Nous remercions le Professeur JP Spatz (Département de chimie biophysique de l&#39;Université de Heidelberg et du ministère des nouveaux matériaux et des biosystèmes, Institut Max Planck pour les systèmes intelligents, Stuttgart) pour son soutien en nature. Le soutien financier de la Max-Planck-Gesellschaft et la Deutsche Forschungsgemeinschaft (DFG SFB/TR79 à EAC-A.) Sont également très reconnu.

Une. Synthèse de 11-Mercaptoundecanoyl-N-hydroxysuccinimide ester (NHS-MU) <ol><li> Ajouter goutte à goutte une solution de 500 mg de N-hydroxysuccinimide et 30 mg de 4 - (diméthylamino) pyridine dans 10 ml d&#39;acétone (PA) pour 1 g d&#39;acide 11-mercaptoundécanoïque dans 40 ml de dichlorométhane (pa) à la température ambiante (RT). </li><li> Refroidir la réaction à 0 ° C et ajouter goutte à goutte 1,1 g de N, N &#39;-dicyclohexylcarbodiimide dans 10 ml de dichlorométhane (sous atmosphère d&#39;azote). Gardez la réaction à basse température pendant 1 heure puis mélanger à température ambiante jusqu&#39;au lendemain. </li><li> Filtrer le précipité et le sécher sous pression réduite. On purifie le produit par Chromatographie éclair avec du benzène de pétrole et d&#39;acétate d&#39;éthyle (ap) dans un rapport de 1:1. </li></ol> 2. Préparation de couches d&#39;or homogènes <ol><li> Des lamelles de verre propres avec des lingettes de précision et soniquer les pendant 5 min dans une solution contenant un mélange 1:1 d&#39;acétate d&#39;éthyle (pa) et de méthanol (pa). Rinse substrats avec du méthanol et sèchent sous flux d&#39;azote. </li><li> Placer les substrats de nettoyage dans le dispositif de revêtement par pulvérisation. Évacuer la chambre. Enlever la couche d&#39;oxyde de chrome plus élevée à partir de la cible de chrome par pulvérisation cathodique pendant 60 sec. Placer les échantillons au-dessous de la poutre métallique et les enrober d&#39;une couche de chrome de 10 nm, suivi par enrobage avec une couche d&#39;or de 40 nm. </li></ol> 3. L&#39;immobilisation de la surface de la BMP-2 <ol><li> Dissoudre MU-NHS dans du N, N-dimethlyformamide (DMF) à une concentration finale d&#39;environ 1 mM et l&#39;incubation des substrats d&#39;or dans la solution MU-NHS à température ambiante pendant 4 heures sous atmosphère d&#39;azote. </li><li> Surfaces soniquer dans le DMF pendant 2 min, rincer avec du DMF et MeOH et sèchent sous flux d&#39;azote. </li><li> Préparer une solution stock de rhBMP-2 dans HCl stérile 4 mm avec une concentration de 100 pg / ml (aliquotes de conserver à -80 ° C). Pour les dilutions de travail (3,5 pg / ml), diluer la solution mère dans du PBS / NaCl (PBS contenant 1 M de NaCl) et undjust à pH 8,5, immédiatement avant utilisation. </li><li> Incuber les surfaces fonctionnalisées MU-NHS dans la solution de travail rhBMP-2 à 4 ° C pendant une nuit. Retirer surnageant d&#39;incubation. Surfaces soniquer dans PBS / NaCl pendant 2 minutes et laver 3 fois avec PBS stérile / NaCl. </li></ol> 4. Caractérisation de surface <ol><li> Pour bloquer l&#39;adsorption non spécifique de l&#39;anticorps, incuber les surfaces avec une BSA à 5% (p / v) dans une solution PBS pendant 1 heure à température ambiante, suivi d&#39;une incubation avec un anticorps anti-BMP-2 (1:100 dans 1% (w / v ) solution de BSA / PBS) pendant 1 heure à température ambiante. Laver les surfaces deux fois avec du PBS et de traitement par ultrasons pendant 30 s. </li><li> Incuber les substrats avec un anticorps secondaire conjugué à HRP (1:1000 dans 1% (p / v) de solution de BSA / PBS) pendant 30 min à température ambiante, puis le laver deux fois avec du PBS. </li><li> Utilisez Ampliflu essai rouge pour mesurer l&#39;activité enzymatique HRP à 570 nm avec un lecteur de plaque. </li></ol> 5. Analyse de l&#39;activité biologique <ol><li> Seed 1 x 10 5 souris C2C12 myoblastes par puits dans 6 puits plates dans un milieu de croissance, consistant en un haut DMEM contenant du pyruvate de glucose supplémenté avec 10% de FBS, 1% de pénicilline / streptomycine et de les incuber pendant 24 heures à 37 ° C / 5% CO 2. </li><li> Affamer les cellules dans du DMEM sans sérum pendant 3-5 heures avant le semis sur les surfaces décorées avec BMP-2 immobilisé. </li><li> Pour l&#39;enquête de court terme signalisation induction, remplacer le milieu par 200 ul DMEM sans sérum frais et placer les immobilisés BMP-2 surfaces au-dessus des cellules. La surface doit être manipulé avec des pincettes fines de pointe pour éviter les rayures. </li><li> Retirez délicatement la surface de et aspirer le milieu avec une pipette, laver les cellules deux fois avec du PBS. Procéder à l&#39;analyse des réponses cellulaires. </li><li> Pour l&#39;analyse de l&#39;activité biologique à long terme, les cellules de la plaque sur les surfaces et leur culture à 37 ° C / 5% CO 2 dans des conditions de sérum faible (2% de FBS) pendant six jours. </li></ol>

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Dans ce protocole, nous décrivons la préparation de surfaces fonctionnalisées avec bioactif rhBMP-2. Cette approche comporte deux étapes: 1) la formation initiale d&#39;une monocouche d&#39;auto-assemblage (SAM) d&#39;un groupe de liaison bifonctionnel sur la surface d&#39;or, 2) l&#39;immobilisation covalente de la protéine de rhBMP-2. Dans des travaux antérieurs, nous avons validé la liaison efficace de l&#39;agent de liaison bifonctionnel, et le facteur de croissance, et démontré que la surface immobilisée ...

Une protéine morphogénétique osseuse 2 (BMP-2) est un élément du facteur de croissance transformant (TGF-β) de la famille et agit comme inducteur de la formation de novo de l&#39;os ainsi que la régulation de plusieurs tissus au cours du développement embryonnaire et adulte homéostasie 3.1. Chaque monomère de l&#39;homodimère protéine biologiquement active BMP-2 contient un motif &quot;cysteine ​​de noeud&quot;, qui est hautement conservée dans toutes les PGB 4. Six des sept résidus de cysteine ​​forment des ponts disulfure intramoléculaires qui stabilisent chaque monomère, tandis que le septième cysteine ​​est impliquée dans la dimérisation, la formation d&#39;une liaison intermoléculaire entre les deux monomères 5,6. Ce nœud de cysteine ​​hautement conservés définit la structure tridimensionnelle de la protéine BMP-2 et détermine ses propriétés uniques, telles que résistance à la chaleur, et des dénaturants acide pH 7-9. BMP-2 se lie à la sérine / thréonine kinase des récepteurs transmembranaires, ce qui induit une transduction de signal <sup&gt; 10 à 12. Selon le mode d&#39;oligomérisation du récepteur, différentes voies de signalisation sont activés: une cascade de signalisation Smad-indépendante mène à la phosphatase alcaline de l&#39;induction par l&#39;intermédiaire de la signalisation de p38, tandis qu&#39;une voie de Smad-dépendant activé par les résultats de la phosphorylation du récepteur à Smad translocation nucléaire complexe et l&#39;activation de la transcription d&#39; gènes cibles spécifiques, tels que l&#39;inhibiteur de la différenciation (Id) de 12 à 14. Dans l&#39;os, la BMP-2 induit la différenciation des cellules souches mésenchymateuses en ostéoblastes, stimulant ainsi la guérison et la formation de novo de l&#39;os. Actuellement, exprimée par recombinaison BMP-2 est appliqué en clinique pour améliorer la guérison des sites fracturés. Une stratégie courante dans l&#39;ingénierie tissulaire osseuse est l&#39;utilisation de facteurs de croissance injectables, ce qui est moins invasive par rapport aux systèmes d&#39;administration locale. Cependant, des études in vivo et des applications cliniques ont montré que la demi-vie biologique courte, loc non spécifiquelisation et domiciliation rapide de BMP-2 peut conduire à plusieurs problèmes locaux, ectopiques et systémiques 15. Par conséquent, pour obtenir une présentation efficace, le piégeage ou l&#39;immobilisation de la BMP-2 à l&#39;intérieur ou sur des matériaux est nécessaire pour sa délivrance locale et prolongée au niveau du site cible. Une distribution prolongée peut être obtenue avec des approches de rétention de non-covalentes, telles que le piégeage physique, l&#39;adsorption ou la complexation d&#39;ions 16. Cependant, il est connu que l&#39;adsorption non spécifique de protéines sur les surfaces peut élément de dénaturation des molécules 17. Pour la liaison des facteurs de croissance covalente, différents types de supports ont été développés au cours de la dernière décennie. L&#39;utilisation de molécules de liaison bifonctionnels qui ciblent des groupes amino ou carboxyle de la protéine, par exemple, est un type d&#39;approche qui ne nécessite pas nécessairement une modification de la protéine pour obtenir son immobilisation. En fait, tandis que la modification des protéines offre l&#39;avantage de contrôler l&#39;orientation de la protéine,l&#39;introduction de domaines artificiels, des étiquettes et des chaînes peptidiques spécifiques à un site peut modifier l&#39;activité biologique de facteurs de croissance 17. Ainsi, pour contourner la dénaturation due à l&#39;interaction avec le matériau de support, les surfaces peuvent être fonctionnalisés à l&#39;avance, par exemple, avec une monocouche auto-assemblée (SAM) d&#39;une molécule de liaison, suivie d&#39;un couplage du facteur désiré 18. Nous avons utilisé une approche fondée sur les SAM pour immobiliser de manière covalente BMP-2 sur une surface en ciblant ses résidus aminés libres et avons montré que la protéine immobilisée conserve à la fois son activité biologique à court et à long terme 19. Ce protocole offre un moyen simple et efficace pour délivrer la BMP-2 dans des cellules pour des études in vitro sur les mécanismes qui se produisent à la membrane cellulaire et régulent la signalisation intracellulaire responsable de la signalisation ostéogénique.

<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

la protéine morphogénétique osseuse 2 (BMP-2) est un facteur de croissance incorporé dans la matrice extracellulaire du tissu osseux. BMP-2 agit comme déclencheur de la différenciation des cellules mésenchymateuses en ostéoblastes, stimulant ainsi la guérison et la formation osseuse de novo. L&#39;utilisation clinique de l&#39;humain recombinant BMP-2 (rhBMP-2), en liaison avec des échafaudages a soulevé controverses récentes, sur la base du mode de présentation et la quantité à délivrer. Le protocole présenté ici fournit un moyen simple et efficace pour délivrer la BMP-2 pour des études in vitro sur des cellules. Nous décrivons comment former une monocouche auto-assemblée constituée d&#39;un segment de liaison hétérobifonctionnel, et on montre l&#39;étape ultérieure de liaison pour obtenir une immobilisation covalente de rhBMP-2. Avec cette approche, il est possible d&#39;obtenir une présentation prolongée de la BMP-2, tout en maintenant l&#39;activité biologique de la protéine. En fait, l&#39;immobilisation de la surface de la BMP-2 permet enquêtes ciblées en empêchant annonces non spécifiquesorption, tout en réduisant la quantité de facteur de croissance et, plus particulièrement, ce qui empêche la libération non contrôlée de la surface. Les deux à court et à long terme des événements de signalisation déclenchées par BMP-2 ont lieu lorsque les cellules sont exposées à des surfaces présentant immobilisé de manière covalente rhBMP-2, ce qui rend cette approche appropriée pour des études in vitro sur les réponses cellulaires à la BMP-2 stimulation.

Dans notre configuration, l&#39;or a été choisi comme excipient car il fournit un système biologique non spécifique mais chimiquement accordable. En outre, l&#39;application de monocouches auto-assemblage comporte de nombreux avantages: SAM adsorber spontanément par leurs «tête-groupes&quot; sur les métaux et forment des monocouches avec quelques défauts, tandis que leurs groupes terminaux fonctionnels peuvent encore être modifiés. Ainsi, ils fournissent une plateforme pour adapter les propriétés de l&#39;i...

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

Nous décrivons un procédé pour réaliser une immobilisation efficace de la BMP-2 sur les surfaces. Notre approche est basée sur la formation d&#39;une monocouche auto-assemblée de réaliser la liaison de BMP-2 par l&#39;intermédiaire de ses résidus amines libres covalente. Cette méthode est un outil utile pour étudier la signalisation à la membrane cellulaire.

Liaison covalente de BMP-2 sur les surfaces en utilisant une approche monocouche auto-assemblée

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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JoVE Journal

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Organic Chemistry

Chemistry

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

13.8K vues.  University of Heidelberg.  Nous décrivons un procédé pour réaliser une immobilisation efficace de la BMP-2 sur les surfaces. Notre approche est basée sur la formation d'une monocouche auto-assemblée de réaliser la liaison de BMP-2 par l'intermédiaire de ses résidus amines libres covalente. Cette méthode est un outil utile pour étudier la signalisation à la membrane cellulaire. 

Vidéo: 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.

Stabiliser hépatocellulaire phénotype en utilisant des surfaces synthétiques optimisés

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

Recherche

Chimie

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.

Une approche d&#39;analyse inverse à la caractérisation des Transports chimique dans les peintures

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.

Préparation Facile d&#39;intérieur auto-assemblées particules lipides stabilisés par des nanotubes de carbone

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.

Très stable, fonctionnelle Hairy Nanoparticules et biopolymères à partir de fibres de bois: Vers la nanotechnologie durable

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.

Synthèse à base de microfluidique des cadres organiques covalents (COF): un outil pour la production continue de fibres COF et l&#39;impression directe sur une surface

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.

Moteurs moléculaires axée sur la lumière sur les Surfaces pour l’imagerie moléculaire unique

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.

Analyse des molécules complexes et de leurs réactions sur les surfaces au moyen de la desorption induite par les grappes/spectrométrie de masse d’ionisation

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.

Attachement covalent de molécules uniques pour la spectroscopie de force basée sur l’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.

Synthèse de fluorophores de pyrazole, d’imidazole et de dipyrrinone isoindolone dépendant du pH à l’aide d’une approche de condensation de 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.

Analyse de pesticides organochlorés dans un échantillon de sol par une approche QuEChERS modifiée utilisant le formate d’ammonium

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