SDC，ACH，和CJ是由通过欧洲研究理事会资助启动（ERC-STG-2012-309509）的支持。 J.-MN是由韩国国家研究基金会（NRF）（2011-0018198）的支持;并通过韩国NRF的先锋研究中心计划（2012-009586）资助由科技部，信息通信技术，与未来规划（MSIP）。这项工作也是由中心由韩国MSIP作为全球前沿项目（H-GUARD_2013-M3A6B2078947）资助的生物纳米健康卫士支持。刘和J.-HH是由首尔市，韩国首尔科技奖学金计划的支持。标记的Rep蛋白是从苏阿MYONG博士一份厚礼。

1，玻片制备及清洗 一种微流体腔是由一个石英载玻片和盖玻片的。在棱镜型全内反射荧光（TIRF）显微镜，在石英片的表面进行成像。因此，要彻底清洗采用H 2 O，丙酮，氢氧化钾，以及食人鱼解决方案的石英片是很重要的。 th是多步清洗消除了在其上用单分子荧光测量干涉表面荧光有机分子。此外，食人鱼蚀刻产生羟基，使石英表面的亲水性。游离羟基基团是在步骤3中的氨基硅烷化反应是必不可少的。 <ol><li>钻井石英幻灯片。钻一对孔与3/4 mm金刚石钻头（ 图2a）一个石英片。如果有多个信道是理想的，更钻年利率孔IRS（ 图2b）。这些孔用来注入的解决方案为在第7步微流体室。 <ol><li>保持钻头在湿H 2 O的钻井过程中。 H 2 O的执行作为润滑剂，从而增加了钻头的使用寿命。 </li><li>偶尔擦钻头的尖有助于钻孔。 </li><li>在聚乙二醇化过程后钻孔，标志着滑动，便于识别的一面。这些标记可以作为参考，以避免以后会混淆。对于标记，金刚石钻头都可以使用。不要使用任何笔，因为油墨可能会在表面上。 </li></ol></li><li>用H 2 O清洗将玻片在玻璃染色缸。通常为5〜15的幻灯片可以被放置在一个单一的罐子。冲洗的幻灯片用MilliQ H 2 O重复3次，超声处理的玻片用MilliQ H 2 O的5分钟，去除污垢。处理水并再次冲洗载玻片3次的MilliQ H 2 O 请注意，130 W是用于该协议的超声功率。较高的超声功率可能会降低石英玻片的寿命。 </li><li>清洁用丙酮。取代的MilliQ H 2 O和丙酮。超声处理的载玻片，用丙酮进行20分钟或更长的时间。 </li><li>用H 2 O漂洗丢弃丙酮和冲洗载玻片用MilliQ H 2 O为了消除任何残留的丙酮重复3次。 </li><li>清洗用KOH。用1 M KOH代替水和超声处理的玻片20分钟或更长的时间。过度蚀刻（ 如过夜KOH处理）会提高表面质量，但会引入划痕，这可能与荧光成像的干扰。 </li><li>用H 2 O漂洗冲洗用MilliQ H 2 O的3倍，幻灯片，除去氢氧化钾的痕迹。 </li><li>食人鱼腐蚀。 <ol><li>转让幻灯片到幻灯片杜兰持有人或一个特制的铁弗龙座和特等王牌它们在烧杯中（1L），其位于化学罩。 </li><li> 450毫升H 2 SO 4填充烧杯中。 </li><li>加入150毫升H 2 O 2为3:1比H 2之间SO 4和H 2 O 2。一旦溶液开始沸腾自发地，温度升高超过90℃。确保该H 2 O 2溶液在室温下开始反应之前。否则，该沸腾的溶液的温度可能低于90℃。 </li><li>搅拌适当的混合溶液，并让烧杯静置20分钟。 </li><li>就拿滑出食人鱼解决方案的幻灯片夹在一起，并把它们含有的MilliQ H 2 O滑动架与此同时，废弃的食人鱼溶液放入指定废物瓶一旦达到室温。 </li><li>漂洗玻片3次用MilliQ H 2 O。要特别注意采取进一步输注液PS避免幻灯片的任何可能的污染。继续执行步骤3。这是应立即着手进行下面的步骤。 *注意:食人鱼的解决方案是非常被动的。在处理这个解决方案要格外小心，应采取。此外，当错误地用有机溶剂如丙酮混合，它可能会引起爆炸。 </li></ol></li></ol> 2，清洗盖玻片 一种微流体腔是由石英片和面黏贴唇。当棱镜类型的TIRF显微镜的情况下，盖玻片的表面上不成像。因此，它是足够的清洗盖玻片仅与H 2 O和KOH。万一盖玻片需要进行成像（例如，通过客观型TIRF显微镜），推荐的治疗盖玻片用食人鱼溶液（步骤1.7）。请注意，如果盖玻片表面的PEG化是一种高品质不是，它可能作为蛋白质水槽的ð引起的变化的蛋白质浓度。 <ol><li>用H 2 O漂洗地方盖玻片（24×30，24×40或24×50毫米2）在一个玻璃染色缸。通常为5〜15的盖玻片可以被放置在一个单一的罐子。冲洗盖玻片3次用的MilliQ H 2 O。 </li><li>清洗用KOH。用1M KOH代替水和超声处理的盖玻片20分钟或更长的时间。 </li><li>用H 2 O漂洗冲洗盖玻片3次，用MilliQ H 2 O的去除KOH的痕迹。继续执行步骤3。 </li></ol> 3，氨基硅烷化的载玻片和盖玻片的 通过氨基硅烷化的化学官能化的石英载玻片，并与胺基团的盖玻片的表面上。使用甲醇作为溶剂和乙酸作为催化剂的氨基硅烷化反应。 <ol><li>漂洗用甲醇。取代的MilliQ H 2 O的在染色菜肴（从sTEPS 1和2）用甲醇。请甲醇的幻灯片和盖玻片，直到步骤3.3。不存储在甲醇中的幻灯片和盖玻片的时间过长的时期（ 例如数小时），因为在甲醇中的杂质会吸附到表面上。 </li><li>准备氨基硅烷化的解决方案。 <ol><li>冲洗一个耐热瓶数次用甲醇。超声处理的烧瓶中，用甲醇为5分钟或更长的时间。这是应具有保持清洁的专用瓶。 </li><li>倒入100ml的甲醇加入到烧瓶中。 </li><li>加入5毫升乙酸。 </li><li> ，加入3 mL APTES（3 - 氨丙基三甲氧基硅烷），并通过摇动它轻轻混匀。 </li></ol></li><li>氨基硅烷化。代替甲醇中包含的幻灯片和盖玻片与aminosilanization反应混合物的染色皿。 <ol><li>孵育20-30分钟。在培养过程中，超声处理一次，持续​​1分钟。 </li></ol></li><li>漂洗用甲醇。更换一个用甲醇minosilanization反应。弃去甲醇，加入新鲜的甲醇溶液。重复此步骤三次。 </li></ol> 4，表面钝化用聚合物（第一轮） 通过偶联NHS-酯的聚乙二醇（PEG）钝化的石英载玻片和盖玻片胺涂覆的表面。此反应是用在pH 8.5的PEG溶液中的饱和浓度过夜。 <ol><li>干燥载玻片和盖玻片。干燥用N 2气的幻灯片和盖玻片并将其放置在清洁吸液管箱以这样的方式，该侧具有被聚乙二醇化的朝上。移液管箱部分地填充用MilliQ H 2 O的这种潮湿的环境中防止聚乙二醇化解决方案，从过夜培养过程中干燥。 </li><li>准备反应缓冲液。通过在10ml的溶解84毫克碳酸氢钠制备的新鲜的碳酸氢钠缓冲液（pH 8.5）的0.1M的MilliQ H 2 O。没有必要调节pH值的。此溶液可以冷冻储存为下一次使用（ 如步骤6.1）。 </li><li>准备聚乙二醇化的解决方案。 <ol><li>制备0.2毫克在1.5毫升管的生物素化的NHS-酯PEG（5000道尔顿）13和8毫克的NHS-酯的mPEG（5000道尔顿）的聚乙二醇混合物。当使用幻灯片的N个 ，在单管中制备的N倍大的PEG混合物的量。 </li><li>加入64微升（或N次64微升为幻灯片的N个 ）新鲜制备的缓冲（步骤4.2）中。移液器向上和向下，以完全溶解它们。 </li><li>以16100×g离心1分钟，离心以除去气泡。不要吸取之后。否则，气泡将形成与PEG化在步骤4.4干扰。 </li></ol></li><li>聚乙二醇化。 <ol><li>从步骤4.1滴70微升的聚乙二醇化混合物至干燥石英片。用90微升的情况下为24×50毫米2 COVerslip。 </li><li>轻轻地放在一个干燥的盖玻片从4.1步在解决方案。 </li><li>有聚乙二醇化的统一和高品质，注意不要引入气泡。由于表面张力，大部分的气泡可能自发地离开5分钟由于表面张力中的反应体积。 </li><li>孵育的幻灯片在一个阴暗潮湿的环境。半衰期的NHS-酯的PEG，我们在pH 8使用的是大约一小时。至少，它们孵育2小时。过夜孵育导致PEG化（数据未示出）的质量更高。 </li></ol></li></ol> 5，长期贮存 存储PEG化幻灯片和的N 2盖玻片在-20ºC。 <ol><li>干燥载玻片和盖玻片。小心拆开载玻片和盖玻片滑动盖玻片一侧，用MilliQ H 2 O的冲洗一下，并用N 2干燥。 </li><li>存储载玻片和盖玻片。佛ř立即使用，遵循第二轮聚乙二醇化（步骤6）的过程。为了保存的时间较长，请遵循下面的步骤。 <ol><li>放置一对在50ml管中，使得所述聚乙二醇化的表面面向远离彼此的载玻片和盖玻片的。 </li><li>局部关闭管，吸尘管，然后用氮气填充。这些步骤有助于维护PEG化表面的很长一段时间。拧紧管，并将其存储在-20°C。幻灯片的质素维持良好长达3个月（ 图3a，右 ）。 </li></ol></li></ol> 6，表面钝化用聚合物（第二轮） 额外轮PEG化的，使PEG层更致密，并骤冷的表面上的任何剩余胺基团。使用短的NHS-酯的PEG分子（333道尔顿）的可有效地穿透到现有的PEG层。这是拍摄勾选的使用幻灯片之前对做这第二轮的聚乙二醇化。 <ol><li>准备反应缓冲液。制备新鲜的碳酸氢钠缓冲液（pH 8.5），0.1M的。从步骤4.2将冷冻的溶液也可使用。 </li><li>准备聚乙二醇化的解决方案。溶解7微升250mM的MS4-PEG在63微升的碳酸氢钠缓冲液中。 </li><li>聚乙二醇化。按照同样的步骤，如步骤4.4。温育30分钟到过夜。 </li><li>干燥载玻片和盖玻片。拆机对幻灯片和盖玻片，冲净，用MilliQ H 2 O，与N 2干他们，并让他们在一个干净的吸管盒。进行组装微流体室中第7步。 </li></ol> 7，装配微流控室 组装微流体室中，利用一对PEG化的石英载玻片和盖玻片。双面胶带被用作间隔物。该腔室是密封的环氧树脂胶和，所以lutions是通过在石英片上的孔引入。 <ol><li>放置在一个平面上的石英片与聚乙二醇化的一面朝上。 </li><li>做一个通道（宽度为5〜7mm）对角线上的聚乙二醇化表面通过将双面胶带粘在幻灯片。确保该孔被设置在通道的中心。小心不要让一个室的过程中破坏聚乙二醇化表面。这是可能的放置和坚持不同的双面粘胶带（ 见图2），使多通道投影片。 </li><li>轻轻地放置在顶部的聚乙二醇化盖玻片完成室。 PEG化的一面应朝下。 </li><li>通过按压盖玻片其中双面胶带放置在密封区域的室中。做到这一点，但轻轻地彻底地使腔体变得水密封。 </li><li>收用环氧胶腔的边缘。 </li><li>链霉亲和固定化或中性亲上生物素化的PEG层用P200移液管加入50μl的链霉亲和或中性亲和素在T50溶液（10mM的Tris-HCl [pH为8.0]，50 mM氯化钠缓冲液）0.1毫克/毫升。后1分钟温育，用100μl的缓冲T50平齐。 </li><li>加入生物素标记的生物分子为您的单分子成像。 </li></ol> 8，滑动回收 回收石英幻灯片。用过的载玻片回收由脱盖玻片和双面粘合胶带。 <ol><li>使用后，存放在自来水室。在水中长期孵化简化的室拆卸。 </li><li>采用微波煮沸自来水中的腔。使用耐热玻璃烧杯中。煮沸10分钟或更长的时间。 </li><li>取下用刀片盖玻片和双面粘性胶带。不要按一个石英片垂直于平面。否则，它打破。保持刀片远离通道。 OTherwise，它引入了通道上的划痕。 </li><li>使用家用洗涤剂用手指揉搓它们冲洗的幻灯片。 </li><li>将玻片在玻璃染色缸。通常为5〜15的幻灯片可以被放置在一个单一的罐子。加入10％的家用清洁剂和超声处理的玻片20分钟或更长的时间。冲洗的幻灯片与大量标签的水。 </li><li>转到步骤1.2。 </li></ol>

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我们什么都没有透露。

本协议中的关键步骤 它是必不可少的氨基硅烷化反应之前，使表面具有亲水性。这是通过在食人鱼蚀刻，产生在玻璃/石英表面上的游离羟基基团来实现。建议不要保留食人鱼蚀刻表面暴露于H 2 O或空气中的时间，因为表面的亲水性长期逐渐下降。 在NHS-酯PEG分子反应。这是应使等份，并将它们存储在氮气中于-20ºC。在化学APTES在室温下的保存期很短。这是应了一个新的每月更换。 本协议的修改 当你不能使用食人鱼蚀刻任何实际的原因，您可能会使用蚀刻KOH一段较长时间（ 如 Ôvernight），这也将使得羟基露出。这种替代方法的缺陷是滑盖后由于严重的划痕数再回收变得不可用。 它经常练习使用丙烷火炬，这是有效地消除荧光有机材料7烧玻璃/石英表面。这个程序是不包括在此协议，因为它是多余的食人鱼腐蚀。注意，该步骤将可能导致的羟基的氧化。因此，这个程序应该不会进行一次表面用KOH或食人鱼溶液蚀刻。 当pH值低于7的缓冲液是用于单分子成像，PEG的构象从"蘑菇"改变为"刷"，这降低了钝化的程度。因此，当pH值低于7.0时，则建议进一步钝化用二琥珀酰亚胺基酒石酸盐&lt;表面SUP&gt; 7。 观点 这项工作提供了强大的协议实现高品质的表面钝化。该协议将是那些涉及蛋白质14以及细胞提取物中的蛋白复合物和免疫沉淀15单分子荧光的研究很有用。它将被广泛用于如力量光谱和扭矩光谱其他16个单分子技术。它也将是用于防止细胞从吸附到表面17是有用的。 在这项工作中提供的协议，要求其多步程序。使用脂质-PEG 8和多聚赖氨酸的PEG 9的表面钝化，可作为一种替代的方法。因为，它们不需要任何化学反应，它是很容易实现。然而，钝化的程度没有那么高，通过化学MODIF实现ication表面的。

进行单分子蛋白质研究时，必须达到表面钝化的高品质，使得实验不受任何表面诱导的蛋白质功能失常或变性1,2。而与表面活性剂，如牛血清白蛋白处理后的玻璃表面，通常用于单分子核酸研究3中 ，其钝化的程度还不够高蛋白质的研究。涂有聚合物（聚乙二醇，PEG）玻璃表面优异的钝化性能4-6。因此，它已成为普遍适用于单分子蛋白质的研究自从它被引入到一个单分子荧光研究7-10。聚合物涂层过程需要多个表面处理7,11,12。因此，难以追随整个过程，而不进行详细说明。根据该协议，我经常上的表面钝化的程度各不相同秒，然后。在这里，我们提出了一个强大的协议有一步一步的指示，这将消除单分子蛋白质研究的主要瓶颈之一。参见图1概述。

<table border="1" cellpadding="0" cellspacing="0" width="662">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>1. Slide preparation and cleaning</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>32201</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>631-0136 631-0144 631-0147</td>
			<td>24x32mm2, 22x40mm2, 
			24x50mm2 &#160;(No.1&#189;, 
			Rectangular)</td>
		</tr>
		<tr>
			<td>Diamond drill bits</td>
			<td>MTN-Giethoorn, The Netherlands</td>
			<td>LA-0564-00DB</td>
			<td>3/4 mm</td>
		</tr>
		<tr>
			<td>Duran slide holder</td>
			<td>LGS, The Netherlands</td>
			<td>213170003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass staining dishes</td>
			<td>Fisher Scientific</td>
			<td>300101</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Boom</td>
			<td>6233905</td>
			<td>Opened bottles should be stored at 4 &#176;C. 
			1 L, hydrogen peroxide 30%</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Boom</td>
			<td>80900627.9010</td>
			<td>Sulfuric acid 95-98%</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>30603</td>
			<td>Potassium hydroxide</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>32213</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td></td>
			<td>Tabletop ultrasonic cleaner, 3510</td>
		</tr>
		<tr>
			<td>Quartz slides</td>
			<td>Finkenbeiner</td>
			<td></td>
			<td>1&#8221; x 3&#8221;, 1 mm thick</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>2. Coverslip cleaning</td>
		</tr>
		<tr>
			<td>Duran slide holder</td>
			<td>LGS, The Netherlands</td>
			<td>213170003</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>30603</td>
			<td>Potassium hydroxide</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td></td>
			<td>Tabletop ultrasonic cleaner, 3510</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>3. Amino-silanization of slides and coverslips</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>320099-500ML</td>
			<td>Acetic acid, glacial</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-Aldrich</td>
			<td>281778-100ML</td>
			<td>Store at the room temperature under N2 . Replace once in a month. 
			3-aminopropyl trimethoxysilane</td>
		</tr>
		<tr>
			<td>Flask (200mL)</td>
			<td>VWR</td>
			<td>Flask (200 mL)</td>
			<td>Have one flask dedicated for aminosilanization. 
			Pyrex flask</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>32213</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson</td>
			<td></td>
			<td>Tabletop ultrasonic cleaner, 3510</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>4. Surface passivation using polymer (the first round)</td>
		</tr>
		<tr>
			<td>Biotin-mPEG</td>
			<td>Laysan Bio</td>
			<td>Biotin-PEG-SVA-5000-100mg</td>
			<td>Store aliquots of 1-2 mg (enough for 10 slides) at -20 &#176;C under N2. 
			Biotin-PEG-SVA, MW 5,000&#160;Da</td>
		</tr>
		<tr>
			<td>mPEG</td>
			<td>Laysan Bio</td>
			<td>MPEG-SVA-5000-1g</td>
			<td>Store aliquots of 80 mg (enough for 10 slides) at -20 &#176;C under N2. 
			mPEG-succinimidyl valerate (SVA), MW 5,000 Da</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>6. Surface passivation (the second round)</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-100ML</td>
			<td>Dimethyl sulfoxide BioReagent, for molecular biology, &#8805;99.9%</td>
		</tr>
		<tr>
			<td>DST</td>
			<td>Pierce</td>
			<td>20589</td>
			<td>Disuccinimidyl tartarate</td>
		</tr>
		<tr>
			<td>MS4-PEG</td>
			<td>Pierce</td>
			<td>22341</td>
			<td>Dissolve 100 mg MS4-PEG in 1.1 mL DMSO. Store 20 mL aliquots at -20 &#176;C. 
			Short NHS-ester PEG, MW 333 Da</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>7. Assembling a microfluidic chamber</td>
		</tr>
		<tr>
			<td>Double sticky tape</td>
			<td>Scotch</td>
			<td></td>
			<td>10mm wide</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Thorlabs</td>
			<td>G14250</td>
			<td>Devcon 5 minute epoxy</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-1 KG</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Pierce</td>
			<td>31000</td>
			<td>Stock in 5 mg/mL in H2O at 4 &#176;C. 
			ImmunoPure NeutrAvidin Biotin-Binding protein</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Invitrogen</td>
			<td>S-888</td>
			<td>Stock 5 mg/mL in H2O or T50 at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503-1 KG</td>
			<td>Tris</td>
		</tr>
	</tbody>
</table>

surface passivation for single-molecule protein studies

单分子荧光光谱法已被证明是有助于理解在纳米级范围广泛的生物现象。什么这种技术可以产生对生物科学的重要例子是在蛋白质 - 蛋白质和蛋白质 - 核酸相互作用的机械见解。当蛋白质的相互作用是探测在单分子水平，蛋白质或它们的底物通常是固定在玻璃表面，使之有一个长期观察。这种固定方式可能会引入不必要的表面假象。因此，有必要以钝化玻璃表面，使之惰性的。使用聚乙二醇（PEG）表面涂层突出其高性能防止蛋白质从与玻璃表面的非特异性相互作用。然而，该聚合物涂层过程是困难的，由于从一系列的表面处理和严格要求的表面需要是产生的并发症是不含任何​​荧光分子在该过程的结束。在这里，我们提供了一个强大的协议有一步一步的指示。它涵盖表面清洁，包括食人鱼蚀刻，表面功能化与胺基团，终于PEG涂层。要获得的PEG层的高密度，我们引入了两轮，显着提高了钝化的质量对表面进行处理与PEG分子的新战略。我们提供有代表性的结果，以及每个关键步骤的实用建议，让任何人都可以实现高品质的表面钝化。

微流体室组件后（步骤7.1 - 7.6）和实施步骤7.7之前，建议进行聚乙二醇化表面的质量控制。 如果表面钝化已成功完成，有小于10的非特异性吸附每个成像区域（25毫米×25毫米）的蛋白质时1-10 nM的荧光标记的蛋白质被添加到腔室（ 图3a，左轴 ）观察到的。 当任何的清洗或反应步骤没有被适当进行的，非特异性的数目吸附蛋白质显著增加，并且对CCD屏幕可通过荧光信号达到饱和。例如，如果在食人鱼蚀刻被跳过，有100倍的较大的观察非特异吸附的量（ 图3a;比较左边中间 ）。当第二聚乙二醇化步将被跳过，有大约3时间SA较大的非特异性吸附量观察到（ 图3b）。当过期化学品（ 如 APTES在室温下贮存数月）用于（数据未示出）的PEG化的程度低是观察。表面的质量也下降下来的时候一个显著的时间走过了，因为它是聚乙二醇化（ 图3a;右向左进行比较）。 据我们所知，那就是两轮聚乙二醇化的引入对单分子研究的第一次。两轮聚乙二醇化的保证PEG层形成（ 图3b）的最高品质。双聚乙二醇化的优秀的自然是在电影里突出显示（比较电影1A与1B电影 ）。在这些电影，从在溶液中的荧光分子的背景信号被观察到时，双聚乙二醇化是弱得多的<html>用，这表明蛋白质是由双聚乙二醇化层更有效地排斥。虽然这两步过程是强烈建议，第二个聚乙二醇化步骤可能，如果你的实验是可以容忍的，以非最佳钝化被跳过。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50549/50549fig1highres.jpg" width="500" /> 图1:管脚的表面处理剂（a）在显微镜载玻片用丙酮清洗，KOH和食人鱼溶液。它是功能化与APTES和聚乙二醇化与NHS-酯PEG。 （b）在盖玻片清洗用KOH，以及用食人鱼溶液如果必要的。它是功能化与APTES和聚乙二醇化与NHS-酯PEG。 fig2highres.jpg"宽度="500"/&gt; 图2:微流控室 （ 一 ）单通道室。显微镜载玻片上有两个洞钻。它被组装在一起使用的双面胶带的两片盖玻片。 （b）一个三通道室。显微镜载玻片有六个孔钻。它是组装使用4片的双面胶带的盖玻片。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/50549/50549fig3highres.jpg" width="500" /> 图3:与溶液中的染料标记的蛋白质的CCD拍摄的图像 。而（a）在溶液中采用60X物镜用10nM Cy3标记labeld代表采取的棱镜型全内反射荧光显微镜CCD图像。 （左）A面是按照本文中的协议。 （中）A面为准备相红按照本文中的协议，但是食人鱼蚀刻被跳过。 （右）A面是按照本文中的协议，并在氮气中-20℃保存3个月。 （b）与10纳米的Cy3 labeld代表在溶液中采取的CCD图像。 （左）A面是按照本文中的协议。 （右）A面是按照本文中的协议，但是第二轮聚乙二醇化被跳过。比例尺= 5微米。 电影1:在溶液中染料标记蛋白的CCD拍摄的电影 。在溶液中使用60X物镜用10nM Cy3标记labeld代表CCD电影是由棱镜型全内反射荧光显微镜。时间分辨率为100毫秒。 （ 一 ）A面是按照本文中的协议。 （b）一个面是按照本文中的协议，但是第二轮PEGyl的通报BULLETIN被跳过。 <a href="https://www.jove.com/files/ftp_upload/50549/Movie1a.avi" target="_blank">点击这里</a>观看电影1a和<a href="https://www.jove.com/files/ftp_upload/50549/Movie1b.avi" target="_blank">点击这里</a>查看电影1B。

Watch this Scientific Journal Video about Surface Passivation for Single-molecule Protein Studies at JoVE.com

Surface Passivation for Single-molecule Protein Studies

我们描述了使用聚乙二醇（PEG）的钝化玻璃表面的方法。该协议涵盖了表面清洗，表面功能化和PEG涂层。我们引进用于治疗表面与PEG分子过两轮，比现有的方法而产生钝化优质的新战略。

表面钝化单分子蛋白质研究

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important ...

surface-passivation-for-single-molecule-protein-studies

Research

JoVE Journal

Chemistry

41.5K 次观看.  Delft University of Technology. 我们描述了使用聚乙二醇（PEG）的钝化玻璃表面的方法。该协议涵盖了表面清洗，表面功能化和PEG涂层。我们引进用于治疗表面与PEG分子过两轮，比现有的方法而产生钝化优质的新战略。 

视频: Surface Passivation for Single-molecule Protein Studies

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

表面修饰碳纳米管通过土柱运输

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

方法通过表面氧化还原操纵液态金属的表面张力

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Surface Functionalization of Metal-Organic Frameworks for Improved Moisture Resistance

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. However, the main issue limiting their applicability is their poor stability in humid conditions. The common methods to overcome this problem involve the formation of strong metal-linker bonds by using highly charged metals, which is limited to a number of structures, the introduction of alkylic groups to the framework by post-synthetic modification (PSM) or chemical vapour deposition (CVD) to enhance overall hydrophobicity of the framework. These last two usually provoke a drastic reduction of the porosity of the material. These strategies do not permit to exploit the properties of the MOF already available and it is imperative to find new methods to enhance the stability of MOFs in water while keeping their properties intact. Herein, we report a novel method to enhance the water stability of MOF crystals featuring Cu2(O2C)4&#160;paddle-wheel units, such as HKUST (where HKUST stands for Hong Kong University of Science &#38; Technology), with the catechols functionalized with alkyl and fluoro-alkyl chains. By taking advantage of the unsaturated metal sites and the catalytic catecholase-like activity of CuII ions, we are able to create robust hydrophobic coatings through the oxidation and subsequent polymerization of the catechol units on the surface of the crystals under anaerobic and water-free conditions without disrupting the underlying structure of the framework. This approach not only affords the material with improved water stability but also provides control over the function of the protective coating, which enables the development of functional coatings for the adsorption and separations of volatile organic compounds. We are confident that this approach could also be extended to other unstable MOFs featuring open metal sites.

Robust functional catechol coatings were produced in one step by direct reaction of the material known as HKUST with synthetic catechols under anaerobic conditions. The formation of homogeneous coatings surrounding the entire crystal is ascribed to the biomimetic catalytic activity of Cu(II) dimers on the external surface of the crystals.

金属-有机框架的表面功能化改善防潮性能

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. ...

Surface Properties of Synthesized Nanoporous Carbon and Silica Matrices

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) with a 4.6 nm pore size, and ordered silica porous matrix, SBA-15, with a 5.3 nm pore size. This work describes the surface properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the differently ordered porous materials with similar pore sizes. For this purpose, OMC and SBA-15 with highly ordered nanoporous structures are synthesized via impregnation of the silica matrix by applying a carbon precursor and by the sol-gel method, respectively. The porous structure of investigated systems is characterized by an N2 adsorption-desorption analysis at 77 K. To determine the electrochemical character of the surface of synthesized materials, potentiometric titration measurements are conducted; the obtained results for OMC shows a significant pHpzc shift toward the higher values of pH, relative to SBA-15. This suggests that investigated OMC has surface properties related to oxygen-based functional groups. To describe the surface properties of the materials, the contact angles of liquids penetrating the studied porous beds are also determined. The capillary rise method has confirmed the increased wettability of the silica walls relative to the carbon walls and an influence of the pore roughness on the fluid/wall interactions, which is much more pronounced for silica than for carbon mesopores. We have also studied the melting behavior of D2O confined in OMC and SBA-15 by applying the dielectric method. The results show that the depression of the melting temperature of D2O in the pores of OMC is about 15 K higher relative to the depression of the melting temperature in SBA-15 pores with a comparable 5 nm size. This is caused by the influence of adsorbate/adsorbent interactions of the studied matrices.

Here we report the synthesis and characterization of ordered nanoporous carbon (with a 4.6 nm pore size) and SBA-15 (with a 5.3 nm pore size). The work describes the surface and textural properties of nanoporous molecular sieves, their wettability, and the melting behavior of D2O confined in the materials.

合成纳米碳和二氧化硅矩阵的表面性能

In this work, we report the synthesis and characterization of ordered nanoporous carbon material (also called ordered mesoporous carbon material [OMC]) ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.