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 Preparación de diferentes soluciones y muestras de proteínas <ol><li> Preparar 100 ml de solución tampón de fosfato (PBS-T) que contiene 50 mM NaH 2 PO 4, NaCl 50 mM, y 10% de glicerol a pH 6,8. </li><li> Preparar 250 ml de solución tampón HEPES que contiene HEPES 10 mM, NaCl 150 mM, 0,005% de Tween 20 a pH 7,4. </li><li> Preparar la solución madre de bloque de construcción 1 (BB1) bloque de la lactosa y la construcción de 2 (BB2) sulfatado lactosa (Figura 1) en el 0,2 mM en PBS-T. </li><li> Preparar 1 ml de las soluciones de proteína en HEPES: una lectina hypogaea raquis (AHL) a 500 nm, la mioglobina en 1 m, y lisozima a 500 nM. </li><li> Preparar 20 ml de SDS al 1% en agua ultrapura. </li></ol> 2 Preparación de la matriz CoCRR <ol><li> Limpie la superficie de oro del prisma de 48 horas antes de su uso con un limpiador de plasma durante 3 minutos bajo estas condiciones: 75% de oxígeno, 25% de argón, 0,6 mbar, potencia de 40 W. </li><li> Preparar once solutio puros y mixtosns de BB1 ​​y BB2 con [BB1] / ([BB1] + [BB2]) proporciones entre 0 y 100% en incrementos de 10% a una concentración total de BB 0,1 mM en PBS-T. </li><li> Depósito 8 gotas nl de estas soluciones puras y mixtas sobre la superficie del prisma utilizando un observador sin contacto por cuadruplicado para cada relación con 44 puntos en total (Figura 2). NOTA: 10% de glicerol agregado en PBS-T es muy importante para reducir la evaporación del disolvente y el cambio de concentración BB después de la deposición. </li><li> Coloque el prisma interior de una placa de Petri que contiene 1 ml de agua ultrapura y dejarlo O / N a temperatura ambiente durante autoensamblaje de BB1 ​​y BB2 en la superficie de oro. Aquí, se supone que la composición media de la superficie en el SAM mixta refleja la composición de la solución mixta de deposición (Figura 3) 11. </li><li> Lavar a fondo el prisma con agua ultrapura y luego secarlo bajo un flujo de argón. </li></ol> 3. Proteína Sensing por SPRI <ol><li> Ajuste el incubator en la que el aparato de SPRI se coloca a 25 ° C para evitar los cambios del índice de refracción inducidos por la variación de la temperatura durante la detección de proteínas. </li><li> Insertar un prisma no funcionalizado de enjuague en el 10 l poliéter éter cetona (PEEK) celular conectado a una bomba de jeringa controlada por ordenador, un desgasificador y una válvula de inyección de presión media de 6 puertos de flujo (Figura 4). Rellene el sistema de flujo de recién filtrada y desgasificada corriendo HEPES tampón. </li><li> Retire el prisma de enjuague e inserte el prisma que contiene la matriz CoCRR en la celda de flujo. Ejecutar HEPES a un / caudal min 100 l. Con la ayuda de la cámara CCD eliminar las burbujas de aire presentes en la superficie del prisma pasando tampón en funcionamiento rápidamente. </li><li> Definir área de estudio para cada terreno dibujando un círculo con el mismo diámetro de la zona de interés sobre la base de una imagen bien contrastada-de la matriz y con la ayuda de software integrado en el sistema de SPRI. </li><li> Trazar las curvas de plasmones, que representan reflejarIVITY curvas en función del ángulo de incidencia, para todos los puntos. </li><li> Elija el ángulo (posición en la que se registrarán las curvas cinéticas) trabajando al más alto pendiente de las curvas de reflectividad. Gire el espejo de exploración para fijar el ángulo de trabajo seleccionado para mediciones cinéticas. </li><li> Continuar para ejecutar HEPES a través del sistema de flujo hasta que la señal de reflectividad para todos los puntos es estable y constante. </li><li> Inyectar 1 ml solución AHL con una jeringa en el bucle de la muestra (500 l) con la válvula de inyección en posición de &quot;carga&quot;. A continuación, poner la válvula de inyección a la posición &quot;inyección&quot;. La tasa de flujo utilizado para toda la inyección de proteína fue de 100 l / min. </li><li> Iniciar mediciones cinéticas por vigilar las variaciones de reflectividad en función del tiempo de forma simultánea en todos los lugares. </li><li> Al final de la inyección de la proteína, enjuagar la matriz con tampón de corrida durante 8 min. Por último, se inyecta 1 ml de SDS al 1% para regenerarla. </li><li> Repita el mismo procedimiento para el otro proteins. </li></ol> 4. Procesamiento y Análisis de Datos <ol><li> Después de la entrada de la solución de proteína en la célula de flujo, de unión molecular se produce e induce un desplazamiento de las curvas de plasmones y una variación de la reflectividad. El software de adquisición de imágenes convierte los valores de intensidad de luz medidos a gris niveles de escala, dando imágenes SPR Figura 5A, y genera la variación de la reflectividad en función del tiempo, dando sensogramas (Figura 5B). </li><li> Utilice un software de cálculo matemático para representar la reflectividad (R%) al final de cada inyección de proteínas en comparación con el [BB1] / ([BB1] + [BB2]) Relación de generar el perfil 2D continua evolución (CEP) para cada muestra (Figura 5C). </li><li> Añadir el [BB1] / ([BB1] + [BB2]) en relación sensogramas (Figura 5B) para generar 3D paisaje continua evolución (CEL) para cada proteína (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> Los pasos más críticos para la construcción de este eT están dedicados a asegurar una buena reproducibilidad del sistema. Por ejemplo, la limpieza de la superficie de oro del prisma con un procedimiento estandarizado antes de su uso, la adición de 10% de glicerol en los once soluciones puras y mixtas de BB1 ​​y BB2 para eliminar la evaporación del disolvente durante BBs auto-montaje en la superficie de oro del prisma, depositar múltiples repeticiones para cada [BB1] / ([BB1] + [BB2]) relación, etc....

Métodos de detección de proteínas precisos y rápidos son muy importantes en el diagnóstico médico y la proteómica. Arrays de proteínas de detección de clásicos, como los biochips, se basan en el principio de reconocimiento &quot;tecla de bloqueo-y-&quot; y requieren receptores específicos como aptámeros, anticuerpos, o miméticos. En los últimos años, la detección diferencial inspirado por el olfato humano y gustation ha surgido como una alternativa 1. Esta nariz electrónica / lengua enfoque (en / eT) se basa en la unión de analitos a una matriz de receptores de reacción cruzada (CRRS), que no necesitan ser muy específica o selectiva de las moléculas diana diferencial por que permitan superar la laboriosa proceso de desarrollo de receptores altamente selectivos. Es la respuesta combinada de todos los receptores que crea un patrón distinto para cada muestra, como una huella digital, lo que permite su identificación. Los dos retos clave para el desarrollo de elecTronic nariz / lengua para la detección de proteína eficaz son la producción de elementos de detección que tienen la capacidad de distinguir entre los analitos estructuralmente similares y el sistema de transducción apropiado para el evento de unión. Hasta ahora, los estudios han reportado varios enfoques para el desarrollo matriz 2. Por ejemplo, en un estudio se desarrolló una identificación basada en matrices de proteínas usando CRRS preparados a partir de derivados de tetra-carboxyphenylporphyrin mediante el acoplamiento de los grupos carboxilo a varios aminoácidos o dipéptidos para proporcionar receptores diferenciales que poseen un núcleo hidrófobo de afinidad por las proteínas y las periferias cargadas para distintas impartir unión diferencial. Usando este sistema, diferentes proteínas y mezclas de proteínas se identificaron mediante la medición de la fluorescencia enfriamiento de los receptores de la interacción con los analitos 3,4. En otro estudio, una biblioteca de 29 CRRS tripéptido que contienen restos de ácido borónico y sintetizado de una manera combinatoria en un hexasubstituido andamio benceno fue desarrollado para la detección de proteínas con una detección colorimétrica-indicador de la absorción de 5,6. Con un diseño de este tipo, cada receptor mostró capacidad de unión diferencial con proteínas sobre la base de la varianza en los brazos de péptidos, y los ácidos borónicos asistidos en la diferenciación de las proteínas de glicoproteínas. Más recientemente, una matriz compuesta de diferentes nanopartículas de oro funcionalizadas catiónicos conjugados con un poli fluorescente aniónico polímero (p-phenyleneethynylene) (PPE) se ha creado para detectar e identificar proteínas 7. La unión competitiva entre analitos de proteínas y se extinguió PPE complejos de nanopartículas / oro fluorescencia regeneradas, produciendo patrones de reconocimiento para proteínas distintas. En este estudio, las interacciones proteína-nanopartículas funcionalizadas se sintonizan mediante la variación de las propiedades fisicoquímicas de grupos terminales de nanopartículas. Además, se ha demostrado que este enfoque es eficaz para el análisis de proteínas en un medio rico en proteína compleja y talescomo suero humano a concentraciones fisiológicamente relevantes, lo cual demuestra el potencial de eT en perfiles de muestras reales para el diagnóstico de enfermedades 8. Aunque muy prometedores, estos sistemas tienen algunas limitaciones inherentes. Requieren el diseño y la síntesis de de 5 a 29 CRRS con estructuras muy complicadas. Además, a diferencia del sistema olfativo que se restablece después de cada medición, detección de proteínas requiere la preparación de una matriz por muestra. Finalmente, vigilancia de los eventos de unión en tiempo real son extremadamente difícil. En este contexto, un enfoque combinatorio se propuso mediante el uso de un pequeño número de moléculas simples y de fácil acceso con diferentes propiedades fisicoquímicas (hidrófilas, hidrófobas, cargado positivamente, cargado negativamente, neutral, etc.) Como bloques de construcción (BBS) 9. Mediante la mezcla de los BB en variables y proporciones controladas y permitiendo que las mezclas que se auto-ensamblan en la superficie de oro de unprisma, se ha creado una serie de superficies combinatorias que ofrece propiedades adecuadas para la unión a proteínas. En particular, las monocapas auto-ensambladas en este sistema permiten una fácil puesta a punto de una gama de propiedades de la superficie de una manera muy divergentes, lo que permite diversos receptores de reacción cruzada combinatorias (CoCRRs) para ser producido con rapidez y eficacia. Detección de proteínas se realizó utilizando un sistema de detección óptica, la superficie de formación de imágenes por resonancia de plasmón (SPRI). En pocas palabras, un amplio haz monocromático de luz polarizada a partir de un LED se ilumina toda la zona CoCRR matriz en la superficie del prisma. Una cámara de video CCD de alta resolución proporciona imágenes de diferencia en tiempo real a través de todos los puntos de la matriz CoCRR. Captura todos los cambios locales en la superficie de la matriz CoCRR proporcionar información detallada sobre los eventos de unión y procesos cinéticos 10. Mientras tanto, con la ayuda de software de imágenes, imágenes SPR correspondientes a los puntos se convierten automáticamente a las variaciones de reflectividad versus tiempo, generando una serie de curvas de unión cinéticos llamados sensogramas. Por lo tanto, SPRI permite una observación libre de etiquetas, síncrono, paralelo y en tiempo real de los eventos de unión. Además, la matriz CoCRR obtenido es regenerable y reutilizable para el análisis de proteínas. Este protocolo describe la construcción de la lengua electrónica mediante el uso de sólo dos moléculas pequeñas como bloques de construcción e ilustra su aplicación para el análisis de proteínas comunes basados ​​en patrones de reconocimiento continuas obtenidas con 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 probar la capacidad de la lengua electrónica para el análisis de proteínas común, se utilizaron tres proteínas: AHL, mioglobina y lisozima. Para cada proteína, un perfil continuo 2D distinta evolución, CEP, fue generada por el ET, como se muestra en la Figura 6. Además, gracias a SPRI, que es capaz de controlar el tiempo real de adsorción y desorción cinética, para cada proteína un tiempo patrón de reconocimiento continuo dependientes, llamados 3D pai...

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.

Lengua electrónica Generar Patrones reconocimiento continuo para el análisis de proteínas

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

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JoVE Journal

Bioengineering

7.8K vistas.  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. 

Video: 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.

El estudio de las trayectorias celular Rolling en los patrones del receptor asimétrica

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

Investigación

Bioingeniería

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.

Reducción de la interfaz de Bio-electrónica con 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.

Fabricación y caracterización de un sistema electrónico conforme de la piel-como para la gestión cuantitativa, cutánea de la herida

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.

Bilayers Individual Molécula microscopía de fluorescencia en planar compatibles

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.

Ingeniería Molecular Recognition con polímeros biomiméticos en una sola pared 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 de microfluidos con multiplexado de detección electrónica para el seguimiento espacial de las 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.

Un método de fabricación simple y escalable para dispositivos electrónicos orgánicos sobre los Textiles

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.

Configuración optimizada y protocolo para la proyección de imagen de dominio magnético con In Situ la medición histéresis

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 Análisis celular multiparamétrico mediante matrices de transistores de efecto de campo moduladas por carga micro orgánica

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

Análisis de Patrones de Expansión de 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 destacado: Desarrollo de un nuevo modelo de análisis de elementos finitos para mejorar las técnicas quirúrgicas ortognáticas 

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 Análisis de Elementos Finitos para Evaluar los Patrones de Expansión a partir de la Expansión Palatina Rápida Asistida Quirúrgicamente

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