Este trabalho foi apoiado por subsídios a SHO dos Institutos Nacionais de Saúde (R01 GM092993), a Fundação Nacional de Ciência (NSF CARREIRA Award e DBI 0.964.216), o Escritório de Pesquisa Naval (ONR) Programa Jovem Pesquisador eo Prêmio Parceria Minnesota de Biotecnologia e Genômica Médica. Fabricação de dispositivos foi realizada na Universidade de Minnesota Nanofabricação Center (NFC), que recebe o apoio da NSF por meio da Rede de Infra-estrutura Nacional de Nanotecnologia. Este trabalho também foi apoiado por subsídios para MR dos Institutos Nacionais de Saúde (NS048357, R21 NS073684), a National Multiple Sclerosis Society (CA1060A11), o Applebaum, Hilton, Peterson e Sanford Fundações ea família McNeilus. Os autores agradecem a Hyungsoon Im de assistência com ilustrações e Shailabh Kumar de assistência com microscopia eletrônica de varredura.

1. Microfabricação de Microwell matriz Substrato <ol><li> Comece com uma pastilha de silício de 4 polegadas com 100 nm de óxido crescido termicamente. </li><li> Gire SPR-955 0.7 photoresist na bolacha a 4.000 rpm por 30 seg. </li><li> Asse numa placa de aquecimento a 115 ° C durante 90 seg. </li><li> Expor photoresist. <ol><li> Use uma máscara que irá criar uma mM buracos dispostos em um arranjo hexagonal com um período de 3 um, onde a matriz de cobre a 2 mm Área x 2 mm. </li><li> Expor bolacha num passo i-line utilizando um tamanho de passo de 6 mm e uma exposição de 200 mJ / cm 2. </li></ol></li><li> Asse numa placa de aquecimento a 115 ° C durante 90 seg. </li><li> Desenvolver usando um desenvolvedor de spin para depositar duas poças de CD-26 no wafer para um tempo de desenvolvimento total de 90 seg. </li><li> Enxaguar abundantemente com água ultrapura DI e seco com N2. </li><li> Etch do óxido em um etcher reativa-ion para 6 min com 50 sccm de Ar, 25 sccm de CF 4 e 50 sccm de CHF 3 floasa, a uma pressão de 75 mTorr. </li><li> Etch silício em um ICP profunda trincheira etcher reativa-ion por 2 min com 63 sccm de C 4 F 8, 27 sccm de SF 6, 40 sccm de Ar, e 10 sccm de O2 que flui a uma pressão de 14 mTorr. </li><li> Enxaguar em acetona, metanol e isopropanol, para remover resistir. </li><li> Coloca-se num banho de H 2 SO 4: H 2 O 2 a 1:01 durante 10 minutos para limpar a superfície. </li><li> Enxaguar abundantemente com água ultrapura DI e seco com N2. </li><li> Deposite 1,080 Å de Al 2 O 3 por deposição de camadas atômicas a 250 ° C. </li></ol> 2. Preparação de Poli (dimetilsiloxano) Rodo <ol><li> Se possível, trabalhar em uma sala limpa. Caso contrário, trabalhar no ambiente mais limpo possível. </li><li> Obter um prato de plástico Petri (ou outro recipiente descartável limpo) com ≥ lados 13 mm. </li><li> Nesse prato, despejar 5 g agente PDMS cura, seguido de 50 g de PDMS base agente (ou qualquer coisa com uma relação de 01:10 que a maioria irá preencher a placa de Petri). Misture bem com uma pipeta de plástico descartável ou vara. </li><li> Retirar as bolhas, colocando o PDMS misturados numa câmara com uma linha de vácuo, a aplicação de vácuo, até sair bolhas de mistura (cerca de 30 min). </li><li> Se ainda não estiver na placa de Petri, despeje PDMS em placa de Petri, evitando a criação de bolhas de ar. </li><li> Curar durante a noite numa placa de aquecimento a&gt; 50 ° C (mais elevada de calor durante menos tempo também funciona). </li><li> Com lâmina de barbear novo / limpo, cuidadosamente cortar um pedaço retangular de aproximadamente 2,5 x 2,5 cm, mantendo a superfície superior o mais limpo possível. Descasque fora com um pano limpo fórceps. Apare uma borda (até 1,5 cm), pressionando para baixo em lâmina de barbear em um movimento, para fazer borda superior tão afiada quanto possível (esta será a borda utilizada no processo de rodo). </li><li> Limpa-se com acetona, metanol, álcool isopropílico, e ultrapura DI água, e, em seguida, secar com azoto gasoso limpo. Guarde em um prato limpo Petri. </li></ol> 3. Preparação de vesículas <ol><li> Recolhe soluções de lipídicos de fosfatidilcolina de 25 mg / ml de ovo (PC) e 1 mg / ml de 1,2-dipalmitoil-sn-glicero-3-fosfoetanolamina-N - (lissamina rodamina B sulfonil) de sal de amónio (Rho-DPPE) em clorofórmio e uma solução a 0,5 mg / ml de mono-sialogangliósido GM1, em clorofórmio / metanol (2:1 v / v). </li><li> Num frasco pequeno de vidro fazer uma mistura que resulta em 97% molar de PC, 2% molar de GM1 e 1% molar de DPPE-Ró, adicionando 18,9 uL de 25 mg / mL de PC, 39,6 ul de 0,5 mg / ml de GM1 e 7,9 ul de 1 mg / mL de Rho-DPPE utilizando seringas de vidro. </li><li> Nota:. O material suplementar para Nair et al 25 contém uma excelente planilha Excel customizáveis ​​para o cálculo das quantidades de lipídios necessários para criar vesículas de qualquer composição desejada. </li><li> Coloque frasco num exsicador de vácuo e remover o solvente deixando o frasco sob vácuo durante 6 horas. </li><li> Adicione uma solução aquosa de 0,1M de NaCl. Adicionar 0,5 ml de solução de NaCl 0,1 M para o frasco contendo a película de lípidos seca. </li><li> Permitir que a mistura aquosa de lípido para descansar durante a noite. </li><li> Resumidamente mistura vortex para criar uma suspensão de vesículas, em seguida, sonicar durante 20 min num banho de ultra-sons à temperatura ambiente. </li><li> Extrude a suspensão vesícula através de uma membrana de policarbonato com 100 nm de tamanho de poros. Passar a suspensão de vesículas através do filtro de 17x. </li><li> Armazenar as vesículas extrusadas em um frasco de vidro a 4 ° C. </li></ol> 4. Formação de esféricas lipídicas Bicamadas suportados <ol><li> Colete 700 nm de diâmetro SiO 2 contas. As contas são fornecidos em água destilada com uma concentração estoque de 1,4 x 10 11 contas / ml. </li><li> Num tubo de Eppendorf de 1,5 ml preparar uma suspensão de esferas de 1 ml, com uma concentração de 1,5 x 10 10 contas / ml pela adição de 107 ul de solução-mãe de talão de 893 ul de 0,1 M de NaCl. </li><li> Vortex a suspensão para garantir que ele está bem mixa. </li><li> Centrifugar a suspensão a 1700 xg durante 20 min, em seguida, descartar o sobrenadante. Ressuspender as pérolas em 1 ml de 0,1 M de NaCl. Repita este passo mais duas vezes para lavar bem as contas. </li><li> Para formar os SSLBs, num tubo Eppendorf de 1,5 ml adicionar 25 ul da suspensão de esferas de SiO 2 para 200 ul de 1 mg / ml de suspensão de vesículas. Vortex a mistura e deixar repousar durante 1 hora à temperatura ambiente. Nesta fase, a concentração deve ser SSLB 1,7 x 10 9 SSLBs / ml. </li><li> Centrifugar a 1700 xg por 20 min para agregar as SSLBs. A pastilha deve aparecer rosa devido à ruptura de vesículas com Rho-DPPE sobre as contas. </li><li> Descartar o sobrenadante e ressuspender em 225 ul de solução salina tamponada com fosfato (PBS), pH = 7,4. Repita os passos de 4,6-4,7 mais duas vezes para remover quaisquer vesículas unruptured da suspensão SSLB. </li></ol> 5. Assembleia da SSLB Matriz <ol><li> Wafer Cleave de matrizes de micropoços, resultando em peças retangulares com 4-6 micmatrizes rowell por peça. </li><li> Vortex misturar a suspensão SSLB, em seguida, coloque 10 ml de suspensão SSLB em cada matriz de micropoços. Para que não descanso para 1 hora para permitir SSLBs para resolver para a superfície. </li><li> Lave cuidadosamente o chip conjunto de micropoços com PBS a partir de um frasco de lavagem, em seguida, mergulhe em um banho de PBS preparada em um prato raso. </li><li> Enquanto submerso, coloque delicadamente o PDMS rubor rodo no chip conjunto de micropoços e gentilmente deslize-o ao longo da superfície de 5x para remover SSLBs que não estão imobilizadas em micropoços. </li><li> Segure o chip conjunto de micropoços, com uma pinça e agite no banho PBS para remover quaisquer SSLBs excesso. </li><li> Remover rapidamente o chip do banho de rodo e coloque em banho-PBS fresco até à sua utilização. Certifique-se que a superfície superior do chip permanece molhado para preservar os SSLBs. </li></ol> Nota: O processo de montagem descrito acima pode ser utilizado para criar matrizes de partículas de membranas naturais, como partículas de mielina isoladas de cérebro de rato, ou phovesículas spholipid sem os SiO 2 suportes talão. Este trabalho é descrita em pormenor em Wittenberg et 21 al. E os resultados representativos são apresentados abaixo. 6. Cholera Toxin Binding Assay <ol><li> Prepara-se uma solução de 2 mg / ml de albumina de soro bovino (BSA) em PBS. </li><li> Prepara-se uma solução com a concentração desejada de Alexa 488 conjugado com a toxina da cólera subunidade B em PBS com 2 mg / ml de BSA. </li><li> Retire o chip conjunto SSLB do banho de PBS e pavio fora a maior parte da solução PBS usando um laboratório de limpar. Deixar simplesmente suficiente PBS no chip para manter a matriz SSLB hidratado. </li><li> Para impedir a ligação não específica, adicionar 200 ul de 2 mg / ml de solução de BSA a matriz do chip SSLB. Deixe descansar por 1 hora em uma caixa umidificado. </li><li> Com uma micropipeta, remover 200 mL de solução a partir do topo do chip de matriz SSLB. </li><li> Adicione 200 ml de solução de toxina da cólera ao chip conjunto SSLB e deixe descansar por 1 hora em uma caixa umidificado. </li><li> Lave cuidadosamente o chip conjunto SSLB com PBS a partir de um frasco de lavagem para remover qualquer toxina da cólera não ligado. </li><li> Wick o excesso de PBS usando um laboratório de limpar. Antes de imagem de chip tampa com um pedaço 24 milímetros tampa x 40 mm. </li><li> Matrizes de imagem com um microscópio vertical usando filtro define apropriado para os fluoróforos de interesse. </li><li> Analisar a intensidade de fluorescência de SSLBs individuais em uma matriz usando a função de análise de partículas automatizada de software ImageJ. </li><li> Compilar intensidades de fluorescência média de SSLBs individuais em histogramas que resumem as intensidades de SSLBs encontrados em uma determinada matriz. </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Spectroscopic+Detections.">Multiplexed Spectroscopic Detections.</a> Annu. Rev. Anal. Chem. 1 (1), 515-547 (2008).</li><li>Kundu, J., Levin, C. S., Halas, 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=Real-Time+Monitoring+of+Lipid+Transfer+Between+Vesicles+and+Hybrid+Bilayers+on+Au+Nanoshells+Using+Surface+Enhanced+Raman+Scattering+(SERS).">Real-Time Monitoring of Lipid Transfer Between Vesicles and Hybrid Bilayers on Au Nanoshells Using Surface Enhanced Raman Scattering (SERS).</a> Nanoscale. 1 (1), 114-117 (2009).</li><li>Junesch, J., Sannomiya, T., Dahlin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Properties+of+Nanohole+Arrays+in+Metal-Dielectric+Double+Films+Prepared+by+Mask-on-Metal+Colloidal+Lithography.">Optical Properties of Nanohole Arrays in Metal-Dielectric Double Films Prepared by Mask-on-Metal Colloidal Lithography.</a> ACS Nano. 6 (11), 10405-10415 (2012).</li><li>Wu, M., Holowka, D., Craighead, H. G., Baird, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Plasma+Membrane+Compartmentalization+with+Patterned+Lipid+Bilayers.">Visualization of Plasma Membrane Compartmentalization with Patterned Lipid Bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (38), 13798-13803 (2004).</li><li>Hatzakis, N. S., Bhatia, V. K., Larsen, J., Madsen, K. L., Bolinger, P. Y., Kunding, A. H., Castillo, J., Gether, U., Hedegard, P., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Curved+Membranes+Recruit+Amphipathic+Helices+and+Protein+Anchoring+Motifs.">How Curved Membranes Recruit Amphipathic Helices and Protein Anchoring Motifs.</a> Nat. Chem. Biol. 5 (11), 835-841 (2009).</li><li>Roizard, S., Danelon, C., Hassaine, G., Piguett, J., Schulze, K., Hovius, R., Tampe, R., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+G-Protein-Coupled+Receptors+in+Cell-Derived+Plasma+Membranes+Supported+on+Porous+Beads.">Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads.</a> J. Am. Chem. Soc. 133 (42), 16868-16874 (2011).</li></ol>

Os autores não têm concorrentes interesses financeiros para divulgar.

Neste trabalho é mostrar que monodispersas SiO 2 contas revestidas com camadas duplas lipídicas suportados podem ser dispostos em conjuntos de micropoços, sem a necessidade de ligandos alvo nas bicamadas lipídicas ou a superfície do substrato, e as matrizes podem ser usadas para caracterizar as interacções lípido-toxina. A nós constante de dissociação calculada para a ligação CTx/GM1 compara favoravelmente, dada a grande disparidade de valores na literatura, com um relatório anterior por Winter ...

Membranas lipídicas bicamada são estruturas essenciais na natureza. Membranas plasmáticas celulares e membranas de organelas são compostas por bicamadas lipídicas que incorporam uma série de moléculas que são necessárias para a vida. Muitos processos que sustentam a vida ocorrem na superfície de células ou são mediadas por moléculas associadas com membranas de bicamada lipídica. De facto, muitos processos farmacêuticos alvo ou moléculas são encontrados sobre ou dentro de membranas de 1,2. Por conseguinte, é necessário investigar analiticamente processos, tais como reacções químicas ou eventos de ligação não covalentes que ocorrem sobre as superfícies da membrana. Porque as membranas naturais podem ser difíceis de isolar e / ou de interface com os sensores, muitos investigadores empregam membranas modelo simplificado para a realização de estudos analíticos. Um certo número de sistemas de membranas modelo são descritos na literatura, variando a partir de vesículas gigantes que podem ser dezenas a centenas de microns de diâmetro até os lipossomas com dimensões nanométricas 3,4. Alterntivamente, bicamadas lipídicas planas depositados em suportes sólidos, isto é, suportadas bicamadas lipídicas (EBSL), pode ser formado de uma série de superfícies diferentes e têm sido amplamente utilizados em biofísicos, bioquímicos, e aplicações analíticas 5. Acoplamento EBSL com materiais eléctricos ou ópticos permite a investigação de bioquímica e biofísica da membrana, através da utilização de diferentes técnicas analíticas. A microscopia de fluorescência 6, 7 eletroquímica, espectroscopia óptica 8, microscopia de varredura por sonda 9, a ressonância de plasma de superfície 10, e espectrometria de massa 11 têm sido utilizados para estudar a estrutura e propriedades de EBSL. Matrizes SLB oferecem versatilidade adicional na concepção de sensores para ensaios multiplex 12,13. Outras aplicações utilizam matrizes SLB para imitar a junção que se forma entre células do sistema imunológico 14. Métodos de preparação para matrizes SLB têm variado de microfluidic se aproxima de 15 a aqueles que empregam as barreiras físicas entre os patches SLB adjacentes. 16 Outros grupos têm usado métodos de impressão 17, padronização fotoquímica 18 e vários nanoengineering se aproxima de 19 a criar matrizes SLB. Neste artigo e vídeo que acompanha demonstramos um método para formar matrizes SLB SLB depositando-revestidos SiO 2 contas em conjuntos ordenados de micropoços 20. Referimo-nos aos SLB-revestidos SiO 2 pérolas esféricas como bicamadas lipídicas suportadas (SSLBs). Esta técnica é uma extensão do trabalho anterior que criou conjuntos de vesículas de fosfolipídios e biomembranas derivados de fontes naturais 21, de onde também mostram exemplos de resultados. Outros métodos para arraying partículas de membranas ou vesículas têm contado com padrões de ligantes específicos de segmentação em superfícies que se associam com ligantes complementares contidas na superfície da vesícula. Exemplos incluem a biotina-associação avidina 22,23 e esquemas de hibridização DNA 24. Nossa abordagem requer apenas uma matriz de micropoços sem partes necessárias de segmentação ou de reconhecimento. O tamanho de SSLBs é definida pelo diâmetro das SiO 2 suportes de talão, que têm baixa poli-dispersividade. Ao ajustar o diâmetro de micropoços apenas maior que o diâmetro SSLB, apenas um único SSLB depositada em cada microplaca. Um poli (dimetilsiloxano) (PDMS) rodo então remove da superfície de todos os SSLBs que não estão imobilizados em micropoços. Os micropoços e matrizes SSLB resultantes têm elevada densidade (~ 10 5 SSLBs / mm 2) com 3 um espaçamento centro-a-centro e periodicidade hexagonal. Por serialmente depositando SSLBs com diferentes composições de lípidos, é possível criar matrizes de múltiplos componentes com SSLBs posicionados aleatoriamente. Para demonstrar a capacidade de detecção de matrizes SSLB, usamos a interação da toxina da cólera (CTx) com um gangliosídeo (GM1) incorporada nas SSLBs. Compartículas de membranas naturais, que eram capazes de detectar lípidos específicos de células em matrizes de multicomponentes que contenham material de membrana a partir de dois tipos de células diferentes.

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			<td height="42" style="height:42px;width:239px;">4-inch silicon wafers</td>
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			<td height="21" style="height:21px;width:239px;">i-line stepper</td>
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			<td height="42" style="height:42px;width:239px;">Vision 320 reactive ion etcher</td>
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			<td height="42" style="height:42px;width:239px;">Deep trench reactive ion etcher</td>
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			<td style="width:119px;">SLR-770</td>
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			<td style="width:91px;">Ellsworth Adhesives</td>
			<td style="width:119px;">184 SIL ELAST KIT 0.5KG</td>
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			<td height="42" style="height:42px;width:239px;">egg phosphatidylcholine</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>840051C</td>
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			<td height="84" style="height:84px;width:239px;">1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>810158C</td>
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			<td height="42" style="height:42px;width:239px;">monosialoganglioside GM1&#160;</td>
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			<td height="42" style="height:42px;width:239px;">Silica beads</td>
			<td style="width:91px;">Bangs Laboratories</td>
			<td style="width:119px;">SS03N/4666</td>
			<td>Packaging on the bead container states the beads are 900 nm in diameter. However, after light-scattering and electron microscopy we determined the beads are roughly 700 nm in diameter.</td>
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			<td height="42" style="height:42px;width:239px;">Cholera toxin B-subunit, Alexa 488 conjugate</td>
			<td style="width:91px;">Molecular Probes</td>
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			<td height="42" style="height:42px;width:239px;">Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate</td>
			<td style="width:91px;">R&#38;D Systems</td>
			<td style="width:119px;">NL1326R</td>
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			<td height="42" style="height:42px;width:239px;">FM1-43</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">T-3136</td>
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			<td height="42" style="height:42px;width:239px;">Eppendorf MiniSpin centrifuge</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td style="width:119px;">05-401-09</td>
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formation of biomembrane microarrays with a squeegee-based assembly method

Membranas de bicamada lipídica formar as membranas plasmáticas de células e definir os limites de organelos subcelulares. Na natureza, estas membranas são misturas heterogéneas de vários tipos de lípidos, contêm proteínas ligadas à membrana, e são decorados com hidratos de carbono. Em algumas experiências, é desejável para dissociar as propriedades biofísicas ou bioquímicas da bicamada lipídica dos da membrana natural. Tais processos requerem o uso de sistemas modelo tais como vesículas gigantes, lipossomas ou bicamadas lipídicas suportados (EBSL). Matrizes de EBSL são particularmente atraentes para aplicações de detecção e imitando interações célula-célula. Aqui nós descrevemos um novo método para a formação de matrizes SLB. Submicron diâmetro SiO 2 grânulos são primeiro revestidas com camadas duplas de lípido para formar EBSL esféricas (SSLBs). Os grânulos são, em seguida, depositada numa matriz de micropoços micro fabricado submícron de diâmetro. A técnica de preparação utiliza um &quot;rodo&quot; para limpar a superfície do substrato, enquanto deixandog trás SSLBs que se instalaram em micropoços. Este método não requer nenhuma modificação química do substrato de micropoços, nem quaisquer ligandos alvo particulares no SSLB. Os micropoços são ocupados por grânulos individuais, porque o diâmetro do poço é ajustado para ser apenas maior do que o diâmetro do cordão. Tipicamente, mais de 75% dos poços estão ocupados, enquanto o restante permanece vazio. Em tampão de matrizes SSLB exibir estabilidade a longo prazo maior do que uma semana. Vários tipos de SSLBs podem ser colocados em uma única matriz pela deposição de série, e as matrizes podem ser usados ​​para a detecção, o que demonstramos ao caracterizar a interação da toxina da cólera com gangliosídeo GM1. Mostramos também que as vesículas de fosfolípidos, sem os suportes de talão e biomembranas de fontes celulares podem ser dispostos com o mesmo método e os lípidos da membrana específicos para células podem ser identificadas.

Quando SiO 2 pérolas são misturadas com uma solução de vesículas compostas por fosfolípidos, lípidos fluorescentes e outros lípidos, tais como gangliósidos, as vesículas se rompem na superfície do grânulo de SiO 2 para formar SSLBs, como mostrado esquematicamente na Figura 1A. Depois de lavar as SSLBs, uma gota de solução SSLB é colocada sobre uma matriz de micropoços, e os grânulos são deixadas assentar à superfície. (Figura 1b1) Isto também p...

Watch this Scientific Journal Video about Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method at JoVE.com

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Bicamadas lipídicas suportadas e partículas de membranas naturais são sistemas convenientes que podem aproximar as propriedades das membranas celulares e ser incorporadas em uma variedade de estratégias de análise. Aqui demonstramos um método para a preparação de compostos de microarrays de suporte revestidas de lípidos em bicamada-SiO 2 grânulos, vesículas de fosfolípidos ou partículas de membranas naturais.

Formação de biomembrana Microarrays com um método de montagem baseada em Rodo

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

formation-biomembrane-microarrays-with-squeegee-based-assembly

Research

JoVE Journal

Bioengineering

13.6K visualizações.  University of Minnesota.  Bicamadas lipídicas suportadas e partículas de membranas naturais são sistemas convenientes que podem aproximar as propriedades das membranas celulares e ser incorporadas em uma variedade de estratégias de análise. Aqui demonstramos um método para a preparação de compostos de microarrays de suporte revestidas de lípidos em bicamada-SiO 2 grânulos, vesículas de fosfolípidos ou partículas de membranas naturais. 

Vídeo: Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Síntese Nanostar ouro com um método de crescimento de sementes de Prata Mediated

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Bioengenharia

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays de polímeros para Discovery High Throughput de Biomateriais

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para a formação de biofilme Streamer em um dispositivo micro com micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescência Biomembrane Força Probe: quantificação simultânea de Kinetics receptor de ligando e induzida Encadernação-Sinalização Intracelular em uma única célula

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication pelo Solvent-assistida Lipid bicamada (SALB) Método

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e Montagem de microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricação de poços de Agarose personalizado para a propagação de células e tecido anel Self-assembly usando moldes 3D-impresso

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificação simultânea de kynureninas selecionadas analisadas por espectrometria de cromatografia líquida-massa em média coletada de culturas celulares cancerosas

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.