用于质谱光度法的表面钝化与功能化
Surface passivation and functionalisation for mass photometry.
作者信息
Sülzle Jenny, Elfeky Laila, Manley Suliana
机构信息
Laboratory of Experimental Biophysics (LEB), Institute of Physics and Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
出版信息
J Microsc. 2024 Jul;295(1):14-20. doi: 10.1111/jmi.13302. Epub 2024 Apr 12.
Interferometric scattering (iSCAT) microscopy enables the label-free observation of biomolecules. Consequently, single-particle imaging and tracking with the iSCAT-based method known as mass photometry (MP) is a growing area of study. However, establishing reliable cover glass passivation and functionalisation methods is crucial to reduce nonspecific binding and prepare surfaces for in vitro single-molecule binding experiments. Existing protocols for fluorescence microscopy can contain strongly scattering or mobile components, which make them impractical for MP-based microscopy. In this study, we characterise several different surface coatings using MP. We present approaches for cover glass passivation using 3-aminopropyltriethoxysilane (APTES) and polyethylene glycol (PEG, 2k) along with functionalisation via a maleimide-thiol linker. These coatings are compatible with water or salt buffers, and show low background scattering; thus, we are able to measure proteins as small as 60 kDa. In this technical note, we offer a surface preparation suitable for in vitro experiments with MP.
干涉散射(iSCAT)显微镜能够对生物分子进行无标记观察。因此,基于iSCAT的称为质量光度法(MP)的单粒子成像和跟踪是一个不断发展的研究领域。然而,建立可靠的盖玻片钝化和功能化方法对于减少非特异性结合并为体外单分子结合实验准备表面至关重要。现有的荧光显微镜实验方案可能包含强散射或可移动成分,这使得它们不适用于基于MP的显微镜。在本研究中,我们使用MP对几种不同的表面涂层进行了表征。我们提出了使用3-氨丙基三乙氧基硅烷(APTES)和聚乙二醇(PEG,2k)进行盖玻片钝化以及通过马来酰亚胺-硫醇连接体进行功能化的方法。这些涂层与水或盐缓冲液兼容,并显示出低背景散射;因此,我们能够测量低至60 kDa的蛋白质。在本技术说明中,我们提供了一种适用于MP体外实验的表面制备方法。