The authors would like to acknowledge the startup fund from SUNY Albany and the access of facilities at the University at Albany Cancer Research Center.

注意:在本协议中使用的几种化学品刺激，而且危险情况下的皮肤接触。咨询材料安全数据表（MSDS），并执行此协议之前，必须穿戴适当的个人防护装备。在步骤（1.1.1）中使用的食人鱼溶液是高度腐蚀性的，并应通过添加过氧化物慢慢搅拌下的酸来制备。处理这种解决方案非常小心在通风橱中。使用适当的眼部保护及耐酸手套。三甲基氯硅烷（TMCS）是后（1.1.6）用于在可选步骤的腐蚀性，可燃性化学。处理这种化学物质在通风橱中。 注意:执行条形码滑动制造和临界流动图案化程序在一个干净的房间，以尽量减少污染颗粒物。灰尘颗粒可能阻塞的端口和PDMS模具的微通道，并干扰流动图案。 1.构造的一dimensiona的微升DNA条形码幻灯片<ol><li>条形码流动模式准备的SU-8大师 注意:使用计算机辅助设计（CAD）软件创建的附图对垂直流动模式（图1A-B）。这些图案呈现在铬光掩模。面膜的透明区域对应于SU-8大师的功能。 <ol><li>清洗的硅晶片（直径100mm）彻底3 的 H 2 SO 4的混合物:1 30％ 的 H 2 O 2（食人鱼溶液）加热到96℃。用去离子水和异丙醇冲洗晶片，接着用氮气吹枪干燥。 </li><li>倒在晶片上约4毫升SU-8 2025光刻胶。使用可编程旋涂机，以在500rpm以3000rpm均匀扩散的光致抗蚀剂的晶片上，持续10秒，然后30秒。这就形成了一个光致抗蚀剂层构成的厚度25〜微米。逐步让纺停止前减速 - 这是迈ntain均匀涂层在晶片的表面上。 </li><li>烘烤的加热板上进行1分钟的涂覆的晶片在65℃下，然后进行5分钟，在95℃下。这个步骤允许该涂层固化。冷却至室温5分钟。 </li><li>放置的铬掩模（图1C）上的光致抗蚀剂涂层。暴露所述掩模特征到近UV光（350-400纳米，曝光能量150-160毫焦/厘米2）20秒。 注意:对铬掩模的设计中包含20个信道，其每一个是20微米宽，50微米的间距。的通道进行的图案的相对端的一端卷绕。总而言之，20个通道覆盖有一个长40毫米的〜并〜20mm的宽度的矩形区域。每个信道由出对应于入口和出口的两个圆形特征两侧。入口和出口是可以互换的。 </li><li>烤在电炉上露出的晶片5分钟，在95℃下。酷逐步RT。 </li><li>沉浸在SU-8开发者搅拌晶圆5分钟。洗晶片的新鲜的SU-8显影剂的一小部分，然后异丙醇。干燥用氮气吹枪的晶片。硬烘烤晶片上的200℃热板上30分钟，并允许该晶片逐渐冷却至室温。 注意:开发可进行较长的时间，如果在白色膜洗涤在该步骤后观察。 可选:通过将其暴露于三甲基氯硅烷的蒸汽在一个封闭的培养皿10分钟Silanize的SU-8大师。 </li></ol></li><li>在PDMS条形码模具的研制<ol><li>结合40.0克硅橡胶基地4.0克固化剂。搅拌该预聚物混合物剧烈10分钟。脱气在真空下20分​​钟。 注:一般来说，使用10:1（基料:固化剂）的质量比。 </li><li>倒在预聚物到含有的硅晶片与条形码图案的SU-8主培养皿。与PDMS混合物的高度应为约7.5毫米或以上。脱气在切赫混合物我菜第二次以除去任何残留的气泡，然后烘烤该混合物1小时在75℃，以允许PDMS固化。 注:重要的是要保持足够厚度的PDMS板的〜7.5毫米或以上，以避免通过在PDMS模具阶梯孔插入时的引脚/管材加太紧张的PDMS玻璃债券是非常重要的（1.3.3.1） </li><li>使用手术刀，小心地围绕PDMS厚板的含有条形码模制特征和剥离从晶片板坯的区域切断。 </li><li>修剪板坯的边缘，以达到与PDMS条形码模具的所需形状。冲20个孔（1.0毫米直径）通过模具用活检穿孔与柱塞。确保该孔与条形码图像的圆形特征对准。这些孔用作入口和出口。 </li></ol></li><li>聚-L-赖氨酸的一维图案形成（PLL）的<ol><li>除去任何灰尘用氮气吹枪的聚L-赖氨酸涂覆的玻璃载片的表面上。 ATTAC小时PDMS模具到干净的载玻片上。确保模具和滑动的边缘对齐。 </li><li>烘烤1.5小时在75℃，以加强与PDMS模具和PLL涂覆幻灯片之间的键。同时，准备20片的柔性聚乙烯管（3- 4英寸件，具有0.5mm的内径和1.5毫米的外径）。 注意:管件的数目对应于在条形码PDMS模具入口的数目。油管用于夫妇在PDMS条形码模具氮气罐配有压力调节器的通道。 </li><li>每个管件的一端，连接一个不锈钢空心销（1毫米直径）。抽吸无菌过滤聚L-赖氨酸通过销子溶液，直到至少1厘米的管填充有溶液。 <ol><li>紧固销（连接到溶液填充管）与PDMS条形码模具（图1E）的入口。附加管的另一端连接到一个压力调节氮气罐设置。使该溶液使用压力范围穿过模具流0.5-1磅为至少6小时。 注:请参考所有流量图形的程序步骤（1.3.3）。 </li></ol></li></ol></li><li>的ssDNA 14的一维图案形成 注意:磷酸盐缓冲盐水（PBS）在随后的步骤中使用从137毫摩尔NaCl，10毫的 Na 2 HPO 4，和2mM KH 2 PO 4缓冲液的pH为7.4制备。 <ol><li>制备2mM的双（磺基）辛二酸酯（BS3）的PBS溶液。在PBS或超纯水制备300微米溶液 A，B 和 C的ssDNA（5&#39;-胺改性，80个核苷酸）的。 注意:使用BS3的溶液中约30分钟的准备后，由于N-羟基酯易发生水解。 </li><li>结合2.5微升300微米，A，B，或C的单链DNA与2.5微升2mM的二（sulfosuccinim牧歌）辛二酸酯在PBS每个通道A，B和C的DNA被指定给信道1，2，和3中。该顺序也适用于3个信道（图2A）剩余集。 </li><li>执行步骤（1.3.3）。这时候，通过不锈钢销和插入聚乙烯管吸出5微升BS3 / DNA溶液，然后耦合的PDMS模具氮源。允许BS3 / DNA溶液流动通过条形码模具约40分钟，或直到在通道被填充。 </li><li>一旦停止所有通道都充满的流动，然后孵育在条形码的BS3 / DNA溶液在RT搅拌2小时。不要让解决方案干涸。烤的PDMS模具1小时，在75℃连接条码幻灯片。 注意:为方便对准在随后的流动图案化步骤，在条形码滑动的通道图形的边缘可被概括。这是通过使用金刚石scrib小心刮擦玻璃表面进行E要生成载玻片上的底部对准标记。对准在该协议的后期阶段可以在显微镜下进行检查。 </li><li>取下条形码幻灯片的PDMS模具。用0.01％轻轻洗滑动SDS一次和三次用超纯水。 <ol><li>干用显微镜载玻片微调条形码幻灯片。存储在一个干净的50毫升离心管中以供后续使用条形码滑动。 </li></ol></li></ol></li></ol> 2.验证的条形码滑动一维模式注意:此验证协议也可在评估后续流动图案化步骤的质量适于使用。 <ol><li>阻断的幻灯片<ol><li>制备的1％牛血清白蛋白（BSA）的PBS。过滤该溶液通过0.45μm针筒过滤器在使用前。使用凝胶加载尖，申请50微升1％BSA溶液到条形码幻灯片的一个边缘。 </li><li>孵育1％BSA SOLUT离子为1小时，在室温。 </li></ol></li><li>孵化与花青3（Cy3标记）结合的互补DNA <ol><li>准备鸡尾酒的&#39;，B&#39;和 C&#39;寡核苷酸共轭Cy3的一端。 A&#39;，B&#39; 和C&#39;的序列互补的A，B 和 C的，分别。所述的Cy3 DNA的工作浓度是0.05μM0.1％BSA中。 </li><li>除去从滑动通过移液的BSA溶液，然后应用在滑动的同一边缘30微升的DNA鸡尾酒溶液。孵育的幻灯片在RT 1小时的的Cy3-DNA的鸡尾酒。请在黑暗中，以保护Cy3的部分从漂白这一培养步骤。 </li></ol></li><li>荧光强度分析<ol><li>吸取出的Cy3-DNA的鸡尾酒和清洗下滑1％BSA，PBS，终于在稀释的PBS（1个P本领域的PBS，用50份超纯水）。干用微调的幻灯片。 </li><li>观察使用荧光显微镜或微阵列扫描仪的荧光（图2B）。当使用微阵列扫描仪，设置激光发射波长532纳米，在5微米的像素尺寸，光电倍增管（PMT）增益在450和功率为15％。 </li></ol></li></ol> 3.制作的2维（3×3）的DNA阵列14的<ol><li>在PDMS条形码模具的研制<ol><li>执行程序（1.1）构建另一个SU-8大师，但这次使用了镀铬面罩与流型垂直于第一设计。执行过程（1.2），以建立一个新的PDMS模具垂直模式。 </li></ol></li><li>单链DNA的二维流动图案形成<ol><li> 对于3×3阵列 ，制备150μM的储备溶液A&#39;-i的，B&#39;-ⅱ的，C&#39;-ⅲ，A&#39;-ⅳ，B&#39;-V，C&#39;-vi中，A&#39;-VII，乙&#39;-VIII，和C&#39;-IX的DNA中的3％BSA / PBS中。这些寡核苷酸作为"桥接序列"（图2A）。结合寡核苷酸溶液（溶液1至3），使得工作浓度是50μM的每个寡核苷酸。 使用表1中提供的指南。 </li><li>流量3％BSA / PBS封闭液到所有的20条通道为1小时，在0.5-1磅。 </li><li>流的解决方案1，2和3为信道1，2，和3中。按照此顺序的3个通道后续集。流在大约40分钟，通常完成。在室温下孵育该DNA溶液2小时，以允许所述DNA杂交。 </li><li>流量3％BSA / PBS封闭液到所有的20条通道为1小时，在0.5-1磅，除去未杂交的DNA。揭去的PDMS平板，并通过浸渍载玻片成的3％BSA洗涤得到的DNA微阵列载玻片/ PBS中一次和两次的PBS，随后稀释的PBS（1份的PBS和50份超纯水）。 ðRY使用显微镜载玻片微调的幻灯片。 </li><li>执行第2，使用寡核苷酸类似于i"键 IX"是的Cy3共轭第二验证步骤（ 图2C）。存储在一个50毫升的离心管中以供后续使用的3×3阵列的幻灯片。 注意:DNA微阵列可以存储在室温在干燥器中数月。 </li></ol></li></ol> 4.转化的3×3的DNA阵列为抗体阵列<ol><li>的抗体 - 寡核苷酸（DEAL）偶联物的制备<ol><li>制备的200mM的琥珀酰亚胺基4-甲酰基苯甲酸（S-4FB）和40毫琥珀酰亚胺基-6- hydrazinonicotinamide（S-HYNIC）在无水N，N-二甲基甲酰胺（DMF）中的解决方案。 </li><li>准备达捕捉抗体在PBS 7分开的溶液（1毫克/毫升）。 注意:如果该抗体原液含有叠氮化钠抑菌剂，使用旋脱盐Ç执行与PBS缓冲液交换olumns 7 kDa的分子量截止（MWCO）。 </li><li>准备单独的400微米的解决方案，I&#39;，II&#39;，III&#39;，IV&#39;，V&#39;六&#39; 和VII"的DNA。将每个捕获抗体一个寡核苷酸序列。结合40微升400微米的DNA与12.25微升DMF的离心管和自旋向下调匀。 <ol><li>到各DNA / DMF溶液，在DMF中添加2.3微升的200mM的S-4FB。对于每100捕获抗体微克，在DMF增加2.25微升40毫米S-HYNIC。 </li></ol></li><li>孵育抗体/ S-HYNIC和DNA / S-4FB溶液4小时，在室温。 </li><li>在准备抗体-DNA偶联反应，通过用柠檬酸盐缓冲液在pH 6洗涤他们准备新的旋转脱盐柱（每个DNA溶液和一个用于每个抗体）。 </li><li> 4小时培养后，要对每个抗体/ S-HYNIC和DNA / S-4FB解决方案缓冲交换。结合在S-4FB共轭<stro纳克&gt; I&#39;，II&#39;，III&#39;，IV&#39;，V&#39;，VI&#39;和VII&#39;寡核苷酸与抗体-S-HYNIC偶联物。孵育混合物2小时，在室温。 </li><li>孵育反应O / N在4℃。 </li></ol></li><li>的抗体 - 寡核苷酸（DEAL）偶联物纯化 注意:为了纯化抗体 - 寡核苷酸缀合物，在配备的Superose 6 10/300 GL柱标准FPLC系统进行快速蛋白液相层析（FPLC）。 <ol><li>设置FPLC系统以280纳米的UV检测器的波长。用PBS（pH 7.4）中的等度流动以0.3毫升/分钟到抗体 - 寡核苷酸缀合物从过量的S-4FB-DNA中分离出来。 </li><li>汇集含有缀合物的级分，浓缩的级分的使用自旋过滤器，用10 kDa的MWCO 150微升的体积。 </li></ol></li><li>抗体的二维图案形成<ol><li>准备另一PDMS模具微室或微孔我们在步骤荷兰国际集团的制造过程（1.2）。在PDMS模具可能含有微升至纳升井免疫。 注:对于一般的生物标记物检测流体样品中，一个PDMS模具多口井的配合与阵列幻灯片。在PDMS模具的功能取决于所研究的系统。例如，从单细胞实验的蛋白质的检测可以用含有微容器用0.15-NL体积的PDMS模具来执行。 </li><li> （可选）主题的PDMS模具等离子清洗（18 W）1.5分钟，以呈现其图案的表面亲水性。之前等离子体清洗，用胶带，以阻止将直接结合到3×3阵列滑动所有其他表面。 注意:步骤（4.3.2）是可选的，但通常执行时的PDMS模具是用于在单细胞实验中使用。 </li><li>交配的PDMS模具利用3×3阵列的幻灯片，则块用在PBS的1％BSA的幻灯片。孵育1小时，在室温。 Meanwhi乐，准备抗体结合物寡核苷酸鸡尾酒（200微升最终体积）。各缀合物的工作浓度为在1％BSA / PBS中的10微克/毫升。 </li><li>添加抗体-寡核苷酸鸡尾酒，然后孵育1小时，在37℃，以允许缀合物的寡核苷酸部分杂交上的3×3阵列的特定点，由此该DNA阵列转换为抗体阵列。 </li><li>清洁和干燥的幻灯片重复步骤（3.2.4）。最高的灵敏度，如果抗体阵列立即使用来实现。长期储存，可能导致灵敏度降低。 </li></ol></li><li>检测蛋白质<ol><li>冻干重组蛋白或准备一个过滤的细胞样本。 </li><li>配合微阵列与定制设计的芯片，并阻断用3％BSA的PBS表面1小时。然后取出BSA溶液，并应用样品，孵育2小时。 </li><li>在PBS洗掉，用3％的BSA样品三次，一个ð然后由产品数据表提供的浓度添加检测抗体。孵育2小时。 </li><li>在PBS洗掉的检测抗体通过的3％BSA三次，然后添加花青-5（Cy5的）-streptavidin在1微克/毫升，然后孵育1小时。 </li><li>清洁和干燥的幻灯片扫描重复步骤（3.2.4）。 </li></ol></li></ol>

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The authors have no competing interests to disclose.

流量模式的设计是在制造的2-D芯片的第一个关键步骤。为了产生在玻璃基板上两个重叠的DNA模式，所述第一设计的信道特征应垂直于与第二（图1A-B）的 。该设计还考虑到芯片的下游应用。在单细胞分析的情况下，微阵列被用于检测来自单个细胞包封在微容器的蛋白质，因此所述通道尺寸都是用，与2-D阵列对准微容器兼容。每个设计呈现在光掩模和标准光刻技术被用于制造硅晶片（图1C-D）上的SU-8的信道的功能。这作为主成型硅橡胶。一旦模具已经制造，它被结合到一个锁相环涂覆滑动，然后再加上一个氮气气流建立（图1E）。在设定了我们使用简单，并具有容易地结合到与压力调节氮气源的任何实验室的优点。我们使用的廉价的多个三通阀连接到一个氮气罐的组件。必须保持的气流为图案形成在低压力（0.5-1磅），以避免从模具载玻片的脱层。与PDMS模具内泄漏可以妥协的图案的完整性。 在这个协议中的DNA流动造型步骤利用单链DNA与精心挑选的正交序列（ 图2A）。前流动图案化，在这些寡核苷酸的独特序列应该为缺乏串扰测试。一个简单的测试串扰是通过修改验证过程中，我们介绍了在我们的协议（步骤2）完成。代替使用的鸡尾酒的Cy3标记的互补DNA，只有一种类型的互补序列被用于在一个时间幻灯片的所选区域上的多个DNA序列被固定。在不存在DNA的交扰，只有一个磁条（1-D的DNA阵列）或一个位置（2-D核酸阵列）应该下一个的Cy3过滤器是荧光。彻底验证正交序列的例子可以材料和设备的表中找到。第一个DNA图案化步骤引入了三个形形色色的"锚"的寡核苷酸。这些80-nt的序列包含了两个独特的20-mer的顺序交替两个20-mer的聚-A地区。这些锚定序列的5&#39;-胺修饰是必需的胺至胺交联17带PLL由BS3介导的。所述第二DNA图案化步骤不需要交联剂。这一步引入"架桥"寡核苷酸部分与锚定序列杂交。每个60-nt的桥接序列由一个20聚体区域互补的一个锚定的序列，一个20聚体聚的隔板中，和一独特的20聚体序列的。该DNA阵列的验证（FIGURË2B-C）的每一个的DNA流入图案化步骤之后执行，以评估的图案化步骤的质量，并确保没有交叉污染14。这是通过引入的Cy3缀合的寡核苷酸探针与所述DNA杂交的模式进行。与用于分析荧光强度在步骤（2.3.2）中规定的参数，所述DNA模式应表现出至少40000盟的荧光强度来执行使用微阵列最大化下游免疫测定的灵敏度。验证过程中战略性地利用不同的Cy3-DNA组产生了另一种模式（ 图2D），从而证明了DNA构图的灵活性步骤 8。未能实现均匀的型态可能造成泄漏或机械障碍物（如粉尘颗粒）在流图案化步骤遇到。 的抗体 - 寡核苷酸缀合物DEAL的制剂为第另一关键步骤是的协议。抗体的选择是由所研究的生物系统所决定的。在偶联中使用的寡核苷酸应具有互补的序列的那些锚定的DNA阵列上。在S-4FB和S-HYNIC接头的贮存液应新鲜配制，并远离湿气，以避免水解。在我们的缀合协议中使用的温育时间足够长介绍关于抗体和DNA所需的功能。最终偶联反应仅通过经由FPLC除去未反应的S-4FB共轭寡淬灭。因此，FPLC纯化应立即完成偶联后进行。当与在我们的协议中描述的系统中，低流速（0.2-0.25毫升/分钟）进行FPLC，也可以使用，以提高分辨率。结合物洗脱的宽峰（约9-15毫升洗脱体积）的较窄和较高的DNA峰（17-20毫升洗脱体积）之前。我们不建议存储解决方案偶联物与过量的S-4FB-DNA的，因为这可能导致在缀合于抗体抗原结合位点与过量官能化的DNA的损失。的敏感性，特异性，再现性和使用DEAL轭合物夹心ELISA中的已被证明在以前的研究中。8,9,11,12,16为了评估抗体-DNA缀合物的质量，所述缀合物可以在DNA阵列，以产生孵育抗体阵列，其随后用于在夹心ELISA实验，以产生校准曲线蛋白质检测（图3F）。生成这些校准曲线，可以使用在传统的夹心ELISA试剂盒中提供的重组蛋白。使用高品质的偶联物应该产生线性范围和检测的下限与这些试剂盒的相媲美。作为一个例子，对INFγ和TNFα的下限为夹心ELISA我们的抗体阵列上（图3F）都〜50-100微克/毫升，并且consisten吨以〜15-1,000微克/毫升的在产品数据表对于常规夹心ELISA试剂盒所指示的线性检测范围。在下游的夹心ELISA实验中获得的差的信号读出可以归因于DNA阵列，过量的DNA中的缀合物溶液的干扰，或可替代地，ssDNAs的不完全偶联的抗体的低质量。 为抗体阵列上进行的夹心ELISA主要关注点包括试剂之间以及是否该信号反映了真实的生物事件的串扰。我们在这个协议中使用正交的DNA已被彻底验证，以不到0.1％的串扰。因此，任何串扰在免疫测定阶段观察到的主要是由抗体和ELISA试剂。阵列中的测试对于串扰抗体中前应进行对实际样品进行检测。一旦抗体面板构造，一些夹心ELISA对照实验应该被法律约束ormed具备以下条件:1.在不抗原，2.在不检测抗体和3.检测抗体和仅标记试剂。如果串扰是在免疫测定阶段观察到的，抗体对和/或ELISA试剂应更换。应当指出的是，使用可商购的抗体与常规的ELISA试剂盒并不能保证适用性的基于阵列的夹心ELISA技术。 该技术的优点包括廉价的制造，微型尺寸和设计的灵活性。我们的制造协议不需要的微阵列点样器，可能无法使用抗体阵列的许多用户。流构图建立能够容易地组装在简单的实验室。对于未配有用于光刻工具的实验室，我们提供的CAD设计中的协议可以被转发至公司提供的微细加工服务。瘦身传统的芯片进入紧凑型阵列制造为我们的协议允许与在单细胞的实验中使用的微芯片的兼容性。我们的协议特性生产数组作为单链DNA阵列首次转换到抗体芯片之前。同的ssDNAs初步图案形成具有许多优点。首先，ssDNAs的是化学上更稳定的相比抗体，因而所述的ssDNA图案化幻灯片可以不显著降解8,16被存储在干燥器中的至少6个月。第二，我们的方法提供了一个最终用户的灵活性通过简单地混合所述选择性偶联物一起在无微阵列的变形例的解决方案，以选择所需要的测定法，;与此相反，常规的蛋白质/抗体阵列是预先设计并固定在基板上，因此测定的改变需要的蛋白质/抗体的从一开始就形成图案/打印。代表性的研究说明了使用在高通量条形码抗体微阵列的detectioN的单个细胞的多个信号蛋白。通过改变流动图案的设计和/或所用的寡核苷酸图案形成方案，众多阵列布局可以探讨。作为这种应用的一个例子，从单个细胞功能的蛋白质可进行研究。该芯片用于单细胞分析包括多达〜8700微容器，每一个完整的3×3的抗体阵列（ 图3H）一致。这样一个建立允许高通量检测。蛋白质通过细胞培养的微容器分泌可以通过该阵列来检测。多功能芯片的设计也是芯片与其它通用组件8,15合并后允许多重检测血清，细胞裂解液和单细胞蛋白。除了 ​​蛋白质检测中，3×3抗体阵列也是在捕获细胞 （图3G）是有用的。 这种方法的主要缺点是其附加并发症exity与常规微阵列的制造相比较。策略用于构图和阵列设计，必须同时考虑所制造的阵列的特定应用仔细概念化。这种方法还需要一些关键的步骤，包括验证过程和测试串扰。的3×3阵列构造使用我们的协议被限制在检测7蛋白的时间。但是，这种限制可以通过创建更大的阵列被超越。这里展示的协议不限于3×3微阵列的制造。我们已经能够成功地制作5×5芯片和数组元素的其他方面。原则上， 另一个 n×m个阵列可以使用类似的技术，只要在创造DNA初步图案化阵列中使用的寡核苷酸集是正交避免阵列元件之间的串扰来制造。扩展阵列的创建是通过流式细胞patterni实现纳克n种类型的ssDNA的锚的第一DNA图案化步骤，然后是n×m个桥接的ssDNA序列用于第二构图步骤。例如，创建一个5×5阵列 ，锚定序列A到E都可以使用，而桥接序列A&#39;，我要E&#39;-XXV被利用。实现自动化的流动图案形成过程中，机器人平台已经开发18虽然这仅利用一种DNA流动图案化步骤。原则上，相同的平台可以扩展到垂直流动图案的第二步骤，而两个步骤之间的切换可以手动需要剥离第一图案化步骤之后的第一PDMS模具，随后洗涤并干燥该滑动（这个步骤将需要大约10分钟），交配滑动与另一模具的PDMS，以促进垂直取向第二图案形成步骤之前。 我们已经讨论了详细的程序制作一个n×m个阵列图案与抗体高密度，持有在蛋白质组学研究和细胞信号应用前景巨大潜力。这里介绍的协议也可以作为更精细的DNA /抗体阵列用于其它目的的结构的引导。

抗体微阵列已被广泛用于蛋白质组学研究了数十年来检查定位的蛋白质，包括蛋白生物标志物1-3的存在。虽然这个领域目前正面临着来自其他高通量技术的巨大挑战，如质谱（MS），仍然有足够的空间用于抗体芯片的效用，主要是因为这些设备提供简单的数据解释和易用的界面与其他实验。近年来，微阵列集成到微芯片的支架提供了抗体微阵列的新契机兴旺4-7。例如，条形码芯片集成到一个单细胞的微芯片已被用于在小区通信研究8,9。该技术具有比其他可用的芯片技术优势明显。它具有数组元素在10-100微米，比典型的150微米大小的常规芯片的Elemen使用更小TS。较小的阵列元件的构造是利用系统的流动图案形成方法来实现，这产生了紧凑的微阵列，可以检测单细胞分泌的蛋白质和细胞内蛋白质。另一个优点是使用简单，仪器免费安装的。这是特别重要的，因为大多数实验室和小公司可能无法访问微阵列核心设备。这种条码抗体微阵列具有增强测定的吞吐量和可用于在单细胞进行高多重分析，同时实现高灵敏度和特异性与常规夹心酶联免疫吸附试验的可比性（ELISA 8）。这项技术已发现许多应用中检测来自成胶质细胞瘤9-11 T细胞的蛋白质，12和循环肿瘤细胞13。或者，条形码DNA微阵列单独已用于神经元和星形胶质细胞对模拟天生的精确定位荷兰国际集团的脑组织14的 体内组装。 这个协议只聚焦于实验步骤和二维（2-D）条码抗体微阵列具有在流体样品中的生物标志物的检测的潜在应用集结块和单个细胞。该技术是基于一个寻址单链的，一维（1-D）的DNA微阵列构造使用被在玻璃基板上的空间图案正交的寡核苷酸。一维图案时形成平行流动通道中的流动图案化步骤中使用，而这样的图像呈现为离散的频带视觉上相似于1-D的通用产品代码（UPC）条形码。的2-D（n×m个 ）的抗体阵列的构成-让人想起的2-D快速响应（QR）码矩阵-需要更复杂的图案化的策略，但允许抗体在较高密度8,15的固定。该制造需要两个DNA图案化步骤，与第一图案垂直于第二。这两种模式的交叉点构成的阵列的n×m个元素。通过策略性选择的单链DNA（ssDNA）的流动图案化利用的序列，在一给定阵列的每个元素被分配一个特定的地址。这个空间参考是必要的，在微阵列载玻片荧光信号之间进行区分。的单链DNA阵列被转换为抗体阵列通过互补DNA抗体偶联物掺入，形成了所谓的DNA编码的抗体库（DEAL 16）的平台。 此视频协议描述了在两个取向创建n×m个抗体阵列，其中包括制备聚二甲基硅氧烷（PDMS）条码模具，流动图案化单链DNA，制备抗体-寡核苷酸缀合物DEAL，和3×3 DNA阵列转换成一个3的关键步骤×3抗体芯片。

<table><tbody><tr><td>Sylgard 184 silicone elastomer base</td><td>Dow Corning</td><td>3097366-1004</td><td></td></tr><tr><td>Sylgard 184 silicone elastomer curing agent</td><td>Dow Corning</td><td>3097358-1004</td><td></td></tr><tr><td>SU-8 2025 photoresist</td><td>MicroChem</td><td>Y111069</td><td></td></tr><tr><td>Silicon wafers&amp;nbsp;</td><td>University Wafers</td><td>452</td><td></td></tr><tr><td>Poly-L-lysine coated glass slides</td><td>Thermo Scientific</td><td>C40-5257-M20</td><td></td></tr><tr><td>Oligonucleotides</td><td>Integrated DNA Technologies</td><td></td><td>*Custom-ordered from Integrated DNA Technologies, see &lt;strong&gt;Table 2&lt;/strong&gt;.</td></tr><tr><td>Poly-L-lysine adhesive stock solution</td><td>Newcomer Supply</td><td>1339</td><td></td></tr><tr><td>Bis (sulfosuccinimidyl) suberate&amp;nbsp; (BS3)</td><td>Thermo Scientific</td><td>21585</td><td></td></tr><tr><td>1x Phosphate buffered saline, pH 7.4</td><td>Quality Biological</td><td>114-058-101</td><td></td></tr><tr><td>&amp;Auml;kta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System</td><td>GE (Amersham Pharmacia)&amp;nbsp;</td><td>18-1112-41</td><td></td></tr><tr><td>Superose 6 10/300 GL column</td><td>GE Healthcare Life Sciences</td><td>17-5172-01</td><td></td></tr><tr><td>Capture antibodies</td><td>various</td><td>various</td><td>*Antibody selection depends on application</td></tr><tr><td>Succinimidyl-6-hydrazino-nicotinamide (S-HyNic)</td><td>Solulink</td><td>S-1002</td><td></td></tr><tr><td>Succinimidyl-4-formylbenzamide (S-4FB)</td><td>Solulink</td><td>S-1004</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylformamide</td><td>Sigma-Aldrich</td><td>227056</td><td></td></tr><tr><td>Citric acid, anhydrous</td><td>Acros</td><td>42356</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318</td><td></td></tr><tr><td>Amicon Ultra spin filter 10 kDa MWCO</td><td>EMD Millipore</td><td>UFC201024</td><td></td></tr><tr><td>Spin coater</td><td>Laurell Technologies</td><td>WS-650-MZ</td><td></td></tr><tr><td>Biopsy punch with plunger (1.0 mm diameter)</td><td>Ted Pella, Inc.</td><td>15110-10</td><td></td></tr><tr><td>Diamond scribe (Style 60)</td><td>SPI supplies</td><td>6004</td><td></td></tr><tr><td>Trimethylchlorosilane</td><td>Sigma Aldrich</td><td>92361</td><td></td></tr></tbody></table>

flow-pattern guided fabrication of high-density barcode antibody microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

对于PDMS模具（图1A-1B）的设计被利用CAD程序（AutoCAD中）绘制。两种设计显示功能的渠道流动图案，一横一纵。每个设计的左，右部分是对称的;无论他们可能是入口或出口。每20个通道是从一端一直到另一端卷绕。每个设计印刷在铬光掩模（图1C）。所制造的SU-8主一个晶片上示于图1D。为了便于流动图案化的PLL或DNA，将PDMS模具偶合到氮气流建立（图1E）。 还有在这个协议中使用了三个流动图案化步骤。第一步固定不变的PLL在玻璃基板上，而随后的步骤都引入寡核苷酸的解决方案。在表1中，oligonucle解1-3 otide组合物给出。各寡核苷酸的工作浓度是50μM和各溶液的总体积是39微升。这些解决方案通过组合13微升每种寡核苷酸原液（150μM）的制备。 图案化的DNA微阵列的单位是重复的，在整个载玻片相邻。对于3×3芯片，最大密度是在一个载玻片约40万点。侧由端与商业DNA微阵列比较表明，我们的技术是约5倍更敏感时结合的Cy3标记的互补DNA。图案化的DNA比较均匀，在多个重复〜5％的变化。从生物样品测定蛋白质含量时，这些功能保证我们技术的高定量能力。此外，DNA的与所述流道设计组合的正交提供flexibili图案化在各种几何形状的（图2D）TY。 通过杂交DNA的标记抗体（图3B）的DNA阵列转换成抗体阵列后，所得抗体面板用于多重检测，主要是通过夹心ELISA平台，如图3C所示。在夹心ELISA，生物素化的检测（二次）抗体结合到捕获的蛋白质，并用荧光团共轭链霉抗随后的标记允许基于荧光的检测（图3C-E）。检测的蛋白示&lt;从一端到载玻片（图3D）的另一端的10％的变化。 利用3×3阵列 ，我们能够检测高达7蛋白质，而分配一个数组元素作为参考（Cy3标记）和另一种元素作为阴性对照。例如，在单细胞实验执行用于肿瘤研究中，蛋白质白细胞介素-6（IL-6），基质金属蛋白酶-9（MMP9），肝细胞生长因子（HGF），血管内皮生长因子（VEGF），白细胞介素-8（IL-8），和巨噬细胞迁移抑制因子（MIF）从单个小区可以同时检测（图3E）。 我们还使用重组蛋白的不同浓度校准该系统。在图3F中，校准曲线的重组蛋白干扰素γ（IFN-γ），肿瘤坏死因子α（TNF-α），白细胞介素-13（IL-13），肿瘤坏死因子β（TNFβ）颗粒酶B，白细胞介素-4（IL-4）和白细胞介素-8（IL-8）被示出。检测的灵敏度非常相似，关于孔板常规夹心ELISA的甚至与高载荷的DNA，并可能在基片上的抗体。 条形码antibodÿ微阵列可用于检测生物标志物的流体样品中，以及在单细胞。自单细胞分析不是此协议的焦点，我们只是例举的3×3抗体微阵列的应用中的细胞因子的检测。通过抗体-表面标记的相互作用，分化8的簇（CD8）阳性细胞被捕获在微阵列元件（ 图3G;在顶部上的PDMS芯片）中的一个，以及所述元素的其余部分被用来检测分泌的细胞因子（ 图3H）。与此细胞捕获方法中，"单元阵列"可以形成。这个阵列捕获技术还允许对细胞进行各ELISA实验的数量控制。将细胞物理隔离，在微容器，使得所检测的蛋白质涉及到特定的单细胞。在图3H中，示出每个微容器配备有16微阵列元素，以确保封装一个完整的3×3芯片集。 <img alt="图1" src="/files/ftp_upload/53644/53644fig1.jpg" /> 图1 的设计与PDMS条形码模具基于流的DNA图案的制造。面板（A）表示 AutoCAD绘图水平和垂直的一维条形码图案。面板（B）示出从（A）的叠加水平和垂直条形码图案。绿框包围图案与离散3×3阵列领域。 （ 三）铬掩模制造的SU-8大师。 （D）的SU-8的主制造在硅晶片上。 （ 五）建立了流动图案。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53644/53644fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" sRC ="/文件/ ftp_upload / 53644 / 53644fig2.jpg"/&gt; 图2. 建筑和一个3×3 的DNA阵列的 验证。这个数字已经被修改"定量细胞相互作用的功能与应用，以胶质母细胞瘤肿瘤细胞，"王，J。等人，2012年， 纳米快报 12 。 版权所有2012年美国化学学会。适于与许可。将DNA流动图案化步骤（A）的方案。 （B）的荧光强度的20个通道中的选定区域（由垂直线跟踪）的一维条码的轮廓。的Cy3结合的寡核苷酸杂交进行验证DNA阵列。在完成第二DNA图案化步骤之后进行验证用Cy3缀合的DNA阵列（C）的荧光强度分布。 （ 四）从不同的FL替代荧光图案OW-图形化的战略。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53644/53644fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53644/53644fig3.jpg" /> 图3. 应用程序的 3×3 芯片 的 。（A）方案的抗体，寡核苷酸DEAL轭合物的合成。 （B）的方案的DNA阵列转化为抗体阵列通过杂交用抗体-寡核苷酸缀合物。 （C）的方案利用3×3微阵列在夹心ELISA平台。这个数字已经被修改"定量细胞相互作用的功能与应用，以胶质母细胞瘤肿瘤细胞，"王，J。等人，2012年， 纳米快报 12。版权所有2012，由美国化学学会。改编许可。面板（D）示出下一个Cy5的信道产生的荧光读数。这对应于该检测到的6蛋白质夹心ELISA实验。面板（E）是一放大的下Cy3和Cy5通道3×3阵列读出的信息。面板（F）示出的夹心ELISA标准曲线的重组蛋白。面板（G）示出了一个"单元阵列"，通过与阵列上的抗体结合捕获细胞形成。面板（H）显示叠加在一个微芯片微容器的检测结果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53644/53644fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 解 </td><td> 寡核苷酸组成 </td></tr><tr&gt; <td> 1 </td><td> A&#39;-i的，B&#39;-ⅱ，C&#39;-ⅲ </td></tr><tr><td> 2 </td><td> A&#39;-ⅳ，B&#39;-V，C&#39;-六</td></tr><tr><td> 3 </td><td> A&#39;-VII，B&#39;-VIII，C&#39;-IX </td></tr></tbody></table> 表1.组合物溶液1-3 DNA流动图案的。 <table fo:keep-together.within-page="1"><tbody><tr><td colspan="2">锚定序列</td></tr><tr><td>一个</td><td> ATCCTGGAGCTAAGTCCGTA-AAAAAAAAAAAAAAAAAAAA-ATCCTGGAGCTAAGTCCGTA-AAAAAAAAAAAAAAAAAAAA </td></tr><tr><td rowspan="2">乙</td><td> GCCTCATTGAATCATGCCTA-AAAAAAAAAAAAAAAAAAAA-GCCTCATTGAATCATGCCTA- </td></tr><tr><td> AAAAAAAAAAAAAAAAAAAA </td></tr><tr><td> C </td><td> GCACTCGTCTACTATCGCTA-AAAAAAAAAAAAAAAAAAAA-GCACTCGTCTACTATCGCTA-AAAAAAAAAAAAAAAAAAAA </td></tr><tr><td colspan="2">桥接序列</td></tr><tr><td> A&#39;-I </td><td> TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-ATGGTCGAGATGTCAGAGTA </td></tr><tr><td> B&#39;-II </td><td> TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-ATGTGAAGTGGCAGTATCTA </td></tr><tr><td> C&#39;-III </td><td> TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA-ATCAGGTAAGGTTCACGGTA </td></tr><tr><td> A&#39;-IV </td><td> TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-GAGTAGCCTTCCCGAGCATT </td></tr><tr><td> B&#39;-V </td><td> TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-ATTGACCAAACTGCGGTGCG </td></tr><tr><td> C&#39;-VI </td><td> TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA-TGCCCTATTGTTGCGTCGGA </td></tr><tr><td> A&#39;-VII </td><td> TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-TCTTCTAGTTGTCGAGCAGG </td></tr><tr><td> B&#39;-VIII </td><td> TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-TAATCTAATTCTGGTCGCGG </td></tr><tr><td> C&#39;-IX </td><td> TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA，GTGATT<html> AAGTCTGCTTCGGC </td></tr><tr><td colspan="2">的Cy3共轭的寡核苷酸进行验证</td></tr><tr><td>一个&#39; </td><td> TACGGACTTAGCTCCAGGAT </td></tr><tr><td> B&#39; </td><td> TAGGCATGATTCAATGAGGC </td></tr><tr><td> C&#39; </td><td> TAGCGATAGTAGACGAGTGC </td></tr><tr><td>一世&#39; </td><td> TACTCTGACATCTCGACCAT </td></tr><tr><td>二&#39; </td><td> TAGATACTGCCACTTCACAT </td></tr><tr><td>三" </td><td> TACCGTGAACCTTACCTGAT </td></tr><tr><td> IV" </td><td> AATGCTGGGGAAGGCTACTC </td></tr><tr><td> V&#39; </td><td> CGCACCGCAGTTTGGTCAAT </td></tr><tr><td>六" </td><td> TCCGACGCAACAATAGGGCA </td></tr><tr><td>七&#39; </td><td> CCTGCTCGACAACTAGAAGA </td></tr><tr><td>八&#39; </td><td> CCGCGACCAGAATTAGATTA </td></tr><tr><td> IX" </td><td> GCCGAAGCAGACTTAATCAC </td></tr></tbody></table>"FO:保持-together.within页="1"&gt; 表2中的DNA流动图案形成中使用的序列。

Watch this Scientific Journal Video about Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray at JoVE.com

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

高密度条码抗体芯片的水流模式的指导下制作

这个协议概括了大规模的制造中，复用的二维DNA或抗体阵列，在细胞信号研究和生物标记物检测的应用潜力。

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

flow-pattern-guided-fabrication-high-density-barcode-antibody

Nanyang Technological University

Research

JoVE Journal

Chemistry

14.6K 次观看.  University at Albany, State University of New York. 这个协议概括了大规模的制造中，复用的二维DNA或抗体阵列，在细胞信号研究和生物标记物检测的应用潜力。 

视频: Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

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

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

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

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 ...

环境科学

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

高密度DNA和RNA微阵列.光刻合成、杂交和制备大型核酸库

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

遗传学

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

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

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

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 ...

免疫与感染

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Glycochips Production

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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

A Peptide Microarray Technology for Specificity Profiling of Antibodies

Source: Cornett, E. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55912">Analysis of Histone Antibody Specificity with Peptide Microarrays.</a> J. Vis. Exp. (2017).
This video demonstrates a technique that applies peptide microarray technology to profile the specificity of antibodies recognizing post-translational modifications (PTMs) on histone peptides. A microarray slide containing immobilized fluorophore-conjugated histone peptides with known PTM combinations is treated with target PTM-specific antibodies. This process helps identify the antibody&#39;s specificity for the target and its potential cross-reactivity with non-target PTMs.

用于抗体特异性分析的肽微阵列技术

1. Designing the Array Slide and Source Plate Layout

	Under the &#39;Plan a slide layout&#39; heading on the ArrayNinja interface, click on the link that ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.