WO2023140732A1

WO2023140732A1 - Method for measuring a linearized protein, a 2d material membrane, a nanopore device comprising the 2d material membrane, a system comprising the nanopore device, and a production method for providing the 2d material membrane

Info

Publication number: WO2023140732A1
Application number: PCT/NL2023/050025
Authority: WO
Inventors: Sabina CANEVA; Dong Hoon Shin; Xiliang YANG
Original assignee: Technische Universiteit Delft
Priority date: 2022-01-21
Filing date: 2023-01-20
Publication date: 2023-07-27

Abstract

The invention provides a measurement method for measuring a linearized protein (10) using a 2D material membrane (100), wherein: the linearized protein (10) is functionally coupled to a protein optical emitter (21); the 2D material membrane (100) comprises a rim (115) defining a membrane nanopore (110), wherein the rim (115) comprises a [room-temperature] membrane optical emitter (22), and wherein the 2D material membrane (100) comprises a defect (120) selected from the group comprising an impurity, a vacancy, and a vacancy complex, wherein the defect (120) provides the membrane optical emitter (22); the protein optical emitter (21) and the membrane optical emitter (22) form FRET donor-acceptor pair emitters (20) having a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range; a measurement stage comprises: (i) passing the linearized protein (10) through the membrane nanopore (110); (ii) providing donor excitation radiation (51) having a wavelength selected from the donor excitation radiation range; and (iii) measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal.

Description

METHOD FOR MEASURING A LINEARIZED PROTEIN, A 2D MATERIAL MEMBRANE, A NANOPORE DEVICE COMPRISING THE 2D MATERIAL MEMBRANE, A SYSTEM COMPRISING THE NANOPORE DEVICE, AND A PRODUCTION METHOD FOR PROVIDING THE 2D MATERIAL MEMBRANE

FIELD OF THE INVENTION

The invention relates to a measurement method for measuring a linearized 5 protein. The invention further relates to a 2D material membrane. The invention further relates to a nanopore device comprising the 2D material membrane. The invention further relates to a system comprising the nanopore device. The invention further relates to a production method for providing the 2D material membrane. 0 BACKGROUND OF THE INVENTION

Methods for measuring proteins are known in the art. For instance, WO2014014347A1 describes a device for determining the type of protein in a liquid, the device comprising (a) an immobilized ATP dependent protease based molecular transporter machine configured to guide a protein that is functionalized with labels through a detection area of a5 detector, (b) said detector, configured to detect a signal as function of the labels of the labelled amino acids, and (c) a processor unit, configured to identify from the detector signal a sequence of amino acids of the functionalized protein, wherein the processor unit is further configured to compare the identified sequence of amino acids with the occurrence of such sequence in a database of proteins and to identify the type of protein. 0 WO2011040996A1 describes methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore,5 energy may be transferred from the excited donor label to the acceptor label of the monomer.

US20050282229A1 describes methods and apparatus for sequencing and/or identifying proteins, polypeptides and/or peptides. Proteins containing labeled amino acid residues may be synthesized and passed through nanopores. A detector operably coupled to a nanopore may detect labeled amino acid residues as they pass through the nanopore. 0 WO2014014347A1 describes a device for determining the type of protein in a liquid. Zhongying Wang, Baoxia Mi., “Environmental Applications of 2D Molybdenum Disulfide (MoS2) Nanosheets”, Environmental Science & Technology, 2017, 51, 8229-8244, describes the use of M0S2 nanosheets for water-related environmental applications such as contaminant adsorption, photocatalysis, membrane-based separation, sensing, and disinfection.

CN113533275A describes a solid nanopore-fluorescence resonance energy transfer composite detection method.

SUMMARY OF THE INVENTION

Molecular signatures may hold crucial information about the role of biomolecules in health and disease. The ability to decode the sequence of biomolecules may therefore be vitally important as it may provide breakthroughs in understanding the link between biological structure and (mal)function. Yet, the decoding of sequences may rapidly become more challenging when moving from the genome to the proteome, due to the staggering number of proteins in individual cells, the larger number of distinct subunits, and the post- translational modifications proteins can be subjected to.

Unlike DNA, proteins can presently not be amplified in vitro. Hence, characterizing, especially identifying, both abundant and rare cellular proteins may require highly sensitive yet high-throughput techniques. Currently, the gold standard for protein sequencing may be mass-spectrometry, which may, however, be limited in terms of sensitivity and dynamic range. Mass-spectrometry may also rely on large amounts of sample to perform ensemble measurements, and is thus not truly a single-molecule technique.

An alternative method may be Edman degradation, where the ordered identification of an amino acid sequence occurs through a sequence of chemical reactions that cyclically label, cleave and identify residues one at the time. Its main shortcomings may be the length of the chain that can be sequenced (typically shorter than ~50 amino acids) and the relatively time-consuming nature of the process.

The prior art may further describe nanogaps and nanopores devices that can access information about single-molecule position, dynamics, mechanics and interaction with the environment. Nanogaps may essentially be tunnelling-based biosensors, where the modulation of a tunnelling current across a nanogap between two electrodes reports on the presence and electronic structure of a trapped biomolecule. On the other hand, nanopores embedded in a membrane may rely on a nanoscale confinement to measure the ionic current modulation during biomolecule translocation, acting as both an active delivery channel and as a localized detector. The considerable success and continued widespread use of nanopores may derive from the simplicity of the basic experimental implementation combined with its versatility and modularity.

In particular, the prior art may describe biological nanopores and solid-state nanopores. For biological nanopores, access to purified protein nanopores and enzymes, as well as stable lipid bilayers may be required. Compared to biological nanopores, solid-state nanopores may lead in terms of robustness, lifetime, parallelization, mass producibility and integration with on-chip optics and electronics.

In a typical nanopore experiment, an insulating membrane containing a nanometer-sized pore may be placed between two electrolyte-filled compartments. When a voltage is applied to the compartments, an ionic current may flow through the nanopore. As a molecule translocates through the nanopore, a modulation of the ionic currents may be observed, which may provide structural information about the molecule. Nanopore-based protein identification may rely on a current trace signal: a measurement of changes in an ionic current across a nanopore as a (single) protein approaches, departs and/or passes through the nanopore. The current trace signal may then in principle be used to identify the protein. However, it may be difficult to distinguish proteins based on their current trace signals, among others due to background noise, particularly with regards to complex samples comprising many proteins and/or similar proteins. In particular, it may be difficult to differentiate between the signal of the different (proteinogenic) amino acids as they pass through the pore.

Plasmonic nanopores may consist of plasmonic nanostructures fabricated on top of a nanopore on a solid-state chip, which can concentrate an incident optical field. Plasmon resonance biosensing may be performed by detecting the presence of a biomolecule from a measurable shift in the plasmonic resonance of the device during illumination. The use of a highly focused laser source for the readout may, however, also invariably lead to local heating, which can lead to degradation of proteins. Furthermore, the plasmon response may be relatively sensitive to small (~1 nm) geometric changes of the nanoantennas, which may shift the resonance and thus decrease the sensitivity of the device. Hence, the reliable nanofabrication of complex plasmonic shapes with well-defined structures may be critical for such approaches, yet may be difficult to control and consistently reproduce. Moreover, the tight focusing of the optical light makes plasmonic sensing less easily scalable as it may require a high power laser beam incident on the nanopore.

The prior art may further describe nanopores integrated with optical tweezers with a combination of high-resolution ionic current measurements and optical forces to control the position and translocation speed of biomolecules, particularly in relation to polynucleotides, attached to polystyrene beads. However, the technique may be low-throughput and limited to observing one molecule at the time. Additionally, it may require a tightly focused laser beam for bead trapping, which may limit the potential integration with optical detection schemes sensitive to photobleaching.

Hence, it is an aspect of the invention to provide an alternative method for measuring a protein, which method preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

In a first aspect, the invention may provide a measurement method (or “method”) for measuring a linearized protein using a 2D material membrane. In particular, the protein may be functionally coupled, such as tagged with, a protein optical emitter. In embodiments, the 2D material membrane may comprise a (layered) material selected from the group comprising a hexagonal boron nitride and a transition metal di chalcogenide. In further embodiments, the 2D material membrane may comprise a rim defining a membrane nanopore, especially wherein the rim comprises a membrane optical emitter (site). The protein optical emitter and the membrane optical emitter may especially form FRET donor-acceptor pair emitters having a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range. The measurement method may further comprise a measurement stage. The measurement stage may comprise passing the linearized protein through the membrane nanopore. The measurement stage may further comprise providing donor excitation radiation having a wavelength selected from the donor excitation radiation range, especially to the membrane nanopore, and especially during the passing of the linearized protein through the nanopore. The measurement stage may further comprise measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal.

Hence, the measurement method may comprise passing a linearized protein associated with a protein optical emitter through a membrane nanopore while providing donor excitation radiation (to the membrane nanopore), wherein a membrane optical emitter is arranged in the vicinity of the membrane nanopore, especially in the rim of the membrane nanopore, and wherein the protein optical emitter and the membrane optical emitter form a FRET pair. Hence, as the protein passes through the membrane nanopore, emission (radiation) from the membrane nanopore may vary as the distance between the protein optical emitter and the membrane optical emitter changes. Thereby, the emission signal as the protein passes through the membrane nanopore may be characteristic of the specific protein. In particular, a (time) trace of the emission as the protein passes through the membrane nanopore may be a protein fingerprint. In embodiments, the protein may comprise a plurality of tagged amino acids, especially (all) amino acids of one or more amino acid types. Thereby, for instance, FRET may occur whenever such amino acid passes through the membrane nanopore, providing the relative locations of such amino acids along the length of the protein.

In particular, the invention may facilitate the characterization, especially the identification, of a biomolecule, especially a protein, through the adoption of a 2D material membrane with an integrated optical emitter. The invention may especially relate to a (monolithic) 2D material membrane comprising a membrane nanopore, the 2D material membrane featuring an optical emitter, especially at the rim of the membrane nanopore. Thereby, the 2D material membrane can both confine the protein and detect (via FRET) labelled residues along the protein chain during biomolecule translocation. This localized interaction may elicit a strong optical response, acting as a sensitive, real-time molecular scanner. Further, the membrane optical emitter in the 2D material membrane may provide a (relatively) high quantum yield, a greater photostability and a longer fluorescent lifetime relative to conventionally used FRET probes.

Further, the labelling of one or a few subsets of amino acid types may be sufficient to provide a protein-specific fingerprint (also see below). The protein fingerprint may enable identification of a protein, with more labelled subsets of amino acid types providing a stronger, less error-prone fingerprint. For instance, 90% of human proteins can be correctly identified with reference to a human proteome database by the order in which labelled cysteine (C) and lysine (K) residues appear, while this number increases to 99% when C, K and methionine (M) residues are labelled. The identification of a protein based on the protein fingerprint may, for instance, be performed such as described in Yao et al., “Single-Molecule Protein Sequencing through Fingerprinting: Computational Assessment”, Physical Biology, 2015, or such as described in Ohayon et al., “Simulation of single-protein nanopore sensing shows feasibility for whole-proteome identification”, PLoS Computational Biology, 2019, which are hereby herein incorporated by reference.

Hence, in specific embodiments, the invention may provide a measurement method for measuring a linearized protein using a 2D material membrane, wherein: the protein is functionally coupled to a protein optical emitter; the 2D material membrane comprises a rim defining a membrane nanopore, wherein the rim comprises a membrane optical emitter; the protein optical emitter and the membrane optical emitter form FRET donor-acceptor pair emitters having a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range; a measurement stage comprises: (i) passing the linearized protein through the membrane nanopore; (ii) providing donor excitation radiation having a wavelength selected from the donor excitation radiation range; and (iii) measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal.

Hence, the invention may provide a measurement method for measuring a protein, especially a linearized protein, and to provide a related emission signal. The measurement method may especially involve measuring a protein to determine a characteristic signal. The term “characteristic signal” may herein refer to a measurement of an interaction between the protein and the membrane, especially between the protein optical emitter and the membrane optical emitter. In particular, the characteristic signal may comprise an emission trace signal, i.e., a measurement over time of the emission (radiation) from the membrane nanopore, wherein the interaction between the protein and the nanopore results in modulation of the emission radiation. For example, the protein may pass through (also: “translocate”) the nanopore, resulting in a modulation of the emission due to FRET as the protein moves. Hence, in embodiments, the characteristic signal may comprise an emission trace signal. In further embodiments, the characteristic signal may comprise a protein fingerprint, such as a protein fingerprint based on the emission trace signal. In further embodiments, the characteristic signal may comprise a protein identity, such as based on the emission trace signal, or such as based on the protein fingerprint.

The term “protein” may refer herein to a chain of amino acids of any length, especially a chain of at least two amino acids, more especially a chain of ten or more amino acids. The term protein may thus also refer to a peptide, oligopeptide, and polypeptide. In embodiments, the protein may comprise a peptide, especially wherein the peptide comprises a number of amino acids selected from the range of 2 - 50. In embodiments, the linearized protein may comprise at least 30 amino acids, such as at least 50 amino acids, especially at least 100 amino acids.

For (high-confidence) protein identification (also see below), it may be beneficial for the linearized protein, especially a plurality of amino acids of the linearized protein, to be functionally coupled to a plurality of protein optical emitters. In particular, when more protein optical emitters are functionally coupled to the linearized protein, especially to specific amino acids of the linearized protein, more information about the linearized protein may be obtained from the measuring of the emission.

Hence, in embodiments, the linearized protein may comprise N1 amino acids, such as wherein N1 is at least 30, especially at least 50, such as at least 100, wherein the linearized protein is functionally coupled to at least 0.05*Nl protein optical emitters, such as at least 0.08*Nl, especially at least 0.1*Nl. In further embodiments, the linearized protein may be functionally coupled to at least 0.13*Nl protein optical emitters, such as at least 0.15*Nl. In further embodiments, at least 0.05*Nl amino acids of the linearized protein may be functionally coupled to a (respective) protein optical emitter, such as at least 0.08*Nl, especially at least 0.1*Nl. In further embodiments, at least 0.13*Nl amino acids of the linearized protein may be functionally coupled to a (respective) protein optical emitter, such as at least 0.15*Nl.

In further embodiments, the linearized protein may be functionally coupled to at most Nl protein optical emitters, such as at most O.9*N1, especially at most O.7*N1. In further embodiments, the linearized protein may be functionally coupled to at most 0.5*Nl protein optical emitters, such as at most 0.3 *N1. In further embodiments, at most N1 amino acids of the linearized protein may be functionally coupled to a (respective) protein optical emitter, such as at most O.9*N1, especially at most O.7*N1. In further embodiments, at most 0.5*Nl amino acids of the linearized protein may be functionally coupled to a (respective) protein optical emitter, such as at most 0.3 *N1.

For instance, in embodiments, 0.05*Nl - 0.3*Nl amino acids of the linearized protein may be functionally coupled to a (respective) protein optical emitter.

In general, the protein may be linear, i.e., a linearized protein. A linearized protein (or “linear protein”) may pass (through) the nanopore in a linear fashion, i.e., the linear protein may pass through the nanopore in an outstretched chain. An initial protein may, for instance, be linearized through denaturation. Denaturation is a process wherein a protein unfolds to its primary structure. Herein, the term “denaturation” refers to both unfolding of the protein and disulfide bond reduction. Methods for denaturation of proteins will be known to the person skilled in the art. In embodiments, the denaturation of an initial protein may comprise exposing the initial protein to one or more denaturants, especially one or more denaturants selected from the group comprising urea, SDS, guanidinium chloride, high salt concentration, and lithium perchlorate.

The term “initial protein” may herein especially refer to a (natural) protein prior to the pretreatment stage. The initial protein may, for instance, be obtained from a biological sample.

The protein may be functionally coupled to a protein optical emitter. In particular, an amino acid of the protein may be functionally coupled to the protein optical emitter. In embodiments, the protein may comprise the protein optical emitter, such as via a covalent bond between the protein optical emitter and an amino acid of the protein. In further embodiments, the protein may be associated with the protein optical emitter, such as via a tag connected to an amino acid of the protein, wherein the tag (non-covalently) associates with the protein optical emitter.

The protein may especially comprise a tagged amino acid, i.e., an amino acid connected to a tag, especially covalently connected to a tag. The term “tag” (also: “label”) may herein refer to a non-natural modification, i.e., a modification that was deliberately applied to the protein for measurement. In embodiments, an amino acid of a specific type, such as a cysteine, in the protein may be deliberately tagged. In further embodiments, especially (essentially) each amino acid of a specific type, such as each cysteine, may be tagged. In such an embodiment, it may be possible to identify the positions of the tagged type of amino acid in the protein as the protein passes (through) the nanopore, which may substantially facilitate protein identification. In particular, the tag may comprise or be associated to a protein optical emitter. The term “tag” may also refer to a plurality of (different) tags. Especially, different (types of) amino acids in the protein may be tagged with different tags, such as wherein the different tags comprise or associate with different protein optical emitters. For instance, each cysteine in the protein may be tagged with a first tag comprising or associating with a first protein optical emitter, and each lysine in the protein may be tagged with a second tag comprising or associating with a second protein optical emitter.

Hence, in embodiments, the linearized protein may comprise a plurality of amino acids comprising a first subset of a first amino acid type and a second subset of a second amino acid type, wherein the protein optical emitter comprises a protein optical emitter of a first type and a protein optical emitter of a second type, wherein amino acids of the first subset are functionally coupled to a (respective) protein optical emitter(s) of the first type, and wherein amino acids of the second subset are functionally coupled to a (respective) protein optical emitter(s) of the second type. In particular, the amino acids of the first subset may be tagged with first tags, wherein the first tags comprise or associate with the (respective) protein optical emitter(s) of the first type, especially wherein the first tags comprise the (respective) protein optical emitter(s) of the first type. Similarly, the amino acids of the second subset may be tagged with second tags, wherein the second tags comprise or associate with the protein optical emitter(s) of the second type, especially wherein the second tags comprise the protein optical emitter(s) of the second type. In particular, the first amino acid type and the second amino acid type may be different (amino acid types).

In further embodiments, the linearized protein comprises n subsets of different amino acid types, and the protein optical emitter may comprise n different protein optical emitter types, wherein amino acids of each of the n subsets are associated to a different protein optical emitter type of the n different optical emitter types. In particular, the different protein optical emitter types may differ in their excitation radiation range and/or their emission radiation range, especially in both. In further embodiments n may be selected from the range of 2 - 10, such as from the range of 2 - 6, especially from the range of 3 - 5, or especially from the range of 2-4. In further embodiments, n=2. In further embodiments, n=3.

Hence, in embodiments, the method of the invention may facilitate multi-color FRET analysis, i.e., an analysis comprising (simultaneously) interrogating a plurality of FRET pairs.

For instance, in embodiments, the linearized protein may comprise 2 subsets of different amino acid types: cysteine residues and lysine residues. In such embodiments, (essentially) all cysteine residues in the linearized protein may be functionally coupled to a first protein optical emitter, and (essentially) all lysine residues in the linearized protein may be functionally coupled to a second protein optical emitter. In particular, both the first protein optical emitter and the second protein optical emitter may form FRET donor-acceptor pair emitters with the membrane optical emitter. For instance, the first protein optical emitter may be a donor optical emitter with respect to the membrane optical emitter, whereas the second protein optical emitter may be an acceptor optical emitter with respect to the membrane optical emitter. Further, the membrane optical emitter may comprise a plurality of membrane optical emitters, such as a first membrane optical emitter that forms a FRET pair with the first protein optical emitter and a second membrane optical emitter that forms a FRET pair with the second protein optical emitter.

It will be clear to the person skilled in the art that the protein may comprise further (types of) amino acids, which are not associated to an optical emitter. In particular, as indicated above, a protein may be successfully identified based on the determination of the order of a subset of the amino acids in the protein.

The phrase “a first subset of a first amino acid type” and similar phrases may herein refer to a subset of the amino acids of the linearized protein, wherein the amino acids in the subset are of the same amino acid type, such as the amino acids in the subset all comprising cysteine. The subset does not necessarily comprise all amino acids of the amino acid type as, for instance, only part of the amino acids of the amino acid type may have been tagged/labelled.

In particular, it will be clear to the person skilled in the art that the tagging of a specific amino acid type may not result in the tagging of 100% of the amino acids of the amino acid type. Hence, in embodiments, the first subset of the first amino acid type may comprise at least 70% of (first) amino acids of the first amino acid type in the linearized protein, such as at least 80%, especially at least 90%, including 100%. Similarly, in embodiments, the second subset of the second amino acid type may comprise at least 70% of (second) amino acids of the second amino acid type in the linearized protein, such as at least 80%, especially at least 90%, including 100%.

Hence, in further embodiments, the linearized protein may comprise n subsets of different amino acid types, wherein each of the n subsets comprises at least 70% of amino acids of the (respective) amino acid type, such as at least 80%, especially at least 90%, including 100%.

In embodiments, the protein optical emitter may comprise a chromophore, especially a fluorophore, or a nanoparticle probe. In further embodiments, the protein optical emitter may comprise a dye compound selected from the group comprising the Cyanine family, the Alexa family, the Atto family, the Dy family, and the Rhodamine family. In further embodiments, the protein optical emitter may be selected from the group comprising Alexa 488, Alexa532, Atto488, Atto647n, Cy3, Cy3b, Cy5, and Cy7, especially from the group comprising Alexa 488, Alexa532 and Cy3.

The term “protein optical emitter” (or “first optical emitter” or “protein- associated optical emitter”) may herein refer to an optical emitter that is associated to the protein, as to distinguish it from the membrane optical emitter (or “second optical emitter” or “membrane-associated optical emitter”). Hence, it will be clear to the person skilled in the art that the term “protein optical emitter” should not be construed to imply that the protein optical emitter is proteinogenic. The term "membrane optical emitter” may herein refer to an optical emitter that is associated to the 2D material membrane. The term “optical emitter” may herein especially refer to a fluorescent or phosphorescent structure, such as a chemical compound, that upon excitation with light (e.g. radiation from a laser), emits light of a different wavelength. The FRET donor-acceptor pair optical emitters (also: “donor-acceptor pair optical emitters”) may especially comprise fluorescent structures and/or phosphorescent structures. The term “FRET donor-acceptor pair optical emitters” may herein especially refer to two optical emitters capable of FRET energy transfer, i.e., energy transfer in a non-radiative distance-dependent fashion, especially through dipole-dipole coupling of the donor optical emitter and the acceptor optical emitter.

In further embodiments, the protein optical emitter may comprise a nanoparticle probe. The nanoparticle probe may especially comprise a nanoparticle defect (providing optical emission characteristics). In particular, in embodiments, the nanoparticle probe may comprise a 2D material, especially a 2D material quantum dot. For instance, 2D material quantum dots may be obtained with the processes described in Duong et al., “Facile Production of Hexagonal Boron Nitride Nanoparticles by Cryogenic Exfoliation”, Nano Letters, 2019, and in Wang et al., “Cryo-mediated exfoliation and fracturing of layered materials into 2D quantum dots”, Science Advances, 2017, which are hereby herein incorporated by reference. For instance, such a nanoparticle probe may be attached to an (initial) protein via maleimide chemistry or click chemistry. The 2D material nanoparticles can be produced by cryogenic exfoliation of commercially available 2D material powders. The term “2D material membrane” (or two-dimensional material membrane”) may herein refer to a crystalline material consisting of single- or few-layer atoms, in which the in-plane interatomic interactions are much stronger than those along the stacking direction.

In particular, in embodiments, the 2D material membrane may comprise an insulator or semiconducting layered material selected from the group comprising hexagonal boron nitride and transition metal dichalcogenides, especially tungsten disulfide. 2D material (or “Single-layer material“) may comprise crystals comprising a single layer of atoms. The atoms in the single layer can be of the same element (e.g. all C in graphene) or compounds of 2 or more elements (e.g. B and N in hBN). The atoms may be arranged in a hexagonal lattice on a single plane (graphene, and hBN) or on three atomic planes as in transition metal dichalcogenides (TMDs). TMDs refer to 2D materials with the chemical formula MX2, wherein M represents a transition metal atom (e.g. Mo, W) and X represents a chalcogen atom (S, Se, Te). In TMDs, a metal layer may be sandwiched between chalcogen layers. 2D materials may be held together by strong in-plane covalent bonds, while weaker out-of-plane van der Waals bonds may exist between layers of 2D material in a vertical multilayer stack. For these reasons, 2D materials may be relatively easily exfoliated into thinner crystals and down to a single layer. Atomically-thin films can also be grown catalytically over large areas (~mm²) typically on metal films and foils (Cu, Fe, Ni, Pt, Pd, Co).

In embodiments, the 2D material membrane may especially comprise a (layered) van der Waals (vdW) material.

In further embodiments, the 2D material membrane may comprise boron nitride, especially hexagonal boron nitride. In further embodiments, the 2D material membrane may comprise a transition metal di chalcogenide, especially tungsten disulfide.

The 2D material membrane may especially comprise a rim defining a membrane nanopore, i.e., the 2D material membrane may comprise a membrane nanopore bordered by a rim. Hence, the 2D material membrane may comprise a membrane nanopore. A membrane nanopore may be a pore with an (equivalent circular) diameter in the nanometer range, for example with a (smallest) circular equivalent diameter selected from the range of 0.01 - 100 nm. The nanopore may be comprised by and may be formed by a solid state nanopore (a hole in a synthetic material), especially by a 2D material membrane. The membrane nanopore may especially comprise a through-hole through the membrane comprising the membrane nanopore. The term “membrane nanopore” may herein also be referred to as “nanopore”. The equivalent circular diameter (or ECD) (or “circular equivalent diameter”) of an (irregularly shaped) two-dimensional shape is the diameter of a circle of equivalent area. For instance, the equivalent circular diameter of a square with side a is 2*a*SQRT(l/7t). For a circle, the diameter is the same as the equivalent circular diameter. Would a circle in an xy- plane with a diameter D be distorted to any other shape (in the xy-plane), without changing the area size, then the equivalent circular diameter of that shape would be D.

Hence, in embodiments, the nanopore may be comprised by or may be formed by a 2D material membrane (or “membrane”). Especially, the 2D material membrane may comprise a channel through which a biomolecule, such as a protein, could pass. In further embodiments, the nanopore may be arranged in a membrane, especially such that a biomolecule, such as the protein, could pass from a first side of the membrane via a channel in the nanopore to a second side of the membrane.

In embodiments, the 2D material membrane, especially the rim, may comprise a membrane optical emitter. In particular, the 2D material membrane may comprise a crystal defect structure (or “defect”), especially an in-plane crystal defect structure, providing (or “resulting in”) a membrane optical emitter (or “membrane optical emitter site”). Defects in 2D materials may substantially influence the electrical, optical, mechanical and/or magnetic properties of the 2D materials, and may result in the introduction of optical emitters. In particular, the defect may be selected from the group comprising an impurity, a vacancy, and a vacancy complex. With regards to hexagonal boron nitride, for instance, the defect may be selected from the group comprising a single point defect (negatively charged boron vacancies (VB~), an N vacancy, an antisite defect (VNNB), and a substitutional defect complex VBC“N.

Prominent (i.e. frequently observed) defects in hBN crystals may have emission peaks at (about) 580 nm and at (about) 623 nm. The former may be tentatively attributed to negatively charged boron monovacancies, while the latter may be tentatively ascribed to nitrogen antisite defects. Carbon implantation may also generate defects in the visible spectral range that may affecting the intensity of the emission peak at (about) 580 nm (e.g. VBC-N).

Hence, in embodiments, the 2D material membrane may comprise hexagonal boron nitride, and the membrane optical emitter may have an emission radiation range with a peak wavelength selected from the range of 500 - 700 nm, such as from the range of 550 - 650 nm, especially from the range of 575 - 585 nm, or such as from the range of 618 - 628 nm. In further embodiments, the membrane optical emitter may have a peak wavelength in the emission radiation range of (about) 580 nm or (about) 623 nm, especially (about) 580 nm, or especially (about) 623 nm.

In further embodiments, the 2D material membrane may comprise hexagonal boron nitride, and the 2D material membrane may comprise a defect structure selected from the group comprising a vacancy, especially a negatively charged boron monovacancy, an antisite, especially a nitrogen antisite, and an impurity, especially a carbon impurity.

In further embodiments, the membrane optical emitter may comprise a roomtemperature membrane optical emitter, i.e., a membrane optical emitter that can emit photons at room-temperature. Such membrane optical emitter may be particularly suitable in the context of the present invention in view of ease of operation and the stability of the protein during measurement.

For instance, a defect structure in hexagonal boron nitride may give rise to a room-temperature emitter, which may form a FRET pair with a protein optical emitter. Hexagonal boron nitride (hBN) may be a wide band-gap 2D material, which can host a broad range of deep-trap crystallographic defects that act as ultrabright (-4000 kcts/s), highly photostable, room-temperature optical emitters. In particular, hBN defects may provide optical emitters with high quantum efficiencies, which may be suitable for FRET. Further, hBN optical defects can display narrow spectral linewidths and maintain their photophysical properties in liquid and in (relatively) harsh chemical environments, making them more robust optical labels with respect to dye molecules for applications in bioimaging/sensing in physiological conditions. The fluorescent lifetime (-3 ns) of the optical emitters may also compare favorably with conventionally used organic dyes (-0.3-1 ns).

Similarly, defects in transition metal di chalcogenide 2D materials can also provide membrane optical emitters with a range of emission wavelengths, such as in the visible wavelength range.

The measurement method may especially relate to the use of FRET donoracceptor pair optical emitters. The term “FRET” (Forster Resonance Energy Transfer) may herein refer to the transfer of the energy of a donor optical emitter to an acceptor optical emitter, which may occur when the donor-acceptor pair optical emitters are within a predetermined distance range, such as within several nanometers. Hence, the FRET donor-acceptor pair optical emitters may comprise a first optical emitter and a second optical emitter, wherein the FRET donor-acceptor pair optical emitters have a donor excitation radiation range (or “donor excitation radiation wavelength range”), an acceptor excitation radiation range (or “acceptor excitation radiation wavelength range”), a donor emission radiation range (or “donor emission radiation wavelength range”) and an acceptor emission radiation range (or “acceptor emission radiation wavelength range”), wherein one of the FRET donor-acceptor pair optical emitters (or “donor emitter”) is excitable by donor excitation radiation in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair optical emitters (or “acceptor emitter” is configured to provide acceptor emission in the FRET acceptor emission radiation range upon excitation with donor excitation radiation in the donor excitation radiation range of the one of the FRET donor-acceptor pair optical emitters when the first optical emitter and the second optical emitter are configured within a predetermined distance range. Hence, if the donor optical emitter and the acceptor optical emitter are arranged within a predetermined distance range, which may vary for different FRET donor-acceptor pairs, the donor optical emitter may upon excitation with donor excitation radiation transfer energy to the acceptor optical emitter, whereupon the acceptor optical emitter may emit acceptor emission radiation. This energy transfer may occur with a specific FRET efficiency depending on the (exact) distance between the donor optical emitter and the acceptor optical emitter. Hence, by measuring the FRET (transfer) efficiency, information regarding the distance between the donor optical emitter and the acceptor optical emitter is obtained. In particular, the FRET transfer efficiency may be sensitive to sub-nanometer distance changes, which may make FRET an outstanding spectroscopic ruler for probing, for example, biological systems.

Energy transfer based sensing of biomolecules may be among the most effective schemes for fast and accurate biodetection. In this field, single-molecule Forster energy transfer (smFRET) may have become a popular tool for dynamic structural biology and a ubiquitous ‘spectroscopic ruler’, providing very accurate information about distances at the singlemolecule level with high spatial (nanometer) and temporal (millisecond) resolution. In smFRET measurements, biomolecules of interest are optically labelled with a pair of probes (donors and acceptors). Energy transfer takes place through dipole-dipole interactions between an excited donor and an (non-excited) acceptor in a non-radiative process provided that: 1) the emission spectrum of the donor partially overlaps with the excitation spectrum of the acceptor, 2) the proximity of the donor and acceptor is less than a critical distance (typically in the range of <10 nm for fluorescent dye molecules), and 3) the transition dipoles of the donor and acceptor are approximately parallel. FRET efficiencies may be monitored by detecting the emission characteristics (e.g. intensity and lifetime) of the donor and acceptor. In practice, the selection criteria for donors and acceptors may include sensitivity, stability, biocompatibility, technical availability, ease of conjugation and cost. Beyond organic dyes, a range of other materials may serve as optical probes, including fluorescent proteins, inorganic nanoparticles and 2D materials.

The protein optical emitter may, in embodiments, be selected in view of their size. In particular, the protein optical emitter may be selected such that the linearized protein functionally coupled with the protein optical emitter can pass through the nanopore. Hence, the protein optical emitter may be (relatively) small.

Hence, in embodiments, the protein optical emitter may comprise one or more of an organic dye and a 2D material (nanoparticle), such as a 2D material quantum dot.

Especially, in embodiments, the protein optical emitter may comprise a 2D material (nanoparticle). 2D materials may be an emerging class of atomically-thin crystals displaying robust and tunable optical properties in the visible region and enhanced energy transfer efficiencies due to the in-plane polarization of the dipole in the material. Furthermore, they may offer ample opportunities for functionalization/conjugation through electrostatic (K- 7t) interactions or click chemistry, low-cost and high-yield production, as well as good biocompatibility. Further, a 2D material optical emitter may have a (relatively) narrow photoluminescence spectrum, which may facilitate the simultaneous use of a plurality of (distinct) protein optical emitters, which may, for instance, facilitate measuring a larger number of subsets of different amino acid types. Hence, in embodiments, the protein optical emitter may especially comprise a 2D material (nanoparticle), such as a 2D material quantum dot.

In embodiments, the protein optical emitter may be the one of the FRET donoracceptor pair optical emitters, wherein the membrane optical emitter is the other of the FRET donor-acceptor pair optical emitters. In further embodiments, the membrane optical emitter may be the one of the FRET donor-acceptor pair optical emitters, wherein the protein optical emitter is the other of the FRET donor-acceptor pair optical emitters.

The FRET excitation and emission ranges may, for example, comprise wavelengths in the UV range, the visible light range, and/or the (N)IR range. Hence, the FRET excitation and emission ranges may, for example, comprises a (sub)range selected from within the range of 200 - 1500 nm, such as from the range of 300 - 900 nm, especially from within the range of 400 - 800 nm. In embodiments, the donor excitation radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, such as from the range of 300 - 900 nm, especially from the range of 400 - 800 nm. In embodiments, the donor emission radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, such as from the range of 300 - 900 nm, especially from the range of 400 - 800 nm. In embodiments, the acceptor excitation radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, such as from the range of 300 - 900 nm, especially from the range of 400 - 800 nm. In embodiments, the acceptor emission radiation range may comprise a (sub)range selected from the range of 200 - 1500 nm, such as from the range of 300 - 900 nm, especially from the range of 400 - 800 nm. The FRET excitation and emission ranges will in general depend on the used FRET pairs.

The term “predetermined distance range” and similar terms may herein especially refer to a distance range wherein FRET energy transfer can occur for the FRET donor-acceptor pair optical emitters, which may vary for different sets of FRET donor-acceptor pair optical emitters.

In embodiments, the method may comprise determining a FRET efficiency pattern based on the related emission signal. The FRET efficiency pattern may be characteristic of the protein. Especially, the FRET efficiency pattern may comprise a protein fingerprint.

It will be clear to the person skilled in the art that FRET donor-acceptor pair optical emitters may be selected that have a predetermined distance suitable for an interaction between the membrane optical emitter and the protein optical emitter as the linearized protein passes through the membrane nanopore. Hence, the FRET donor-acceptor pair optical emitters may, for instance, be selected based on the size of the membrane nanopore, such as based on the circularly equivalent diameter of the membrane nanopore. In particular, the protein optical emitter may be selected based on the size of the membrane nanopore and the absorption and emission ranges of the membrane optical emitter.

The measurement method may further comprise a measurement stage. The measurement stage may especially comprise passing the linearized protein through the membrane nanopore. The person skilled in the art will be familiar with methods for passing a protein through a nanopore, such as through a solid-state nanopore. For instance, in embodiments, the measurement method may comprise passing the linearized protein through the nanopore by imposing a voltage across the membrane nanopore, especially wherein the voltage is selected from the range of -500 - 500 mV, such as selected from the range of -200 - 200 mV. In particular, in such embodiments, the protein may be (relatively) uniformly charged to promote the translocation. The movement of the linearized protein through the membrane nanopore may be influenced not only by the voltage, but also, for instance, by tweezer techniques and/or the choice of electrolyte. For instance, ionic liquids may slow down the translocation speed of a linearized protein through a membrane nanopore. In further embodiments, the measurement method may comprise controlling the passing of the linearized protein through the membrane nanopore with tweezers, such as optical tweezers, or such as acoustic tweezers. The use of optical tweezers may result in photobleaching of (some) optical emitters. Hence, in embodiments involving optical tweezers, the measurement method may comprise spatially and/or temporally separating the provision of trapping radiation and fluorescence excitation radiation, such as donor excitation radiation.

In further embodiments, a combination of methods may be used for passing the linearized protein through the membrane nanopore, such as a combination of a current and tweezers.

In embodiments, the linearized protein may be tethered to a bead. The tethering to the bead may facilitate controlling the passage of the linearized protein through the membrane nanopore. For instance, the measurement method may comprise controlling the bead position using tweezers and electrophoretic force (to control the position of the linearized protein with respect to the bead). Such embodiments may also facilitate multi-pass recordings, i.e., measuring multiple passages of the linearized protein through the membrane nanopore by moving the linearized protein back and forth (such as by moving the bead).

Hence, in embodiments, the measurement stage may comprise passing the linearized protein through the membrane nanopore multiple times, such as at least twice, especially at least thrice.

In further embodiments, the measurement method, especially the pretreatment stage, may comprise tethering the linearized protein to a bead. It will be clear to the person skilled in the art how a protein may be tethered to a bead. For instance, the protein may be tethered to a bead via biotin-streptavidin bonding.

The measurement stage may further comprise providing donor excitation radiation having a wavelength selected from the donor excitation radiation range, especially to the membrane nanopore, or especially to the one of the FRET donor-acceptor pair emitters. In particular, the one of the FRET donor-acceptor pair emitters may absorb the donor excitation radiation and emit donor emission radiation (in the donor emission radiation range), or, if the other of the FRET donor-acceptor pair emitters is sufficiently close, may transfer energy to the other of the FRET donor-acceptor pair emitters, which may subsequently emit acceptor emission radiation (in the acceptor emission radiation range). The emission of donor emission radiation and acceptor emission radiation in a given timeframe may thus be indicative of the distance between the protein optical emitter and the membrane optical emitter.

Hence, the measurement stage may further comprise measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal. In particular, in general, the measurement stage may comprise measuring emission in the donor emission radiation range and the acceptor emission radiation range to provide the related emission signal.

The term “related emission signal” may herein refer to a signal that is related to the detected emission (radiation). In particular, the related emission signal may comprise raw and/or processed data related to the (detected) emission (radiation). In embodiments, the related emission signal may comprise a time trace of detected radiation in the donor emission radiation range and the acceptor emission radiation range.

In embodiments, the measurement stage may further comprise providing acceptor excitation radiation to the membrane nanopore, especially according to an alternativelaser excitation (ALEX) scheme. Such embodiments may provide for more reliable singlemolecule FRET detection, as described in Van Ginkel et al., “Single-molecule peptide fingerprinting”, PNAS, 2017, which is hereby herein incorporated by reference.

In embodiments, the measurement method may further comprise a pretreatment stage. In particular, the pretreatment stage may comprise providing the linearized protein from an initial protein.

For instance, in embodiments, the pretreatment stage may comprise denaturating the initial protein, such as by exposing the protein to sodium dodecyl sulfate (SDS). Exposure of the protein to SDS may further provide the benefit that the protein may be (relatively) uniformly charged, which may facilitate passing the linearized protein across the membrane nanopore via imposing a current (see above).

In further embodiments, the pretreatment stage may comprise providing a (first) tag to an amino acid in the initial protein, wherein the first tag comprises or is associated to the protein optical emitter. In particular, the pretreatment stage may comprise functionally coupling the protein to the protein optical emitter.

Hence, in embodiments, the initial protein may comprise a plurality of amino acids comprising a first subset of a first amino acid type and a second subset of a second amino acid type, and the pretreatment stage may comprise providing first tags to the first subset of the first amino acid type and second tags to the second subset of the second amino acid type. In such embodiments, the first tags may comprise or associate with protein optical emitters of a first type, especially wherein the first tags comprise the protein optical emitters of the first type. Similarly, in such embodiments, the second tags may comprise or associate with protein optical emitters of the second type, especially wherein the second tags comprise protein optical emitters of the second type.

In further embodiments, the pretreatment stage may comprise providing n different tags to n subsets of different amino acid types in the initial protein, wherein the n different tags comprise n different protein optical emitter types. Hence, in such embodiments, each amino acid of a given amino acid type may be associated to a protein optical emitter of the same protein optical emitter types, but two amino acids of two different amino acid types may be associated with different protein optical emitter types.

During the passing of the linearized protein through the membrane nanopore, the membrane nanopore may be arranged in a liquid solution. In embodiments, the solution may comprise a salt concentration selected from the range of 1 mM - 5M, especially of a salt selected from the group comprising NaCl, KC1, and LiCl. In further embodiments, the solution may have a pH selected from the range of 6 - 9. In further embodiments, the solution may comprise Tris and/or EDTA.

In embodiments, the measurement method may further comprise a fingerprint provision stage. The fingerprint provision stage may comprise providing a protein fingerprint based on the emission signal. The term “protein fingerprint” may herein refer to a proteinspecific (unique) signal, especially wherein the protein fingerprint is suitable for identification of the protein. Herein, the term “protein fingerprint” may especially refer to one or more of the related emission signal, (therefrom derived) FRET efficiency values, and an order of amino acids (based on the related emission signal).

It will be clear to the person skilled in the art that the protein fingerprint may vary in dependence on, for example, the used FRET donor-acceptor pair emitters and the membrane nanopore, i.e., there may be a plurality of (possible) protein fingerprints (unique) for the linearized protein in dependence on, for example, the selected FRET donor-acceptor pair emitters. Hence, in embodiments, the measurement method may comprise providing a plurality of protein fingerprints of the linearized protein by varying one or more of the FRET donor-acceptor pair emitters, such as by successively passing the linearized protein through different 2D material membranes. In further embodiments, the protein fingerprint may comprise data related to emission signals (independently) obtained using a plurality of (different) FRET donor-acceptor pair emitters.

In further embodiments, the measurement method may comprise a protein identification stage comprising identifying the linearized protein (or the initial protein) by comparing the protein fingerprint to protein-related information in reference data. Especially, the protein-related information may comprise predetermined protein fingerprints, and the protein may be identified based on a comparison between the protein fingerprint and the predetermined protein fingerprints in the reference data. In further embodiments, the protein- related information may comprise predicted predetermined protein fingerprints, which may each be predicted based on a corresponding (known) protein structure of a protein (isoform), especially wherein each is predicted based on a corresponding (known) protein sequence of a protein (isoform).

The (measurement) method according to the invention may be particularly suitable to identify different proteoforms of a protein, especially due to alternative splicing, as the method may not rely on protein fragmentation, may be versatile in the amino acids that can be tagged, and may be sensitive towards identifying “missing” amino acids due to single nucleotide polymorphisms (SNPs) and alternative splicing. The term “missing amino acid” may especially refer to an amino acid that is typically present in a certain location in the protein, but has been replaced due to a mutation or is absent due to alternative splicing. The identification of proteoforms may be important as several diseases, such as cystic fibrosis, cancer and Parkinson disease have been associated with mutations in their splicing elements that lead to alternative splicing and abnormal protein production. Similarly, the post- translational modification of proteins may be a key signature of several diseases in neurology, oncology and immunology, and post-translationally modified amino acids may be identified via the method of the invention (such as by specifically tagging post-translationally modified amino acids). The term “proteoform” may herein refer to all different forms of a protein that may be transcribed from a single protein encoding gene, wherein the difference may be due to alternative splicing and/or differences in post-translational modifications, as well as to different forms of a protein resulting from gene variations such as SNPs, i.e., the term “proteoform” may herein also refer to two proteins transcribed from two different alleles of the same gene in two different individuals.

Hence, in embodiments, the protein identification stage may comprise identifying a proteoform of the protein, especially wherein the reference data comprises protein-related information pertaining to the proteoforms. In further embodiments, the protein identification stage may comprise identifying an alternatively spliced form of the protein, especially wherein the reference data comprises protein-related information pertaining to alternative splicing.

In embodiments, the reference data may comprise an (online) database. Hence, the (measurement) method may comprise retrieving protein-related information from the reference data, especially from the (online) database.

In further embodiments, the fingerprint provision stage may comprise predicting a (partial) amino acid sequence based on the related emission signal, and may provide a protein fingerprint comprising the predicted (partial) amino acid sequence. In such embodiments, the protein-related information may comprise amino acid sequences. The amino acid sequences may especially comprise translated nucleotide sequences, and/or amino acid sequences resulting from alternative splicing.

Hence, in embodiments, the protein fingerprint may comprise a deduced (or “predicted”) (partial) amino acid sequence, wherein the protein-related information comprises reference amino acid sequences.

In a further aspect, the invention may provide a 2D material membrane as such. In particular, the 2D material membrane may be suitable for the measurement method according to the invention.

The 2D material membrane may comprise a (layered) material selected from the group comprising a hexagonal boron nitride and a transition metal di chalcogenide, such as tungsten disulfide, tungsten diselenide, molybdenum disulfide and molybdenum diselenide, especially a material selected from the group comprising hexagonal boron nitride and tungsten disulfide. In embodiments, the 2D material membrane may comprise a rim, wherein the rim defines (at least part of) a membrane nanopore. Hence, the 2D material membrane may comprise a membrane nanopore, which may be (at least partially) bordered by the rim.

The 2D material membrane may further comprise a membrane optical emitter. In particular, the 2D material membrane may comprise a defect, especially an (in-plane) crystal defect structure, providing (or “resulting in”) a membrane optical emitter. In embodiments, the membrane optical emitter may be arranged in the rim, i.e., the rim may comprise the membrane optical emitter. In particular, in embodiments, the rim may comprise the defect providing the membrane optical emitter.

As will be clear to the person skilled in the art, the linearized protein may pass through the membrane nanopore in different orientations, i.e., the linearized protein may rotate along a longitudinal axis of the linearized protein. As FRET is extremely sensitive to slight changes in distance, the occurrence of FRET, and thus the emitted radiation from the membrane nanopore, may vary depending on the orientation of the linearized protein as the protein optical emitter passes through the membrane nanopore. It may thus be beneficial to provide a plurality of membrane optical emitters in the rim, or generally in the vicinity of the membrane nanopore, to reduce the impact of the orientation of the linearized protein. In particular, it may be beneficial to have the membrane optical emitters be regularly arranged around at the rim, such as in an equidistant manner.

Hence, in embodiments, the 2D material membrane may comprise a plurality of (same) membrane optical emitters. Especially, the 2D material membrane, especially the rim, may comprise a plurality of defects providing a plurality of (respective) membrane optical emitters. Hence, in embodiments, the rim may comprise (a plurality of defects providing) a plurality of membrane optical emitters. In further embodiments, the plurality of membrane optical emitters may be regularly arranged around the rim, such as (essentially) equidistant to one another.

Similarly, it may be beneficial to provide a plurality of different membrane optical emitters in the vicinity of the membrane nanopore, especially in the rim, such that more protein optical emitters can be identified via respective FRET pairs.

Hence, in embodiments, the 2D material membrane may comprise a plurality of different membrane optical emitters. Especially, the 2D material membrane, especially the rim, may comprise a plurality of different defects providing a plurality of (respective) different membrane optical emitters. Hence, in embodiments, the rim may comprise (a plurality of defects providing) a plurality of different membrane optical emitters.

The membrane optical emitter may especially have an emission radiation range with a peak wavelength selected from the range of 200 - 1500 nm, such as from the range of 300 nm - 900 nm, especially from the range of 400 - 800 nm.

For instance, in embodiments, the (layered) material may comprise hexagonal boron nitride, and the membrane optical emitter may have an emission radiation range with a peak wavelength selected from the range of 300 - 900 nm. There may be several fluorophores that also span this range and could serve as a complementary probe for FRET.

The membrane nanopore may have an (in-plane) size suitable for a linearized protein to pass through the membrane nanopore, while also facilitating an interaction between the protein optical emitter and the membrane optical emitter. In particular, the size of the membrane nanopore may be sufficient that the linearized protein fits through, but is not so large that the linearized protein may pass through without the protein optical emitter coming close enough to the membrane optical emitter for FRET.

Hence, in embodiments, the membrane nanopore may have a circularly equivalent diameter selected from the range of 0.1 - 20 nm, such as from the range of 5 - 20 nm, especially from the range of 10 - 20 nm, such as from the range of 10 - 15 nm.

Generally, the membrane nanopore may have a circular shape (in-plane with the 2D material membrane). However, other shapes are not excluded. For instance, in embodiments, the membrane nanopore may have a shape selected from the group comprising a triangular shape, a square shape, and a hexagonal shape.

The inclusion of the membrane optical emitter in the rim of the membrane nanopore may be particularly convenient, as thereby the membrane optical emitter is close to the channel through which the linearized protein passes, which facilitates FRET, but also as the membrane nanopore and the defect resulting in the membrane optical emitter may be provided in a single step (see below). However, in embodiments, the membrane optical emitter may also be arranged outside of the rim, i.e., further removed from the membrane nanopore. The membrane optical emitter may especially be arranged at a distance from the nanopore suitable for FRET to occur as a linearized protein (functionally coupled with a protein optical emitter) passes through the membrane nanopore. Hence, in embodiments, the membrane optical emitter may, such as in-plane of the 2D material membrane, be arranged at a first distance dl from a center of the membrane nanopore, especially wherein the first distance dl < 20 nm, such as < 15 nm, especially < 10 nm, such as < 8 nm. In further embodiments, the first distance di may be at least 3 nm, such as at least 4 nm, especially at least 5 nm, such as at least 6 nm.

In embodiments, the 2D material membrane may be a monolayer material. However, in further embodiments, the 2D material membrane may be a multilayer material, such as comprising a plurality of layers of the same (layered) material, or such as comprising a plurality of layers of different materials. For instance, in embodiments, the 2D material membrane may have a thickness of at most 200 nm, such as at most 100 nm, especially at most 60 nm. In further embodiments, the 2D material membrane may have a thickness of at least one monolayer.

In further embodiments, the 2D material membrane may be a monolayer obtained with chemical vapor deposition (CVD).

In embodiments, the 2D material membrane may comprise a plurality of rims defining a plurality of membrane nanopores, especially wherein each rim comprises a membrane optical emitter, such as each rim comprising a plurality of (regularly arranged) membrane optical emitters. Thereby, a plurality of linearized proteins may be analyzed simultaneously.

In a further aspect, the invention may provide a nanopore device. The nanopore device may especially comprise the 2D material membrane according to the invention.

In embodiments, the nanopore device may comprise a flow cell, especially wherein the 2D material membrane is arranged in the flow cell. In particular, the 2D material membrane may be arranged between two flow cell chambers, i.e., the two flow cell chambers may share the 2D material membrane. In particular, the two flow cell chambers may be fluidically separated but for the membrane nanopore in the 2D material membrane.

In embodiments, the two flow cell chambers may be configured for hosting an electrolyte, especially the (liquid) solution (see above).

In embodiments, the nanopore device may comprise a first electrode arranged in a first of the two flow cell chambers and a second electrode arranged in a second of the two flow cell chambers, wherein the electrodes are configured for providing a voltage over the 2D material membrane. In particular, by applying the voltage, the linearized protein may be passed through the membrane nanopore. In a further aspect, the invention may provide a system for measuring a linearized protein. The system may especially comprise one or more of the nanopore device according to the invention, a sample handler, a radiation source, a single-molecule fluorescence microscope, and a control system. Especially, in embodiments, the system comprises the nanopore device, the sample handler, the radiation source, the single-molecule fluorescence microscope, and the control system.

In embodiments, the sample handler may be configured to provide the linearized protein to the nanopore device, especially to a first of the two flow cell chambers, such as to a cis side of the membrane nanopore.

In further embodiments, the radiation source may be configured to provide donor excitation radiation to the membrane nanopore (of the 2D material membrane), especially to the rim of the membrane nanopore.

In further embodiments, the single-molecule fluorescence microscope may be configured to measure emission (radiation) in a donor emission radiation range and/or in an acceptor emission radiation range and to provide a related emission signal to the control system. In general, the single-molecule fluorescence microscope may be configured to measure emission (radiation) in the donor emission radiation range and in the acceptor emission radiation range. For instance, the single-molecule fluorescence microscope may be configured to detect emission (radiation) in the range of 200 - 1500 nm, such as in the range of 300 - 900 nm, especially in the range of 400 - 800 nm. The single-molecule fluorescence microscope may especially be configured to detect the emission from the membrane nanopore (in the 2D material membrane in the nanopore device).

In embodiments, the single-molecule fluorescence microscope may especially comprise a single point scanning confocal microscope or a spinning disk confocal microscope, especially a spinning disk confocal microscope. A spinning disk confocal microscope may be particularly suitable for multiplexing, such as for embodiments wherein the 2D material membrane comprises a plurality of membrane nanopores.

In further embodiments, the radiation source may also be configured to provide acceptor excitation radiation to the membrane nanopore, especially according to the alternativelaser excitation (ALEX) scheme (see above). The control system may especially be configured to control one or more of the nanopore device, the sample handler, the radiation source, and the single-molecule fluorescence microscope.

The term “controlling” and similar terms herein may especially refer at least to determining the behavior or supervising the running of an element. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc.. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element. The controlling of the element can be done with a control system. The control system and the element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise the control system. In embodiments, the control system and the element may not be physically coupled. Control can be done via wired and/or wireless control. The term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one master control system may be a control system and one or more others may be slave control systems.

In embodiments, the control system may be configured to determine a protein fingerprint based on the emission signal. In particular, in embodiments, the control system may be configured to determine a (partial) amino acid sequence based on the emission signal. Hence, in embodiments, the protein fingerprint may comprise the (partial) amino acid sequence.

In further embodiments, the control system may be configured to identify the protein by comparing the emission signal or the protein fingerprint, especially the emission signal, or especially the protein fingerprint, to protein-related information in reference data.

In particular, in embodiments, the control system may be configured to execute, in an operational mode, the measurement method according to the invention.

Hence, the system, especially the control system, may have an operational mode. The term “operational mode” may also be indicated as “controlling mode”. The system, or apparatus, or device (see further also below) may execute an action in a “mode” or “operational mode” or “mode of operation”. Likewise, in a method an action, stage, or step may be executed in a “mode” or “operation mode” or “mode of operation”. This does not exclude that the system, or apparatus, or device may also be adapted for providing another operational mode, or a plurality of other operational modes. Likewise, this does not exclude that before executing the mode and/or after executing the mode one or more other modes may be executed. However, in embodiments a control system (see further also below) may be available, that is adapted to provide at least the operational mode. Would other modes be available, the choice of such modes may especially be executed via a user interface, though other options, like executing a mode in dependence of a sensor signal or a (time) scheme, may also be possible. The operational mode may in embodiments also refer to a system, or apparatus, or device, that can only operate in a single operation mode (i.e. “on”, without further tunability).

In embodiments, the system may further comprise a pretreatment device for providing the linearized protein from an initial protein.

In further embodiments, the pretreatment device may be configured to denature the initial protein, such as by exposing the initial protein to a denaturant, such as SDS (see above).

In further embodiments, the pretreatment device may be configured to provide a first tag to an amino acid of the initial protein, especially wherein the first tag comprises or is associated with a protein optical emitter. In particular, in embodiments, the pretreatment device may be configured to provide first tags to a first subset of a first amino acid type and second tags to a second subset of a second amino acid type. In such embodiments, the first tags may comprise or associate with protein optical emitters of a first type, especially wherein the first tags comprise the protein optical emitters of the first type. Similarly, in such embodiments, the second tags may comprise or associate with protein optical emitters of the second type, especially wherein the second tags comprise protein optical emitters of the second type.

In further embodiments, the pretreatment device may be configured to provide n different tags to n subsets of different amino acid types in the initial protein, wherein the n different tags comprise n different protein optical emitter types. Hence, in such embodiment, each amino acid of a given amino acid type may be associated to a protein optical emitter of the same protein optical emitter types, but two amino acids of two different amino acid types may be associated with different protein optical emitter types. In further embodiments n may be selected from the range of 2 - 10, such as from the range of 2 - 6, especially from the range of 3 - 5, or especially from the range of 2-4. In further embodiments, n=2. In further embodiments, n=3. In a further aspect, the invention may provide a production method for providing the 2D material membrane according to the invention from a 2D material. In embodiments, the production method may comprise providing a membrane nanopore and a membrane optical emitter in the 2D material by exposing the 2D material to one or more of ion beam milling, dielectric breakdown, electron beam milling, reactive ion etching, chemical etching, and femtosecond laser milling.

For instance, hBN nanopores may be fabricated via focused ion beam (FIB) milling with gallium ions and subsequently spatially-mapped and characterized using photoluminescence (PL) confocal microscopy. The milling process may deterministically provide quantum emitters through the formation of edges in hBN. In particular, FIB milling can lead to ion implantation but may also result in ejection of B and/or N atoms from the hBN lattice, either single atoms or more complex defect structures. The nature of the defect may depend on FIB milling parameters, such as ion source, milling time, and dose.

Further, hBN and other 2D material emitters can also come in the form of nanoparticles, which may be produced by a cryo-exfoliation process. The technique may generate nanoparticles with diameters as small as 0.7 nm that exhibit sharp spectral lines, with high yields and without requiring specialized equipment.

The production method may especially comprise providing the membrane nanopore and an associated rim such that the rim comprises an optical membrane emitter.

Hence, the production method may comprise introducing a defect, especially an (in-plane) crystal defect structure, in the 2D-material membrane, especially in the rim, wherein the defect results in a membrane optical emitter.

In embodiments, the production method may further comprise treating the defect for one or more of further activating, purifying and/or stabilizing the defect. In particular, the production method may comprise exposing the defect to high temperature annealing (in vacuum, argon, hydrogen atmospheres) or to oxygen/ozone plasma. Such treatments may beneficially also reduce unwanted carbon deposits on the membrane surface.

The method and/or system may be applied in or may be part of analysis methods/sy stems of biological samples, such as protein samples, particularly in relation to (partial) protein sequencing and/or protein identification.

BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1 schematically depicts an embodiment of the measurement method. Fig. 2 schematically depicts a further embodiment of the measurement method. Fig. 3 schematically depicts an embodiment of the nanopore device. Fig. 4 schematically depicts an embodiment of the system. Fig. 5A-C and 6 schematically depict further aspects of the invention. The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Fig. 1 schematically depicts an embodiment of the measurement method for measuring a linearized protein 10 using a 2D material membrane 100. In the depicted embodiment, the protein 10 is functionally coupled to a protein optical emitter 21, such as via a first tag 31. The 2D material membrane 100 may comprise a (layered) material selected from the group comprising a hexagonal boron nitride and a transition metal di chalcogenide. In particular, in the depicted embodiment, the 2D material membrane 100 comprises a rim 115 defining a membrane nanopore 110, wherein the rim 115 comprises a (room -temperature) membrane optical emitter (site) 22. Especially, the 2D material membrane 100 may comprise a defect 120 providing the membrane optical emitter 22.

The protein optical emitter 21 and the membrane optical emitter 22 may especially form FRET donor-acceptor pair emitters 20 having a donor excitation radiation range, an acceptor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range.

The measurement method may especially comprise a measurement stage. The measurement stage may comprise passing the linearized protein 10 through the membrane nanopore 110. Fig. 1 schematically depicts the linearized protein 10 entering the membrane nanopore 110 (left) and having partially passed through the membrane nanopore 110 (right).

The measurement stage may further comprise proving providing donor excitation radiation 51, especially to the membrane nanopore 110. In particular, the donor excitation radiation 51 may have a wavelength selected from the donor excitation radiation range. Hence, one of the FRET donor-acceptor pair optical emitters 23 (or “donor emitter”) may be excitable by donor excitation radiation in the donor excitation radiation range, wherein the other of the FRET donor-acceptor pair optical emitters 24 (or “acceptor emitter”) may be configured to provide acceptor emission in the FRET acceptor emission radiation range upon excitation with donor excitation radiation in the donor excitation radiation range of the one of the FRET donor-acceptor pair optical emitters 23 when the protein optical emitter 21 and the membrane optical emitter 22 are configured within a predetermined distance range. In particular, FRET may be very sensitive to the distance between the donor and the acceptor emitter as it may scale with the 6^th power of the distance. For instance, in Fig. 1, little or no FRET may occur with emitter distance d2 depicted on the left side of the figure, but substantial FRET may occur with the emitter distance d2 depicted on the right side of the figure.

The measurement stage may further comprise measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal. As the energy transfer of FRET, and thus the emission radiation, depends on the distance between the optical emitters 21,22 as the linearized protein passes through the membrane nanopore 110, the related emission signal may provide a time trace of tagged amino acids passing through the membrane nanopore. Thereby, the related emission signal may be indicative of the order of the tagged amino acids in the linearized protein, and especially on the distances between tagged amino acids in the linearized protein.

In particular, the emission signal may be indicative of a (partial) amino acid sequence of the protein. In further embodiments, the method may comprise determining a (partial) amino acid sequence of the protein based on the emission signal, and especially providing a corresponding protein fingerprint. Hence, the protein fingerprint may comprise the (partial) amino acid sequence.

In further embodiments, the measurement method may comprise identifying the linearized protein 10 (or the initial protein 5) based on the emission signal or based on a protein fingerprint. In particular, in embodiments, the measurement method may comprise identifying the linearized protein 10 based on the deduced (partial) amino acid sequence. In further embodiments, the measurement method comprises comparing the emission signal (or the protein fingerprint) to protein-related information in reference data, such as in a reference database.

In the depicted embodiment, the material may comprise hexagonal boron nitride. In further embodiments, the layered material may, for instance, comprise a transition metal dichalcogenide. In embodiments, the measurement stage may comprise passing the linearized protein 10 through the membrane nanopore 110 via one or more of (i) imposing a voltage across the membrane nanopore 110; controlling a motion of the linearized protein 10 with optical tweezers; and controlling a motion of the linearized protein 10 with acoustic tweezers.

In Fig. 1, the 2D material membrane 100 is supported by a solid-state chip 213.

In embodiments, the protein optical emitter 21 may comprise a dye compound selected from the group comprising the Cyanine (or “Cy™) family, the Alexa (or “Alexa Fluor™”) family, the Atto (or “Atto™”) family, the Dy family, and the Rhodamine family.

Fig. 2 schematically depicts an embodiment of the measurement method, wherein the measurement method comprises a pretreatment stage for providing the linearized protein 10 from an initial protein 5. In particular, in the depicted embodiment, the initial protein 5 may be folded and untagged. In the depicted embodiment, the pretreatment stage comprises (i) subjecting the initial protein 5 to denaturation, and (ii) providing a tag 31 to an amino acid in the initial protein 5, wherein the first tag 31 comprises or is associated to the protein optical emitter 21.

In particular, in the depicted embodiment, the linearized protein 10 comprises a plurality of amino acids 11 comprising a first subset 15a of a first amino acid type and a second subset 15b of a second amino acid type, wherein the protein optical emitter 21 comprises protein optical emitters of a first type 25a and protein optical emitters of a second type 25b, wherein amino acids of the first subset 15a are functionally coupled to the protein optical emitters of the first type 25a, and wherein amino acids of the second subset 15b are functionally coupled to the protein optical emitters of the second type 25b. For visualization purposes, a relatively high number of protein optical emitters 21 may be depicted relative to the depicted amino acids. For instance, in embodiments, 5-20% of the amino acids in the linearized protein 10 may be functionally coupled to a (respective) protein optical emitters.

Hence, in the depicted embodiment, the pretreatment stage comprises providing first tags to amino acids of the first subset 15a, wherein the first tags comprise or associate with protein optical emitters of the first type 25a, and providing second tags to the amino acids of the second subset 15b, wherein the second tags comprise or associate with protein optical emitters of the second type 25b.

Fig. 3 schematically depicts an embodiment of the nanopore device 210. In the depicted embodiment, the nanopore device comprises the 2D material membrane 100. In particular, the nanopore device 210 comprises a flow cell 211, wherein the 2D material membrane 100 is arranged in the flow cell 211, especially between two flow cell chambers 212. During operation of the nanopore device 210, a biomolecule, especially the linearized protein 10, may be provided to one of the two flow cell chambers 212, especially to a cis flow cell chamber 212C, and may pass through the membrane nanopore 110 to the other of the two flow cell chambers 212, especially to a trans flow cell chamber 212T.

In embodiments, the nanopore device 210, especially the flow cell chambers 212, may be configured to host a (liquid) solution, especially an electrolyte. In embodiments, the solution may comprise a salt concentration selected from the range of 1 mM - 5M. In further embodiments, the solution may have a pH selected from the range of 6 - 9.

In embodiments, the nanopore device 210 may comprise a solid-state chip 213 configured to host the 2D material membrane 100.

Fig. 3 further schematically depicts an embodiment of the 2D material membrane 100. The 2D material membrane 100 may comprise a rim 115 defining a membrane nanopore 110, wherein the rim 115 comprises a defect 120 providing a membrane optical emitter 22. In embodiments, the membrane optical emitter 22 has an emission radiation range with a peak wavelength selected from the range of 300 nm - 900 nm. In further embodiments, the membrane nanopore 110 may have a circularly equivalent diameter selected from the range of 0.1 - 20 nm, such as from the range of 5 - 20 nm.

In particular, the enlarged view of the 2D material membrane 100 schematically depicts an example of a defect 120, which may be in the vicinity of a membrane nanopore 110.

In further embodiments, the rim 115 may comprise a plurality of defects 120 providing a plurality of (respective) membrane optical emitters 22.

In further embodiments, the 2D material membrane 100 may comprise a plurality of rims 115 defining a plurality of membrane nanopores 110, especially wherein each rim 115 comprises a (plurality of) membrane optical emitter(s) 22.

Fig. 4 schematically depicts an embodiment of a system 200 for measuring a linearized protein 10. In the depicted embodiment, the system 200 comprises a nanopore device 210, a sample handler 220, a radiation source 230, a single-molecule fluorescence microscope 240, and a control system 300. In particular, the sample handler 220 may be configured to provide the linearized protein 10 to the nanopore device 210, such as to a cis flow cell chamber 212 of the nanopore device 210. The radiation source 230 may be configured to provide donor excitation radiation 51, especially to the membrane nanopore 110 (of the 2D material membrane 100). In embodiments, the radiation source 230 may further be configured to provide acceptor excitation radiation to the membrane nanopore 110 (see above), especially by alternating between providing donor excitation radiation 51 and acceptor excitation radiation. The single-molecule fluorescence microscope 240 may be configured to measure emission radiation in a donor emission radiation range and/or in an acceptor emission radiation range from the membrane nanopore 110, and especially to provide a related emission signal to the control system 300.

In embodiments, the control system 300 may be configured to determine a protein fingerprint based on the emission signal, and especially to identify the linearized protein 10 by comparing the protein fingerprint to protein-related information in reference data.

In the depicted embodiment, the system 200 further comprises a pretreatment device 250. The pretreatment device 250 may especially be configured for providing the protein 10 from an initial protein 5. In embodiments, the pretreatment device 250 may be configured to denature the initial protein 5. In further embodiments, the pretreatment device 250 may be configured to provide a tag 31 to an amino acid 11 of the initial protein 5, especially wherein the tag 31 comprises or is associated with a protein optical emitter 21. In further embodiments, the pretreatment device 250 may be configured to tether the initial protein 5 to a bead.

In the depicted embodiment, the single-molecule fluorescence microscope 240 may especially comprise a single point scanning confocal microscope. In further embodiments, the single-molecule fluorescence microscope 240 may comprise a spinning disk confocal microscope, especially wherein the spinning disk confocal microscope is configured to detect emission (radiation) from a plurality of membrane nanopores 110.

Experiments

Unless explicitly specified otherwise, the experiments described below are performed using the following materials and methods.

Bulk hBN crystals are exfoliated using the scotch tape method to a final thickness in the 10-200 nm range. The tape with crystals is directly applied to the SiC>2/Si substrate, yielding clean surfaces with negligible polymer residues. The hBN crystals on SiC>2/Si are introduced in the focused ion beam (FIB) milling system and arrays or cavities are generated in the crystal by irradiation with a gallium (Ga) beam. After patterning, the hBN crystals are inspected with an optical microscope and photoluminescence spectra are acquired in a Raman/PL system. Subsequently the crystal substrates are immersed in MilliQ water and imaged in an inverted fluorescence microscope. Fluorescence images are acquired under 525 nm illumination.

Fig. 5A schematically depicts photoluminescence spectra obtained using following exposure of a 2D material to excitation radiation having an excitation wavelength of 514 nm. Specifically, Fig. 5 A schematically depicts intensity I (in a.u.) vs. emission wavelength X (in nm) of an hBN crystal on a SiC>2/Si substrate, wherein line LI corresponds to measurements at the milled hBN regions, and wherein line L2 corresponds to measurements of the SiC>2/Si support. As depicted in Fig. 5A, the hBN crystal has a clear emission peak at a wavelength of about 552 nm. The peak wavelength of 552 nm may be due to the gallium milling, such as due to Ga implantation. The peak at -525 nm in line L2 corresponds to the first order optical Si characteristic mode.

The used illumination conditions are compatible with single-molecule FRET with conventional fluorophore probes, such as, for example, with Atto488 or Cy3 as donor, or with Cy5 as acceptor.

Fig. 5B schematically depicts an optical image of a 2D material, specifically an hBN crystal on SiC>2/Si substrate, wherein an array of defects 120, especially cavities, has been milled into the hBN crystal. The scale bar indicates a length of 50 pm.

Fig. 5C schematically depicts the hBN crystal of Fig. 5B imaged with an epifluorescence microscope with an excitation wavelength of 525 nm. In particular, Fig. 5C depicts the detection of emission radiation from the defects 120, demonstrating that upon excitation with the excitation radiation, the defects emit emission radiation (of a different wavelength), i.e., the defects 120 are optical emitters.

Fig. 6 schematically depicts in the top panel optical transition energies E (in eV) of hBN defects calculated using spin-polarized density functional theory (DFT) with the Heyd- Scuseria-Emzerhof functional, as described in Cholsuk et al., “Tailoring the Emission Wavelength of Color Centers in Hexagonal Boron Nitride for Quantum Applications”, Nanomaterials (Basel) 2022, 12 (14), which is hereby herein incorporated by reference. The two vertical stripes represent the most frequently reported ZPL wavelengths, i.e. 585±10 nm and 623±10 nm. Specifically, line L3 corresponds to native defects, line L4 corresponds to Si- based defects, line L5 corresponds to O-based defects, and line L6 corresponds to C-based defects. Fig. 6 further schematically depicts in the bottom panel the excitation and emission spectra for various types of fluorophores in normalized intensity I vs wavelength X (in nm). The excitation and emission wavelength maxima are indicated by the vertical dotted lines. Specifically, lines L7 and L8 correspond to the excitation and emission wavelength maxima of GFP, lines L9 and LIO correspond to the excitation and emission wavelength maxima of AF488, lines Li l and L12 correspond to the excitation and emission wavelength maxima of Cy3, lines L13 and L14 correspond to the excitation and emission wavelength maxima of Cy3.5, lines L15 and L16 correspond to the excitation and emission wavelength maxima of Cy5, and lines L17 and L18 correspond to the excitation and emission wavelength maxima of AF647.

Hence, as will be clear to the skilled person, various combinations of hBn defects and fluorophores can be selected to form suitable FRET pairs.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species". Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A measurement method for measuring a linearized protein (10) using a 2D material membrane (100), wherein: the protein (10) is functionally coupled to a protein optical emitter (21); the 2D material membrane (100) comprises a rim (115) defining a membrane nanopore (110), wherein the rim (115) comprises a membrane optical emitter (22), and wherein the 2D material membrane (100) comprises a defect (120) selected from the group comprising an impurity, a vacancy, and a vacancy complex, wherein the defect (120) provides the membrane optical emitter (22); the protein optical emitter (21) and the membrane optical emitter (22) form FRET donor-acceptor pair emitters (20) having a donor excitation radiation range, a donor emission radiation range and an acceptor emission radiation range; the measurement method comprises a measurement stage comprising: (i) passing the linearized protein (10) through the membrane nanopore (110); (ii) providing donor excitation radiation (51) having a wavelength selected from the donor excitation radiation range; and (iii) measuring emission in the donor emission radiation range and/or the acceptor emission radiation range to provide a related emission signal.

2. The measurement method according to claim 1, wherein the 2D material membrane comprises a material selected from the group comprising a hexagonal boron nitride and a transition metal dichalcogenide.

3. The measurement method according to any one of the preceding claims, wherein the linearized protein (10) comprises a plurality of amino acids (11) comprising a first subset (15a) of a first amino acid type and a second subset (15b) of a second amino acid type, wherein the protein optical emitter (21) comprises a protein optical emitter of a first type (25a) and a protein optical emitter of a second type (25b), wherein amino acids of the first subset (15a) are functionally coupled to a protein optical emitter of the first type (25a), and wherein amino acids of the second subset (15b) are functionally coupled to a protein optical emitter of the second type (25b).

4. The measurement method according to any one of the preceding claims, wherein the measurement method comprises a pretreatment stage for providing the linearized protein (10) from an initial protein (5), wherein the pretreatment stage comprises one or more of: (i) subjecting the initial protein (5) to denaturation, and (ii) providing a first tag (31) to an amino acid in the initial protein (5), wherein the first tag (31) comprises or is associated to the protein optical emitter (21).

5. The measurement method according to any one of the preceding claims, wherein the measurement stage comprises passing the linearized protein (10) through the membrane nanopore (110) via one or more of: imposing a voltage across the membrane nanopore (110); controlling motion of the linearized protein (10) with optical tweezers; and controlling motion of the linearized protein (10) with acoustic tweezers.

6. The measurement method according to any one of the preceding claims, wherein the membrane nanopore (110) is arranged in a liquid solution, wherein the solution comprises a salt concentration selected from the range of 1 mM - 5M, and wherein the solution has a pH selected from the range of 6 - 9.

7. The measurement method according to any one of the preceding claims, wherein the membrane nanopore (110) has a circularly equivalent diameter selected from the range of 0.1 - 20 nm, and wherein the membrane optical emitter (22) has an emission radiation range with a peak wavelength selected from the range of 300 nm - 900 nm.

8. The measurement method according to any one of the preceding claims, wherein the protein optical emitter (21) comprises a dye compound selected from the group comprising the Cyanine family, the Alexa family, the Atto family, the Dy family, and the Rhodamine family.

9. The measurement method according to any one of the preceding claims, wherein the measurement method further comprises: a fingerprint provision stage comprising providing a protein fingerprint based on the emission signal; a protein identification stage comprising identifying the linearized protein (10) by comparing the protein fingerprint to protein-related information in reference data.

10. The measurement method according to claim 9, wherein the protein fingerprint comprises a deduced amino acid sequence, and wherein the protein-related information comprises reference amino acid sequences.

11. A 2D material membrane (100) for the measurement method according to any one of the preceding claims, wherein the 2D material membrane (100) comprises a material selected from the group comprising a hexagonal boron nitride and a transition metal dichalcogenide, wherein the 2D material membrane (100) comprises a rim (115) defining a membrane nanopore (HO), and wherein the rim (115) comprises a membrane optical emitter (22), wherein the 2D material membrane comprises a defect (120) selected from the group comprising an impurity, a vacancy, and a vacancy complex, wherein the defect (120) provides the membrane optical emitter (22), wherein the membrane nanopore (110) has a circularly equivalent diameter selected from the range of 0.1 - 20 nm, and wherein the membrane optical emitter (22) has an emission radiation range with a peak wavelength selected from the range of 300 nm - 900 nm.

12. The 2D material membrane (100) according to claim 11, wherein the rim (115) comprises a plurality of defects (120) providing a plurality of membrane optical emitters (22).

13. The 2D material membrane (100) according to any one of the preceding claims 11-12, wherein the 2D material membrane (100) comprises a plurality of rims (115) defining a plurality of membrane nanopores (110), wherein each rim (115) comprises a membrane optical emitter (22).

14. A nanopore device (210) comprising the 2D material membrane (100) according to any one of the preceding claims 11-13, wherein the nanopore device (210) comprises a flow cell (211), wherein the 2D material membrane (100) is arranged in the flow cell (211).

15. A system (200) for measuring a linearized protein (10), wherein the system (200) comprises the nanopore device (210) according to claim 14, a sample handler (220), a radiation source (230), a single-molecule fluorescence microscope (240), and a control system (300), wherein the sample handler (220) is configured to provide the linearized protein (10) to the nanopore device (210), wherein the radiation source (230) is configured to provide donor excitation radiation (51), wherein the single-molecule fluorescence microscope (240) is configured to measure emission radiation in a donor emission radiation range and/or in an acceptor emission radiation range and to provide a related emission signal to the control system (300).

16. The system (200) according to claim 15, wherein the control system (300) is configured to determine a protein fingerprint based on the emission signal, and to identify the linearized protein (10) by comparing the protein fingerprint to protein-related information in reference data.

17. The system (200) according to any one of the preceding claims 15-16, wherein the system (200) comprises a pretreatment device (250) for providing the linearized protein (10) from an initial protein (5), wherein the pretreatment device (250) is configured to: denature the initial protein (5); and/or provide a first tag (31) to an amino acid (11) of the initial protein (5), wherein the first tag (31) comprises or is associated with a protein optical emitter (21).

18. The system (200) according to any one of the preceding claims 15-17, wherein the control system (300) is configured to execute in an operational mode the measurement method according to any one of the preceding claims 1-10.

19. A production method for providing the 2D material membrane (100) according to any one of the preceding claims 12-14 from a 2D material, wherein the production method comprises providing a membrane nanopore (110) and a membrane optical emitter (22) in the 2D material by exposing the 2D material to one or more of ion beam milling, dielectric breakdown, electron beam milling, reactive ion etching, chemical etching, and femtosecond laser milling.