对于DNA折纸分析和实验准备云母和硅衬底
Summary
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.
Abstract
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.
Introduction
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'脏'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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Dr. Gary Bernstein for use of the AFM.
Materials
| Eppendorf epT.I.P.S. Reloads, capacity 2-200 μL | VWR International, LLC | 22491733 | 10 reload tray of 96 tips |
| Microcentrifuge Tubes, Polypropylene | VWR International, LLC | 87003-290 | 0.65 mL, natural |
| Research Plus Pippete – Single Channel – 20-200 μL | A. Daigger & Company, Inc. | EF8960F-3120000054 EACH | Adjustable Volume |
| Research Plus Pippete – Single Channel – 2-20 μL | A. Daigger & Company, Inc. | EF8960D-3120000038 EACH | Adjustable Volume |
| Scotch 237 Permanent Double-Sided Tape | Office Depot, Inc. | 602710 | 3/4" x 300", Pack of 2 |
| Vortex Mixer | Thermo Scientific | M37610-33Q | |
| Wafer container single, 2" (50 mm), 60 mm x 11 mm | Electron Microscopy Sciences | 64917-2 | 6 per pack |
| 6" Wafer, P-type, <100> orientation, w/ primary flat | Nova Electronic Materials, Ltd. | GC49266 | |
| Powder-Free Nitrile Examination Gloves | VWR International, LLC | 82062-428 | Catalog number is for size large |
| High Accuracy Noncontact probes with Au reflective coating | K-Tek Nanotechnology, Inc. | HA_NC/15 | |
| Autoclave Pan | A. Daigger & Company, Inc. | NAL692-5000 EF25341C | |
| Sol-Vex II Aggressive Gloves, Size: 9-9.5; 15 mil, 13 inch – 1 dz | Spectrum Chemical Mfg. Corp. | 106-15055 | Before use, rinse with water and scrub together until no bubbles form on the gloves. |
| Tweezers PTFE 200 mm Square | Dynalon Corp. | 316504-0002 | |
| Muscovite Mica Sheets V-5 Quality | Electron Microscopy Sciences | 71850-01 | 10 per pack |
| Mica Disc, 10 mm | Ted Pella, Inc | 50 | Mica discs are optional |
| Scriber Diamon Pen for Glassware | VWR International, LLC | 52865-005 | |
| Scintillation Vials, Borosilicate Glass, with Screw Cap – 20 mL | VWR International, LLC | 66022-060 | Case of 500, with attached polypropylene cap and pulp foil liner |
| 4 x 5 Inch Top PC-200 Hot Plate, 120 V/60 Hz | Dot Scientific, Inc. | 6759-200 | |
| Straight-Sided Glass Jars, Wide Mouth | VWR International, LLC | 89043-554 | Case of 254, caps with pulp/vinyl liner attached |
| Standar-Grade Glass Beaker, 250 mL Capacity | VWR International, LLC | 173506 | |
| Beakers, PTFE | VWR International, LLC | 89026-022 | For use with HF |
| Shallow form watch glass, 3" | VWR International, LLC | 66112-107 | Case of 12 |
| Plastic Storage Container | VWR International, LLC | 470195-354 | For secondary container |
| General-Purpose Liquid-In-Glass Thermometers | VWR International, LLC | 89095-564 | |
| High precision and ultra fine tweezers | Electron Microscopy Sciences | 78310-0 | |
| Polycarbonate Faceshield | Fisher Scientific, Inc. | 18-999-4542 | |
| Neoprene Apron | Fisher Scientific, Inc. | 19-810-609 | |
| Calcium Gluconate, Calgonate | W.W Grainger, Inc. | 13W861 | Tube, 25 g |
| Hydrogen Peroxide 30 % CR ACS 500 mL | Fisher Scientific, Inc. | H325 500 | HARMFUL, TOXIC |
| 3-Aminopropyltriethoxysilane | Gelest Inc. | SIA0610.0-25GM | Let warm to room temperature before use. |
| Ammonium hydroxide, 2.5 L | Fisher Scientific, Inc. | A669-212 | HARMFUL, TOXIC |
| Hydrochloric acid | Fisher Scientific, Inc. | A144-212 | HARMFUL, TOXIC |
| Hydrofluoric acid | Fisher Scientific, Inc. | A147-1LB | HARMFUL, TOXIC |
| MultiMode Nanoscope IIIa | Veeco Instruments, Inc. | n/a | Any AFM capable of tapping mode is suitable for analysis |
| Dunk basket | Made in lab | Made in lab | The dunk basket was made using the bottom of a PTFE bottle with holes drilled in, PTFE handle, and all PTFE screws. |
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