Wir danken Prof. JP Spatz (Institut für Biophysikalische Chemie, Universität Heidelberg und der Abteilung Neue Materialien und Biosysteme, Max-Planck-Institut für Intelligente Systeme, Stuttgart) für die freundliche Unterstützung. Die finanzielle Unterstützung von der Max-Planck-Gesellschaft und der Deutschen Forschungsgemeinschaft (DFG SFB/TR79 EAC-A.) Sind ebenfalls sehr anerkannt.

1. Synthese von 11-Mercaptoundecanoyl-N-hydroxysuccinimidester (MU-NHS) <ol><li> (Dimethylamino) pyridin in 10 ml Aceton (pa) und 1 g 11-Mercaptoundecansäure in 40 ml Dichlormethan (Pa) bei Raumtemperatur (RT) - eine Lösung von 500 mg N-Hydroxysuccinimid und 30 mg 4 hinzuzufügen. </li><li> Die Reaktion wird auf 0 ° C gekühlt und tropfenweise mit 1,1 g N, N &#39;-Dicyclohexylcarbodiimid in 10 ml Dichlormethan (unter Stickstoffatmosphäre). Halten Sie die Reaktion bei niedriger Temperatur für 1 h und dann über Nacht bei RT rühren. </li><li> Der Niederschlag abfiltriert und trocknet es im Vakuum. Reinige das Produkt durch Flash-Chromatographie mit Petrolether Benzol und Ethylacetat (PA) in einem Verhältnis von 1:1. </li></ol> 2. Herstellung von homogenen Gold-Schichten <ol><li> Saubere Deckgläser mit Präzision Tücher und beschallen sie für 5 Minuten in einer Lösung, die eine 1:1-Mischung von Ethylacetat (pa) und Methanol (pa). RinsE Substrate mit Methanol und Trocknen unter Stickstoffstrom. </li><li> Legen Sie die sauberen Substrate in der Sputter-Beschichtungsvorrichtung. Kammer evakuieren. Entfernen der obersten Schicht aus Chromoxid Chromtargets durch Sputtern für 60 sek. Die Proben werden unter der Metallstrahl und überziehen sie mit einer 10 nm Chromschicht, gefolgt von der Beschichtung mit einer 40 nm dicken Goldschicht. </li></ol> 3. Oberflächen Immobilisierung von BMP-2 <ol><li> Auflösen MU-NHS in N, N-dimethlyformamide (DMF) zu einer Endkonzentration von etwa 1 mM und Inkubation Goldsubstrate in der MU-NHS-Lösung bei RT für 4 h unter Stickstoffatmosphäre. </li><li> Ultraschallbad Flächen in DMF für 2 Minuten gründlich mit DMF und MeOH und trocknen unter Stickstoffstrom. </li><li> Bereiten Sie eine Stammlösung von rhBMP-2 in sterile 4 mM HCl mit einer Konzentration von 100 ug / ml (store-Aliquots bei -80 ° C). Für die Arbeit Verdünnungen (3,5 ug / ml), verdünnt das Stammlösung in PBS / NaCl (PBS mit 1 M NaCl) und eineP assen auf pH 8,5 unmittelbar vor der Verwendung. </li><li> Inkubieren funktionalisierten Oberflächen MU-NHS in der rhBMP-2 Arbeitslösung bei 4 ° C über Nacht. Überstand entfernen Inkubation. Ultraschallbad Flächen in PBS / NaCl für 2 Minuten und waschen 3x mit sterilem PBS / NaCl. </li></ol> 4. Oberflächencharakterisierung <ol><li> Unspezifische Adsorption von Antikörpern zu blockieren, Inkubation Oberflächen mit einer 5% BSA (w / v) in PBS-Lösung für 1 h bei RT, gefolgt von einer Inkubation mit einem Anti-BMP-2-Antikörper (1:100 in 1% (w / v ) BSA / PBS-Lösung) für 1 h bei RT. Zweimal waschen mit PBS Flächen beschallen und für 30 Sekunden. </li><li> Substrate inkubieren mit HRP-konjugierten sekundären Antikörper (1:1000 in 1% (w / v) BSA / PBS-Lösung) für 30 min bei RT, dann zweimal mit PBS waschen. </li><li> Verwenden Ampliflu Red Assay HRP enzymatische Aktivität messen bei 570 nm mit einem Plattenlesegerät. </li></ol> 5. Analyse der biologischen Aktivität <ol><li> Seed 1 x 10 5 Maus C2C12 Myoblasten pro Well in 6-Well-plAtes in Wachstumsmedium, bestehend aus DMEM high glucose Pyruvat mit 10% FBS, 1% Penicillin / Streptomycin und inkubieren für 24 h bei 37 ° C / 5% CO 2 enthält, ergänzt. </li><li> Hungern Zellen in Serum-freien DMEM für 3-5 Stunden vor auf die mit immobilisiertem BMP-2 verziert Oberflächen Aussaat. </li><li> Für die Untersuchung der kurzfristigen Signal Induktion, ersetzen Sie das Medium durch 200 ul frisches Serum-freien DMEM und legen Sie die immobilisierten BMP-2-Flächen über den Zellen. Die Oberfläche sollte mit feiner Spitze Pinzette gehandhabt werden, um Kratzer zu vermeiden. </li><li> Oberflächen sanft entfernen und saugen das Medium mit einer Pipette, waschen Sie die Zellen zweimal mit PBS. Gehen Sie zur Analyse von Zell-Antworten. </li><li> Für die Analyse der Langzeit biologische Aktivität, Platte Zellen auf Oberflächen-und Kultur sie bei 37 ° C / 5% CO 2 in niedrigen Serumbedingungen (2% FBS) für sechs Tage. </li></ol>

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In diesem Protokoll beschreiben wir die Herstellung von Oberflächen mit bioaktiven rhBMP-2 funktionalisiert ist. Dieser Ansatz weist zwei Schritte auf: 1) die anfängliche Bildung einer selbstorganisierenden Monoschicht (SAM) eines bifunktionellen Linkers an der Goldoberfläche, 2) kovalente Immobilisierung von rhBMP-2-Proteins. In früheren Arbeiten, validiert man die effektive Bindung des bifunktionellen Linker und dem Wachstumsfaktor, und zeigten, dass auf der Oberfläche immobilisierten rhBMP-2 behält seine biolog...

Morphogenetisches Knochenprotein 2 (BMP-2) ist ein Mitglied der transformierenden Wachstumsfaktor (TGF-β)-Familie und als Induktor der de novo Knochenbildung sowie Regulator mehreren Geweben während der embryonalen Entwicklung und Homöostase Erwachsenen 1-3. Jedes Monomer des biologisch aktiven homodimeren BMP-2-Protein enthält eine &quot;Cystein-Knoten&quot;-Motiv, das in allen hoch BMPs 4 konserviert ist. Sechs der sieben Cysteinreste eine intramolekulare Disulfidbindungen bilden, die jedes Monomer zu stabilisieren, während der siebte Cystein an der Dimerisierung beteiligt sind, die einen intermolekularen Bindung zwischen den beiden Monomeren 5,6. Diese hochkonservierten Cystein-Knoten definiert, die die dreidimensionale Struktur des BMP-2-Protein bestimmt und seine einzigartigen Eigenschaften, wie Beständigkeit gegen Hitze, Denaturierungsmittel und sauren pH 7-9. BMP-2-Bindung an Serin / Threonin-Kinase-Transmembran-Rezeptoren, wodurch die Signaltransduktion zu induzieren <sup&gt; 12.10. Abhängig von der Art der Rezeptoroligomerisation werden unterschiedliche Signalwege aktiviert: ein Smad-unabhängige Signalkaskade führt zu der alkalischen Phosphatase-Induktions über p38-Signalisierung, während ein Smad-abhängigen Weg durch Rezeptor-Phosphorylierung führt Smad-Komplex nukleäre Translokation und Aktivierung der Transkription von Aktiv spezifischer Zielgene, wie z. B. der Inhibitor der Differenzierung (Id) 12-14. In Knochen, BMP-2 induziert die Differenzierung von mesenchymalen Stammzellen zu Osteoblasten, wodurch die Stimulierung der Heilung und de novo-Knochenbildung. Derzeit rekombinant exprimierten BMP-2 ist klinisch angewendet, um die Heilung von gebrochenen Seiten zu verbessern. Eine übliche Strategie im Knochengewebe ist der Einsatz von injizierbaren Wachstumsfaktoren, die zum lokalen Abgabesystemen weniger invasiv ist. In vivo-Studien und klinische Anwendungen haben gezeigt, dass die kurze biologische Halbwertszeit, unspezifische locsierung und schnelle lokale Clearance von BMP-2 kann sich auf mehrere lokale und systemische Probleme Eileiter 15 führen. Daher ist notwendig für die örtliche verzögerte Abgabe an der Zielstelle, um eine effektive Präsentation, das Einschließen oder Immobilisierung von BMP-2 in oder an Materialien zu erhalten. Anhaltende Abgabe kann mit nicht-kovalente Retentions Ansätze wie physikalischen Einschluß, Adsorption oder Ionenkomplex 16 erreicht werden. Es ist jedoch bekannt, dass unspezifische Adsorption von Proteinen an Oberflächen können die Ergebnisse in eine Denaturierung der Moleküle 17. Für die kovalente Bindung von Wachstumsfaktoren, wurden verschiedene Typen von Trägern in den letzten zehn Jahren entwickelt worden. Die Verwendung von bifunktionellen Verknüpfungsmoleküle, die Amino-oder Carboxylgruppen des Proteins beispiels Ziel ist eine Art von Ansatz, der nicht unbedingt erforderlich ist Proteinmodifikation seiner Immobilisierung zu erreichen. In der Tat bietet, während die Proteinmodifikation Vorteil der Steuerung Proteinorientierung,die Einführung der künstlichen Domänen-, Peptid-Tags und ortsspezifische Ketten verändern die biologische Aktivität von Wachstumsfaktoren 17. Somit, um die Denaturierung aufgrund der Wechselwirkung mit dem Trägermaterial zu umgehen, Oberflächen vorher funktionalisiert werden, beispielsweise mit einer selbstorganisierten Monoschicht (SAM) eines Verbindungsmoleküls, gefolgt von der Kopplung des gewünschten Faktor 18. Wir haben eine SAM-Ansatz, indem sie auf seiner freien Aminreste verwendet werden, um kovalent immobilisieren BMP-2 auf eine Oberfläche und haben gezeigt, dass das Protein immobilisiert sowohl die kurz-und langfristigen biologischen Aktivität 19 behält. Dieses Protokoll stellt eine einfache und effiziente Möglichkeit, BMP-2-Zellen für in vitro-Untersuchungen über die Mechanismen, die an der Zellmembran auf und regulieren intrazellulären Signalisierung für osteogene Signalisierung verantwortlich zu liefern.

<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

Morphogenetisches Knochenprotein 2 (BMP-2) ist ein Wachstumsfaktor in der extrazellulären Matrix von Knochengewebe eingebettet ist. BMP-2 fungiert als Auslöser von mesenchymalen Zelldifferenzierung zu Osteoblasten und stimulieren damit Heilung und de novo Knochenbildung. Der klinische Einsatz von rekombinanten humanen BMP-2 (rhBMP-2) in Verbindung mit den letzten Gerüsten hat Kontroversen angehoben, bezogen auf die Darstellungsweise und die Menge geliefert werden. Die hier vorgestellten Protokoll bietet eine einfache und effiziente Möglichkeit, BMP-2 in vitro-Untersuchungen an Zellen zu liefern. Wir beschreiben, wie eine selbstorganisierte Monoschicht, die aus einem heterobifunktionellen Linkers zu bilden, und zeigen die nachfolgende Bindungsschritt, um eine kovalente Immobilisierung von rhBMP-2 zu erhalten. Mit diesem Ansatz ist es möglich, eine anhaltende Präsentation von BMP-2 bei gleichzeitiger Beibehaltung der biologischen Aktivität des Proteins. In der Tat wird die Oberfläche Immobilisierung von BMP-2 können gezielte Untersuchungen durch Verhinderung unspezifischer AnzeigenDesorption, während die Verringerung der Menge an Wachstumsfaktor und, vor allem, behindern unkontrollierten Freisetzung von der Oberfläche. Beide kurz-und langfristigen Signalereignisse von BMP-2 ausgelöst stattfinden, wenn die Zellen auf Oberflächen präsentiert kovalent immobilisierten rhBMP-2 ausgesetzt, so dass dieser Ansatz geeignet für in-vitro-Studien an Zell-Antworten auf BMP-2 Stimulation.

In unserem Aufbau wurde Gold als Hilfsstoff gewählt, da es eine biologisch unspezifische aber chemisch abstimmbaren System. Darüber hinaus ist die Anwendung von selbstorganisierenden Monoschichten bringt viele Vorteile: SAMs adsorbieren spontan über ihre &quot;head-Gruppen&quot; auf Metalle und Form Monoschichten mit wenigen Fehlern, während ihre funktionellen Endgruppen kann weiter modifiziert werden. Somit stellen sie eine Plattform, um die Eigenschaften der Oberfläche in einer kontrollierten Art und Weise aber s...

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

Wir beschreiben ein Verfahren zum Erreichen effizienten Immobilisierung von BMP-2 auf die Oberflächen. Unser Ansatz beruht auf der Bildung einer selbstorganisierten Monoschicht, um die kovalente Bindung des BMP-2 mit seinem freien Aminresten zu erreichen basiert. Dieses Verfahren ist ein nützliches Werkzeug, um Signalisierung auf der Zellmembran zu studieren.

Die kovalente Bindung von BMP-2 auf Oberflächen mit einem selbstorganisierten Monoschicht-Ansatz

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

Research

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JoVE Journal

JoVE Core

Organic Chemistry

Chemistry

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

13.8K Aufrufe.  University of Heidelberg.  Wir beschreiben ein Verfahren zum Erreichen effizienten Immobilisierung von BMP-2 auf die Oberflächen. Unser Ansatz beruht auf der Bildung einer selbstorganisierten Monoschicht, um die kovalente Bindung des BMP-2 mit seinem freien Aminresten zu erreichen basiert. Dieses Verfahren ist ein nützliches Werkzeug, um Signalisierung auf der Zellmembran zu studieren. 

Serie: 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.

Stabilisierung Leberzell Phänotyp mit optimierten Surfaces

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

Forschung

Chemie

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.

Eine inverse Analyse Ansatz zur Charakterisierung von chemischen Transport in Farben

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.

Facile Herstellung von Intern Selbstorganisierte Lipidpartikel stabilisiert durch Kohlenstoff-Nanoröhren

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.

Hochstabile, funktionelle Hairy Nanopartikel und Biopolymere aus Holzfasern für eine nachhaltige Nanotechnologie

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.

Mikrofluidische Synthese von kovalenten organischen Frameworks (COFs): Ein Werkzeug zur kontinuierlichen Produktion von COF-Fasern und Direktdruck auf einer Oberfläche

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.

Lichtgetriebenen molekularen Motoren auf Oberflächen für einzelne molekulare Bildgebung

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 komplexer Moleküle und ihrer Reaktionen auf Oberflächen mittels Cluster-induzierter Desorption/Ionisationsmassspektrometrie

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.

Kovalente Befestigung einzelner Moleküle für die AFM-basierte Kraftspektroskopie

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.

Synthese von pH-abhängigem Pyrazol, Imidazol und Isoindolon Dipyrrinon Fluorophoren mit einem Claisen-Schmidt-Kondensationsansatz

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 von Organochlorpestiziden in einer Bodenprobe mit einem modifizierten QuEChERS-Ansatz unter Verwendung von Ammoniumformiat

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