Nanogel surface coatings for improved single-molecule imaging substrates
Nanogel surface coatings for improved single-molecule imaging substrates
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DOI：
10.1098/rsif.2010.0669
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发表时间：
2011-10-07
影响因子：
3.9
通讯作者：
Mitra, Robi D.
中科院分区：
文献类型：
--
作者：
Tessler, Lee A.;Donahoe, Casey D.;Mitra, Robi D.
抗蛋白质吸附的表面对于许多生物分析应用是重要的。牛血清白蛋白(BSA)涂层和多臂聚乙二醇(PEG)涂层表现出低水平的非特异性蛋白质吸附，并使高定量的单分子(SM)蛋白质研究成为可能。最近，一种用聚乙二醇-牛血清白蛋白纳米凝胶涂覆玻璃的方法被开发出来，这种纳米凝胶是这两种低背景涂层的一种很有前途的混合物。我们对纳米凝胶涂层进行了表征，以确定其是否适合SM蛋白质实验。SM吸附计数表明，与共价偶联的BSA表面和多臂聚乙二醇单分子膜相比，纳米凝胶涂层表面对蛋白质的吸附能力较低，这是迄今观察到的蛋白质吸附程度最低的表面之一。此外，纳米凝胶涂层能够抵抗DNA吸附，这突显了该涂层在各种SM实验中的实用性。纳米凝胶涂层与表面活性剂相容，而牛血清白蛋白涂层不相容。最后，将涂层应用到真实世界的研究中，我们发现单配体分子可以被拴在这个表面上，并通过数字免疫分析以高灵敏度和高特异性进行检测。这些结果表明，聚乙二醇-牛血清白蛋白纳米凝胶涂层在蛋白质的SM分析中具有很高的应用价值。
Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG-BSA nanogel coatings will be highly useful for the SM analysis of proteins.