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

Abstract

Solid‐state nanopores are an emerging technology used as a high‐throughput, label‐free analytical method for the characterization of protein aggregation in an aqueous solution. In this work, we used Levodopamine to coat a silicon nitride nanopore surface that was fabricated through a dielectric breakdown in order to reduce the unspecific adsorption. The coating of inner nanopore wall by investigation of the translocation of heparin. The functionalized nanopore was used to investigate the aggregation of amyloid‐β and α‐synuclein, two biomarkers of degenerative diseases. In the first application, we demonstrate that the α‐synuclein WT is more prone to form dimers than the variant A53T. In the second one, we show for the Aβ(42)‐E22Δ (Osaka mutant) that the addition of Aβ(42)‐WT monomers increases the polymorphism of oligomers, while the incubation with Aβ(42)‐WT fibrils generates larger aggregates.

Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution

“…We propose that one attractive approach to address this need is to monitor aggregation using nanopores. − This approach takes advantage of a transient increase in electrical resistance when particles move through electrolyte-filled nanopores, a mode of detection that does not require labeling of particles. ,,,,, Three types of nanopores are commonly employed for the detection of oligomers of amyloid-forming proteins: First are protein nanopores (so-called biological nanopores), which have the advantage that they can be produced with exquisite reproducibility and that they typically do not clog during measurements, while having the disadvantage that, thus far, their pore diameters are typically smaller than 5 nm, limiting them to the detection of monomers or the smallest oligomers of amyloid-forming proteins. , Second, nanopores at the tip of glass capillaries or in polymer films have the advantage that they can be readily produced, while their conical and elongated pore shape hinders the estimation of particle shape. ,, And third, nanopores in solid-state SiNx membranes have the advantage that they can be fabricated with a large range in diameters in order to adjust it to the size of oligomers of interest. , Possible disadvantages of these pores are that their fabrication is typically a serial process, that it requires specialized equipment, that their diameters tend to grow during experiments due to slow etching in aqueous electrolyte, and that coatings must be employed to minimize nonspecific interactions of proteins with the walls of the nanopore. ,− So far, most studies using nanopores in the context of analyzing amyloid oligomers focused either on the detection of oligomers or on monitoring the aggregation process over time; typically these studies could not determine the size of amyloid oligomers, and even fewer studies were able to interrogate oligomer shapes. ,,− For reviews of progress in this field, please see Houghtaling et al, Meyer et al, and Chen et al, as well as recent reports. ,,,,, In addition, nanopore-based methods are also being used to follow ligand-induced conformational changes in proteins and to study the enzymatic process on a single-molecule level. , …”

“…42,44,49−51 For reviews of progress in this field, please see Houghtaling et al, 49 Meyer et al, 52 and Chen et al, 58 as well as recent reports. 43,44,46,47,59,60 In addition, nanopore-based methods are also being used to follow ligand-induced conformational changes in proteins 61 and to study the enzymatic process 62 on a single-molecule level. 63,64 Here, we apply nanopore-based resistive pulse recordings to the single-particle characterization of oligomers of αSyn and reveal the size and shape of αSyn oligomers.…”

Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution

“…We suggest that one attractive approach to address this need may be to monitor aggregation using nanopores. [36][37][38][39][40][41][42][43][44] So far most studies using nanopores focused on either detecting protein aggregates or on following the aggregation process over time. 37,39,44 For instance, we have used nanopores to characterize amyloid-β (Aβ-40 and Aβ-42) aggregates to differentiate different aggregates including oligomers, protofibrils, and fibrils.…”

“…We propose that one attractive approach to address this need is to monitor aggregation using nanopores. [38][39][40][41][42][43][44][45][46] So far, most studies using nanopores focused on either the detection of particles or on monitoring the aggregation process over time, typically these studies did not determine the size of amyloid oligomers and even fever studies interrogated oligomer shapes. 39,41,[46][47][48] For a review of progress in this field, please see Houghtaling et al 46 as well as recent reports.…”

“…39,41,[46][47][48] For a review of progress in this field, please see Houghtaling et al 46 as well as recent reports. 40,41,43,44,[49][50][51][52][53] Here, we apply this approach to single-particle characterization of oligomers of αSyn and reveal their size and shape. We demonstrate that the size distribution of αSyn oligomers contains ten sub-populations of oligomers with distinct and stable number of aggregated monomers.…”