Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation Revealed with Single Conical Nanopore

Abstract

Amyloid fibrils are involved in several neurodegenerative diseases. However, because of their polymorphism and low concentration, they are challenging to assess in real-time with conventional techniques. Here, we present a new approach for the characterization of the intermediates: protofibrils and "end-off" aggregates which are produced during the amyloid formation. To do so, we have fashioned conical track-etched nanopores that are functionalized to prevent the fouling. Using these nanopores, we have followed the kinetic of amyloid growth to discriminate the different intermediates protofibrils and "end-off. Then, the nanopore was used to characterize the effect of promoter and inhibitor of the fibrillation process. Finally, we have followed in real-time the degradation of amyloid with peptase. Compare with the SiN nanopore, the track-etched one features exceptionally high success rate via functionalization and detection in "one-pot". Our results demonstrate the potential for a conical nanopore to be used as a routine technique for the characterization of the amyloid growth and/or degradation.

Track‐Etched Nanopore/Membrane: From Fundamental to Applications

“…[ 262 ] The analysis of the formation of amyloid intermediates is achieved using PEG‐functionalized conical nanopores where PEG chains are used as antifouling coatings (Figure 16b). [ 258 ] By continuous translocating solutions containing initially only monomers, a change in event distributions is observed as shown in Figure 16c. The method allows detecting several populations of protein aggregates in the same sample.…”

“…Our group has developed continuous real‐time analysis of protein aggregation and their degradation based on PEG‐functionalized conical track‐etched nanopore. [ 258–260 ] It has been proved that amyloids are involved in many age‐related degenerative diseases, including Alzheimer's and Parkinson's by their misfolding and aggregation. [ 261 ] Figure a shows the aggregation process of amyloid proteins, including four steps among which the intermediates are the most toxic.…”

“…Reproduced with permission. [ 258 ] Copyright 2018, American Chemical Society. c) The histogram of relative current blockade for β‐lactoglobulin amyloids at 3, 6, 16, 24, and 34 h measured in a nanopore of D tip = 7.6 nm, D base = 600 nm, and L = 6 µm.…”

Surface coatings for solid-state nanopores

“…200,201 Other covalent surface modifications Nanopore surfaces that expose carboxyl groups are often coated by reaction with carbodiimide moieties for coupling molecules of interest. 123,127,237,238,[240][241][242][243][244][245][246][247]254 This technique works well on the walls of nanopores in polymer films and is robust and versatile, particularly for imparting specificity for detection of specific DNA strands or other biomolecules. 217,218,223,224,227,229,232,244,254 The same coupling chemistry has been employed to engineer nanopore systems that can be gated 210,212,[214][215][216]219,221,235,238,241,246 or to generate nanopore diodes.…”

“…213,214 Changes in pH resulted in changes in the resistance of the nanopore or, after functionalization of the biotin-PEG layer with the proteins avidin or streptavidin, this system detected biotinylated IgG, and biotinylated BSA. 213 Finally, the same group applied a PEG layer on the walls of nanopores in a PET film through linking to carboxylate groups on the pore surface to enable the detection of amyloids without clogging 247 or they attached PEG-spiropyrans to the same pores to generate a light-and pH-responsive nanopore. 246 Inspired by biological ion channels, Brunsen et al functionalized a mesoporous thin film of silica with polymer brushes composed of poly(methacryloyl ethylene phosphate) (PMEP) to modulate ion transport by changing pH.…”