调查单分子粘附的原子力谱
Summary
A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.
Abstract
原子力谱是研究分子在表面和界面的理想工具。一个试验性协议，以夫妻种类繁多的单分子的共价键上的原子力显微镜针尖呈现。同时针尖被钝化，以防止尖端和基板，这是一个先决条件，研究附着在针尖单个分子之间的非特异性相互作用。分析，以确定的粘合力，粘合长度，并在固体表面上和生物界面这些分子不久提出并提供用于进一步阅读外部引用的自由能。实施例的分子是聚（氨基酸）聚酪氨酸，接枝聚合物PI- 克 -PS和磷脂POPE（1-棕榈酰-2-油酰- SN -glycero -3-磷酸）。这些分子是从不同的表面，如CH 3 -SAMs，氢封端的金刚石解吸并支持的各种溶剂的条件下的脂质双层。最后，该力分光单分子实验的优点进行了讨论，包括装置，以决定是否确实是一个单一分子已经研究了实验。
Introduction
在过去的30年中，原子力显微镜（AFM）已被证明是一个有价值的成像技术来研究生物学1,2和3的合成材料和表面，因为它提供了分子的空间分辨率在三个维度，可以在不同的溶剂进行操作环境。此外，AFM单分子力谱（SMFS）使测量力范围从PN为μN政权，并给予前所未有的洞察到例如蛋白质折叠4,5，高分子物理6 – 8，和单分子表面相互作用9 –学习背后的单个分子，而不是分子的集合体12 .The理由是为了避免影响平均，往往掩盖了罕见的事件或隐藏的分子状态。此外，众多的分子参数，如轮廓长度，库恩长度，粘合自由能等可以获得。此详述在下面的实施例。在一个典型的AFM-SMFS实验，探针分子通过接头分子偶联到一个非常尖锐的尖端。尖端本身位于一可弯曲的悬臂的端部。如果针尖被带入与该表面接触的探针分子将与该表面相互作用。通过在尖端，力，因此自由能缩回观察悬臂的偏转，以从表面分离的分子可以被确定。以获得有意义的统计数据，大量的所谓的力 – 距离曲线已被收购。此外，能有真正的单分子实验（ 即 ，使用一个和在整个实验期间相同的探针分子）探针分子应当共价偶联到所述针尖。这里，一个实验协议悬臂官能经由共价键的单个分子呈现。单分子既可以通过氨基或硫基耦L组的针尖。缀合方法可以在广泛的各种溶剂（有机相和水）占所用的聚合物的溶剂化性质来进行。
在第一部分中，一个通用的方案来共价通过接头分子与AFM针尖描述附加一个单一的分子（“探针分子”）。为此目的，有机NHS-或马来酰亚胺化学过程中使用13。随着协议3例如分子，数据采集和数据分析的方法描述和提供了用于进一步阅读参考。的例子的分子是：（线性）聚合物酪氨酸，接枝聚合物PI- 克 -PS和脂质POPE。这包括协议的微小变化，例如共价连接半胱氨酸。另外，一个段是专用不同的表面的制备诸如金刚石表面，CH 3 -self组装单层和脂质双层。这些接口都省恩是很好的参考和实例。
Protocol
Representative Results
Discussion
在过去的几十年中，单分子实验提供了前所未有的见解的分子机制，竟然是在生命科学及以后的宝贵途径。以实现从SMFS实验，理想地1，并在同一分子用在实验的全过程良好的和有意义的统计数据。与此相反，以与分子的合奏实验，SMFS实验能够检测罕见事件和隐藏的分子状态。的单分子实验的另一个优点是，它们可以通过分子动力学模拟24,25手段进行建模。
上述悬臂?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank the DFG (Hu 997/2-2) for financial support. FS acknowledges the Hanns-Seidel-Stiftung (HSS). SKr was supported by the Elitenetzwerk Bayern in the framework of the doctorate program Material Science of Complex Interfaces. SKi thanks the SFB 863 for financial support.
Materials
| Materials | |||
| Hellmanex III alkaline liquid concentrate (detergent solution) | Hellma | ||
| RCA (ultrapure water, hydrogen peroxide (35 %), ammonia (32%); 5:1:1(v/v/v)) | Sigma | ||
| Vectabond reagent / APTES (3-Aminopropyl)triethoxysilane | Vectorlabs | ||
| Dry acetone (< 50 ppm H2O) | Sigma | ||
| Dry chloroform (> 99.9 %) | Sigma | ||
| Triethylamine | Sigma | ||
| Ultrapure water | Biochrom, Germany | ||
| Di-sodium tetraborate (> 99.5 %) | Biochrom, Germany | ||
| Boric Acid | Biochrom, Germany | ||
| Monofunctional α-methoxy-ω-NHS PEG, 5kDa, “methyl-PEG-NHS” | Rapp, Germany | ||
| Heterobifunctional α,ω-bis-NHS PEG, 6 kDa, “NHS-PEG-NHS” | Rapp, Germany | ||
| Heterobifunctional α-maleimidohexanoic- ω-NHS PEG, 5 kDa, “Mal-PEG-NHS” | Rapp, Germany | ||
| Probe molecule (polymer, lipid, etc.) | |||
| Equipment | |||
| Sufficient amount of glass crystallising dishes with spout (10 ml), glass petri dishes (500 µl) and glass lids | VWR International GmbH, Germany | ||
| Laboratory oven model UF30 | Memmert, Germany | ||
| Temperature controlled sonicator | VWR International GmbH, Germany | ||
| Plasma system "Femto", 100 W | Diener, Germany | ||
| One separate glass syringe for each organic solvent | VWR International GmbH, Germany | ||
| Vortex mixer | VWR International GmbH, Germany | ||
| Microcentrifuge tubes (0.5 ml or 1.5 ml) | Eppendorf | ||
| Pipettes: 10-100 µl, 50-200 µl and 100-1000 µl | Eppendorf | ||
| AFM with temperature controlled fluid cell (e.g. MFP-3D with BioHeater) | Asylulm Research, Santa Barbara | ||
| Soft SiN cantilevers cantilever, typically made from silicon nitride (SiN) (spring constant less than 100 pN/nm, e.g. MLCT) | Bruker AXS, Santa Barbara |
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