O. Kurtulus e D. Dimlioglu sono supportati da una Università della California Laboratorio Commissioni Research Award Program 12-LR-237197. P. Daggumati è supportato da una University of California Davis Research Investimenti in Scienze e Ingegneria (RISE) Award. CA Chapman è supportato da un Dipartimento di istruzione universitaria di Assistenza Aree di Fellowship bisogno nazionale. Questo lavoro è stato sostenuto da UC Lab Commissioni Programma di ricerca, UC Davis RISE, e UC Davis College of Engineering di start-up fondi.

1. Nanoporoso Oro Fabrication <ol><li> Substrati puliti in soluzione Piranha <ol><li> Aggiungere 25 ml di perossido di idrogeno (30%) di acido solforico 100 ml (96%) in un piatto di cristallizzazione e scaldare la miscela a 65 ° C su una piastra. ATTENZIONE: I liquidi sono estremamente corrosivi e devono essere maneggiati con cura. La soluzione esausta non deve essere conservata in un contenitore sigillato, come può esplodere. </li><li> Luogo di 1 pollice da vetrini da 3 pollici nella miscela con pinze resistenti agli acidi e quindi ripulirli per 10 min. Utilizzare una barca immunoistochimica porcellana per batch pulizia piccole lamelle. Trattare piccole coprioggetto con plasma ad aria a 10 W per 30 secondi prima di immergerlo nella soluzione. </li><li> Sciacquare i campioni puliti in piatti di cristallizzazione sotto corrente (DI) di acqua deionizzata per 3 min. Campioni in piega con una pistola di azoto oltre privi di pelucchi asciugamani. </li></ol></li><li> Preparare maschera stencil (Metodo 1: utilizzare questa per la creazione di modelli di millimetro scala) <ol><li> Punch 250 micron di spessore lastre in elastomero di silicone con biopsia pugni e / o incidere fuori regioni con un bisturi su una superficie di plastica semi-dura. Pulire le lastre in elastomero trasformati in 70% di isopropanolo e asciugare con pistola azoto. </li><li> Posizionare il foglio perforato su una sfilacciato asciugamano camera bianca e allineare i coprioggetti piranha-puliti sopra lo stencil con la superficie del campione verso lo stencil. </li></ol></li><li> Modello lift-off photoresist (Metodo 2: utilizzare questo per creare pattern sub-millimetrica scala) <ol><li> Posizionare un piranha-pulito vetrino da microscopio a girare Chuck e soffiare via le particelle con la pistola di azoto. Pipettare 1,5 ml di promotore di adesione (esametildisilazano) sul vetrino utilizzando una pipetta di plastica. Stendere il promotore facendo girare la slitta successivamente a 500 rpm per 5 sec e 1.500 rpm per 30 sec. Cuocere il vetrino su una piastra riscaldante a 115 ° C per 7 minuti e lasciate raffreddare per 5 minuti. </li><li> Dispensare 4 ml di photoresist sul vetrino (3 pollici by 1 pollice). Stendere il photoresist facendo girare la diapositiva con lo stesso protocollo per il promotore di adesione. Cuocere il photoresist su una piastra riscaldante a 115 ° C per 1,5 minuti e lasciate raffreddare per 10 minuti. </li><li> Esporre il photoresist vetrino rivestito con luce UV (intensità: 22 mW / cm 2) attraverso una maschera di trasparenza per 15 sec. Cuocere il fotoresist su una piastra riscaldante a 115 ° C per 1,5 minuti e attendere 45 min. Sciogliere il photoresist esposto nel sviluppatore per almeno 3,5 min. Sciacquare abbondantemente con acqua deionizzata. Ispezionare i modelli sviluppati sotto un microscopio ottico. </li></ol></li><li> Deposito metalli precursori per la produzione di film sottili np-Au <ol><li> Caricare i campioni in una macchina di sputtering che può depositare autonomamente oro, argento e cromo. Deposito a pulire i campioni per 90 sec a 50 W con 25 mTorr argon atmosfera di elaborazione prima di iniziare la deposizione di metallo. </li><li> Sputtering cromo per 10 minuti a 300 W fino a 10 anni mTorr argon. Polverizzazione catodica oro per 90 secondi a 400 W ONUder 10 mTorr argon. Co-sputtering oro e d&#39;argento per 10 minuti con l&#39;argento a 200 W e di potenza Au a 100 W. Spegnere oro polverizzazione catodica fonte di circa 10 secondi prima di spegnere l&#39;argento sputtering fonte. </li></ol></li><li> Ottenere microdisegnature metallici precursori <ol><li> Sonicare campioni photoresist rivestite in ~ 180 ml di spogliarellista photoresist per 10 cicli di 20 sec sonicazione e 2 minuti di pausa tra i cicli. Lavare i campioni con acqua deionizzata e asciugare con una pistola di azoto. Ispezionare i modelli in metallo sotto un microscopio. </li><li> Sbucciate lo stencil elastomero da campioni rivestiti non photoresist con due pinzette per rivelare il metallo depositato. </li></ol></li><li> Dealloy precursore metallico e modificare nanostruttura tramite trattamento termico <ol><li> Riempire un bicchiere di vetro da 200 ml con 170 ml di acido nitrico (70%) e mantenere la temperatura della soluzione a 55 ° C su una piastra. ATTENZIONE: L&#39;acido nitrico è estremamente corrosivo e deve essere maneggiato con equipaggiamento protettivo. </li&gt; <li> Trattare piccole coprioggetto con plasma ad aria a 10 W per 30 secondi prima di immersione in acido nitrico. Luogo di 1 pollice da vetrini da 3 pollici nel bicchiere con pinze resistenti agli acidi e dealloy loro per 15 min. Utilizzare una barca immunoistochimica porcellana per batch dealloying piccole lamelle. </li><li> Sciacquare i campioni dealloyed da successivamente immergendole in due bicchieri pieni di acqua DI fresca tre volte. Conservare i campioni in acqua deionizzata e sostituire l&#39;acqua con acqua DI fresca ogni giorno per almeno una settimana. Campioni in piega con una pistola di azoto oltre asciugamani filacciosi prima dell&#39;uso. </li><li> Caricare campioni su un wafer di silicio pulita in un rapido apparecchiature per il trattamento termico. Regolare la temperatura tra 200 ° C e 450 ° C e la velocità di rampa di 10 ° C / sec. Esporre i campioni alla temperatura prescritta per 10 min sotto azoto ambiente. Lasciare la camera di fresco (&lt;100 ° C) e rimuovere i campioni. In alternativa, inserire lentamente i campioni sulla piastra per il trattam termicoent. </li></ol></li></ol> 2. Colture Cellulari <ol><li> Preparare i campioni np-Au per la coltura cellulare <ol><li> Luogo np-Au campioni in piatti di polistirolo e il trattamento con plasma ad aria a 10 W per 30 sec e trasferire i campioni di piastre di coltura tissutale a 24 pozzetti. </li><li> Aggiungere 500 microlitri mezzi di coltura completi (Modified Eagle Medium di Dulbecco con il 10% di siero fetale bovino e 1% di penicillina / streptomicina) in ciascun pozzetto. Conservare in un incubatore umidificato a 37 ° C e 5% di CO 2 fino semina delle cellule (&lt;1 ora). </li></ol></li><li> Mantenere, di passaggio, e di semi di cellule <ol><li> Mantenere le cellule di interesse (in questo caso 3T3-NIH fibroblasti o astrociti murini) a T75 palloni con terreni di coltura e di passaggio quando le cellule sono il 70% confluenti. </li><li> Per passaging le cellule, rimuovere i supporti dal pallone, lavare due volte con 10 ml di tampone fosfato (PBS), aggiungere 2 ml di 1x tripsina / EDTA e incubare fino a quando il distacco delle cellule (~ 5 min). Aggiungere 3 ml di mezzi freschi ecentrifugare a 1200 rpm per 3 min. Aspirare il surnatante e sospendere il pellet in 2 mezzi ml. </li><li> Rimuovere il supporto spesi dai pozzetti e seme le cellule sulla vetrini ad una densità di 25.000 cellule / cm 2 con un volume finale di 1 ml. Agitare la piastra di coltura avanti-indietro e destra-sinistra per garantire un rivestimento uniforme delle cellule sugli esemplari. Incubare le cellule fino al momento dell&#39;analisi durante il controllo quotidiano. </li></ol></li></ol> 3. Cellulare e Analisi Materiale <ol><li> Colorare le cellule di visualizzare citoscheletro e nuclei <ol><li> Rimuovere i supporti spesi dai pozzetti e lavare le cellule due volte con PBS. Fissare le cellule in paraformaldeide 4% in PBS per 15 min. </li><li> Preparare la soluzione di colorazione 300 nM Alexa Fluor 488 phalloidin in PBS con 1% di albumina di siero bovino. </li><li> Lavare le cellule due volte con PBS, e li permeabilize in 500 microlitri 0,1% Triton X-100 in PBS per 5 min. </li><li> Lavare le cellule due volte con PBS e trasferirli da pulire pozzetti. Blot 50 - 200 ml soluzione di colorazione sui campioni e conservare al buio per 20 min. </li><li> Lavare le cellule con PBS e contro-macchia con 3 Nm di DAPI in PBS per 5 min. </li><li> Lavare le cellule con PBS, e tuffo in acqua deionizzata prima di montare su vetrini di copertura in vetro con supporti di montaggio e sigillare con smalto trasparente. </li></ol></li><li> Acquisire immagini di campioni np-Au e colture cellulari su superfici np-Au <ol><li> Superfici Immagine np-Au con microscopio elettronico a scansione (SEM) con 50.000 X ingrandimento a 10 kV elettrone energia utilizzando rivelatore di elettroni secondari. </li><li> Cattura immagini cellulari composite in punti diversi i campioni utilizzando un microscopio invertito a fluorescenza con ingrandimento 10X con i cubetti di filtro appropriate. </li></ol></li><li> Le immagini del processo per determinare la morfologia dei pori e delle cellule <ol><li> Aprire le immagini in ImageJ e suddivisa in singoli canali se applicabile. Convertire le immagini a 8-bit, sottrarre il fondo, e lisciare loro dai filtri d&#39;medianang. Regolare la soglia manualmente o con built-in di soglia algoritmi per evidenziare i pori (vuoti) e corpi cellulari / nuclei. </li><li> Utilizzare il comando spartiacque per separare i pori unite o cellule. Impostare i parametri di analisi delle particelle ed eseguire il comando per estrarre il numero di particelle, area media, e la copertura per cento di particelle. Usare le immagini di cellule DAPI macchiati per il conteggio delle cellule e le immagini di cellule falloidina macchiati per quantificare copertura cellulare per cento. </li><li> Modificare i file di macro inclusi per eseguire analisi in batch di immagini multiple. </li></ol></li></ol>

<ol>
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Gli autori non hanno alcun interesse finanziario in conflitto.

Dimostriamo due tecniche diverse per micropattern np-Au film per espandere l&#39;uso di questi film in microsistemi e studi biologici. Deposito a spruzzo oro e argento è un metodo versatile per creare modelli np-Au, come sputtering è compatibile con i processi convenzionali di microfabbricazione e la composizione della lega e lo spessore può essere facilmente controllato variando i poteri individuali pistola sputtering (per obiettivi d&#39;oro e d&#39;argento) e il tempo di deposizione rispettivamente. Tipico film di...

<p class="jove_content"&gt; Materiali con caratteristiche nano-scala hanno mostrato risultati promettenti nel migliorare diverse applicazioni, tra cui le celle a combustibile<sup&gt; 1</sup&gt; Sensori,<sup&gt; 2,3</sup&gt; E dispositivi biomedicali<sup&gt; 4,5</sup&gt;. Un materiale relativamente nuovo è oro nanoporous (np-Au), che è prodotto da un processo di auto-assemblaggio nanoscala. Il precursore di np-Au è una lega d&#39;oro che più comunemente è costituito da argento al 60% a 80% in percentuale atomica. Brevemente, la caratteristica nanostruttura poro aperto è il risultato di riarrangiamento di atomi di oro in cluster come argento viene sciolto con un acido forte (<em&gt; Esempio</em&gt; Acido nitrico 70%) o sotto un potenziale elettrochimico<sup&gt; 6-8</sup&gt;. Np-Au benefici da vari attributi desiderabili, tra cui ampia superficie effettiva, elevata conducibilità elettrica, ben consolidate tecniche di funzionalizzazione superficiale, e biocompatibilità<sup&gt; 9</sup&gt;. Anche se vi è stata una rapida espansione di studi su np-Au, la maggior parte di loro concentrarsi sulle proprietà meccaniche di np-Au<sup&gt; 10,11</sup&gt;, Attività catalitica<sup&gt; 12</sup&gt;, E biomolecolare prestazioni di rilevamento<sup&gt; 13-15</sup&gt;. Mentre gli attributi desiderabili sono molto utili per diversi strumenti biomedici<sup&gt; 16,17</sup&gt;, Le applicazioni in questo settore sono stati limitati. Una possibile ragione per questo è che la maggior parte degli studi si sono prevalentemente utilizzati campioni macro-scala (<em&gt; Esempio</em&gt; Film, lastre, lingotti) e le tecniche per incorporare np-Au in sistemi miniaturizzati sono rimasti inadeguati. In realtà ci sono solo una manciata di esempi di utilizzo di tecniche di microfabbricazione convenzionali che impiegano film np-Au<sup&gt; 16-20</sup&gt;. Con l&#39;avvento della tecnologia miniaturizzazione e la necessità di nuovi strumenti biomedici, è diventato cruciale per poter integrare nuovi materiali nelle apparecchiature. Ciò richiede tipicamente che i materiali possono essere depositati e modellati con tecniche convenzionali di microfabbricazione. Inoltre, la rapida quantificazione delle interazioni cellula-materiale è comunemente necessario valutare la biocompatibilità di un nuovo materiale. L&#39;obiettivo di questo lavoro è quello di dimostrare le tecniche di base per micropattern np-Au film e quantificare sia nanostruttura e le interazioni cellula-materiale tramite l&#39;elaborazione di immagini digitali.</p

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Gold target</td>
 <td>Lesker</td>
 <td>EJTAUXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Chrome target</td>
 <td>Lesker</td>
 <td>EJTCRXX353A2</td>
 <td >Adhesive layer</td>
 </tr>
 <tr>
 <td>Silver target</td>
 <td>Lesker</td>
 <td>EJTAGXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Porcelain boat</td>
 <td>Thomas Scientific </td>
 <td>8542E40</td>
 <td >Used for processing small samples</td>
 </tr>
 <tr>
 <td>Nitric acid</td>
 <td>Sigma-Aldrich</td>
 <td>43873</td>
 <td >Used at 70% for dealloying</td>
 </tr>
 <tr>
 <td>Sulfuric acid</td>
 <td>J.T Baker </td>
 <td>7664-93-9</td>
 <td >Used at 96% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>J.T Baker </td>
 <td>7722-84-1</td>
 <td >Used at 30% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Ted Pella</td>
 <td>150xx</td>
 <td >Available in several sizes</td>
 </tr>
 <tr>
 <td>Silicone elastomer sheets</td>
 <td>Rogers Corporation </td>
 <td>HT 6240</td>
 <td >Available in several thicknesses</td>
 </tr>
 <tr>
 <td>Hexamethyldisilazane</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td >Used as adhesion promoter for positive resist</td>
 </tr>
 <tr>
 <td>Microposit MF CD26</td>
 <td>Shipley</td>
 <td>38490</td>
 <td >Positive photoresist developer</td>
 </tr>
 <tr>
 <td>PRS 3000</td>
 <td>J.T Baker</td>
 <td>JT6403-5</td>
 <td >Positive photoresist stripper</td>
 </tr>
 <tr>
 <td>Circular glass coverslips (12 mm)</td>
 <td>Ted Pella</td>
 <td>26023</td>
 <td >Used as substrate for metal patterns and cell culture</td>
 </tr>
 <tr>
 <td>Glass slides (1 x 3 inch)</td>
 <td>Ted Pella</td>
 <td>26007</td>
 <td >Used as substrate for metal patterns</td>
 </tr>
 <tr>
 <td>Kapton polyimide tape</td>
 <td>VWR</td>
 <td>82030-950</td>
 <td >Used for securing elastomer</td>
 </tr>
 <tr>
 <td>Transparency masks</td>
 <td>Output City</td>
 <td></td>
 <td>Used in photolithography <a href="http://www.outputcity.com/" target="_blank">http://www.outputcity.com/</a></td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td >Used for activating glass surfaces</td>
 </tr>
 <tr>
 <td>Sputtering machine</td>
 <td>Kurt J. Lesker</td>
 <td>LAB18</td>
 <td >Used for depositing metals</td>
 </tr>
 </tbody>
 </table>

microfabrication of nanoporous gold patterns for cell-material interaction studies

Materiali nanostrutturati con dimensioni caratteristiche di decine di nanometri hanno migliorato le prestazioni delle diverse tecnologie, tra cui le celle a combustibile, biosensori, rivestimenti dei dispositivi biomedicali e strumenti di consegna della droga. Oro nanoporous (np-Au), prodotto da un processo di auto-assemblaggio nano-scala, è un materiale relativamente nuovo che presenta un&#39;ampia area superficiale effettiva, elevata conducibilità elettrica, e l&#39;attività catalitica. Queste proprietà hanno reso np-Au un materiale interessante per la comunità scientifica. Maggior parte degli studi su NP-Au impiegano campioni macro scala e concentrarsi sulla scienza fondamentale della materia e delle sue applicazioni catalitiche e sensore. Gli esemplari macro scala limitano il potenziale di NP-Au in sistemi miniaturizzati, inclusi i dispositivi biomedici. Al fine di risolvere questi problemi, abbiamo inizialmente descriviamo due metodi diversi per micropattern np-Au film sottili su substrati rigidi. Il primo metodo utilizza mascherine stencil manualmente-prodotti per la creazione di millimetro scala modelli NP-Au, while il secondo metodo utilizza decollo fotolitografia per modelli modello sub-millimetrica scala. Come i film sottili np-Au sono ottenute tramite il processo sputtering-deposizione, che siano compatibili con le tecniche convenzionali di microfabbricazione, quindi suscettibili di integrazione facile in microsistemi. Questi sistemi includono elettricamente indirizzabili piattaforme biosensore che beneficiano di elevata superficie effettiva superficie, conducibilità elettrica, e l&#39;oro-tiolo-based bioconjugation superficie. Descriviamo coltura cellulare, immunostaining, e le tecniche di elaborazione di immagini per quantificare l&#39;interazione del np-Au con cellule di mammifero, che è un parametro importante per alcune prestazioni biosensori. Ci aspettiamo che le tecniche qui illustrate dovranno favorire l&#39;integrazione delle np-Au in piattaforme a diverse scale di lunghezza e in numerose applicazioni, tra cui biosensori, sistemi di stoccaggio di energia, e catalizzatori.

La figura 1 illustra le principali fasi procedurali, tra cui la creazione dei modelli NP-Au, coltura delle cellule, quantificando la nanostruttura, e la caratterizzazione morfologie cellulari. Lo stencil elastomero mostrato in Figura 2a (in alto) è utilizzato per la creazione dei modelli NP-Au mostrati nelle immagini sottostanti. Figura 2b è una fotografia della barca in porcellana per i campioni di elaborazione batch. Figura 2c mostra il cambiamento ...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Segnaliamo su tecniche per micropattern nanoporosi film sottili di oro tramite la stampa stencil e fotolitografia, così come i metodi di cellule di coltura, su modelli di microfabbricati. Inoltre, si descrivono metodi di analisi di immagine per caratterizzare morfologia del materiale e le cellule in coltura mediante scansione elettronica e tecniche di microscopia a fluorescenza.

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, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Research

JoVE Journal

Bioengineering

11.3K Views.  University of California, Davis. Segnaliamo su tecniche per micropattern nanoporosi film sottili di oro tramite la stampa stencil e fotolitografia, così come i metodi di cellule di coltura, su modelli di microfabbricati. Inoltre, si descrivono metodi di analisi di immagine per caratterizzare morfologia del materiale e le cellule in coltura mediante scansione elettronica e tecniche di microscopia a fluorescenza.

Video: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

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 ...

Ricerca

Bioingegneria

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Sintesi, assemblaggio e caratterizzazione di film monostrato Protetta delle nanoparticelle d&#39;oro per Elettrochimica proteine ​​monostrato

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

A Novel tridimensionale del flusso Camera dispositivo per studiare Chemokine-diretto stravaso di cellule circolanti in condizioni di flusso fisiologici

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

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.

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 ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Sintesi di particelle d'oro rivestite di polimeri 10-nm funzionalizzate per il Targeting di endotelio e Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabbricazione di una matrice di multiplex artificiale microambiente cellulare

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In Vivo a due colori immagini fotone di cellule reporter geneticamente taggate nella pelle

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Sintesi di nanocluster d'oro a emissione di infrarossi nel vicino infrarosso per applicazioni biologiche

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

Frazionamento asimmetrico del flusso campo-flusso per il dimensionamento delle nanoparticelle d'oro in sospensione

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...