Este trabalho foi apoiado pela Marie Curie Internacional Reintegração Grant e pela Fundação Alemã-Israel. Reconhecemos Mr. Yair Razvag para análise AFM.

1. Auto-montagem de Homoaromatic Dipeptídeos <ol><li> Pesa-se o péptido desejado na sua forma liofilizada (por exemplo, NH2-Phe-Phe-OH, Boc-Fen-Fen-COOH) e preparar uma solução de reserva dissolvendo o péptido em 1,1,1,3,3,3-hexafluoro -2-propanol (HFP) à concentração adequada (por exemplo, 100 mg / ml para o NH2-Phe-Phe-OH e Boc-Fen-Fen-COOH) 7,17,46. </li><li> Misture a solução com vórtice e colocados sobre o banco até que o péptido seja dissolvido completamente e a solução parece claro (alguns minutos). </li><li> Dilui-se a solução-mãe de péptido, com um solvente adequado, a uma concentração adequada (por exemplo, 2 mg / ml de NH2-Phe-Phe-OH em ​​água destilada triplo (TDW) para a formação de nanotubos; por adição de 2 ul de péptido solução de estoque de 98 ul TDW, 5 mg / ml de Boc-Fen-Fen-COOH em etanol para a formação de estruturas esféricas). </li><li> Manter a solução à TA durante 24hr. </li><li> A fim de evitar qualquer preaggregation, preparar soluções de fresco para cada experiência. </li></ol> 2. Coassembly of Two Homoaromatic Dipeptídeos <ol><li> Prepara-se uma solução de etanol a 50% por mistura de volumes iguais de TDW e etanol absoluto. Use vortex para misturar as duas soluções. </li><li> Pesar 2 mg do péptido NH2-Phe-Phe-OH e 1 mg do péptido de Boc-Phe-Phe-OH. Dissolve-se cada péptido em HFP para uma concentração de 100 mg / ml. </li><li> Misturar as soluções de peptídeos usando vórtice e colocá-los no banco até os péptidos estão completamente dissolvidos e as soluções parecem claras. </li><li> Misturar as soluções de péptidos para a relação desejada. Nesta experiência específica mistura de 10 ul de péptido NH2-Phe-Phe-OH com 6 ul de péptido Boc-Phe-Phe-OH (para uma razão final de 05:03, respectivamente). Devido a elevada volatilidade do solvente HFP, recomenda-se para preparar uma grande quantidade desta solução (em leaSão 10 mL). </li><li> Use vórtice para misturar a solução estoque péptidos misturados. </li><li> Dilui-se a solução estoque péptidos misturadas com etanol a 50% para a concentração final desejada. Nesta experiência específica, a fim de obter uma concentração final de 5 mg / ml para o NH2-Phe-Phe-OH e 3 mg / ml em vez do Boc-Phe-Phe-OH, respectivamente, adicionar 8 mL da solução de estoque péptidos misturados de 92 ul da solução de etanol a 50%. Use uma pipeta para misturar suavemente a solução. </li><li> Manter a solução à TA durante 24 horas. </li><li> Deve notar-se que, devido à natureza altamente volátil do solvente, as experiências são sensíveis a pequenas mudanças na concentração dos péptidos. Portanto, as soluções de reserva frescas devem estar preparados para cada experimento. </li></ol> 3. Caracterização das auto montado estruturas utilizando Microscopia Eletrônica de Varredura (MEV) <ol><li> Após 24 horas de incubação, aplicar uma gota de 10 ul da solução de péptidos em enseada vidror deslizamento e seco à temperatura ambiente. </li><li> Revestir a amostra sobre o vidro com uma camada fina de ouro (alguns nanómetros) utilizando um revestidor por crepitação durante 90 seg. </li><li> Imagem assembléias usando SEM operando a 10-20 kV. </li></ol> 4. Caracterização das auto montado estruturas utilizando Microscopia Eletrônica de Transmissão (TEM) <ol><li> Coloque uma gota de 10 ul da solução de péptidos numa grelha de cobre de malha 200 coberta com carvão e estabilizada por um suporte de película de polímero. </li><li> Após 1 min remover o excesso de líquido usando papel de filtro. </li><li> Prepara-se uma solução de 2% de acetato de uranilo em TDW. Filtrar a solução através da unidade de filtro de 0,22 um. </li><li> Para corar a amostra (coloração negativa), colocar uma gota de 10 ul de solução de acetato de uranilo na grelha. </li><li> Depois de 30 seg remover o excesso de fluido utilizando papel de filtro. Deve notar-se que, apesar de coloração negativa melhora o contraste das imagens, ele não é essencial em todos os casos. </li><li> Imagem da amostra no the grade por TEM operando a 120 kV. </li></ol> 5. Caracterização tridimensional das Assembléias por Microscopia de Força Atômica (AFM) <ol><li> Prepara-se uma amostra para a análise de AFM utilizando o procedimento descrito no ponto 3.1. </li><li> Analisar a amostra sobre o vidro usando um instrumento AFM trabalhar no modo AC. Use vigas de silício com uma constante de mola de 3 N / m e uma freqüência de ressonância de 75 kHz. </li><li> Comece a digitalização de uma grande área da grelha, a fim de encontrar a estrutura desejada. Em seguida, se concentrar em uma área menor específico e digitalizá-lo (o tamanho da digitalização foi de 2,5 mM x 2,5 mm para a imagem incluída no manuscrito). </li></ol> 6. Caracterização da Estrutura Secundária de FT-IR <ol><li> Aplicar uma gota de 30 ul da solução de péptidos para uma janela de CaF 2. </li><li> Permitir que a solução a secar à temperatura ambiente. </li><li> A adsorção de água no espectro de IR é a 1,650 cm1.Este pico é, no centro da banda de amida I da ligação peptídica. É também um pico característico para as estruturas helicoidais-α de péptidos e proteínas 47. A fim de ultrapassar este problema e evitar o sinal da água, uma troca de hidrogénio para deutério, deve ser realizada. Colocar uma gota de óxido de deutério (D 2 O), sobre a amostra de peptídeo seca. A queda deve ser grande o suficiente para cobrir completamente o depósito de peptídeo na janela. </li><li> Deixar a amostra seca sob vácuo. </li><li> Repita os passos 6.3 e 6.4 2x para assegurar a troca de hidrogênio-to-deutério máxima. Salve a amostra sob vácuo até a sua análise. </li><li> Grave os espectros FT-IR utilizando um sulfato triglycine deuterado (DTGS) detector. O sistema de FT-IV inclui um gerador de gás de purga, a fim de evitar a humidade no ambiente da amostra. Para amostras de peptídeos curtos, é melhor para fazer a varredura da amostra de 2.000 x com uma resolução de 4 cm -1. Os valores mínimos de transmitância pode ser determinada pelo modoftware fornecido com o instrumento. </li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Em resumo, este estudo demonstra a facilidade com que os conjuntos com base em peptídeos podem ser formados in vitro. O processo envolve a péptidos e solventes comercialmente disponíveis, e isto ocorre espontaneamente sob as condições ambientes, mediante a adição de um solvente polar para o tubo de ensaio. É fundamental utilizar HFP como um solvente dos peptídeos, devido à baixa solubilidade dos péptidos em outros solventes orgânicos. Além disso, devido à alta volatilidade da HFP, é necessário p...

Formas Natureza ordenado e funcional das estruturas pelo processo de auto-montagem biomolecular. Compreender as forças que governam este processo espontâneo pode levar à capacidade de imitar a auto-montagem in vitro e, consequentemente, grandes avanços na área de ciências de materiais 10,11. Peptídeos, especificamente, são uma grande promessa como um bloco de construção biomolecular, pois apresentam grande diversidade estrutural, a facilidade de síntese química, e pode facilmente ser funcionalizadas com entidades biológicas e químicas. O campo de peptídeo auto-montagem foi iniciada por Ghadiri e seus colegas, que demonstraram a auto-montagem de nanotubos de peptídeo por peptídeos cíclicos com a alternância de D-e L-aminoácidos 12. Outras abordagens de sucesso para o projeto de conjuntos de peptídeos incluem peptídeos lineares bolaamphiphile 5, amphiphiles (AP) 6, peptídeos auto-complementares não conjugados iônicos 13, peptídeos surfactantes-like <sup&gt; 4,14, e dibloco copolypeptides 15. Uma abordagem mais recente envolve a auto-montagem de peptídeos aromáticos curtas, denominado dipeptides homoaromatic. Estes péptidos compreendem apenas dois aminoácidos com carácter aromático (por exemplo, Fen-Fen, terc-butilo (Boc)-Phe-Phe) 7,8,16-21. As estruturas formadas por estes péptidos homoaromatic incluem estruturas tubulares, esferas, os conjuntos do tipo folha e fibras 6,8,15,21-32. As fibras em alguns casos, gerar uma malha de fibrila que produz um hidrogel 33-37. Estes conjuntos foram explorados para aplicações de biosensing, distribuição de medicamentos, a eletrônica molecular, etc 38-45. Este artigo descreve os passos experimentais necessários para iniciar o auto-montagem espontânea de peptídeos homoaromatic. Além disso, apresenta-se o processo de péptido coassembly. Este processo envolve a auto-montagem de mais do que um tipo de péptidomonómero. A nossa demonstração inclui o coassembly de dois péptidos disponíveis comercialmente: o péptido difenilalanina (NH2-Phe-Phe-COOH) e o seu análogo Boc protegido (Boc-Fen-Fen-OH). Cada um dos péptidos auto-monta para uma estrutura supermolecular: as formas peptídicas difenilalanina conjuntos tubulares e os péptidos auto-montagem de Boc-Phe-Phe-OH em ​​ambos esferas ou fibras de acordo com o solvente 7,17,46. Nós misturado os dois peptídeos em certas proporções e caracterizadas as assembleias resultaram por microscopia eletrônica, microscopia de força e espectroscopia FT-IR. Os métodos demonstrou a formação de uma estrutura à base de péptido, que é constituída por elementos esféricos com um diâmetro de vários microns (1-4 mm), que são ligados por conjuntos alongados com um diâmetro de algumas centenas de nanómetros (~ 300-800 nm) . Os conjuntos de cadeias assemelham frisado na sua morfologia, como as estruturas esféricas parecem ser enfiada sobre oassembléias alongados. Nós, portanto, classificou essas assembléias &quot;colares biomolecular&quot;. Os &quot;colares biomoleculares&quot; poderá servir como um novo biomaterial, como um agente de entrega de drogas ou como suporte para aplicações eletrônicas. Além disso, o processo que conduz à auto-montagem de péptidos podem ser utilizadas com outras classes de péptidos e biomoléculas. Isso pode levar a uma melhor compreensão das forças envolvidas na auto-montagem e a formação de novas estruturas 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

Na natureza, complexas estruturas funcionais são formadas por auto-montagem de biomoléculas em condições suaves. Compreender as forças que controlam a auto-montagem e imitando este processo in vitro vai trazer grandes avanços nas áreas de ciência e nanotecnologia de materiais. Entre os blocos de construção biológica disponíveis, os péptidos têm várias vantagens uma vez que apresentam diversidade substancial, a sua síntese em grande escala é simples, e podem ser facilmente modificados com entidades biológicas e químicas 1,2. Várias classes de péptidos concebidos, tais como péptidos cíclicos, péptidos de anfifilo e peptídeo-conjugados de auto-montam em estruturas ordenadas em solução. Dipeptides Homoaromatic, são uma classe de peptídeos auto-organizados curtas que contenham toda a informação molecular necessário para formar estruturas ordenadas, como nanotubos, esferas e fibrilas 3-8. Uma grande variedade destes péptidos estão disponíveis comercialmente. <pclass = &quot;jove_content&quot;&gt; Este artigo apresenta um procedimento que leva à formação de estruturas ordenadas pela auto-montagem de peptídeos homoaromatic. O protocolo requer apenas reagentes comerciais e equipamentos básicos de laboratório. Além disso, o documento descreve alguns dos métodos disponíveis para a caracterização de conjuntos com base em peptídeos. Estes métodos incluem elétrons e microscopia de força atômica e Fourier Transform Infrared Spectroscopy-(FT-IR). Além disso, o manuscrito demonstra a mistura de peptídeos (coassembly) e a formação de um &quot;pérolas num colar&quot; estrutura semelhante por este processo. 9 Os protocolos aqui apresentados podem ser adaptadas a outras classes de péptidos ou blocos de construção biológica e pode potencialmente levar à descoberta de novas estruturas à base de péptidos e para um melhor controlo da sua montagem.

Este documento descreve um método para a formação de estruturas ordenadas na escala nano e micrómetro pelo auto-montagem de peptídeos. De modo a demonstrar este processo simples que apresentar e caracterizar o coassembly de dois péptidos aromáticos simples (Figura 1). Um dos péptidos é o NH2-Phe-Phe-OH (difenilalanina) péptido, que pode auto-montar em uma solução aquosa em estruturas tubulares ocas com dimensões nanométricas 7. O outro péptido é o seu análogo Boc p...

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

Este documento descreve a formação de estruturas com base em peptídeos altamente ordenada pelo processo espontâneo de auto-montagem. O método utiliza peptídeos comercialmente disponíveis e equipamento de laboratório comum. Esta técnica pode ser aplicada a uma grande variedade de péptidos e podem conduzir à descoberta de novos conjuntos com base em peptídeos.

A formação de estruturas biomoleculares ordenada pelo auto-montagem de pequenos peptídeos

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 visualizações.  The Hebrew University of Jerusalem.  Este documento descreve a formação de estruturas com base em peptídeos altamente ordenada pelo processo espontâneo de auto-montagem. O método utiliza peptídeos comercialmente disponíveis e equipamento de laboratório comum. Esta técnica pode ser aplicada a uma grande variedade de péptidos e podem conduzir à descoberta de novos conjuntos com base em peptídeos. 

Vídeo: 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 Inspirado Auto-montagem de Partículas estampados e Reconfigurável

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

Química

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.

A funcionalização assistida por microondas, de poli (etileno glicol) e Peptídeos On-resina para uso em Polimerizações cadeia e formação 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.

A auto-montagem de formas bidimensionais complexos de ADN de cadeia simples Azulejos

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.

Insights sobre as interações de aminoácidos e peptídeos com Materiais Inorgânicos Usando Single-Molecule Força Spectroscopy

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.

Enxertia de nanotubos de carbono de Multiwalled com poliestireno para habilitar auto-montagem e anisotrópica Patchiness

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 eletrônica de transmissão de líquido-celular para rastreamento auto-montagem de 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.

Fabricação de nanocristais micélica esférico e em forma de verme, combinando Electrospray, auto-montagem e controle de estrutura de base 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.

Síntese e caracterização de 1,2-Ditiolano modificado peptídeos auto-montagem

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.

Lumíforo formação em várias conformações de albumina de soro bovino por ligação 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 ...

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.

Formação de carbonato de cálcio na presença de aditivos Biopoliméricos

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