Wogelred Louise, Höök Fredrik, Agnarsson Björn, Sjövall Peter
RISE Research Institutes of Sweden, P.O. Box 857, SE-50115 Borås, Sweden and Department of Physics, Chalmers University of Technology, SE-41296 Göteborg, Sweden.
Department of Physics, Chalmers University of Technology, SE-41296 Göteborg, Sweden.
Biointerphases. 2018 Mar 15;13(3):03B413. doi: 10.1116/1.5019749.
Accurate detection and quantification of individual molecules is important for the development of improved diagnostic methods as well as biochemical characterization of disease progression and treatments. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface analysis technique capable of imaging the distribution of specific molecules on surfaces with a high spatial resolution (<1 μm) and high sensitivity. ToF-SIMS is particularly suitable for detection of molecules up to ∼2 kDa, including lipids, whereas larger molecules, such as peptides and proteins, are fragmented during analysis, which makes them difficult to identify. In this study, an approach for extending the molecular detection capability of ToF-SIMS is presented, based on the specific binding of functionalized liposomes to molecular targets on the sample surface and subsequent detection of the liposomes by ToF-SIMS. Furthermore, by using different recognition elements conjugated to liposomes with different lipid compositions, simultaneous detection of different targets was accomplished. This multiplexing capability was investigated for two types of recognition elements (antibodies and cholera toxin) and for target molecules immobilized on surfaces using two frequently applied surface functionalization strategies: a supported lipid bilayer aimed to mimic a cell membrane and a polyethylene glycol modified surface, commonly employed in bioanalytical sensor applications. The efficacy of the conjugation protocols and the specificity of the recognition mechanism were confirmed using quartz crystal microbalance with dissipation monitoring, while fluorescence microscopy was used to validate the ToF-SIMS data and the reliability of the freeze-drying step required for ToF-SIMS analysis. The results demonstrated specific binding of the two types of liposomes to each target and showed a concentration-dependent binding to the targets on the different model surfaces. In particular, the possibility to use the contrasts in the mass spectra of SIMS to identify the concentration dependent coverage of different liposomes opens up new opportunities for multiplexed detection and quantification of molecules at biotechnology relevant interfaces.
准确检测和定量单个分子对于改进诊断方法的开发以及疾病进展和治疗的生化特征分析至关重要。飞行时间二次离子质谱（ToF-SIMS）是一种表面分析技术，能够以高空间分辨率（<1μm）和高灵敏度对表面特定分子的分布进行成像。ToF-SIMS特别适用于检测分子量高达约2 kDa的分子，包括脂质，而较大的分子，如肽和蛋白质，在分析过程中会碎片化，这使得它们难以识别。在本研究中，提出了一种扩展ToF-SIMS分子检测能力的方法，该方法基于功能化脂质体与样品表面分子靶标的特异性结合，随后通过ToF-SIMS检测脂质体。此外，通过使用与具有不同脂质组成的脂质体偶联的不同识别元件，实现了对不同靶标的同时检测。针对两种类型的识别元件（抗体和霍乱毒素）以及使用两种常用的表面功能化策略固定在表面上的靶标分子，研究了这种多重检测能力：一种旨在模拟细胞膜的支持脂质双层和一种聚乙二醇修饰的表面，常用于生物分析传感器应用。使用具有耗散监测功能的石英晶体微天平确认了偶联方案的有效性和识别机制的特异性，同时使用荧光显微镜验证了ToF-SIMS数据以及ToF-SIMS分析所需的冻干步骤的可靠性。结果表明两种类型的脂质体与每个靶标均有特异性结合，并显示出与不同模型表面上的靶标呈浓度依赖性结合。特别是，利用SIMS质谱中的对比度来识别不同脂质体的浓度依赖性覆盖的可能性，为在生物技术相关界面上对分子进行多重检测和定量开辟了新机会。
