Questo lavoro è stato supportato dal internazionali Marie Curie per il reinserimento Grant e dalla Fondazione tedesca-Israele. Riconosciamo Mr. Yair Razvag per l&#39;analisi AFM.

1. Auto-assemblaggio di Homoaromatic dipeptidi <ol><li> Pesare il peptide desiderato nella sua forma liofilizzata (es. NH 2-Phe-Phe-OH, Boc-Phe-Phe-COOH) e preparare una soluzione madre sciogliendo il peptide in 1,1,1,3,3,3-esafluoro -2-propanolo (HFP) alla concentrazione appropriata (ad esempio 100 mg / ml di NH 2-Phe-Phe-OH e Boc-Phe-Phe-COOH) 7,17,46. </li><li> Mescolare la soluzione con vortex e posto in panchina fino a che il peptide è completamente sciolto e la soluzione sembra chiaro (pochi minuti). </li><li> Diluire la soluzione peptide magazzino, con un solvente adatto, per la concentrazione appropriata (ad esempio 2 mg / ml di NH 2-Phe-Phe-OH in acqua distillata tripla (TDW) per la formazione di nanotubi; aggiungendo 2 ml di peptide soluzione stock da 98 microlitri TDW, 5 mg / ml di Boc-Phe-Phe-COOH in etanolo per la formazione di strutture sferiche). </li><li> Mantenere la soluzione a temperatura ambiente per 24hr. </li><li> Per evitare qualsiasi preaggregation, preparare le soluzioni madre fresche per ogni esperimento. </li></ol> 2. Coassembly of Two Homoaromatic dipeptidi <ol><li> Preparare una soluzione di etanolo al 50% mescolando volumi uguali di TDW e etanolo assoluto. Utilizzare agitare per miscelare le due soluzioni. </li><li> Pesare 2 mg di peptide NH 2-Phe-Phe-OH e 1 mg del peptide Boc-Phe-Phe-OH. Sciogliere ciascun peptide in HFP ad una concentrazione di 100 mg / ml. </li><li> Mescolare i peptidi soluzioni madre con vortex e metterli in panchina fino a quando i peptidi sono completamente sciolti e le soluzioni sembrano chiare. </li><li> Frullare i peptidi soluzioni madre per il rapporto desiderato. In questo esperimento si fondono 10 ml di peptide NH 2-Phe-Phe-OH con 6 ml di peptide Boc-Phe-Phe-OH (in un rapporto finale di 05:03 rispettivamente). A causa della elevata volatilità del solvente HFP, si raccomanda di preparare una grande quantità di questa soluzione madre (almst 10 microlitri). </li><li> Usa vortex per miscelare la soluzione stock peptidi misti. </li><li> Diluire la soluzione stock peptidi miscelato con il 50% di etanolo alla concentrazione finale desiderata. In questo esperimento, in modo da ottenere una concentrazione finale di 5 mg / ml di NH 2-Phe-Phe-OH e 3 mg / ml di Boc-Phe-Phe-OH, rispettivamente, aggiungere 8 microlitri della soluzione madre peptidi miscelati a 92 microlitri della soluzione di etanolo al 50%. Utilizzare una pipetta per mescolare delicatamente la soluzione. </li><li> Conservare la soluzione a temperatura ambiente per 24 ore. </li><li> Va notato che a causa della natura altamente volatile del solvente, gli esperimenti sono sensibili a piccoli cambiamenti nella concentrazione dei peptidi. Pertanto, le soluzioni di archivi freschi dovrebbero essere preparati per ogni esperimento. </li></ol> 3. Caratterizzazione del Sé assemblato strutture con microscopia elettronica a scansione (SEM) <ol><li> Dopo 24 ore di incubazione, applicare una goccia 10 ml della soluzione di peptidi su una baia di vetroslittamento r e asciugare a temperatura ambiente. </li><li> Coat campione sul vetro con un sottile strato di oro (alcuni nanometri) utilizzando un dispositivo di rivestimento a polverizzazione per 90 sec. </li><li> Immagine assemblee utilizzando SEM operante a 10-20 kV. </li></ol> 4. Caratterizzazione del Sé assemblato strutture con microscopia elettronica a trasmissione (TEM) <ol><li> Una goccia 10 ml della soluzione di peptidi su una griglia di rame 200 mesh rivestito con carbonio e stabilizzato da un supporto film polimerico. </li><li> Dopo 1 min rimuovere il liquido in eccesso con carta da filtro. </li><li> Preparare una soluzione di 2% acetato di uranile in TDW. Filtrare la soluzione con unità filtro 0,22 micron. </li><li> Per macchiare il campione (colorazione negativa), una goccia di soluzione di acetato di uranile 10 microlitri sulla griglia. </li><li> Dopo 30 sec rimuovere il liquido in eccesso con carta da filtro. Va notato che sebbene colorazione negativa migliora il contrasto delle immagini, non è essenziale in tutti i casi. </li><li> Immagine del campione su °e griglia TEM operando a 120 kV. </li></ol> 5. Caratterizzazione tridimensionale delle Assemblee dalla microscopia a forza atomica (AFM) <ol><li> Preparare un campione per l&#39;analisi AFM utilizzando la procedura descritta nel paragrafo 3.1. </li><li> Analizzare il campione sul vetro con uno strumento AFM lavora in modalità AC. Utilizzare cantilever silicio con costante elastica 3 N / m ed una frequenza di risonanza di 75 kHz. </li><li> Inizia con un&#39;analisi un&#39;ampia area di rete, al fine di trovare la struttura desiderata. Poi concentrarsi su una specifica area più piccola e la scansione (il formato di scansione è stata del 2,5 micron x 2.5 micron per l&#39;immagine incluso in questo manoscritto). </li></ol> 6. Caratterizzazione della struttura secondaria da FT-IR <ol><li> Applicare una goccia 30 ml della soluzione di peptidi ad una finestra CaF 2. </li><li> Lasciare che la soluzione per asciugare a temperatura ambiente. </li><li> L&#39;adsorbimento di acqua nello spettro IR è di 1650 cm-1.Questo picco è al centro della banda ammide I del legame peptidico. E &#39;anche un picco tipico per strutture α-elica di peptidi e proteine ​​47. Per superare questo problema ed evitare il segnale di acqua, uno scambio idrogeno-a-deuterio deve essere eseguita. Mettere una goccia di ossido di deuterio (D 2 O) sul campione peptide essiccato. Il calo dovrebbe essere sufficiente a coprire completamente i deposito peptide sulla finestra. </li><li> Lasciare il campione a secco sotto vuoto. </li><li> Ripetere i punti 6.3 e 6.4 2x per garantire lo scambio massimo idrogeno-to-deuterio. Salvare il campione sotto vuoto fino alla sua analisi. </li><li> Registrare gli spettri FT-IR utilizzando un triglicina solfato di deuterio (DTGS) rivelatore. Il sistema FT-IR comprende un generatore di gas di lavaggio, al fine di impedire all&#39;umidità nelle frazioni del campione. Per i campioni di piccoli peptidi, è preferibile eseguire la scansione del campione 2.000 x con una risoluzione di 4 cm -1. I valori minimi di trasmittanza possono essere determinati dal modoftware fornito con lo strumento. </li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

In sintesi, questo documento dimostra la facilità con cui le assemblee a base di peptidi possono essere formati in vitro. Il processo prevede peptidi disponibili in commercio e solventi, e si verifica spontaneamente in condizioni ambientali, per aggiunta di un solvente polare nella provetta. È fondamentale utilizzare HFP come solvente dei peptidi, a causa della bassa solubilità dei peptidi in altri solventi organici. Inoltre, a causa della elevata volatilità di HFP è necessario preparare soluzione madre fr...

Natura forma strutture ordinate e funzionale dal processo di auto-assemblaggio biomolecolare. Comprendere le forze che governano questo processo spontaneo può condurre alla capacità di imitare auto-assemblaggio in vitro e di conseguenza a maggiori progressi nel campo della scienza dei materiali 10,11. Peptidi, in particolare, molto promettenti come un blocco di costruzione biomolecolare, dal momento che presentano grandi diversità strutturale, facilità di sintesi chimica, e può essere facilmente funzionalizzati con entità biologiche e chimiche. Il campo di peptide auto-assemblaggio è stato lanciato da Ghadiri e dei suoi colleghi, che hanno dimostrato l&#39;auto-assemblaggio di nanotubi peptidici da peptidi ciclici con alternanza di D-e L-aminoacidi 12. Altri approcci di successo per la progettazione di assiemi peptidici includono peptidi lineari bolaamphiphile 5, amphiphiles (AP) 6, non coniugati auto-complementari peptidi ionici 13, peptidi tensioattivi simile <sup&gt; 4,14, e diblock copolypeptides 15. Un approccio più recente prevede l&#39;auto-assemblaggio di piccoli peptidi aromatici, definito dipeptidi homoaromatic. Questi peptidi comprendono solo due amminoacidi con la natura aromatica (ad es Phe-Phe, terz-butil dicarbonato (Boc)-Phe-Phe) 7,8,16-21. Le strutture formate da questi peptidi homoaromatic includono strutture tubolari, sfere, assemblee lastriformi e fibre 6,8,15,21-32. Le fibre in alcuni casi generano una maglia fibrille che produce un idrogel 33-37. Queste assemblee sono state sfruttate per applicazioni di biosensori, somministrazione di farmaci, elettronica molecolare, ecc. 38-45 Questo documento descrive i passi sperimentali necessarie per avviare la spontanea auto-assemblaggio di peptidi homoaromatic. Inoltre, esso presenta il processo di peptide coassembly. Questo processo implica l&#39;auto-assemblaggio di più di un tipo di peptidemonomero. La nostra dimostrazione include la coassembly di due peptidi disponibili in commercio: il peptide diphenylalanine (NH 2-Phe-Phe-COOH) e il suo Boc protetto analogico (Boc-Phe-Phe-OH). Ciascuno dei peptidi auto-assembla in una struttura supermolecolare: i formulari diphenylalanine peptide assemblee tubolari e le Boc-Phe-Phe-OH peptide auto-assembla in entrambi sfere o fibre a seconda del solvente 7,17,46. Abbiamo mescolato i due peptidi in determinati rapporti e caratterizzato le assemblee portato mediante microscopia elettronica, la forza microscopia e la spettroscopia FT-IR. I metodi hanno dimostrato la formazione di una struttura basata peptide che comprende elementi sferiche con un diametro di alcuni micron (1-4 micron) che sono collegati da assembly allungati con un diametro di poche centinaia di nanometri (~ 300-800 nm) . Le assemblee assomigliano stringhe di perline nella loro morfologia, le strutture sferiche sembrano essere filettato sulassiemi allungati. Abbiamo quindi chiamato queste assemblee &quot;collane biomolecolari&quot;. Le &quot;collane biomolecolari&quot; potrebbero servire come un nuovo biomateriale, come agente di rilascio di farmaci o come impalcatura per applicazioni elettroniche. Inoltre, la procedura che porta alla auto-assemblaggio di peptidi può essere utilizzato con altre classi di peptidi e biomolecole. Essa può portare ad una migliore comprensione delle forze coinvolte in auto-assemblaggio e la formazione di nuove strutture ordinate.

<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

In natura, strutture funzionali complesse sono formate dalla auto-assemblaggio di biomolecole in condizioni blande. Comprendere le forze che controllano l&#39;auto-assemblaggio e imita questo processo in vitro porterà importanti progressi nei settori della scienza dei materiali e delle nanotecnologie. Tra i mattoni biologico disponibili, peptidi hanno diversi vantaggi in quanto presentano sostanziali diversità, la loro sintesi in grande scala è semplice, e possono essere facilmente modificati con entità biologiche e chimiche 1,2. Diverse classi di peptidi progettati come peptidi ciclici, peptidi amphiphile e peptide-coniugati auto-assemblano in strutture ordinate in soluzione. Dipeptidi Homoaromatic, sono una classe di piccoli peptidi auto-assemblati che contengono tutte le informazioni molecolari necessarie per formare strutture ordinate come i nanotubi, sfere e fibrille 3-8. Una grande varietà di questi peptidi è disponibile in commercio. <pclass = &quot;jove_content&quot;&gt; Questo articolo presenta una procedura che porta alla formazione di strutture ordinate dalla auto-assemblaggio di peptidi homoaromatic. Il protocollo richiede soltanto reagenti commerciali e attrezzature di laboratorio di base. Inoltre, il documento descrive alcuni dei metodi disponibili per la caratterizzazione di assiemi a base di peptidi. Questi metodi comprendono elettrone e microscopia a forza atomica e Fourier-Transform Infrared Spectroscopy (FT-IR). Inoltre, il manoscritto dimostra la miscela di peptidi (coassembly) e la formazione di un &quot;perle su un filo&quot; struttura simile da questo processo. 9 I protocolli qui presentati possono essere adattati ad altre classi di peptidi o blocchi biologica e possono potenzialmente portare alla scoperta di nuove strutture a base di peptidi e per un migliore controllo del loro assemblaggio.

Questo documento descrive un metodo per la formazione di strutture ordinate su nano-e micrometro scala dalla auto-assemblaggio di peptidi. Per dimostrare questo semplice processo presentiamo e caratterizzare il coassembly di due semplici peptidi aromatici (Figura 1). Uno dei peptidi è il NH 2-Phe-Phe-OH (diphenylalanine) peptide, che può auto-assemblano in una soluzione acquosa in strutture tubolari cave di dimensioni nanometriche 7. L&#39;altro peptide è suo analogo protetto Bo...

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

Questo documento descrive la formazione di strutture a base di peptidi altamente ordinati dal processo spontaneo di auto-assemblaggio. Il metodo utilizza peptidi disponibili sul mercato e le apparecchiature di laboratorio comune. Questa tecnica può essere applicata a una grande varietà di peptidi e può portare alla scoperta di nuovi assemblaggi a base di peptidi.

Formazione di strutture biomolecolari ordinate dalla Self-assemblaggio di piccoli peptidi

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

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12.7K Views.  The Hebrew University of Jerusalem.  Questo documento descrive la formazione di strutture a base di peptidi altamente ordinati dal processo spontaneo di auto-assemblaggio. Il metodo utilizza peptidi disponibili sul mercato e le apparecchiature di laboratorio comune. Questa tecnica può essere applicata a una grande varietà di peptidi e può portare alla scoperta di nuovi assemblaggi a base di peptidi. 

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 Ispirato auto-assemblaggio di particelle Fantasia e riconfigurabile

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

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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.

Forno a microonde assistita Funzionalizzazione di poli (etilene glicole) e On-resina Peptidi per l&#39;uso a polimerizzazione a catena ed idrogel Formazione

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.

Auto-assemblaggio di forme complesse bidimensionali da Campagna DNA a singolo filamento

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

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.

Intuizioni le interazioni di aminoacidi e peptidi con materiali inorganici Uso singola molecola Forza Spettroscopia

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.

L'innesto Multiwalled Carbon Nanotubes con polistirolo per abilitare l'auto-assemblaggio e discromie anisotropo

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 elettronica in trasmissione di liquidi-cellula per l'inseguimento auto-assemblaggio delle nanoparticelle

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.

Fabbricazione di nanocristalli micellare sferica e a forma di verme combinando Electrospray, auto-assemblaggio e controllo di struttura a base di solvente

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

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

Sintesi e caratterizzazione di 1,2-Dithiolane per volta peptidi autoassemblanti

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

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 formazione in varie conformazioni di albumina di siero bovino di legame dell'oro

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 ...

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Formazione carbonato di calcio in presenza di additivi biopolimerici

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living ...