Este trabalho foi apoiado por Ogden Faculdade de Ciências e Engenharia, incluindo suporte interno do Departamento de Química e do Escritório de Investigação (PACR 13-8032) da Western Kentucky University. A assistência do Dr. John Andersland no Centro de Microscopia WKU (MEV) e Professor Associado Yan Cao do Instituto WKU para Ciência de Combustão e Engenharia (análise BET) tem sido fundamental para a realização deste trabalho.

1. Preparação da Emulsão <ol><li> Índice de emulsão <ol><li> Massa uma quantidade adequada de sal para produzir 10 ml de solução de 0,03-M. Para platina (IV) medida cloreto de 0,101 g, para o zinco (II) cloreto (ZnCl2) medida 0,032 g, e cloreto de sódio (NaCl) medida 0,018 g. </li><li> Em tubos de ensaio individuais, cada dissolver o sal em 10 ml de água Dl. Reservada para uso posterior. </li><li> Utilize a 20 mL, frasco de vidro selado para o conteúdo ao longo deste procedimento. Tare um equilíbrio para o frasco de vidro. </li><li> Pesar polidimetilsiloxano terminado em vinilo por lentamente despejando-a sobre uma haste de agitação e para dentro do frasco de vidro que descansa na escala zerada. Pesar 1,02 g (equivalente a 1.080 mL). Nota: A elevada viscosidade deste polímero torna impraticável pipetagem. </li><li> Pipetar 1,02 ml de n-heptano para o frasco. Adicionar 2 gotas de agente tensioactivo não iónico (monooleato de sorbitano) ao frasco. Pipetar 0,3 ml de solução salina e 0,45 ml de di-água ao frasco. </li><li> Selar o frasco de vidro por estragar a tampa firmemente. Agita-se vigorosamente durante 60 segundos para iniciar a emulsão antes de iniciar sonicação. </li></ol></li><li> Construção de água-banho Sonicator Aparelho <ol><li> Preencha sonicador com água até a linha de preenchimento mínimo. Adicione 250 ml de água da torneira para um copo de 400 ml. Encha o copo de 400 ml com gelo, de modo que o nível da água é apenas na borda. </li><li> Coloque este recipiente no interior do banho de água sonicador. Verifique a linha de preenchimento-no sonicator, ajustando, se necessário. Coloque um anel de ficar junto ao banho-maria sonicator. </li><li> Usando dois anel de suporte grampos, posicioná-los de modo a que o braço se estende para fora, perpendicularmente ao anel de suporte e uma outra está estendido para o banho de água de modo que está a apontar para baixo para dentro do copo cheio com água gelada. Anexar outro grampo para o anel de suporte com um termómetro para baixo no copo de 400 ml de modo a que a temperatura pode ser monitorizada ao longo de sonicação. </li></ol></li><li> Emulsificação Procedimento <ol><li> Colocar o frasco contendo emulsão de modo que ele é inteiramente submerso no copo de 400 ml, ao garantir que na braçadeira saliente para dentro da proveta. </li><li> Assegure-se que o frasco de vidro contendo a mistura de PDMS não estiver em contacto com as paredes do copo para eliminar o calor provocado pelo atrito. </li><li> Ligue o sonicator e definir o tempo de ultra-sons durante 7 min. Uma vez que a emulsão é muito sensível ao calor, assegurar que a temperatura no interior da taça é entre 0 e 5 ° C ao longo de sonicação. Comece sonicação uma vez que a temperatura no interior do copo é desejável. </li><li> Após 7 minutos de ultra-sons, remover o frasco e agitar suavemente / redemoinho durante 1 min, mantendo a parte superior do frasco para eliminar quaisquer aglomerados que possam formar na emulsão. </li><li> Elimine o conteúdo da proveta. Recarga com 250 ml de água e adicionar gelo ao preenchimento para dentro de 1 cm do topo do copo. </li><li> Coloque o frasco de volta no gancho metálico. Mergulhe-o sob a água gelada para resume ultra-sons durante 7 min. </li><li> Repita os passos 1.3.3 a 1.3.6 para um total de oito períodos de sonicação, 7-mínimo, ou até que a sonicação parece ser homogénea e sem aglomerados estão presentes. Armazenar à temperatura ambiente. NOTA: A emulsificação deve ser estável durante vários dias, mas pode ser recuperado por sonicação como acima em intervalos de 7 min até que apareça homogénea. Armazenar à temperatura ambiente. </li></ol></li></ol> 2. A reticulação <ol><li> Instalação para Adição de trietoxissilano / Solução surfactante <ol><li> Pipetar 5,4 ml trietoxissilano num tubo de ensaio. Coloque em um rack de tubo de ensaio sob o capô para uso posterior. </li><li> Encha um copo de 400 ml com água gelada e coloque-o sob o capô. Próximo ao local copo de 400 ml um anel de ficar com uma braçadeira anexado, estendido diretamente sobre a abertura do copo. Este será o banho de gelo durante a adição de silano. </li><li> Colocar uma placa quente sobre o outro lado do anel de suporte. Encha um copo de 800 ml com cerca de 700 ml deagua de torneira. Coloque-o sobre a placa de aquecimento. </li><li> Ligue a placa quente e manter uma temperatura de 75 a 85 ° C no interior do copo de 800 ml. Anexar uma braçadeira com um termómetro para o suporte anelar de modo que a temperatura da água dentro da proveta de 800 ml, pode ser monitorizada. </li><li> Produzir a solução de agente tensioactivo por dissolução de 0,5 g de sulfato de dodecilo de sódio a 375 ml de água (4,62 mm). Adicionar cerca de 10 ml de solução de surfactante a um tubo de ensaio limpo vazio. </li><li> Anexar outro grampo para o anel de suporte sob o capô com o tubo de ensaio surfactante fixadas de modo que o seu nível de líquido está abaixo da superfície da água dentro da proveta de 800 ml. Permita 10 minutos para o equilíbrio térmico. </li><li> Molhar um pedaço de papel de filtro e colocá-lo no topo de um pequeno funil. Coloque a haste do funil para dentro de um balão de Erlenmeyer de 250 ml e colocar o balão sob o capô. </li></ol></li><li> A adição de silano <ol><li> Coloque o frasco de emulsão no grampo sobre o gelo-água bicoer no interior do exaustor. Coloque a mistura de PDMS na pinça de modo que o conteúdo do frasco estão abaixo da superfície da água. A adição do silano provoca uma reacção exotérmica, portanto, a emulsão deve ser mantido frio, a fim de manter a sua estrutura. </li><li> Remover a tampa do frasco de vidro para evitar a acumulação gasoso. </li><li> Lentamente o tubo de ensaio contendo silano para dentro do frasco de vidro numa corrente contínua durante um período de cerca de 10 seg (cerca de 0,5 ml / seg). ATENÇÃO: A adição de silano inicia uma reacção exotérmica e a libertação de cloreto de hidrogénio cáustico (HCl) de gás. O frasco vai se tornar extremamente quente e um gás tóxico irá evoluir a partir da mistura. Não mexa o conteúdo ao adicionar o silano. </li><li> Depois de adicionar trietoxissilano completamente, mexa delicadamente o conteúdo com uma haste de agitação de vidro, enquanto vestindo uma luva de proteção de calor. Aguarde 2 min ou até paradas de gás evolução do frasco. </li><li> Folldevido a reticulação, não há separação de fases visível na amostra. Se aglomerados estão presentes, selar o frasco e agite durante 20 segundos rigorosamente, mantendo o frasco pela tampa. </li></ol></li><li> Produção Bead <ol><li> Use, uma pipeta de Pasteur de vidro limpo para desenhar a emulsão reticulada a partir do frasco de vidro. Adicionar a emulsão reticulada gota a gota à solução de tensioactivo (o qual deve ser mantida entre 75 e 85 ° C) para dentro do tubo de ensaio, tendo o mínimo de tempo possível de entre as gotas. </li><li> 30 seg 1 min depois da adição da emulsão, a solução de agente tensioactivo irá começar a desenvolver-se lentamente o gás como os sólidos comecem a formar-se dentro do tubo de ensaio. </li><li> Enquanto vestindo luvas de protecção térmica, pegue o tubo de ensaio fora da braçadeira e despeje todo o seu conteúdo para o aparelho de filtração sob o capô. Filtrar durante 5 min. Remover o papel de filtro a partir do filtro. </li><li> Transfira os sólidos filtrados em um vidro de relógio e pérolas separadas para O / N secagem sob o hood. Limpeza das esferas pode ser adiada indefinidamente. Loja grânulos à TA seco num frasco de vidro selado até ser necessário, e limpo imediatamente antes da utilização. </li></ol></li><li> Bead Limpeza <ol><li> Criar um outro aparelho de filtração sob o capô e colocar as pérolas secas dentro do funil na parte superior do papel de filtro. </li><li> Use um frasco de lavagem de plástico cheio de DI-água para enxaguar as contas suavemente, movendo-os ligeiramente para garantir que todas as contas são lavados. </li><li> Deixe as contas secar durante 1 hora, colocando-os em um vidro de relógio sob o capô. Utilizar um frasco de lavagem cheio com hexanos para enxaguar os grânulos utilizando o mesmo método para a lavagem com água. </li><li> Coloque as contas em um vidro de relógio. Coloque o vidro de relógio e as contas sob o capô para secar. </li><li> Depois as contas estão completamente secos, coloque-os em um pequeno frasco de vidro selado e armazenar a RT para uso futuro. </li></ol></li><li> Montagem dos grânulos para análise em MEV e SEM Configurações <ol><li> Coloque uma tira de afazeresfita condutiva uble lados de carbono na parte superior do topo para o qual os grânulos irão ser montadas. Com uma tesoura, cortar ao redor do topo para garantir que não trava fita sobre as bordas. </li><li> Coloque um pedaço de papel de filtro sob a ponta sobre uma superfície plana. Remover a camada superior da fita de modo que a parte inferior de adesivo é exposto. </li><li> Delicadamente despeje os grânulos sobre o topo. Algumas pérolas vai ficar com a fita, mas a maioria vai saltar fora e terra sobre o papel de filtro. Derrame-los de volta para o frasco se ficassem no papel de filtro. Repita, se necessário, lavando todas as pérolas (de acordo com 2.4.2 a 2.4.5) que se tornam contaminados. </li><li> Para garantir as contas estão seguros no canhoto, use uma seringa e levemente soprar muito estreitamente à superfície do topo. Despeje mais contas sobre o stub, se apenas alguns aderida à fita. Certifique-se de que todas as contas são seguros antes de colocar o stub para a câmara de SEM e evacuando-lo. </li><li> Uma vez que as amostras foram montadas adequadamente eles já estão prontos para sofrer SEM analysis 15. Coletar imagens no modo LOW VAC em 15 keV para optimizar a resolução das características da superfície do talão. </li></ol></li></ol>

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The authors have nothing to disclose.

Os grânulos produzidos utilizando este protocolo (e ajustando a concentração de eletrólitos e identidade) são fundamentalmente diferentes daquelas produzidas com uma emulsão de baixa força iónica, como pode ser visto através da comparação das Figura 1A a outras imagens SEM na Figura 1. Nosso relatório inicial usado PtCl 4, com a intenção de catalisar a polimerização ulterior reticulação na interface aquosa-14 alifático. Nesse relatório, grandes ...

Polydimethylsiloxane (PDMS) is one of the most widely used silicone compounds. Its biocompatibility has led to widespread use in implant and other biomedical engineering structures1,2. It is trivially cross-linked into elastic structures using an organosilyl compound (such as triethoxysilane), a simple and reliable procedure which has made it useful for cast polymer applications where some flexibility is required3. Once cross-linked, PDMS is largely inert, particularly in biological conditions, and is therefore useful for a variety of food and medical applications4,5. Ease of casting, chemical inertness, and hydrophobicity have made it a natural choice for microfluidic devices6,7. Its affinity for non-halogenated, non-polar organic compounds has made it a popular stationary phase in separations chemistry8-10. 
Recently, microbubble fabrications have been used to generate porous beads for use as catalyst structural substrates or in chemical separations11,12. In both applications, ideal materials will have a maximized surface-area-to-volume (SAV) ratio for best efficiency. In a microbubble fabrication process, microstructuring of materials is typically accomplished by isolating the polymer in aliphatic &#8220;microbubbles&#8221; by emulsification in an aqueous continuous phase. The initial report of microporous PDMS beads produced them by mechanical emulsification of two phases (aliphatic and aqueous)13. The stock PDMS liquid (and its cross-linking agent) is dissolved into the aliphatic phase, which is structured into microscopic beads by being forced to cavitate within the (continuous) aqueous phase. The emulsification is stabilized by the addition of a non-ionic surfactant. When the emulsion is added dropwise to a heated bath, solid beads form by agglomeration of the microbubbles into clusters of tiny spheres of cross-linked PDMS. Our goal in this protocol is to modify this procedure to develop beads with an inverted porosity to improve the SAV ratio of the material.
As reported previously, control of the beads can be directed to some extent by the aliphatic:aqueous ratios in the emulsion. However, we have reported recently that addition of platinum(IV) chloride (PtCl4) inverts the porosity: materials are formed in which the PDMS is riddled with concave pores14. This indicates that the aqueous layer cavitates inside the aliphatic one, despite having similar aliphatic:aqueous ratios to those published in the original work13. The primary advantage of our method is that this concave porosity should naturally result in an increased SAV ratio, and thus, improved efficiency for analytical chemistry applications. While we are continuing to explore the specific effects of the addition of the platinum compound, we show here that the same effect can be accomplished using any aqueously soluble ionic compound, though perhaps to a reduced extent. Because our techniques also differ in some key aspects from what has been previously reported, we present our protocols here as a video to encourage others to extend our methods. Most notably, we use a common bath sonicator of the type used to clean glassware or other equipment, rather than the (considerably more expensive) probe sonicator often used in microbubble fabrication. This adjusted approach to the microbubble fabrication procedure could potentially be extended for the production of large quantities of bulk materials as well, creating porous sheets or slabs which could have applications for biomedical devices, aerospace and automotive industry, or substrates for chemical catalysis. Users seeking to generate high-SAV-ratio, microstructured materials using other similar polymers for such analyses may find that our protocols can be extended to any polymer for which the microbubble emulsion technique can be applied.

<table><tbody><tr><td>Poly(dimethylsiloxane), vinyl terminated</td><td>Sigma-Aldrich</td><td>68083-19-2</td><td></td></tr><tr><td>n-Heptane</td><td>Sigma-Aldrich</td><td>142-82-5</td><td>Flammable</td></tr><tr><td>Triethoxysilane</td><td>Sigma-Aldrich</td><td>998-30-1</td><td>Flammable, Accutely Toxic</td></tr><tr><td>Sorbitan Monoleate (Span-80)</td><td>Fluker</td><td>1338-43-8</td><td></td></tr><tr><td>Platinum(IV) Chloride</td><td>Sigma-Aldrich</td><td>13454-96-1</td><td>Accutely Toxic</td></tr><tr><td>Zinc(II) Chloride</td><td>Sigma-Aldrich</td><td>7646-85-7</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>2.8 L Water Bath Sonicator</td><td>VWR</td><td>97043-964</td><td></td></tr></tbody></table>

microbubble fabrication of concave-porosity pdms beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer&#8211;Emmett&#8211;Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.

Imagens representativas de SEM de grânulos resultantes a partir de emulsões com diferentes condições electrolíticas são mostrados na Figura 1. A Figura 1A mostra um grânulo semelhantes aos obtidos por Dufaud, et al. 13, produzido utilizando os nossos procedimentos, sem a adição de qualquer electrólito. Grânulos mostrado na Figura 1B-D, resultando em diferentes morfologias para cada ião de metal. Para todas as imagens apre...

Watch this Scientific Journal Video about Microbubble Fabrication of Concave-porosity PDMS Beads at JoVE.com

Microbubble Fabrication of Concave-porosity PDMS Beads

Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized.

Fabricação de microbolhas de Côncavo-porosidade Beads PDMS

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is ...

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8.3K visualizações.  Western Kentucky University. Procedures used to generate microstructured concave-porosity polydimethylsiloxane beads are presented. Effects of electrolyte concentration and identity within the aqueous phase are particularly emphasized. 

Vídeo: Microbubble Fabrication of Concave-porosity PDMS Beads

Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7. The fabricated microstructures of conducting polymers have been used as humidity8, chemical8, and glucose sensors9. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15.

A micropunching lithography approach is developed to generate micro- and submicron-patterns on top, sidewall and bottom surfaces of polymer substrates. It overcomes the obstacles of patterning conducting polymers and generating sidewall patterns. This method allows rapid fabrication of multiple features and is free of aggressive chemistry.

Micropunching Litografia de Geração de Micro e submicrométricas-padrões sobre substratos poliméricos

Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771. They offer the advantages of ...

Engenharia

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biologia

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Chips microfluídicos controlados com Arrays microválvula elastoméricos

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Alta taxa de transferência de célula única e múltipla de células Micro-encapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Bioengenharia

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Criação de Sub-50 nm nanofluidic Junções em PDMS Microfluidic Chip via processo de automontagem de coloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

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 ...

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

Fabricação de dispositivos de refração-índice-combinada para microfluídica biomédica

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Duplo Emulsion Geração de utilizar um dispositivo de polidimetilsiloxano (PDMS) Co-Axial Flow Foco

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Janela em um Microworld: Simple sistemas microfluídicos para estudar o transporte microbiana em Meios Porosos

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.