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 Préparation des différentes solutions et échantillons de protéines <ol><li> Préparer 100 ml d&#39;une solution tampon phosphate (PBS-G) contenant 50 mM de NaH 2 PO 4, NaCl 50 mM, glycerol à 10% et à un pH de 6,8. </li><li> Préparer 250 ml d&#39;une solution tampon HEPES contenant 10 mM d&#39;HEPES, 150 mM de NaCl, 0,005% de Tween 20 à pH 7,4. </li><li> Préparer une solution stock de bloc de construction 1 (BB1) lactose et le renforcement bloc 2 (BB2) sulfaté lactose (Figure 1) à 0,2 mM dans du PBS-G. </li><li> Préparer 1 ml de solutions de protéines dans HEPES: Une lectine rachis hypogaea (AHL) à 500 nm, la myoglobine à 1 uM, et lysozyme à 500 nM. </li><li> Préparer 20 ml de SDS à 1% dans de l&#39;eau ultrapure. </li></ol> 2 Préparation de la matrice CoCRR <ol><li> Nettoyer la surface d&#39;or du prisme 48 heures avant de l&#39;utiliser avec un nettoyeur à plasma pendant 3 min dans ces conditions: 75% d&#39;oxygène, 25% d&#39;argon, 0,6 mbar, puissance 40 W. </li><li> Préparer solutio pur et mixte onzens de BB1 ​​et BB2 avec [BB1] / ([BB1] + [BB2]) rapports entre 0 et 100% par incréments de 10% à une concentration BB total de 0,1 mM dans du PBS-G. </li><li> Dépôt 8 gouttelettes nl de ces solutions pures et mixtes sur la surface du prisme à l&#39;aide d&#39;un observateur sans contact en quatre pour chaque rapport avec 44 points au total (figure 2). NOTE: 10% de glycerol ajoutée dans du PBS-G est très important pour réduire l&#39;évaporation du solvant et le changement de concentration BB après le dépôt. </li><li> Placer le prisme à l&#39;intérieur d&#39;une boîte de Pétri contenant 1 ml d&#39;eau ultra-pure et laisser O / N à la température ambiante pour l&#39;auto-assemblage de BB1 ​​BB2 et sur la surface d&#39;or. Ici, on suppose que la composition de surface moyenne dans le SAM mixte reflète la composition de la solution mixte de dépôt (figure 3) 11. </li><li> Laver soigneusement le prisme avec de l&#39;eau ultra pure et puis on sèche sous un flux d&#39;argon. </li></ol> 3 protéines de détection par SPRi <ol><li> Réglez le incubator dans laquelle l&#39;appareil est placé SPRi à 25 ° C afin d&#39;éviter les changements d&#39;indice de réfraction induits par la variation de température au cours de la détection de protéines. </li><li> Insérer un prisme non fonctionnalisé rinçage dans le 10 ul éther cétone de polyéther (PEEK) cellule connectée à une pompe à seringue commandé par ordinateur, un dégazeur et une soupape d&#39;injection de fluide sous pression 6 ports écoulement (figure 4). Remplir dans le système d&#39;écoulement avec fraîchement filtré et dégazé course de tampon HEPES. </li><li> Retirer le prisme de rinçage et insérer le prisme contenant le réseau CoCRR dans la cellule d&#39;écoulement. Exécuter HEPES à un débit de 100 ul / min. Avec l&#39;aide de la caméra CCD éliminer les bulles d&#39;air présentes sur la surface du prisme en passant rapidement tampon de migration. </li><li> Définir la zone d&#39;étude pour chaque tache en dessinant un cercle de même diamètre pour la zone d&#39;intérêt basé sur une image bien contrastée de la gamme et avec l&#39;aide d&#39;un logiciel intégré dans le système SPRi. </li><li> Tracer des courbes de plasmon, qui représentent le refletLes courbes de ivité en fonction de l&#39;angle d&#39;incidence pour tous les spots. </li><li> Choisir l&#39;angle (position à laquelle les courbes cinétiques seront enregistrées) de travail au plus haut de la pente des courbes de réflectivité. Tourner le miroir de balayage pour fixer l&#39;angle de travail choisi pour les mesures cinétiques. </li><li> Continuez à faire fonctionner HEPES à travers le système d&#39;écoulement jusqu&#39;à ce que le signal de réflectivité pour toutes les taches est stable et constant. </li><li> Injecter 1 ml de solution de LAH avec une seringue dans la boucle d&#39;échantillon (500 ul) à la vanne d&#39;injection en position &quot;charge&quot;. Ensuite, mettre la soupape d&#39;injection en position &quot;injection&quot;. Le débit utilisé pour toutes les injections de la protéine était de 100 pl / min. </li><li> Lancer des mesures cinétiques en surveillant les variations de la réflectivité en fonction du temps à la fois sur tous les points. </li><li> A la fin de l&#39;injection de la protéine, rincer la matrice avec un tampon en cours d&#39;exécution pendant 8 min. Enfin, injecter 1 ml 1% de SDS pour le régénérer. </li><li> Répétez la même procédure pour l&#39;autre proteins. </li></ol> 4 Traitement et analyse des données <ol><li> Suite à l&#39;entrée de la solution de protéine dans la cellule d&#39;écoulement, moléculaire liaison se produit et induit un décalage des courbes de plasmon et une variation de la réflectivité. Le logiciel d&#39;acquisition d&#39;image convertit les valeurs d&#39;intensité lumineuse mesurées à des niveaux d&#39;échelle de gris, ce qui donne des images SPR Figure 5A, et génère la variation de la réflectivité en fonction du temps, ce qui donne sensorgrammes (Figure 5B). </li><li> Utilisation d&#39;un logiciel mathématique de calcul pour tracer la réflectivité (R%) à la fin de chaque injection de la protéine par rapport à la [BB1] / ([BB1] + [BB2]) rapport pour générer 2D profil d&#39;évolution continue (PEC) pour chaque échantillon (Figure 5C). </li><li> Ajouter le [BB1] / ([BB1] + [BB2]) dans le rapport sensorgrammes (figure 5B) pour générer paysage 3D continu de l&#39;évolution (CEL) pour chaque protéine (figure 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> Les étapes les plus critiques pour la construction de cette eT sont dédiés à assurer une bonne reproductibilité du système. Par exemple, le nettoyage de la surface d&#39;or du prisme avec une procédure normalisée avant utilisation, en ajoutant 10% de glycerol dans les onze solutions pures et mixtes de BB1 ​​et BB2 pour éliminer l&#39;évaporation du solvant pendant BB auto-assemblage sur la surface d&#39;or du prisme, dépôt de plusieurs répétitions pour chaque [BB1] / ([BB1] + [BB2]) Ratio, et...

Méthodes de détection des protéines précises et rapides sont très importants dans le diagnostic médical et de la protéomique. Puces à protéines de détection classiques, tels que les biopuces, sont basés sur le principe de la reconnaissance &quot;clé lock-et-&quot; et nécessitent des récepteurs spécifiques tels que les aptamères, des anticorps ou mimétiques. Au cours des dernières années, la détection différentielle inspiré par le olfaction humaine et gustation a émergé comme une alternative 1. Ce nez / langue électronique (fr / eT) approche est basée sur la liaison d&#39;analytes à un réseau de récepteurs à réaction croisée (SDRC), qui n&#39;ont pas besoin d&#39;être très spécifique ou sélective pour les molécules cibles différentielle permet donc de surmonter le laborieux processus de développement de récepteurs hautement sélectifs. Il s&#39;agit de la réponse combinée de tous les récepteurs qui crée un motif distinct pour chaque échantillon, comme une empreinte digitale, permettant son identification. Les deux principaux défis pour le développement des electronic nez / langue pour la détection de la protéine efficace sont la production d&#39;éléments de détection qui ont la capacité de faire la distinction entre les analytes structurellement similaires et le système de transduction approprié pour l&#39;événement de liaison. Jusqu&#39;à présent, les études ont rapporté diverses approches du développement du tableau 2. Par exemple, dans une étude d&#39;une identification basée sur la baie de protéines a été développé en utilisant CRC qui préparés à partir des dérivés tétra-carboxyphenylporphyrin par couplage de groupes carboxyle de différents acides aminés ou de dipeptides à fournir des récepteurs différentiels possédant un noyau hydrophobe de l&#39;affinité pour les protéines et les périphéries chargées distinctes pour transmettre liaison différentielle. En utilisant ce système, les différentes protéines et des mélanges de protéines ont été identifiées par la mesure de l&#39;extinction de fluorescence des récepteurs de l&#39;interaction avec les analytes 3,4. Dans une autre étude, une bibliothèque de 29 CRC qui tripeptide contenant et fragments d&#39;acide boronique synthétisé d&#39;une manière combinatoire sur un hexasubstil&#39;ar- échafaudage de benzène a été mis au point pour détecter les protéines avec une détection colorimétrique indicateur d&#39;absorption de 5,6. Avec une telle conception, chaque récepteur différentiel a montré une capacité de liaison de protéines sur la base de la variance dans les bras et les peptides, les acides boroniques ont aidé à la différenciation de protéines à partir de glycoprotéines. Plus récemment, un tableau composé de différents cationiques fonctionnalisés nanoparticules d&#39;or conjugués avec un poly anionique polymère fluorescente (p-phénylèneéthynylène) (PPE) a été créé pour détecter et identifier les protéines 7. La liaison de concurrence entre les analytes de protéines et trempé complexes de nanoparticules / or EPI fluorescence régénérées, la production de modèles de reconnaissance distinctes pour les protéines. Dans cette étude, les interactions protéines-nanoparticules fonctionnalisées ont été réglées en faisant varier les propriétés physico-chimiques des groupes terminaux de nanoparticules. En outre, il a été montré que cette approche est efficace pour l&#39;analyse des protéines dans un milieu complexe et riche en protéines telleque le sérum humain à des concentrations physiologiquement pertinents, montrant ainsi le potentiel de eT dans le profilage des échantillons réels pour diagnostiquer des états pathologiques 8. Bien que très prometteur, ces systèmes ont des limites inhérentes. Ils nécessitent la conception et la synthèse du 5 au 29 CRC qui avec des structures très compliquées. En outre, contrairement au système olfactif qui est remis à zéro après chaque mesure, de la protéine de détection nécessite la préparation d&#39;une matrice par exemple. Enfin, le suivi des événements de liaison en temps réel sont extrêmement difficiles. Dans ce contexte, une approche combinatoire a été proposée en utilisant un petit nombre de molécules simples et facilement accessibles avec différentes propriétés physico-chimiques (hydrophile, hydrophobe, chargé positivement, chargé négativement, neutre, etc.) En tant que blocs de construction (BBS) 9. En mélangeant les BB en variant et des proportions contrôlées et en permettant aux mélanges à s&#39;auto-assembler sur la surface d&#39;or d&#39;unprisme, un réseau de surfaces combinatoires comportant des propriétés appropriées pour la protéine de liaison a été créé. Notamment, les monocouches auto-assemblées sur ce système permettent le réglage facile d&#39;une gamme de propriétés de surface de manière très divergente, ce qui permet divers récepteurs à réaction croisée combinatoires (CoCRRs) pour être rapidement et efficacement produite. Protéine de détection a été effectuée en utilisant un système de détection optique, la surface d&#39;imagerie par résonance de plasmon (SPRi). En bref, un large faisceau monochromatique de lumière polarisée à partir d&#39;une LED illumine toute la surface de la matrice CoCRR sur la surface du prisme. Une caméra CCD haute résolution de la vidéo fournit des images de différence en temps réel sur tous les spots de la gamme CoCRR. Il saisit toutes les modifications locales à la surface de la matrice CoCRR fournissant des informations détaillées sur les événements de liaison et les processus cinétiques 10. Pendant ce temps, à l&#39;aide de logiciels d&#39;imagerie, images SPR correspondant aux taches sont automatiquement converties en variations de réflectivité versus le temps, générer une série de courbes de liaison cinétiques appelés sensorgrammes. Ainsi, SPRi permet une observation, synchrone, parallèle, sans étiquette et en temps réel des événements de liaison. En outre, le tableau CoCRR obtenu est régénérable et réutilisable pour l&#39;analyse des protéines. Ce protocole décrit la construction de la langue électronique en utilisant seulement deux petites molécules comme des blocs de construction et illustre son application pour l&#39;analyse des protéines communes basées sur des modèles de reconnaissance continus obtenus avec 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> Pour sonder la capacité de la languette électronique pour l&#39;analyse des protéines commun, trois protéines ont été utilisées: AHL, la myoglobine et le lysozyme. Pour chaque protéine, un profil 2D distinct continu de l&#39;évolution de la CEP, a été générée par l&#39;ET, comme le montre la figure 6. En outre, grâce à SPRi, qui est capable de surveiller les adsorption et de désorption cinétique en temps réel, pour chaque protéine un temps de modèl...

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.

Langue Electronique Génération Schémas de reconnaissance continue pour l&#39;analyse des protéines

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

Research

JoVE Journal

Bioengineering

7.8K vues.  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. 

Vidéo: 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.

Etudier Trajectoires Cellule roulant sur les Patterns récepteur asymétrique

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

Recherche

Bio-ingénierie

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.

Combler l&#39;interface bio-électronique avec 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.

Fabrication et caractérisation d&#39;un système électronique conforme de la peau comme pour la gestion quantitative, cutanée plaies

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 seule molécule microscopie à fluorescence sur Planar pris en charge

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.

Reconnaissance moléculaire Ingénierie avec Polymères Bio-mimétiques sur simple 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 ...

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.

Plate-forme microfluidique avec multiplexée Détection électronique pour le suivi spatial des particules

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.

Une méthode de fabrication simple et évolutive pour les dispositifs électroniques organiques sur les 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.

Installation optimisée et protocole d’imagerie de domaines magnétiques sur mesure In Situ de hystérésis

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 Analyse cellulaire multiparamétrique par des réseaux de transistors à effet de champ à modulation de charge micro organique

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

Analyse des modèles d’expansion de SARPE à l’aide de modèles d’éléments finis

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.

Pleins feux sur l’auteur : Développement d’un nouveau modèle d’analyse par éléments finis pour améliorer les techniques chirurgicales orthognathiques 

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.

Modèle d’analyse par éléments finis pour l’évaluation des modèles d’expansion à partir d’une expansion palatine rapide assistée chirurgicalement

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