Nanopore sensor using a swellable substrate for detecting molecules, use of the device and manufacturing method

Abstract

A device (1) for the detection of a molecule comprises: a substrate (2) having a predefined thickness and comprising a first upper surface (2a), an opposite first lower surface (2b) and a plurality of pores (3) extending between the first upper surface (2a) and the first lower surface (2b), and a coating layer (4) of plasmonic material, having a respective thickness, disposed on the first upper surface (2a), extending at least partly into at least one pore (3) of the substrate (2). The coating layer (4) comprises at least one passage (5) placed in communication with the at least one pore (3) of the substrate (2), such that, in use of the device (1), said passage (5) defines a path for the passage of a molecule and is configured to amplify an electromagnetic radiation and consequently generate a hotspot, so that a molecule flowing in the passage emits an electrically or optically detectable detection signal when it passes through the hotspot.

Description

[0001] NANOPORE SENSOR USING A SWELLABLE SUBSTRATE FOR DETECTING MOLECULES, USE OF THE DEVICE AND MANUFACTURING METHOD

[0002] DESCRIPTION

[0003] Technical Field

[0004] 5 The present invention relates to a device for the detection of molecules, and in particular for the identification of molecules, such as, for example, proteins and nucleic acids. The present invention also relates to a method for manufacturing said device, and to a system and method for the detection of molecules through said device. The device, system, method for detection and method for manufacturing the device of the 10 present invention are applicable in the field of personalized medicine.

[0005] Background Art

[0006] In the known art there is the need to identify certain molecules present in the organism, for example the human proteome, in order to prevent, diagnose and treat many of the existing pathologies.

[0007] 15 Traditional approaches for the detection and characterization of molecules, for example of proteins, such as mass spectrometry, X-ray crystallography, nuclear magnetic resonance spectroscopy and biochemical assays, including the ELISA assay, are associated with very high times and costs and with low yields. Moreover, such approaches require the presence of expert operators.

[0008] 20 It is also observed that the known protein labeling techniques allow, with a certain complexity, to identify only a reduced presence of proteins in a complex matrix.

[0009] For these reasons, the known methods are poorly applicable to the single person and are not applicable to personalized medicine, thus affecting its development and diffusion. It follows that the development of a simple, effective and low-cost technique for the identification of molecules and their respective sequencing could open a new paradigm in the study of the proteome, thus leading to the diffusion of preventive diagnostic assays and personalized treatments.

[0010] In the state of the art, methods based on nanopore devices have therefore been developed. Such devices comprise a substrate of plasmonic material, for example gold or silver, which has a funnel-shaped channel for the passage of the molecules. By applying a potential difference, and in particular by means of electrophoresis, it is possible to move a molecule to be detected inside said channel.

[0011] In further detail, the devices of the known art provide for the formation of a hotspot inside the channel by means of illumination with an electromagnetic radiation with an appropriate wavelength. During the passage of the molecule inside the channel, the hotspot stimulates the molecule at a certain time instant, in which the molecule emits an optically or electrically detectable signal, for example a Raman signal, configured to generate a Raman spectrum when acquired by means of Raman spectroscopy.

[0012] By way of example, it should be noted that in the detection of proteins it is possible to identify the amino acids when they are stimulated in the hotspot and, once at least part of a sequence of amino acids has been identified, it is possible to identify the specific protein present in the channel.

[0013] An example of a method for the detection and sequencing of molecules is shown in the document “Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing, ZHAO YINGQI et Al,”, in which the molecules to be detected are absorbed by a particle blocked in a channel. Examples of a device for the detection of molecules are shown, instead, in the documents US 2022 / 291194 Al, US 2012 / 142016 Al, WO 2024 / 102081 and in the document “Stochastic sensing of proteins with receptor-modified solid-state nanotechnology, Ruoshan Wei et Al”.

[0014] Problem of the prior art

[0015] The devices and methods of the prior art have a low quality of detection, and consequently of sequencing, due to an inadequate sliding of the molecules to be detected inside the channel, which causes a low-accuracy detection of the molecules. This is mainly due to the dimensional limitations of the nanopores, which therefore have dimensions that are too large with respect to the molecules to be detected and / or to the sliding speed of the molecules, which is excessively high to allow for optimal detection.

[0016] It is also observed that, in the known detection techniques that solely exploit electrical measurements to detect the signal of the molecule, the electrical measurement alone does not allow for an accurate determination of complex molecules. For example, proteins are composed of 20 different amino acids, each of which can undergo particular chemical modifications. To date, the total number of these detected modifications, called post-translational modifications, is about 400. The determination of these modifications would allow for considerable advantages in personalized medicine. However, the known devices in the state of the art do not allow to study and detect said modifications in a simple way.

[0017] In addition, devices with known plasm onic pores are highly subject to manufacturing defects. Indeed, such devices require the use of light radiation at a specific wavelength in order to generate a resonance phenomenon at a specific frequency. In the presence of a small manufacturing defect in the plasmonic device, this resonance frequency could vary, thus affecting the performance of the device and the quality of detection. It is observed that also the angle of incidence or the polarization of electromagnetic radiation constitutes an important role in the detection of molecules, and if not optimized they could also lead to the reduction of the quality of detection.

[0018] Finally, the high costs necessary to produce nanopores of specific dimensions make the known devices difficult to access.

[0019] In this regard, it should be noted that the devices described in documents US 2022 / 291194 Al, US 2012 / 142016 Al, WO 2024 / 102081 and “Stochastic sensing of proteins with receptor-modified solid-state nanotechnology, Ruoshan Wei et Al” require the artificial manufacturing of nanopores by means of costly and complex techniques, in order to allow a precise and stringent control of their size and shape. As anticipated, this results in extremely high manufacturing costs and complexity.

[0020] Summary of the invention

[0021] In this context, the technical task underlying the present invention is to propose a device for the detection of molecules, a method for manufacturing the device, a system and a method using said device that overcome the drawbacks of the known art cited above.

[0022] In particular, it is purpose of the present invention to provide a device for the detection of molecules, a system and related method capable of optimizing the size of the passage for the molecules, with reduced costs and simple manufacture.

[0023] It is also purpose of the present invention to provide a device for the detection of molecules, related system and method having a high quality of detection and sensing of molecules, and in particular of single amino acids, nucleotides or low molecular weight molecules.

[0024] It is still purpose of the present invention to provide a method for manufacturing a device for the detection of molecules that allows optimizing the size of the passage for the molecules, while maintaining low costs and high simplicity of manufacturing.

[0025] The stated technical task and the specified purposes are substantially achieved by a device, system and method for the detection of molecules and by a method for manufacturing said device comprising the technical features set forth in one or more of the appended claims.

[0026] Advantages of the invention

[0027] The device of the present invention allows to obtain at least one passage for the molecules having a size such as slowing down the translocation speed of the molecules and allowing the detection and sequencing of some constituents of the molecules, for example nucleotides of nucleic acids and amino acids of proteins.

[0028] Advantageously, the size is modifiable and adaptable to specific needs.

[0029] A further advantage of the device is that it can be used for the detection of molecules having a broad spectral response, which can be associated with an appropriate angle of incidence of the electromagnetic radiation and an appropriate polarization.

[0030] It is observed that the passage for the molecules of this device is obtainable thanks to the particular combination of a porous substrate, commonly used for example in electrophoresis techniques, and a coating layer superimposed thereon. In particular, the substrate is advantageously manufacturable with known materials, compatible with existing protocols and easily adoptable, in such a way as to reduce the costs associated with detection and facilitate the formation of the device.

[0031] Thanks to an embodiment, the substrate is able to further slow down the passage of the molecules, allowing for a more accurate and precise detection.

[0032] This device can also be combined with known labeling techniques, in order to further optimize the identification of the detected molecules.

[0033] The method for manufacturing the device is advantageously able to optimize the formation of the device itself according to the needs, and in particular according to the thickness of the coating layer.

[0034] Thanks to an embodiment, the manufacturing method is able to direct the passage for the molecules in order to have a more accurate and controlled detection thereof.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Further characteristics and advantages of the present invention will appear more clearly from the indicative, and therefore non-limiting, description of an embodiment of the present invention, as illustrated in the accompanying drawings in which:

[0037] - Figure 1 is a schematic sectional view of a first embodiment of a device according to the present invention integrated in a system according to the present invention;

[0038] - Figure 2 is a schematic sectional view of a second embodiment of a device according to the present invention in use, integrated in a system according to the present invention; - Figure 3 is a schematic sectional view of the device of Figure 2 comprising a base layer;

[0039] - Figure 4 is a schematic sectional view of a third embodiment of the device according to the present invention.

[0040] DETAILED DESCRIPTION

[0041] The present invention relates to a device 1 for the detection of molecules. The molecules to be detected are, for example, polymeric biomolecules, such as proteins of the human or animal proteome, metabolites, nucleic acids such as DNA or RNA.

[0042] It is noted that this device 1 is for example suitable to be integrated into a system for the detection of molecules, which by applying an electromagnetic radiation on the device 1 and by means of an electrophoresis process allows the translocation of a molecule to be detected through the device 1.

[0043] Such a system, also an object of the present invention, will be better detailed in the continuation of the present description. However, for ease of reading, it is anticipated that the system 100 of the present description comprises a container 10 configured to contain a swelling solution 9, for example a saline solution, and the molecule to be detected, within which the device 1 is immersed in use, a light source 11 configured to emit an electromagnetic radiation suitable to illuminate the device 1 in use, and a generator 12 configured to generate a potential difference on opposite sides of the device 1 in use.

[0044] According to a preferred embodiment, the device 1 is made integrally with the container 10 and is adapted to divide the container 10 into two distinct chambers, each containing the swelling solution 9, placed in fluid communication with each other. Preferably, the system 100 is embodied in a microfluidic device. The device 1 of the present invention comprises a substrate 2 which has a predetermined thickness along a main direction Z-Z, and comprises a first upper surface 2a and an opposite first lower surface 2b, which are preferably spaced from each other along the main direction Z-Z. In greater detail, the first upper and lower surfaces 2a, 2b are preferably transverse, and more preferably orthogonal, to the main direction Z-Z.

[0045] Within the scope of the present description, the main direction Z-Z and the further directions described below are to be considered defined as a function of the Cartesian axes system, as commonly known in the technical field.

[0046] The substrate 2 comprises a plurality of pores 3 that extend through the thickness of the substrate 2 between the first upper surface 2a and the first lower surface 2b.

[0047] According to one aspect, the pores 3 can be through-pores, thus extending along the entire thickness of the substrate 2, or blind pores, extending only partially through the thickness of the substrate 2.

[0048] At least one pore 3 of the substrate 2 extends along the entire thickness of the substrate 2 itself, placing an environment external to the first lower surface 2b in communication with an environment external to the first upper surface 2a.

[0049] The device 1 further comprises a coating layer 4, made of plasmonic material, which has a respective thickness along the main direction Z-Z and comprises a second upper surface 4a and an opposite second lower surface 4b.

[0050] Within the scope of the present description, by plasmonic material is meant a conductive material with a plasma frequency greater than the frequency of electromagnetic radiation used to illuminate the device 1 in use. Typical examples of plasmonic materials in the visible spectrum are noble metals.

[0051] The coating layer 4 is disposed on the first upper surface 2a of the substrate 2, so that the second lower surface 4b of the coating layer 4 is placed in contact with the first upper surface 2a of the substrate 2.

[0052] The coating layer 4 extends at least partially inside at least one pore 3 of the substrate 2. For the purposes of the present invention, the at least one pore 3, through which the coating layer 4 extends, extends for the entire thickness of the substrate 2 along the main direction Z-Z.

[0053] It is noted that the coating layer 4 occludes to a greater or lesser extent the at least one pore 3 depending on its own thickness. In greater detail, for a reduced thickness, the coating layer 4 only partially occludes the pore 3, while for greater thicknesses the coating layer 4 completely or almost completely occludes the pore 3, as better detailed in the continuation of the present description.

[0054] The coating layer 4 comprises at least one passage 5 placed in communication with the at least one pore 3 of the substrate 2. Preferably, the passage 5 extends at least along the main direction Z-Z through the thickness of the coating layer 4.

[0055] In use of the device 1, said passage 5 defines a path for the passage of a molecule to be detected. The passage 5 is configured to amplify an electromagnetic radiation and generate a hotspot inside the passage 5 itself when it is illuminated by said electromagnetic radiation. In this way, the molecule to be detected, by flowing into the passage 5, emits a detection signal, which can be electrically or optically detectable, when it passes through the hotspot. Preferably, the detection signal of the molecule is of an optical type. Always preferably, said signal is detectable by means of Raman spectroscopy or, alternatively, by means of fluorescence or luminescence techniques.

[0056] Alternatively, the detection signal of the molecule is of an electrical type, preferably a low-intensity current. In this case, the signal is electrically detectable, for example by means of appropriate electronic components.

[0057] Optionally, the detection signal of the molecule is detectable by means of electrical measurements.

[0058] It is noted that the molecules are configured to possess a specific charge such that, upon application of a potential difference on the device 1, they are moved through it. Optionally, should the molecules not possess an intrinsic charge, they are previously treated to acquire the required charge.

[0059] It is noted that the passage 5 of the device 1 is adapted to be excited by means of an electromagnetic radiation having a wavelength in the visible spectrum, in particular indicatively comprised between 300 nm and 1000 nm, with a respective angle of incidence and polarization. The device 1 thus described is able to work over a wide spectrum, thus having a high robustness. It is good to note that, depending on the application and the properties of the layers of the device 1, it is possible to identify an optimal combination of wavelength of the electromagnetic radiation, angle of incidence and possible polarization.

[0060] When excited, the optical energy is concentrated in the passage 5, thus entailing an increase in the electric field. Advantageously, the increase in the intensity of the electric field improves the resolution of the detection of the molecule. For simplicity of description, in the following reference will be made to a single pore 3 and to a single passage 5 in communication with said pore 3, however, embodiments in which multiple passages 5 are formed in the coating layer 4 and placed in communication with respective pores 3 are also to be considered included.

[0061] According to one aspect, the passage 5 extends for the entire thickness of the coating layer 4 along the main direction Z-Z. Alternatively, the passage 5 extends for a longitudinal extension smaller than the thickness of the coating layer 4.

[0062] According to a further aspect, in use the passage 5 extends along a first direction X-X orthogonal to the main direction Z-Z of a first extension. In addition, the passage 5 extends along a second direction Y-Y orthogonal to the main direction Z-Z and to the first direction X-X of a second extension.

[0063] According to the same aspect, the at least one pore 3 of the substrate 2, through which the coating layer 4 extends, has a first pore extension along the first direction X-X, and preferably a second pore extension along the second direction Y-Y.

[0064] Preferably, the first extension of the passage 5 is smaller than the first pore extension. Similarly, the second extension of the passage 5 is smaller than the second pore extension.

[0065] In accordance with a preferred embodiment, the first extension of the passage 5 has a maximum dimension, preferably comprised between 2 and 20 nm, more preferably between 2 and 10 nm, even more preferably between 2 and 5 nm. Preferably, the second extension of the passages has a minimum dimension equal to or greater than the maximum dimension of the first extension, more preferably equal to or greater than 2 nm. It is worth noting that, in this way, the passage 5 has a dimension sufficient to allow the passage of the molecules of interest, while maintaining a sufficiently small dimension to allow for reducing the translocation speed of the molecules.

[0066] It is worth also noting that the passage 5 thus defined, when hit by an electromagnetic radiation, is capable of generating a hotspot of extremely small dimensions, equal to or less than 1 nm.

[0067] It is observed that the dimension of the passage 5 is modifiable and adaptable according to specific needs, in particular by modifying the thickness of the coating layer 4, as better detailed in the continuation of the present description.

[0068] According to the preferred embodiment of the invention, the substrate 2 is made of gel, preferably agarose gel.

[0069] It is worth noting that that the gel naturally has pores of a specific conformation, capable of allowing the passage of molecules inside them.

[0070] In more detail, agarose gel is commonly used in the electrophoresis process and is therefore compatible with existing protocols in the known art for defining its porosity, and in particular the arrangement, frequency, size and shape of the pores. In this way, it is possible to optimize the porosity of the substrate 2 and consequently adapt the dimension of the passage 5 to the specific needs.

[0071] It is observed that the substrate 2 in gel thus acts as a template, or model, for the formation of nanopores, i.e., the passage 5 for the molecules. In this way, it is possible to avoid the use of targeted nanopore manufacturing techniques, which would significantly increase the costs and complexity of manufacturing the device.

[0072] Still advantageously, the gel is configured to slow down the molecule, which is unfolded and elongated at the entrance of the passage 5, thus improving the detection of the molecule itself and obtaining a more accurate detection of the signal emitted by each portion of the molecule.

[0073] It is noted, in fact, that by unfolding and elongating the molecule, each detection signal emitted by the molecule at a specific time instant is the one related to a single portion of the molecule itself. By slowing down the sliding of the molecule, it is therefore possible to stimulate it for a longer time, obtaining a more accurate detection signal. For example, in the case where the molecule is a protein, it is possible to detect with greater accuracy the detection signals emitted by different amino acids.

[0074] In addition, the gel substrate allows distributing the potential drop over a greater distance compared to the plasmonic layer alone and reducing the electric field applied in use, thus further reducing the speed of the molecule.

[0075] A further advantage of the agarose gel is that of having an excellent permeability to biomolecules. Therefore, it is possible to combine the excellent movement properties of the biomolecules of the agarose gel with a reduced dimension of the nanopore given by the coating layer 4.

[0076] According to an alternative embodiment, the substrate 2 is made of an intrinsically porous metallic material, for example porous aluminum.

[0077] It should be noted that any intrinsically porous material, for example commonly commercial materials, can act as the substrate 2, as it allows the creation of plasmonic passages aligned with the pores of the material itself.

[0078] According to an embodiment of the invention, the coating layer 4 is made of a metallic material, preferably one of gold, silver, gold-silver alloys. Alternatively, the coating layer 4 can be made of plasmonic compounds, possibly combinable with the aforementioned metals, including compounds based on molybdenum dichloride (MoC12). Advantageously, such compounds allow performing both electrical and optical measurements to detect the signal generated by the molecule. Always advantageously, these compounds belong to the family of two-dimensional materials, which allow creating passages having nanometric diameters and thicknesses of a few Angstroms. These types of passages have optimal properties for performing electrical measurements and, at the same time, are able to generate plasmons in the visible spectrum thanks to the optical properties of the two-dimensional MoC12.

[0079] In accordance with a first embodiment, shown in Figure 1, the thickness of the coating layer 4 is comprised between 1 and 20 nm, more preferably between 1 and 15 nm, more preferably equal to or greater than 1 nm and less than 15 nm.

[0080] According to this embodiment, the coating layer 4 only partially occludes the pore 3 of the substrate 2. It is observed that, thanks to the porous nature of the substrate 2, the coating layer 4 has discontinuities that materialize in the passage 5, formed in correspondence with the pore 3, capable of allowing the passage of the molecules.

[0081] According to an aspect of this first embodiment, the passage 5 of the coating layer 4 extends inside the at least one pore 3 of the substrate 2. In this way, the dimension of the pore 3 is considerably reduced by the presence of the coating layer 4. Thanks to the reduced dimension of the passage 5, the molecule is forced to slow down and arrange itself in such a way that only small portions can pass at a time, and in the case of proteins only one amino acid at a time.

[0082] In accordance with the first embodiment, when the substrate 2 is made of gel, it is preferably subjected to "baking", that is to say to dehydration, drying or controlled desiccation, before the coating with the coating layer 4. For the manufacturing of the device 1, the deposition of the coating layer 4 is therefore provided, for example by sputtering or by evaporator. This deposition preferably takes place in a vacuum environment, thus also allowing the last residues of liquid present in the substrate 2 to be removed.

[0083] It is good to note that the above allows the determination of the configuration of the pores 3, which are further modified by the deposition of the metal.

[0084] It is observed in fact that the gel, in contact with the metal, undergoes some deformations in the interface area between metal and gel and, above all, in correspondence with the pores 3, following the local alteration of the temperature and surface tension. This results in a further modification of the conformation of the pores naturally present in the gel. By modifying the deposition parameters, including, for example, the temperature, it is therefore possible to give rise to different conformations of the pores at the gel-metal interface.

[0085] In accordance with a second embodiment, shown in Figure 2 in use condition, the thickness of the coating layer 4 is comprised between 15 and 50 nm, preferably greater than 15 nm and less than or equal to 50 nm.

[0086] According to this embodiment, the coating layer 4 almost completely occludes the pore 3 of the substrate 2. Furthermore, the coating layer 4 is less deformable than the substrate 2.

[0087] According to an aspect of this second embodiment, the passage 5 comprises a slit that extends through the coating layer 4 and is disposed externally to the at least one pore 3, adjacent to it.

[0088] It is observed that plasmonic materials, and in particular metallic materials, when deposited on a porous substrate, such as the substrate 2, have the tendency to grow as polycrystalline materials. As is known in the state of the art, the coating layer 4, being made of a plasmonic material, for example metal, naturally has one or more slits, or cracks formed in correspondence with the junction between the grain boundaries, that is, between the single crystals. It is observed that these cracks or slits act as plasmonic nanopores.

[0089] It is worth noting that these slits materialize in the passage 5 of the coating layer 4, and in use of the device 1, as better detailed below, are able to define the path for the molecules. In detail, in use, and therefore in the presence of a buffer solution, for example a saline solution, these slits widen due to the swelling of the substrate 2, thus allowing a molecule to pass.

[0090] In accordance with an alternative, non-claimed embodiment, the coating layer 4 is substantially continuous, devoid of slits or cracks. In this embodiment, the slits, and therefore the passage 5 resulting therefrom, are formed in the coating layer 4 upon swelling of the substrate 2, i.e., when the device 1 is immersed in the swelling solution in use.

[0091] It is observed that the molecule, in use, passes through the pore 3 of the substrate 2 and directs itself inside the slit of the passage 5, which has a smaller dimension with respect to the pore 3 and thus allows reducing the translocation speed of the molecule.

[0092] In accordance with the second embodiment, when the substrate 2 is made of gel, it is preferably subjected to controlled dehydration, drying or desiccation before the coating with the coating layer 4. For the manufacturing of the device 1, the deposition of the coating layer 4 is therefore provided, for example by sputtering or by evaporator. This deposition preferably takes place in a vacuum environment. The same considerations set forth for the first embodiment regarding the deposition of the coating layer 4 on a gel substrate also apply to this second embodiment.

[0093] However, it is good to note that the device 1 of the second embodiment is subjected, following the deposition, to rehydration by immersion in a swelling solution 9. This rehydration allows restoring the nature of the gel and, as better detailed below, defining the path for the molecules.

[0094] In other words, the device 1 according to the second embodiment is obtained by deposition on the substrate 2 of the coating layer 4 and insertion of the device 1 into a swelling solution 9 to allow the substrate 2 to swell, absorbing the swelling solution 9, and the pore 3 to expand, entailing the widening of the slit defined by the passage 5, thus generating the path for the molecules. The mode of definition of the passage 5 will be better detailed in the continuation of the present description.

[0095] It should be noted that the substrate 2 can be stored both in "dry" conditions and in "swollen" conditions by means of the swelling solution 9, thus allowing the storage of the device 1 both in its first and in its second embodiment, and also in preswelling conditions of the second embodiment.

[0096] It is good to note that in both embodiments, that is, both following the swelling of the substrate 2 and of the sole deposition of the coating layer 4 on the substrate 2, the coating layer 4, and in general, the metal -gel interface, maintains the same overall optical properties, regardless of the variation in the configuration of the pores 3.

[0097] According to the first and / or the second embodiment, the device 1 can optionally comprise a particle disposed in proximity to the passage 5, internally or externally to it. In greater detail, the particle is configured to at least partially obstruct the passage 5 and / or an inlet of the passage 5.

[0098] According to this optional aspect, in use, the coating layer 4 is configured to generate the hotspot between the particle and a side wall of the passage 5 when the latter is illuminated by the electromagnetic radiation. In more detail, the particle is spaced from the side wall of the passage 5 so that the molecule flows between the particle and the side wall of the passage 5 in correspondence with the hotspot. Advantageously, this allows to further decrease the dimensions of the passage 5 through which the molecule flows.

[0099] Still optionally, the particle and / or the wall of the passage 5 can undergo a surface treatment of deposition of aluminum oxide or of organic molecules having the same sign as the molecule to be detected, in order to prevent the adhesion of the molecule and / or repel said molecule.

[0100] Still optionally, the particle is inserted into the substrate 2 and disposed in correspondence with the passage 5 by means of electrophoresis. Alternatively, the particle is inserted into the passage 5 through the second upper surface 4a of the coating layer 4.

[0101] According to an aspect of the invention, shown in Figure 4, the device 1 may comprise an outer layer 6 superimposed on the second upper surface 4a of the coating layer 4. Preferably, said outer layer 6 is made of gel. It is observed that said outer layer, when a potential difference is applied, is able to further improve the slowing effect. In fact, this layer allows distributing the potential drop over a greater distance, reducing the electrophoretic field and, consequently, the speed of the molecule. According to a further aspect, shown in Figure 3, the device 1 comprises a base layer 7 superimposed on the first upper surface 2a of the substrate 2.

[0102] Preferably, the base layer 7 is a membrane. More preferably, the base layer 7 is made of silicon nitride. Still more preferably, the base layer 7 has a thickness along the main direction Z-Z comprised between 50 and 500 nm, more preferably around 100 nm.

[0103] Always preferably, the base layer 7 comprises one or more through openings 7a, such that the coating layer 4 is disposed in at least one through opening 7a in contact with the first upper surface 2a of the substrate 2. Still preferably, each through opening 7a extends at least along the first direction X-X of an opening extension, which is greater than the pore extension of one or more adjacent pores 3. Still preferably, the opening extension of each through opening 7a has a dimension comprised between 1 and 50 micrometers.

[0104] It is observed that each of the through openings 7a are preferably aligned with at least one pore, and preferably with a plurality of pores 3 of the substrate 2, such that the passage 5 of the coating layer 4 is in communication with the at least one pore 3 of the substrate 2.

[0105] According to a further aspect of the invention, the device 1 comprises an intermediate layer, not illustrated, disposed at least in part between the substrate 2 and the coating layer 4. This intermediate layer is preferably made of a metallic material, such as for example titanium or chromium. Alternatively, the intermediate layer is made of any opaque material. Preferably, the intermediate layer has a thickness along the main direction Z-Z comprised between 2 and 10 nm. It should be noted that the intermediate layer acts as a filter during the detection of the signal generated by the molecule. In fact, this layer is adapted to filter components coming from a zone below said layer, and to prevent the generation of plasmons at the interface between the first upper surface 2a of the substrate 2 and the second lower surface 4b of the coating layer 4, which, in the case of Raman spectroscopy, would otherwise generate a Raman signal interfering with the signal to be detected.

[0106] Having described the device 1, its manufacturing method, also an object of the present invention, will now be described.

[0107] In detail, the steps of the method described below, with which the device 1 is obtained, are to be considered common to all the embodiments cited above, unless specific indications refer to a single embodiment.

[0108] The method for manufacturing the device 1 initially provides for preparing the substrate 2.

[0109] Preferably, the step of preparing the substrate 2 provides for providing a substrate 2 in gel. Always preferably, the step of preparing the substrate 2 provides for subjecting the substrate 2 to controlled dehydration, drying or desiccation for a specific time interval at a controlled temperature.

[0110] The dehydration, drying or desiccation of the gel is known in the reference sector and will not be further described.

[0111] The manufacturing method therefore comprises the step of depositing the coating layer 4 on the first upper surface 2a of the substrate 2, such that the coating layer 4 extends at least partially inside the at least one pore 3 of the substrate 2, and such that the at least one passage 5 of the coating layer 5 is in communication with the pore 3.

[0112] Preferably, the step of depositing the coating layer 4 occurs by means of a sputtering process, or sputter coating or by means of an evaporator, known in the state of the art.

[0113] According to a preferred aspect related to the second embodiment of the device 1, the method therefore provides for immersing the device 1 in a container 10 containing a swelling solution 9 to swell the substrate 2 by absorption by the substrate 2 of at least part of the solution.

[0114] With particular reference to the second embodiment of the device 1, the substrate 2, by swelling, places the coating layer 4 in a state of tension, since, as previously anticipated, such coating layer 4 is less deformable than the substrate 2. This entails the widening of the slit of the passage 5 so as to define the passage for a molecule. In other words, the grains of the coating layer 4 in correspondence with the slit separate, thus allowing the passage of the molecule.

[0115] According to an aspect of the invention, with particular reference to the second embodiment of the device, it is observed that the step of preparing the substrate provides for providing the support base 7, in accordance with what is described above.

[0116] According to the same aspect, the method provides for forming one or more through openings 7a in the support base 7. Preferably, said openings are formed by irradiation with a focused ion beam or by photolithography and the so-called reactive ion-etching technique.

[0117] Always according to the same aspect, the method therefore provides for treating a surface of the support base 7 to favor the adhesion of the substrate 2 to the support base 7. Preferably, the treatment of the support base 7 occurs by means of plasma cleaning, using a gas comprising oxygen in a percentage preferably equal to 100%.

[0118] The method therefore provides for depositing the substrate 2 on the support base 7 so that the first upper surface 2a of the substrate 2 is in contact with the support base 7. Preferably, the deposition of the substrate 2 on the support base 7 occurs by means of a spin coating technique. Always preferably, the deposition step provides for waiting for the drying of the substrate 2 at temperature for a period of time of at least 5 minutes and at 60°C for a period of time of at least 5 minutes.

[0119] According to the same aspect, the step of depositing the coating layer 4 preferably provides for depositing an adhesion layer, or molecular primer, for example a silane layer, preferably hexamethyldisilazane (HMDS), preferably by means of chemical photolithography, on the upper surface 2a of the substrate 2 and on the base layer 7, to favor the adhesion of the coating layer 4.

[0120] Always preferably, the step of depositing the coating layer 4 therefore provides for drying the adhesion layer at 60° for a period of time of at least 5 minutes and at room temperature for at least 6 hours. It is observed that in this way the substrate 2 is mechanically stabilized.

[0121] It is observed that the adhesion layer can alternatively comprise an intermediate layer of metallic material, such as titanium or chromium, or of a different opaque material. Said layer preferably has a thickness comprised between 2 and 10 nm.

[0122] It should be noted that, in the case where the intermediate layer is made of titanium or chromium, there is an increase in the signal-to-noise ratio if Raman spectroscopy is applied for the detection of the signal generated by the molecule. In greater detail, this intermediate layer allows filtering any components coming from the components placed below said layer, obtaining a signal as free as possible from unwanted components. Advantageously, it is noted that neither titanium nor chromium generate plasmons. Their use therefore avoids the generation of an unwanted Raman signal.

[0123] Always according to this aspect, the step of depositing the coating layer 4 therefore provides for depositing the coating layer 4 on the support base 7 in correspondence with at least one through opening 7a. In other words, the coating layer 4 is inserted into the through openings 7a and is placed in contact with the substrate 2. It is observed that an outermost coating layer is deposited on the base layer 7. Preferably, the deposition of the coating layer 4 occurs by means of sputtering.

[0124] It is worth noting that, with particular reference to the second embodiment of the device 1, the base layer 7 allows directing the swelling of the substrate 2, such that the pores expand along the main direction Z-Z following the absorption of the swelling solution 9 along a path defined by the through openings 7a. This allows to better direct and align the slit of the passage 5 to the pore 3, allowing a more precise formation of the passage for the molecules.

[0125] It is therefore observed that the first embodiment of the device 1 is made by means of a manufacturing method that provides for the preparation of a substrate and deposition of a coating layer on the substrate, while the second embodiment of the device 1 of the present invention is instead made by means of a manufacturing method that provides for the preparation of a substrate, the deposition of a coating layer and the immersion of the device 1 in a swelling solution 9. In greater detail, the immersion in the swelling solution 9 causes the substrate 2 to swell by absorbing the swelling solution 9, the pore 3 expands, entailing the widening of the slit defined by the passage 5, thus generating the path for the molecules.

[0126] As anticipated, a further object of the present invention is a system 100 for the detection of a molecule, which comprises a device 1 according to the present description.

[0127] The system 100 further comprises a container 10 configured to contain a swelling solution 9, preferably a buffer solution or saline solution, such as a mixture of water and sodium chloride NaCl 100 mM, and the molecule to be detected. In use, the device 1 is immersed in the swelling solution 9 inside the container 10. In this way, the substrate 2 is able to absorb at least part of the swelling solution 9 and the molecule to be detected. It is observed that, as anticipated, the substrate 2 contained in the container 10 defines two separate chambers, placed in fluid communication only through the at least one pore 3 and the respective passage 5. The container 10 thus configured allows the movement of the molecules only through said pore 3 and said passage 5, by applying a potential difference on the device 1, as better detailed below.

[0128] The system 100 also comprises a light source 11 configured to emit an electromagnetic radiation and illuminate the passage 5 of the device, so that a hotspot is generated inside the passage 5 following the amplification of the electromagnetic radiation by the latter.

[0129] Preferably, the light source 11 is a laser. More preferably, the electromagnetic radiation is monochromatic.

[0130] Advantageously, the electromagnetic radiation can have any wavelength in the visible or infrared light spectrum. Still advantageously, the electromagnetic radiation can be linearly polarized, circularly polarized (chiral polarization), i.e., endowed with angular momentum, or non-polarized. It is observed that an electromagnetic radiation with chiral polarization allows amplifying the Raman signal, thus allowing greater accuracy in the detection of the molecule.

[0131] It should be noted, in fact, that the fractal conformation of the passage 5 of the device 1 allows using any wavelength in the visible spectrum, i.e., indicatively comprised between 300 nm and 1000 nm, and respective polarization of the electromagnetic radiation.

[0132] The system 100 also comprises a generator 12 configured to generate a potential difference between the first lower surface 2b of the substrate 2 and the second upper surface 4a of the coating layer 4, so as to move the molecule in use through the at least one pore 3 and the passage 5. In more detail, the potential difference is configured to move the molecule by means of electrophoresis.

[0133] According to an aspect, it is possible to use a pulsed-type electrophoresis technique, with a radiation emitted by the light source 11 which is also pulsed, having out-of-phase pulsation times, in order to avoid interference between the two solicitations.

[0134] According to an embodiment, the electrophoresis can be performed through a two-electrode configuration. In more detail, the generator 12 comprises two electrodes 12a, 12b, preferably of silver / silver chloride (Ag / AgCl), each immersed in the swelling solution 9 in a respective chamber of the container 10, i.e., on opposite sides of the device 1. According to this configuration, one of the two electrodes is grounded, while the other electrode 12b is set to a voltage of, for example, 200 mV. The application of this potential difference allows moving the molecule through the pore 3 of the substrate 2 and through the passage 5. According to an alternative embodiment, the electrophoresis can be performed by means of a 3-electrode configuration. In more detail, the generator 12 comprises three electrodes immersed in the solution, wherein one electrode is grounded, one electrode is set to a voltage of 50 mV in proximity to the substrate 2, and the third electrode is set to 200 mV.

[0135] It should be noted that the three-electrode configuration allows controlling the movement of the molecule through the device 1 in a more accurate and precise manner.

[0136] It should be noted that by regulating the potential difference, it is possible to regulate the translocation time of the molecule, and therefore obtain more precise and accurate detections thereof.

[0137] It is noted that, should the outer layer 6, the base layer 7 or any further coating be present, the generator 12 applies a potential difference between the first lower surface 2b of the substrate 2 and the upper surface of the outermost layer, i.e., of the layer most distant from the substrate along the main direction Z-Z.

[0138] Finally, a further object of the present description is a method for the detection of molecules by means of a system 100 according to the present description.

[0139] The detection method comprises the step of inserting the device 1 into the container 10. It is observed that the device 1 can be external to the container, or integrally formed therewith.

[0140] With particular reference to the second embodiment of the device 1, the detection method therefore provides for inserting the swelling solution 9 into the container 10. In this way, the device 1 is immersed in the swelling solution 9.

[0141] Preferably, the method therefore provides for waiting a predetermined period of time, such that the substrate 2 absorbs at least part of the swelling solution 9. It is noted that for the second embodiment, this step allows the expansion of the passage 5 and the formation of the path for the molecules inside the coating layer 4.

[0142] The method therefore provides for supplying one or more molecules to be detected in the swelling solution 9.

[0143] The detection method therefore provides for illuminating the passage 5 with an electromagnetic radiation emitted by the light source 11, such as to generate a hotspot inside the passage 5 when said radiation is amplified by the passage 5.

[0144] The method therefore comprises the step of generating a potential difference between the first lower surface 2b of the substrate 2 and the second upper surface 4a of the coating layer 4, or any upper surface of the outermost layer of the device 1, by means of the generator 12 to move the molecule through the at least one pore 3 and through the passage 5 in correspondence with the hotspot.

[0145] The method therefore provides for detecting the detection signal generated by the molecule localized in correspondence with the generated hotspot.

[0146] In case the detection signal is an optical signal, said signal is detectable with instruments adapted to detect the Raman spectrum or alternatively, by means of fluorescence or luminescence spectroscopy.

[0147] In case the signal is of an electrical type, said signal is detectable by means of appropriate electronic components, for example current detection instruments.

[0148] Optionally, it is possible to detect both optical signals and electrical signals within the same experiment.

[0149] It is observed that the device 1, and therefore the system 100, of the present invention is combinable with labeling techniques to optimize the identification process. Always optionally, therefore, the method comprises the further step of labeling by means of marking the portions of detected molecule, for example the amino acids of the proteins, in order to simplify the identification process.

[0150] By way of example, it is noted that to identify a protein, it is not necessary to measure the sequence of all the amino acids. In fact, the sequence of two or three amino acids, with respect to the total number of twenty, is sufficient to identify most of the known proteins.

[0151] By means of advanced data analysis and artificial intelligence techniques, it is therefore possible to identify which and how many amino acids are necessary for a correct identification of the entire proteome or a portion of it. In combination with known labeling techniques, it is possible to increase the speed of identification of the amino acids and therefore of the protein.

[0152] It is observed that the device 1, the system 100 and the methods described above can be applied, by way of example, for the identification of a DNA sequence. In more detail, it is possible to use a double-stranded DNA molecule, in order to reduce the number of bases to be detected, from four bases, known as bases A, T, C, G, to two base pairs, namely the AT and CG pairs. This results in a considerable advantage in the identification of the DNA sequence, especially with respect to the known techniques, which use single DNA strands.

Claims

CLAIMS1. A device (1) for the detection of a molecule configured to emit an electrically or optically detectable detection signal, comprising:- a substrate (2) made of gel having a predefined thickness and comprising a first upper surface (2a) and an opposite first lower surface (2b), the substrate (2) in gel having a plurality of pores (3) extending between the first upper surface (2a) and the first lower surface (2b) of the substrate (2);- a coating layer (4) of plasmonic material, which has a thickness comprised between 15 and 50 nm and comprises a second upper surface (4a) and an opposite second lower surface (4b), the coating layer (4) being disposed on the first upper surface (2a) with the second lower surface (4b) in contact with the first upper surface (2a), and extending at least partly into at least one pore (3) of the substrate (2),wherein the coating layer (4) comprises at least one passage (5) placed in communication with the at least one pore (3) of the substrate (2),wherein, when the device (1) is being used, said passage (5) defines a path for the passage of a molecule and is configured to amplify an electromagnetic radiation and generate a hotspot inside the passage (5) when illuminated by the electromagnetic radiation, such that a molecule flowing in the passage emits an electrically or optically detectable detection signal when passing through the hotspot,the device (1) being made by means of a manufacturing method which provides for the preparation of the substrate (2) in gel, the deposition of the plasmonic coating layer (4) on the first upper surface (2a) of the substrate (2) so that the coating layer (4) extends at least partly inside at least one pore (3) and the at least one passage (5) is incommunication with said pore (3), and the immersion of the device (1) in a swelling solution (9), the substrate (2) acting as a template for the formation of said passage (5).

2. A device (1) for the detection of a molecule configured to emit an electrically or optically detectable detection signal, comprising:- a substrate (2) made of gel having a predefined thickness and comprising a first upper surface (2a) and an opposite first lower surface (2b), the substrate (2) in gel having a plurality of pores (3) extending between the first upper surface (2a) and the first lower surface (2b) of the substrate (2);- a coating layer (4) of plasmonic material, which has a thickness comprised between 1 and 15 nm and comprises a second upper surface (4a) and an opposite second lower surface (4b), the coating layer (4) being disposed on the first upper surface (2a) with the second lower surface (4b) in contact with the first upper surface (2a), and extending at least partly into at least one pore (3) of the substrate (2),wherein the coating layer (4) comprises at least one passage (5) placed in communication with the at least one pore (3) of the substrate (2),wherein, when the device (1) is being used, said passage (5) defines a path for the passage of a molecule and is configured to amplify an electromagnetic radiation and generate a hotspot inside the passage (5) when illuminated by the electromagnetic radiation, such that a molecule flowing in the passage emits an electrically or optically detectable detection signal when passing through the hotspot,the device (1) being made by means of a manufacturing method which provides for the preparation of the substrate (2) in gel and the deposition of the plasmonic coatinglayer (4) on the first upper surface (2a) of the substrate (2), the substrate (2) acting as a template for the formation of said passage (5).

3. The device (1) according to claim 1 or 2, wherein:- the passage (5) comprises a slit extending through the coating layer (4) and disposed externally to the at least one pore (3) of the substrate (2) and adjacent to said pore (3), when in combination with claim 1;- the passage (5) extends within the at least one pore (3) of the substrate (2), when in combination with claim 2.

4. The device (1) according to any one of claims 1 to 3, wherein the first upper surface (2a) and the first lower surface (2b) are spaced from each other along a main direction (Z-Z), and wherein the passage (5) extends at least along the main direction (Z-Z), and along a first direction (X-X) orthogonal to the main direction (Z-Z) of a first extension, the at least one pore (3) of the substrate (2) having a first pore extension along the first direction (X-X) greater than the first extension of the passage (5).

5. The device (1) according to claim 4, wherein the passage (5) extends along a second direction (Y-Y) orthogonal to the main direction (Z-Z) and to the first direction (X-X) of a second extension, the first extension of the passage (5) having a maximum dimension, preferably comprised between 2 and 20 nm, the second extension of the passage having a minimum dimension equal to or greater than the maximum dimension of the first extension, preferably equal to or greater than 2 nm.

6. The device (1) according to any one of claims 1 to 5, wherein:- the substrate (2) is made of gel, preferably agarose gel;- the coating layer (4) is made of a metallic material, preferably one of gold, silver, gold and silver alloys and / or of a plasmonic composite material, preferably based on molybdenum dichloride.

7. The device (1) according to any one of claims 1 to 6, comprising a particle arranged near the passage (5), the coating layer (4) being configured to generate the hotspot between the particle and a side wall of the passage (5) when illuminated by an electromagnetic radiation, the particle being spaced from the side wall of the passage (5) so that the molecule flows between the particle and the side wall of the passage (5) at the hotspot.

8. The device (1) according to any one of claims 1 to 7, comprising an outer layer (6), preferably in gel, superimposed on the second upper surface (4a) of the coating layer (4).

9. The device (1) according to any one of claims 1 to 8, comprising a base layer (7) superimposed on the first upper surface (2a) of the substrate (2), the base layer (7) comprising one or more through openings (7a), the coating layer (4) being disposed in at least one through opening (7a) in contact with the first upper surface (2a) of the substrate (2).

10. A method for manufacturing a device (1) according to any one of claims 1 to 9, comprising the steps of:- preparing the substrate (2) in gel having a plurality of pores (3);- depositing the coating layer (4) on the first upper surface (2a) of the substrate (2) so that the coating layer (4) extends at least partly inside at least one pore (3) of the substrate (2) and the at least one passage (5) of the coating layer (4) is in communication with the at least one pore (3),the substrate (2) in gel acting as a template for the formation of said passage (5).

11. The method according to claim 10, when dependent on claim 1, comprising the further step of:- immersing the device (1) in a swelling solution (9) to swell the substrate (2) by absorption of at least part of the swelling solution (9), the passage (5) of the coating layer (4) expanding upon the occurrence of the swelling of the substrate (2) to define the path for the passage of a molecule.

12. The method according to claim 10 or 11, wherein the step of preparing the substrate (2) comprises the following sub-steps:- providing a support base (7);- forming one or more through openings (7a) in the support base (7), preferably by means of a focused ion beam;- treating a surface of the support base (7) to favor the adhesion of the substrate (2) to the support base (7);- depositing the substrate (2) on the support base (7) so that the first upper surface (2a) of the substrate (2) is in contact with the support base (7);and wherein the step of depositing the coating layer (4) comprises depositing the coating layer (4) on the substrate (2) at least in correspondence with a through opening (7a) of the support base (7).

13. The method according to claim 12, wherein the step of depositing the coating layer (4) provides for depositing an adhesion layer on the upper surface (2a) of the substrate (2) to promote adhesion of the coating layer (4), the adhesion layer comprising one of - a silane layer, preferably hexamethyldisilazane, or- an intermediate layer of metallic material and / or of an opaque material.

14. A system (100) for the detection of a molecule configured to generate an electrically or optically detectable detection signal, comprising:- a device (1) according to any one of claims 1 to 9,- a container (10) configured to contain a swelling solution (9) and the molecule to be detected and to receive the device (1), the substrate (2) being configured to absorb at least part of the swelling solution (9) when immersed in the container containing said solution;- a light source (11) configured to illuminate the passage (5) with an electromagnetic radiation such as to generate a hotspot when the electromagnetic radiation is amplified by the passage (5);- a generator (12) configured to generate a potential difference on opposite sides of the device (1) between the first lower surface (2b) of the substrate (2) and the second uppersurface (4a) of the coating layer (4) to move the molecule through the at least one pore (3) of the substrate (2) and through the passage (5).

15. A method for the detection of molecules by means of a system (100) according to claim 14, comprising the steps of- inserting the device (1) into the container (10);- inserting the swelling solution (9) into the container (10);- providing one or more molecules to be detected in the swelling solution (9);- illuminating the passage (5) with an electromagnetic radiation emitted by the light source (11) to generate a hotspot in the passage (5);- generating a potential difference between the first lower surface (2b) of the substrate (2) and the second upper surface (4a) of the coating layer (4) with the generator (12) to move the molecule through the at least one pore (3) of the substrate (2) and through the passage (5) in correspondence with the hotspot;- detecting the detection signal generated by the molecule localized in correspondence with the generated hotspot.

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