Name: fabricating high-viscosity droplets using microfluidic capillary device with phase-inversion co-flow structure
Uploaded: 2018-04-17T14:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure at JoVE.com

Este trabalho foi apoiado pela Fundação Nacional de ciências naturais da China (n. 51420105006 e 51322501). Agradecemos o Daniel por sua discussão útil sobre as ideias de alta viscosidade.

<ol>
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Fluids. 20, 053302 (2008).</li><li>Shestopalov, I., Tice, J. D., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+synthesis+of+nanoparticles+performed+on+millisecond+time+scale+in+a+microfluidic+droplet-based+system.">Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system.</a> Lab Chip. 4, 316-321 (2004).</li><li>Zheng, B., Roach, L. S., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+protein+crystallization+conditions+on+a+microfluidic+chip+using+nanoliter-size+droplets.">Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets.</a> J. Am. Chem. Soc. 125, 11170-11171 (2003).</li><li>Nie, Z. H., Xu, S. Q., Seo, M., Lewis, P. C., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+production+of+biopolymer+microcapsules+with+controlled+morphology.">Microfluidic production of biopolymer microcapsules with controlled morphology.</a> J. Am. Chem. Soc. 127, 8058-8063 (2005).</li><li>Abate, A. R., Kutsovsky, M., Seiffert, S., Windbergs, M., Pinto, L. F., Rotem, A., Utada, A. S., Weitz, D. A. <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+monodisperse+microparticles+from+non-Newtonian+polymer+solutions+with+microfluidic+devices.">Synthesis of monodisperse microparticles from non-Newtonian polymer solutions with microfluidic devices.</a> Adv. Mater. 23, 1757-1760 (2011).</li><li>Seo, M., Nie, Z., Xu, S., Mok, M., Lewis, P. C., Graham, R., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+microfluidic+reactors+for+polymer+particles.">Continuous microfluidic reactors for polymer particles.</a> Langmuir. 21, 11614-11622 (2005).</li><li>Duncanson, W. J., Lin, T., Abate, A. R., Seiffert, S., Shah, R. K., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+synthesis+of+advanced+microparticles+for+encapsulation+and+controlled+release.">Microfluidic synthesis of advanced microparticles for encapsulation and controlled release.</a> Lab Chip. 12, 2135-2145 (2012).</li><li>Song, H., Chen, D. L., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+in+droplets+in+microfluidic+channels.">Reactions in droplets in microfluidic channels.</a> Angew. Chem. Int. Ed. 45, 7336-7356 (2006).</li><li>Chen, H., Zhao, Y., Li, J., Guo, M., Wan, J., Weitz, D. A., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+in+double+emulsions+by+flow-controlled+coalescence+of+encapsulated+drops.">Reactions in double emulsions by flow-controlled coalescence of encapsulated drops.</a> Lab Chip. 11, 2312-2315 (2011).</li><li>Wang, P., Li, J., Nunes, J., Hao, S., Liu, B., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+micro-reactor+for+internal+gelation+to+fabricate+ZrO2+ceramic+microspheres.">Droplet micro-reactor for internal gelation to fabricate ZrO2 ceramic microspheres.</a> J. Am. Ceram. Soc. 100, 41-48 (2017).</li><li>Chen, H., Man, J., Li, Z., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+high-viscosity+droplets+by+surface-controlled+breakup+of+segment+flow.">Microfluidic generation of high-viscosity droplets by surface-controlled breakup of segment flow.</a> ACS Appl. Mater. Interfaces. 9, 21059-21064 (2017).</li><li>Man, J., Li, Z., Li, J., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+inversion+of+slug+flow+on+step+surface+to+form+high+viscosity+droplets+in+microchannel.">Phase inversion of slug flow on step surface to form high viscosity droplets in microchannel.</a> Appl. Phys. Lett. 110, 181601 (2017).</li></ol>

Os autores não têm nada para divulgar.

O dispositivo de fase-inversão de fluxo co fornece um método simples e direto para a frente para gerar monodisperso gotículas de alta viscosidade. Este dispositivo tem uma estrutura similar aos dispositivos de fluxo co comum, como a estrutura de fluxo de co básico consiste de um tubo interno inserido no tubo do meio, a saída dos quais está ligada ao tubo de saída. No entanto, existem duas diferenças principais entre o dispositivo de fluxo de co de inversão de fase e o dispositivo de fluxo co comum para a geraç?...

A geração de gotículas está se tornando uma tecnologia-chave em uma variedade de aplicações, tais como a entrega da droga, síntese de materiais, bioprinting 3D, ensaios de célula e comida engenharia1,2,3,4 , 5 , 6. dispositivos microfluídicos com entroncamento7,8, co1,9de fluxo, ou fluxo de focagem10,11 estruturas são amplamente utilizadas para gerar monodisperso gotículas de emulsão simples. Seleção de uma fase contínua mais viscosa irá facilitar a formação de gotículas12e as viscosidades de ambos os fluidos, contínuas e dispersas são comumente inferiores a 0,1 Pas em gotículas microfluídica13. No entanto, em muitas aplicações, a fase dispersa pode ter uma viscosidade várias centenas de vezes superior da água, tais como o glicerol14, soluções contendo nanopartículas15, proteínas16ou polímeros17 , 18 , 19, apesar de ser difícil conseguir monodisperso gotas diretamente de fluidos de alta viscosidade em um estábulo pingando regime11 em dispositivos microfluídicos, especialmente para os líquidos com uma viscosidade η > 1 Pa · s14 ,17,18,19. Além disso, tem sido relatado13,18 que métodos microfluidic típico para a formação de gotículas exigem líquidos com uma viscosidade relativamente baixa e moderada tensão interfacial para formar gotículas uniformes em um gotejamento estável regime.
Para uma fase dispersa com uma viscosidade ligeiramente maior do que 0,1 Pas, existem várias abordagens possíveis para facilitar a formação de gotículas com entroncamento típico, co fluxo ou fluxo de focagem microfluidic dispositivos: (1) diminuição da viscosidade do dispersos fase, diluindo-a em um solvente volátil11,20; (2) diminuir a relação de viscosidade dispersos-para-contínua aumentando a viscosidade da fase contínua1,11; (3) diminua a taxa de fluxo da fase dispersa a um valor extremamente baixo, mantendo um alto fluxo contínuo de-dispersa taxa relação 14,19. No entanto, essas abordagens não são práticas para líquidos com viscosidade muito maior, como eles abaixarão significativamente a taxa de produção enquanto a elevar drasticamente o consumo de solvente volátil ou a fase contínua. Além disso, tem sido relatado que algumas soluções de polímero de alta viscosidade com η > 1 Pa · s ainda não quebraram em gotas com abordagens mencionadas acima de17,19.
Existem também diversos projetos de melhoria de dispositivos microfluídicos que introduzir uma terceira fase de fluido no sistema, que facilita a geração de gotículas de alta viscosidade. As inovações incluem: bolhas introduzidas para cortar um segmento jorrando gotas21, um líquido imiscível parentes com viscosidade moderada, introduzido como fase intermediária entre a fase de dipsersed e a fase contínua18, e microreactors introduzido para gerar gotículas de alta viscosidade de dois precursores de baixa viscosidade21,22,23. No entanto, como um fluido mais está envolvido no processo, o sistema torna-se mais complicado, e os dispositivos normalmente trabalham em um regime de fluxo muito mais estreito do que os dispositivos típicos para a geração de gotículas de emulsão simples.
Para gerar monodisperso gotas diretamente de um fluido de alta viscosidade com η > 1 Pa · s, métodos de superfície controlada fase-inversão tem sido investigado24. Como a geração de gotículas de baixa viscosidade é muito mais fácil do que as gotas de alta viscosidade12, alongadas gotículas de baixa viscosidade em uma fase contínua de alta viscosidade são geradas pela primeira vez usando uma estrutura de fluxo típico de co e então terminaram devido para a mudança de superfície molhabilidade a jusante da estrutura de fluxo de co. O fluido de baixa viscosidade lançado inversamente encapsula o fluido de alta viscosidade a jusante em gotas para que a inversão de fase está concluída. De acordo com o mecanismo de inversão de fase, gotículas de alta viscosidade monodisperso podem ser geradas com base em um dispositivo de fluxo típico de co, enquanto a saída do dispositivo co fluxo é tratada para ser molhadas pelo fluido de baixa viscosidade e então conectada a um tubo mais largo24 ,25.

<table><tbody><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>8240</td><td>Square - Miniature Hollow Glass Tubing, I.D.=0.4mm, OD=0.8mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>CV2033</td><td>Round - Miniature Hollow Glass Tubing, I.D.=0.2mm, O.D.=0.33mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>CV1017</td><td>Round - Miniature Hollow Glass Tubing, I.D.=0.1mm, O.D.=0.17mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>Q14606</td><td>Square - Miniature Hollow Glass Tubing, I.D.=1.05mm+0.1/-0, OD=1.5mm</td></tr><tr><td>Standard Glass Capillaries</td><td>WPI</td><td>1B100-6</td><td>Round - Glass Tubing, I.D.=0.58mm, O.D.=1.00mm</td></tr><tr><td>Glycerol</td><td>Sinopharm Chemical Reagent Beijing</td><td>10010618</td><td></td></tr><tr><td>Paraffin Liquid</td><td>Sinopharm Chemical Reagent Beijing</td><td>30139828</td><td></td></tr><tr><td>Poly(vinyl alcohol), PVA-124</td><td>Sinopharm Chemical Reagent Beijing</td><td>30153084</td><td></td></tr><tr><td>Span 80</td><td>Sigma-Aldrich</td><td>85548</td><td></td></tr><tr><td>Starch</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Trichloro(octadecyl)silane</td><td>Sigma-Aldrich</td><td>104817</td><td></td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Honey</td><td></td><td></td><td>Chaste tree honey, common food product purchased from supermarket</td></tr><tr><td>DEVCON 5 Minute Epoxy</td><td>ITW&amp;nbsp;</td><td></td><td>Epoxy glue</td></tr><tr><td>Blunt Tip Stainless Steel Dispensing Needles (Luer Lock)</td><td>Suzhou Lanbo Needle, China</td><td>LTA820050</td><td>20G x 1/2&quot;&amp;nbsp;</td></tr><tr><td>Tungsten/Carbide Scriber</td><td>Ullman</td><td>1830</td><td>For cutting glass tubing</td></tr><tr><td>Microscope Slides</td><td>Sail Brand</td><td>7101</td><td>76.2 mm x 25.4 mm, Thickness 1 - 1.2 mm</td></tr><tr><td>Polyethylene Tubing</td><td>Scientific Commodities</td><td>BB31695-PE/5</td><td>I.D. = 0.86 mm, O.D. = 1.32 mm</td></tr><tr><td>Syringe Pumps</td><td>Longer Pump, China</td><td>LSP01-1A</td><td>3 pumps needed for the experiments</td></tr></tbody></table>

fabricating high-viscosity droplets using microfluidic capillary device with phase-inversion co-flow structure

A geração de gotículas monodisperso com alta viscosidade sempre foi um desafio em microfluídica da gota. Aqui, demonstramos um dispositivo de fluxo de co de inversão de fase para gerar gotículas uniformes de alta viscosidade em um líquido de baixa viscosidade. O dispositivo capilar microfluidic tem uma estrutura de fluxo co comum com sua saída, conectando a um tubo mais largo. Alongada gotículas de líquido de baixa viscosidade primeiro são encapsuladas pelo fluido de alta viscosidade na estrutura de fluxo de co. Como as gotas de baixa viscosidade alongadas fluem através da saída, que é tratada para ser molhadas pelo fluido de baixa viscosidade, inversão de fase é então induzida pela adesão das gotas de baixa viscosidade para a ponta da saída, o que resulta no inverso subsequente encapsulamento do fluido de alta viscosidade. O tamanho de gotas alta viscosidade resultantes pode ser ajustado alterando a relação de taxa de fluxo do fluido de baixa viscosidade para o fluido de alta viscosidade. Vamos demonstrar vários exemplos típicos da geração de gotículas de alta viscosidade com uma viscosidade até 11,9 Pas, tais como solução de glicerol, mel, amido e polímero. O método fornece uma abordagem simples e direta para gerar monodisperso gotículas de alta viscosidade, que podem ser usadas em uma variedade de aplicações baseadas em gotículas, como síntese de materiais, administração de medicamentos, ensaio de células, bioengenharia e comida de engenharia.

Um dispositivo capilar microfluidic com uma inversão de fase, estrutura de fluxo de co foi projetado para gerar monodisperso aquosa gotículas de alta viscosidade, como mostrado na figura 1A. Na Figura 1, a fase aquosa de alta viscosidade foi o glicerol, que tem uma viscosidade ηw = 1,4 Pas; a fase de óleo de baixa viscosidade foi parafina líquida, que tem uma viscosidade ηó = 0,029 P...

Watch this Scientific Journal Video about Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure at JoVE.com

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

Fabricação de gotículas de alta viscosidade usando dispositivo capilar microfluídico com estrutura de co-fluxo de inversão de fase

Um dispositivo de inversão de fase de fluxo co é demonstrado para gerar gotículas de alta viscosidade monodisperso acima 1 Pas, que é difícil perceber em microfluídica da gota.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

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10.3K visualizações.  University of Science and Technology Beijing. Um dispositivo de inversão de fase de fluxo co é demonstrado para gerar gotículas de alta viscosidade monodisperso acima 1 Pas, que é difícil perceber em microfluídica da gota.

Vídeo: Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

Fluorescence detection methods for microfluidic droplet platforms

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

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

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

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

Bioengenharia

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.

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Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

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Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

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Autor em destaque: Integrando abordagens computacionais e experimentais em oncologia de precisão

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

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