The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

1: Preparação de várias soluções e as amostras de proteínas <ol><li> Preparar 100 ml de solução tampão de fosfato (PBS-G), que contém 50 mM de NaH 2 PO 4, 50 mM de NaCl, e 10% de glicerol a pH 6,8. </li><li> Preparar 250 ml de solução tampão HEPES contendo 10 mM de HEPES, 150 mM de NaCl, 0,005% de Tween 20 a pH 7,4. </li><li> Preparar a solução estoque de bloco 1 (BB1) lactose e bloco 2 (BB2) sulfatada lactose (Figura 1) em 0,2 mM em PBS-G. </li><li> Prepare 1 ml de soluções de proteína em HEPES: Uma lectina raque hypogaea (AHL) a 500 nm, a mioglobina a 1 uM, e lisozima em 500 nM. </li><li> Preparar 20 ml de SDS a 1% em água ultrapura. </li></ol> 2: Preparação da Matriz CoCRR <ol><li> Limpar a superfície de ouro do prisma 48 h antes da utilização com um líquido de limpeza de plasma durante 3 min nas seguintes condições: 75% de oxigénio, 25% de árgon, 0,6 mbar, potência a 40 W. </li><li> Prepare onze solutio puro e misturadons de BB1 ​​e BB2 com [BB1] / ([BB1] + [BB2]) relações entre 0 e 100%, em incrementos de 10% numa concentração total BB de 0,1 mM em PBS-G. </li><li> Depósito 8 gotas nl destas soluções puras e mistas na superfície do prisma através de um observador não-contato em quadruplicado para cada relação, com 44 pontos no total (Figura 2). NOTA: 10% de glicerol em PBS-G é muito importante para reduzir a evaporação do solvente e a alteração da concentração de BB após a deposição. </li><li> Inserir o prisma dentro de um prato de Petri contendo 1 ml de água ultrapura e deixá-lo O / N à temperatura ambiente durante a auto-montagem de BB1 ​​e BB2 sobre a superfície de ouro. Aqui, assume-se que a composição média de superfície na mistura SAM reflecte a composição da solução mista, a deposição de (Figura 3) 11. </li><li> Lave bem o prisma com água ultrapura e seque-a sob um fluxo de argônio. </li></ol> 3. Protein Sensing por SPRI <ol><li> Defina o incubator em que o aparelho SPRI é colocado a 25 ° C, para evitar alterações de índice de refracção induzidas por variação da temperatura durante a detecção da proteína. </li><li> Inserir um prisma não-funcionalizado de lavagem em 10 mL de éter do poliéter-cetona (PEEK), célula de fluxo ligado a uma bomba de seringa controlada por computador, um desgaseificador e uma válvula de injecção de média pressão 6-porta (Figura 4). Preencha o sistema de fluxo com recém filtrados e desgaseificados correndo HEPES tampão. </li><li> Remover o prisma enxaguamento e inserir o prisma que contém a matriz CoCRR na célula de fluxo. Executar HEPES a uma taxa de fluxo de 100 mL / min. Com a ajuda da câmara CCD de remover as bolhas de ar presentes na superfície do prisma, através da passagem de tampão rodando rapidamente. </li><li> Definir a área de estudo para cada ponto de desenho de um círculo com o mesmo diâmetro para a zona de interesse com base numa imagem de bom contraste a matriz e com a ajuda do software integrado no sistema SPRI. </li><li> Traçar curvas de plasmons, que representam refletirivity curvas em função do ângulo de incidência, para todos os pontos. </li><li> Escolha o ângulo (posição na qual curvas cinéticas será gravado) que trabalham na maior inclinação das curvas de refletividade. Gire o espelho de varredura para corrigir o ângulo de trabalho selecionado para medições cinéticas. </li><li> Continuar a correr através do sistema de HEPES fluxo até que a reflectividade do sinal para todos os pontos é estável e constante. </li><li> Injectar 1 mL de solução de AHL com uma seringa para dentro do circuito da amostra (500 ul) com a válvula de injecção na posição de &quot;carga&quot;. Em seguida, coloque a válvula de injeção para a posição &quot;injeção&quot;. A taxa de fluxo utilizada para toda a injecção de proteína foi de 100 ul / min. </li><li> Comece medidas cinéticas por monitorar variações de refletividade contra o tempo simultaneamente em todos os pontos. </li><li> No final da injeção de proteína, lavar a matriz com tampão de corrida para 8 min. Finalmente, injectam-se 1 ml de 1% de SDS a regenerá-lo. </li><li> Repetir o mesmo procedimento para a outra proteins. </li></ol> 4 Processamento de Dados e Análise <ol><li> Após a entrada da solução de proteína na célula de fluxo, ligação molecular ocorre e induz uma mudança das curvas de plasmon e uma variação da reflectividade. O software de aquisição de imagem converte os valores de intensidade de luz medidos os níveis de cinzento da escala, dando imagens SPR Figura 5A, e gera a variação da ref lectividade em função do tempo, dando sensorgramas (Figura 5B). </li><li> Use um software de computação matemática para traçar a refletividade (R%) no final de cada injeção de proteína em relação ao [BB1] / ([BB1] + [BB2]) Relação de gerar perfil 2D contínua evolução (CEP) para cada amostra (Figura 5C). </li><li> Adicionar o [BB1] / ([BB1] + [BB2]) em relação sensorgramas (Figura 5B) para gerar 3D paisagem evolução contínua (CEL) para cada uma das proteínas (Figura 5D). </li></ol>

<ol>
	<li>Margulies, D., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+protein+recognition+as+an+alternative+approach+to+antibody-mimetics.">Combinatorial protein recognition as an alternative approach to antibody-mimetics.</a> Current Opinion in Chemical Biology. 14, 705-712 (2010).</li><li>Umali, A. P., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+approach+to+differential+sensing+using+synthetic+molecular+receptors.">A general approach to differential sensing using synthetic molecular receptors.</a> Current Opinion in Chemical Biology. 14, 685-692 (2010).</li><li>Baldini, L., Wilson, A. J., Hong, J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-based+detection+of+different+proteins+using+an+array+of+fluorescent+protein+surface+receptors.">Pattern-based detection of different proteins using an array of fluorescent protein surface receptors.</a> J. Am. Chem. Soc. 126, 5656-5657 (2004).</li><li>Zhou, H. C., Baldini, L., Hong, J., Wilson, A. J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+of+proteins+based+on+an+array+of+functionalized+porphyrins.">Pattern recognition of proteins based on an array of functionalized porphyrins.</a> J. Am. Chem. Soc. 128, 2421-2425 (2006).</li><li>Wright, A. T., Griffin, M. J., Zhong, Z., McCleskey, S. C., Anslyn, E. V., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptors+create+patterns+that+distinguish+various+proteins.">Differential receptors create patterns that distinguish various proteins.</a> Angew. Chem. Int. Ed. 44, 6375-6378 (2005).</li><li>Wright, A. T., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptor+arrays+and+assays+for+solution-based+molecular+recognition.">Differential receptor arrays and assays for solution-based molecular recognition.</a> Chem. Soc. Rev. 35, 14-28 (2006).</li><li>You, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+proteins+using+nanoparticle&#8211;fluorescent+polymer+&#8216;chemical+nose&#8217;+sensors.">Detection and identification of proteins using nanoparticle&#8211;fluorescent polymer &#8216;chemical nose&#8217; sensors.</a> Nature Nanotechnology. 2, 318-323 (2007).</li><li>De, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+proteins+in+human+serum+using+conjugates+of+nanoparticles+and+green+fluorescent+protein.">Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein.</a> Nature Chemistry. 1, 461-465 (2009).</li><li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+evolution+profiles+for+electronic-tongue-based+analysis.">Continuous evolution profiles for electronic-tongue-based analysis.</a> Angew. Chem. Int. Ed. 51, 10394-10398 (2012).</li><li>Campbell, C. T., Kim, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPR+microscopy+and+its+applications+to+high-throughput+analyses+of+biomolecular+binding+events+and+their+kinetics.">SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics.</a> Biomaterials. 28, 2380-2392 (2007).</li><li>Stranick, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer-scale+phase+separation+in+mixed+composition+self-assembled+monolayers.">Nanometer-scale phase separation in mixed composition self-assembled monolayers.</a> Nanotechnology. 7, 438-442 (1996).</li></ol>

The authors have no conflicts of interest to declare.

<html> Os passos mais críticos para a construção deste eT são dedicados a garantir uma boa reprodutibilidade do sistema. Por exemplo, a limpeza da superfície de ouro do prisma com um procedimento normalizado antes da utilização, a adição de 10% de glicerol em onze soluções puras e mistas de BB1 ​​e BB2 para eliminar a evaporação do solvente durante o BBS de auto-montagem em ouro na superfície do prisma, depositando várias repetições para cada [BB1] / ([BB1] + [BB2]) Relação, etc. Tal como ...

Métodos de detecção de proteínas precisas e rápidas são muito importantes no diagnóstico e proteômica médicos. Matrizes de detecção de proteínas clássicos, tais como biochips, baseiam-se no princípio do reconhecimento &quot;lock-e-chave&quot; e requerem receptores específicos, tais como anticorpos, aptâmeros, ou miméticos. Nos últimos anos, o diferencial de sensoriamento inspirado pelo olfato humano e gustação surgiu como uma alternativa 1. Este nariz / língua eletrônica (EN / ET) abordagem é baseada na ligação dos analitos a uma série de receptores de reação cruzada (CRRs), que não precisam ser altamente específica ou selectiva para as moléculas-alvo diferencial, assim, permitir a superar o laborioso processo de desenvolvimento altamente selectivos receptores. É a resposta combinada de todos os receptores que cria um padrão distinto para cada uma das amostras, como uma impressão digital, permitindo a sua identificação. Os dois principais desafios para o desenvolvimento de electronic nariz / lingueta para a detecção de proteína são eficazes na produção de elementos sensores que têm a capacidade de distinguir entre os analitos estruturalmente semelhantes, e o sistema de transdução apropriado para o evento de ligação. Até agora, os estudos relataram várias abordagens para o desenvolvimento de matriz 2. Por exemplo, num estudo de uma identificação à base de proteínas de matriz foi desenvolvido usando CRRs preparados a partir de derivados de tetra-carboxyphenylporphyrin por acoplamento de grupos carboxilo para vários aminoácidos ou dipéptidos para proporcionar receptores diferenciais possuindo um núcleo hidrofóbico de afinidade para as proteínas e as periferias carregadas para distintos transmitir diferencial obrigatório. Usando este sistema, as proteínas diferentes e misturas de proteínas foram identificadas através da medição da fluorescência de têmpera dos receptores após interacção com os analitos 3,4. Em outro estudo, uma biblioteca de 29 CRRs contendo tripéptido e radicais de ácidos borónicos sintetizado de uma forma combinatória num hexasubstituído andaime benzeno foi desenvolvido para a detecção de proteínas com uma detecção colorimétrica indicador de absorção de 5,6. Com uma tal concepção, cada receptor demonstrou a capacidade de ligação diferencial com proteínas com base na variância nos braços de péptidos, e dos ácidos borónicos assistidas em diferenciação de proteínas de glicoproteínas. Mais recentemente, uma matriz composta de diferentes catiónicos funcionalizado nanopartículas de ouro conjugadas com um poli aniónico fluorescente polímero (p-phenyleneethynylene) (PPE) foi desenvolvido para detectar e identificar proteínas 7. A ligação competitiva entre analitos de proteína e temperada de EPI complexos de nanopartículas / ouro regeneradas fluorescência, produzindo padrões de reconhecimento distintos para as proteínas. Neste estudo, as interacções proteína-nanopartícula funcionalizados foram ajustadas variando as propriedades físico-químicas dos grupos terminais de nanopartículas. Além disso, demonstrou-se que esta abordagem é eficaz para análise de proteínas em meio complexo rico e proteína talcomo soro humano em concentrações fisiologicamente relevantes, demonstrando assim o potencial de eT em perfis de amostras reais para o diagnóstico de estados de doença 8. Apesar de muito promissora, estes sistemas têm algumas limitações inerentes. Eles exigem projetar e sintetizar 5-29 CRRs com estruturas bastante complexas. Além disso, ao contrário do sistema olfactivo que é reposto depois de cada medição, a detecção de proteínas requer a preparação de uma matriz por amostra. Finalmente, monitorando eventos de ligação em tempo real são extremamente difíceis. Neste contexto, uma abordagem combinatória foi proposta por meio de um pequeno número de moléculas simples e facilmente acessíveis com diferentes propriedades físico-químicas (hidrofílicas, hidrofóbicas, de carga positiva, carregada negativamente, neutro, etc.) Como blocos de construção (BBS) 9. Ao misturar BBs em diferentes proporções e controladas e permitindo que as misturas para auto-montar sobre a superfície de ouro de umprisma, uma disposição de superfícies combinatórias que caracterizam as propriedades adequadas para a proteína de ligação foi criado. Notavelmente, as monocamadas auto-montadas sobre este sistema de permitir ajuste fácil de uma variedade de propriedades de superfície de uma forma altamente divergente, permitindo diversos receptores de reacção cruzada (CoCRRs combinatórias) para ser rápida e eficientemente produzido. Detecção das proteínas foi realizada utilizando um sistema de detecção óptica, a superfície de ressonância de plasmon (SPRI). Resumidamente, uma ampla do feixe de luz polarizada monocromática a partir de um diodo emissor de luz ilumina toda a área de matriz CoCRR sobre a superfície do prisma. A câmera de alta resolução de vídeo CCD fornece imagens diferença em tempo real em todos os pontos da matriz CoCRR. Ele captura todas as alterações locais na superfície da matriz CoCRR fornecendo informações detalhadas sobre os eventos de ligação e processos cinéticos 10. Enquanto isso, com a ajuda de software de imagem, imagens SPR correspondentes aos pontos são automaticamente convertidos em variações de refletividade versus de tempo, gerando uma série de curvas de ligação cinética chamados sensorgramas. Assim, SPRI permite uma observação, síncrona, em paralelo, livre de marcador e em tempo real de eventos de ligação. Além disso, a matriz CoCRR obtido é regenerável e reutilizável para análise de proteínas. Este protocolo descreve a construção da língua electrónico através de apenas duas pequenas moléculas como blocos de construção e ilustra a sua aplicação para a análise de proteínas comuns com base em padrões de reconhecimento contínuos obtidos com SPRI.

<table border="0" cellpadding="0" cellspacing="0" width="1511">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td style="width:799px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRi apparatus&#160;</td>
			<td>Horiba Scientific-GenOptics</td>
			<td style="width:225px;"></td>
			<td>SPRi apparatus is placed in a temperature regulated incubator at 25&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Memmert</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump&#160;</td>
			<td>Cavro scientific instruments</td>
			<td style="width:225px;">Cavro XLP 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Micro Elite Degasser&#160;</td>
			<td style="width:203px;">Alltech</td>
			<td style="width:225px;">AT590507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-port medium pressure injection valve</td>
			<td>Upchurch Scientific</td>
			<td style="width:225px;"></td>
			<td>The volume of injection loop used is 500 &#181;l.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Femto plasma cleaner (version 7)</td>
			<td>Diener Electronic</td>
			<td style="width:225px;"></td>
			<td>On-line degassing system with 2 channel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spotter</td>
			<td style="width:203px;">Siliflow</td>
			<td style="width:225px;"></td>
			<td>&#160;It is a non-contact piezoelectric spotter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SPRi-Biochip</td>
			<td>Horiba Scientific-GenOptics</td>
			<td align="right" style="width:225px;">36000067</td>
			<td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">erythrina cristagalli lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L5390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">arachis hypogaea lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L0881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">myoglobin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lysozyme</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#945;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#947;&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sulfated lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">G5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SDS</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">tween 20</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td align="right" style="width:225px;">274348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">HEPES</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium chloride</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S3014</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<html> Para investigar a possibilidade de a lingueta electrónica para análise de proteína comum, foram usadas três proteínas: AHL, mioglobina e lisozima. Para cada proteína, um perfil 2D distinta contínua evolução, PEC foi gerado pelo eT, como mostrado na Figura 6. Além disso, graças à SPRI, que é capaz de controlar os adsorção e dessorção cinética em tempo real, para cada uma proteína padrão de reconhecimento contínuo dependente do tempo, denominado 3D p...

Watch this Scientific Journal Video about Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis at JoVE.com

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

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 ...

electronic-tongue-generating-continuous-recognition-patterns-for

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Bioengineering

7.8K visualizações.  Institut Nanosciences et Cryogénie, CEA-Grenoble. 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. 

Vídeo: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

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

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Colmatar a interface Bio-eletrônico com Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Fabricação e caracterização de um Sistema Eletrônico Conformal-pele como para a gestão quantitativa, Ferida Cutânea

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Bicamadas única molécula de microscopia de fluorescência em Planar Apoiado

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Reconhecimento Molecular de engenharia com Polymers Bio-mimético em únicos Walled nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

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 Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Um método de fabricação simples e escalável para dispositivos eletrônicos orgânicos sobre os têxteis

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

Configuração otimizada e protocolo para a imagem latente magnética domínio em Situ histerese medicão

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In vitro Análise celular multiparamétrica por micro matrizes transistores moduladas por carga orgânica

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

Análise de padrões de expansão do SARPE usando modelos de elementos finitos

Finite Element Analysis Model for Assessing Expansion Patterns from Surgically Assisted Rapid Palatal Expansion

Surgically assisted rapid palatal expansion (SARPE) was introduced to release bony resistance to facilitate skeletal expansion in skeletally mature patients. However, asymmetric expansion between the left and right sides has been reported in 7.52% of all SARPE patients, of which 12.90% had to undergo a second surgery for correction. The etiologies leading to asymmetric expansion remain unclear. Finite element analysis has been used to evaluate the stress associated with SARPE in the maxillofacial structures. However, as a collision of the bone at the LeFort I osteotomy sites occurs only after a certain amount of expansion, most of the existing models do not truly represent the force distribution, given that the expansion amount of these existing models rarely exceeds 1 mm. Therefore, there is a need to create a novel finite element model of SARPE that could perform a clinically required amount of expander activation for further analysis of the expansion patterns of the hemimaxillae in all three dimensions. A three-dimensional (3D) skull model from cone beam computed tomography (CBCT) was imported into Mimics and converted into mathematical entities to segment the maxillary complex, maxillary first premolars, and maxillary first molars. These structures were transferred into Geomagic for surface smoothing and cancellous bone and periodontal ligament creation. The right half of the maxillary complex was then retained and mirrored to create a perfectly symmetrical model in SolidWorks. A Haas expander was constructed and banded to the maxillary first premolars and first molars. Finite element analysis of various combinations of buccal osteotomies at different angles with 1 mm clearance was performed in Ansys. A convergence test was conducted until the desired amount of expansion on both sides (at least 6 mm in total) was achieved. This study lays the foundation for evaluating how buccal osteotomy angulation influences the expansion patterns of SARPE.

Autor em destaque: Desenvolvimento de um novo modelo de análise de elementos finitos para técnicas cirúrgicas ortognáticas aprimoradas 

A set of novel finite element models of surgically assisted rapid palatal expansion (SARPE) that could perform a clinically required amount of expander activation with various angles of buccal osteotomy was created for further analysis of the expansion patterns of the hemimaxillae in all three dimensions.

Modelo de Análise por Elementos Finitos para Avaliação de Padrões de Expansão da Expansão Rápida do Palato Assistida Cirurgicamente

Surgically assisted rapid palatal expansion (SARPE) was introduced to release bony resistance to facilitate skeletal expansion in skeletally mature ...