Reconhecemos Melissa Grunlan para discussões úteis sobre a formação de SAM. Esta pesquisa foi suportada em parte pela A. Robert Welch Foundation (A-1585).

Preparação de reagentes Prepare TUBOS tampão (80 mM PIPES, 1mM MgCl 2, 1 mM EGTA, ajustado para pH 6,8 com KOH). Alíquotas e armazenar glicose oxidase, catalase e glicose a -70 ° C e taxol a -20 ° C de acordo com protocolos existentes. 1,2 alíquotas e tubulina armazenar a -70 ° C de acordo com as instruções fornecidas pelo fornecedor. Microeletrodos padrão de Au utilizando litografia <ol><li> Corte em pastilhas de silício 1,5 1 cm &#39;substratos cm, utilizando um escriba ou cortador de bolacha. Limpe os substratos, colocando-os num copo com acetona o suficiente para cobri-los e sonicate por 5 minutos. Sem permitir que os substratos para secar, lave-os em acetona fresco, logo em seguida enxágüe em isopropanol e seco usando nitrogênio. </li><li> Au fabricar microeletrodos nos substratos limpos usando o padrão, fotolitografia tom positivo ou litografia por feixe de elétrons, dependendo do tamanho das características desejadas. 3 Ao projetar a geometria padrão, manter o diâmetro de cada padrão de eletrodo inferior a 100 microns para garantir a cobertura pela PEG SAM (descrito na próxima seção). Incluem suportes de contato (~ 300 mm 2) posicionados 300-500 mm do padrão com uma linha de 1 milímetro de conectar o teclado com o eletrodo (Fig. 1). Coloque os padrões litográficos perto do meio do substrato para permitir o espaço em torno do padrão para a montagem da câmara de fluxo. <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="figura 1" /> Figura 1. Geometria da amostra. Por favor, <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1390/1390fig1.jpg">clique aqui</a> para ver uma versão ampliada da figura 1. </li><li> Após o desenvolvimento do padrão na camada de resistir, um arranhão na linha de resistir a partir do bloco de contato para a borda do substrato usando uma pinça (Fig. 1). Após a deposição de metal, do zero vai permitir o contato elétrico entre o eletrodo ea borda do substrato. A câmara de deposição, de preferência com duas fontes, é utilizado para depositar a camada metálica. Os eletrodos são formados pela deposição de 2 nm de Cr (por adesão), seguido de 6 nm de Au, sem quebra de vácuo. A microbalança de cristal de quartzo equipado dentro da câmara pode ser usada para medir a espessura dos filmes durante a deposição. </li></ol> Preparar o SAM <ol><li> Pré-aqueça o forno e em uma área bem ventilada a 75 ° C. Preparar a solução de silanização, adicionando 20 mL de tolueno para um copo de vidro de 250 ml. Em seguida, adicione 1 mL PEG-silano (ou seja, 5% v / v) para o tolueno, utilizando uma pipeta de plástico. Misture o PEG-silano bem pipetando cima e para baixo 5 a 10 vezes e depois mexendo com a pipeta de 30 segundos. </li><li> Coloque as amostras padrão no recipiente com o lado virado para cima padrão, certificando-se que eles são completamente submersas na solução de PEG-silano tolueno e são espaçadas ao longo do fundo do copo. Colocar o copo no forno e asse 18-21 horas a 75 ° C. Enquanto o SAM está se formando, preparar o counterelectrode (veja abaixo) e montar os componentes adicionais necessários para as etapas restantes. </li><li> Após 18-21 horas no forno, o tolueno deve evaporar deixando as amostras padrão em um resíduo PEG-silano viscoso. Adicionar 20 mL de tolueno para o copo e mergulhar as amostras por 1-2 minutos. Remove as amostras do copo e enxaguar com tolueno, em seguida, isopropanol, e finalmente seco com nitrogênio. Neste ponto, as amostras devem olhar limpo ea camada de SAM deve ser invisível a olho nu. SAMs preparado desta maneira são utilizáveis ​​por até dois dias, quando armazenados em local seco (25 ° C, umidade moderada). Sem sucesso SAM resultados a formação de um revestimento de resíduo nublado ou irregular hued na superfície. </li></ol> Fabricar o counterelectrode Na mesma configuração de câmara de deposição utilizado para deposição de microeletrodos, faça uma counterelectrode depositando 1 nm de Cr, seguido por 4 nm de Au para um 22 x 50 mm N º 1 ou lamela de vidro n º 0. O counterelectrode deve ser quase transparente com um tom cinzento-claro ou rosa. Armazenar os counterelectrode revestido virado para cima em uma placa de Petri para mantê-lo limpo. Tubulina polimerizar em MTs <ol><li> Descongelar unlabeled tubulina e tubulina rodamina e imediatamente se combinam em uma proporção de 1:2 rotulados para unlabeled obter MTs brilhante. Mix pipetando cima e para baixo várias vezes. Polimerizar a tubulina para 20-40 minutos em um banho de água aquecida a 37 ° C. O comprimento do MTs será maior por mais tempo de polimerização. Enquanto a polimerização da tubulina é, prepare um tampão PIPES-taxol, adicionando taxol a 20 mM em um tubo de microcentrífuga contendo tampão PIPES. Misture o taxol bem pipetando cima e para baixo várias vezes e vórtex, e depois colocar o tubo no banho aquecida a 37 ° C. Após a polimerização, diluir a MTs por 1 / 100 em tampão PIPES-taxol quente e escudoda luz. MTs preparada desta forma alongada ao longo do tempo e pacote, que pode ser reduzido pela diluição. </li><li> Prepare uma mistura de oxigênio 10x de limpeza (OS) através da combinação de 30 mL tampão PIPES fria (0-4 ° C), 5 mL de 50% 2-mercaptoetanol, 5 mL de glicose oxidase, catalase 5 mL e 5 mL de glicose. 2 A glicose deve ser adicionado por último. Mix pipetando cima e para baixo 3-5 vezes após a adição de cada componente. Coloque a mistura OS no gelo, que permanecerá em vigor durante cerca de duas horas. </li><li> Verifique se a polimerização MT foi bem-sucedida e que o MTs são estáveis ​​sob vários minutos de excitação de fluorescência pela imagem da MTs preparado com 1x mix OS. Para geração de imagens a MTs preparado desta forma, não se esqueça de usar uma combinação de excitação / emissão de fluorescência filtro adequado para rodamina. Determinar a melhor diluição de trabalho para filamentos de imagens individuais por mais diluir a MTs por 1 / 10 a 1 / 100 com tampão PIPES e imagem com 1x mix OS. </li><li> Preparar uma câmara de fluxo, imprensando uma lamela de vidro de grandes dimensões (22 x 50 mm) e pequeno (22 x 22 mm) com dois pedaços de fita dupla-face colocada 2-3 mm. A fita é facilmente cortado em tiras, colocando-o sobre uma lâmina de vidro e corte com uma lâmina de barbear. Introduzir alguns microlitros de MTs na célula de fluxo, utilizando uma micropipeta. Para imagens de alta ampliação, os objectivos de longa distância de trabalho podem ser necessárias para geração de imagens da superfície superior da câmara de fluxo. A distância entre a superfície lamela / amostra até a superfície superior da câmara de fluxo é determinado pela espessura da fita e deve ser 60-100 mm. </li></ol> Padrão de MTs por eletroforese <ol><li> Prepare uma câmara de eletroforese, colocando duas tiras finas de fita dupla face em toda a amostra padrão, SAM-revestido de tal forma que o eletrodo está entre as tiras com o caminho de ligação perpendicular à fita (Fig. 2). Coloque o counterelectrode na fita dupla-face com o lado do metal tocar a fita de modo que a borda da amostra overhangs o counterelectrode por cerca de 3 mm (Fig. 2). Cortar vários comprimentos de 15 centímetros do fio de cobre isolados ímã e tira um centímetro do isolamento fora de ambas as extremidades. Anexar um fio para a área de Au exposta do substrato usando uma pequena gota de tinta prata tendo o cuidado de evitar fazer contato elétrico com o counterelectrode (isso pode ser verificado com um multímetro). Anexar um segundo fio à counterelectrode usando a pintura prata. Fios ligados com tinta de prata pode ser ainda mais segura para as amostras com fita. <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="figura 2" /> Figura 2. Montagem de células de fluxo para um microscópio invertido de fluorescência. Por favor, <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1390/1390fig2.jpg">clique aqui</a> para ver uma versão ampliada da figura 2. </li><li> Introduzir MTs na diluição desejada usando tampão PIPES combinado com OS em 1x de concentração final. Imagem com ampliação de 20x e localize o modelo sobre o wafer de silício antes da aplicação de uma voltagem. Opcionalmente, a câmara de fluxo pode ser selada com unha polonês para impedir fluxo de fluido e migração MT no plano da câmara de fluxo. </li><li> Aplicar uma tensão de até 1 V e observar a migração das MTs. Tensões mais baixas (até cerca de 0,5 V) pode ser usado se prendendo reversível de MTs é desejado, porém isto requer selando a câmara de fluxo para evitar desvio lateral. Unha polonês funciona bem para selar as extremidades abertas da câmara de fluxo. Em tensões acima de cerca de 0,7 V, cerca de adesão MT pode ser visto sobre o padrão, embora seja fraco. Em tensões acima de cerca de 0,9 V, a migração MT será mais rápido, ea adesão será mais forte. Acima de cerca de 1,2 V a probabilidade de indução de eletrólise (formação de bolhas de gás) e também para destruir o microeletrodos aumenta significativamente. A migração pode ser rastreado, ajustando o plano vertical do foco do microscópio. Se realizado corretamente, o MTs vai localizar na superfície do eletrodo. </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

A migração dos MTs é facilmente visualizado através de microscopia de fluorescência por causa de seu brilho e da fluorescência de fundo baixa. O método é geralmente aplicável a padronização biomolécula, uma vez que só exige que as biomoléculas ser induzido a levar uma carga líquida pelo ajuste adequado do pH. Eletroosmose é evitada nesta configuração, porque não há superfícies carregadas como as paredes de sílica usada em eletroforese capilar. Várias modificações des...

No. 1 vidro borossilicato lamelas (22 X 50 mm) foram adquiridos da VWR. Polido de grade eletrônico do tipo p &lt;111&gt; wafers de silício foram adquiridos da Addison Engineering, Inc. alta pureza pintura prata foi obtida a partir Estrutura Probe, Inc. (Catalog # 5001 AB). Duplas multa bitola imã cobre (~ 0,08 milímetros (0,003) de diâmetro) podem ser obtidos a partir de empresas de abastecimento eletrônicos.

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

Nós apresentamos um procedimento para a formação de um poli (etileno glicol) (PEG) monocamada auto-montados trimetoxissilano (SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formado em uma etapa única montagem e impede a incrustação em superfícies de silício e ouro. A SAM é usada para microeletrodos casaco estampados com a norma, litografia tom positivo. Usando o microtúbulos como um exemplo, nós aplicamos uma tensão DC para induzir a migração eletroforética para o eletrodo revestido SAM de forma reversível. A câmara de fluxo é usado para geração de imagens a migração eletroforética e padronização de microtúbulos<em&gt; In situ</em&gt; Meio de microscopia de epifluorescência. Este método é geralmente aplicável a padronização biomolécula, como se emprega a eletroforese para imobilizar moléculas-alvo e, portanto, não requer específicas interações moleculares. Além disso, evita problemas encontrados ao tentar padrão as moléculas SAM diretamente usando técnicas litográficas. A compatibilidade com litografia por feixe de elétrons permite que este método seja usado para biomoléculas padrão em nanoescala.

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Nós apresentamos um procedimento para a formação de um poli (etileno glicol) monocamada auto-montados (PEG-SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formada em uma única etapa e impede a incrustação em superfícies de silício e ouro. Eletroforese é então utilizado para padronização biomoléculas até a nanoescala.

Antifouling monocamadas auto-organizadas em Microeletrodos de Biomoléculas Patterning

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.4K visualizações.  Texas A&M University (TAMU). Nós apresentamos um procedimento para a formação de um poli (etileno glicol) monocamada auto-montados (PEG-SAM) em um substrato de silício com microeletrodos de ouro. O PEG-SAM é formada em uma única etapa e impede a incrustação em superfícies de silício e ouro. Eletroforese é então utilizado para padronização biomoléculas até a nanoescala.

Vídeo: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Bioengenharia

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Células padronização na opticamente transparentes Indium Tin Oxide Eletrodos

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Biologia

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Investigando única molécula de adesão de Força Atômica Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engenharia

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Padronização celular em photolithographically Definido Parylene-C: SiO<sub&gt; 2</sub&gt; Substratos

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

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 ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Preparação de Neuronal co-culturas com precisão única célula

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Neurociência

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Padronização de Microrganismos e Micropartículas através da Montagem Sequencial assistida pela Capilaridade

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plataforma microfluídico com multiplexada detecção electrónica do Ordenamento do Rastreamento de Partículas

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Um método versátil de padronização proteínas e células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Robô macio bioinspirado com microeletrodos incorporados

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...