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. Herstellung der verschiedenen Lösungen und Protein-Proben <ol><li> Planen 100 ml Phosphatpufferlösung (PBS-G), der 50 mM NaH 2 PO 4, 50 mM NaCl und 10% Glycerin bei pH 6,8. </li><li> Planen 250 ml HEPES-Pufferlösung, enthaltend 10 mM HEPES, 150 mM NaCl, 0,005% Tween 20 bei pH 7,4. </li><li> Bereiten Stammlösung von Baustein 1 (BB1) Laktose und Baustein 2 (BB2) sulfatierte Laktose (Abbildung 1) bei 0,2 mM in PBS-G. </li><li> Vorbereitung 1 ml der Proteinlösung in HEPES: A rachis hypogaea Lectin (AHL) bei 500 nm, Myoglobin bei 1 uM, und Lysozym bei 500 nM. </li><li> Es werden 20 ml 1% SDS in ultrareinem Wasser. </li></ol> 2. Herstellung der CoCRR Array <ol><li> 75% Sauerstoff, 25% Argon, 0,6 mbar, Leistung 40 W.: die Goldoberfläche des Prismas 48 Stunden vor der mit einem Plasma-Reiniger für 3 min unter diesen Bedingungen Gebrauch zu reinigen </li><li> Bereiten elf reinen und gemischten solutions BB1 und BB2 mit [BB1] / ([BB1] + [BB2]) Verhältnisse zwischen 0 und 100% in Schritten von 10% bei einem Gesamt BB Konzentration von 0,1 mM in PBS-G. </li><li> Hinterlegung 8 nl Tröpfchen dieser reinen und gemischten Lösungen auf der Prismenoberfläche unter Verwendung eines berührungs Spotter vierfach für jede Verhältnis mit 44 Vertiefungen insgesamt (Abbildung 2). HINWEIS: 10% Glycerin in PBS-g ist sehr wichtig, um die Lösungsmittelverdampfung und die Änderung der Konzentration BB nach der Abscheidung zu reduzieren. </li><li> Legen Sie das Prisma in einer Petrischale mit 1 ml hochreinem Wasser und lassen Sie es O / N bei RT Selbstorganisation von BB1 und BB2 auf der Goldoberfläche. Hierin wird angenommen, dass die durchschnittliche Oberflächenzusammensetzung in der gemischten SAMs spiegelt die Zusammensetzung der Abscheidungslösung gemischt (Figur 3) 11. </li><li> Gründlich waschen das Prisma mit hochreinem Wasser und trocknen Sie sie unter einem Strom von Argon. </li></ol> 3. Proteinerkennung durch SPRi <ol><li> Stellen Sie die incubator, in dem die SPRi Vorrichtung wird bei 25 ° C platziert, um Änderungen des Brechungsindex durch die Temperaturänderung während der Proteinerfassungs induzierte vermeiden. </li><li> Legen Sie eine nicht funktionalisierte Spülen Prisma in der 10 ul Polyether-Ether-Keton (PEEK), die Durchflusszelle mit einem Computer gesteuert Spritzenpumpe, einem Entgaser und einem 6-Tor-Mitteldruck-Einspritzventil (4). Füllen Sie das Flusssystem mit frisch gefiltert und entgast Laufpuffer HEPES. </li><li> Entfernen Sie die Spülung Prisma und legen das Prisma, das die CoCRR Array in der Durchflusszelle. Run HEPES bei einer 100 ml / min Volumenstrom. Mit Hilfe der CCD-Kamera entfernen alle auf der Prismenoberfläche vorliegenden Luftblasen, indem Laufpuffer schnell. </li><li> Definieren Arbeitsbereich für jeden Punkt durch Zeichnen eines Kreises mit dem gleichen Durchmesser für den Bereich von Interesse auf der Basis eines kontrastreichen Bildes der Anordnung und mit Hilfe der integrierten Software SPRi System. </li><li> Spurenplasmonenkurven, die reflektieren darstellenduktivität Kurven in Abhängigkeit von dem Einfallswinkel für alle Flecken. </li><li> Wählen Sie die Arbeitswinkel (Position, an der kinetischen Kurven werden aufgezeichnet) auf der höchsten Steigung der Reflexionskurven. Drehen Sie den Scanspiegel, um den ausgewählten Arbeitswinkel für kinetische Messungen zu beheben. </li><li> Weiterhin HEPES durch den Strömungssystem laufen, bis Reflexionssignal für alle Flecken stabil und konstant ist. </li><li> Injizieren von 1 ml AHL-Lösung mit einer Spritze in die Probenschleife (500 ul) mit dem Einspritzventil auf Position &quot;Last&quot;. Dann legen Sie das Einspritzventil zu &quot;Injektion&quot; zu positionieren. Die Flussrate für die gesamte Protein Injektion verwendet wurden 100 ul / min. </li><li> Starten kinetische Messungen durch Überwachung Reflektivität Variationen gegen die Zeit gleichzeitig auf allen Flecken. </li><li> Am Ende des Protein-Injektion, Spülung der Anordnung mit Laufpuffer für 8 min. Schließlich injizieren 1 ml 1% SDS, um es zu regenerieren. </li><li> Wiederholen Sie den Vorgang für die andere proteins. </li></ol> 4. Datenverarbeitung und Analyse <ol><li> Nach Eingabe der Proteinlösung in der Strömungszelle, molekulare Bindung auftritt, und bewirkt eine Verschiebung des Plasmonen Kurven und eine Veränderung des Reflexionsvermögens. Die Bildaufnahme-Software wandelt die gemessenen Lichtintensitätswerte Grauskalapegel, wodurch SPR Bilder 5a und erzeugt die Veränderung des Reflexionsvermögens als Funktion der Zeit, so dass Sensorgramme (5B). </li><li> Verwenden Sie ein Mathe-Computing-Software, um die Reflektivität (R%) am Ende jedes Proteins Injektion gegenüber dem [BB1] Grundstück / ([BB1] + [BB2]) Verhältnis zu 2D kontinuierliche Entwicklung Profil (CEP) für jede Probe zu erzeugen (Abbildung 5C). </li><li> Fügen Sie die [BB1] / ([BB1] + [BB2])-Verhältnis im Sensorgramme (5B), um 3D-Landschaft kontinuierliche Entwicklung (CEL) für jedes Protein (5D) zu erzeugen. </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> Die wichtigsten Schritte zur Konstruktion dieser eT sind bemüht, eine gute Reproduzierbarkeit des Systems zu gewährleisten. Beispielsweise die Reinigung der Goldoberfläche des Prismas mit einem standardisierten Verfahren vor Verwendung Zugabe von 10% Glycerin in den elf reinen und gemischten Lösungen BB1 und BB2 zur Lösungsmittelverdampfung während BBs selbstorganisierende auf der Goldoberfläche des Prismas zu vermeiden, Abscheiden mehrerer Wiederholungen für jede [BB1] / ([BB1] + [BB2])-Verhältnis, e...

Präzise und schnelle Proteinerkundungsmethoden sind in der medizinischen Diagnostik und der Proteomik sehr wichtig. Klassische Proteindetektions Arrays, wie Biochips, auf der &quot;Schloss-und-Schlüssel&quot; Anerkennung Prinzip und erfordern spezifische Rezeptoren wie Aptamere, Antikörper oder Mimetika. In den letzten Jahren hat Differenzmess inspiriert durch den menschlichen Geruchssinn und gustation als Alternative 1 entstanden. Diese elektronische Nase / Zunge (DE / ET) Ansatz basiert auf unterschiedlichen Bindung von Analyten auf ein Array von kreuzreaktiven Rezeptoren (CRRs), die nicht brauchen, sehr spezifisch oder selektiv für die Zielmoleküle zu sein auf der Basis ermöglichen so die mühsame zu überwinden Prozess der Entwicklung hochselektiver Rezeptoren. Es ist die kombinierte Antwort aller Rezeptoren, die ein eindeutiges Muster für jede Probe erstellt, wie ein Fingerabdruck, so dass ihre Identifikation. Die beiden wichtigsten Herausforderungen für die Entwicklung von elektronischen Nase / Lasche wirksame Protein Erfassungs sind die Herstellung von Sensorelementen, die die Fähigkeit zu strukturell ähnlichen Analyten und das entsprechende Übertragungssystem für das Bindungsereignis zu unterscheiden. Bisher wurden Untersuchungen verschiedene Ansätze zur Entwicklung Array 2 angegeben. Beispielsweise in einer Studie einer Array-basierten Identifizierung von Proteinen wurde unter Verwendung von Tetra-CRRs carboxyphenylporphyrin Derivaten hergestellt durch Kopplung der Carboxygruppen an verschiedene Aminosäuren oder Dipeptide Differenz Rezeptoren besitzen einen hydrophoben Kern auf Affinität für Proteine ​​und verschiedene Umfänge berechnet vorsehen entwickelten Vermittlung differentielle Bindung. Mit diesem System wurden verschiedene Proteine ​​und Proteingemische durch Messen der Fluoreszenzlöschung von den Rezeptoren durch die Wechselwirkung mit den Analyten 3,4 identifiziert. In einer anderen Studie wurde eine Bibliothek von 29 CRRs Tripeptid und Boronsäurereste enthalten in einer kombinatorischen Art und Weise synthetisiert auf einem hexasubstituierte Benzolgerüst wurde zur Erfassung Proteine ​​mit einem Indikator-Aufnahme kolorimetrischen Nachweis 5,6 entwickelt. Mit einer solchen Konstruktion, zeigte jeder Rezeptor Differenzbindungskapazität mit Proteinen auf der Basis der Abweichung in den Peptid Arme und der Boronsäuren der Differenzierung von Proteinen aus Glycoproteinen begleitet. In jüngerer Zeit eine Reihe von verschiedenen kationischen funktionalisierten Goldnanopartikel mit einem anionischen fluoreszierenden Polymer Poly (p-phenylenethinylen) (PPE) konjugiert zusammen wurde geschaffen, um zu detektieren und zu identifizieren Proteine ​​7. Die kompetitive Bindung zwischen Protein Analyten und abgeschreckt PPE / Gold-Nanopartikel-Komplexen regeneriert Fluoreszenz, Herstellung verschiedene Erkennungsmuster für Proteine. In dieser Studie wurden die funktionalisierten Nanopartikel-Protein-Wechselwirkungen durch Variieren physikochemischen Eigenschaften der Nanopartikel-Endgruppen abgestimmt. Weiterhin wurde gezeigt, dass dieser Ansatz wirksam ist für die Proteinanalyse in komplexen und eiweißreichen Medium, wie beispielsals Humanserum bei physiologisch relevanten Konzentrationen, was zeigt, das Potenzial der eT in Profi realen Proben für die Diagnose von Krankheitszuständen 8. Wenn auch sehr vielversprechend, haben diese Systeme einige inhärente Einschränkungen. Sie erfordern Planung und Synthese von 5-29 CRRs mit recht komplizierten Strukturen. Darüber hinaus, im Gegensatz zu dem olfaktorischen System, der sich mit folgenden jeder Messung Protein Erkundung erfordert Herstellung eines Arrays pro Probe. Schließlich sind die Überwachung in Echtzeit Bindungsereignisse extrem schwierig. In diesem Zusammenhang wurde ein kombinatorischer Ansatz unter Verwendung einer kleinen Anzahl von einfachen und leicht zugänglichen Molekülen mit unterschiedlichen physikalisch-chemischen Eigenschaften als Bausteine ​​(BBS) 9 vorgeschlagen (hydrophile, hydrophobe und positiv geladene, negativ geladene, neutrale, etc.). Durch Mischen BBs in unterschiedlichen und kontrollierten Anteilen und man die Mischungen zur Selbstorganisation an der Goldoberfläche einerPrisma, ein Array von Flächen mit entsprechenden kombinatorischen Eigenschaften zum Binden von Protein wurde erstellt. Bemerkenswert ist, die selbstorganisierte Monolagen auf diesem System ermöglichen eine einfache Abstimmung der eine Reihe von Oberflächeneigenschaften in einem stark divergent, so dass diverse kombikreuzreaktive Rezeptoren (CoCRRs), um schnell und effizient produziert. Protein Erfassungs wurde unter Verwendung eines optischen Erfassungssystems, Oberflächenplasmonresonanz-Bildgebung (SPRi). Kurz gesagt, beleuchtet ein Breitstrahl monochromatisch polarisierte Licht von einem LED-Array das gesamte CoCRR Bereich auf der Oberfläche des Prismas. Eine hochauflösende CCD-Videokamera bietet Echtzeit-Differenzbilder über alle Orte der CoCRR Array. Es erfasst alle lokalen Änderungen an der Oberfläche des CoCRR Array mit detaillierten Informationen über die Bindungsereignisse und kinetischen Prozessen 10. Dessen mit Hilfe der Bildbearbeitungssoftware SPR entsprechenden Bilder Flecken werden automatisch Variationen der Reflektivität Versu umgewandelts Zeit, Erzeugen einer Reihe von kinetischen Bindungskurven genannte Sensorgrammen. So erlaubt SPRi ein Etikett frei, synchrone, parallele und Echtzeit-Beobachtung von Bindungsereignissen. Zusätzlich wird die erhaltene CoCRR Array regenerierbar und wiederverwendbar für die Proteinanalyse. Dieses Protokoll beschreibt die Konstruktion des elektronischen Zunge mit nur zwei kleine Moleküle als Bausteine ​​und zeigt seine Anwendung zur Analyse von Proteinen gemeinsam, die auf kontinuierlichen Erkennungsmuster mit SPRi erhalten.

<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> Um die Fähigkeit der elektronischen Zunge für gemeinsame Protein-Analyse zu untersuchen, wurden drei Proteine ​​verwendet: AHL, Myoglobin und Lysozym. Für jedes Protein eine deutliche 2D kontinuierliche Entwicklung Profil CEP wurde die ET erzeugt wird, wie in 6 gezeigt. Darüber hinaus dank SPRi, die in der Lage, um die Echtzeit-Adsorption und Desorption Kinetik zu überwachen, für jedes Protein eine zeitabhängige kontinuierliche Erkennungsmuster ist, die so g...

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.

Elektronische Zunge generieren Continuous Erkennungsmuster für die Protein-Analyse

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 Aufrufe.  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. 

Serie: 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.

Studieren Zellrollen Trajectories auf Asymmetric Rezeptor Patterns

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

Forschung

Biotechnik

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.

Bridging the Bio-elektronische Schnittstelle mit 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.

Herstellung und Charakterisierung eines Conformal-Haut-like Electronic System for Quantitative, kutane Wundmanagement

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.

Einzelmolekülfluoreszenzmikroskopie auf Planar Unterstützte 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 ...

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.

Technik Molekulare Erkennung mit Bio-Mimetikum Polymers auf einwandigen Kohlenstoff-Nanoröhrchen

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.

Mikrofluidik-Plattform mit Multiplexed elektronische Sensoren für Raumverfolgung von Partikeln

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.

Eine einfache und skalierbare Herstellungsverfahren für organische elektronische Vorrichtungen auf Textilien

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.

Optimierte Setup und ein Protokoll für magnetische Domäne Bildgebung mit In Situ Hysterese-Messung

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 Multiparametrische Zellanalyse durch mikroorganisch ladungsmodulierte Feldeffekt-Transistorarrays

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

Analyse von Expansionsmustern aus SARPE mit Hilfe von Finite-Elemente-Modellen

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 im Rampenlicht: Entwicklung eines neuartigen Finite-Elemente-Analysemodells für verbesserte orthognathe Operationstechniken 

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.

Finite-Elemente-Analysemodell zur Beurteilung von Expansionsmustern bei chirurgisch unterstützter Gaumenschnellexpansion

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