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 border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">0.22 mm pore diameter filters</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">GSWP04700</td>
			<td style="width:427px;">For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Eppendorf&#160; 5424R microcentrifuge</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Phosphate buffer saline (PBS) pH 7.4 (10X)</td>
			<td style="width:248px;">Thermofisher Scientific</td>
			<td style="width:119px;">70011036</td>
			<td>50 mM potassium phosphate, 150 mM NaCl, pH 7.4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Ultrasonic processor UP50H</td>
			<td style="width:248px;">Hielscher</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Complete Freund&#39;s adjuvant&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">F5881</td>
			<td>Immunopotentiator</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Incomplete Freud&#39;s adjuvant</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">F5506</td>
			<td>For boost injections</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein A antibody purification kit&#160;&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">PURE1A&#160;</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Centrifugal filter devices MWCO&#60;100 KDa</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">UFC510096-96K</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Dialysis tubings, benzoylated</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7884-10FT</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">GE-HealthCare</td>
			<td style="width:119px;">GE27-5330-02</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Bradford reagent</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">B6916-500 mL</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MicroBCA protein assay kit&#160;</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;">23235</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein arraying buffer 2X</td>
			<td style="width:248px;">Whatman (Sigma Aldrich)&#160;</td>
			<td style="width:119px;">S00537</td>
			<td>Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Tween 20&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P9416</td>
			<td>Non-ionic detergent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Bovine serum albumin (BSA)</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">A9418</td>
			<td>Control&#160; for printing; blocking reagent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">384-wells microplate</td>
			<td style="width:248px;">Genetix</td>
			<td style="width:119px;">X6004</td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Robot arrayer for multiple slides</td>
			<td style="width:248px;">MicroGrid II TAS arrayer from Digilab</td>
			<td style="width:119px;"></td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Epoxy substrate glass slides</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td style="width:119px;">VEPO25C</td>
			<td>Solid support for antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Alexa Fluor-647 Succinimidyl-ester&#160;</td>
			<td style="width:248px;">Molecular probes</td>
			<td style="width:119px;">A20006</td>
			<td>Fluorochrome</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">D8418</td>
			<td>Fluorochrome dissolvent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Titramax vibrating platform shaker</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For antibody labeling&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
			<td>For purification of labeled antibodies</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Safe seal brown 0,5 ml tubes</td>
			<td style="width:248px;">Sarstedt</td>
			<td style="width:119px;">72,704,001</td>
			<td>For labeled antibodies storage&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nanodrop 1000 spectrophotometer</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>To quantify antibody concentration and labeling efficiency</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">3 &#181;m pore size polycarbonate 47 mm diameter filter</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">TMTP04700</td>
			<td>To concentrate cells</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 8</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T3038</td>
			<td>For TBSTRR preparation; to block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium chloride</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">S7653</td>
			<td>For TBSTRR preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">20 &#181;m nylon filters&#160;</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">NY2004700</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">10-12 mm filter holders</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">SX0001300</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P8340</td>
			<td>For environmental extract storage</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 9</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T2819</td>
			<td>To block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Duomax 1030 rocking platform shaker</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;"></td>
			<td>To block slides; for incubation processes&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">VWR Galaxy miniarray microcentrifuge</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;">C1403-VWR</td>
			<td>To dry slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Multi-Well microarray hybridization cassette</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td>AHC1X24</td>
			<td>Cassette for 24 assays per slide</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix 4100A microarray scanner&#160;</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Scanner for fluorescence</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix Pro Software</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></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

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

科研

环境科学

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

实验协议生产生物柴油与饱和酮的分离从商业余积<em&gt;等鞭金藻</em&gt;藻类生物量

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Experimental System of Solar Adsorption Refrigeration with Concentrated Collector

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The main components of the system were the adsorbent bed, the condenser, the evaporator, the cooling sub-system, and the solar collector. In the first step of the experiment, the vapor-saturated bed was heated by the solar radiation under closed conditions, which caused the bed temperature and pressure to increase. When the bed pressure became high enough, the bed was switched to connect to the condenser, thus water vapor flowed continually from the bed to the condenser to be liquefied. Next, the bed needed to cool down after the desorption. In the solar-shielded condition, achieved by aluminum foil, the circulating water loop was opened to the bed. With the water continually circulating in the bed, the stored heat in the bed was took out and the bed pressure decreased accordingly. When the bed pressure dropped below the saturation pressure at the evaporation temperature, the valve to the evaporator was opened. A mass of water vapor rushed into the bed and was adsorbed by the zeolite material. With the massive vaporization of the water in the evaporator, the refrigeration effect was generated finally. The experimental result has revealed that both the COP (coefficient of the performance of the system) and the SCP (specific cooling power of the system) of the SAPO-34 zeolite was greater than that of the ZSM-5 zeolite, no matter whether the adsorption time was longer or shorter. The system of the SAPO-34 zeolite generated a maximum COP of 0.169.

With solar energy as the driving force, a novel adsorption refrigeration system has been developed and experimentally investigated. Water vapor and zeolite formed the working pair of the adsorption system. This manuscript describes the setup of the experimental rig, the operation procedure, and the important results.

聚光集热器太阳能吸附制冷实验系统

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

紫外辐射或等离子体照射表面的粉尘充电和动员实验方法

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

用于检测实验方案

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