Name: taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics
Uploaded: 2014-11-10T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

Agradecemos à Fundação Lindback de apoio financeiro, e Dr. Alexandre Sidorenko e Elza Chu para discussões frutíferas e assistência técnica, eo professor Robert Smith para a assistência com o C. Os ensaios de elegans.

NOTA: Nos procedimentos descritos a seguir, as diretrizes de segurança do laboratório deve ser sempre seguido. De particular importância é a segurança quando se lida com alta voltagem (&gt; 500 V) e de manuseamento de produtos químicos. 1. revestimento de um substrato condutor com vela Fuligem <ol><li> Cobre corte de metal em retângulos (75 x 43 mm, 0,5 mm de espessura). Limpe cada substrato de cobre por imersão em etchant cobre por cerca de 30 segundos, lavar com água corrente por cerca de 20 segundos, e seque com papel. NOTA: Se estiver usando o método 1 a seguir, alterar as dimensões de 75 x 25 mm para caber na máquina. </li><li> Varrer uma vela de parafina iluminada sob o substrato de cobre durante 30-45 segundos, para se obter um revestimento de fuligem aproximadamente uniforme (cerca de 40 um de espessura). Mantenha o substrato em ~ 1 cm para dentro da chama. Não toque na superfície fuligem frágil. </li></ol> 2. Proteger a camada de fuligem com Revestimento NOTA: A camada de fuligem é muito frágil, E devem ser revestidos para protecção. Duas alternativas simples (métodos 1 e 2 abaixo) são sugeridos aqui, mas protocolos mais robustos estão atualmente em desenvolvimento. <ol><li> Método 1 <ol><li> Coloque a amostra para o evaporador de metal ou de sistema de pulverização. Seguindo os procedimentos de funcionamento do sistema, evacuar a câmara, e começar a deposição controlada de ouro sobre a camada de fuligem (150-200 nm). Deixe o dispositivo de arrefecer até à temperatura ambiente. </li><li> Dip-revestir o substrato metalizado em uma solução de 1-dodecanotiol (1% v / v, em etanol a 95%, ACS / grau USP), durante 10 minutos dentro de uma capa química. Então, segurando o aparelho em um ângulo perto de 60 °, lavar cuidadosamente a superfície com várias gotas de etanol só. Deixe os dispositivos seca, durante a noite. </li></ol></li><li> Método 2 <ol><li> Em uma capa de química, imediatamente depois do revestimento do substrato com a fuligem e ao mesmo tempo o substrato ainda está quente da chama da vela, depositar algumas gotas de líquido fluorado sobre um lado dosubstrato, e inclinar o substrato a um ângulo próximo de 90 °. Deposite mais gotas, e deixá-los rolar sobre a superfície inteira fuligem. NOTA: Quando a gota cai em um ponto, a fuligem serão lavados daquela área. Deixe as gotículas de líquido propagação fluorado, tanto quanto possível. </li><li> Asse o substrato sobre uma placa quente (160 ° C durante 15 min) no interior de uma capa química. </li><li> Deixe o substrato se sentar durante a noite à temperatura ambiente antes do uso. Armazenar indefinidamente. </li></ol></li></ol> (12 Adaptado de Abdelgawad et al.) 3. Fabricação de Top Eletrodos <ol><li> Desenhe os eletrodos utilizando software de design gráfico. Cada eletrodo é de 2 mm de comprimento, 0,3 mm de largura, ea diferença entre os eletrodos é de 0,3 mm. A diferença entre contatos (para encaixar no conector, veja abaixo) é de 2,3 mm (Figura 1). </li><li> Apare um laminado de cobre flexível (35 mm de espessura) para o formato Monarch (3,87 x 7,5 polegadas). Use outros tamanhos if compatível com a impressora. Coloque o laminado na bandeja de alimentação manual de uma impressora colorida. </li><li> Certifique-se de usar o &quot;preto rico&quot;, ou &quot;Registro de preto&quot;, ao imprimir na folha de cobre (ver 12 Abdelgawad et al. Para mais detalhes) para permitir que uma camada mais densa de tinta preta sobre o substrato de cobre, protegendo o padrão impresso durante gravura . Deixe o substrato seco impresso completamente, durante a noite. </li><li> Dentro de uma capa química, aquecer (40 ° C) num copo com 50 ml de produto corrosivo de cobre. Mergulhe o laminado impresso no copo e agite-o na solução por cerca de 10 min. Tempo de condicionamento depende da solução de produto corrosivo de cobre. A cada poucos minutos, verifique a corrosão e ver se o padrão está intacta. </li><li> Cuidadosamente lavar o laminado com água, e remover o revestimento com acetona e etanol na capa química. Lavar uma vez, e seca suavemente com a toalha de papel laminado. </li><li> Cuidadosamente anexar o laminado com eletrodos a uma ABLslides ss (75 x 25 mm, ~ 1 mm de espessura), usando fita dupla face. Evite bolsas de ar. </li><li> Anexar um filme de perfluoroalcoxi PFA aos eletrodos utilizando fita. Isso serve para evitar o contato acidental dos eletrodos com a gota, o que prejudica top eletrodos devido a curto-circuito. </li></ol> 4. A interface eletrônica (circuito na Figura 2) <ol><li> Soldar os relés e os capacitores C a uma placa de circuito universal. </li><li> Monte o restante dos 10 pilotos de revezamento em uma placa de ensaio sem solda para circuitos eletrônicos. </li><li> Fio de entrada de cada motorista relé para um canal na placa de controle. </li><li> Tirar com cuidado os melhores eletrodos em um conector (Figura 3). Fio condutor de saída de cada retransmissão a um eléctrodo de topo, como mostrado na figura. Note que existe um contato do conector aterrado entre um par de fios de relés, para minimizar o ruído elétrico. NOTA: O conector fica em uma plataforma ajustável para controlar tele distância (0,1-0,5 mm) entre o substrato superior e inferior (fuligem-revestido). </li><li> Utilizar um programa para controlar a temporização para a aplicação de alta voltagem (HV) (cerca de 0,8 seg) para quatro eléctrodos, ao mesmo tempo, deslocando um eléctrodo na direcção de movimento (ou seja, durante 0,8 seg, accionar 1234; em seguida, 2345, 3456, etc. ., 0,8 segundos para cada grupo, e depois para trás, move-se de forma de gotículas na direcção oposta, como bem). </li></ol> 5. Visualização Gota e Manuseio <ol><li> Para gravar gotícula de movimento, utilizar o sistema de visualização, o qual é composto por uma 24X - montagem de ampliação 96X combinado com uma câmara CCD. Ligue o gravador de vídeo para a câmera usando S-video. </li><li> Pipetar uma gota de 4 ul contendo C. elegans em meios na parte inferior do substrato revestido de fuligem. </li><li> Trazer os melhores eletrodos para ~ 0,3 milímetros acima da gota. A gota deve ser próximo ao centro, logo abaixo do quinto eletrodo, para facilitar a operação. </li><li> Ligue a interface eletrônica e alta tensão (500 V RMS), e ajustar a distância do eléctrodo superior à gota, até que começa a se mover. Não deixe que os melhores eletrodos tocar a gota. </li><li> Recolha de dados de registo do número de transferências de gotículas de sucesso no dispositivo em resposta a impulsos eléctricos. Uma experiência de sucesso é caracterizado por, pelo menos, 700 transferências de gotículas, ou seja, uma transferência após cada pulso eléctrico. </li><li> Recolha de dados continuamente, até que a gotícula não mais mover-se em resposta a 5 a 10 pulsos. NOTA: Quando a superfície começa a degradar-se, o movimento pode ser restaurado, trazendo os melhores eléctrodos mais perto da gotícula. </li></ol>

<ol>
	<li>Fair, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics:+is+a+true+lab-on-a-chip+possible.">Digital microfluidics: is a true lab-on-a-chip possible.</a> Microfluid Nanofluid. 3 (3), 245-281 (2007).</li><li>Gupta, S., Alargova, R. G., Kilpatrick, P. K., Velev, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-Chip+Dielectrophoretic+Coassembly+of+Live+Cells+and+Particles+into+Responsive+Biomaterials.">On-Chip Dielectrophoretic Coassembly of Live Cells and Particles into Responsive Biomaterials.</a> Langmuir. 26 (5), 3441-3452 (2009).</li><li>Shih, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spot+analysis+by+digital+microfluidics+coupled+to+nanoelectrospray+ionization+mass+spectrometry.">Dried blood spot analysis by digital microfluidics coupled to nanoelectrospray ionization mass spectrometry.</a> Anal Chem. 84 (8), 3731-3738 (2012).</li><li>Gorbatsova, J., Borissova, M., Kaljurand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-on-dielectric+actuation+of+droplets+with+capillary+electrophoretic+zones+for+off-line+mass+spectrometric+analysis.">Electrowetting-on-dielectric actuation of droplets with capillary electrophoretic zones for off-line mass spectrometric analysis.</a> J Chromatogr. 1234, 9-15 (2012).</li><li>Qin, J., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maze+exploration+and+learning+in+C.+elegans.">Maze exploration and learning in C. elegans.</a> Lab Chip. 7 (2), 186-192 (2007).</li><li>Koc, Y., de Mello, A. J., McHale, G., Newton, M. I., Roach, P., Shirtcliffe, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-scale+superhydrophobicity:+suppression+of+protein+adsorption+and+promotion+of+flow-induced+detachment.">Nano-scale superhydrophobicity: suppression of protein adsorption and promotion of flow-induced detachment.</a> Lab Chip. 8 (4), 582-586 (2008).</li><li>Perry, G., Thomy, V., Das, M. R., Coffinier, Y., Boukherroub, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+protein+biofouling+using+graphene+oxide+in+droplet-based+microfluidic+microsystems.">Inhibiting protein biofouling using graphene oxide in droplet-based microfluidic microsystems.</a> Lab Chip. 12 (9), 1601-1604 (2012).</li><li>Kumari, N., Garimella, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrowetting-Induced+Dewetting+Transitions+on+Superhydrophobic+Surfaces.">Electrowetting-Induced Dewetting Transitions on Superhydrophobic Surfaces.</a> Langmuir. 27 (17), 10342-10346 (2011).</li><li>Freire, S. L. S., Tanner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive-Free+Digital+Microfluidics.">Additive-Free Digital Microfluidics.</a> Langmuir. 29 (28), 9024-9030 (2013).</li><li>Deng, X., Mammen, L., Butt, H. -. J., Vollmer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candle+Soot+as+a+Template+for+a+Transparent+Robust+Superamphiphobic+Coating.">Candle Soot as a Template for a Transparent Robust Superamphiphobic Coating.</a> Science. 335, 67-70 (2011).</li><li>Kang, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Electrostatic+Fields+Change+Contact+Angle+in+Electrowetting.">How Electrostatic Fields Change Contact Angle in Electrowetting.</a> Langmuir. 18 (26), 10318-10322 (2002).</li><li>Abdelgawad, M., Watson, M. W. L., Young, E. W. K., Mudrik, J. M., Ungrin, M. D., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography:+masters+on+demand.">Soft lithography: masters on demand.</a> Lab Chip. 8 (8), 1379-1385 (2008).</li><li>Barbulovic-Nad, I., Yang, H., Park, P. S., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+microfluidics+for+cell-based+assays.">Digital microfluidics for cell-based assays.</a> Lab Chip. 8 (4), 519-526 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li></ol>

The authors declare that they have no competing financial interests.

A etapa mais crítica do protocolo é a proteção da camada de fuligem, diretamente associado ao sucesso em gotas em movimento. Metalização da camada de fuligem (método 1 acima) permite que perto de 100% de sucesso de fabricação. No entanto, o tempo máximo de operação é de cerca de 10 min; possivelmente, fracções de gotículas são molhar a fuligem através de furos na camada de metal. Revestimento da camada de fuligem com o líquido fluorado é a alternativa mais fácil e rápida, e exige o mínimo de recur...

A miniaturização dos dispositivos que funcionam com líquidos é de suma importância para o desenvolvimento de plataformas &quot;lab-on-a-chip&quot;. Nesse sentido, as duas últimas décadas têm testemunhado um progresso significativo na área da microfluídica, com uma variedade de aplicações. 1-5 Contrastando com o transporte de fluidos em canais fechados (canal microfluídicos), DMF manipula gotas em matrizes de eletrodos. Um dos méritos mais atraentes desta técnica é a ausência de partes móveis para o transporte de fluidos, e movimento é imediatamente interrompida ao desligar sinais elétricos. No entanto, o movimento das gotículas é dependente de conteúdos das gotículas, certamente uma característica indesejável para uma plataforma universal &quot;lab-on-a-chip&quot;. Gotículas contendo proteínas e outros analitos aderir a superfícies do dispositivo, tornando-se inabalável. Indiscutivelmente, esta tem sido a principal limitação para o alargamento do âmbito de aplicações DMF; 6-8alternativas para minimizar a proliferação indesejada superfície envolvem a adição de espécies químicas extras para a gota ou seus arredores, o que poderia afetar o conteúdo da gota. Anteriormente, o nosso grupo desenvolveu um dispositivo para permitir o transporte de células e proteínas em DMF, sem aditivos adicionais (dispositivos Campo-DW). 9 Isto foi conseguido através da combinação de uma superfície com base na fuligem vela, 10 com uma geometria do dispositivo que favorece a gotícula de rolamento e leva a uma força ascendente sobre a gotícula, diminuindo ainda mais a interacção gotícula-superfície. Nesta abordagem, gota movimento não está associado a molhar a superfície 11. O objectivo do método descrito abaixo é detalhado para produzir um dispositivo de DMF capaz de transportar gotas contendo proteínas, células e organismos inteiros, sem aditivos adicionais. Os dispositivos de campo-DW pavimentar o caminho para as plataformas totalmente controlados que trabalham em grande parte de forma independente da gota de químicory. Aqui, nós também apresentam simulações mostra que, apesar da alta tensão necessária para o funcionamento do dispositivo, a queda de tensão através da gotícula é uma pequena fracção da tensão aplicada, indicando efeitos insignificantes sobre bioanalytes no interior da gotícula. Na verdade, os testes preliminares com Caenorhabditis elegans (C. elegans), um nemátodo usado para uma variedade de estudos em biologia, mostram que os vermes nadar sem perturbações como as tensões são aplicadas.

<table><tbody><tr><td>Paraffin candle</td><td></td><td></td><td>Any paraffin candle</td></tr><tr><td>Sputtering system</td><td>Denton Vacuum, Moorestown, NJ</td><td></td><td>Sputter coater Desk V HP equipped with an Au target.&amp;nbsp;</td></tr><tr><td>1-dodecanethiol</td><td>Sigma-Aldrich</td><td>471364</td><td></td></tr><tr><td>Teflon</td><td>Dupont</td><td>AF-1600</td><td></td></tr><tr><td>Fluorinert FC-40</td><td>Sigma-Aldrich</td><td>F9755</td><td>Fluorinated liquid: Prepare Teflon-AF resin in Fluorinert FC-40, 1:100 (w/w), to create the hydrophobic coating.</td></tr><tr><td>Graphic design software -Adobe Illustrator</td><td>Adobe Systems</td><td></td><td>Other softwares might be used as well.</td></tr><tr><td>Copper laminate</td><td>Dupont</td><td>LF9110</td><td></td></tr><tr><td>Laser Printer</td><td>Xerox</td><td>Phaser 6360 or similar</td><td>Check for the compatibility with &quot;rich black&quot; or &quot;registration black&quot; (see text).</td></tr><tr><td>Copper Etchant</td><td>Transene</td><td>CE-100</td><td></td></tr><tr><td>Perfluoroalkoxy (PFA) film</td><td>McMaster-Carr</td><td>84955K22</td><td></td></tr><tr><td>Breadboard</td><td>Allied Electronics</td><td>70012450 or similar</td><td>Large enough to allow the assemble of 10 drivers.</td></tr><tr><td>Universal circuit board</td><td>Allied Electronics</td><td>70219535 or similar</td><td></td></tr><tr><td>Connector</td><td>Allied Electronics</td><td>5145154-8 or similar</td><td></td></tr><tr><td>Control board and control program (LabView software)</td><td>National Instruments</td><td>NI-6229 or similar</td><td></td></tr><tr><td>High-voltage amplifier</td><td>Trek</td><td>PZD700</td><td></td></tr><tr><td>Capacitors C and C1, 100 nF, 60 V</td><td>Allied&amp;nbsp;</td><td>8817183</td><td></td></tr><tr><td>Transistor T, NPN</td><td>Allied&amp;nbsp;</td><td>9350289</td><td></td></tr><tr><td>Diode D, 1N4007</td><td>Allied&amp;nbsp;</td><td>2660007</td><td></td></tr><tr><td>Relay&amp;nbsp;</td><td>Allied&amp;nbsp;</td><td>8862527</td><td></td></tr><tr><td>Visualization system</td><td>Edmund Optics</td><td>VZM 200i or similar</td><td>System magnification 24X &amp;#8211; 96X. It is combined with a Hitachi KP-D20B 1/2 in CCD Color Camera.</td></tr><tr><td>Recorder</td><td>Sony</td><td>GV-D1000 NTSC or similar</td><td>It is connected to the camera by an S-video cable.</td></tr><tr><td>Simulations</td><td>COMSOL Multiphysics</td><td>V. 4.4</td><td></td></tr></tbody></table>

taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

Anteriormente, foram utilizados dispositivos Campo-DW para permitir o movimento de proteínas em DMF. Em particular, as gotas com albumina de soro bovino (BSA) pode ser movido a uma concentração 2000 vezes mais elevada do que o relatado anteriormente por outros autores (sem aditivos). Isto era devido à interacção reduzida entre as gotículas e a superfície; A Figura 4 mostra uma gota contendo fluorescentemente marcado com BSA (ver Freire et al 9 para obter mais informação sob...

Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Aproveitando-se de Redução Interação gota-superfície para otimizar o transporte de Bioanalytes em microfluídica Digital

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

taking-advantage-reduced-droplet-surface-interaction-to-optimize

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Engineering

Unit 1

The Laplace Transform

SCIENTISTS IN ACTION

7.8K visualizações.  University of the Sciences. The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Vídeo: Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

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

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Um chip microfluídico para ICPMS Amostra Introdução

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

A Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

Método baseado em uma ponta da pipeta para semeadura pilhas Droplet Microfluidic plataformas

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and ...

Engenharia

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

A microfluídica digital para processamento automatizado Proteômica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biologia

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

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Microfluídico troca de tampão para Micro / nanopartículas celular Engenharia sem interferências

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Plataforma de Microfluidos Digitais baseada em eletrowetting para ensaio imunosorbent automatizado ligado à enzima

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

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 ...

Aproveitando-se de Redução Interação gota-superfície para otimizar o transporte de Bioanalytes em microfluídica Digital

Resumo

Explore mais vídeos

Capítulos neste vídeo

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

Um chip microfluídico para ICPMS Amostra Introdução

Método baseado em uma ponta da pipeta para semeadura pilhas Droplet Microfluidic plataformas

A microfluídica digital para processamento automatizado Proteômica

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

Microfluídico troca de tampão para Micro / nanopartículas celular Engenharia sem interferências

Plataforma de Microfluidos Digitais baseada em eletrowetting para ensaio imunosorbent automatizado ligado à enzima

Dispositivo microfluídico bicamada para produção de plugue combinatório

Dispositivo microfluídico bicamada para produção de plugue combinatório

Dispositivo microfluídico bicamada para produção de plugue combinatório