Name: microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications
Uploaded: 2013-09-23T18:15:00
Description: Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

我们承认从美国国立卫生研究院（NHLBI合约编号HHSN268201000044C到RW，上海和SYS）的资助。

1。 GST和纳米粒子（NP）结合物的制备<ol><li> GST-TCO准备： <ol><li>添加8微升的TCO-NHS溶液（50毫米DMSO）100μL商品及服务税（1毫克/毫升的PBS）和摇动混合物在室温下1小时。 </li><li>使用Zeba旋转脱盐柱除去过量的试剂。存储包含GST-TCO共轭回收的滤液在4℃后才能使用。 </li></ol></li><li> GST-TZ准备： <ol><li>加入6微升TZ-NHS溶液（25mM的在DMF中），以75微升的GST（1毫克/毫升在PBS中），并摇动混合物在室温下1小?...

<ol>
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这里所描述的捕获环加成方法使改性纳米粒子和小分子无标记芯片为基础的互动和动力学研究的快速，可逆固定。固定化的协议可在要求&lt;10μM浓度的小分子配体分钟来进行。通过调节配体浓度和接触时间的固定化密度可以严格控制。我们的数据显示，芯片上的生物正交反应保持原位功能化的纳米颗粒或固定化小分子与他们的目标交互的能力。我们还常表现为两种小分子，（总拥有成本和T...

高效的偶联反应是用于将生物活性分子到表面上用于各种生物技术应用有价值的工具。最近，非常快的生物正交反式 -环辛烯（TCO）之间[4 +2]环加成反应和1,2,4,5 -四嗪（TZ）已被用于标记细胞的表面，亚细胞结构，抗体和纳米粒子1 - 7在这里，我们结合使用的[4 +2]环加成反应与抗原/抗体捕获（GST /抗-GST）为可逆的片上合成的表面等离子体共振（SPR）的相互作用分析多组分结构及监察过程中的实时（ 图1）。8,9值得注意的是，使用一个既定的协议捕获环加成策略使表面再生。8作为稳定的传感器表面有超过配体的方向和密度控制各种新的实验的结果，组装格式现在是可能的。运用这一战略，我们展示的TCO / TZ-衍生的小分子的固定化和在各种缓冲液条件表征环加成率。我们选择了FKBP12和分子AP1497之间的公知的相互作用是FKBP12 10-12结合，例如，以验证该捕集环加成策略保留了小分子与其靶相互作用的能力时，无论是直接连接到固定的GST抗原或与固定化的纳米颗粒（纳米）。 该方法提供了几个优点。首先，小分子上的传感器芯片中的可逆固定化成为可能。第二，小分子的TCO / TZ的固定化也使无标记相互作用的研究，反向规范化SPR研究的取向，并且可以提供一种结合相互作用的互补视图。第三，这种方法可以使微流体合成靶向纳米粒子，以及它们的结合蛋白的直接评定克属性。这有望改善的评价或筛选靶向纳米粒子的效率，也减少了所需的纳米颗粒的量。13-15第四，这种方法可以在连续流动测量实时的生物正交环加成反应的反应动力学。最后，在TCO / TZ固定化化学是健壮的血清中的存在。综上所述，我们预计，这种多功能的方法将广泛地施工方便稳定的传感器表面的各种与微流控相关研究，以在体外和体内细胞的应用。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sensor Chip CM5</td>
			<td>GE Healthcare</td>
			<td>BR-1005-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coupling kit</td>
			<td>GE Healthcare</td>
			<td>BR-1000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>GST capture kit</td>
			<td>GE Healthcare</td>
			<td>BR-1002-23</td>
			<td></td>
		</tr>
		<tr>
			<td>NAP-10 Columns</td>
			<td>GE Healthcare</td>
			<td>17-0854-01</td>
			<td></td>
		</tr>
		<tr>
			<td>GST, lyophilized in 1X PBS</td>
			<td>Genscript</td>
			<td>Z02039</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>rhFKBP12</td>
			<td>R&#38;D Systems</td>
			<td>3777-FK</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant P-20</td>
			<td>GE Healthcare</td>
			<td>BR-1000-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine 2.0</td>
			<td>GE Healthcare</td>
			<td>BR-1003-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba spin desalting column</td>
			<td>Thermo</td>
			<td>89882</td>
			<td>7 K MWCO</td>
		</tr>
		<tr>
			<td>Amicon Ultra 4</td>
			<td>Fisher</td>
			<td>UFC810096</td>
			<td>100 K centrifugal filter</td>
		</tr>
		<tr>
			<td>TCO-OH</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>TCO-NHS</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house, *Commercially available from Click Chemistry Tools # 1016-25</td>
		</tr>
		<tr>
			<td>Tz-BnNH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>Tz-NHS</td>
			<td>Ref. 8</td>
			<td>764701</td>
			<td>Synthesized in-house, *Commercially available from Sigma Aldrich # 764701</td>
		</tr>
		<tr>
			<td>NP-NH2 = CLIO-NH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>AP1497, AP1497-Tz</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>SPR Biosensor</td>
			<td>GE Healthcare</td>
			<td>Biacore T100</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications

方法有超过取向和固定化密度控制生物活性小分子的快速表面固定有用于生物传感器和微阵列应用非常理想。在这项研究中，我们使用了高效共价生物正交反式 -环辛烯（TCO）的[4 +2]环加成反应和1,2,4,5 -四嗪（TZ），使TCO / TZ衍生分子的固定化微流控。我们监测使用表面等离子体共振（SPR）在连续流动条件下的过程中的实时性。以使可逆固定化，并延长了实验范围传感器表面的，我们结合一个非共价的抗原 - 抗体捕获组分与环加成反应。通过交替呈现的TCO或TZ部分到传感器表面，多个捕获环的过程，现在可能在一个传感器表面的各种多组分结构的芯片组装和相互作用的研究。我们我一种小分子，AP1497结合FK506结合蛋白12（FKBP12），与上一个生物传感器芯片2的不同的固定化实验llustrate这种方法和相同的小分子作为固定化和原位功能化的纳米颗粒的一部分。

数据和数字已经改编自参考8。 生物活性小分子与在方向和密度控制高效的可逆固定在新的生物传感器应用的发展起着关键的作用。使用TCO与Tz之间的快速生物正交反应中，我们描述了一种用于逐步组装和配位体的表面与保持生物活性的再生。 图2显示的TZ-BnNH 2固定化的实时监测。 GST-TCO的溶液注射到产生〜400 RU上升响应预先固定化的抗-GST表面。第二次注?...

Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

我们提出了一种小分子的快速，可逆的固定化和功能化的纳米粒子组合件表面等离子体共振（SPR）的研究中，使用连续的片上生物正交环化学和抗体 - 抗原捕获。

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

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Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

由小圆形 DNA 分子构建的稳定 DNA 分子、一维和二维纳米结构

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...