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.

This paper describes the analysis of pore formation and detection of a single protein molecule using a large nanopore among five different pore-forming proteins. We demonstrate that the identification of appropriate pores for nanopore sensing can be achieved by classifying the channel current signals and performing noise analysis. Through these analyses, we selected a perforin nanopore from the membrane attack complex/perforin superfamily and attempted to use it to detect the granzyme B protein, a serine protease. As a result, we found that granzyme B might pass through the perforin nanopore if it adopts an unfolded structure. Our proposed analytical approach should be useful for exploring several types of nanopore as large biological nanopores other than α-hemolysin.

from β-lactoglobulin show several interesting features, such as the high aspect ratio (length vs diameter) making them a structuring agent in foods, stabilizers of foam and in emulsions as well as building blocks of microcapsules. [8] The applications of β-lactoglobulin fibrils in food products require a deep understanding of fibril digestion processes occurring during passage through the stomach and the intestine. [9] Indeed, certain amyloids such as tau, α-synuclein or β-peptides are involved in neurodegenerative diseases. [10] The protein fibril digestion can cleave the fibrils to oligomers with a potential increased pathogenic [11,12] or contribute to the formation of new fibrils as seeds. [13] To date, the investigation of the nondisease-related amyloid-like fibrils degradation combining kinetic and identification of species are difficult due to the difficulty to obtain both information using the same technique. Nanopore technology is one of the most promising approaches to protein sensing. [14,15] It allows single molecule detection providing information about the size, shape as well as charge. [16,17,18] Nanopores are also a platform to characterize the protein conformational change including folding/ unfolding. [19] Biological nanopores take advantage of their precise structure, allowing the characterization of polypeptide length and the identification of amino-acids, [20] opening a new avenue for protein sequencing. [21,22] On the other hand, they were used to detect small oligomers of peptides [23] and antibody prion interactions. [24] Solid-state nanopores and nanopipettes allow the characterization of the protein and peptide oligomerization giving also information on their morphology. [25] This makes them a candidate to develop new analytical tools for amyloid sensing. [26] However, their diameter and their short lifetime don't allow protofibril detection nor long time analysis. We recently reported that conical track-etched nanopore is likely the most suitable candidate to investigate the protein aggregation from small aggregates to protofibrils scale. [27] In this report, β-lactoglobulin aggregation was studied, discriminating between oligomer and protofibril populations. In addition, the long nanopore lifetime and the possibility to perform long time experiments, is suitable to analyze the digestion of β-lactoglobulin aggregates. The most relevant physiological proteases in digestion process are pepsin (stomach) and trypsin (small intestine). β-lactoglobulin aggregates are used as structuring agents in food processing. Their enzymatic degradation follows different pathways depending on the enzyme and the pH. The transient species produced during the degradation process are difficult to characterize under continuous measurement. Here, conical track-etched nanopores are used to investigate the β-lactoglobulin degradation by pepsin (pH 2) and trypsin (pH 9). Before enzyme addition, two distinct populations, oligomers and protofibrils, are identified. Just after enzyme addition, the aggregate size decr...

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