Esta investigación fue financiada por los Institutos Nacionales de Salud (NIH) de subvención R01 EB008268.

1. Chip de fabricación 12 <ol><li> Hacer dos placas de Zeonex 690R gránulos: distribuir 8-9 gramos gránulos Zeonex uniformemente en el centro de una placa de metal, precalentar en la prensa calentada a 198 ° C durante 5 min, y luego aplicar presión lentamente a 2.500 psi durante otros 5 min. Para completar este paso, se utilizó una prensa Carver caliente. </li><li> Graba en relieve el canal microfluídico en la placa con un molde de epoxi. Los detalles sobre la fabricación de moldes se describen en otra parte 12 (diseño de canal en la figura 2b): ponga una placa en el molde de epoxi; precalentarlos en la prensa calentada a 157 ° C durante 10 min y después aplicar presión lentamente a 1.000 psi durante otros 10 min . Retire la placa del molde a mano o con una pinza antes de que se enfríe. (Nota: use guantes térmicos durante este paso.) </li><li> Perforar los agujeros en el chip en relieve en la entrada, el puerto de residuos, y la salida del canal microfluídico. Se utilizó un diámetro de 1,25 mmeternos broca para hacer los tres agujeros. </li><li> Bond el chip en relieve con otra placa (en la parte superior): Lavar las patatas fritas con IPA, RNAse Away y agua desionizada en secuencia; aire seco y precalentar ellos en la prensa calentada a 131 ° C durante 10 min y después se presiona a 350 psi para otro 10 min. </li><li> Adjuntar Nanoports en la entrada, los residuos, y los puertos de salida por separado usando JB Weld Epoxy; curar durante 15 a 24 horas según las instrucciones del fabricante. </li><li> Enjuague el canal SPE con 50 l de RNasa lejos, seguidos con 100 ml de agua libre de nucleasas. </li><li> Cargar el canal SPE con 4 l de la solución de injerto (metacrilato de metilo con 3% w / v benzofenona) y luego reticular el metacrilato por incubación durante 10 min en un horno de radiación UV, en longitud de onda 365 nm UV y 2.000 2 mJ / cm. Retire la solución residual injerto con vacío. Se utilizó el vacío de la pared. Para obtener los mejores resultados, una solución fresca se debe utilizar. Por lo general es aceptable, sin embargo, para almacenar unsolución preparada a 4 ° C durante hasta una semana. </li><li> Hacer la solución columna SPE (todos los% v / v): 16% dimetacrilato de etileno, 24% de metacrilato de butilo, 42% de 1-dodecanol, 18% de ciclohexanol, y añadir 1% de 2-dimetilamino-4-(metil-fenilamino)-fenol como el foto-iniciador. </li><li> Sacar el mismo volumen de solución de sílice como microesferas de la solución columna SPE (para hacer una mezcla 1:1), y que se seque completamente a 70 ° C durante la noche en un horno de vacío. Romper la pastilla tocando en el tubo con la tapa cerrada. Añadir la solución de la columna SPE en el tubo de sílice de microesferas secas y vortex para mezclar. </li><li> Carga 4 l de la solución SPE en el mismo canal y después reticular por irradiación UV durante 2,5 min. A continuación, darle la vuelta al chip de una y otra para irradiar min 2,5. La columna SPE polimerizado debería ser un sólido de color blanco opaco. </li><li> Lavar el canal con 500 l de metanol al 100% para enjuagar el exceso de reactivos. Este lavado también elimina intra-po polímerodisolventes rogenic, creando así la estructura de poro abierto de la columna SPE cuando se seca en condiciones ambientales. </li><li> Acople dos calentadores de película delgada a la parte inferior del chip con cinta térmicamente conductor. Los lados de los calentadores deben estar alineadas con el borde de los canales anchos para la desnaturalización y el recocido por separado (véase la Figura 1). </li><li> Coloque cinco termopares en el chip a través de los muertos terminó abiertos los canales laterales de cada chip (véase la Figura 2b). Los termopares permanecer en estos canales mediante ajuste a presión. Cinta se utiliza para fijar aún más la ubicación de los termopares. </li></ol> 2. Método de extracción en fase sólida <ol><li> Preparar 96 l de etanol al 70% con agua tratada con DEPC y se autoclave (Millipore filtrada desionizada o-). </li><li> Añadir 4 l de 25X ARN Asegure a 96 l de 70% de etanol en un tubo, y duplicar la misma mezcla con 100% de etanol en un tubo separado. </li><li> Preparar 300l de tampón canal mezclando los siguientes: 75 l de 6 M de tiocianato de guanidina (GuSCN), 150 l de 2-propanol, 63 l de agua libre de nucleasa, y 12 l de ARN Aseguren 25X. </li><li> Preparar 312 l de tampón de lisis mediante la mezcla: 100 l de 6 M de GuSCN, 200 l de 2-propanol, y 12 mu l de ARN Aseguren 25X. </li><li> Calentar el etanol al 70%, etanol al 100%, tampón de canal y tampones de lisis a 60 º C durante 10-20 min para activar el seguro de ARN. </li><li> Para equilibrar el canal microfluídico, ejecute el buffer del canal a través del canal de SPE a un caudal de 0,8 ml / hr. </li><li> Rápida y descongelar la muestra en VTM a 37 ° C en baño de agua y retire inmediatamente la muestra del baño de agua tan pronto como se descongela. </li><li> Se centrifuga la muestra a 13.000 rpm durante 10 min, y transferir 100 l del sobrenadante en 300 l de tampón de lisis. Completar la mezcla mediante la adición de 6 l de 1 mg / ARN mu l portadoras. </li><li> Vortex para mezclar, y después de centrifugado5 segundos, cargue el lisado completo en una jeringa Luer-lok cc 1. </li><li> Ejecutar el lisado a través del canal de SPE a un caudal de 0,8 ml / hr. </li><li> Lavar el canal de SPE con 100 l de etanol al 70%, seguido por 100 l de etanol of100% a 1 ml / hr. (Hay un volumen de 50 l muertos en la punta de la jeringa que no será expulsado, por lo que en realidad sólo 50 l pasa por el canal.) </li><li> Coloque una jeringa vacía en la marca de 0,5 ml y empujar el aire a través del canal para que se seque a 1 ml / hr. </li><li> Ejecutar 67,5 l de agua libre de nucleasa a través del canal para eluir los ácidos nucleicos unidos a 0,5 ml / hr, y recoger 13,5 l de la NanoPort en el puerto de residuos. (La pérdida de fluido se debe al volumen de 54 l muerto en la punta de la jeringa y el canal de SPE.) </li></ol> 3. RT-PCR <ol><li> Preparar 36,5 l de la mezcla de RT-PCR maestro durante el paso de aire seco (2,12) utilizando los reactivos en un Qiagen OneStep RT-PCR kits. </li><li> Cargar RT-PCR reactivo en la NanoPort en el puerto de residuos, y se mezcla con el ARN para obtener el siguiente 50 l de reacción RT-PCR: 13,5 mu l de ARN se eluyó en agua libre de nucleasa, 4 l de RT-PCR de mezcla de enzimas, 10 l solución Q, 10 μl1X uno tope graduado, 2 l 25 mM MgCl 2, 1 uL 50 mM adelante primer 5&#39;-GAC CRA TCC TGT CAC CTC TGA &#39;C-3, 1 l 50 mM primers revertir 5&#39;-AGG ACG TTY TGG ACA AAK CGT CTA-3 &#39;, 2 l dNTP, 0,75 l 1,0% w / v de BSA, 0,73 l PEG8000 y 5,02 l de agua libre de nucleasas. </li><li> Cargar la mezcla de RT-PCR en el canal RT por vacío suave (se utilizó el vacío de la pared) y sellar la NanoPort con un accesorio cerrado. </li><li> Aplicar 35 V al calentador 1. Mantener los reactivos en el canal TA durante 30 min después del calentador 1 se equilibra a 50 º C. Mantener los reactivos en el canal TA durante 15 min después del calentador 1 se equilibra a 95 º C. </li><li> Aplicar 27 V al calentador 1 y 50 V al calentador 2, esperar alrededor de tres minutos o hasta que el calentador 1 se equilibra a 60 y deg; C y el calentador 2 se equilibra a 95 º C. </li><li> Empuje los reactivos en el canal de PCR a 0,5 l / min. Se debe tomar alrededor de 20 min para los reactivos fluyan a través del canal de serpentina. </li><li> Cuando la muestra llega a la final del canal de serpentina, recoger los productos de PCR en la toma de corriente mediante una pipeta y la punta de tamaño apropiado. </li></ol> 4. Productos de detección de PCR <ol><li> Usamos un Agilent DNA de prueba de alta sensibilidad para detectar los productos. Para ejecutar esta prueba saque el kit de ADN Agilent alta sensibilidad del refrigerador 30 minutos antes de la prueba. </li><li> Encienda el Bioanalyzer y abra el software Expert 2100. </li><li> Cargar el 1 l de los productos de PCR en la prueba y siga el protocolo de Agilent ( <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">20Manual.pdf http://gcf.pbrc.edu/docs/Agilent/Agilent%</a> ). </li><li> Analizar y registrar los datos. </li></ol>

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Los autores han declarado conflictos de intereses financieros.

El método de diagnóstico presentados aquí demuestran la capacidad de un sistema integrado de chip de plástico microfluídico para amplificar ARN influenza A partir de muestras de pacientes con alta especificidad y un bajo límite de detección 13 Se diseñó este chip para el punto de potencial de las pruebas de atención:. (A) la temperatura y fluídico el control se simplifica, (b) el chip es de bajo costo y adecuadas para la fabricación de alto rendimiento utilizando moldeo por inyección, y (c) el ch...

Millones de muertes han sido reportadas durante las tres pandemias de influenza en el siglo 20 1. Por otra parte, la pandemia de influenza más reciente fue declarada por la Organización Mundial de la Salud (OMS), dos en 2009, y el 1 de agosto de 2010, 18.449 muertes fueron reportadas por la OMS 3. Esta pandemia ha demostrado una vez más la elevada carga de enfermedades infecciosas, y la necesidad de una detección rápida y exacta de la gripe a activar la confirmación de la enfermedad rápidamente, la respuesta de salud pública apropiada y un tratamiento efectivo 4. Hay varios métodos utilizados para el diagnóstico de la gripe, que incluyen inmunoensayos rápidos, pruebas de antígeno fluorescente directa (DFA) y los métodos de cultivo viral. Inmunoensayos rápidos dramáticamente carecen de sensibilidad 5-8, mientras que los otros dos métodos son de trabajo intensivo y requiere mucho tiempo 9. Las pruebas moleculares ofrecen múltiples ventajas, incluyendo un vuelco corto tiempo, Sensit altoividad, y una mayor especificidad. Varias entidades comerciales han estado trabajando para pruebas moleculares rápidas (también llamadas pruebas de ácido nucleico o NAT) para las enfermedades infecciosas, y varios tienen pruebas de influenza en sus tuberías. Sin embargo la mayoría de ellos requieren fuera de chip preparación de la muestra. Ninguna de las Enmiendas de Mejoras de Laboratorio Clínico (CLIA) renunciaron pruebas moleculares incorporar preparación de la muestra en el cartucho de ensayo o módulo. Lab-on-a-chip desempeña un papel importante en el desarrollo de dispositivos de prueba en el punto de atención. Después de la introducción de la primera chip de PCR en 1993 10, numerosos esfuerzos se han puesto en el desarrollo de chips de prueba de ácidos nucleicos. Sin embargo, sólo unos pocos de estos tienen preparación integrada muestra en bruto con la amplificación aguas abajo. Hemos demostrado anteriormente la miniaturización de una columna de extracción en fase sólida (SPE) en un chip microfluídico de plástico 11 y desarrollar lación y optimización de un flujo continuo de chip PCR 12. Este sentido, ampliar el trabajo previo para integrar la SPE con RT y PCR medidas en un solo chip para el diagnóstico clínico y demostrar su capacidad para amplificar los ácidos nucleicos de los pacientes nasofaríngeo (NP) hisopados y aspirados.

<table border="1"><tbody><tr><td> Nombre del reactivo / material </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> 1-dodecanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 443816-500G </td></tr><tr><td> 2,2-dimetoxi-2-fenilacetofenona </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 196118-50G </td></tr><tr><td> 2100 Bioanalyzer </td><td> Agilent Technologies, Santa Clara, CA </td><td> G2943CA </td></tr><tr><td> 2-propanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 19516 </td></tr><tr><td> Benzofenona </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 239852-50G </td></tr><tr><td> BSA </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> A7979-50ML </td></tr><tr><td> Butil metacrilato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 235865-100 ml </td></tr><tr><td> Carrier RNA </td&gt; <td> Qiagen, Valencia, CA </td><td> 1017647 </td></tr><tr><td> Ciclohexanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 105899-1L </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> E7023 </td></tr><tr><td> Dimetacrilato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335861 </td></tr><tr><td> Dimetacrilato de etilenglicol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335681-100ML </td></tr><tr><td> Jeringa de vidrio 250 l </td><td> Hamilton, Reno, NV </td><td> 81127 </td></tr><tr><td> Tiocianato de guanidina </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 50981 </td></tr><tr><td> Kit de ADN de alta sensibilidad </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Prensa caliente </td><td> Carver, Wabash, IN </td><td> 4386 </td></tr><tr><td> JB Weld Epoxies </td><td> Mcmaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Jeringas Luer-Lok </td><td> BD-Medical, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Cloruro de Magnesio </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> AB-0359 </td></tr><tr><td> Metanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 494437 </td></tr><tr><td> Metil metacrilato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> M55909 </td></tr><tr><td> NanoPort </td><td> Upchurch Scientific </td><td> N-333 a 01 </td></tr><tr><td> NanoPort montaje </td><td> Upchurch Scientific </td><td> F-120x </td></tr><tr><td> Nucleasa libre de agua </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> PR-P1193 </td></tr><tr><td> OneStep RT-PCR kit </td><td> Qiagen, Valencia, CA </td><td> 210210 </td></tr><tr><td> PEG8000 </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 41009 </td></tr&gt; <tr><td> Fuente de alimentación </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> Lejos RNAsa </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 83931-250ML </td></tr><tr><td> RNASecure </td><td> Applied Biosystems, Foster City, CA </td><td> AM7005 </td></tr><tr><td> Las microesferas de sílice </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Bomba de jeringa </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Cinta térmicamente conductora </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Par termoeléctrico </td><td> Omega Engineering, Stamford, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> Calentadores de película fina </td><td> Minco, Minneapolis, MN </td><td> HK5166R529L12A </td></tr><tr><td> Crosslinker Ultravioleta </td><td> UPV, Upland, CA </td><td> CL-1000 </td></tr><tr><td> Zeonex </td> &lt;td&gt; Zeon Chemicals, Louisville, KY </td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

Diagnósticos rápidos y eficaces desempeñan un papel importante en el control de enfermedades infecciosas al permitir la gestión eficaz del paciente y el tratamiento. Aquí, se presenta un sistema integrado de chip microfluídico termoplástico con la capacidad de amplificar el virus en pacientes nasofaríngeas (NP) frotis y aspirados. Después de cargar la muestra del paciente, el dispositivo de microfluidos secuencialmente lleva a cabo en el chip lisis celular, la purificación del ARN y otra de concentración dentro de la extracción en fase sólida (SPE), transcripción inversa (RT) y reacción en cadena de la polimerasa (PCR) en RT-PCR cámaras, respectivamente. Punto final de la detección se realizó usando un Bioanalyzer fuera de chip (Agilent Technologies, Santa Clara, CA). Por periféricos, se utilizó una bomba de jeringa de un solo reactivo para conducir y muestras, mientras que dos calentadores de película fina se utilizaron como fuentes de calor para la RT y PCR cámaras. El chip está diseñado para ser de una sola capa y adecuado para la fabricación de alto rendimiento para reducir la fabricatien tiempo y costo. El chip microfluídico proporciona una plataforma para analizar una amplia variedad de virus y bacterias, limitado sólo por cambios en el diseño de reactivos necesarios para detectar nuevos patógenos de interés.

<html> Un resultado típico se muestra en la Figura 3 para una gripe A muestra infectada lavado nasofaríngeo. Debido a las diferentes cantidades de virus de la gripe en cada muestra de paciente, la concentración final del producto de PCR puede variar. Un buen resultado debe tener bajo nivel de ruido, dos picos claros de escalera (35 y 10380 pb) y un pico de producto único en el tamaño del producto diseñado (107 pb) para la muestra positiva. Mientras que el pico del producto teóricamente debería es...

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Microfluidic Chip Fabrication and Method to Detect Influenza

Un sistema integrado de chip microfluídico termoplástico ha sido desarrollado para su uso como un diagnóstico molecular. El chip realiza la extracción de ácido nucleico, la transcriptasa inversa y la PCR. Los métodos para fabricar y ejecutar el chip se describen.

Fabricación de chips de microfluidos y método para detectar la influenza

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

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14.8K vistas.  Boston University. Un sistema integrado de chip microfluídico termoplástico ha sido desarrollado para su uso como un diagnóstico molecular. El chip realiza la extracción de ácido nucleico, la transcriptasa inversa y la PCR. Los métodos para fabricar y ejecutar el chip se describen.

Video: Microfluidic Chip Fabrication and Method to Detect Influenza

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detección de fluorescencia para las plataformas de las gotas de microfluidos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Investigación

Bioingeniería

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Un chip de microfluidos para el análisis químico versátil de células individuales

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricación Microfluídica De Fibras Poliméricas Y Biohíbridas Con Tamaño y Forma Prediseñados

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip de microfluidos para ICPMS Introducción Muestra

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Guiada por imagen, fabricación basada en láser de Redes de microfluidos derivados-vascular

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

La fabricación de dispositivos de microfluidos basados ​​en papel tridimensional para inmunoensayos

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Seca de la película basada en la fotoresistencia el Biosensor electroquímico de microfluidos plataforma: Fabricación de dispositivo, preparación del análisis de la en-viruta y el funcionamiento del sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabricación y validación de un sistema de órganos-en-viruta con electrodos integrados directamente cuantificar Transendothelial resistencia eléctrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabricación escalable de elástico, doble canal, Chips microfluídicos órgano

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...