Questo lavoro è stato sostenuto da Western Kentucky University Ogden College of Science and Engineering, incluso il supporto interno del Dipartimento di Chimica e dall&#39;Ufficio di Ricerca (PACR 13-8032). L&#39;assistenza del dottor John Andersland presso l&#39;impianto di WKU Microscopia (immagini SEM) e Professore Associato Yan Cao dell&#39;Istituto WKU per Combustion Scienza e Ingegneria (analisi BET) è stata fondamentale per lo svolgimento di questo lavoro.

1. Preparazione di emulsioni <ol><li> Contenuti emulsione <ol><li> Messa una quantità appropriata di sale per la produzione di 10 ml di soluzione 0,03-M. Per platino (IV) misura cloruro 0,101 g, per lo zinco (II) cloruro (ZnCl 2) misura 0,032 g, e cloruro di sodio (NaCl) misura 0,018 g. </li><li> In provette singoli, dissolvere ogni sale in 10 ml di acqua deionizzata. Mettere da parte per un uso successivo. </li><li> Utilizzare a 20 ml, flaconcino di vetro richiudibile per i contenuti durante tutta la procedura. Tarare un equilibrio per la fiala di vetro. </li><li> Pesare polidimetilsilossano vinil-denuncia da parte lentamente versando sopra un bastoncino per mescolare e nel flacone di vetro appoggiata sulla scala azzerato. Pesare 1,02 g (equivalente a 1.080 ml). NOTA: L&#39;alta viscosità di questo polimero rende impraticabile pipettamento. </li><li> Pipettare 1,02 ml di n-eptano flaconcino. Aggiungere 2 gocce di tensioattivo non ionico (sorbitan monooleato) alla fiala. Pipettare 0,3 ml di soluzione salina e 0,45 ml di acqua demineralizzata al flacone. </li><li> Sigillare la fiala di vetro avvitando il coperchio ermeticamente. Agitare energicamente per 60 secondi per avviare l&#39;emulsione prima di iniziare ultrasuoni. </li></ol></li><li> Costruzione di Bagnomaria Sonicator Apparatus <ol><li> Riempire sonicatore con acqua fino alla linea di riempimento minimo. Aggiungere 250 ml di acqua di rubinetto in un bicchiere da 400 ml. Riempire il becher da 400 ml con ghiaccio così che il livello dell&#39;acqua è solo sul bordo. </li><li> Inserire questa coppa all&#39;interno del sonicatore bagnomaria. Controllare il fill-line sul sonicatore, regolando se necessario. Posizionare un anello supporto direttamente sul sonicatore bagnomaria. </li><li> Utilizzando due anello basamento morsetti, posizionarli in modo che un braccio viene estesa, perpendicolare alla gradinata e un altro è esteso verso il bagno di acqua in modo che sia rivolta verso il basso nel contenitore riempito con acqua ghiacciata. Fissare un&#39;altra fascetta all&#39;anello stand con un termometro nel becher da 400 ml in modo che la temperatura può essere monitorato durante sonicazione. </li></ol></li><li> Emulsificazione Procedura <ol><li> Posizionare la fiala-emulsione contenente in modo che sia completamente immerso nel becher da 400 ml, fissandolo nel morsetto sporgente verso il basso nel becher. </li><li> Assicurarsi che la fiala di vetro contenente la miscela PDMS non tocca le pareti del becher per eliminare il calore per attrito. </li><li> Accendere il sonicatore e impostare il tempo di ultrasuoni per 7 minuti. Poiché l&#39;emulsione è molto sensibile al calore, assicurare che la temperatura all&#39;interno del recipiente è compreso tra 0 e 5 ° C durante sonicazione. Avviare sonicazione quando la temperatura all&#39;interno del bicchiere è desiderabile. </li><li> Dopo 7 minuti di sonicazione, togliere la fiala e scuotere delicatamente / turbolenza per 1 min, tenendo la parte superiore del flaconcino per eliminare eventuali grumi che possono formarsi in emulsione. </li><li> Eliminare il contenuto del becher. Riempire con 250 ml di acqua e ghiaccio per aggiungere il riempimento da 1 cm nella parte superiore della coppa. </li><li> Posizionare la fiala nel morsetto. Immergere sotto l&#39;acqua ghiacciata per risommaree sonicazione per 7 minuti. </li><li> Ripetere i punti 1.3.3 a 1.3.6 per un totale di otto periodi sonicazione, 7 min, o fino a quando la sonicazione appare omogeneo e senza grumi sono presenti. Conservare a temperatura ambiente. NOTA: L&#39;emulsione deve essere stabile per diversi giorni, ma può essere recuperato mediante sonicazione come sopra in 7-min intervalli finché non appare omogeneo. Conservare a temperatura ambiente. </li></ol></li></ol> 2. La reticolazione <ol><li> Configurazione per l&#39;aggiunta di trietossisilano / Tensioattivo Soluzione <ol><li> Pipettare 5.4 ml trietossisilano in una provetta. Mettere in un rack provetta sotto il cofano per un uso successivo. </li><li> Riempire un bicchiere da 400 ml con acqua ghiacciata e posizionarlo sotto il cofano. Accanto al posto becher da 400 ml basamento un anello con un morsetto attaccato, estesa direttamente sull&#39;apertura del bicchiere. Questo sarà il bagno di ghiaccio per l&#39;aggiunta di trietossisilano. </li><li> Posizionare una piastra calda sull&#39;altro lato del supporto ad anello. Riempire un bicchiere di 800 ml con circa 700 ml diacqua di rubinetto. Mettere sul piatto caldo. </li><li> Accendere la piastra riscaldante e mantenere una temperatura di 75 a 85 ° C all&#39;interno del becher da 800 ml. Fissare un morsetto con un termometro per il supporto ad anello in modo che la temperatura dell&#39;acqua all&#39;interno del becher da 800 ml può essere monitorato. </li><li> Produrre la soluzione tensioattivo sciogliendo 0,5 g di sodio dodecilsolfato a 375 ml di acqua (4,62 mM). Aggiungere circa 10 ml di soluzione di tensioattivo a una provetta vuota pulita. </li><li> Fissare un&#39;altra fascetta all&#39;anello stare sotto il cofano con la provetta tensioattivo fissate in modo che il suo livello del liquido è al di sotto della superficie dell&#39;acqua all&#39;interno del becher da 800 ml. Lasciare 10 minuti per il raggiungimento dell&#39;equilibrio termico. </li><li> Bagnare un pezzo di carta da filtro e posizionarlo nella parte superiore di un piccolo imbuto. Posizionare il gambo dell&#39;imbuto all&#39;interno di una beuta da 250 ml e il recipiente sotto il cofano. </li></ol></li><li> Aggiunta di trietossisilano <ol><li> Posizionare il flaconcino dell&#39;emulsione nel morsetto sopra il becco acqua ghiacciataer all&#39;interno della cappa. Posizionare la miscela PDMS nel morsetto in modo che il contenuto della fiala sono sotto la superficie dell&#39;acqua. L&#39;aggiunta di triethoxysilane provoca una reazione esotermica, quindi l&#39;emulsione deve essere mantenuto freddo per mantenere la sua struttura. </li><li> Togliere il coperchio dalla fiala di vetro per evitare accumulo di gas. </li><li> Versare lentamente il triethoxysilane provetta contenente nella fiala di vetro in un flusso continuo per un periodo di circa 10 secondi (circa 0,5 ml / sec). ATTENZIONE: L&#39;aggiunta di triethoxysilane avvia una reazione esotermica e il rilascio di caustica acido cloridrico (HCl) gas. La fiala diventa estremamente caldo e un gas tossico evolverà dalla miscela. Non mescolare il contenuto, mentre l&#39;aggiunta del trietossisilano. </li><li> Dopo aver aggiunto completamente trietossisilano, mescolare delicatamente il contenuto con una bacchetta di vetro scalpore, mentre indossa un guanto protettivo termico. Attendere per 2 minuti o fino a quando si ferma di gas in continua evoluzione dal flaconcino. </li><li> Follcausa reticolazione, non vi è alcuna separazione di fase visibile nel campione. Se sono presenti grumi, sigillare la fiala e agitare rigorosamente per 20 secondi mentre si tiene la fiala dal coperchio. </li></ol></li><li> Produzione Bead <ol><li> Utilizzare un ambiente pulito, Pasteur pipetta di vetro per disegnare l&#39;emulsione reticolato dal flaconcino di vetro. Aggiungere l&#39;emulsione reticolato goccia a goccia alla soluzione di tensioattivo (che dovrebbe essere mantenuta tra 75 e 85 ° C) nella provetta prendendo il minor tempo possibile tra gocce. </li><li> 30 sec a 1 min dopo l&#39;aggiunta dell&#39;emulsione, la soluzione tensioattivo inizierà lentamente ad evolversi gas come solidi cominciano a formarsi all&#39;interno della provetta. </li><li> Mentre indossa guanti protettivi calore, prendere la provetta dalla pinza e versare l&#39;intero contenuto nel dispositivo di filtrazione sotto il cofano. Filtro per 5 min. Rimuovere la carta da filtro dal filtro. </li><li> Trasferire i solidi filtrati su un vetro da orologio e perline separate per O / N asciugatura sotto la hood. Pulizia delle perline può essere rinviata a tempo indeterminato. Conservare le sfere a RT essiccato in una fiala di vetro sigillato fino al momento, e pulire immediatamente prima dell&#39;uso. </li></ol></li><li> Bead Pulizia <ol><li> Creare un altro apparato di filtrazione sotto il cofano e posizionare i talloni secchi all&#39;interno dell&#39;imbuto sopra la carta da filtro. </li><li> Utilizzare una bottiglia di lavaggio di plastica riempita con acqua demineralizzata per lavare le perline delicatamente, li muoversi leggermente al fine di garantire tutte le perle vengono risciacquati. </li><li> Lasciate che le perline asciugare per 1 ora mettendoli su un vetro da orologio sotto il cofano. Utilizzare una bottiglia di lavaggio riempita con esani per sciacquare le perline con lo stesso metodo per il risciacquo con acqua. </li><li> Mettere le perline su un vetro da orologio. Posizionare il vetro d&#39;orologio e le perline sotto il cofano a secco. </li><li> Dopo le perle sono completamente asciutti, metterli in una piccola fiala di vetro sigillato e conservare a temperatura ambiente per un utilizzo futuro. </li></ol></li><li> Montaggio delle Perline per impostazioni di analisi SEM e SEM <ol><li> Posizionare una striscia di donastro conduttivo di carbonio uble lati sopra l&#39;stub su cui saranno montate le perline. Utilizzando le forbici, tagliare intorno lo stub per garantire che non si blocca a nastro sopra i bordi. </li><li> Posizionare un pezzo di carta da filtro sotto lo stub su una superficie piana. Rimuovere lo strato superiore dal nastro in modo che la parte inferiore adesiva è esposta. </li><li> Versare delicatamente le perle sulla stub. Alcune perle che si attacchi al nastro, ma la maggior parte si rimbalzano e atterrare sulla carta da filtro. Versare questi indietro nel flaconcino se sono rimasti sulla carta da filtro. Ripetere se necessario, lavare eventuali perline (secondo 2.4.2 a 2.4.5), che diventano contaminati. </li><li> Per garantire le perle sono sicuro sul stub, utilizzare una siringa a bulbo e leggermente soffiare molto da vicino alla superficie stub. Versare più perline sopra lo stub se solo pochi aderito al nastro. Assicurarsi che tutti i cordoni siano sicuri prima di mettere il mozzicone nella camera SEM ed evacuare esso. </li><li> Una volta che i campioni sono stati montati correttamente sono ora pronti a subire SEM analeYsis 15. Raccogliere le immagini in modalità LOW VAC a 15 keV per ottimizzare la risoluzione delle caratteristiche superficiali del tallone. </li></ol></li></ol>

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	<li>Pedraza, E., Brady, A. C., Fraker, C. A., Stabler, 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=Synthesis+of+macroporous+poly(dimethylsiloxane)+scaffolds+for+tissue+engineering+applications.">Synthesis of macroporous poly(dimethylsiloxane) scaffolds for tissue engineering applications.</a> J. Biomater. Sci., Polym. Ed. 24 (9), 1041-1056 (2013).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+Where+we+have+been+and+where+we+are+going.">Biomaterials: Where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>B&#233;langer, M. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+for+PDMS-based+microfluidic+devices.">Surface modification for PDMS-based microfluidic devices.</a> Electrophoresis. 33 (1), 89-104 (2012).</li><li>Spietelun, A., Pilarczyk, M., Kloskowski, A., Namie&#347;nik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylene+glycol-coated+solid-phase+microextraction+fibres+for+the+extraction+of+polar+analytes&#8212;A+review.">Polyethylene glycol-coated solid-phase microextraction fibres for the extraction of polar analytes&#8212;A review.</a> Talanta. 87, 1-7 (2011).</li><li>Vas, G., V&#233;key, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+microextraction:+a+powerful+sample+preparation+tool+prior+to+mass+spectrometric+analysis.">Solid-phase microextraction: a powerful sample preparation tool prior to mass spectrometric analysis.</a> J. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+porous+materials:+past,+present+and+future.">Characterization of porous materials: past, present and future.</a> Colloids Surf. A. 241 (1), 3-7 (2004).</li></ol>

The authors have nothing to disclose.

Le perle prodotte usando questo protocollo (e regolando la concentrazione di elettroliti e l&#39;identità) sono fondamentalmente diversi da quelli prodotti con una emulsione di bassa forza ionica, come visto dal confronto della figura 1A alle altre immagini SEM di Figura 1. La relazione iniziale usati PtCl 4 con l&#39;intenzione di catalizzare ulteriormente la polimerizzazione reticolazione all&#39;interfaccia acquosa-alifatico 14.</sup...

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.

Immagini rappresentative SEM di perline derivanti da emulsioni con condizioni elettroliti diverse sono mostrati in figura 1. La figura 1A mostra una perlina simili a quelli ottenuti da Dufaud, et al. 13, prodotta con le nostre procedure, senza l&#39;aggiunta di qualsiasi elettrolita. Perline mostrati nella Figura 1B-D, con conseguente diverse morfologie per ogni ione metallico. Per tutte le immagini mostrate, 300 ml di soluzioni elettrolitiche 0,03-M...

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.

Fabbricazione microbolle di Perline PDMS Concavo-porosità

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 ...

microbubble-fabrication-of-concave-porosity-pdms-beads

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8.3K Views.  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. 

Video: 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 per la generazione di Micro-e submicroniche modelli sul Polymer Substrati

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

Ricerca

Ingegneria

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.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

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 Microfluidic controllati con le matrici elastomeriche microvalvola

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.

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

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

Bioingegneria

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.

Creazione di Sub-50 Nm nanofluidic giunzioni in PDMS Microfluidic Chip tramite processo di auto-assemblaggio di particelle colloidale

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.

Una tecnica Microfluidic per sondare deformabilità cellulare

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.

Fabbricazione di dispositivi rifrangenti-Indice-abbinati per biomedica microfluidica

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.

Doppia Emulsione Generazione Utilizzando un Polidimetilsilossano (PDMS) coassiale flusso fuoco Dispositivo

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.

Finestra su un micromondo: Sistemi Microfluidic semplice per studiare il trasporto microbica in mezzi porosi

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.