这项工作是由美国国立卫生研究院（补助08UDE017788-05）的支持。 EBP也承认支持西班牙基金会科学与技术（FECYT）。作者感谢人Shonda T.盖洛德和Pratyusha Mogalisetti的手稿的批判性阅读。

<img alt="图1" width="600px" fo:content-width="5.5in" src="/files/ftp_upload/50726/50726fig1.jpg" /> 图1。 。工作流应用的光纤微球体抗体阵列唾液剖析 （1）微球的内部编码的两种荧光染料，（2）所编码的微球在外部修改与蛋白特异性的单克隆抗体，（3）复用的微球混合，和（4）随机地沉积在微孔蚀刻在光纤束的近端，（5）唾液蛋白是通过夹心免疫捕获的微球，和（6）使用荧光显微镜进行定量。 1。编码微球<ol><li>称取钠铕（Ⅲ）thenoyltrifluoroacetonate三水（欧盟TTA，MW =869.54克/摩尔）在琥珀色玻璃瓶中，并在四氢呋喃（THF）准备一个200毫米原液。用移液枪轻轻混匀;直观地验证染料完全溶解。 </li><li>称取香豆素30（C30，MW =347.41克/摩尔）在琥珀色玻璃瓶中，并准备一个12毫米原液在THF。用移液枪轻轻混匀，直观地验证了染料完全溶解。 </li><li>制备700微升使用储备溶液和THF中以达到表1中列出的最终浓度为每微球型工作液。 </li><li>确保微球（10％重量/体积）深受涡旋暂停，再换乘60微升悬浮液1.5 ml离心管中。 </li><li>加入600微升PBS，以微球悬浮和20倍吹打混匀。离心管在10,000 rpm 3分钟，小心取出上清液。重复此过程，另2个洗球。 </li><li>使用相同的程序，步骤1.5十二烷基硫酸钠洗涤微球沉淀3次。 </li><li>离心微球/ THF悬浮液以10,000 rpm离心3分钟，除去上清液，并重新悬浮于600微升的沉淀工作溶液列于表1。将混合物转移至新的1.5 ml微量离心管中。 </li><li>密封离心管封口膜，并把它放在摇床。摇在3,000 rpm孵育24小时，用一个盒子上面有铝箔覆盖安装避光。 </li><li>离心微球悬浮液在10,000 rpm下3分钟，以形成颗粒。 </li><li>除去上清液染料溶液，冲洗微球6X与600微升甲醇中，然后用600微升PBS含0.01％Tween-20的另一种6倍，如在步骤1.5中描述。 </li><li>存储编码微球在600微升PBS含0.01％吐温20在4°C，直到使用，避光。 </li></ol><table border="1"><tbody><tr><td> 微球型名称: </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </STRONG&gt; </td><td> 7 </td></tr><tr><td> EU-TTA（MM） </td><td> 100 </td><td> 100 </td><td> 10 </td><td> 100 </td><td> 10 </td><td></td><td></td></tr><tr><td> C30（MM） </td><td></td><td> 1 </td><td> 1 </td><td> 6 </td><td> 6 </td><td> 1 </td><td> 6 </td></tr></tbody></table> 表1中。的Eu-TTA和C30浓度的工作溶液。 2。蛋白质捕获微球的制备<ol><li>制备的MES缓冲液[2 - （N-吗啉）乙磺酸（MES）的0.1M，氯化钠0.9％，SDS 0.01％，pH值为5.7]和PBS / SDS缓冲液（磷酸钠0.01 M，氯化钠0.154 M，SDS 0.01％，pH值7.4）预先在室温下。 </li><li>加入200μl编码微球悬浮液（含〜2毫克微球）到一个安全锁1.5 ml离心管。使用这种类型的离心管中，以避免在步骤2.6中温育过程中泄漏是很重要的。 </li> &lt;LI&gt;清洗微球600微升的MES按照步骤1.5中所述缓冲3倍。加入700μl的MES在微球颗粒和吹打拌匀。 </li><li>准备0.105克/ ml的1 - 乙基-3 - [3 - 二甲基氨基丙基]碳二亚胺盐酸盐（EDC，MW =191.7克/摩尔）和0.163克/毫升磺基-N-羟基硫代琥珀酰亚胺（磺基-NHS，MW =217.13克/ mol）的在MES缓冲溶液中分离管。 </li><li>加入新鲜配制的EDC解决方案， 一滴一滴进入微球悬浮液150微升。 EDC的水解速度非常快，因此，它必须使用新鲜的溶液，以确保固定化效率。紧接在添加EDC后，加入150μl的磺基-NHS溶液的微球，通过移液混合均匀，并验证该悬浮液是均匀的。 </li><li>盖上管，用封口膜密封，并将其放置在摇床。摇在1,500 rpm 4小时，用一个盒子上面有铝箔避光。 </li><li>离心微球suspens离子以10,000 rpm离心3分钟，以形成沉淀物，并除去上清液。如在步骤1.5中所述，用500μL的PBS / SDS缓冲液3X冲洗微球。 </li><li>加入200μl的PBS / SDS缓冲液的颗粒，混合微球以及后转移到300微升含60微克的捕获抗体的PBS / SDS缓冲液。它首先暂停微球的颗粒，然后加入微球悬浮液到抗体溶液中是很重要的。 </li><li>通过摇动在1,500 rpm 4小时在摇床上孵育抗体 - 微球混合物中，用框有铝箔避光。 </li><li>离心微球悬浮液在10,000 rpm下3分钟，以形成沉淀物，并除去上清液。用500μlStartingBlock（TBS）封闭缓冲液3次，如在步骤1.5中所述洗涤微球。 </li><li>混合性微球用1ml TBS的封闭缓冲液孵育微球在1,500 rpm进行1小时的摇床上，用一个框科夫避光红色的有铝箔。 </li><li>离心微球悬浮液在7,000 rpm离心3分钟，以形成沉淀物，并除去上清液。用500μLTBS封闭缓冲液，如在步骤1.5中所述洗涤沉淀3次。 </li><li>离心微球溶液以7,000 rpm离心3分钟，以形成沉淀物，并除去上清液。加入500μlTBS封闭液并吹打混匀。 </li><li>存储修改后的微球悬浮在4°C，直到使用，避光。 </li><li>重复此步骤，以使其他类型使用相应抗体的微球。 </li></ol> 3。光纤芯片组装<ol><li>混合50μl的每种类型的蛋白捕获微球的成新的高压灭菌的离心管中。离心机股票微球池在7000 rpm下3分钟，除去上清，使剩余的体积为约100微升。 </li><li>手工切割的光纤束至大约5厘米的长度和依次用30，15，9,6，3，1，0.5和0.05微米尺寸的金刚石研磨膜的抛光机上抛光的两端。超声处理在去离子水中2分钟，在抛光束以除去任何颗粒。 </li><li>把小电磁搅拌棒加入0.5 ml离心管中，加入400微升的无蛋白质的PBS缓冲液中，并搅拌上磁性搅拌板30分钟以除去气泡。 </li><li>在150秒10,16 0.025 1N HCl溶液蚀刻抛光纤维束的一端。立即浸没和超声处理的蚀刻结束在去离子水中1分钟。干燥该纤维束的压缩空气。 </li><li>装载在纤维束上的纤维托座和阻止蚀刻端在无气泡，无蛋白的PBS缓冲液1小时。 </li><li>关于光纤束的蚀刻端存储的微球悬浮液的存款1微升等份。保护设置从光与铝箔盒，并等待15分钟。悬浮液的量将通过蒸发减少，微球强行进入蚀刻的微孔。重复此步加载一次。 </li><li>使用与样品溶液饱和棉签除去未被困在微孔微球。蛋白质微阵列现在可以使用了。请立刻步骤4.1唾液分析。 </li></ol> 4。唾液样本分析使用微芯片<ol><li>加入200μl样品溶液（100微升唾液上清液稀释StartingBlock T20（PBS）等体积的封闭液。唾液上清液中收集协议先前已公布的10）。入0.5ml的灭菌离心管中。沾在纤维进入溶液中加载的蛋白质微阵列孵育在摇床上以600rpm搅拌2小时，用的是盒覆盖有铝箔避光。 </li><li>准备20毫升洗涤缓冲液，加入200μl的10％BSA溶液和200μl的10％Tween-20的成19.6毫升PBS，英里x深受轻轻晃动。 </li><li>浸蛋白质微阵列到含有200μl的洗涤缓冲液的新高压灭菌的离心管中，并摇动以600rpm持续2分钟。重复此洗3次。 </li><li>孵育微阵列中含有抗-VEGF，IP-10，IL-8，EGF，MMP-9的5μg/ ml的检测抗体鸡尾酒的100微升溶液中，在StartingBlock T20 IL-1β的生物素化的检测抗体（PBS）封闭缓冲液30分钟，在室温下在摇床上以600rpm，从光利用箱用铝箔覆盖保护。 </li><li>按照步骤4.3中所述用洗涤缓冲液清洗芯片3倍。 </li><li>孵育微阵列在200μl的PBS中20μg/ ml的链霉亲和R-藻红蛋白缀合物（SAPE）的溶液在600rpm的摇床上10分钟，用盒子覆盖有铝箔避光。 </li><li>按照步骤4.3中所述用洗涤缓冲液洗芯片5倍。 </li><li>清洁纤维（ 即 ，端部远离的远端从微球），捆绑用无水乙醇饱和棉签。 </li><li>干燥压缩空气的光纤的两端。通过纤维的前端安装的光纤上的落射荧光显微镜和图像。 </li><li>获取对应的Eu-TTA，C30，和SAPE荧光发射强度的微球的图像。光过滤器的设置和曝光时间的Eu-TTA，C30，和SAPE的荧光成像被描述在表2中。 </li></ol><table border="1"><tbody><tr><td> 渠道 </td><td> EU-TTA </td><td> C30 </td><td> SAPE </td></tr><tr><td>激励</td><td> 365/10x </td><td> 350/50x </td><td> 546/10x </td></tr><tr><td>分光镜</td><td> 525DCLP </td><td> 400DCLP </td><td> 560LP </td></tr><tr><td>发射器</td><td> 620/60m </td><td> 460/50m </td><td> 580/30m </td></tr><tr><td>曝光时间</td><td> 1秒</td><td> 0.3秒</td><td> 1秒</td></tr></tbody></table> 表2。参数用于微阵列的三个荧光图像。

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作者宣称，他们有没有竞争的财务权益。

研究者应该格外注意以下步骤:为更好的解码精度，有必要验证微球均匀地悬浮在所有孵育和微球编码过程中的洗涤步骤。此外，经编码的微球需要在整个实验中，以避光。下列正确的编码和储存程序中，我们发现，总体解码准确率为99％以上。该编码微球应保存在4℃保存，避免冷冻和避光。在适当的储存条件下，编码微球是稳定超过六个月。在编码和修改步骤，需要格外小心，以减?...

由于报道由Mark Schena并在90年代中期的同事第一芯片，这个强大的工具已被用于生物研究1的许多领域。能够同时检测多种蛋白质在诊断液，如血液的抗体微阵列，具有在临床诊断和生物标志物筛选2-10重要的应用。唾液，含有许多相同的分析物如血，一直被认为是一个优选的替代方案，因为血液唾液收集是安全的，非侵入性的，并且可以进行通过微创医疗人员11-13。目前，使用唾液样本复用蛋白分析是由几个重要的因素，其中包括目标分析物14的低浓度和不同的生物标记物15的宽浓度范围的限制。 。 在此，我们证明6种蛋白的分析:人血管内皮生长因子（VEGF），干扰素γ-诱导蛋白10（IP-10），白细胞介素-8（IL-8），表皮生长因子（EGF），基质金属蛋白酶9（MMP-9），和白细胞介素-1β（IL-1β） 。该方法的性能用标准溶液构成的重组蛋白分析物和封闭缓冲液中最初验证。从不同的慢性呼吸系统疾病的患者以及健康对照真正的唾液样本进行了测试表现令人满意。该协议应当适用于其他蛋白质的分析物和其他基于微球的实验。该平台提供了相当大的优势，在分析化学领域，因为它使低浓度的几种蛋白质具有广泛的动态范围，最小的非特异性相互作用，减少样品消耗和低成本的快速，精确，可重复的和同时分析在比较的类似的酶联免疫吸附试验（ELISA）。

<table><tbody><tr><td>Eu-TTA dye</td><td>Fisher Scientific</td><td>AC42319-0010</td><td></td></tr><tr><td>THF</td><td>Sigma-Aldrich</td><td>34865-100ML</td><td></td></tr><tr><td>Amber glass vial</td><td>Fisher Scientific</td><td>03-339-23B</td><td></td></tr><tr><td>Coumarin 30 dye</td><td>Sigma-Aldrich</td><td>546127-100MG</td><td></td></tr><tr><td>Microspheres</td><td>Bangslabs</td><td>PC05N/6698</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>PBS 10x concentrate</td><td>Sigma-Aldrich</td><td>P5493-1L</td><td></td></tr><tr><td>Water</td><td>Sigma-Aldrich</td><td>W4502-1L</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860-100ML</td><td></td></tr><tr><td>Tw-20</td><td>Sigma-Aldrich</td><td>P7949-100 ml</td><td></td></tr><tr><td>BupH MES buffered saline</td><td>Thermo Scientific</td><td>28390</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>05030-500ML-F</td><td></td></tr><tr><td>NaOH solution</td><td>Fisher Scientific</td><td>SS256-500</td><td></td></tr><tr><td>Safe-lock microcentrifuge tube</td><td>VWR labshop</td><td>53511-997</td><td></td></tr><tr><td>EDC</td><td>Thermo Scientific</td><td>22980</td><td></td></tr><tr><td>Sulfo-NHS</td><td>Thermo Scientific</td><td>24510</td><td></td></tr><tr><td>Human VEGF capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB293</td><td></td></tr><tr><td>Human IP-10 capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB266</td><td></td></tr><tr><td>Human IL-8 capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB208</td><td></td></tr><tr><td>Human EGF capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB636</td><td></td></tr><tr><td>Human MMP-9 capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB936</td><td></td></tr><tr><td>Human IL-1&amp;beta; capture antibody</td><td>R&amp;amp;D Systems</td><td>MAB601</td><td></td></tr><tr><td>Mouse IgG1 isotype control antibody</td><td>R&amp;amp;D Systems</td><td>MAB002</td><td></td></tr><tr><td>StartingBlock (TBS) buffer</td><td>Thermo Scientific</td><td>37542</td><td></td></tr><tr><td>HCl standard solution 1.0 N</td><td>Sigma-Aldrich</td><td>318949-500 ml</td><td></td></tr><tr><td>0.5 ml microcentrifuge tubes</td><td>Fisher Scientific</td><td>05-408-120</td><td></td></tr><tr><td>Protein-free (PBS) buffer</td><td>Thermo Scientific</td><td>37572</td><td></td></tr><tr><td>Recombinant human VEGF 165</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td></td></tr><tr><td>Recombinant human IP-10</td><td>R&amp;amp;D Systems</td><td>266-IP</td><td></td></tr><tr><td>Recombinant human IL-8</td><td>R&amp;amp;D Systems</td><td>208-IL</td><td></td></tr><tr><td>Recombinant human EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td></td></tr><tr><td>Recombinant human MMP-9</td><td>R&amp;amp;D Systems</td><td>911-MP</td><td></td></tr><tr><td>Recombinant human IL-1&amp;beta;</td><td>R&amp;amp;D Systems</td><td>201-LB</td><td></td></tr><tr><td>StartingBlock T20 (PBS) buffer</td><td>Thermo Scientific</td><td>37539</td><td></td></tr><tr><td>Blocker BSA in PBS</td><td>Thermo Scientific</td><td>37525</td><td></td></tr><tr><td>Biotinylated VEGF detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF293</td><td></td></tr><tr><td>Biotinylated IP-10 detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF266</td><td></td></tr><tr><td>Biotinylated IL-8 detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF208</td><td></td></tr><tr><td>Biotinylated EGF detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF236</td><td></td></tr><tr><td>Biotinylated MMP-9 detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF911</td><td></td></tr><tr><td>Biotinylated IL-1&amp;beta; detection antibody</td><td>R&amp;amp;D Systems</td><td>BAF201</td><td></td></tr><tr><td>Streptavidin, R-phycoerythrin</td><td>Invitrogen</td><td>S-21388</td><td></td></tr><tr><td>Ethanol (200 proof)</td><td>Sigma-Aldrich</td><td>E7023-500ML</td><td></td></tr></tbody></table>

multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles

本文中，我们描述了一种协议，用于同时测量6蛋白在唾液中使用的光纤基于微球的抗体阵列。所采用的免疫阵列技术结合基于微球的悬浮阵列制造与使用荧光显微镜的优点。如在视频协议描述的，可商购的4.5微米的聚合物微球进行编码成七种不同类型的，由两个荧光染料物理截留在微球内的浓度分化。表面含羧基的编码微球通过EDC / NHS偶联化学进行了修改，用单克隆捕获抗体。组装的蛋白质微阵列，不同类型的编码和官能化的微球混合，并随机地沉积在4.5微米的微孔，从而进行化学蚀刻在一光纤束的近端。光纤束被用来同时作为载体和用于成像的米icrospheres。一旦组装后，该微阵列用于捕获蛋白在从诊所收集唾液上清液。该检测是基于使用生物素化的检测抗体的混合物为不同的分析物与链霉亲和结合的荧光探针，R-藻红蛋白的夹心免疫测定。微阵列是在三个不同的通道，两个对微球的注册和一个用于检测信号成像，荧光显微镜。的荧光显微照片，然后解码，并在MATLAB中使用自制的算法进行分析。

从三个通道表示光纤束的一小部分的荧光图像示于图2A-C。使用MATLAB写的一个算法这些图像进行分析（如在讨论部分更详细地描述）。该分析采用从铕-TTA编码图像（ 图2A）中的信息和C30的编码图像（ 图2B）来解码的微球，并计算信号的图像（ 图2C）在不同的微球的荧光强度。与相关蛋白的标准中获得的校准曲线，计算蛋白的唾液样品中的浓度。 </...

Watch this Scientific Journal Video about Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles at JoVE.com

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

我们描述了使用多路复用基于微球的抗体阵列分析唾液蛋白的过程。单克隆抗体共价连接到荧光染料编码的4.5微米聚合物用碳二亚胺化学性微球。修改后的微球沉积在光纤的微孔用荧光夹心免疫测定蛋白水平在唾液。

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

multiplexed-fluorescent-microarray-for-human-salivary-protein

Research

JoVE Journal

Chemistry

11.6K 次观看.  Tufts University. 我们描述了使用多路复用基于微球的抗体阵列分析唾液蛋白的过程。单克隆抗体共价连接到荧光染料编码的4.5微米聚合物用碳二亚胺化学性微球。修改后的微球沉积在光纤的微孔用荧光夹心免疫测定蛋白水平在唾液。 

视频: Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

科研

生物工程

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

免疫与感染

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.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

高密度条码抗体芯片的水流模式的指导下制作

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

化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

生物化学

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

无交叉反应和无免疫多路复用夹芯芯片

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

复用荧光免疫检测方法和故障排除

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

生物学

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

多路复用表面等离子体共振成像芯片上的多模态分析平台，用于分析细胞外囊泡亚群

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

A Bead-Based Multiplex Immunoassay to Identify Pathogen-Specific Antibodies in Saliva

Source: Augustine, S. A. J., et al. <a href="https://app.jove.com/v/54415">Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens.</a> J. Vis. Exp. (2016).
This video demonstrates a bead-based multiplex immunoassay technique to simultaneously detect antibodies for multiple environmental pathogens in human saliva. Fluorescent beads were coupled to antigens from different pathogens, which were captured using antibodies in the human saliva sample. Upon labeling the antibodies using a fluorescent reporter, the beads were analyzed using flow cytometry to assess the presence of different pathogen-specific antibodies in the saliva.

一种基于微珠的多重免疫测定法，用于鉴定唾液中的病原体特异性抗体

1. Bead Coupling

	Couple the antigens to the bead sets using the concentrations shown in Table 1.
	Add each antigen to the activated beads and bring the ...