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Abstract
Introduction
Protocol
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Chemistry

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

Published: March 06, 2017
doi:
10.3791/54975
, ,
1Institute of Chemistry and The Center for Nanoscience and Nanotechnology,The Hebrew University of Jerusalem

Summary

在这里，我们提出了一个协议使用原子力显微镜（AFM）测量由单分子力光谱测量相互作用的定义明确的无机表面和任一的肽或氨基酸之间的力。从测量中获得的信息是重要的，以便更好地了解肽 – 无机材料相间。

Abstract

蛋白质或肽和无机材料之间的相互作用导致了一些有趣的流程。例如，含矿物质结合蛋白导致具有独特性能的复合材料的形成。此外，生物结垢的不希望的过程是由生物分子，主要是蛋白质，表面上的吸附开始。该有机层是细菌的粘合层，并允许他们与表面相互作用。理解支配在有机 – 无机界面的相互作用的基本力因此对于研究许多领域重要，并可能导致新的材料设计用于光学，机械和生物医学应用。本文展示了单分子力光谱技术，其利用原子力显微镜测量或肽或氨基酸和良好定义的无机表面之间的粘附力。该技术包括一个协议，用于生物分子附着到AFM小费通过原子力显微镜的共价柔性接头和单分子力谱的测量。此外，这些测量值的分析是包括在内。

Introduction

蛋白质和无机矿物质之间的相互作用导致具有独特性能的复合材料结构。这包括与高的机械强度或独特的光学性质的材料。 1，2例如，与矿物羟磷灰石的胶原蛋白的结合产生任一软或硬的骨骼为不同的功能。 3短肽还可以结合具有高度特异性无机材料。 4，5，6，这些肽的特异性已用于设计新的磁性和电子材料，7，8，9的制造纳米结构材料，生长晶体， 10纳米粒子合成。 11了解底层肽或蛋白质和无机材料之间的相互作用，因此使我们能够具有改进的吸附性能设计新型复合材料的机制。另外，由于植入物具有免疫应答相间是由蛋白质介导，更好地理解与无机材料的蛋白质的相互作用会提高我们的设计的植入物的能力。涉及蛋白质与无机表面相互作用的另一个重要领域是防污材料的制造。 12，13，14，15生物污染是不希望的过程，其中生物附着到表面。这对我们的生活的许多不利影响。例如，在医疗设备的细菌生物污损导致医院获得性感染。海洋生物对船和大型船舶生物污染增加燃料的消耗。 12，16，17，18

单分子力谱（SMFS），使用AFM，可以直接测量的氨基酸或与基板的肽之间的相互作用。 19，20，21，22，23，24，25，26其他方法，如噬菌体展示，27，28 石英晶体微天平（QCM）29或表面等离子体共振（SPR）29，30，31，32，裁判“> 33衡量肽和蛋白质来无机表面的散装的相互作用。34，35，36 这意味着，通过这些方法获得的结果涉及分子或聚集体的合奏。在SMFS，一个或非常少的分子被固定在针尖和它们与所需底物相互作用进行测量。这种方法可以扩展通过从表面拉动蛋白质研究蛋白质折叠。此外，它可以被用来测量细胞和蛋白质与抗体与其配体的结合之间的相互作用。 37，38，39，40本文详细介绍了如何可以肽或氨基酸连接到使用硅醇化学针尖。此外，本文介绍如何执行力测量，以及如何分析结果。

Protocol

1.修改提示购买氮化硅（ 的 Si 3 N 4）的AFM悬臂用硅提示（〜2纳米的标称悬臂半径）。 通过在无水乙醇中浸泡20分钟，清洁每个AFM悬臂。干燥在常温下。然后通过暴露于O 2等离子体5分钟治疗悬臂。 暂停清洁提示上述（3厘米）的含甲基三乙氧基硅烷溶液和3-（氨丙基）三乙氧基硅烷中的15：1的比例（V / V）在惰性气氛（氮气或氩气）下一个干燥?…

Representative Results

图1展示尖端修改程序。在第一步骤中，等离子体处理改变氮化硅尖端的表面上。尖端呈现OH基团。然后这些基团将与硅烷反应。在此步骤结束时，尖端的表面将由自由-NH 2基团所覆盖。然后，这些游离胺将用Fmoc -PEG-NHS，共价连接物反应。 Fmoc基团的PEG连接的是由pipyridine，脱保护试剂除去。最后，该检查的氨基酸或肽分子通过使用偶联试剂HBTU PEG的游离…

Discussion

步骤1.3，1.4和1.7中的协议，应广泛护理和在一个非常温和的方式进行。在步骤1.3中，将尖端不应该在用硅烷混合物和硅烷化过程中应在惰性气氛（无水分）进行接触。 45这样做是为了防止多层形成和因硅烷分子容易进行水解在水分存在完成。 45

在步骤1.4中，温度和时间应妥善保管。开始步骤1.5之前，尖端应冷却到室温;否则会被损坏。在偶?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Marie Curie International Reintegration Grant (EP7). P. D. acknowledges the support of the Israel Council for Higher Education.

Materials

Silicon nitride (Si3N4) AFM cantilevers with silicon tips Bruker (Camarilo, CA, USA) MSNL10, nominal cantilevers radius ~2 nm 
Methyltriethoxysilane  Acros Organics (New Jersey, USA) For Silaylation of the AFM tip 
3-(Aminopropyl) triethoxysilane Sigma-Aldrich (Jerusalem, Israel) Used for tip modification 
Triisopropylsilane Sigma-Aldrich (Jerusalem, Israel) Used for tip modification
N-Ethyldiisopropylamine Alfa-Aesar (Lancashire, UK) Used for tip modification
Triethylamine Alfa-Aesar (Lancashire, UK) Used for tip modification
Piperidine Alfa-Aesar (Lancashire, UK) Used for tip modification
Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide  (Fmoc-PEG-NHS) Iris Biotech GmbH (Deutschland, Germany) Used as the covalent flexible linker  (MW = 5000 Da)
2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) Alfa Aser (Heysham, England) Used as a coupling reagent. 
N-methyl-2-pyrrolidone (NMP) Acros Organics (New Jersey, USA) Used as Solvent in Tip modification procedure
DMF (dimethylformamide) Merck (Darmstadt, Germany) Used as Solvent in Tip modification procedure
Trifluoro acetic acid (TFA) Merck (Darmstadt, Germany)
Acetic anhydride Merck (Darmstadt, Germany)
Peptides GL Biochem (Shanghai, China).
Phenylalanine and Tyrosine  Biochem (Darmstadt, Germany) 
30% TiO2 dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP) Applied Vision Laboratories (Jerusalem, Israel) (30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)
Mica substrates TED PELLA, INC. (Redding, California, USA) 9.9 mm diameter
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Tags

Single-molecule Force SpectroscopyAmino AcidsPeptidesInorganic MaterialsSurface ChemistryCompositesChordatesAFM CantileversSilane CompoundsFmoc-PEG-NHSPiperidineN-Methyl-2-pyrrolidoneAmino Acid Coupling

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Das, P., Duanias-Assaf, T., Reches, M. Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy. J. Vis. Exp. (121), e54975, doi:10.3791/54975 (2017).
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