WO2025120010A1

WO2025120010A1 - Ply nanopores

Info

Publication number: WO2025120010A1
Application number: PCT/EP2024/084743
Authority: WO
Inventors: Michael Mayer; Alessandro Ianiro; Anasua Mukhopadhyay; Wachara Chanakul; Yu-Noel LARPIN; Saurabh AWASTHI
Original assignee: Adolphe Merkle Institute University of Fribourg
Current assignee: Adolphe Merkle Institute University of Fribourg
Priority date: 2023-12-04
Filing date: 2024-12-04
Publication date: 2025-06-12
Anticipated expiration: 2026-06-04

Abstract

The invention relates to a nanopore-based sensing device. In one aspect of the invention, a nanopore-based sensing device comprises at least one membrane, wherein the membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode, the membrane comprising at least one nanopore comprising pneumolysin (PLY) monomers.

Description

PLY nanopores

Field of the invention

The invention relates to the field of nanopore-based sensing and sensing devices based on biological nanopores. These sensors can be used for analyzing biopolymers and nanoparticles.

Background

Sensing technology based on biological nanopores has become an increasingly important analytical tool as it allows the characterization of unlabeled synthetic and biological molecules such as polymers, DNA, proteins, peptides and small molecular analytes. Biological nanopore sensors typically consist of two electrolyte-filled compartments (called cis- and trans-compartments, respectively) separated by a phospholipid membrane bearing a single nanometric pore. When a potential difference is applied across the membrane, the electrolyte ions flow through the nanopore, resulting in a net ionic current. The translocation or entry of a (macro)molecule through the pore hinders the passage of ions, resulting in transient decrease of the ionic current. This signal, called resistive pulse, can be analyzed in real-time or offline to determine structural properties of the analyte.

If the nanopore diameter is larger than the largest dimension of the translocating molecule, resisting pulse sensing provides information on the volume and shape of the analyte. To reliably determine volume and shape, a protein has to sample different orientations while translocating through the nanopore. Biological nanopores employed in resistive pulse sensing are typically too small for volume or shape determination. The largest biological nanopore to date is a two-component pleurotolysin (PlyAB) toxin, which can form cylindrical pores with an internal diameter of 5.5 nm and length of 10 nm. Such a pore enables the detection of small folded proteins up to approximately 64.5 kDa, while translocation of larger proteins such as human albumin (HSA) and transferrin (HTr) necessitates specific engineering of the lumen of the PlyAB pore. Solid-state nanopores can be used as an alternative to biological nanopores for determining the volume and shape of folded proteins within a size range of approximately 50 to 970 kDa. Nevertheless, solid-state nanopores have several drawbacks. First, proteins tend to absorb onto the inner surface of the nanopore. Second, achieving uniformity in the manufacturing of solid-state nanopores is crucial for reliable detection yet is still challenging. Third, compared to biological nanopores, modifying the internal pore surface to optimize detection is not straightforward: the surface charge, which governs the nanofluidic properties of the pore, cannot be modified with atomic precision, and binding elements cannot be introduced with controlled stoichiometry. Fourth, solid-state nanopores often grow in diameter due to the slow etching of the material that bears the pore. This etching changes the shape of the pore as well as its size, thereby introducing uncertainty in the structural characterization of translocating macromolecules.

Because of the limitations of solid-state nanopores, the development of large biological nanopores enabling volume and shape determination of a wide range of proteins is crucial.

Objective technical problem to be solved

There is therefore a need in the art for large-diameter biological nanopores that can be used to determine volume and shape of proteins and other nanoparticles.

Summary of the invention

The problem is solved by a nanopore-based sensing device comprising at least one membrane, wherein the at least one membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode, the membrane comprising at least one nanopore comprising pneumolysin (PLY) monomers, wherein the at least one nanopore does not comprise a DNA scaffold.

The problem is also solved by a kit for assembling a nanopore-based sensing device, the kit comprising

• a device comprising at least one membrane, wherein the at least one membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode, pre-conditioned PLY monomers and/or at least one pre-assembled nanopore comprising PLY monomers, wherein the at least one pre-assembled nanopore does not comprise a DNA scaffold. means to facilitate assembly of the pre-conditioned PLY monomers into a nanopore and/or insertion of the assembled or pre-assembled nanopore into the membrane.

Furthermore, the problem is solved by the use of a nanopore comprising PLY monomers, wherein the at least one nanopore does not comprise a DNA scaffold, for sensing proteins, macromolecules, nanoparticles, and/or assemblies of nanoparticles.

Furthermore, the problem is solved by the use of a nanopore comprising PLY monomers, wherein the at least one nanopore does not comprise a DNA scaffold, for controlling the translocation probability of a molecule or particle of interest.

Brief description of the figures

Figure 1 shows the assembly and characterization of the PLY nanopore, a) Representation of a section of pneumolysin (PLY) nanopore (PDB: 5aoe). The section was constructed using UCSF Chimera software, b) Typical single-step insertion to form single pore for PLY nanopores under +100 mV applied potential (200 kHz sampling rate and a 10 kHz low-pass Gaussian filter) in 500 mM NaCI, 50 mM Tris-HCI, at pH 7.5. The inset shows a TEM micrograph of PLY pore formation with a 25 nm inner diameter, c) Current-voltage diagram for a typical PLY pore, d) Histogram showing the distribution of pore diameters of PLY in cholesterol-containing planar lipid bilayers under an applied potential of +100 mV (n = 50). The top panel in (d) represents the calculated number of monomers of PLY corresponding to the pore diameter values.

Figure 2 shows the noise analysis of the PLY nanopore. Typical current power spectral density (PSD) of open-state pores of PLY nanopore (red) and baseline (grey) measured at +100 mV applied voltage and 500 mM NaCI, 50 mM Tris-HCI, at pH 7.5 buffer solution.

Figure 3 shows protein sensing through the PLY pore, (a) Schematic depiction of a cut- through of the PLY nanopore for bio-analyte sensing, (b) Baseline-corrected current recording, showing resistive pulses due to the translocation of protein (Concanavalin A) (upward spikes) through PLY nanopore. Python was used to perform baseline correction on the provided data. Data was filtered with an additional 20 kHz low-pass Gaussian filter for visualization, (c) Examples of two resistive pulses are shown with expected time and Al/I axis, d) Shape approximation determined by modelling the crystal structure of five proteins from the Protein Data Bank (red dots) with an ellipsoid (grey) of rotation with the same volume of the proteins (anti-biotin Fab fragment (FAB; 7fab), concanavalin A dimer (CA dimer, 1gkb), Hemoglobin (Hb, 1a3n), Human serum albumin (HSA; 1AO6), concanavalin A tetramer (CA tetra, 5cna), and concanavalin A tetramer (104 kDa, 5cna)) e) Distribution of excluded volumes (A) and f) length-to-diameter ratios (m) determined from individual resistive pulses from the translocation of five different proteins. Horizontal dotted lines represent expected reference values from ellipsoid theoretical modelling. The whisker range displayed is the 10th to 90th percentile (g, h) Median values of (e) the excluded volume (A), and (f) the length-to-diameter ratio (m) determined from single event analyses of five proteins plotted against the expected theoretical values. Error bars show the first and third quartiles. The black dotted lines represent the ideal 1 :1 agreement (slope = 1), and the solid lines are the linear regressions performed imposing a zero intercept. Error bars show the first and third quartiles. The red solid line is the linear fit through all the proteins.

Figure 4 shows a comparison of the measured (median) excluded volumes (A) of single protein translocations through the PLY pore with and the corresponding molecular weight of the protein.

Figure 5 shows the detection of Tau oligomers with PLY nanopores in 500 mM NaCI, Tris HCI, pH 7.5. (a) Illustration of tau 441 amino acid sequence showing the positions of amino acids, acidic domains (N and C terminal), and tubulin binding repeats, (b) Surface representation of Tau 441 monomer, (c) Examples of two resistive pulses are shown with expected time and Al/I axis, (d) Baseline-corrected current recording, showing resistive pulses due to the translocation of Tau oligomers (upward spikes) at different time points, (e-g) Excluded volume, A, distributions of tau oligomers with varying time points (0 minutes (e) to 2 hours (g)) during the protein aggregation as determined by PLY nanopores. Tau oligomer sub-populations are marked in colored shaded curves as monomers to hexamers with sizes from » 70 nm3 (monomer; 1-mer) to » 420 nm3 (hexamer; 6-mer).

Figure 6 shows a single-molecule shape estimation of Tau oligomers in solution. Comparison of length-to-diameter ratios (m) determined from the resistive pulses from the translocation of tau samples through PLY nanopore in 500 mM NaCI, Tris-HCI, pH 7.5 buffer solution, (a-c) Measured length-to-diameter ratio (m; Figure 5e-g) of tau monomer and oligomers during aggregation at varying time points (0 to 2h) as determined by PLY nanopores, d) Combined values of measured length-to-diameter ratio of tau oligomers to characterize the different sub-populations.

Figure 7 shows a) the amino acid sequence of PLY monomer (pdb: 5LY6). The possible mutation sites were the amino acids inside the pore lumen (R147 to 1198 and G243 to L313), top region (M1 to G25, T57 to A146 and Y199 to Y242, 1314 to Y350), side region (E26 to N56 and V351 to D471); b) the corresponding cartoon representation of the PLY monomer (pdb: 5LY6) depicts the possible mutation regions.

Figure 8 shows detection of Httexl oligomers with PLY nanopores in 500 mM NaCI, Tris HCI, pH 7.5. (a) Schematic representation of MBP-httexon1 protein and removal of an N-terminal MBP affinity tag. The MBP and Httexonl domains are separated by a factor Xa cleavage site. Note that MBP is not depicted to scale, (b) Representative example of electrical recordings of httexonl protein and its oligomer translocations through PLY nanopore. Python was used to perform baseline correction on the provided data. Data was filtered with an additional 20 kHz low-pass Gaussian filter for visualization, (c) Excluded volume, A, distributions of the oligomers with varying time points (up to 9 days) during the protein aggregation as determined by PLY nanopores. Httexonl oligomer subpopulations are shown as dimers (2-mers; 02) to 36-mers (036) with sizes from » 23 nm3 (02) to « 414 nm3 (036).

Figure 9 shows a comparison of length-to-diameter ratios (m) determined from the resistive pulses from the translocation of Httexonl samples through PLY nanopores in 500 mM NaCI, Tris-HCI, pH 7.5 buffer solution. For different oligomer sizes, OX = Oligomer size where X is the number of monomers. The plot indicates combined values of measured length-to-diameter ratio (m) of Httexonl protein and its oligomers during aggregation at different time points (2 to 9 days) as determined by PLY nanopores.

Detailed description

In one aspect, the invention relates to a nanopore-based sensing device comprising at least one membrane, wherein the at least one membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode, the membrane comprising at least one nanopore comprising or consisting of pneumolysin (PLY) monomers, wherein the at least one nanopore does not comprise a DNA scaffold. A nanopore-based sensing device or nanopore sensor is herein defined as a system, e.g., a flow cell, comprising at least one membrane, the membrane comprising at least one nanopore, that is capable of detecting the presence and/or determining the volume and shape of an analyte. The nanopore-based sensing device derives its sensing capability from the nanopore used therein, wherein the nanopore allows the translocation of the analyte.

Preferably, the sensing properties of the device are based on measuring ionic current. Specifically, they are based on measuring changes in current due to the translocation of an analyte through the nanopore. A net ionic current occurs when a potential difference is applied across the membrane and electrolyte ions flow through the nanopore. The translocation or entry of an analyte through the pore hinders the passage of ions, resulting in a transient decrease of the ionic current, also called resistive pulse, which is measured to determine properties of the analyte.

In order to be able to measure the translocation of an analyte through the pore by measuring, it is therefore necessary that the nanopore is comprised in a membrane that is arranged in a way that separates two compartments within the device, with both compartments being accessible by an electrode. Only such a configuration allows the sensing device to use ionic current for sensing an analyte. A membrane that separates two compartments, with both compartments being accessible by an electrode, is called a planar membrane.

The nanopore-based sensing device may comprise additional elements such as chambers, scaffolds, electrodes, circuits, sensor chips, liquid medium, stabilizers or buffers that are necessary to support and stabilize the membrane and/or the nanopore and to enable the generation and read-out of an electric signal.

The nanopore-based sensing device according to the invention is capable of sensing proteins, e.g., native or denatured proteins, (bio)macromolecules, e.g., DNA or RNA molecules including DNA origami, exosomes or liposomes, nanoparticles and assemblies of nanoparticles and colloids.

In a preferred embodiment, the sensing properties, e.g., translocation frequency, residence time in the pore, wall interactions, of the nanopore-based sensing device are adjustable via the applied electric field, properties of the ions in the electrolyte solution, concentration of ions in the electrolyte solution, and/or the pH of the electrolyte solution. The translocation of proteins through nanopores takes place under the influence of two main forces: electrophoresis and hydrodynamic drag by electroosmotic flow. These phenomena depend on the applied potential V, the charge of the proteins, the charge of the inner surface of the nanopore, and the concentration of ions. The charge state of a protein, as well as that of a surface, depends on the nature of the charged groups, the pH of the medium, and the concentration of the ions. Without wanting to be bound by a mechanism, it is believed that for the PLY nanopores of the invention, selecting a specific pH value, specific ionic strength, as well as a specific applied potential makes it possible to control the balance between electrophoresis and electroosmotic flow, thereby favoring or disfavoring the translocation of a certain analyte of interest versus other molecules or analytes in solution.

The membrane in the nanopore-based sensing device according to the invention may be any membrane used in the art for embedding a biological nanopore. In some embodiments, the membrane is a planar membrane. Preferably, the membrane is a lipid bilayer membrane or a block-copolymer membrane.

In one embodiment, the lipid bilayer membrane in the nanopore-based sensing device according to the invention is a diphytanoyl-phosphatidylcholine and cholesterol (DiPhyPC : Chi) bilayer, preferably with a ratio of DiPhyPC : Chi of about 70 : 30. As demonstrated in the examples below, a ratio of DiPhyPC : Chi of about 70 : 30 provides a high success rate of nanopore formation in the bilayer. Additionally, the lipid bilayer has high stability and quality as shown by baseline current and capacitance measurements of the bilayer and current-voltage relationship (l-V curve) of the inserted nanopore over a wide range of applied potentials. A lipid bilayer membrane of high quality and high stability typically shows a baseline current with low noise (i.e., the standard deviation of the measured baseline current is small) and the baseline current is free of any sporadic current fluctuations (i.e., no erratic current spikes). In contrast, a lipid bilayer of low quality and stability would show a large standard deviation of the recorded baseline current and, because of the membrane instability, several erratic current fluctuations would be measured because transient openings would form. Also, unstable membranes typically degrade after a few minutes of recording, whereas stable, high quality membranes provide current baseline with low noise for longer periods of time, e.g. 30 minutes. According to the invention, the at least one membrane of the nanopore-based sensing device comprises at least one nanopore. In some embodiments, the membrane comprises exactly one nanopore or several nanopores. In some embodiments, the membrane divides a chamber into two compartments, with the nanopore being the only connection between the two compartments.

In a preferred embodiment, the nanopore-based sensing device according to the invention comprises a multitude of membranes each comprising exactly one nanopore.

The nanopores used in the nanopore-based sensing device according to the invention comprise pneumolysin (PLY) monomers. Pneumolysin is naturally occurring in Streptococcus pneumoniae, a potent human pathogen, and is a cholesterol-dependent cytolysin (CDC) that belongs to the pore-forming toxins (PFTs) family which oligomerize into the plasma membrane by forming trans-membrane pores and finally damage and kill host cells. As a bacterial exotoxin, PLY is instrumental in the breach of epithelial and endothelial barriers and the incapacitation of the host’s immune system.

Naturally occurring, wild-type PLY monomer has the amino acid sequence depicted in SEQ ID NO: 1. As used herein, the term PLY monomer comprises not only proteins or polypeptides having exactly the sequence of SEQ ID NO: 1, but also proteins or polypeptides having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, the identity being determined over the whole length of the sequence by any method known in the art. The PLY monomers may also be shortened or lengthened compared to the wild-type PLY monomer.

Preferably, the nanopore used in the nanopore-based sensing device consists only of PLY monomers and/or does not comprise a DNA scaffold, more preferably the nanopore does not comprise any type of scaffold. The nanopores used in the context of the invention are capable of assembling into a membrane and maintaining stable structure, size and functionality without the need of a DNA scaffold, such as a DNA scaffold or DNA-origami techniques in order to control or stabilize the physical and biochemical characteristics of the pore. As used herein, “scaffold” refers to any molecule used to provide structural support to a pore, whether during assembly or afterwards. Preferably, the nanopores used in the nanopore-based sensing device according to the invention are made up of and consist entirely of PLY monomers. In some embodiments, the nanopore comprises between 18 and 63 PLY monomers. In preferred embodiment, the nanopore is an assembly of and thus consists of 35 PLY monomers.

In the context of the invention, the fact that a nanopore is made up of and consisting entirely of PLY monomers does not exclude that, prior to pore assembly or during the assembly phase, the PLY proteins form dimers, trimers, or oligomers, and that the PLY oligomers react with one another in that form in order to form the nanopore.

In a preferred embodiment, the nanopores used in the context of the invention are cylindrically shaped.

In some embodiments, the nanopores have an inner diameter in the range of 10 to 40 nm, preferably 15 to 30 nm. In a preferred embodiment, the nanopores have a diameter of about or exactly 20 nm or about. In another preferred embodiment, the nanopores have a diameter of at least 20 nm, preferably about or exactly 25 nm.

The nanopore diameter may be calculated by calculating the equivalent pore diameter of a cylindrical pore based on the conductance values of the difference between baseline current (V = +100 mV) and the current after single-pore insertion of PLY (V = +100 mV), taking into account the channel and access resistances.

Determination of the inner diameter with the following equations:

Where; d_p (m) = Inner diameter of nanopore e_I (O. in) = Electrical resistivity of the electrolyte buffer

G (O^-1) = Conductance difference between baseline and open-state pore current

(in) = Effective pore length of 9.5 nm, as estimated from the crystal structure of PLY. Then the number of PLY monomers within PLY nanopores using the geometric model is calculated.

Determination of the inner diameter with the following equations:

Where; d_p (m) = Inner diameter of the nanopore, taking these values from equation (1) n = Number of monomers in PLY nanopore s = Diameter of a PLY monomer in PLY nanopore. The diameter of the cylinder is calculated based on equation (2) where the inner diameter of the nanopore is 25 nm and PLY pore consists of 42 PLY monomers, according to the previous cryo-EM studies. The diameter of a PLY monomer in a PLY nanopore is approximately 2.07nm.

In some embodiments, the nanopores have an effective length in the range of 5 to 20 nm. In a preferred embodiment, the nanopores have an effective length of about or exactly 9.5 nm.

The nanopores used in the context of the invention have a larger internal diameter than any other biological nanopore previously described. This makes them particularly useful for determining the volume and shape of large analytes such as proteins, macromolecules and nanoparticles since the larger the nanopore, the larger the analyte that can be analyzed with the help of said nanopore. For example, analytes with a size of up to 20 nm can be analyzed.

In addition, the nanopores used in the context of the invention are capable of selfincorporating into a membrane and forming pores over a wide range of pH and ionic strengths that remain stable over a long period of time. Their easy assembly makes the nanopores of the invention ideal for the use in nanopore-based sensing devices. Furthermore, the nanopores do not show gating, i.e. , no spontaneous opening and closing of the pore, or clogging, i.e., no permanent current reduction due to proteins sticking to the lumen wall.

The nanopores of the invention have a cylindrical pore shape, which is favorable, as the electric field inside the pore is constant along its length, making it possible to use modulations in current due to rotations of an analyte to determine its shape. Additionally, their effective length is also comparably long, making it suitable for the determination of details of shape, volume, dipole moment, etc.

In some embodiments, at least one PLY monomer used in the nanopores of the Nanopore-based sensing device according to the invention is modified compared to a naturally occurring monomer, the modification being an amino acid exchange, deletion and/or insertion and/or a chemical derivatization of an amino acid. For the discussion of mutations in PLY subunits for use in the invention herein below, the numbering of the sequence as shown in Fig. 7 is adhered to. This numbering corresponds to the residue numbering in naturally occurring PLY of SEQ ID NO: 1.

The term “chemical derivatization of an amino acid” is herein defined as any chemical modification that maintains the basic chemical structure and properties of the amino acid. Examples thereof include the addition of functional groups such as maleimides or hydroxysuccinimide (NHS) to amine or thiol groups or modification of carboxylic groups, e.g., by N-ethyl-3-N', N'-dimethyl aminopropyl carbodiimide (EDC) coupling.

In some embodiments, at least one PLY monomer is modified by first replacing all existing cysteines with another amino acid and then incorporating a cysteine (either by amino acid exchange or amino acid addition) at a desired position (for instance in the lumen of the pore). This cysteine can then be used for site-selective attachment of desired chemical moieties or functionalities. In preferred embodiments, cysteine between V351 to D471 is modified.

In a preferred embodiment, all cysteine residues are replaced with other amino acids, preferably an amino acid that does not affect the pore-forming function of the protein, e.g., alanine. In other embodiments, at least one cysteine residue is modified by adding a functional group such as an azide group or a maleimide. The addition of functional groups allows for the attachment of further functional groups, e.g., nanobodies capable of recognizing particular analytes.

Cysteine residues generally show a low abundance and have the highest nucleophilicity of all naturally occurring amino acids. By replacing and/or modifying individual or all cysteine residues, a high selectivity of functionalization is therefore achieved.

In some embodiments, at least one PLY monomer is modified by replacing at least one amino acid carrying a charge with an amino acid carrying the opposite or no charge. Amino acids carrying a negative charge at pH 7 are aspartic acid and glutamic acid. Amino acids carrying a positive charge at pH 7 are lysine, arginine and histidine. At pH 7.5, the lumen of the PLY nanopores used in the invention is negatively charged. This allows positively charged and somewhat negatively charged proteins to translocate from the cis to the trans compartment under negative applied potential (on the trans side) via the electroosmotic flow (EOF) as the dominating force. This condition also allows capture of large protein analytes (with a size exceeding the diameter of the PLY pore) at the entrance of the nanopore. Changing the charge of the amino acids within the pore lumen, changes the direction of the EOF (if the trans compartment remains negatively polarized) and thus allows the translocation of highly positively charged analytes if the electrophoretic force exceeds the drag force of the opposing EOF.

In preferred embodiments, at least one amino acid selected from the group comprising residues 147 to 198 and 243 to 313 of SEQ ID NO: 1 is modified. These amino acids are inside the pore lumen of the PLY nanopores described herein. Modifying these amino acids allows to change and adjust the EOF as well as the electrostatic properties of the nanopore. This may be used to either attract certain analytes or to prevent them from entering the pore.

In other preferred embodiments, at least one amino acid selected from the group comprising residues 1 to 25, 57 to 146, 199 to 242, and 314 to 350 of SEQ ID NO: 1 is modified. These amino acids are outside of the membrane bound regions of the PLY nanopores described herein. Modifying these amino acids therefore allows to attach a functional group before the lumen of the nanopore, regions, e. g., an antibody for selective detection of its antigen. In one embodiment, a PLY monomer to be used in the nanopore of the nanopore-based sensing device comprises modified amino acids both inside the pore lumen and outside the membrane bound region.

In some embodiment, the PLY monomer is truncated from the N-terminus. This enhances pore formation. In a preferred embodiment, the first 16 amino acids from the N-terminus of the PLY monomer are deleted.

In another preferred embodiment, the PLY monomers are crosslinked with each other.

In addition to the mutations described above, the PLY monomers for use in a nanoporebased sensing device according to the invention may comprise one or more conservative mutations. Typically, conservative mutations wherein an amino acid is replaced by a residue with very similar properties are anticipated to have no or only a limited effect on nanopore function. Examples of conservative mutations include S to T, R to K, D to E, N to Q, A to V, I to L, F to Y and vice versa.

In some embodiments, only some PLY monomers of the nanopore are modified. By mixing wild-type PLY monomers and modified monomers with each other, it is possible to e.g., selectively introduce individual binding sites into the pore lumen.

In other embodiments, all PLY monomers of the nanopore are modified.

In some embodiments, the PLY monomers carry different mutations. In other embodiments, the PLY monomers carry the same mutation or mutations.

In some embodiments, the at least one nanopore of the nanopore-based sensing device is stabilized with a polymeric surfactant. In other embodiments, the at least one nanopore of the nanopore-based sensing device has been assembled in the presence of a polymeric surfactant. The polymeric surfactant is preferably an amphipol. Amphipols are a class of surfactants that substitute detergents and keep membrane proteins soluble in detergent-free aqueous solution by stabilizing them biochemically. Amphipols are commercially available, for example, Amphipol A8-35 (Anatrace, OH). Preferably, the amphipol is present at a concentration of 0.2 pM.

In one embodiment, the at least one nanopore of the nanopore-based sensing device is stabilized with a solution comprising amphipol, a salt and a buffer. In a preferred embodiment, the at least one nanopore of the nanopore-based sensing device is stabilized with an aqueous solution comprising amphipol at a concentration of 0.2 pM, NaCI or a similar salt at a concentration of between about 200 mM and 500 mM, and a Tris-HCI buffer at a concentration of 10 mM, the solution havingpH in a range of between about 6.5 and about 8.0. Such a solution enables PLY monomers to form nanopores that remain highly stable after assembly.

In another aspect, the invention relates to a kit for assembling a nanopore-based sensing device, the kit comprising

• a device comprising at least one membrane, wherein the at least one membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode,

• pre-conditioned PLY monomers and/or at least pre-assembled nanopore consisting of PLY monomers, wherein the at least one pre-assembled nanopore does not comprise a DNA scaffold,

• means to facilitate assembly of the pre-conditioned PLY monomers into a nanopore and/or insertion of the assembled or pre-assembled nanopore into the membrane.

In one embodiment, the PLY monomers of the kit are pre-conditioned. Pre-conditioned as used herein means that the PLY monomers have been purified and/or solubilized with a polymeric surfactant. In another embodiment, the kit comprises a pre-assembled nanopore comprising or consisting of PLY monomers, wherein the pre-assembled nanopore consists only of PLY monomers and does not comprise a DNA scaffold. The pre-assembled nanopore may be stabilized.

The means to facilitate assembly of the pre-conditioned PLY monomers into a nanopore and/or insertion of the assembled or pre-assembled nanopore into the membrane may be a buffer comprising further stabilizers. In one embodiment, the means are a buffer comprising a polymeric surfactant, preferably amphipol.

The kit of the invention allows a user to assemble a PLY nanopore-based sensing device ad hoc. This circumvents the problem of having to store the nanopore-based sensing device for a prolonged time which requires stabilizing the nanopore and the membrane. In another aspect, the invention relates to the use of a nanopore comprising or consisting of PLY monomers, wherein the at least one nanopore does not comprise a DNA scaffold, for sensing proteins, macromolecules, nanoparticles and/or assemblies of nanoparticles, based on ionic current measurement. In a preferred embodiment, the nanopore is used for determining the volume and/or shape of a protein, macromolecule, nanoparticle and/or assembly of nanoparticles. For example, the nanopore can be used to detect particular antibodies, differentiate between single nanoparticles and nanopore assemblies or verify DNA origami structures.

In yet another aspect, the invention relates to the use of a nanopore comprising or consisting of PLY monomers, wherein the at least one nanopore does not comprise a DNA scaffold, for controlling the translocation probability of a molecule or particle of interest, i.e., favoring or disfavoring the translocation of a certain analyte of interest. Therefore, a nanopore comprising or consisting of PLY monomers may be used sorting and filtering analytes. The use may comprise tuning pH and/or applied potential or attaching particles to the nanopore.

Examples

Example 1 - In-situ PLY pore assembly formation in planar lipid bilayer

Pneumolysin (PLY) monomers purification. Pneumolysin (PLY) monomers were expressed in E. coli and purified. Briefly, the PLY gene was amplified from Streptococcus pneumoniae D39, and the PCR products as well as the pET28a vector were cut with the complementing restriction enzymes (BamHI, Xhol) and ligated using T4-DNA ligase. Vector were transformed in BL21 (DE3) pLysS competent cells. Positive clones were confirmed by sequencing. Protein expression was induced with 1 mM IPTG (Sigma- Aldrich) at the bacterial culture OD₅₀₀ = 0.5-0.7. Bacterial cells were incubated for 4 hours at 37 °C. The recombinant proteins were purified with Ni-NTA 1 ml columns (MACHEREY- NAGEL, Oensingen, Switzerland), and in the final step the sample was dialyzed against 50 mM Tris-HCI buffer (pH = 7.0).

Activation of PLY monomers and treatment with amphipol. A stock solution of DTT (20 mM) in 50 mM Tris-HCI (pH 7.5) 500 mM NaCI buffer was prepared. The purified PLY solution (1 mg ml’¹, 5 pL) was first activated with 7.5 mM DTT in the above buffer for 10 mins at 37 °C in ThermoMixer (Eppendorf). The activated PLY monomers were incubated with Amphipol solution maintaining PLY: Amphipol = 1:2 molar ratio in 50 mM Tris-HCI (pH 7.5) buffer solution for 30 minutes at 37 °C in ThermoMixer (Eppendorf).

Planar lipid bilayer (membrane) formation: A lipid mixture containing 70 mol-% 1,2- diphytanoyl-sn-glycero-3-phosphocholine (DiphyPC) and 30 mol-% cholesterol in octane was prepared to maintain 15 mg ml’¹ final concentration of DiphyPC. Planar lipid bilayer current measurements were performed using an integrated chip-based, four-channel parallel bilayer recording setup (Orbit Mini; Nanion Technologies) and EDR3 software with multielectrode cavity-array (MECA) chips (lonera, Germany). MECA chips bearing four channels with a diameter of 150 pm were used throughout our experiments to support the lipid bilayers. The lipid bilayers were formed using the technique described by Wang et al. Briefly, the recording buffer (50 mM Tris-HCI (pH 7.5) 500 mM NaCI; 150 pl) was added to the cis compartment of the MECA chip and all four channels were wetted by applying a gentle pressure using a syringe plunger. The bilayers were then formed by painting the lipid solution (0.3 -0.4 pL) close to the cavity of the chips. The quality of the lipid bilayer was confirmed by measuring a baseline current (-0.3 < I < 0.3 nA) and capacitance of 30 ± 5 nF. The recording software (EDR) automatically estimates the membrane capacitance by analyzing the current response to an applied triangular potential. The stability of the bilayers (absence of leak currents, expected noise level) was analyzed by applying transmembrane voltages of up to 100 mV for 1 min at both polarities.

In-situ PLY pore assembly formation in planar lipid bilayer. The bilayers formed using the above-mentioned technique were allowed to stabilize for 5 minutes. Using a pipet tip, 2.5 pL of amphipol-incubated PLY monomer stock solution (0.2 mg ml’¹) was added to the cis chamber of the MECA chip. An applied potential of +100 mV was maintained until a single-step current jump was observed, indicating successful assembly of PLY pore into the lipid bilayer. During a typical experiment, a pore would be inserted within 10-15 minutes of PLY amphipol incubated solution addition. The inner diameter and the number of PLY monomers were calculated from the pore conductance values. The pore conductance was obtained from the difference between baseline and open-state pore current. This diameter of the pore was estimated using the following equation, which assumes the pore to be perfectly cylindrical and accounts for both the channel and the access resistances:

In equation 1, d_p [m] is the inner diameter of the nanopore, I_p [m] is the pore length, p_rf [ m] is the electrical resistivity of the electrolyte buffer and G [S] is the conductance of the pore. The channel length

= 9.5- 10^-9 m was estimated from the crystal structure of PLY (PDB: 5aoe). Then the number of PLY monomers composing the nanopore was calculated using a previously published geometric model. All electrical measurements were conducted with a sampling rate of 200 kHz and 20 nA range.

Example 2 - Pneumolysin (PLY) nanopore characterization

The ability of Pneumolysin (PLY) pores in lipid bilayer inspired by the crystal structures of PLY (PDB: 5aoe) membrane pores (Figure 1a) to detect medium to large-sized proteins was investigated, which proteins were predicted to fit within the lumen of the 42-meric pore complex. The PLY monomers (53.7 kDa) were expressed in E. coli cells and purified by Ni-NTA affinity chromatography as described in the methods section. PLY pore complex assembly on cholesterol-containing liposomes has shown a sufficiently homogenous and stable population in the presence of a polymeric surfactant, Amphipol A8-35 (amphipol). Hence, amphipol was used to improve the solubility and oligomerization of PLY monomers in the experiments. The PLY monomers were incubated with the amphipol solution (1 :2 molar ratio; see methods for details) in 50 mM Tris-HCI at pH 7.5 at 37 °C for 30 minutes to facilitate the insertion of PLY pore into the lipid bilayer. The amphipol incubated solution in the absence of a cholesterol-containing lipid bilayer shows a few occurrences of PLY pore complex formation. The negatively stained TEM sample featured (Figure 1b; inset) isolated ring-shaped PLY pore complexes with a ca. ~25 nm inner diameter of the pore which is in agreement with the literature reported dimension of the pores. However, it was observed that the probability of finding a PLY pore complex in the absence of a cholesterol-lipid bilayer is low since PLY belongs to a family of CDCs. Amphipol incubated PLY solution was used to study the incorporation of large pores into the cholesterol-containing artificial planar lipid bilayer nanopore recordings. Planar lipid bilayers were formed using a lipid mixture containing 70 mol-% 1 ,2-diphytanoyl-sn- glycero-3-phosphocholine (DiphyPC) and 30 mol-% cholesterol (Chi.) in octane (see methods section for details) in 500 mM NaCI buffered with 50 mM Tris-HCI at pH 7.5. It was found that PLY pores were inserted in situ into planar lipid bilayers by adding the amphipol incubated PLY solution (final PLY concentration 0.003 mg ml’¹) to the cis compartment of the lipid bilayer recording setup under +100 mV applied potential (Figure 1b). Figure 1b displays typical electrical recordings of PLY single-pore insertions, where the magnitude of the current jump caused by the pore insertion enables the estimation of the nanopore inner diameter (see method section for details). Figure 1d shows the calculated diameters of inserted PLY pores from single insertion events (N=50). The histogram suggests that PLY forms 20.5±1.02 nm pores that correspond to 35±2 PLY monomers under the above-mentioned condition. The majority of the inserted pores were formed by spontaneous single-step insertions within 10-15 minutes of PLY incubated solution addition into the setup and remained stable for over 30 minutes after insertion under ±100 mV applied potential. The mechanism of membrane insertion of the PLY large pore complex involves a multistage process where in the first step the oligomers tether into a ring-shaped complex to the bilayer without puncturing and in the next step the pore inserts into the membrane. The current-voltage relationship (l-V curve) of the inserted PLY pore is shown in Figure 1c. The symmetric nature of the l-V curve under varying applied potentials (ranging from -100 mV to +100 mV) indicates the cylindrical nature of the PLY pore lumen and its stability over a wide range of applied potentials. The power spectral densities (PSD) of the recordings before (baseline) and after nanopore insertion were compared to assess the stability of the PLY pores under 500 mM NaCI buffered with 50 mM Tris-HCI at pH 7.5 (Figure 2). Figure 2 depicts an increase in noise in the low- frequency domain of the spectra (noise) hinting at fluctuations in the membrane in analogy to the PLY pore formation mechanism during assembly of the ~35 PLY monomers to form the large diameter nanopore.

Several variables, including ionic strength, pH, and voltage, can influence the sensitivity and specificity of biological nanopores which could be exploited for protein analyte sensing purposes. It was found that the PLY nanopores could be inserted efficiently into phospholipid bilayers in the neutral pH range of 7 to 7.5 (approx. 70 % success rate) with median pore diameter values 20±1 nm corresponding to 34 ± 2 PLY monomer complex assembly formation. Amphipol incubated PLY solution did not show significant pore insertion probability in phospholipid bilayer under acidic/basic pH (6.5, 8, and 8.5) conditions. However, it was found occasional pore insertions at pH 6.5, 8, and 8.5 and the calculated median diameters of the PLY pores at these pH values are 16 ± 2 nm.

Example 3 - Protein translocations

After the successful insertion of a PLY pore, a stepwise potential sweep from -100 mV to +100 mV was performed to ensure the pore stability and to measure the offset current at 0 applied potential. Subsequently, 5 pl of analyte (protein) solutions at a concentration of 1mg ml’¹ were added into the cis compartment. Translocation of the analyte is promoted by applying constant potentials of either +100 or -100 mV across the PLY nanopore. Every 5 minutes, the potential sweep is repeated to verify the stability of the nanopore and to measure the offset current. The acquired data were filtered with a Gaussian low-pass filter at a cut-off frequency of 20 kHz and performed a threshold search (5 x the standard deviation of the baseline current) for resistive pulses within the current recording using the procedure described in Data analysis software.

Example 4 - PLY nanopore to estimate the volume and the shape of translocating analytes

Biological nanopores with varying diameters have been used in the literature to detect small proteins in the molecular weight range of 5-25 kDa whereas proteins in the molecular weight range of 30-125 kDa have been shown recently to be trapped within the conical lumen of YaxAB pore. However, the narrow width of the YaxAB pore lumen is not effective for large protein translocations. The transport of protein bio-analytes along the large pore lumen of PLY (Figure 3) was examined with single-channel current recordings. Five folded proteins with varying sizes and charge (Figure 3d): Human IgG Fragment antigen-binding (FAB, 45 kDa, 7fab), concanavalin A dimer (CA dimer, 53 kDa, 1gkb), Hemoglobin (Hb, 64.5 kDa, 1a3n), Human Serum Albumin (HSA, 66.5 kDa, 1ao6) and concanavalin A tetramer (CA tetramer, 104 kDa, 5cna) were selected as model proteins to investigate the translocation at an electrophysiological buffer of pH 7.5. Figure 3a shows the schematic representation of protein sensing through PLY nanopore under an applied potential. It was found that upon the addition of protein analytes to the cis chamber at a concentration final concentration of 32 pg ml-1, current blockade events occurred (Figure 3b-c). Control experiments performed in the absence of any protein analytes showed no such current blockade event. A representative example of electrophysiology traces of individual proteins at -100 mV is shown in Figure 3b (Concanavalin A protein). When a single protein analyte passes through the electrolyte-filled PLY nanopore, there will be a transient increase in electrical resistance that results in characteristic resistive pulse signals. The upward spikes in Figure 3b indicate individual resistive current pulses towards zero current due to the translocation of a single protein analyte (the raw data was filtered with an additional 20 kHz low-pass Gaussian filter for visualization). The resistive pulses of single protein translocation events contain information about their physical properties including volume (i.e., size “A”) and shape (length-to-diameter ratio “m”). Ellipsoid modeling (see methods for details) of the proteins provided the estimated theoretical (reference) volume (A = 69 to 310 nm³), and length-to-diameter ratio (m = 0.60 to 1.32) (Figure. 3d) of the proteins. Figure 3 (e, f) shows the approximate excluded volume (A, nm³) and length-to-diameter ratio (m) of five translocating proteins (FAB, Hb, HSA, and Concanavalin dimer, and tetramer) through the PLY pore. The dotted lines in Figure 3 (e, f) indicate the approximate theoretical values of volume and length-to- diameter ratio. The volume and shape of the selected proteins was determined by relating the amplitude of the resistive pulse to the volume of the particle and the shape of the signal to the particle’s shape. Figure 3g, h shows the distribution of experimental volumes and length-to-diameter ratios compared with reference values for each protein, illustrating that this analysis yields good estimates of A and m (Figure 3e, f). The events were analyzed with a dwell time exceeding 300 ps (the data was digitally low-pass filtered at 20 kHz). Figure 4 shows the overall comparison of estimated experimental volumes (median values) with the molecular weight of the corresponding protein. The volume of each protein at a given voltage increases with the molecular weight of the protein, with the larger protein CA tetramer showing the highest volume, and the smaller FAB showing the lowest volume (Figure 4). This behavior is in agreement with the analogy that larger proteins tend to show a larger current blockade in nanopore resistive pulse signals.

Example 5 - PLY nanopore for Tau protein oligomer characterization at the singleparticle level

To demonstrate the potential of PLY nanopore, the selectivity of the pore in the singleparticle characterization of protein oligomer to reveal the size and shape of Tau oligomers was investigated. Aggregates of Tau are characteristic features in a wide range of neurodegenerative diseases, including Alzheimer’s disease. Though large fibrillar aggregates of amyloid proteins are prominent pathological features, in recent years oligomers formed early during the aggregation process are in many cases believed to be more toxic species. Efforts to inhibit the aggregation could offer new targets for therapeutic developments. By contrast, the technical difficulties in efficiently studying and characterizing small oligomers still persist since the small oligomers exist in transient populations in a heterogeneous mixture of oligomers. Single-molecule measurements through PLY nanopore can in principle overcome the challenge of studying protein aggregation in real time. Recently, solid-state nanopores have been employed for the detection and size and shape determination of oligomers of amyloid-forming protein a-Synuclein in solution. Giamblanco et al. have shown oligomeric and fibrillar particle distributions during the tau aggregation process using single conical nanopores. However, a quantitative analysis of the soluble early Tau oligomers is not well studied. Here the ability of PLY pores to quantify the populations of Tau early oligomers of different sizes is demonstrated for the first time to date using stable biological nanopores.

Tau aggregation protocol: i) Reaction buffer preparation: 20 mL PBS buffer solution (pH = 7.1) was filtered through a sterile 0.22 pm pore size PES membrane filter (syringe) and dry powder TCEP was added to prepare 0.5mM TCEP in PBS buffer solution. pH of the final solution was maintained at pH = 6.7 (by adding a few uL 0.1M KOH solution) and the solution was filtered through a 0.22 pm pore size PES membrane filter. ii) Heparin solution preparation: Fresh heparin solution (194 pM) was prepared by dissolving 3.5 mg of HMW heparin dry powder (MW = 17-19 kDa) in 1 mL reaction buffer at room temperature. The solution was shaken vigorously and vortexed 2 times for 5 seconds and filtered through a sterile 0.20 pm pore size PES membrane filter.

Hi) Tau solution preparation: Tau441 (dry powder) was dissolved in reaction buffer (0.33 mg ml’¹) vortexed for a few seconds and then centrifuged for 1 min at 10,000 rpm at 20°C to eliminate air bubbles. The final solution was up-concentrated (0.6 mg ml^-1) and buffer exchange using a 30kDa amicon filter with reaction buffer at 10,000 rpm. Heparin stock (194 pM) solution was added to the Tau solution in a 1 :2 molar ratio and kept for aggregation at 37 °C and 500 rpm in ThermoMixer. The collection of samples was done at different times and the aliquots were flash frozen (10 pL portions) and stored at -80 °C freezer for future experiments.

Results Tau-441 full-length amino acid sequence is shown in Figure 5a consisting of an N-terminal acidic domain (1 to 103), a proline-rich domain (197 to 244), and a C-terminal domain with tubulin binding repeats which are believed to drive Tau fibrilization. Figure 5d indicates the original current traces of electrical recordings through PLY nanopores, featuring translocation events of individual oligomers at constant -100 mV applied potential under 500 mM NaCI, 50 mM Tris-HCI, at pH 7.5 buffer. Several hundreds of individual translocation events (representative examples of single events shown in Figure 5c) from tau oligomer samples were recorded to estimate the size distribution and the oligomers were classified in terms of monomer numbers. The volume of monomeric tau protein was estimated to be 70 nm³ using nanopore experiments (Figure 5e-g). Mass photometry quantification of native wild-type Tau solution revealed a monomer-dimer- trimeric equilibrium with a dominant monomeric presence. The nanopore recordings with the native Tau solution (Tau 0m) under non-aggregation conditions show two major subpopulations of Tau (Figure 5e) with estimated volumes of ~70 nm³ and ~140 nm³ consisting of monomers and dimers which is in good agreement with the earlier reports. Figure 5e-g represents a single oligomer analysis of Tau samples under aggregation conditions (see methods for details) using PLY nanopore. To quantify the volumes of oligomers in the sample solution as a function of time the areas of the volume distribution peaks corresponding to monomers were fitted. As the aggregation proceeded a distinct change in the oligomer populations was observed in the volume distribution from native Tau (Tau 0m) to the 2 hours sample (Figure 5e-g), with the appearance of higher order oligomers having volumes up to an estimated ~ 420 nm³ corresponding to 6-mers. The single-particle level nanopore results indicate 1-6-mer populations in the Tau aggregation samples with the major sub-population of the 3-mer in the 2-hour sample. Surprisingly, it was observed a distinct shift in the position of 3-mer volume in the volume distribution fits in the 2-hour sample. The volume of Tau oligomer sub-populations was approximated based on the volume of monomers. However, previous literature reports with a combination of NMR spectroscopy, electron paramagnetic resonance, and small-angle X- ray scattering reveal that the soluble tau oligomers contain a dynamic, noncooperatively stabilized core with a variable diameter. The reported dynamic structure of soluble tau oligomers could be a possible rationale for the observed shift in the 3-mer population. The results of estimated oligomer volumes from PLY nanopore indicate the potential resolution of nanopore-based fingerprinting of small subpopulations of tau oligomers in comparison to TEM imaging-based size analysis. Next, the shape (length-to-diameter ratio, m; Figure 6) of each oligomeric subpopulation was determined from the heterogeneous Tau aggregation samples in solution by analyzing minimum and maximum current blockade (i.e., 2H_min and 21l_max) values from the nanopore recordings. The AFM-based morphology study of Tau oligomers revealed a sphere-shaped dynamic structure of the oligomers that turns into p-sheet rich upon aggregation and the TEM analysis of the Tau oligomers did not show any occurrence of fibrillar/protofibrilar structure. Therefore, the shape of each oligomer in the Tau samples was modeled as an oblate ellipsoid i.e. m < 1. To achieve high precision in the determination of shape, the m values of the oligomers were quantified from their corresponding volume distributions presented in Figure 6 with a cutoff range of ±10 nm³. Figure 6 shows the distribution of m values obtained for the oblate model of native Tau solution and Tau samples under aggregation conditions. The native Tau solution under no aggregation condition shows two distributions of the population (Figure 6a), 1-mer, and 2-mer that correspond to the oblate shape of m (± SD) = 0.46 (± 0.11) and 0.51 (± 0.12). The m values represent the median numbers from the events analyzed for shape estimation. Tau 1-hour aggregated sample solution showed (Figure 6b) 1 to 4-mer populations of oligomers with m (± SD) = 0.43 (± 0.09), 0.60 (± 0.12), 0.66 (± 0.10), and 0.75 (± 0.15). Characterization of Tau 2-hour sample (Figure 6b) revealed 1 to 5-mer populations of oligomers with m (± SD) = 0.45 (± 0.09), 0.53 (± 0.13), 0.62 (± 0.10), 0.76 (± 0.08) and 0.77 (± 0.07). The low abundance of 6-mers in the samples limited the approximate shape determination of the oligomeric subpopulation. Figure 6d shows the combined length-to-diameter ratio (m) of the different sub-populations of Tau 1- mer to 5-mer during the aggregation process. The nanopore results indicate that the length-to-diameter ratio (m) of individual oligomers from 1-mer to 5-mer is consistently increasing from -0.45 to - 0.77 and approaching towards a more spherical shape (i.e. -1). This enables us to characterize the approximate size and shape of each Tau oligomer population through PLY nanopore in real time. Nanopore quantification of specific subpopulations of protein aggregates through PLY pore could provide important insights on potential biomarker developments for neurodegenerative diseases.

Example 6 - PLY nanopore for Htt protein oligomer characterisation at the singleparticle level

To demonstrate the potential of PLY nanopore, the selectivity of the pore in the single particle characterization of protein oligomers and the size and shape of Httexonl oligomers were investigated.

N-terminal MBP affinity tag removal and Httexonl protein aggregation. Human HTT Protein, MBP, His Tag (HTT-H51M5, MBP-Httexon1) was purchased from ACRO Biosystems. Factor Xa protease was purchased from Promega (V5581).

The MBP-Httexon1 protein has the amino acid sequence depicted in SEQ ID NO: 2:

MBP(KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDG PDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYN KDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGY

AFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN

GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTD

EGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITK)-Plus(LEVLFQGP) - Target - Linker

(GGGSGGGS)— 10*His (HHHHHHHHHH),

Target(exonl), as depicted in SEQ ID NO: 3:

MATLEKLMKAFESLKSFQQQQQQQQQQQQQQQQQQQQQPPPPPPPPPPPQLPQPPP QAQPLLPQPQPPPPPPPPPPGPAVAE

The commercial fusion proteins were dialyzed against a buffer containing 50 mM Tris-CI (pH 7.5) and 100 mM NaCI, and the protein aggregation was performed following the protocol according to [Poirier MA, Li H, Macosko J, Cai S, Amzel M, Ross CA. Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization. J Biol. Chem. 2002; 277(43): 41032-41037. doi:10.1074/jbc.M205809200],

To cleave the MBP affinity tag and promote aggregation of the exonl, the dialyzed fusion protein (0.25 mg/ml) was treated with Factor Xa protease (the enzyme-to-protein ratio of 1 :25) at room temperature (indicated in figure 8a).

Characterisation of Htt aggregation: Aliquots were collected at different time intervals, and the cleavage reaction was then analyzed using PLY nanopore experiments (indicated in figure 8b-c), as discussed before in the tau aggregation experiment.

As demonstrated in Figure 9, this approach makes it possible to determine the shape of oligomers of various sizes.

Claims

1. A nanopore-based sensing device comprising at least one membrane, wherein the membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode, the membrane comprising at least one nanopore comprising pneumolysin (PLY) monomers, wherein the at least one nanopore does not comprise a DNA scaffold.

2. The nanopore-based sensing device according to claim 1, wherein the nanopore has diameter in the range of 10 to 40 nm, preferably 20 or 25 nm, and/or an effective length in the range of 5 to 20 nm, preferably 9.5 nm.

3. The nanopore-based sensing device according to claim 1 or 2, wherein the sensing properties of the device are based on measuring ionic currents.

4. The nanopore-based sensing device according to claim 1 to 3, wherein the device is capable of sensing proteins, macromolecules, nanoparticles and/or assemblies of nanoparticles.

5. The nanopore-based sensing device according to any of claims 1 to 4, wherein the nanopore comprises between 18 to 63 PLY monomers, preferably 35 PLY monomers.

6. The nanopore-based sensing device according to any of claims 1 to 5, wherein at least one PLY monomer is modified compared to a naturally occurring monomer, the modification being an amino acid exchange, deletion and/or insertion and/or a chemical derivatization of an amino acid.

7. The nanopore-based sensing device according to claim 6, wherein at least one PLY monomer is modified by replacing at least one cysteine residue with another amino acid and/or by adding a functional group to at least one cysteine residue.

8. The nanopore-based sensing device according to 6 or 7, wherein the at least one PLY monomer is modified by replacing at least one amino acid carrying a charge with an amino acid carrying the opposite or no charge.

9. The nanopore-based sensing device according to 7 or 8, wherein at least one amino acid selected from the group comprising residues 147 to 198 and 243 to 313 of SEQ ID NO: 1 and/or at least one amino acid selected from the group comprising residues 1 to 25, 57 to 146, 199 to 242, and 314 to 350 of SEQ ID NO: 1 is modified.

10. The nanopore-based sensing device according to claim 6, wherein the first 16 amino acids of the N-terminus of the PLY monomers are deleted and/or the PLY monomers are crosslinked with each other.

11. The nanopore-based sensing device according to any of claims 1 to 10, wherein the at least one nanopore is stabilized with a polymeric surfactant and/or has been assembled in the presence of a polymeric surfactant, wherein the polymeric surfactant is preferably an amphipol.

12. The nanopore-based sensing device according to any of claims 1 to 11 , wherein the sensing properties of the device are adjustable via the applied electric field, properties of the ions in the electrolyte solution, concentration of ions in the electrolyte solution, and/or the pH of the electrolyte solution.

13. The nanopore-based sensing device according to any of claims 1 to 12, wherein the membrane is a planar lipid bilayer membrane or a block-copolymer membrane.

14. A kit for assembling a nanopore-based sensing device, the kit comprising

• a device comprising at least one membrane, wherein the membrane is arranged in a way that it separates two compartments within the device, both of which are accessible by an electrode,

• pre-conditioned PLY monomers and/or at least one pre-assembled nanopore comprising PLY monomers, wherein the PLY monomers or the pre-assembled nanopore do not comprise a DNA scaffold, means to facilitate assembly of the pre-conditioned PLY monomers into a nanopore and/or insertion of the assembled or pre-assembled nanopore into the membrane.

15. The kit according to claim 14, wherein the means to facilitate assembly of the preconditioned PLY monomers into a nanopore and/or insertion of the assembled or pre-assembled nanopore into the membrane comprises a buffer comprising a polymeric surfactant, preferably an amphipol.

16. Use of a nanopore comprising PLY monomers, wherein the nanopore does not comprise a DNA scaffold, for sensing, based on ionic current measurementproteins, macromolecules, nanoparticles and/or assemblies of nanoparticles.

17. Use of a nanopore comprising PLY monomers, wherein the nanopore does not comprise a DNA scaffold, for controlling the translocation probability of a molecule or particle of interest, based on ionic current measurement.