Institut Européen des Membranes, UMR5635 UM ENCSM CNRS, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
INM UM, CNRS, INSERM, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.
Anal Chem. 2023 Aug 29;95(34):12623-12630. doi: 10.1021/acs.analchem.3c00017. Epub 2023 Aug 16.
In this work, early-stage Aβ42 aggregates were detected using a real-time fast amyloid seeding and translocation (RT-FAST) assay. Specifically, Aβ42 monomers were incubated in buffer solution with and without preformed Aβ42 seeds in a quartz nanopipette coated with L-DOPA. Then, formed Aβ42 aggregates were analyzed on flyby resistive pulse sensing at various incubation time points. Aβ42 aggregates were detected only in the sample with Aβ42 seeds after 180 min of incubation, giving an on/off readout of the presence of preformed seeds. Moreover, this RT-FAST assay could detect preformed seeds spiked in 4% cerebrospinal fluid/buffer solution. However, in this condition, the time to detect the first aggregates was increased. Analysis of Cy3-labeled Aβ42 monomer adsorption on a quartz substrate after L-DOPA coating by confocal fluorescence spectroscopy and molecular dynamics simulation showed the huge influence of Aβ42 adsorption on the aggregation process.
在这项工作中，使用实时快速淀粉样蛋白成核和转位（RT-FAST）测定法检测了早期 Aβ42 聚集物。具体而言，将 Aβ42 单体在含有和不含有预先形成的 Aβ42 种子的缓冲液中孵育，在涂有 L-DOPA 的石英纳米管中孵育。然后，在各种孵育时间点上通过 flyby 电阻脉冲感应分析形成的 Aβ42 聚集物。仅在孵育 180 分钟后含有 Aβ42 种子的样品中检测到 Aβ42 聚集物，给出了存在预先形成的种子的开/关读出。此外，该 RT-FAST 测定法可以检测到在 4％脑脊液/缓冲液中添加的预先形成的种子。但是，在这种情况下，检测到第一个聚集物的时间增加了。通过共焦荧光光谱和分子动力学模拟分析 L-DOPA 涂覆后石英基质上 Cy3 标记的 Aβ42 单体的吸附，表明 Aβ42 吸附对聚集过程有巨大影响。
