Biophysics, Institute of Molecular Biosciences (IMB), NAWI Graz, University of Graz, Humboldtstr. 50/III, Graz 8010, Austria.
Field of Excellence BioHealth, University of Graz, Graz 8010, Austria.
Anal Chem. 2023 Jan 17;95(2):587-593. doi: 10.1021/acs.analchem.2c03168. Epub 2022 Dec 27.
Microfluidic diffusional sizing (MDS) is a recent and powerful method for determining the hydrodynamic sizes and interactions of biomolecules and nanoparticles. A major benefit of MDS is that it can report the size of a fluorescently labeled target even in mixtures with complex, unpurified samples. However, a limitation of MDS is that the target itself has to be purified and covalently labeled with a fluorescent dye. Such covalent labeling is not suitable for crude extracts such as native nanodiscs directly obtained from cellular membranes. In this study, we introduce fluorescent universal lipid labeling for MDS (FULL-MDS) as a sparse, noncovalent labeling method for determining particle size. We first demonstrate that the inexpensive and well-characterized fluorophore, Nile blue, spontaneously partitions into lipid nanoparticles without disrupting their structure. We then highlight the key advantage of FULL-MDS by showing that it yields robust size information on lipid nanoparticles in crude cell extracts that are not amenable to other sizing methods. Furthermore, even for synthetic nanodiscs, FULL-MDS is faster, cheaper, and simpler than existing labeling schemes.
微流控扩散尺寸分析（MDS）是一种最近发展起来的强大方法，可用于确定生物分子和纳米颗粒的流体动力学尺寸和相互作用。MDS 的一个主要优点是，即使在与复杂、未纯化的样品混合的情况下，它也可以报告荧光标记靶标的大小。然而，MDS 的一个限制是，目标本身必须经过纯化，并与荧光染料共价标记。这种共价标记不适合直接从细胞膜获得的天然纳米盘等粗提取物。在本研究中，我们引入了用于 MDS 的荧光通用脂质标记（FULL-MDS）作为一种稀疏的、非共价标记方法，用于确定颗粒大小。我们首先证明，廉价且特征明确的荧光染料尼罗蓝会自发分配到脂质纳米颗粒中，而不会破坏其结构。然后，我们通过展示 FULL-MDS 可在其他尺寸测定方法不可行的粗细胞提取物中提供脂质纳米颗粒的稳健尺寸信息，突出了 FULL-MDS 的关键优势。此外，即使对于合成的纳米盘，FULL-MDS 也比现有的标记方案更快、更便宜、更简单。
