximity to targeted cells and tissues and could be considered as a starting point for other fiber-based OEIP and "iontronic" technologies enabling favorable implantable device geometries. While characterizing capillary OEIPs we observed deviations from the typical linear current-voltage behavior. Here we report a systematic investigation of these irregularities by performing experimental characterizations in combination with computational modelling. The cause of the observed irregularities is due to concentration polarization established at the OEIP inlet, which in turn causes electric field-enhanced water dissociation at the inlet. Water dissociation generates protons and is typically problematic for many applications. By adding an ion-selective cap that separates the inlet from the source reservoir this effect is then, to a large extent, suppressed. By increasing the surface area of the inlet with the addition of the cap, the concentration polarization is reduced which thereby allows for significantly higher delivery rates. These results demonstrate a useful approach to optimize transport and delivery of therapeutic substances at low concentrations via miniaturized electrophoretic delivery devices, thus considerably broadening the opportunities for implantable OEIP applications.


Assuntos
Eletroforese/instrumentação , Dispositivos Lab-On-A-Chip
14.
Eur Biophys J ; 48(3): 261-266, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30826854

RESUMO

Solid-state nanopores are considered an attractive basis for single-molecule DNA sequencing. At present, one obstacle to be overcome is the improvement of their temporal resolution, with the DNA molecules remaining in the sensing volume of the nanopore for a long period of time. Here, we used a composite system of a concentration gradient of LiCl in solution and a nanofiber mesh to slow the DNA perforation speed. Compared to different alkali metal solutions with the same concentration, LiCl can extend the dwell time to 20 ms, five times longer than NaCl and KCl. Moreover, as the concentration gradient increases, the dwell time can be tuned from dozens of milliseconds to more than 100 ms. When we introduce a nanofiber mesh layer on top of the pore in the asymmetric solution, the DNA molecules get retarded by 162-185 [Formula: see text]s/nt, which is three orders of magnitude slower than the bare nanopore. At the same time, because the molecule absorption region becomes larger at the pore vicinity, the higher molecule capture rate improves the detection efficiency.


Assuntos
DNA/química , Eletroforese/instrumentação , Cloreto de Lítio/química , Movimento (Física) , Nanofibras , DNA/genética , Cinética , Análise de Sequência de DNA
15.
Electrophoresis ; 40(10): 1417-1425, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30830963

RESUMO

The performance of conventional surface plasmon resonance (SPR) biosensors can be limited by the diffusion of the target analyte to the sensor surface. This work presents an SPR biosensor that incorporates an active mass-transport mechanism based on dielectrophoresis and electroosmotic flow to enhance analyte transport to the sensor surface and reduce the time required for detection. Both these phenomena rely on the generation of AC electric fields that can be tailored by shaping the electrodes that also serve as the SPR sensing areas. Numerical simulations of electric field distribution and microparticle trajectories were performed to choose an optimal electrode design. The proposed design improves on previous work combining SPR with DEP by using face-to-face electrodes, rather than a planar interdigitated design. Two different top-bottom electrode designs were experimentally tested to concentrate firstly latex beads and secondly biological cells onto the SPR sensing area. SPR measurements were then performed by varying the target concentrations. The electrohydrodynamic flow enabled efficient concentration of small objects (3 µm beads, yeasts) onto the SPR sensing area, which resulted in an order of magnitude increased SPR response. Negative dielectrophoresis was also used to concentrate HEK293 cells onto the metal electrodes surrounded by insulating areas, where the SPR response was improved by one order of magnitude.


Assuntos
Eletroforese/instrumentação , Eletroforese/métodos , Ressonância de Plasmônio de Superfície/métodos , Difusão , Eletrodos , Eletro-Osmose , Desenho de Equipamento , Células HEK293 , Humanos , Dispositivos Lab-On-A-Chip , Látex , Ressonância de Plasmônio de Superfície/instrumentação
16.
Electrophoresis ; 40(10): 1436-1445, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30706494

RESUMO

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single-cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 µm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 µm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP-release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single-cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP-release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single-cell analysis.


Assuntos
Impedância Elétrica , Eletroforese/instrumentação , Eletroforese/métodos , Dispositivos Lab-On-A-Chip , Microeletrodos , Calibragem , Desenho de Equipamento , Hidrodinâmica , Técnicas Analíticas Microfluídicas/instrumentação , Poliestirenos , Saccharomyces cerevisiae/citologia , Sensibilidade e Especificidade , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
17.
Electrophoresis ; 40(10): 1478-1485, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30701577

RESUMO

Here, we report a microfluidic same-single-cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single-cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P-glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control on both fluorescence detection systems due to the inhibition by CsA.


Assuntos
Separação Celular/métodos , Eletroforese/métodos , Leucemia Mieloide Aguda/patologia , Análise de Célula Única/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Separação Celular/instrumentação , Forma Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Eletroforese/instrumentação , Desenho de Equipamento , Fluorescência , Corantes Fluorescentes/farmacologia , Humanos , Dispositivos Lab-On-A-Chip , Leucemia Mieloide Aguda/tratamento farmacológico , Microscopia de Fluorescência/instrumentação , Rodamina 123/farmacologia , Análise de Célula Única/instrumentação
18.
Electrophoresis ; 40(10): 1498-1509, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30706961

RESUMO

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.


Assuntos
Comunicação Celular , Eletroforese/instrumentação , Eletroforese/métodos , Agregação Celular , Linhagem Celular , Eletrodos , Desenho de Equipamento , Humanos , Rim/citologia , Rim/embriologia , Dispositivos Lab-On-A-Chip , Poliestirenos
19.
Methods Mol Biol ; 1914: 131-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729463

RESUMO

This chapter describes the analysis of signaling pathways in bone cells by the use of western blotting and immunoprecipitation, including a step-by-step guide to cell culture techniques, cellular and subcellular fractionation, protein isolation, purification, measurement, electrophoretic transfer, and detection.


Assuntos
Western Blotting/métodos , Osso e Ossos/citologia , Imunoprecipitação/métodos , Proteínas/análise , Transdução de Sinais , Animais , Western Blotting/instrumentação , Osso e Ossos/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Eletroforese/instrumentação , Eletroforese/métodos , Humanos , Imunoprecipitação/instrumentação , Proteínas/isolamento & purificação , Proteínas/metabolismo
20.
Electrophoresis ; 40(10): 1426-1435, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30786069

RESUMO

Analytes concentration techniques are being developed with the appealing expectation to boost the performance of biosensors. One promising method lies in the use of electrokinetic forces. We present hereafter a new design for a microstructured plasmonic sensor which is obtained by conventional microfabrication techniques, and which can easily be adapted on a classical surface plasmon resonance imaging (SPRI) system without further significant modification. Dielectrophoretic trapping and electro-osmotic displacement of the targets in the scanned fluid are performed through interdigitated 200 µm wide gold electrodes that also act as the SPR-sensing substrate. We demonstrate the efficiency of our device's collection capabilities for objects of different sizes (200 nm and 1 µm PS beads, as well as 5-10 µm yeast cells). SPRI is relevant for the spatial analysis of the mass accumulation at the electrode surface. We demonstrate that our device overcomes the diffusion limit encountered in classical SPR sensors thanks to rapid collection capabilities (<1 min) and we show a consequent improvement of the detection limit, by a factor >300. This study of an original device combining SPRI and electrokinetic forces paves the way to the development of fully integrated active plasmonic sensors with direct applications in life sciences, electrochemistry, environmental monitoring and agri-food industry.


Assuntos
Eletroforese/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Difusão , Eletrodos , Eletro-Osmose , Eletroforese/métodos , Desenho de Equipamento , Processamento de Imagem Assistida por Computador , Limite de Detecção , Saccharomyces cerevisiae/citologia , Fluxo de Trabalho
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