Real-Time Fast Amyloid Seeding and Translocation of α-Synuclein with a Nanopipette

Abstract

The detection to α-synuclein (αS) assemblies as a biomarker of synucleinopathies is an important challenge for further development of an early diagnosis tool. Here, we present proof of concept real-time fast amyloid seeding and translocation (RT-FAST) based on a nanopipette that combines in one unique system a reaction vessel to accelerate the seed amplification and nanopore sensor for single-molecule αS assembly detection. RT-FAST allows the detection of the presence αS seeds WT and A53T variant in a given sample in only 90 min by adding a low quantity (35 μL at 100 nM) of recombinant αS for amplification. It also shows cross-seeding aggregation by adding mixing seeds A53T with WT monomers. Finally, we establish the dependence between the capture rate of aggregates by the nanopore sensor and the initial seed concentration from 200 pM to 2 pM, which promises further development toward a quantitative analysis of the initial seed concentration.

Investigation of α‐Synuclein and Amyloid‐β(42)‐E22Δ Oligomers Using SiN Nanopore Functionalized with L‐Dopa

“…In addition, polymer nanopores were used to provide evidence for the promoting role of pyrimethanil for Aβ(42) aggregation during the lag‐phase [13] and Aβ(42) protofibril growth [14] . Recently, nanopipette‐type SSN was used to amplify the αS misfolding, thus opening a new opportunity to develop diagnosis tools for Parkinson's disease [15] …”

“…[14] Recently, nanopipette-type SSN was used to amplify the αS misfolding, thus opening a new opportunity to develop diagnosis tools for Parkinson's disease. [15] From an experimental point of view, raw Silicon base nanopore requires oxidative treatment by piranha, ozone or plasma to increase its wettability to enable its filling electrolyte solution. For protein measurements, this treatment, combined with their low concentrations, limits the interfacial concentration of protein at the equilibrium.…”

Fast Decoding of the First Steps of Protein Aggregation Using a Nanopipette

“…In this issue of ACS Central Science , Meyer et al reports on the development of new nanopipette reservoirs to detect at the single-molecule level the first steps of amyloid protein aggregation related to neurodegenerative pathologies. 1 Many diseases are caused by misfolding of proteins, which often lead to aggregate formation. 2 These diseases reduce life quality and expectancy.…”

“…Meyer et al pave the way to the early detection in vitro of α-synuclein (αS) assemblies with a nanopipette. 1 The team developed a real-time fast amyloid seeding and translocation (RT-FAST) technique to detect and quantify this neural presynaptic biomarker involved in Parkinson’s disease using nanopipettes and electrical detection. The study shows the detection of α-synuclein seeding in real time over 90 min, much faster than the traditional methods proposed (days scale).…”

“…(d) Distribution of the amplitude of the current blockade recorded for the sample seeded with 200 pM of α-synuclein A53T as a function of incubation time. Reproduced with permission from ref ( 1 ). Copyright 2022 The Authors.…”