This work was supported by NSF/DBI-1255991 grant to IML.

1. Preparación del capilar Microreactor <ol><li> Cortar el diámetro interior 100 mm (ID) x capilar 360 micras de diámetro exterior (OD) a una longitud de 7-8 cm, y el 20 micras ID x 90 micras OD capilar a 3-5 cm con la cuchilla de capilar de vidrio; verificar en el microscopio que ambos extremos capilares tienen un corte limpio y recto, sin las rebabas que sobresalen. </li><li> Insertar el 20 micras ID x 90 micras capilar OD en un extremo de la 100 micras ID x 360 micras capilar OD, para una longitud de ~ 6 mm; en caso de necesidad, observar esta operación bajo un microscopio. </li><li> Aplique una pequeña gota de pegamento E6000 alrededor de la unión de los capilares 90 micras OD / ID 100 micras con el fin de un Q-tip, y dejar que la cura pegamento O / N a temperatura ambiente. </li><li> Cortar el 20 micras ID x 90 micras capilar OD insertado a una longitud de ~ 10-15 mm; este será el emisor de ionización electrospray. </li><li> -Pre cortar el 1/32 &quot;OD PEEK, 1/16&quot; PEEK OD y OD bañera de PTFE 1/16 &quot;ing en trozos de 4-5 cm de longitud; estos serán los manguitos utilizados en las uniones capilares para proporcionar una conexión libre de fugas. </li><li> Conectar el extremo opuesto de la 100 micras ID x 360 micras OD capilar a una unión PEEK; utilizar un manguito de 1/32 &quot;PEEK para proporcionar una conexión libre de fugas. </li><li> Insertar / apretar un pedazo de ~ 5 cm de largo tubo de PTFE 1/16 &quot;en el extremo opuesto de la unión. </li><li> Pesar ~ 4 mg de C18 (5 micras) partículas en un pre-limpiado / secado 2 ml vial de vidrio; añadir 0,5 ml de isopropanol, cerrar el vial y dispersar la suspensión en el baño de ultrasonidos. </li><li> Retirar con una jeringa de 250 l, aproximadamente 200 l de suspensión, insertar la aguja de la jeringa en el &quot;tubo de PTFE 1/16 de la unión PEEK, y dispensar lentamente la suspensión en el 100 micras DI x 360 micras capilar OD; observar bajo el microscopio como el capilar se llena con partículas de hasta una longitud de mm embalaje 2-3 se consigue, lo que será el microrreactor (Figura 1) el 20 μ.capilar m Identificación retiene las partículas en el micro-reactor a través de un efecto de distorsión trapezoidal. 15 </li><li> Enjuague el micro-reactor con solución de ~ 50 l de H2O / CH3CN 50:50 v / v, y luego con solución de ~ 50 l de H2O / CH3CN 98: 2 v / v. </li></ol> 2. Preparación de las soluciones de muestra Nota: Las operaciones que impliquen la manipulación de disolventes y ácidos orgánicos y preparación de la solución se van a realizar en una campana extractora. Llevar gafas, guantes y ropa de protección. <ol><li> Enjuague con CH 3 OH y secar unos viales de vidrio (4 ml) y los tubos de polipropileno (15 ml). </li><li> Preparar 10 ml de una solución de H 2 O / CH 3 CN (98: 2 v / v) en un tubo de 15 ml de polipropileno por mezcla de 9,8 ml de H 2 O con 200 l CH 3 CN; mezclar mediante ultrasonidos. </li><li> Preparar 10 ml de una solución de H 2 O / CH 3 CN (50:50 v / v) en un tubo de 15 ml de polipropileno por mezcla de 5 ml H 2 &lt;/ sub&gt; O con 5 ml de CH3CN; mezclar mediante ultrasonidos. </li><li> Preparar 4 ml de una solución acuosa acidificada (TFA 0,01% v / v) mediante la adición de 4 l de TFA (10%) a 4 ml H 2 O / CH 3 CN (98: 2 v / v) en un vial de vidrio de 4 ml; mezclar mediante ultrasonidos. </li><li> Preparar 4 ml de una solución orgánica acidificada (TFA 0,01% v / v) mediante la adición de 4 l de TFA (10%) a 4 ml H 2 O / CH 3 CN (50:50 v / v) en un vial de vidrio de 4 ml; mezclar mediante ultrasonidos. </li><li> Pesar 16 mg NH 4 HCO 3 con una microbalanza analítica en un vial de vidrio de 4 ml; añadir 4 ml de la H 2 O / CH 3 CN: solución (98 / v 2 v) y se dispersan por sonicación; esto se traduce en una solución 50 mM de NH 4 HCO 3 (pH ~ 7,8) en tampón acuoso. </li><li> Pesar 5 mg de una proteína estándar (por ejemplo, albúmina de suero bovino, la hemoglobina α / β, el citocromo C, anhidrasa carbónica, α-2-HS-glicoproteína, etc.) con una microbalanza analítica en un vial de vidrio de 4 ml; añadir 4 ml finales de DIr y dispersar por sonicación; esto típicamente resulta en una solución de concentración de alto nivel M. </li><li> Preparar 1 ml de solución de proteína (1-2 M) diluyendo la solución de concentración de alto nivel mu M con un volumen apropiado de NH 4 HCO 3 (50 mM) de tampón acuoso; mezclar mediante ultrasonidos. </li><li> Preparar la solución de tripsina (5 mM) disolviendo 20 g de tripsina de secuenciación grado en 170 l NH 4 HCO 3 (50 mM) de tampón acuoso; dispersar mediante ultrasonidos. </li></ol> 3. Configuración Experimental Nota: El sistema LTQ-MS está provisto de una fuente ESI modificado que incluye una casa construida XYZ-etapa que permite la interconexión del espectrómetro de masas a diversos enfoques de entrada de muestra. <ol><li> Desconectar el capilar micro-reactor de la unión PEEK y PEEK conectarse a la camiseta. </li><li> Inserte el cable a ~ 2 cm de largo Pt en el brazo lateral del PEEK T; utilizar un &quot;manga 1/32 PEEK para proporcionar unaLibre de fugas y conexiones para el aislamiento del alambre de Pt. </li><li> Conectar un 50 micras ID x 360 micras OD (~ 0,5 m de largo) de transferencia de muestras capilar en el extremo opuesto de la PEEK Tee; utilizar un manguito de 1/32 &quot;PEEK para proporcionar una conexión libre de fugas. </li><li> Asegurar el Tee PEEK en el XYZ-etapa y la posición del emisor ESI ~ 2 mm de distancia del capilar de entrada de espectrómetro de masas. </li><li> Conectar el cable de Pt a la fuente de alimentación ESI del espectrómetro de masas. </li><li> Conectar el extremo opuesto del capilar de transferencia de muestra a la unión de acero inoxidable; utilizar un manguito de 1/16 &quot;PEEK para proporcionar una conexión libre de fugas. </li><li> Conectar un 4-5 cm de largo tubo de PTFE 1/16 &quot;hasta el extremo opuesto de la unión de acero inoxidable e inserte la aguja de la jeringa 250 l en el tubo de PTFE; encender la bomba y ajustar el caudal al valor deseado (por ejemplo, 2 l / min). </li><li> parámetros de adquisición de datos de MS en tándem de entrada en el paquete de software que controla el instrumento EMd guardar como un archivo de método para preparar la instalación para la realización del análisis; para el sistema LTQ-EM, utilice los parámetros de análisis de datos dependientes siguientes: dinámica de exclusión habilitado para 180 seg con un ancho de masas exclusión ± 1,5 m / z; uno miembros de exploración seguido de uno de zoom y MS 2 scans en los 5 primeros picos más intensos, zoom ancho de barrido ± 5 m / z; la disociación inducida por colisión (CID) los parámetros establecidos en el ancho de separación de 3 m / z, la energía de colisión normalizada del 35%, la activación Q 0.25, y el tiempo de activación de 30 ms. </li></ol> 4. Configuración de microfluidos <ol><li> Preparar un dispositivo microfluídico de vidrio o sustratos poliméricos; 16-18 el diseño del dispositivo se proporciona en la Figura 2A, que consiste en un microrreactor (1) (0,13 mm de ancho x 2 mm de longitud x 50 m de profundidad) conectado a una entrada (2 ), salida (3) y un puerto lateral (4); los canales de interconexión para manipulaciones de fluidos son ~110 micras de ancho y 50 m de profundidad ~; una estructura de múltiples canales (5), que consta de ~ 1,5 a 2 micras de profundidad x 10 m de ancho x 100 micras microcanales largos colocados 25 m de distancia, actuará como un filtro para retener las partículas en el microrreactor. </li><li> Habilitar conexiones de fluidos a la entrega al chip por encolado conectores especializados a los puertos de entrada, o mediante la elaboración de un soporte polimérico que tiene capacidad para accesorios de suministro de fluido (Figura 2B). 16 </li><li> Preparar una suspensión de partículas C18 (5 micras) tal como se describe en el paso 1.8; entregar la suspensión para el microrreactor con una jeringa mediante el uso de 1/16 &quot;tubo de PTFE conectado al puerto de lado de chip (4); sellar el puerto después de la carga de partículas con pegamento epoxi y cura para 1 hr en un horno a 90 ºC 16. </li><li> Inserte un capilar (20 micras x 90 micras ID DE x 10 mm de longitud) en el orificio de salida, sellar con pegamento E6000, y curemos O / N; esto se convertirá en el emisor ESI ( <strong&gt; 6). </li><li> Conectar un 50 micras ID x 360 OD m (~ 0,5 m de largo) de transferencia de muestras capilar al puerto de entrada de chip (2); conectar el extremo opuesto del tubo capilar a una jeringa de 250 l y la bomba de jeringa, como se describe en 3.6 a 3.7. </li><li> Colocar un peso ligero PEEK Tee conector de la línea de transferencia de muestra justo antes de la abertura de entrada (2) e insertar un alambre de Pt en el lado del brazo de la T, como se describe en 3.2; este será el electrodo ESI. </li><li> Fijar el dispositivo de microfluidos en el escenario XYZ con el emisor ESI posicionado ~ 2 mm de distancia del capilar de entrada de espectrómetro de masas; preparar el MS para el análisis como se describe en 3.8. </li></ol> 5. Carga de la muestra, de digestión proteolítica y elución para MS Análisis Nota: Todos los pasos de transferencia de solución / muestra se llevan a cabo con la ayuda de una bomba de jeringa y debe permitir por unos minutos adicionales para completar, para compensar los muertos-volumES asociados a las líneas de transferencia desde y hacia el micro-reactor; el tiempo necesario dependerá de las velocidades de flujo implicadas. <ol><li> Enjuague el microrreactor y prepararlo para el análisis mediante la infusión de una solución acuosa de NH 4 HCO 3 (50 mM) en H2O / CH3CN (98: 2 v / v) a 2 l / min durante 5 min; utilizar una jeringa de 250 microlitros. </li><li> Cargar la muestra de proteína (1 M) en el microreactor mediante la infusión de la solución de muestra a 2 l / min durante 5 min; utilizar una jeringa de 250 microlitros. </li><li> Infundir la solución de tripsina (5 mM) sobre la proteína adsorbida en el microrreactor a 2 l / min para 1-3 min. </li><li> Se detiene el proceso de digestión proteolítica enjuagando el microrreactor con una solución acuosa acidificada de TFA (0,01% v / v) en H 2 O / CH 3 CN (98: 2 v / v) a 2 l / min durante 5 min; utilizar una jeringa de 250 microlitros. </li><li> Iniciar la elución de la proteína digerir del microrreactor mediante la infusión de una solución orgánica acidificada de TFA (0,01%v / v) en H 2 O / CH 3 CN (50:50 v / v) a 300 nl / min. </li><li> Encienda el proceso de adquisición de datos de MS y la tensión de ESI (~ 2.000 V); adquirir datos de MS utilizando los parámetros de adquisición de los datos proporcionados en el paso 3.8; observar la elución de los péptidos trípticos. </li></ol> 6. Proceso de Datos <ol><li> Procesar los datos de MS en tándem con un motor de búsqueda compatible con el formato de datos MS prima. <ol><li> Procesar los archivos RAW LTQ-MS utilizando un organismo específico de la base de datos de proteínas mínimamente redundante; cargar los datos en bruto en el motor de búsqueda, establecer los límites de tolerancia de masa de los padres y de iones fragmento a 2 y 1 Da, respectivamente, y el uso de fragmentos única plenamente tríptico con hasta dos divisiones perdidas para la búsqueda; no permiten modificaciones posteriores a la traducción; establecer las tasas de descubrimiento de alta y media confianza falsos (FDR) a 1% y 3%, respectivamente, y no permiten modificaciones posteriores a la traducción. </li></ol></li><li> Filtrar los datos para seleccionar únicos de alta confianzapéptido coincide con las proteínas en la base de datos; evaluar la proteína y a nivel de péptido resultados. </li></ol>

<ol>
	<li>Petersen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Bioreactors.">Microfluidic Bioreactors.</a> Encyclopedia of Microfluidics and Nanofluidics. , (2014).</li><li>Matosevic, S., Szita, N., Baganz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+and+applications+of+immobilized+microfluidic+enzymatic+reactors.">Fundamentals and applications of immobilized microfluidic enzymatic reactors.</a> J. Chem. Technol. Biotechnol. 86 (3), 325-334 (2011).</li><li>Liu, Y., Liu, B., Yang, P., Girault, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+enzymatic+reactors+for+proteome+research.">Microfluidic enzymatic reactors for proteome research.</a> Anal. Bioanal. Chem. 390 (1), 227-229 (2008).</li><li>Wu, H., Zhai, J., Tian, Y., Lu, H., Wang, X., Jia, W., Liu, B., Yang, P., Xu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+enzymatic-reactors+for+peptide+mapping:+strategy,+characterization,+and+performance.">Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.</a> LabChip. 4 (6), 588-597 (2004).</li><li>Jin, L. J., Ferrance, J., Sanders, J. C., Landers, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchip-based+proteolytic+digestion+system+driven+by+electroosmotic+pumping.">A microchip-based proteolytic digestion system driven by electroosmotic pumping.</a> LabChip. 3 (1), 11-18 (2003).</li><li>Asanomi, Y., Yamaguchi, H., Miyazaki, M., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-immobilized+microfluidic+process+reactors.">Enzyme-immobilized microfluidic process reactors.</a> Molecules. 7 (16), 6041-6059 (2011).</li><li>Aravamudhan, S., Joseph, P. J., Kuklenyik, Z., Boyer, A. E., Barr, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+microfluidic+enzyme+reactor+mass+spectrometry+platform+for+detection+of+anthrax+lethal+factor.">Integrated microfluidic enzyme reactor mass spectrometry platform for detection of anthrax lethal factor.</a> , 1071-1074 (2009).</li><li>Liu, X., Lo, R. C., Gomez, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+a+microfluidic+enzyme+reactor+utilizing+magnetic+beads.">Fabrication of a microfluidic enzyme reactor utilizing magnetic beads.</a> Electrophoresis. 30 (12), 2129-2133 (2009).</li><li>Liu, Y., Xue, Y., Ji, J., Chen, X., Kong, J., Yang, P., Girault, H. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticle+assembly+microfluidic+reactor+for+efficient+on-line+proteolysis.">Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis.</a> Mol. Cell. Proteomics. 6 (8), 1428-1436 (2007).</li><li>Wu, H., Tian, Y., Liu, B., Lu, H., Wang, X., Zhai, J., Jin, H., Yang, P., Xu, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titania+and+alumina+sol-gel-derived+microfluidics+enzymatic-reactors+for+peptide+mapping:+design,+characterization,+and+performance.">Titania and alumina sol-gel-derived microfluidics enzymatic-reactors for peptide mapping: design, characterization, and performance.</a> J. Proteome Res. 3 (6), 1201-1209 (2004).</li><li>Ji, J., Zhang, Y., Zhou, X., Kong, J., Tang, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+protein+digestion+through+the+confinement+of+nanozeolite-assembled+microchip+reactors.">Enhanced protein digestion through the confinement of nanozeolite-assembled microchip reactors.</a> Anal. Chem. 80 (7), 2457-2463 (2008).</li><li>Hustoft, H. K., Brandtzaeg, O. K., Rogeberg, M., Misaghian, D., Torsetnes, S. B., Greibrokk, T., Reubsaet, L., Wilson, S. R., Lundanes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+enzyme+reactor+and+high+resolving+chromatography+in+&amp;#34;sub-chip&amp;#34;+dimensions+for+sensitive+protein+mass+spectrometry.">Integrated enzyme reactor and high resolving chromatography in &amp;#34;sub-chip&amp;#34; dimensions for sensitive protein mass spectrometry.</a> Scientific Reports. 3 (3511), 1-7 (2013).</li><li>Pramanik, B. N., Mirza, U. A., Ing, Y. H., Liu, Y. H., Bartner, P. L., Weber, P. C., Bose, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-enhanced+enzyme+reaction+for+protein+mapping+by+mass+spectrometry:+A+new+approach+to+protein+digestion+in+minutes.">Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry: A new approach to protein digestion in minutes.</a> Protein Sci. 11 (11), 2676-2687 (2002).</li><li>Olszowy, P. P., Burns, A., Ciborowski, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-assisted+sample+preparation+for+proteomic+analysis.">Pressure-assisted sample preparation for proteomic analysis.</a> Anal. Biochem. 438 (1), 67-72 (2013).</li><li>Lord, G. A., Gordon, D. B., Myers, P., King, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tapers+and+restrictors+for+capillary+electrochromatography+and+capillary+electrochromatography-mass+spectrometry.">Tapers and restrictors for capillary electrochromatography and capillary electrochromatography-mass spectrometry.</a> J. Chromatogr. A. 768, 9-16 (1997).</li><li>Lazar, I. M., Kabulski, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+LC+Device+with+Orthogonal+Sample+Extraction+for+On-Chip+MALDI-MS+Detection.">Microfluidic LC Device with Orthogonal Sample Extraction for On-Chip MALDI-MS Detection.</a> Lab Chip. 13 (11), 2055-2065 (2013).</li><li>Harrison, D. J., Manz, A., Fan, Z. H., Ludi, H., Widmer, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+and+sample+injection+systems+on+a+planar+glass+chip.">Capillary electrophoresis and sample injection systems on a planar glass chip.</a> Anal. Chem. 64 (17), 1926-1932 (1992).</li><li>Jacobson, S. C., Hergenroder, R., Koutny, L. B., Warmack, R. J., Ramsey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Injection+Schemes+and+Column+Geometry+on+the+Performance+of+Microchip+Electrophoresis+Devices.">Effects of Injection Schemes and Column Geometry on the Performance of Microchip Electrophoresis Devices.</a> Anal. Chem. 66 (7), 1107-1113 (1994).</li><li>Armenta, J. M., Perez, M. J., Yang, X., Shapiro, D., Reed, D., Tuli, L., Finkielstein, C. V., Lazar, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Proteomic+Protocol+for+Biomarker+Fingerprinting+in+Cancerous+Cells.">Fast Proteomic Protocol for Biomarker Fingerprinting in Cancerous Cells.</a> J. Chromatogr. A. 1217, 2862-2870 (2010).</li><li>Doucette, A., Craft, D., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometric+Study+of+the+Effects+of+Hydrophobic+Surface+Chemistry+and+Morphology+on+the+Digestion+of+Surface-Bound+Proteins.">Mass Spectrometric Study of the Effects of Hydrophobic Surface Chemistry and Morphology on the Digestion of Surface-Bound Proteins.</a> J. Am. Soc. Mass Spectrom. 14, 203-214 (2003).</li><li>Lazar, I. M., Trisiripisal, P., Sarvaiya, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Liquid+Chromatography+System+for+Proteomic+Applications+and+Biomarker+Screening.">Microfluidic Liquid Chromatography System for Proteomic Applications and Biomarker Screening.</a> Anal. Chem. 78 (15), 5513-5524 (2006).</li><li>Lazar, I. M., Karger, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Open-Channel+Electroosmotic+Pumping+System+for+Microfluidic+Sample+Handling,&amp;#34.">Multiple Open-Channel Electroosmotic Pumping System for Microfluidic Sample Handling,&amp;#34.</a> Anal. Chem. 74 (24), 6259-6268 (2002).</li><li>Lazar, I. M., Rockwood, A. L., Lee, E. D., Sin, J. C. H., Lee, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+TOFMS+Detection+for+Capillary+Electrophoresis.">High-speed TOFMS Detection for Capillary Electrophoresis.</a> Anal. Chem. 71 (13), 2578-2581 (1999).</li></ol>

The authors declare no competing financial interests.

El micro-reactor descrito en este trabajo proporciona una forma fácil de implementar montaje experimental para llevar a cabo la digestión enzimática de las proteínas para permitir el análisis de MS y la identificación en menos de 30 minutos. Las ventajas de este sistema, en comparación con los enfoques convencionales, incluyen la simplicidad, velocidad, bajo consumo de reactivo y bajos costos. En particular, no hay necesidad de perlas de tripsina inmovilizada caros y cartuchos. La preparación del microrreactor c...

La identificación y caracterización de las proteínas purificadas se consigue frecuentemente mediante el uso de técnicas de EM. La proteína se digiere con una enzima y sus péptidos se analizan además por MS mediante una sencilla configuración experimental de infusión. La digestión proteolítica es necesaria para la generación de pequeños fragmentos de péptidos que se encuentran en el rango de masa útil de la mayoría de los analizadores de MS, y que puede ser fácilmente fragmentado a través de baja energía de disociación inducida por colisión para generar información de la secuencia de aminoácidos. Para las proteínas aisladas o mezclas de proteínas simples, no hay más necesidad de la separación cromatográfica de péptidos antes de la detección EM. Una mezcla de 25-50 péptidos se puede analizar fácilmente mediante la infusión de la muestra con una bomba de jeringa directamente en la fuente de MS de iones. El espectrómetro de masas puede llevar a cabo el análisis y confirmar la secuencia de una proteína en un breve espacio de tiempo. Con los métodos de adquisición de datos modernos, este proceso puede llevarse a cabo wentro de unos minutos o incluso segundos. El factor limitante en la realización de todo el proceso en una corta escala de tiempo es la etapa de digestión proteolítica. Por lo general, esto se realiza a través de un par de horas (o S / N), en solución, a 37 ºC, usando sustrato: proporciones de enzimas (50-100): 1. Para reducir el tiempo de digestión enzimática para minutos o segundos, microrreactores enzima inmovilizada, en forma de reactores de microfluidos o cartuchos disponibles en el mercado, se han descrito. 1-6 Típicamente, la enzima se inmoviliza por covalente, no covalente de adsorción / física, complejo la formación o la encapsulación, 3,6 la eficiencia mejorada del proceso enzimático ser habilitado por la gran superficie a volumen y las proporciones de enzima a sustrato. Otras ventajas de los reactores inmovilizados incluyen la autólisis y la interferencia reducidos de la enzima en el análisis de MS, la mejora de la estabilidad de la enzima y la reutilización. Se ha descrito una variedad de enfoques, el uso de vidrio o dispositivos microfabricados poliméricos,el uso de enzimas inmovilizadas sobre perlas magnéticas por las interacciones antígeno-anticuerpo, 7,8 atrapados en las redes de nanopartículas de oro, 9 encapsulado en titania-alúmina sol-geles 10 y nanozeolites, 11 o capturados a través de Ni-NTA o His-Tag la formación del complejo. 6 Alternativamente , se han desarrollado capilares abiertos-tubulares con enzimas inmovilizadas, también. 12 por otra parte, el aumento de la escisión proteolítica se ha demostrado mediante el uso de irradiación de microondas controlado 13 o tecnología bicicleta asistida por presión o presión (PCT) para la reducción de los tiempos de reacción a 30-120 min. 14 A pesar de las múltiples ventajas de los reactores de enzimas inmovilizados, los costos de los cartuchos comerciales es alta, la disponibilidad de dispositivos de microfluidos para uso rutinario es limitado, y el uso de los resultados de tecnologías de microondas o PCT en necesidad de instrumentación adicional. El objetivo de este trabajo fue desarrollar un método que circumveNTS estas desventajas, y que se puede implementar fácilmente en cada laboratorio para capacitar a los investigadores un método sencillo y eficaz para llevar a cabo la escisión enzimática de las proteínas en la preparación para el análisis de MS en cuestión de minutos. El enfoque se basa en el uso de hidrófobos, C18-partículas que son pre-cargados en un capilar o un dispositivo de microfluidos, y la adsorción de la proteína (s) de interés en estas partículas, seguido por digestión enzimática durante la infusión de la enzima sobre el lecho compacto y proteína (s) capturada. En este enfoque, el sustrato se inmoviliza a través de interacciones no covalentes, y la enzima se infunde a través de la proteína inmovilizada. La eficiencia de la digestión proteolítica se incrementa por las grandes áreas de superficie de partículas que exponen a la proteína para el procesamiento enzimático, distancias y tiempos de difusión hacia y desde la superficie de partículas reducido, una mejor transferencia de masa, no unión covalente que pueden afectar a la actividad de la enzima, la capacidad de forma rápida evaluate combinaciones de diferentes enzimas, disponibilidad de acceso, y la multiplexación si el proceso se ejecuta en un formato de microfluidos. Este enfoque se demuestra con el uso de una mezcla de proteínas estándar y de tipo tripsina de la enzima más utilizada para la digestión proteolítica antes de la detección ESI-MS. El espectrómetro de masas utilizado para la detección en este estudio fue un instrumento lineal trampa cuadrupolo (LTQ).

<table>
	<tbody>
		<tr>
			<td>Ion trap ESI-MS</td>
			<td>Thermo Electron</td>
			<td>LTQ</td>
			<td>The LTQ mass spectrometer is used for acquiring tandem MS data</td>
		</tr>
		<tr>
			<td>XYZ stage</td>
			<td>Newport</td>
			<td>Multiple parts</td>
			<td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Edmund optics</td>
			<td>G81-278</td>
			<td>The microscope is used to observe the microreactor packing process</td>
		</tr>
		<tr>
			<td>Analytical balance/Metler</td>
			<td>VWR</td>
			<td>46600-204</td>
			<td>The balance is used to weigh the protein samples</td>
		</tr>
		<tr>
			<td>Ultrasonic bath/Branson</td>
			<td>VWR</td>
			<td>33995-540</td>
			<td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td>
		</tr>
		<tr>
			<td>Syringe pump 22</td>
			<td>Harvard Apparatus</td>
			<td>552222</td>
			<td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td>
		</tr>
		<tr>
			<td>Milli-Q ultrapure water system&#160;</td>
			<td>EMD Millipore</td>
			<td>ZD5311595&#160;</td>
			<td>The MilliQ water system is used to prepare purified DI water</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (1000 &#181;L)</td>
			<td>VWR</td>
			<td>53513-410</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (100 &#181;L)</td>
			<td>VWR</td>
			<td>53513-406</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (10 &#181;L)</td>
			<td>VWR</td>
			<td>53513-402</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Fused silica capillary (100 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP100375</td>
			<td>This capillary is used for the fabrication of the microreactor</td>
		</tr>
		<tr>
			<td>Fused silica capillary (20 &#181;m ID x 100 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP020090</td>
			<td>This capillary is used for the fabrication of the ESI emitter</td>
		</tr>
		<tr>
			<td>Fused silica capillary (50 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP050375</td>
			<td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td>
		</tr>
		<tr>
			<td>Glass capillary cleaver</td>
			<td>Supelco</td>
			<td>23740-U</td>
			<td>This is a tool for cutting fused silica capillaries at the desired length</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Eclectic Products</td>
			<td>E6000 Craft</td>
			<td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Epo-Tek</td>
			<td>353NDT</td>
			<td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td>
		</tr>
		<tr>
			<td>Reversed phase C18 particles (5 &#181;m)</td>
			<td>Agilent Technologies</td>
			<td>Zorbax 300SB-C18</td>
			<td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4 mm x 20 cm C18 LC column from Agilent</td>
		</tr>
		<tr>
			<td>Syringe/glass (250 &#181;L)</td>
			<td>Hamilton</td>
			<td>81130-1725RN</td>
			<td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td>
		</tr>
		<tr>
			<td>Internal reducing PEEK Union (1/16&#8221; to 1/32&#8221;)</td>
			<td>Valco</td>
			<td>ZRU1.5FPK</td>
			<td>This union is used to connect the 250 &#181;L syringe to the microreactor for loading the 5 &#181;m particle slurry</td>
		</tr>
		<tr>
			<td>Stainless steel union (1/16&#8221;)</td>
			<td>Valco</td>
			<td>ZU1XC</td>
			<td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td>
		</tr>
		<tr>
			<td>Microvolume PEEK Tee connector (1/32&#8221;)</td>
			<td>Valco</td>
			<td>MT.5XCPK</td>
			<td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td>
		</tr>
		<tr>
			<td>Tee connector (light weight)</td>
			<td>Valco</td>
			<td>C-NTXFPK</td>
			<td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td>
		</tr>
		<tr>
			<td>Pt wire (0.404 mm)</td>
			<td>VWR</td>
			<td>66260-126</td>
			<td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td>
		</tr>
		<tr>
			<td>PTFE tubing (1/16&#8221; OD)</td>
			<td>Valco</td>
			<td>TTF115-10FT</td>
			<td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&#160;</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8220; ID x 1/16&#8221; OD)</td>
			<td>Upchurch Scientific</td>
			<td>1565</td>
			<td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50 &#181;m ID transfer capillary</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8221; ID x 1/32&#8221; OD)</td>
			<td>Valco</td>
			<td>TPK.515-25</td>
			<td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td>
		</tr>
		<tr>
			<td>Clean-cut polymer tubing cutter</td>
			<td>Valco</td>
			<td>JR-797</td>
			<td>This cutter is used to pre-cut the 1/16&#8221; and 1/32&#8217; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td>
		</tr>
		<tr>
			<td>Amber vial (2 mL)</td>
			<td>Agilent&#160;</td>
			<td>HP-5183-2069</td>
			<td>The vials are used to prepare sample solutions and the C18 particle slurry&#160;</td>
		</tr>
		<tr>
			<td>Amber vial (4 mL)</td>
			<td>VWR</td>
			<td>66011-948</td>
			<td>The vials are used to prepare sample solutions</td>
		</tr>
		<tr>
			<td>Polypropylene tube (15 mL)</td>
			<td>Fisher</td>
			<td>12-565-286D</td>
			<td>The vials are used to prepare buffer solutions</td>
		</tr>
		<tr>
			<td>Cylinder (100 mL)</td>
			<td>VWR</td>
			<td>24710-463</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Cylinder (10 mL)</td>
			<td>VWR</td>
			<td>24710-441</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>83007-386</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (100 &#181;L)</td>
			<td>VWR</td>
			<td>53503-781</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (10 &#181;L)</td>
			<td>VWR</td>
			<td>53511-681</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Glass substrates</td>
			<td>Nanofilm</td>
			<td>B270 white crown, 3&#8221; x 3&#8221;</td>
			<td>These are glass substrates for microchip fabrication</td>
		</tr>
		<tr>
			<td>Male nut fitting (1/16&#8221;)</td>
			<td>Upchurch</td>
			<td>P203X</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td>Nanoport assembly</td>
			<td>Upchurch</td>
			<td>N-122H</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein standards</td>
			<td>Sigma</td>
			<td>Multiple #</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC grade</td>
			<td>Fisher</td>
			<td>A955</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC grade</td>
			<td>Fisher</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, HPLC grade</td>
			<td>Sigma</td>
			<td>650447</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Aldrich</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

La gran mayoría de la espectrometría de masas (MS) a base de los métodos de análisis de proteínas implican una etapa de digestión enzimática antes de la detección, por lo general con tripsina. Este paso es necesario para la generación de péptidos de bajo peso molecular, generalmente con MW &lt;3.000-4.000 Da, que caen dentro del rango de escaneado efectivo de la instrumentación de espectrometría de masas. protocolos convencionales implican la digestión enzimática O / N a 37 ºC. Los avances recientes han llevado al desarrollo de una variedad de estrategias, típicamente implica el uso de un microrreactor con enzimas inmovilizadas o de una serie de procesos físicos complementarios que reducen el tiempo necesario para la digestión proteolítica a unos pocos minutos (por ejemplo, de microondas o de alta presión). En este trabajo se describe un método sencillo y rentable que puede ser implementado en cualquier laboratorio para lograr la rápida digestión enzimática de una proteína. La proteína (o mezcla de proteínas) se adsorbe en alta perf de fase inversa C18-unidoormance partículas de sílice líquidos cromatografía (HPLC) precargados en una columna capilar, y tripsina en tampón acuoso se infunde a través de las partículas para un corto período de tiempo. Para habilitar la detección MS en línea, los péptidos trípticos se eluyen con un sistema disolvente con el aumento de contenido orgánico directamente en la fuente de MS de iones. Este enfoque evita el uso de partículas de alto precio enzima inmovilizada y no necesita ninguna ayuda para completar el proceso. La digestión de proteínas y análisis de la muestra completa se puede lograr en menos de 3 min y ~ ~ 30 min, respectivamente.

Un resultado representativo de un proceso de digestión proteolítica lleva a cabo de forma simultánea en una mezcla de proteínas, con los microrreactores descritos anteriormente (Figuras 1 o 2), se proporciona en la Tabla 1. La tabla comprende las secuencias de péptidos únicos que identifican una proteína particular, la cruz puntuación de correlación (Xcorr) (es decir, una puntuación que caracteriza la calidad del partido-experimental p...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Tratamiento enzimático rápido de proteínas para la detección MS con un microrreactor de flujo continuo

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K vistas.  Virginia Tech. A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

Video: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricación de ADN electroquímico biosensores para la detección Reagentless de ácidos nucleicos, proteínas y moléculas pequeñas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Investigación

Bioingeniería

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

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 ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

Una plataforma de microfluidos para el procesamiento de precisión de pequeño volumen de la muestra y su uso para Tamaño partículas biológicas separadas con un Microdevice acústica

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Detección Sensible de Proteopathic Siembra Actividad con FRET Citometría de Flujo

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

Síntesis microondas impulsada de nanopartículas de óxido de hierro para la detección rápida de la aterosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Síntesis de cationizada Magnetoferritin para la magnetización ultra-rápido de las células

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Plataforma de microfluidos con multiplexado de detección electrónica para el seguimiento espacial de las partículas

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Tratamiento enzimático rápido de proteínas para la detección MS con un microrreactor de flujo continuo

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