Michael Henry, Dublin City University, amablemente ayudó con la recopilación de datos y el análisis se muestra en la Figura 5. Este trabajo fue apoyado en parte por (1) George Mason University, (2) el italiano IstitutoSuperiore di Sanita &#39;en el marco de la Italia / EE.UU. convenio de colaboración entre el Departamento de Salud y Servicios Humanos, la Universidad George Mason, y el Ministerio de Salud Pública italiana, (3) de los NIH, subvenciones del programa IMAT 1R21CA137706-01 y 1R33CA173359-01 a LAL, y (4) Nanociencias Ceres, Inc .

El plasma humano y se recogió la orina de donantes voluntarios sanos, con el consentimiento informado por escrito, después de George Mason University Institutional Review Board aprobó protocolos. Los donantes se distribuyeron por igual entre hombres caucásicos y mujeres entre las edades de 25 y 42 muestras fueron analizadas de forma individual y no se agruparon. 1. Nanoparticle Procesamiento de suero o plasma Muestras Biomarcadores abundantes bajas potenciales en plasma son capturados, en solución, con nanopartículas de hidrogel. Las partículas se añaden al plasma, se incubaron, separadas por centrifugación, se lavaron, y las proteínas capturadas se eluyen. Las proteínas eluidas se secan bajo flujo de nitrógeno durante aguas abajo secuenciación de espectrometría de masas y la identificación. <ol><li> Diluir 500 l de suero de 1: 2 con 50 mM Tris HCl pH 7 (500 l de suero + 500 l Tris HCl) en un tubo de microcentrífuga. </li><li> Añadir 500 l de poli (Nipam / AAC) nanop núcleoartículos. Incubar durante 15 min a TA. </li><li> Girar la muestra a 16.100 xg, 25 ° C durante 10 min en una centrífuga equipada con un rotor de ángulo fijo. Retire y deseche el sobrenadante. </li><li> Añadir 500 l tiocianato de sodio (25 mM) al sedimento. Resuspender las nanopartículas con la pipeta vigorosamente hacia arriba y abajo varias veces. </li><li> Girar la muestra a 16.100 xg, 25 ° C durante 10 min en una centrífuga con un rotor de ángulo fijo. Retire y deseche el sobrenadante. </li><li> Añadir 500 l de agua MilliQ al sedimento de nanopartículas. Resuspender las nanopartículas con la pipeta vigorosamente hacia arriba y abajo varias veces. </li><li> Girar la muestra a 16.100 xg, 25 ° C durante 10 min en una centrífuga con un rotor de ángulo fijo. Retire y deseche el sobrenadante. </li><li> Prepare el buffer de elución fresca: Añadir 700 l de acetonitrilo a 300 l de hidróxido de amonio en un tubo limpio. Precaución: El hidróxido de amonio es corrosivo. Utilizar con una ventilación adecuada. Nota: El tampón de elución debe ser prepared inmediatamente antes del uso. No guarde el tampón de elución. </li><li> Añadir 300 l de tampón de elución al sedimento de nanopartículas. Resuspender las nanopartículas con la pipeta vigorosamente hacia arriba y abajo varias veces. Incubar durante 15 min a TA. </li><li> Girar la muestra a 16.100 xg, 25 ° C durante 10 min en una centrífuga con un rotor de ángulo fijo. Retire y guarde el eluido en un tubo de microcentrífuga limpio, etiquetado. </li><li> Repita los pasos 1.9 a 1.10. Combinar los dos eluidos en un tubo de microcentrífuga. </li><li> Secar los eluidos bajo flujo de nitrógeno en un colector del evaporador de nitrógeno a 42,1 ° C, con flujo de aire se establece en 8 (Figura 2F). </li><li> Almacenar el producto eluido se secó a temperatura ambiente durante O / N de almacenamiento, o a -20 ° C para almacenamiento a largo plazo, antes de la espectrometría de masas, el Western Blot, o ensayos ELISA (opcional). </li></ol> 2. Procesamiento de nanopartículas de Muestras de orina La orina normal contiene menos de 30 mg / dl de proteína ymenos de 1+ sangre. Sin embargo, muchas enfermedades / condiciones pueden alterar los niveles normales de proteína en la orina y la sangre. Para ayudar en la determinación del volumen óptimo de nanopartículas para añadir a la muestra de orina, un análisis de orina se lleva a cabo antes de la recolección de nanopartículas. Pueden existir biomarcadores de orina en concentraciones extremadamente bajas, que pueden requerir la optimización de la relación de las nanopartículas con el volumen de orina. Este procedimiento describe nanopartículas recolección de muestras de orina para el análisis de transferencia de Western aguas abajo. <ol><li> Recoger muestras de orina en un vaso de plástico limpio y seco. Se requiere un volumen mínimo de 22 ml. Orina conserve las muestras a -80 ° C hasta el momento de analizar. </li><li> Descongele la orina congelada a temperatura ambiente oa 4 ° CO / N. Mezclar brevemente en un vórtex. Vierta al menos 22 ml de orina en un tubo de polipropileno de fondo cónico de 50 ml. </li><li> Gira la orina en una centrífuga con un rotor oscilante en 3700 xg durante 15 min. Decantar la orina, sin perturbar el sedimento, en un polypr fondo cónico de 50 ml limpiotubo opylene. Deseche el sedimento. </li><li> Realice un análisis de orina usando un multi-analito &quot;tira reactiva de orina&quot; tira reactiva. Nota: almacene las tiras reactivas en un recipiente herméticamente cerrado, protegido de la luz solar directa y la humedad 17, 18. <ol><li> Coloque la tira reactiva, boca arriba, sobre una toalla de papel limpia y seca. </li><li> Usar una pipeta desechable para aspirar 1 ml de la orina preparado en la etapa 2.3. </li><li> Establecer un temporizador durante 2 minutos, pero no lo inicie. Dispensar rápidamente 1 - 2 gotas de orina en cada almohadilla de prueba de la tira de reactivo. Iniciar inmediatamente el temporizador. Nota: No sumerja la tira reactiva directamente en el recipiente de la orina. Los indicadores de colorante / químicas en la tira de reactivo potencialmente pueden filtrarse fuera de la tira de reactivo en el recipiente de orina. </li><li> A la hora indicada en el envase tira de reactivo, registrar los resultados cualitativos para los diversos analitos en la tira de reactivo mediante la comparación del color de las tiras reactivas individuales en los correspondientes indica un código de coloresres en el recipiente de reactivo. Resultados de orina normales típicos son: (normal o negativo) de sangre, proteínas, leucocitos, nitrito, glucosa, cetona, bilirrubina y urobilinógeno; PH de la orina (5,5 a 7,0); gravedad específica (1,001-1,020). Nota: Los niveles altos de proteína en una muestra de orina, pueden competir con la proteína de interés en los puntos de las nanopartículas vinculante. Para maximizar la cosecha proteína con nanopartículas, la relación de volumen de volumen de nanopartículas / orina se puede ajustar a cualquiera de limitar la competencia de las proteínas de alta abundancia, o puede ser optimizado para cosechar las proteínas que existen en concentraciones muy bajas. Si el valor de proteína en la orina es 1 + o mayor, añadir volúmenes 2x de las nanopartículas en el paso 2.5 a continuación. Si el analito de interés es una proteína abundante muy bajo, puede ser necesario aumentar el volumen de las nanopartículas hasta 2 ml (Figura 3). </li></ol></li><li> Transferir 20 ml de la orina aclarado desde el paso 2.3 en un tubo de polipropileno de 50 ml de fondo cónico limpio. Noperturbar cualquier residuo / pellet que puede estar en la parte inferior del tubo de la orina. Añadir 200 l de nanopartículas a la muestra 20 ml de orina. Mezclar brevemente en un vórtex. Nota: Si el valor de proteína en la orina es 1 + o mayor, añadir 400 l de nanopartículas a 20 ml de orina. </li><li> Incubar la mezcla de orina / nanopartícula durante 30 min a RT, sin balanceo / mezcla. </li><li> Haga girar la suspensión orina / nanopartícula en una centrífuga equipada con un rotor oscilante en 3700 xg durante 10 min. Nota: Si un pellet no es visible después de la centrifugación, girar las muestras para un adicional de 2 - 7 min. </li><li> Retire y deseche el sobrenadante. Añadir 500 l de agua MilliQ al sedimento de nanopartículas. </li><li> Resuspender las nanopartículas con la pipeta vigorosamente hacia arriba y abajo varias veces. Transferir la solución de nanopartículas a un tubo de microcentrífuga de 1,5 ml limpio. </li><li> Girar las nanopartículas en una centrífuga equipada con un rotor de ángulo fijo a 16.100 xg durante 10 min. Nota: Si un pellet no es visible folLowing centrifugación, girar las muestras para un adicional de 2 - 7 min. </li><li> Retire y deseche el sobrenadante. </li><li> Repita los pasos 2,8 y 2,11 (dos veces) con 200 l 18 MilliQ agua para lavar las nanopartículas. </li><li> Prepare el buffer de elución fresca: Añadir 970 l de acetonitrilo a 30 l de hidróxido de amonio en un tubo limpio. Precaución: El hidróxido de amonio es corrosivo. Utilizar con una ventilación adecuada. Nota: El tampón de elución debe prepararse inmediatamente antes de su uso. No guarde el tampón de elución. </li><li> Añadir 20 l de tampón de elución al sedimento de nanopartículas. Resuspender las nanopartículas con la pipeta vigorosamente hacia arriba y abajo varias veces. Incubar durante 15 min a TA. </li><li> Haga girar las muestras de nanopartículas a 16.100 xg durante 10 min. Retire y guarde el sobrenadante en un tubo de microcentrífuga de 1,5 ml limpio. No perturbar el sedimento de nanopartículas. Deseche el sedimento en la basura de riesgo biológico. </li><li> Colocar las muestras en un estante en una campana para vapores químicos. Abra las tapas y aumUbaté a TA durante 30 min. Alternativamente, colocar las muestras bajo flujo de nitrógeno a 40 ° C hasta que se seque (1 - 2 h). </li><li> Añadir 15 l de Tris-glicina SDS tampón de muestra (2x) a las muestras. Se calienta a 100 ° C en un bloque de calor seco durante 5 minutos con las tapas abiertas o hasta que el volumen que queda en el tubo no es más de 20 l. Nota: no utilizar un baño de agua hirviendo. La humedad del baño de agua será evitar la evaporación de la memoria intermedia. </li><li> Coloque la tapa en los tubos. Sacar los tubos del bloque de calor. La muestra puede ser almacenada a -80 ° C o utilizarse inmediatamente para el análisis de transferencia de western aguas abajo. </li></ol>

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F., Refson, J., Demoliou-Mason, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor+(PDGF):+actions+and+mechanisms+in+vascular+smooth+muscle.">Platelet-derived growth factor (PDGF): actions and mechanisms in vascular smooth muscle.</a> Gen Pharmacol. 27 (7), 1079-1089 (1996).</li><li>Izycki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+levels+of+IGF-I+and+IGF-II+in+patients+with+lung+cancer+during+chemotherapy.">Serum levels of IGF-I and IGF-II in patients with lung cancer during chemotherapy.</a> Exp Oncol. 26 (4), 316-319 (2004).</li><li>Jalosinski, M., Karolczak, K., Mazurek, A., Glabinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+methylprednisolone+and+mitoxantrone+on+CCL5-induced+migration+of+lymphocytes+in+multiple+sclerosis.">The effects of methylprednisolone and mitoxantrone on CCL5-induced migration of lymphocytes in multiple sclerosis.</a> Acta Neurol Scand. 118 (2), 120-125 (2008).</li><li>Kitazume, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+insights+into+beta-galactoside+alpha2,6-sialyltransferase+secretion+in+vivo.">Molecular insights into beta-galactoside alpha2,6-sialyltransferase secretion in vivo.</a> Glycobiology. 19 (5), 479-487 (2009).</li><li>Saito, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+hepatic+NKT+cells+in+leukocyte+cell-derived+chemotaxin+2-deficient+mice+contributes+to+severe+concanavalin+A-induced+hepatitis.">Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.</a> J Immunol. 173 (1), 579-585 (2004).</li><li>Struyf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARC/CCL18+is+a+plasma+CC+chemokine+with+increased+levels+in+childhood+acute+lymphoblastic+leukemia.">PARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia.</a> Am J Pathol. 163 (5), 2065-2075 (2003).</li><li>Michael, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tissue+kallikrein+5+is+a+member+of+a+proteolytic+cascade+pathway+involved+in+seminal+clot+liquefaction+and+potentially+in+prostate+cancer+progression.">Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression.</a> J Biol Chem. 281 (18), 12743-12750 (2006).</li></ol>

Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

Importancia clínica Se cree que una muestra de suero o plasma para contener baja abundancia de proteínas y péptidos que pueden proporcionar una rica fuente de información sobre el estado del organismo en su conjunto en circulación. A pesar de la promesa de la proteómica en suero, hay tres barreras fisiológicas fundamentales y serios que frenan el descubrimiento de biomarcadores y la traducción al beneficio clínico 10, 11, 16, 25. <p class="jove_conten...

A pesar de la finalización de la secuenciación del genoma humano, no se ha logrado avances significativos en la identificación de biomarcadores predictivos de la enfermedad en etapa temprana, o que se correlacionan con el resultado terapéutico o pronóstico 1. Una razón para esta falta de progreso es que existen muchas biomarcadores potencialmente significativos a una concentración por debajo del límite de detección de espectrometría de masas convencional y otras plataformas de descubrimiento de biomarcadores. Espectrometría de masas (MS) y Monitoreo de Reacción Múltiple (MRM) tienen una sensibilidad de detección típicamente mayor que 50 ng / ml, mientras que la mayoría de los analitos medidos por inmunoensayos en una caída de laboratorio clínico en el intervalo entre 50 pg / ml y 10 ng / ml . Esto significa que muchos biomarcadores, en particular en la etapa temprana de una enfermedad no pueden ser detectados por MS convencional y MRM 2. Además, la presencia de proteínas de alta abundancia tales como la albúmina y la inmunoglobulina en fluidos biológicos complejos, con frecuencia máscara por exc mil millones de vecesess de baja abundancia, proteínas de bajo peso molecular y péptidos 3, 4. Por esta razón se requieren varias etapas preparatorias de la muestra antes de la secuenciación de espectrometría de masas y la identificación. Uno de tales paso preparatorio emplea el agotamiento de proteínas de alta abundancia con columnas de agotamiento disponibles comercialmente 5-8. Desafortunadamente este paso conduce a la reducción del rendimiento de biomarcadores candidatos porque son a menudo asociadas no covalentemente con proteínas portadoras que están siendo eliminados. Otro desafío está representado por la estabilidad de biomarcadores candidatos ex vivo una vez que se recogieron las muestras. Las proteínas están sujetos a la degradación por proteasas endógenas o exógenas 9. Nanopartículas hidrogel puede superar estos retos críticos mediante la amplificación de la concentración de biomarcador putativo a un nivel dentro del rango del ensayo, mientras que la protección de la proteína de la degradación 10-13. Es importante señalar ªLMW en proteínas en la sangre son una mezcla de proteínas intactas pequeñas, así como fragmentos de proteínas grandes. Proteínas del tejido derivado de más de 60 kDa son demasiado grandes para entrar pasivamente el flujo de sangre a través de la membrana basal vascular, pero pueden ser representados en la sangre como péptidos o fragmentos de proteínas 14. Nuestro objetivo es medir los biomarcadores novedosos de circulación que pueden ser candidatos para la detección temprana de la enfermedad, estratificación de los pacientes para la terapia y el seguimiento de la respuesta al tratamiento. Nuestros nanopartículas se crean para excluir selectivamente inmunoglobulinas alta abundancia y albúmina, mientras que la captura simultáneamente proteínas y péptidos más pequeños y concentrándolos hasta 100 veces, dependiendo del volumen de partida. Nuestro grupo identificó un conjunto de pequeñas colorantes orgánicos que pueden actuar con éxito como de alta afinidad cebos moleculares para proteínas y péptidos. Colorante de unión a proteínas se cree que es debido a una combinación de interacción hidrófoba y electrostáticas. Los anillos aromáticos en el tinte se intercalan con las proteínas a través de bolsillos hidrófobos sobre la superficie de la proteína 11. Los cebos, dependiendo de su química, muestran una afinidad particular para las clases de analitos seleccionados. Los cebos compiten con las proteínas portadoras, como la albúmina, para las proteínas o péptidos. El peso de proteínas / péptidos de bajo peso molecular se quedan atrapados en la partícula. Proteínas de alto peso molecular tales como la albúmina y la inmunoglobulina se impide la entrada de la partícula debido a la capacidad de tamizado debido a la restrictiva de poro del hidrogel 11 (Figura 1). Nanopartículas de hidrogel son sintetizados por polimerización por precipitación iniciada por persulfato de amonio 11. N-isopropilacrilamida (Nipam), co-monómeros de ácido acrílico (AAC) y alilamina (AA) y el agente de reticulación N, N &#39;-Methylenebisacrylamide (BIS) se dejó reaccionar a 70 ° C durante 6 horas en condiciones diluidas 11, 13. La afinidad de unión de alta proteína de poli (N-isopropilacrilamida-co-ácido acrílico) (poli (NIPAM-co-AAC) nanoparticlesis logrado mediante la incorporación de covalentemente colorantes que contienen grupos amino (es decir., Colorantes sulfonatedanthraquinonetriazine) en las nanopartículas a través una reacción de amidación se realiza en disolventes acuosos u orgánicos dependiendo de las características hidrófilas / hidrófobas de los colorantes 11, se utiliza 13. sustitución nucleofílica de los grupos amina en la nanopartícula con el átomo de cloruro de un colorante anthraquinonetriazine para crear poli contiene el colorante (Nipam -CO-alilamina) (AA) nanopartículas 11, 12. Un procedimiento de polimerización de dos etapas se utiliza para crear nanopartículas de hidrogel que contiene una capa exterior de ácido vinilsulfónico (VSA) 11, 13. Nanopartículas de hidrogel se pueden aplicar a diversos fluidos biológicos, incluyendo sangre entera, plasma, suero, fluido cerebroespinal, sudor, y orina. En un solo paso, en solución, la nanoparticles realizan una rápida (en minutos) el secuestro y la concentración de analitos de bajo peso molecular 10, 11, 13, 15-18. Las proteínas se eluyeron posteriormente de las nanopartículas y se detectaron usando Western Blot 19-21, espectrometría de masas 10, 11, 13, 15, 18, ​​22, 23, inmunoensayos / ELISA 10, 11, 15, 18, ​​o microarrays de proteínas de fase inversa 16, 24 ensayos. Las nanopartículas funcionalizadas con el cebo químico, y la presentación de un núcleo o núcleo de la arquitectura cáscara, captura y se concentran las proteínas sobre la base de las propiedades fisicoquímicas de cebo / corteza. Diferentes colorantes incorporados en las nanopartículas, por lo tanto capturar diferentes subconjuntos de proteínas con eficiencia basado en la afinidad de tinte, el pH de la solución, y la presencia / ausencia de competir proteínas de alto abundante 13 variable. Además, la cantidad de nanopartículas en relación con el volumen de la solución afectará a la producción de proteína a partir de las nanopartículas. Estos aspectos dehidrogel nanopartícula cosecha se demostró utilizando tres cebos nanopartículas diferentes para las proteínas de recolección de muestras de plasma que contienen altas cantidades de proteínas, y de las muestras de orina que normalmente no contienen grandes cantidades de proteína. En este protocolo se demuestra la recolección y la concentración de factor de necrosis tumoral alfa (TNFa) a partir de muestras de plasma utilizando poli (NIPAM-co-AAC), poli (NIPAM / tinte), y las nanopartículas core-shell (poli (NIPAM-co-VSA)) . Poli (NIPAM / colorante) nanopartículas se muestran para concentrar el antígeno especies Mycobacterium que se añadió a las muestras de orina humanos, para imitar los individuos infectados por el Mycobacterium tuberculosis.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

hydrogel nanoparticle harvesting of plasma or urine for detecting low abundance proteins

El descubrimiento de biomarcadores Novela juega un papel crucial en la prestación de detección de la enfermedad más sensible y específica. Desafortunadamente muchos biomarcadores de baja abundancia que hay en fluidos biológicos no pueden ser fácilmente detectados con espectrometría de masas o inmunoensayos, ya que están presentes en muy baja concentración, son lábiles, y son a menudo enmascarados por proteínas de alta abundancia tales como albúmina o inmunoglobulina. Bait contiene poli (N-isopropilacrilamida) (Nipam) nanopartículas a base son capaces de superar estas barreras fisiológicas. En un paso que son capaces de captar, concentrar y preservar biomarcadores de fluidos corporales. -Analitos de bajo peso molecular entran en el núcleo de la nanopartícula y son capturados por diferentes tintes químicos orgánicos, que actúan como cebos de proteína de alta afinidad. Las nanopartículas son capaces de concentrar las proteínas de interés en varios órdenes de magnitud. Este factor de concentración es suficiente para aumentar el nivel de proteína de tal manera que las proteínas están dentro de lalímite de detección de los espectrómetros de masas actuales, el Western Blot, e inmunoensayos. Las nanopartículas se pueden incubar con una plétora de fluidos biológicos y que son capaces de enriquecer en gran medida la concentración de proteínas y péptidos de bajo peso molecular mientras que excluye la albúmina y otras proteínas de alto peso molecular. Nuestros datos muestran que una amplificación de 10.000 veces en la concentración de un analito particular se puede lograr, lo que permite espectrometría de masas y los inmunoensayos para detectar biomarcadores previamente indetectables.

Hidrogel Nanoparticle Tamaño y Uniformidad Poli (Nipam-AAC) partículas se han producido con muy alto rendimiento y reproducibilidad entre y dentro de los lotes. Las partículas tienen muy buena estabilidad coloidal a temperatura ambiente durante el tiempo necesario para la captura, almacenamiento, y la elución de las proteínas (por lo menos 48 h), y la precipitación de nanopartículas no se ha observado (Figura 1) 11. La estabilidad coloidal ...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Hidrogel Nanoparticle recolección de plasma o de orina para la detección de proteínas de baja abundancia

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

hydrogel-nanoparticle-harvesting-plasma-or-urine-for-detecting-low

Research

JoVE Journal

Bioengineering

13.8K vistas.  George Mason University. Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Video: Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

La litografía de plasma superficial Patrones de Creación de redes de telefonía

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Investigación

Bioingeniería

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Diseño y Construcción de Artificial matriz extracelular (AECM) Las proteínas de<em&gt; Escherichia coli</em&gt; Para la piel Ingeniería de Tejidos

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D hidrogel andamios para articular condrocitos cultura y cartílago Generación

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

a base de varilla de fabricación de dispositivos robóticos adaptables pinza neumática suaves para la manipulación del tejido delicado

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted membrana plasmática Entrega de cargo encapsulado hidrofóbico en un cristal líquido portador de nanopartículas

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Patrones bioactivas proteínas o péptidos en hidrogel con fotoquímica para usos biológicos

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Inyección de nanopartículas de Retroductal a la glándula submaxilar de ratón

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

Aparato para la recolección de tejido Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabricación de un hidrogel de derivados de matriz extracelular renal corteza

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...