Name: experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip
Uploaded: 2017-02-07T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip at JoVE.com

我们感谢来自马德里自治大学安东尼·克萨达博士提供蓝藻菌。这项工作是由西班牙部:EconomíaŸCompetitividad的Subdirección一般德PROYECTOS德Investigación（MINECO）资助，授予没有。 AYA2011-24803和ESP2014-58494-R。

1.免疫原的制备<ol><li>生长在表1所述条件下在相应的培养基中每蓝藻菌株。 注:生长培养基和培养每个蓝藻应变条件列于表1。所有的蓝藻菌株，用K17外，属于从自治大学（马德里，西班牙）安东尼奥的Quesada的基团。针对Planktothrix冬凌草的抗体从Vilasouto水库（西班牙北部）此蓝藻的单特异性绽放的自然样品产生。 </li><li>量化使用细胞计数室通过光学显微镜从晚指数或静止生长期培养得到约10 8个细胞/ mL的细胞的数量。 </li><li>收获细胞从5毫升培养物离心以2,000×g离心5分钟。 </li><li>弃上清，悬浮细胞在10毫升浴缸e为5毫升1×磷酸盐缓冲盐水的（1×PBS）中，以获得约10 8细胞/ mL。 </li><li>均质化和裂解悬浮液通过超声处理的细胞为5个循环，每个30秒，以在冰上-第60暂停30-到，使用一种便携式，手持型超声波处理器或由管浸入的水浴在最大振幅（30 kHz）的细胞破碎。 </li><li>重复步骤1.3 - 1.5蓝藻的每一株。 注:超声产生细胞分裂和细胞内容发布。而蛋白质和多糖是良好的免疫原，具体分子，例如脂类和核酸，不诱发本身体液免疫原性应答。因此，它们必须结合于载体，如多糖或蛋白质，以增加分子的复杂性。此外，超声处理释放可以触发抗体产生细胞内物质。 </li></ol> 2.生产多克隆抗体的<ol><li>准备第一IMM通过混合在步骤1.5用0.5ml完全弗氏佐剂所获得的超声波处理细胞裂解液的0.5毫升unogen剂量。它递送给动物设施为多克隆兔抗体产生的操作员。 </li><li>通过混合在步骤1.5与0.5毫升弗氏不完全佐剂所获得的相同匀浆/裂解物的0.5毫升如前制备三个剂量中的抗体生产过程如存储器提升进一步使用。它们给动物设施满足所述抗体的生产过程。 </li><li>重复步骤2.1和2.2为每个新的抗体的产生过程。 注:通常，抗体的产生是委托专门的动物设施或者公司，因为相应的许可和培训要求与动物一起工作。该公司供应存在于血清样品中的抗原特异性抗体的量的相对酶联免疫吸附测定（ELISA）的测量。 </li></ol> 3.抗体PURIFication <ol><li>净化的免疫球蛋白G（IgG）的同时从免疫和通过蛋白质A亲和层析在步骤2中收集的免疫前血清级分。一些商业纯化试剂盒的基础上盒系统，做工精良;按照供应商的说明。 </li><li>当纯化试剂盒不提供脱盐系统，无论是通过透析或通过使用离心过滤装置用100 kDa的（或更低）膜的孔径的纯化的抗体的洗脱到0.1X PBS后更改缓冲器。 </li><li>通过测量280nm处的吸光率或通过使用比色的方法，如布拉德福德23，洛瑞24，或二喹啉甲酸（BCA）25确定抗体浓度。 注:重要的是要避免包括在洗脱缓冲液（ 例如，Tris缓冲液），因为它们与抗体竞争结合与环氧基活化的固体表面胺基。经过PUrification，测试用ELISA中抗体活性是重要的。 </li></ol> 4.荧光抗体标记<ol><li>通过将含染料用于标记1mg蛋白质的入100微升二甲基亚砜（DMSO）商业小瓶标签在被荧光（ 例如，远红荧光染料）第3获得的纯化的抗体。以2mg / mL的浓度添加溶解的染料各抗体制备的2微升在50微升的在50mM磷酸盐缓冲盐水（pH 8.5）的最终体积。 </li><li>保持在连续搅拌下的标记反应在环境温度下1小时，1200转的振动平台上。 </li><li>纯化通过尺寸排阻色谱的标记的抗体（ 例如，使用凝胶用捕集到柱1.5和30 kDa的之间的分馏范围），以下供应商的建议。 </li><li>测量在280nm的吸光度和在洗脱液650nm和计算拉贝下面供应商的建议玲效率。 注:最多50×50μL的抗体 - 标记反应可以用荧光染料用于标记1mg蛋白质的单一小瓶完成。建议用铝箔覆盖，或者该管，以使用不透明0.5毫升管，以避免步骤4.3之后淬火处理。对于IgG抗体，最佳标记与3-7摩尔每抗体的摩尔染料而实现的。 </li></ol> 5. CYANOCHIP生产<ol><li> 抗体和控制打印解决方案 <ol><li>由每种抗体以1mg / mL的在商业1×蛋白质印刷缓冲器含0.01％（体积/体积）吐温20（一种非离子型清洁剂），所有这些最终浓度混合制备印刷溶液各纯化抗体的30微升。 注意:可替换地，抗体溶液可以含有20％甘油，1％（重量/体积）蔗糖（或海藻糖，作为防腐剂），和0.01％（体积/体积）Tween 20的碳酸盐缓冲液（pH 8.5）。 </li><li>制备印刷溶液作为对照的30微升:（一）1×蛋白质打印缓冲器含0.01％（体积/体积）吐温20，（B）蛋白A纯化的免疫前血清在1毫克/毫升在1×蛋白质打印缓冲器与0.01％（体积/体积）吐温20，和（c）的牛血清白蛋白/（BSA）以1mg含0.01％毫升在1×蛋白印刷缓冲液（v / v）吐温20。 </li><li>制备不同浓度（ 例如，从50微克/毫升至1微克/毫升）在用吐温20 1×蛋白打印缓冲器，如在步骤5.1.1荧光标记的，纯化的免疫前血清的30微升。这些样品将用作荧光帧标记物和用于点样后的相对荧光定量。 </li><li>添加每步骤5.1.1，5.1.2制备的打印解决方案的井30微升，和5.1.3的最高品质的384孔微孔板用于微阵列的制造，如聚丙烯。 注:蛋白质打印缓冲区增加抗体的质量和稳定性，吐温20均质现货月rphology并建立蛋白偶联起来。建议保持在使用前4℃下的384孔微量。在-20℃保存以备长时间。聚丙烯具有低的DNA，蛋白质，细胞提取物，和小分子的内在结合。 </li></ol></li><li> 抗体印刷到载玻片 注意:通过使用不同的阵列平台，像接触，分针，或非接触设备，诸如压电打印机或"喷墨"技术的打印抗体上激活的显微镜载玻片。在这项工作中，CYANOCHIP已经常规地接触使用机器人系统（点样仪）能够在微米尺度点样抗体的NL量的打印。 <ol><li>设置印刷室的环境条件下，以20℃，40 - 50％的相对湿度。 </li><li>设置幻灯片基板（ 例如，75〜×25毫米环氧活化显微镜载玻片）执行几个相同的抗体ARRA每张幻灯片YS。 </li><li>发现每种纯化抗体，包括对照和参照帧，在一个一式三份点图案;在这些条件下，将斑点180 - 200微米的直径。 </li><li>打印后，留下的幻灯片至少30分钟在室温下让他们干，然后将它们保存在4℃;用于现场工作时，幻灯片可以被运输和储存在环境温度下数月。 注:CYANOCHIP在每张幻灯片3×8个相同的芯片或一个1×9格式的芯片完全相同9一个数组一式三份点的微阵列格式打印。每个数组的大小不能大于反应室尺寸24孔密封垫（通常是7.5×6.5毫米，3×8的杂交室）更高。 </li><li>使用微阵列分析环境样品之前，使用FSMI确定在滴定曲线的每个抗体工作稀释度。对于每个抗体，使用相应的IMM的标准浓度unogen（3月10日至4月10日细胞/ mL）和荧光抗体的系列稀释液（1之间:和1 500:32,000）。最佳抗体浓度对应于在滴定曲线获得的最大信号强度的50％。此外，对于每种抗体的敏感性和特异性必须确定，如在布兰科等人所述。 22。 </li></ol></li></ol> 6.准备环境多分析提取物的荧光夹心免疫芯片的（FSMI） <ol><li> 从液体样品多分析提取 <ol><li>取1 - 100毫升的液体试样的用无菌注射器（ 例如，水从储水器的岸）;样品的量很大程度上取决于靶的潜在浓度。 </li><li>通过使水样通过3微米孔径，47毫米直径的聚碳酸酯过滤器集中的细胞;一个细胞浓度10之间3 -细胞10 8 / mL的期望正检测，但实际的浓度是未知的。 </li><li>通过用刮它;恢复收集在过滤用1mL一个改良的Tris缓冲盐水，吐温20增强缓冲液（0.4M的Tris-盐酸（pH值8），0.3M NaCl和0.1％吐温20 TBSTRR）所述生物质刮铲入15mL的管中。 </li><li>均质化，并通过使用一个手持型超声波处理器分解，如，或者仅仅通过多次上下吹打在步骤1.5中所述;这个准备由微阵列分析样品。 </li></ol></li><li> 从固体样品多分析提取物 <ol><li>重达0.5克固体样品（ 例如，岩石，土壤，或沉淀物）成10毫升的管中，加入高达2毫升TBSTRR的。 </li><li>通过在管浸入超声波仪探头，由管浸入强大超声波仪喇叭的水浴，或通过使用一个手持式超声发生器声处理。执行至少5×30秒周期在30 kHz时，停在冰冷30秒。 </li><li>过滤除去砂，粘土和其它粗物料用10毫升的注射器联接到一个直径10到12毫米，5至20微米的孔尺寸的尼龙过滤器保持器。通过过滤器推样品到1.5毫升管中。如果过滤器饱和，搅拌在注射器中的悬浮液，并把它一个新的;此准备滤液材料用于免疫测定（步骤7）。 注:TBSTRR的缓冲能力取决于样品的类型。它的环境提取物的制备后立即进行的免疫测定法，以避免酶降解的分析物的效果是很重要的。可替代地，在-80℃添加蛋白酶抑制剂进入环境提取并冷冻，直到下一个步骤。 </li></ol></li></ol> 7.荧光夹心免疫芯片（FSMI） <ol><li> 阻断CYANOCHIP 注:立即使用前，治疗为Printed滑动到方框上滑动的所有自由环氧基团和以除去过量的非共价结合的抗体组成。 <ol><li>浸入该微阵列成的0.5M的Tris-HCl（pH为9），用5％（重量/体积）在一个干净的表面适度搅拌5分钟BSA溶液（ 例如，培养皿或者一个50毫升管）从摇臂平台。另外，躺下滑动到100到200μL下降与面临的芯片斑点上述溶液中。请假3 - 5分钟，然后继续。 </li><li>用塑料尖镊子小心地拿起滑板;尽量避免接触芯片区域。通过轻轻敲击滑动到纸巾去除多余的液体。沉浸它在0.5M的Tris-HCl（pH8）中，用2％（重量/体积），用于与从一个摇杆平台温和搅拌30分钟BSA溶液。 </li><li>使用适用于显微镜载玻片商业离心 - （300 XG 1分钟200）通过执行一个短暂离心干燥的幻灯片。可替代地，通过轻轻敲击ONT干燥该滑动OA纸巾。 </li></ol></li><li> 与微阵列的多分析样品提取液的培养 <ol><li>设置在与24孔为多个微阵列市售微阵列杂交盒中滑动;按照供应商的说明。 </li><li>滑动和盒式组件后，吸移管到样本提取物或它在TBSTRR稀释到盒的各孔的50μL的。 </li><li>对每个样品重复步骤7.2.2至进行分析。 </li><li>作为空白对照，吸管50 TBSTRRμL的缓冲到盒中的至少两个分开的井。 </li><li>孵育在室温下1小时以吹打每15分钟或通过在温和摇动离开它混合。可替代地，孵育在4℃下12小时。 注意:使用其他孵育垫片作为微阵列图案的功能。的时间和步骤7.2.5孵化温度是通常取决于亲和力和结合き经验参数每个代动力学成对抗原抗体。 </li></ol></li><li> 洗涤 <ol><li>通过将带盒向下，并仔细敲它放到一个干净的，吸收纸取出样品。 </li><li>通过加入TBSTRR 150μL到每一个洗井，并消除缓冲器，如上述。 </li><li>重复步骤7.3.2三次。 </li></ol></li><li> 孵化与荧光检测抗体 <ol><li>添加含有17抗蓝藻应变抗体的抗体混合物的50微升，每个标记在TBSTRR荧光染料用1％（重量/体积）BSA中。确定每个荧光抗体的浓度在混合物中（从0.7至2微克/毫升） 通过执行每种抗原/抗体对22的滴定实验。 </li><li>孵育在室温下1小时后，如在步骤7.2.5所述，或为在4℃下12小时。 </li></ol></li><li> 洗出的荧光抗体&lt;/ STRONG&gt; <ol><li>除去荧光未结合的抗体，如在步骤7.3。 </li><li>拆开纸盒，沉浸于快速冲洗滑入0.1X PBS（ 例如，在一个50毫升管）。 </li><li>干滑如步骤7.1.3。 </li></ol></li></ol> 8.扫描荧光<ol><li>扫描荧光滑动在荧光扫描仪对远红荧光染料的最大发射荧光峰。就拿芯片的多个图像在不同的扫描参数，一般通过降低激光增益值。 注:避免饱和点（&gt; 65000荧光计数） - 他们是从规模，并可能引入量化误差。 </li></ol> 9.图像处理和数据分析<ol><li>使用图像分析和定量的商业软件;该软件提供的荧光强度的测量（FI;从单个点的所有像素的中值或平均值）为电子整个芯片的现货阿赫。减去周围的景点当地背景:FI = FI 现货 - FI 本地背景 。 </li><li>保存Fi数据，并使用电子表格程序打开。 </li><li>使用的空白阵列作为阴性对照以识别并丢弃误报获得的值。应用下面的公式来计算每个抗体现场FI:FI =（FI 样本 - FI 空白 ），其中FI 样品 = Fi 热点 -在芯片FI 当地背景 ，样品提取物和FI 空白 = FI 点运行- FI 本地背景在空白微阵列。 </li><li>适用的2-到附加截止阈整个微阵列的FI误报的概率最小化的平均3倍;这是为劣质微阵列的图像和低信号对噪声的比率尤其相关。 </li><li>创建图和/或在必要时进行进一步的分析。 </LI&gt; </ol>

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The authors have nothing to disclose.

这里，使用CYANOCHIP，17抗体微阵列为广泛蓝藻属的检测和鉴定，多路复用荧光夹心免疫测定中描述22。这些蓝藻代表最频繁的海底和浮游属淡水栖息地，其中一些是毒素生产者。最近，荧光夹心免疫测定格式已被用来鉴别在环境应用26，27，28的微生物和/或bioanalytes。该协议主要是基于两个步骤:（i）固定...

的检测和在复杂的天然微生物群落的微生物的监测在许多领域，包括生物医学，环境生态，天体生物学至关重要。 蓝藻是公知的用于其以形成细胞的繁殖（过度增殖）在淡水能力的原核微生物。它们是普遍存在的，并且许多物种都能够产生毒素，导致不仅对人类健康的潜在危险，同时也向生态影响。在这方面，它是开发的早期检测蓝藻和/或它们在土壤和水中的毒素的快速和灵敏的方法是至关重要的。为了这个目的，多重荧光夹心免疫芯片（FSMI）已经发展成为一个工具，水管理人员，帮助他们做出决策，因此，在实施适当的水管理方案。 的方法的多元化已经发展到检测和识别青色obacterial细胞和藻毒素在土壤和水，包括光学显微镜，分子生物学，和免疫学技术。这些方法可以在所提供的信息相差很大。显微技术是基于细胞形态和体内荧光从蓝藻颜料，如藻蓝蛋白检测或叶绿素a 1。虽然它们是用于实时和频繁的监测快速和廉价的方法是通知有关的类型和样品中存在的蓝藻数目，它们不给有关的潜在毒性信息。此外，它们需要专门知识的一定水平，考虑到它往往是很困难的密切相关的物种2之间进行区分。为了克服这些限制，光镜必须由生物和生物化学筛选测定法和用于藻毒素的鉴定和定量物理化学方法陪同。 <p类="jove_content"&gt;酶联免疫吸附测定（ELISA），蛋白质磷酸抑制测定（PPIA），以及在小鼠中的神经化学测试是生物化学筛选测定法用于检测藻毒素的例子。而前两个是快速和灵敏的方法，当使用ELISA和PPIA测试限于三种类型的毒素已描述了误报。鼠标生物测定是一种定性的技术具有低灵敏度和精度，以及特殊许可和培训是必需的。此外，它不提供有关的样品中存在的毒素的类型的信息。藻毒素可以识别并通过其它分析方法，例如高效液相色谱（HPLC），液相色谱 - 质谱法进行定量（LC-MS），气相色谱（GC），气相色谱 - 质谱（GC-MS），或飞行的基质辅助激光解吸/电离飞行时间（MALDI-TOF）。然而，这仅仅是可能的，如果参考标准，这是需要编确定复杂样品中个体毒素浓度，可用3,4。此外，这些方法费时;需要昂贵的设备，耗材，和样品制备;而且必须由经验丰富和专业的工作人员来完成。 基于分子的方法已经应用于数十年来检测，识别和量化蓝藻和其相应的藻毒素由于发表在基因组数据库的序列信息（ 例如，国家生物技术信息中心，NCBI）。在这些方法是那些基于聚合酶链反应（PCR），它需要的套的用于DNA扩增的引物的设计，并取决于不同蓝藻物种的DNA序列的先前知识。虽然基因检测，如藻蓝蛋白操纵子，导致在属的水平准确识别，一些物种或菌株与未被发现此方法。然而，毒素编码基因，如那些属于微囊操纵子，促进毒素的样品中鉴定，其中生产者稀少5。然而，通过PCR检测毒素标记不一定意味着在环境中的毒性。此外，该组的开发来分析样品中存在的蓝藻和毒素生产者物种的整个范围的引物仍是不完整的，和进一步的研究必须完成识别未知物种。其他分子技术非基于PCR的， 如原位杂交（FISH）和DNA微阵列的荧光。 在过去的二十年中，微阵列技术已经获得了重要性在许多应用领域，尤其是在环境监测。 DNA微阵列允许7物种和分析4之间的歧视，6， </SUP&gt;，8，9，10，但它们被认为是非常费力和耗时的涉及多个步骤（ 例如，微阵列性能，DNA提取，PCR扩增，以及杂交）的任务。出于这个原因，基于抗体，如夹层和竞争性免疫微阵列费时测定以下，已成为用于检测多个环境样品11，12，13的一个重要且可靠的高通量方法。抗体的能力特异性地识别其靶化合物，并检测少量分析物和蛋白质，以产生针对几乎任何物质的抗体的可能性沿，使抗体微阵列为环境目的的强大技术。此外，实现了多种的能力分析中作为英格尔测定法中，用检测从ppb至ppm的的限制，是本方法14的主要优点之一。 基于抗体的生物传感器已被证明是用于环境监测15，16，17，18，19，20，21的检测范围广泛的病原体和毒素的敏感和快速的工具。而DNA的方法涉及多个步骤，所述基于抗体的微阵列只要求主要是基于在合适的溶液中缓冲一小段裂解步骤的小样品制备。 Delehanty和Ligler 15报告基于能够检测4 ppb的一个的蛋白质浓度的抗体夹心免疫测定的蛋白质和复杂混合物细菌分析物的同时检测ð10 4 CFU /细胞中的溶液。 Szkola 等。 21已经开发proteotoxins和小毒素，化合物的同时检测可能在生物战中使用的廉价而可靠的多重微阵列。他们检测蓖麻毒素的浓度，以检测3 ppb的，在不到20分钟的上限。最近，CYANOCHIP，一种基于微阵列抗体的生物传感器用于原位检测有毒和无毒的蓝藻 ，已经描述了22。该芯片可为潜在的蓝藻水华的鉴定，主要是在水环境中，这是很难辨别微观。检测微阵列的的限制为10 2 - 10 3个细胞的大多数物种，转动该生物传感器为用于多重检测和蓝藻鉴定具有成本有效的工具，即使在种的水平。所有这些特性使antiboDY微阵列技术，尤其是在这项工作中提出的方法中，更快和更简单的方法相比，上述技术。 这项工作提出的使用的抗体微阵列的生物传感器，以检测土壤和水样中蓝藻的存在下的实验的两个例子。它是基于夹心免疫测定形式的简单和可靠的方法，该方法需要非常小的样品体积和非常基本的样品制备。该方法需要很短的时间，并可以在现场容易地进行。

<table><tbody><tr><td>0.22 mm pore diameter filters</td><td>Millipore</td><td>GSWP04700</td><td>For preparation of immunogens</td></tr><tr><td>Eppendorf&amp;nbsp; 5424R microcentrifuge</td><td>Fisher Scientific</td><td></td><td>For preparation of immunogens</td></tr><tr><td>Phosphate buffer saline (PBS) pH 7.4 (10x)</td><td>Thermo Fisher Scientific</td><td>70011036</td><td>50 mM potassium phosphate, 150 mM NaCl, pH 7.4</td></tr><tr><td>Ultrasonic processor UP50H</td><td>Hielscher</td><td></td><td>For preparation of immunogens</td></tr><tr><td>Complete Freund&#039;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5881</td><td>Immunopotentiator</td></tr><tr><td>Incomplete Freud&#039;s adjuvant</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F5506</td><td>For boost injections</td></tr><tr><td>Protein A antibody purification kit&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>PURE1A&amp;nbsp;</td><td>For isolation of IgG</td></tr><tr><td>Centrifugal filter devices MWCO&amp;lt;100 kDa</td><td>Millipore</td><td>UFC510096-96K</td><td>For isolation of IgG</td></tr><tr><td>Dialysis tubings, benzoylated</td><td>Sigma-Aldrich</td><td>D7884-10FT</td><td>For isolation of IgG</td></tr><tr><td>Illustra Microspin G-50 columns&amp;nbsp;</td><td>GE-HealthCare</td><td>GE27-5330-02</td><td>For isolation of IgG</td></tr><tr><td>Bradford reagent</td><td>Sigma-Aldrich</td><td>B6916-500 mL</td><td>To quantify the antibody concentration</td></tr><tr><td>MicroBCA protein assay kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23235</td><td>To quantify the antibody concentration</td></tr><tr><td>Protein arraying buffer 2x</td><td>Whatman (Sigma Aldrich)&amp;nbsp;</td><td>S00537</td><td>Printing buffer; 30 - 40% glycerol in 1x PBS with 0.01% Tween 20</td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P9416</td><td>Non-ionic detergent</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A9418</td><td>Control&amp;nbsp; for printing; blocking reagent</td></tr><tr><td>384-wells microplate</td><td>Genetix</td><td>X6004</td><td>For antibody printing</td></tr><tr><td>Robot arrayer for multiple slides</td><td>MicroGrid II TAS arrayer from Digilab</td><td></td><td>For antibody printing</td></tr><tr><td>Epoxy substrate glass slides</td><td>Arrayit corporation</td><td>VEPO25C</td><td>Solid support for antibody printing</td></tr><tr><td>Alexa Fluor-647 Succinimidyl-ester&amp;nbsp;</td><td>Molecular probes</td><td>A20006</td><td>Fluorochrome</td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8418</td><td>Fluorochrome dissolvent</td></tr><tr><td>Heidolph Titramax vibrating platform shaker</td><td>Fisher Scientific</td><td></td><td>For antibody labeling&amp;nbsp;</td></tr><tr><td>Illustra Microspin G-50 columns&amp;nbsp;</td><td>Healthcare</td><td>27-5330-01</td><td>For purification of labeled antibodies</td></tr><tr><td>Safe seal brown 0.5 mL tubes</td><td>Sarstedt</td><td>72,704,001</td><td>For labeled antibodies storage&amp;nbsp;</td></tr><tr><td>Nanodrop 1000 spectrophotometer</td><td>Thermo Scientific</td><td></td><td>To quantify antibody concentration and labeling efficiency</td></tr><tr><td>3 &amp;micro;m pore size polycarbonate 47 mm diameter filter</td><td>Millipore</td><td>TMTP04700</td><td>To concentrate cells</td></tr><tr><td>1 M Trizma hydrochloride solution pH 8</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T3038</td><td>For TBSTRR preparation; to block slides</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S7653</td><td>For TBSTRR preparation</td></tr><tr><td>20 &amp;micro;m nylon filters&amp;nbsp;</td><td>Millipore</td><td>NY2004700</td><td>For environmental extract preparation</td></tr><tr><td>10 - 12 mm filter holders</td><td>Millipore</td><td>SX0001300</td><td>For environmental extract preparation</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P8340</td><td>For environmental extract storage</td></tr><tr><td>1 M Trizma hydrochloride solution pH 9</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T2819</td><td>To block slides</td></tr><tr><td>Heidolph Duomax 1030 rocking platform shaker</td><td>VWR</td><td></td><td>To block slides; for incubation processes&amp;nbsp;</td></tr><tr><td>VWR Galaxy miniarray microcentrifuge</td><td>VWR</td><td>C1403-VWR</td><td>To dry slides</td></tr><tr><td>Multi-Well microarray hybridization cassette</td><td>Arrayit corporation</td><td>AHC1X24</td><td>Cassette for 24 assays per slide</td></tr><tr><td>GenePix 4100A microarray scanner&amp;nbsp;</td><td>Molecular Devices</td><td></td><td>Scanner for fluorescence</td></tr><tr><td>GenePix Pro Software</td><td>Molecular Devices</td><td></td><td>Software for image analysis and quantification</td></tr></tbody></table>

experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip

全球变暖和富营养化使一些水生生态系统的行为触发快速和大规模的蓝藻生长为真生物反应器;这有相关的健康和经济后果。许多蓝藻菌株毒素生产者，只有少数细胞是必要的，以诱导对环境造成不可挽回的损失。因此，水体当局和管理部门需要快速，高效提供了可靠的数据来支持他们的预防或治疗的决定早期预警系统。该原稿通过使用抗体微阵列芯片17的抗体（ABS）与分类的分辨率（CYANOCHIP）报告用于在现场检测毒素的蓝藻菌的实验方案。在这里，多重荧光夹心免疫芯片（FSMI）为17株蓝藻同步监测经常发现在淡水生态系统中脱颖而出，他们中的一些毒素生产者，描述。多微阵列PLE相同重复的CYANOCHIP（最多24）被印刷到单个载玻片同时测试了类似的样本数。液体样品可以通过用抗体（ABS）直接培养或通过1〜3微米的过滤器过滤细胞浓度后进行测试。固体样品，如沉积物和地岩石，首先匀浆并通过在孵育缓冲液中的手持式超声发生器分散。然后，他们将被过滤（5 - 20微米）以除去粗料，并将滤液孵育腹肌。免疫反应是由终培养揭示与17荧光标记腹肌的混合物和由便携式荧光检测器被读出。整个过程大约需要3小时，其中大部分是对应于孵化的两个1小时周期。输出的是一个图像，其中亮点对应蓝藻标志物阳性检出。

这个工作介绍使用CYANOCHIP抗体微阵列的最相关淡水蓝藻物种的同时鉴定（ 表1）一个多重免疫测定试验。微阵列可以是印刷到显微镜载玻片上一个3×8微阵列格式。每个微阵列是由一组中一个一式三份点图案印刷17的抗体，其相应的免疫前抗体和BSA作为阴性对照。所述微阵列还包含一种荧光帧，使用荧光标记的免疫前抗体容易地定位微阵列图案22（<...

Watch this Scientific Journal Video about Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip at JoVE.com

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

experimental-protocol-for-detecting-cyanobacteria-liquid-solid

Research

JoVE Journal

Environment

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

9.0K 次观看.  Centro de Astrobiología (CAB, INTA-CSIC). The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

视频: Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

科研

癌症研究

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

生物工程

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

使用快速检测策略早期检测蓝藻花和相关氰毒素

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

化学

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

生物学

Human Serum Anti-aquaporin-4 Immunoglobulin G Detection by Cell-based Assay

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay (CBA) is a widely used method to detect anti-AQP4 IgG in human serum with high sensitivity and specificity. Briefly, serum anti-AQP4 IgG is captured by AQP4-transfected cell that is fixed on the biochip then detected by a fluorescein-labelled secondary antibody. Fluorescence microscopy is utilized to visualize the fluorescence, and the intensity of fluorescence is evaluated by at least two experienced clinicians. A final diagnosis of NMOSD can be made based on the combination of anti-AQP4 IgG detection results, clinical manifestations, and neuroradiological findings. According to previous studies, CBA is more sensitive and specific than other anti-AQP4 IgG detection methods, and it can be applied to both clinical diagnosis and studies of NMOSD. The method has limitations; for example, an international scale to evaluate serum anti-AQP4 IgG titers is still lacking. Here, a detailed protocol for human serum anti-AQP4 IgG detection using CBA is described.

Cell-based assay is a widely used method to detect serum anti-aquaporin-4 immunoglobulin G. This method could be applied to clinical diagnosis and scientific researches of neuromyelitis optical spectrum disorders.

人血清抗水药物蛋白-4 免疫球蛋白 g 的细胞检测

Anti-aquaporin-4 (AQP4) immunoglobulin G (IgG) is the core diagnostic biomarker for neuromyelitis optica spectrum disorders (NMOSD). The cell-based assay ...

免疫与感染

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

DNA的亲和纯化的芯片（DAP芯片）方法来确定目标基因的细菌双组分调控系统

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

用于检测实验方案

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