WO2025120150A1

WO2025120150A1 - Methods and apparatus for forming apertures, methods and apparatus for unblocking apertures, methods of sensing molecular entities in apertures, and measurement systems

Info

Publication number: WO2025120150A1
Application number: PCT/EP2024/085059
Authority: WO
Inventors: Ping Xie; Ken Healy; Justin Millis
Original assignee: Oxford Nanopore Technologies PLC
Current assignee: Oxford Nanopore Technologies PLC
Priority date: 2023-12-08
Filing date: 2024-12-06
Publication date: 2025-06-12
Anticipated expiration: 2026-06-08

Abstract

Methods and apparatus for forming and/or unblocking apertures are provided, as well as measurement methods and systems. In one arrangement, dielectric breakdown is used to form apertures in a membrane. A potential difference is applied between a first common electrode and a second common electrode. The first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane. The method comprises creating and/or controlling growth of apertures through a plurality of target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of a plurality of control electrodes. Each control electrode contacts the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode.

Description

METHODS AND APPARATUS FOR FORMING APERTURES, METHODS AND APPARATUS FOR UNBLOCKING APERTURES, METHODS OF SENSING MOLECULAR ENTITIES IN APERTURES, AND MEASUREMENT SYSTEMS

The disclosure relates to methods and apparatus for forming and/or unblocking apertures and/or for sensing molecular entities in apertures. The apertures may be nanoscale apertures, which may be referred to as nanopores, each having dimensions at the nanometre scale, for example a length and/or diameter of less than about 100 nm. The resultant porous membrane may be used in a wide number of applications.

Nanopores may be used in various devices where operations at the nanoscale are required. One important application is in localising, detecting and/or characterising molecules such as polynucleotides or polypeptides. Nanopore fdters and nanoscale porous membranes are likewise important for many critical biological separation and characterization procedures, as well as fdtration processes. Many other microfluidic and nano-fluidic processing and control applications similarly rely on nanoscale features in nanometric materials.

To produce a nanoscale structure such as a nanopore in a nanometrically-thin material, it is in general required to manipulate with the precision of single atoms. This is in contrast to most conventional microelectronic fabrication processes, which characteristically only require precision down to 10s of nanometres. Without feature resolution and fabrication precision at the atomic level, it is challenging to manipulate nanometrically-thin materials in a manner that exploits the particular characteristics which emerge at the nanoscale. Numerous methods for preparing nanopores in solid state membranes have been proposed, such as for example the methods disclosed in W003003446 A3.

High precision nanoscale processing has historically required a one-at-a-time fabrication paradigm that is often costly and inefficient. Generally, the high-volume, batch fabrication techniques of conventional microelectronic production have been incompatible with nanoscale feature production and material manipulation. This has impeded commercial implementation of many important nanoscale applications.

Dielectric breakdown has been explored as an alternative approach for forming nanoscale apertures. However, controlling the dielectric process has been found to be challenging. Individual electronic control of the breakdown process for each aperture was found to be necessary to avoid damage to the membrane in which the apertures were formed and/or to achieve a desired aperture size. Forming apertures in thicker membranes was difficult because larger voltages were necessary. Larger voltages increase the risk of damage to the membrane or the formation of irregular apertures. Apertures of a precise desired size between a given solution chamber could only be produced one at a time, unless complex microfluidic arrangements were provided for forming multiple, mutually isolated fluid chambers at different positions, limiting the possibilities of commercial application.

During use of apertures after their formation, for example in sensing applications where measurements are made on molecular entities in the apertures, apertures can become blocked, which may interrupt the measurements. If blockages are not removed promptly, apertures can become permanently unusable.

It is an object of the present disclosure to at least partially address one or more of the issues discussed above.

According to an aspect of the invention, there is provided a method of forming apertures in a membrane using dielectric breakdown, comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and creating and/or controlling growth of apertures through a plurality of target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of a plurality of control electrodes, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode.

Thus, a method is provided that improves formation of apertures in membranes by using control electrodes in addition to first and second common electrodes to create and/or control growth of the apertures. The control electrodes may be considered as being connected electrically in parallel between the first and second common electrodes (while also being electrically in series between respective target portions and the second common electrode). In comparison to where potential differences across target portions are controlled exclusively using common electrodes, the additional use of the control electrodes allows for more granular control of aperture creation and/or growth, improving flexibility and/or performance.

In an embodiment, each target portion defines a respective first target surface on the one side of the membrane and a respective second target surface on the other side of the membrane, the first target surface being in contact with the first body of liquid, the second target surface being in contact with the second body of liquid. The second body of liquid may be contained in a bath defining a bulk region and a plurality of fluidic passages. The second common electrode contacts the second body of liquid in the bulk region. Each fluidic passage extends from a respective one of the second target surfaces to the bulk region and opens out into the bulk region at a distal end of the fluidic passage. Each control electrode contacts the second body of liquid at a position electrically in series between a respective one of the second target surfaces and the distal end of the fluidic passage corresponding to that second target surface. Each fluidic passage thus acts as an electrical resistor between a respective control electrode and the second common electrode, allowing potentials of control electrodes to differ from each other and from that of the second common electrode.

In an embodiment, the method comprises creating apertures through a selected subset of the target portions by using control electrodes to cause potential differences to be applied across the target portions in the selected subset that are larger than potential differences applied across target portions that are not in the selected subset, thereby selectively initiating dielectric breakdown in the selected subset of the target portions. This approach allows apertures to be formed as and when they are needed, thereby allowing fresh apertures to be made available from the same membrane for measurement runs performed at different times. Freshly formed apertures may reduce or eliminate measurement problems associated with storage of membranes with apertures, such as surface contamination or clogging that may occur during storage.

In an embodiment, the target portions are configured to have a range of different thicknesses and the method comprises using the control electrodes to apply a corresponding range of different potential differences across the target portions to promote creation of apertures. Thus, the granular control provided by control electrodes can be used to simultaneously create apertures having a range of different lengths (determined by the membrane thicknesses) in the same membrane without changing potentials applied by the common first and second electrodes or needing to apply global voltages that are excessively high for the thinnest target portions.

In an embodiment, the method comprises operating each control electrode in an aperture creation mode and an aperture monitoring mode, wherein: the aperture creation mode comprises applying a voltage to the control electrode that promotes aperture creation through the target portion corresponding to the control electrode; and the aperture monitoring mode comprises using the control electrode to monitor a state of the aperture by measuring an electrical characteristic associated with the aperture. The electrical characteristic may be a voltage of the liquid adjacent to the control electrode. Using the control electrodes in the two different modes enhances control of aperture formation without requiring additional electrodes.

In an embodiment, the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode. The method may comprise operating one of the control electrodes in the aperture creation mode and the aperture detection mode in an alternating sequence in each of one or more of the target portions until creation of an aperture is detected in the target portion. After aperture creation, the control electrode can be set to stay in the aperture detection mode, either immediately, after a few further cycles of the aperture creation mode, or after the aperture size reaches a target size. A maximum potential difference applied to the target portion during each operation in the aperture creation mode in the alternating sequence may be progressively increased during the alternating sequence. This approach controls aperture formation on a target portion by target portion basis, promoting reliable formation of apertures without requiring undesirably large over-voltages.

In an embodiment, the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform. Arranging for the aperture promoting waveform to be balanced in this way reduces or eliminates depletion of the electroactive species (e.g., a mediator) adjacent to the control electrodes by the aperture promoting waveform. This reduces negative impacts of the aperture promoting waveform, such as unwanted offsets, on measurements performed at a later time

In an embodiment, the aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture through the target portion corresponding to the control electrode. The control electrodes allow aperture blocking to be detected without requiring separate apparatus elements, allowing corrective action to be taken promptly and minimising system complexity.

In an embodiment, the method comprises controlling a voltage of the control electrode to unblock the aperture in response to detection of blocking by the blocking detection mode. Thus, detection of blocking and corrective action can be implemented using the same electrodes, thereby further minimizing system complexity and the need for additional components and/or electrical connections in areas where limited space is available. This is particularly beneficial in contexts where a large number of apertures are present in close proximity.

In an embodiment, the controlling of the voltage of the control electrode to unblock the aperture comprises applying an unblocking voltage waveform, the unblocking voltage waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform. Arranging for the unblocking voltage waveform to be balanced in this way reduces or eliminates depletion of the electron mediator redox couple (e.g., a mediator) in the liquid adjacent to the control electrodes by the unblocking voltage waveform. This reduces negative impacts of the unblocking voltage waveform, such as unwanted offsets, on measurements performed at a later time as well as allowing the unblocking voltage waveform to be applied more frequently and/or for longer periods, thereby enhancing a capability of the system to deal with stubborn blockages.

In an embodiment, the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform; the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode; the method comprises generating a combined driving signal, the combined driving signal comprising an alternating sequence of the aperture promoting waveform and the unblocking voltage waveform; and the method comprises alternately switching between applying the aperture promoting waveform from the combined driving signal and applying the aperture detection mode, thereby applying the aperture creation mode and the aperture detection mode in an alternating sequence, until creation of an aperture is detected in the target portion. Using a combined driving signal that provides portions suitable both for creating apertures and unblocking apertures allows this functionality to be achieved with fewer distinct voltage supplies and/or electrical connections, as well as simpler switching arrangements.

In an embodiment, voltages applied to the first and second common electrodes and the control electrodes during the creation of apertures through the plural target portions are configured such that differences between the voltages applied by the control electrodes and the second common electrode do not exceed 30% of a voltage difference between the first and second common electrodes. Keeping the voltages of the control electrodes relatively close to the voltage of the second common electrode ensures that ionic currents along the fluidic passages remain relatively low. Depletion of the electrode and/or adjacent electron mediator redox couple (e.g., a mediator) in the liquid is thereby reduced.

In an embodiment, the control electrodes are used after creation of apertures in respective target portions to control growth of the apertures in the target portions. The control of growth comprises monitoring an electrical characteristic dependent on a size of an aperture being grown and using the control electrode to stop growth of the aperture in response to the monitoring indicating that a target size has been attained. Controlling growth by monitoring the electrical characteristic provides flexibility for creating apertures of a range of desired sizes, as well as improving reliability. The resistance through an aperture decreases as the aperture grows, which in the absence of control may favour formation of apertures having a size that is similar to a thickness of the membrane directly adjacent to the aperture. The control electrodes provide flexibility for selecting different aperture sizes. For example, smaller aperture sizes can be created by stopping the aperture growth process early. Alternatively, the control electrodes can be used to apply larger potential differences across the target portions than is typically possible via the first and second common electrodes to form larger apertures than would otherwise be possible.

According to an alternative aspect, there is provided a method of unblocking apertures in a membrane of a measurement system configured to sense a molecular entity in each of the apertures by performing a measurement that is dependent on an interaction between the molecular entity and the aperture, the method comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and unblocking apertures through plural target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of a plurality of control electrodes, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode, wherein the controlling of the voltages of the control electrodes comprises applying an unblocking voltage waveform to each control electrode, the unblocking voltage waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

Thus, a method is provided in which control electrodes are used to unblock apertures. The control electrodes are also positioned to allow the measurements of the molecular entities to be performed using the same control electrodes. Unblocking and measurements can thus be performed using the same apparatus elements, thereby minimizing system complexity. The control electrodes allow corrective action to be performed promptly when blocking of an aperture is detected. Arranging for the unblocking voltage waveform to be balanced in the manner specified above reduces or eliminates depletion of the liquid (e.g., a mediator) adjacent to the control electrodes by the unblocking voltage waveform. This reduces negative impacts of the unblocking voltage waveform, such as unwanted offsets, on measurements performed at a later time as well as allowing the unblocking voltage waveform to be applied more frequently and/or for longer periods, thereby enhancing a capability of the system to deal with stubborn blockages.

According to an alternative aspect, there is provided a method of sensing molecular entities in apertures in a membrane, the method comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and using a plurality of control electrodes to sense molecular entities in the apertures by performing measurements with the control electrodes that are dependent on interactions between the molecular entities and the apertures, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode, wherein the method further comprises preconditioning respective portions of the second body of liquid adjacent to the control electrodes prior to the measurements with the control electrodes, the preconditioning being performing by driving current across interfaces between the control electrodes and the portions of the second body liquid to adjust compositions of the portions of the second body of liquid.

Thus, a method is provided in which compositions of the liquid adjacent to the control electrodes can be adjusted prior to measurements using the control electrodes. The adjustment of the compositions may be such as to reduce or prevent erroneous drift of the subsequent measurements. Thus, the preconditioning can drive the system to a steady state condition more quickly than would be the case through normal operation, thereby saving time (by reducing or avoiding the need to wait to reach the steady state condition) and/or improving accuracy (by reducing or avoiding signal drift during measurements).

According to an alternative aspect, there is provided an apparatus for forming apertures in a membrane using dielectric breakdown, comprising: a membrane having a plurality of target portions; a first common electrode and a second common electrode, the first common electrode being configured to contact a first body of liquid on one side of the membrane and the second common electrode being configured to contact a second body of liquid on the other side of the membrane; a plurality of control electrodes, each control electrode configured to contact the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode; and a controller configured to: apply a potential difference between the first common electrode and the second common electrode; and create and/or control growth of apertures through the plurality of target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of the plurality of control electrodes and the first common electrode.

According to an alternative aspect, there is provided a measurement system configured to sense a molecular entity, comprising: a membrane having a plurality of target portions and respective apertures through the target portions; a first common electrode and a second common electrode, the first common electrode being configured to contact a first body of liquid on one side of the membrane and the second common electrode being configured to contact a second body of liquid on the other side of the membrane; a plurality of control electrodes, each control electrode configured to contact the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode; and a controller configured to: apply a potential difference between the first common electrode and the second common electrode; use the control electrodes to sense molecular entities in the apertures by performing measurements that are dependent on interactions between molecular entities and the apertures; and unblock apertures through plural target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of the plurality of control electrodes and the first common electrode, the controller being configured to control the voltages of the control electrodes by applying an unblocking voltage waveform to each control electrode, the unblocking voltage waveform configured such that current flows from the control electrode into liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

Embodiments of the disclosure will now be described, by way of example only, with reference to the accompanying drawings in which corresponding reference symbols indicate corresponding parts.

Figure 1 schematically depicts an apparatus for performing methods of the disclosure.

Figure 2 schematically depicts a portion of a membrane assembly supporting a membrane in the apparatus of Figure 2.

Figure 3 depicts an equivalent circuit for an example three of the target regions of the membrane of Figure 2.

Figure 4 is a graph schematically depicting how aperture formation can be controlled using only first and second common electrodes.

Figure 5 depicts an example circuit arrangement for allowing a control electrode to be selectively connected to a first voltage supply and a sensing circuit. Figure 6 is a graph depicting a first driving signal generated by the first voltage supply of Figure 5.

Figure 7 is a graph depicting example operation of a control electrode controlled using the circuit arrangement of Figure 5 and the first driving signal of Figure 6.

Figure 8 depicts an example circuit arrangement for allowing a control electrode to be selectively connected to a first voltage supply, a sensing circuit, and a second voltage supply.

Figure 9 is a graph depicting a first driving signal generated by the first voltage supply of Figure 8.

Figure 10 is a graph depicting a second driving signal generated by the second voltage supply of Figure 8.

Figure 11 is a graph depicted example operation of a control electrode controlled using the circuit arrangement of Figure 8 and the first and second driving signals of Figures 9 and 10.

Figure 12 is a graph depicting a combined driving signal generated by the first voltage supply of Figure 5.

Figure 13 is a graph depicting example operation of a control electrode controlled using the circuit arrangement of Figure 5 and the combined driving signal of Figure 12.

Figure 1 depicts an apparatus 2 that can be used to implement various methods of the present disclosure.

In one class of embodiment, the apparatus 2 is configured to form apertures 20 (see Figure 2) in a membrane 4. In another class of embodiment, the apparatus 2 is configured to, additionally or alternatively, unblock apertures 20 in a membrane 4. The membrane 4 may be a solid-state membrane. The apertures may be nanoscale apertures, for example apertures having a characteristic dimension (e.g., diameter or depth or both) of the order of lOOnm or less, optionally 50nm or less, optionally 20nm or less, optionally lOnm or less, optionally 5nm or less, optionally 2nm or less, optionally Inm or less. Each aperture 20 provides a conduit extending from one side of the membrane 4 to the other side of the membrane 4, thereby fully traversing the membrane 4. The apertures may be referred to as nanopores. Different sizes may be selected for different applications. For example, for direct DNA/RNA sequencing (ssDNA, RNA), relatively small apertures may typically be preferred, such as apertures in the range of about 1 to 2nm diameter. For other applications, such as counting of larger entities (which may be referred to as blob counting), larger apertures may be preferred, depending on the target entity size. For example apertures having diameters in the range of a few nm to 10s of nm may be used for such applications.

In an embodiment, as exemplified in Figure 2, the apparatus 2 comprises a membrane assembly 6 for supporting the membrane 4. In an embodiment, the membrane assembly 6 comprises the membrane 4 and a support structure 8-10. The support structure 8-10 is attached to and/or supports the membrane 4.

The membrane 4 and/or membrane assembly 6 may be composed of various materials and combinations of materials. Typically, the membrane 4 comprises a dielectric material. The membrane 4 may comprise, consist essentially of, or consist of, one or more of the following in any combination: silicon nitride; silicon oxide; a two-dimensional material; a material formable using atomic layer deposition (ALD). The two-dimensional material may for example comprise graphene, an MXene, M0S2, and/or h-BN. The material formable using ALD may for example comprise one or more of the following: HfO_x, ZrO_x, and/or A1O_X. Materials formable using ALD are desirable because their thicknesses can be controlled with high precision.

The thickness of the membrane 4 is not particularly limited. Typically, the thickness of the membrane 4 will be in the range of from about 0.3nm to about 50nm.

In an embodiment, the membrane assembly 6 is formed from one or more silicon wafers or similar, for example by lithographic manufacturing techniques. In the example shown, the membrane 4 and layer 8 of the support structure are formed from a first silicon wafer and layer 10 of the support structure is formed from a second silicon wafer. The first and second silicon wafers are bonded together by a glue layer 9. The resistivity of the membrane 4 is sufficiently high to allow dielectric breakdown. If the resistivity is anisotropic, the resistivity should be sufficiently high in the direction perpendicular to the membrane 4 to allow dielectric breakdown. The membrane 4 may comprise a single layer or a plurality of layers.

The membrane 4 comprises a plurality of target portions 15A-C. The apparatus 2 is configured to apply a potential difference across the membrane 4 to form apertures 20 in the target portions 15A-C by dielectric breakdown. The target portions 15A-C may be spaced apart sufficiently that a reduction in local resistance that occurs when dielectric breakdown is initiated in one target portion 15A-C does not prevent dielectric breakdown from being initiated in a neighbouring target portion 15A-C.

In some embodiments, as exemplified in Figure 2, each of the target portions 15A-C comprises a recess 16. The recesses 16 may be formed for example using lithography and a reactive ion etch. The recesses 16 may be formed in either or both sides of the membrane 4. A recess 16 in a given target portion 15 provides a local path of reduced thickness through the membrane, thereby favouring initiation of aperture formation in the recess of the target portion 15A-C. A region of the membrane 4 at the base of the recess 16 is thinner than regions of the membrane 4 outside of the recess 16.

A shape in plan view of the recesses 16 may take any form, for example circular. The recesses 16 may have a cross-section parallel to a plane of the membrane 4 that is the same at all depths. The recess 16 may, for example, define a cylindrical void. Alternatively, the recesses 16 may have cross -sections parallel to the plane of the membrane 4 that vary in size as a function of depth, for example getting smaller as depth increases. The recess may, for example, define a hemispherical void. The depth of the recess 16 may progressively increase from an edge of an opening of the recess 16 towards a central region of the recess 16. The recess 16 may have a curved surface facing Into the void defined by the recess 16, such as a spherical surface surface. The area of the thinnest portion of the membrane 4 in plan view in a recess 16 may be significantly smaller than the area in plan view of the whole of the recess 16. The area in which dielectric breakdown will be most favoured (i.e., the thinnest region) may therefore be smaller in such a recess 16 than in other arrangements. The likelihood of more than one separate aperture forming in the same recess 16 is therefore reduced because there is less space available for such a process to occur.

As depicted in Figures 1 and 2, in some embodiments the apparatus 2 comprises a first bath configured to hold a first body of liquid 11 on one side of the membrane 4. The first body of liquid 11 may comprise an ionic solution. The ionic solution may container electron mediator redox couple (e.g., a mediator) and/or be referred to as a mediator. The apparatus 2 further comprises a second bath configured to hold a second body of liquid 12 on the other side of the membrane 4 (i.e., on the opposite side of the membrane 4 to the first body of liquid 11). The second body of liquid 12 may comprise an ionic solution. The ionic solution may container and/or be referred to as a mediator. The composition of the second body of liquid 12 may be the same as or different from the composition of the first body of liquid 11. Example compositions for the first and second bodies of liquid 11 are provided below towards the end of the description. The liquids may be referred to as sensing solutions. As described below, the sensing solutions may comprise a buffer.

The apparatus 2 comprises a controller 14 for controlling application of potential differences across the membrane 4. The controller 14 may comprise any suitable combination of hardware, software, firmware, electrical connections, voltage supplies, switching arrangements, etc. required to achieve the desired functionality. The controller 14 may be configured to control potentials applied to a range of electrodes by controlling associated voltage supplies and/or electrical connections between the voltage supplies and the electrodes. The electrodes contact the first and second bodies of liquid 11, 12 and apply potential differences across the membrane 4 via the first and second bodies of liquid 11, 12.

In an embodiment, the apparatus 2 comprises a first common electrode 41 and a second common electrode 42. The first common electrode 41 contacts the first body of liquid 11 on one side of the membrane 4. The second common electrode 42 contacts the second body of liquid 12 on the other side of the membrane 4. The apparatus 2 further comprises a plurality of control electrodes 43A-C.

As depicted in Figure 2, each target portion 15A-C defines a respective first target surface 21A-C on one side of the membrane 4 (the upper side in the orientation of the figures) and a respective second target surface 22A-C on the other side of the membrane 4 (the lower side in the orientation of the figures). The first target surface 21 A-C is in contact with the first body of liquid 11. The second target surface 22A-C is in contact with the second body of liquid 12. The second body of liquid 12 is contained in the second bath. The second bath defines a bulk region 121 and a plurality of fluidic passages 122 A-C. In the example shown, the fluidic passages 122A-C open out at proximal ends into respective well regions 123A-C adjacent to the target portions 15 A-C of the membrane 4. The second body of liquid 12 extends continuously through the bulk region 121 and the fluidic passages 122A-C (including, where provided, the well regions 123A-C).

The second common electrode 42 contacts the second body of liquid 12 in the bulk region 121. Each fluidic passage 122A-C extends from a respective one of the second target surfaces 22A-C to the bulk region 121 and opens out into the bulk region 121 at a distal end 31A-C of the fluidic passage 122A-C. Each control electrode 43A-C contacts the second body of liquid 12 at a position electrically in series between a respective one of the second target surfaces 22A-C and the distal end 31A-C of the fluidic passage 122A-C corresponding to that second target surface 22A-C. Each control electrode 43A-C is thus positioned along a path passing through the fluid (which conducts the electrical current) at a position between the respective one of the second target surfaces 22A-C and the distal end 31A-C of the fluidic passage 122A-C corresponding to that second target surface 22A-C. The fluidic passages 122A-C are associated with different respective second target surfaces 22A-C and are fluidically isolated from each other between the second target surfaces 22A-C and the bulk region 121. Each second target surface 22A-C is fluidically connected to the bulk region 121 solely by the fluidic passage 22A-C associated with the second target surface 22A-C. Thus, second target surface 22A is fluidically connected to the bulk region 121 solely by fluidic passage 22A. There is no fluidic route from the second target surface 22 A to the bulk region 121 passing through any portion of any fluidic passage 22B, 22C other than fluidic passage 22A. Similarly, second target surface 22B is fluidically connected to the bulk region 121 solely by fluidic passage 22B, and second target surface 22C is fluidically connected to the bulk region 121 solely by fluidic passage 22C.

Figure 3 depicts an equivalent circuit for an example three of the target portions 15A-C of the membrane of Figure 2.

Figure 4 is a graph schematically depicting how aperture formation can be controlled using only the first and second common electrodes 41, 42. The voltages applied by the first and second common electrodes 41, 42 may be referred to as global insertion voltages in this case as they are applied equally to all of the target portions 15A-C. In this example, the first common electrode 41 is set to ground voltage and a voltage V2 is applied to the second common electrode 42. The vertical axis represents voltage relative to ground voltage. The thicker line curve labelled 52 represents a variation in a potential difference across the membrane 4 as a function of time for a representative one of the target portions 15A-C. In the geometry of Figure 2, the potential difference across the membrane 4 in each target portion 15A-C is approximately equal to the voltage at the control electrode 43A-C corresponding to that target portion 15A-C.

In a period before creation of an aperture 20 in the representative target portion 15A-C (before time 50 in the graph), the potential difference across the membrane 4 in the target portion 15A-C is very close to the potential difference between the first and second common electrodes 41, 42 (which is V2 in this example). This high voltage drives creation of the aperture 20, which in this example occurs at time 50. Creation of the aperture 20 causes the resistance across the target portion 15A-C to fall, which leads to a corresponding drop in the voltage across the membrane 4. The size of the voltage drop, dV, depends on the resistances of the aperture 20 and the fluidic passage 122A-C associated with the target portion 15A-C. These resistances determine how the voltage is divided according to the equivalent circuit shown in Figure 3 . Ideally, the voltage drop dV is large enough to prevent a second aperture 20 being created in the same target portion 15A-C.

In practice, imperfections in a manufacturing process used to create the membrane 4 and/or surrounding structures may lead to variations in the voltage at which dielectric breakdown occurs in different target portions 15A-C. For example, the imperfections may lead to variations in thickness and/or composition of the membrane. Where deposited thin fdm is used, process imperfections may lead to stoichiometry variations that affect the dielectric breakdown voltage. Where the aperture creation is driven only by the first and second common electrodes 41, 42, the variations in structure and/or composition may be allowed for by applying a voltage across the membrane 4 that is higher than a designed breakdown voltage to ensure that apertures 20 are formed in a majority of the target portions 15A-C. However, in practice it has been found that significant numbers of the target portions 15A-C may withstand the voltage without breakdown unless relatively large over-voltages (i.e., voltages that are higher than a designed breakdown voltage by a large amount) are applied. Excessively large over-voltages may risk creating multiple apertures in some target portions 15A-C, damaging the membrane 4, or inhibiting optimal control of aperture growth in target portions 15A-C where apertures 20 have been formed. On the other hand, if the applied voltage is too low, dielectric breakdown may not occur at all in a large number of the target portions 15A-C. The overall effect may be to reduce yield of target portions having a single aperture. The reduction in yield may be particularly significant where a range of voltages at which dielectric breakdown occurs is commensurate with or larger than the voltage drop expected when an aperture 20 is created.

Arrangements of the present disclosure address the above issues by using control electrodes 43A-C in addition to the first and second common electrodes 41, 42 to control the aperture creation and/or growth process. In particular, the apparatus 2 applies a potential difference between the first common electrode 41 and the second common electrode 42 and is additionally configured to create and/or control growth of apertures 20 through the target portions 15A-C by controlling the potential difference across each target portion 15A-C by controlling a respective control electrode 43A-C.

In one class of embodiment, the control electrodes 43A-C are used to detect where apertures 20 have been formed and allow appropriate corrective action to be taken in response. The ability to apply the corrective action makes it possible to apply a smaller potential difference initially and only step up the voltage gradually and only for target portions where it is needed. This reduces or avoids the need for undesirably large over-voltages and/or improves yield.

In an embodiment, each control electrode 43A-C is used to detect when a dielectric breakdown event occurs by monitoring a voltage at the control electrode 43A-C while a voltage is applied between the first and second common electrodes 41, 42. The voltage may be applied as a constant DC voltage or as a slowly ramping voltage. When breakdown happens, the resistance across the membrane 4 suddenly reduces, which can be detected as a change in current flow or voltage. For example, where the first common electrode 41 is held at ground, the voltage would drop from a value near a voltage V2 of the second common electrode 42 to a voltage between the voltages of the first and second common electrodes 41, 42. Voltages to the control electrodes 43A-C can be cut off as soon as formation of apertures 20 in the respective target portions 15A-C is detected.

In an embodiment, voltages applied to the first and second common electrodes 41, 42 and the control electrodes 43A-C during the creation of apertures 20 through the plural target portions 15A-C are configured such that differences between the voltages applied by the control electrodes 43A-C and the second common electrode 42 are relatively small, for example not exceeding 30%, optionally 25%, optionally 20%, optionally 15%, optionally 10%, optionally 8%, optionally 6%, optionally 4%, optionally 2%, optionally 1%, of a voltage difference between the first and second common electrodes 41, 42. Keeping the voltages of the control electrodes 43A-C relatively close to the voltage of the second common electrode 42 ensures that ionic currents along the fluidic passages 122A-C remain relatively low. Depletion of the electrode and/or mediator can thereby be kept acceptably low.

After it is determined where apertures 20 have not yet been formed, the control electrodes 43A-C may be used to take targeted corrective action. For example, the control electrodes 43A-C may be used to apply a potential difference across the membrane 4 selectively in those target portions 15A-C (where apertures 20 have not yet been formed) that is higher than the potential difference applied previously to those target portions 15A-C. The control electrodes 43A-C are individually controllable so the potential difference can be applied selectively only to those target portions 15A-C where apertures 20 have not yet been formed. The potential difference may be progressively ramped up for each target portion 15A-C until an aperture 20 is formed in the target portion 15A-C.

The voltage in the bulk region 121 of the second body of liquid 12 is predominantly determined by the voltage of the second common electrode 42, while each individual control electrode 43A-C is separated from the second common electrode 42 by volumes of liquid constrained within the fluidic passages 122A-C, which act as fluidic resistors. Each target portion 15A-C is thus relatively unaffected by (e.g., protected from) voltages applied to control electrodes 43A-C that do not correspond to that target portion 15A-C. After breakdown has been initiated successfully in target portions 15A-C, for example using one or more of the methods described above, control electrodes 43A-C can be used to control growth of apertures 20 in respective target portions 15A-C independently.

In one arrangement, for each target portion 15A-C, the control of growth comprises using the respective control electrode 43A-C to apply a different potential difference across the target portion 15A-C than would result solely from voltages of the first and second common electrodes 41 and 42. Alternatively or additionally, the control of growth comprises monitoring an electrical characteristic dependent on a size of an aperture 20 being grown and using the control electrode 43A-C to stop growth of the aperture 20 in response to the monitoring indicating that a target size has been attained. Controlling growth by monitoring the electrical characteristic provides flexibility for creating apertures 20 of a range of desired sizes, as well as improving reliability. The resistance through an aperture 20 decreases as the aperture 20 grows, which in the absence of control may favour formation of apertures 20 having a size (e.g., diameter) that is similar to a thickness of the membrane 4 directly adjacent to the aperture 20. The control electrodes 43A-C provide flexibility for selecting different aperture sizes. For example, smaller aperture sizes can be created by stopping the aperture growth process early. Alternatively, the control electrodes 43A-C can be used to apply larger potential differences across the target portions 15A-C than is typically possible via the first and second common electrodes 41 and 42 (which are constrained by the presence of the fluidic passages 122A-C between the second common electrode 42 and the membrane 4) to form larger apertures 20 than would otherwise be possible.

In an embodiment, as depicted schematically in Figure 2, the control electrodes 43A-C are used to selectively create apertures 20 in a desired subset of the target portions 15A and 15C (i.e., not in all of the target portions). The method may comprise creating apertures 20 through the selected subset of the target portions 15A and 15C by using control electrodes 43A and 43C to cause potential differences to be applied across the target portions 15 A and 15C in the selected subset that are larger than potential differences applied across target portions 15B that are not in the selected subset. The method thereby selectively initiates dielectric breakdown in the selected subset of the target portions 15A and 15C.

This functionality may be desirable in cases where it is not desired to use all possible apertures 20 in a membrane 4 in the same measurement run (e.g., in measurements involving using the apertures 20 to sense molecular entities in the apertures 20 by performing measurements that are dependent on interactions between the molecular entities and the apertures 20). Thus, apertures 20 in a first fraction of the membrane 4 can be created and used in a first measurement run and, at later time, apertures 20 in a second fraction of the membrane 4 can be created and used in a second measurement run. This approach ensures that the apertures 20 are as freshly formed as possible before measurement runs, which may advantageously reduce or eliminate problems associated with storage of membranes with apertures such as surface contamination or clogging during storage.

In an embodiment, the creating of the apertures 20 through the selected subset of the target portions 15A and 15C comprises applying voltages to control electrodes 43A and 43C corresponding to the selected subset of target portions 15A and 15C such that potential differences between the control electrodes 43 A and 43 C and the first common electrode 41 are larger than a potential difference between the first and second common electrodes 41 and 42. Thus, the control electrodes 43A and 43C are used to increase potential differences across the membrane 4 in selected target portions 15A and 15C relative to what would be the case using only the first and second common electrodes 41 and 42.

In a variation on the above embodiment, the creating of the apertures 20 through the selected subset of the target portions 15A and 15C may comprise applying voltages to control electrodes 43B other than control electrodes 43A and 43B corresponding to the selected subset of target portions 15A and 15C such that potential differences between the control electrodes 43B and the first common electrode 41 are smaller than a potential difference between the first and second common electrodes 41 and 42. Thus, control electrodes 43B in this case are used to suppress potential differences across the membrane 4 in selected target regions 43B relative to what would be the case using only the first and second common electrodes 41 and 42. The effect is again that potential differences across the membrane 4 in selected target portions 15A and 15C where it is desired to create apertures 20 are larger than in target portions 15B where it is not desired to create apertures 20.

The flexibility provided by the control electrodes 43A-C may additionally or alternatively be exploited to facilitate formation of apertures in target regions 15A-15C configured to have different nominal structures, such as different thicknesses. Thus, the target portions 15A-15C may be configured to have a range of different thicknesses (e.g., two or more different thicknesses) and the method comprises using the control electrodes 43A-C to apply a corresponding range of different potential differences across the target portions 15A-C to promote creation of apertures 20. For example, the control electrodes 43A-C may be used to apply larger potential differences across target portions 15A-C having higher thickness. Forming apertures 20 in target portions 15A-C having different thicknesses provides pores having different lengths, which may be useful for different kinds of measurements. Providing target portions 15A-C with different thicknesses may also provide additional design freedom for the fluidic passages 122A-C.

In some embodiments, the control electrodes 43A-C are selectively used in two modes, an aperture creation mode and an aperture monitoring mode. The aperture creation mode comprises applying a voltage to the control electrode 43A-C that promotes aperture creation through the target portion 15A-C corresponding to the control electrode 43A-C. The aperture monitoring mode comprises using the control electrode 43A-C to monitor a state of the aperture 20 by measuring an electrical characteristic associated with the aperture 20. The electrical characteristic may be, or may be dependent on, an electrical resistance through the aperture 20. The electrical characteristic may be a voltage of the liquid adjacent to the control electrode. As described above, a voltage measured at a control electrode 43A-C may change when an electrical resistance associated with an aperture 20 changes. A relatively abrupt change may be observed when an aperture 20 is created. A more gradual change may occur during growth of an aperture 20 after creation of the aperture 20.

In an embodiment, the aperture monitoring mode for a given control electrode 43A-C comprises an aperture detection mode configured to detect whether an aperture 20 has been created through the target portion 15A-C corresponding to the control electrode 43A-C. The aperture detection mode may comprise monitoring a signal (e.g., voltage) from the control electrode 43A-C to detect abrupt changes in the voltage, for example to detect when the signal changes by more than a predetermined threshold amount in a predetermined time period. The controller 14 may be configured to operate one of the control electrodes 43A-C in the aperture creation mode and the aperture detection mode in an alternating sequence in each of one or more of the target portions 15A-C until creation of an aperture 20 is detected in the target portion 15A-C. A maximum potential difference applied to the target portion 15A-C during each operation in the aperture creation mode in the alternating sequence may be progressively increased during the alternating sequence. This approach controls aperture formation on a target portion by target portion basis, promoting reliable formation of apertures without requiring undesirably large over-voltages.

In some configurations, especially where volumes of well regions 123A-C adjacent to the membrane 4 are relatively small and/or flow resistances associated with the fluidic passages 122A-C are relatively large, it is desirable to minimize depletion of the control electrodes 43A-C and/or mediator during aperture creation. If depletion of the control electrodes 43A-C and/or mediator is not controlling sufficiently, it can become difficult to make a reliable judgement about when an aperture 20 has been created because changes in voltage and/or current associated with aperture creation become less pronounced. The above-described approach of alternating between the aperture creation mode and the aperture detection mode assists with this by allowing short pulses to be applied in the aperture creation mode. This provides time between the pulses to allow recovery of the mediator. In a time between applications of the aperture creation mode, mediator may be partially or completely replenished by diffusion and/or a voltage (e.g., a smaller voltage than that applied via the second common electrode 42) may be applied to the control electrode 43 A-C to reverse the electrode/mediator consumption. If a pulse of the aperture creation mode is not successful, multiple cycles of the aperture creation mode and the aperture detection mode can be used with either the same voltage or increasingly higher voltages in the aperture creation mode until the aperture detection mode detects that an aperture 20 has been formed.

Figure 5 schematically depicts an example circuit arrangement for implementing the alternating between the aperture creation mode and the aperture detection mode . A switching arrangement 63 is provided that can selectively connect the control electrode 43A-C to a first voltage supply 64 to implement the aperture creation mode. The switching arrangement 63 can also selectively connect the control electrode 43 A-C to a sensing circuit 66 to implement the aperture detection mode. The alternating between the aperture creation mode and the aperture detection mode can be implemented by driving the switching arrangement 63 to alternately switch the connection from the control electrode 43 A-C to one or the other of the first voltage supply 64 and the sensing circuit 66.

In this example, the aperture creation mode comprises applying an aperture promoting waveform 60. The first voltage supply 64 may be configured to output a first driving signal comprising a sequence of the aperture promoting waveforms 60. The first driving signal can be applied simultaneously, as needed, to any number of the control electrodes 43A-C or, indeed, to other electrodes if needed. The first driving signal may thus be referred to as a first global signal or first global waveform. An example of such a first driving signal is shown in Figure 6, where the vertical axis represents voltage (V) relative to ground voltage and the horizontal axis represents time (t). In this example, the first common electrode 41 is set to ground voltage and the voltage of the second common electrode 42 is labelled V2. The aperture promoting waveforms 60 in the first driving signal are separated from each other to provide time slots to implement the aperture detection mode.

As exemplified in Figure 6, each aperture promoting waveform 60 may be configured such that current flows from the control electrode 43A-C into the liquid adjacent to the control electrode 43A-C (corresponding to a flow mainly towards the second common electrode 42 before aperture creation) during a first portion 61 of the aperture promoting waveform 60 and from liquid adjacent to the control electrode 43A-C back into the control electrode 43A-C (corresponding to a flow mainly from the second common electrode 42 back towards the control electrode 43A-C before aperture creation) during a second portion 62 of the aperture promoting waveform 60. Balancing the aperture promoting waveform 60 in this way may advantageously reduce mediator consumption by the control electrode 43A-C, thereby avoiding degradation of the ionic solution, reducing the need for corrective action, and/or reducing or avoiding negative impacts on measurements that may be performed at a subsequent time using the created apertures 20.

The aperture promoting waveform 60 may be configured such that a total amount of charge flowing (e.g., across an interface between a respective control electrode and the adjacent ionic liquid) during the first portion 61 of the aperture promoting waveform 60 is substantially the same as the total amount of charge flowing during the second portion 62 of the aperture promoting waveform 60. For example, an average magnitude of a difference between the voltage of the aperture promoting waveform 60 and a voltage of the second common electrode 42 may be substantially the same in the first and second portions 61, 62 of the aperture promoting waveform 60 with a duration of the first portion 61 being substantially the same as a duration of the second portion 62. In other embodiments, the aperture promoting waveform 60 may be configured such that a total amount of charge flowing during the first portion 61 of the aperture promoting waveform 60 is substantially the same as the total amount of charge flowing during the second portion 62 of the aperture promoting waveform 60 using an asymmetric waveform. For example, an average magnitude of a difference between the voltage of the aperture promoting waveform 60 and a voltage of the second common electrode 42 may be arranged to be different in the first and second portions 61, 62 of the aperture promoting waveform 60 with a corresponding difference between durations of the first portion 61 and the second portion 62.

In some embodiments, the aperture promoting waveform 60 varies symmetrically relative to an average value. The aperture promoting waveform 60 may comprise one or more cycles of an oscillatory function, such as a square-wave, saw-tooth, sinusoid, etc. In the example of Figure 6, each aperture promoting waveform 60 has a square-wave form. A voltage of each aperture promoting waveform 60 is centred on the voltage V2 of the second common electrode 42. Three examples of an aperture promoting waveform 60 are shown in Figure 6.

The aperture detection mode is configured to detect whether an aperture 20 has been created through the target portion 15A-C corresponding to the control electrode 43A-C. The aperture detection mode may comprise operating the control electrode 43A-C in a passive state in which the control electrode 43A-C does not actively apply a voltage. A voltage of the control electrode 43A-C in the aperture detection mode may thus be substantially equal to a voltage across the membrane 4.

Figure 7 is a graph depicting example operation of a control electrode 43A-C in which the aperture creation mode and the aperture detection mode are applied in an alternating sequence to a target portion 15A- C until creation of an aperture 20 is detected in the target portion 15A-C. The thicker line curve labelled 52, which comprises segments 52A-52F, represents a variation in a potential difference across the membrane 4 as a function of time. In the geometry of Figure 2, the potential difference across the membrane 4 in each target portion 15A-C is approximately equal to the voltage at the control electrode 43A-C of the target portion 15A-C.

Each operation in the aperture creation mode may comprise applying one or more instances of the aperture promoting waveform 60. In the example of Figure 7, the first voltage supply 64 provides the first driving signal of Figure 6. The switching arrangement 63 is controlled to connect the control electrode 43A- C to the first voltage supply 64 during each aperture promoting waveform 60 of the first driving signal and to connect the control electrode 43A-C to the sensing circuit 66 in between the aperture promoting waveforms 60. An alternating sequence is thus applied that comprises single instances of the aperture promoting waveform 60 interleaved by instances of the aperture detection mode. Segments 52A and 52B correspond to a first instance of the aperture promoting waveform 60 in the first driving signal of Figure 6. Segment 52C corresponds to a first instance of the aperture detection mode. The control electrode 43A-C is put into a passive state in the aperture detection mode. As discussed above with reference to Figure 4, the voltage of the control electrode 43A-C in such a passive state will become equal to the voltage V2 of the second common electrode 42 while an aperture 20 has not yet been formed in the target portion 15A-C corresponding to the control electrode 43A-C. Segments 52D and 52E correspond to a second instance of the aperture promoting waveform 60.

The voltage range labelled 70 in Figure 7 represents a range of voltages of the control electrode 43A- C at which it is expected that an aperture 20 will be created. In the example of Figure 7, an aperture 20 is created during segment 52D. The created aperture 20 is detected as soon as the subsequent aperture detection mode starts at time 50 and the switching arrangement 63 is controlled to prevent switching back in of the first driving signal from the first voltage supply 64. The voltage of the control electrode 43A-C will thus remain (segment 52F) at the voltage corresponding to the voltage across the membrane 4 in the target portion 15A-C. This voltage will be lower than V2 because of the reduction in resistance across the target portion 15A-C caused by the creation of the aperture 20 (e.g., see “dV” in Figure 4). This reduces the chances of a second aperture 20 being created in the same target portion 15A-C. Furthermore, due to the use of the first driving signal to drive creation of the apertures 20 (rather than purely the potential difference provided by the first and second common electrodes 41 and 42), the voltage of the second common electrode 42 can be kept well below a level that would risk unwanted creation of apertures 20 due to a potential difference applied across a target portion 15A-C purely by the first and second common electrodes 41 and 42.

As depicted in Figures 6 and 7, a maximum potential difference applied to the target portion 15A-C during each operation in the aperture creation mode in the alternating sequence may be progressively increased during the alternating sequence. For example, where the aperture promoting waveform 60 comprises one or more cycles of an oscillatory function, an amplitude of the oscillatory function (e.g., height of square wave) may be progressively increased. Progressively increasing the maximum potential difference progressively increases the chances of creating an aperture 20. In the first driving signal of Figure 6, the amplitude of the square wave corresponding to the second instance of the aperture promoting waveform 60 (middle waveform) is larger than the amplitude of the square wave corresponding to the first instance of the aperture promoting waveform 60 (leftmost waveform). The amplitude of the square wave corresponding to the third instance of the aperture promoting waveform 60 (rightmost waveform) is larger than the amplitude of the square wave corresponding to the second instance of the aperture promoting waveform 60 (middle waveform).

After apertures 20 have been created in the membrane 4, the apertures 20 may be used to perform measurements. The measurements may comprise sensing molecular entities in the apertures, for example by performing measurements that depend on an interaction between the molecular entity and the aperture 20. The measurements may be performed by a measurement system configured to sense a molecular entity in each of the apertures by performing a measurement that is dependent on an interaction between the molecular entity and the aperture. During such use, which may involve maintaining a relatively large potential difference across the target portion 15A-C in which the aperture 20 is formed, the aperture 20 may become obstructed (clogged). If the obstruction is not cleared promptly, the aperture 20 may become permanently obstructed.

It is possible to unblock apertures 20 by reversing a polarity of the voltage across the target portion 43A-C containing the blocked aperture 20. For example, a typical implementation might apply a potential difference across a membrane of 200mV by applying a voltage at the first common electrode 41 of lOOmV and a voltage at the second common electrode 42 of 300mV. A direct DC voltage of OV could then be applied at a control electrode 43A-C to drive unblocking of an aperture 20 in the target portion 15A-C corresponding to the control electrode 43A-C. The DC voltage of 0V would drive about 1.56nA of current across the interface between the control electrode 43A-C and the adjacent ionic solution. For a typical well region having 30 micron diameter and 30 micron depth, filled with standard 150mM ferri/ferro mediator, it would take roughly 200 seconds to completely deplete the mediator. If we consider a 10% level of mediator consumption as the limit of what can be tolerated before a resulting voltage offset becomes too large, it would be necessary to limit application of the unblocking voltage to about 20 seconds, which may not be enough to achieve unblocking reliably. Furthermore, even mediator depletion levels lower than 10% may still undesirably affect measurements. Other redox pair mechanisms such as Ag/AgCl electron mediator redox couples are envisaged.

Arrangements of the present disclosure may be configured to address the clogging problem using the control electrodes 43A-C in a way that improves on applying a DC unblocking voltage as described above. In the examples described below with reference to Figures 8-13, methods to implement unblocking of apertures 20 are described in the context of arrangements that are also capable of creating the apertures 20. However, it will be understood that the aperture unblocking functionality can be applied more generally, including in contexts where methods of the present disclosure are not necessarily used to create the apertures 20 in the first place. In this context, the apertures 20 may comprise apertures formed in a solid state membrane or apertures formed in other membranes, such as nanopores inserted in lipid bilayers.

In one class of embodiment, the aperture monitoring mode is configured to comprise a blocking detection mode. The blocking detection mode is configured to detect blocking of a previously created aperture 20 through a target portion 15A-C using a control electrode 43A-C corresponding to the target portion 15A-C. The blocking of the aperture 20 may be detected, for example, by detecting an increase in electrical resistance associated with the aperture 20. A voltage of the control electrode 43A-C may be controlled to unblock the aperture 20 in response to detection of blocking by the blocking detection mode.

The unblocking functionality may be combined with the aperture creation functionality described above. Figures 8 to 11 depicts an example implementation based on expanding the functionality of the example described above with reference to Figures 5 to 7.

Figure 8 depicts a circuit arrangement corresponding to that of Figure 5 except that the switching arrangement 63 is additionally configured to allow the control electrode 43A-C to be selectively connected to a second voltage supply 68. Thus, at any given time the control electrode 43A-C can be connected to any one of the first voltage supply 64, second voltage supply 68, and sensing circuit 66.

Figure 9 is a graph showing an example first driving signal generated by the first voltage supply 64 for use in the context of the example of Figures 8 to 11. The vertical axis represents voltage (V) relative to ground voltage and the horizontal axis represents time (t). In this example, the first common electrode 41 is set to ground voltage and the voltage of the second common electrode 42 is labelled V2. The aperture promoting waveforms 60 in the first driving signal are separated from each other to provide time slots for implementing an aperture detection mode. The first driving signal comprises a sequence of aperture promoting waveforms 60 similar to those of Figure 6. The aperture promoting waveforms 60 may take any of the forms discussed above with reference to the example of Figures 5 to 7.

In an embodiment, the controlling of the voltage of the control electrode 43A-C to unblock the aperture 20 comprises applying an unblocking voltage waveform 70. The second voltage supply 68 may be configured to output a second driving signal comprising a sequence of the unblocking voltage waveforms 70. Like the first driving signal, the second driving signal can be applied simultaneously, as needed, to any number of the control electrode 43A-C or, indeed, to other electrodes if needed. The second driving signal may thus be referred to as a second global signal or second global waveform. An example of such a second driving signal is shown in Figure 10, where the vertical axis represents voltage (V) relative to ground voltage and the horizontal axis represents time (t). In this example, the first common electrode 41 is set to ground voltage and the voltage of the second common electrode 42 is labelled V2.

As exemplified in Figure 10, each unblocking voltage waveform 70 may be configured such that current flows from the control electrode 43A-C into the liquid adjacent to the control electrode 43A-C (e.g., accompanied by flow towards the second common electrode 42) during a first portion 71 of the unblocking voltage waveform 70 and from the liquid adjacent to the control electrode 43A-C back into the control electrode 43A-C (e.g., accompanied by flow from the second common electrode 42 towards the control electrode 43A-C) during a second portion 72 of the unblocking voltage waveform 70. Thus, the unblocking voltage waveform 70 improves on the alternative approach discussed above of applying a DC unblocking voltage. The voltage applied during the portion 71 promotes unblocking of the aperture 20 but may deplete the mediator. The voltage applied during portion 72 is in the opposite sense, however, and provides an at least partial cancellation of the mediator depletion of portion 71. The portion 72 thus at least partially reverses the mediator depletion caused by portion 71. At least partially reversing the mediation depletion in this manner may also allow unblocking voltages to be applied for longer periods and more cycles without excessive mediator depletion, thereby facilitating removal of even the most stubborn blockages.

The unblocking voltage waveform may be configured such that a total amount of charge flowing during the first portion of the unblocking voltage waveform is substantially the same as the total amount of charge flowing during the second portion of the unblocking voltage waveform. Arranging for the total amount of charge to be the same may provide optimal balancing (cancelling) of mediator consumption. The first and second portions 71 and 72 of the unblocking voltage waveform 70 may in principle have the same duration or different durations. However, typically there is a limit to how much the voltage applied by a control electrode 43A-C can be raised above the voltage V2 at the second common electrode 43 before risking damage to the membrane 4. This may mean that, as exemplified in Figure 10, an average magnitude of a difference between the voltage of the unblocking voltage waveform 70 and the voltage V2 of the second common electrode 42 is different (e.g., higher, as in the example shown) in the first portion 71 of the unblocking voltage waveform 70 than in the second portion 72 of the unblocking voltage waveform 70. To compensate for this difference, as also exemplified in Figure 10, the duration of the first portion 71 may be made shorter than the duration of the second portion 72.

The unblocking voltage waveform may be selectively applied only to a subset of apertures that need unblocking while other apertures are operated normally, for example to sense molecular entities, optionally without reversing of a polarity of voltage applied across them. Thus, the method may simultaneously use: a first subset of the control electrodes to apply the unblocking voltage waveform to a corresponding first subset of the apertures in the membrane; and a second subset of the control electrodes to sense molecular entities in a corresponding second subset of the apertures in the membrane by performing measurements with the control electrodes that are dependent on interactions between the molecular entities and the apertures. The sensing of the molecular entities may be performed by the second subset of control electrodes without applying the unblocking voltage waveform via the second subset of control electrodes. The first and second subsets of electrodes may be selected for example by detecting whether apertures are blocked or unblocked and assigning apertures to the first and second subsets accordingly.

Figure 11 is a graph depicting example operation of the control electrode 43A-C using the circuit arrangement of Figure 8 and the first and second driving signals of Figures 9 and 10. The control electrode 43A-C is operated to apply the aperture creation mode and the aperture detection mode in an alternating sequence until creation of an aperture is detected in the target portion 15A-C corresponding to the control electrode 43A-C. The thicker line curve labelled 52, which comprises segments 52A-J, represents a variation in a potential difference across the membrane 4 as a function of time. Segments 52A-F in Figure 11 correspond to segments 52A-F in Figure 7. The switching arrangement 63 is controlled to switch connection from the control electrode 43A-C alternately to the first voltage supply 64 and the sensing circuit 66 to apply the sequence of aperture promoting waveforms 60 (segments 52A-B and 52D-E) interleaved with application of the aperture detection mode (segments 52C and 52F) to detect when an aperture 20 has been created. Like in the example of Figure 7, an aperture 20 is created during the high voltage segment 52D and subsequently detected at time 50 at the start of the next aperture detection mode.

Subsequently to time 50, during the segment 52F, the control electrode 43A-C is operated in an aperture monitoring mode. The aperture monitoring mode comprises the blocking detection mode. The blocking detection mode may be implemented by the sensing circuit 66 to which the control electrode 43A-C is connected during segment 52F. Blocking of the recently created aperture 20 in the respective target portion 15A-C occurs at time 80, leading to a change in voltage of the control electrode 43A-C (segment 52G). The change in voltage is detected during segment 52G and identified as indicative of blocking of the aperture 20. At time 82, the switching arrangement 63 responds to the detection of the blocking by controlling the control electrode 43A-C to unblock the aperture 20. The switching arrangement 63 achieves this in this example by switching connection of the control electrode 43A-C from the sensing circuit 66 to the second voltage supply 68, thereby causing the unblocking voltage waveform of Figure 7 to be applied to the control electrode 43A- C (segments 52H and 521). Unblocking of the pore is detected at time 84 and the switching arrangement 63 responds by switching the connection of the control electrode 43A-C from the second voltage supply 68 back to the sensing circuit 66. Subsequently to time 84, during the segment 52J, the control electrode 43A-C may be operated again in the aperture monitoring mode to detect and respond to any further blocking events. In a variation on the approach of Figures 8-11, the first voltage supply 64 can be configured to generate a combined driving signal. Like the first and second driving signals discussed above, the combined driving signal can be applied simultaneously, as needed, to any number of the control electrode 43A-C or, indeed, to other electrodes if needed. The combined driving signal may thus be referred to as a combined global signal or combined global waveform. An example of such a combined driving signal is shown in Figure 12. The combined driving signal comprises an alternating sequence of the aperture promoting waveform 60 and the unblocking voltage waveform 70. The switching arrangement 63 may be controlled to alternately switch between applying the aperture promoting waveform 60 from the combined driving signal and applying the aperture detection mode, thereby applying the aperture creation mode and the aperture detection mode in an alternating sequence, until creation of an aperture 20 is detected in the target portion 15A-C. The aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture 20 through the target portion 15A-C corresponding to the control electrode 43A-C. The method comprises applying the unblocking waveform from the combined driving signal to unblock the aperture in response to detection of blocking by the blocking detection mode.

The switching arrangement 63 may be configured to implement this arrangement using the circuit arrangement shown in Figure 5. Figure 13 is a graph depicting example operation of the control electrode 15A-C using the circuit arrangement of Figure 5 and the combined driving signal of Figure 12. The combined driving signal of Figure 12 is superimposed as a thinner solid line in Figure 13 for ease of comparison. This implementation provides similar functionality to that described with reference to Figures 8-11 except that a single combined driving signal is used (as shown in Figure 12) instead of two separate driving signals (as shown in Figures 9 and 10). This may simplify hardware requirements significantly by reducing the number of electrical connections that need to be made to each control electrode 43A-C and/or reducing the complexity of the switching arrangement 63.

In the present implementation, the control electrode 43A-C is operated to apply the aperture creation mode and the aperture detection mode in an alternating sequence until creation of an aperture is detected in the target portion 15A-C corresponding to the control electrode 43A-C. The thicker line curve labelled 52, which comprises segments 52A-J, represents a variation in a potential difference across the membrane 4 as a function of time. Segments 52A-F in Figure 11 correspond to segments 52A-F in Figures 7 and 11. The switching arrangement 63 is controlled to switch connection from the control electrode 43A-C alternately to the first voltage supply 64 and the sensing circuit 66 to apply the sequence of aperture promoting waveforms 60 (segments 52A-B and 52D-E) interleaved with application of the aperture detection mode (segments 52C and 52F) to detect when an aperture 20 has been created. Thus, at time 90 the switching arrangement 63 switches connection from the first voltage supply 64 to the sensing circuit 66. At time 91, the switching arrangement 63 switches connection from the sensing circuit 66 back to the voltage supply 64. The timing of the switching is such that the combined driving signal has by time 91 reached the start of the next instance of the aperture promoting waveform 60 (i.e., skipping over an instance of the unblocking waveform that occurred between times 90 and 91). At time 92, after completion of the aperture promoting waveform 60, the switching arrangement 63 switches connection from the first voltage supply 64 to the sensing circuit 66. In the same way as in the examples of Figures 7 and 11, an aperture 20 is created during the high voltage segment 52D and subsequently detected at time 92 by the aperture detection mode.

Subsequently to time 92, during the segment 52F, the control electrode 43A-C is operated in an aperture monitoring mode. The aperture monitoring mode comprises the blocking detection mode. The blocking detection mode may be implemented by the sensing circuit 66 to which the control electrode 43A-C is connected during segment 52F. Blocking of the recently created aperture 20 in the respective target portion 15A-C occurs at time 93, leading to a change in voltage of the control electrode 43A-C (segment 52G). The change in voltage is detected during segment 52G and identified as indicative of blocking of the aperture 20. At time 94, the switching arrangement 63 responds to the detection of the blocking by controlling the control electrode 43A-C to unblock the aperture 20. The switching arrangement 63 achieves this in this example by switching connection of the control electrode 43A-C from the sensing circuit 66 to the first voltage supply 64 at a time point corresponding to the start of one of the unblocking waveforms 70 of the combined driving signal (see Figure 12). This causing the unblocking waveform 70 to be applied to the control electrode 43A- C (segments 52H and 521). Unblocking of the pore is detected at time 95 and the switching arrangement 63 responds by switching the connection of the control electrode 43A-C from the first voltage supply 64 back to the sensing circuit 66. Subsequently to time 95, during the segment 52J, the control electrode 43A-C may be operated again in the aperture monitoring mode to detect and respond to any further blocking events. The functionality of Figure 13 is thus similar to that described above with reference to Figure 11 except that only a single driving signal needs to be generated (the combined driving signal) and implementation can be achieved with a simpler circuit arrangement (e.g., as shown in Figure 5 rather than as shown in Figure 8).

Using embodiments of the present disclosure to reduce or avoid mediator consumption during unblocking of apertures 20 may improve performance by reducing or avoiding drifts or offsets in measurement signals that might otherwise occur.

In principle, measurements of entities in the apertures 20 using the control electrodes 43A-C (e.g., using voltage sensing) could be performed without needing significant net current flow through any of the control electrode 43A-C. When this is strictly the case, there would be no mediator consumption adjacent to the control electrodes 43A-C and no corresponding voltage drifting caused by mediator concentration changes. However, in practice perfectly zero net current is not easily achieved. For example, an input to electronics used for the measurements may have a finite leakage current, which will drive a small but not zero current through the control electrode 43A-C and continuously consume mediator. Various other imperfections or defects may also lead to variations in mediator concentrations. Usually, the currents involved are small enough that diffusion from the fluidic passages 122A-C will eventually compensate the mediator consumption and the system will reach a steady state. In the steady state, a measurement voltage may have an offset but the offset will be constant and is therefore less problematic than a drifting signal. However, it may take many hours to reach the steady state and the measurement voltage may steadily drift in the meantime. In some cases, when DNA enters an aperture 20, the aperture 20 may become charge selective. Due to the charge difference of ferri and ferro, they will experience different electrodialysis in the fluidic channel. If the DNA remains in the aperture 20 for long enough, the ferri and ferro concentrations in the well will eventually settle down to a different value from the bulk concentration and cause a constant voltage offset. Again, this process may take many hours, and the voltage offset will keep drifting before it reaches the steady state.

In a voltage sensing system, by measuring the drifting rate, one can estimate the eventual steady state mediator concentration. Apparatus and methods of the present disclosure can then be used to deliberately inject net current to drive the mediator in the well into the steady state condition, and thereby actively stop the drifting. Alternatively or additionally, batches of charge can be progressively injected into the control electrode 43A-C until it is detected that the drifting has stopped. For example, a trial and error approach can be used in which a cycle comprising charge injection followed by drift measurement is used. This cycle can be repeated, optionally with progressively reduced amounts of charge injection, until the drift is found to drop below a predetermined threshold level. Once the system reaches the steady state, the voltage reading will be off by a constant offset, which will not affect most or all downstream data analysis, such as basecall.

Based on the above insight, a method may be provided for sensing molecular entities in apertures 20 in a membrane 4. The method comprises applying a potential difference between a first common electrode 41 and a second common electrode 42. The first common electrode 41 contacts a first body of liquid 11 on one side of the membrane 4 and the second common electrode 42 contacts a second body of liquid 12 on the other side of the membrane 4. The electrodes, first and second bodies of liquid and/or membrane 4 may take any of the forms discussed above with reference to the figures. The method further comprises using a plurality of control electrodes 43A-C to sense molecular entities in the apertures 20 by performing measurements with the control electrodes 43A-C that are dependent on interactions between the molecular entities and the apertures 20. Each control electrode 43A-C contacts the second body of liquid 12 at a position electrically in series between a respective one of the target portions 15A-C and the second common electrode 42. The control electrodes 43A-C may take any of the forms described above with reference to the figures. The method further comprises preconditioning respective portions of the second body of liquid 12 adjacent to the control electrodes 43A-C (e.g., the well regions 123A-C where an arrangement of the type shown in Figure 2 is used) prior to the measurements with the control electrodes 43A-C. The preconditioning is performing by driving current across interfaces between the control electrodes 43A-C and the portions of the second body liquid 12 to adjust compositions of the portions of the second body of liquid 12. The adjustment of the compositions of the portions of the second body of liquid by the preconditioning is such as to reduce or prevent erroneous drift of the subsequent measurements. Thus, the preconditioning can drive the system to the steady state condition more quickly than would be the case through normal operation, thereby saving time (by reducing or avoiding the need to wait to reach the steady state condition) and/or improving accuracy (by reducing or avoiding signal drift during measurements).

More generally, one or more of the apertures 20 formed using the methods and apparatus discussed above, or according to other embodiments, can be used to sense a molecular entity by performing a measurement (e.g. an electrical measurement or an optical measurement) that is dependent on an interaction between the molecular entity and the aperture. An apparatus may be provided that has a plurality of the apertures 20 thus formed and a measurement system configured to sense a molecular entity in each of the apertures 20 by performing a measurement that is dependent on an interaction between the molecular entity and the aperture 20.

Sensing of molecular entities can provide the basis for identifying single molecules and molecular entities. There are a wide range of possible applications, such as sequencing of DNA or other nucleic acids; sensing of chemical or biological molecules for security and defence; detection of biological markers for diagnostics; ion channel screening for drug development; and label free analysis of interactions between biological molecules.

The molecular entity may be polymeric entity such as an amino acid, peptide, polypeptide, a protein or a polynucleotide. The polynucleotide may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the polynucleotide can be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For instance, the polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light and are the primary cause of skin melanomas. One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide can comprise one strand of RNA hybridised to one strand of DNA. The molecular entity may comprise a single stranded or double stranded polynucleotide. The polynucleotide may be partially double stranded. The polynucleotide may be labelled with one of more of a fluorescent label, an optical label, a magnetic species or a chemical species, wherein detection of the species or label is indicative of the polynucleotide. Nucleic acid probes may be hybridised to the polynucleotide and resultant structure detected by translocation through an aperture of the array, such as disclosed in published application W02007/041621. The polynucleotide may be labelled with one or more acceptor labels, which interact with one or more donor labels attached to an aperture of the array, such as disclosed by published application WO2011/040996. The polynucleotide may be any synthetic nucleic acid known in the art. The molecular entity may be an aptamer. The molecular entity is caused to translocate the aperture and the interactions between the molecular entity and the aperture measured.

Translocation of the molecular entity through the aperture may be assisted by a motor protein such as a polynucleotide handling enzyme, or a polypeptide handing enzyme such as disclosed in published application WO2013/123379 Preferred enzymes are polymerases, exonucleases, helicases and topoisomerases, such as gyrases. Any helicase may be used in the invention. The helicase may be or be derived from a Hel308 helicase, a RecD helicase, such as Tral helicase or a TrwC helicase, a XPD helicase or a Dda helicase. The helicase may be any of the helicases, modified helicases or helicase constructs disclosed in WO2013/057495, WO2013/098562, and WO2013098561. Alternatively translocation of the molecular entity through the pore may also be assisted by voltage control, such as disclosed by W02008/124107.

The characteristic to be determined may be a sequence characteristic of the polymer.

A solid state membrane may comprise either or both of organic and inorganic materials, including, but not limited to, microelectronic materials, whether electrically conducting, electrically semiconducting, or electrically insulating, including materials such as II -IV and III-V materials, oxides and nitrides, such as silicon nitride, AI2O3, and SiC>2, Si, M0S2, solid state organic and inorganic polymers such as polyamide, plastics such as Teflon®, or elastomers such as two-component addition-cure silicone rubber, and glasses. A membrane may be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick such as those disclosed in U.S. Patent No. 8,698,481, and U.S. Patent Application Publication 2014/174927, both hereby incorporated by reference. More than one layer of material can be included, such as more than one graphene layer, as disclosed in US Patent Application Publication 2013/309776, incorporated herein by reference. Suitable silicon nitride membranes are disclosed in U.S. Patent No. 6,627,067, and the membrane may be chemically functionalized, such as disclosed in U.S. Patent Application Publication 2011/053284, both hereby incorporated by reference. The internal walls of the apertures may be coated with a functionalised coating, such as disclosed in published application W02009/020682.

In a further embodiment, a biological nanopore may be provided within a solid state aperture. Such a structure is disclosed for example in U.S. Patent No. 8,828,211, hereby incorporated by reference.

The biological pore may be a transmembrane protein pore. Transmembrane protein pores for use in accordance with embodiments of the disclosure can be derived from beta-barrel pores or alpha-helix bundle pores, beta-barrel pores comprise a barrel or channel that is formed from beta-strands. Suitable beta-barrel pores include, but are not limited to, alpha-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP). alpha-helix bundle pores comprise a barrel or channel that is formed from alpha-helices. Suitable alpha-helix bundle pores include, but are not limited to, inner membrane proteins and outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from Msp or from a-hemolysin (a-HL). The transmembrane pore may be derived from lysenin. Suitable pores derived from lysenin are disclosed in WO 2013/153359. The nanopore may be CsgG such as disclosed in WO 2016/034591.

The measurement may for example be electrical, optical or both. The electrical measurement may comprise measurement of ion flow through the apertures under a potential difference or concentration gradient. Electrical measurements may be made using standard single channel recording equipment as 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 WO-2000/28312. Alternatively, electrical measurements may be made using a multi-channel system, for example as described in International Application WO-2009/077734 and International Application WO-2011/067559. Optical measurements may be combined with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan;81(l):014301).

The sensing apparatus may comprise a measurement system arranged as disclosed in any of WO- 2008/102210, WO-2009/07734, WO-2010/122293, WO-2011/067559 or WO2014/04443. The sensing apparatus may comprise electrodes arranged on each side of the membrane in order to measure an ion current through an aperture under a potential difference. The electrodes may be connected to an electrical circuit which includes a control circuit arranged to supply a voltage to the electrodes and a measurement circuit, arranged to measure the ion flow. A common electrode may be provided to measure ion flow through the apertures between the common electrode and electrodes provided on the opposite side of the membrane.

Fluid chambers provided on either side of the nanopore array may be referred to as the cis and trans chambers. The molecular entity to be determined by the array of nanopores is typically added to the cis chamber comprising the common electrode. Separate trans chambers may be provided on the opposite side of the array, each trans chamber comprising an electrode wherein ion flow through each aperture is measured between an electrode of the trans chamber and the common electrode.

Depending upon the aperture length (the distance between the two sides of the membrane), one or more polymer units may be present in the pore at any particular time and the measurements may generate a complex signal, potentially influenced by multiple polymer units at a time. Machine learning can be used to extract information from the signal, as discussed for example in WO2018/203084, herein incorporated in its entirety by reference.

Any measurement system used may be linked to or comprise a processor such as an ASIC, FPGA, or computer. Analysis of the measurements may be carried out in the sensing apparatus, alternatively it may be done remotely, such as by a cloud based system.

Suitable conditions for measuring ionic currents through aperture pores are known in the art. The method is typically carried out with a voltage applied across the membrane and aperture. The voltage used is typically from +5 V to -5 V, such as from +4 V to -4 V, +3 V to -3 V or +2 V to -2 V. The voltage used is typically from -600 mV to +600mV or -400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range lOOmV to 2V. It is possible to increase discrimination between different nucleotides by an aperture by using an increased applied potential. As an alternative to measurement of an ionic current, measurement of a conductance or resistance may be carried out.

Alternative or additional measurements associated with movement of the molecular entity with respect to the aperture may be carried out, such as measurement of a tunnelling current across the aperture (Ivanov AP et al., Nano Lett. 2011 Jan 12; 1 l(l):279-85), or a field effect transistor (FET) device, such as disclosed by WO 2005/124888, US8828138, WO 2009/035647, or Xie et al, Nat Nanotechnol. 2011 Dec 11; 7(2): 119-125. The measurement device may be an FET nanopore device comprising source and drain electrodes to determine the presence or passage of a molecular entity in the apertures. An advantage of employing an FET nanopore device, namely one employing FET measurements across the apertures, or one employing measurement of a tunnelling current across the aperture, is that the measurement signal is very local to a particular aperture and therefore a device comprising a shared trans chamber may be employed. This greatly simplifies the construction of the device without the need to provide separate trans chambers for each aperture, such as one for the measurement of ion flow through the apertures, as described above. As a result, very high densities of apertures in the array may be conveniently provided, for example an array comprising apertures having a pitch of less than 10pm and a density of 10⁶apertures/cm².

Sensing methods, particularly those involving measurement of an ionic current, may be performed in a sensing solution comprising various different charge carriers, including for example metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1 -ethyl -3 -methyl imidazolium chloride. Potassium chloride (KC1), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is typically used. KC1, NaCl and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric across the membrane. For instance, the type and/or concentration of the charge carriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration may be 3 M or lower and is typically from 0. 1 to 2.5 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations.

The sensing solution may comprise a buffer. Any buffer may be used. Typically, the buffer is phosphate buffer. The sensing solution may comprise a buffer to regulate the pH. Any buffer suitable for the desired pH may be used. Maintaining a particular pH may be desirable for a variety of reasons, including maintaining consistent motor protein and biological nanopore performance, maintaining a consistent surface charge on solid-state membranes, and maintaining a consistent charge (and thus a consistent driving force and capture rate) on target analytes such as DNA.

Either or both of the first and second ionic solutions used for forming the apertures may also be used as the sensing solution. Either or both of the first and second ionic solutions may comprise a biological fluid containing ions (e.g. from salt), such as blood or plasma.

The ability to be able to form porous structures comprising arrays of apertures enables a large range of potential applications, such as for example the provision of structures that are initially present in a non- porous state but which may be activated in situ by dielectric breakdown to make them porous. As nanopores may be fragile and/or have a limited lifetime, generating pores in situ by dielectric breakdown enables a much longer shelflife when the structures are stored and distributed in the non-porous state. The non-porous structures may be used for example to initially contain a species which is subsequently released through the porous structure created by dielectric breakdown. Example uses are filter membranes, drug delivery and printing applications. The species to be delivered may comprise an ion or molecule. The molecule may be any such as a drug. The porous membrane may act an electrochemical frit, wherein formation of the apertures provides an ionic connection between the baths. The ability to form a porous membrane in situ enables a species to be contained within either the first or second bath until required or is able to limit the ability of a species provided in one bath to interfere with a species present in the second bath. For example, the first bath may contain a reference electrode such as Ag/AgCl, wherein it is desirable to limit the interaction of silver ions with a biochemical reagent present in the second bath.

The features defined in the claims may be used together in any combination.

Claims

1. A method of forming apertures in a membrane using dielectric breakdown, comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and creating and/or controlling growth of apertures through a plurality of target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of a plurality of control electrodes, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode.

2. The method of claim 1, wherein each target portion defines a respective first target surface on the one side of the membrane and a respective second target surface on the other side of the membrane, the first target surface being in contact with the first body of liquid, the second target surface being in contact with the second body of liquid.

3. The method of claim 2, wherein the second body of liquid is contained in a bath defining a bulk region and a plurality of fluidic passages.

4. The method of claim 3, wherein the second common electrode contacts the second body of liquid in the bulk region.

5. The method of claim 4, wherein each fluidic passage extends from a respective one of the second target surfaces to the bulk region and opens out into the bulk region at a distal end of the fluidic passage.

6. The method of claim 5, wherein each control electrode contacts the second body of liquid at a position electrically in series between a respective one of the second target surfaces and the distal end of the fluidic passage corresponding to that second target surface.

7. The method of any of claims 3 to 6, wherein the fluidic passages are associated with different respective second target surfaces and are fluidically isolated from each other between the second target surfaces and the bulk region, such that each second target surface is fluidically connected to the bulk region solely by the fluidic passage associated with the second target surface.

8. The method of any preceding claim, comprising creating apertures through a selected subset of the target portions by using control electrodes to cause potential differences to be applied across the target portions in the selected subset that are larger than potential differences applied across target portions that are not in the selected subset, thereby selectively initiating dielectric breakdown in the selected subset of the target portions.

9. The method claim 8, wherein the creating of the apertures through the selected subset of the target portions comprises: applying voltages to control electrodes corresponding to the selected subset of target portions such that potential differences between the control electrodes and the first common electrode are larger than a potential difference between the first and second common electrodes; or applying voltages to control electrodes other than control electrodes corresponding to the selected subset of target portions such that potential differences between the control electrodes and the first common electrode are smaller than a potential difference between the first and second common electrodes.

10. The method of any preceding claim, wherein the target portions are configured to have a range of different thicknesses and the method comprises using the control electrodes to apply a corresponding range of different potential differences across the target portions to promote creation of apertures.

11. The method of any preceding claim, comprising: operating each control electrode in an aperture creation mode and an aperture monitoring mode, wherein: the aperture creation mode comprises applying a voltage to the control electrode that promotes aperture creation through the target portion corresponding to the control electrode; and the aperture monitoring mode comprises using the control electrode to monitor a state of the aperture by measuring an electrical characteristic associated with the aperture.

12. The method of claim 11, wherein the electrical characteristic is, or is dependent on, an electrical resistance through the aperture, optionally the electrical characteristic being a voltage of the liquid adjacent to the control electrode.

13. The method of claim 11 or 12, wherein the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode.

14. The method of claim 13, comprising operating one of the control electrodes in the aperture creation mode and the aperture detection mode in an alternating sequence in each of one or more of the target portions until creation of an aperture is detected in the target portion.

15. The method of claim 14, wherein a maximum potential difference applied to the target portion during each operation in the aperture creation mode in the alternating sequence is progressively increased during the alternating sequence.

16. The method of any of claims 11 to 13, wherein the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform.

17. The method of claim 16, wherein the aperture promoting waveform is configured such that a total amount of charge flowing during the first portion of the aperture promoting waveform is substantially the same as the total amount of charge flowing during the second portion of the aperture promoting waveform.

18. The method of claim 17, wherein an average magnitude of a difference between the voltage of the aperture promoting waveform and a voltage of the second common electrode is substantially the same in the first and second portions of the aperture promoting waveform.

19. The method of any of claims 16 to 18, wherein: the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode; the method comprises operating each of one or more of the control electrodes in the aperture creation mode and the aperture detection mode in an alternating sequence in a respective target portion until creation of an aperture is detected in the target portion; and each operation in the aperture creation mode comprises applying one or more instances of the aperture promoting waveform.

20. The method of claim 19, wherein a maximum potential difference applied to the target portion during each operation in the aperture creation mode in the alternating sequence is progressively increased during the alternating sequence.

21. The method of any of claims 11 to 13, wherein the aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture through the target portion corresponding to the control electrode.

22. The method of claim 21, comprising controlling a voltage of the control electrode to unblock the aperture in response to detection of blocking by the blocking detection mode.

23. The method of claim 22, wherein the controlling of the voltage of the control electrode to unblock the aperture comprises applying an unblocking voltage waveform, the unblocking voltage waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

24. The method of claim 23, wherein the unblocking voltage waveform is configured such that a total amount of charge flowing during the first portion of the unblocking voltage waveform is substantially the same as the total amount of charge flowing during the second portion of the unblocking voltage waveform.

25. The method of claim 24, wherein an average magnitude of a difference between the voltage of the unblocking voltage waveform and a voltage of the second common electrode is different in the first portion of the unblocking voltage waveform than in the second portion of the unblocking voltage waveform.

26. The method of any of claims 23 to 25, wherein: the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform; the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode; the method comprises generating a combined driving signal, the combined driving signal comprising an alternating sequence of the aperture promoting waveform and the unblocking voltage waveform; and the method comprises alternately switching between applying the aperture promoting waveform from the combined driving signal and applying the aperture detection mode, thereby applying the aperture creation mode and the aperture detection mode in an alternating sequence, until creation of an aperture is detected in the target portion.

27. The method of claim 26, wherein: the aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture through the target portion corresponding to the control electrode; and the method comprises applying the unblocking waveform from the combined driving signal to unblock the aperture in response to detection of blocking by the blocking detection mode.

28. The method of any preceding claim, wherein voltages applied to the first and second common electrodes and the control electrodes during the creation of apertures through the plural target portions are configured such that differences between the voltages applied by the control electrodes and the second common electrode do not exceed 30% of a voltage difference between the first and second common electrodes.

29. The method of any preceding claim, wherein the control electrodes are used after creation of apertures in respective target portions to control growth of the apertures in the target portions.

30. The method of claim 29, wherein for each target portion the control of growth comprises using the control electrode to apply a different potential difference across the target portion than would result solely from voltages of the first and second common electrodes.

31. The method of claim 29 or 30, wherein the control of growth comprises monitoring an electrical characteristic dependent on a size of an aperture being grown and using the control electrode to stop growth of the aperture in response to the monitoring indicating that a target size has been attained.

32. A method of unblocking apertures in a membrane of a measurement system configured to sense a molecular entity in each of the apertures by performing a measurement that is dependent on an interaction between the molecular entity and the aperture, the method comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and unblocking apertures through plural target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of a plurality of control electrodes and the first common electrode, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode, wherein the controlling of the voltages of the control electrodes comprises applying an unblocking voltage waveform to each control electrode, the unblocking voltage waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

33. The method of claim 32, wherein the unblocking voltage waveform is configured such that a total amount of charge flowing during the first portion of the unblocking voltage waveform is substantially the same as the total amount of charge flowing during the second portion of the unblocking voltage waveform.

34. The method of claim 33, wherein an average magnitude of a difference between the voltage of the unblocking voltage waveform and a voltage of the second common electrode is higher in the first portion of the unblocking voltage waveform than in the second portion of the unblocking voltage waveform.

35. The method of any of claims 32 to 34, wherein each target portion defines a respective first target surface on the one side of the membrane and a respective second target surface on the other side of the membrane, the first target surface being in contact with the first body of liquid, the second target surface being in contact with the second body of liquid.

36. The method of claim 35, wherein the second body of liquid is contained in a bath defining a bulk region and a plurality of fluidic passages.

37. The method of claim 36, wherein the second common electrode contacts the second body of liquid in the bulk region.

38. The method of claim 37, wherein each fluidic passage extends from a respective one of the second target surfaces to the bulk region and opens out into the bulk region at a distal end of the fluidic passage.

39. The method of claim 38, wherein each control electrode contacts the second body of liquid at a position electrically in series between a respective one of the second target surfaces and the distal end of the fluidic passage corresponding to that second target surface .

40. The method of any of claims 36 to 39, wherein the fluidic passages are associated with different respective second target surfaces and are fluidically isolated from each other between the second target surfaces and the bulk region, such that each second target surface is fluidically connected to the bulk region solely by the fluidic passage associated with the second target surface.

41. The method of any of claims 32 to 40, comprising simultaneously using: a first subset of the control electrodes to apply the unblocking voltage waveform to a corresponding first subset of the apertures in the membrane; and a second subset of the control electrodes to sense molecular entities in a corresponding second subset of the apertures in the membrane by performing measurements with the control electrodes that are dependent on interactions between the molecular entities and the apertures, the sensing of the molecular entities being optionally performed by the second subset of control electrodes without applying the unblocking voltage waveform via the second subset of control electrodes.

42. A method of sensing molecular entities in apertures in a membrane, the method comprising: applying a potential difference between a first common electrode and a second common electrode, wherein the first common electrode contacts a first body of liquid on one side of the membrane and the second common electrode contacts a second body of liquid on the other side of the membrane; and using a plurality of control electrodes to sense molecular entities in the apertures by performing measurements with the control electrodes that are dependent on interactions between the molecular entities and the apertures, each control electrode contacting the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode, wherein the method further comprises preconditioning respective portions of the second body of liquid adjacent to the control electrodes prior to the measurements with the control electrodes, the preconditioning being performing by driving current across interfaces between the control electrodes and the portions of the second body liquid to adjust compositions of the portions of the second body of liquid.

43. The method of claim 42, wherein the adjustment of the compositions of the portions of the second body of liquid by the preconditioning is such as to reduce or prevent erroneous drift of the subsequent measurements.

44. The method of any preceding claim, wherein: the membrane comprises one or more of the following in any combination: silicon nitride; silicon oxide; a two-dimensional material, optionally graphene, an MXene, M0S2, and/or h-BN; and a material formable using atomic layer deposition, optionally HfO_x, ZrO_x, and/or A1O_X; the membrane has a thickness in the range of from about 0.3nm to about 50nm; and/or the apertures in the membrane have a diameter less than about lOOnm.

45. An apparatus comprising a membrane in which a plurality of apertures have been created using the method of any of claims 1-31.

46. The apparatus of claim 45, further comprising a measurement system configured to sense a molecular entity in each of the apertures by performing a measurement that is dependent on an interaction between the molecular entity and the aperture.

47. An apparatus for forming apertures in a membrane using dielectric breakdown, comprising: a membrane having a plurality of target portions; a first common electrode and a second common electrode, the first common electrode being configured to contact a first body of liquid on one side of the membrane and the second common electrode being configured to contact a second body of liquid on the other side of the membrane; a plurality of control electrodes, each control electrode configured to contact the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode; and a controller configured to: apply a potential difference between the first common electrode and the second common electrode; and create and/or control growth of apertures through the plurality of target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of the plurality of control electrodes and the first common electrode.

48. The apparatus of claim 47, wherein each target portion defines a respective first target surface on the one side of the membrane and a respective second target surface on the other side of the membrane, the first target surface configured to be in contact with the first body of liquid, the second target surface configured to be in contact with the second body of liquid.

49. The apparatus of claim 48, comprising a bath configured to contain the second body of liquid, the bath defining a bulk region and a plurality of fluidic passages.

50. The apparatus of claim 49, wherein the second common electrode is configured to contact the second body of liquid in the bulk region.

51. The apparatus of claim 50, wherein each fluidic passage extends from a respective one of the second target surfaces to the bulk region and opens out into the bulk region at a distal end of the fluidic passage.

52. The apparatus of claim 51, wherein each control electrode is configured to contact the second body of liquid at a position electrically in series between a respective one of the second target surfaces and the distal end of the fluidic passage corresponding to that second target surface.

53. The apparatus of any of claims 49 to 52, wherein the fluidic passages are associated with different respective second target surfaces and are fluidically isolated from each other between the second target surfaces and the bulk region, such that each second target surface is fluidically connected to the bulk region solely by the fluidic passage associated with the second target surface.

54. The apparatus of any of claims 47 to 53, wherein the controller is configured to create apertures through a selected subset of the target portions by using control electrodes to cause potential differences to be applied across the target portions in the selected subset that are larger than potential differences applied across target portions that are not in the selected subset, thereby selectively initiating dielectric breakdown in the selected subset of the target portions.

55. The apparatus of claim 54, wherein the creating of the apertures through the selected subset of the target portions comprises: applying voltages to control electrodes corresponding to the selected subset of target portions such that potential differences between the control electrodes and the first common electrode are larger than a potential difference between the first and second common electrodes; or applying voltages to control electrodes other than control electrodes corresponding to the selected subset of target portions such that potential differences between the control electrodes and the first common electrode are smaller than a potential difference between the first and second common electrodes.

56. The apparatus of any of claims 47 to 55, wherein the target portions are configured to have a range of different thicknesses and the controller is configured to use the control electrodes to apply a corresponding range of different potential differences across the target portions to promote creation of apertures.

57. The apparatus of any of claims 47 to 56, wherein the controller is configured to: operate each control electrode in an aperture creation mode and an aperture monitoring mode, wherein: the aperture creation mode comprises applying a voltage to the control electrode that promotes aperture creation through the target portion corresponding to the control electrode; and the aperture monitoring mode comprises using the control electrode to monitor a state of the aperture by measuring an electrical characteristic associated with the aperture.

58. The apparatus of claim 57, wherein the electrical characteristic is, or is dependent on, an electrical resistance through the aperture, optionally the electrical characteristic being a voltage of the liquid adjacent to the control electrode.

59. The apparatus of claim 57 or 58, wherein the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode.

60. The apparatus of claim 59, wherein the controller is configured to operate one of the control electrodes in the aperture creation mode and the aperture detection mode in an alternating sequence in each of one or more of the target portions until creation of an aperture is detected in the target portion.

61. The apparatus of claim 60, wherein the controller is configured such that a maximum potential difference applied to the target portion during each operation in the aperture creation mode in the alternating sequence is progressively increased during the alternating sequence.

62. The apparatus of any of claims 57 to 59, wherein the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform.

63. The apparatus of claim 62, wherein the aperture promoting waveform is configured such that a total amount of charge flowing during the first portion of the aperture promoting waveform is substantially the same as the total amount of charge flowing during the second portion of the aperture promoting waveform.

64. The apparatus of claim 63, wherein an average magnitude of a difference between the voltage of the aperture promoting waveform and a voltage of the second common electrode is substantially the same in the first and second portions of the aperture promoting waveform.

65. The apparatus of any of claims 62 to 64, wherein: the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode; the controller is configured to operate each of one or more of the control electrodes in the aperture creation mode and the aperture detection mode in an alternating sequence in a respective target portion until creation of an aperture is detected in the target portion; and each operation in the aperture creation mode comprises applying one or more instances of the aperture promoting waveform.

66. The apparatus of claim 65, wherein the controller is configured such that a maximum potential difference applied to the target portion during each operation in the aperture creation mode in the alternating sequence is progressively increased during the alternating sequence.

67. The apparatus of any of claims 57 to 59, wherein the aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture through the target portion corresponding to the control electrode.

68. The apparatus of claim 67, wherein the controller is configured to control a voltage of the control electrode to unblock the aperture in response to detection of blocking by the blocking detection mode.

69. The apparatus of claim 68, wherein the controlling of the voltage of the control electrode to unblock the aperture comprises applying an unblocking voltage waveform, the unblocking voltage waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

70. The apparatus of claim 69, wherein the unblocking voltage waveform is configured such that a total amount of charge flowing during the first portion of the unblocking voltage waveform is substantially the same as the total amount of charge flowing during the second portion of the unblocking voltage waveform.

71. The apparatus of claim 70, wherein an average magnitude of a difference between the voltage of the unblocking voltage waveform and a voltage of the second common electrode is different in the first portion of the unblocking voltage waveform than in the second portion of the unblocking voltage waveform.

72. The apparatus of any of claims 69 to 71, wherein: the aperture creation mode comprises applying an aperture promoting waveform, the aperture promoting waveform configured such that current flows from the control electrode into the liquid adjacent to the control electrode during a first portion of the aperture promoting waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the aperture promoting waveform; the aperture monitoring mode comprises an aperture detection mode configured to detect whether an aperture has been created through the target portion corresponding to the control electrode; the controller is configured to generate a combined driving signal, the combined driving signal comprising an alternating sequence of the aperture promoting waveform and the unblocking voltage waveform; and the controller is configured to alternately switch between applying the aperture promoting waveform from the combined driving signal and applying the aperture detection mode, thereby applying the aperture creation mode and the aperture detection mode in an alternating sequence, until creation of an aperture is detected in the target portion.

73. The apparatus of claim 72, wherein: the aperture monitoring mode comprises a blocking detection mode configured to detect blocking of a previously created aperture through the target portion corresponding to the control electrode; and the controller is configured to apply the unblocking waveform from the combined driving signal to unblock the aperture in response to detection of blocking by the blocking detection mode.

74. The apparatus of any of claims 47 to 73, wherein voltages applied to the first and second common electrodes and the control electrodes during the creation of apertures through the plural target portions are configured such that differences between the voltages applied by the control electrodes and the second common electrode do not exceed 30% of a voltage difference between the first and second common electrodes.

75. The apparatus of any of claims 47 to 74, wherein the controller is configured to use control electrodes after creation of apertures in respective target portions to control growth of the apertures in the target portions.

76. The apparatus of claim 75, wherein for each target portion the control of growth comprises using the control electrode to apply a different potential difference across the target portion than would result solely from voltages of the first and second common electrodes.

77. The apparatus of claim 75 or 76, wherein the control of growth comprises monitoring an electrical characteristic dependent on a size of an aperture being grown and using the control electrode to stop growth of the aperture in response to the monitoring indicating that a target size has been attained.

78. A measurement system configured to sense a molecular entity, comprising: a membrane having a plurality of target portions and respective apertures through the target portions; a first common electrode and a second common electrode, the first common electrode being configured to contact a first body of liquid on one side of the membrane and the second common electrode being configured to contact a second body of liquid on the other side of the membrane; a plurality of control electrodes, each control electrode configured to contact the second body of liquid at a position electrically in series between a respective one of the target portions and the second common electrode; and a controller configured to: apply a potential difference between the first common electrode and the second common electrode; use the control electrodes to sense molecular entities in the apertures by performing measurements that are dependent on interactions between molecular entities and the apertures; and unblock apertures through plural target portions of the membrane by controlling the potential difference across each target portion by controlling a respective control electrode of the plurality of control electrodes and the first common electrode, the controller being configured to control the voltages of the control electrodes by applying an unblocking voltage waveform to each control electrode, the unblocking voltage waveform configured such that current flows from the control electrode into liquid adjacent to the control electrode during a first portion of the unblocking voltage waveform and from the liquid adjacent to the control electrode back into the control electrode during a second portion of the unblocking voltage waveform.

79. The system of claim 78, wherein the unblocking voltage waveform is configured such that a total amount of charge flowing during the first portion of the unblocking voltage waveform is substantially the same as the total amount of charge flowing during the second portion of the unblocking voltage waveform.

80. The system of claim 79, wherein an average magnitude of a difference between the voltage of the unblocking voltage waveform and a voltage of the second common electrode is higher in the first portion of the unblocking voltage waveform than in the second portion of the unblocking voltage waveform.

81. The system of any of claims 78 to 80, wherein each target portion defines a respective first target surface on the one side of the membrane and a respective second target surface on the other side of the membrane, the first target surface configured to be in contact with the first body of liquid, the second target surface configured to be in contact with the second body of liquid.

82. The system of claim 81 , comprising a bath configured to contain the second body of liquid, the bath defining a bulk region and a plurality of fluidic passages.

83. The system of claim 82, wherein the second common electrode is configured to contact the second body of liquid in the bulk region.

84. The system of claim 83, wherein each fluidic passage extends from a respective one of the second target surfaces to the bulk region and opens out into the bulk region at a distal end of the fluidic passage.

85. The system of claim 84, wherein each control electrode is configured to contact the second body of liquid at a position electrically in series between a respective one of the second target surfaces and the distal end of the fluidic passage corresponding to that second target surface.

86. The system of any of claims 82 to 85, wherein the fluidic passages are associated with different respective second target surfaces and are fluidically isolated from each other between the second target surfaces and the bulk region, such that each second target surface is fluidically connected to the bulk region solely by the fluidic passage associated with the second target surface.

87. The system of any of claims 78 to 86, wherein the controller is configured to simultaneously use: a first subset of the control electrodes to apply the unblocking voltage waveform to a corresponding first subset of the apertures in the membrane; and a second subset of the control electrodes to sense molecular entities in a corresponding second subset of the apertures in the membrane by performing measurements with the control electrodes that are dependent on interactions between the molecular entities and the apertures, the sensing of the molecular entities being optionally performed by the second subset of control electrodes without applying the unblocking voltage waveform via the second subset of control electrodes.

88. The apparatus of any of claims 45 to 77 or the system of any of claims 78 to 87, wherein: the membrane comprises one or more of the following in any combination: silicon nitride; silicon oxide; a two-dimensional material, optionally graphene, an MXene, M0S2, and/or h-BN; and a material formable using atomic layer deposition, optionally HfO_x, ZrO_x, and/or A1O_X; the membrane has a thickness in the range of from about 0.3nm to about 50nm; and/or the apertures in the membrane have a diameter less than about lOOnm.