O. Kurtulus e D. Dimlioglu são suportados por um laboratório taxas Research Award Programa Universidade da Califórnia 12-LR-237197. P. Daggumati é apoiado por uma Universidade da Califórnia Davis investimentos em pesquisa no (RISE) Prêmio de Ciência e Engenharia. CA Chapman é apoiado por um departamento de pós-graduação Áreas de Assistência de Fellowship Nacional Need. Este trabalho foi apoiado pelo Programa Lab UC taxas Research, UC Davis RISE, e UC Davis Faculdade de Engenharia fundos start-up.

1. Nanoporous Fabrication Ouro <ol><li> Substratos limpos em solução Piranha <ol><li> Adicionar 25 ml de peróxido de hidrogénio (30%) a 100 ml de ácido sulfúrico (96%) em um prato de cristalização e aquecer a mistura a 65 ° C numa placa de aquecimento. AVISO: Os líquidos são extremamente corrosivo e deve ser manuseado com cuidado. A solução passou não deve ser armazenado num recipiente fechado, como pode explodir. </li><li> Coloque 1 polegada por lâminas de microscópio de 3 polegadas na mistura usando uma pinça anti-ácido e limpá-los por 10 min. Use um barco immunostaining porcelana para lote limpeza pequenas lamelas. Tratar com pequenas lamelas de plasma de ar de 10 W durante 30 segundos antes da imersão na solução. </li><li> Lavar as amostras limpas em pratos de cristalização sob água corrente deionizada (DI) por 3 min. Amostras secar com uma arma de nitrogênio mais sem fiapos toalhas. </li></ol></li><li> Prepare máscara stencil (Método 1: Use este para a criação de padrões milímetros escala) <ol><li> Perfurador 250 folhas de elastômero de silicone m de espessura com biópsia socos e / ou fora inciso regiões com um bisturi sobre uma superfície de plástico semi-rígido. Limpe as folhas de elastômero processados ​​em 70% isopropanol e seco com arma de nitrogênio. </li><li> Coloque a folha de soco em uma toalha que não solte fiapos sala limpa e alinhar as lamelas piranha-limpos sobre o estêncil com a superfície da amostra de frente para o stencil. </li></ol></li><li> Padrão lift-off photoresist (Método 2: Usar isso para a criação de padrões sub-milímetro escala) <ol><li> Coloque uma piranha-limpa lâmina de microscópio girando chuck e explodir a qualquer partículas com arma de nitrogênio. Pipetar 1,5 ml de promotor de aderência (hexametildisilazano) sobre a lâmina de vidro usando uma pipeta de plástico. Espalhar o promotor girando a corrediça, sucessivamente, a 500 rpm durante 5 segundos e 1500 rpm durante 30 segundos. Asse o slide numa placa de aquecimento a 115 ° C por 7 min e deixe esfriar por 5 min. </li><li> Dispensar 4 ml de material fotosensitivo sobre a lâmina de vidro (de 3 polegadas by 1 polegada). Espalhar o material fotosensitivo, girando o diapositivo com o mesmo protocolo como para o promotor de adesão. Asse o fotorresiste numa placa de aquecimento a 115 ° C por 1,5 min e deixe esfriar por 10 min. </li><li> Expor o slide photoresist revestido com luz UV (intensidade: 22 mW / cm 2) por meio de uma máscara de transparência por 15 s. Coza a fotorresistência sobre uma placa quente a 115 ° C durante 1,5 min e esperar durante 45 min. Dissolve-se o foto-resistente exposta no revelador durante pelo menos 3,5 minutos. Enxaguar abundantemente com água DI. Inspecione os padrões desenvolvidos sob um microscópio óptico. </li></ol></li><li> Metais precursores de depósito para produzir np-Au filmes finos <ol><li> Coloque as amostras em uma máquina de pulverização catódica, que pode depositar independentemente de ouro, prata e cromo. Sputter-limpar as amostras por 90 segundos a 50 W com menos de 25 mTorr atmosfera processamento argônio antes de iniciar a deposição de metal. </li><li> Sputter chrome por 10 min a 300 W em 10 mTorr argônio. Sputter ouro por 90 segundos a 400 W under 10 mTorr árgon. Co-pulverização catódica ouro e prata por 10 minutos com a prata em 200 W e potência de 100 W. Au Desligue ouro pulverização catódica fonte de cerca de 10 segundos antes de desligar a fonte de prata por pulverização catódica. </li></ol></li><li> Obter precursoras micropadrões de metal <ol><li> Sonicar amostras fotosensitivo revestidos em ~ 180 ml de extractor fotorresistência durante 10 ciclos de 20 segundos de sonicação e 2 minutos de pausa entre os ciclos. Lavar as amostras com água DI e seque com um arma de nitrogênio. Inspecionar os padrões de metal sob um microscópio. </li><li> Descasque o stencil elastômero a partir de amostras não-fotossensíveis revestidos utilizando duas pinças para revelar o metal depositado. </li></ol></li><li> Dealloy precursor de metal e nanoestrutura modify via tratamento térmico <ol><li> Encher um copo de vidro de 200 ml com 170 ml de ácido nítrico (70%) e manter a temperatura da solução a 55 ° C numa placa de aquecimento. CUIDADO: O ácido nítrico é extremamente corrosivo e deve ser manuseado com equipamentos de proteção adequados. </li&gt; <li> Tratar com pequenas lamelas de plasma de ar de 10 W durante 30 segundos antes da imersão em ácido nítrico. Local 1 polegada por lâminas de microscópio de 3 polegadas no copo usando fórceps ácido-resistentes e dealloy los durante 15 min. Use um barco immunostaining porcelana para batch-dealloying pequenas lamelas. </li><li> Lavar as amostras dealloyed por imersão sucessivamente em duas provetas cheias com água DI fresco três vezes. Armazenar as amostras em água DI e substituir a água com água DI fresco todos os dias por pelo menos uma semana. Amostras secar com uma arma de nitrogênio sobre toalhas que não soltem fiapos antes do uso. </li><li> Coloque as amostras em uma pastilha de silício limpa em um equipamento de processamento térmico rápido. Ajustar a temperatura para valores entre 200 ° C e 450 ° C e a taxa de rampa de 10 ° C / seg. Expor as amostras à temperatura prescrita durante 10 min sob azoto ambiente. Deixe a câmara fria (&lt;100 ° C) e retirar as amostras. Alternativamente, lentamente colocar as amostras sobre a placa para tratam térmicaent. </li></ol></li></ol> 2. Cultura de Células <ol><li> Prepare amostras np-Au para cultura de células <ol><li> Coloque np-Au amostras em pratos de poliestireno e tratar com plasma de ar de 10 W durante 30 segundos e transferir as amostras para placas de cultura de tecidos de 24 poços. </li><li> Adicionar 500 ul de meio de cultura completo (Meio Eagle Modificado por Dulbecco com 10% de soro fetal bovino e 1% de penicilina / estreptomicina) a cada poço. Armazenar num incubador humidificado a 37 ° C e 5% de CO 2 até semeando as células (&lt;1 hr). </li></ol></li><li> Células manter, de passagem, e semente <ol><li> Manter as células de interesse (neste caso, os fibroblastos 3T3-NIH ou astrócitos murinos) em frascos T75 com meios de cultura e de passagem, quando as células são de 70% confluentes. </li><li> Para passaging das células, remove material da garrafa, lavar duas vezes com 10 ml de solução salina tamponada com fosfato (PBS), adicionar 2 ml de 1x tripsina / EDTA e as células a incubar até separar (~ 5 minutos). Adicionar 3 ml novas mídias ecentrifugar a 1200 rpm durante 3 min. Aspirar o sobrenadante e suspender o sedimento em media de 2 ml. </li><li> Remova o material gasto a partir dos poços e as células das sementes para as lamelas de vidro com uma densidade de 25.000 células / cm 2 com um volume final de 1 ml. Agite a placa de cultura voltada para trás e direita-esquerda para garantir um revestimento uniforme de células sobre as amostras. Incubar as células até a análise ao inspecionar diariamente. </li></ol></li></ol> 3. Celular e Análise de Material <ol><li> Stain células para visualizar citoesqueleto e núcleos <ol><li> Remova a mídia passou dos poços e lavar as células duas vezes com PBS. Fixar as células em 4% de paraformaldeído em PBS, durante 15 min. </li><li> Preparar a solução de coloração a 300 nm com Alexa Fluor 488 faloidina em PBS com 1% de albumina de soro bovino. </li><li> Lave as células duas vezes com PBS, e elas permeabilizar em 500 ul 0.1% Triton X-100 em PBS durante 5 min. </li><li> Lave as células duas vezes com PBS e transferi-los para limpar poços. Blot 50 - 200 mL de solução de coloração para as amostras e armazenar em escuro por 20 min. </li><li> Lavar as células com PBS e de contra-coloração com 3 nM de DAPI em PBS durante 5 min. </li><li> Lavar as células com PBS, e mergulhe em água deionizada antes da montagem em lâminas de vidro com tampa meios de montagem e vedação com claro unha polonês. </li></ol></li><li> Aquisição de imagens de amostras np-Au e culturas de células em superfícies np-Au <ol><li> Superfícies imagem np-Au com microscópio eletrônico de varredura (MEV) com ampliação de 50.000 X em 10 kV de energia do elétron usando detector de elétrons secundários. </li><li> Capturar imagens de células compostas em diferentes pontos nas amostras utilizando um microscópio de fluorescência invertido em aumento de 10x com os cubos de filtros adequados. </li></ol></li><li> Processamento de imagens para determinar a morfologia dos poros e celular <ol><li> Abertura de imagens no ImageJ e dividida em canais individuais se aplicável. Converta as imagens em 8 bits, subtrair o fundo, e alisá-los por filteri medianang. Ajuste o limiar de forma manual ou por built-in limiarizar algoritmos para destacar os poros (vazios) e corpos celulares / núcleos. </li><li> Use o comando divisor de águas para separar poros incorporadas ou células. Definir parâmetros de análise de partículas e executar o comando para extrair o número de partículas, área média e cobertura por cento de partículas. Use imagens de células DAPI manchadas para contagem celular e imagens de células phalloidin manchadas para quantificar a cobertura de celular por cento. </li><li> Modifique os arquivos de macro incluídos para realizar a análise de múltiplas imagens em lote. </li></ol></li></ol>

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Os autores não têm interesse financeiro conflitantes.

Nós demonstramos duas técnicas diferentes para micropattern np-Au filmes para a expansão do uso destes filmes em microssistemas e estudos biológicos. Sputter ouro e prata-revestimento é um método versátil para criar padrões de np-Au, como pulverização catódica é compatível com os processos convencionais de microfabricação e da composição da liga e a espessura pode ser facilmente controlada através da variação das potências individuais pistola de pulverização (para objectivos de ouro e prata) e o t...

<p class="jove_content"&gt; Materiais com características nano-escala têm se mostrado promissora no reforço várias aplicações, incluindo as células de combustível<sup&gt; 1</sup&gt;, Sensores<sup&gt; 2,3</sup&gt; E dispositivos biomédicos<sup&gt; 4,5</sup&gt;. Um material relativamente novo é ouro nanoporoso (np-Au), a qual é produzida por um processo de auto-montagem de nano-escala. O precursor de NP-Au é uma liga de ouro que mais comumente consiste em prata de 60% a 80% em percentagem atómica. Resumidamente, a característica de nanoestrutura de poros abertos, é o resultado do rearranjo de átomos de ouro em aglomerados como a prata é dissolvido por um ácido forte (<em&gt; Por exemplo</em&gt; Ácido nítrico 70%) ou sob um potencial eletroquímico<sup&gt; 6-8</sup&gt;. Np-Au benefícios de vários atributos desejáveis, incluindo a grande área de superfície eficaz, de alta condutividade elétrica, técnicas de funcionalização de superfície bem estabelecidas, e biocompatibilidade<sup&gt; 9</sup&gt;. Embora tenha havido uma rápida expansão dos estudos sobre NP-Au, a maioria deles concentrar em propriedades mecânicas do np-Au<sup&gt; 10,11</sup&gt;, A atividade catalítica<sup&gt; 12</sup&gt; E desempenho de detecção biomolecular<sup&gt; 13-15</sup&gt;. Embora os atributos desejáveis ​​são altamente úteis para várias ferramentas biomédicas<sup&gt; 16,17</sup&gt;, As aplicações nesta área têm sido limitados. Uma possível razão para isto é que a maioria dos estudos têm predominantemente utilizadas amostras de macro-escala (<em&gt; Por exemplo</em&gt; Chapas, folhas, e lingotes) e as técnicas para a incorporação np-Au em sistemas miniaturizados permaneceram inadequada. Na verdade, há apenas um punhado de exemplos do uso de técnicas de microfabricação convencionais que utilizam filmes np-Au<sup&gt; 16-20</sup&gt;. Com o advento da tecnologia de miniaturização e a necessidade de novas ferramentas biomédicas, tornou-se essencial para ser capaz de se integrar novos materiais para dispositivos. Isso normalmente requer que os materiais podem ser depositados e modelado com técnicas convencionais de microfabricação. Além disso, a rápida quantificação das interacções célula-prima é geralmente necessária para avaliar a biocompatibilidade de um material novo. O objetivo deste artigo é demonstrar as técnicas básicas para micropattern np-Au filmes e quantificar tanto a nanoestrutura e as interações célula-material através de processamento de imagem digital.</p

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Gold target</td>
 <td>Lesker</td>
 <td>EJTAUXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Chrome target</td>
 <td>Lesker</td>
 <td>EJTCRXX353A2</td>
 <td >Adhesive layer</td>
 </tr>
 <tr>
 <td>Silver target</td>
 <td>Lesker</td>
 <td>EJTAGXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Porcelain boat</td>
 <td>Thomas Scientific </td>
 <td>8542E40</td>
 <td >Used for processing small samples</td>
 </tr>
 <tr>
 <td>Nitric acid</td>
 <td>Sigma-Aldrich</td>
 <td>43873</td>
 <td >Used at 70% for dealloying</td>
 </tr>
 <tr>
 <td>Sulfuric acid</td>
 <td>J.T Baker </td>
 <td>7664-93-9</td>
 <td >Used at 96% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>J.T Baker </td>
 <td>7722-84-1</td>
 <td >Used at 30% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Ted Pella</td>
 <td>150xx</td>
 <td >Available in several sizes</td>
 </tr>
 <tr>
 <td>Silicone elastomer sheets</td>
 <td>Rogers Corporation </td>
 <td>HT 6240</td>
 <td >Available in several thicknesses</td>
 </tr>
 <tr>
 <td>Hexamethyldisilazane</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td >Used as adhesion promoter for positive resist</td>
 </tr>
 <tr>
 <td>Microposit MF CD26</td>
 <td>Shipley</td>
 <td>38490</td>
 <td >Positive photoresist developer</td>
 </tr>
 <tr>
 <td>PRS 3000</td>
 <td>J.T Baker</td>
 <td>JT6403-5</td>
 <td >Positive photoresist stripper</td>
 </tr>
 <tr>
 <td>Circular glass coverslips (12 mm)</td>
 <td>Ted Pella</td>
 <td>26023</td>
 <td >Used as substrate for metal patterns and cell culture</td>
 </tr>
 <tr>
 <td>Glass slides (1 x 3 inch)</td>
 <td>Ted Pella</td>
 <td>26007</td>
 <td >Used as substrate for metal patterns</td>
 </tr>
 <tr>
 <td>Kapton polyimide tape</td>
 <td>VWR</td>
 <td>82030-950</td>
 <td >Used for securing elastomer</td>
 </tr>
 <tr>
 <td>Transparency masks</td>
 <td>Output City</td>
 <td></td>
 <td>Used in photolithography <a href="http://www.outputcity.com/" target="_blank">http://www.outputcity.com/</a></td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td >Used for activating glass surfaces</td>
 </tr>
 <tr>
 <td>Sputtering machine</td>
 <td>Kurt J. Lesker</td>
 <td>LAB18</td>
 <td >Used for depositing metals</td>
 </tr>
 </tbody>
 </table>

microfabrication of nanoporous gold patterns for cell-material interaction studies

Materiais nanoestruturados com tamanhos recurso em dezenas de nanômetros têm melhorado o desempenho de várias tecnologias, incluindo as células de combustível, biossensores, revestimentos de dispositivos biomédicos, e ferramentas de administração de medicamentos. Ouro nanoporos (np-Au), produzido por um processo de auto-montagem nano-escala, é um material relativamente novo que apresenta grande área de superfície eficaz, de alta condutividade elétrica, e atividade catalítica. Estas propriedades tornaram-np Au um material atraente para a comunidade científica. A maioria dos estudos sobre a np-Au utilizar espécimes macro-escala e foco em ciência fundamental da suas aplicações catalíticas e sensor de materiais. Os espécimes macro-escala limitar o potencial de np-Au em sistemas miniaturizados, incluindo dispositivos biomédicos. A fim de abordar estas questões, inicialmente descrever dois métodos diferentes para micropattern np-Au filmes finos sobre substratos rígidos. O primeiro método utiliza máscaras de estêncil manualmente produzidos para a criação de milímetro escala padrões np-Au, while o segundo método usa lift-off fotolitografia para padrões sub-milímetro escala padrão. À medida que os filmes finos de NP-Au são obtidos por um processo de deposição por borrifamento, eles são compatíveis com as técnicas convencionais de microfabricação, assim passíveis de integração fácil em microssistemas. Estes sistemas incluem plataformas biossensor eletricamente endereçáveis ​​que beneficiam de área de alto eficaz superfície, condutividade elétrica, e bioconjugation superfície tiol à base de ouro. Descrevemos de cultura de células, a imunocoloração, e as técnicas de processamento de imagem para quantificar a interacção do np-Au com células de mamíferos, o que é um parâmetro de desempenho importante para algumas biossensores. Esperamos que as técnicas ilustradas aqui vai facilitar a integração das np-Au em plataformas em várias escalas de comprimento e em inúmeras aplicações, incluindo biossensores, sistemas de armazenamento de energia, e catalisadores.

A Figura 1 apresenta os principais passos processuais, incluindo a criação de padrões de np-Au, cultura de células, quantificando a nanoestrutura, e caracterizar a morfologia celular. A matriz de elastómero mostrado na Figura 2a (superior) é usada para criar os padrões de np-Au mostrados nas imagens subjacentes. Figura 2b é uma fotografia do barco de porcelana para amostras de processamento em lote. Figura 2c mostra a mudança de cor dos padrõe...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nós relatamos em técnicas para micropattern nanoporous filmes finos de ouro através de impressão stencil e fotolitografia, bem como métodos para cultura de células sobre os padrões microfabricados. Além disso, descrevemos métodos de análise de imagem para caracterizar a morfologia do material e as células cultivadas utilizando electrónica de varrimento e técnicas de microscopia de fluorescência.

Microfabricação de Padrões de Ouro Nanoporosos para celular-primas estudos de interacção

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Research

JoVE Journal

Bioengineering

11.3K visualizações.  University of California, Davis. Nós relatamos em técnicas para micropattern nanoporous filmes finos de ouro através de impressão stencil e fotolitografia, bem como métodos para cultura de células sobre os padrões microfabricados. Além disso, descrevemos métodos de análise de imagem para caracterizar a morfologia do material e as células cultivadas utilizando electrónica de varrimento e técnicas de microscopia de fluorescência.

Vídeo: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Estudando Trajetórias celular Rolamento no Patterns Receptor Asymmetric

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Bioengenharia

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Síntese, Assembléia e Caracterização de monocamada Protected Films nanopartículas de ouro para Eletroquímica monocamada Protein

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Um dispositivo de câmara de fluxo tridimensional Novel para estudar Chemokine dirigido extravasamento de células circulantes fisiológicos sob condições de fluxo

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

Língua Eletrônica Geração de padrões contínuos reconhecimento para análise de proteínas

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparação e Fotoacústica Análise de Veículos celulares contendo ouro nanobastões

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Síntese de funcionalizados 10 nm polímero-revestida com partículas de ouro para o direcionamento do endotélio e entrega da droga

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricação de uma matriz de microambiente celular Artificial multiplexado

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Síntese de nanoaglomerados de ouro emissor próximos para aplicações biológicas

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Fracionamento de fluxo de fluxo assimétrico para dimensionamento de nanopartículas de ouro em suspensão

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...