Esta pesquisa foi suportada pelo National Institutes of Health (NIH) concessão R01 EB008268.

1. Fabricação de chips 12 <ol><li> Fazer duas placas de aglomerados Zeonex 690R: distribuir 8-9 gramas pelotas Zeonex uniformemente no centro de uma placa de metal, o pré-aquecimento na prensa aquecida a 198 ° C durante 5 min e, em seguida, aplicar a pressão lentamente até 2500 psi, durante mais 5 min. Para concluir esta etapa, foi utilizado um Carver imprensa quente. </li><li> Grava o canal microfluídico na placa com um molde de epóxi. Os detalhes sobre a fabricação do molde são descritos em outra parte 12 (canal de desenho na Figura 2b): colocar uma placa sobre o molde epóxi; preaquecimento los na prensa aquecida a 157 ° C durante 10 min e, em seguida, aplicar a pressão lentamente até 1000 psi por mais 10 min . Remova a placa do molde à mão ou usando uma pinça, antes que esfrie. (Nota: usar luvas térmicas durante esta etapa.) </li><li> Perfura-se o chip em relevo, na entrada, a porta de resíduos, e a saída do canal microfluídico. Usamos um diâmetro de 1,25 mmeter broca para fazer os três orifícios. </li><li> Ligação do chip em relevo com uma outra placa (em cima): Lavar as fichas com IPA, RNAse Fora água desionizada e em sequência; ar seco e pré-aquecer-los na prensa aquecida a 131 ° C durante 10 min e em seguida pressionar a 350 psi para uma outra 10 min. </li><li> Anexar Nanoports na entrada, de resíduos, e as portas de saída separadamente usando JB Weld Epoxy; curar durante 15 a 24 horas de acordo com as instruções do fabricante. </li><li> Lavar o canal SPE com 50 ul RNAse Away, seguiu com 100 ul de água nuclease livre. </li><li> Carregar o canal de SPE com 4 ul de solução de enxerto (metacrilato de metilo, de 3% w / v de benzofenona) e, em seguida, a ligação cruzada de metacrilato, por incubação durante 10 minutos num forno de UV de comprimento de onda 365 nm sob UV e 2.000 mJ / cm 2. Remover a solução residual com enxerto de vácuo. Utilizou-se o vácuo de parede. Para obter os melhores resultados, uma solução fresca deve ser usado. É geralmente aceitável, no entanto, para armazenar umsolução preparada a 4 ° C durante até uma semana. </li><li> Tornar a solução coluna SPE (todos% v / v): dimetacrilato de etileno de 16%, 24% de metacrilato de butilo, 42% de 1-dodecanol, ciclohexanol 18%, e adicionar 1% de 2-dimetilamino-4-(metil-f enilamino)-fenol como o foto-iniciador. </li><li> Retire o mesmo volume de solução de sílica microesferas como sendo a solução da coluna SPE (para fazer uma mistura a 1:1), e secá-la completamente, a 70 ° C durante a noite num forno de vácuo. Quebra-se o pellet tocando o tubo com a tampa fechada. Adicionar a solução da coluna SPE dentro do tubo de sílica seco de microsferas e vortex para misturar. </li><li> Carregar 4 ul de solução de SPE no mesmo canal e, em seguida, formar ligações cruzadas por radiação UV é de 2,5 min. Em seguida, vire o chip de uma e outra irradiar para 2,5 min. A coluna de SPE polimerizada deve ser um sólido opaco branco. </li><li> Lavar o canal com 500 ul de metanol a 100% para enxaguar o excesso de reagentes. Esta lavagem remove também intra-polímero posolventes rogenic, criando desse modo a estrutura de poros abertos da coluna SPE, após secagem em condições ambientes. </li><li> Anexar dois aquecedores de película fina para o fundo do chip com a fita condutora térmica. Os lados dos aquecedores têm de estar alinhadas com a borda dos canais largos para desnaturação e recozimento separadamente (ver Figura 1). </li><li> Coloque cinco termopares no chip através da morte terminou canais laterais abertas de cada chip (ver Figura 2b). Os termopares ficar nestes canais via ajuste de imprensa. Fita foi utilizada para continuar a fixar a localização dos termopares. </li></ol> 2. Método de Extracção em Fase Sólida <ol><li> Preparar 96 ul de etanol a 70%, com água tratada com DEPC e autoclavadas (desionizada ou Millipore-filtrada). </li><li> Adicionar 4 de ul de RNA 25X Fixe a 96 ul de etanol a 70% em um tubo, e duplicar a mesma mistura com 100% de etanol, num tubo separado. </li><li> Prepare 300ul de tampão de canal através da mistura do seguinte: 75 jul de tiocianato de guanidina 6 M (GuSCN), 150 ul de 2-propanol, 63 ul de água livre de nuclease, e 12 ul de RNA 25X seguros. </li><li> Preparar 312 ul de tampão de lise por mistura de: 100 uL de 6 M GuSCN, 200 ul de 2-propanol, e 12 ul de RNA 25X seguros. </li><li> Aquece-se a etanol a 70%, etanol a 100% de tampão de canal, e tampões de lise a 60 ° C durante 10-20 min para ativar o Secure RNA. </li><li> Para equilibrar o canal microfluídico, execute o buffer de canal através do canal de SPE com um caudal de 0,8 ml / hr. </li><li> Quick-descongelar a amostra de teste em VTM a 37 ° C em banho de água, e remover a amostra do banho de água imediatamente, logo que ele é descongelado. </li><li> Centrifugar amostra a 13.000 rpm durante 10 min, e transferir 100 ul do sobrenadante para 300 ul de tampão de lise. Completa a mistura por adição de 6 ul de 1 ug / ul de ARN transportador. </li><li> Vortex para misturar, e após centrifugação para5 seg, carregar o lisado inteiro para uma seringa Luer-lok cc 1. </li><li> Executar lisado através do canal de SPE com um caudal de 0,8 ml / hr. </li><li> Lava-se a canal de SPE com 100 ul de etanol a 70%, seguido de 100 ul de etanol of100% a 1 ml / hr. (Há um volume de 50 ul morto na ponta da seringa que não será empurrada para fora, então na verdade apenas 50 ul está a atravessar o canal.) </li><li> Posicionar uma seringa vazia na marca de 0,5 ml e empurrar o ar através do canal para secá-la, a 1 ml / hr. </li><li> Executar 67,5 ul de água livre de nuclease através do canal para eluir os ácidos nucleicos ligados a 0,5 ml / h, e recolher 13,5 uL da nanoport no porto de resíduos. (A perda de líquidos é devido ao volume de 54 ul morto na ponta da seringa e o canal de SPE.) </li></ol> 3. RT-PCR <ol><li> Prepare 36,5 uL da mistura principal de RT-PCR durante o passo de ar seco (2,12), utilizando os reagentes em um OneStep RT-PCR Qiagen kits. </li><li> Carregar RT-PCR reagente no nanoport no porto de resíduos, e mistura-se com o RNA para obter a seguinte reacção 50 uL de RT-PCR: 13,5 ul de RNA eluído em água livre de nuclease, 4 ul de mistura de RT-PCR de enzima, solução de 10 ul de Q, 10 μl1X um tampão passo, 2 mM de MgCl 2 25 uL, 1 ul de 50 mM de primer forward 5&#39;-GAC CRA TGT TCC CAC CTC TGA C-3 &#39;, 1 ul de 50 mM de iniciadores inverso 5&#39;-TGG AGG GCA TTY ACA AAK CGT CTA-3 &#39;, 2 ul de dNTPs, 0,75 ul de 1,0% w / v de BSA, 0,73 e 5,02 ul de água PEG8000 nuclease ul livre. </li><li> Coloque a mistura de RT-PCR para o canal de RT por vácuo suave (utilizou-se o vácuo de parede) e selar o Nanoport com um encaixe fechado. </li><li> Aplicar 35 V para o aquecedor 1. Manter os reagentes no canal de RT, durante 30 min, após o aquecedor 1 equilibra a 50 ° C. Manter os reagentes no canal de RT, durante 15 min, após o aquecedor uma equilibra a 95 ° C. </li><li> Aplicar 27 V para o aquecedor 1 e 50 V para o aquecedor 2, esperar cerca de três minutos ou até que um aquecedor equilibra a 60 e deg, C e aquecimento 2 equilibra a 95 ° C. </li><li> Empurrar os reagentes para dentro do canal de PCR com 0,5 ul / min. Deve ter cerca de 20 min para os reagentes fluir através do canal de serpentina. </li><li> Como a amostra torna-o no final do canal de serpentina, recolher os produtos de PCR na saída utilizando um pipetador de tamanho adequado e de ponta. </li></ol> 4. Detecção de Produtos PCR <ol><li> Usamos um Agilent teste de DNA de alta sensibilidade para detectar os produtos. Para executar este teste tirar a Agilent Kit DNA Alta Sensibilidade da geladeira 30 minutos antes do teste. </li><li> Ligue o Bioanalyzer e abra o software especialista 2100. </li><li> Carregar o 1 uL dos produtos de PCR para o ensaio e seguir o protocolo Agilent ( <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent 20Manual.pdf%</a> ). </li><li> Analisar e registrar os dados. </li></ol>

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Os autores declararam não interesses concorrentes financeiros.

O método de diagnóstico aqui apresentados demonstram a capacidade de um chip microfluídico integrado plástico para amplificar influenza A RNA a partir de amostras de pacientes com elevada especificidade e um limite de detecção de baixo 13 Concebemos este chip para o ponto de teste de potencial cuidado:. (A) a temperatura do fluido e controle foram simplificados, (b) o chip é de baixo custo e conveniente para a fabricação de alto rendimento usando moldagem por injecção, e (c) o chip é descartável ...

Milhões de mortes foram relatadas durante os três pandemias de gripe do século 20 1. Além disso, a pandemia de gripe mais recente foi declarada pela Organização Mundial da Saúde (OMS) 2, em 2009, e em 1 de agosto de 2010, 18.449 mortes foram registradas pela OMS 3. Esta pandemia demonstrou mais uma vez a alta carga de doenças infecciosas, e da necessidade de rápida e precisa detecção de gripe para ativar a confirmação da doença rápida, resposta adequada de saúde pública e tratamento eficaz 4. Existem vários métodos utilizados para o diagnóstico da gripe, que incluem imunoensaios, ensaios rápidos antigénio directa fluorescente (DFA) e os métodos de cultura viral. Imunoensaios rápidos dramaticamente falta de sensibilidade 5-8, enquanto que os outros dois métodos são muito trabalhosa e demorada 9. Os testes moleculares oferecem múltiplas vantagens, incluindo um curto tempo de rotação SensIT, de altaivity, e maior especificidade. Várias entidades comerciais têm vindo a trabalhar no sentido rápidos testes moleculares (também chamados de testes de ácidos nucleicos ou NAT) para doenças infecciosas, e vários ensaios têm influenza em suas condutas. No entanto a maior parte deles necessitam off-chip de preparação da amostra. Nenhuma das alterações Clinical Laboratory Improvement (CLIA) renunciou testes moleculares incorporar a preparação da amostra para o cartucho de ensaio ou módulo. Lab-on-a-chip desempenha um papel importante no desenvolvimento de dispositivos de point-of-care de teste. Após a introdução do chip primeira PCR, em 1993, 10, numerosos esforços têm sido feito para desenvolver chips de teste de ácido nucleico. No entanto, apenas alguns deles têm a preparação da amostra integrado bruto com amplificação a jusante. Temos anteriormente demonstrada a miniaturização de uma coluna de extracção em fase sólida (SPE) em um chip microfluídico plástico 11 ea desenvolvermento e otimização de um contínuo fluxo de chip de PCR 12. Aqui, nós estendemos o trabalho anterior para integrar a SPE com RT e as etapas de PCR em um único chip para diagnósticos clínicos e demonstrar a sua capacidade para amplificar ácidos nucléicos de paciente nasofaringe (NP), esfregaços e aspirados.

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </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> Metacrilato de butilo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 235865-100 ml </td></tr><tr><td> RNA Transportador </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 de etileno </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335861 </td></tr><tr><td> Dimetacrilato de etilenoglicol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335.681-100ML </td></tr><tr><td> Seringa de vidro de 250 jil </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 DNA Alta Sensibilidade </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Prensagem a quente </td><td> Carver, Wabash, EM </td><td> 4386 </td></tr><tr><td> JB Weld Epóxis </td><td> McMaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Luer-Lok seringas </td><td> BD-Medical, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Cloreto de Magnésio </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> Metacrilato de metilo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch Científico </td><td> N-333-01 </td></tr><tr><td> Nanoport Fitting </td><td> Upchurch Científico </td><td> F-120x </td></tr><tr><td> Água livre de nuclease </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> Fonte de alimentação </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> RNAse Fora </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> Sílica microesferas </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Bomba de seringa </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Tape termicamente condutiva </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Par termoelétrico </td><td> Omega Engenharia, Stamford, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> Película fina Aquecedores </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

Diagnóstico rápidos e eficazes têm um papel importante no controle de doenças infecciosas, permitindo que a gestão do paciente e tratamento eficazes. Aqui, apresentamos um chip microfluídico integrado termoplástico com a capacidade de amplificar vírus influenza A em paciente nasofaringe (NP), esfregaços e aspirados. Durante o carregamento da amostra do paciente, o dispositivo micro sequencialmente realiza on-chip a lise das células, purificação e concentração de RNA dentro da extracção em fase sólida (SPE), a transcrição reversa (RT) e de reacção em cadeia da polimerase (PCR), em câmaras de RT-PCR, respectivamente. Detecção do ponto final é realizada usando um Bioanalyzer off-chip (Agilent Technologies, Santa Clara, CA). Para os periféricos, utilizou-se uma bomba de seringa para dirigir reagentes e amostras, ao passo que dois aquecedores de película fina foram utilizadas como fontes de calor para a temperatura ambiente e as câmaras de PCR. O chip é desenhado para ser de camada única e adequado para o fabrico de elevado rendimento para reduzir o Fabricatiem tempo e custo. O chip microfluídico proporciona uma plataforma para analisar uma grande variedade de vírus e bactérias, limitado apenas pelas mudanças na concepção reagente necessários para detectar novos agentes patogénicos de interesse.

<html> Um resultado típico é mostrado na Figura 3, para uma amostra de influenza A lavagem nasofaríngeo infectado. Devido às diferentes quantidades de vírus influenza em cada amostra de paciente, a concentração final de produto de PCR pode variar. Um bom resultado deve ter baixo ruído, dois picos escada claras (35 e 10380 pb) e um pico único produto com o tamanho do produto concebido (107 pb) para a amostra positiva. Enquanto que o pico do produto deveria, teoricamente, estar ausente dos control...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

Um chip microfluídico integrado termoplástico tem sido desenvolvido para utilização como um diagnóstico molecular. O chip realiza a extracção dos ácidos nucleicos, a transcriptase reversa, e PCR. Os métodos para fabricar e executando o chip encontram-se descritos.

Fabricação de chips microfluídicos e método para detectar a gripe

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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14.9K visualizações.  Boston University. Um chip microfluídico integrado termoplástico tem sido desenvolvido para utilização como um diagnóstico molecular. O chip realiza a extracção dos ácidos nucleicos, a transcriptase reversa, e PCR. Os métodos para fabricar e executando o chip encontram-se descritos.

Vídeo: 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 detecção de fluorescência para as plataformas de gotículas microfluídicos

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

Bioengenharia

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;

Um chip microfluídico para a Versátil Análise Química de células individuais

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.

Fabricação microfluida de fibras poliméricas e biohibridas com tamanho e forma predesigned

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.

Um chip microfluídico para ICPMS Amostra Introdução

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.

guiado por imagem, fabricação à base de laser de Redes microfluídicos Vascular derivadas

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.

Fabricação de dispositivos microfluídicos tridimensional baseados em papel para imunoensaios

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.

Película seca baseada em fotorresiste eletroquímica Microfluidic Biosensor plataforma: Fabricação de dispositivo, ensaio em-microplaqueta preparação e operação de 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.

Fabricação e validação de um sistema de órgãos-em-microplaqueta com eléctrodos integrados para quantificar diretamente a resistência elétrica de Transendothelial

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.

Fabricação escalável de Stretchable, Dual Channel, órgão Microfluidic Chips

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