迈克尔·亨利，都柏林城市大学，请协助与图5所示的数据收集和分析，这项工作得到了部分支持（1）乔治·梅森大学，（2）意大利IstitutoSuperiore二SANITA"在意大利/美国框架健康的乔治·梅森大学的美国能源部和人类服务部，以及公共卫生意大利外交部，（3）美国国立卫生研究院，IMAT计划资助1R21CA137706-01和1R33CA173359-01对上湖人，以及（4）谷神星纳米科学公司之间的合作协议。

人血浆和尿液是从健康志愿捐献者采集，并以书面的知情同意书，下面的乔治·梅森大学机构审查委员会批准的协议。捐助者平均分配白人男性和女性之间的25和42样品进行单独分析岁之间，并没有合并。 血清或血浆样品1纳米工艺在等离子体电势低丰度生物标记物被捕获，在溶液中，用凝胶粒子。将粒子加入到等离子体中，温育，通过离心分离，洗涤，和所捕获的蛋白被洗脱。将洗脱的蛋白质根据用于下游质谱测序和鉴定的氮气流中干燥。 <ol><li>在离心管2与50毫米TrisHCl pH值为7（500微升血+500微升TrisHCl）的稀释:500微升血清1。 </li><li>加入500μl聚（异丙基丙烯酰胺/ AAC）核心nanop的文章。孵育15分钟，在室温。 </li><li>在装有固定角转子的离心旋转的样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>加入500μl加入硫氰酸钠（25毫摩尔）的粒料。通过剧烈上下吹打多次重悬的纳米颗粒。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>加入500微升的MilliQ水的纳米颗粒沉淀。通过剧烈上下吹打多次重悬的纳米颗粒。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。拆下并丢弃上清液。 </li><li>准备新鲜的洗脱缓冲液:加入700μL乙腈300μL氢氧化铵在一个干净的试管中。注意:氢氧化铵具有腐蚀性。使用适当的通风。注意:洗脱缓冲液必须准备ARED即将使用前。请勿将洗脱缓冲液。 </li><li>加入300微升的洗脱缓冲液中的纳米颗粒。通过剧烈上下吹打多次重悬的纳米颗粒。孵育15分钟，在室温。 </li><li>在具有固定角度转子离心机旋转样品在16,100 XG，25℃下进行10分钟。取出并保存洗脱液在干净，标记的离心管。 </li><li>重复步骤1.9 - 1.10。二者结合起来洗脱液为一体的离心管。 </li><li>干燥下，在氮气蒸发器歧管的氮气流的洗脱液在42.1℃，用空气流设置为8（ 图2F）。 </li><li>储存在室温下干燥洗脱液为O / N个存储，或在-20℃下长期贮存，前质谱法，免疫印迹，或酶联免疫吸附试验（可选）。 </li></ol>尿液样本2纳米工艺正常尿液中含有低于30毫克/分升的蛋白质和比1 +血少。然而，很多疾病/状况可能改变尿蛋白和血液的正常水平。为了帮助确定纳米颗粒的最佳量添加到尿样，尿分析之前进行纳米粒子的收获进行。尿液生物标志物可以存在于非常低的浓度，这可能需要优化的纳米颗粒的比对尿量。此过程说明了下游Western blot分析尿液样本的纳米粒子收获。 <ol><li>收集尿液样本中的清洁，干燥的塑料杯。 à22毫升最小的体积是必需的。商店尿液标本在-80°C，直到准备分析。 </li><li>解冻的冷冻尿液在室温或4°CO / N。在旋涡混合器简要组合。倾至少22毫升尿入50毫升的锥形底聚丙烯管中。 </li><li>旋尿中有水平转子离心机在3700 XG 15分钟。倒出尿液，而不会干扰颗粒，放入干净的50ml锥形底polypropylene管。弃沉淀。 </li><li>使用多分析物"尿液试纸"的试剂条进行尿检。注意事项:避免阳光直射，湿度17，18的试纸存放在密闭的容器了。 <ol><li>莱试剂条，面朝上，放在干净，干燥的纸巾。 </li><li>使用一次性吸管吸1毫升步骤2.3准备的尿液。 </li><li> 2分钟设置一个计时器，但不启动它。快速分配1 - 在试剂条的每个测试点2滴尿。立即启动定时器。注意:不要直接在尿液的容器浸入试剂条。在试剂条上的染​​料/化学指标可以潜在浸出试剂条的进入尿液的容器中。 </li><li>在使用的试剂条容器所指示的时间，由单独的试剂条的颜色进行比较，对相应的颜色编码的籼稻记录定性结果的试剂条上的各种分析物器试剂容器上。典型的正常尿的结果是:（正常的或负的）的血液，蛋白质，白细胞，亚硝酸盐，葡萄糖，酮，胆红素和尿胆素原;尿pH值（5.5 - 7.0）;比重（1.001 - 1.020）。注意:高蛋白质水平在尿液样本可以与感兴趣的蛋白质竞争结合于所述纳米颗粒的站点。为了最大限度地提高蛋白质的收获与纳米颗粒，该纳米颗粒体积/尿体积比率可以调整为从高丰度蛋白质或者限制竞争，或者可以被优化到收获中存在的浓度非常低的蛋白质。如果尿蛋白值是1 +或更高，在下面的步骤中添加2.5纳米的2倍量。如果感兴趣的分析物是非常低丰度蛋白质，它可能是必要的，以增加纳米颗粒的量最多至2ml（图3）。 </li></ol></li><li>将20毫升的尿液澄清步骤2.3到一个干净的50ml锥形底聚丙烯管。别扰乱任何碎片/丸粒可能在尿液管的底部。加入200μl纳米颗粒的20毫升尿样。在旋涡混合器简要组合。注意:如果尿蛋白值是1 +或更高，加入400μl的纳米颗粒到20ml的尿液。 </li><li> 30分钟，在室温下孵育尿/纳米颗粒的混合物，无摆动/混合。 </li><li>旋尿/纳米颗粒悬浮液在配备水平转子在3700离心10 min的离心机。注意:如果粒料是不可见的离心后，旋转试样进行一个额外的2 - 7分钟。 </li><li>拆下并丢弃上清液。加入500μl的MilliQ水的纳米颗粒沉淀。 </li><li>通过剧烈上下吹打多次重悬的纳米颗粒。纳米粒子溶液转移到一个干净的1.5 ml离心管。 </li><li>纺纳米颗粒在装备有固定角度转子以16,100 xg离心10分钟，离心。注意:如果小球是不可见的甲级降脂离心，旋转试样进行一个额外的2 - 7分钟。 </li><li>拆下并丢弃上清液。 </li><li>重复步骤2.8和2.11（两次），用200μl18的MilliQ水冲洗该纳米颗粒。 </li><li>准备新鲜的洗脱缓冲液:加入970μL乙腈至30微升氢氧化铵在一个干净的试管中。注意:氢氧化铵具有腐蚀性。使用适当的通风。注意:洗脱缓冲液必须临用前制备。请勿将洗脱缓冲液。 </li><li>加入20微升的洗脱缓冲液的纳米颗粒沉淀。通过剧烈上下吹打多次重悬的纳米颗粒。孵育15分钟，在室温。 </li><li>纺纳米颗粒样品在16100×g离心10分钟。取出并保存在上清干净，1.5 ml离心管。不破坏纳米颗粒。丢弃在生物危害的垃圾沉淀。 </li><li>将样品在通风橱机架。打开瓶盖和公司ubate在室温30分钟。或者，将下在40℃的氮气流中的样品直至干燥（1 - 2小时）。 </li><li>加入15微升的Tris-甘氨酸SDS样品缓冲液（2×）的样品。热火100 ℃，在5分钟的盖子，直至留在管内的体积的干热块是不超过20微升。注意:不要用沸水浴中。从水浴中的湿度会防止缓冲器的蒸发。 </li><li>广场上的管帽。从加热块中取出试管。可将样品保存在-80℃下或立即用于下游免疫印迹分析。 </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.

临床意义 血清或血浆样品被认为含有低丰度循环的蛋白质和多肽可提供的信息有关的生物体作为一个整体的国家的丰富来源。尽管血清蛋白质组学的承诺，有三个基本的和严重的生理障碍，阻碍生物标志物的发现和翻译临床获益10，11，16，25。 一，重要诊断标志物可能存在于非常低丰度（浓度）在血液中。早期病变组织，例如预转?...

尽管完成了人类基因组测序，显著进展尚未作出确定的生物标志物预测的早期阶段的疾病，或与治疗结果，或预测1相关。其中一个原因缺乏进展是，很多潜在的生物标志物显著以低于传统质谱和其他生物标志物发现平台的检测极限浓度存在。质谱（MS）和多反应监测（MRM）具有典型的检测灵敏度大于50纳克/毫升，而在50 pg / ml和10毫微克/毫升在临床实验室下降了免疫测定的范围广大的分析物的。这意味着许多生物标志物，特别是在疾病的早期阶段无法通过常规的MS和MRM 2被检测到。除了高丰度蛋白质，如白蛋白和免疫球蛋白的复合物的生物流体的存在往往掩盖由十亿倍货存ESS的低丰度，低分子量蛋白质和肽3，4。为此几个样品的准备步骤之前所需要质谱测序和鉴定。一个这样的预备步骤中采用的高丰度蛋白质的与市售的耗尽列5-8的耗尽。不幸的是这一步骤导致候选生物标志物的产率的降低，因为它们通常是非共价地与该被除去的载体蛋白相关联。另一个挑战是候选生物标志物前体的稳定性表示，一旦样品的采集。蛋白质是发生降解内源性或外源性蛋白酶9。水凝胶的纳米颗粒可以通过扩增推定的生物标志物的浓度，以测定的范围内的电平超越这些关键的挑战，同时保护蛋白免受降解10-13。 要注意个很重要在LMW在血液中的蛋白质是小完整蛋白质以及大蛋白的片段的混合物。组织来源的蛋白质比60 kDa的较大的过大而不能穿过血管基底膜被动地进入血流，但它们可以在血液中作为肽或蛋白质片段14来表示。我们的目标是测量新颖循环生物标志物，可以是候选疾病的早期发现，患者分层为治疗，并监测对治疗的反应。在创建了纳米颗粒选择性地排除高丰度的免疫球蛋白和白蛋白，同时捕获较小的蛋白质和肽，并根据初始体积上集中起来，以100倍。 我们的团队确定了一套小的有机染料，可成功地用作高亲和力的分子饵料蛋白质和多肽。蛋白质 - 染料的结合被认为是由于疏水性和静电相互作用的结合第对染料的芳环交织经由蛋白质上的表面11的疏水口袋的蛋白质。 的毒饵，这取决于它们的化学性质，表现出分析物的选择的类的特定亲和力。诱饵与载体蛋白竞争，如白蛋白，用于蛋白质或肽。低分子量蛋白/肽被截留在颗粒中。高分子量的蛋白质，如白蛋白和球蛋白是从进入的，因为筛分能力的粒子可防止因水凝胶11（图1）的限制孔。 水凝胶粒子是通过由过硫酸铵11开始沉淀聚合合成。异丙基丙烯酰胺（NIPAM），丙烯酸（AAC）和烯丙基胺（AA）和交联剂N，N&#39;-亚甲基双（BIS）的共聚单体被允许在70℃下反应6小时，在稀释条件11,13。聚的高蛋白结合亲和性（N-异丙基丙烯酰胺-共聚-丙烯酸）（聚（NIPAM-CO-AAC）nanoparticlesis通过共价掺入含氨基的染料（ 即 ，sulfonatedanthraquinonetriazine染料）在纳米粒子，通过实现在这取决于染料的亲水/疏水特性的水性或有机溶剂中进行的酰胺化反应如图11所示，用一个anthraquinonetriazine染料的氯原子在纳米颗粒中的胺基团的13。亲核取代被用于创建聚含染料（NIPAM -共聚-丙烯胺）（AA）的纳米颗粒11，12。的两步聚合方法被用来创建含有乙烯基磺酸（VSA）11，13的外壳的水凝胶粒子。 水凝胶纳米粒子可用于各种生物流体，包括全血，血浆，血清，脑脊液，汗液，尿等。在一个步骤中，在溶液中，nanoparticles进行快速（几分钟内）封存和低分子量的分析物浓度10，11，13，15-18。蛋白质随后从纳米颗粒洗脱和检测用蛋白质印迹19-21，质谱法10，11，13，15，18，22，23，免疫/ ELISA 10，11，15，18，或反相蛋白质微阵列16， 24测定。纳米功能化化学饵料，并提出核或核 - 壳结构，捕捉和集中的基础上饵/壳的物理化学性质的蛋白质。因此，掺入纳米颗粒不同的染料将捕获的蛋白质的不同亚群具有不同的基础上，染料亲和效率，溶液的pH，并且存在/不存在竞争的高丰度蛋白13。此外，纳米颗粒的相对量，以将溶液的体积将影响从纳米颗粒中的蛋白质的产率。这些方面水凝胶的纳米粒子的收获是使用三种不同的纳米颗粒饵收割蛋白是由含有高含量的蛋白质的血浆样品显示出，并从尿样品通常不包含大量的蛋白质。在这个协议中，我们证明收获和浓缩使用聚（NIPAM-CO-的AAc），聚（NIPAM /染料），以及核 - 壳纳米颗粒的血浆样品的肿瘤坏死因子α（TNFα）（聚（NIPAM-CO-VSA）） 。聚（NIPAM /染料）的纳米颗粒被示为集中于加入人尿样品分枝杆菌抗原，以模仿结核分枝杆菌感染的个体。

<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

新型生物标志物的发现在提供更多的敏感性和特异性疾病检测至关重要的作用。不幸的是许多存在于生物流体中低丰度生物标记物不能容易地与质谱法或免疫测定法，因为它们存在于非常低的浓度，是不稳定的，并且往往是由高丰度蛋白质，如白蛋白或免疫球蛋白掩蔽检测。诱饵含聚（N-异丙基丙烯酰胺）（NIPAM）基于纳米颗粒是能够克服这些生理屏障。在一个步骤中，他们能够捕捉，集中和保持体液的生物标记物。低分子量的分析物进入所述纳米颗粒的核心，由不同的有机的化学染料，它作为高亲和力蛋白诱饵被捕获。该纳米颗粒可以由几个数量级集中感兴趣的蛋白质。这个浓度因子是足以增加蛋白质水平，使得蛋白质内的目前质谱仪，免疫印迹和免疫检测限。纳米颗粒可以一起孵育过多的生物体液和它们能够极大丰富的低分子量蛋白质和肽的浓度，同时排除白蛋白和其它高分子量的蛋白质。我们的数据表明，在特定分析物的浓度的10,000倍放大，可以实现，使质谱和免疫检测以前无法检测的生物标志物。

水凝胶纳米粒子大小和均匀度 聚（NIPAM-AAC）颗粒已产生极高的收益率和可重复性的和批次内。所述颗粒具有在RT非常好的胶体稳定性期间需要用于捕获，存储，和蛋白质的洗脱（至少48小时）的时间，以及纳米颗粒沉淀没有被观察到（ 图1）11。胶体稳定性可能是由该纳米颗粒快速蛋白质/肽的摄取很重要。 从血浆?...

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.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

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 次观看.  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.

视频: 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.

等离子体表面图案光刻技术的蜂窝网络创作

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

科研

生物工程

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

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

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

心肌在心脏组织工程中的纤维蛋白凝胶的封装

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

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

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

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

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

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

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

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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

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

3D水凝胶支架的关节软骨细胞培养及软骨生成

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

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

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

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

娇嫩的组织操纵定制的软机器人气动设备手爪基于杆制造

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.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

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.

生物活性蛋白或多肽在水凝胶上的应用

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

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

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

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

Retroductal 纳米微粒注射液对小鼠颌下腺的治疗

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

Apparatus for Harvesting Tissue Microcolumns

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

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

水样组织的采集装置

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.

肾皮质细胞外基质衍生水凝胶的制备

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