Michael Henry, Dublin City University, gentilmente ajudou na coleta e análise de dados mostrado na Figura 5. Este trabalho foi financiado parcialmente por (1) George Mason University, (2) o italiano IstitutoSuperiore di Sanita », no âmbito da Itália / EUA acordo de cooperação entre o Departamento de Saúde e Serviços Humanos, George Mason University, e do Ministério da Saúde Pública italiano, (3) NIH, bolsas do programa IMAT 1R21CA137706-01 e 1R33CA173359-01 a LAL, e (4) Ceres Nanociências, Inc .

O plasma humano e urina foi coletada a partir de doadores voluntários saudáveis, com consentimento informado por escrito, seguindo de Revisão Institucional da Universidade George Mason Conselho aprovou protocolos. Os doadores foram distribuídos igualmente entre homens brancos e mulheres entre as idades de 25 e 42 amostras foram analisadas individualmente e não foram reunidas. 1. nanopartículas de Processamento de amostras de soro ou plasma Baixas biomarcadores potenciais abundante no plasma são capturados, em solução, com nanopartículas de hidrogel. As partículas são adicionadas ao plasma, incubadas, separadas por centrifugação, lavadas, e as proteínas capturadas são eluídas. As proteínas eluidas são secos sob um fluxo de azoto durante a jusante sequenciação e espectrometria de massa de identificação. <ol><li> Dilui-se 500 ul de soro de 1: 2, com 50 mM Tris-HCl de pH 7 (500 mL de soro + 500 ul Tris-HCl), em um tubo de microcentrífuga. </li><li> Adicionar 500 mL de poli (NiPAM / AAC) núcleo NanOpartigos. Incubar durante 15 minutos à temperatura ambiente. </li><li> Girar a amostra a 16100 x g, 25 ° C durante 10 min numa centrífuga equipada com um rotor de ângulo fixo. Remova e descarte o sobrenadante. </li><li> Adicionar 500 ul de tiocianato de sódio (25 mM) para o sedimento. Volte a suspender as nanopartículas por vigorosamente pipetando para cima e para baixo várias vezes. </li><li> Girar a amostra a 16100 x g, 25 ° C durante 10 min numa centrífuga com um rotor de ângulo fixo. Remova e descarte o sobrenadante. </li><li> Adicionar 500 mL de água MilliQ para o sedimento de nanopartículas. Volte a suspender as nanopartículas por vigorosamente pipetando para cima e para baixo várias vezes. </li><li> Girar a amostra a 16100 x g, 25 ° C durante 10 min numa centrífuga com um rotor de ângulo fixo. Remova e descarte o sobrenadante. </li><li> Preparar tampão de eluição fresco: Adicionar 700 ul de acetonitrilo e 300 ul de hidróxido de amónio num tubo limpo. Atenção: hidróxido de amônio é corrosivo. Utilize com ventilação adequada. Nota: O tampão de eluição deve ser prepared imediatamente antes da utilização. Não guarde o tampão de eluição. </li><li> Adicionar 300 ul de tampão de eluição à pelete de nanopartículas. Volte a suspender as nanopartículas por vigorosamente pipetando para cima e para baixo várias vezes. Incubar durante 15 minutos à temperatura ambiente. </li><li> Girar a amostra a 16100 x g, 25 ° C durante 10 min numa centrífuga com um rotor de ângulo fixo. Remova e guarde o fluido em um tubo de microcentrífuga rotulado limpo. </li><li> Repita os passos 1,9-1,10. Combine as duas eluatos em um tubo de microcentrífuga. </li><li> Secam-se os produtos de eluição sob fluxo de azoto, em um evaporador colector de azoto a 42,1 ° C, com um fluxo de ar ajustado para 8 (Figura 2F). </li><li> Armazenar o eluato secas à temperatura ambiente durante ó armazenamento / N, ou a -20 ° C para armazenamento a longo prazo, antes da espectrometria de massa, transferência de western, ensaios de ELISA ou (opcional). </li></ol> 2. nanopartículas Processamento das amostras de urina A urina normal contém menos do que 30 mg / dl e proteínamenos do que 1 + no sangue. No entanto, muitas doenças / condições podem alterar os níveis normais de proteína na urina e no sangue. Para ajudar a determinar o volume óptimo de nanopartículas para adicionar à amostra de urina, uma análise de urina é realizada antes da colheita de nanopartículas. Biomarcadores de urina podem existir em concentrações extremamente baixas, o que pode exigir optimizar a proporção de nanopartículas com o volume de urina. Este processo descreve a colheita de amostras de urina de nanopartículas para análise de Western blot a jusante. <ol><li> Coletar amostras de urina em um ambiente limpo, copo de plástico seco. Um volume mínimo de 22 ml é necessária. Urina loja amostras à temperatura de -80 ° C até estar pronto para a análise. </li><li> Descongele a urina congelada à temperatura ambiente ou a 4 ° CO / N. Misture brevemente em um misturador de vórtice. Despeje pelo menos 22 ml de urina para um tubo de polipropileno de fundo cónico de 50 ml. </li><li> Girar a urina numa centrífuga com um rotor basculante para fora a 3.700 xg durante 15 min. Decantar a urina, sem perturbar o sedimento, em um POLYPR fundo cónico de 50 ml limpotubo opylene. Descarte o sedimento. </li><li> Realizar exame de urina usando uma multi-analito &quot;vareta de urina&quot; tira reagente. Nota: guarde as tiras reagentes em um recipiente bem fechado longe da luz solar e umidade 17, 18 direto. <ol><li> Coloque a tira reagente, virada para cima, sobre uma toalha de papel limpo e seco. </li><li> Usar uma pipeta descartável para aspirar 1 ml de urina, preparado no passo 2.3. </li><li> Defina um temporizador para 2 min, mas não iniciá-lo. Dispensar rapidamente 1-2 gotas de urina em cada almofada de teste da tira reagente. Iniciar imediatamente o timer. Nota: Não mergulhe a tira reagente diretamente no recipiente de urina. Os indicadores de tintura / química na fita reagente pode, potencialmente, vaza da tira reagente para o recipiente de urina. </li><li> No tempo indicado na embalagem tira reagente, gravar os resultados qualitativos para os vários analitos sobre a tira reagente, comparando a cor das fitas reagentes individuais indica o código de cores correspondentedores no recipiente de reagente. Resultados de urina normais típicas são: (normal ou negativa) para o sangue, proteína, leucócitos, nitrito, glucose, cetona, bilirrubina, e urobilinogénio; Urina pH (5,5-7,0); gravidade específica (1,001-1,020). Nota: Os níveis elevados de proteínas numa amostra de urina, podendo competir com a proteína de interesse para os locais de ligação sobre as nanopartículas. Para maximizar a colheita de proteínas com nanopartículas, a / volume de urina de volume de nanopartículas pode ser ajustado para qualquer competição limite de proteínas de alta abundância, ou pode ser otimizado para colher as proteínas que existem em concentrações muito baixas. Se o valor de proteínas na urina é 1 + ou maior, adicionar volumes 2x de nanopartículas no passo 2.5 abaixo. Se o analito de interesse é uma proteína muito baixo abundante, pode ser necessário aumentar o volume de nanopartículas até 2 ml (Figura 3). </li></ol></li><li> Transferir 20 ml de urina clarificado do passo 2.3 em 50 mL de um tubo de polipropileno de fundo cónico limpo. Nãoperturbar quaisquer detritos / sedimento que pode ser no fundo do tubo de urina. Adicionar 200 ul de nanopartículas para os 20 ml da amostra de urina. Misture brevemente em um misturador de vórtice. Nota: Se o valor de proteínas na urina é 1 + ou maior, adicionar 400 mL de nanopartículas a 20 ml de urina. </li><li> Incubar a mistura de urina / nanopartículas durante 30 min à temperatura ambiente, sem balançar / mistura. </li><li> Girar a suspensão de urina / nanopartículas numa centrífuga equipada com um rotor basculante para fora a 3.700 xg durante 10 min. Nota: Se uma bolinha não é seguir a centrifugação visível, girar as amostras para um adicional de 2-7 min. </li><li> Remova e descarte o sobrenadante. Adicionar 500 mL de água MilliQ para o sedimento de nanopartículas. </li><li> Volte a suspender as nanopartículas por vigorosamente pipetando para cima e para baixo várias vezes. Transferir a solução de nanopartículas de um tubo de microcentrífuga de 1,5 ml limpo. </li><li> Rodar as nanopartículas numa centrífuga equipada com um rotor de ângulo fixo a 16.100 xg durante 10 min. Nota: Se uma bolinha não é visível folmugido centrifugação, girar as amostras para um adicional de 2-7 min. </li><li> Remova e descarte o sobrenadante. </li><li> Repita os passos 2.8 e 2.11 (duas vezes) com 200 ul de 18 MiliQ de água para lavar as nanopartículas. </li><li> Preparar tampão de eluição fresco: Adicionar 970 ul de acetonitrilo a 30 mL de hidróxido de amónio num tubo limpo. Atenção: hidróxido de amônio é corrosivo. Utilize com ventilação adequada. Nota: O tampão de eluição deve ser preparado imediatamente antes da utilização. Não guarde o tampão de eluição. </li><li> Adicionar 20 ul de tampão de eluição à pelete de nanopartículas. Volte a suspender as nanopartículas por vigorosamente pipetando para cima e para baixo várias vezes. Incubar durante 15 minutos à temperatura ambiente. </li><li> Gire as amostras de nanopartículas a 16.100 xg por 10 min. Retire e guarde o sobrenadante em um, 1,5 ml tubo de microcentrífuga limpo. Não perturbar o pellet de nanopartículas. Descarte a pelota no lixo de risco biológico. </li><li> Colocar as amostras em um rack em um exaustor química. Abra as tampas e incUbaté à TA durante 30 min. Em alternativa, colocar as amostras de acordo com o fluxo de azoto a 40 ° C até secar (1 - 2 horas). </li><li> Adicionar 15 ul de tampão de amostra de Tris-glicina SDS (2x) para as amostras. Aquece-se a 100 ° C num bloco de aquecimento a seco durante 5 minutos com as tampas abertas, ou até que o volume deixado no tubo não é mais de 20 ul. Nota: não use um banho de água fervente. A humidade do banho de água irá evitar a evaporação do tampão. </li><li> Coloque a tampa sobre os tubos. Retire os tubos do bloco de aquecimento. A amostra pode ser armazenada a -80 ° C ou usado imediatamente para a análise de Western blot a jusante. </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.

Relevância Clínica Uma amostra de soro ou plasma é pensado para conter baixa abundância de proteínas e peptídeos que podem proporcionar uma fonte rica de informações sobre o estado do organismo como um todo em circulação. Apesar da promessa de proteômica de soro, há três barreiras fisiológicas fundamentais e graves dificultando a descoberta de biomarcadores e tradução para o benefício clínico 10, 11, 16, 25. 1. biomar...

Apesar da conclusão do seqüenciamento do genoma humano, um progresso significativo não foi alcançado na identificação de biomarcadores preditivos de doença em estágio inicial, ou que se correlacionam com os resultados terapêuticos, ou prognóstico 1. Uma das razões para esta falta de progresso é que muitos biomarcadores potencialmente significativas existem em uma concentração abaixo do limite de detecção de espectrometria de massa convencional e outras plataformas de descoberta de biomarcadores. Espectrometria de massa (MS) e monitorização de reacções múltiplas (MRM) tem uma sensibilidade de detecção, tipicamente maior do que 50 ng / ml, enquanto a maioria dos analitos medido por imunoensaios em uma queda de laboratório clínico no intervalo entre 50 pg / ml e 10 ng / mL . Isto significa que muitos biomarcadores, particularmente na fase inicial de uma doença que não pode ser detectado por MS convencional e MRM 2. Além disso, a presença de proteínas de elevada abundância, tais como albumina e imunoglobulina em fluidos biológicos complexos, frequentemente mascarar por mil vezes excess baixa abundância, proteínas de baixo peso molecular e de péptidos 3, 4. Por esta razão várias etapas de preparação da amostra é necessária antes da espectrometria de massa e sequenciação de identificação. Um tal passo preparatório emprega a depleção de proteínas de alta abundância com colunas de depleção disponíveis comercialmente 5-8. Infelizmente, este passo leva à diminuição do rendimento de biomarcadores candidatos porque eles são muitas vezes não covalentemente associada com proteínas transportadoras que estão a ser removidos. Um outro desafio é representado pela estabilidade dos biomarcadores candidatos ex-vivo, uma vez que as amostras são recolhidas. As proteínas são sujeitas a degradação por proteases endógenas ou exógenas 9. Nanopartículas de hidrogel podem ultrapassar estes desafios críticos, amplificando a concentração biomarcador putativo para um nível dentro da gama do ensaio, ao mesmo tempo proteger a proteína de degradação 10-13. É importante notar ªLMW em proteínas no sangue são uma mistura de proteínas intactas pequenas, bem como fragmentos de proteínas grandes. Proteínas derivadas de tecido superior a 60 kDa são demasiado grandes para entrar passivamente o fluxo de sangue através da membrana basal vascular, mas eles podem ser representados no sangue como péptidos ou fragmentos de proteínas 14. Nosso objetivo é medir novos circulando biomarcadores que podem ser candidatos para a detecção precoce da doença, a estratificação dos pacientes para a terapia, e monitorar a resposta à terapia. Nossos nanopartículas são criados para excluir selectivamente imunoglobulinas e albumina de abundância alta e, simultaneamente, a captura de proteínas e péptidos mais pequenos e concentrá-los até 100 vezes dependendo do volume de partida. O nosso grupo identificado um conjunto de pequenas corantes orgânicos que podem funcionar com sucesso como engodos moleculares de elevada afinidade para proteínas e péptidos. Ligação da proteína-corante é provavelmente devido a uma combinação de interacção hidrofóbica e electrostáticos. Os anéis aromáticos do corante intercalar com proteínas através de bolsas hidrofóbicas na superfície de proteínas 11. Os iscos de acordo com a química, mostram uma afinidade específica para as classes de analitos seleccionados. Os iscos competir com as proteínas transportadoras, tais como albumina, para as proteínas ou péptidos. As de baixo peso molecular de proteínas / péptidos tornam-se aprisionados na partícula. Proteínas de alto peso molecular tais como albumina e imunoglobulinas são impedidas de entrar na partícula, devido à capacidade de crivagem, devido ao poro restritiva do hidrogel 11 (Figura 1). Nanopartículas de hidrogel são sintetizados por polimerização a precipitação iniciada por persulfato de amónio 11. N-isopropilacrilamida (NiPAM), co-monómeros de ácido acrílico (AAC) e alilamina (AA) e N, N&#39;-metilenobisacrilamida (BIS) de reticulação são deixados reagir a 70 ° C durante 6 h em condições de diluição 11, 13. A afinidade de ligação da proteína de poli (N-isopropilacrilamida-co-ácido acrílico) (poli (NiPAM-co-AAc) nanoparticlesis conseguida por ligação covalente incorporando corantes contendo amino (isto é. Sulfonatedanthraquinonetriazine, corantes) em que as nanopartículas através uma reacção de amidação realizada em solventes aquosos ou orgânicos, dependendo das características hidrofóbicas / hidrofílicas dos corantes 11, 13. substituição nucleofílica dos grupos amina na nanopartículas com o átomo de cloro de um corante anthraquinonetriazine é utilizado para criar poli que contém o corante (NiPAM -CO-alilamina) (AA) nanopartículas 11, 12. Um processo de polimerização em duas etapas é utilizado para criar as nanopartículas de hidrogel contendo um invólucro exterior de ácido vinilsulfónico (VSA) 11, 13. Nanopartículas de hidrogel pode ser aplicado a vários fluidos biológicos, incluindo o sangue total, plasma, soro, fluido cefalorraquidiano, suor e urina. Em uma etapa, em solução, o nanoparticles realizar uma rápida (dentro de minutos), a fixação ea concentração de analitos de baixo peso molecular de 10, 11, 13, 15-18. As proteínas são seguidamente eluídas das nanopartículas e detectado utilizando a técnica Western Blot 19-21, espectrometria de massa de 10, 11, 13, 15, 18, ​​22, 23, imunoensaios / ELISA 10, 11, 15, 18, ​​ou proteína de fase inversa microarray 16, 24 ensaios. As nanopartículas funcionalizadas com isca química, e apresentando um núcleo ou núcleo shell arquitetura, captura e concentrar as proteínas com base nas propriedades físico-químicas isca / shell. Diferentes corantes incorporados nas nanopartículas, por conseguinte, irá capturar diferentes subconjuntos de proteínas com base na eficiência de afinidade de corante, pH da solução, e a presença / ausência de proteínas de alto-13 competem variando abundante. Além disso, a quantidade de nanopartículas em relação ao volume da solução irá afectar a produção de proteína a partir das nanopartículas. Estes aspectoscolheita das nanopartículas de hidrogel são demonstradas utilizando três iscas nanopartículas diferentes para proteínas de colheita de amostras de plasma que contêm quantidades elevadas de proteína, e a partir de amostras de urina, que tipicamente não contêm grandes quantidades de proteínas. Neste protocolo, demonstrar a colheita concentrando e factor de necrose tumoral alfa (TNFa) a partir de amostras de plasma utilizando poli (NiPAM-co-AAc), poli (NiPAM / corante), e nanopartículas de casca Core- (poli (NiPAM-co-VSA)) . Poli (NiPAM / corante) nanopartículas são mostrados para concentrar antigénio espécies de Mycobacterium que foi adicionado a amostras de urina humana, para imitar os indivíduos infectados pelo bacilo 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

Descoberta de biomarcadores Novel desempenha um papel crucial no fornecimento de detecção mais sensível e específico para a doença. Infelizmente muitos biomarcadores baixa abundância que existem em fluidos biológicos não pode ser facilmente detectada com a espectrometria de massa ou imunoensaios, pois eles estão presentes em concentrações muito baixas, são instáveis, e muitas vezes são mascarados por proteínas de abundância elevada, tais como albumina ou imunoglobulina. Bait contendo poli (N-isopropilacrilamida) (NiPAM) nanopartículas à base são capazes de superar essas barreiras fisiológicas. Num passo que eles são capazes de captar, concentrar e preservar biomarcadores de fluidos corporais. Analitos de baixo peso molecular entrar no núcleo da nanopartícula e são capturados por diferentes corantes químicos orgânicos, os quais actuam como iscos de proteína com elevada afinidade. As nanopartículas são capazes de se concentrar as proteínas de interesse em várias ordens de magnitude. Este factor de concentração é suficiente para aumentar o nível de proteína de tal modo que as proteínas estão dentro dolimite de detecção de corrente de espectrómetros de massa, transferência de western, e imunoensaios. As nanopartículas podem ser incubadas com uma variedade de fluidos biológicos e que são capazes de enriquecer grandemente a concentração de proteínas de baixo peso molecular e de péptidos e excluindo albumina e outras proteínas de alto peso molecular. Os nossos dados mostram que uma amplificação de 10.000 vezes na concentração de um analito particular, pode ser conseguido, permitindo que a espectrometria de massa e ensaios imunológicos para detectar biomarcadores anteriormente indetectáveis.

Hidrogel nanopartículas tamanho e uniformidade Poli (NiPAM-AAC) partículas foram produzidas com um rendimento extremamente elevado e reprodutibilidade entre e dentro dos grupos. As partículas têm muito boa estabilidade coloidal à TA durante o tempo necessário para a recolha, o armazenamento, e a eluição das proteínas (pelo menos 48 horas), e precipitação das nanopartículas não foi observado (Figura 1) 11. A estabilidade coloidal pode ...

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 nanopartículas colheita de plasma ou urina para detectar proteínas de baixa abundância

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 visualizações.  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.

Vídeo: 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.

Padronização de plasma de superfície litografia para a Criação de Redes de celular

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

Bioengenharia

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de células progenitoras neurais em um hidrogel Peptide 3-dimensional de auto-montagem

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

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.

Design e Construção de Matriz Extracelular Artificial (MECa) Proteínas de<em&gt; Escherichia coli</em&gt; Para engenharia de tecidos da pele

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

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.

Fabricação baseada em vara de customizáveis ​​macias robóticos pneumáticas Gripper Aparelhos para a manipulação de tecidos delicados

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 Plasma Membrane entrega de um hidrofóbica Carga encapsulado em um cristal líquido nanopartículas Transportador

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.

Padronização bioativas proteínas ou peptídeos em hidrogel usando fotoquímica para aplicações biológicas

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

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.

Aparelhos para colheita de tecido 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.

Fabricando um rim córtex Extracellular Matrix-derivado hidrogel

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

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Interfaces de hidrogel fotodegradável para triagem, seleção e isolamento de bactérias

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Síntese de Hidrogel Adesivo Forte, Gelatina O-Nitrosobenzaldeído

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...