Nanopore supporting structure and its fabrication

Abstract

A nanopore support structure is disclosed comprising a wall layer having walls defining a plurality of wells and an overhang extending from the wall across each of the wells, the overhang defining an aperture configured to support a membrane suitable for insertion of a nanopore. Further disclosed are a nanopore sensing device comprising the nanopore support structure, and methods of fabricating the nanopore support structure and the nanopore sensing device.

Description

[Technical Field]

[0001] The present invention relates to nanopore support structures and methods for making nanopore support structures.

[0002] Nanopore sensors have been developed to sense a wide range of species, including single molecules such as polymer molecules. A known nanopore sensor device is the MinION™, manufactured and sold by Oxford Nanopore Technologies Ltd. Nanopore-based sensing therein utilizes measurement of ionic current flow through a biological nanopore located in a highly resistive amphiphilic membrane. The MinION™ has an array of nanopore sensors. For example, when a molecule, such as a polymeric analyte such as DNA, is translocated through the nanopore, measurement of the fluctuation in ionic current can be used to determine the sequence of the DNA strand. Nanopore devices for detecting analytes other than polynucleotides, such as proteins, are also known from WO 2013 / 123379, the entire contents of which are incorporated herein by reference.

[0003] The amphiphilic membrane in which the nanopore is located is typically supported on a support structure. Many designs of such structures are known. For example, US2015 / 265994, the entire contents of which are incorporated herein by reference, discloses a support design for an array of membranes. US2015 / 265994 discloses a nanopore support structure comprising an inner portion defining a solid inner recess that functions as a well, and an outer portion resembling a strut extending outward from the inner portion and having a gap between them. In such a nanopore support structure, the outer portion (strut) is outside the footprint of the inner recess (well) of the inner portion. The supported membrane extends across the opening of the inner recess. Therefore, the actual membrane is relatively large, which creates a large membrane capacitance and reduces the mechanical robustness of the membrane. However, if the size of the inner recess is reduced, the volume of the fluid reservoir within the inner recess is reduced and therefore drains more quickly. Thus, the size of the inner recess is balanced between the membrane properties and the volume of fluid available within the inner recess.

[0004] For example, it is known from the nanoporous support structure disclosed in US 2015 / 265994 that the balance between membrane properties and the volume of available fluid in the inner recess is not only important, but it has been found that these influence the membrane, and when using this type of nanoporous support structure in which access holes open at the bottom of each well, the membrane is able to move throughout the support structure. Due to the excess pressure caused by the surface tension of the membrane, the solution in the wells tends to slowly drain through the access holes, and the membrane eventually collapses.

[0005] Reliably forming films with desirable properties is always difficult. It would be advantageous to provide improved support structures that facilitate the formation of more stable films and have properties better suited to the sensing applications in which they are used.

[0006] The present invention relates to such improved nanopore support structures and methods for making them.

[0007] According to a first aspect of the present invention, there is provided a nanopore support structure comprising a wall layer having walls defining a plurality of wells, and an overhang extending from the wall across each of the wells, the overhang defining an aperture configured to support a membrane suitable for insertion of a nanopore.

[0008] The inclusion of an overhang defines an aperture through which a membrane can be formed. This allows the size of the well to be increased while reducing the size and area of ​​the actual membrane. This further reduces the capacitance of the membrane and improves membrane stability. The size of the aperture can also be selected independently of the size of the well, providing greater flexibility in the design parameters of the nanopore support structure. The size of the well can be increased in depth and / or cross-sectional area. Increasing the size of the well allows more buffer solution to be stored within the well, allowing nanopore sensing devices containing such nanopore support structures to operate for longer periods before the buffer solution is depleted.

[0009] In some embodiments, the walls can be provided without features, such as depressions or protrusions, that retain non-polar media, e.g., oil. The walls can be featureless. The walls can be substantially smooth. Providing walls without non-polar media-retaining features has the advantage that wells having electrodes on their walls or their respective bases can be provided with greater depths and aspect ratios without non-polar media infiltrating onto the electrodes in the wells. This allows for larger well volumes and greater amounts of redox electrode mediator to be provided within the wells, thereby extending the life of the device during operation.

[0010] In other embodiments, the wall may be provided with a non-polar media control feature, such as a protrusion, that may extend across the entire wall or a portion of the wall extending from the overhang.

[0011] In some embodiments, the nanopore support structure further comprises protrusions extending laterally beyond the extent of the overhang, which allow for further control over the surface and structural properties of the overhang, thereby improving the performance of the nanopore support structure and the sensing device within which it functions.

[0012] In some embodiments, the protrusions include inner protrusions that extend laterally from the inner overhang of each well and / or outer protrusions that extend laterally from the outer overhang of each well, the inner and outer protrusions providing control over the surface and structural properties of both the inner and outer overhangs of the wells.

[0013] In some embodiments, the protrusions are positioned to increase retention of non-polar media by the overhangs. Retention of non-polar media on the surfaces of the overhangs of the nanopore support structure promotes membrane formation on the nanopore support structure. Alternatively or additionally, in some embodiments, the nanopore support structure comprises outward protrusions that extend laterally beyond the extent of the outer surface of the wall layer between the wells. Some outward protrusions may extend from both the outer surface of the wall layer and one or more overhangs.

[0014] In some embodiments, for each aperture, the outer protrusion defines a respective protrusion wall that at least partially surrounds and is recessed from the aperture. Providing the protrusion wall may allow a meniscus to form across each aperture such that the meniscus extends at least partially within the respective aperture. This greater control of the meniscus may be used to aid in the formation of a film within the aperture and may avoid the need for pre-treatment to promote film formation.

[0015] In some embodiments, the nanopore support structure comprises a plurality of peripheral outer protrusions, for each aperture, comprising an inner surface facing toward the aperture and an outer surface facing away from the aperture that define the protrusion walls, wherein the inner and / or outer surfaces of one or more of the peripheral outer protrusions may comprise a micropatterned structure.

[0016] In some embodiments, the outer protrusions further include intermediate outer protrusions disposed in regions between the peripheral outer protrusions. The intermediate outer protrusions may aid in delivering and controlling a non-polar medium used to form the membrane across the aperture. One or more of the intermediate outer protrusions may include a micropatterned structure. In particular, one or more surfaces of the one or more intermediate outer protrusions may include a micropatterned structure.

[0017] In some embodiments, the protrusions are positioned to increase the stiffness of the overhang, which reduces the likelihood of damage, thereby improving the longevity of the structure and improving the consistency of device performance.

[0018] In some embodiments, the wells have respective bases that can be used to support additional structures important in the nanopore sensing device, such as electrodes or additional apertures.

[0019] In some embodiments, the wall layer further defines a base, which may be advantageous in some applications because the base layer may be formed as part of the same process that defines the wall layer.

[0020] In some embodiments, the nanopore support structure further comprises a substrate, the wall layer being secured to the substrate, and the substrate defining the base of the well. Using a separate substrate to define the base may be advantageous in some manufacturing methods, in which case it may be desirable to define the base using a different material than that used for the wall layer.

[0021] In some embodiments, the overhang and wall layers comprise a hardened negative photoresist material, which is advantageous because the photoresist material can be formed into a desired structure by controlling the exposure of the resist.

[0022] In some embodiments, the nanopore support structure further comprises a barrier disposed within the well, the barrier capable of reducing scattering of electromagnetic radiation at a wavelength for curing a negative tone photoresist material. Scattering can cause exposure of the photoresist material in unwanted areas, thereby altering the structure of the nanopore support structure from its intended design. The barrier reduces this effect by reducing scattering of light.

[0023] In some embodiments where the wells have respective bases, the barriers extend from the bases to the overhangs, which improves the scattering reduction provided by the barriers and in some embodiments also allows the barriers to provide structural support to the overhangs.

[0024] In some embodiments, the barriers extend inwardly into the well from the wall, and optionally the barriers curve inwardly into the well along their extent, which can improve the ability of the barriers to provide structural support, particularly in some embodiments of the method of manufacture.

[0025] In some embodiments, the wall layer and overhang are respective molded components that are secured together. Molding is an alternative manufacturing method to the use of photoresist and may be preferred depending on the requirements of the material from which the nanopore support structure is formed.

[0026] In some embodiments, the nanopore support structure further comprises membranes extending across each aperture, and optionally also comprises nanopores inserted in at least some of the membranes.

[0027] According to a second aspect of the present invention, there is provided a nanopore sensing device comprising a planar structure comprising first and second chambers and a nanopore support structure according to the first aspect of the present invention, the planar structure being provided with a plurality of fluid passages extending between the first and second chambers and including respective wells and apertures in the nanopore support structure, the apertures opening into the first chamber; and an electrode arranged to sense fluid potential in each passage between the nanopore and the second chamber.

[0028] The nanopore sensing device may include an array of membranes, each membrane provided across a respective aperture, and each membrane may include a nanopore.

[0029] As discussed above, the nanopore support structures of the first aspect of the present invention offer advantages in terms of their structural and electrical properties. Accordingly, nanopore sensing devices equipped with these nanopore support structures have improved reliability and flexibility in their design. For example, the nanopore support structure helps balance pressure across the membrane and pin the membrane to the aperture. This makes them compatible with wells having bottom openings or through-holes that can be used to provide a passage between the nanopore and a second chamber.

[0030] In some embodiments, the passageway can include a fluidic resistor portion between the electrode and the second chamber, which can create a fluidic resistance between the electrode and the second chamber comparable to the resistance through the nanopore between the electrode and the first chamber, thereby enabling an advantageous configuration for measuring fluidic potential.

[0031] In some embodiments, the planar structure comprises an additional layer, and the fluidic resistor portion is formed within the additional layer. Placing the fluidic resistor portion in the additional layer allows for additional design flexibility compared to having to form the fluidic resistor portion within the wall layer or substrate of the nanopore support structure.

[0032] In some embodiments, the first and second chambers are on opposite sides of a planar structure and a passageway extends through the planar structure, thereby allowing the flow of ions through the nanopore and enabling the measurement of properties of molecules passing through the nanopore.

[0033] In some embodiments, the nanopore sensing device further comprises drive electrodes in the first and second chambers that allow a voltage to be applied to drive an ionic current between the two chambers, thereby enabling measurement of the properties of molecules passing through the nanopore.

[0034] According to a third aspect of the present invention, there is provided a method for manufacturing a nanopore support structure, the method comprising: forming a wall layer having walls defining a plurality of wells; and forming overhangs extending from the walls across the wells, the overhangs defining apertures configured to support a membrane suitable for insertion of a nanopore.

[0035] As discussed above, forming a nanopore support structure with an overhang defines an aperture through which a membrane can be formed. This allows the well size to remain large while reducing the size and area of ​​the actual membrane. This also reduces membrane capacitance and improves membrane stability. The size of the aperture can also be selected independently of the well size, providing greater flexibility in the design parameters of the nanopore support structure.

[0036] In some embodiments, the method further comprises forming protrusions extending laterally beyond the extent of the overhang, which allow for further control over the surface and structural properties of the overhang, thereby improving the performance of the nanopore support structure.

[0037] In some embodiments, the protrusions include inner protrusions that extend laterally from the inner overhang of each well and / or outer protrusions that extend laterally from the outer overhang of each well, the inner and outer protrusions providing control over the surface and structural properties of both the inner and outer overhangs of the wells.

[0038] In some embodiments, the protrusions are positioned to increase retention of non-polar media by the overhangs, which retention of non-polar media on the surface of the nanopore support structure promotes membrane formation on the nanopore support structure.

[0039] In some embodiments, the protrusions are positioned to increase the stiffness of the overhang, which reduces the likelihood of damage, thereby improving the longevity of the structure and improving the consistency of device performance.

[0040] In some embodiments, the method further comprises forming a base of the well, which, as discussed in connection with the nanopore sensing device above, may be used to support further structures important in the nanopore sensing device, such as electrodes or further apertures.

[0041] In some embodiments, the wall layer comprises the base, which can be advantageous in some applications because the base layer can be formed simultaneously with the wall layer.

[0042] In some embodiments, the method includes depositing an uncured negative photoresist material, exposing the negative photoresist material to light so as to harden the negative photoresist material in the form of a nanopore support structure, and removing the uncured negative photoresist material. Forming structures using photoresist material can be advantageous because the structures can be precisely patterned using optical techniques and the structure in different regions can be controlled using multiple exposure and / or deposition and removal steps.

[0043] In some embodiments, the method includes depositing an uncured negative photoresist material, exposing the negative photoresist material using a multiple exposure technique to harden the negative photoresist material in the form of nanopore support structures, and removing the uncured negative photoresist material. Using a multiple exposure technique with a single removal step is advantageous because it allows multiple photoresist layers to be deposited with the underlying exposed layer supporting the other layers to prevent undesired movement of the resist structures.

[0044] In some embodiments, the step of exposing the negative photoresist material using a multiple exposure technique is performed to harden the negative photoresist material in the form of overhangs and at least the upper section of the wall layer to a level deeper than the overhangs. Exposing to different depths allows both structures to be formed without the need to deposit additional layers of photoresist, which can reduce manufacturing time.

[0045] In some embodiments, exposing the negative photoresist material using a multiple exposure technique comprises exposing the negative photoresist material in separate exposure steps, which may be preferred depending on the capabilities of the available optics, for example, when only uniform illumination over a large area is possible.

[0046] In some embodiments, exposing the negative photoresist material using a multiple exposure technique includes exposing the negative photoresist material with spatial modulation of intensity, which can be advantageous because, for example, it can eliminate the need to create multiple masks for separate exposure steps.

[0047] In some embodiments, the method further includes, before depositing an uncured negative photoresist material and exposing the negative photoresist material using a multiple exposure technique, performing an initial stage including depositing an initial layer of uncured negative photoresist material and exposing the initial layer of negative photoresist material to harden the negative photoresist material in the form of a lower section of a wall layer, wherein the uncured negative photoresist material exposed using the multiple exposure technique is deposited as a further layer on the initial layer of uncured negative photoresist material. This multiple deposition technique can enable the formation of deeper wells, which is advantageous in some applications.

[0048] In some embodiments, the method includes exposing the negative photoresist material to light such that the negative photoresist material hardens in the form of overhangs and laterally extending protrusions in the areas of the overhangs, which allow for further control over the surface and structural properties of the overhangs, thereby improving the performance of the nanopore support structure.

[0049] In some embodiments, the method includes exposing the negative photoresist material using a multiple exposure technique to harden the negative photoresist material in the form of overhangs and laterally protruding inner protrusions within the extent of the overhangs inside each well.

[0050] In some embodiments, the method includes depositing an overhanging layer of uncured negative photoresist material; exposing the first layer of negative photoresist material to light so as to harden the negative photoresist material in the form of overhangs; depositing a top layer of uncured negative photoresist material on the overhanging layer of uncured negative photoresist material; and exposing the top layer of negative photoresist material to light so as to harden the negative photoresist material in the form of laterally protruding outward protrusions outside the extent of each well.

[0051] The inner and outer protrusions provide control over the surface and structural properties of the overhangs both inside and outside the well. Forming the outer protrusions using photoresist requires an additional deposition step to ensure proper formation of the overhangs below the protrusions.

[0052] In some embodiments, the step of removing uncured negative photoresist material is performed only after the negative photoresist has hardened in the form of overhangs and protrusions. In embodiments involving multiple deposition steps, removing all uncured photoresist in a single step after all curing steps improves the efficiency of the manufacturing process and allows lower layers of photoresist to support upper layers during curing.

[0053] In some embodiments, a method includes depositing a first layer of uncured negative photoresist material, exposing the first layer of negative photoresist material to light so as to harden the negative photoresist material in the form of a wall layer and a barrier disposed within the wells, removing the uncured negative photoresist material of the first layer to form the wells and the barrier, depositing a second layer of uncured negative photoresist material on the first layer of uncured negative photoresist material, exposing the second layer of negative photoresist material to light so as to harden the negative photoresist material in the form of an overhang, the barrier being shaped to reduce scattering of electromagnetic radiation applied in the exposure, and removing the uncured negative photoresist material of the second layer. The use of a barrier prevents scattering that could cause hardening of the photoresist in undesired areas and lead to structural defects. The barrier also provides structural support to higher layers in embodiments involving multiple deposition steps when at least a portion of the uncured photoresist is removed between steps.

[0054] In some embodiments, the method includes forming a wall layer having walls defining a plurality of wells and forming overhangs in a separate step, the overhangs being secured to the wall layer so as to extend from the walls across the wells. Performing separate steps can improve flexibility of the manufacturing method.

[0055] In some embodiments, forming the wall layer comprises molding the wall layer onto a substrate. Molding can be advantageous in some circumstances, depending, for example, on the material used for the nanopore support structure. Molding the wall layer onto a substrate advantageously bonds the wall layer to the substrate.

[0056] In some embodiments, the substrate has an electrode formed thereon and the wall layer is molded onto the substrate to position the electrode within the well. The electrode can be implemented such that the substrate is formed from a different material than the wall layer. This improves flexibility in the design and manufacture of the electrode.

[0057] In some embodiments, forming the overhangs includes molding the overhangs with protrusions that extend laterally beyond the extent of the overhangs. In some embodiments, the protrusions include outward protrusions that extend away from the respective wells, and forming the overhangs includes molding the overhangs with the outward protrusions on the wall layer. In some embodiments, forming the overhangs includes molding the overhangs on the wall layer. The protrusions have advantages as discussed above. The protrusions can be configured concentrically around the aperture. Molding the overhangs separately from the wall layer and molding them on the wall layer allows for more flexibility in the selection of materials for the wall layer and the overhangs, which may be necessary depending on the exact molding process used.

[0058] In some embodiments, the method further includes forming membranes extending across each aperture and, optionally, inserting nanopores into at least some of the membranes, thereby preparing the nanopore support structure for incorporation into a nanopore sensing device. In some embodiments, the membranes extend in a planar direction.

[0059] According to a fourth aspect of the present invention, there is provided a method for manufacturing a nanopore sensing device, the method comprising: fabricating a nanopore support structure by the method according to the third aspect of the present invention; and fabricating a nanopore sensing device, the nanopore sensing device comprising a planar structure having first and second chambers and a nanopore support structure, the planar structure being provided with a plurality of fluid passages extending between the first and second chambers and including respective wells and apertures in the nanopore support structure, the apertures opening into the first chamber; and an electrode arranged to sense fluid potential in each passage between the nanopore and the second chamber.

[0060] According to a fifth aspect, there is provided a method of making a nanopore array support structure, the method comprising the steps of: providing a planar substrate comprising an array of electrodes; providing a wall layer on the substrate to form wells, each well comprising an electrode; washing the electrodes to remove any residue from the step of providing the wall layer; and providing a top layer on the wall layer defining an array of corresponding apertures configured to support membranes suitable for insertion of nanopores.

[0061] The wall layer and / or top layer may be formed by a molding process.

[0062] The top layer may include an overhang extending from the wall across each of the wells, the overhang defining an aperture configured to support a membrane suitable for insertion of a nanopore.

[0063] Cleaning the electrode may include exposing a surface of the electrode to at least one of a plasma treatment, a chemical treatment, a UV treatment, a heat treatment, and a laser treatment.

[0064] The method may further include forming an array of membranes over a corresponding array of apertures.

[0065] The method may still further comprise the step of providing a nanopore in each of the membranes of the array.

[0066] Providing a cleaning process before adding the top layer allows the electrode to be cleaned while ensuring that the top layer is not subjected to a cleaning process that could damage the surface and make it less than optimal for forming a film.

[0067] To enable a better understanding, embodiments of the invention will now be described, by way of non-limiting example, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0068] [Figure 1a] FIG. 1 is a cross-sectional side view of a nanopore support structure. [Figure 1b] FIG. 1b is a plan view of the array of nanopore support structures of FIG. 1a. [Figure 1c] illustrates a bilayer within a nanopore support structure. [Figure 2] 10A-10C are cross-sectional side views of nanopore support structures having alternative configurations. [Figure 3] FIG. 1 is a cross-sectional side view of a nanopore sensing device incorporating a nanopore support structure. [Figure 4] FIG. 10 is a cross-sectional side view of a well overhang with a protrusion. [Figure 5a] 1A-1C are cross-sectional side views of a nanopore support structure during steps of a first fabrication method using photoresist. [Figure 5b] 1A-1C are cross-sectional side views of a nanopore support structure during steps of a first fabrication method using photoresist. [Figure 5c]1A-1C are cross-sectional side views of a nanopore support structure during steps of a first fabrication method using photoresist. [Figure 5d] 1A-1C are cross-sectional side views of a nanopore support structure during steps of a first fabrication method using photoresist. [Figure 5e] 1A-1C are cross-sectional side views of a nanopore support structure during steps of a first fabrication method using photoresist. [Figure 6a] 1A-1C are side views of a nanopore support structure during steps of a second fabrication method using photoresist. [Figure 6b] 1A-1C are side views of a nanopore support structure during steps of a second fabrication method using photoresist. [Figure 6c] 1A-1C are side views of a nanopore support structure during steps of a second fabrication method using photoresist. [Figure 6d] FIG. 1 is a side view of the resulting nanopore support structure with a membrane formed therein. [Figure 7a] FIG. 10 is a side view of a comparative example of a nanopore support structure without overhangs. [Figure 7b] FIG. 1 is a side view of a nanopore support structure fabricated using a second fabrication method with a membrane formed therein. [Figure 8a] 10A-10C are side views of a nanopore support structure during steps of a third fabrication method using photoresist. [Figure 8b] 10A-10C are side views of a nanopore support structure during steps of a third fabrication method using photoresist. [Figure 9a] 10A-10C are side views of a nanopore support structure during steps of a fourth fabrication method using photoresist. [Figure 9b] 10A-10C are side views of a nanopore support structure during steps of a fourth fabrication method using photoresist. [Figure 9c] FIG. 1 is a plan view of the resulting nanopore support structure. [Figure 10a] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10b] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10c] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10d] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10e] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10f] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10g] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10h] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10i] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10j] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10k] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 10l] 10A-10C are side views of a nanopore support structure during steps of a fourth manufacturing method using molding. [Figure 11] 1 is a graph of open pore current versus time for a conventional nanopore support structure and a nanopore support structure. [Figure 12] 1 shows possible shapes of the aperture of the nanopore support structure. [Figure 13] 1 is a schematic cross-sectional view of a sensing device or component thereof having a turret-like well array structure connected to a substrate having electrodes at the base of the walls. [Figure 14a] 14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 14b] 14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 14c]14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 14d] 14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 14e] 14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 14f] 14A-14C are cross-sectional views of process steps that can be used to fabricate the components of FIG. 13. [Figure 15] FIG. 10 is a perspective view of a mold having protrusions with shortened fins. [Figure 16] FIG. 12 is a perspective view of a single compartment of an improved well array structure, where a recess is provided in the base of the compartment. [Figure 17a] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device or component thereof having a turret-like well array structure connected to a coated, drilled, filled, and etched substrate, before the well array structure is connected. [Figure 17b] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device or component thereof having a turret-like well array structure connected to a coated, drilled, filled, and etched substrate, before the well array structure is connected. [Figure 17c] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device or component thereof having a turret-like well array structure connected to a coated, drilled, filled, and etched substrate, before the well array structure is connected. [Figure 17d] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device or component thereof having a turret-like well array structure connected to a coated, drilled, filled, and etched substrate, before the well array structure is connected. [Figure 17e] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device or component thereof having a turret-like well array structure connected to a coated, drilled, filled, and etched substrate, before the well array structure is connected. [Figure 18] 1 is a cross-sectional view of a conductive via in a substrate. [Figure 19a] 1 is a cross-sectional view of a via through a substrate before coating with a metal film. [Figure 19b] 1 is a cross-sectional view of a via through a substrate before coating with a metal film. [Figure 20a] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20b] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20c] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20d] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20e] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20f] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 20g] 1A-1C are schematic cross-sectional views of method steps for forming a sensing device from a sheet having a metallized coating on one side, including forming and filling vias and forming a well array structure in the sheet on the other side. [Figure 21]FIG. 1 is a screenshot of current measurements obtained during blocking of a pore by an analyte (TBA), the pore being inserted into a membrane supported on a sensing structure of the present invention. [Figure 22a] FIG. 1 is a cross-sectional view of a conductive via through a sheet having metal films on both sides. [Figure 22b] FIG. 1 is a cross-sectional view of a conductive via through a sheet having a metal film on one side and a screen-printed pad on the other. [Figure 23a] 1 is a schematic plan view of tracks and pads on a substrate surface aligned with a cross-sectional view of conductive vias connected to the pads; [Figure 23b] 1 is a schematic cross-sectional view and a plan view of a pad with a capillary stop. [Figure 24] 1 is a table of sheet materials and corresponding properties. [Figure 25] 1 is a cross-sectional view of the layers forming a sensor on a substrate formed using the techniques disclosed herein. [Figure 26a] This is a process performed to fabricate the overhang in a single layer of material. [Figure 26b] This is a process performed to fabricate the overhang in a single layer of material. [Figure 26c] This is a process performed to fabricate the overhang in a single layer of material. [Figure 26d] This is a process performed to fabricate the overhang in a single layer of material. [Figure 27] 1 is a cross-sectional view of a well formed in a substrate with an overhang layer attached to the well in place to create the well. [Figure 28] FIG. 1 is a schematic top view of a nanopore support structure with outward protrusions. [Figure 29a] FIG. 1 is a schematic top view of a nanopore support structure with outward protrusions. [Figure 29b] 1 is a cross-sectional side view of a nanopore support structure having outward protrusions. [Figure 30]FIG. 10 is a top view of a nanopore support structure having an alternative form of outer protrusion.

[0069] 1a and 1b show a nanopore support structure 1 in which known problems are reduced. The nanopore support structure 1 is designed to support a membrane 7, which in turn supports a nanopore 8 inserted into the membrane 7. The nanopore 8 is used to enable nanopore-based sensing, which may employ measurement of ionic current flow through the nanopore 8. When a molecule, such as DNA, translocates through the nanopore 8, a change in ionic current flow is generated that allows the molecule to be characterized. Examples of suitable membranes 7 and nanopores 8 are discussed further below.

[0070] The nanopore support structure 1 comprises a wall layer 2 comprising walls 3 that define a plurality of wells 4. While a single well is illustrated in FIG. 1a for clarity, as shown in FIG. 1b, a plurality of wells 4 can be arranged in a plane in a regular planar array having a repeating structure when viewed along the longitudinal axis of the wells 4 perpendicular to the plane of the array (the vertical direction in FIG. 1). The array can be arranged in a rectilinear grid or a hexagonal layout. While FIG. 1b shows only nine wells 4 for simplicity, in general, the nanopore support structure 1 can have any number of wells, typically much more than nine, for example, on the order of 1,000 or more, or even up to 1,000,000 or more.

[0071] The walls 3 of the well 4 can extend perpendicularly from the base 11 of the well. The footprint of the well 4 can be circular, but this is not required and other shapes can be used, such as square, rectangular, oval, or hexagonal. In some embodiments, the well 4 has a substantially constant cross-section along at least one axis, for example, having the shape of a prism. In some embodiments, the well 4 may not have a constant cross-section. For example, the well 4 can be in the form of a hemisphere or a "dimple."

[0072] The nanopore support structure 1 includes an overhang 5 extending from the wall 3 across each of the wells 4. The overhang 5 defines the upper extent of the well 4 along the longitudinal axis of the well 4. The overhang 5 extends in a plane parallel to the plane of the array of wells 4 and perpendicular to the longitudinal axis of the well 4. In FIG. 1 , where the wells 4 are cylindrical, the overhang 5 extends perpendicular to the wall 3, but this may not be the case if the wells 4 have other shapes. The overhang 5 extends inward from the wall 3 toward the center of the well 4. The overhang 5 may be substantially symmetrical about the longitudinal axis of the well 4, thereby extending the same distance from the wall 3 at all points around the periphery of the well 4, although this is not required; the wells 4 may have different shapes that are not symmetrical about the longitudinal axis of the well 4.

[0073] The overhang 5 defines an aperture 6 configured to support a membrane 7 suitable for insertion of a nanopore 8. The aperture 6 may be circular, but in other embodiments may have other shapes, such as a square. The shape of the aperture 6 may be independent of the shape of the well 4, such as the cross-section of the well.

[0074] Aperture 6 may also have more complex shapes, for example, having one or more portions of its periphery extending inward toward the center of aperture 6. Examples of such shapes are shown in Figure 12, where the black portions represent aperture 6. These may take the form of finger structures around the periphery of aperture 6, or regularly spaced triangular features that give aperture 6 the appearance of a gear. Such structures help to better deliver and control the oil used to promote film formation.

[0075] The use of aperture 6 defined by overhang 5 allows for a decoupling of the size of well 4 from the size of membrane 7 required to cover well 4. In particular, the size of aperture 6 can be limited relative to the diameter of well 4. This limitation on aperture 6 can reduce the size and area of ​​the actual membrane, thereby reducing the capacitance of membrane 7 and improving the stability of membrane 7.

[0076] It may also eliminate the need for a pretreatment step when the membrane 7 is formed on the nanopore support structure 1. Such a pretreatment step may include flowing a non-polar medium over the nanopore support structure 1 to promote adhesion of the membrane 7 to the nanopore support structure 1. Having a limited aperture 6 may make this step unnecessary.

[0077] The limited aperture 6 can further equalize pressure across the membrane 7, pinning the membrane 7 in position at the aperture 6. In particular, the top and bottom surfaces of the membrane 7 can extend from the same plane (i.e., the end faces of the overhangs 5 ​​that define the aperture 6). This reduces the depth of the membrane 7 and improves the symmetry of the membrane 7 along the longitudinal axis of the well, which further reduces any bias that could lead to movement of the membrane 7 and ejection of the well 4 during use, as described below. This enables the use of wells 4 having openings or through-holes in the base 10 of the well 4.

[0078] The bilayer in the well can be configured as shown in FIG. 1c, where the meniscus of the fluid in the well protrudes at least partially through the aperture 6, while the meniscus of the fluid in the cis-chamber extends toward the aperture, forming a bilayer interface. The bilayer interface forming the membrane can be adjacent to a region of the aperture defined by a plane defined by the surfaces of the overhang 5. The bilayer interface can be flush with the upper edge of the aperture. The meniscus forming the membrane can be asymmetric. FIG. 1c shows an outward protrusion 51a, discussed below in connection with FIGS. 28 and 29.

[0079] Furthermore, the size of the well 4 can be increased without requiring a larger membrane 7, which is advantageous in many situations. In particular, this can allow a higher ionic current to be maintained through the nanopore 8 for a longer period of time. Figure 11 compares the open pore current measured through (i) a conventional design of nanopore support structure 1 lacking an overhang 5, as shown in the bottom trace, and (ii) a nanopore support structure 1 having an overhang 5 defining an aperture 6 according to the present invention, as shown in the top trace. In the conventional design, the open pore current drops more rapidly, dropping abruptly to zero after less than 25 hours as the mediator solution is depleted. In contrast, the open pore current through the nanopore support structure 1 with the overhang 5 drops more slowly, remaining at approximately 50% of its original value even after 40 hours. This solution depletion issue can also be addressed by providing a second chamber 32 separate from the well 4, as discussed further below.

[0080] The size of the wells 4 can generally be increased by increasing the depth and / or increasing the cross-sectional area (i.e., footprint) transverse to the depth, however, increasing the depth is preferred to provide a high density array, as increasing the cross-section reduces the density of apertures 6 across the array.

[0081] Typically, the aspect ratio of width to depth of the wells may be at least 1:3, preferably at least 1:5, and more preferably at least 1:10. "Width" in this context may be the diameter in the case of wells 4 that are circular, or may be a characteristic width such as the square root of the cross-sectional area.

[0082] Typically, the aspect ratio of aperture width to well width may be at least 2:3, preferably in the range of 1:2 to 1:5, but may be up to 1:10. "Width" in this context may be the diameter in the case of wells 4 that are circular, or may be a characteristic width such as the square root of the cross-sectional area.

[0083] Typically, the wells 4 may have a depth of at least 50 μm, preferably at least 120 μm, although this is not limiting and the wells may for example have a depth of up to 1 mm.

[0084] The height of the pillars can range from 5 μm to 30 μm, such that the aspect ratio of pillar height to well depth can range from 1:10 to 1:200.

[0085] The wells 4 have respective bases 10. The bases 10 define the lower extent of the wells 4 along the longitudinal axis of the wells 4. The bases 10 extend in a plane parallel to the plane of the array of wells 4. In FIG. 1, the bases 10 are perpendicular to the walls 3 of the nanopore support structure 1, although in general this may not be the case depending on the shape of the wells 4.

[0086] The nanopore support structure 1 further comprises a substrate 20. The substrate 20 may be formed from a different material than the wall layer 2. For example, the substrate 20 may be formed from silicon or a plastic, such as Kapton™. The wall layer 2 is secured to the substrate 20, which defines the base 10 of the well 4. The wall layer 2 may be formed directly on the substrate 20, or may be formed separately and secured to the substrate 20 in a subsequent step.

[0087] The nanopore support structure 1 can include an electrode 11 formed on the base 10 of a well 4, which can be filled with an ionic solution, which can be an aqueous solution. The ionic solution can include a soluble electrode mediator, such as a ferri / ferrocyanide compound.

[0088] In instances where the well 4 is closed, the electrode 11 can be used to measure the ionic current within the well 4 relative to a reference outside the well 4. In instances where the well forms part of a passageway, for example of the type shown in Figure 3, the electrode 11 can be used to measure the fluid potential within the well 4.

[0089] 1a shows a membrane 7 extending across each aperture 6 and a nanopore 8 inserted into the membrane 7. The nanopore 8 can be a biological nanopore, and the membrane 7 can have a biological nanopore inserted therein. The membrane 7 can be an amphiphilic membrane formed, for example, from a phospholipid bilayer. However, a nanopore sensing device 30 comprising a nanopore support structure 1 according to the present invention can be provided without the membrane 7 and nanopore 8. In this case, the end user performs the steps of forming the membrane 7 and inserting the nanopore 8 therein.

[0090] 2 shows a nanopore support structure 1 having an alternative configuration in which the wells 4 have respective bases 10, but the wall layer 2 further defines the bases 10. In this case, the bases 10 and the wall layer 2 are formed from the same material and may, but need not, be formed in the same manufacturing process. Even when the wall layer 2 defines the bases 10, the nanopore support structure 1 may still comprise a substrate 20, with the wall layer 2 being secured to the substrate 20.

[0091] A nanopore support structure 1 such as that shown in Figures 1 or 2 can be incorporated into a nanopore sensing device. When the well 4 is closed, one possible approach is to provide the nanopore support structure 1 with an aperture 6 that opens into the chamber 25, as shown, for example, in Figure 1.

[0092] Another approach is to place the nanopore support structure 1 between two chambers and position the well 4 to form part of the passageway between the chambers, an example of which is described here.

[0093] FIG. 3 illustrates a nanopore sensing device 30 configured as follows: The nanopore sensing device 30 includes a first chamber 31 and a second chamber 32, with a planar structure 40 between the first chamber 31 and the second chamber 32. The first and second chambers 31, 32 are filled with fluid. While the first and second chambers 31, 32 are shown schematically in FIG. 3, they may be arranged in any suitable configuration. The first and second chambers 31, 32 may be closed or may be arranged as part of a flow cell that allows for the flow of solutions therethrough. In FIG. 3, the well 4 is separate from both the first chamber 31 and the second chamber 32. However, in some embodiments, such as those shown in FIGS. 1 and 2, the second chamber 32 is provided by the well 4 itself. When the well 4 is separate from the second chamber 32, each well 4 may have its own second chamber 32, or alternatively, one or more wells 4 may share a common second chamber 32. In some embodiments, all of the wells 4 of the nanopore sensing device 30 may share a single second chamber 32 .

[0094] The planar structure 40 is provided with a plurality of fluid passages 41 extending between the first chamber 31 and the second chamber 32. The fluid passages 41 are therefore filled with fluid and fluidly connect the first and second chambers 31, 32. Because each of the fluid passages 41 is connected to the first and second chambers 31, 32, the nanopores 8 are in parallel paths of fluid communication. The plurality of fluid passages 41 may be arranged in a two-dimensional array across the planar structure 40.

[0095] 3, the configuration of the planar structure 40 is not shown, and the fluid passages 41 are shown only diagrammatically. The configuration of the planar structure 40 and the fluid passages 41 will be described in detail below.

[0096] In this example, the first and second chambers 31, 32 are on opposite sides of the planar structure 40, so that the fluid passageway 41 extends through the planar structure 40. However, the first and second chambers 31, 32 may alternatively be located at different positions on the same side of the planar structure 40.

[0097] In this example, drive electrodes 33, 34 are provided in the first and second chambers 31, 32. In use, a potential difference may be applied across the drive electrodes 33, 34, and thus across each fluid passage 41, to induce the flow of analyte between the first chamber 31 and the second chamber 32. The drive electrodes 33, 34 may be configured to apply substantially the same potential difference across all of the fluid passages 41. Additionally or alternatively, the nanopore sensing device 30 may be configured to induce the flow of analyte through the fluid passages 41 using other techniques.

[0098] The first chamber 31 may function as a cis chamber and hold an analyte to be analyzed by the nanopore sensing device 30. The second chamber 32 may function as a trans chamber and receive the analyte from the first chamber 31.

[0099] The planar structure 40 therefore supports nanopores 8 in the membrane 7 that extend across each fluid passage 41 .

[0100] Each fluid passage 41 is provided with an electrode 11 positioned to sense the fluid potential within the respective fluid passage 41 between the nanopore 8 and the second chamber 32. When an analyte passes through the nanopore 8, a fluctuation in potential caused by a change in ionic current flow is detected by the electrode 11. Thus, the nanopore 8 and electrode 11 in a given fluid passage 41 act as respective sensors in the nanopore sensor device 30. The planar structure 40 comprises a nanopore support structure 1 and a base layer 42 fixed together. As shown in FIG. 3, the base layer 42 replaces the substrate 20 of the nanopore support structure 1 in this example, although alternatively, the entire nanopore support structure 1 as shown in FIGS. 1 or 2 may be fixed to the base layer 42.

[0101] The nanopore support structure 1 is configured to support a nanopore 8 in a membrane 7 that extends across a fluid passageway 41. In particular, the nanopore support structure 1 is provided with wells 4 that open into the first chamber 31. The wells 4 form part of the fluid passageway 41 and therefore have openings in their bases 10.

[0102] As previously described, the well 4 is configured to support a membrane 7 that extends across the fluid passageway 41, specifically across the opening of the well 4, thereby supporting a nanopore 8. The nanopore support structure 1 includes a wall layer 2 with walls 3 that define the well 4.

[0103] The base layer 42 is between the nanopore support structure 1 and the second chamber 32 and includes the following layers: The base layer 42 includes a semiconductor wafer 43, which in this example is formed by the substrate 20 of the nanopore support structure 1. The semiconductor wafer 43 supports a circuit layer 44. The circuit layer 44 includes circuit components connected to the electrodes 11. The circuit components can allow the fluid potential at the electrodes 11 to be sensed by an external circuit.

[0104] Fluidic passage 41 can provide the function of a fluidic resistor between electrode 11 and second chamber 32. "Between" is used in the sense of fluid movement, and therefore the flow path between electrode 11 and second chamber 32 includes fluidic resistor portion 50. Fluidic resistor portion 50 may not be physically located entirely between electrode 11 and second chamber 32 in all embodiments. More specifically, fluidic passage 41 includes well 4, as described above, extending through base layer 42 and fluidically connected in series with fluidic resistor portion 50 formed in base layer 42. The fluidic resistor portion includes circuit layer access hole 80 extending through circuit layer 44 and wafer access hole 82 extending through semiconductor wafer 43 and fluidically connected to circuit layer access hole 80.

[0105] The fluidic resistor portion 50 is configured to form a fluidic resistor. In this manner, the fluidic passage 41 is configured to provide a fluidic resistor between the electrode 11 and the second chamber 32.

[0106] The wafer access holes 82 form part of the fluidic resistor portion 50 and the fluid passage 41, and their dimensions can be configured to determine the fluidic resistance of the fluidic resistor portion 50. The wafer access holes 82 are structurally and geometrically configured to function as a fluidic resistor. This can be achieved by defining the aspect ratio of the wafer access holes 82. Additionally or alternatively, other techniques for implementing fluidic resistance in the wafer access holes 82 can be used, such as configuring the dimensions of the circuit layer access holes 80 to function as a fluidic resistor.

[0107] The fluid resistance of the wafer access hole 82 can be varied by modifying its dimensions, particularly its aspect ratio, as well as by modifying the ion concentration of the fluid in the first and second chambers 31, 32. For example, the wafer access hole 82 can be configured with a high aspect ratio to increase the resistance. Additionally or alternatively, the fluid in the wafer access hole 82 can have a lower ion concentration compared to the fluid in the first and second chambers 31, 32 to increase the resistance of the wafer access hole 82. Maintaining a higher ion concentration in the first and second chambers 31, 32 improves the signal-to-noise ratio.

[0108] As a result, the fluid passage 41 forms a voltage divider across the electrode 11 .

[0109] The resistance of the nanopore 8 resides in the first leg of the voltage divider between the electrode 11 and the first chamber 31. The well 4 is designed to have minimal fluid resistance compared to the nanopore 8.

[0110] The fluidic resistance of fluidic resistor portion 50 exists in the second leg of the voltage divider between electrode 11 and second chamber 32. Circuit layer access hole 80 and wafer access hole 82 may be designed to provide negligible fluidic resistance compared to the fluidic resistance of fluidic resistor portion 50, although this is not required and they may be designed to provide additional fluidic resistance.

[0111] As a result of the voltage divider, the fluid potential sensed within the fluid passage 41 by the electrode 11 allows for sensing of the current flowing through the fluid passage 41 and thus the nanopore 8. This allows for nanopore sensing.

[0112] The fluid passage 41, and in particular the fluid resistor portion 50, can be configured so that when the fluid passage 41 is filled with fluid, the resistance of the two legs of the voltage divider is substantially matched and relatively high compared to the resistance of the fluid in the first and second chambers 31, 32, thereby causing the resistance of the first and second chambers 31, 32 to have little effect on the measurement.

[0113] The signal-to-noise ratio can be optimized by selecting the fluid resistances of the two legs of the voltage divider to be equal. However, this is not required, and the fluid resistance of the fluid resistor portion 50 can be varied to take other factors into account while still obtaining an acceptable signal-to-noise ratio. An acceptable signal-to-noise ratio can be achieved if the fluid resistance of the second leg of the voltage divider is significantly lower than the resistance of the first leg of the voltage divider, for example, if the fluid resistance of the second leg of the voltage divider is 10% or less, e.g., 2%, of the resistance of the first leg of the voltage divider. In some embodiments, the lower limit of the fluid resistance of the second leg of the voltage divider can be set by the desired signal-to-noise ratio.

[0114] Other factors that may be considered in selecting the fluidic resistance of the second leg of the voltage divider, specifically the fluidic resistor portion 50, are as follows.

[0115] As the fluidic resistance of the second leg increases, ion diffusion decreases and ion depletion near the pore increases, causing signal attenuation over the timescale of a typical event for which a signal is acquired. To increase the available read time, which is limited by this effect, the fluidic resistance of the fluidic resistor portion 150 can be reduced. In many embodiments, this factor can impose an upper limit on the fluidic resistance of the fluidic resistor portion 50.

[0116] As the fluidic resistance of the fluidic resistor portion 50 increases, the voltage fluctuation across the nanopore 8 increases, which can complicate signal processing. To limit this effect, the fluidic resistance of the fluidic resistor portion 50 can be reduced. Reducing the fluidic resistance of the fluidic resistor portion 50 can increase bandwidth or provide room for additional capacitance in the fluid passage 41.

[0117] Taking these factors into consideration, the fluidic resistance of the second leg of the voltage divider can typically be up to 50%, or up to 25%, lower than the resistance of the nanopore 8. In some embodiments, the optimal fluidic resistance of the second leg of the voltage divider can be about 10% of the resistance of the nanopore 8.

[0118] When the ratio of the fluidic resistance of the second leg of the voltage divider to the resistance of the first leg of the voltage divider is reduced, the signal-to-noise ratio is not directly proportional to the resistance ratio. For example, in some embodiments, when the fluidic resistance of the second leg of the voltage divider is about 10% of the resistance of the nanopore 8, the signal-to-noise ratio is about 30% of its optimal value.

[0119] The overhang illustrated in Figure 3 can be implemented in the sensing device described and shown in WO2020 / 183172, the entire contents of which are incorporated herein by reference.

[0120] We will now describe various methods for fabricating the above-mentioned nanopore support structure 1. These methods refer specifically to the nanopore support structure 1 shown in FIG. 1 in which the base 10 of the well 4 is formed by a separate substrate 20, but can be equally adapted to the nanopore support structure 1 shown in FIG. 2 in which the base 10 of the well is formed by a portion of the wall layer 2.

[0121] As shown in the example of FIG. 4 , some of the methods result in overhang 5 further comprising protrusions 51, 52 that extend laterally beyond the extent of overhang 5. As discussed further below, in some embodiments, this may be achieved by depositing (or laminating) an additional layer of photoresist material on top of overhang 5. Protrusions 51, 52 may act to support membrane 7 or to control where oil (or more generally, non-polar media) is retained on nanopore support structure 1. For this reason, protrusions 51, 52 may also be referred to as oil control features (or more generally, non-polar media control features). In some embodiments, the oil retention properties of protrusions 51, 52 may help prevent oil from covering electrode 11, which could reduce the electrical performance of electrode 11.

[0122] The walls 3 of the well 4 may also be designed with oil (or more generally non-polar media) control in mind.

[0123] In some embodiments, wall 3 may be featureless. This may be because the wall is not intentionally exposed to non-polar media during the processes of filling the wells, forming the membrane, and inserting the nanopores into the membrane, and therefore control features are not necessary. This may reduce collection of non-polar media on wall 3, which may be advantageous in some situations, for example, to reduce collection of non-polar media on electrode 11.

[0124] In other embodiments, wall 3 may be provided with a non-polar media control feature, such as a protrusion. Such a non-polar media control feature may extend across the entire wall 3, from overhang 5 to base 11, or may be provided only in a portion of wall 3 adjacent overhang 5, e.g., region 60 in the example of FIG. 4. By way of example, protrusion 52 may be provided at the interface between overhang 5 and the wall of well 4. By way of example, the wall of well 3 may incorporate fin-like features such as those disclosed in US 2015 / 265994.

[0125] The protrusions 51, 52 comprise an inner protrusion 52 that protrudes laterally to the extent of the inner overhang 5 of each well 4, and an outer protrusion 51 that protrudes laterally to the extent of the outer overhang 5 of each well 4. The protrusions 51, 52 extend in opposite directions along the same axis, which is the same as the longitudinal axis of the wells 4. However, this is not required, and generally the outer and inner protrusions 51, 52 may extend along different axes from each other and / or along an axis that is different from the longitudinal axis of the wells 4. The protrusions 51, 52 in FIG. 4 have a substantially square shape, although other shapes are possible depending on the particular application or intended function of the protrusions 51, 52.

[0126] Typical protrusions 51, 52 are arrays of small posts or posts with small fingers. In particular, protrusions 51, 52 can be used to increase the retention of non-polar media by overhang 5, which is advantageous for promoting accurate formation of membrane 7 in aperture 6. Additionally or alternatively, protrusions 51, 52 can be used to increase the rigidity of overhang 5. Rigidity can be increased, for example, by forming straight or curved ridges on overhang 5 that extend from wall 3 onto overhang 5 (in the case of inner protrusions 52) or from above wall 3 onto overhang 5 (in the case of outer protrusions 51).

[0127] Although the embodiment of FIG. 4 includes both inner and outer protrusions 52, 51, the overhang 5 may alternatively include only inner protrusions 52 or only outer protrusions 51.

[0128] In some embodiments, the outward protrusions 51 may be formed to define protrusion walls 101 that at least partially surround each aperture 6. Figure 28 illustrates such an embodiment. In such embodiments, the outward protrusions 51 may protrude laterally beyond the extent of the overhangs 5 ​​outside each well 4 and / or beyond the extent of the outer surface of the wall layer 2 between the wells 4 (i.e., the surface of the wall layer 31 facing the first chamber 31). The outward protrusions 51 formed on either the wall layer 2 or the overhangs 5 ​​may be formed using processes described in more detail below.

[0129] 28 shows a top view of a nanopore support structure 1 comprising multiple wells 4, each with an overhang 4 defining an aperture 6. For clarity, only one of the illustrated wells 4 is fully labeled in the drawing. While only four wells 4 are illustrated, it should be understood that any number of wells 4 and corresponding features may be used.

[0130] For each aperture 6, the outer protrusions 51 comprise a plurality of peripheral outer protrusions 51 at least partially surrounding the aperture 6 and defining respective protrusion walls 101 recessed therefrom. In the illustrated embodiment, the outer protrusions 51 comprise a plurality of peripheral outer protrusions 51 a per aperture 6. The peripheral outer protrusions 51 a are disposed partially around their respective apertures 6, leaving gaps between the peripheral outer protrusions 51 a. The peripheral outer protrusions 51 a comprise an inner surface facing towards the aperture and an outer surface facing away from the aperture, which define the protrusion walls 101. The peripheral outer protrusions 51 a may be disposed concentrically around the aperture.

[0131] In the illustrated embodiment, three peripheral outer protrusions 51 a are provided for each aperture 6. Any other number of peripheral outer protrusions 51 a, including one, may be used to define the peripheral wall 101 around each aperture. The number of peripheral outer protrusions 51 a may vary between 2 and 20. One or more of the peripheral outer protrusions 51 a may be common to multiple apertures 6.

[0132] The protruding wall 101 is configured to allow a meniscus to form across each aperture 6 such that the meniscus extends at least partially into the respective aperture 6. In particular, the height of the protruding wall 101 above the aperture and / or the distance between the aperture 6 and the protruding wall (i.e., how far the protruding wall 101 is set back from the edge of the aperture 6) can be configured to allow a meniscus to form that extends at least partially into the aperture 6. Generally, the protruding wall 6 can be such that the interface between the upper and lower fluids is in a Fakir state. Conditions for forming a Fakir state are discussed in Afferrante et al., J. Phys.: Condens. Matter 22 (2010) 325107 (incorporated herein by reference). This control of the meniscus can be used to assist in the formation of a film at the aperture, avoiding the need for pre-treatment to promote film formation.

[0133] In the illustrated embodiment, the peripheral outer protrusions 51a are positioned with gaps between them so that the protrusion wall 101 only partially surrounds the aperture 6. While the protrusion wall 101 is shown in FIG. 28 as surrounding approximately 85% of the aperture, the gaps within the wall can be sized so that the protrusion wall surrounds 75% to 95%, including the aperture 6. Here, the surrounded percentage is equal to the ratio of the total azimuthal extent of the protrusion wall 101 (considered in polar coordinates relative to the center of the aperture 6) to a full circle (i.e., 360°). Such coverage provides a balance between controlling the meniscus within the peripheral wall 101 and allowing the flow of nonpolar media through the gaps toward the aperture 6.

[0134] The outer protrusions 51 in the illustrated embodiment further comprise intermediate outer protrusions 51b, which are located in the areas between the peripheral outer protrusions 51a. The intermediate outer protrusions 51b further aid in the flow of non-polar media and limit wetting of buffer solution into the protrusion wall 101. The illustrated intermediate protrusions 51b have a substantially triangular cross-section, although other cross-section shapes can be used.

[0135] Figures 29a and 29b illustrate further views of the nanopore support structure 1 of Figure 28. Figure 29a shows outer protrusions 51a, 51b around a single aperture 6. As can be seen, both the peripheral outer protrusion 51a and the intermediate outer protrusion 51b are arranged concentrically around the aperture 6. The intermediate outer protrusion 51b may be shared between multiple apertures 6, as in Figure 28, but when viewed from the perspective of a single aperture 6, a set of intermediate outer protrusions 51b may be arranged around the aperture 6 concentrically with the aperture 6 and the peripheral outer wall 51a.

[0136] Figure 29b shows a cross section of an individual well 4, illustrating the peripheral outward protrusion 51b of that well 4. Figures 29a and 29b illustrate exemplary relative dimensions of the various components of the nanopore support structure 1. It should be understood that these dimensions are provided by way of example only and are not intended to be limiting.

[0137] In the embodiment of FIG. 28 , the inner and outer surfaces of the peripheral outer protrusion 51 a are substantially smooth. In other embodiments, the inner and / or outer surfaces may include a micropatterned structure. FIG. 30 illustrates an embodiment in which the outer surface of the peripheral outer protrusion 51 a has a micropatterned structure 102. The micropatterned structure 102 can further aid in the flow of non-polar media and limit wetting away from the aperture 6. The micropatterning can be formed by processing a molding structure, which is used to form the outer protrusion 51, as described in more detail below. The micropatterning can also be used on one or more surfaces of one or more intermediate outer protrusions 51 a. By way of example, the micropatterned structure is provided on the intermediate outer protrusion 51 b in FIG. 28 as teeth having a substantially square outline, although other shapes can be implemented. The micropatterned structure 102 can retain oil used in membrane formation. The gaps between the peripheral outer protrusions 51 a can facilitate the flow of oil used in membrane formation across the surface of the well array. To prevent the membrane from descending into the gap and rupturing the membrane, micro-patterned structures 102 can be configured in registration with the gap and act to support the membrane while allowing oil to flow across the surface between the structures. Thus, in some embodiments, one or more intermediate outer protrusions 51 b can include micro-patterned structures 102 on one or more surfaces of the intermediate outer protrusions 51 b, the micro-patterned structures extending toward the gap between the peripheral outer protrusions 51 a.

[0138] One type of method involves using multiple exposure steps with controlled exposure times, or using grayscale exposure techniques to form the overhang structure with only a single final development step. First, we describe an example in which the nanopore support structure 1 is formed by a method using exposure and hardening of a negative photoresist material, whereby the overhang 5 and wall layer 2 comprise the hardened negative photoresist material. Such a method generally includes depositing an unhardened negative photoresist material, exposing the negative photoresist material to light so as to harden it in the form of the nanopore support structure 1, and removing the unhardened negative photoresist material.

[0139] In some embodiments, the method includes depositing an uncured negative photoresist material, exposing the negative photoresist material using a multiple exposure technique to harden the negative photoresist material in the form of a nanopore support structure 1, and removing the uncured negative photoresist material. An example of this type of multiple exposure method is shown in Figure 5. In this example, the nanopore support structure 1 is formed by multiple exposures of the overhang structure, with only one final development step.

[0140] 5, prior to performing the steps of depositing an uncured negative photoresist material and exposing the negative photoresist material using a multiple exposure technique, the method includes performing an initial stage including depositing an initial layer of uncured negative photoresist material and exposing the initial layer of negative photoresist material to light so as to harden the negative photoresist material in the form of a lower section of wall layer 2. This is shown in FIG. 5a where the initial layer is exposed.

[0141] 5b, an uncured negative photoresist material exposed using a multiple exposure technique is deposited as a further layer on the initial layer of uncured negative photoresist material. Subsequently, a step of exposing the negative photoresist material using a multiple exposure technique is carried out so as to harden the negative photoresist material in the form of overhangs 5 ​​and at least the upper section of the wall layer 2 to a level deeper than the overhangs 5.

[0142] In FIG. 5, exposing the negative photoresist material using a multiple exposure technique involves exposing the negative photoresist material in separate exposure steps. This is shown in FIGS. 5c and 5d. In FIG. 5c, the negative photoresist material is hardened in the form of overhangs 5. In FIG. 5d, in a separate exposure step, the negative photoresist material is hardened in the form of at least the upper section of the wall layer 2 to a level deeper than the overhangs 5. In FIG. 5e, a single development step is performed on the entire stack of layers of negative photoresist material to remove all unhardened photoresist material. Hardening the photoresist material to a deeper level in FIG. 5d than in FIG. 5c can be achieved in various ways. For example, the photoresist material can be exposed for a longer time in FIG. 5d than in FIG. 5c, or the intensity of the light used to expose the photoresist material can be higher in FIG. 5d than in FIG. 5c.

[0143] This type of technique can be facilitated by the specific selection of photoresist material. For example, an advantageous property of permanent photoresist materials, such as thick SU-8, is that the exposure light gradually penetrates from top to bottom during the exposure process due to the change in the material's absorption coefficient during and after exposure. This makes it easy to properly select the exposure timing to expose only the top of the resist layer. This, combined with multiple exposures, allows for the exposure of overhangs while leaving unexposed photoresist material underneath. This means that there is no need to develop the initial layer before depositing the uncured negative photoresist material that will be exposed using the multiple exposure technique; the uncured photoresist material can naturally act as a support for the upper photoresist material during subsequent steps.

[0144] The multiple exposure technique is not limited to two different exposure levels provided in two separate exposure steps. An alternative approach is grayscale lithography / exposure. In this technique, the step of exposing a negative photoresist material using multiple exposure techniques involves exposing the negative photoresist material with spatial modulation of intensity. This can be used to create various thicknesses of hardened portions of the photoresist material (up to the total thickness of the resist) depending on the exposure dose. In this way, the cross-section of the aperture 6 can be easily adjusted to promote the formation of the correct film 7.

[0145] As noted above, in some embodiments, the method includes exposing a negative photoresist material to light so as to harden the negative photoresist material in the form of overhangs 5 ​​and protrusions 51, 52 extending laterally beyond the overhangs 5. The protrusions may be inner protrusions 52 and / or outer protrusions 51.

[0146] In some embodiments in which medial protrusions are formed, the method includes exposing the negative photoresist material using a multiple exposure technique to harden the negative photoresist material in the form of overhangs 5 ​​and medial protrusions 52 extending laterally beyond the extent of the overhangs 5 ​​within each well 4. The medial protrusions 52 can be formed simultaneously with the overhangs 5 ​​in the multiple exposure technique. For example, in the process shown in FIG. 5c, greater exposure intensity or exposure time can be provided at some points along the overhangs 5 ​​to expose the photoresist material deeper and form the medial protrusions 52. Alternatively, the exposure intensity can be spatially modulated in a single exposure step. The multiple exposure or grayscale exposure technique allows these protrusions to form not only on top of the overhang surface but also below the overhangs 5, using the support of unexposed photoresist material below the overhangs 5. This can result in better film formation, better oil control, and overall film stability.

[0147] 6 illustrates an embodiment of a method in which both outer protrusions 51 and inner protrusions 52 are formed. In this embodiment, the method includes depositing an overhanging layer of uncured negative photoresist material and exposing the first layer of negative photoresist material to light so as to harden the negative photoresist material in the form of overhangs 5. This corresponds to the steps illustrated in FIGS. 5b-5d, although it is not essential that the tops of walls 3 be formed at this point, similar to that shown in FIG. 5d. The tops of walls 3 may be formed after overhangs 5 ​​and protrusions 51, 52 are fully formed.

[0148] Following the formation of the overhangs, the method includes depositing a top layer of uncured negative photoresist material on the overhang layer of uncured negative photoresist material, as shown in FIG. 6a. Next, a step of exposing the second layer of negative photoresist material is performed so as to harden the negative photoresist material in the form of outward protrusions 51 that extend laterally outside the extent of each well 4, as shown in FIG. 6b. In FIG. 6b, inner protrusions 52 are formed simultaneously with the outer protrusions 51. This can be achieved by exposing the photoresist material to a higher intensity or for a longer time in the areas where inner protrusions 52 will be formed.

[0149] In some embodiments, as shown in FIG. 6b, outer protrusion 51 is directly above inner protrusion 52. This is because when inner protrusion 52 is formed, the top layer of photoresist material is also exposed. If outer protrusion 51 is not directly above inner protrusion 52, the inner protrusion must be formed before the top layer of uncured negative photoresist material is deposited. The step of removing the uncured negative photoresist material is performed only after the negative photoresist has hardened in the form of overhang 5 and protrusions 51, 52. This final development step is shown in FIG. 6c and removes all unexposed (and therefore uncured) photoresist material. Following this, a membrane 7 can be formed over aperture 6 using oil and polymer, as shown in FIG. 6d. However, as noted above, because membrane formation can be performed by the end user, the step shown in FIG. 6d may not be performed before shipping the product.

[0150] The outer protrusions 51 can be formed coincident with, offset from, or a combination of the inner protrusions 52. This can be achieved by separate lamination and exposure processes. When the nanopore support structure 1 includes electrodes 11 to enable voltage sensing, each well 4 has an access hole in the bottom in the form of a fluid passage 41 for connecting to a fluidic resistor portion 50. If a membrane is formed over the wells with access holes using known structures such as those disclosed in US 2015 / 265994, additional pressure from the surface tension of the membrane, or even a pressure perturbation within the fluid, can push the liquid through the fluid passage 41 and slowly drain the buffer solution from the well 4 until the membrane 7 collapses to the bottom of the well 4.

[0151] In contrast, the membrane 7 formed over the aperture 6 by the overhang 5 has one of the following characteristics: (i) a reduced size, thus increasing its stability; (ii) minimal surface tension in its steady state; (iii) a mostly planar surface; and (iv) a shallow depth that can be controlled by the thickness of the overhang. Any small perturbation that pushes the membrane 7 away from the aperture 6 will increase the membrane's size, which is energetically unfavorable. As illustrated in FIG. 7 , one or more of these features can function to effectively anchor the membrane 7 in place at the aperture 6 and help form a long-term stable membrane 7 for voltage-sensing applications.

[0152] Figure 7a shows a nanopore support structure without overhangs 5. The large size of membrane 7 means that membrane 7 is less stable over time. In contrast, Figure 7b shows a nanopore support structure 1 according to the present invention. The smaller aperture 6 means that membrane 7 is much smaller and more stable over time. In all examples discussed herein, the overhang 5 and aperture 6 structure eliminates the need for an oil / membrane surface coating to penetrate the bottom of well 4 during use, as was necessary in previous nanopore support structures. As long as the oil / membrane mixture covers overhangs 5 ​​(and any existing protrusions, e.g., in the form of posts / fingers as seen in Figure 7b), nanopore support structure 1 provides the necessary oil control and membrane support functions. This allows for greater flexibility in the selection of materials for the well structure, since wells are not limited to hydrophobic materials that constitute wall layer 2, which was previously required to promote oil adhesion. Hydrophilic wall layer 2 and / or base 10 may be useful for fluidic resistor filling and bubble resistance. This can also eliminate the pre-treatment step in which oil is applied, thereby simplifying the process of assembling a nanopore sensing device 30 using the nanopore support structure 1. All that is required is to evacuate or pressure-fill the well 4, drain and remove the buffer solution from the front, add the oil / membrane mixture to the top of the well 4 where the overhang 5 and possibly the protrusions 51, 52 are located, and allow the solution to flow through the cis-chamber.

[0153] Attempting to create overhang 5 using an additional layer of photoresist material deposited after the initial layer has been exposed and developed can present challenges that make the exposure process less reliable than necessary. First, the lack of support beneath the additional layer of photoresist material suspended above well 4 causes the additional layer to sag and bow across well 4. Second, the developed structure of well 4 creates an optical cavity, which causes light from subsequent exposure steps to scatter from the boundaries of the hardened photoresist material. As a result, a partially exposed thin film of hardened photoresist material remains across the intended aperture 6 after the final development step due to light refracted / scattered toward the unexposed areas. These effects are illustrated in Figure 8. Figure 8a shows the subsequent layer of unexposed photoresist material sagging within well 4. In addition to the undesired sagging, light is refracted / reflected toward the under-mask portion of the layer of photoresist material, exposing undesired areas of the photoresist material. As a result, following a subsequent development step, the partially exposed film of hardened photoresist material seals the aperture 6 as shown in Figure 8b.

[0154] One way to minimize these effects is the multiple exposure technique already mentioned above. Alternatively or additionally, additional support structures within the wells 4 can be provided for the first layer of photoresist material, thereby reducing sagging of subsequent layers of photoresist material. These support structures can also be referred to as barriers 70. In such embodiments, the nanopore support structure 1 further comprises a barrier 70 disposed within the wells 4, which is capable of reducing scattering of electromagnetic radiation at wavelengths for curing the negative photoresist material.

[0155] A method of forming a nanopore support structure 1 including a barrier 70 includes depositing a first layer of uncured negative photoresist material, exposing the first layer of negative photoresist material to light so as to harden the negative photoresist material in the form of a wall layer 2 and a barrier 70 disposed within a well 4, removing the uncured negative photoresist material of the first layer to form the well 4 and the barrier 70, depositing a second layer of uncured negative photoresist material on the first layer of uncured negative photoresist material, exposing the second layer of negative photoresist material to light so as to harden the negative photoresist material in the form of an overhang 5, the barrier 70 being shaped to reduce scattering of electromagnetic radiation applied in the exposure, and removing the uncured negative photoresist material of the second layer.

[0156] As shown in Figure 9a, the barrier 70 can reduce refraction / reflection of light within the well 4 and also reduce sagging of the suspended photoresist material layer. Figure 9b shows that the aperture 6 is free after development and removal of the uncured photoresist material. In Figures 9a and 9b, the wells 4 have respective bases 10 and the barrier 70 extends from the bases 10 to the overhangs 5.

[0157] For wells 4 that do not have a fluid passage 41 at their bottom, the volume of the well 4 is very important and is proportional to the run time of the nanopore support structure 1. Therefore, it is desirable to minimize the volume of the barrier 70 without significantly affecting its rigidity. There are many ways to design a small, rigid barrier 70 to hold the suspended layer of photoresist material. The barrier 70 means that additional layers of suspended photoresist material traverse a much shorter distance and therefore do not sag. Figure 9c shows an example of such a barrier 70 in a top view of the nanopore support structure 1. The darker areas indicate the first layer of photoresist material, and the five-leaf, curved barrier 70 is clearly visible. The barrier 70 extends inward from the wall 3 into the well 4. The barrier 70 is designed to minimize the volume it occupies; in Figure 9c, the barrier 70 curves inward into the well 4 along its length. The curvature increases rigidity and helps reflect / block light from the center of the well 4. A second layer of photoresist material covers both the dark green and light green areas, leaving a much smaller aperture 6 for forming membrane 7 .

[0158] To date, all methods for forming the nanopore support structure 1 involve exposing a layer of photoresist material. Alternatively, the nanopore support structure 1 can be formed by molding. In this case, the wall layer 2 and overhangs 5 ​​are respective molded components that are secured together. An example of a method for fabricating the nanopore support structure 1 using molding is shown in FIG. 10. This method involves forming a wall layer 2 that includes walls 3 that define a plurality of wells 4, and forming overhangs 5 ​​in a separate step, where the overhangs 5 ​​are secured to the wall layer 2 so as to extend from the walls 3 across the wells 4.

[0159] The step of forming the wall layer 2 involves casting the wall layer 2 on a substrate 20, which in this example is made of silicon. This is shown in Figures 10a-10d. In Figure 10a, an uncured molding agent 102 is vacuum-filled into a wall layer mold 100 made of polydimethylsiloxane (PDMS). In this example, the height H of the walls 3 is about 90 μm. The substrate 20 has an electrode 11 formed thereon, which in this example is made of platinum. In Figure 10b, the wall layer mold 100 is aligned with the electrode 11 so that the wall layer 2 is cast onto the substrate 20 and positions the electrode 11 within the well 4. Pressure is applied to compress the uncured molding agent 102 onto the substrate 20 and into the wall layer mold.

[0160] In FIG. 10c, the former is cured by exposure to ultraviolet (UV) light, resulting in the hardened former 104 taking the shape of the wall layer 2. In this example, UV light with a wavelength of approximately 365 nm is used. Depending on the choice of former, other methods, such as heating the former, may be used to harden the former 102. In FIG. 10d, the wall layer mold 100 is removed. In some cases, this may leave a thin layer of hardened former 104 across the surface of the electrode 11, as shown in FIG. 10d. This is undesirable because it can interfere with the electrical performance of the nanopore sensing device 30 in which the nanopore support structure 1 is incorporated. To remove this layer, a process known as "descumming" is performed. This is illustrated in FIG. 10e, where an oxygen plasma or laser can be applied to clean the surface of the electrode 11. This step may not be necessary in all embodiments of the method, depending on whether a layer of hardened former 104 is left on the electrode 11.

[0161] After forming the wall layer 2 on the substrate 20, the overhangs 5 ​​are separately formed. This is illustrated in FIGS. 10f-10i. In FIG. 10f, uncured molding agent 102 is vacuum-filled into an overhang mold 110 formed of PDMS. In this example, the overhangs have a thickness of approximately 30 μm and are configured so that, when the overhangs are fixed to the wall layer 2, the apertures 6 of the wells 4 are approximately 10 μm to approximately 60 μm, preferably approximately 40 μm. The overhang mold 110 is configured so that multiple overhangs 5 ​​are formed simultaneously and positioned directly on corresponding walls 3 with correct alignment and spacing to define the apertures 6 of multiple wells 4. This means that multiple wells 4 can be formed simultaneously, thereby increasing production speed compared to having to separately form the overhangs 5 ​​and individually align them with the corresponding walls 3.

[0162] In FIG. 10g, the overhang mold 110 is compressed onto a base 112, which in this example is made of the same PDMS material as the overhang mold 110. Pressure is applied to compress the uncured molding agent 102 into the overhang mold 110. Optionally, as shown in FIG. 10h, a partial curing step is performed. This promotes retention of the correct shape of the overhangs 5 ​​while still allowing some deformation of the overhangs 5, which can be used to secure the overhangs 5 ​​to the wall layer 2. In FIG. 10i, the overhang mold 110 is removed from the base 112, and the overhangs 5 ​​remain with the overhang mold 110, as illustrated by the overhang portions 103. This is achieved by peeling away the base 112, leaving the pre-aligned overhang mold 110 with the partially cured molded array of overhangs 5 ​​still present and held in a state ready to be aligned and secured to the wall layer 2.

[0163] 10j-10l illustrate how the overhangs 5 ​​are secured to the wall layer 2 during the step of forming the overhangs 5. Forming the overhangs 5 ​​involves molding the overhangs 5 ​​onto the wall layer 2. In FIG. 10j, an overhang mold 110 with the overhangs 5 ​​is aligned with the wall layer 2 on the substrate 20. Once the overhang mold 110 is grounded, pressure is applied to compress the overhangs 5 ​​against the top of the wall layer 2. In FIG. 10k, UV light is applied to harden the overhangs 5 ​​and bond them to the wall layer 2. In FIG. 10l, the overhang mold 110 is removed, leaving the completed nanopore support structure 1. Optionally, an additional heating step can be performed to further harden the molding agent, although this can be reduced or omitted entirely. Additionally or alternatively, an oxygen plasma cleaning step can be applied between steps 10i and 10j. Not only does this serve to clean the surface of the electrode 11, but the process also leaves adhesive properties on the surface of the hardened molding compound 104 that enhance its bond to the overhang portion 103.

[0164] 10f-10i, the step of forming the overhangs 5 ​​includes molding the overhangs 5 ​​with protrusions that protrude laterally beyond the extent of the overhangs 5. In particular, the protrusions include outer protrusions 51 that protrude away from each well 4, and the step of forming the overhangs 5 ​​includes molding the overhangs 5 ​​with the outer protrusions on the wall layer 2. In this example, the outer protrusions 51 have a height above the overhangs 5 ​​of about 15 μm. The protrusions may also include inner protrusions that protrude laterally beyond the extent of the overhangs 5 ​​inside each well 4.

[0165] Additionally or alternatively to plasma cleaning of any residue on the surfaces of molded components, for example, PDMS stamps can leave residual oligomeric DMS material on the surface of the mold that leads to variations in the surface energy of the molded chips; solvent extraction can be used to remove unwanted residue, such as unreacted DMS oligomers.

[0166] In the above example, the well array structure 2 is attached to a substrate 20 having electrodes 11. The substrate can include one of: a printed circuit board, a plastic interposer made from a laser-ablated plastic sheet, a silicon interposer, and the surface of an application-specific integrated circuit (ASIC) having exposed terminals on one side that function as electrodes. The substrate 20 can be a plastic sheet having a laser-ablated well array structure on one surface. The plastic sheet can be coated with a metal film.

[0167] The well array structure 2 can be formed from a single component, which can include one of: a molded material such as a molded polymer 102, a three-dimensional print, or a patterned photoresist material. Three-dimensional prints can be made from materials including dielectric inks (UV curable acrylates), pseudo-polyimide inks, polyetherketones, polylactic acid, dielectric ceramics such as Li2MoO4 and aqueous solutions of ceramic particles, polyetherimides, and polyimides.

[0168] Alternatively, the well array structure 2 may comprise two separate layers, i.e., the well array structure may have a well layer 2, e.g., a base layer that forms the hardened molding agent 104, and an overhang layer 5. The overhang layer 5 may thus be said to extend at another level or layer from the well array structure 2.

[0169] The base layer 2 may be configured as a permanent layer of the device 100, and the support layer 5 defining the overhang may be removably attached thereto, allowing the device to be processed for reuse or repositioning.

[0170] The base layer 2 that defines the wells 4 can include at least one of: a molded polymer 102, a three-dimensional printed layer, a plastic interposer made from a laser ablated plastic sheet, a patterned photoresist material, glass, silicon, or silicon dioxide. The base layer has an array of walls 3 that define through-holes with apertures to define the wells 4 when connected to a substrate 20.

[0171] The support layer 5 that defines the overhang can comprise at least one of: a molded polymer 102, a three-dimensional printed layer, a plastic interposer made from a laser-ablated plastic sheet, a patterned photoresist material, glass, or silicon dioxide. The support layer has support posts in the form of protrusions 51, 52 that support the retention of oil to form an amphiphilic membrane over the well 4. Alternatively, the support layer can comprise a solid-state nanopore or a hybrid nanopore (a biological nanopore positioned in a solid-state aperture).

[0172] In light of the teachings herein, the fabrication methods and examples below, various combinations of substrate 20, base layer 2 defining the well array, and support layer 5 defining the overhangs are possible.

[0173] FIG. 13 shows an example of a nanopore support structure 1 that can be used to fabricate a nanopore sensing device 30, or portion thereof, for supporting a plurality of nanopores on an array of wells 4, only three of which are shown. The sensing device has a substrate 20 having a surface 10 on which an array of electrodes 11 is located. The electrodes 11 are exposed at apertures or open areas 158 at the bottom of the wells, as can be seen. The electrodes can be connected to or configured on electronic circuitry. On the substrate 20 is a well array structure 2 having an array of walls 3 that define through-holes 160 with apertures 158 to define wells 4 when connected to the substrate 20. The well array structure can be directly connected to the substrate 20, this connection being shown with an adhesive layer 162. Additionally or alternatively, the connection can use an adhesive surface or other such bonding material or method. When connected to substrate 20, the through-holes defining wells 4 and apertures 158 are aligned with the array of electrodes 11 such that the bases of the wells of the well array structure are at least partially defined by the electrodes. The bases of the wells may also be at least partially defined by surface 10 of substrate 20.

[0174] The substrate 20 can be provided prior to supporting the plurality of nanopores on the array of wells to form the sensing device 30. In other words, after the substrate is connected to the well array structure, no further processing or manufacturing steps are required on the substrate or well array structure that would adversely affect the performance of the sensing device 30.

[0175] Also provided separately is a well array structure 2 having posts 164 on one side and apertures 158 on the other side. By providing the substrate and well array structure separately, they can be manufactured without the conditions under which they are manufactured causing any harm to the other components.

[0176] The substrate 20 may have electronic circuitry on its surface 10, or the substrate may be a printed circuit board. The electronic circuitry and electrodes may be provided on the surface of an application specific integrated circuit (ASIC).

[0177] Separately providing the well array structure can include forming the well array structure 2 on the surface of the substrate 10; i.e., after the substrate 20 is provided, the surface 10 can be used as a base on which the well array structure is formed. The well array structure can be molded and then applied to the substrate, or can be molded directly onto the substrate surface 10.

[0178] Electrode 11 is shown in Figure 13 as part of surface 10 of substrate 202. In an alternative example, through holes can be provided in the substrate and filled with a conductive material to provide conductive vias, the exposed portions of the vias functioning as electrodes that at least partially form the bases of wells 4 in apertures 158 of the well array structure.

[0179] The well array structure can have a thickness of 10 microns to 1000 microns, preferably 10 microns to 500 microns, and more preferably 100 microns to 500 microns. The well depth can be about 5 microns to about 1500 microns, preferably 10 microns to 500 microns, and more preferably 100 microns to 500 microns. The pitch between wells in the well array structure can be about 10 to about 1000 microns, preferably 10 microns to 500 microns, and more preferably 20 microns to 100 microns. The through-holes can have a diameter of less than 200 microns, preferably 10 microns to 200 microns.

[0180] After the sensing device 30 is formed, a liquid can be introduced into the well 4 to form a membrane across the well before inserting the nanopore into the membrane. The membrane can be (i) an amphiphilic membrane supporting a biological nanopore, (ii) a solid membrane having a solid-state nanopore, or (iii) a solid membrane supporting a biological nanopore in a pore within the solid membrane.

[0181] Sensing device 30 can be fabricated in many ways. The functionality of the sensing device can also be implemented by incorporating a mixture of different modular components described herein, such as substrate 20 and well array structure 2, which can themselves be fabricated in different ways using different materials. Overall, however, the key elements, i.e., substrate 20 and well array 2, are connected together at a later stage in sensor fabrication. In many of the examples herein, well array structure 2 can be removably attached to a substrate such that it can be removed and replaced with a new substrate, thus making the sensing device recyclable. In one example, forming sensing device 30 includes forming well array structure 2 having through-holes 160 and further providing a patterned adhesive surface, which can be adhesive layer 162, on at least one of well array structure 2 or substrate 20, the patterned adhesive surface defining a plurality of adhesive-free portions.

[0182] The adhesive-free portions can be areas such as holes or voids in the patterned adhesive surface that expose the electrodes 11 to the through-holes of the well array structure. The patterned adhesive surface can also be one of the surfaces 10 of the well array structure 2 or substrate 20 that has been prepared or treated to impart adhesive properties to the connecting surface, for example, by exposing the surface to UV light or plasma treatment.

[0183] The well array structure 2 is aligned and connected to the substrate 20 using an adhesive layer 162 so that the array of electrodes 6 located on its surface at least partially defines a portion of a single well.

[0184] Substrate 20 supports or incorporates electronic circuitry having electrodes 11 and is in contact with the electrodes or formed independently of the well array structure that includes the electrodes. The adhesive surface of adhesive layer 162 can be heated to aid in bonding the well array structure to the substrate. Heating can be limited to a temperature below the outgassing temperature of the components of the electronic circuitry. Additionally or alternatively, electromagnetic radiation can be used to bond the well array structure to substrate 20. Adhesive layer 162 can be a photopatternable adhesive that is exposed to light prior to bonding. Additionally or alternatively, the well array structure and / or electrodes located at the base of the wells can be exposed to a plasma treatment that serves to at least one of: clean the exposed surfaces and temporarily change the properties of the exposed surfaces to make them adhesive, e.g., tacky, in nature.

[0185] 13, adhesive layer 162 can be a separate layer relative to the well array structure or substrate. However, adhesive layer 162 can be incorporated into one of the well array structure or substrate. Alternatively, adhesive layer 162 can be a region of the well array and / or substrate that has been modified, such as by heating, to have adhesive properties.

[0186] One exemplary method of the present invention allows for the well array structure 2 to be fabricated separately from the substrate 20 and then combined after optimal processing. This method comprises: (i) fabricating a free-standing microfluidic well array structure 2 from thick-film photoresist approximately 120 microns thick, available from TOKYO OHKA KOGYO CO., LTD. (TOK); (ii) fabricating a free-standing patterned adhesive layer 162 using at least one of a photosensitive adhesive (also available from TOK), a laser-cut adhesive, or a liquid adhesive to control wetting of the substrate / resist interface or prepare the surface of the well array structure or substrate to have adhesive properties; (iii) aligning and placing, e.g., by a robot, the adhesive onto the substrate 20; (iv) aligning and placing, e.g., by a robot, the microfluidic structure 2; and (v) post-processing to crosslink the material, e.g., using UV exposure or heating.

[0187] More specifically, the photoresist can take the form of a thick film laminate that is deposited onto a sacrificial layer. The sacrificial layer can be, for example, Omnicoat (RTM) or polystyrene, which can be spin-coated onto a Si wafer. The use of a silicon layer is just one technique that allows the well array structure to be fabricated independently of the substrate to which it is connected. As described herein, fabricating the components separately avoids the manufacturing process of one component from adversely affecting the other.

[0188] To reduce adhesion to the mask during the lithography process, a post-process heating process can be employed or the mask can be pre-treated to avoid adhesion.

[0189] Post-processing temperature control is employed to minimize stresses in the laminate caused by connections to other surfaces.

[0190] If the adhesive layer is made from a photosensitive adhesive, a plurality of vias are provided which align with the apertures 158 of the microfluidic structure to function as extending through-holes 160. The adhesive layer 162 can be selected to have the same surface energy as the well array structure 2. By way of example, the adhesive layer 162 can be obtained from TOK (NC-00075S, thicknesses of 20 um and 50 um).

[0191] Other methods of forming a freestanding patterned adhesive layer include (but are not limited to): using a laser machine to ablate appropriate vias in an existing adhesive material such as a pressure-sensitive adhesive (PSA), using a liquid adhesive and controlled wetting at the interface of the well array structure and the substrate, using an aerosol spray or microdrop dispenser, or using capillary action with the liquid adhesive. The adhesive material can have the same surface energy as the well array structure.

[0192] Baking can be accomplished to ensure complete cross-linking, with the baking temperature being limited to be below the outgassing temperature of the substrate 20, which may be, for example, a polymer present on a PCB.

[0193] For example, after forming the well array structure 2 independently on a Si wafer, the Si wafer is removed. The removal step can include lift-off with toluene before attaching to a substrate using adhesive.

[0194] The adhesive layer can be adhesive, i.e., the well array structure has adhesive properties on the surface adjacent the aperture 158. This can be achieved by not hard-baking the well array structure, leaving residual solvent and / or uncrosslinked material. Heat and pressure can be used to enable adhesion.

[0195] The well array structure can have a thin film of adhesive transferred to its surface in a stamp transfer process. The stamp can be made of polydimethylsiloxane (PDMS). The stamp can have thin bristles to control the amount of adhesive transferred. PDMS has a low surface energy, making it suitable for controlling the amount of adhesive that remains on the bristles. Stamp transfer is preferentially performed on the well array structure 2. This is because the substrate can have electrodes thereon, while the layout and density of the bristles can be arranged to prevent any transfer of adhesive to the electrode surface on the substrate 20, and applying adhesive to the well array structure 2 mitigates this transfer from occurring.

[0196] In another example, forming the sensing device 30 includes using a molding technique to form the well array structure 2. This can be accomplished independently of the fabrication of the substrate 20. The independent formation of the well array structure can be performed on a reference surface before subsequent removal and application to the substrate, or alternatively, molding can be performed directly on the substrate.

[0197] The basic steps of a molding example are illustrated in Figures 14a-14f. First, a dispenser 166 deposits a molding agent 102, hereafter referred to as polymer 102, onto the surface of a substrate 20, which illustratively includes an electrode 11. The polymer 102 is dispersed evenly across the surface of the substrate.

[0198] Separately, in a second step, polymer 102 is dispensed into mold 100, which is a negative of the well array structure 2 to be formed. Mold 100 can be fabricated from a master 168, which is identical to the well array structure to be formed. During research and development, the inventors chose to use the current MinION (RTM) well array structure, although examples of the present invention are not limited thereto. Alternatively, mold 100 can be fabricated from scratch without a master. In this way, defects can be reduced when creating the mold.

[0199] In a third step, as shown in Figure 14c, the mold 100, now filled and evenly covered with polymer 102, is aligned with the substrate 20, which is also covered with polymer 102, so that no part of the mold is exposed. The alignment brings the mold 100 and substrate 20 together so that the protrusions 170 align and correspond to the electrodes 11.

[0200] In a fourth step, the mold 100 and substrate 20 are brought together under pressure, and the protrusions 170 squeeze out excess polymer from the surface of the electrode. The elastic properties of the mold 100 allow it to conform and adapt to variations in the surface of the substrate 20, such as dome-shaped electrodes, uneven surface features, or curvature in the surface of the substrate 20. In a fifth step, the polymer 102 is cured before the mold is separated from the substrate, leaving the polymer adhered to the substrate 20 in a sixth step. The polymer is formed into a well array structure 2.

[0201] Figures 15 and 16 show the mold 100 and the resulting shape of the polymer 102 it can produce. The protrusion 170 is shown with fins 172 and extensions 174 that remove the polymer from the electrodes during polymer formation. The purpose of the well array structure 2 is clearly described in WO 2014 / 064443. What cannot be appreciated from the known art is the adaptation of molding techniques and hardware necessary to optimize the fabrication process and subsequent performance of nanopore sensing devices with improved well array structures. Figure 16 shows features, such as three recesses 176, that can control the distribution of pretreatment oil before forming a film on the well array structure. In this example, the recesses are shown as rectangular notches or grooves. The recesses can be equally spaced, but there can be any number or arrangement of recesses or pockets for holding pretreatment oil, etc. The recesses function to prevent oil from pooling at the bottom of the compartment 4 and covering the electrodes 11.

[0202] The protrusions 170 form through-holes in each compartment 4 and function to displace the polymer from the electrode 11. Partial contamination of a flat electrode can occur, leaving a film of molding material covering part of the electrode surface. To counter this, the protrusions 170 are provided with curved or domed extensions at the end of the protrusion, as shown in Figure 15, or stepped extensions with gradually decreasing disks. The purpose of these extensions 174 at the end of the protrusions in the mold 100 is to create a pressure gradient in the liquid molding material polymer as the elastomer in the mold is compressed. This functions to push the liquid out as the mold conforms to the electrode surface and makes contact with the electrode. In Figure 15, a dimple is present in the center due to a photolithographic defect. This defect allows molding material to accumulate in the dimple, while the surrounding area of ​​the electrode is empty. One or more dimples can be provided on the top of the protrusion to allow molding material to accumulate in the dimple. The dimples can be, for example, star-shaped, e.g., a five-pointed star.

[0203] Alternatively, the mold 100 can be placed against a substrate, a molding agent 102 can be injected between the mold and the substrate, and then the molding agent can be treated to harden and fix to the substrate. The mold can then be removed, leaving the well array structure on the substrate.

[0204] The molding agent 102 has been described as a polymer such as poly(methyl methacrylate). The polymer can be dissolved in a solvent before being deposited on the underside of the mold 100. For example, PS (35 KDa) can be dissolved in 500 mg / mL of chloroform. After molding, the polymer can be heated to evaporate the solvent before the mold is removed from the substrate.

[0205] The amount of solvent in the molding agent can be selected to prevent any change in the dimensions of mold 100, which would otherwise tend to absorb some of the solvent and lose some structural rigidity.

[0206] The mold can be thermoplastic or can be made of a silicone material such as polydimethylsiloxane (PDMS). The master 168 can be made from a sturdy, dimensionally stable material such as a silicon wafer.

[0207] The molding agent can be a two-part heat-cured epoxy or a UV-cured material. It has been found that heat-cured epoxies cure slower than UV-cured epoxies, requiring the mold to be held in place for a longer period of time.

[0208] The molding material may include one or more of epoxy, acrylamide / methacrylamide, polyester, styrene copolymer, vinyl, polyurethane (isocyanate / amine chemistry), or polyimide.

[0209] Numerous molding materials are available from various manufacturers: DYMAX (RTM) materials, including parts 6-621, 431, 3069, 3069-GEL, 3099, and 1072-M; LOCTITE materials, including parts AA 3321 LC, AA 3936, 3922, 3921, 3311, 3301, and 3211; EPOXIES materials, including parts 7108, 7156, and 7159; NORLAND PRODUCTS materials, including parts NOA81, NOA61, NOA88, and NOA83H. Materials from Epoxy Technology, including parts UV10MED, UV22DC80-1MED, UV10, UV10TKLO-2, UV16, and UV15LV; materials from MasterBond, including parts OG198-54, MED-OG198-54, OG142-87, and OG-112; materials from Permabond, including parts UV80 and UV605, and materials from Ostemer, including part "322." As an example, two suitable materials were from Masterbond (RTM), part UN10MED, and EpoTek (RTM), part OG198-54. The connection between the well array structure 2 and the substrate 20 can be semi-permanent. In other words, the well array structure 2 and the substrate 20 can be peeled apart so that the formed well array structure can be removed and reapplied to another substrate. Alternatively, after removal, a new well array structure can be formed in its place. In this way, the substrate and associated device or component can be recycled.

[0210] Although the above process describes forming or attaching a well array structure to a substrate so that the electrodes can be accessed through the through-holes, the electrodes 11 can be subjected to further treatment to clean their surfaces. Cleaning can include at least one of chemical cleaning or plasma treatment.

[0211] The mold 100 of the present invention can be formed independently on a known TSV 4, which minimizes the effect of processing on the surface energy of the well array structure 2, or the mold can be formed independently on a substrate such as a printed circuit board.

[0212] Although well arrays with fine features for oil control, as shown in FIG. 16, can be molded, the shapes of the wells 4 can be formed similar to those shown in FIGS. 1-4, i.e., wells 4 with featureless walls 3 can be formed adjacent to the electrodes 11, while an upper overhang 5 layer with protrusions 51, 52 resembling fins 172 can be formed. The molding examples herein demonstrate that wells with high aspect ratios in the range of 20:1 and features with complex structures, as shown in FIG. 16, can be molded. Thus, molding can create high aspect ratio wells and / or overhangs 5 ​​with lower aspect ratios in the range of 3:1 to 2:1.

[0213] In a further example of the present invention, the improved substrate 20 can use alternative materials and manufacturing methods to those described above. This can enable cost-effective, consumable, high-throughput, and low-material-cost sensor manufacturing. In this example, laser machining and screen printing methods are used to provide the integrated electrodes. This method is compatible with reel-to-reel or panel-to-panel processing.

[0214] By way of example, Figures 17a-e illustrate a step-by-step process that can be performed to provide electrodes 11 on a substrate 20 on which a well array structure 2 can be formed. The method involves taking a sheet 180 of thermoplastic foil, which can be, for example, polyimide, polyetherimide, polyetheretherketone (PEEK), polycarbonate, or polyethylene terephthalate (PET). The thickness is about 0.5 mm or less. A metal film 182 is formed on the top and bottom surfaces, as can be seen, using a metallization process. The metallization process can be sputtering, and the applied metal can be, for example, silver, gold, or platinum. The metallization provides redistribution lines (RDLs) formed on the surface of the sheet.

[0215] FIG. 17a shows such a sheet 180 having a metal film 182. As shown in FIG. 17b, a laser is used to machine multiple vias 184 through the metallized film. The vias 184 are then filled with a conductive paste, such as a carbon or silver paste, before being cured to provide conductive vias 186. Each conductive via 186 has a low resistance of about 200 to about 300 ohms. The paste can be filled using screen printing. Screen printing increases contact between the conductive vias 186 and the metal film 182 by forming dome-shaped mushroom-like regions of excess paste at the openings of the vias 184, as shown in FIG. 17c. This overflow can serve to provide connections between the metal films on each side of the sheet 180. Then, as shown in FIG. 17d, a RDL pattern is formed on the top and bottom metal films 182 to provide a circuit with features 188, such as contact tracks 188, tracks 188, or contact pads 188, including electrodes 11 on one or both sides. FIG. 17 e shows how portions of the metal film 142 can be retained to define the electrodes 11 , close the apertures 158 and define the bottoms of the compartments 4 of the well array structure 2 .

[0216] As mentioned above, the well array structure 2 can be formed independently using (i) a photoresist layer with or without a separate adhesive layer, or (ii) a mold 100. The use of a mold is particularly well suited to accommodating uneven surfaces caused by the domed ends of the tracks 188 and / or conductive vias 186, and to accommodating variations in the height of the surface of the substrate 20.

[0217] To implement the metal film 182, the sheet 180 can be formed by sputtering a metal onto the sheet before later using laser ablation to define the electrodes 11 and any circuitry. As an example, platinum can be sputtered onto a PEEK sheet to provide a high purity electrode 11. A high quality electrode can be provided to maximize electron transfer when the sensor device 30 is operating.

[0218] FIG. 18 illustrates the effect of the shape of the via 184 on the dimensions of the interface of the metal film 182 with an electrode or other circuitry. It is clear that the size of the via aperture formed in the surface of the sheet 180 by laser drilling affects the size of the contact area of ​​the conductive via. The via formed in FIG. 17b has parallel sides and a high aspect ratio, which creates a small aperture in the surface, which then minimizes exposure to the surface of the sheet 180. When filled by screen printing, a small, dome-shaped contact is formed. In practice, the shape of the via 184 is determined by the thickness of the sheet 180 and the drilling geometry, i.e., the laser drilling process. To maximize utilization of the surface of the sheet 180, the footprints of the vias 184 and conductive vias 186 should be minimized.

[0219] As an alternative to coating sheet 180 with a metallized film 182, drilling, and then filling the formed vias 184 with a conductive paste, a bare sheet without a metallized surface can be first drilled and then coated using a physical vapor deposition (PVD) process. The PVD metal coating can be formed to extend from the plane of the sheet into the vias 184, thereby forming an alternative conductive via 186 to that shown in FIG.

[0220] In practice, as shown in Figure 17b, high aspect ratio vias 184 that extend vertically from one side of the sheet to the other have very small apertures and 90 degree taper angles in the surface, making it difficult to PVD coat the walls of the vias furthest from the surface of the sheet due to limited access.

[0221] Figure 19a shows the dimensions of the metal coating 182 coating, forming a sheet perforated from one side and a via with flat sides, while Figure 19b shows the subsequently applied PVD coating 182, forming a sheet perforated from both sides and a via with raised or waisted sides. The angle between the plane of the sheet and the side of the via extending therefrom is the taper angle. Thus, the taper angle on one or both sides is greater than 90 degrees.

[0222] 19a, if only one side of sheet 180 has a taper angle greater than 90 degrees, the size of the opening of one of the vias will be larger than the other. In this case, the ratio of aperture size or diameter on one side to the other may be about 1:4, or preferably about 1:3, or more preferably about 1:2. In this configuration, one side has an aperture to receive the PVD so that the entire wall of the via is coated.

[0223] As shown in FIG. 19b, drilling from both sides results in a taper angle of greater than 90 degrees on both sides of the sheet 180. While the openings can have different sizes, the via can be substantially symmetrical and, as viewed, appears to have two lines of symmetry, one horizontal and one vertical. In this case, the ratio of aperture size or diameter on one side compared to the other side can be approximately 1:1. This minimizes the footprint of the via as viewed from the surface of the sheet and from both sides. Furthermore, the configuration of FIG. 19b allows both sides of the via to receive PVD so that the entire via wall is coated.

[0224] 20a-20g illustrate the fabrication process for a hybrid well array 190 that combines the functionality of the substrate 20 and well array structure 2 described above into a single-piece component for nanopore sensing. Like reference numerals refer to like features. Fabrication of the hybrid well array 190 begins with a sheet 180 having a metal film 182 formed on only one side or deposited on only one side to provide the sheet as shown in FIG. 20a. As shown in FIG. 20b, vent holes 192 are drilled on the side of the sheet opposite the metal film, and then partial vias 184 are drilled through the metal film on the side of sheet 180 opposite the vent holes 192. The resulting structure can be seen in FIG. 20c with vias 184 extending from one side of the sheet to the other, with a wide perforated portion and a narrow vent hole 192. In Figures 20d and 20e, the wide ends of vias 184 are filled with conductive paste before the metal film is trimmed to form circuits connected to conductive vias 186. Note that the vents are too narrow to allow for the conductive paste to fill or completely fill them. The purpose of the vents is to allow for the wide portions of the vias to be completely filled by preventing trapped air from preventing the vias from filling. The vent 192 side of sheet 180 is then processed to form posts 164, as shown in Figure 20f, and then the material around those posts is removed to define walls 3 of compartment 4 and expose conductive vias 186, as shown in Figure 20g.

[0225] The characteristics of the electrodes 11 on the substrate 20 affect the performance of the sensing device 30. As described above, the sheet 180 can be sputtered with metal followed by laser ablation. Optimal performance has been achieved by sputtering platinum onto a poly(ether ether ketone) PEEK sheet. The vias 184 can then be filled. The base of the compartment 4 can be defined by a sputtered metal film 182 connected to a conductive via 186, as shown by way of example in FIG. 17e. The conductive via 186 is configured to provide a low-resistance electrical contact with a resistance between 150 ohms and 300 ohms, or between 200 ohms and 300 ohms.

[0226] The material selected for sheet 180 was PEEK. In the example, the sheet had a double gloss finish and a thickness of 0.25 mm. PEEK was selected based on at least one of many factors, including its mechanical stability to provide a reliable connection with connectors, such as Samtec z-array connectors (https: / / www.samtec.com / connectors / high-speed-board-to-board / high-density-arrays / zray), its good adhesion properties with sputtered platinum layers, allowing for high-resolution track and gap formation without delamination, and its suitability for achieving high-aspect ratio laser-drilled via holes. Other materials found to be suitable with comparable performance include polyetherimide (PEI).

[0227] Prior to sputtering, the sheet 180 is cleaned, for example with ethanol, before loading into the sputtering chamber. In the example, 50 nm of platinum was coated on both sides of the film.

[0228] A laser was used to pattern and perforate the metallized sheet 180. The pattern laser can be, for example, an SMI laser (MSV-301). The perforation laser can be, for example, a direct-write laser (MSV-101UV).

[0229] Filling of vias 184 was performed using a DEK™ screen printing process. After filling the vias from the backside using silver ink, the panel was flipped over to cap the vias on the front side. The ink was then cured in an oven at 110°C.

[0230] In each example, the performance of the sensing device 30 was evaluated by fabricating a known MinION (RTM) device with a combined well array structure 2 and substrate 20, or hybrid well array 190, instead of the known structure and wafer. After assembly, the well array structure was filled with fluid, and a biological nanopore was attached to the membrane formed across compartment 2. During evaluation, indicators of suitable performance included: an open pore current of approximately 180-200 pA with 180 mV applied across the pore, a noise level with a standard deviation of approximately 2-3 pA for the open pore current, and a clear thrombin binding aptamer (TBA) event, indicating the absence of additional high RC time constants in the setup. Details of the use of TBAs are described in WO 2014 / 064443. Ion channel recordings with TBA in each of the examples revealed results as shown in FIG. 21 , which indicate that the ionic current measured through the pores is blocked by the analyte (TBA), the pores being inserted into a membrane supported on a well array structure 2 made of photoresist on a substrate 20 made of plastic sheet 180, as described above.

[0231] Many examples are described as having a sheet 180 with metal films 182 on both sides, with tracks and / or contact pads formed in the or each metal film. As a lower-cost alternative, one side of the sheet can be coated with a metal film, while the contact pads are screen-printed on the non-metallized side, eliminating the need for metal patterning. The vias 140 can be filled and the contact pads formed in a single step. Figure 22a shows a schematic diagram of a sheet with metal films on both sides, with conductive vias 186 passing through them and connecting to contact pads 188. The surfaces of both metal films 182 have been trimmed, and this schematic diagram is shown over an image of the actual pads. In the example of Figure 22a, the metal film is platinum.

[0232] 22b is a schematic diagram of a sheet having a metal film on one side, where conductive vias 186 are screen printed onto the sheet 180, simultaneously forming contact pads 188. Both conductive vias 186 and contact pads 188 have the same material, which in this example is silver.

[0233] The density of the vias 184, tracks 188, or contact pads 188 formed during the screen printing process is affected by controlled paste spreading. Channel density is defined by a combination of the track or pad size, their spacing, and print size. Preventing ink spreading during printing is important for precisely defining the print size. This also prevents shorts by controlling the deposition and spreading of the conductive paste. To prevent screen-printed ink from spreading, capillary stops 194 can be formed adjacent to the vias 184. A top view of the conductive vias 186 adjacent to the tracks is shown in FIG. 23a, aligned with a cross-sectional view of the sheet 180 with the conductive vias 186 and a PCB underneath the sheet. While the diameter of the conductive vias 186 forming the electrodes is 100 microns, the capillary stops are shown extending around the apertures created by the vias 184, limiting the print diameter to 110 microns. FIG. 23b shows the geometry of the capillary stops 194 in more detail. A groove is formed adjacent to but spaced from the aperture of the via 184 in the sheet 180. The height of the aperture is in the same plane as the sheet 180, and the groove extends into the sheet. In the example shown, the groove does not surround the via to allow the track to extend along the plane of the sheet 180. The capillary stop groove is an effective way to provide a pinning point to prevent ink spreading.

[0234] The material selected for the sheet 180 took into consideration its use as a well array structure in the nanopore sensing device 30. The required dispersive component of the sheet material is 40-55 mN / m, while the polar component must be less than 3 mN / m. The contact values ​​of PEI, PEEK, PC, and PET were evaluated and their properties tabulated, as shown in Figure 24. During the evaluation of these materials, their inherent surface properties due to the laser ablation process were also important. These surface properties can be adjusted based on the atmosphere during the ablation process to minimize oxidation.

[0235] To obtain the required surface energy without relying on maintaining bulk properties, the process can involve: surface coating using functional groups (silanes) that remain on the surface after ablation, and plasma treatment / thin film deposition, which can be modified to achieve the required surface properties.

[0236] As described in the device and method of forming the device above, the sensing device 30 can have a well array structure 2 attached to a substrate 20 having electrodes 11. The well array structure 2 can be formed from a single component. Alternatively, the well array structure 2 can include two separate layers: a base layer and an overhang layer 5 that form the well layer 2.

[0237] An example of a sensing device 30 incorporating a combination of layers fabricated using different techniques as described above is shown in FIG.

[0238] FIG. 25 includes a substrate 20 formed using a sheet 180 having a metal film 182 laser-etched to have a patterned surface defining vias 184 and electrodes 11. The electrodes connect to the opposite side of the substrate through conductive vias 186 and contact pads 188. Alternatively, the sheet and metal film may be implemented by a printed circuit board or provided on the surface of an ASIC device with exposed electrodes. A well array structure 2 is formed on the substrate 20 with wells 4 defined by walls and located over the electrodes 11. The well array structure may be formed from 3D printing, molding, laser-etched plastic, or an etched laminate sheet.

[0239] The well array structure 2 and electrodes 11 can be plasma cleaned prior to attachment of the overhang 5 layer provided on the well array structure 2, which in turn can be formed from 3D printed, molded, laser etched plastic, or an etched laminate sheet. The overhang layer 5 can be removably attached to the well array structure 2. Protrusions 51 are shown on the overhangs outside the wells, but can similarly be applied to other sides of the overhangs, i.e., sides facing the electrodes and / or extending within the plane defined by the overhang layer.

[0240] The techniques used to fabricate the well array structure 2 and overhang layer can be made from different techniques taught herein. As an example, the well array structure 2 has high aspect ratio wells formed from a mold. The aspect ratio of the wells is defined by the depth and width of the wells, which is influenced by the well array structure material 2 and can exceed approximately 3:1. The overhang layer can also be formed from a mold, or alternatively from a laser-ablated plastic sheet 180. The aspect ratio between the film thickness and the height of the protrusions 51, 52 is lower than the aspect ratio of the wells, and can be as low as approximately 2:1.

[0241] 26a-26d show the steps taken to fabricate overhangs in a single bare sheet 180. A low-aspect-ratio protrusion, which may exceed approximately 2:1, can be laser-formed on the top surface, as shown. The sheet is then drilled to create the walls 3 that define the wells 4. Note that the drilling creates tapered walls, as described in connection with FIG. 19 above, so that the overhangs are formed in a single layer. After drilling, the surfaces of the walls 3 are coated, preferably with platinum sputtering, to form the electrodes 11 and contact pads 188. The platinum coating can be trimmed using a laser. Finally, a closure 196 can be applied over the surface of the substrate 20 bearing the electrodes 11 and contact pads to define the base of the wells 4. The closure can be a further substrate 20.

[0242] Any electrode material can be applied, with platinum being proposed as an example because it is particularly suitable for sensing purposes. Allowing the laser to pass straight through the sheet 180 improves the manufacturing process because electrodes or other such structures do not block the performance of the laser drilling. The use of laser drilling is suitable for angled holes. Laser drilling is also suitable for forming low aspect ratio features, such as protrusions. Incorporating an overhanging material layer into a single part can simplify the manufacturing process. Laser drilling through the sheet and subsequent electrode coating can result in an electrode with an increased surface area compared to the electrode at the base of the cylindrical well 4.

[0243] FIG. 27 shows a portion of a sensing device 30 formed on a substrate, which may be a laser-etched sheet 180, or a PCB with blind vias 198 defining wells 4. The wells can have tapered sides to increase the area of ​​the electrodes 11 within the wells, which also makes coverage easier. Laser drilling has no shape constraints, allowing for well profiles of various shapes to be formed. The substrate is treated with laser etching before filling the vias 184, 186 and creating contact pads 186 on one side of the substrate 20. Wells 4 are also preferably coated with platinum to define the electrodes 11 and contact pads within the wells. The electrodes extend from the wells for connection to the vias 186. The wells are capped by an overhang 5 layer, shown on the substrate prior to attachment, with protrusions 51.

[0244] Alternative methods for forming the nanopore support structure 1 are also possible. In some embodiments, the UV radiation is UVA radiation. In some embodiments, the UV radiation is provided by a UV laser (e.g., a gas laser, a laser diode, or a solid-state laser). In some embodiments, the UV radiation is provided using a UV lamp. In some embodiments, the UV radiation is high-intensity UV radiation. A combination of methods may be used for different components. For example, molding may be used for some components, while other components may be formed from a photoresist material. Some components, such as electrodes, may be formed by etching, optionally chemical etching.

[0245] After fabricating the nanopore support structure 1, for example using any of the methods described above, the nanopore support structure 1 may be used to fabricate a nanopore sensing device 30, for example as shown in Figure 3. In this case, the nanopore support structure 1 is incorporated into a planar structure 40, and the nanopore sensing device 30 is assembled incorporating the planar structure 40.

[0246] 1 and 2 show a membrane 7 extending across the aperture 6 and a nanopore 8 inserted in the membrane 7, some types of nanopore support structure 1 may be provided without the membrane 7 and nanopore 8. In that case, the end user of the nanopore support structure 1 performs the steps of forming the membrane 7 and inserting the nanopore 8 therein.

[0247] Examples of membranes 7 and nanopores 8 are as follows:

[0248] In one type of nanopore support structure 1, the nanopore 8 is a biological nanopore and the membrane 7 can have the biological nanopore 8 inserted therein.

[0249] Membrane 7 may be an amphiphilic layer, which is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. Amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and monolayer-forming amphiphiles are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Membrane 7 may be a triblock or diblock copolymer membrane.

[0250] Membrane 7 may be one of the membranes disclosed in WO2014 / 064443 or WO2014 / 064444, which are incorporated herein by reference in their entirety, and which also disclose suitable polymers.

[0251] The amphiphilic molecule may be chemically modified or functionalized to facilitate binding of the polynucleotide.

[0252] The amphiphilic layer can be a monolayer or a bilayer.

[0253] The membrane 7 can be a lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734, and WO2006 / 100484, which are incorporated by reference in their entireties. Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69:3561-3566).

[0254] The nanopore 8 can be any transmembrane pore. The nanopore 8 can be biological or artificial. Suitable nanopores 8 include, but are not limited to, protein pores, polynucleotide pores, and solid-state pores. The nanopore 8 can be a DNA origami pore (Langecker et al., Science, 2012;338:932-936).

[0255] A transmembrane protein pore can comprise a barrel or channel through which ions can flow. The barrel or channel of a transmembrane protein pore typically comprises amino acids that facilitate interactions with nucleotides, polynucleotides, or nucleic acids.

[0256] The transmembrane protein pore for use in accordance with the present invention may be derived from a β-barrel pore or an α-helix bundle pore. The transmembrane pore may be derived from or based on, for example, Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, and the hemolytic protein fragacetoxin C (FraC). The transmembrane protein pore may be derived from CsgG. A suitable pore derived from CsgG is disclosed in WO 2016 / 034591. The transmembrane pore may be derived from lysenin. A suitable pore derived from lysenin is disclosed in WO 2013 / 153359.

[0257] The analyte (including, for example, a protein, peptide, small molecule, polypeptide, or polynucleotide) may be present in a sample. The analyte may be any suitable sample. The analyte may be a biological sample. Any embodiment of the methods described herein may be performed in vitro on an analyte obtained or extracted from any organism or microorganism. The organism or microorganism is typically an archaea, prokaryote, or eukaryote, and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera, and protista. In some embodiments, the methods of various aspects described herein may be performed in vitro on an analyte obtained or extracted from any virus.

[0258] The analyte may be a liquid sample from any source, such as biological, industrial, or environmental. The analyte may include a bodily fluid. The bodily fluid may be obtained from a human or an animal. The human or animal may have, be suspected of having, or be at risk for a disease. The analyte may be urine, lymph, saliva, mucus, semen, or amniotic fluid, but may also be whole blood, plasma, or serum. Typically, the analyte is from a human, but may alternatively be from another mammal, for example, from commercial livestock such as a horse, cow, sheep, or pig, or alternatively, from a pet animal such as a cat or dog. Alternatively, the analyte may be from a plant.

[0259] The analyte can be a non-biological sample. The non-biological sample can be a fluid sample. An ionic salt, such as potassium chloride, can be added to the sample to achieve ionic flow through the nanopore.

[0260] A polynucleotide can be single-stranded or double-stranded. At least a portion of the polynucleotide can be double-stranded.

[0261] A polynucleotide can be a nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide can comprise one strand of RNA hybridized to one strand of DNA. A polynucleotide can be any synthetic nucleic acid known in the art.

[0262] Polynucleotides can be naturally occurring or artificial.

[0263] The method can involve measuring two, three, four, five, or more properties of the polynucleotide. The one or more properties can be selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified.

[0264] The nanopore can be part of an array containing a plurality of nanopores, as disclosed in WO2014 / 064443. Polynucleotides can be translocated through the nanopore array, as disclosed in WO2013 / 041878 and WO2014 / 135838, and parameters such as current can be measured over time, from which the sequence of the polynucleotide can be determined. Suitable electrical measurements are described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7; Lieberman KR et al., J Am Chem Soc. 2010;132(50):17961-72, and International Application WO2000 / 28312. Other types of measurements can be performed, such as optical measurements, such as fluorescence measurements and FET measurements. Optical and electrical measurements can be performed simultaneously. See, for example, Heron AJ et al., J Am Chem Soc. 2009;131(5):1652-3).

[0265] The polynucleotides can be labeled and measurements can be performed to determine the polynucleotide sequence from measurements of the label. The analyte can be an Expandomer as disclosed in WO2014 / 074922.

[0266] Secondary structure can be measured in a variety of ways. For example, if the method involves electrical measurements, the secondary structure can be measured using changes in residence time or changes in the ionic current flowing through the pore. This makes it possible to distinguish between regions of single-stranded and double-stranded polynucleotides.

[0267] The presence or absence of any modification can be measured. The method can include determining whether the polynucleotide is modified by methylation, by oxidation, by damage, by one or more proteins, or by one or more labels, tags, or spacers. Specific modifications will result in specific interactions with the pore, which can be measured using the methods described below.

[0268] In some embodiments of the various aspects described herein, the method can involve further characterizing the target polynucleotide. When the target polynucleotide contacts the pore, one or more measurements indicative of one or more properties of the target polynucleotide are taken as the polynucleotide moves relative to the pore.

[0269] The method may involve determining whether the polynucleotide is modified. The presence or absence of any modification may be measured. The method may include determining whether the polynucleotide is modified by methylation, oxidation, damage, one or more proteins, or one or more labels, tags, or spacers.

[0270] The present invention also provides a device for characterizing a target analyte, such as a labeled polynucleotide. The device comprises a plurality of pores as disclosed herein and a plurality of membranes. The plurality of pores may be present in the plurality of membranes. The number of pores and membranes may be equal. A single pore may be present in each membrane.

[0271] A device for characterizing a target analyte may include an array of pores as disclosed herein within a plurality of membranes.

[0272] The device may further comprise instructions for carrying out the method. The device may be any conventional device for analyte analysis, such as an array or chip. Any of the embodiments discussed above in relation to the method are equally applicable to the device of the present invention. The device may further comprise any of the features present in the kit as disclosed herein.

[0273] The device may be configured to carry out the methods as disclosed herein.

[0274] The apparatus may comprise a nanopore sensing device 30 capable of supporting a plurality of pores and membranes and operable to perform characterization of an analyte using the pores and membranes, and at least one port for delivering material for performing the characterization.

[0275] Alternatively, the apparatus may comprise a nanopore sensing device 30 capable of supporting a plurality of pores and membranes and operable to perform characterization of an analyte using the pores and membranes, and at least one reservoir for holding material for performing the characterization.

[0276] The apparatus may comprise a sensor device capable of supporting a membrane and a plurality of pores and membranes and operable to perform characterization of an analyte using the pores and membranes, at least one reservoir holding material for performing the characterization, a fluidic system configured to controllably supply material from the at least one reservoir to the sensor device, and one or more containers for receiving respective samples, the fluidic system configured to selectively supply analytes from the one or more containers to the sensor device.

[0277] The device may be any of those described in WO2009 / 077734, WO2010 / 122293, WO2011 / 067559, or WO00 / 28312.

[0278] Control of analyte movement through the nanopore, e.g., rate of translocation, analyte rejection, etc., can be managed by the systems and methods disclosed in WO2016 / 059427, which is incorporated herein by reference in its entirety. Rejection of the analyte by the nanopore sensor can include expulsion of the analyte from the nanopore.

[0279] The features in the above description and drawings are interchangeable and compatible in light of the teachings herein. The present invention has been described above purely by way of example, and modifications can be made within the spirit and scope of the invention, which extend to equivalents of the described features and combinations of one or more features described herein. The invention also resides in any individual feature described or suggested herein.

Claims

1. 1. A nanopore support structure comprising: a wall layer comprising walls defining a plurality of wells; an overhang extending from the wall across each well, the overhang defining an aperture configured to support a membrane for insertion of a nanopore; a protruding portion protruding laterally in the range of the overhang; The protrusions include a plurality of inner protrusions protruding laterally in the range of the overhang inside each well, and a plurality of outer protrusions protruding laterally in the range of the overhang outside each well, A nanopore support structure, each of said outer projections being directly above each of said inner projections.

2. 10. The nanopore support structure of claim 1, wherein said nanopore support structure comprises wall layer outward projections that project laterally of an area of ​​an outer surface of said wall layer between said plurality of wells.

3. 3. The nanopore support structure of claim 2, wherein for each aperture, said wall layer outward projection defines a respective projection wall at least partially surrounding and recessed from said aperture.

4. The nanopore support structure of claim 3 , wherein said protrusion wall partially surrounds said aperture and has a gap therein.

5. 5. The nanopore support structure of claim 3 or 4, wherein each protrusion wall is configured such that a meniscus is formed at each aperture such that the meniscus extends at least partially within each aperture.

6. 6. The nanopore support structure of claim 3 , comprising, for each aperture, a plurality of peripheral outer protrusions defining said protrusion walls and comprising an inner surface facing towards said aperture and an outer surface facing away from said aperture.

7. 7. The nanopore support structure of claim 1, wherein the protrusions are positioned to increase retention of non-polar media by the overhangs.

8. The nanopore support structure of claim 1 , wherein the protrusions are arranged to increase the stiffness of the overhang.

9. The nanopore support structure of claim 1 , wherein each of said plurality of wells has a base.

10. The nanopore support structure of claim 9 , wherein said wall layer further defines said base.

11. 10. The nanopore support structure of claim 9, wherein said nanopore support structure further comprises a substrate, said wall layer being secured to said substrate, said substrate defining said base of said well.

12. The nanopore support structure of claim 1 , wherein the overhang and wall layer comprise a hardened negative photoresist material.

13. a barrier disposed within the well, the barrier reducing scattering of electromagnetic radiation at a wavelength for curing the negative photoresist material; 13. The nanopore support structure of claim 12, wherein each of said plurality of wells has a base, said barrier extending from said base to said overhang, said barrier extending inwardly from said wall into said well.

14. 12. The nanopore support structure of claim 1, wherein said wall layer and said overhang are molded components fixed together.

15. 15. The nanopore support structure of claim 1, further comprising a plurality of membranes extending across each aperture, and optionally also comprising nanopores inserted in at least some of said plurality of membranes.

16. 1. A nanopore sensing device comprising: a first chamber and a second chamber; 16. A planar structure comprising the nanopore support structure of claim 1, wherein the planar structure is provided with a plurality of fluid passages extending between the first chamber and the second chamber and including respective wells and apertures of the nanopore support structure, the apertures opening into the first chamber; and an electrode positioned to sense a fluid potential in each fluid passage between the nanopore and the second chamber.

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