Farzadfard Azad, Mason Thomas O, Kunka Antonin, Mohammad-Beigi Hossein, Bjerregaard-Andersen Kaare, Folke Jonas, Aznar Susana, Kallunki Pekka, Buell Alexander K
Protein Biophysics group, Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 227, 2800, Kgs., Lyngby.
H. Lundbeck A/S, Carl Jacobsens Vej 22, 2500, København, Denmark.
Angew Chem Int Ed Engl. 2025 Feb 10;64(7):e202419173. doi: 10.1002/anie.202419173. Epub 2025 Jan 2.
Seed amplification assays (SAAs) are a promising avenue for the early diagnosis of neurodegenerative diseases. However, when amplifying fibrils from patient-derived samples in multiwell plates, it is currently highly challenging to accurately quantify the aggregates. It is therefore desirable to transfer such assays into a digital format in microemulsion droplets to enable direct quantification of aggregate numbers. To achieve transfer from conventional plate-based to the microfluidic digital format, effective seed amplification needs to be achieved inside the microdroplets. Therefore, we establish a new set of assay conditions that enable highly efficient seed amplification in plates without any shaking. However, the same set of conditions displayed a very different behavior upon transfer to a microfluidic platform where no amplification was observed. We demonstrate that this is caused by the suppression of all secondary processes that could amplify the seeds in the complete absence of mechanical perturbations inside the microdroplets. We further show that the amplification inside droplets can be achieved by subjecting the microemulsions to high-frequency vibrations using a piezo device. Taken together, our results provide novel insights into the physical requirements of alpha-synuclein seed amplification and demonstrate a pathway towards the development of effective digital SAAs.
种子扩增分析（SAA）是神经退行性疾病早期诊断的一个有前景的途径。然而，当在多孔板中从患者来源的样本扩增纤维时，目前要准确量化聚集体极具挑战性。因此，期望将此类分析转化为微乳液液滴中的数字形式，以实现对聚集体数量的直接量化。为了实现从传统基于平板的形式向微流控数字形式的转化，需要在微液滴内部实现有效的种子扩增。因此，我们建立了一组新的分析条件，能够在无需任何振荡的平板中实现高效的种子扩增。然而，当转移到微流控平台时，相同的条件表现出非常不同的行为，在该平台上未观察到扩增。我们证明这是由于在微液滴内部完全没有机械扰动的情况下，所有可能扩增种子的二级过程受到抑制所致。我们进一步表明，通过使用压电装置使微乳液受到高频振动，可以在液滴内部实现扩增。综上所述，我们的结果为α-突触核蛋白种子扩增的物理要求提供了新的见解，并展示了开发有效的数字SAA的途径。
