Name: preparation of mica and silicon substrates for dna origami analysis and experimentation
Uploaded: 2015-07-23T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation at JoVE.com

The authors thank Dr. Gary Bernstein for use of the AFM.

1.实验计划和备料<ol><li>确定将在实验中使用的DNA折纸的设计，浓度，和功能性。14-16这里，我们使用在1×TAE制备/ 镁离子的溶液（40毫摩尔Tris-碱，20mM的一种DNA折纸矩形设计乙酸，2毫摩尔EDTA和12mM的乙酸镁，pH值8.0）。17 </li><li>高压灭菌所有的提示，管和容器使用。这些材料都必须兼容釜。 </li><li>准备无菌水供应用于冲洗。填写无菌罐约500毫升18MΩx厘米的水，放在一个烤盘，煮沸5分钟，盖关闭，并采取罐子离开烤盘中，放凉的盖子放置在容器上，但没有拧紧。存放在冰箱和每个月或在必要时准备一个新的供应。 </li></ol> 2.准备云母基材<ol><li>切割衬底，以适当的大小（1厘米×1cm的正方形），用剪刀。云母是薄和脆。可替换地，购买云母中，不需要切割圆盘形式。 </li><li>切割用双面胶带的云母。云母是由矿物由嵌入离子分离的层，每一层可以当粘附到双面胶带上剥离。18 <ol><li>将云母正方形的双面胶带仍处于胶带分配器，确保它被牢固地附着于磁带上。小心地将云母和磁带之间的镊子，最上面的层将被删除，并保持在磁带上。立即拆除后，云母方将拥有干净的一面朝下。确保存储在容器之前翻转云母过来。 </li><li>重复此三至四次，以确保完全除去最上层和充足的清洗。 </li></ol></li><li>可替代地，粘附云母到容器或台式用一块双面胶带和使用第二片送剔和剥离顶端最云母层。云母将在两种情况下被适当地清洁，虽然替代方法使沉积DNA和漂洗和干燥样品困难的，因为在第二粘附在载体上。 </li></ol> 3.存入DNA折纸上云母<ol><li>简要地说，使用涡旋混合器，以确保即使在溶液的纳米结构分散混合的DNA折纸的小瓶中。 </li><li>吸管4微升溶液涂布的云母，确保枪头不接触衬底。留在云母的DNA折纸约10分钟，以确保足够的覆盖范围。沉积时间将根据DNA的折纸的使用，以及所期望的覆盖范围的浓度（ 图2和3）会发生变化。 <ol><li>冲洗DNA折纸术的解决方案还是使用100微升无菌水通过水槽或其他液体容器云母基板。拿起用镊子云母。吸取上的水基材，具有朝向镊子的末端的液滴的流动。摇用锋利的运动向下的云母以除去多余的水。握住镊子直立，使得水将流向镊子，以避免样品污染。 </li></ol></li><li>干燥氮气源源不断（N 2），持续1分钟的基板上。确保任何多余的水被除去。一个额外的100微升无菌水反复冲洗。干燥用N 2衬底另外3分钟。完全干性基板是必要的成功原子力显微镜（AFM）分析（ 图4）。 </li><li>分析使用AFM或储存于密闭容器中的基板。 </li></ol> 4. CMOS /硅清洁套装式<ol><li>注意:当使用CMOS设置，使用个人防护装备在任何时候。该试剂包括强酸，强碱，氢氟酸（HF）和强氧化剂而CAn，其中废溶剂发生反应试剂，如果不妥善处理。坚持以下安全防范措施: <ol><li>众议院CMOS板凳在化学罩，没有其他进程或调校。 </li><li>使用CMOS替补席上的时候，要戴上丁腈手套，实验室外套，护目镜，大型工业丁腈手套，围裙溢出，并在任何时候都面罩。 </li><li>用塑料桶作为二级防护解决方案时准备。 </li><li>使用的惰性氟化聚合物测量烧杯用于处理浓HF。 </li><li>使葡萄糖酸钙软膏可作为任何皮肤接触急救。 </li><li>只允许经过适当培训的人员进行处理。 </li><li>始终确保实验室的另一名成员是目前在紧急情况下。 </li><li>保持MSDS信息引擎盖附近的所有化学品。 </li><li>熟悉的机构或公司的化学品泄漏和暴露的政策。 注:HF容易渗透皮肤并且是钙清道夫，如果发生接触骨骼的影响和损坏的神经。皮肤暴露于浓氢氟酸几毫升可能是危险的，甚至是致命的。建立必要的防范措施，以确保不发生接触。 </li></ol></li><li>在单独的电炉单独250ml玻璃烧杯进行RCA1和RCA2。每个烧杯中应该包含一个搅拌棒。监视使用温度计夹紧所以搅拌棒不爆炸到灯泡的溶液的温度。覆盖用表玻璃，以减少蒸发的影响的烧杯中。 </li><li> RCA1准备<ol><li>放置加入50ml 18MΩx厘米水成使用测量烧杯指定RCA1烧杯中。 </li><li>加入15mL浓氢氧化铵（NH 4 OH）的烧杯中。冲洗，用25毫升水的测量烧杯和漂洗水添加到RCA1烧杯中。 </li><li>打开上热板的热量和搅拌器并使RCA1浴至70℃。 </l我&gt; <li>加入15mL 30％过氧化氢 ​​（H 2 O 2）至RCA1烧杯中。使用在1小时内的RCA1溶液中的 H 2 O 2后已被添加。镀液可以三天的跨度内多次使用如果15毫升过氧化物每次加入到浴中。 </li><li>用清水彻底冲洗测量烧杯中，将清洗液倒掉在适当的RCA1废液瓶。 </li></ol></li><li> RCA2准备<ol><li>加70毫升18MΩx厘米水使用彻底冲洗测量烧杯指定RCA2烧杯中。 </li><li>添加15ml浓盐酸（HCl）的。冲洗用20毫升水的测量烧杯中，并把它添加到RCA2烧杯中。 </li><li>增加热量，搅拌热板的速度，直到溶液达到70℃。 </li><li>添加15毫升30％H 2 O 2的。像RCA1浴，使用该溶液在1小时内从当H 2 O 2的加入;另外，在浴可以三天的跨度内重复使用多次，如果将15ml的H 2 O 2的每次使用前加入。 </li></ol></li><li> HF溶液的制备<ol><li>放置50ml水在惰性含氟聚合物烧杯中。 </li><li>措施4毫升的塑料测量烧杯浓氢氟酸（49％）的，并把它添加到惰性含氟聚合物烧杯中。 </li><li>冲洗出来的塑料测量烧杯中总共50毫升的水，加入的清洗水，以高频烧杯中。用清水彻底洗出测量烧杯中，并丢弃在指定HF废弃物容器洗涤。 </li></ol></li></ol> 5.准备和清洁硅衬底<ol><li>切割硅晶片到晶片<ol><li>识别在硅晶片的平坦研磨面的垂直和平行晶格方向。这些方向来帮助裂解广场更容易。下面的说明适用于cleav荷兰国际集团的硅&lt;110&gt;，可能不适合于其他结晶取向。 </li><li>放置在硅晶片上的软表面抛光的正面朝上，如卫生巾。使用钻石尖笔抄写，沿主平边晶圆轻轻缺口底部。放置一个小金属丝，例如回形针，下面的切口，并轻轻放置手指或镊子上切口的任一侧并压下压力施加到晶片。这样做将分开晶圆成两半沿着自然裂开方向晶格线。 </li><li>在另一卫生巾，用铅笔和直尺，量出的平方的期望的宽度由在顶层和卫生巾的底部标示的点。连接这些点用直线。这将作为一个指导方针，甚至方形的形状。 </li><li>放置晶片的一个半部边先测量的线之间的冲洗对线卫生巾和在步骤5.1.2重复将新鲜破碎垂直PIEC现在上课应的切割正方形的宽度。转晶片水平上的卫生巾。把它放在垂直线之间以及在步骤5.1.2重复这个过程。 </li><li>存储新鲜切割晶圆芯片在干净的小瓶中装满去离子水，以防止刮伤。硅芯片可以无限期地存储，但在开始之前，实验应清洗。 </li></ol></li><li>硅CMOS清洁<ol><li>当RCA1溶液达到适当的温度，用惰性含氟聚合物篮子"直径的2淹没八到十为1cm×4cm的硅晶片中的溶液中。氧的气泡将形成在芯片和烧杯壁。如果没有气泡产生时，H 2 O 2的劣化。离开芯片在10到20分钟的溶液中，搅拌该筐上下每隔几分钟，以保持芯片粘在一起。 </li><li>提起含有硅芯片组成的篮子，沥干。移动篮下牛逼多Ø废物烧杯中，用18MΩx厘米的水彻底冲洗。沉浸在洗涤烧杯中，并轻摇上下持续20秒。排水篮下用清水彻底冲洗过的废烧杯中。清空废烧杯存入指定RCA1废液瓶，并重新注入水。 </li><li>后RCA1清洗完成后，将篮放入1:50的HF烧杯为10至20秒。使用温和的向上和向下运动的混合芯片和高频。提起水桶扣篮让HF排完全离开。 注:该芯片的表面应该是疏水的;水不会弄湿芯片，而是形成具有高的接触角的液滴。这表明，在氧化硅已被蚀刻掉，并在芯片现在由Si-H键终止。 </li><li>将洗烧杯和冲洗烧杯在一个塑料桶，将篮子在漂洗烧杯中，用清水冲洗18MΩx厘米的水。浸没篮到洗涤烧杯并搅拌20秒。 </li><li>完成第二漏极和RINSË周期18MΩx厘米的水。倾倒的洗涤水进入漂洗烧杯中，并重新填充洗涤烧杯中的水。倒入所有废物到指定的塑料HF废液瓶。 </li><li>当RCA2溶液达到适当的温度，淹没用筐的硅芯片中的解决方案。留在10至20分钟的溶液中。现在该芯片将亲水由于薄（1-2纳米）的氧化膜的生长。 </li><li>时间适量后删除并遵循同样的程序冲洗为RCA1。处置废弃物相应的RCA2废物容器中。从篮子用塑料镊子取出每块芯片，用清水冲洗干净，吹干氮气。 </li><li>储屑在一个塑料晶片盒或以18MΩ的x厘米水的小瓶。当清洗后为3天左右储存在水中的硅将保持清洁。确保工作区域进行适当的清理和CMOS手套的外观已经冲。离开CM在引擎盖操作系统手套干燥。 </li></ol></li></ol> 6.存入DNA折纸上APTES官能硅<ol><li>硅自组装单分子层形成<ol><li>温馨APTES到RT开幕前。如果瓶子太冷，可能会出现冷凝，在储存期间引起APTES的水解。加入1980微升18MΩx厘米的水和20微升的APTES到一个干净的闪烁瓶和漩涡混合。立即使用此解决方案。 </li><li>放置一个清洁的硅芯片的反射侧上放入闪烁瓶中，盖住它，并让坐20分钟。使用镊子取下芯片并冲洗用200μl水和干燥1分钟以N 2流。 </li></ol></li><li>沉积DNA折纸上APTES功能化硅 注意:该步骤上官能硅沉积的DNA折纸类似于那些用于沉积在云母上。 <ol><li>简言之混合DNA折纸瓶和吸管4微升解决方案到硅衬底上。如果必要的话，增加用于覆盖整个衬底作为官能硅比云母基材更加疏水的DNA折纸溶液的体积。使用玻璃盖玻片按沉积溶液向下和防止在长期沉积的蒸发。 </li><li>让该溶液静置的时间所必需的量为所用的浓度和期望的覆盖（ 见图2和3的时间和浓度对表面覆盖的效果）。冲洗基板用100μl无菌18MΩ的x厘米水和干燥用N 2 1分钟。 </li><li>重复该漂洗用另外100微升无菌水和干燥用N 2基板3分钟。 </li><li>样品储存在一个干净的容器中，直到进一步的实验或成像可以执行。样品开始后约一到两周的存储，显示微粒积累取决于如何亩CH处理它们。 </li></ol></li></ol> 7. AFM成像和DNA折纸样品图像分析<ol><li>在空气震动模式下成像的目的使用原子力显微镜。轻敲模式可以确保最小的力将被施加到脆弱纳米结构相比，接触模式。 注意:摄像参数将取决于仪器。提出的所有图像采用多模纳秒示波器IIIa受体抓获。 </li><li>选择AFM探针用于非接触/轻敲在空气模式，具有金反射涂层，〜300 kHz的谐振频率（标称值），力常数为40N / m和尖端半径&lt;10纳米的。 </li><li>处理和分析的NanoScope使用分析软件原子力显微镜图像。使用ImageJ执行覆盖率计算。19 </li></ol>

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(77), e50268 (2013).</li><li>Woo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+molecular+recognition+based+on+the+geometry+of+DNA+nanostructures.">Programmable molecular recognition based on the geometry of DNA nanostructures.</a> Nature Chemistry. 3, 620-627 (2011).</li><li>Schlegel, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation+sorption+on+the+muscovite+(001)+surface+in+chloride+solutions+using+high-resolution+X-ray+reflectivity.">Cation sorption on the muscovite (001) surface in chloride solutions using high-resolution X-ray reflectivity.</a> Geochim. Cosmochim. Acta. 70, 3549-3565 (2006).</li><li>Rasband, W. S., Howarter, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health.">National Institutes of Health.</a> Langmuir. 22, 11142-11147 (2006).</li><li>Kershner, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placement+and+orientation+of+individual+DNA+shapes+on+lithographically+patterned+surfaces.">Placement and orientation of individual DNA shapes on lithographically patterned surfaces.</a> Nature Nanotechnology. 4, 557-561 (2009).</li><li>Hung, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-area+spatially+ordered+arrays+of+gold+nanoparticles+directed+by+lithographically+confined+DNA+origami.">Large-area spatially ordered arrays of gold nanoparticles directed by lithographically confined DNA origami.</a> Nature Nanotechnology. 5, 121-126 (2010).</li><li>Sarveswaran, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.Adhesion+of+DNA+nanostructure+and+DNA+origami+to+lithographically+patterned+self-assembled+monolayers+on+Si[100.">et al.Adhesion of DNA nanostructure and DNA origami to lithographically patterned self-assembled monolayers on Si[100.</a> Proc. SPIE-Int. Soc. Opt. Eng. 7637, 76370M (2010).</li><li>Pillers, M. A., Lieberman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+stability+of+DNA+origami+on+mica.">Thermal stability of DNA origami on mica.</a> J. Vac. Sci. Technol. B. 32, 040602 (2014).</li><li>Song, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Visualization+of+Transient+Thermal+Response+of+a+DNA.">Direct Visualization of Transient Thermal Response of a DNA.</a> Origami. J. Am. Chem. Soc. 134, 9844 (2012).</li><li>Wei, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+thermal+behavior+of+DNA+origami+nanostructures.">Mapping the thermal behavior of DNA origami nanostructures.</a> J. Am. Chem. Soc. 135 (16), 6165-6176 (2013).</li><li>Hyojeong Kim, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+DNA+Origami+Nanostructures+under+Diverse+Chemical+Environments.">Stability of DNA Origami Nanostructures under Diverse Chemical Environments.</a> Chem. Mater. 26, 5265-5273 (2014).</li></ol>

The authors have nothing to disclose.

有迹象表明，需要强调的实现一致的和理想的结果几个步骤。对云母的样品，按照严格的和彻底的清洗和干燥制度，如在步骤3.3和3.4，将确保各DNA折纸的高品质的图片可使用原子力显微镜，而不在代表结果部分中概述的各种问题而获得。硅样品最重要的是在基片的清洁。继在深入细致的步骤概述5.2清洗程序将确保一个适当清洁氧化硅表面就会实现。此外，监测的质量和化学品，如过氧化氢，氢氟?...

于2006年首次推出，脱氧核糖核酸折纸利用DNA寡核苷酸的自组装性质，以产生可设计和高度有序的纳米结构。1结构的无数已经报道，从笑脸到锁存三维盒2的DNA折纸可以被官能与各种生物分子和纳米结构，从而引起研究应用在纳米电子学，医学，和量子计算。3然而，分析和许多未来的应用不仅取决于结构设计，而且还取决于该DNA折纸纳米结构表面的粘合性。在这个手稿中描述的方法涉及的DNA折纸样品对两种类型的衬底的制备:云母和官能化的氧化硅。 云母是，选择的基板为脱氧核糖核酸折纸的研究，因为它是原子级平坦，有一层0.37高度毫微米±0.02纳米。4也EAS随手清理，使样品制备和原子力显微镜（AFM）的研究简单。白云母含有钾在各解理面的高密度，但这些离子扩散远离云母表面在水中时。以介导的DNA折纸的结合到云母基板， 镁离子被用来反转云母的负电荷和静电结合的DNA磷酸骨架到基板（ 图1A）。5混合物退火的DNA中的大的存在订书钉链过度给高覆盖和良好的图像上云母因为DNA的折纸与Mg 2+ -terminated表面的粘附力比单链寡核苷酸（短链）的粘合更强。其它带正电的离子，包括镍2+和Co 2+可以用来控制DNA对云母的粘合。6,7-改变在溶液中可以介导阿德一价和二价阳离子的浓度DNA的折纸锡永和表面扩散速率。8然而，该协议用 ​​于制备云母基板和沉积并冲洗折纸通常没有明确发表的手稿。9描述没有一个明确的协议，可重复的结果可能是很难获得的。 云母是绝缘体，因此它是不适合作为底物在纳米电子学的一些应用。硅钝化的薄同质氧化物具有所需的电特性，包括与现有互补金属氧化物半导体（CMOS）处理的兼容性来创建输入/输出结构和地形特征。存储在空中硅片与钝化无论是厚的热氧化物或薄的自然氧化膜是比较脏的，具有较高的微粒计数。氧化硅具有低得多的表面电荷密度比云母，和电荷密度是高度依赖于氧化物制备和历史。在镁离子浓度ABO已经150mM的，矩形的DNA折纸的良好覆盖范围（最多4个/μm2）可以在氧等离子体处理的硅衬底来实现;然而，该浓度和覆盖可以根据尺寸和纳米结构的设计被用来改变。10的另一种协议用 ​​于调谐表面电荷是3-氨基丙基硅烷（APTES）（ 图1B）的阳离子自组装单层附着到氧化物。上APTES伯胺可以在低于9的pH值11对于APTES的完整单层可以成功存入被质子化，修改衬底的电荷和疏水性，对硅必须适当使用美国无线电公司（RCA）的协议清洁。这些协议包括在氢氧化铵和过氧化氢溶液（RCA1）处理以去除有机残留物和颗粒污染物。简要蚀刻在氢氟酸水溶液中移除沿与原生氧化层即坚持以任何氧化离子污染物。最后，将样品暴露于盐酸和过氧化氢 ​​溶液（RCA2）以除去金属和离子污染物，并形成薄，均匀的氧化层12最洁净室已指定罩在CMOS清洗协议，与什么可以使用严格的规定在这些区域。一个常见的问题出现在离子如钠，它可以通过创建midbandgap陷阱扰乱的CMOS结构的电子特性的形式。13离子常用于DNA的折纸制备和沉积缓冲器可能污染的CMOS浴和会导致使用其他研究者的问题洁净室。出于这个原因，我们组采用的是&#39;脏&#39;的CMOS清洗替补安排专门用于DNA折纸研究的小样本。这个过程是一个很好的替代传统的洁净室的设置和可以适于没有获得洁净室的CMOS长凳实验室。

<table><tbody><tr><td>Eppendorf epT.I.P.S. Reloads, capacity 2-200&amp;nbsp;&amp;mu;l</td><td>VWR International, LLC</td><td>22491733</td><td>10 reload tray of 96 tips</td></tr><tr><td>Microcentrifuge Tubes, Polypropylene</td><td>VWR International, LLC</td><td>87003-290</td><td>0.65 ml, natural</td></tr><tr><td>Research Plus Pippete - Single Channel - 20-200&amp;nbsp;&amp;mu;l</td><td>A. Daigger &amp;amp; Company, Inc.</td><td>EF8960F-3120000054 EACH</td><td>Adjustable Volume</td></tr><tr><td>Research Plus Pippete - Single Channel - 2-20&amp;nbsp;&amp;mu;l</td><td>A. Daigger &amp;amp; Company, Inc.</td><td>EF8960D-3120000038 EACH</td><td>Adjustable Volume</td></tr><tr><td>Scotch 237 Permanent Double-Sided Tape</td><td>Office Depot, Inc.</td><td>602710</td><td>3/4&quot; x 300&quot;, Pack of 2</td></tr><tr><td>Vortex Mixer</td><td>Thermo Scientific</td><td>M37610-33Q</td><td></td></tr><tr><td>Wafer container single, 2&quot; (50 mm), 60 mm x 11&amp;nbsp;mm</td><td>Electron Microscopy Sciences</td><td>64917-2</td><td>6 per pack</td></tr><tr><td>6&quot; Wafer, P-type, &amp;lt;100&gt; orientation, w/ primary flat</td><td>Nova Electronic Materials, Ltd.</td><td>GC49266</td><td></td></tr><tr><td>Powder-Free Nitrile Examination Gloves</td><td>VWR International, LLC</td><td>82062-428</td><td>Catalog number is for size large</td></tr><tr><td>High Accuracy Noncontact probes with Au reflective coating</td><td>K-Tek Nanotechnology, Inc.</td><td>HA_NC/15</td><td></td></tr><tr><td>Autoclave Pan</td><td>A. Daigger &amp;amp; Company, Inc.</td><td>NAL692-5000 EF25341C</td><td></td></tr><tr><td>Sol-Vex II Aggressive Gloves, Size: 9-9.5; 15 mil, 13 inch - 1 dz</td><td>Spectrum Chemical Mfg. Corp.</td><td>106-15055</td><td>Before use, rinse with water and scrub together until no bubbles form on the gloves.</td></tr><tr><td>Tweezers PTFE 200 mm Square</td><td>Dynalon Corp.</td><td>316504-0002</td><td></td></tr><tr><td>Muscovite Mica Sheets V-5 Quality</td><td>Electron Microscopy Sciences</td><td>71850-01</td><td>10 per pack</td></tr><tr><td>Mica Disc, 10 mm</td><td>Ted Pella, Inc</td><td>50</td><td>Mica discs are optional</td></tr><tr><td>Scriber Diamon Pen for Glassware</td><td>VWR International, LLC</td><td>52865-005</td><td></td></tr><tr><td>Scintillation Vials, Borosilicate Glass, with Screw Cap - 20 ml</td><td>VWR International, LLC</td><td>66022-060</td><td>Case of 500, with attached polypropylene cap and pulp foil liner</td></tr><tr><td>4 x 5 Inch Top PC-200 Hot Plate, 120 V/60 Hz</td><td>Dot Scientific, Inc.</td><td>6759-200</td><td></td></tr><tr><td>Straight-Sided Glass Jars, Wide Mouth</td><td>VWR International, LLC</td><td>89043-554</td><td>Case of 254, caps with pulp/vinyl liner attached</td></tr><tr><td>Standar-Grade Glass Beaker, 250 ml Capacity</td><td>VWR International, LLC</td><td>173506</td><td></td></tr><tr><td>Beakers, PTFE</td><td>VWR International, LLC</td><td>89026-022</td><td>For use with HF</td></tr><tr><td>Shallow form watch glass, 3&quot;</td><td>VWR International, LLC</td><td>66112-107</td><td>Case of 12</td></tr><tr><td>Plastic Storage Container</td><td>VWR International, LLC</td><td>470195-354</td><td>For secondary container</td></tr><tr><td>General-Purpose Liquid-In-Glass Thermometers</td><td>VWR International, LLC</td><td>89095-564</td><td></td></tr><tr><td>High precision and ultra fine tweezers</td><td>Electron Microscopy Sciences</td><td>78310-0</td><td></td></tr><tr><td>Polycarbonate Faceshield</td><td>Fisher Scientific, Inc.</td><td>18-999-4542</td><td></td></tr><tr><td>Neoprene Apron</td><td>Fisher Scientific, Inc.</td><td>19-810-609</td><td></td></tr><tr><td>Calcium Gluconate, Calgonate</td><td>W.W Grainger, Inc.</td><td>13W861</td><td>Tube, 25 g</td></tr><tr><td>Hydrogen Peroxide 30% CR ACS 500 ml</td><td>Fisher Scientific, Inc.</td><td>H325 500</td><td>HARMFUL, TOXIC</td></tr><tr><td>3-Aminopropyltriethoxysilane</td><td>Gelest Inc.</td><td>SIA0610.0-25GM</td><td>Let warm to room temperature before use.</td></tr><tr><td>Ammonium hydroxide, 2.5 L</td><td>Fisher Scientific, Inc.</td><td>A669-212</td><td>HARMFUL, TOXIC</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific, Inc.</td><td>A144-212</td><td>HARMFUL, TOXIC</td></tr><tr><td>Hydrofluoric acid</td><td>Fisher Scientific, Inc.</td><td>A147-1LB</td><td>HARMFUL, TOXIC</td></tr><tr><td>MultiMode Nanoscope IIIa</td><td>Veeco Instruments, Inc.</td><td></td><td>Any AFM capable of tapping mode is suitable for analysis</td></tr><tr><td>Dunk basket</td><td>Made in lab</td><td>Made in lab</td><td>The dunk basket was made using the bottom of a PTFE bottle with holes drilled in, PTFE handle, and all PTFE screws.</td></tr></tbody></table>

preparation of mica and silicon substrates for dna origami analysis and experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

两个变量决定了DNA折纸术的覆盖面在基板上:溶液浓度和接触时间。之前已经报道了DNA的折纸对云母的吸附特性和APTES官能硅氧化物13在沉积溶液和云母的最终覆盖范围的DNA折纸的浓度之间的关系总结于表1和图2，示出浓度增加的结果在更大的覆盖范围。结合的时间依赖性见于图3。表面覆盖先前研究，以量化的DNA折纸云母的结合行为和改性氧化硅的表面...

Watch this Scientific Journal Video about Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation at JoVE.com

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

preparation-mica-silicon-substrates-for-dna-origami-analysis

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

14.2K 次观看.  University of Notre Dame. Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

视频: Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

科研

生物工程

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

复杂的二维形状的从单链DNA的瓷砖自组装

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

化学

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

生物学

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

生物化学

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

遗传学

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 ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

基因治疗诱导聚化涂层保护 dna 折纸纳米结构

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

脱氧核糖核酸介导基板无机结构纳米图案用于传感应用

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

通过等离子体 DNA 折纸纳米天线实现单分子 SERS 测量

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

作者聚焦:通过等离子体 DNA 折纸纳米天线实现单分子表面增强拉曼散射测量  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

等离子体DNA折纸纳米天线实现的单分子表面增强拉曼散射测量

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...