Este trabajo fue apoyado por el Marie Curie Internacional Reintegración Grant y por la Fundación Alemana-Israel. Reconocemos el Sr. Yair Razvag para el análisis de AFM.

1. El autoensamblaje de homoaromáticos Dipéptidos <ol><li> Pesar el péptido deseado en su forma liofilizada (por ejemplo, NH2-Phe-Phe-OH, Boc-Phe-Phe-COOH) y preparar una solución madre disolviendo el péptido en 1,1,1,3,3,3-hexafluoro -2-propanol (HFP) a la concentración apropiada (por ejemplo, 100 mg / ml de NH 2-Phe-Phe-OH y Boc-Phe-Phe-COOH) 7,17,46. </li><li> Mezclar la solución con vórtice y el lugar en el banco hasta que el péptido se disuelva completamente y la solución parece clara (unos pocos minutos). </li><li> Diluir la solución de péptido de stock, con un disolvente adecuado, a la concentración apropiada (por ejemplo, 2 mg / ml de NH 2-Phe-Phe-OH en agua destilada Triple (PST) para la formación de nanotubos; mediante la adición de 2 l de péptido solución madre a 98 l TDW, 5 mg / ml de Boc-Phe-Phe-COOH en etanol para la formación de estructuras esféricas). </li><li> Mantenga la solución a temperatura ambiente durante 24hora </li><li> Con el fin de evitar cualquier preaggregation, preparar soluciones madre frescos para cada experimento. </li></ol> 2. Coassembly de Dos homoaromáticos Dipéptidos <ol><li> Preparar una solución de etanol al 50% mediante la mezcla de volúmenes iguales de TDW y etanol absoluto. Utilice vórtice para mezclar las dos soluciones. </li><li> Pesar 2 mg del péptido NH 2-Phe-Phe-OH y 1 mg del péptido Boc-Phe-Phe-OH. Disolver cada péptido en HFP a una concentración de 100 mg / ml. </li><li> Mezclar los péptidos soluciones madre utilizando vórtice y colocarlos en el banco hasta que los péptidos se disolvieron por completo y las soluciones parecen claras. </li><li> Mezcla los péptidos soluciones madre a la relación deseada. En este experimento específico mezclar 10 l de péptido NH 2-Phe-Phe-OH con 6 l de péptido Boc-Phe-Phe-OH (hasta una relación final de 05:03, respectivamente). Debido a la alta volatilidad del disolvente HFP, se recomienda preparar una gran cantidad de esta solución madre (por lo leao 10 l). </li><li> Utilice vortex para mezclar la solución de péptidos mezclados. </li><li> Diluir la solución madre de péptidos mezclados con etanol al 50% a la concentración final deseada. En este experimento específico, con el fin de obtener una concentración final de 5 mg / ml de NH 2-Phe-Phe-OH y 3 mg / ml para Boc-Phe-Phe-OH, respectivamente, añadir 8 l de la solución madre de péptidos mezclados a 92 l de la solución de etanol al 50%. Utilice una pipeta para mezclar suavemente la solución. </li><li> Mantenga la solución a temperatura ambiente durante 24 horas. </li><li> Cabe señalar que debido a la naturaleza altamente volátil del disolvente, los experimentos son sensibles a pequeños cambios en la concentración de los péptidos. Por lo tanto, nuevas soluciones madre deben prepararse para cada experimento. </li></ol> 3. Caracterización de los auto-ensambladas Estructuras Usando microscopía electrónica de barrido (SEM) <ol><li> Después de 24 horas de incubación, se aplica una gota de 10 l de la solución de los péptidos en una cala de vidrior resbalón y seco a temperatura ambiente. </li><li> Escudo de la muestra en el vaso con una fina capa de oro (de unos pocos nanómetros) usando un revestidor por bombardeo iónico durante 90 segundos. </li><li> Imagen de los ensamblados mediante SEM funciona a 10-20 kV. </li></ol> 4. Caracterización de los auto-ensambladas Estructuras Utilizando Microscopía Electrónica de Transmisión (TEM) <ol><li> Coloque una gota de 10 l de la solución de los péptidos sobre una rejilla de cobre de malla 200 cubierto de carbono y estabilizado por un soporte de película de polímero. </li><li> Después de 1 min retirar el exceso de líquido con papel de filtro. </li><li> Preparar una solución de 2% de acetato de uranilo en TDW. Se filtra la solución con unidad de filtro de 0,22 micras. </li><li> Para la tinción de la muestra (tinción negativa), coloque una gota de 10 l de solución de acetato de uranilo en la parrilla. </li><li> Después de 30 segundos quitar el exceso de líquido con papel de filtro. Cabe señalar que aunque la tinción negativa mejora el contraste de las imágenes, no es esencial en todos los casos. </li><li> Imagen de la muestra en el the rejilla por TEM que opera a 120 kV. </li></ol> 5. Caracterización tridimensional de las Asambleas por Microscopía de Fuerza Atómica (AFM) <ol><li> Preparar una muestra para el análisis AFM usando el procedimiento descrito en el apartado 3.1. </li><li> Analizar la muestra sobre el vidrio utilizando un instrumento AFM se trabaja en modo de CA. Utilice palancas de silicio con una constante de resorte de 3 N / m y una frecuencia de resonancia de 75 kHz. </li><li> Iniciar mediante el escaneo de un área grande de la red, con el fin de encontrar la estructura deseada. Luego concéntrese en un área más pequeña específico y escanearlo (el tamaño de escaneo fue de 2,5 m x 2,5 m para la imagen incluida en este manuscrito). </li></ol> 6. Caracterización de la estructura secundaria por FT-IR <ol><li> Aplique una gota de 30 l de la solución de los péptidos a una ventana de CaF 2. </li><li> Deje que la solución se seque a temperatura ambiente. </li><li> La adsorción de agua en el espectro IR es a 1650 cm -1.Este pico está en el centro de la banda de amida I del enlace peptídico. También es un pico típico para estructuras α-helicoidal de péptidos y proteínas 47. Con el fin de superar este problema y evitar la señal de agua, se debe realizar un intercambio de hidrógeno a deuterio. Coloque una gota de óxido de deuterio (D2O) en la muestra de péptido se secó. La caída debe ser lo suficientemente grande como para cubrir completamente el depósito de péptido en la ventana. </li><li> Permita que la muestra se seque al vacío. </li><li> Repita los pasos 6.3 y 6.4 2x para asegurar el intercambio máxima-hidrógeno-deuterio. Guarde la muestra bajo vacío hasta su análisis. </li><li> Registre los espectros FT-IR usando una triglicina sulfato deuterado detector (DTGS). El sistema FT-IR incluye un generador de gas de purga, con el fin de evitar que la humedad en los alrededores de la muestra. Para las muestras de péptidos cortos, lo mejor es escanear la muestra 2000 x con una resolución de 4 cm -1. Los valores mínimos de transmitancia se pueden determinar por el modoftware suministrado con el instrumento. </li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

En resumen, este trabajo demuestra la facilidad con la que conjuntos basados ​​en péptidos se pueden formar in vitro. El proceso consiste en péptidos y disolventes disponibles comercialmente, y se produce espontáneamente en condiciones ambientales, después de la adición de un disolvente polar para el tubo de ensayo. Es crucial usar HFP como disolvente de los péptidos, debido a la baja solubilidad de los péptidos en otros disolventes orgánicos. Además, debido a la alta volatilidad de HFP es necesari...

Formas Naturaleza estructuras ordenadas y funcionales en el proceso de auto-ensamblaje biomolecular. La comprensión de las fuerzas que rigen este proceso espontáneo puede conducir a la capacidad de imitar auto-ensamblaje in vitro y en consecuencia a los grandes avances en el área de ciencias de los materiales 10,11. Los péptidos, específicamente, una gran promesa como un bloque de construcción biomolecular, ya que presentan gran diversidad estructural, facilidad de síntesis química, y pueden ser fácilmente funcionalizados con entidades biológicas y químicas. El campo de los péptidos auto-ensamblaje fue iniciado por Ghadiri y sus colegas, quienes demostraron el auto-ensamblaje de nanotubos peptídicos mediante péptidos cíclicos con ácidos y D-12 L-aminoácidos alterna. Otros enfoques exitosos para el diseño de ensamblajes de péptidos incluyen péptidos lineales bolaamphiphile 5, amphiphiles (AP) 6, péptidos iónicos autocomplementarias no conjugados 13, péptidos similares a tensioactivos <sup&gt; 4,14, y dibloque copolypeptides 15. Un enfoque más reciente es el de la auto-ensamblaje de péptidos cortos aromáticos, denominado dipéptidos homoaromáticos. Estos péptidos comprenden sólo dos aminoácidos con la naturaleza aromático (por ejemplo, Phe-Phe, dicarbonato de terc-butilo (Boc)-Phe-Phe) 7,8,16-21. Las estructuras formadas por estos péptidos homoaromáticos incluyen estructuras tubulares, esferas, conjuntos laminares y fibras 6,8,15,21-32. Las fibras en algunos casos generar una malla de fibrillas que se obtiene un hidrogel 33-37. Estos conjuntos han sido explotados para aplicaciones de biosensores, la administración de fármacos, la electrónica molecular, etc. 38-45 Este documento describe los pasos experimentales necesarios para poner en marcha el auto-ensamblaje espontáneo de péptidos homoaromáticos. Además, se presenta el proceso de péptido coassembly. Este proceso implica el auto-ensamblaje de más de un tipo de péptidomonómero. Nuestra demostración incluye la coassembly de dos péptidos disponibles comercialmente: el péptido difenilalanina (NH 2-Phe-Phe-COOH) y su análogo protegido con Boc (Boc-Phe-Phe-OH). Cada uno de los péptidos auto-ensambla en una estructura supramolecular: los formularios de péptidos difenilalanina conjuntos tubulares y el péptido auto-ensambla Boc-Phe-Phe-OH en cualquiera de esferas o fibras dependiendo del disolvente 7,17,46. Mezclamos los dos péptidos en ciertas proporciones y caracterizaron los conjuntos resultantes mediante microscopía electrónica, microscopía de fuerza y ​​la espectroscopia FT-IR. Los métodos demostraron la formación de una estructura basada en péptidos que se compone de elementos esféricos con un diámetro de varias micras (1-4 micras) que están conectados por conjuntos alargados con un diámetro de unos pocos cientos de nanómetros (~ 300-800 nm) . Los conjuntos de cadenas de cuentas parecen en su morfología, como las estructuras esféricas parecen ser roscado en laconjuntos alargados. Por ello denominamos estas asambleas &quot;collares biomoleculares&quot;. Los &quot;collares biomoleculares&quot; pueden servir como un nuevo biomaterial, como un agente de administración de fármacos o como un andamio para aplicaciones electrónicas. Por otra parte, el procedimiento que conduce a la auto-ensamblaje de péptidos se puede utilizar con otras clases de péptidos y biomoléculas. Todo ello puede conducir a una mejor comprensión de las fuerzas que intervienen en el autoensamblaje y la formación de nuevas estructuras ordenadas.

<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>NH2-Phe-Phe-OH</td>
			<td>Bachem</td>
			<td>G-2925.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boc-Phe-Phe-OH</td>
			<td>Bachem</td>
			<td>A-3205.0005</td>
			<td></td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td>Sigma-Aldrich</td>
			<td>52512-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute (Dehydrated) AR sterile</td>
			<td>Bio-Lab Ltd.</td>
			<td>52555</td>
			<td>Blending with TDW for the preparation of 50% solution</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>73943</td>
			<td>For negative staining. It is possible to work without it.</td>
		</tr>
		<tr>
			<td>glass cover slip</td>
			<td>Marienfeld Laboratory Glassware</td>
			<td>110590</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Electron Microscopy Sciences</td>
			<td>FCF200-Cu-50</td>
			<td>Formvar/Carbon 200 Mesh, Cu</td>
		</tr>
		<tr>
			<td>Quantitive filter paper</td>
			<td>Whatman</td>
			<td>1001055</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium Oxide (D2O)</td>
			<td>Sigma-Aldrich</td>
			<td>151882-100G</td>
			<td>99.9 atom % D</td>
		</tr>
		<tr>
			<td>CaF2 window</td>
			<td>PIKE Technologies</td>
			<td>160-1212</td>
			<td>25 mm x 2 mm window. For FT-IR measurments</td>
		</tr>
		<tr>
			<td>AFM tips</td>
			<td>NanoScience Instruments</td>
			<td>CFMR</td>
			<td>Aspire probes, CFMR-25 series</td>
		</tr>
		<tr>
			<td>Filter units</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td>Millex-GV, 0.22 &#956;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>SEM</td>
			<td>FEI</td>
			<td></td>
			<td>Quanta 200 ESEM</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>FEI</td>
			<td></td>
			<td>Tecnai T12 G2 Spirit</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>JPK Instruments</td>
			<td></td>
			<td>A JPK NanoWizard3</td>
		</tr>
		<tr>
			<td>FT-IR</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nicolet 6700 advanced gold spectrometer</td>
		</tr>
		<tr>
			<td>FT-IR Purge</td>
			<td>Parker</td>
			<td></td>
			<td>BALSTON FT-IR Purge Gas Generator model 75-52</td>
		</tr>
		<tr>
			<td>OMNIC (Nicolet) software</td>
			<td>Thermo Nicolet Corporation</td>
			<td></td>
			<td>For FT-IR spectra analysis</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Wisd Laboratory Equipment</td>
			<td></td>
			<td>ViseMix VM</td>
		</tr>
		<tr>
			<td>Weight</td>
			<td>Mettler Toledo</td>
			<td></td>
			<td>NewClassic MS</td>
		</tr>
		<tr>
			<td>Sputter coater</td>
			<td>Polaron</td>
			<td></td>
			<td>SC7640 Sputter Coater</td>
		</tr>
	</tbody>
</table>

formation of ordered biomolecular structures by the self-assembly of short peptides

En la naturaleza, las estructuras funcionales complejos se forman por el auto-ensamblaje de biomoléculas en condiciones suaves. La comprensión de las fuerzas que controlan auto-ensamblaje e imitando este proceso in vitro traerá consigo importantes avances en las áreas de ciencias de los materiales y la nanotecnología. Entre los bloques de construcción biológicos disponibles, los péptidos tienen varias ventajas ya que presentan una diversidad sustancial, su síntesis en gran escala es sencillo, y que pueden ser fácilmente modificados con entidades biológicas y químicas 1,2. Varias clases de péptidos diseñados tales como péptidos cíclicos, péptidos de anfífilo y-péptido conjugados de auto-ensamblarse en estructuras ordenadas en solución. Dipéptidos homoaromáticos, son una clase de péptidos auto-ensambladas cortos que contienen toda la información molecular necesaria para formar estructuras ordenadas tales como nanotubos, esferas y fibrillas de 3-8. Una gran variedad de estos péptidos está disponible comercialmente. <pclass = &quot;jove_content&quot;&gt; Este artículo presenta un procedimiento que conduce a la formación de estructuras ordenadas por el auto-ensamblaje de péptidos homoaromáticos. El protocolo requiere sólo reactivos comerciales y equipamiento básico de laboratorio. Además, el artículo describe algunos de los métodos disponibles para la caracterización de conjuntos basados ​​en péptidos. Estos métodos incluyen de electrones y microscopía de fuerza atómica y de Fourier-Transform espectroscopia infrarroja (FT-IR). Por otra parte, el manuscrito demuestra la mezcla de péptidos (coassembly) y la formación de un &quot;cuentas de un collar&quot; estructura similar por este proceso. 9 Los protocolos presentados aquí pueden adaptarse a otras clases de péptidos o bloques de construcción biológica y pueden potencialmente conducir al descubrimiento de nuevas estructuras a base de péptidos y para un mejor control de su montaje.

En este trabajo se describe un método para la formación de estructuras ordenadas en la nano-y micrométrico escala por el auto-ensamblaje de péptidos. Con el fin de demostrar este sencillo proceso presentamos y caracterizar la coassembly de dos péptidos aromáticos simples (Figura 1). Uno de los péptidos es el 2-Phe-Phe-OH (difenilalanina) péptido NH, que puede auto-ensamblan en una solución acuosa en estructuras tubulares huecos con dimensiones nanométricas 7. El otro pép...

Watch this Scientific Journal Video about Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides at JoVE.com

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

En este trabajo se describe la formación de estructuras a base de péptidos altamente ordenadas por el proceso espontáneo de auto-ensamblaje. El método utiliza péptidos disponibles comercialmente y equipo de laboratorio común. Esta técnica se puede aplicar a una gran variedad de péptidos y puede conducir al descubrimiento de nuevos conjuntos basados ​​en péptidos.

Formación de estructuras biomoleculares ordenada por el auto-ensamblaje de péptidos cortos

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

formation-ordered-biomolecular-structures-self-assembly-short

Research

Education

JoVE Journal

JoVE Core

Chemistry

Thermodynamics

SCIENTISTS IN ACTION

12.8K vistas.  The Hebrew University of Jerusalem.  En este trabajo se describe la formación de estructuras a base de péptidos altamente ordenadas por el proceso espontáneo de auto-ensamblaje. El método utiliza péptidos disponibles comercialmente y equipo de laboratorio común. Esta técnica se puede aplicar a una gran variedad de péptidos y puede conducir al descubrimiento de nuevos conjuntos basados ​​en péptidos. 

Video: Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

Origami Inspired autoensamblaje de partículas modeladas y Reconfigurable

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Investigación

Química

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Rapid ensayos colorimétricos para distinguir cualitativamente ARN y ADN en muestras Biomolecular

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

Funcionalización asistida por microondas de poli (etilenglicol) y los péptidos sobre resina para uso en polimerizaciones de cadena y Formación de hidrogel

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Autoensamblaje de formas bidimensionales complejas de una sola hebra de ADN Azulejos

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

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 estable, funcional Hairy nanopartículas y biopolímeros con fibras de madera: Hacia Nanotecnología Sostenible

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

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

Miradas en torno a las interacciones de los aminoácidos y péptidos con materiales inorgánicos Uso de una sola molécula de la Fuerza Espectroscopia

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

Injerto de nanotubos de carbono pirólisis con poliestireno para permitir uno mismo-Asamblea y pliegues anisotrópico

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Microscopia electrónica de transmisión de líquido de la célula para el rastreo de autoensamblaje de las nanopartículas

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Fabricación de nanocristales micelar esférica y en forma de gusano combinando Electrospray, uno mismo-Asamblea y Control de la estructura de base solvente

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

Luminophore formación en diversas conformaciones de albúmina sérica bovina mediante la Unión de Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...