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 Preparazione di varie soluzioni e proteine ​​Campioni <ol><li> Preparare 100 ml di tampone fosfato (PBS-G) contenente 50 mM di NaH 2 PO 4, 50 mM NaCl, e 10% glicerolo a pH 6,8. </li><li> Preparare 250 ml di soluzione tampone HEPES contenente HEPES 10 mM, NaCl 150 mM, 0,005% di Tween 20 a pH 7,4. </li><li> Preparare la soluzione madre di building block 1 (BB1), lattosio e la costruzione blocco 2 (BB2) solfato lattosio (Figura 1) a 0.2 mM in PBS-G. </li><li> Preparare 1 ml di soluzioni di proteine ​​in HEPES: Un lectine rachide hypogaea (AHL) a 500 nm, la mioglobina a 1 micron, e lisozima a 500 nm. </li><li> Preparare 20 ml di 1% SDS in acqua ultrapura. </li></ol> 2 Preparazione del Array CoCRR <ol><li> Pulire la superficie d&#39;oro del prisma 48 ore prima dell&#39;uso con un detergente plasma per 3 min in queste condizioni: 75% ossigeno, 25% di Argon, 0,6 mbar, potenza 40 W. </li><li> Preparare undici SOLUTIO puri e mistins di BB1 e BB2 con [BB1] / ([BB1] + [BB2]) rapporti tra 0 e 100% con incrementi del 10% ad una concentrazione totale di BB di 0,1 mM in PBS-G. </li><li> Cassetta di 8 gocce nl di queste soluzioni puri e misti sulla superficie del prisma con un spotter senza contatto in quadruplice copia per ciascun rapporto con 44 punti in totale (Figura 2). NOTA: 10% glicerolo aggiunto in PBS-G è molto importante per ridurre l&#39;evaporazione del solvente e la variazione della concentrazione di BB dopo la deposizione. </li><li> Posizionare il prisma all&#39;interno di una capsula Petri contenente 1 ml di acqua ultrapura e lasciarlo O / N a RT per l&#39;auto-assemblaggio di BB1 e BB2 sulla superficie di oro. Qui, si ipotizza che la composizione superficiale media nel misto SAM riflette la composizione della soluzione mista di deposizione (Figura 3) 11. </li><li> Lavare accuratamente il prisma con acqua ultrapura e quindi asciugare sotto un flusso di argon. </li></ol> 3. proteine ​​rilevamento da spri <ol><li> Impostare il incubator in cui l&#39;apparato SPRI è posto a 25 ° C per evitare variazioni dell&#39;indice di rifrazione indotte da variazioni di temperatura durante il rilevamento di proteine. </li><li> Inserire un prisma non funzionalizzata risciacquo in polietere etere chetone 10 microlitri (PEEK) cella di flusso collegato ad una pompa a siringa controllata dal computer, un sistema di degasaggio e una valvola di iniezione media pressione 6 porte (Figura 4). Compilare il sistema di flusso con appena filtrata e degassificati esecuzione HEPES tampone. </li><li> Rimuovere il prisma risciacquo e inserire il prisma contenente la matrice CoCRR nella cella di flusso. Esegui HEPES a / flusso min 100 ml. Con l&#39;aiuto della camera CCD eliminare eventuali bolle d&#39;aria presenti sulla superficie del prisma passando tampone di corsa rapidamente. </li><li> Definire area di studio per ogni spot disegnando un cerchio con lo stesso diametro per l&#39;area di interesse sulla base di un&#39;immagine ben contrastata, della matrice e con l&#39;aiuto di un software integrato nel sistema di spri. </li><li> Tracciare le curve di plasmoni, che rappresentano rifletterecurve ivity in funzione dell&#39;angolo incidente, per tutti i punti. </li><li> Scegliere l&#39;angolo (posizione in cui verranno registrate le curve cinetiche) che lavorano alla massima pendenza delle curve di riflettività. Ruotare lo specchio di scansione per fissare l&#39;angolo di lavoro selezionato per misure cinetiche. </li><li> Continuare a eseguire HEPES attraverso il sistema di flusso fino a quando il segnale di riflessione per tutti i punti è stabile e costante. </li><li> Iniettare 1 ml di soluzione AHL con una siringa nel loop del campione (500 microlitri) con la valvola di iniezione in posizione &quot;carico&quot;. Poi mettere la valvola di iniezione in posizione &quot;iniezione&quot;. La portata utilizzata per tutti l&#39;iniezione proteina era di 100 microlitri / min. </li><li> Inizia misure cinetiche monitorando le variazioni di riflettività contro il tempo simultaneamente su tutti i punti. </li><li> Al termine dell&#39;iniezione proteine, sciacquare la matrice con tampone di corsa per 8 min. Infine, iniettare 1 ml di 1% SDS per rigenerarlo. </li><li> Ripetere la stessa procedura per l&#39;altro proteins. </li></ol> 4 Elaborazione dati e analisi <ol><li> Dopo l&#39;entrata di soluzione proteica nella cella di flusso, molecolare di legame si verifica e induce uno spostamento delle curve plasmon e una variazione di riflettività. Il software di acquisizione delle immagini converte i valori di intensità luminosa misurata al grigio livelli di scala, dando immagini SPR Figura 5A, e genera la variazione di riflettività in funzione del tempo, dando sensorgrams (Figura 5B). </li><li> Utilizzare un software di calcolo matematico per tracciare la riflettività (R%) alla fine di ogni iniezione proteina contro il [BB1] / ([BB1] + [BB2]) rapporto di generare 2D profilo evoluzione continua (CEP) per ogni campione (Figura 5C). </li><li> Aggiungere il [BB1] / ([BB1] + [BB2]) in rapporto sensorgrams (Figura 5B) per generare 3D continua evoluzione del paesaggio (CEL) per ogni proteina (Figura 5D). </li></ol>

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

The authors have no conflicts of interest to declare.

<html> Le fasi più critiche per la costruzione di questa eT sono dedicati per garantire una buona riproducibilità del sistema. Ad esempio, la pulizia della superficie d&#39;oro del prisma con una procedura standardizzata prima dell&#39;uso, aggiungendo il 10% di glicerolo nelle undici soluzioni puri e misti di BB1 e BB2 per eliminare l&#39;evaporazione del solvente durante BB autoassemblante sulla superficie di oro del prisma, depositando più replicati per ogni [BB1] / ([BB1] + [BB2]) rapporto, ecc. Come per ...

Metodi di rilevamento proteine ​​precisi e rapidi sono molto importanti nella diagnostica medica e della proteomica. Array di proteine ​​rilevamento classici, come biochip, si basano sul &quot;tasto lock-e-&quot; principio di riconoscimento e richiedono recettori specifici come aptameri, anticorpi, o mimetici. Negli ultimi anni, il rilevamento differenziale ispirato alla olfatto umano e gustation è emerso come alternativa 1. Questo naso elettronico / lingua (/ eT eN) approccio si basa sul legame di analiti ad una serie di recettori cross-reattivi (CRRS), che non hanno bisogno di essere altamente specifico e selettivo per le molecole target differenziale permettere così a superare il laborioso processo di sviluppo di recettori altamente selettivi. È la risposta combinata di tutti i recettori che crea un modello distinto per ogni campione, come un&#39;impronta digitale, permettendo la sua identificazione. Le due sfide fondamentali per lo sviluppo di elettronic naso / lingua per il rilevamento della proteina efficace sono la produzione di elementi sensibili che hanno la capacità di distinguere tra analiti strutturalmente simili e il sistema di trasduzione appropriata per l&#39;evento vincolante. Fino ad oggi, gli studi hanno riportato vari approcci allo sviluppo di matrice 2. Ad esempio, in uno studio di una identificazione basata su array di proteine ​​è stato sviluppato utilizzando CRRS preparati derivati ​​tetra-carboxyphenylporphyrin accoppiando i gruppi carbossilici ai vari amminoacidi o dipeptidi per fornire recettori differenziali in possesso di un nucleo idrofobico per affinità con le proteine ​​e periferie cariche distinte per impartire differenziale vincolante. Utilizzando questo sistema, diverse proteine ​​e miscele proteiche sono stati identificati mediante misurazione della fluorescenza dei recettori seguito all&#39;interazione con gli analiti 3,4. In un altro studio, una biblioteca di 29 CRRS contenenti tripeptide e frazioni di acido boronici sintetizzato in modo combinatorio su una hexasubstistituito patibolo benzene è stato sviluppato per il rilevamento di proteine ​​con un rilevamento colorimetrico indicatore-assorbimento 5,6. Con un tale disegno, ogni recettore ha mostrato capacità di legame differenziale con le proteine ​​in base alla varianza tra le braccia peptide, e gli acidi boronici assistiti nella differenziazione delle proteine ​​da glicoproteine. Più di recente, una serie composta da diversi cationici funzionalizzati nanoparticelle d&#39;oro coniugati con un poli anionico polimero fluorescente (p-phenyleneethynylene) (PPE) è stato creato per rilevare ed identificare le proteine ​​7. Il legame concorrenziale tra analiti proteici e bonificato PPE / oro complessi nanoparticelle artificiali fluorescenza, producendo modelli di riconoscimento distinti per le proteine. In questo studio, le interazioni proteina-nanoparticelle funzionalizzate sono stati sintonizzati variando le proprietà fisico-chimiche dei gruppi terminali nanoparticelle. Inoltre, è stato dimostrato che questo approccio è efficace per l&#39;analisi delle proteine ​​in complessi e proteine ​​ricche supporto, adcome siero umano a concentrazioni fisiologicamente rilevanti, mostrando in tal modo il potenziale di eT in profilatura campioni reali per la diagnosi di stati di malattia 8. Anche se molto promettente, questi sistemi hanno alcune limitazioni intrinseche. Essi richiedono la progettazione e la sintesi di 5-29 CRRS con strutture molto complesse. Inoltre, a differenza del sistema olfattivo che è resettato dopo ciascuna misurazione, rilevamento proteina richiede la preparazione di una matrice per campione. Infine, monitoraggio eventi vincolanti in tempo reale sono estremamente difficili. In questo contesto, un approccio combinatorio è stato proposto con un piccolo numero di molecole semplici e facilmente accessibili con diverse proprietà fisico-chimiche (idrofili, idrofobici, carica positiva, carica negativa, neutra, ecc.) Come blocchi da costruzione (BBS) 9. Mescolando bbs di variabili e proporzioni controllate e permettendo le miscele di auto-assemblarsi sulla superficie d&#39;oro di unprisma, una vasta gamma di superfici combinatorie che caratterizzano le proprietà appropriate per la proteina di legame è stato creato. In particolare, i monostrati auto-assemblati su questo sistema consentono una facile messa a punto di una serie di proprietà di superficie in modo altamente divergente, consentendo diversi recettori cross-reattivi combinatorie (CoCRRs) per essere prodotta rapido ed efficiente. Rilevamento delle proteine ​​è stata effettuata utilizzando un sistema di rilevamento ottico, risonanza plasmonica di superficie per immagini (SPRI). Brevemente, un ampio fascio monocromatico luce polarizzata da un LED illumina l&#39;intera area matrice CoCRR sulla superficie del prisma. Una videocamera CCD ad alta risoluzione offre immagini di differenza in tempo reale in tutti i punti della matrice CoCRR. Cattura tutte le modifiche locali alla superficie della matrice CoCRR fornire informazioni dettagliate sugli eventi di legame e processi cinetici 10. Nel frattempo, con l&#39;aiuto di software di imaging, immagini SPR corrispondenti a punti vengono convertiti automaticamente alle variazioni di riflettività versus tempo, generando una serie di curve cinetiche di legame chiamato sensorgrams. Così, SPRI permette un&#39;osservazione, sincrono, in parallelo, privo di etichetta e in tempo reale degli eventi vincolanti. Inoltre, la matrice CoCRR ottenuto è rigenerabile e riutilizzabile per analisi delle proteine. Questo protocollo descrive la costruzione della lingua elettronica utilizzando solo due piccole molecole come blocchi di costruzione e illustra la sua applicazione per l&#39;analisi delle proteine ​​comuni basate su modelli di riconoscimento continui ottenuti con spri.

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

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

<html> Per sondare la capacità della lingua elettronica per analisi comune proteine, sono stati usati tre proteine: AHL, mioglobina e lisozima. Per ogni proteina, un profilo distinto 2D continua evoluzione, CEP, è stato generato dal eT, come mostrato nella Figura 6. Inoltre, grazie alla SPRI, che è in grado di monitorare in tempo reale i adsorbimento e desorbimento cinetica, per ogni proteina un tempo dipendente modello riconoscimento continuo, chiamati 3D continua evoluzi...

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.

Lingua elettronica Generazione di modelli continui di rilevazione delle analisi delle proteine

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 Views.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

Video: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Studiare Traiettorie cellulare Rolling Patterns Receptor asimmetrica

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

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Bioingegneria

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.

Colmare il Bio-Electronic Interface con Biofabrication

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

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Microfabrication di Patterns Oro nanoporosi per Cell-materiali Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

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.

Fabbricazione e la caratterizzazione di un Conformal pelle simile sistema elettronico di gestione quantitativa, cutanea Ferita

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.

Singola molecola microscopia a fluorescenza su Planar bilayers supportati

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 ...

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.

Piattaforma microfluidica con multiplex di rilevamento elettronico per spaziale monitoraggio di particelle

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

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

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

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

Un metodo semplice e scalabile di fabbricazione per dispositivi elettronici organici sui tessili

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.

Installazione ottimizzata e protocollo per l'Imaging di dominio magnetico con In Situ isteresi misura

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 Analisi cellulare multiparametrica mediante array di transistor a effetto di campo modulati con carica organica micro

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

Analisi dei modelli di espansione da SARPE utilizzando modelli agli elementi finiti

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.

Autore in primo piano: Sviluppo di un nuovo modello di analisi agli elementi finiti per il miglioramento delle tecniche chirurgiche ortognatiche 

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.

Modello di analisi agli elementi finiti per la valutazione dei modelli di espansione da espansione palatale rapida assistita chirurgicamente

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