Este trabalho foi financiado pelo Ministério de Ciência e Tecnologia de Taiwan sob Grant Número 101-2221-E-002-064-MY3.

1. A fabricação de dispositivos microfluídicos <ol><li> Use um software de layout gráfico (por exemplo, AutoCAD) para desenhar o contorno de um T-microcanais. Para o t-microcanais, os dois canais de alimentação são de 90 m de largura e 2500 uM de comprimento, e o canal de confluência 180 é um de largura e 3000 uM de comprimento. Ligar a extremidade de cada canal para um indivíduo círculo com um diâmetro de 1100 um. </li><li> Mark &quot;claro&quot; e &quot;escuro&quot; para a exposição e áreas cobertas, respectivamente. Para um fotorresistente negativo (por exemplo, SU-8), a forma do T-microcanal é &quot;claro&quot; e torno é &#39;escuro.&#39; </li><li> Usar um gerador de padrão de laser com um comprimento de onda de 442 nm e um tamanho mínimo de funcionalidade de 2 uM para transferir o padrão de microcanais o t-fotomáscara para um cromo-em-vidro. </li><li> Use a photomask, um substrato (por exemplo, de um lado só pastilha de silício polido) e um photoresist epóxi permanente (por exemplo,., SU-8) para fazer um molde através de um processo de litografia padrão. A camada foto-resistente é de 55,2 mm de espessura. Em geral, a espessura fotorresistência deve ser mais fino do que a profundidade da correlação da lente objectiva 17-19. </li><li> Use o molde e de um material transparente, tal como polidimetilsiloxano (PDMS), para fabricar o t-microcanal 20. </li><li> Para a ligação fluídica, utilizar um tubo de aço inox de 2 mm de diâmetro exterior para perfurar furos de passagem alinhados com os padrões circulares nas PDMS. </li><li> Tratar as superfícies dos PDMS e uma lâmina de vidro com plasma de oxigénio a 60 W durante 30 segundos. Prenda as PDMS para a lâmina de vidro. As superfícies oxidadas dos dois materiais de criar uma ligação forte. Coloque a estrutura PDMS colada sobre uma placa quente durante 5 minutos a 120 ° C. </li><li> Insira tubos de teflon para os buracos perfurados para conexão fluídica. </li></ol> 2. Setup Experimental <ol><li> Construir o sistema schlieren microescala de um Hoffman modulação microscópio de contraste através da remoção da placa de ranhura no plano focal da frente do condensador e substituindo o modulador com uma faca de ponta no plano focal posterior da objectiva 5X 3. A profundidade de correlação, o que depende da abertura numérica da objectiva 17-19, deve ser suficiente para cobrir toda a profundidade do dispositivo de microfluidos. A superfície do fio da navalha é enegrecida por anódica de óxido de alumínio para reduzir a sua reflectividade. </li><li> Monte a câmera de alta velocidade para o tubo trinocular do microscópio através de um adaptador C-mount. Já a câmara enfrentar o caminho óptico do microscópio através de um divisor de feixe. Ligue a câmara a um computador desktop através de um cabo Ethernet. Defina a correção de gama a 1 para a câmera de modo que a sua leitura em tons de cinza é proporcional à luminância de entrada. </li><li> Ligar a fonte de luz. A fim de evitar o excesso de calor, use LED (díodo emissor de luz) de iluminação. </li><li> Use um software de processamento de imagem (por exemplo, &lt;/ Em&gt; função imread em MATLAB) para obter os valores de tons de cinza de uma imagem adquirida. Remover o gume de faca, ajustar a iluminação, a abertura e o tempo de exposição de modo que a leitura média escala de cinzentos da imagem é aproximadamente 10% menor do que o valor máximo. Isto denota a intensidade do fundo para um corte 0% e nós usamos um valor de 230 para uma imagem de 8-bit. </li><li> Insira o fio da navalha para bloquear completamente a luz incidente. Grave a leitura média em tons de cinza da imagem. Isto indica a intensidade do fundo para um corte de 100% e o valor é de cerca de 15 para uma imagem de 8-bit. </li><li> Ajustar a posição da faca de ponta de modo a que a leitura média de escala de cinzentos da imagem obtida encontra-se no meio dos valores de 0% e 100% de corte. Agora o grau de corte é ajustada para 50%. </li><li> Prepare dois fluidos transparentes com índices de refração 21 conhecidas que são completamente miscíveis uns com os outros como os constituintes. Para avaliar a dependência da ind refraçãoex sobre a concentração da mistura, verifique a literatura 21 ou usar a equação Gladstone-Dale 22. Se a curva é linear ao longo de toda a gama, escolher outros componentes do fluido. Em seguida, escolhe uma composição designada abaixo do qual o índice de refracção da solução varia linearmente com a concentração. Por exemplo, usar diluir etanol aquoso com uma fracção de massa de 0,05 e água como os fluidos de trabalho. </li><li> Colocar a t-microcanais na fase espécime. Dispor o t-microcanal assim com o canal confluente paralelo ao gume de faca (Figura 1). </li><li> Prepare duas seringas idênticas: Uma seringa é enchida com o fluido de trabalho que serve como fluido de referência (água), e a seringa B é preenchido com o outro fluido de trabalho (diluir o etanol aquoso). O tamanho da seringa Q depende da taxa de fluxo desejada e as especificações da bomba de seringa: Q = πd 2 V / 4, onde d é o diâmetro interno do syrInge e V é a velocidade do êmbolo. Fluxo de pulsação geralmente pode ser impedido por escolha de uma pequena seringa para aumentar V 23. </li><li> Recolhe-se o fluido a partir da saída de t-microcanais num copo. Assegurar o tubo de saída de Teflon é fixada à parede do recipiente e a sua extremidade está abaixo do nível do líquido no copo de modo a evitar a vibração que seria causada pela gota breakoff. </li></ol> 3. Calibração <ol><li> Adquirir as imagens dos dois fluidos de mistura e as imagens de referência. <ol><li> Em um dado número de Reynolds Re, ajustar a taxa de fluxo das bombas de seringa, Q. Q é calculado a partir de Q = μ (W + D) Re / 4ρ, onde μ e ρ são a viscosidade e densidade do fluido de trabalho, e W e D são a largura e profundidade do canal de afluência do t-microcanal, respectivamente. </li><li> Carregar uma bomba de seringa com uma bomba e a outra com syringe B. Ligue as duas entradas do T-microcanais a seringa A e B via seringa Teflon tubulação. Iniciar as bombas de seringa para fornecer os fluidos de trabalho para o t-microcanal a taxas de fluxo de volume idênticas. </li><li> Aguarde até que o fluxo constante estabelece. A condição de fluxo constante é definida pelo aparecimento de um padrão estacionário schlieren. </li><li> Use o software da câmera controlada para gravar vinte quadros de mistura de fluidos a uma taxa de quadros de 30 fps. </li><li> Parar a bomba que é carregado com seringa B. Apenas bombear o fluido de referência (água) através de uma entrada para o canal de afluência do t-microcanal a uma taxa constante. </li><li> Espere até que a condição de fluxo constante é atingido e nenhum padrão schlieren é observado. </li><li> Use o software da câmera controlada para tomar a imagem de referência, quando nenhuma falta de homogeneidade óptica está presente na T-microcanais. Grave vinte quadros a uma taxa de quadros de 30 fps. </li><li> Repita 3.1.1 a 3.1.7 em diferentes número de Reynolds: Re = 1, 5, 10, 20 e 50 de modo a que nenhuma estrutura fluxo complexo emerge na região de confluência do T-24 de microcanais. </li></ol></li><li> Usar um software de processamento de imagem para dividir a imagem adquirida I (i, j) na imagem de referência I 0 (i, j) 25, em que i e j são os índices de pixel por. </li><li> Empregar um pacote de CFD (Computational Fluid Dynamics) para simular a mistura dos fluidos designados na T-microcanais. <ol><li> Construir o modelo tridimensional para a geometria do t-microcanal. Discretizar o domínio do fluxo em redes estruturadas. Para aumentar a precisão, empregar malha mais fina na confluência ea região central da T-microcanais. </li><li> Atribuir as propriedades físicas dos fluidos e estabelecer as condições de contorno para o domínio do fluxo. Durante o processo de resolução de problemas, determinar o coeficiente de difusão dependente da concentração, a partir da concentração obtida na últimaiteração 26, a fim de atualizar a concentração local. </li><li> Examine as sensibilidades dos resultados computados pela realização do estudo grade 27. </li></ol></li><li> Para cada nó (x i, y i) na -plane xy, empregar a ferramenta de pós-processamento de CFD para tomar os valores médios do campo de concentração através da profundidade do canal pela regra trapezoidal: W (x i, y j) = {Σ K [W (x i, j y, z k) + w (x i, j y, z k 1)] ∙ (z k 1 - z k) / 2} / D 28, em que D é a profundidade do canal. Use o esquema diferencial central para calcular a derivativa de concentração em relação ao sentido transversal de fluxo: (∂ w / ∂ y) i, j = [W (x i, y j 1) - w (x i, y j -1)] / (y j 1 - y j -1). </li><li> Para ambos os gradientes positivos e negativos, extrair a razão dos valores de escala de cinzentos I / I 0 (obtido em 3.2) e o gradiente de fracção de massa ∂ w / y ∂ (obtido em 3.4) em locais específicos, tais como y = 0 (linha de centro, streamwise direção) ou várias dado x (direção cross-stream). </li><li> Traçar os resultados e determinar a relação entre a I / I 0 e ∂ w / ∂ y, I / I0 </sub&gt; = C 1 ∂ w / ∂ y + C 2 (C 1 e C 2 são constantes), com regressão linear. </li></ol> 4. Quantificação <ol><li> Repita os passos 3.1 a 3.2 para a mistura no dispositivo de microfluidos alvo. A profundidade do dispositivo de microfluidos de destino deve ser idêntico ou próximo daquele do t-microcanal. Se fenômeno instável é esperado, adquirir um clipe de vídeo (sequência de imagens) na etapa 3.1.4 vez. A taxa de quadros deve ser suficientemente alto para resolver a dinâmica do fluxo transiente claramente, enquanto que o tempo de exposição deve ser idêntico ao valor utilizado em 2.4, 2.5, 3.1.4 e 3.1.7. </li><li> Utilize a relação obtida no passo 3.6 para converter a razão dos valores de escala de cinzentos para a fracção de gradiente de massa no dispositivo de microfluidos alvo. </li></ol>

<ol>
	<li>Merzkirch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Flow Visualization. , (1974).</li><li>Settles, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Schlieren and Shadowgraph Techniques. , (2001).</li><li>Sun, C. -. l., Hsiao, T. -. h. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+microfluidic+mixing+using+microscale+schlieren+technique.">Quantitative analysis of microfluidic mixing using microscale schlieren technique.</a> Microfluidics and Nanofluidics. 15 (2), 253-265 (2013).</li><li>Sun, C. -. l., Sun, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+mixing+in+a+microfluidic+oscillator+using+an+impinging+jet+on+a+concave+surface.">Effective mixing in a microfluidic oscillator using an impinging jet on a concave surface.</a> Microsystem Technologies. , (2011).</li><li>Strook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaotic+mixer+for+microchannels.">Chaotic mixer for microchannels.</a> Science. 295 (5555), 647-651 (2002).</li><li>Wheat, P. M., Posner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+mixing+using+equilibrium+reactions.">Quantifying mixing using equilibrium reactions.</a> Physics of Fluids. 21 (3), 037101 (2009).</li><li>Liu, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+mixing+in+a+three-dimensional+serpentine+microchannel.">Passive mixing in a three-dimensional serpentine microchannel.</a> Journal of Microelectromechanical Systems. 9 (2), 190-197 (2000).</li><li>Munson, M. S., Yager, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+quantitative+optical+method+for+monitoring+the+extent+of+mixing+applied+to+a+novel+microfluidic+mixer.">Simple quantitative optical method for monitoring the extent of mixing applied to a novel microfluidic mixer.</a> Analytica Chimica Acta. 507 (1), 63-71 (2004).</li><li>Bradfield, W. S., Sheppard, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microschlieren-a+technique+for+the+study+of+details+in+compressible+flow.">Microschlieren-a technique for the study of details in compressible flow.</a> Aero/Space Engineering. 5 (18), 37-56 (1959).</li><li>Scroggs, S. D., Settles, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+study+of+supersonic+microjets.">An experimental study of supersonic microjets.</a> Experiments in Fluids. 21, 401-409 (1996).</li><li>Phalnikar, K. A., Alvi, F. S., Shih, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 31st AIAA Fluid Dynamics Conference &amp; Exhibit. , (2001).</li><li>Phalnikar, K. A., Kumar, R., Alvi, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+free+and+impinging+supersonic+microjets.">Experiments on free and impinging supersonic microjets.</a> Experiments in Fluids. 44 (5), 819-830 (2008).</li><li>Naughton, J. W., Bishop, D. S., Walrath, D. E., Lindberg, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-schlieren+characterization+of+a+high+momentum+micro-fluidic+actuator.">Micro-schlieren characterization of a high momentum micro-fluidic actuator.</a> , (2002).</li><li>Chen, H. -. M., Wu, H. -. W., Lee, J. -. N., Ting, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 23th National Conference of the Chinese Society of Mechanical Engineers. , (2006).</li><li>Davidson, G. P., Emmony, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schlieren+probe+method+for+the+measurement+of+the+refractive+index+profile+of+a+shock+wave+in+a+fluid.">A schlieren probe method for the measurement of the refractive index profile of a shock wave in a fluid.</a> Journal of Physics E: Scientific Instruments. 13, 92-97 (1980).</li><li>Xie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schlieren+confocal+microscopy+for+phase-relief+imaging.">Schlieren confocal microscopy for phase-relief imaging.</a> Optics Letters. 39, 1238-1241 (2014).</li><li>Inou&#233;, S., Spring, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Video Microscopy: The Fundamentals. , (1997).</li><li>Meinhart, C. D., Wereley, S. T., Gray, M. H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+illumination+for+two-dimensional+particle+image+velocimetry.">Volume illumination for two-dimensional particle image velocimetry.</a> Measurement Science and Technology. 11 (6), 809-814 (2000).</li><li>Olsen, M. G., Adrian, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out-of-focus+effects+on+particle+image+visibility+and+correlation+in+microscopic+particle+image+velocimetry.">Out-of-focus effects on particle image visibility and correlation in microscopic particle image velocimetry.</a> Experiments in Fluids. 29 (1), S166-S174 (2000).</li><li>Friend, J., Yeo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microfluidic+devices+using+polydimethylsiloxane.">Fabrication of microfluidic devices using polydimethylsiloxane.</a> Biomicrofluidics. 4 (2), 026502 (2010).</li><li>Wohlfarth, C., Lechner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landolt-B&#246;rnstein:+Numerical+Data+and+Functional+Relationships+in+Science+and+Technology.">Landolt-B&#246;rnstein: Numerical Data and Functional Relationships in Science and Technology.</a> Optical Constants: Refractive Indices of Pure Liquids and Binary Liquid Mixtures (Supplement to III/38). 47, (2008).</li><li>Heller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remarks+on+refractive+index+mixture+roles.">Remarks on refractive index mixture roles.</a> Journal of Physical Chemistry. 69 (4), 1123-1129 (1965).</li><li>Korczyk, P. M., Cybulski, O., Makulska, S., Garstecki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unsteadiness+of+the+rates+of+flow+on+the+dynamics+of+formation+of+droplets+in+microfluidic+systems.">Effects of unsteadiness of the rates of flow on the dynamics of formation of droplets in microfluidic systems.</a> Lab on a Chip. 11 (1), 173-175 (2011).</li><li>Dreher, S., Engler, M., Kockmann, N., Woias, P., Bockhorn, H., Mewes, D., Peukert, W., Warnecke, H. .. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+and+experimental+investigations+of+convective+micromixers+and+microreactors+for+chemical+reactions.">Theoretical and experimental investigations of convective micromixers and microreactors for chemical reactions.</a> Micro and Macro Mixing: Analysis, Simulation and Numerical Calculation. , (2010).</li><li>Huang, C. Y., Gregory, J. W., Sullivan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modified+schlieren+technique+for+micro+flow+visualization.">A Modified schlieren technique for micro flow visualization.</a> Measurement Science &amp; Technology. 18 (5), N32-N34 (2007).</li><li>Tyn, M. T., Calus, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+concentration+dependence+of+mutual+diffusion+coefficients+of+some+binary+liquid+systems.">Temperature and concentration dependence of mutual diffusion coefficients of some binary liquid systems.</a> Journal of Chemicaland Engineering Data. 20 (3), 310-316 (1975).</li><li>Celik, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+estimation+and+reporting+of+uncertainty+due+to+discretization+in+CFD+applications.">Procedure for estimation and reporting of uncertainty due to discretization in CFD applications.</a> Journal of Fluids Engineering. 130 (7), 078001 (2008).</li><li>Tasi&#263;, A. &. #. 3. 8. 1. ;., Djordjevi&#263;, B. D., Grozdani&#263;, D. K., Radojkovi&#263;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mixing+rules+in+predicting+refractive+indices+and+specific+refractivities+for+some+binary+liquid+mixtures.">Use of mixing rules in predicting refractive indices and specific refractivities for some binary liquid mixtures.</a> Journal of Chemical and Engineering Data. 37 (3), 310-313 (1992).</li><li>Hsiao, P. -. J., Chen, S. -. T., Hsiao, T. -. H., Sun, C. -. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+coarse-resolution+numerical+simulation+with+experimental+measurements+of+wind+turbine+aerodynamic+performance.">A comparison of coarse-resolution numerical simulation with experimental measurements of wind turbine aerodynamic performance.</a> , (2013).</li><li>Sun, C. -. l., Huang, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+schlieren+visualization+of+near-bubble+mass+transport+during+boiling+of+2-propanol/water+mixtures+in+a+square+capillary.">Microscale schlieren visualization of near-bubble mass transport during boiling of 2-propanol/water mixtures in a square capillary.</a> Experiments in Fluids. 55 (7), 1778 (2014).</li><li>Panigrahi, P. K., Muralidhar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Schlieren and Shadowgraph Methods in Heat and Mass Transfer. , (2012).</li></ol>

Os autores não têm nada a revelar.

Para uma mistura de fluidos em um dispositivo de microfluidos, a técnica schlieren microescala é capaz de medir a magnitude do gradiente de concentração através de quantificar as alterações na intensidade da luz. Uma vez que o princípio desta técnica baseia-se na detecção da alternância de propagação da luz, os fluidos de trabalho e o dispositivo de microfluidos tem que ser transparente para a luz incidente. Além disso, o protocolo requer uma relação linear entre o índice de refracção da solução e ...

Mistura de fluido é uma questão importante que se encontra em muitos processos industriais e sistemas biológicos. Com o surgimento de microfluídica, misturando em microescala trouxe muita atenção devido ao seu desafio no domínio da difusão entre os mecanismos de transporte de massa. Desde a concepção de uma validação quantitativa micromisturador efetiva exigida, vários métodos de medição foram desenvolvidos 5-7. No entanto, a estrutura tridimensional, geralmente encontrado em micromisturadores eficientes 5, exige uma representação mais precisa da área de concentração que as técnicas de medição comuns não conseguem fornecer. Devido ao limite do ângulo de visão ou de reacção cinética 8 6, os métodos acima mencionados pode produzir resultados incorrectos que não representam correctamente a homogeneidade da mistura. Para fluidos opticamente transparentes misturando em microestruturas opticamente transparentes, técnica schlieren microescala 3,9-14 </sup&gt; fornece uma alternativa atraente para analisar a mistura não homogeneidade. No passado, a técnica schlieren microescala tem sido principalmente utilizada para visualizar o fluxo compressível 9-13, 15 ou 16 gradiente de fase. Microscópicos benefícios técnica schlieren de ambos uma disposição óptica simples e alta sensibilidade e permite não só a investigação não-invasiva de fluxo característica específica que causa distúrbio óptico, mas é adequado para utilização na avaliação da mistura. Neste trabalho, construímos o sistema schlieren microescala, inserindo uma faca de ponta no plano focal de trás da objetiva de um microscópio, descrevem um procedimento de calibração para realizar análise quantitativa, e relatar uma medida de validação em um oscilador microfluídico 4. Para implementar as medições, os fluidos de trabalho são adequadamente seleccionados de modo a que o índice de refracção dos fluidos misturados varia linearmente com a composição, e a espessura do dispositivo de microfluidos alvo é idêntico ao deE utilizada na calibração. Além concentração de espécies, técnica schlieren microescala pode ser estendido para medir o gradiente de outra quantidade escalar que está linearmente correlacionada com o índice de refracção, tal como a temperatura ou a salinidade.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Permanent Epoxy Negative Photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2150</td>
			<td></td>
		</tr>
		<tr>
			<td>single side polished silicon wafer</td>
			<td>Light Technology</td>
			<td>S4W1PP5SABUP1</td>
			<td>p-type, diameter: 100&#177;0.5 mm, thickness: 525 &#177;25 mm, orientation: (1 0 0)</td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>kdScientific</td>
			<td>kds210</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, i.e. 10 ml</td>
			<td>Terumo</td>
			<td>SS-10L2138</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoffman modulation contrast microscope</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>5X objective lens</td>
			<td>Leica Microsystems</td>
			<td>N PLAN</td>
			<td>NA = 0.12</td>
		</tr>
		<tr>
			<td>knife-edge</td>
			<td></td>
			<td></td>
			<td>custom made part</td>
		</tr>
		<tr>
			<td>camera, i.e. high speed</td>
			<td>Integrated Design Tools</td>
			<td>NX7-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>C-mount adapter HC 0.63x</td>
			<td>Leica Microsystems</td>
			<td>541537</td>
			<td></td>
		</tr>
		<tr>
			<td>camera operating software</td>
			<td>Integrated Design Tools</td>
			<td>MotionPro X Studio 2.02.01</td>
			<td></td>
		</tr>
		<tr>
			<td>polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard-184 silicone elastomer kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon tubing, i.e. O.D. x I.D. 1/16 in. x 0.031 in.</td>
			<td>Supelco</td>
			<td>58700-U</td>
			<td></td>
		</tr>
		<tr>
			<td>micro glass slide</td>
			<td>Matsunami Glass</td>
			<td>S2215</td>
			<td></td>
		</tr>
		<tr>
			<td>hot plate</td>
			<td>Yeong-Shin</td>
			<td>HP-303DN</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled water, i.e. HPLC grade</td>
			<td>Alps Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl alcohol, i.e. reagent grade</td>
			<td>Nihon Shiyaku Reagent</td>
			<td>EA448652</td>
			<td></td>
		</tr>
		<tr>
			<td>image processing software</td>
			<td>Mathworks</td>
			<td>MATLAB R2009a</td>
			<td></td>
		</tr>
		<tr>
			<td>computational fluid dynamics package</td>
			<td>ESI group</td>
			<td>CFD-ACE+ 2008</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing mixing inhomogeneity in a microfluidic device by microscale schlieren technique

Neste artigo, apresentamos o uso da técnica schlieren microescala para medir a mistura não homogeneidade em um dispositivo microfluídico. O sistema schlieren microescala é construído a partir de um microscópio de contraste de modulação Hoffman, que proporciona fácil acesso para o plano focal posterior da lente da objectiva, através da remoção da placa de fenda e substituindo o modulador com uma lâmina. O princípio de funcionamento de técnica schlieren microescala assenta na detecção de luz desvio provocado pela variação do índice de refracção 1-3. A luz desviada ou escapa ou está obstruído pela faca de ponta para produzir uma brilhante ou uma faixa escura, respectivamente. Se o índice de refracção da mistura varia de forma linear com a sua composição, a mudança local na intensidade da luz no plano da imagem é proporcional ao gradiente de concentração normal em relação ao eixo óptico. A imagem de micro-schlieren dá uma projecção bidimensional da luz produzida perturbado por falta de homogeneidade tridimensional. Para realizar a análise quantitativa, é descrito um procedimento de calibração que se mistura dois líquidos em um t-microcanal. Nós realizamos uma simulação numérica para obter o gradiente de concentração na T-microcanais que correlaciona-se estreitamente com a imagem micro-schlieren correspondente. Por comparação, uma relação entre as leituras de escala de cinzentos da imagem micro-schlieren e os gradientes de concentração apresentadas num dispositivo de microfluidos é estabelecida. Usando essa relação, somos capazes de analisar a falta de homogeneidade de mistura imagem micro-schlieren associado de e demonstrar a capacidade técnica de schlieren microescala com medições em um oscilador microfluídico 4. Para fluidos opticamente transparentes, a técnica schlieren microescala é uma ferramenta de diagnóstico atraente para fornecer informação instantânea de campo total que mantém as características tridimensionais do processo de mistura.

A proporção em tons de cinza de I / I 0 sob diferente número de Reynolds para ambos os gradientes positivos e negativos da fracção de massa é mostrado (Figura 2) com uma banda simétrica que aparece no meio do t-microcanal. Em baixo número de Reynolds, a cauda da banda schlieren é expandido e turva devido à dispersão através da interface de mistura. À medida que o número de Reynolds aumenta, o comprimento de difusão encurta levando a uma banda mais estreita. Em...

Watch this Scientific Journal Video about Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique at JoVE.com

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analisando falta de homogeneidade de mistura em um dispositivo micro, pela técnica Microscale Schlieren

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

analyzing-mixing-inhomogeneity-microfluidic-device-microscale

Research

JoVE Journal

Bioengineering

8.9K visualizações.  National Taiwan University. Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image. 

Vídeo: Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo microfluídicos para Recriando um microambiente do tumor<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengenharia

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Um dispositivo micro para o Estudo múltiplas linhagens distintas

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

A técnica de microfluídica para sondar Deformabilidade celular

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para a formação de biofilme Streamer em um dispositivo micro com micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Camada por camada de colágeno Deposição em microfluídicos para Microtissue Estabilização

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable rede Vascular com um modelo de tecido em um dispositivo Microfluidic

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Geração de Condições Ambientais Dinâmicas usando um dispositivo microfluido de alto rendimento

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulando e caracterizando nanopartículas lipídicas para entrega de genes usando uma plataforma de mistura microfluida

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

Preparação e avaliação estrutural de monocamadas de células epiteliais em um dispositivo de cultura microfluídica de tamanho fisiológico

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...