WO2017211995A1

WO2017211995A1 - Nanopore structure

Info

Publication number: WO2017211995A1
Application number: PCT/EP2017/064040
Authority: WO
Inventors: Grégory F SCHNEIDER; Hadi Arjmandi TASH; Amedeo BELLUNATO
Original assignee: Universiteit Leiden
Priority date: 2016-06-10
Filing date: 2017-06-08
Publication date: 2017-12-14
Also published as: GB201610190D0

Abstract

A nanopore structure comprising a first layer having an upper and lower surface, comprising at least one channel extending through the layer from the upper to the lower surface. A second layer, having an upper and lower surface, is disposed over the first layer, wherein the second layer also comprises at least one channel extending through the layer from the upper to lower surface. The two layers are orientated so that the at least one channel of the first and second layers intersect, resulting in the structure comprising a nanopore at the intersection between the channels.

Description

NANOPORE STRUCTURE

BACKGROUND The present invention relates to structures, and methods of making such structures, which comprise a nanocapillary/nanopore/nanogap and their use as platforms for biomolecule detection/sequencing and electron tomography.

The mono-atomic thickness of two-dimensional materials is comparable with the spacing between the building blocks composing biomolecules. Hence, such materials may potentially provide adequate resolution for single building block identification.

The fast translocation of biomolecules, which is not traceable via the monitoring of ionic current through the nanocapillary, is an important limitation for the development of nanocapillary systems for sequencing purposes.

Two graphene electrodes positioned very close to each other with a nanoscale gap in-between is a model system to achieve biomolecule sequencing. An electrical current tunnelling between the electrodes depends on the bases travelling through the gap, i.e. the tunnelling current will fluctuate depending on the chemistry, orbital configuration, geometry and/or configuration of the base passing through the gap at any one time.

Fabrication of nanogap devices employing conventional nanofabrication techniques, such as transmission electron microscopy, high temperature transmission electron microscopy, electron beam lithography, helium ion beam lithography and focused ion beam, requires extremely high levels of resolution and alignment accuracy. The complexity of fabrication through such methods is one of the reasons why such systems have not yet been realised.

The present invention addresses the problems currently faced in the production of structures comprising nanocapillaries/nanopores/nanogaps.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The present invention provides a structure and method for preparing such structures for use in biomolecule detection, electron tomography and sequencing. According to the present invention, there is provided a structure comprising: a first layer having an upper and lower surface, comprising at least one channel extending through the layer from the upper to the lower surface;

a second layer having an upper and lower surface disposed over the first layer, and comprising at least one channel extending through the layer from the upper to lower surface; wherein the at least one channel of the first and second layers intersect, resulting in the structure comprising an aperture at the intersection between the channels. In an embodiment, the structure comprises multiple layers having an upper and lower surface disposed over one another, wherein each of the layers comprise at least one channel extending through the layer from the upper to the lower surface, wherein the channels of each of the layers intersect with the channels of the layers directly above and/or below each layer so as to result in the formation of a connecting fluid pathway through the structure.

Preferably, the structure further comprises a sandwich layer comprising a two-dimensional electrically conductive material disposed between the first and second layers, wherein the sandwich layer also comprises channels corresponding to the channels of the first and second layers resulting in the sandwich layer being divided into one or more pairs of electrodes.

Conveniently, the channels of the first, second and sandwich layer each independently have a width of from about 0.3 nm to about 500 nm, such as from about 0.3 nm to about 200 nm, for example from about 0.3 nm to about 100 nm, preferably from about 0.3 nm to about 1.0 nm and most preferably about 0.3 nm.

Advantageously, the aperture has a size of from about 0.3 nm x 0.3 nm to about 500 nm x 500 nm, such as from about 0.3 nm x 0.3 nm to about 200 nm x 200 nm, for example from about 0.3 nm x 0.3 nm to about 100 nm x 100 nm, preferably from about 0.3 nm x 0.3 nm to about 1.0 nm x 1.0 nm and most preferably about 0.3 nm x 0.3 nm.

Preferably, the first and second layer each have a thickness of from about 10 nm to about 1000 nm, such as from about 15 nm to about 500 nm, for example from about 20 nm to about 50 nm.

Conveniently, the sandwich layer has a size of from about 20 pm x 20 pm to about 500 pm x 500 pm, such as from about 30 μιη x 30 pm to about 200 μητι x 200 pm, preferably from about 40 pm x 40 pm to about 50 pm x 50 pm. Advantageously, the structure is deposited on the surface of a support comprising a hole having a size of from about 20 pm x 20 pm to about 500 μητι x 500 pm, such as from about 30 pm x 30 pm to about 200 pm x 200 pm, preferably from about 40 pm x 40 pm to about 50 pm x 50 pm.

Preferably, one or more metallic electrodes are connected to the sandwich two-dimensional material comprising layer, preferably wherein the electrodes connect to external equipment using silver paste or wire bonding.

Conveniently, the sandwich layer comprises a material selected from the group consisting of graphene, borophene, germanene, silicene, stanene, boron nitride, molybdenum disulphide, tungsten diselenide, tungsten disulphide, fluorographene, phosphorene and thin films of polythiophenes, conducting polymers and metals, such as gold.

Advantageously, the material comprising the first and second layers is a polymer, preferably wherein the polymer is an epoxy resin, methacrylate polymer or thiol-ene polymer.

Preferably, the structure comprises multiple layers comprising a two-dimensional electrically conductive material positioned between layers comprising intersecting channels.

Conveniently, the sandwich layer comprising the electrically conductive two-dimensional material is divided into multiple pairs of electrodes. According to another aspect of the present invention, there is provided a method of fabricating a structure as detailed above comprising the steps of:

depositing a first layer having an upper and lower surface over a second layer having an upper and lower surface, wherein the first and second layers comprise at least one length of sacrificial material extending from the upper to lower surface;

orientating the first and second layers so that the lengths of sacrificial material intersect; and subjecting the structure to at least one etching procedure to remove the sacrificial material wherein removal of the intersecting channels results in the formation of an aperture through the structure. Preferably, the method further comprises sandwiching a layer comprising a two-dimensional material between a first and second layers and wherein the at least one etching procedure to also removes the portion of the sandwiched layer covered by the sacrificial material of the first and second layers, wherein the portion of the sandwiched layer etched away results in the sandwich layer being divided into one or more pairs of electrodes.

Conveniently, the method comprises two separate etching procedures, wherein the first etching procedure removes the sacrificial material of the first and second layers, and the second etching procedure removes the portion of the sandwiched layer exposed upon the first etching procedure.

Advantageously, the first etching procedure comprises the use of an aqueous solution comprising an etchant selected from the group consisting of ammonium persulfate, ferric chloride, hydrofluoric acid, ethylene diamine pyrocatechol, aqua regia, tetramethylammonium hydroxide, potassium hydroxide, potassium cyanide, potassium iodide/iodine and hydrogen peroxide. Preferably, the second etching procedure is a dry etching procedure, wherein the dry etching procedure comprises the use of a plasma comprising fluorocarbons, oxygen, chlorine, boron trichloride, air, methane, ammonia or a mixture thereof.

Conveniently, the sacrificial material of the first and second layer comprises a material selected from gold, copper, aluminium, graphene and combinations thereof.

Advantageously, wherein the length of sacrificial material of the first and second layers has a thickness of from about 0.3 to about 500 nm, such as from about 0.3 nm to about 200 nm, for example from about 0.3 nm to about 100 nm, preferably from about 0.3 nm to about 0.3 nm and most preferably about 0.3 nm.

Preferably, the method comprises the deposition of one of more metallic electrodes onto the sandwich layer. Conveniently, the one or more metallic electrodes are deposited via the use of a mechanical mask or electron beam lithography.

Advantageously, a micromanipulator is used to control the rotation angle of the first and second layers so as to position the intersection point of the sacrificial material. Preferably, the first and second layers are prepared using the nanoskiving process by slicing layers from a polymer block at a non-parallel angle to the horizontal length of a sacrificial material embedded within the polymer block. The skilled person will be aware of the definition of the term nanoskiving. Briefly, the term "nanoskiving" refers to any method suitable for thinly slicing layers from a polymer block as detail above, such as mechanical cutting, microtomy, ultramicrotomy and other comparable methods. According to another aspect of the invention, there is provided an apparatus for biomolecule detection, electron tomography and sequencing, wherein the apparatus comprises a structure as detailed above.

According to a further aspect of the invention, there is provided the use of a structure or an apparatus as detailed above for biomolecule detection, electron tomography and sequencing.

According to another aspect of the invention, there is provided a structure obtainable by the methods as detailed above. As used herein, the term "two-dimensional material" refers to a thin film material having a thickness of less than about 100 nanometers, such as a thin film of gold having a thickness of less than about 100 nanometers. Indeed, other metals may be used for the formation of thin two-dimensional films along with thin films of polythiophenes and conducting polymers. Furthermore, the term "two-dimensional material" also encompasses materials comprised of a single layer of atoms as well as to a plurality of such layers having a thickness of less than about 100 nanometers. Examples of such materials are graphene, borophene, germanene, silicene, stanene and phosphorene, boron nitride, molybdenum disulphide, tungsten diselenide, tungsten disulphide and fluorographene. As used herein, the term "graphene" refers to a molecule in which a plurality of carbon atoms (e.g., in the form of five-membered rings, six-membered rings, and/or seven-membered rings) are covalently bound to each other to form a (typically sheet-like) polycyclic aromatic molecule. Consequently, and at least from one perspective, a graphene may be viewed as a single layer of carbon atoms that are covalently bound to each other (most typically sp² bonded). It should be noted that under the scope of this definition, the term "graphene" also includes molecules in which several (e.g., two, three, four, five to ten, one to twenty, one to fifty, or one to hundred) single layers of carbon atoms are stacked on top of each other to a maximum thickness of about 100 nanometers. Consequently, the term "graphene" as used herein refers to a single layer of aromatic polycyclic carbon as well as to a plurality of such layers having a thickness of less than about 100 nanometers. The two-dimensional material preferably has a thickness of from about 0.3 nm to about 100 nm, such as about 0.3 nm to about 90, 80, 70, 60, 50, 40, 30, 20 or 10 nm, preferably about 0.3 nm to about 5 nm and most preferably about 0.3 nm.

As used herein, the term "aperture" refers to any hole in the structure of the invention through which molecules, for example, but not limited to, biopolymers and single stranded DNA, may pass. Throughout this specification the term "aperture" is used interchangeably with the terms "nanocapillary", "nanopore" and "nanogap".

The present invention will now be described, by way of example, with reference to the accompanying figures, in which:

Figure 1 is a schematic detailing the concept of preparing an aperture through two polymeric layers. Figure 2 is a schematic detailing the concept of preparing a fluid pathway (channel) through multiple polymeric layers.

Figure 3 is a schematic view of a structure comprising a nano-sized aperture within an electrically conductive two-dimensional material according to the invention and a method to make such a structure.

Figure 4 is a schematic view of the method employed to arrive at a sacrificial layer embedded between two epoxy resin layers. Figure 5 is a detailed representation of a graphene sacrificial ribbon embedded within two polymeric layers.

Figure 6 is a schematic view of the use of multiple electrodes and/or multiple detection stages to probe the characteristics of a DNA molecule passing through the nano-sized gap between the electrodes. Figure 7 is a micrograph of a structure of the invention along with a graph detailing the transmembrane potential of the electrically conductive two-dimensional layer relative to the applied current. Figure 8 is an image of a polymerase chain reaction product of DNA showing the passage of DNA through a nanopore of a structure according to the invention.

Figure 9 is a graph detailing translocation events through a nanopore of a structure according to the invention. In particular, Figure 9 shows the interracial nanopore acting as a single molecule sensor.

Figure 10 is a power spectral density graph showing the noise analysis across a nanopore of a structure according to the invention. DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, there is provided a structure comprising:

a first layer having an upper and lower surface, comprising at least one channel extending through the layer from the upper to the lower surface;

a second layer having an upper and lower surface disposed over the first layer, and comprising at least one channel extending through the layer from the upper to lower surface; wherein the at least one channel of the first and second layers intersect, resulting in the structure comprising an aperture at the intersection between the channels. Preferably, the structure comprises multiple layers having an upper and lower surface disposed over one another, wherein each of the layers comprise at least one channel extending through the layer from the upper to the lower surface, wherein the channels of each of the layers intersect with the channels of the layers directly above and/or below each layer so as to result in the formation of a connecting fluid pathway through the structure.

According to another aspect of the present invention, there is provided a method of fabricating a structure as detailed above comprising the steps of:

orientating the first and second layers so that the lengths of sacrificial material intersect; and subjecting the structure to at least one etching procedure to remove the sacrificial material wherein removal of the intersecting channels results in the formation of an aperture through the structure.

Preferably, the method further comprises sandwiching a layer comprising a two-dimensional material between a first and second layers and wherein the at least one etching procedure to also removes the portion of the sandwiched layer covered by the sacrificial material of the first and second layers, wherein the portion of the sandwiched layer etched away results in the sandwich layer being divided into one or more pairs of electrodes. For example, the etching away of the sandwiched two-dimensional material layer may result in the layer being divided into at least one pair of opposing two-dimensional layers, which may serve as a pair of the electrodes for measuring the properties of a (bio-)molecule translocating through the gap. Figure 1 is a schematic detailing the concept employed to produce an aperture in the structure of the invention. Figure 1 (d) shows two polymeric layers with one on top of the other. Each of the polymeric layers has a length of sacrificial material running through its length. In this embodiment the sacrificial material is Au, although any suitable sacrificial material may be used.

The two polymeric slabs have been arranged so that the two lengths of sacrificial material cross one another to form an intersect (this is easily visualised in Figures 1(a-c)). The polymeric layers are then subjected to an etching procedure to remove the sacrificial material. In this embodiment, where the sacrificial material is Au, the etchant may be a potassium cyanide or ammonium persulfate solution, although any other etchant suitable for removing the Au sacrificial material may be used.

After etching away the lengths of sacrificial material, an aperture (labelled in Figure 1 (e) as a nano-capillary) is formed through the structure. The size of the aperture is dependent on the width of the length of sacrificial material running through each of the polymeric slabs. Figures 1 (a) and 1 (b) detail how the size of the aperture at the intersect point may be controlled by altering the width of the length of sacrificial material running through each of the polymeric layers. To summarise Figure 1 , Figures 1 (a) to 1 (c) show the geometrical concept of a zero-thickness nanopore. A zero dimensional point resulting from two crossing lines is detailed in Figure 1 (a). The addition of a dimensionality forms a surface at the intersection as shown in Figure 1 (b). The same surface can be preserved without any additional thickness over a three-dimensional structure by the intersection of the upper and lower surfaces of two nanorods. Figures 1(d) to 1 (e) illustrate the realization steps. Au nanorods are embedded inside polymer carriers, Figure 1(d), and placed one on top of each other in a twisted configuration, Figure 1 (e). The Au nanorods are etched creating a hole (a zero thickness nanopore) at the interface of the two layers.

It is envisaged that the structure of the invention is not to be limited to only two polymeric layers disposed on top of one another. Indeed, the structure of the invention may comprise multiple polymeric layers disposed over one another with a fluidic pathway extending through the structure.

Such an embodiment is detailed in Figures 2(a) to (c). Figure 2(a) details the positioning of three polymeric layers disposed on top of one another, wherein each polymeric layer comprises a channel extending from its upper to lower surface. The width of this channel may be the same as detailed elsewhere within this specification.

The channels of each of the polymeric layers are orientated so as to intersect the channels of the polymeric layers lying directly above and/or below each respective layer which results in the formation of a fluidic channel (pathway), preferably a nano-fluidic channel (pathway) through the structure.

This fluidic pathway is highlighted in Figure 2(b) which is a side view of the three polymeric layers of Figure 2(a) disposed on top of one another. For clarity, the second polymeric layer is not shown as if this was included, only the side of the polymer block would be seen with the channel running through the polymeric layer from left to right. In this figure it can be seen that the orientation of the channels of each of the layers results in the formation of a fluidic channel (pathway) through the structure. Figure 2(c) exemplifies how that an even greater number of polymeric layers comprising a channel may be disposed on top of one another so as to form a fluidic channel (pathway) through the structure.

Although not shown in these figures, it is contemplated that the structures detailed in Figure 2 may comprise sandwiched two-dimensional material layers between at least one pair of polymeric layers. Alternatively, two-dimensional material layers may be sandwiched between each pair of polymeric layers or a selection of these polymeric layers. The two-dimensional material may be any material as detailed herein.

These sandwiched two-dimensional materials may comprise an aperture and the resulting structures may be used in various apparatuses for different sensing applications as detailed herein.

These structures may be made by any of the methods detailed herein. For example, each of the polymeric layers may comprise a length of sacrificial material, such as gold, running through its length. After disposing each of the layers on top of one another and orientating the layers so that the lengths of sacrificial material in each polymeric layer intersect that of the polymeric layer(s) directly above and/or below each polymeric layer, the structure may be subjected to an etching procedure to remove the lengths of sacrificial material so as to result in the formation of a fluidic channel (pathway) through the structure. Any etching procedure as detailed herein may be used to remove the sacrificial material. In this embodiment, where the sacrificial material is Au, the etchant may be a potassium cyanide or ammonium persulfate solution, although any other etchant suitable for removing the Au sacrificial material may be used. Figure 3 shows a schematic of a preferred method employed to arrive at a preferred embodiment of the structure of the invention.

In Figure 3(a) a support substrate is provided which comprises a hole of a size of about 50 pm x 50 pm. Any sufficiently flat material can be use as this support. This material should be thin enough to be able to drill a hole close to its centre, yet thick enough to provide the required mechanical stability. Preferably, the substrate is a glass disk with a diameter of about 1 cm and a thickness of about 500 pm. In this embodiment, a laser may be used to drill the hole.

In Figure 3(b) a first polymeric layer is placed on top of the support substrate. This layer comprises a length of sacrificial material extending from the upper to lower surface. In this embodiment, the polymeric layer is an epoxy resin layer, specifically an EPON™ Resin 828 or 812 layer, although any other suitable polymer resins may be used such as methacrylate polymers or thiol-ene polymers. Figures 4 and 5 detail the method employed for obtaining an epoxy resin polymeric layer with an embedded sacrificial material and this method is described below. As shown in Figure 4, graphene that has been grown on a copper support is deposited on the surface of an epoxy resin layer which has been stained with a staining agent selected from pyrene derivatives, oxazine derivatives, acridine derivatives, fluorescein and any other fluorophores that fluoresce in the visible spectrum preferably, Rhodamine B is used as the staining agent. The graphene film is placed so as to be in contact with the surface of the epoxy resin layer so that the copper support is on top of the graphene. Therefore, the graphene film is sandwiched between the epoxy resin layer and the copper support. In this embodiment, graphene is the sacrificial material referred to above, although graphene may be substituted for any other sacrificial material as detailed herein. For example, the sacrificial material may comprise a material selected from gold, copper, aluminium, graphene and combinations thereof.

Although the graphene used in this particular example has been grown on copper, graphene, or indeed any other electrically conductive two-dimensional material grown on any other suitable support, such as nickel, ruthenium and iridium, may be used. Additionally, graphene from any other sources, such as exfoliated graphite or chemically reduced graphene.

A wire is then deposited on the surface of the copper support and is fixed to the support using a metallic paint, preferably a silver paint. The wire is present so that the sacrificial (graphene) layer may be connected to an external circuit as required.

The structure is then subjected to an etching procedure to remove the copper support via the use of an etchant solution. Suitable etchants include, but are not limited to, ammonium persulfate, ferric chloride, hydrofluoric acid, ethylene diamine pyrocatechol, aqua regia, tetramethylammonium hydroxide, potassium hydroxide and hydrogen peroxide. Preferably, ammonium persulfate is used as the etchant. After which, a second epoxy resin layer is deposited on top of the sacrificial layer thus sandwiching the sacrificial layer between the two epoxy resin layers. This second layer is not stained with Rhodamine B. Therefore, as one epoxy layer is stained and the other is not, this allows easy visualisation of the interface between the two layers, thus allowing identification of the position at which the sacrificial layer lies between the two epoxy layers.

After the sacrificial layer has been sandwiched between these epoxy resin layers, the resulting block is sliced at a perpendicular angle to the basal plane of the sacrificial layer using ultramicrotomy methods discussed herein to arrive at a polymer sheet comprising a sacrificial layer extending from the upper surface to the lower surface of the polymer sheet. This may be better visualised in Figure 5, which shows a thin epoxy resin layer, with a thickness of around 20 nm to 1000 nm, comprising a graphene ribbon, which is the sacrificial layer in this embodiment, extending from the upper to the lower surface of the epoxy resin layer.

The polymer layer (slab) produced by this microtoming method is deposited on the surface of the substrate as depicted in Figure 3(b) with the sacrificial layer (graphene layer in the embodiment described above) extending vertically through the polymer layer. A sandwich layer comprising a two-dimensional electrically conductive material is then deposited over the upper surface of the first layer as detailed in Figure 3(c). The size of this sandwich layer is ideally smaller than the hole within the support substrate. If the size of the sandwich layer is larger than the hole within the support substrate, a lithography step followed by chemical etching is used to remove the portion of the sandwich layer lying outside the area of the opening of the substrate. Preferably, oxygen plasma is the best choice for the etching of the unnecessary areas of the two-dimensional material. Any two-dimensional electrically conductive material may be used in this layer, for example the material may be selected from the group consisting of graphene, borophene, germanene, silicene, stanene, boron nitride, molybdenum disulphide, tungsten diselenide, tungsten disulphide, fluorographene, phosphorene and thin films of polythiophenes, conducting polymers and metals, such as gold. Preferably, the two-dimensional electrically conductive material is graphene.

As depicted in Figure 3(d), metallic electrodes are deposited onto the sandwiched two- dimensional electrically conductive material layer. The metallic electrodes may be made from chromium, gold, aluminium, palladium, titanium or any other conductive material. Preferably, the metallic electrodes are deposited via the use of a mechanical mask or electron beam lithography. Ball bonding, wedge bonding or silver paste can be used to connect the electrodes to external electronic equipment. In Figure 3(e) a second epoxy resin layer comprising a sacrificial layer extending from the upper to the lower surface of the layer is deposited over the sandwiched layer, thus resulting in the two-dimensional electrically conductive material layer being sandwiched between the first and second polymeric layers. The first and second polymer layers are orientated in the structure so that the lengths of sacrificial material cross one another to form an intersect. A micromanipulator may be used to control the rotation angle of the first and second polymeric layers so as to position the intersection point of the sacrificial material. After its formation, the structure is then subjected to at least one etching procedure to remove the sacrificial material and the portion of the sandwiched layer covered by the sacrificial material. The portion of the sandwiched layer etched away results in the formation of intersecting channels with an aperture at the intersection of the channels and the sandwiched layer being divided into one or more pairs of electrodes, preferably two pairs of electrodes. Due to the presence of the hole in the substrate detailed in Figure 3(a), the underside of the first epoxy resin polymer layer is accessible for the purposes of etching away the sacrificial layer. In the embodiment where the sacrificial material of the polymeric layers is graphene and the sandwich layer comprises a two-dimensional electrically conductive material layer, the sacrificial material and the portion of the sandwiched layer covered by the sacrificial material may be etched away in the same step. For example, a dry etching procedure may be used, wherein the dry etching procedure comprises the use of a plasma comprising fluorocarbons, oxygen, chlorine, boron trichloride, air, methane, ammonia or a mixture thereof.

Alternatively, where the sacrificial material is a material other than graphene, for example gold, the structure may be exposed to a first etching procedure using an potassium cyanide solution to etch away the gold sacrificial material, which is then followed by a dry etching procedure as detailed above to etch away the exposed portions of the sandwiched two-dimensional layer. Any other etching procedure as described herein may be used in either of these etching steps.

After this procedure, the resulting structure comprises a nano-sized aperture, the size of which is dictated by the width of the sacrificial material comprised within the first and second epoxy resin polymer layers. The etching procedure(s) also results in the sandwich layer being divided into two pairs of opposing electrodes comprising a cross point, or aperture, which acts as a nanofluidic channel for the migration of biomolecules in the final setup.

Each pair of electrodes may work independently or in conjunction with one another. Indeed, although this method results in the formation of a structure comprising two pairs of electrodes, the structure would work with a single pair of electrodes as well as multiple pairs of electrodes, such as 3, 4, 5, 6, 7, 8, 9 or 10 or more pairs of electrodes. In fact, there is no limit to the number of pairs of electrode that may be comprised within the structure. It is possible to connect the structure to an external circuit via the use of the metallic electrodes deposited onto the sandwiched layer detailed above. The structure obtained by the method described above may be incorporated into an apparatus suitable for, but not limited to, biomolecule detection, electron tomography and sequencing.

As mentioned above, the size of the aperture within the structure is dictated by the width of the sacrificial material comprised within the first and second epoxy resin polymer layers. Therefore, through the use of atomically thin sacrificial layers, such as graphene, the size of the resulting aperture may be finely controlled and apertures corresponding to the width of a single DNA strand may be obtained. In such an apparatus, an electrical current may be tunnelled across at least one pair of the two-dimensional material electrodes. When a single strand of DNA passes through the nanopore, the electrical current will fluctuate depending on the DNA base passing through the gap between the two electrodes. Therefore, this allows for fine resolution sequencing of the DNA strand.

A visual example of a DNA molecule passing through a nanogap between two electrodes comprised of an electrically conductive two-dimensional material is depicted in Figure 6(a). In order to obtain a more accurate sequencing of the DNA molecule, a two-stage detection method may be employed by having two two-dimensional layers comprising a nanopore disposed over, but not in contact with, one another. This may be achieved by depositing another two-dimensional layer on top of the second polymeric layer of the structure and placing a further epoxy resin layer on top of the structure to arrive at a structure comprising two layers of an electrically conductive two-dimensional material comprising a nanopore. It is known in current systems that when a string of molecules, such as a single strand of DNA, passes through an aperture for sequencing, it can occur that the DNA strand alternates backwards and forwards due to Brownian motion which results in the DNA being sequenced as having a repeating set of bases. For example, if the DNA strand comprises a base sequence of ...ACGT... and the molecule alternates backwards and forwards through an aperture of a sequencer between the C and G moieties, the sequencer would read multiple repeats of CGCGCG etc.

However, by disposing multiple electrodes of the invention over one another so as to sequence the same DNA molecule at different locations along the strand, it will be possible to isolate the instances where the DNA strand alternates backwards and forwards through the sequencer and thus remove this error in reading from the resulting sequence. Figure 7 shows two micrographs of a preferred embodiment of the structure of the invention. The horizontal two-dimensional layer can be seen, which is embedded within two epoxy resin polymer slabs. Each of the polymer slabs has a length of sacrificial material running through them with the sacrificial material in this particular embodiment being gold. The epoxy resin slabs are orientated so that the lengths of sacrificial material cross one another to form an intersect. Upon etching away the sacrificial material and the subsequently exposed material of the two-dimensional layer, as detailed above, an aperture is formed within the structure.

The nanocapillaries/nanopores/nanogaps of the structures of the invention have resistances ranging from between about 0.5ΜΩ to about 150ΜΩ at salt concentrations ranging between 1 mM up to 1 M.

The graph of Figure 7 details the transmembrane potential of the electrically conductive two- dimensional layer relative to the applied current across the aperture in the structure. These results show that the nanocapillaries/nanopores/nanogaps show a resistivity of around 2.5ΜΩ at a KCI concentration 1.0M, a resistivity of around 5ΜΩ at a KCI concentration 0.5M and a resistivity of around 10ΜΩ at a KCI concentration 0.1 M.

EXAMPLES

The structures of the invention are suitable for biomolecule detection/sequencing and electron tomography. Specifically, the structures are ideal for sequencing biomolecules, such as DNA, which is able to pass through the nanocapillary of the sandwiched two-dimensional electrically conductive layer.

Example 1

Graphene having a thickness of about 0.3 nm was sandwiched between two polymeric slabs, each having a thickness of about 200 nm. The structure was provided with a nanocapillary of a size of about 10 nm x 10 nm utilising the method detailed above.

The structure was placed into a vessel containing a solution so as to separate the solution into two parts with the only contact point between the two solutions being the nanocapillary of the structure. Lambda DNA was provided to one of the solution chambers (cis chamber) and was electrophoretically driven using a 20 mV transmembrane potential through the nanocapillary of the structure into the opposing chamber (trans chamber). Figure 8 shows a stained agarose gel obtained through the electrophoresis of samples of solutions within the cis and trans chamber before and after the application of the transmembrane potential as detailed above.

Lane 1 shows the results of the electrophoresis of the solution in the cis chamber before application of the potential and a distinct DNA band may be seen.

Lane 2 shows the results of the electrophoresis of the solution in the trans chamber before application of the potential and no distinct DNA band is present. Lane 3 shows the results of the electrophoresis of the solution in the cis chamber after application of the potential and the distinct DNA band is present.

Lane 4 shows the results of the electrophoresis of the solution in the trans chamber after application of the potential and now the distinct DNA band, which corresponds to that seen in the cis chamber, is present. Therefore, at least a portion of the DNA has passed through the nanocapillary from the cis chamber to the trans chamber upon application of the transmembrane potential confirming the capability of this structure to be used in systems for sequencing DNA. Example 2

Graphene having a thickness of about 0.3 nm was sandwiched between two polymeric slabs, each having a thickness of about 200nm. The structure was provided with a nanocapillary of a size of about 10 nm x 10 nm utilising the method detailed above.

The structure was placed into an ionic solution comprising lambda DNA and a transmembrane potential of 50mV was applied. As the DNA passes through the nanocapillary the number of ions located between the nanocapillary contact points varies depending on the size of the base passing through the nanocapillary, which leads to a fluctuation in the current across the nanocapillary. The current across the nanocapillary of the structure was monitored whilst the DNA was passed through the nanocapillary.

Figure 9a shows a typical time trace of the ionic current through an interfacial nanopore (a = 70 nm, h = 50 nm) immersed in a 5 mM LiCI buffer solution. Upon addition of 48.5 kbp λ-DNA molecules, a series of drops in the conductance of the nanopore appears, manifesting the translocation of the DNA molecules through the nanopore.

The duration and blockade current of the translocation events (approximately 400 events) are plotted in Figure 9b. Two highly-populated components with Gaussian distributions are identified in both histograms (solid line and dashed-line curves) that can be attributed to the translocation of DNA molecules with different foldings. The more populated component exhibits an average translocation duration of approximately 22ms which corresponds to approximately 450 ns/bp. Interestingly, the measured dwell time is 1.5 to approximately 100 times longer than the reports for two-dimensional (5 ns/bp - 56 ns/bp), biological (30 ns/bp) and solid-state (40 ns/bp - 300 ns/bp) nanopores.

Several observations suggest the presence of a strong interaction between DNA and the walls of the trench, which eventually slows down the translocation of molecules: First, the majority of the translocation events in interfacial nanopores start sharply but end smoothly (Figure 9a). This observation can be well explained considering a binding mechanism between DNA and the walls of the trench; in fact, the binding requires time and energy to break, in order to let the DNA exit the nanopore. Second, increasing the salt concentration lowers the dwell time through interfacial nanopores. This observation is in striking contrast to the reported behavior of DNA in SiNx nanopores in which the strong binding between Li⁺ to DNA suppresses the translocation speed in high salt concentrations and can be well explained by considering the DNA-nanopore interaction (Kpwa;czyk, S. W. et al., Nano Lett., 2012, 12(2), 1038-1044). Third, the widely spread event duration, ranging from less than approximately 14 ms to over 80 ms (Figure 9b), is another signature of the DNA-nanopore interaction: in the absence of such interaction, dsDNA molecules are expected to exhibit uniform translocations. Hydrophobic interaction between DNA and the trench walls or cross-over from base-base pi- stacking to base-polymer pi-stacking may govern the DNA-wall interaction (Garaj et al., Proceeding Natl. Acad. Sci, PNAS 2013, 1 10 (30), 12192-12196; and Akca ef al., PLoS One, 2011 , 6(4), e18442.).

Example 3 Graphene having a thickness of about 0.3 nm was sandwiched between two polymeric slabs, each having a thickness of about 250nm. The structure was provided with a nanocapillary of a size of about 10 nm x 10 nm utilising the method detailed above.

The structure was placed into an ionic structure and a transmembrane potential of 100 mV was applied. The migration of the ions from the cis chamber to the trans chamber was measured by monitoring the fluctuation in the electrical current across the aperture. The ionic current in nanocapillary systems suffers from finite levels of noise, which depends on several parameters including the type of nanocapillary.

Figure 10 details the power spectral density (PSD) of the nanocapillary of the structure as a function of the frequency of the measured signal. The noise level of these structures is several orders of magnitude lower than existing nanocapillary.

Claims

A structure comprising:

a second layer having an upper and lower surface disposed over the first layer, and comprising at least one channel extending through the layer from the upper to lower surface;

wherein the at least one channel of the first and second layers intersect, resulting in the structure comprising an aperture at the intersection between the channels.

The structure according to Claim 1 , further comprising a sandwich layer comprising a two-dimensional electrically conductive material disposed between the first and second layers, wherein the sandwich layer also comprises channels corresponding to the channels of the first and second layers resulting in the sandwich layer being divided into one or more pairs of electrodes.

The structure according to Claim 1 or Claim 2, wherein the channels of the first, second and sandwich layer each independently have a width of from about 0.3 nm to about 500 nm, such as from about 0.3 nm to about 200 nm, for example from about 0.3 nm to about 100 nm, preferably from about 0.3 nm to about 1.0 and most preferably about 0.3 nm.

The structure according to any preceding claim, wherein the aperture has a size of from about 0.3 nm x 0.3 nm to about 500 nm x 500 nm, such as from about 0.3 nm x 0.3 nm to about 200 nm x 200 nm, for example from about 0.3 nm x 0.3 nm to about 100 nm x 100 nm, preferably from about 0.3 nm x 0.3 nm to about 1.0 nm x 1.0 nm and most preferably about 0.3 nm x 0.3 nm.

The structure according to any preceding claim, wherein the first and second layer each have a thickness of from about 10 nm to about 1000 nm, such as from about 15 nm to about 500 nm, for example from about 20 nm to about 50 nm.

The structure according to any of Claims 2 to 5, wherein the sandwich layer has a size of from about 20 pm x 20 pm to about 500 pm x 500 pm, such as from about 30 pm x 30 μηι to about 200 Mm x 200 Mm, preferably from about 40 pm X 40 Mm to about 50 m x 50 M .

The structure according to any preceding claim, wherein the structure is deposited on the surface of a support comprising a hole having a size of from about 20 pm x 20 Mm to about 500 x 500 , such as from about 30 Mm x 30 Mm to about 200 M x 200 Mm, preferably from about 40 pm x 40 Mm to about 50 Mm x 50 Mm.

The structure according to any of Claims 2 to 7, wherein one or more metallic electrodes are connected to the sandwich two-dimensional material comprising layer, preferably wherein the electrodes connect to external equipment using silver paste or wire bonding.

The structure according to any of Claims 2 to 8, wherein the sandwich layer comprises a material selected from the group consisting of graphene, borophene, germanene, silicene, stanene, boron nitride, molybdenum disulphide, tungsten diselenide, tungsten disulphide, fluorographene, phosphorene and thin films of polythiophenes, conducting polymers and metals, such as gold.

The structure according to any preceding claim, wherein the material comprising the first and second layers is a polymer, preferably wherein the polymer is an epoxy resin, methacrylate polymer or thiol-ene polymer.

The structure according to any of Claims 2 to 10, wherein the structure comprises multiple layers comprising a two-dimensional electrically conductive material positioned between layers comprising intersecting channels.

The structure according to any of Claims 2 to 11 , wherein the sandwich layer comprising the electrically conductive two-dimensional material is divided into multiple pairs of electrodes.

A method of fabricating a structure according to any preceding claim comprising the steps of:

depositing a first layer having an upper and lower surface over a second layer having an upper and lower surface, wherein the first and second layers comprise at least one length of sacrificial material extending from the upper to lower surface; orientating the first and second layers so that the lengths of sacrificial material intersect; and subjecting the structure to at least one etching procedure to remove the sacrificial material wherein removal of the intersecting channels results in the formation of an aperture through the structure.

The method of Claim 13, wherein the method further comprises sandwiching a layer comprising a two-dimensional material between a first and second layers and wherein the at least one etching procedure to also removes the portion of the sandwiched layer covered by the sacrificial material of the first and second layers, wherein the portion of the sandwiched layer etched away results in the sandwich layer being divided into one or more pairs of electrodes.

The method according to Claim 14, wherein the method comprises two separate etching procedures, wherein the first etching procedure removes the sacrificial material of the first and second layers, and the second etching procedure removes the portion of the sandwiched layer exposed upon the first etching procedure.

The method according to Claim 15, wherein the first etching procedure comprises the use of an aqueous solution comprising an etchant selected from the group consisting of ammonium persulfate, ferric chloride, hydrofluoric acid, ethylene diamine pyrocatechol, aqua regia, tetramethylammonium hydroxide, potassium hydroxide, potassium cyanide, potassium iodide/iodine and hydrogen peroxide.

The method according to Claim 15 or Claim 16, wherein the second etching procedure is a dry etching procedure, wherein the dry etching procedure comprises the use of a plasma comprising fluorocarbons, oxygen, chlorine, boron trichloride, air, methane, ammonia or a mixture thereof.

The method according to any of Claims 14 to 17, wherein the sacrificial material of the first and second layer comprises a material selected from gold, copper, aluminium, graphene and combinations thereof.

The method according to any of Claims 14 to 18, wherein the length of sacrificial material of the first and second layers has a thickness of from about 0.3 to about 500 nm, such as from about 0.3 nm to about 200 nm, for example from about 0.3 nm to about 100 nm, preferably from about 0.3 nm to about 0.3 nm and most preferably about 0.3 nm.

20. The method according to any of Claims 14 to 19, wherein the method comprises the deposition of one of more metallic electrodes onto the sandwich layer. 21. The method according to Claim 20, wherein the one or more metallic electrodes are deposited via the use of a mechanical mask or electron beam lithography.

22. The method according to any of Claims 13 to 21 , wherein a micromanipulator is used to control the rotation angle of the first and second layers so as to position the intersection point of the sacrificial material.

23. The method according to any of Claims 13 to 22, wherein the first and second layers are prepared using the nanoskiving process by slicing layers from a polymer block at a non-parallel angle to the horizontal length of a sacrificial material embedded within the polymer block.

24. An apparatus for biomolecule detection, electron tomography and sequencing, wherein the apparatus comprises a structure according to any of Claims 1 to 12. 25. The use of a structure according to any of Claims 1 to 12 or an apparatus according to Claim 24 for biomolecule detection, electron tomography and sequencing.

26. A structure obtainable by the method according to any of Claims 13 to 23.