DCH, MB e FK sono stati sostenuti da un EPSRC Segui su Fund. BL-V e DS sono stati supportati da ogni MRC borse di dottorato di ricerca. KC è stato sostenuto da un finanziamento della piattaforma di Medicina Rigenerativa Regno Unito.

1. Sintesi di PHNGAD (Poly [1,6-hexanodiol / neopentilglicole / di (glicole etilenico) Acido -ALt-adipico] diol) <img alt="Schema 1" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme1.jpg" width="500" /> Schema 1: Sintesi di PHNAGD Rappresentazione schematica della sintesi di PHNAGD.. PHNAGD è stato preparato dalla reazione di 1,6-Hexanodiol, dietilenglicole, neoppentyl glicole e acido adipico. PHNAGD, Poly [1,6-hexanodiol / neopentilglicole / di (glicole etilenico) Acido -ALt-adipico] diolo. <ol><li> Applicare trattamento termico ai monomeri 1,6-esandiolo, di (etilenglicole) e neopentilglicole a 40 ° C per 48 ore in stufa sotto vuoto per rimuovere l&#39;acqua residua. Lasciare al freddo fino a RT sotto vuoto. </li><li> Aggiungere 22 mmol di ciascun monomero e acido adipico (55 mmol) in un pallone a fondo tondo a due colli munito di una barra di agitazione e connesso ad un apparecchio di Dean-Stark. </li><li> Mettere tutta l&#39;assemblea sotto vuoto e scaldare delicatamente il glassware a 40 ° C per 6 ore, in modo da evitare l&#39;assorbimento di umidità durante l&#39;aggiunta della sostanza chimica nel pallone. </li><li> Aggiungi tramite siringa, goccia a goccia, 0,0055 moli di titanio catalizzatore (IV) butossido. </li><li> Mescolare la miscela di reazione a 180 ° C, in atmosfera N 2 per 24 ore, e raccogliere l&#39;acqua residua nella trappola di Dean-Stark. Lasciare raffreddare il prodotto a temperatura ambiente. </li></ol> 2 Sintesi di PU134 <img alt="Schema 2" fo:content-width="5in" src="/files/ftp_upload/51723/51723scheme2.jpg" width="500" /> Schema 2: Sintesi di PU134 Rappresentazione schematica della sintesi del poliuretano 134 PU134 è stato preparato per reazione di 1,0 equiv di un PHNGAD con 2,0 equiv di un Metilenebis 4,4-(fenil isocianato), seguita dall&#39;aggiunta di 1,0. equiv di una catena extender 1,4-butandiolo. <ol><li> Mescolare un equivalente della PHNGAD poliolo (Mn ~ 1.800 Da, 3.2 mmol) con due equivalenti di 4&#39;4-Metilenebis (fenil isocianato) (6,4 mmol) in anidra N, N -Dimethylformamide (12 ml). </li><li> Mescolare la miscela di reazione a 70 ° C, in atmosfera di N 2. </li><li> Aggiungi tramite siringa, goccia a goccia il titanio catalizzatore (IV) butossido (0,8% in peso). </li><li> Dopo 1 ora, aggiungere uno equivalente della catena extender 1,4-butandiolo (3,2 mmol). Aumentare la temperatura a 90 ° C e agitare per 24 h in atmosfera di N 2. </li><li> Dopo la reazione, raccogliere il poliuretano per precipitazione con l&#39;aggiunta di etere etilico (esano o acqua potrebbe essere utilizzato anche) gocciolamento nella soluzione di reazione fino a quando si verifica la precipitazione. </li><li> Centrifugare la soluzione a 5.300 xg per 5 min. </li><li> Decantare il surnatante e asciugare a 40 ° C in un forno a vuoto fino a quando il solvente evapora. Nota: Al fine di garantire che il prodotto finale della reazione possedere i corretti parametri relativi alla distribuzione dei pesi molecolari, il Grou funzionaleps del polimero, e le temperature di fusione e di transizione vetrosa, può essere utilizzato varie tecniche e metodi analitici, quali la cromatografia a permeazione di gel o spettroscopia FITR. </li></ol> 3 Preparazione di PU134 Solutions <ol><li> Pesare 200 mg di PU134 in una bottiglia di vetro. </li><li> Diluire PU134 ad una concentrazione finale del 2% in diversi solventi: cloroformio, una combinazione di cloroformio e toluene in rapporto 1: 1, tetraidrofurano, e la combinazione di tetraidrofurano e diclorometano nel rapporto 1: 1. Nota: L&#39;elezione del solvente destra varia a seconda del polimero da utilizzare. Solventi diversi possiedono diversi punti di ebollizione che possono influenzare la solubilizzazione del polimero. </li><li> La soluzione viene agitata per 20 minuti a temperatura ambiente usando un agitatore ad una velocità di 200 mot / min, fino a quando la soluzione diventa omogenea e nessun precipitato viene osservata. </li></ol> 4 Rivestimento di diapositive di vetro con PU134 <ol><li> Posizionare un rouDN 15 mm 2 coprioggetto sulla verniciatura giro. </li><li> Applicare 50 ml di soluzione di PU134 per ciascuno utilizzando una pipetta. Regolare il volume della soluzione PU134 conseguenza per il formato coprioggetto desiderato mantenendo il volume di superficie rapporto proporzionale. </li><li> Spin ogni coprioggetto per 7 secondi a 23 x g. </li><li> Aria coprioggetto asciutto a temperatura ambiente per almeno 24 ore prima della sterilizzazione. </li></ol> 5. irradiazione di Coprioggetti <ol><li> Gamma-irradiare coprioggetto rivestito di polimero, applicando una dose di 10 Grays utilizzando un irradiatore laboratorio per 12 min. </li><li> UV irradiare polimero rivestito coprioggetto utilizzando un 30 W, lampadina UV per 16 min ogni lato. </li><li> Posizionare il coprioggetto polimero rivestito in una piastra di coltura tissutale idonea a seconda della dimensione di diapositiva. </li></ol> 6. Microscopia Elettronica a Scansione <ol><li> Oro cappotto polimero coprioggetto per sputtering per 200 sec in un clima di 5 x 10 -1 millibar di pressione. </li><li> Cattura la micrographs del coprioggetto polimero rivestito utilizzando un microscopio elettronico a scansione ad una tensione di accelerazione di 20 kV in modalità di imaging elettroni secondari. </li></ol> 7. Osservazioni Microscopia a Forza Atomica <ol><li> Scansione un&#39;area di 20 x 20 micron della superficie del polimero. </li><li> Impostare una frequenza di scansione da 1,32 Hz a 1,60 Hz. </li><li> Impostare una risoluzione di 512 x 512 pixel nella regione scansionata. </li><li> Calcolare il Root Mean Square (RMS o Rq) del rivestimento, utilizzando la media delle deviazioni di altezza tratti dal piano dati dell&#39;immagine media, espressa come: <img fo:content-width="1in" src="/files/ftp_upload/51723/51723eq1.jpg" width="123" /> Dove Zi è il valore corrente Z, e N è il numero di punti all&#39;interno dell&#39;area dato. </li><li> 7.5 Calcolare la deviazione o media rugosità superficiale (Ra) dell&#39;immagine utilizzando, <img fo:content-width="1.5in" src="/files/ftp_upload/51723/51723eq2.jpg" width="154" /> Dove Z (x) è la funzione che descrive tegli superficie profilo analizzato in termini di altezza (Z) e la posizione (x) del campione sulla lunghezza valutazione &quot;L&quot;. Ra rappresenta il valore medio della superficie rispetto al piano di mezzeria. </li></ol> 8 coltura cellulare e la differenziazione <ol><li> Cultura e differenziare l&#39;umano linea di cellule staminali embrionali (hESC) H9 come descritto in Hay 17. </li><li> Staccare cellule utilizzando un reagente dissociazione e replate su vetrini rivestiti PU134 al giorno 9 del processo di differenziazione, in presenza di un mezzo privo di siero come descritto in Szkolnicka 18,19. Nota: L&#39;uso di una cella di dissociazione enzimatica è preferito al distacco delle cellule fisica. </li></ol> 9. citocromo P450 saggio funzionale <ol><li> Misurare l&#39;attività del CYP3A seguendo le istruzioni del produttore. </li><li> Incubare hESC epatociti derivati ​​al giorno 13 o il giorno 19, e dei media, senza cellule - come controllo negativo, con il substr appropriatomangiato per 5 ore a 37 ° C. </li><li> Raccogliere surnatanti di cellule e dei media, eseguire il test secondo le istruzioni del produttore. </li><li> Misurare il livello di attività CYP e relativa normalizzata alla superficie (cm 2). </li></ol> 10 Immunostaining <ol><li> Lavare hESC derivato epatociti con PBS, per 1 min, ripetere due volte. </li><li> Aggiungere il ghiaccio freddo 100% di metanolo per riparare le cellule, posto in -20 ° C per 10 min. </li><li> Lavare le cellule con PBS per 5 minuti e ripetere due volte. </li><li> Incubare le cellule con PBS / T (0,1% Tween) / 10% BSA per 1 ora a RT. </li><li> Aspirare la soluzione PBST e aggiungere l&#39;anticorpo primario appropriato diluito in PBS / T (0,1% di Tween) / 1% BSA, incubare a 4 ° C con agitazione O / N. </li><li> Lavare le cellule con PBS / T (0,1% Tween) / 1% BSA per 5 min e ripetuto tre volte. </li><li> Diluire l&#39;anticorpo appropriato in PBS / T (0,1% di Tween) / 1% BSA, aggiungere alle cellule e incubare al buio a temperatura ambiente per 1 ora con agitazione. </li><li> Lavare le cellule con PBS per 5 minuti e ripetere per tre volte. </li><li> Aggiungi Mowiol 488 (contenente DAPI 1: 1.000) per ciascun bene, aggiungere un coprioggetto delicatamente per ridurre il numero di bolle d&#39;aria. Conservare le cellule fissato a 4 ° C al buio. Osservare colorazione utilizzando un microscopio con appositi filtri e lampada fluorescente. Gli anticorpi primari e secondari ottimizzati sono elencati nella Tabella 1. </li></ol>

<ol>
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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+and+gamma+irradiation+effects+on+surface+properties+of+polyurethane+derivate+from+castor+oil.">UV and gamma irradiation effects on surface properties of polyurethane derivate from castor oil.</a> Pol&#237;meros. 23 (3), 305-311 (2013).</li><li>Rosu, L., Cascaval, C. N., Ciobanu, C., Rosu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+UV+radiation+on+the+semi-interpenetrating+polymer+networks+based+on+polyurethane+and+epoxy+maleate+of+bisphenol+A.">Effect of UV radiation on the semi-interpenetrating polymer networks based on polyurethane and epoxy maleate of bisphenol A.</a> Journal of Photochemistry and Photobilogy A: Chemistry. 169 (2), 177-185 (2005).</li><li>Yang, X. F., Tallman, D. E., Bierwagen, G. P., Croll, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blistering+and+degradation+of+polyurethane+coatings+under+different+accelerated+weathering+tests.">Blistering and degradation of polyurethane coatings under different accelerated weathering tests.</a> Polymer Degradation and Stability. 77 (1), 103-109 (2002).</li></ol>

DCH è CSO, direttore, fondatore e azionista di FibromEd Products Ltd. MB e JPI sono soci fondatori in FibromEd Products Ltd.

Molti dei metodi attualmente utilizzati per generare epatociti da cellule staminali si basano su matrici indefinite di origine animale. Questi supporti possono essere costosi e molto variabile, che colpisce la funzione delle cellule e la stabilità, che rappresentano una barriera significativa applicazione. Pertanto, abbiamo effettuato una schermata per materiali sintetici che supportano la cultura di cellule staminali epatociti derivati. Abbiamo identificato, un semplice poliuretano (PU134), formata da polimerizzazione...

I materiali biologici sono stati ampiamente utilizzati per la manutenzione e la differenziazione delle cellule staminali pluripotenti 1. Mentre permettendo, questi substrati biologici spesso contengono una miriade di componenti non definiti. Matrigel è un substrato comunemente usato per la cultura e la differenziazione delle cellule staminali. Purtroppo, la sua composizione variabile influenza la funzione delle cellule e fenotipo. Anche se una serie di matrici biologiche alternative, più definite sono state utilizzate 2-7, la loro origine animale o scarsa scalabilità li rende i candidati non idonei per la produzione industriale. Pertanto, l&#39;individuazione di alternative sintetiche, a composizione definita e prestazioni affidabili, sono obiettivi chiave nella ricerca sulle cellule staminali. Nel tentativo di superare i limiti dei indefiniti substrati di coltura cellulare, collaborazioni interdisciplinari tra chimica e biologia hanno identificato materiali sintetici con la capacità di supportare fenotipo cellulare. Synthsubstrati ETIC sono scalabili, economiche, e possono essere realizzati in strutture 3D complesse, mimando l&#39;ambiente in vivo. Grazie a queste proprietà substrati sintetici sono stati ampiamente utilizzati per sostenere e guidare la differenziazione di molti tipi di cellule 8-10. Test di throughput avanzate e alte hanno facilitato la rapida proiezione di materiali sintetici, dai grandi librerie, e consegnati nuovi materiali con proprietà flessibili con ampie applicazioni nella ricerca e nello sviluppo 11-13 biomedico. Utilizzando un elevato throughput, polimero tecnologia di screening micro-array, abbiamo rapidamente identificato un semplice poliuretano (PU134), adatto per la manutenzione di cellule staminali derivate epatociti umani. Questo polimero è risultato essere superiore a substrati derivati ​​di origine animale per quanto riguarda la differenziazione e la funzione 14-16 epatociti. Abbiamo quindi ottimizzato il processo di rivestimento in condizioni, la topografia e la sterilizzazione per accedere effettisulle prestazioni del polimero nella stabilizzazione funzione degli epatociti e la durata della vita. Ciò ha implicazioni significative per quanto riguarda la comprensione fondamenti della biologia degli epatociti per la modellazione basata su cellule e applicazioni di medicina rigenerativa. La tecnologia qui descritta costituisce un esempio di come la superficie di un polimero sintetico può essere ottimizzato per preservare fenotipo cellulare. Crediamo che la combinazione di questa tecnologia con un protocollo di differenziazione degli epatociti privo di siero efficace ha il potenziale per fornire una produzione scalabile di epatociti per l&#39;utilizzo nella modellazione in vitro e della medicina rigenerativa.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Synthesis, preparation, coating and characterization of polymer PU134 coated coverslips</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Edmun B&#252;hler</td>
			<td>KS-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Irradiator</td>
			<td>CIS Biointernational</td>
			<td>IBL 637&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Specialty Coating System</td>
			<td>&#160;P-6708</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope&#160;</td>
			<td>Philips</td>
			<td>XL30CPSEM</td>
			<td></td>
		</tr>
		<tr>
			<td>Atomic Force Microscope</td>
			<td>DimensionV Nanoscope, VEECO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p4-GLO CYP3A4</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>UV bulb</td>
			<td>ESCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoScope analysis software</td>
			<td>VEECO</td>
			<td></td>
			<td>version 1.20</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>TH45200</td>
			<td>Use Volocity 4 Software</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Corning, UK&#160;</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>Scientific Laboratory Supplies</td>
			<td>MIC3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylene glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>93171</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;1,6-hexanediol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>240117</td>
			<td></td>
		</tr>
		<tr>
			<td>Neopentyl glycol</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>408255</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipic acid</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>9582</td>
			<td></td>
		</tr>
		<tr>
			<td>anhydrous N,N-Dimethylformamide</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>676845</td>
			<td></td>
		</tr>
		<tr>
			<td>titanium (IV) butoxide&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>244112</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-butanediol&#160;</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>493732</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum oven</td>
			<td>Thermoscientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#8217;-Methylenebis(phenyl isocyanate)</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>101688</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofurane</td>
			<td>Sigma&#8211;Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Bal-Tec SCD 050</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Inmunostaining</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (-MgCl2, -CaCl2)</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>&#160;Store at room temperature</td>
		</tr>
		<tr>
			<td>PBST, PBS made up with 0.1% TWEEN 20&#160;</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>EC607&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>&#160; Scientific Laboratory Supplies Ltd</td>
			<td>CHE5010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich, UK</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>MOWIOL 488 DAPI</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td>Made up in Tris HCL and glycerol as per manufacturers instructions</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell culture and Functional assay</td>
		</tr>
		<tr>
			<td>CYP3A activity pGLO kit</td>
			<td>Promega</td>
			<td>V8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepatozyme</td>
			<td>Gibco</td>
			<td>17705021</td>
			<td></td>
		</tr>
		<tr>
			<td>TryLE express</td>
			<td>Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/12604013">12604013</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

stabilizing hepatocellular phenotype using optimized synthetic surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Solvente del polimero influenza la topografia della superficie del polimero rivestito Poliuretano 134 è stato solubilizzato in cloroformio, da solo o in combinazione con toluene o tetraidrofurano o diclorometano ei vetrini spin-rivestiti con le diverse formulazioni. Microscopia elettronica a scansione (SEM) e microscopia a forza atomica (AFM) sono stati usati per caratterizzare le proprietà fisiche dei rivestimenti polimerici (Figura 1). Il rivestimento ot...

Watch this Scientific Journal Video about Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces at JoVE.com

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Questo articolo si concentrerà sullo sviluppo di superfici polimeriche rivestite a lungo termine, coltura stabile di cellule staminali derivate epatociti umani.

Stabilizzazione epatocellulare Fenotipo Utilizzando superfici sintetiche ottimizzati

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

stabilizing-hepatocellular-phenotype-using-optimized-synthetic

Research

JoVE Journal

Chemistry

10.2K Views.  University of Edinburgh.  Questo articolo si concentrerà sullo sviluppo di superfici polimeriche rivestite a lungo termine, coltura stabile di cellule staminali derivate epatociti umani. 

Video: Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Legame covalente di BMP-2 su superfici utilizzando un auto-assemblato Monolayer Approccio

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Ricerca

Chimica

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Verso Biomimicking Legno: Fabbricato Free-standing Films di Nanocellulose, lignina, e policatione sintetico

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

In Situ SIMS e IR Spettroscopia di superfici ben definite Preparato da atterraggio morbido di ioni Mass-selezionati

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. The most common techniques utilized are imaging modalities such as Magnetic Resonance Imaging (MRI), Positron Emission Topography (PET), and Computed Topography (CT) and are optimal for understanding the physical structure of the disease but can only be performed once every four to six weeks due to the use of imaging agents and overall cost. With this in mind, the development of &#8220;point of care&#8221; techniques, such as biosensors, which evaluate the stage of disease and/or efficacy of treatment in the clinician&#8217;s office and do so in a timely manner, would revolutionize treatment protocols.1 As a means to exploring ferrocene based biosensors for the detection of biologically relevant molecules2, methods were developed to produce ferrocene-biotin bio-conjugates described herein. This report will focus on a biotin-ferrocene-cysteine system that can be immobilized on a gold surface.

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodologia sintetico per Asymmetric Ferrocene derivati ​​sistemi bio-coniugati attraverso Solid Metodologia Resina a base di fase

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Ottimizzazione di proteine ​​sintetiche: identificazione di interposizione Dipendenze Indicazione Strutturalmente e / o residui funzionalmente collegate

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un<sup&gt; 125</sup&gt; Azide gruppo I-etichettati protesico

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Colore Spot Test come strumento presuntivo per la rilevazione rapida di Cathinones sintetico

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Caratterizzazione di polimeri sintetici tramite matrice assistita Laser desorbimento ionizzazione tempo di spettrometria di massa (MALDI-TOF) di volo

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces are demonstrated. This work requires careful design, considerable synthetic effort, and proper analysis. The rotary motion of the molecular motor in solution is shown by 1H NMR and UV-vis absorption spectroscopy techniques. In addition, the method to graft the motor onto an amine-coated quartz is described. This method helps to gain more insight into molecular machines.

The manuscript describes how to synthesize and graft a molecular motor on surfaces for single molecular imaging.

Motori molecolari basati su luce sulle superfici per l'Imaging molecolare singola

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of materials is highly related to its photocatalytic performance, it is necessary to standardize the synthetic methods in order to obtain the most functional architectures and, thus, the highest photocatalytic efficiency. Here, we report a reliable route to obtain BiOI microspheres via the solvothermal process, using Bi(NO3)3 and potassium iodide (KI) as precursors, and ethylene glycol as a template. The synthesis is standardized in a 150 mL autoclave, at 126 &#176;C for 18 h. This results in 2-3 &#181;m-sized mesoporous microspheres, with a relevant specific surface area (61.3 m2/g). Shortening the reaction times in the synthesis results in amorphous structures, while higher temperatures lead to a slight increase in the porosity of the microspheres, with no effect in the photocatalytic performance. The materials are photo-active under UV-A/visible light irradiation for the degradation of the antibiotic ciprofloxacin in water. This method has demonstrated to be effective in interlaboratory tests, obtaining similar BiOI microspheres in Mexican and Chilean research groups.

This article describes a synthetic method to obtain bismuth oxyiodide microspheres, which are highly functional to perform the photocatalytic removal of organic pollutants, such as ciprofloxacin, in water under UV-A/visible light irradiation.

Un Facile metodo sintetico per ottenere bismuto Oxyiodide microsfere altamente funzionale per i processi fotocatalitici di depurazione delle acque

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...