Este trabalho foi apoiado pela pesquisa fundamental Shenzhen financiamento (Grant no. JCYJ 20150630170146829, JCYJ20160531195439665 e JCYJ20160317152359560). Os autores que gostaria de agradecer a Prof Y. Chen no Shenzhen institutos de avançada tecnologia, da Academia Chinesa de ciências para suporta.

1. fabricação do molde
<ol><li>Duas máscaras usando um software de desenho de projeto. Descrever o contorno da estrutura microchannel e use duas camadas separadas para máscara 1 e 2 no mesmo arquivo de desenho, por isso, verifique todas as conexões entre os diferentes canais. Imprima diferentes camadas independentemente para placa no vidro por um vendedor com resolução de 1 µm de cromo. Certifique-se de que as máscaras são escuras com estruturas projetadas transparentes, como uma polaridade negativa. Nota: A máscara 1 contém o canal de entrada de fase dispersa e um orifício. A máscara 2 contém o canal de entrada da fase contínua, o filtro e a tomada.</li>
	<li>No laboratório designado fotolitografia, limpe uma bolacha de silicone de 3 polegadas de diâmetro. Coloque a bolacha em um aplicador de rotação, ligue o aspirador para fixar a hóstia para o chuck de volta. Rotação-casaco 2-3 mL de SU-8 2025 fotorresiste negativo para a bolacha de 10 s a 1.000 rpm, então 30 s a 3.000 rpm, fornecendo a espessura da primeira camada de 20 µm.</li>
	<li>Asse macio em uma chapa de 95 ° C por 6min. Depois a bolacha revestida esfria à temperatura ambiente (RT), expô-lo através da máscara 1 sob um colimado 15 mW/cm2, 365 nm de UV para 18 s. pós-exposição, asse em uma chapa de 95 ° C por 6min e, em seguida, permitir a bolacha arrefecer à RT</li>
	<li>Repita o processo de revestimento de rotação. Aplicar 2-3 mL de SU-8 2100 fotorresiste negativo para a bolacha de 10 s a 1.000 rpm, então 30 s a 2.000 rpm, fornecendo a espessura da segunda camada de 130 µm. Depois Asse macio em uma chapa de 95 ° C por 35 min, coloque a máscara 2 sobre a segunda camada fotorresiste para expor por 30 s, que foi alinhado com a camada de canal de fase dispersa por um alinhador de UV. Pós-exposição, asse em uma chapa de 95 ° C por 7 min.</li>
	<li>Desenvolva a bolacha por imersão num banho de 50 mL de propileno glicol acetato de éter metílico mexido até características tornam-se claras sobre a bolacha e, em seguida, lavagem-a com álcool etílico. Finalmente, coloque a bolacha na plataforma termostática para assar por 2 h.</li>
</ol>2. fabricação de Chip Microfluidic Flow-foco semi-3D
<ol><li>Monômero de PDMS Mix e seu agente de cura em uma relação de peso ligeiramente diferentes para a parte superior e as camadas de fundo, normalmente de 10:1 para a camada superior e 8:1 para a camada inferior, usando agitador automático pomada para 4 min. Nota: As lajes PDMS superior e inferior são preparadas em uma relação um pouco diferente (10:1 e 8:1 respectivamente) da base PDMS para agente de cura, aumentando a força de ligação. Quando a proporção de 5:1 é escolhida para a camada inferior, a laje PDMS inferior mais aguerrida dificulta o alinhamento e reduz a força de ligação, devido à falta de flexibilidade. Além disso, a espessura da laje inferior PDMS é cerca de 1 mm para adaptar a distância de trabalho de microscópio. O chip deforma-se facilmente sob a alta pressão quando é escolhida a proporção de 1:15.</li>
	<li>Despeje a mistura no molde silicone concluído em um prato de petri de 90 mm e fornecer uma espessura de 2 ~ 3 mm. Coloque-o em uma câmara de vácuo e desgaseificar até todas as bolhas de ar desaparecem. Curar a 80 ° C, durante 1 h no forno.</li>
	<li>Permita o PDMS arrefecer RT. uso um bisturi para cortar o dispositivo pelo menos 3 mm longe as características e lentamente retire a camada PDMS do wafer de silício. Socar a entrada da fase dispersa, a entrada da fase contínua e a saída na camada superior de PDMS usando um soco de diâmetro de 0,75 mm.</li>
	<li>Limpe o PDMS com fita adesiva para remover partículas de poeira. Plasma tratar a parte superior e inferior camadas PDMS simultaneamente por 2 min em um plasma W 300 limpador. Coloque a camada superior da superfície da camada inferior e deslize as superfícies relativamente até características estão alinhadas a visualização através de um microscópio estéreo.</li>
	<li>Cure o dispositivo em um forno a 120 ° C por um dia aumentar a força e completar a ligação.</li>
</ol>3. reagentes preparação
<ol><li>Preparar a solução da fase contínua: hexadecano dissolvendo-se 18 vol % à base de silicone não iónico surfactante.</li>
	<li>Preparar a solução da fase dispersa: hidrofílico poli (etileno glicol) diacrylate (PEGDA, 255 MW,) contendo 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (98%, 224 MW) na concentração de 5 mg/mL como o foto-iniciador e rodamina B ( 95%, 479.01 MW) em uma concentração de 1 mmol/L, como os corantes fluorescentes.</li>
	<li>Encha os reservatórios de 1ml de controlador de pressão pneumática com a fase contínua. Encha a ponta de carregamento de gel de 200 µ l com a fase dispersa.</li>
</ol>4. sistema preparação
<ol><li>Coloque o dispositivo microfluidic semi-3D no palco do microscópio óptico invertido, visualização com uma câmera de alta velocidade.</li>
	<li>Conectar o tubo de propileno (FEP) de etileno fluorado no buraco perfurado da fase contínua, anexando um curto tubo de aço inoxidável e, em seguida, insira a extremidade de uma carga de gel de ponta no orifício perfurado da fase dispersa. Insira um comprimento de 20 cm de tubo de FEP na saída no aparelho e coloque a extremidade de um tubo de centrífuga de 15 mL. Nota: A configuração do sistema é ilustrada na Figura 1.</li>
</ol>5. formação de gotas
<ol><li>Coloque o dispositivo na bancada de trabalho de um microscópio invertido e certifique-se de que a junção de diferentes canais aproximadamente está localizada na posição da fonte de luz do microscópio. Foco do microscópio invertido em uma região que contém a interseção de duas fases, a região do orifício e o canal a jusante.</li>
	<li>Conjunto de pressões de duas fases, usando o controlador de pressão pneumática para entregar o líquido lentamente para a região de interseção, com 15 mbar para a fase dispersa e 30 mbar para a fase contínua. Espere 3 min para estabilização e equilíbrio até o fluxo de fluido estável carregando sem bolhas e resíduo PDMS.</li>
	<li>Os parâmetros na interface de usuário do software de controle de entrada. Ajustar a pressão da fase dispersa como a pressão de base-nível do sistema para uma constante, por exemplo, mantê-lo em 45 mbar. Aumentar a pressão da fase contínua até que o modo de separação de emulsão é alterado da jorrar para o modo streaming de ponta e, em seguida, espere 5 min para estabilização.</li>
	<li>Coloque a extremidade do tubo de FEP conectando o furo de saída para o tubo de centrifugação para recolher as gotas.</li>
</ol>6. PEGDA partículas coleta e caracterização
<ol><li>Fixar o tubo de centrifugação obliquamente a fixação. Obter as partículas PEGDA através da solidificação rápida pela exposição aos raios UV quando fase líquida de fluxo para o tubo.</li>
	<li>Ao finalizar o processo de coletando, a amostra e observar as partículas PEGDA através do microscópio fluorescente com 20 X ou 60 objetos X, respectivamente. Nota. As imagens digitais fluorescentes capturadas pela câmera são analisadas por uma rotina de software sob medida. A imagem é primeira deconvolução baseada o algoritmo R-L17 para eliminar o efeito de desfocagem do fora-de-foco de luz e a esfera de objetos na imagem são extraídos com base no método de deteção de borda sagaz; Finalmente, o diâmetro de cada objeto da esfera pode ser calculado usando Hough transformação18. Como resultado, média e desvio padrão dos diâmetros dos objetos da esfera em cada imagem podem ser estimados.</li>
</ol>

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Os autores não têm nada para divulgar.

A geração de gotículas no modo de focagem fluxo usando 2D e semi-3D microfluidic dispositivo anteriormente foi desenvolvida em uma variedade de relatórios8,9,15,19,20, 21. Nestes sistemas, o líquido aquoso que não poderia ser solidificado foi escolhido como a fase dispersa, tais como água desionizada...

Sistemas baseados em gotículas microfluidic têm a capacidade de produzir altamente monodisperso gotículas de nanômetros de micrômetro de diâmetro faixa1 e mantenha o grande potencial no elevado-throughput droga descoberta2, síntese de biomoléculas3 ,4e o diagnóstico teste5. Devido as vantagens únicas de gotículas menores, tais como a maior área de superfície a relação entre o volume e as aplicações em grande escala com consumo de alguns microlitros de amostra, a tecnologia tem atraído interesse extensa em uma ampla gama de campos. A emulsificação de dois líquidos imiscíveis é um dos métodos mais comuns para gerar gotículas. Em relatórios anteriores no campo, os pesquisadores desenvolveram uma variedade de geometrias de formação diferente da gota, incluindo entroncamento, fluxo de focagem e co fluindo geometrias. A geometria do entroncamento, a fase dispersa é entregue através de um canal perpendicular para o canal principal, na qual a fase contínua flui6,7. A típica bidimensional (2D) fluxo de focagem8,9 , na geometria do fluxo de fase dispersa é cortado de lateral; e para o co fluindo geometria10,11, por outro lado, um capilar introduzindo o fluxo da fase dispersa é colocado co-axial dentro de um capilar maior para a geometria co fluindo, para que o fluxo da fase dispersa é cortado de todas as direções.
O tamanho da gota é controlado ajustando a relação de taxa de tamanho e fluxo de canal, e o tamanho mínimo produzido por co fluindo ou entroncamento é limitado a dezenas de micrômetros. Para o sistema de formação de gotículas fluxo de focagem, três modos de separação da gota formam ajustando a relação de pressão de duas fases e a concentração de surfactante, incluindo o gotejamento regime, o regime de jorrando e streaming de ponta15. Dica-fluxo contínuo modo também é chamado de formação do segmento e a aparência de uma fina será observado o segmento de desenho da ponta do cone de fluxo da fase dispersa. Estudos anteriores demonstraram gotículas menor que alguns micrômetros poderiam ser gerados que ponta-fluxo contínuo processo em 2D ou 3D semi fluxo-foco dispositivo8,12. No entanto, como uma solução aquosa contendo uma concentração muito baixa de PEGDA foi usada como a fase dispersa, a taxa de encolhimento de partículas PEGDA era cerca de 60% de gotas de diâmetro originais após foto-polimerização, enquanto PEGDA sem diluição como o fase dispersa levou ao modo instável streaming de ponta12. Tensão interfacial é um parâmetro importante do processo de emulsão e diminuirá devido à adição de tensoativo para o líquido da fase contínua, levando à diminuição do tamanho das gotas, maior frequência de geração13, ponta altamente curvada, e prevenção de instabilidade14. Além disso, quando a concentração de surfactante em massa é muito maior do que a concentração crítica micelle, a tensão interfacial é aproximadamente invariável no estado saturado13 e o modo streaming de ponta pode ocorrer15.
Baseia as observações acima, neste trabalho, desenvolvemos uma abordagem superficial para geração de gotículas PEGDA usando um semi-3D fluxo de focagem microfluidic dispositivo, fabricado pelo método de litografia macia multi-camada. Diferentes do dispositivo de fluxo de focagem 2D típico, o dispositivo de fluxo de focagem semi-3D tem um canal raso fase dispersa e um canal de fase profunda contínua, para que a fase dispersa pode ser cortada de cima e para baixo ao lado lateral. Isso fornece maior gama de ajuste para o modo de fluxo-foco, reduzindo a energia e a pressão necessária para a separação da gota. Diferente do anterior relatório12, a fase dispersa é puro PEGDAcontaining foto-iniciador, certificando-se que a taxa de encolhimento de partículas PEGDA é inferior a 10%,16; e a fase contínua é a mistura de hexadecano dissolvendo-se com uma concentração alta em massa de tensoativo não iônico à base de silicone. Gotículas de tamanho controlável e uniformes foram produzidas, ajustando a relação de pressão de duas fases. O diâmetro de gotas muda de 80 µm a 1 µm, como a separação da gota processa alterações do modo jorrando para o modo streaming de ponta. Além disso, a partícula PEGDA foi sintetizada através do processo de foto-polimerização sob exposição aos raios UV. O sistema de microfluidic de geração de gotículas com facilidade de fabricação irá fornecer mais possibilidades para aplicações biológicas.

<table><tbody><tr><td>Silicon wafer</td><td>Huashi Co., Ltd</td><td></td><td></td></tr><tr><td>SU-8 2025, 2100</td><td>Microchem Co.</td><td>Y111069</td><td></td></tr><tr><td>SU-8 developer</td><td>Microchem Co.</td><td>Y020100</td><td></td></tr><tr><td>Chromium mask</td><td>Qingyi Precision Mask Making Co., Ltd</td><td></td><td></td></tr><tr><td>polydimethylsiloxane(PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>poly(ethylene glycol) diacrylate (PEGDA)</td><td>Sigma</td><td>26570-48-9</td><td></td></tr><tr><td>2-hydroxy-40-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>TCI</td><td>H1361-5G</td><td>photoinitiator</td></tr><tr><td>Hexadecane</td><td>Sigma</td><td>544-76- 3</td><td></td></tr><tr><td>ABIL EM 90</td><td>CHT</td><td>144243-53-8</td><td>surfactants</td></tr><tr><td>Rhodamine B</td><td>Aladdin</td><td>81-88-9</td><td>fluorescent dye</td></tr><tr><td>Spin Coater</td><td></td><td></td><td></td></tr><tr><td>Lithography machine</td><td></td><td></td><td></td></tr><tr><td>Automatic ointment agitator</td><td>Thinky</td><td>ARV-310</td><td></td></tr><tr><td>Oven</td><td>BluePard</td><td></td><td></td></tr><tr><td>Optical microscope</td><td>OLYMPUS</td><td>IX71</td><td></td></tr><tr><td>High-speed camera</td><td>Hamamatsu, Japan</td><td>ORCA-flash</td><td></td></tr><tr><td>MAESFLO Microfluidic Fluid Control System</td><td>FLUIGENT</td><td>MFCS-EZ</td><td></td></tr><tr><td>UV lamp</td><td>FUTANSI</td><td></td><td>365 nm UV light, 8000 MW/CM&lt;sup&gt;2&lt;/sup&gt;</td></tr></tbody></table>

generation of size-controlled poly (ethylene glycol) diacrylate droplets via semi-3-dimensional flow focusing microfluidic devices

Gotículas de diacrylate (PEGDA.) uniforme e tamanho-controláveis poli (etileno glicol) poderiam ser produzidas através do fluxo focando o processo em um dispositivo microfluidic. Este trabalho propõe um semi-three-dimensional (semi-3D) fluxo de focagem microfluidic chip para formação de gotículas. O chip do polydimethylsiloxane (PDMS) foi fabricado usando o método de litografia macia multi-camada. Hexadecano contendo surfactante foi usado como a fase contínua e PEGDA com iniciador-foto do ultravioleta (UV) foi a fase dispersa. Surfactantes permitiu a tensão de superfície local soltar e formaram uma dica mais cúspides que promoveu quebrando em microgotículas. Como a pressão da fase dispersa era constante, o tamanho das gotas de tornou-se menor com o aumento da pressão da fase contínua antes de fase dispersa fluxo foi interrompido. Como resultado, as gotas com variação de tamanho de 1 µm a 80 µm de diâmetro poderiam ser alcançadas seletivamente, alterando a relação de pressão em dois canais de entrada, e o coeficiente médio de variação foi estimado para ser abaixo de 7%. Além disso, as gotas se transformava em microgrânulos pela exposição aos raios UV para foto-polimerização. Conjugação de biomoléculas superfície tão microgrânulos têm muitas aplicações potenciais nas áreas de biologia e química.

O chip de microfluidic fluxo de focagem semi-3D foi fabricado usando técnicas de litografia macia multi-camada conforme descrito acima. O processo de fabricação e os resultados para o molde mestre no protocolare mostrado na Figura 2. A primeira camada, que proporciona um canal de 65 µm de largura por introduzir a fase dispersa e uma 50 µm Largura do orifício (Figura 2um), é de 20 µm de espessura. Uma cama...

Watch this Scientific Journal Video about Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices at JoVE.com

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Aqui, apresentamos um protocolo para ilustrar os processos de fabricação e verificando as experiências de um semi-three-dimensional (semi-3D) fluxo de focagem microfluidic chip para formação de gotículas.

Geração de tamanho controlado poli (etileno glicol) Diacrylate gotículas através de Semi-3-Dimensional Flow focando Microfluidic dispositivos

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

generation-size-controlled-poly-ethylene-glycol-diacrylate-droplets

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

7.7K visualizações.  Tsinghua University. Aqui, apresentamos um protocolo para ilustrar os processos de fabricação e verificando as experiências de um semi-three-dimensional (semi-3D) fluxo de focagem microfluidic chip para formação de gotículas.

Vídeo: Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

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

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Dispositivo micro centrífuga baseada capilar para a Formação tamanho controlável de Monodisperse microgotas

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

Engenharia

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 ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Uma matriz de gotícula femtoliter para síntese de proteínas massivamente paralelas de moléculas de DNA único

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Química

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.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

Fabricando degradáveis hidrogel de Thermoresponsive em várias escalas de comprimento através da extrusão reativa, microfluídica, auto-montagem e eletrofiação

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

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

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

Dispositivos baseados em vidro para gerar gotas e emulsões

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

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.

Multi-passo fotolitografia Variável Altura para Valved multicamada microfluídicos Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

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

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

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

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

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

Configuração de um dispositivo microfluídico para produção combinatória de plugues

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

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.

Dispositivo microfluídico bicamada para produção de plugue combinatório

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...