Name: real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer
Uploaded: 2012-08-20T12:00:00
Description: Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

通过了美国国家科学基金会，美国国立卫生研究院（NIH），材料技术中心（MTC）和ORDA在SIUC提供了财政支持这项工作。我们感谢美国国家科学基金会的资助（CHE-0959568）购买一个FE-SEM。我们希望，感谢马修麦卡罗尔的有益讨论。朱莉娅·雷耶斯会喜欢她的奖学金和财政支持，感谢COLCIENCIAS，哥伦比亚机构和大学Pedagogica的的ŸTecnologica哥伦比亚。

A. PDA脂质体4,5,6的合成与表征注1：保护PDA解决方案，光在所有的实验步骤，每一容器上使用的铝箔包装。 注2：两个不同的脂质体溶液（B和C）以下步骤A（PDA脂质体的合成与表征）。 1。合成N-羟基Diacteylene（NHS-PCDA） <ol><li>为了准备脂质体，PCDA-NHS的一个重要成分是必需的。我们已经合成PCDA-NHS使用下列程序： </li><li>添加10,12 ...

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	<li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Fluorescence Spectroscopy. , (1999).</li><li>Charych, D. H., Nagy, J. O., Spevak, W., Bednarski, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Colorimetric+Detection+of+a+Receptor-Ligand+Interaction+by+a+Polymerized+Bilayer+Assembly.">Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly.</a> Science. 261, 585-588 (1993).</li><li>Yoon, B., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Conceptual+and+Technological+Advances+in+Polydiacetylene-based+Supramolecular+Chemosensors.">Recent Conceptual and Technological Advances in Polydiacetylene-based Supramolecular Chemosensors.</a> Chem. Soc. Rev. 38, 1958-1968 (2009).</li><li>Xuelian, L. i., Kohli, P. u. n. i. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+Molecular+Interactions+in+Biosensors+Based+on+Fluorescence+Resonance+Energy+Transfer.">Investigating Molecular Interactions in Biosensors Based on Fluorescence Resonance Energy Transfer.</a> J. Phys. Chem. C. 114, 6255-6264 (2010).</li><li>Li, X., Matthews, S., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Resonance+Energy+Transfer+in+Polydiacetylene+Liposomes.">Fluorescence Resonance Energy Transfer in Polydiacetylene Liposomes.</a> J. Phys. Chem. B. 112, 13263-13272 (2008).</li><li>Li, X., McCarroll, M., Kohli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+Fluorescence+Resonance+Energy+Transfer+in+Conjugated+Liposomes.">Modulating Fluorescence Resonance Energy Transfer in Conjugated Liposomes.</a> Langmuir. 22, (2006).</li><li>New, R. R. C., New, R. 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=.">.</a> Liposomes: A Practical Approach. , 33-104 (1990).</li><li>Chen, X., Lee, J., Jou, M. J., Kim, J. -. M., Yoon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+and+Fluorometric+Detection+of+Cationic+Surfactants+Based+on+Conjugated+Polydiacetylene+Supramolecules.">Colorimetric and Fluorometric Detection of Cationic Surfactants Based on Conjugated Polydiacetylene Supramolecules.</a> Chem. Commun. , 3434-3436 (2009).</li><li>Yarimaga, O., Im, M., Choi, Y. -. K., Kim, T. W., Jung, Y. K., Park, H. G., Lee, S., Kim, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Color+Display+System+Based+on+Thermochromic+Conjugated+Polydiacetylene.">A Color Display System Based on Thermochromic Conjugated Polydiacetylene.</a> Macromolecular Research. 18, 404-407 (2010).</li></ol>

我们已经进行了选择性结合脂质体的表面上的蛋白质的赖氨酸残基的使用NHS-胺反应。 FRET为基础的方法是能够进行实时监控，生物素 - 链霉亲和素结合蛋白（BSA）结合到脂质体表面。类似的过程可以被应用到与他们的选择性受体的各种蛋白质相互作用的结合动力学研究。有灵活性是在选择的荧光基团，将提供的荧光的光谱特性，根据J值的变化。 PDA是一个普遍的接受。因此，随着多个荧光?...

<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>的10,12 pentacosadiynoic酸（PCDA） </td><td>政府飞行服务队的化学品</td><td> 3261 </td><td>光敏感</td></tr><tr><td> N-羟基琥珀酰亚胺（NHS） </td><td> Acros Organics公司</td><td> 157270250 </td><td>湿度敏感</td></tr><tr><td> 1 - （3 - （二甲基氨基）丙基）-3 - 乙基碳二亚胺盐酸盐（EDC） </td><td> CHEM-IMPEX国际</td><td> 00050 </td><td></td></tr><tr><td> 1,2 -二肉豆蔻酰-SN-甘油-3 -磷酸胆碱（DMPC） </td><td>阿凡提极性脂质</td><td> 850345P </td><td></td></tr><tr><td>罗丹明标记牛丝氨酸的嗯白蛋白（BSA-RH） </td><td> Sigma Aldrich公司</td><td> A4537 </td><td></td></tr><tr><td> （1,2 -二油酰-sn-甘油-3 -磷酸乙醇胺-N-（生物素基）（生物素-DOPE） </td><td>阿凡提极性脂质</td><td> 870282 </td><td></td></tr></tbody></table>

real-time monitoring of ligand-receptor interactions with fluorescence resonance energy transfer

荧光共振能量转移是一个过程，非辐射能量是从激发供体分子转移到基态的受体分子通过远程偶极-偶极相互作用1。我们在本传感检测，利用PDA的一个有趣的特性：蓝移后的UV-Vis的PDA的电子吸收光谱（ 图1）中的分析物相互作用与受体连接到PDA 2,3,4,7。这种在PDA中的吸收光谱的移位提供了在光谱重叠（J），PDA（受体）和若丹明（供体）之间的变化导致在FRET效率的变化。因此，分析物（配体）和受体之间的相互作用是通过FRET检测到供体荧光和PDA之间。特别是，我们显示了一个的模型蛋白质分子链霉亲和素的检测。我们还证明了牛血清白蛋白（BSA）的脂质体的表面用共价键结合的FRET机制。这些t之间的相互作用他双分子层脂质体和蛋白质分子可以在实时感测到。所提出的方法是用于感测小的化学和大型生物化学分子的一般方法。由于荧光本质上是更敏感比比色法，测定的检出限，可以在子纳摩尔范围或低8。另外，PDA可以作为一个通用的受体，在FRET，这意味着，多个传感器可以开发PDA（受体）官能化与捐助者和PDA的脂质体的表面上附着的不同的受体。

Watch this Scientific Journal Video about Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer at JoVE.com

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

我们表明FRET之间的共轭聚合物的聚二乙炔（PDA）和荧光基团连接到PDA脂质体的生物分子的传感表面。 PDA脂质体也包含在其表面上的生物分子的受体分子可以用作探针。配体 - 受体相互作用导致这是传感机构的基础的荧光团和PDA之间的FRET效率的变化。

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

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