Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation

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Amyloid growth, inhibition and real-time enzymatic degradation revealed with single conical nanopore Nicoletta Giamblanco, Diego Coglitore, Alberto Gubbiotti, Tianji Ma, Emmanuel Balanzat, Jean-Marc Janot, Mauro Chinappi, and Sebastien Balme Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03523 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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Analytical Chemistry

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Amyloid growth, inhibition and real-time enzymatic

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degradation revealed with single conical nanopore

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Nicoletta Giamblanco1, Diego Coglitore1, Alberto Gubbiotti2, Tianji Ma1, Emmanuel Balanzat3,

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Jean-Marc Janot1, Mauro Chinappi4, Sebastien Balme1*

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1

Institut Européen des Membranes, UMR5635 université de Montpellier ENSCM CNRS-, Place

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Eugène Bataillon, 34095 Montpellier cedex 5, France 2

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Dipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma , Via

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Eudossiana 18, 00184 Roma, Italia. 3

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Centre de recherche sur les Ions, les Matériaux et la Photonique, UMR6252 CEA-CNRSENSICAEN, 6 Boulevard du Maréchal Juin, 14050 Caen Cedex 4, France

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Dipartmento di Ingegneria Industriale, Università di Roma Tor Vergata , Via del Politecnico 1,

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00133 Roma, Italia.

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*Correspondance to Sebastien Balme : [email protected]

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ABSTRACT

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Amyloid fibrils are involved in several neurodegenerative diseases. However due to their

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polymorphism and low concentration, they are challenging to assess in real-time with

ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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conventional techniques. Here, we present a new approach for the characterization of the

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intermediates: protofibrils and “end-off” aggregates which are produced during the amyloid

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formation. To do so, we have fashion conical track-etched nanopores functionalized to prevent

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the fouling. Using these nanopore we have follow the kinetic of amyloid growth discriminated

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the different intermediates protofibrils and “end-off. Then, nanopore was used to characterize the

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effect of promoter and inhibitor of fibrillation process. Finally, we have followed in real time the

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degradation of amyloid with peptase. Compare to SiN nanopore, the track-etched one features

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exceptional high success rate via functionalization and detection operated “one-pot”. Our results

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demonstrate the potential for a conical nanopore to be used as a routine technique for the

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characterization of the amyloid growth and/or degradation.

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Keywords : amyloid, nanopore, track-etched


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1. INTRODUCTION

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Protein misfolding and aggregates cause numerous physiological disorders. Among the various

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structures of protein aggregates, the amyloids are involved in many age-related degenerative

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diseases including Alzheimer's, Parkinson's, type II diabetes and cataract1–3. The mechanism of

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fibrillation involves several intermediate steps from the less ordered molecular arrangement to

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the highest as follows: at first the misfolded protein, then the aggregates and the protofibril, and

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finally the fibril

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parallel pathways, in which low concentrations structurally heterogeneous and intermediates

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(aggregates and protofibrils) are generated3–5. Diagnosis of diseases is usually associated with

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the amyloid fibril. However, the intermediates (the aggregates and protofibrils) are known to be

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the most toxic entities6. Thus all intermediates which co-exist during the fibrillation process have

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to be characterize 5.

1,4

. The amyloid fibrillogenesis is a complex process, characterized by multiple

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Significant advances on the understanding of fibrillogenesis were achieved by usual methods2.

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The characterization of the amyloid size, shape and the co-existence of different intermediates

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are only allowed by ex-situ analysis using Cryo Transmission Electron Microscopy (Cryo-TEM)

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or atomic force microscopy (AFM)7. The characterization the amyloid growth can be performed

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using intercalated agents (ThioflavineT or congo red), diffusion light scattering (DLS),

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fluorescence anisotropy, fluorescence correlation spectroscopy (FCS) and the increase of β-sheet

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structures using circular dichroism (CD)8–10. Despite the effort to optimize the sample

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preparation and the characterization methods, the main challenge is to propose the real-time

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monitoring of the amyloid growth, the morphology and the heterogeneity of the intermediates

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(i.e “end-off” aggregate and protofibrils) with a minimum of sample preparation steps to

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overcome the limits of commonly used techniques11. To reach this goal, we propose to use solid-


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state nanopore12–14 technology that is a versatile and label-free method for single molecule

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detection. It allows the determination of DNA sub-structure15, protein shape16, conformational

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changes17,18 and unfolding19. Protein oligomers have been detected by glass nanopipette20, raw21–

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23

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functionalization and the filling with buffer solution, make it not the proper tool for the amyloid

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investigation. Only one approach using nanopores has allowed characterizing the different

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intermediate of αβ-peptide aggregates. It consists to coat a SiN nanopore with a phospholipid

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bilayer in order to avoid the adsorption and the nanopore fouling24. This elegant strategy is

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nevertheless limited by the time resolution for small aggregate due to their fast translocation. In

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addition the coating with phospholipid have to be performed before each experiment26.

and functionalized SiN24–26. However for SiN, its short lifetime27, the difficulties in its

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This study aims to provide a new analysis tool to characterize the amyloid growth able to

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differentiate “end-off” aggregate and protofibrils. To do so we have considered the conical

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nanopore obtained by track-etched technic functionalized with PEG to prevent the fouling

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(Figure SI-1). This strategy was chosen to tackle the main problems of SiN nanopore (short

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lifetime and the low resolution of protein monomer). The β-lactoglobulin amyloids were

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characterized using the usual diffusion light scattering (DLS) and circular dichroism (CD)

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methods. The suitability of our method was evaluated to characterize the amyloid growth under

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different condition such as presence of promotor or inhibitor as well as to follow the enzymatic

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degradation.

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2. RESULTS AND DISCUSSION

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2.1. Conical Nanopore design and functionalization

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We produced conical nanopores obtained by single track irradiation followed by a chemical

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etching by electrostopping technique13,28. After the chemical etching, the current-voltage (I-V)


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curve was measured at pH 6.0 and 2.7. At pH 6.0 The I–V curves recorded (KCl 1 M) are not

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linear (Figure SI-1 and SI-2) due to the negative charge (pKa 3.8) of the carboxylate groups

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induced by the chemical etching process. At pH 2.6, the wall of the PET conical pore becomes

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weakly positively charged, resulting in an inversion of ionic current rectification. The nanopore

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was grafted inside with PEG 5kD for its antifouling properties to prevent non-specific adsorption

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of protein. The success of the grafting was confirmed by the loss of the ionic current

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rectification, and the lower conductance values at low salt concentration due to the replacement

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of COO- by the PEG which diminished surface charge of the nanopore. For the different

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nanopores, a thickness of the PEG layer were estimated about 3±1.5 nm from conductance

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decrease which suggests a mushroom conformation25. This agrees with the low density of

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carboxylate groups reported for PET nanopore (~ 1e nm-2)29. The geometrical properties of

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nanopore used in this study are reported on Table SI-1.

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2.2. Amyloids detection and characterization by conical nanopore

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The β-lactoglobulin amyloids were produced by incubation at pH 2, 70°C, NaCl 150 mM.

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Having confirmed the formation of β-lactoglobulin amyloids, at different heating times, i.e. 0

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min, 3 h, 6 h, 16 h, 24 h, 34 h, by diffusion light scattering (DLS) (Figure SI-3) and atomic force

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microscopy (AFM) (Figure SI-4) we investigated their translocation by resistive pulse

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method30,31. We performed the detection from the base to the tip side of the nanopore to ensure

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the capture of protofibril. Indeed this strategy allows to prevent the fouling by the protofibrils

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larger than the nanopore tip diameter (Figure SI-1). We also chose to perform the experiments at

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1M KCl and pH 2.7 which avoid the current rectification in conical nanopore. In the absence of

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amyloids, no current-pulse events were observed in the PEG-functionalized nanopore. After the

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addition of the β-lactoglobulin amyloid, (800 ± 70 nM monomeric concentrations) resulted after


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incubating for 24h, we observed the characteristic current blockades in all the nanopores. The

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events are observed only when the amyloid sample is present (Figure SI-5). After every

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experiments, the nanopore was rinsed with water until the current was constant and stable (no

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current blockade occurred), so that it could be re-used for other amyloid sensing experiments.

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The histograms of the relative current ∆I/I0 (Figure 1, Table SI-2) reveal two populations

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centered on 0.039, 0.061 for NP1 (117.5 nm base, 12.1 nm tip), 0.017, 0.022 for NP2 (300 nm

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base, 3.8 nm tip) and 0.065, 0.11 for smallest nanopore NP3 (92.5 nm base, 2.7 nm tip). The two

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populations can be also seen in an AFM micrograph (Figure SI-4) and are likely to correspond to

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the two distributions of ∆I/I0. The mechanism of β-lactoglobulin fibrillogenesis is complex. The

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intermediates of partially unfolded proteins can form aggregates leading to fibrils or low

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molecular weight, “dead-end” species, which hinder the fibrils formation4.

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For the dwell time (Figure SI-5), the distribution histograms are fitted to a single exponential

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with mean time 16.8 ± 0.32 ms for NP1 at a biased voltage of -200 mV. At the same voltage the

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dwell time is 3 times slower (51.2 ± 1.65 ms) for NP2. For NP3 at -150 mV the mean time is

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127.3 ± 9.97 ms. For smaller nanopores (NP2 and NP3) the translocation was found to be slower.

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This can be explained by the increase of escape energy due to the confinement32,33.

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We were detected the β-lactoglobulin monomer through the NP3. The ∆I/I0 distribution is

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narrow, monomodal and centered on 0.034 confirming the homogeneity of the sample (Figure

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SI-6). The dwell time distributions are fitted to a single exponential with mean time 56.3 ms

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which is faster than the amyloid (127 ms). Interestingly, monomers and amyloids can be

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discriminated in the events map (Figure 1d).

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Figure 1. Translocation of protein fibrils trough conical nanopore. (a) Histograms of relative

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current blockade (∆I/I0) induced by β -lactoglobulin amyloid (prepared from 5.4 µM in 150 mM

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NaCl pH 2.0, 24 h at 70°C) concentration 800 ± 70 nM nM in 1M KCl at pH 2.6. The fibrils

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detection are performed with (a) NP1 (rt 12.1 nm and rB 117.5 nm) under -200 mV, (b) NP2

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nanopore (rt 3.8 nm, rB 300 nm) under -200 mV and (c) NP3 (rt 2.7 nm, rB 92.5 nm) under -150

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mV applied potential. (d) Event map for native β-lactoglobulin monomer (pink) and amyloid

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prepared from 5.4 µM in 150 mM NaCl pH 2.0 24 h at 70°C (blue) detected with nanopore NP3

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(rtip 2.7 nm, rbase 92.5 nm). N = number of events for each pore during 300 s for NP1, 360 s for

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NP2 and 300 s for NP3.


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To go further, we modeled the expected range of relative current blockades. The pore is

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modeled as a truncated cone of length Lp , tip radius rT, base radius rB. The charges due to PEG

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grafting are neglected. The amyloid structure is described as a cylinder of radius ramy and length

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Lamy which translocates from the base to the tip side along the nanopore axis. This approximation

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does not take into account the possible differences in current blockade due to the amyloid

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orientation. However, for narrow pores (NP2, NP3 and NP4) and very small cone angle α, when

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the amyloid entered from the base side reaches the tip, it can explore only very small

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orientations. Under these assumptions, the total resistance of the pore portion between quote ξ

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and quote ξ can be calculated with a 1-D approximation:

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=

,

(1)

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where ξ is the coordinate along the pore axis, ξ is the area of the pore section available for

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the electrolyte and κ is the conductivity. When ξ1 and ξ2 are quotes of the two pore entrances,

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equation (1) provide the total resistance or the system where, due to the high aspect ratio of the

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nanopore, the access resistance is neglected. In the presence of the amyloid, the latter contributes

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to divide the total integral into the sum of three resistances in series, see figure 2a,

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= + +

(2)

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Indicated with x the position of the forward end of the amyloid, is the resistance of the

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portion of the pore from the narrow aperture until the position x, is the resistance

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corresponding to the pore region occupied by the amyloid and is the resistance of the

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nanopore between the end of the fibril and the wide aperture (see details on the SI section :

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Models for Relative current blockade).

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The maximum resistance occurs when the fibril is located at the nanopore tip = 0 . This resistance can be written as :


8

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=

!"#

= $ %

&!'

,-&!' ./ 0&!' 1,/ .&!' 1

log +,-

&!' ./ .&!' 1,/ 0&!' 1

-3 0-&!'

2 + , ./

&!' 14

5.

(3)

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Equation (3) allows to calculate the maximum relative current blockades as ∆6/68 = 1 −

;
and length ? = 2> .

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Using the geometrical parameter for NP3 and the experimental data

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obtained > = 2.12 ± 0.04 FG which is in good agreement with the structure of the β-

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lactoglobulin monomer4. Then, we used this value of ramy= 2 nm to calculate the maximum

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current blockade as a function of the amyloid length. The results are reported in figure 2b and,

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for NP3, are in good agreement with the observed current blockades. Indeed, the first distribution

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centered on 0.065 corresponds to aggregate smaller than 20 nm while the second distribution

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centered is assigned to larger protofibrils until 70 nm length. These values are consistent with the

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AFM characterization (Figure SI-4). On the contrary, for the NP1, our model is not suitable

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because the assumption of amyloid aligned with the nanopore axis fails. Indeed, due to the large

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tip radius, the amyloid can adopt different orientation also at the pore tip. In this case, the

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amyloid clogs a larger portion of the tip section and, consequently, a large blockade is expected.

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Hence, the incoherence between experiment and model when comparing NP2 and NP1, is not

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surprising. It should be noticed here that compared to solid-state nanopores, the size is not

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obtained by imaging but deduced by ionic current measurements. To calculate the tip diameter,

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we assume that the nanopore does not present irregularities. We used PET with biaxial

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orientation, which limits these irregularities but cannot guaranty their absence. This is the main

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problem of such PET nanopores.

ΔA A

Amyloid Growth, Inhibition, and Real-Time Enzymatic Degradation

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