Ringraziamo il Prof. JP Spatz (Dipartimento di chimica biofisica, Università di Heidelberg e attrezzature dei nuovi materiali e dei Biosistemi, Istituto Max Planck per Sistemi Intelligenti, Stuttgart) per il suo supporto gentile. Il sostegno finanziario della Max-Planck-Gesellschaft e la Deutsche Forschungsgemeinschaft (DFG SFB/TR79 a EAC-A.) Sono notevolmente riconosciuto.

1. Sintesi di 11-Mercaptoundecanoyl-N-idrossisuccinimmide Ester (MU-NHS) <ol><li> Aggiungere goccia a goccia una soluzione di 500 mg di N-idrossisuccinimmide e 30 mg 4 - (dimetilammino) piridina in 10 ml di acetone (pa) a 1 g di acido 11-mercaptoundecanoic in 40 ml di diclorometano (pa) a temperatura ambiente (RT). </li><li> Raffreddare la reazione a 0 ° C e aggiungere goccia a goccia 1,1 g N, N &#39;-dicicloesilcarbodiimmide in 10 ml di diclorometano (in atmosfera di azoto). Mantenere la reazione a bassa temperatura per 1 ora e poi agitare a temperatura ambiente durante la notte. </li><li> Filtrare il precipitato e asciugare sotto pressione ridotta. Purificare il prodotto mediante cromatografia flash con benzene petrolio e acetato di etile (pa) in un rapporto di 1:1. </li></ol> 2. Preparazione di strati d&#39;oro omogenei <ol><li> Vetrini pulite con panni di precisione e sonicare per 5 minuti in una soluzione contenente una miscela 1:1 di acetato di etile (pa) e metanolo (pa). Rinse substrati con metanolo e asciugare sotto flusso di azoto. </li><li> Posizionare i supporti puliti nel dispositivo di rivestimento polverizzazione. Evacuare la camera. Rimuovere lo strato di ossido di cromo superiore dal bersaglio di cromo sputtering per 60 sec. Porre i campioni sotto la trave di metallo e ricoprirli con uno strato di cromo 10 nm, seguita da rivestimento con uno strato di oro 40 nm. </li></ol> 3. Immobilizzazione superficie di BMP-2 <ol><li> Sciogliere MU-NHS in N, N-dimethlyformamide (DMF) a una concentrazione finale di circa 1 mM e incubare substrati d&#39;oro nella soluzione MU-NHS a temperatura ambiente per 4 ore sotto atmosfera di azoto. </li><li> Superfici Sonicare in DMF per 2 minuti, sciacquare con DMF e MeOH e asciugare sotto flusso di azoto. </li><li> Preparare una soluzione stock di rhBMP-2 in sterile HCl 4 mm, con una concentrazione di 100 pg / ml (negozio aliquote a -80 ° C). Per diluizioni di lavoro (3,5 mcg / ml), diluire la soluzione madre in PBS / NaCl (PBS contenente 1 M NaCl) e unegolare a pH 8,5 immediatamente prima dell&#39;uso. </li><li> Incubare superfici funzionalizzate MU-NHS nella soluzione rhBMP-2 ore a 4 ° C per una notte. Rimuovere il surnatante incubazione. Superfici Sonicare in PBS / NaCl per 2 minuti e lavare 3x con PBS sterile / NaCl. </li></ol> 4. Caratterizzazione superficiale <ol><li> Per bloccare l&#39;adsorbimento non specifico di anticorpo, incubare superfici con BSA 5% (w / v) in PBS per 1 ora a RT, seguita da incubazione con un anti-BMP-2 anticorpi (1:100 in 1% (w / v ) soluzione di BSA / PBS) per 1 ora a RT. Lavare le superfici due volte con PBS e ultrasuoni per 30 sec. </li><li> Incubare substrati con HRP-anticorpo secondario coniugato (1:1000 in 1% (w / v) di BSA / PBS) per 30 min a temperatura ambiente, poi lavare due volte con PBS. </li><li> Utilizzare Ampliflu test Red per misurare l&#39;attività enzimatica HRP a 570 nm con un lettore di piastre. </li></ol> 5. Analisi dell&#39;attività biologica <ol><li> Seed 1 x 10 5 topo C2C12 mioblasti per pozzetto in 6 pozzetti plAtes in terreno di coltura, costituiti da un elevato glucosio DMEM contenente piruvato supplementato con 10% FBS, 1% di penicillina / streptomicina e incubare per 24 ore a 37 ° C / 5% di CO 2. </li><li> Morire di fame cellule in DMEM senza siero per 3-5 ore prima di semina sulle superfici decorate con immobilizzata BMP-2. </li><li> Per le indagini dell&#39;induzione segnalazione breve termine, sostituire il mezzo da 200 microlitri DMEM fresco senza siero e posizionare le immobilizzati BMP-2 superfici sopra le celle. La superficie deve essere maneggiato con le pinzette punta fine per evitare di graffiare. </li><li> Rimuovere delicatamente le superfici e aspirare il terreno con una pipetta, lavare le cellule due volte con PBS. Procedere all&#39;analisi delle risposte delle cellule. </li><li> Per l&#39;analisi dell&#39;attività biologica a lungo termine, cellule piastra su superfici e cultura loro a 37 ° C / 5% di CO 2 in condizioni di siero bassi (2% FBS) per sei giorni. </li></ol>

<ol>
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In questo protocollo si descrive la preparazione di superfici funzionalizzate con bioattivo rhBMP-2. Questo approccio comprende due fasi: 1) la formazione iniziale di una auto-assemblaggio monostrato (SAM) di un linker bifunzionale sulla superficie di oro; 2) immobilizzazione covalente della proteina rhBMP-2. Nel lavoro precedente, abbiamo convalidato l&#39;effettivo legame del linker bifunzionale e il fattore di crescita, e dimostrato che in superficie immobilizzato rhBMP-2 mantiene la sua attività biologica 19.<...

Proteina morfogenetica ossea 2 (BMP-2) è un membro del fattore di crescita trasformante (TGF-β) famiglia e agisce come induttore del de novo formazione ossea e regolatore di diversi tessuti durante lo sviluppo embrionale e adulta omeostasi 1-3. Ogni monomero del omodimerica BMP-2 proteina biologicamente attiva contiene un motivo &quot;cisteina nodo&quot;, che è altamente conservata in tutti BMP 4. Sei dei sette residui di cisteina formano legami disolfuro intramolecolari che stabilizzano ciascun monomero, mentre la settima cisteina è coinvolto nella dimerizzazione, formando un legame intermolecolare tra i due monomeri 5,6. Questo nodo di cisteina altamente conservati definisce la struttura tridimensionale della proteina BMP-2 e determina le proprietà uniche, come la resistenza al calore, denaturanti e acido pH 7-9. BMP-2 si lega a serina / treonina chinasi recettori transmembrana, inducendo così la trasduzione del segnale <sup&gt; 10-12. A seconda della modalità di oligomerizzazione recettoriale, diverse vie di segnalazione vengono attivati: una cascata di segnalazione Smad-indipendente conduce ad induzione fosfatasi alcalina tramite segnalazione p38, mentre un percorso Smad-dipendente attivata risultati fosforilazione recettoriale in Smad traslocazione nucleare complesso e l&#39;attivazione della trascrizione dei specifici geni bersaglio, come l&#39;inibitore di differenziazione (Id) 12-14. In osso, BMP-2 induce la differenziazione delle cellule staminali mesenchimali in osteoblasti, stimolando così la guarigione e de novo formazione di osso. Attualmente, ricombinante espresso BMP-2 è applicata clinicamente per migliorare la guarigione di siti fratturati. Una strategia comune in ingegneria del tessuto osseo è l&#39;uso di fattori di crescita iniettabili, che è meno invasivo rispetto a sistemi di applicazione locale. Tuttavia, studi in vivo ed applicazioni cliniche hanno dimostrato che la breve emivita biologica, loc aspecificalizzazione e rapida domiciliazione del BMP-2 può portare a diversi problemi locali, ectopiche e sistemiche 15. Quindi, per ottenere una presentazione efficace, l&#39;intrappolamento o immobilizzazione di BMP-2 all&#39;interno o sul materiale necessario alla sua consegna locale e costante a sito bersaglio. Consegna sostenuta può essere ottenuto con metodi di conservazione non covalenti, come intrappolamento fisico, adsorbimento o complessazione di ioni 16. Tuttavia, è noto che adsorbimento non specifico di proteine ​​su superfici può causarne la denaturazione delle molecole 17. Per il legame di fattori di crescita covalente, diversi tipi di supporti sono stati sviluppati negli ultimi dieci anni. L&#39;uso di molecole bifunzionali che mirano linking amminici o carbossilici gruppi della proteina per esempio, è un tipo di approccio che non richiede necessariamente modificazione delle proteine ​​per raggiungere la sua immobilizzazione. Infatti, mentre modificazione delle proteine ​​offre il vantaggio di controllare l&#39;orientamento proteine,l&#39;introduzione di domini artificiali, tag e catene peptidiche specifiche del sito può alterare l&#39;attività biologica di fattori di crescita 17. Così, per aggirare denaturazione dovuta all&#39;interazione con il materiale di supporto, le superfici possono essere funzionalizzati anticipo, per esempio, con un monostrato auto-assemblato (SAM) di una molecola di collegamento, seguito da accoppiamento del fattore desiderato 18. Abbiamo usato un approccio basato SAM per immobilizzare covalentemente BMP-2 su una superficie di mira i suoi residui amminici liberi e abbiamo dimostrato che la proteina immobilizzata mantiene sia la sua attività biologica a breve e lungo termine 19. Questo protocollo fornisce un modo semplice ed efficace per fornire BMP-2 alle cellule per studi in vitro sui meccanismi che si verificano a livello della membrana cellulare e regolano segnalazione intracellulare responsabili per la segnalazione osteogenico.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(dimethylamino)pyridin</td>
			<td>Sigma-Aldrich</td>
			<td>522805</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>AppliChem</td>
			<td>A2282</td>
			<td></td>
		</tr>
		<tr>
			<td>11-mercaptoundecanoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>674427</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichlormethane</td>
			<td>Merck</td>
			<td>106050</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-dicyclohexylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D80002</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum benzene</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Carl Roth</td>
			<td>M 875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylacetate</td>
			<td>AppliChem</td>
			<td>A3550</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>4627</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Carl Roth</td>
			<td>T921</td>
			<td></td>
		</tr>
		<tr>
			<td>rhBMP-2</td>
			<td>R&#38;D Systems</td>
			<td>355-BM</td>
			<td>Carrier-free; expressed in E.coli</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(dimethyl siloxane) (PDMS)</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rhBMP-2</td>
			<td>Sigma</td>
			<td>B9553</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ampliflu Red assay</td>
			<td>Sigma</td>
			<td>90101</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) (1x), liquid</td>
			<td>Gibco</td>
			<td>41966</td>
			<td>High glucose</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td>Sterile filtered, cell culture tested</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05% (1x) with EDTA 4Na</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine (0.1 M)</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>33226</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (1%)</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Lysis buffer (ALP assay)19</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)(1 mM)</td>
			<td>Carl Roth</td>
			<td>HNO3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc chloride (ZnCl2) (1 mM)</td>
			<td>Carl Roth</td>
			<td>3533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>p-nitrophenylphosphate (pNPP)</td>
			<td>Sigma</td>
			<td>S0942</td>
			<td>Phosphatase substrate</td>
		</tr>
		<tr>
			<td>Anti-mysin heavy chain (MHC)</td>
			<td>Developmental Studies Hybridoma Bank, University of Iowa</td>
			<td>MF20</td>
			<td>Monoclonal antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ultrsonic bath (Sonorex Super RK 102H), Frequency 35 kHz</td>
			<td>BANDELIN electronic GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MED 020 Sputtercoating system</td>
			<td>BAL-TEC AG</td>
			<td></td>
			<td>Coating conditions 
			Cr: 120 mA, 1.3 x 10-2 mbar, 30 sec 
			Au: 60 mA, 5.0 x 10-2 mbar, 45 sec</td>
		</tr>
		<tr>
			<td>Tecan Infinite M200 Plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

covalent binding of bmp-2 on surfaces using a self-assembled monolayer approach

Proteina morfogenetica 2 (BMP-2) è un fattore di crescita incorporato nella matrice extracellulare del tessuto osseo. BMP-2 funge da innesco della differenziazione delle cellule mesenchimali in osteoblasti, stimolando così la guarigione e la formazione ossea novo de. L&#39;uso clinico di ricombinante umano BMP-2 (rhBMP-2) in combinazione con ponteggi ha sollevato controversie recenti, in base alla modalità di presentazione e l&#39;importo da erogare. Il protocollo qui presentato fornisce un modo semplice ed efficace per fornire BMP-2 per studi in vitro su cellule. Si descrive come formare un monostrato auto-assemblato composto da un linker eterobifunzionale, e mostriamo la successiva fase vincolante per ottenere l&#39;immobilizzazione covalente di rhBMP-2. Con questo approccio è possibile ottenere una presentazione sostenuta di BMP-2, pur mantenendo l&#39;attività biologica della proteina. Infatti, l&#39;immobilizzazione superficie di BMP-2 consente indagini mirate impedendo annunci aspecificheorption, riducendo la quantità di fattore di crescita e, in particolare, ostacolando rilascio incontrollato dalla superficie. Entrambi gli eventi di segnalazione a breve e lungo termine, innescato da BMP-2 si svolgono quando le cellule sono esposte a superfici che presentano covalentemente immobilizzato rhBMP-2, rendendo questo approccio adatto per studi in vitro sulle risposte cellulari ai BMP-2 stimolazione.

Nella nostra configurazione, l&#39;oro è stato scelto come eccipiente in quanto fornisce un sistema biologicamente aspecifica ma chimicamente sintonizzabile. Inoltre, l&#39;applicazione di monostrati auto-assemblaggio comporta molti vantaggi: SAM adsorbe spontaneamente tramite loro &quot;testa-gruppi&quot; a metalli e formare monostrati con pochi difetti, mentre i loro gruppi terminali funzionali possono essere ulteriormente modificati. Così prevedono la piattaforma per adattare le proprietà dell&#39;interfaccia cont...

Watch this Scientific Journal Video about Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach at JoVE.com

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

Noi descriviamo un metodo per realizzare immobilizzazione efficiente di BMP-2 sulle superfici. Il nostro approccio si basa sulla formazione di un monostrato auto-assemblato per realizzare il legame di BMP-2 attraverso i suoi residui amminici liberi covalente. Questo metodo è un utile strumento per studiare la segnalazione alla membrana cellulare.

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 ...

covalent-binding-bmp-2-on-surfaces-using-self-assembled-monolayer

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Organic Chemistry

Chemistry

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

13.8K Views.  University of Heidelberg.  Noi descriviamo un metodo per realizzare immobilizzazione efficiente di BMP-2 sulle superfici. Il nostro approccio si basa sulla formazione di un monostrato auto-assemblato per realizzare il legame di BMP-2 attraverso i suoi residui amminici liberi covalente. Questo metodo è un utile strumento per studiare la segnalazione alla membrana cellulare. 

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

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.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

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 ...

Ricerca

Chimica

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

Un approccio di analisi inversa per la caratterizzazione dei Trasporti Chemical nelle vernici

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

Preparazione facile di Internamente auto-assemblato Lipid particelle stabilizzati da nanotubi di carbonio

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Altamente stabile, funzionale Peloso nanoparticelle e biopolimeri da fibre di legno: verso nanotecnologia sostenibile

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The first COF was reported in 2005 with much effort centered on the establishment of new synthetic routes for its preparation. To date, most available synthetic methods for COF synthesis are based on bulk mixing under solvothermal conditions. Therefore, there is increasing interest in developing systematic protocols for COF synthesis that provide for fine control over reaction conditions and improve COF processability on surfaces, which is essential for their use in practical applications. Herein, we present a novel microfluidic-based method for COF synthesis where the reaction between two constituent building blocks, 1,3,5-benzenetricarbaldehyde (BTCA) and 1,3,5-tris(4-aminophenyl)benzene (TAPB), takes place under controlled diffusion conditions and at room temperature. Using such an approach yields sponge-like, crystalline fibers of a COF material, hereafter called MF-COF. The mechanical properties of MF-COF and the dynamic nature of the approach allow the continuous production of MF-COF fibers and their direct printing onto surfaces. The general method opens new potential applications requiring advanced printing of 2D or 3D COF structures on flexible or rigid surfaces.

Microfluidic-based Synthesis of Covalent Organic Frameworks COFs: A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

We present a novel microfluidic-based method for synthesis of covalent organic frameworks (COFs). We demonstrate how this approach can be used to produce continuous COF fibers, and also 2D or 3D COF structures on surfaces.

Sintesi microfluidica a base di strutture organiche COF (COF): uno strumento per la produzione continua di fibre COF e stampa diretta su una superficie

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The ...

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 ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Analisi delle molecole complesse e delle loro reazioni sulle superfici mediante mezzi di desorpazione/spettrometria di massa indotta da cluster

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Attacco covalente di singole molecole per la spettroscopia della forza basata su AFM

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, dipyrrinones, and pharmaceuticals. Additionally, restricted rotation of these systems often results in highly to moderately fluorescent systems as observed in 3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidin-3-ones, xanthoglows, pyrroloindolizinedione analogs, BODIPY analogs, and the phenolic and imidazolinone ring systems of Green Fluorescent Protein (GFP). This manuscript describes an inexpensive and operationally simple method of performing a Claisen-Schmidt condensation to generate a series of fluorescent pH dependent pyrazole/imidazole/isoindolone dipyrrinone analogs. While the methodology illustrates the synthesis of dipyrrinone analogs, it can be translated to produce a wide range of conjugated bicyclic aromatic compounds. The Claisen-Schmidt condensation reaction utilized in this method is limited in scope to nucleophiles and electrophiles that are enolizable under basic conditions (nucleophile component) and non-enolizable aldehydes (electrophile component). Additionally, both the nucleophilic and electrophilic reactants must contain functional groups that will not inadvertently react with hydroxide. Despite these limitations, this methodology offers access to completely novel systems that can be employed as biological or molecular probes.

The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.

Sintesi di pirazolo dipendente dal pH, imidazolo e fluorofori di Isoindolone Dipyrrinone usando un approccio di condensazione claisen-schmidt

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, ...

Analysis of Organochlorine Pesticides in a Soil Sample by a Modified QuEChERS Approach Using Ammonium Formate

Currently, the QuEChERS method represents the most widely used sample preparation protocol worldwide for analyzing pesticide residues in a broad variety of matrices both in official and non-official laboratories. The QuEChERS method using ammonium formate has previously proven to be advantageous compared to the original and the two official versions. On the one hand, the simple addition of 0.5 g of ammonium formate per gram of sample is sufficient to induce phase separation and achieve good analytical performance. On the other hand, ammonium formate reduces the need for maintenance in routine analyses. Here, a modified QuEChERS method using ammonium formate was applied for the simultaneous analysis of organochlorine pesticide (OCP) residues in agricultural soil. Specifically, 10 g of the sample was hydrated with 10 mL of water and then extracted with 10 mL of acetonitrile. Next, phase separation was carried out using 5 g of ammonium formate. After centrifugation, the supernatant was subjected to a dispersive solid-phase extraction clean-up step with anhydrous magnesium sulfate, primary-secondary amine, and octadecylsilane. Gas chromatography-mass spectrometry was used as the analytical technique. The QuEChERS method using ammonium formate is demonstrated as a successful alternative for extracting OCP residues from a soil sample.

The present protocol describes the utilization of ammonium formate for phase partitioning in QuEChERS, together with gas chromatography-mass spectrometry, to successfully determine organochlorine pesticide residues in a soil sample.

Analisi di pesticidi organoclorurati in un campione di terreno mediante un approccio QuEChERS modificato utilizzando formiato di ammonio

Currently, the QuEChERS method represents the most widely used sample preparation protocol worldwide for analyzing pesticide residues in a broad variety ...