KG foi apoiada pela concessão P-1760 RA Fundação Welch, TCU Andrews Instituto de Matemática e Ciências da Educação (para KG), TCU pesquisa e criatividade Atividade Grant (a KG) e TCU SERC Grant (a JHS).

1. Síntese de biotina-Fc-cisteína (1) <ol><li> Métodos em fase sólida para produzir resina-1 ligado. <ol><li> Coloque biotina resina carregada (250 mg, 0,145 mmol) para uma seringa de vidro sinterizado e inchar a resina através da elaboração de dimetilformamida (5 mL) e agitando a seringa num agitador de laboratório durante 20 min. Expelir a solução e repetir dimetilformamida inchaço mais uma vez. </li><li> Remover o grupo protector Fmoc por adição de 4-6 ml de 20% de piperidina em dimetilformamida para a seringa, seguido por 10-15 min de agitação. Repetir o processo de desprotecção com mais de 4-6 ml de piperidina. Lava-se a resina com uma sequência de 3x dimetilformamida, 3x dimetilformamida: metanol (1: 1), metanol 3x: diclorometano (1: 1), diclorometano 3x, ~ 5 ml cada. Fazer um teste de ninidrina (+) sobre uma pequena amostra (~ 10) dos grânulos para confirmar a desprotecção bem sucedida pela presença de azul após aquecimento. </li><li> Misturar uma solução contendo ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico(203,3 mg, 0,4350 mmol), hidrato de 1-hidroxibenzotriazole (58,8 mg, 0,413 mmol), carbodiimida de diisopropilo (0,0673 ml, 0,435 mmol), diisopropil etil amina (0,0757 ml, 0,435 mmol), e uma mistura 4: 1 de diclorometano e dimetilformamida. Desenhe esta dentro da seringa de frita e agitar suavemente num agitador de laboratório durante 6 horas. Em seguida expelir a solução a partir da seringa e lavagem conforme descrito anteriormente. </li><li> Realizar o teste da ninidrina (-), tal como descrito acima para confirmar acoplamento. O teste de ninidrina ainda podem ser úteis na confirmação de acoplamento apesar de a cor laranja do cordão de derivados de a fixação da porção contendo ferro. </li><li> Em seguida, remover o grupo Fmoc através da adição de 20% de piperidina em dimetilformamida e lavado como descrito acima. O teste de ninidrina (+) deve ser utilizado para confirmar a remoção do Fmoc. </li><li> Prepara-se uma solução composta por Fmoc-Cys (Trt) -OH (254,8 mg, 0,4350 mmol), hidrato de 1-hidroxibenzotriazole (58,8 mg, 0,4125 mmol), carbodiimida de diisopropilo (0,0673 ml, 0,4350mmol), diisopropiletilamina (0,0757 mL, 0,4350 mmol), e uma mistura 4: 1 de diclorometano e dimetilformamida. Adicione este cocktail acoplamento cisteína a seringa frita e agitar suavemente durante 6 horas. Lavar utilizando o protocolo descrito anteriormente. </li><li> Confirmar acoplamento utilizando o teste de ninidrina (-), seguida por remoção do componente de Fmoc com 20% de piperidina e lavagem. Verifique se o terminal de acesso gratuito amina usando o teste de ninidrina (+). </li></ol></li><li> A clivagem de um a partir da resina. <ol><li> Adicione uma solução de TFA (9,45 ml), água (0,25 ml), 1,2-etanoditiol (0,25 ml), e silano de triisopropilo (0,1 ml), adicioná-lo à seringa e agitar suavemente durante 4 h. </li><li> Recolher a solução vermelha-castanha resultante num tubo de Eppendorf e evapora-se o TFA lentamente com uma corrente de ar. </li><li> Adicionar éter dietílico frio (~ 15 ml) para o tubo de Eppendorf para precipitar 1, que irá formar uma ligeira agitação. Isolar o produto por meio de centrifugação (1 g, 5 min). Then ciclos repetidos de lavagens de éter etílico (~ 60 ml total) e centrífuga para obter 1 como um sólido vermelho / marrom. </li></ol></li></ol> 2. Caracterização e Análise de 1 <ol><li> Confirmar que a identidade corresponder a conectividade e composição mostrada na Figura 2, usando 1 H (16 varrimentos) e 13 C RMN (512 scans) em metanol deuterado (300 ul) e análise de ESI-MS. Espere os seguintes resultados: 1 H RMN Espectro (CD 3 OD) δ / ppm: 1,407-1,684 (m, 6H), 2,245 (t, 2H), 2,665-3,150 (m, 12H), 4,015 (t, 1H), 4,104 (d, 2H ), 4,274 (q, 1H), 4.426 (d, 2H), 4,479 (q, 1H), 4,595 (t, 1H) e espectro de 13 C RMN (CD 3 OD) δ / ppm: 24,644 (CH2), 25,472 (CH 2), 28,051 (CH2), 28,300 (CH2), 35,474 (CH2), 38,698 (CH2), 39,241 (CH2), 39,717 (CH2), 55,340 / 55,538 (Cp-anel), 60,286 (CH), 61,964 (CH), 62,521 / 62,821 (Cp-ring), 66,038 / 66,170 (Cp-ring), 69,153 / 69,328 (Cp-ring), 71,468 / 71,593 (Cp-ring), 76,466 (CH), 171,770 (C = O), 175,361 (C = O). ESI-MS (m / z): Encontrado: 639,00 [1 + Na] +, teórico: 639,1 [1 + Na] + e HR-MS (m / z): Encontrado: 617,2049 [1 + H] +, Teórico: 617.1622 [1 + H] +. </li><li> Realizar HPLC, análise elementar para confirmar a composição de um isolado. Realizar cromatogramas de HPLC utilizando uma coluna de fase reversa C8 com MeOH a 100% a uma taxa de fluxo de 0,5 ml / min. Nota: tempos de retenção de HPLC foram: 3,198-4,674 min. </li></ol> 3. Imobilização de 1 sobre uma superfície de ouro <ol><li> Cut polímero apoiado lâminas de ouro em quadrados de ~ 0,25 em 2. </li><li> Encher um copo de 50 ml com uma solução de água Dl de 1 (~ 1 mM). </li><li> Adicionar a lâmina de ouro para o copo e cubra com um vidro de relógio. Tudoow da lâmina de vidro a incubar O / N à temperatura ambiente sem agitação. </li><li> Remover o slide de ouro a partir da solução e permitir que ela seca ao ar. </li><li> Obter imagens de microscopia eletrônica de varredura, utilizando um microscópio eletrônico de varredura (ou equivalente) para observar imobilizada 1. </li></ol>

The authors have nothing to disclose.

A síntese de derivados de ferroceno assimétricas é um desafio em solução. Por exemplo, as tentativas para produzir uma solução em resultou em baixos rendimentos do produto desejado (menos de 20%). Do mesmo modo, reacções que utilizam ácido carboxílico 1&#39;-amino-ferroceno (sans Fmoc) e biotina ligada resina resultou em um produto insolúvel consistente com o produto polimerizado relatado por Baristic et al. e produto mínima. 47 Isto é ainda mais complicada pel...

Biossensores são pequenos dispositivos que utilizam a tecnologia de reconhecimento biomolecular como a plataforma para análise seletiva e são utilizados para a sua especificidade, velocidade e baixo custo. Os biossensores eletroquímicos para a detecção de biomoléculas estão na vanguarda deste campo, devido à sua simplicidade, custo-benefício, e alta sensibilidade. 1,3 A anatomia geral destes sensores é um eletrodo equipado com uma molécula de reconhecimento específico para o marcador biológico de interesse . A ligação do marcador por molécula de reconhecimento resulta de uma alteração local do potencial ou da corrente que pode ser detectada por uma simples medição. Até à data, a porção reconhecimento pode variar de enzimas, anticorpos, 4-8 9-12 células inteiras, 13-16, 17-20 receptores peptídeos 21-23 e 24 e de DNA em grande parte com foco em moléculas maiores, biológicos. 25-28 Research esforços nessa arena têm se concentrado principalmente na imunossensores where uma imunoglobulina é imobilizada com um núcleo activo redox (tais como ferroceno) e utilizados para detectar um anticorpo de interesse. Estes estudos foram excluídos aplicações clínicas devido à baixa precisão e tempo de consumo decorrentes das complicações decorrentes do uso de antígeno / anticorpos. 1,3 crescente atenção centrou-se na detecção de moléculas pequenas (menos de 1 kg / mol) de biomédica , alimentos e interesse ambiental, além de segurança nacional. 29 Os exemplos mais conhecidos de dispositivos biossensores são monitores de glicose de auto-teste, que têm tela impressa eletrodos enzimáticos acoplados a um metro amperometric bolso. Estes sistemas utilizam tipicamente um método coulométrico onde a quantidade total de carga gerada pela reacção de oxidação da glicose é medida ao longo de um período de tempo. Dispositivos mobiliários deve ser portátil, robusto e de mão de fazer uso fácil para a população em geral. Tag Redox como ferrocene são necessry para proporcionar a detecção eletroquímica de biomarcadores ou pequenas moléculas em solução como a maioria dos biomarcadores não são intrinsecamente eletroquimicamente ativa. 30-38 Ferroceno é uma molécula organometallic que é um padrão-ouro para eletroquímica, o que o torna uma excelente opção para a integração em biossensores eletroquímicos. Espécies ativas redox à base Ferroceno já acumulou considerável atenção devido ao seu pequeno tamanho, boa estabilidade, acesso sintético conveniente, modificação química fácil, lipofilicidade relativa, e facilidade de ajuste redox. 3,30-42 Pequenas moléculas baseado no núcleo ferrocene tem sido amplamente utilizado como detectores de íons metálicos e pequenas moléculas. 32-38,43 Sistemas dirigidas a espécies maiores, como biomoléculas têm utilizado a ligação de anticorpos grandes ou imunoglobulinas para derivados do ferroceno que foram incorporados em uma superfície eletroquímica. 1,3,39 , 44 Em cada caso, o potencial de corrente e intensity do par redox Fe III / Fe II foi alterado por acoplamento molecular, produzindo, assim, um novo identificador espectroscópica indicando a presença da molécula de analito. Esta mudança surge a partir da sobreposição extensiva que ocorre entre o sistema-pi dos anéis ciclopentadienilo e os de ferro orbitais d. Se o sistema pi é modificado, ou seja, derivado ou reagir, em seguida, a interacção orbital, por sua vez, a mudança. Isto irá afectar o núcleo Fe e pode ser observada como uma mudança no potencial do par Fe III / Fe II. 40,45,46 Estas propriedades tornam um tal sistema atractivo para uso como um agente de quantificação em um imunoensaio electroquímico ou biossensor. De modo a produzir sistemas de ferroceno contendo capacidades específicas para biossensores é óptima para modificar um anel de Cp com a bio-receptor específico para uma molécula alvo e utilizar o outro anel de Cp como uma amarra molecular para a leitura electroquímica ou electrode (Figura 1). Síntese destes derivados do ferroceno assimétricos é desafiado por reações colaterais e a formação de espécies diméricas e poliméricas formadas sobre intermolecular cross-linking. 47 No entanto, o acoplamento química produzindo uma ligação amida é o caminho mais direto para fornecer derivados simples de ferrocene envolvendo componentes biológicos tais como péptidos e seus metabolitos. Portanto, as técnicas de fase sólida primeiro desenvolvidos na década de 1950 por Merrifield de síntese de péptidos podem ser aplicados a compostos organometálicos contendo ferroceno. Através da utilização da molécula de ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico ortogonalmente substituído, um sistema de ferroceno, que pode incluir uma porção do receptor (biotina), leitura electroquímica (ferroceno), e o componente de imobilização-ligante (cisteína) possui Foram construídas aqui detalhado. A síntese deste bio-conjugado é discutido bem como prova para a imobilização sobre uma superfície de ouro. Este trabalho represenst a primeira apresentação de um sistema composto de biotina, ferroceno e um aminoácido para a imobilização sobre uma superfície de ouro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Biotin Novatag Resin</td>
			<td>NovaBiochem</td>
			<td>8550510001</td>
			<td></td>
		</tr>
		<tr>
			<td>TORVIQ 10ML LUER LOCK FRITTED SYRINGE</td>
			<td>Fisher</td>
			<td>NC9299151</td>
			<td></td>
		</tr>
		<tr>
			<td>piperdine</td>
			<td>Acros</td>
			<td>P/3520/PB05</td>
			<td></td>
		</tr>
		<tr>
			<td>ninhydrin test</td>
			<td>Sigma-Aldrich</td>
			<td>60017-1ea</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#8217;-Fmoc-amino-ferrocene-1-carboxylic acid</td>
			<td>Omm Scientific</td>
			<td>Special Order</td>
			<td></td>
		</tr>
		<tr>
			<td>1-&#8203;Hydroxybenzotriazole hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>157260-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Diisopropylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D125407-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-&#8203;Diisopropylethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>496219-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Cys(Trt)-OH</td>
			<td>Novabiochem</td>
			<td>8520080025</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T5408</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-ethanedithiol</td>
			<td>Sigma-Aldrich</td>
			<td>2930</td>
			<td></td>
		</tr>
		<tr>
			<td>triisopropyl silane</td>
			<td>Sigma-Aldrich</td>
			<td>233781</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes (20 mL)</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>any source</td>
			<td></td>
			<td>dry with molecular sieves prior to use &#38; store in 100 mL media bottle for easy usage.</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>any source</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

A detecção precoce é uma chave para o sucesso do tratamento da maioria das doenças, e é especialmente indispensável para o diagnóstico e tratamento de vários tipos de cancro. As técnicas mais comuns utilizados são métodos de imagem, como a ressonância magnética (MRI), Positron Emission Topografia (PET), e Topografia Computadorizada (CT) e são ideais para a compreensão da estrutura física da doença, mas só pode ser realizada uma vez a cada quatro a seis semanas, devido à utilização de agentes de imagiologia e de custo global. Com isto em mente, o desenvolvimento de &quot;point of care&quot; técnicas, como biossensores, que avaliam o estágio da doença e / ou a eficácia do tratamento no consultório do médico e fazê-lo em tempo hábil, iria revolucionar os protocolos de tratamento. 1 Como um meio para explorar biossensores ferroceno base para a detecção de moléculas biologicamente relevantes 2, foram desenvolvidos métodos para produzir conjugados de bio-ferroceno-biotina aqui descritos. Este relatório vai se concentrar em um sistema-biotina-ferrocene cisteína que pode ser imobilizada em uma superfície de ouro.

A forma ligada resina de 1 é mostrada na Figura 2. A ligação covalente do componente ferroceno dá origem a uma coloração laranja para as pérolas de resina que é persistente com lavagem contínua e indicativo de um complexo de ferro contendo imobilizada em oposição a absorção de ferro pela PEG componente da esfera de resina. A forma livre da resina-1 é idêntica em cor para as pérolas de resina. A seguir à remoção do composto a partir da resina-esferas, ...

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodologia sintética para Asymmetric Ferroceno Derivados Sistemas Bio-conjugadas via Metodologia base de resina em Fase Sólida

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

Research

JoVE Journal

Chemistry

9.6K visualizações.  Texas Christian University. The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Vídeo: Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

Sapphire celular de alta pressão para medições de Equilíbrio de Fases de CO<sub&gt; 2</sub&gt; / Orgânicos / Sistemas de Água

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

Química

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Estabilização hepatocelular Fenótipo Usando superfícies sintéticas otimizados

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

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

Difração de raios-X em Ciência da Conservação: Rumo a rotina determinação da estrutura cristalina de corrosão produtos na arte Heritage Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

Medição do Tamanho de Partículas Distribuição em turvas Solutions pela Dynamic dispersão de luz Microscopia

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Integração da Extração de Fase Sólida Miniaturizada e Detecção de LC-MS / MS de 3-Nitrotirosina em Urina Humana para Aplicações Clínicas

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Aplicação e metodologia da técnica não-destrutiva 19F no domínio do tempo NMR para medir o teor em flúor-contendo produtos de droga

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to investigate the lipid raft hypothesis. Numerous studies, however, neglect the impact of physiological solution conditions. On that account, the current work presents the effect of high-salinity buffer and trans-membrane solution asymmetry on liquid-liquid phase separation in charged GUVs grown from dioleylphosphatidylglycerol, egg sphingomyelin, and cholesterol. The effects were studied under isothermal and varying temperature conditions.
We describe equipment and experimental strategies applicable for monitoring the stability of coexisting liquid domains in charged vesicles under symmetric and asymmetric high-salinity solution conditions. This includes an approach to prepare charged multicomponent GUVs in high-salinity buffer at high temperatures. The protocol entails the option to perform a partial exchange of the external solution by a simple dilution step while minimizing the vesicle dilution. An alternative approach is presented utilizing a microfluidic device that allows for a complete external solution exchange. The solution effects on phase separation were also studied under varying temperatures. To this end, we present the basic design and utility of an in-house built temperature control chamber. Furthermore, we reflect on the assessment of the GUV phase state, pitfalls associated with it and how to circumvent them.

Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

Comportamento de fase de vesículas carregadas em solução simétrica e assimétrica condições monitoradas com microscopia de fluorescência

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2&#039;-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Protocolo para síntese de fase sólida de oligómeros de ARN contendo um 2&#39;-<em&gt; O</em&gt; -tiofenilmetilo Modificação e Caracterização através do Dicroísmo Circular

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Caracterização de Polímeros Sintéticos via Espectrometria de Massa por Ionização a Laser com Matriz Assistida por Tempo de Voo (MALDI-TOF)

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

Fase sólida síntese de [4,4] Spirocyclic oximas

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...