Apoio financeiro fornecido pelo NIH conceder HL89764, NSF concede 0546012, 0730503 e 0725528.

A. Biotinilação de DNA DNA plasmidial foram covalentemente biotinilado guanina com etiquetas específicas biotina, conforme descrito pelo fabricante (Mirus Bio, Madison, WI), mas dimensionada para ter ~ 1-2 rótulos biotina por DNA. DNA plasmídeo (pEGFP-C1, 4,9 kb, Clontech, Mountain View, CA) foi rotulada neste protocolo. <ol><li> Dissolver quantidade desejada de PDNA em tampão TE de DNase e livre de RNase-free (qualidade de biologia molecular-grade) de água para fazer uma solução de DNA 1μg/μL. </li><li> Realizar a reação rotulagem usando as misturas de reacção seguinte. Adicione o rótulo de TI último reagente. </li></ol> DNA para a reação 100μg: <table border="1"><tbody><tr><td> DNase-RNase livre e sem água </td><td> 75 mL </td></tr><tr><td> Tampão 10X Labeling A </td><td> 20 mL </td></tr><tr><td> 1μg/μL DNA </td><td> 100 L </td></tr><tr><td> Rotulá-la de reagente </td><td> 5 mL </td></tr><tr><td> Volume Total </td><td> 200 mL </td></tr></tbody></table><ol start="3"><li> Incubar a reação a 37 ° C por 1 hora. </li><li> Purificar a amostra rotulada por etanol ou isopropanol precipitação seguintes protocolos padrão. </li></ol> Nota: as colunas de filtração em gel com base pode levar a absorbância UV alto ou o fundo de fluorescência, que pode afetar a quantificação de DNA ou a caracterização de fluorescência. Nota: O nível de Biotinilação pode ser determinada por Haba baseado em testes. B. Rotulagem da Polymer Cy5-Catiônicos Quitosana (390 kDa, 83,5% desacetilado, Vanson, Redmond, WA) foi usado como um polímero catiônico modelo neste estudo. As aminas primária gratuita na espinha dorsal do polímero quitosana foram marcados com Cy5-NHS (Amersham Biosciences, Piscataway, NJ). <ol><li> Para facilitar a conjugação completa dos Cy5 corante, calcular a quantidade necessária de Cy5-NHS tal que a relação molar de Cy5: aminas primárias é de 1: 200. </li><li> Ajustar o pH da solução de quitosana (em tampão acetato 25mM) para ~ 6,5 pela adição de NaOH. Note-se que a reação do SNS é mais eficiente em pH básico, mas a solubilidade da quitosana aqui limita a faixa de pH de trabalho. </li><li> Agitando, lentamente adicione o montante calculado de Cy5-NHS (1 mg / ml DMSO) para a solução de quitosana de forma gota a gota. </li><li> Agitar a mistura no escuro à temperatura ambiente durante a noite. </li><li> Para purificar, diálise com 10k MWCO Slide-a-Lyzer (Pierce) por 2 horas contra tampão acetato de 1% à temperatura ambiente no escuro. </li><li> Substituir buffer e diálise outra hr 2 à temperatura ambiente no escuro. </li><li> Substituir buffer e diálise overnight a 4 ° C no escuro. </li><li> Loja purificada rotulados de polímero a -20 ° C. </li></ol> Nota: Neste estudo, uma curva padrão é construída através da medição da intensidade de emissão de Cy5 NHS-éster a 670 nm. Caracterizar a densidade de rotulagem, pela medição da emissão obtidos a 670 nm de Cy5 marcado com quitosana na curva padrão. Absorbância também pode ser usado para determinar a eficiência de marcação, mas não foi realizado aqui. C. Preparação do QD-rotulados de DNA e Cy5-Polymer A razão molar de PDNA para QD foi mantido em excesso (PDNA: QD ≈ 1, 2) para garantir a conjugação completa dos QDs para PDNA. O número de QDs rotulados em cada PDNA pode ser estimado através TEM imagens ou outros recursos equivalentes. Em nosso estudo, o número de QDs por PDNA é estimado em ~ 1-3 por TEM e espectroscopia única molécula. Use um Millipore Milli-Q de água gradiente (&gt; 18,0 MW, 0.2um filtrada) durante a preparação. <ol><li> Calcular a quantidade necessária de quitosana para 10μg PDNA desejado de acordo com relação N / P, a relação teórica de aminas protonadas na solução de quitosana para os fosfatos negativa na solução de DNA. </li><li> Adicionar estreptavidina-funcionalizados 605QDs (Qdot 605 ITK, Invitrogen, Carlsbad, CA) para a solução PDNA biotinilado. </li><li> Incubar a solução à temperatura ambiente no escuro por 15 min. </li><li> Adicione o DNA-QD rotulados em 50 solução de sulfato de sódio mM para fazer a 200μL volume final. </li><li> Diluir Cy5-quitosana, de acordo com a desejada relação N / P, com água Milli-Q para fazer a 200μL volume final. </li></ol> Nota: Mantenha a reação no escuro para evitar fotobranqueamento possível. Importante: Tenha cuidado ao usar o Qdot 605 ITK ™ estreptavidina conjugada (a série ITK), como pontos quânticos neste catálogo são projetados com a finalidade de FRET. A série Qdot regulares são conjugados com uma camada de PEG para evitar ligações não específicas, especialmente para a rotulagem celular. No entanto, essa camada adicional aumenta a distância doador receptor, resultando em reduced eficiência da transferência de energia. D. Fabricação do SU-8 Mestres usando fotolitografia padrão <ol><li> Wafer Si é piranha limpo e cozido a 200 ° C por 5 min. </li><li> Para a espessura de 25μm mestre designado, casaco girar o fotorresiste negativo (SU-8 2025, Microchem, Newton, MA), em Si wafer a 2000 rpm por 30 seg. </li><li> Bake suave a bolacha numa placa de aquecimento com uma rampa de 65 ° C / hr a 95 ° C. </li><li> Exponha à luz UV (365 nm) para 250mJ/cm 2 a máscara de um filme (CAD / Art Services, Bandon, OR), contendo o projeto de microcanais. </li><li> Pós-exposição cozer a bolacha numa placa de aquecimento com uma rampa de 65 ° C / hr a 95 ° C. </li><li> Desenvolver o wafer usando SU-8 desenvolvedor fotorresiste. </li><li> O wafer é difícil cozido estampados numa placa de aquecimento com uma rampa de 65 ° C / h até 200 ° C. Manter o wafer de 200 ° C por pelo menos 5 horas, então, gradualmente, esfriar a bolacha até à temperatura ambiente. </li></ol> Importante: rampa gradual durante o SU-8 processo de cozimento mestre é necessário, caso contrário, o SU-8 estrutura pode destacada do wafer de silício ou fissuras na estrutura do SU-8 pode ser induzida pelo estresse de liberação. E. Moldagem Réplica de PDMS com os mestres e União para a tampa de vidro <ol><li> O mestre SU-8 é colocado em um barco de pesagem. </li><li> Mix de silicone elastômero e agente de cura (Poli (dimetilsiloxano), PDMS, Sylgard 184, Dow Corning, Midland, MI), em um 10: 1 ratio. </li><li> Despeje a mistura sobre o PDMS mestre SU-8 e deixar o barco pesa num exsicador de vácuo para remover as bolhas. </li><li> Curar o PDMS a 65 ° C por 1-2 horas. </li><li> Descasque a tira PDMS do molde mestre Si. </li><li> Entradas e saídas de soco canal do dispositivo fluídico. </li><li> Limpe a tira de PDMS e tampa de vidro com etanol e, em seguida, secar ao ar. </li><li> Tratar a limpo PDMS tiras e tampa de vidro com plasma de oxigênio (20W por 1min). </li><li> Imediatamente tira o vínculo PDMS com tampa de vidro. </li><li> Deixe o chip colado microfluídicos no forno a 95 ° C para pernoite. </li></ol> Importante: O tratamento do plasma e assar durante a noite são essenciais para a força de ligação reforçada. F. Monitorar a formação de DNA Nanocomplexes No dispositivo micro <ol><li> Preencha o canal microfluídicos com água (para garantir que não haja bolhas dentro do canal microfluídicos), antes de carregar os reagentes para garantir fluidez durante o experimento. </li><li> Carregar o DNA QD-rotulados e Cy5 marcado quitosana soluções em duas seringas de vidro individuais, através da tubulação descrito no vídeo. </li><li> Conecte o tubo com as duas entradas de dispositivos microfluídicos. Ser cautelosos para não introduzir qualquer ar durante o processo. Definir a taxa de fluxo em 20nL/min (PHD-2000 bomba de seringa, Holliston, MA), sob condições de fluxo laminar. </li><li> Verifique os microcanais sob o microscópio. </li><li> Quando o fluxo é estável (~ 15 a 20 minutos), QD-mediada FRET devem ser observadas no centro do canal. </li><li> Tirar fotos de fluorescência (CCD refrigerado, Qimaging, BC, Canadá) em diferentes locais ao longo do canal. </li><li> Analisar as imagens de fluorescência com ImageJ e OriginLab. </li></ol><img src="/files/ftp_upload/1432/1432fig1.jpg" alt="Figura 1" /> Figura 1. QD-FRET fornece uma indicação sensível do início da Nanocomplexes DNA auto-montagem <ol style="list-style-type:lower-alpha;"><li> Quantum dot-mediada transferência de energia de ressonância de fluorescência (QD-FRET) pode fornecer uma indicação quantitativa e altamente sensível de estabilidade Polyplex em qualquer ambientes extra-ou intra-celular, permitindo a detecção inequívoca do início das interações entre o DNA eo portador do gene. O par FRET, 605QD e Cy5, foi escolhido com base na maximização sobreposição espectral entre o doador eo receptor e minimizar o potencial cross-talk. Para este par, a distância é 69.4Å Förster. 3 </li><li> Auto-montagem do nanocomplexes QD-FRET DNA. DNA plasmídeo aniônicos (PDNA) ea transportadora gene catiônicos foram marcadas com QD (doador de energia) e Cy5 (energia aceitante), respectivamente. QD-FRET nanocomplexes foram formados através de coacervação complexa eletrostática. Após a excitação a 488 nm, QD-FRET mediada emissão Cy5 indicado formação de um nanocomplex compacto e intacta. O tempo de permanência (t R) pode ser calculado de acordo com a distância (x) que mede a partir de onde as duas correntes se encontram para a posição de reação sob investigação, ea velocidade média do fluxo (v). Devido à natureza do fluxo laminar, mistura só ocorre na interface (centro de cada imagem), permitindo o cálculo preciso de transporte de massa em função de t R. A resolução temporal pode ser ajustada pela variação da applietaxas de fluxo d. (Inset) FRET mediada por sinal foi observada imediatamente na interface, quando as duas correntes atendidas, indicando que a ligação foi rápida, ocorrendo dentro de alguns milissegundos de acordo com as taxas de fluxo aplicadas. Bar escala: 100μm. </li></ol>

<ol>
	<li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> J Control Release. 116, 83-89 (2006).</li><li>Chen, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.</a> Mol. Ther. 16, 324-332 (2008).</li><li>Zhang, C. Y., Yeh, H. C., Kuroki, M., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Quantum-Dot-Based+DNA+Nanosensor.">Single-Quantum-Dot-Based DNA Nanosensor.</a> Nat Mat. 4, 826-831 (2005).</li></ol>

The authors have nothing to disclose.

<ul><li> Importância do nosso trabalho: <ol><li> Esta é a primeira tentativa de monitorar poliméricos DNA nanocomplex auto-montagem cinética em tempo real (milissegundo resolução) através QD-FRET respostas dentro de um chip simples microfluídicos. </li><li> QD-mediada FRET fornece uma indicação altamente sensível e quantitativa do aparecimento de interações moleculares e em todo o processo de auto-montagem, enquanto que a microfluídica oferece um microambiente bem controlados para espacialmente analisar o processo durante a síntese nanocomplex DNA. </li><li> A integração de microfluídica e nanofotônica sugere uma abordagem nova e interessante investigar qualquer tipo de reações de complexação. </li><li> O resultado QD-FRET nanocomplexes DNA poliméricos podem ser facilmente aplicadas para o estabelecimento de relações estrutura-função 1,2. </li></ol></li></ul>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-8</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU-8 2025</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>pEGFP-C1</td>
<td>4.9 kb, MW = 3.3 x 106</td>
</tr>
<tr>
<td>LabelIT Biotin Labeling Kit</td>
<td>&nbsp;</td>
<td>Mirus Bio LLC</td>
<td>MIR 3400</td>
<td>Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work.</td>
</tr>
<tr>
<td>Streptavidin 605QD</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Qdot&reg; 605 ITK&trade; Streptavidin Conjugate</td>
<td>&nbsp;</td>
<tr>
<td>Cy5-NHS Ester</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>PA15101</td>
<td>&nbsp;</td>
<tr>
<td>Chitosan</td>
<td>&nbsp;</td>
<td>Vanson</td>
<td>390 kDa </td>
<td>83.5% deacetylated</td>
</tr>
<tr>
<td>Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545C</td>
<td>No. 1; Size: 40 x 22mm</td>
</tr>
<tr>
<td>Gastight Glass Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>TLL series</td>
<td>50&mu;L to 500&mu;L depending on sample volume</td>
</tr>
<tr>
<td>Tygon Tubing</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>0.02 ID, 100ft</td>
<td>Tygon Tubes Microbore, 0.02 ID, 100ft</td>
</tr>
<tr>
<td>Connector</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>HTX-23R</td>
<td>Customized in length of 0.750"</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD-2000</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Qimaging</td>
<td>Intensified Retiga Cooled</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX-51</td>
<td>100W mercury arc lamp</td>
</tr>
<tr>
<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>v1.36b</td>
<td>http://rsb.info.nih.gov/ij</td>
</tr>
<tr>
<td>Origin Pro8</td>
<td>&nbsp;</td>
<td>OriginLab</td>
<td>Student Version</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>475AF40</td>
<td>Excitation filter in both channels</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595AF60</td>
<td>Emission filter in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>670DF40</td>
<td>Emission filter in QD-FRET channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>500 DRLP</td>
<td>Long pass dichroic in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595DRLP</td>
<td>Long pass dichroic in QD-FRET channel</td>
</tr>		</tbody>
		</table>
		</div>

combining qd-fret and microfluidics to monitor dna nanocomplex self-assembly in real-time

Avanços na genômica continuar a alimentar o desenvolvimento de terapêuticas que possam alvo patogenia a nível celular e molecular. Tipicamente funcional dentro da célula, baseados em ácido nucléico terapêuticas requerem um sistema de entrega eficiente intracelular. Uma abordagem amplamente adotado é o de DNA complexo com um portador do gene para formar nanocomplexes via eletrostática auto-montagem, facilitando a captação celular de DNA, protegendo-o contra a degradação. O desafio consiste no desenho racional de portadores do gene eficiente, uma vez que a dissociação prematura ou excessivamente ligação estável seria prejudicial para a absorção celular e eficácia terapêutica. Nanocomplexes sintetizados através da mistura de massa mostrou uma grande diversidade de comportamento intracelular desembalar e tráfico, o que foi atribuído à heterogeneidade no tamanho e na estabilidade do nanocomplexes. Essa heterogeneidade dificulta a avaliação exata da cinética de auto-montagem e contribui para a dificuldade em correlacionar suas propriedades físico para a eficiência de transfecção ou bioativos. Nós apresentamos uma convergência romance de nanofotônica (ie QD-FRET) e microfluídica para caracterizar a cinética em tempo real do nanocomplex auto-montagem sob fluxo laminar. QD-FRET fornece uma indicação altamente sensível do início de interações moleculares e medida quantitativa em todo o processo de síntese, enquanto a microfluídica oferece um microambiente bem controlados para espacialmente analisar o processo com alta resolução temporal (~ milissegundos). Para o sistema modelo de nanocomplexes poliméricos, duas etapas distintas no processo de auto-montagem foram capturados por esta plataforma analítica. O aspecto cinética do processo de auto-montagem obtidos em microescala seria particularmente valiosos para microreactor baseado reações que são relevantes para muitas aplicações de micro e nano-escala. Além disso, nanocomplexes pode ser personalizada através de um design adequado dos dispositivos microfludic, ea conseqüente QD-FRET nanocomplexes DNA poliméricos podem ser facilmente aplicadas para o estabelecimento de relações estrutura-função.

Watch this Scientific Journal Video about Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time at JoVE.com

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Nós apresentamos uma integração inovadora e poderosa de nanofotônica (QD-FRET) e microfluídica para investigar a formação de polyplexes polieletrólito, que é esperado para prestar um melhor controle e síntese de polyplexes uniforme e personalizável para à base de ácido nucléico futuro terapêutica.

Combinando QD-FRET e microfluídica para monitorar Nanocomplex DNA Automontagem-in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

Universidad San Sebastian

Research

JoVE Journal

Biology

11.0K visualizações.  Johns Hopkins University. Nós apresentamos uma integração inovadora e poderosa de nanofotônica (QD-FRET) e microfluídica para investigar a formação de polyplexes polieletrólito, que é esperado para prestar um melhor controle e síntese de polyplexes uniforme e personalizável para à base de ácido nucléico futuro terapêutica.

Vídeo: Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

Resposta quimiotática de microorganismos marinhos para Micro-Escala de Camadas de nutrientes

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Biologia

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

Estudos de Quimiotaxia bacteriana Usando microfluídica - Entrevista

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

Aplicando a microfluídica Eletrofisiologia

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

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 ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determinar perfis de expressão genética em C. elegans Usando Microarray e PCR em tempo real

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando uma única molécula de Manipulação e imagem para o estudo das interações proteína-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Baseado-Probe-PCR em tempo real Abordagens para a medição quantitativa de microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...