KG fue apoyado por la subvención P-1760 Fundación RA Welch, TCU Instituto Andrews de Matemáticas y Educación la Ciencia (a KG), TCU Investigación y Creatividad Actividad Grant (a KG) y TCU SERC Grant (a JHS).

1. Síntesis de biotina-Fc-cisteína (1) <ol><li> Métodos en fase sólida para producir unidos a la resina 1. <ol><li> Coloque resina cargada biotina (250 mg, 0,145 mmol) en una jeringa de vidrio fritado y se hinchan la resina mediante la elaboración de dimetilformamida (5 ml) y agitando la jeringa en un agitador de laboratorio durante 20 min. Expulsar a la solución y repita dimetilformamida hinchazón una vez más. </li><li> Eliminar el grupo protector Fmoc mediante la adición de 4-6 ml de 20% de piperidina en dimetilformamida a la jeringa seguido de 10-15 min de agitación. Repita el proceso de desprotección con otro 4-6 ml de piperidina. Lavar la resina con una secuencia de 3x dimetilformamida, 3x dimetilformamida: metanol (1: 1), metanol 3x: diclorometano (1: 1), 3x diclorometano, ~ 5 ml cada uno. Hacer una prueba de la ninhidrina (+) en una pequeña muestra (~ 10) de las perlas para confirmar la desprotección con éxito por la presencia de azul después del calentamiento. </li><li> Mezcle una solución que contiene ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico(203,3 mg, 0,4350 mmol), 1-hidroxibenzotriazol hidrato (58,8 mg, 0,413 mmol), diisopropil carbodiimida (0,0673 ml, 0,435 mmol), diisopropiletilamina (0,0757 ml, 0,435 mmol), y una mezcla 4: 1 de diclorometano y dimetilformamida. Dibuje esta en la jeringa poroso y agite suavemente en un agitador de laboratorio durante 6 horas. Entonces expulsar la solución de la jeringa y lavar como se describió anteriormente. </li><li> Realizar la prueba de la ninhidrina (-) como se describe anteriormente para confirmar acoplamiento. La prueba de la ninhidrina todavía puede ser útil para confirmar acoplamiento a pesar del color anaranjado de la perla derivado de la unión del resto que contiene hierro. </li><li> A continuación, eliminar el grupo Fmoc mediante la adición de 20% de piperidina en dimetilformamida y se lavó como se describe anteriormente. El ensayo de ninhidrina (+) se debe utilizar para confirmar la eliminación de Fmoc. </li><li> Preparar una solución compuesta de Fmoc-Cys (Trt) -OH (254,8 mg, 0,4350 mmol), 1-hidroxibenzotriazol hidrato (58,8 mg, 0,4125 mmol), diisopropil carbodiimida (0,0673 ml, 0,4350mmol), diisopropiletilamina (0,0757 ml, 0,4350 mmol), y una mezcla 4: 1 de diclorometano y dimetilformamida. Añadir este cóctel acoplamiento cisteína la jeringa poroso y agite suavemente durante 6 horas. Lave usando el protocolo descrito anteriormente. </li><li> Confirmar el acoplamiento mediante la prueba de la ninhidrina (-), seguido de la eliminación del componente de Fmoc con piperidina al 20% y lavado. Verifique el terminal libre amina mediante la prueba de la ninhidrina (+). </li></ol></li><li> La escisión de 1 de la resina. <ol><li> Prepare una solución de TFA (9,45 ml), agua (0,25 ml), 1,2-etanoditiol (0,25 ml), y silano triisopropilo (0,1 ml), añadirlo a la jeringa y agite suavemente durante 4 horas. </li><li> Recoger la solución rojo-marrón resultante en un tubo Eppendorf y se evapora el TFA lentamente usando una corriente de aire. </li><li> Añadir éter dietílico frío (~ 15 ml) al tubo Eppendorf para precipitar 1, que formará agitación suave. Aislar el producto a través de centrifugación (1 g, 5 min). Trepetición de gallina ciclos de lavados de éter de dietilo (~ 60 ml total) y centrifugar para obtener 1 como un sólido rojo / marrón. </li></ol></li></ol> 2. Caracterización y Análisis de 1 <ol><li> Confirmar que la identidad coincide con la conectividad y la composición que se muestra en la Figura 2 usando 1 H (16 scans) y 13 C NMR (512 exploraciones) en metanol deuterado (300 l) y el análisis ESI-MS. Cuente con los siguientes resultados: Espectro de RMN 1H (CD3OD) 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) y 13 C espectro de RMN (CD3OD) δ / ppm: 24,644 (CH 2), 25.472 (CH 2), 28,051 (CH 2), 28,300 (CH 2), 35,474 (CH 2), 38,698 (CH 2), 39,241 (CH 2), 39,717 (CH 2), 55,340 / 55,538 (anillo Cp), 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 (anillo Cp), 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] + y HR-MS (m / z): Encontrado: 617,2049 [1 + H] +, teórico: 617.1622 [1 + H] +. </li><li> Realizar HPLC, análisis elemental para confirmar la composición de aislado 1. Llevar a cabo cromatogramas de HPLC usando una columna de fase inversa C8 con 100% de MeOH a un caudal de 0,5 ml / min. Nota: los tiempos de retención de HPLC fueron: 3,198 a 4,674 min. </li></ol> 3. Inmovilización de 1 sobre una superficie de oro <ol><li> Polímero Cut respaldado diapositivas de oro en cuadrados de ~ 0,25 en 2. </li><li> Llenar un vaso de precipitados de 50 ml con una solución de agua DI de 1 (~ 1 mM). </li><li> Añadir la diapositiva de oro al vaso de precipitados y cubrir con un vidrio de reloj. Todosow el portaobjetos de vidrio para incubar O / N a temperatura ambiente sin agitación. </li><li> Retire la diapositiva oro de la solución y permitir que se seque al aire. </li><li> Obtener la digitalización de imágenes de microscopía electrónica utilizando un microscopio electrónico de barrido (o equivalente) para observar inmovilizado 1. </li></ol>

The authors have nothing to disclose.

La síntesis de derivados del ferroceno asimétricas es un reto en solución. Por ejemplo, los intentos para producir 1 en solución resultó en bajos rendimientos del producto deseado (menos de 20%). Asimismo, las reacciones que utilizan el ácido carboxílico 1&#39;-amino-ferroceno (sans Fmoc) y resina de biotina unido resultaron en producto insoluble consistente con el producto polimerizado reportado por Baristic et al. y el producto mínimo 47. Esto se complica aún más...

Los biosensores son dispositivos pequeños que emplean la tecnología de reconocimiento biomolecular como la plataforma para el análisis selectivo y se utilizan por su especificidad, velocidad y bajo costo. Biosensores electroquímicos para la detección de biomoléculas están a la vanguardia de este campo debido a su simplicidad, eficacia de costes, y alta sensibilidad. 1,3 La anatomía general de estos sensores es un electrodo equipado con una molécula de reconocimiento específica para el marcador biológico de interés . La unión del biomarcador por la molécula de reconocimiento se traduce en un cambio local de potencial o corriente que puede ser detectado por medición simple. Hasta la fecha, el fragmento de reconocimiento puede variar de enzimas, anticuerpos 4-8, 9-12 células enteras, 13-16, 17-20 receptores péptidos 21-23 y 24 de ADN y se han centrado en gran medida de mayor tamaño, las moléculas biológicas. 25-28 Investigación los esfuerzos en este campo se han concentrado principalmente en inmunosensores where una inmunoglobulina es inmovilizado con un núcleo activo redox (tal como ferroceno) y se utiliza para detectar un anticuerpo de interés. Estos estudios han sido excluidos de las aplicaciones clínicas debido a la escasa precisión y tiempo de consumo derivados de las complicaciones derivadas de la utilización de antígenos / anticuerpos. 1,3 creciente atención se ha centrado en la detección de moléculas pequeñas (menos de 1 kg / mol) de biomédica , la comida y el interés ambiental, además de la seguridad nacional. 29 Los ejemplos más conocidos de dispositivos biosensores son monitores de glucosa en la auto-prueba, que tienen serigrafiadas electrodos enzimáticos acoplados a un metro amperométrica bolsillo. Estos sistemas utilizan típicamente un método coulométrica donde se mide la cantidad total de carga generada por la reacción de oxidación de la glucosa durante un período de tiempo. Dispositivos negociables deben ser portátil, robusto y de mano para hacer un uso fácil para la población en general. Etiquetas redox tales como ferroceno son necessary para proporcionar la detección electroquímica de biomarcadores o pequeñas moléculas en solución como la mayoría de los biomarcadores no son intrínsecamente electroquímicamente activo. 30-38 ferroceno es una molécula organometálica que es un estándar de oro para la electroquímica, lo que hace que sea una excelente opción para la integración en biosensores electroquímicos. Especies activas redox a base de ferroceno ya han ganado considerable atención debido a su pequeño tamaño, buena estabilidad, el acceso sintético conveniente, modificación química fácil, lipofilicidad relativa, y la facilidad de sintonización redox. 3,30-42 moléculas pequeñas basado en el núcleo ferroceno tiene han utilizado ampliamente como detectores de iones metálicos y moléculas pequeñas. 32-38,43 sistemas de focalización especies más grandes, tales como biomoléculas han utilizado la fijación de grandes anticuerpos o inmunoglobulinas a derivados del ferroceno que han sido incrustados en una superficie electroquímica. 1,3,39 , 44 En cada caso, la intensi potencial y actualty de la pareja redox Fe III / Fe II se alteró tras el acoplamiento molecular, produciendo de este modo un nuevo mango espectroscópica que indica la presencia de la molécula de analito. Este cambio se debe a la amplia superposición que se produce entre el sistema pi de los anillos de ciclopentadienilo y las de hierro orbitales d. Si el sistema pi se modifica, es decir, derivado o se hace reaccionar, a continuación, la interacción orbital, a su vez, el cambio. Esto afectará el núcleo Fe y puede ser observada como un cambio en el potencial del par Fe III / Fe II. 40,45,46 Estas propiedades hacen que un sistema de este tipo atractivo para su uso como un agente de cuantificación en un inmunoensayo electroquímico o biosensor. Con el fin de producir sistemas de ferroceno que contiene específicas para capacidades de biosensores es óptimo para modificar un anillo de Cp con el específico para una molécula diana bio-receptor y utilizar el otro anillo Cp como una correa de sujeción molecular para la lectura electroquímica o elecelectrodo (Figura 1). La síntesis de estos derivados de ferroceno asimétricas es desafiada por reacciones secundarias y la formación de especies diméricas y poliméricas formadas sobre la reticulación intermolecular. 47 Sin embargo, la química de acoplamiento de la producción de un enlace amida es la ruta más directa para proporcionar derivados simples de ferroceno que implican componentes biológicos tales como péptidos y sus metabolitos. Por lo tanto, las técnicas de fase sólida desarrollados por primera vez en la década de 1950 por Merrifield de síntesis de péptidos se pueden aplicar a los compuestos organometálicos que contienen ferroceno. Mediante el uso de la molécula de ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico ortogonalmente sustituido, un sistema de ferroceno que puede contener un resto receptor (biotina), lectura electroquímica (ferroceno), y el componente de inmovilización-enlazador (cisteína) tiene ha construido y se detalla en este documento. La síntesis de este bio-conjugado se discute, así como pruebas para la inmovilización en una superficie de oro. Este represen trabajots la primera presentación de un sistema compuesto de biotina, ferroceno y un aminoácido para la inmovilización en una superficie de oro.

<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

La detección temprana es la clave para el éxito del tratamiento de la mayoría de las enfermedades, y es particularmente imprescindible para el diagnóstico y el tratamiento de muchos tipos de cáncer. Las técnicas más comunes utilizados son Imaging modalidades tales como imágenes por resonancia magnética (MRI), tomografía por emisión de Topografía (PET), y computarizada Topografía (TC) y son óptimas para la comprensión de la estructura física de la enfermedad, pero sólo se pueden realizar una vez cada cuatro a seis semanas debido a la utilización de agentes de formación de imágenes y el coste total. Con esto en mente, el desarrollo de &quot;punto de atención&quot; técnicas, como biosensores, que evalúan el estado de la enfermedad y / o la eficacia del tratamiento en el consultorio del médico y lo hacen de una manera oportuna, revolucionaría protocolos de tratamiento. 1 Como un medio para explorar biosensores basados ​​ferroceno para la detección de moléculas biológicamente relevantes 2, se han desarrollado métodos para producir bio-conjugados ferroceno-biotina descritos en este documento. Este informe se centrará en un sistema biotina-ferroceno-cisteína que puede ser inmovilizada sobre una superficie de oro.

La forma unida a resina de 1 se muestra en la Figura 2. La unión covalente del componente de ferroceno da lugar a una coloración naranja a las perlas de resina que es persistente con lavado continuo e indicativo de una plancha inmovilizada que contiene el complejo en comparación con la absorción de hierro por el componente PEG de la perla de resina. La forma libre de resina de 1 es idéntico en color a las perlas de resina. Después de la eliminación del compuesto ...

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.

Metodología sintética para asimétrica Ferroceno derivados Sistemas de Bio-conjugadas vía sólida metodología a base de resina de fase

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 vistas.  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.

Video: 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.

De alta presión de la célula de zafiro para la Fase Equilibrios mediciones de CO<sub&gt; 2</sub&gt; / Orgánicos / Sistemas de Agua

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

Investigación

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.

Estabilizar hepatocelular Fenotipo Usando superficies sintéticas optimizadas

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.

Difracción de polvo de rayos X en Ciencias de la Conservación: Hacia rutina de determinación de estructura cristalina de los productos de corrosión de objetos de arte del patrimonio

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.

Medición de Tamaño de Partícula de distribución en turbias Soluciones por Dynamic Light Scattering Microscopía

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.

Integración de la Extracción de Fase Sólida Miniaturizada y la Detección de LC-MS / MS de 3-Nitrotirosina en Orina Humana para Aplicaciones 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.

Aplicación y metodología de la técnica de RMN no destructivos 19F dominio del tiempo para medir el contenido en medicamentos que contienen flúor

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.

Comportamiento de la fase de vesículas cargadas en solución simétrica y asimétrica condiciones monitoreadas con microscopía de fluorescencia

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 la síntesis en fase sólida de oligómeros de ARN que contiene un 2&#39;-<emO</em&gt; -tiofenilmetilo Modificación y caracterización mediante 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.

Caracterización de polímeros sintéticos vía matriz asistida por láser desorción de ionización tiempo de vuelo (MALDI-TOF) espectrometría de masas

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.

Síntesis en fase sólida de oximas espirocícliclos [4,4]

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 ...