作者非常感谢在南加州大学教授安德烈·阿玛尼在此协议的支持。由国家科学基金会为这项工作的初步发展提供了资金[085281和1028440]和国立卫生研究所通过美国国立卫生研究院主任的新的创新奖励计划1DP2OD007391-01]。其他信息可在<a href="http://web.missouri.edu/~hunthk/" target="_blank">http://web.missouri.edu/〜hunthk /</a> 。

1。背景通过一个简单的三个步骤（ 图1），生物素连接到这些设备的表面。首先，我们将表面清洗干净，并填充羟基暴露要么氧等离子体或食人鱼解决方案的设备，它。第二，我们使用气相沉积重视终止与伯胺通过水解和缩合反应的羟基硅烷偶联剂。第三，我们重视通过N-羟基琥珀酰亚胺（NHS）酯化学表面的生物素。我们直接选择这些技术有兴趣的读者，我们以前更多有关这些技术的发展，以及我们的动机的解释工作。 这些反应的成功可以通过光镜和荧光显微镜进行评估后，伯胺此外，以及后加入生物素。虽然光米icroscopy可以用来确定是否表面功能化协议，在微球表面，甚至污染损害，荧光显微镜被用来验证生物素分子的表面覆盖的质量和均匀性，以及生物素的能力（链霉）亲和表面结合。来评估胺的覆盖范围，表面上我们用异硫氰酸荧光素（FITC标记）荧光染料，与伯胺反应形成稳定的共价硫脲联动的微。得克萨斯红一直以抗生物素​​蛋白结合的荧光染料用于标记通过生物素 - 抗生物素蛋白相互作用的表面上的生物素组。在这两种情况下，染料的选择，可以互动与表面上的官能团（或者通过受体 - 配体相互作用或共价键）。 在这里，硅烷偶联剂，其中有三个离开组和一个功能组，提供了桥梁的赌注WEEN表面无机和有机探针分子。伯胺的功能与NHS酯反应定量，以形成稳定的酰胺键。在这种情况下，我们使用生物素探针分子，其戊酸侧链已修改与NHS酯组。实际上，任何探针分子NHS酯组可以添加到表面采用以下协议。此外，这些协议是通用的，可用于任何硅器件表面功能化与生物素。 这些协议的主要挑战实际上不是化学本身，而是在硅胶微球的处理。请注意，整个协议，微球应茎，由grapsing用锋利的尖镊子轻轻处理。这使微球本身远离镊子，允许步骤之间的便捷的交通。下面的协议中的许多步骤是专为解决这一事实或。 2。微球的制备<ol><li>微球的构建存储住房。 <ol><li>使用exacto刀，切成1一块厚纸板¼×1平方米;磁带这到正常大小的玻片，并附加满足其两端，形成一卷透明胶带1节，纸板。 </li><li>这将创建一个平台，微球上，可以从周围环境的提升和隔离，并防止损坏其表面。 </li></ol></li><li>用剪刀，剪光纤从光纤线轴第3英寸。 </li><li>使用无聂纤维的，汽提塔，从过去的0.5英寸的切割件的纤维年底剥夺保护的聚合物涂层，只留下硅芯。清理剩余的任何一个Kimwipe的聚合物表面挫伤与甲醇轻轻擦拭与Kimwipe的的纤维。 </li><li>使用裸光纤切割刀，修剪剥离的结束使日只有约1毫米剥离纤维仍然对光纤末端。 </li><li>放入一个CO 2激光的路径剥去光纤的一端，照顾到纤维等，其剥离的一端朝下，垂直对齐。 </li><li>打开CO 2激光，剥离年底光纤表面。激光将使用2秒约3.5％的电力领域融入纤维中剥离的结束。 </li><li>使用镊子，认真把握的微干（非精简部分光纤）。将干到磁带辊微住房。载玻片可存储在培养皿中。这使得微球的安全存储。 </li></ol> 3。填充表面羟基<ol><li>使用食人鱼解决方案： <ol><li>在一个铰链盖60毫升的聚丙烯小瓶，准备一个Piranha溶液（70:30发烟H体积<sub&gt; 2 SO 4：H 2 O 2（30％）），加入5毫升的双氧水，发烟硫酸11.6毫升的小瓶。注意：穿耐酸手套。 </li><li>转让载玻片上，持有至少1球，小瓶。微球应该是在与液体接触，但液体不应该接触的硬纸板。调整溶液的体积，如果需要的话。 </li><li>轻轻从药瓶中取出玻片，并把它插入到另一个塑料瓶含有DDI的H 2 O让样品在坐5分钟的解决方案。 </li><li>轻轻从药瓶中取出玻片，并把它放在一个烤箱在80°C 10分钟，晾干表面。 </li></ol></li><li>使用氧等离子体处理： <ol><li>转移至少含有一个微氧离子室的玻璃幻灯片。 </li><li>设置200毫托氧气压力，功率为120 W。样品的氧等离子体暴露2英里nutes。 </li><li>等离子室中取出玻片。 </li></ol></li></ol> 4。将硅烷偶联剂表面<ol><li>置于载玻片上，至少含有一个微球，成真空干燥器在通风柜。 </li><li>也通风柜，打开硅烷偶联剂瓶（（APTMS）在这种情况下，aminopropyltrimethoxysilane使用），开瓶放置在干燥器。 </li><li>更换真空干燥器的盖子，吸气或房子真空线连接插座端口。 </li><li>打开房子真空或水行，撤离的真空干燥器。一旦真空密封盖子和底座之间形成，开始计时反应。这将存入到薄膜表面的硅烷偶联剂。 </li><li> 15分钟后，关闭真空，并慢慢打开的端口，让空气进入干燥器。对于这种偶联剂，15分钟是足以形成unifo的表面上的RM单层。对于其他偶联剂，时间可能需要进行调整。 </li></ol> 5。附加到表面的生物素<ol><li>约一小时前重视生物素，准备在60毫升的聚丙烯小瓶的N-hydroxylsuccinimide素（NHS-生物素）10毫米的解决方案的一个铰链盖，无水二甲基亚砜（DMSO）。 <ol><li>如果NHS-生物素已储存的冷，让前集结平衡至室温。 </li><li>超声充分溶解在溶剂中的NHS-生物素粉1个小时的解决方案。 </li></ol></li><li>玻片包含到另一个塑料瓶底部的微球，微球与转移，在茎顶端小瓶。 </li><li>转让，玻璃后面的幻灯片（这样的解决方案不触及微球，因为它被添加到小瓶），使用下来一侧的小瓶塑料吸管，NHS-生物素的解决方案的一个适当的音量。一DD足够的解决方案，包括微球表面。 </li><li> 30分钟，在室温下放置小瓶现在包含在DMSO溶液到一个摇摆孵化器的微球和NHS-生物素（倾斜托盘），分别与5转5度的倾斜角度和速度，。 </li><li>轻轻地取出玻片从小瓶，轻轻地滑入另一个充满DDI的H 2 O小瓶像以前一样以相同的速度和倾斜角度为10分钟，倾斜托盘上放置小瓶。淡水重复此步骤两次，每次。这有助于消除多余的二甲基亚砜，从表面和消除任何物理吸附的生物素，实际上并没有移植到表面。 </li><li>从瓶中取出玻片，放置在烘箱在80°C下10分钟，或者直到所有水滴从表面移除。 </li></ol> 6。胺结尾的二氧化硅荧光标记<ol><li>到标记后treatm的胺基与APTMS耳鼻喉科，准备在一间漆黑的房间，由溶解在1 mL无水二甲基亚砜1毫克FITC标记的FITC标记的解决方案。 </li><li>加入50μLFITC标记的解决方案，以1毫升0.1 M的碳酸氢钠缓冲稀释在6.1的解决方案。 </li><li>聚丙烯小瓶60毫升的溶液放置在一个铰链盖，轻轻地滑入胺结尾的球，重新安置在载玻片上，小瓶。 </li><li>小瓶放置在冰浴，让样品反应，至少4个小时在黑暗中的FITC标记的解决方案。 </li><li>通过两个10分钟的球碳酸钠缓冲冲洗，去除多余的荧光。像以前一样，充满了一个缓冲的铰链盖60毫升的聚丙烯小瓶，轻轻滑动到解决方案中的玻片上，该解决方案只覆盖球，而不是纸板住房照顾。用铝箔的小瓶，和以前一样，在5度5 RPM倾斜托盘放在小瓶。 </li><li>轻轻从小瓶中取出玻片，轻轻滑入充满了DDI的H 2 O，并用铝箔覆盖另一个小瓶。像以前一样以相同的速度和倾斜角度为10分钟，倾斜托盘上放置小瓶。淡水重复此步骤两次，每次。这从表面上看，有助于消除多余的染料。 </li><li>从小瓶轻轻地取出玻片，干在成像前10分钟在80ç烤箱。 </li></ol> 7。荧光标记的生物素终止二氧化硅<ol><li>准备一个10μg/ mL的溶液，得克萨斯红抗生物素蛋白磷酸缓冲液。 </li><li>加入一个铰链盖的解决方案，以60毫升的聚丙烯小瓶，轻轻滑动到溶液中含有生物素终止微玻璃幻灯片，使微球只是覆盖解决方案。 </li><li>室温在黑暗中反应30分钟。 </li><li>删除多余的荧光，通过两个10分钟的冲洗s的微球在PBS缓冲。 <ol><li>像以前一样，充满了一个缓冲的铰链盖60毫升的聚丙烯小瓶，轻轻滑动到解决方案中的玻片上，该解决方案只覆盖球，而不是纸板住房照顾。 </li><li>用铝箔的小瓶，和以前一样，在5度5 RPM倾斜托盘放在小瓶。 </li></ol></li><li>轻轻从小瓶中取出玻片，轻轻滑入充满了DDI的H 2 O，并用铝箔覆盖另一个小瓶。像以前一样以相同的速度和倾斜角度为10分钟，倾斜托盘上放置小瓶。淡水重复此步骤两次，每次。这从表面上看，有助于消除多余的染料。 </li><li>从小瓶轻轻地取出玻片，干在烤箱在80°Ç成像前10分钟。 </li></ol> 8。代表结果正确功能微球s可以通过光学和荧光显微镜鉴定。如果做正确的表面功能，它应该导致生物素分子在表面上的一致密集覆盖，表面应保持功能化后的缺陷和无污染，以维持他们的高灵敏度检测实验。光学显微镜可以用来探测后者，而荧光显微镜可以探测表面覆盖的质量和均匀。在图2中，我们显示正确的功能微球的例子。这些图像表明，有因官能（ 图2a）没有表面损坏或污染，微显示表面上均匀一致的覆盖面，要么胺组（ 图2b）或生物素组（ 图2c） 。 如果球都没有被正确的功能，光学微图像无线会出现表面污染，结块或非均匀覆盖，并在表面的明显缺陷，如表面裂纹，（ 图3）。在这里，我们看到了一个常见的​​例子表面污染，造成表面上聚集试剂。 <img alt="图1" src="/files/ftp_upload/3866/3866fig1.jpg" /> 图1 3步附加硅胶微球表面探针分子的反应计划。 <a href="http://www.jove.com/files/ftp_upload/3866/3866fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3866/3866fig2.jpg" /> 图2。二氧化硅微球。一）硅微光学显微镜通过接触氧等离子体填充羟基二）填充二氧化硅微球的荧光显微镜与伯胺，用FITC染料标记; C）荧光显微镜硅公里crosphere填充用生物素和德克萨斯红抗生物素蛋白共轭标记。从Soteropulos许可转载，CE，亨特，香港与阿玛尼，使用回音壁模式微腔的结合动力学测定;物理学。 LETT。99 103703-103703（2011年）。版权所有2011年，美国物理研究所17。 <img alt="图3" src="/files/ftp_upload/3866/3866fig3.jpg" /> 图3。不当功能领域的光学显微镜。在这里，你可以看到右上角的表面上的灰尘，以及污染扩大表面。此外，右侧显示的光学微表面上的小草皮。

<ol>
	<li>Datar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cantilever+Sensors:+Nanomechanical+Tools+for+Diagnostics.">Cantilever Sensors: Nanomechanical Tools for Diagnostics.</a> MRS Bull. 34, 449-454 (2009).</li><li>Hunt, H. K., Armani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+biological+and+chemical+sensors.">Label-free biological and chemical sensors.</a> Nanoscale. 2, 1544-1559 (2010).</li><li>Sundberg, F., Karlsson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+characterization+of+immune+responses+using+label-free+biacore+immunoassays.">Rapid detection and characterization of immune responses using label-free biacore immunoassays.</a> Immunology. 120, 46-47 (2007).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , (2008).</li><li>Bernards, M. T., Cheng, G., Zhang, Z., Chen, S. F., Jiang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonfouling+polymer+brushes+via+surface-initiated,+two-component+atom+transfer+radical+polymerization.">Nonfouling polymer brushes via surface-initiated, two-component atom transfer radical polymerization.</a> Macromolecules. 41, 4216-4219 (2008).</li><li>Fan, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+optical+biosensors+for+unlabeled+targets:+A+review.">Sensitive optical biosensors for unlabeled targets: A review.</a> Anal. Chim. Acta. 620, 8-26 (2008).</li><li>Qavi, A. J., Washburn, A. L., Byeon, J. Y., Bailey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+technologies+for+quantitative+multiparameter+biological+analysis.">Label-free technologies for quantitative multiparameter biological analysis.</a> Analytical and Bioanalytical Chemistry. 394, 121-135 (2009).</li><li>Hunt, H. K., Soteropulos, C., Armani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioconjugation+Strategies+for+Microtoroidal+Optical+Resonators.">Bioconjugation Strategies for Microtoroidal Optical Resonators.</a> Sensors. 10, 9317-9336 (2010).</li><li>Kalia, J., Raines, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Bioconjugation.">Advances in Bioconjugation.</a> Curr. Org. Chem. 14, 138-147 (2010).</li><li>Matsko, A. B., Savchenkov, A. A., Strekalov, D., Ilchenko, V. S., Maleki, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Applications+of+Whispering-Gallery+Mode+Resonators+in+Photonics+and+Nonlinear+Optics.">Review of Applications of Whispering-Gallery Mode Resonators in Photonics and Nonlinear Optics.</a> IPN Progress Report. , 42-162 (2005).</li><li>Armani, A. M., Kulkarni, R. P., Fraser, S. E., Flagan, R. C., Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free,+single-molecule+detection+with+optical+microcavities.">Label-free, single-molecule detection with optical microcavities.</a> Science. 317, 783-787 (2007).</li><li>Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+single+nanoparticle+detection+and+sizing+by+mode+splitting+in+an+ultrahigh-Q+microresonator.">On-chip single nanoparticle detection and sizing by mode splitting in an ultrahigh-Q microresonator.</a> Nat. Photon. 4, 122-122 (2010).</li><li>Armani, D. K., Kippenberg, T. J., Spillane, S. M., Vahala, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high-Q+toroid+microcavity+on+a+chip.">Ultra-high-Q toroid microcavity on a chip.</a> Nature. 421, 925-928 (2003).</li><li>Vollmer, F., Arnold, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whispering-gallery-mode+biosensing:+label-free+detection+down+to+single+molecules.">Whispering-gallery-mode biosensing: label-free detection down to single molecules.</a> Nat. Methods. 5, 591-596 (2008).</li><li>Vollmer, F., Arnold, S., Keng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+virus+detection+from+the+reactive+shift+of+a+whispering+gallery+mode.">Single virus detection from the reactive shift of a whispering gallery mode.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20701-20704 (2008).</li><li>Hunt, H. K., Armani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+microcavity+optical+biosensors.">Recycling microcavity optical biosensors.</a> Opt. Lett. 36, 1092-1094 (2011).</li><li>Soteropulos, C. E., Hunt, H. K., Armani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+binding+kinetics+using+whispering+gallery+mode+microcavities.">Determination of binding kinetics using whispering gallery mode microcavities.</a> Appl. Phys. Lett. 99, 103703-103703 (2011).</li></ol>

没有利益冲突的声明。

在协议中所述，我们创建了一个平台，住房，运输整个功能化过程中的硅胶微球其茎。这房屋平台创建作为解决表面污染和破坏，导致微接触的整个官能过程中使用的各种容器的墙壁。我们意识到，不断地安装和拆卸功能化过程中个人微不同的容器中出现的主要困难。这是难以避免的打击对象刷牙微用镊子移动时，每个单独持有到茎。稳定在载玻片上的每一个微球的位置，我们可以简单地在不同?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>甲醇</td><td>费舍尔</td><td> 67-56-1 </td><td> ACS级</td></tr><tr><td>硫酸</td><td>费舍尔</td><td> 8014-95-7 </td><td>复明</td></tr><tr><td>过氧化氢</td><td>费舍尔</td><td> 7722-84-1 </td><td> 30 wt％的</td></tr><tr><td> aminopropyltrimethoxysilane </td><td>费舍尔</td><td> 13822-56-5 </td><td></td></tr><tr><td> NHS-生物素的EZ连接器</td><td>刺穿</td><td> 20217 </td><td></td></tr><tr><td>二甲基亚砜</td><td>费舍尔</td><td> 67-68-5 </td><td>无水</td></tr><tr><td>异硫氰酸荧光素</td><td>刺穿</td><td> 46425 </td><td></td></tr><tr><td>磷酸盐缓冲液</td><td>费舍尔</td><td> 7647-14-5 </td><td>粉精矿</td></tr><tr><td>碳酸氢钠缓冲</td><td>费舍尔</td><td> NC0099321 </td><td></td></tr><tr><td>德州红 - 抗生物素蛋白共轭</td><td>刺穿</td><td> A820 </td><td></td></tr><tr><td>光纤</td><td>纽波特</td><td> F - 资深大律师</td><td></td></tr><tr><td>光纤剥</td><td>光纤仪器销售</td><td> NN - 175 </td><td>无，聂175微米脱衣舞</td></tr><tr><td> kimwipes </td><td>费舍尔</td><td> 06666A </td><td></td></tr><tr><td>纯光纤切割刀</td><td> ilsintech </td><td> CL-03A </td><td></td></tr><tr><td>玻璃载玻片</td><td>费舍尔</td><td> 12-550B </td><td></td></tr><tr><td>聚丙烯瓶</td><td>费舍尔</td><td> 03-341-75A </td><td>铰链盖60毫升， </td></tr><tr><td>孵化跷</td><td&gt;厂商VWR </td><td> 12620-910 </td><td></td></tr><tr><td>真空干燥器</td><td>费舍尔</td><td> 08-594-15B </td><td></td></tr></tbody></table>

attaching biological probes to silica optical biosensors using silane coupling agents

为了与生物环境，生物传感器平台，如流行的Biacore系统（基于表面等离子体共振（SPR）技术），接口，利用各种表面改性技术，例如，可以防止表面污垢，调整表面的疏水/亲水性，适应各种电子环境，最频繁，诱导特异性对利益的目标。1-5这些技术其他高度敏感的生物传感器的功能扩展到现实世界的复杂环境中应用，例如虽然商业化的生物传感平台，如Biacore，如血液，尿液和废水分析。2,6-7很好地理解，标准执行等表面改性技术，这些技术还没有被翻译在一个标准化的方式向其他标签免费的生物传感平台，如回音壁模式（WGM公司）光学谐振器。8-9 &lt;<html> / P&gt; WGM公司的光学谐振腔的有前途的技术代表进行无标签的超低浓度的物种种类繁多的检测6,10-12灵敏度高，这些平台是其独特的几何光学的结果：WGM公司的光学谐振腔局限循环13一样的SPR平台在具体的，整体的共振频率的光，光场未完全密闭的感应装置，但evanesces;这个“消逝的尾巴”，可以与周围环境中的物种相互作用。这种相互作用导致改变光场的有效折射率，造成轻微的，但检测，转向装置的共振频率。由于光场的循环，它可以与环境互动多次，导致固有的信号放大，非常高的敏感性，环境中的细微变化。2,14-15 帐篷“要在复杂环境中进行有针对性的检测，这些平台必须与探针分子（通常是一个半有约束力的对，如抗体/抗原）通过表面改性。配对虽然WGM公司光学谐振器，可以在几个几何捏造各种材料系统，是最常见的二氧化硅微球，这些球一般制作光纤，它提供了一个“干”的微可以在官能和检测实验处理结束。二氧化硅表面化学适用于附加到其表面的探针分子，然而，为平面的基板上生成的传统技术往往没有足够的这些三维结构，微球表面的任何变化（灰尘，污染，表面缺陷，不均匀涂料）可以有严重的消极后果，对他们的检测能力。在这里，我们展示了一个浅显的方法WGM公司使用硅烷偶联剂，无机表面和生物环境，弥合素连接到硅表面硅微光学谐振腔的表面官能。8,16，虽然我们使用本报告中的传感器系统的硅微WGM公司谐振器，协议是通用的，可用于任何硅器件表面的功能化与生物素。

Watch this Scientific Journal Video about Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents at JoVE.com

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

传感器接口与复杂，生物环境，并进行有针对性的结合具有高度特异性的探针，通过表面改性传感器的高灵敏度的传感器检测。在这里，我们展示了硅光传感器使用硅烷偶联剂弥合传感器和生物环境与生物素的表面功能。

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

attaching-biological-probes-to-silica-optical-biosensors-using-silane

Research

JoVE Journal

Bioengineering

12.8K 次观看.  University of Missouri. 传感器接口与复杂，生物环境，并进行有针对性的结合具有高度特异性的探针，通过表面改性传感器的高灵敏度的传感器检测。在这里，我们展示了硅光传感器使用硅烷偶联剂弥合传感器和生物环境与生物素的表面功能。

视频: Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

柔性神经微探针的使用附加与Biodissolvable胶粘剂刚性加劲肋插入

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.