在水中水合蛋白原位表征SALVI和TOF-SIMS
Summary
这项工作提出了液体处理和样品引入用于原位时间飞行二次离子质谱法的蛋白质的生物分子的水溶液中分析一个协议到一个微通道。
Abstract
使用分析系统在液体真空接口（SALVI）和时间飞行二次离子质谱法（TOF-SIMS）的水溶液中的蛋白质的生物分子的原位表征这项工作表明。纤连蛋白的蛋白质膜被固定在形成SALVI检测区域的氮化硅（SIN）膜。在TOF-SIMS分析中，进行了分析的三种模式包括高空间分辨率质谱，二维（2D）成像和深度剖析。质谱在积极和消极模式获得的。还分析去离子水作为参比样品。我们的结果表明，在水中的纤连蛋白膜具有单独与水相比更明显的和更强的水簇的峰。的氨基酸的片段的特征峰，可以在水合蛋白质TOF-SIMS光谱观察到。这些结果表明，在表面上的蛋白分子的吸附，可以研究动感中LLY使用SALVI和TOF-SIMS在首次的液体环境。
Introduction
水化是该结构，1构象，2和蛋白质的生物活性的3至关重要。他们周围的蛋白质没有水分子不会有可行的生物活性。具体地，水分子与表面和蛋白质的内部结构进行交互，和蛋白质的不同水合状态使这种相互作用是不同的。4蛋白与固体表面的相互作用是在纳米技术，生物材料和组织工程的过程影响的基本现象。研究早就表明，作为蛋白质遇到表面，可能会发生的构象变化。 TOF-SIMS已经设想作为具有研究蛋白质-固体界面的电位的技术。5-7理解在固体表面上，这可能提供了它们的结构的机构的基本理解蛋白的水合是重要的，构象和生物人的活动。
然而，主要的表面分析技术大多是基于真空和挥发性液体直接的研究应用，由于挥发性液体的真空环境下迅速蒸发是困难的。我们开发了一种真空兼容微流体接口，系统分析在液体真空接口（SALVI），以使液体表面，并使用时间飞行二次离子质谱法（TOF-SIMS）液-固相互作用的直接观察。8- 11独特的方面包括以下内容：1）的检测窗的直径为使液体表面的直接成像的2-3微米的孔，2）表面张力被用于保持所述孔中的液体，以及3）SALVI是在多个平台上的分析的便携式11,12
SALVI由氮化硅（SIN）膜作为检测区域，并提出聚二甲基硅氧烷（PDMS）的微通道。这是FABRicated在洁净室，并在制造和关键设计因素已在先前的论文和专利被详细描述。8-12 TOF-SIMS的作为分析工具中的应用进行了使用各种水溶液和复杂液态混合物，其中一些的证明含有纳米颗粒。13-17具体地说，SALVI液体TOF-SIMS允许动态活生物系统 （即生物膜），单细胞，和固体电解质界面的液-固界面的探测，原位冷凝阶段打开新的机会研究包括使用TOF-SIMS液体。然而，目前的设计不允许气液相互作用爱好。这是未来发展的一个方向。 SALVI已被用于研究水合蛋白质膜在这项工作中，第一次。
纤连蛋白是一种常用的蛋白二聚体，由18由一对的二硫键连接的两个几乎相同的单体，的我旗下著名其结合细胞的能力。它19,20被选择作为模型系统来说明水合蛋白薄膜可以使用SALVI液体TOF-SIMS方法来探测动态。该蛋白质溶液引入到微通道。 12小时温育后，形成在SiN膜的背面侧水合蛋白质膜。去离子水是用来冲洗掉蛋白质出台后的通道。信息来自于使用动态TOF-SIMS的SALVI微纤连蛋白水合蛋白质分子收集。 DI水还研究了作为控制与水合纤维连接蛋白薄膜得到的结果进行比较。水合蛋白膜和DI水之间观察明显的差异。这项工作表明，关于在液体环境表面蛋白吸附可以使用该新型SALVI和液体TOF-SIMS方法进行研究。该视频协议的目的是人谁是感兴趣的人士提供技术指导在利用与TOF-SIMS SALVI的各种应用此新的分析工具，并减少在液体处理不必要的错误以及TOF-SIMS数据采集和分析。
Protocol
Representative Results
Discussion
SALVI是微流体接口，允许动态液面与液 – 固界面的分析通过基于真空手段，如TOF-SIMS和扫描电子显微镜（SEM）。由于小孔的使用情况，以直接在真空暴露液体，SALVI是适用于许多精密聚焦光谱和成像技术没有任何修改; 22可移植性和微流体的多功能性使之成为真正的多模成像平台。不同的特性和相对于其他现有技术SALVI的意义归纳在最近几个评价22-25作为SALVI技术的开发者，我们小组?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.
Materials
| ToF-SIMS | IONTOF | TOF.SIMS 5 | Resolution: > 10,000 m/Δm for mass resolution; > 4,000 m/Δm for high spatial resolution |
| System for Analysis at the Liquid Vacuum Interface (SALVI) | Pacific Northwest National Laboratory | N/A | SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level. |
| PEEK Union | Valco | ZU1TPK | for connecting the inlet and outlet of SALVI |
| 5 Axes Sample Stage | IONTOF | N/A | Stage is self-made for mounting SALVI in ToF-SIMS |
| Barnstead Nanopure Water Purification System | Thermo Fisher Scientific | D11921 | ROpure LP Reverse Osmosis filtration module (D2716) |
| Syringe | BD | 309659 | 1 mL |
| Pipette | Thermo Fisher Scientific | 21-377-821 | Range: 100 to 1000 mL |
| Pipette Tip | Neptune | 2112.96.BS | 1000 µL |
| Centrifuge Tube | Corning | 430791 | 15 mL |
| Fibronectin | Sigma-Aldrich | F1141 | 1 mg/mL |
| Ethanol | Thermo Fisher Scientific | S25310A | 95% Denatured |
| Gibco PBS | Thermo Fisher Scientific | 10010-023 | pH 7.4 |
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