Questo lavoro è stato sostenuto da sovvenzioni a SHO dal National Institutes of Health (R01 GM092993), la National Science Foundation (NSF CARRIERA Award e DBI 0.964.216), l&#39;Office of Naval Research (ONR) Giovane Programma Investigator Award e il Minnesota Partnership for Biotechnology e genomica medica. Fabbricazione del dispositivo è stata eseguita presso l&#39;Università del Minnesota Nanofabbricazione Center (NFC), che riceve il sostegno della NSF attraverso l&#39;infrastruttura di rete National Nanotechnology. Questo lavoro è stato supportato anche da sovvenzioni di MR dal National Institutes of Health (NS048357, R21 NS073684), la National Multiple Sclerosis Society (CA1060A11), la Applebaum, Hilton, Peterson e Sanford Fondazioni e la famiglia McNeilus. Gli autori desiderano ringraziare Hyungsoon Im per l&#39;assistenza con illustrazioni e Shailabh Kumar per l&#39;assistenza con il microscopio elettronico a scansione.

1. Microfabrication di Microwell Array substrato <ol><li> Iniziare con un wafer di silicio da 4 pollici con 100 nm di ossido cresciuto termicamente. </li><li> Spin SPR-955 0,7 photoresist sul wafer a 4.000 rpm per 30 sec. </li><li> Cuocere su una piastra riscaldante a 115 ° C per 90 sec. </li><li> Esporre photoresist. <ol><li> Utilizzare una maschera che creerà 1 micron fori disposti in una matrice esagonale con un periodo di 3 micron in cui l&#39;array copre un&#39;area mm x 2 2 mm. </li><li> Esporre wafer in un passo-passo i-line utilizzando un passo di 6 mm e una esposizione di 200 mJ / cm 2. </li></ol></li><li> Cuocere su una piastra riscaldante a 115 ° C per 90 sec. </li><li> Sviluppare con uno sviluppatore di selezione per depositare 2 pozze d&#39;CD-26 sul wafer per un tempo di sviluppo totale di 90 sec. </li><li> Sciacquare abbondantemente con acqua ultrapura DI e asciugare con N 2. </li><li> Etch l&#39;ossido in un incisore reattiva ioni di litio per 6 min con 50 sccm di Ar, 25 sccm di CF 4 e 50 sccm di CHF 3 floala ad una pressione di 75 mTorr. </li><li> Etch silicio in una profonda ICP-trincea incisore reattiva di litio per 2 min con 63 sccm di C 4 F 8, 27 sccm di SF 6, 40 sccm di Ar, e 10 sccm di O 2 che scorre ad una pressione di 14 mTorr. </li><li> Sciacquare in acetone, metanolo e isopropanolo per rimuovere resistere. </li><li> Posto in un bagno di H 2 SO 4: H 2 O 2 1:1 10 min per pulire la superficie. </li><li> Sciacquare abbondantemente con acqua ultrapura DI e asciugare con N 2. </li><li> Deposita 1.080 Å di Al 2 O 3 da deposizione di strati atomici a 250 ° C. </li></ol> 2. Preparazione di Poli (dimetilsiloxano) seccatoio <ol><li> Se possibile, lavorare in una camera sterile. In caso contrario, lavorare in un ambiente più pulito possibile. </li><li> Ottenere un piatto di plastica Petri (o altro contenitore monouso pulito) con ≥ 13 millimetri lati. </li><li> In quel piatto, versare il 5 g agente indurente PDMS, seguita da 50 g di PDMS base l&#39;agente (o qualsiasi cosa con un rapporto 1:10 che per lo più riempire il piatto di Petri). Mescolare accuratamente con una pipetta di plastica usa e getta o asta. </li><li> Rimuovere le bolle posizionando il PDMS misti in una camera con una linea di vuoto, applicando vuoto fino bolle fuoriescono miscela (circa 30 min). </li><li> Se non è già nel piatto di Petri, versare PDMS in piatto di Petri, evitando la creazione di bolle d&#39;aria. </li><li> Curare durante la notte su una piastra riscaldante a&gt; 50 ° C (maggiore calore per meno tempo funziona anche). </li><li> Con la nuova / lama di rasoio pulito, accuratamente tagliato un pezzo rettangolare di circa 2,5 x 2,5 centimetri, mantenendo superficie superiore più pulito possibile. Sbucciare fuori con una pinza pulita. Tagliare un bordo (fino a 1,5 cm) premendo sulla lama di rasoio in un unico movimento, per rendere bordo superiore più nitido possibile (questo sarà il bordo utilizzato nel processo tergipavimento). </li><li> Pulire con acetone, metanolo, alcol isopropilico e acqua deionizzata ultrapura, e poi asciugare con azoto pulito. Conservare in un piatto pulito Petri. </li></ol> 3. Preparazione di vescicole <ol><li> Raccogliere soluzioni stock lipidici di fosfatidilcolina 25 mg / ml uovo (PC) e 1 mg / ml di 1,2-dipalmitoil-sn-glycero-3-fosfoetanolamina-N - (lissamina rodamina B sulfonyl) sale di ammonio (Rho-PDPE) in cloroformio e / soluzione madre ml 0,5 mg di monosialoganglioside GM1 in cloroformio / metanolo (2:1 v / v). </li><li> In un flaconcino piccolo bicchiere fare una miscela che si traduce nel 97% molare PC, 2 mol% GM1 e 1 mole% Rho-PDPE aggiungendo 18,9 ml di 25 mg / ml PC, 39,6 ml di 0,5 mg / ml GM1 e 7,9 ml di 1 mg / ml Rho-PDPE usando siringhe di vetro. </li><li> Nota:. Il materiale supplementare per Nair et al 25 contiene un ottimo foglio di calcolo Excel personalizzabile per il calcolo degli importi di lipidi necessari per creare vescicole di qualsiasi composizione desiderata. </li><li> Posizionare flaconcino in un essiccatore sotto vuoto e rimuovere il solvente lasciando il flaconcino sotto vuoto per 6 ore. </li><li> Preparare una soluzione acquosa di 0,1M NaCl. Aggiungere 0,5 ml di soluzione 0,1 M di NaCl al flaconcino contenente il film lipidico essiccato. </li><li> Lasciare la miscela lipidica acquosa per riposare durante la notte. </li><li> Brevemente mix vortice per creare una sospensione vescicola, poi con ultrasuoni per 20 minuti in un bagno sonicatore a temperatura ambiente. </li><li> Prelevare la sospensione vescicola attraverso una membrana di policarbonato con 100 nm dimensione dei pori. Passare la sospensione vescicola attraverso il filtro di 17x. </li><li> Conservare le vescicole estrusi in un flacone di vetro a 4 ° C. </li></ol> 4. Formazione di sferici supportati doppi strati lipidici <ol><li> Raccogliere 700 nm di diametro SiO 2 perline. Le perle sono forniti in acqua distillata con una concentrazione scorta di 1,4 x 10 11 perline / ml. </li><li> In una provetta Eppendorf da 1,5 ml preparare una sospensione di sferette 1 ml con una concentrazione di 1,5 x 10 10 perline / ml aggiungendo 107 ml di soluzione tallone stock a 893 ml di 0,1 M NaCl. </li><li> Agitare la sospensione per assicurarsi che sia ben missa. </li><li> Centrifugare la sospensione a 1.700 xg per 20 minuti poi scartare il surnatante. Risospendere le sfere in 1 ml di 0,1 M di NaCl. Ripetere questa operazione altre due volte di lavare accuratamente le perline. </li><li> Per formare i SSLBs, in una provetta Eppendorf da 1,5 ml aggiungere 25 microlitri della sospensione di sferette SiO 2 a 200 ml di 1 mg / ml di sospensione di vescicole. Vortex il composto e lasciate riposare per 1 ora a temperatura ambiente. In questa fase la concentrazione SSLB dovrebbe essere 1,7 x 10 9 SSLBs / ml. </li><li> Centrifugare a 1.700 xg per 20 min a pellet i SSLBs. Il pellet dovrebbe apparire rosa a causa della rottura di vescicole con Rho-PDPE sulle perline. </li><li> Eliminare il surnatante e risospendere in 225 microlitri tampone fosfato salino (PBS), pH = 7.4. Ripetere i passaggi 4,6-4,7 altre due volte per rimuovere eventuali vescicole unruptured dalla sospensione SSLB. </li></ol> 5. Assemblea dei SSLB Array <ol><li> Wafer Cleave di matrici micropozzetti con conseguente pezzi rettangolari con 4-6 micRowell array per pezzo. </li><li> Vortex mescolare la sospensione SSLB, quindi versare 10 ml di sospensione SSLB su ogni matrice microwell. Per timore di riposo per 1 ora per consentire SSLBs di depositarsi sulla superficie. </li><li> Lavare delicatamente il chip matrice pozzetto con PBS da una bottiglia di lavaggio, poi immergere in un bagno PBS preparato in un piatto fondo. </li><li> Mentre sommerso, inserire delicatamente il PDMS filo seccatoio sul chip matrice microwell e farlo scorrere delicatamente lungo la superficie 5x per rimuovere SSLBs che non sono immobilizzati in pozzetti. </li><li> Afferrare il chip matrice pozzetto con una pinzetta e delicatamente agitare nel bagno PBS per rimuovere eventuali SSLBs eccesso. </li><li> Rimuovere rapidamente il chip dal bagno spatola e mettere in un bagno PBS fresco fino a nuovo uso. Assicurarsi che la superficie superiore del chip rimane bagnato per preservare le SSLBs. </li></ol> Nota: Il metodo di assemblaggio sopra descritto può essere usato per creare matrici di particelle di membrana naturali, come particelle mielina isolato da cervello di topo, o phovescicole spholipid che non SiO 2 supporti tallone. Questo lavoro è descritto in dettaglio in Wittenberg et al. 21 e risultati rappresentativi sono mostrati di seguito. 6. Tossina colerica Binding Assay <ol><li> Preparare una soluzione di 2 mg / ml di sieroalbumina bovina (BSA) in PBS. </li><li> Preparare una soluzione con concentrazione desiderata di Alexa 488 coniugato tossina colerica B-subunità in PBS con 2 mg / ml di BSA. </li><li> Rimuovere il chip matrice SSLB dal bagno PBS ed evacuare la maggior parte della soluzione PBS usando un laboratorio pulire. Lasciare basta PBS sul chip per mantenere la matrice SSLB idratata. </li><li> Per prevenire il legame non specifico, aggiungere 200 ml di 2 mg / ml BSA al chip matrice SSLB. Lasciare riposare per 1 ora in una scatola umidificata. </li><li> Con una micropipetta, rimuovere 200 ml di soluzione dalla parte superiore del chip matrice SSLB. </li><li> Aggiungere 200 ml di soluzione di tossina del colera al chip matrice SSLB e lasciare riposare per 1 ora in una scatola umidificata. </li><li> Lavare delicatamente il chip matrice SSLB con PBS da una bottiglia di lavaggio per eliminare ogni tossina del colera non legato. </li><li> Wick via l&#39;eccesso di PBS utilizzando un laboratorio wipe. Prima di imaging chip di copertura con una scivolata 24 millimetri di copertura x 40 mm. </li><li> Array di immagine con un microscopio verticale con filtro imposta appropriato per i fluorofori di interesse. </li><li> Analizzare l&#39;intensità di fluorescenza dei singoli SSLBs di un array utilizzando la funzione di analisi delle particelle automatizzata del software ImageJ. </li><li> Compilare intensità di fluorescenza medi dei singoli SSLBs in istogrammi che riassumono le intensità di SSLBs trovate su un dato array. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Spectroscopic+Detections.">Multiplexed Spectroscopic Detections.</a> Annu. Rev. Anal. Chem. 1 (1), 515-547 (2008).</li><li>Kundu, J., Levin, C. S., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Monitoring+of+Lipid+Transfer+Between+Vesicles+and+Hybrid+Bilayers+on+Au+Nanoshells+Using+Surface+Enhanced+Raman+Scattering+(SERS).">Real-Time Monitoring of Lipid Transfer Between Vesicles and Hybrid Bilayers on Au Nanoshells Using Surface Enhanced Raman Scattering (SERS).</a> Nanoscale. 1 (1), 114-117 (2009).</li><li>Junesch, J., Sannomiya, T., Dahlin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Properties+of+Nanohole+Arrays+in+Metal-Dielectric+Double+Films+Prepared+by+Mask-on-Metal+Colloidal+Lithography.">Optical Properties of Nanohole Arrays in Metal-Dielectric Double Films Prepared by Mask-on-Metal Colloidal Lithography.</a> ACS Nano. 6 (11), 10405-10415 (2012).</li><li>Wu, M., Holowka, D., Craighead, H. G., Baird, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Plasma+Membrane+Compartmentalization+with+Patterned+Lipid+Bilayers.">Visualization of Plasma Membrane Compartmentalization with Patterned Lipid Bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (38), 13798-13803 (2004).</li><li>Hatzakis, N. S., Bhatia, V. K., Larsen, J., Madsen, K. L., Bolinger, P. Y., Kunding, A. H., Castillo, J., Gether, U., Hedegard, P., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Curved+Membranes+Recruit+Amphipathic+Helices+and+Protein+Anchoring+Motifs.">How Curved Membranes Recruit Amphipathic Helices and Protein Anchoring Motifs.</a> Nat. Chem. Biol. 5 (11), 835-841 (2009).</li><li>Roizard, S., Danelon, C., Hassaine, G., Piguett, J., Schulze, K., Hovius, R., Tampe, R., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+G-Protein-Coupled+Receptors+in+Cell-Derived+Plasma+Membranes+Supported+on+Porous+Beads.">Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads.</a> J. Am. Chem. Soc. 133 (42), 16868-16874 (2011).</li></ol>

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In questo lavoro mostriamo che monodisperse SiO 2 perle rivestite con doppi strati lipidici supportati possono essere disposte in array micropozzetti senza la necessità per il targeting ligandi sulle membrane lipidiche o la superficie del substrato, e le matrici possono essere utilizzati per caratterizzare le interazioni tossina-lipidico. La costante di dissociazione abbiamo calcolato per CTx/GM1 vincolante confronta favorevolmente, data l&#39;ampia disparità di valori in letteratura, con una precedente rel...

Le membrane doppio strato lipidico sono strutture essenziali in natura. Membrane plasmatiche cellulare e membrane degli organelli sono composti di bistrati lipidici che incorporano un numero di molecole che sono necessarie per la vita. Molti processi di sostegno vitale verificano sulla superficie delle cellule o sono mediati da molecole associate con membrane a doppio strato lipidico. Infatti, molti prodotti farmaceutici processi o molecole bersaglio si trovano su o nelle membrane 1,2. È pertanto necessario indagare analiticamente processi, come reazioni chimiche o eventi di legame covalenti che si verificano sulla superficie della membrana. Poiché le membrane naturali possono essere difficili da isolare e / o interfaccia con i sensori, molti ricercatori utilizzano membrane modello semplificato di effettuare studi analitici. Un certo numero di sistemi a membrana modello sono descritti in letteratura, che vanno da vescicole giganti che possono essere decine a centinaia di micron di diametro a liposomi con dimensioni nanometriche 3,4. Alterntivamente, bistrati lipidici planari depositati su supporti solidi, cioè, supportati bistrati lipidici (SLB), possono essere formati su un numero di superfici diverse e sono stati ampiamente utilizzati in biofisici, biochimici e applicazioni analitiche 5. Accoppiamento SLB con materiali elettrici o ottici permette lo studio di membrana biochimica e biofisica attraverso l&#39;uso di diverse tecniche analitiche. Microscopia a fluorescenza 6, elettrochimica 7, 8 spettroscopia ottica, microscopia a scansione di sonda 9, risonanza plasmonica di superficie 10, 11 e spettrometria di massa sono stati tutti impiegati per studiare la struttura e le proprietà di SLB. Array SLB offrono maggiore versatilità nella progettazione di sensori per il test multiplex 12,13. Altre applicazioni utilizzano matrici SLB di imitare il bivio che si forma tra le cellule immunitarie 14. Metodi di preparazione per gli array SLB hanno variato dal microfluIDIC avvicina 15 a quelli che impiegano le barriere fisiche tra patch SLB adiacenti. sedici Altri gruppi hanno utilizzato metodi di stampa 17, patterning fotochimico 18 e vari approcci nanoingegneria 19 per creare array SLB. In questo lavoro e video allegato si dimostra un metodo per formare matrici SLB depositando SLB-SiO 2 perle rivestite in array ordinati di micropozzetti 20. Ci riferiamo alle SLB-rivestite SiO 2 perle come sferici doppi strati lipidici supportati (SSLBs). Questa tecnica è un&#39;estensione del precedente lavoro che ha creato schiere di vescicole fosfolipidiche e biomembrane derivati ​​da fonti naturali 21, da cui mostriamo anche Esempio risultati. Altri metodi per arraying particelle biomembrane o vescicole hanno fatto affidamento su modelli di specifici ligandi di targeting su superfici che si associano con ligandi complementari presenti sulla superficie delle vescicole. Gli esempi includono biotina-avidina associazione 22,23 e schemi di ibridazione DNA 24. Il nostro approccio richiede solo un array di micropozzetti senza di targeting o di riconoscimento porzioni necessarie. La dimensione di SSLBs è definita dal diametro delle SiO 2 supporti tallone, che hanno un basso poli-dispersity. Sintonizzando il diametro micropozzetti a poco più grande del diametro SSLB, un solo SSLB deposita in ciascun pozzetto. A poli (dimetilsiloxano) (PDMS) spatola rimuove poi dalla superficie tutti i SSLBs che non sono immobilizzati in pozzetti. I pozzetti e array SSLB risultanti hanno alta densità (~ 10 5 SSLBs / mm 2) con 3 micron spaziatura da centro a centro e la periodicità esagonale. Seriale i depositando SSLBs con differenti composizioni lipidiche, è possibile creare array multicomponenti con SSLBs posizionati in modo casuale. Per dimostrare la capacità di rilevamento di array SSLB, abbiamo usato l&#39;interazione della tossina colerica (CTx) con un ganglioside (GM1) incorporato nei SSLBs. Conparticelle di membrana naturali, siamo stati in grado di rilevare lipidi cellula-specifici in matrici multicomponente contenenti materiale di membrana da due tipi di cellule differenti.

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			<td height="42" style="height:42px;width:239px;">4-inch silicon wafers</td>
			<td style="width:91px;">University Wafer</td>
			<td align="right" style="width:119px;">425</td>
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			<td height="42" style="height:42px;width:239px;">Shipley MEGAPOSIT SPR955-CM 0.7 photoresist</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">SPR955-CM</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;">Shipley MICROPOSIT CD-26 developer</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">CD-26</td>
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			<td height="21" style="height:21px;width:239px;">i-line stepper</td>
			<td style="width:91px;">Canon</td>
			<td style="width:119px;">2500 i3 stepper</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vision 320 reactive ion etcher</td>
			<td style="width:91px;">Advanced Vacuum</td>
			<td style="width:119px;">Vision 320 RIE</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Deep trench reactive ion etcher</td>
			<td style="width:91px;">Plasma Therm</td>
			<td style="width:119px;">SLR-770</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;">Atomic layer depostion system</td>
			<td style="width:91px;">Cambridge NanoTech</td>
			<td style="width:119px;">Savannah</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;">Dow Corning Sylgard 184 poly(dimethylsiloxane) kit</td>
			<td style="width:91px;">Ellsworth Adhesives</td>
			<td style="width:119px;">184 SIL ELAST KIT 0.5KG</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;">egg phosphatidylcholine</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>840051C</td>
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			<td height="84" style="height:84px;width:239px;">1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>810158C</td>
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			<td height="42" style="height:42px;width:239px;">monosialoganglioside GM1&#160;</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td style="width:119px;">860065P</td>
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			<td height="42" style="height:42px;width:239px;">Silica beads</td>
			<td style="width:91px;">Bangs Laboratories</td>
			<td style="width:119px;">SS03N/4666</td>
			<td>Packaging on the bead container states the beads are 900 nm in diameter. However, after light-scattering and electron microscopy we determined the beads are roughly 700 nm in diameter.</td>
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			<td height="42" style="height:42px;width:239px;">Cholera toxin B-subunit, Alexa 488 conjugate</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">C-34775</td>
			<td></td>
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			<td height="42" style="height:42px;width:239px;">Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate</td>
			<td style="width:91px;">R&#38;D Systems</td>
			<td style="width:119px;">NL1326R</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">FM1-43</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">T-3136</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Eppendorf MiniSpin centrifuge</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td style="width:119px;">05-401-09</td>
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formation of biomembrane microarrays with a squeegee-based assembly method

Doppio strato lipidico delle membrane formano le membrane plasmatiche delle cellule e definiscono i confini di organelli subcellulari. In natura, queste membrane sono miscele eterogenee di molti tipi di lipidi, contengono proteine ​​di membrana e sono decorate con i carboidrati. In alcuni esperimenti, è opportuno disaccoppiare le proprietà biofisiche o biochimiche del doppio strato lipidico da quelle della membrana naturale. Tali casi richiedono l&#39;uso di sistemi modello come vescicole giganti, liposomi o doppi strati lipidici supportati (SLB). Matrici di SLB sono particolarmente interessanti per le applicazioni di rilevamento e mimando interazioni cellula-cellula. Qui si descrive un nuovo metodo per la formazione di matrici SLB. Inferiori al micron di diametro SiO 2 perle sono in primo luogo rivestiti con doppi strati lipidici per formare SLB sferici (SSLBs). Le perle sono poi depositati in una matrice di pozzetti inferiori al micron di diametro micro-fabbricato. La tecnica di preparazione utilizza un &quot;seccatoio&quot; per pulire la superficie del substrato, mentre leaving dietro SSLBs che si sono insediati nei pozzetti. Questo metodo non richiede alcuna modificazione chimica del substrato micropozzetti, né alcun particolare ligandi di targeting sul SSLB. I micropozzetti sono occupate da singole perline perché il diametro bene sia sintonizzato essere appena superiore al diametro del branello. Tipicamente, più il 75% dei pozzi sono occupati, mentre il resto rimane vuoto. In tampone array SSLB mostrano stabilità a lungo termine maggiore di una settimana. Diversi tipi di SSLBs possono essere collocati in una singola matrice mediante deposizione seriale, e le matrici possono essere usate per il rilevamento, che dimostriamo caratterizzando l&#39;interazione di tossina colerica con ganglioside GM1. Mostriamo anche che vescicole fosfolipidiche che non supporti cerchietti e biomembrane da fonti cellulari possono essere disposte con lo stesso metodo e lipidi di membrana cellula-specifici possono essere identificati.

Quando SiO 2 perle sono mescolati con una soluzione di vescicole composte da fosfolipidi, lipidi fluorescenti e altri lipidi come gangliosidi, le vescicole si rompono sulle superfici SiO 2 cerchietti per formare SSLBs, come mostrato schematicamente in figura 1a. Dopo aver lavato i SSLBs, una goccia di soluzione SSLB è posto su una matrice microwell, e le perle sono autorizzati a stabilirsi in superficie. (Figura 1b1) Questo può essere fatto anche con una sospensi...

Watch this Scientific Journal Video about Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method at JoVE.com

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Bistrati lipidici supportati e particelle di membrana naturali sono sistemi convenienti che può approssimare le proprietà delle membrane cellulari e di essere incorporati in una varietà di strategie analitiche. Qui mostriamo un metodo per preparare microarray composto da supportati doppio strato lipidico rivestite SiO 2 perline, vescicole di fosfolipidi di membrana o particelle naturali.

Formazione di microarray di membrana con un metodo di assemblaggio con sede a seccatoio

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

formation-biomembrane-microarrays-with-squeegee-based-assembly

Research

JoVE Journal

Bioengineering

13.6K Views.  University of Minnesota.  Bistrati lipidici supportati e particelle di membrana naturali sono sistemi convenienti che può approssimare le proprietà delle membrane cellulari e di essere incorporati in una varietà di strategie analitiche. Qui mostriamo un metodo per preparare microarray composto da supportati doppio strato lipidico rivestite SiO 2 perline, vescicole di fosfolipidi di membrana o particelle naturali. 

Video: Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Sintesi Nanostar oro con un metodo d&#39;argento crescita Seed Mediated

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Ricerca

Bioingegneria

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays polimerici per Discovery High Throughput di Biomateriali

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocollo per il Biofilm Streamer formazione in un dispositivo a microfluidi con Micro-pilastri

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Fluorescenza Biomembrane Forza Probe: quantificazione simultanea di Cinetica recettore-ligando e Binding-indotta intracellulare segnalazione su una singola cella

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrane Fabrication dal solvente assistita doppio strato lipidico (SALB) Metodo

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e montaggio dei microvasi 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabbricazione di pozzi di agarosio personalizzato per la semina delle cellule e tessuto anello auto-assemblaggio utilizzando stampi 3D-stampato

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificazione simultanea di kynurenine selezionate analizzate dalla cromatografia liquida-spettrometria di massa in mezzo raccolto da colture cellulari tumorali

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.