Protein sequencing apparatus and method

Abstract

The present invention relates to a device (2) for sequencing a protein (4) or part thereof, comprising a substrate (6) comprising a first pore (16), the pore providing or adjacent a plasmonic hotspot (22) such that a protein (4) passing through the hotspot (22) can be sequenced using Raman spectroscopy in use. A second pore (18) fluidly connected to and spaced from the first pore (16) and configured to receive the protein (4). The device (2) comprises a means (34A,B) to bias said protein (4) toward the first pore (16) and / or second pore (18), such that the protein (4) passing through the hotspot (22) is at least partially uncoiled, unfolded or otherwise linearised.

Description

[0001] Protein sequencing apparatus and method

[0002] The present invention relates to an apparatus or method of sequencing a single protein or part thereof.

[0003] Background

[0004] Current protein sequencing methods typically use mass spectroscopy. Such a method requires relatively large sample sizes due to low sensitivity of the apparatus. Mass spectroscopy apparatuses are bulky in size and are therefore not portable. Low detection sensitivity of proteins means said proteins cannot be easily characterised at single-molecule level, and so this has led to a technological lag in protein sequencing, for example, compared genome sequencing. Additionally, single-molecule DNA sequencing methods that work well in reading four DNA bases are unable to discriminate up to twenty kinds of amino acids that form the building blocks of proteins. Sequencing and / or characterisation of proteins at single-molecule sensitivity has thus remained difficult.

[0005] An improvement would be welcome. It is an aim to overcome and / or ameliorate one or more of the above problems.

[0006] Statement of Invention

[0007] According to a first aspect, there is provided: a device for sequencing a protein or part thereof, comprising: a substrate comprising a first pore, the pore providing or adjacent a plasmonic hotspot such that a protein passing through the hotspot can be sequenced using Raman spectroscopy in use; and a second pore fluidly connected to and spaced from the first pore and configured to receive the protein, and where the device comprises a means to bias said protein toward the first pore and / or second pore, such that the protein passing through the hotspot is at least partially uncoiled, unfolded or otherwise linearised.

[0008] The device may comprise a first chamber configured to receive the protein. The first chamber may be fluidly connected to the first pore. The device may comprise a second chamber. The second chamber may be fluidly connected to the second pore.

[0009] A barrier may be interposed the first and second chamber to prevent fluid transfer therebetween (e.g. other than through the first and second pore). The barrier may comprise an inert material. The barrier may comprise glass.

[0010] The first and / or second chamber comprise a hydrogel. The hydrogel may help to uncoil, unfold or otherwise linearise the protein. The hydrogel may be provided adjacent the first pore and / or second pore.

[0011] A nanoparticle is provided within the first pore. The nanoparticle may provide the plasmonic hotspot in concert with the first pore. The nanoparticle may comprise a magnetic material (e.g. magnetite). The nanoparticle may be movable toward / away from the pore. The nanoparticle may be movable via an external magnetic field to move the nanoparticle toward / away from the pore. The external magnetic field may be provided by an electromagnet.

[0012] The nanoparticle comprises a coating. The coating may comprise one or more noble metal. The noble metal may comprise one or more of: gold; silver; and / or platinum.

[0013] The first pore may comprise a concavity to provide the hotspot. The concavity may be bowl shaped and / or curved. The concavity may face toward the Raman spectroscopy laser in use. The biasing means may comprise an electrode. The electrode may be configured to generate an electric field to bias the protein toward the second pore. The biasing means may apply a tension (e.g. electrophoretic force) to the protein.

[0014] A biasing means may bias the protein toward the first pore. A second electrode may be provided to bias the protein toward the first pore. The electrodes (i.e. the first and second electrode) act in concert to control movement or the position of the protein with respect to first and / or second pore (i.e. the electrodes control the force applied to the protein toward / away from the first and second pore).

[0015] The electrodes may provide an electrophoretic / biasing force to the protein. The force may be applied to one or both ends of the protein. The electrodes may be provided in the first and / or second chambers, respectively. The electrodes may be independently controllable. The biasing means may comprise sufficient energy or force to overcome the energy barrier to uncoil or unfold the protein. The electrodes may provide said force.

[0016] The substrate may comprise an inert material. The substrate may comprise a coating. The coating may comprise one or more noble metal. The noble metal may comprise one or more of: gold; silver; and / or platinum.

[0017] The first and second pore may be provided in a common plane (e.g. parallel to one another). The protein may follow a U-shaped path in use.

[0018] The device may comprise a controller. The controller may control the voltage of the one or both electrodes and / or the electromagnet. The device may comprise a communications interface (e.g. wired or wireless).

[0019] The device comprises a microfluidic chip. The first and / or second chamber may provide a microfluidic channel. Control circuitry may control said bias. The control circuitry may comprise the electrode voltage(s). The control circuitry may automatically control said bias (e.g. to automatically control movement of the protein). Said bias may be controlled based on the collected Raman signals of the protein in the plasmonic hotspot. The control circuitry may be configured to move the protein forward and / or backward (i.e. toward / away from the second pore). The control circuitry may communicate or be synchronised with Raman spectrometer such that a Raman spectrum for a sequential positions on the protein can be captured.

[0020] According to a further aspect, there is provided: a pipette comprising the sequencing device of the previous aspect. The pipette may comprise at least two cavities. The cavities may define respective chambers. The cavities may open at an of the pipette to define pores. The end of the pipette may be tapered. At least two of the cavities may comprise an electrode.

[0021] According to a further aspect, there is provided: a system for sequencing a protein or a part thereof comprising: the device of the first aspect; a Raman spectroscopy device configured to use a laser to illuminate the nanoparticle or concavity to generate the hotspot and measure the Raman scattering signals of the protein and / or a power supply to provide electrical power and vary the voltage applied to the biasing means.

[0022] According to a further aspect, there is provided: a method of sequencing a protein or part thereof, comprising: providing a substrate comprising a first pore, the pore providing or adjacent a plasmonic hotspot; providing a second pore fluidly connected to and spaced from the first pore and configured to receive the protein, and biasing said protein toward the second pore, such that the protein passing through the hotspot is at least partially uncoiled, unfolded or otherwise linearised; and using Raman spectroscopy to determine one or more amino acid of the protein passing through the hotspot. The method may comprise moving the position of the nanoparticle. Movement of the nanoparticle may change the shape and / or position of the hotspot. Movement of the nanoparticle may be controlled using a magnetic device (e.g. an electromagnet).

[0023] The method may comprise controlling the position and / or speed of the protein through the hotspot. The position and / or speed of the protein may be controlled via electrodes in first and / or second chamber. The electrodes may vary the electrical force experienced by the protein the respective chambers. The position and / or speed of the protein may be controlled automatically. The position and / or speed of the protein may be controlled synchronously with a Raman spectroscopy apparatus.

[0024] The proteins may be denatured and / or otherwise electrically charged.

[0025] Any aspect of the invention may be combined with any other aspect of the invention where practicable.

[0026] List of drawings

[0027] Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings:

[0028] Figure 1 shows a schematic view of a first embodiment of the sequencing device;

[0029] Figure 2 a top down view of the sequencing device;

[0030] Figure 3 shows a schematic view of a control system of the device;

[0031] Figure 4 shows a schematic view of a second embodiment of the sequencing device;

[0032] Figure 5 shows a schematic view of a control system of the sequencing device; Figure 6 shows a schematic view of a user interface of the sequencing device;

[0033] Figure 7 shows a sectional view of a nanopipette comprising the sequencing device;

[0034] Figure 8 shows an electron microscope images of the nanopipette.

[0035] Description

[0036] The following embodiments are only examples. Although the specification may refer to “an” embodiment in several locations, this does not necessarily mean that each such reference is to the same embodiment(s), or that the feature only applies to a single embodiment.

[0037] The articles “a” and “an” give a general sense of entities, structures, components, compositions, operations, functions, connections or the like in this document. Note also that singular terms may include pluralities.

[0038] Single features of different embodiments may also be combined to provide other embodiments. Furthermore, words "comprising" and "including" should be understood as not limiting the described embodiments to consist of only those features that have been mentioned and such embodiments may also contain features / structures that have not been specifically mentioned. All combinations of the embodiments are considered possible if their combination does not lead to structural or logical contradiction.

[0039] A protein sequencing device 2 is shown in figure 1 . The device 2 is configured to determine an amino acid sequence of all or part of a protein 4 and / or peptide. The device 2 may therefore characterise the protein 4 or peptide, for example, for medical monitoring or diagnosis.

[0040] The device 2 comprises a substrate 6. The substrate 6 forms a base or body of the device 2. The substrate 6 may comprise an inert material. The substrate may comprise glass. For example, the substrate may comprise Silicon-nitride (SisO4). The substrate 6 may provide an electrical insulator and / or chemical barrier. The substrate 6 is generally flat / planar.

[0041] A layer 8 is provided on the substrate 6. The layer 8 coats or is provided planar to the substrate 6. The layer 8 comprises a metallic material configured to provide a plasmonic effect during Raman spectroscopic measurements. The layer 8 may comprises platinum, gold and / or silver. The layer 8 may be deposited on the substrate 6 using any suitable technique (e.g. physical vapor deposition or other vacuum deposition techniques).

[0042] A barrier 10 extends from the substrate 6. The barrier 10 comprises an impermeable material. For example, the barrier may comprise glass. The barrier effectively divides the device 2 into two different chambers 12A,B. The chambers 12A,B may form cis chambers. The two chambers 12A,B are fluidly linked by a third chamber 14. The third chamber 14 may form a trans chamber.

[0043] A first pore 16 extends though the substrate 6 and layer 8. The first pore 16 fluidly connects a first cis chamber 12A with the trans chamber 14. A second pore 18 extends though the substrate 6 and layer 8. The second pore 18 fluidly connects a second cis chamber 12B with the trans chamber 14. The first cis chamber 12A and the second cis chamber 12B are therefore fluidly connected via the trans chamber 14. The substrate 6 therefore comprises a plurality of pores 16,18. The substrate 6 may provide a porous membrane or nanopore arrangement.

[0044] The first pore 16 may comprises a diameter of between 30nm and 300nm; preferably, between 30nm and 200nm; preferably between 30nm and 70nm. The second pore 18 may comprises a diameter of between 10nm and 100nm; preferably, between 20nm and 80nm; preferably between 25nm and 50nm. The first pore 16 and / or second pore 18 may comprise nanopores (e.g. with a diameter less than 500nm; preferably, less than 250nm). The first pore 16 is larger in diameter than the second pore 18. The first pore 16 may be between 10% and 600% larger than the second pore 18; preferably, between 20% and 500%; preferably, between 25% and 70%.

[0045] The first pore 16 is spaced from the second pore 18. The first pore 16 and second pore 18 are spaced about the plane of the substrate 6 (i.e. the pores 16,18 are spaced by a direction perpendicular to the axial lengths thereof). The pores 16, 18 are provided in the same plane. The pores 16,18 are parallel. The pores 16,18 may be spaced by less than or equal to 500nm; preferably, less than or equal to 250nm.

[0046] The first pore 16 is configured to receive a particle 20. A laser 54 illuminates the particle 20 on the first pore 16 that together is configured to induce the localized surface plasmon resonance (LSPR) to generate strong localized electromagnetic field, often referred to as a plasmonic “hotspot” 22. This creates an area in which Raman scattering from a molecule is greatly enhanced, thereby increasing the detectability of Raman scattering within said molecule. The hotspot 22 is typically provided in the vicinity of the particle 20 and an edge 24 of the layer 8. The particle comprises a core 26. The core 26 may comprise a magnetic material, for example, magnetite. An outer layer 28 is provide on the core 26. Typically, the outer layer 28 comprises gold. In other embodiments, the outer layer 28 comprises platinum and / or silver.

[0047] The particle 20 is sized to be received within the first pore 16, but not pass therethrough. The particle 20 may comprise a diameter between 10% and 100% larger than that of the first pore 16. The particle 20 may comprise a nanoparticle.

[0048] A hydrogel 30 is provided in the first cis chamber 12A. The hydrogel 30 is placed adjacent or near the first pore 16. The hydrogel may comprise a network of polymers. The hydrogel 30 may occupy substantially all of the first cis chamber 12A. The hydrogel may comprise polymer materials such as agarose. A casing 32 may enclose all of the chambers 12A,12B,14. This ensures the contents thereof do not escape or are contained as desired. The chamber 14 is connected to a refence electrode 38 (e.g. ground). An electrode 34A,B is provided in each of the cis chambers 12A,B. The electrodes 34A,B provide an potential or electric field within the chambers 12A,B, as will be described in detail later. The electrodes 34A,B provide a voltage 36A,B relative to a refence electrode 38 (e.g. ground). The electrodes 34A,B are independently controllable.

[0049] The device 2 comprises a magnetic device 40. The magnetic device 40 may comprises an electromagnet. Additionally or alternatively, the magnetic device comprises a movable permanent magnet. The magnetic device 40 is operable to induce a magnetic force on the particle 20. As the particle 20 may be magnetic, the position and / or force on the particle 20 may be controlled. For example, the particle 20 may positioned toward or away from the pore 16.

[0050] The device 2 is shown in plan in figure 2. The chambers 12A,12B may define channels 42A,B or inlets / outlets to the device. For example, microfluidic channels may be defined. The channels 42A,B are separated by the barrier 10 and / or other components of the device. The particle 20 is located in the first pore 16.

[0051] Figure 3 schematically shows the electronic components of the device 2. The device 2 comprises a controller 44. The controller 44 may comprise one or more of: a processor; microprocessor; microcontroller; SoC; RAM; non-volatile memory and / or any other processing apparatus. The device 2 may be powered by a power supply 46, for example, a battery or external supply. The device may comprise a communications interface 48. This may allow transfer of data to and / or from the device. For example, this may allow remote control and / or monitoring of the device 2. The communications interface 48 may comprise a wired and / or wireless interface. The communications interface 48 may comprise one or more of: USB; Bluetooth (RTM); Wifi; cellular telephony (e.g. GSM, 3G, 4G, 5G etc.); NFC or other suitable technologies. The controller 44 is operatively connected to the electrodes 34A,B and / or magnetic device 40 to control operation thereof. The controller 44 may optionally be configured to be operatively connected a flow control unit 50. The flow control unit 50 may control flow of samples other components within, in or out of the device 2 (e.g. within or through channels 42A,B).

[0052] The device 2 typically comprises a single piece (i.e. provides a microfluid chip). This allows the device 2 to be portable. In some embodiments one or more of the controller 44, the magnetic device 40 and / or the power supply are provided integrally with the device. In other embodiments, the controller 44, the magnetic device 40 and / or the power supply 46 are form separate devices or modules for the device 2. The controller 44, the magnetic device 40 and / or the power supply 46 may then be operatively connectable via one or more interface. For example, a patch clamp amplifier may be used to control the voltages in the chambers 12A,B. In some embodiments the Raman spectroscopy apparatus may be provide with the device 2.

[0053] Operation of the device

[0054] Referring back the figure 1 , the protein 4 may be initially prepared as required according to the specific purpose and as it typically provided in the art. For example, the protein 4 may be denatured (e.g. the proteins are provided in their primary or secondary structure). The proteins 4 are therefore electrically charged. Both ends of the protein 4 are then cross-linked to a suitable DNA molecule, such that the DNA-protein-DNA construct is longer than the spacing between the first pore 16 and the second pore 18. The DNA-protein-DNA construct is placed in the first cis chamber 12A. An electrophoretic force is generated in the first cis chamber 12A by the electrode 34A to push the protein out the first pore 16 into chamber 14. Another electrophoretic force is generated by the electrode 34B to pull the protein in the chamber 14 through the second pore 18 back into the second cis chamber 12B. The hydrogel 30 causes the protein 4 to uncoil, unfold or otherwise linearise as it passes through the first pore 16. This may help to further denature the protein 4.

[0055] An electric field is applied via the electrodes 34A,B in both the first cis chamber 12A and the second cis chamber 12B, causing a electrophoretic force 52A,B for the protein into the respective chambers 12A,B. The respective forces 52A,B create an effective tensile force in the protein, helping the protein to further uncoil / linearise. The voltages 36A,B are configured to overcome the energy barrier to uncoil the protein (i.e. provide sufficient tension to overcome the coiling force). The voltages 36A,B are independently controllable, thus allowing independent control of the forces 52A,B accordingly.

[0056] Control of the forces 52A,B on the protein 4 may allow movement of the protein 4 accordingly (e.g. in a controllable, reversible and / or slow manner). For example, if force 52B is greater than force 52A, then the protein 4 will move toward the second cis chamber 12B. If force 52A is greater than force 52B, then the protein 4 will move toward the first cis chamber 12A. The controller 44 may therefore control movement of the period by driving the electrodes 34A,B accordingly. The difference between the forces 52A,B may determine the speed / acceleration of the protein 4. Similarly, control of the forces 52A,B on the protein 4 may allow control of the effective tension and / or extent of linearisation of the protein 4. Control will be typically automated to ensure accurate measurement, however, in some embodiments the user may be able to manually control the position of the protein 4.

[0057] In operation, the protein 4 is configured to pass the hotspot 22. Raman spectroscopy can then be used to characterise the protein 4 as it passes the hotspot 22. A laser 54 is used to excite the protein 4 and the emitted Raman signals are measured accordingly. A laser may be used with a circular polarisation to generate a ring-shaped hotspot. The wavelength of the laser depends on the materials of the layer 8 and the nanoparticle coating 28; for example, 785 nm for gold. Optical radiation (i.e. the laser 54) is directed toward the protein, causing the protein to emit Raman radiation. Such Raman radiation may be the result of inelastic scattering via one or more vibrational mode. This radiation can then be measured to determine a characteristic vibrational spectrum of a molecule or functional group etc. The Raman spectroscopy method is conventional and the apparatus may be a conventional or portable Raman spectrometer, which will not be described in detail.

[0058] Position of the particle 20 can be controlled via the magnetic device 40. This generates a force 55 on the particle 20. This can control the position and / or shape of the hotspot 22. This allows the device 2 to “tune” the hotspot for the appropriate protein 4 and / or device configuration. The particle 20 may be displaced away from the pore 16 during initialisation of the device 2 (e.g. where the protein 4 is initially introduced into the second cis chamber 12B). The particle 20 may then be brought into a position to generate the hotspot 22 during a sequencing stage. Typically, the particle 20 is brought close the pore 16 to provide the ring-shaped hotspot between the particle 20 and edge of the pore 16.

[0059] The data from the Raman spectroscopy may be used to characterise the protein 4, in particular to sequence the protein (i.e. determine the amino acids and their order). The measured Rama spectra may be compared with known or reference values for the desired amino acids etc.

[0060] A second embodiment of the invention is shown in figure 4. The second embodiment is on a double-barrel glass nanopipette in which the two chambers is separated by a glass septum and applied electric bias independently. In this embodiment, hydrogel 20 is provided in both the first cis chamber 12A and the second cis chamber 12B. This allows linearising of the protein 4 in both directions.

[0061] In this embodiment, a particle 20 is not provided. The substrate 6 and / or the layer 8 comprise a concavity 56 or bowl-shape. The concavity 56 faces toward the Raman spectroscopy laser 54 in use (i.e. away from the first cis chamber 12A). The concavity 56 is provided on top of the first pore 16. The concavity may extend only through the layer 8, such that the concavity 56 comprises the same material as the layer 8 (e.g. gold). The concavity 56 comprises a curved / arcuate surface. The base / centre of the concavity 56 may be aligned with the pore 16 (i.e. such that the concavity 56 is symmetric about the pore 16). The geometry of the concavity 56 is configured to provide a hotspot 22 without the use of a particle 20. This mitigates the need for the particle 20 and the corresponding magnetic device 40. Operation of device 2 is as previously described.

[0062] Referring to figure 5, control circuitry 58 is provided. The control circuitry 58 is configured to control the voltage applied to one or both of the electrodes 34A.34B. This controls the forces 52A.52B accordingly, and therefore controls the passage of the protein through the pores 16,18. The control circuitry 58 is configured to automatically control the voltage of the electrodes 34A,34B. The control circuitry 58 automatically provides the desired voltage differences between the electrodes 34A,34B to help with unfolding and to drive the protein through the pores 16,18.

[0063] The controller 44 is operatively connected to a user interface 60 to provide feedback / information to the user. The user interface 60 is shown in detail in figure 6. The user interface 60 comprise an input 62 to provide input for the desired voltage of the sensor system. A Raman spectrum 64 is provided in real time or at incremental times. The Raman spectra 66 for the protein is provided. The spectra 66 comprises a stacked image of the Raman spectrum 64 for each pass or “count”. Each pass typically represents a position along length of the molecule. The stacked image provides an intensity “heat map”, thereby allowing identification of certain elements of the protein (e.g. bases, functional groups etc.). Each pass / count is synchronised with the control circuitry 58. The control circuity 58 thus controls position of the protein based on the collected Raman spectrum of the protein in real-time such that the Raman spectrum for each position can be captured selectively and repeatedly. This process controls the movement of the protein back and / or forth in the plasmonic hot spot automatically to collect sufficient Raman signals of each amino acids of the protein in order to sequence the protein.

[0064] The applied voltage 68 for each count / pass may be shown. The data may be provided to the controller 44 from the control circuitry 58. The applied voltage 68 may provide an indication of the movement of the protein.

[0065] Referring to figures 7 and 8, the device 2 may be provided on a nano-device, for example, a nanopipette. The nanopipette comprises two cavities. The cavities extend along the length of a body 70 of the pipette (e.g. to form a channel or like). The cavities open at the end of device. The cavities are therefore fluidly connected the external environment. The open ends of the channels provide the pores 16,18 of the sequencing device. The pore / channels within the body 70 define the respective chambers 12A,12B. The pipette is tapered toward the end 72 thereof. The provides a constriction to define the pores 16,18. The end 72 may comprise a width of between 50nm and 150nm.

[0066] The cavities are separated via a barrier / septum 10. The body 70 comprise a glass material. The end 72 of the body 70 comprises a gold coating. Each chamber 12A,12B is connected respective electrodes 34A,34B, which can apply respective voltages 36A,36B as described above. The sequencing device is thus incorporated into a nanopipette. Although the device in figure 7 is shown having a bowl shaped cavity, any suitable plasmonic arrangement may be used (e.g. as shown in figure 1)

[0067] In general terms, the device 2 is configured to unfold / uncoil a protein 4 by restraining or biasing the protein 4 between two pores. In the present embodiment, the pores 16,18 are provided in the same / common plane. The protein 4 thus takes a U-shaped or concave path through the device 2. In other embodiments, the pores could be provided in a colinear arrangement (i.e. such that the protein follows a linear path) or the pores are angled relative to one another (e.g. such that the proteins follow an angled or L-shaped path).

[0068] The present arrangement allows more accurate characterisation of proteins or peptides, and allows the use of a single protein in contrast to prior art techniques, which require a large sample. The system is small and therefore may be portable.

[0069] Linearisation of the protein allows using Raman spectroscopy to characterise proteins, as the amino acid sidechains can be separated out (i.e. they do not overlap, as occurs in folded proteins).

[0070] The present arrangement provides a label-free Single-molecule protein sequencing apparatus and method. The apparatus or method uses a surface enhanced Raman spectroscopic method for sequencing a single protein or part thereof.

Claims

Claims:1 . A device (2) for sequencing a protein (4) or part thereof, comprising: a substrate (6) comprising a first pore (16), the pore providing or adjacent a plasmonic hotspot (22) such that a protein (4) passing through the hotspot (22) can be sequenced using Raman spectroscopy in use; and a second pore (18) fluidly connected to and spaced from the first pore (16) and configured to receive the protein (4), and where the device (2) comprises a means (34A,B) to bias said protein (4) toward the first pore (16) and / or second pore (18), such that the protein (4) passing through the hotspot (22) is at least partially uncoiled, unfolded or otherwise linearised.

2. A device (2) according to claim 1 , comprising a first chamber (12A) configured to receive the protein (4) and fluidly connected to the first pore (16) and a second chamber (12B) fluidly connected to the second pore (18), and a barrier (10) interposed the first and second chamber (12A,B) to prevent fluid transfer therebetween, other than through the first and second pore (16,18).

3. A device (2) according to claim 2, where the first and / or second chamber (12A,B) comprise a hydrogel (30).

4. A device (2) according to any preceding claim, where a nanoparticle (20) is provided within the first pore (16), the nanoparticle (20) providing the hotspot (22) in concert with the first pore (16).

5. A device (2) according to claim 4, where the nanoparticle (20) comprises a magnetic material and is movable via an external magnetic field to move the nanoparticle (20) toward / away from the pore (16).

6. A device (2) according to claim 4 or 5, where the nanoparticle (20) comprises a coating (28) comprising one or more noble metal.

7. A device (2) according to any preceding claim, where the biasing means comprises electrode (34B), the electrode (34B) configured to generate an electric field to bias the protein (6) toward the second pore (18).

8. A device (2) according to any preceding claim, where the first pore (16) comprises a concavity (56) to provide the hotspot (22).

9. A device (2) according to any preceding claim, where an electrode (34A) is provided to bias the protein (4) toward the first pore (16).

10. A device (2) according to claim 9, where the electrodes (34A,B) act in concert to control movement and / or the position of the protein (4) with respect to first and / or second pore (16,18).

11. A device (2) according to any preceding claim, where the substrate (6) comprises an inert material and a coating (8), the coating (8) comprising one or more noble metals.

12. A device (2) according to any preceding claim, where the first and second pore (16,18) are provided in a common plane, such the protein (4) follows a U-shaped path in use.

13. A device (2) according to any preceding claim, where the device comprises a microfluidic chip.

14. A system for sequencing a protein (4) or a part thereof comprising: the device (2) of any preceding claim; a Raman spectroscopy device configured to provide a laser to generate a hotspot (22) and measure Raman spectra of the protein (4); and a power supply (46) to provide electrical power and vary the voltage applied to the biasing means (34B).

15. A method of sequencing a protein (4) or part thereof, comprising: providing a substrate (6) comprising a first pore (16), the pore (16) providing or adjacent a plasmonic hotspot (22); providing a second pore (18) fluidly connected to and spaced from the first pore (16) and configured to receive the protein (4), and biasing said protein toward the second pore (18), such that the protein passing through the hotspot (22) is at least partially uncoiled, unfolded or otherwise linearised; andusing Raman spectroscopy to determine one or more amino acid of the protein (4) passing through the hotspot (22).5

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