Os autores reconhecem (1) apoios financeiros do Departamento de Ciência e NanoBioMedicina BK21 PLUS NBM Global Research Center de Medicina Regenerativa na Universidade Dankook, e da prioridade do programa Centros de Pesquisa (n º 2009-0093829) financiado pela NRF, República da Coreia, ( 2) as instalações, bem como a assistência técnica e científica, do Centro Australiano de Microscopia e Microanálise da Universidade de Sydney. HKC é grato ao Conselho de Pesquisa Australiano para os apoios financeiros através de uma bolsa de Descoberta de Projetos (DP0985367 &amp; DP120102778). HAB é grato ao Conselho de Pesquisa Australiano para os apoios financeiros através de uma bolsa de ligação do projeto (LP120200489, LP110200316).

1. Preparando o coloidal Probe e AFM Substrato <ol><li> Para preparar sondas coloidais, utilizar um método desenvolvido anteriormente pelos autores. 19 <ol><li> Em resumo, utiliza um suporte de ângulo de 45 ° para fixar uma consola sem pontas, com o ângulo específico de 45 ° (Figura 1A). </li><li> Prepara-se uma corrediça de epóxi manchando uma fina camada de epóxi sobre uma lâmina de microscópio. Usar uma espátula limpo ou uma corrente lenta de azoto para assegurar que a camada de epóxi adicionado à lâmina do microscópio é de altura mínima. </li><li> Apor o slide epóxi para uma lente de microscópio óptico de 40X zoom usando um suporte de design personalizado (Figura 1B). Em seguida, use o cantilever se aproximar do slide epóxi e adquirir uma pequena quantidade de epóxi sobre o cantilever. </li><li> Repita essas etapas para anexar também uma única partícula de interesse no ápice do cantilever (Figura 1C). </li></ol></li><li> Prepare o substrato AFM pela aposição par coloidaltigos para uma lamela de AFM utilizando um adesivo de montagem termoplástico. <ol><li> Aquecer de 35 mm em volta da tampa deslizante para 120 ° C, e aplicar uma pequena quantidade de adesivo para a lamela. A temperatura elevada é necessária para fundir o adesivo termoplástico para aplicação. </li><li> Depois arrefecer, a lamela de 40 ° C, antes de limpar o pó das partículas coloidais para a cola. NOTA: A 40 ° C, a cola é suficientemente definido que as partículas não se tornará incorporado na cola, mas a cola é pegajoso suficiente para garantir que as partículas vão aderir ao substrato. </li><li> Além disso esfriar a lamela para RT e usar um fluxo suave de nitrogênio para explodir a qualquer excesso de partículas desagregadas. </li><li> Lavar várias vezes de substrato com o meio líquido que vai ser usado para medições da sonda coloidal para assegurar que todas as partículas não ligadas são removidas do substrato. NOTA: É importante reduzir os efeitos de partículas de fluxo livre durante a medição, que pode, interagir com o braço de suporte e introduzir erros nos resultados. </li></ol></li></ol> 2. Montar o coloidal Probe, alinhamento a laser, eo equilíbrio do sistema <ol><li> Colocar a tampa com as partículas coloidais para a metade inferior de uma célula de líquido, certificando-se de que o O-ring está encaixado corretamente para evitar qualquer vazamento. </li><li> Coloque uma folha transparente hidrofóbico para o palco microscópio para se proteger contra qualquer líquido que possa vazar durante a experiência, especialmente se usando apenas a metade inferior de uma célula de líquido para a medição, e coloque a célula de líquido para o palco microscópio. NOTA: Para simplicidade pode-se usar apenas a metade inferior de uma célula de líquido, uma vez que o sistema pode ser equilibrada de forma adequada; dica - a evaporação muda a condição da medida e os impactos de resultados / leitura. </li><li> Fixe a sonda coloidal para a cabeça de varredura AFM e montar para a AFM. Com o software de instrumento em AFM, use os botões nocabeça de leitura para trazer a ponta do cantilever em foco. NOTA: Todas as etapas processuais e as medições foram realizadas utilizando um MFP-3D-Bio AFM com software Asylum Research. </li><li> Para maximizar a intensidade, alinhar o laser na ponta do cantilever usando os botões de ajuste apropriadas na cabeça de digitalização. </li><li> Permitir que o sistema atinja o equilíbrio durante 5-10 minutos ou até que o valor da deflexão estabiliza. Use o botão de ajuste de deflexão para trazer o desvio de zero ou ligeiramente negativa. </li><li> Depois que o sistema foi equilibrada no ar, use o software AFM (Painel térmica na janela do Painel Master) para calcular as InvOLS termicamente (sensibilidade) e constante da sonda coloidal primavera. NOTA: Esta sensibilidade será utilizado temporariamente até que a verdadeira sensibilidade é medida após a conclusão da medição (veja o passo 4). <ol><li> Selecione &quot;Cal Primavera Constant&quot; ou &quot;Cal InvOLS&quot; e depois clique em &quot;capturar dados térmica&quot;. </li><li> Uma vezum pico proeminente é aparente, parar a captura de dados, e clique para aumentar ao longo do pico principal. </li><li> Clique em &quot;Inicializar Fit&quot; seguido de &quot;ajustar os dados de térmicas,&quot; para obter a constante da mola calculada automaticamente ou valores InvOLS. </li></ol></li><li> Lentamente, adicionar 2 ml de meio líquido para a célula de líquido através de uma seringa e assegurar que não há bolhas de ar em torno do braço de suporte. Re-alinhar o laser, uma vez que o índice de refracção do meio mudou agora, e uma vez novamente equilibrar o sistema, permitindo que o valor de desvio para a estabilizar antes de se ajustar o desvio de volta a zero. NOTA: Se uma grande diferença de temperatura existente entre o ambiente eo líquido, o equilíbrio vai demorar mais tempo. </li></ol> 3. Imaging and Data Acquisition <ol><li> Defina o tamanho da digitalização inicial a 20 mM, velocidade de varredura de 1 Hz, ângulo de leitura para 90 º, definir o ponto para 0,2 V e obter uma varredura da amostra. Ajuste o ganho conforme necessário para obter traço sobrepostoe refazer curvas. </li><li> Uma vez que uma partícula de interesse for encontrado, zoom imediatamente para que partículas de limitar as interações sonda prolongados com o substrato antes de obter medições de volume vigor. </li><li> Uma vez ampliada, adquirir uma imagem suficiente de uma única partícula ou porção de uma única partícula. Então mude para o Painel de Força no software. Traga a barra de posição vermelho para a posição mais alta, defina a distância vigor a 5 mm, velocidade de varredura de 0,1 Hz, canal de gatilho para nenhum e realizar uma medição de força única. Certifique-se que a sonda não entre em contato com o substrato. </li><li> A partir do gráfico única medida obtida, calcular a linha de deflexão virtuais com o botão direito na janela de gráfico, e selecionar a opção &quot;Calcular Virtual Def Line&quot;. Isto irá calcular automaticamente a deflexão virtual e atualizar o valor conforme necessário dentro do software. </li><li> Mude o canal de gatilho para deflexão e definir o ponto de disparo de 20 nm. Defina a dist vigorance de 1 mícron e ajustar a velocidade de varredura se for o caso, dependendo das forças medidas de interesse. </li><li> Ajustar manualmente o valor para a deflexão Inverse Optical Lever Sensibilidade (InvOLS) no Painel de Revisão da Força após a realização de 2-3 medidas de força único preliminares consecutivos. <ol><li> Realizar uma medição de força única, em seguida, clique no botão &quot;comentário&quot; no painel de força que abre um Painel Mestre Force. </li><li> Destaque a medição de força mais recentemente concluída. De acordo com o &quot;Eixo&quot; título garantir que apenas &quot;DeflV&quot; está marcada. Altere o &quot;eixo X&quot; campo de entrada para &quot;Setembro&quot;, usando o menu suspenso e clique em &quot;fazer gráfico.&quot; </li><li> Clique na aba &quot;Parm&quot; no Painel de Mestre Força e ajustar o valor de &quot;InvOLS&quot; até a região de contato do gráfico é completamente vertical. Em seguida, preencher esse valor no campo &quot;DEFL InvOLS&quot; localizado sob o Cal60; sub-guia na guia Força localizada na janela do Painel de mestre principal. </li><li> Repetir este 2-3 vezes para garantir que o valor InvOLS não se altera significativamente. </li></ol></li><li> Agora que todos os parâmetros foram estabelecidos, garantir que o nível médio líquido ainda é suficiente e que a deflexão ainda é estável. NOTA: Neste momento, curvas de força única ou mapas de força pode ser obtida. Se medidas de força de permanência são desejados, as opções de permanência pode ser acessado no painel de força. </li></ol> 4. Pós-ajuste de sensibilidade para Análise <ol><li> Após a conclusão da aquisição de medição, medir a verdadeira sensibilidade da sonda coloidal. Para fazer isso, realizar uma medição de força através de um relativamente grande deflexão / força com a sonda coloidal no mesmo meio líquido contra um &quot;infinitamente&quot; superfície dura, tal como a mica. NOTA: A sensibilidade foi obtido após a conclusão dos experimentos, pois a grande deflexão / force pode danificar collosondas Idal preparados com colóides porosos ou frágeis. </li><li> O declive da região de contacto é usado pelo software para calcular automaticamente a sensibilidade (Figura 2). Utilize este verdadeiro valor da sensibilidade durante a análise de todas as curvas obtidas com essa sonda coloidal específico de dados. </li></ol>

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The authors declare that they have no competing financial interests.

Várias fontes de instabilidade do sistema presente durante líquido Nanoscopia sonda coloidal podem ser facilmente mitigados por meio de procedimentos de equilíbrio adequados. Instabilidades como discutido anteriormente resultar em resultados errôneos e curvas de força que são mais difíceis de analisar objetivamente. Se todas as fontes de instabilidade foram cuidadas e gráficos semelhantes ao que é mostrado na figura 4 estão ainda presentes, outro parâmetro de medição pode ser o motivo. Outr...

Microscopia de força atómica (AFM) é uma técnica que permite a imagiologia quantitativa e qualitativa e sondagem de uma superfície do material. 4-6 Tradicionalmente, a AFM é utilizado para a avaliação da topografia da superfície, morfologia e estrutura dos materiais multi-fásicos. AFM tem a capacidade de avaliar quantitativamente as interações nano-escala, tais como carga, atração, repulsão e de adesão forças entre uma sonda e substrato específico no ar e meios líquidos 7,8 A AFM originalmente desenvolvido pela Binning, Quate e Gerber 9 usos. uma sonda de conhecida determinada sensibilidade / e constante da mola para se aproximar e / ou digitalização de uma amostra. Devido às interacções físicas entre a sonda e a amostra, o braço de suporte é desviado durante o contacto ou a proximidade e, dependendo do modo de operação, este desvio pode ser traduzido para adquirir a topografia das forças de amostra ou medir presentes entre a sonda e amostra. Modificações para o téc AFMOpaco, como Nanoscopia sonda coloidal, 10 têm permitido cientista para avaliar diretamente as interações nano-força entre dois materiais presentes em um sistema coloidal de interesse. Em Nanoscopia sonda coloidal, uma partícula esférica de escolha é anexado ao ápice de um cantilever, substituindo os tradicionais cônicos e piramidais dicas. Uma partícula esférica é ideal para permitir a comparação com modelos teóricos, tais como a Johnson, Kendal, Roberts (JK) 11 e Derjaguin, Landau, Vervwey, Overbeek (DLVO) 12-14 teorias e para minimizar a influência da rugosidade da superfície na medição. 15 Estas teorias são utilizadas para definir o mecanismo de contacto e das forças inter-partículas esperados dentro de um sistema coloidal. A teoria DLVO combina as forças de van der Waal atraente e forças eletrostáticas repulsivas (devido a camadas duplas elétricas) para explicar quantitativamente o comportamento de agregação de sistemas coloidais aquosas, enquanto a JTeoria KR incorpora o efeito da pressão de contacto e a adesão ao modelo de contacto elástica entre dois componentes. Uma vez que uma sonda apropriada é produzida, que é usado para se aproximar de qualquer outro material / partículas para avaliar as forças entre os dois componentes. Usando uma ponta de um padrão fabricado será capaz de medir forças interativas entre a ponta e um material de escolha, mas a vantagem de usar uma sonda coloidal custom made permite a medição de forças presentes entre os materiais presentes no sistema estudado. Interacções mensuráveis ​​incluem:.. Adesiva, atractivo, repulsivo, carga, e as forças electrostáticas presentes mesmo entre as partículas 16 Além disso, a técnica de sonda coloidal pode ser utilizada para explorar as forças tangenciais presentes entre as partículas e material de elasticidade 17,18 A capacidade de realizar medições em vários meios é uma das principais vantagens da Nanoscopia sonda coloidal. Condições ambientais, líquido mEDIA, ou condições controladas de umidade podem ser usados ​​para simular as condições ambientais do sistema estudado. A capacidade de realizar medições em um meio líquido permite o estudo dos sistemas coloidais em um ambiente que ocorre naturalmente; assim, ser capaz de adquirir quantitativamente dados que é directamente transponível para o sistema no seu estado natural. Por exemplo, as interacções de partículas presentes dentro de inaladores de dose calibrada (MDI) podem ser estudados utilizando um modelo de propulsor líquido com propriedades semelhantes às do propulsor usado no IDC. As mesmas interacções medidos no ar não seria representativo do sistema existente no inalador. Além disso, o meio líquido pode ser modificado para se avaliar o efeito da entrada de humidade, um agente tensioactivo secundário, ou de temperatura sobre as interacções de partículas em um MDI. A capacidade de controlar a temperatura pode ser utilizado para imitar certos passos na fabricação de sistemas coloidais para avaliar o modo como a temperatura, quer na fabricação de ouarmazenamento dos sistemas coloidais podem ter um impacto sobre as interações de partículas. Medidas que podem ser obtidos utilizando as sondas coloidais incluem; Digitalização Topografia, curvas individuais força-distância, mapas de adesão força-distância, e habita medições força-distância. Principais parâmetros que são medidos utilizando o método Nanoscopia sonda coloidal apresentada neste artigo incluem os valores de energia de separação snap-in, carga máxima, e. Snap-in é uma medida das forças atrativas, máx carregar o valor da força máxima aderência, ea energia separação transmite a energia necessária para retirar a partícula de contato. Estes valores podem ser medidos através de medições instantâneas ou de força habitar. Dois tipos diferentes de medidas de parada incluem deflexão e recuo. O comprimento e tipo de medição de permanência pode ser especificamente escolhido para imitar as interacções específicas que estão presentes no interior de um sistema de interesse. Um exemplo é o uso de deflexão de permanência - que detémas amostras em contato em um valor deflexão desejada - para avaliar as ligações adesivas que se desenvolvem em agregados formados em dispersões. As ligações adesivas formadas podem ser medidos como uma função do tempo, e pode fornecer informações sobre as forças necessárias para redispersar os agregados após armazenamento prolongado. A multiplicidade de dados que podem ser obtidos com este método é uma prova da versatilidade do método.

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Double-Bubble Epoxy</td>
			<td style="width:179px;">Hardman</td>
			<td style="width:119px;">4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Veeco Tipless Probes</td>
			<td style="width:179px;">Veeco</td>
			<td style="width:119px;">NP-O10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Porous Particles</td>
			<td style="width:179px;">Pearl Therapeutics</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Atomic Force Microscope (MFP)</td>
			<td style="width:179px;">Asylum&#160;</td>
			<td style="width:119px;">MFP-3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SPIP Scanning Probe Image Processor Software</td>
			<td style="width:179px;">NanoScience&#160; Instruments</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">35 mm Coverslips</td>
			<td style="width:179px;">Asylum</td>
			<td style="width:119px;">504.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tempfix</td>
			<td style="width:179px;">Ted Pella. Inc.</td>
			<td style="width:119px;">16030</td>
			<td></td>
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quantitative and qualitative examination of particle-particle interactions using colloidal probe nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Sistemas coloidais líquidos são usados ​​para vários sistemas de entrega de drogas farmacêuticas. Para a entrega de drogas de inalação, um sistema coloidal comum é a suspensão de inalador de dose medida pressurizada (pMDI). Interacções de partículas presentes no pMDI desempenham um papel vital na formulação de estabilidade física, o armazenamento, e uniformidade de administração de fármaco. Neste artigo, as forças inter-partículas entre as partículas porosas à base de lípidos (~ 2 mm de diâmet...

Watch this Scientific Journal Video about Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy at JoVE.com

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

O exame quantitativo e qualitativo dos Interações partícula-partícula coloidal Usando Probe Nanoscopia

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

quantitative-qualitative-examination-particle-particle-interactions

Research

JoVE Journal

Chemistry

10.9K visualizações.  University of Sydney. Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems. 

Vídeo: Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

A identificação qualitativa dos ácidos carboxílicos, ácidos borónicos e aminas Usando fluoróforos cruciforme

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Química

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Proteômica quantitativa utilizando redutora dimetilação para Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

Síntese e Purificação de Iodoaziridines Envolvendo Quantitative Seleção do Optimal fase estacionária para cromatografia

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Síntese e esfoliação da discotic Zircônio Fosfatos obter cristais coloidais líquidos

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

A Quantitative Glycomics e Proteomics Combinada estratégia de purificação

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Normas para Análise Quantitativa Metalloproteomic Usando Tamanho Exclusão ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

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 ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Faceta-para-faceta vinculando de forma anisotrópica cádmio coloidal calcogênio nanoestruturas

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Photodeposition of Pd onto Colloidal Au Nanorods by Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot electrons upon SPR irradiation drive reductive deposition of Pd on colloidal AuNR in the presence of [PdCl4]2-. Plasmon-driven reduction of secondary metals potentiates covalent, sub-wavelength deposition at targeted locations coinciding with electric field &#8220;hot-spots&#8221; of the plasmonic substrate using an external field (e.g., laser). The process described herein details a solution-phase deposition of a catalytically-active noble metal (Pd) from a transition metal halide salt (H2PdCl4) onto aqueously-suspended, anisotropic plasmonic structures (AuNR). The solution-phase process is amenable to making other bimetallic architectures. Transmission UV-vis monitoring of the photochemical reaction, coupled with ex situ XPS and statistical TEM analysis, provide immediate experimental feedback to evaluate properties of the bimetallic structures as they evolve during the photocatalytic reaction. Resonant plasmon irradiation of AuNR in the presence of [PdCl4]2- creates a thin, covalently-bound Pd0 shell without any significant dampening effect on its plasmonic behavior in this representative experiment/batch. Overall, plasmonic photodeposition offers an alternative route for high-volume, economical synthesis of optoelectronic materials with sub-5 nm features (e.g., heterometallic photocatalysts or optoelectronic interconnects).

A protocol for anisotropic photodeposition of Pd onto aqueously-suspended Au nanorods via localized surface plasmon excitation is presented.

Fotodeposição de PD em au Nanorods coloidal pela excitação de superfície Plasmon

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot ...

Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation

In this work, we demonstrate a facile method for colloidal lead halide perovskite nanoplatelet synthesis (Chemical formula: L2[ABX3]n-1BX4, L: butylammonium and octylammonium, A: methylammonium or formamidinium, B: lead, X: bromide and iodide, n: number of [BX6]4- octahedral layers in the direction of nanoplatelet thickness) via ligand-assisted reprecipitation. Individual perovskite precursor solutions are prepared by dissolving each nanoplatelet constituent salt in N,N-dimethylformamide (DMF), which is a polar organic solvent, and then mixing in specific ratios for targeted nanoplatelet thickness and composition. Once the mixed precursor solution is dropped into nonpolar toluene, the abrupt change in the solubility induces the instantaneous crystallization of nanoplatelets with surface-bound alkylammonium halide ligands providing colloidal stability. Photoluminescence and absorption spectra reveal emissive and strongly quantum-confined features. X-ray diffraction and transmission electron microscopy confirm the two-dimensional structure of the nanoplatelets. Furthermore, we demonstrate that the band gap of perovskite nanoplatelets can be continuously tuned in the visible range by varying the stoichiometry of the halide ion(s). Lastly, we demonstrate the flexibility of the ligand-assisted reprecipitation method by introducing multiple species as surface-capping ligands. This methodology represents a simple procedure for preparing dispersions of emissive 2D colloidal semiconductors.

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.