METHOD OF FABRICATING ARRAYS OF SOLID-STATE NANOPORES

FIELD OF THE INVENTION

[0001] The invention relates to a method of fabricating a nanopore in a membrane of a solid-state device. The invention also relates to a solid-state nanopore device, an apparatus comprising the solid-state nanopore devices and their uses. A further aspect of the invention relates to a kit for fabricating a nanopore in a membrane of a solid-state device.

BACKGROUND

[0002] Solid-state nanopore devices (also known as solid-state nanopores) have emerged as highly promising biosensors that are capable of analyte detection at the single-molecule level. These devices each consist of a nanometre sized hole (also known as a pore or nanopore) formed in a thin impermeable membrane that separates two chambers of electrolyte solution. When an electric field is applied across the solid-state nanopore, ions flow through the pore resulting in a measurable ionic current.

[0003] Biosensing is typically achieved by detecting changes in the ionic current as a biomolecule translocates through the pore. When a biomolecule is drawn into and through the pore (typically via electrophoretic forces), it affects the passage of ions resulting in a change in the ionic current. The duration and amplitude of the change in the ionic current can be used to infer properties of the molecule, such as the size, charge, and interactions with the pore surface. Matching the size of the nanopore to be close to, but slightly larger than, the size of the analyte generally maximises the measured signal. It is therefore desirable to be able to control the size of the nanopore during fabrication.

[0004] Despite the success of ionic current based nanopore sensing, this technique does suffer from several limitations. This includes limited operating bandwidth and low device densities due to the need to fluidically isolate each nanopore for its signal to be read out independently. To overcome these issues, there is interest in developing alternate readout mechanisms to ionic current based sensing. These techniques often require integrating the nanopore with additional nanostructures such as field-effect sensors, tunnelling nanogaps, and plasmonic nanostructures.

[0005] Interest in nanopore sensors has often focused on DNA sequencing applications. However, there are many other applications for nanopore sensors. These devices are, for example, being developed for protein fingerprinting, polymer data storage, biomarker detection, enzymology, ultra-sensitive ion sensors, and nanoscale chemical reactors. For such a wide range of applications to be achieved, it is important to be able to fabricate nanopores of variable diameters that operate in a wide range of environmental conditions.

[0006] A major challenge in developing solid-state nanopore sensors is the ability to fabricate these devices in a scalable and accurate fashion. Unfortunately, the fabrication of such pores has often relied on expensive, time consuming processes. For example, nanopores have been fabricated by using a focussed beam of high energy particles, which has been produced by either a dedicated ion beam machine or a transmission electron microscope. Consequently, the availability of these devices has been limited to the research community and has restricted their commercial viability. This is particularly true for advanced nanopore devices with additional sensing modalities where the nanopore is integrated with additional nanostructures.

SUMMARY OF THE INVENTION

[0007] The invention provides a method of fabricating a nanopore in a membrane of a solid-state device having an electrode. The method comprises: (a) applying an electric field between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore.

[0008] The method of the invention provides several advantages over existing methods for fabricating nanopores. It does not require the use of specialised equipment, such as an ion beam machine or a transmission electron microscope, or trained operators for using such equipment. The method is low cost, easy and quick. It can also be automated for the industrial production of solid-state nanopore devices.

[0009] Solid-state devices having on-chip electrodes are currently manufactured on an industrial scale using, for example, existing semiconductor fabrication processes. The method of the invention can utilise the existing on-chip electrode of such a device to fabricate a nanopore that is self-aligned with the electrode. The location of the nanopore formation can therefore be controlled, unlike in many other existing methods where the location of nanopore formation is random. The method of the invention also permits control of the diameter of the nanopore that is fabricated, so that the nanopore size can be precisely tuned for a particular sensing application.

[0010] The method of fabricating a nanopore in a membrane of a solid-state device having an electrode is also a method for manufacturing a solid-state nanopore device.

[0011] The invention also provides a solid-state nanopore device. [0012] Typically, the solid-state nanopore device comprises: (i) a membrane comprising a dielectric material; and (ii) an electrode on a surface of the membrane, which has a nanopore.

[0013] The solid-state nanopore device may be obtained or is obtainable from the method of the invention.

[0014] The method of the invention may permit the manufacture of solid-state nanopore devices having arrangements or geometries that could not be prepared on an industrial scale before. Furthermore, it may not have been possible to manufacture some of these devices because of their complex architecture.

[0015] A further aspect of the invention is an apparatus. The apparatus comprises a plurality of solid-state nanopore devices of the invention.

[0016] The invention further provides a kit for fabricating a nanopore in a membrane of a solid-state device. The kit comprises (i) a membrane comprising a dielectric material, and an electrode on a surface of the membrane. The electrode is for use in fabricating the nanopore. The kit may further comprise (ii) instructions for performing the method of fabricating the nanopore of the invention.

[0017] The invention also relates to uses of the solid-state nanopore device.

[0018] An aspect of the invention relates to the use of the solid-state nanopore device or the apparatus of the invention as a biosensor for detecting an analyte.

[0019] A further aspect of the invention is the use of the solid-state nanopore device or the apparatus of the invention for sequencing DNA or RNA.

[0020] Another aspect of the invention relates to a method for measuring uniformity and/or consistency of a membrane. The method comprises (i) providing a membrane comprising a dielectric material, wherein a plurality of electrodes are disposed on a surface of the membrane, (ii) applying an electric field between an electrode on the surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane, (iii) measuring a response of the membrane to the electric field, (iv) repeating steps (ii) and (iii) with a different electrode to the electrode(s) already used to apply an electric field and using the responses to measure the uniformity and/or consistency of a membrane.

BRIEF DESCRIPTION OF THE FIGURES

[0021] The invention is described below with reference to the accompanying figures.

[0022] Figure 1 is a schematic diagram of the device geometry without an electrode.

[0023] Figure 2 is a schematic diagram of the device (with electrodes) in accordance with the invention. [0024] Figure 3 is a false-colour scanning electron micrograph in greyscale of the electrode configuration over the suspended region of SiN _x (the suspended SiN _x is the black rectangle). The electrode labels A1 to A4 and B5 to B8 were added to the micrograph and are not part of the device geometry.

[0025] Figure 4 is a schematic diagram showing the measurement setup for performing controlled breakdown (CBD) experiments by applying a voltage between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane.

[0026] Figure 5 is a plot showing the voltage protocol and the resulting measured current (top line) when performing CBD by applying a voltage (line marked with asterisks) between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane.

[0027] Figure 6 shows plots of CBD measurements performed by applying a voltage (line marked with asterisks) between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane. The four measurements were performed on the same membrane by applying a voltage to a different on-chip electrode. The on-chip electrode that the voltage was applied to is shown in the bottom right hand corner of each plot.

[0028] Figure 7 shows fluorescence micrographs of nanopores created when a voltage is applied between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane. The white dashed box depicts the edge of the suspended region of SiN _x . The positions of the electrodes are shown (asterisks for the electrodes upon which CBD was performed and white lines for electrodes where CBD was not performed). The three images represent a time series of data, with an image before, during, and after the application of a voltage that drive Ca ²⁺ ions through the nanopore.

[0029] Figure 8 shows scanning transmission electron microscope images of a nanopore formed using the CBD method. The top row shows bright field images and the bottom row shows high-angle annual dark field images. Images of the nanopore are shown at various magnifications.

[0030] Figure 9 is a series of plots showing CBD measurements (top row) and the resulting conductance (bottom row) of the created nanopore for six different devices.

[0031] Figure 10 shows two CBD plots. On the left is a typical CDB trace. On the right is a zoom in of the CBD trace after breakdown. The grey shaded area represents the area that was integrated to quantify the charge passed after breakdown.

[0032] Figure 11 is a plot of the charge passed after breakdown and the resulting nanopore diameter for the six membranes that were studied. [0033] Figure 12 shows schematics of two different configurations. A forward-biased configuration is shown in (a), whereas (b) shows a reverse-biased configuration.

[0034] Figure 13 shows four images relating to the device used in the experiment, (a) shows a schematic of the device geometry. The images in (b) are false-colour scanning electron micrographs in greyscale of (i) the electrode configuration over the suspended region of SiN _x and (ii) the narrowest part of the metal nanoconstriction, (c) shows a schematic of the experimental setup that was used.

[0035] Figure 14 shows graphs illustrating the temperature dependence of the breakdown voltage when the electric field is applied between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane. This is shown for devices in the (a) reverse-biased configuration and (b) forward-biased configuration.

[0036] Figure 15 shows the measurement traces when performing CBD simultaneous to passing a large current density though an on-chip metal nanoconstriction. The voltage is the line marked with asterisks, (a) shows the transmembrane current (i.e. measured between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane) during CBD. (b) shows the transverse current (i.e. measured across the on-chip nanoconstriction) during CBD. In (b), the line for the voltage lies on the line for the current.

[0037] Figure 16 shows a series of fluorescence micrographs of nanopores created when performing CBD simultaneous to passing a large current density through an on-chip metal nanoconstriction. Images are shown for two separate chips. The white dashed box represents the edge of the suspended region of SiN _x , while the solid white lines depict the electrode edges. For each chip, three micrographs are shown representing a time series of data with a frame before, during and after the application of a voltage that Ca ²⁺ ions through the nanopore.

DETAILED DESCRIPTION

[0038] The invention concerns the fabrication of a nanopore in a membrane of a solid- state device and a solid-state nanopore device.

[0039] The term “nanopore” as used herein refers to an opening or a pore in a surface of a solid-state device to a channel through the solid-state device. The opening or pore is typically in a surface of a membrane of the solid-state device and the channel is through the membrane. The opening or pore typically has a diameter of from 0.5 nm to 20.0 nm or as further described herein. The method of the invention is for fabricating a channel through the membrane of the solid-state device, with an opening or a pore to the channel on a surface of the membrane. [0040] The term “diameter” as used herein in the context of a nanopore refers to either (a) the diameter of a nanopore having a circular shape or (b) for a nanopore having a noncircular shape, the diameter of an equivalent circular nanopore that has the same area as the area of the non-circular nanopore. The diameter of the nanopore can be determined based on the ionic current measured through the nanopore when an electrolyte solution is present on both sides of the membrane and a voltage is applied across the membrane as described in Example 3. Alternatively, the diameter can be determined by directly imaging the nanopore using a transmission electron microscope or other methods of microscopy.

[0041] When a nanopore is fabricated in accordance with a method of the invention, then the nanopore is in an electrode on a surface of the membrane. The method of the invention involves the fabrication of a nanopore using the electrode to create a channel through both the electrode and the membrane. The nanopore is formed at the electrode (e.g. the nanopore is self-aligned with the electrode).

[0042] The term “solid-state device” as used herein refers to any device having a membrane comprising a dielectric material, and an electrode on a surface of the membrane. The electrode is for use in fabricating the nanopore. The solid-state device is the precursor device upon which the method of the invention for fabricating a nanopore is performed. The solid-state device may or may not comprise a nanopore. When the solid- state device comprises a nanopore, then the nanopore may have previously been fabricated by a method of the invention or some other method, preferably a method of the invention. In general, however, it is preferred that the solid-state device does not comprise a nanopore.

[0043] The term “solid-state nanopore device” as used herein refers to a solid-state device, where the electrode on the surface of the membrane has a nanopore, preferably a nanopore fabricated in accordance with a method of the invention.

[0044] The method of the invention comprises (a) applying an electric field between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore.

[0045] For the avoidance of doubt, the electrode is in direct contact with the surface of the membrane. The electrode and the electrolyte solution are on opposite sides of the membrane.

[0046] For ease of reference, the side of the membrane where there is an electrode on the surface of the membrane shall be referred to herein as the “upper surface” of the membrane. The opposing surface of the membrane in contact with an electrolyte solution shall be referred to herein as the “lower surface”. The use of the terms “upper” and “lower” are used as labels that relate to a certain orientation of the solid-state device or the solid-state nanopore device. The use of these terms should not be construed as limiting with respect to the orientation of the membrane or the way in which the method is performed.

[0047] The electrode on the surface of the membrane is an electrode for fabricating a nanopore. Thus, in step (a) of the method of the invention, an electric field is applied between the electrode for fabricating a nanopore on a surface of the membrane and the electrolyte solution.

[0048] Typically, a single nanopore is fabricated in or at the electrode. In the method of the invention, the step of (a) applying an electric field between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane is to fabricate a single nanopore.

[0049] It may be preferable that the electric field in the step of (a) applying an electric field between an electrode for fabricating a nanopore on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane is only applied to the electrode for fabricating the nanopore. The electric field is not applied to any other electrode(s) of the solid-state device.

[0050] Generally, when performing the method of the invention, an electrolyte solution, particularly the electrolyte solution in contact with the lower surface of the membrane, is not in contact with the upper surface of the membrane. Thus, the electrode for fabricating the nanopore is not in contact with an electrolyte solution, particularly the electrolyte solution in contact with the lower surface of the membrane.

[0051] The membrane typically separates (e.g. physically separates) the electrode for fabricating the nanopore from the electrolyte solution.

[0052] In step (a), the electric field is applied between the electrode for fabricating the nanopore and the electrolyte solution to breakdown the dielectric material therebetween, such as to fabricate a nanopore, preferably a single nanopore. In addition to the breakdown of the dielectric material, part of the electrode may breakdown.

[0053] The nanopore is fabricated or formed in the electrode. In other words, the nanopore may pass through the electrode.

[0054] Advantageously, the nanopore that is fabricated by the method is self-aligned with the electrode. The location of the nanopore formation can therefore be controlled and can be incorporated into devices having complex arrangements. [0055] The breakdown is preferably a controlled breakdown (CBD). By controlling the electric field that is applied between the electrode and the electrolyte solution, the breakdown can be controlled.

[0056] The breakdown or the controlled breakdown is preferably by Joule heating. When the electric field is applied between the electrode for fabricating the nanopore and the electrolyte solution, localised Joule heating occurs at the electrode. The localised Joule heating is used to perform a controlled breakdown of the membrane.

[0057] In principle, the electric field could be applied to the electrode and the electrolyte solution in any manner, provided it can be controlled and, preferably, if there is a suitable feedback mechanism for detecting the fabrication of a nanopore. The magnitude and the way in which the electric field that is applied to the electrode and the electrolyte to cause breakdown of the membrane will depend on factors, such as the nature of the membrane, the electrode and the electrolyte solution. This information can be determined by performing routine measurements.

[0058] The electric field may be an alternating electric field (e.g. alternative voltage or alternative current) or a direct electric field (e.g. direct voltage or direct current). It is preferred that the electric field is a direct electric field.

[0059] Typically, the electric field may be an electric potential (e.g. voltage) or an electric current. Thus, the method of the invention may comprise (a) applying a voltage and/or a current between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore. It is preferred that the electric field is an electric potential (e.g. voltage).

[0060] The electric field may, in general, be applied to the electrode and the electrolyte solution as a constant voltage, a constant current, a voltage ramp or a voltage pulse.

[0061] An arbitrary waveform may be applied under either voltage or current control to further optimise the fabrication of the nanopore.

[0062] When the electric field is applied as a constant voltage, then the same voltage is applied (e.g. continuously applied) to the electrode and the electrolyte, such as until fabrication of a nanopore is detected. Similarly, when the electric field is applied as a constant current, then the same current is applied (e.g. continuously applied) to the electrode and the electrolyte, such as until fabrication of a nanopore is detected.

[0063] When the electric field is applied as a voltage ramp, then the voltage may increase or decrease, preferably increase, from a starting voltage to a target voltage. The increase or decrease in the voltage from the starting voltage to a target voltage may be linear or non-linear (e.g. stepwise), preferably linear. [0064] When the electric field is applied as a voltage ramp, then the voltage may increase or decrease, preferably increase, from a starting voltage to a target voltage. The increase or decrease in the voltage from the starting voltage to a target voltage may be linear. When the increase or decrease in the voltage is linear, then the voltage ramp is referred to herein as a “linear voltage ramp”.

[0065] When the electric field is applied as a voltage pulse, then the voltage may be applied as a series of pulses. For each pulse, the voltage is switched from a first state to a second state for a first time period and then the voltage is then switched from the second state to the first state for a second time period. The pulse is then repeated. The voltages of the first state and the second state are different.

[0066] The pulses are typically applied until breakdown and/or fabrication of the nanopore is/are detected.

[0067] It is preferred that the electric field is applied as a voltage ramp. Thus, the method comprises (a) applying a voltage ramp field between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore. When applying the voltage ramp, then preferably there is a linear increase or a linear decrease from a starting voltage to a target voltage.

[0068] When a voltage is applied between the electrode and the electrolyte solution, particularly when a voltage ramp is applied, then a negative voltage may be applied (e.g. as the starting voltage) to the electrode and a positive voltage is applied to the electrolyte solution or the electrolyte solution is held at ground voltage. This configuration is referred to herein as the forward-biased configuration. An advantage of the forward-biased configuration is that breakdown has been found to occur at relatively low voltages.

[0069] Alternatively, when a voltage is applied between the electrode and the electrolyte solution, particularly when a voltage ramp is applied, then a positive voltage may be applied (e.g. as the starting voltage) to the electrode and a negative voltage is applied to the electrolyte solution or the electrolyte solution is held at ground voltage. This configuration is referred to herein as the reverse-biased configuration. It has been found that the reverse-biased configuration has a larger temperature dependence than the forward-biased configuration, which can be advantageous in certain methods.

[0070] The method of the invention may further comprise (b) measuring a response, preferably an electrical response, of the solid-state device to the electric field, preferably to detect the fabrication of a nanopore. This step is performed when applying the electric field in step (a). By obtaining feedback on the effect of the electric field on the solid-state device it is possible to ensure that the breakdown of the membrane is controlled until a nanopore is fabricated.

[0071] The measuring a response of the solid-state device may be measuring a change in the current or the electric potential (e.g. voltage) of the solid-state device.

[0072] When the electric field applied between the electrode and the electrolyte solution is an electric potential (e.g. voltage), then the current of the solid-state device may be measured. When the electric field applied between the electrode and the electrolyte solution is an electric current, then the electric potential (e.g. voltage) of the solid-state device may be measured.

[0073] When a voltage is applied between the electrode and the electrolyte solution in step (a) of the method, then the method may further comprise (b) measuring a current of the solid-state device in response to the voltage applied between the electrode and the electrolyte solution in contact with an opposing surface of the membrane, preferably to detect the fabrication of a nanopore. The electrode is the electrode on a surface of the membrane and the electrolyte solution is the electrolyte solution in contact with an opposing surface of the membrane.

[0074] The method of the invention may further comprise (c) detecting the fabrication of a nanopore, such as from the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution.

[0075] It is possible to detect the fabrication of a nanopore by measuring the current of the solid-state device as an electric potential (e.g. voltage) is applied between the electrode and the electrolyte solution or by measuring the electric potential of the solid- state device as a current is applied between the electrode and electrolyte solution.

[0076] To detect the fabrication of a nanopore, the response of the solid-state device can be compared to a predetermined threshold, such as a predetermined value for the electric potential (e.g. voltage) or the current of the solid-state device.

[0077] Additionally or alternatively, when a uniform electric field is applied between the electrode and the electrolyte solution, then the fabrication of a nanopore may be detected when there is a sudden change or a spike in the response of the solid-state device. The sudden change or spike may be a sudden change or spike in the electric potential (e.g. voltage) or current of the solid-state device. A uniform electric field is, for example, a constant voltage, a constant current or a linear voltage ramp applied between the electrode and the electrolyte solution.

[0078] The method of the invention may further comprise (c) detecting the fabrication of a nanopore, such as from the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution, and then removing, maintaining or reducing the electric field between the electrode on the surface of the membrane and the electrolyte solution in contact with an opposing surface of the membrane. It is preferred that the electric field is removed or reduced between the electrode and the electrolyte solution.

[0079] The removal or reduction of the electric field between the electrode and the electrolyte solution may occur within a short time period of detecting the fabrication of the nanopore. The short time period may be 2 seconds, preferably 1 second, more preferably 0.5 seconds.

[0080] After detecting the fabrication of a nanopore, the removal or the reduction of the electric field between the electrode and the electrolyte solution can be performed in a variety of ways, depending on the desired outcome.

[0081] The electric field applied between the electrode and the electrolyte solution may be removed. When the electric field is removed, there is no electric field applied between the electrode and the electrolyte solution. The removal of the electric field in this way may prevent further breakdown of the membrane or enlargement of the nanopore that has been fabricated.

[0082] Advantageously, the method of the invention can be used to fabricate nanopores having diameters of a desired size. It is possible to control the diameter of the nanopore that is formed in several ways.

[0083] One way is by controlling the electric field that is initially applied between the electrode and the electrolyte solution to fabricate a nanopore having a certain diameter. The nature of the electric field that should be applied to fabricate a nanopore having a specific diameter can be determined by performing routine experiments.

[0084] Accordingly, the method of the invention may comprise (a) applying an electric field between the electrode on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore having a predetermined diameter. This method may further comprise (c) detecting the fabrication of the nanopore, such as from the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution, and then removing the electric field between the electrode and the electrolyte solution. The electric field is removed to prevent enlargement of the nanopore that has been fabricated.

[0085] Another way of controlling the diameter of the nanopore is to reduce the electric field applied between the electrode and the electrolyte solution in a controlled manner to ensure continued breakdown of the membrane until the diameter of the nanopore reaches a certain size. The way in which the electric field is reduced to obtain a nanopore having a specific diameter can be determined by performing routine experiments.

[0086] Typically, the electric field is controllably reduced.

[0087] The method of the invention may comprise (c) detecting the fabrication of the nanopore, such as from the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution, and then reducing the electric field between the electrode and the electrolyte solution, such as to fabricate a nanopore having a predetermined diameter.

[0088] It has been found that there is an increase in the nanopore diameter with increasing current through the nanopore after the initial breakdown of the membrane to form the nanopore. For example, the rate at which the applied voltage is reduced after breakdown may depend on the current measured from the solid-state device.

[0089] The method of the invention may comprise:

(c) (i) detecting the fabrication of the nanopore, such as from the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution, and

(ii) reducing the electric field between the electrode and the electrolyte solution while measuring the response of the solid-state device (e.g. to reducing the electric field applied between the electrode and the electrolyte solution) to fabricate a nanopore having a predetermined diameter.

[0090] The electric field may, for example, be reduced by a stepwise reducing the electric field or linearly reducing the electric field.

[0091] The reducing the electric field may be by reducing the electric potential (e.g. voltage) or current applied between the electrode and the electrolyte solution.

[0092] For convenience, the response of the solid-state device to the electric field applied between the electrode and the electrolyte solution that is measured to detect the fabrication of a nanopore shall be referred to herein as the “first response”. The response of the solid-state device that is measured when reducing the electric field to fabricate a nanopore having a predetermined diameter shall be referred to herein as the “second response”. The use of the labels “first” and “second” in the context of these “responses” should not be construed as limiting. Thus, the reference to a “second response” does not require the measurement of a “first response”.

[0093] The measuring a second response of the solid-state device may be measuring the current or the electric potential (e.g. voltage) of the solid-state device, such as until the current or the electric potential (e.g. voltage) reaches a predetermined threshold or value. [0094] When the electric field applied between the electrode and the electrolyte solution is an electric potential (e.g. voltage), then the current of the solid-state device may be measured. When the electric field applied between the electrode and the electrolyte solution is an electric current, then the electric potential (e.g. voltage) of the solid-state device may be measured.

[0095] The method of the invention may therefore comprise:

(c) (i) detecting the fabrication of the nanopore, such as from a first response of the solid-state device to the electric field applied between the electrode and the electrolyte solution, and

(ii) reducing the electric field between the electrode and the electrolyte solution while measuring a second response of the solid-state device (e.g. to reducing the electric field applied between the electrode and the electrolyte solution) to fabricate a nanopore having a predetermined diameter.

[0096] To determine if a nanopore having a predetermined diameter has been fabricated, the second response of the solid-state device can be compared to a predetermined threshold, such as a predetermined value for the electric potential (e.g. voltage) or the current of the solid-state device. The predetermined threshold for fabricating a nanopore having a predetermined diameter is different to the predetermined threshold (as defined hereinabove) for fabricating the nanopore. When the electric potential or the current of the solid-state device reaches the predetermined value, then a nanopore having the desired diameter has been fabricated.

[0097] The predetermined threshold is for fabricating a nanopore having a predetermined diameter. It is preferred that the predetermined threshold is a predetermined current of the solid-state device.

[0098] Step (c)(ii) of the method may comprise reducing the electric field between the electrode and the electrolyte solution while measuring the second response of the solid- state device (e.g. to reducing the electric field applied between the electrode and the electrolyte solution) until the second response reaches a predetermined threshold, such as to fabricate a nanopore having a predetermined diameter.

[0099] It is preferred that the electric field is an electric potential (e.g. a voltage), and the method of the invention comprises:

(c) (i) detecting the fabrication of the nanopore from a first current response of the solid-state device to the electric potential applied between the electrode and the electrolyte solution, and (ii) reducing the electric potential between the electrode and the electrolyte solution while measuring a second current response of the solid-state device (e.g. to reducing the electric potential applied between the electrode and the electrolyte solution) until the second current response reaches a predetermined current, such as to fabricate a nanopore having a predetermined diameter.

[0100] The method of the invention may further comprise (c)(iii) removal of the electric field (e.g. applied between the electrode and the electrolyte solution), such as when the nanopore having a predetermined diameter has been fabricated.

[0101] It is preferred that the method further comprises (c)(iii) removal of the electric field when the second response has reached a predetermined threshold. The predetermined threshold is for fabricating a nanopore having a predetermined diameter. More preferably, the method further comprises (c)(iii) removal of the electric potential when the second current response has reached the predetermined current (e.g. for fabricating a nanopore having a predetermined diameter).

[0102] The solid-state device may have a plurality of electrodes. Each electrode of the plurality of electrodes may be for fabricating a nanopore.

[0103] Advantageously, the method of the invention of the invention can be repeated using different electrodes on a surface of the membrane for fabricating a plurality of nanopores, such as an array of nanopores. It has been found that the creation of a nanopore in a first electrode and membrane of the solid-state device does not affect the fabrication of a second nanopore in a second electrode and the membrane.

[0104] The term “electrode” as used herein refers to any electrical conductor disposed on a surface of a membrane of the solid-state device. The electrode may be referred to herein as an on-chip electrode.

[0105] In principle, any electrode may be used in the invention.

[0106] Typically, the electrode comprises a metal (e.g. is metallic), a semimetal material or a semiconductor material.

[0107] The or each electrode may comprise a metal, preferably a metal selected from Ag, Pt, Fe, Ti, Cr, Au and a combination of any two or more thereof.

[0108] The or each electrode may comprise a semimetal material or a semiconductor material, such as a semimetal material or a semiconductor material where the current through the electrode depends on the local electric field. Such materials include graphene, M0S2, silicon, WS2.

[0109] The solid-state device may comprise a plurality of electrodes on a surface of the membrane. The plurality of electrodes is on the same surface of the membrane. Thus, a first electrode of the plurality of electrodes is on the surface of the membrane. A second electrode of the plurality of electrodes is on the surface of the membrane. A third electrode, for example, of the plurality of electrodes may be on the surface of the membrane etc.

[0110] The terms “first”, “second” and “third” etc in the context of the electrodes are used as labels for convenience to differentiate between the electrodes on the surface of the membrane. The use of these terms should not be interpreted as limiting with respect to the order for performing the method (e.g. a nanopore does not have to be fabricated using the first electrode before a nanopore is fabricated with the second electrode) or the total number of electrodes that are present (e.g. reference to a third electrode does not require the presence of a second electrode).

[0111] Typically, there is no fluidic connection between each electrode of the plurality of electrodes on the surface of the membrane. The absence of a fluidic connection between the electrodes ensures that the method for fabricating a nanopore at, say, the first electrode does not affect the fabrication of a nanopore at the second electrode.

[0112] In the method of the invention described above, the electrode on a surface of the membrane is preferably the first electrode.

[0113] The invention therefore relates to a method of fabricating a nanopore in a membrane of a solid-state device having a plurality of electrodes. More preferably, the method is a method of fabrication a plurality of nanopores in a membrane of a solid-state device having a plurality of electrodes. Typically, a single nanopore is fabricated in or at each electrode.

[0114] The method comprises: (a) applying an electric field between a first electrode (e.g. of the plurality of electrodes) on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane to fabricate a nanopore. Step (a) of the method may be as further defined hereinabove.

[0115] The method may further comprise steps (b) and/or (c) as defined above, where the electrode is the first electrode.

[0116] When the solid-state device has a plurality of electrodes, then the method may comprise (d) repeating step (a) for a second electrode (e.g. of the plurality of electrodes) on a surface of the membrane. The electric field that is applied between the second electrode and the electrolyte solution may be the same as or different to the electric field that was applied between the first electrode and the electrolyte solution. It is preferred that the electric field that is applied between the second electrode and the electrolyte solution is the same as the electric field that was applied between the first electrode and the electrolyte solution.

[0117] It is preferred that the method comprises (d) repeating steps (a) to (c) for a second electrode (e.g. of the plurality of electrodes) on a surface of the membrane. Each of steps (b) and (c) may be as defined hereinabove.

[0118] When step (b) is repeated for the second electrode, the response (e.g. the first response) of the solid-state device to the electric field to detect the fabrication of a second nanopore may be the same as or different to the response (e.g. the first response) of the solid-state device to the electric filed to detect the fabrication of the first nanopore. It is preferred that the response of the solid-state device to the electric field to detect the fabrication of a second nanopore is the same as the response of the solid-state device to the electric filed to detect the fabrication of the first nanopore.

[0119] When step (c) is repeated for a second electrode, the second response of the solid-state device to reducing the electric field applied between the first electrode and the electrolyte solution (e.g. to fabricate a first nanopore having a predetermined diameter) may be the same as or different to the second response of the solid-state device to reducing the electric field applied between the second electrode and the electrolyte solution (e.g. to fabricate a second nanopore having a predetermined diameter). The predetermined diameter of the first nanopore may be the same or different to the predetermined diameter of the second nanopore. In other words, the method can be used to fabricate nanopores having diameters of different sizes in the same solid-state device.

[0120] In general, the method of fabricating a plurality of nanopores in a membrane of a solid-state device having a plurality of electrodes comprises:

(a) applying an electric field between a first electrode (e.g. of the plurality of electrodes) on a surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane;

(b) measuring a first response of the solid-state device to the electric field (e.g. to detect the fabrication of a first nanopore), preferably when applying the electric field in step (a);

(c) detecting the fabrication of a first nanopore, such as from a second response of the solid-state device to the electric field applied between the electrode and the electrolyte solution; and

(d) repeating each of steps (a) to (c), preferably sequentially repeating each of steps (a) to (c), for each electrode of the plurality of electrodes on a surface of the membrane, such as to fabricate a nanopore in each electrode. [0121] Step (c) of the method may preferably comprise:

(c) (i) detecting the fabrication of the first nanopore, such as from a first response of the solid-state device to the electric field applied between the first electrode and the electrolyte solution, and

(ii) reducing the electric field between the first electrode and the electrolyte solution while measuring a second response of the solid-state device (e.g. to reducing the electric field applied between the first electrode and the electrolyte solution) to fabricate a first nanopore having a predetermined diameter.

[0122] Step (c) of the method may further comprise (c)(iii) removal of the electric field (e.g. applied between the first electrode and the electrolyte solution), such as when the first nanopore having a predetermined diameter has been fabricated.

[0123] It is preferred that step (c) of the method further comprises (c)(iii) removal of the electric field when the second response has reached a predetermined threshold.

[0124] The method of the invention can be used to fabricate a nanopore in a solid-state device that already comprises a nanopore. The nanopore may have been fabricated by a method of the invention or by a different method.

[0125] The solid-state device may comprise a pre-existing nanopore on a surface of the membrane. The pre-existing nanopore is spatially separated on the surface of the membrane from the or each electrode for fabricating a nanopore on the surface of the membrane.

[0126] It is preferred that there is no fluidic connection between the pre-existing nanopore and the or each electrode on the surface of the membrane.

[0127] The method of the invention can then be performed as described herein using the solid-state device comprising a pre-existing nanopore.

[0128] As a general feature of the invention, the or each electrode of the solid-state device may have a sacrificial part. The sacrificial part is for fabricating a nanopore, preferably a single nanopore. More specifically, the sacrificial part is for controlling the location of nanopore fabrication.

[0129] The sacrificial part of the electrode may have a higher resistance (e.g. electrical resistance) than the resistance of the remainder of the electrode.

[0130] By passing a current through a sacrificial part of the electrode, localised Joule heating of the membrane occurs under the sacrificial part of the electrode due to its higher resistance. The location of nanopore fabrication is therefore determined by the location of the sacrificial part and can therefore be controlled. [0131] Typically, the sacrificial part is a portion of the electrode that has a geometry to provide a higher resistance than the resistance of the remainder of the electrode. The geometry of the portion of the electrode may be an end (e.g. a pointed end) of the electrode or thin part of the electrode compared to the remainder of the electrode. The sacrificial part may be a constriction in the electrode.

[0132] The solid-state nanopore device may comprise the remaining part of the electrode and the nanopore (e.g. formed from the sacrificial part of the electrode). The remaining part of the electrode and the nanopore may form part of an on-chip sensing modality, such as a field-effect sensor, a tunnelling nanogap, a plasmonic nanostructure or a radiofrequency antenna. The remaining part of the electrode, in the context of the solid-state nanopore device, may be referred to herein as the “electrode”.

[0133] The sacrificial electrode may remain as part of the final device geometry and can provide additional functionality to the nanopore sensor. This may include providing additional read-out mechanisms based on field effect sensors, tunnelling nanogaps, or RF electrodes, or may provide a mechanism to control the translocation dynamics by changing the local electric field.

[0134] Two or more electrodes, preferably two electrodes, may be arranged on a surface of the membrane to provide a sacrificial region for fabricating a nanopore. The sacrificial region is for fabricating a nanopore, preferably a single nanopore. More specifically, the sacrificial region is for controlling the location of nanopore fabrication. The electrodes may, for example, be arranged in a way in which the nanopore formed in each electrode can form a single nanopore. This can happen when the nanopores formed in each electrode are closely spaced to one another.

[0135] The sacrificial part of each electrode may be arranged to provide the sacrificial region. By arranging the sacrificial parts of the electrodes in this way, the localised Joule heating can be directed to a specific area of the membrane for controlled breakdown.

[0136] Typically, a single nanopore is fabricated in the electrode at the sacrificial part of the electrode. When there is a sacrificial region, then a single nanopore is fabricated in the electrodes at the sacrificial region, e.g. the sacrificial region provided by the sacrificial parts of the electrodes.

[0137] When a sacrificial region is provided for the fabrication of a nanopore, then step (a) of the method of the invention may comprise (a) applying an electric field between (i) two or more electrodes arranged on a surface of the membrane to provide a sacrificial region for fabrication a nanopore and (ii) an electrolyte solution in contact with an opposing surface of the membrane, preferably to fabricate a nanopore at the sacrificial region, more preferably to fabricate a single nanopore at the sacrificial region.

[0138] A general feature of the method of the invention is that it can be used to fabricate a nanopore having a diameter of from about 0.5 nm to about 50 nm, preferably from about 1 .0 nm to about 30.0 nm, more preferably from about 1.5 nm to about 20 nm.

[0139] The method of the invention is typically performed when the solid-state device is supported on a substrate. Substrates are normally used during the manufacture of solid- state nanopore devices.

[0140] The substrate may be a silicon substrate or a glass substrate. Glass substrates have been used to reduce the device capacitance. It is preferred that the substrate is a silicon substrate, particularly when the dielectric material of the membrane is SiN _x .

[0141] The substrate may have a thickness of from about 50 pm to about 750 pm, preferably from about 100 pm to about 500 pm, more preferably from about 200 pm to about 400 pm.

[0142] The electrolyte solution is for fabricating a nanopore. When the solid-state device comprises a plurality of electrodes, then the electrolyte solution may be for fabricating a plurality of nanopores.

[0143] In principle, any electrolyte solution can be used in the method of the invention, provided it is chemically compatible (i.e. it does not react with) the solid-state device or its constituent components.

[0144] The electrolyte solution may be a chloride-based salt solution, such as a solution of an alkali metal chloride. It is preferred that the electrolyte solution is a solution of lithium chloride or potassium chloride.

[0145] In the present invention, the solid-state device, the solid-state nanopore device and the kit each comprise a membrane.

[0146] Typically, the membrane comprises, or consists essentially of, a dielectric material.

[0147] The dielectric material may be selected from SiN _x , AI2O3, TiC>2, HfC>2, hexagonal boron nitride (h-BN) and an MXene.

[0148] SiN _x refers to a silicon-nitride. When the dielectric material is SiN _x , then x may be from about 0.70 to about 1.50. It is preferred that x is from about 1.00 to about 1.30, particularly about 1.05 to about 1.25.

[0149] MXene refers to a class of two-dimensional inorganic compounds, which consist of layers of transition metal carbides, nitrides or carbonitrides. The layers are a few atoms in thickness. [0150] It is preferred that the dielectric material is SiN _x .

[0151] The membrane may comprise, or consist essentially of, a single layer of the dielectric material. Alternatively, the membrane may comprise a plurality of layers of the dielectric material. It is preferred that the membrane comprises, or consists essentially of, a single layer of the dielectric material.

[0152] Typically, the membrane has a thickness (e.g. a total thickness) the membrane has a thickness of from about 5.0 nm to about 50.0 nm, preferably from about 7.5 nm to about 40.0 nm, more preferably from about 10.0 nm to about 30.0 nm.

[0153] The membrane may be supported on an under layer, such as a SiC>2 under layer. The membrane and this under layer may be supported on the substrate when performing the method of the invention. The presence of the under layer is advantageous because it reduces the noise of the final solid-state nanopore device.

[0154] The under layer may have a thickness (e.g. a total thickness) of from about 100 nm to about 5000 nm, preferably from about 250 nm to about 2000 nm, more preferably from about 400 nm to about 750 nm (e.g. from about 400 nm to about 600 nm).

[0155] The solid-state device or the solid-state nanopore device of the invention may further comprise a dielectric layer disposed on the membrane (e.g. an upper surface of the membrane) and the electrode(s). The dielectric layer can passivate the surface of the device and reduce the capacitance of the electrodes.

[0156] The solid-state device or the solid-state nanopore device of the invention may comprise an on-chip sensing modality.

[0157] The on-chip sensing modality may include or be a field-effect sensor, a tunnelling nanogap, a plasmonic nanostructure or a radiofrequency antenna.

[0158] A field-effect sensor, such as a field-effect transistor (FET), is a device where its conductance is sensitive to the local electric potential. When the on-chip sensing modality is a field-effect sensor, then the on-chip sensing modality may include a nanopore, such as a nanopore as described herein. Integrating a field-effect sensor with a nanopore provides an alternative sensing modality to ionic current based sensing. This is because the translocation of biomolecules through a nanopore modifies the local electric potential and can therefore be detected by changes in the conductance of the field-effect sensor.

[0159] Integrating a nanopore with a field-effect sensor has two major advantages over ionic current based sensing. Firstly, these devices can be fabricated with high density and the signal from each field-effect sensor read out independently. This is in contrast to ionic current based sensing where each pore must be fluidically isolated thus placing a limit on the device density that can be obtained. Secondly, these devices typically have a high conductance and therefore don't require sensitive amplifiers to measure the current through the device. The result of this is that capacitive noise no longer dominates the signal at high frequencies (>10 kHz).

[0160] A tunnelling nanogap consists of a nanogap (e.g. < 5 nm) between two conductive electrodes. When an electric potential is applied between the electrodes, electrons can tunnel through the potential barrier resulting in a measurable current. Placing a molecule of interest within the gap results in a change in the measured current as electrons now tunnel through the molecule.

[0161] An advantage of a device comprising a tunnelling nanogap is its sensitivity to the electronic structure of the translocating analyte, which can, for example, be used to detect and distinguish between different nucleobases, amino acids and carbohydrates. The inclusion of a plurality of tunnelling nanogaps would enable high device densities as each nanogap can be measured independently without the need for fluidic isolation.

[0162] A plasmonic nanostructure can be integrated with a nanopore to enable optical based sensing. These nanostructures can produce strong, localised optical fields resulting from the oscillation of free electrons in a metal (i.e. the plasmon effect). The highly localised optical signal created by a plasmonic nanostructure can be used to significantly increase the fluorescence intensity of labelled molecules thus providing a higher signal-to-noise ratio in these devices.

[0163] In general, the on-chip sensing modality may include and/or be electrically coupled to an electrode, such as an electrode described above.

[0164] The electrode may also be used to modify the local electric field and therefore control the translocation dynamics of biomolecules through the nanopore.

[0165] When the device is a solid-state nanopore device, then the on-chip sensing modality may include the nanopore.

[0166] The solid-state nanopore device is typically obtained or obtained from the solid- state device using a method of the invention.

[0167] The solid-state nanopore device comprises an electrode on a surface of the membrane, which has a nanopore. The nanopore is fabricated or formed in the electrode. In other words, the nanopore may pass through the electrode and/or is self-aligned with the electrode.

[0168] The invention also provides an apparatus. The apparatus comprises a plurality of solid-state nanopore devices of the invention. The solid-state nanopore devices may be arranged in an array. [0169] The invention further provides a kit for fabricating a nanopore in a membrane of a solid-state device. The kit comprises (i) a membrane comprising a dielectric material, and an electrode on a surface of the membrane, such as described herein. The membrane may be a pre-fabricated membrane.

[0170] The membrane comprising a dielectric material, and an electrode on a surface of the membrane may be a solid-state device as described herein, wherein the electrode is for use in fabricating the nanopore.

[0171] The kit may further comprise (ii) instructions for performing the method of fabricating the nanopore of the invention.

[0172] The instructions are for the method of fabricating a nanopore in a membrane of a solid-state device having an electrode, in accordance with the invention.

[0173] The instructions may be in the form of a paper document, a computer storage medium, a hyperlink to a website or a QR code with a hyperlink to a website.

[0174] The invention also relates to uses of the solid-state nanopore device or the apparatus of the invention.

[0175] The solid-state nanopore device or the apparatus can be used as a biosensor for detecting an analyte, such as described in the art (Reference 1: J.K. Rosenstein et al).

[0176] The solid-state nanopore device or the apparatus of the invention can be used for sequencing DNA or RNA. Such uses are described in the art (Reference 2: Y. Feng et al).

[0177] Another aspect of the invention relates to a method for measuring the uniformity and/or consistency of a membrane. The method may be a method for determining that the membrane meets a minimum requirement.

[0178] The method comprises (i) providing a membrane comprising a dielectric material, wherein a plurality of electrodes are disposed on a surface of the membrane, (ii) applying an electric field between an electrode (e.g. of the plurality of electrodes) on the surface of the membrane and an electrolyte solution in contact with an opposing surface of the membrane, (iii) measuring a response of the membrane to the electric field, (iv) repeating steps (ii) and (iii) with a different electrode (e.g. of the plurality of electrodes) to the electrode(s) already used to apply an electric field and using the responses to measure the uniformity and/or consistency of a membrane.

[0179] The electrode, membrane, electric field and the electrolyte solution may each independently be as described herein.

[0180] The electrode that has or the electrodes that have already been used to apply an electric is/are the electrode(s) used in step (ii) of the method. [0181] The uniformity and/or consistency of the membrane may be determined by comparing the responses of the membrane to the electric field obtained from each electrode.

[0182] If the responses fall within a predetermined range, then the membrane may be uniform and/or may meet a consistency requirement. If a response falls outside the predetermine range, then the membrane is not uniform and/or does not meet the consistency requirement.

[0183] Additionally or alternatively, the membrane may be uniform and/or may meet a consistency requirement if the responses of the membrane satisfy a predetermined requirement. The predetermined requirement may be the responses of the membrane having a current or an electric potential (e.g. voltage) that reaches a predetermined threshold or value.

[0184] The electric field may be a predefined electric field, such as for producing a predetermined or an expected response.

[0185] Typically, the plurality of electrodes is disposed on a single surface of the membrane.

[0186] In the method of the invention, the step of (i) providing a membrane comprising a dielectric material, wherein a plurality of electrodes are disposed on a surface of the membrane may comprise, or consist essentially of, depositing a plurality of electrodes onto a surface of a membrane comprising a dielectric material.

[0187] As used in the present disclosure, the term “comprises” has an open meaning, which allows other, unspecified features to be present. This term embraces, but is not limited to, the semi-closed term “consisting essentially of” and the closed term “consisting of”. Unless the context indicates otherwise, the term “comprises” may be replaced with either “consisting essentially of” or “consists of’. The term “consisting essentially of” may also be replaced with “consists of”.

Examples

[0188] The invention will now be illustrated by the following non-limiting examples.

Fabrication Methods

General method for device fabrication

[0189] Substrates were prepared by conventional methods. Wet thermal oxidation was used to create the SiC>2 layer and low-pressure chemical vapour deposition was used to fabricate the SiN _x layer. Suspended SiN _x membranes

[0190] All of the following fabrication steps were performed on the wafer scale to manufacture suspended SiN _x membranes with no on chip electrodes.

1. Spin coat photoresist: Spin coat MCC Primer 80/20 at 3000 rpm followed by Shipley 18-18 at 3000 rpm. Bake the wafer at 115°C for 60 sec.

2. Photolithography and development: Expose to UV light using a mask aligner with a dose of 60 mJ/cm ² . Develop in CD-26 for 60 s followed by DI water rinse.

3. Reactive Ion Etching (RIE) of SiNx/SiC Hard Mask: Etch SiN _x and SiC>2 layers using reactive ion etching with gas flows of C4F8 at 45 seem and O2 at 2 seem.

4. Resist Strip: Leave wafer in acetone for 15 minutes to remove the resist.

5. Frontside Protect with Resist: Spin MCC Primer 80/20 on the wafer at 3000 rpm followed by PMMA 950k A5 at 2000 rpm. Bake at 190° for 10 minutes.

6. Wafer Etch: Load wafer into a single sided wafer holder sealed with o-rings so that only the back side of the wafer is exposed to solution. Place wafer in 30% KOH at 80°C for 8 hours. Wash wafer in DI wafer and dry with N2.

Electrodes on suspended SiN _x membranes

[0191] The following steps can be performed either before or after etching of the substrate to create suspended SiN _x membranes, i.e. step 5 in the procedure above. An advantage of performing this step prior to etching the substrate is that it reduces handling of the suspended SiN _x which can result in damage to the membrane. However, it means that the electrode configuration of the entire wafer must be written at this stage which can be wasteful for iterative research purposes.

1. Spin coat photoresist primer: Spin coat Ti primer onto chip at 3000 rmp and bake at 120°C for 2 minutes.

2. Spin coat photoresist: Spin coat ma-N 1420 at 3000 rpm and bake at 100°C for 2 minutes.

3. Photolithography and development: Expose to UV light using a mask aligner with a dose of 550 mJ/cm ² . Develop in ma-D 533/S for 80 seconds followed by DI water.

4. Thermal Evaporation: Evaporate 5nm Cr followed by 75nm Au.

5. Liftoff: Leave in warm (50°C) acetone for until liftoff is complete (approximately! hour). 6. Spin coat e-beam resist primer: Spin coat PMGI SF6 at 4000 rmp and bake at 190°C.

7. Spin coat e-beam resist: Spin coat 950k PMMA A8 at 6000 rpm for 60 s. Bake on hotplate at 175°C for 60 s.

8. Spin coat e-spacer: Spin coat e-spacer 300Z onto device at 15000 rpm. Bake on hotplate at 80°C for 60 s.

9. Electron beam lithography: Expose using electron beam lithography with a dose of about 950 C/cm ² .

10. Develop: Place chip in DI water for 60 s to remove e-spacer and dry with N2. Place chip in MIBKJPA 1:3 for 4 minutes followed by an IPA rinse and dry with N2. Place chip in MF 319 for 7-15 s followed by DI water rinse and dry with N2.

11. Thermal evaporation: Evaporate 5nm Cr followed by 30nm Au.

12. Liftoff: Place in Microposit 1165 at 50°C until liftoff is complete (approximately 4 hours).

[0192] The devices in Example 6 were fabricated after substrate etching and electrode fabrication. The devices in Examples 1 to 5 below were fabricated prior to substrate etching.

Example 1

Device geometry and experimental setup

[0193] A general schematic of the device (1) geometry that was used in the controlled breakdown (CBD) experiments is shown in Figure 1 without an electrode. In general, the device consists of a 20 nm thick Si-rich SiN _x membrane (10) suspended on 500 nm of SiC>2 (30) on a 300 pm thick Si substrate (50). The suspended section of SiN _x membrane (20) was about 50 x 50 pm ² . The Si-rich SiN _x membrane (10) had a value of x = 1.14. The stoichiometry of nitrogen to silicon was estimated from the measured refractive index of the film (Reference 3: T. Gilboa et al). The thickness of the film was estimated from ellipsometry.

[0194] The SiO2 layer (30) was used to reduce the capacitance and therefore the high frequency noise. It also ensures that the leakage current is only through the suspended region of SiN _x (20). Without this layer, charge could be transported from the electrolyte solution, to the Si substrate, and through the SiN _x . [0195] A schematic of the device (1) geometry with an electrode (100) on the surface of the SiN _x membrane (10) is shown in Figure 2. Eight independently addressable metal electrodes (5/95/10 nm Ti/Pt/Ti) were patterned on the membrane surface via photolithography. Finally, a 5nm thick SiC>2 layer was deposited over the electrodes via atomic layer deposition (not shown in Figure 2). A false-colour scanning electron micrograph of the electrode configuration over the suspended region of SiN _x is shown in greyscale in Figure 4. For convenience, in the following discussion the electrode labels A1-A4 and B5-B8 were added to the micrograph in Figure 3. The device shown in Figures 2 and 3 was used to perform the controlled breakdown experiments (CBD).

[0196] A schematic diagram showing the measurement setup for performing CBD is shown in Figure 4. CBD was performed by applying a voltage between an electrode (100) on the membrane surface (10) and an electrolyte solution (200) in contact with the other side of the membrane (10). In this experimental setup the electrolyte (200) is only present on one side of the membrane (10) in the device (1). The electrolyte (200) was 3.6 M LiCI.

Controlled breakdown (CBD)

[0197] CBD was performed by applying a negative voltage to an on-chip electrode (100) while the electrolyte solution (200) in contact with the other side of the membrane (10) is held at ground. This is a forward-biased configuration. The inventors have studied this process and found that an oxidation reaction does not need to occur to inject electrons into the dielectric since they can be supplied by the metal electrode. This results in breakdown occurring at a relatively low voltage (~5-6 V for this device geometry).

[0198] To perform CBD, the amplitude of the voltage applied to the on-chip electrode (100) was increased in steps of 100 mV every 4 s while simultaneously measuring the resulting current. When a spike in the current was observed, the voltage was immediately reduced to a pre-defined value of 3.8 V and held at this value for 20 s. The voltage was then reduced in steps of 100 mV every 15 s to a value of 2.5 V, and then to 0 V in steps of 100 mV every 2 s. This voltage protocol was found to consistently fabricate nanopores with diameters in the range of 1-10 nm. The voltage protocol for the nanopore fabrication experiment is shown in Figure 5. The top line represents the resulting measured current. The presence of a nanopore was confirmed by fluorescence imaging.

[0199] The breakdown protocol described above is different from protocols that have been used previously, such as when those typically used during CBD where the voltage is applied between electrolyte solutions in contact with either side of the membrane. In such measurements, the voltage is typically immediately reduced to 0 V following breakdown. However, when performing CBD on devices (1) with on-chip electrodes (100) in the forward-biased configuration, breakdown occurs at relatively low voltages (5-6 V for this device geometry). If the voltage is immediately reduced following breakdown at such low voltages, “soft-breakdown” will typically occur. This is where the membrane material is not fully removed during breakdown to form a nanopore. As such, it is necessary to slowly reduce the voltage following breakdown to create nanopores using this CBD configuration.

Example 2

Fabrication of multiple nanopores

[0200] The same type of device and measurement setup as described in Example 1 above were used in this experiment. The electrodes (100) were labelled as shown in Figure 3.

[0201] Four CBD experiments (as shown in Figure 6) were performed on a single membrane where the breakdown voltage was sequentially applied to a different on-chip electrode for each measurement. See Figure 6, where the top line of each plot is the measured current.

[0202] Each CBD measurement was quantitatively similar, with breakdown occurring at - 5.8, -5.5, -5.7, and -5.6 V. The leakage current prior to breakdown was similar for each CBD measurement, with values of -110, -125, -106 and -124 pA measured at 5 V.

[0203] The fact that the current prior to breakdown was independent of previous CBD measurements indicates that there is no fluidic connection between the on-chip electrodes after nanopore formation. If there was a fluidic connection between the on-chip electrodes, redox reactions would occur due to the large voltage difference between the electrodes which would modify the measured current. This is an important result as it enables the independent fabrication of multiple nanopores in a single membrane without affecting the previously created pores.

[0204] An advantage of the CBD configuration involving an electrode on one side of the membrane and an electrolyte on the other side of the membrane is that it enables the independent fabrication of multiple nanopores in a single membrane without affecting previously created pores. If electrolyte was present on both sides of the membrane, previously created nanopores would be enlarged during each new breakdown event since a significant ionic current would flow through the pore. Position and number of nanopores

[0205] To determine the position and number of nanopores created using this CBD protocol, fluorescence imaging of the nanopores was performed with Ca ²⁺ and Ca ²⁺ indicators. To do this, the devices were cleaned in DI water, followed by acetone and O2 plasma etching. Prior to measurement, the devices were again cleaned via UV-ozone treatment. The devices were then loaded into a device holder that was mounted in an inverted microscope. For fluorescence imaging, the device was illuminated by a fibre- coupled 488nm tunable Argon ion laser. A 498 nm dichroic mirror reflected the incoming light towards the sample where a 60x objective was used to illuminate the sample and collect the emitted fluorescence. The cis and trans chambers (in contact with the upper and lower side of the membrane, respectively) were filled with CaCh solution (50 pM CaCh, 100 mM KCI in DI water) and Fluo-4 solution (5 pM Fluo-4, 100 mM KCI in DI water) respectively. Ag/AgCI electrodes were inserted into each chamber and a negative voltage applied to the trans chamber to drive Ca ²⁺ ions through the nanopore. Transport of Ca ²⁺ ions into the trans chamber activates the Ca ²⁺ dependent Fluo-4 resulting in a highly localised fluorescence hotspot at the nanopore which was recorded by an electron multiplying charge coupled device.

[0206] Figure 7 shows fluorescence micrographs of the nanopores formed from the CBD measurements shown in Figure 6. Three fluorescence micrographs are shown representing a time series of data with a frame before, during, and after the application of a voltage that drives Ca ²⁺ ions through the nanopore. The white dashed box represents the edge of the suspended region of SiN _x . CBD was performed on the electrodes labelled A1, A3, B5, and B7.

[0207] A single fluorescence signal was observed in each electrode that CBD was performed on (e.g. A1, A3, B5 and B7). See Figure 7, middle image. No fluorescence signals were observed in electrodes that CBD was not performed on. This result demonstrates that the CBD protocol used enables the independent fabrication of a single nanopore in each electrode on the membrane surface.

[0208] To further characterise the nanopores, TEM imaging of the nanopores was performed. Figure 8 shows bright-field (top row) and high-angle annular dark field (bottom row) scanning transmission electron micrographs of one of the nanopores that was produced at various magnifications. A nanopore with diameter ~20nm was observed in the SiN _x membrane (see right most images). This is surrounded by a larger region (200 nm) where the metal has been removed. Example 3

Nanopores with different diameters

[0209] The same type of devices and measurement setups as described in Example 1 above were used in this experiment. To characterise the size of the nanopores created by the CBD protocol, a single nanopore was fabricated in six different membranes. After fabricating the nanopore, reservoirs on both sides of the membrane were filled with 3.6M LiCI and left for one hour to allow the ionic current through the pore to stabilise. Figure 9 shows the CBD curve (top row) along with a measurement of the resulting nanopore conductance (bottom row) for each device.

[0210] The resulting nanopore diameters were estimated from the following equation. where R is the measured resistance, a is the conductivity of the electrolyte, d is the nanopore diameter and t is the membrane thickness.

[0211] The resulting nanopore diameters were determined to be 1.4, 2.8, 4.8, 9.1 and 6.3 nm. One device underwent soft breakdown and no appreciable nanopore was created.

[0212] By comparing the CBD curves and the resulting nanopore conductance, a correlation between the current measured while ramping the voltage to zero following breakdown and the resulting nanopore diameter was observed. Measuring a greater current when ramping the voltage to zero after breakdown generally results in the formation of a larger nanopore (e.g. compare the CBD curves and resulting pore diameter of chip 5 and chip 6 in Figure 9).

[0213] With regard to the correlation between the current measured after breakdown and the resulting nanopore diameter, the charge passed after breakdown while the voltage is being decreased to zero has been quantified. Specifically, the current between - 3.6V and 0V while the voltage is reduced after breakdown was integrated (see the shaded area in Figure 10). As shown in Figure 11 , in general there is an increase in the nanopore diameter with increasing charge passed after breakdown. This result indicates that the nanopore diameter can be precisely controlled by using an active feedback where the rate at which the voltage is ramped down after breakdown depends on the measured current. Example 4

Fabrication of nanopores using a reverse biased configuration

[0214] The same type of devices as described in Example 1 above were used in this experiment. The measurement setups in Examples 1 to 3 above used a forward biased configuration, as shown in Figure 12(a). This experiment was performed where the electric field was applied in the reverse biased configuration. See Figure 12(b).

[0215] The reverse-biased configuration is achieved when the electric field is applied so that an oxidation reaction must occur to inject electrons into the membrane (i.e. electrons cannot be supplied by the on-chip electrode). Since breakdown occurs at a much higher voltage for this electric field configuration, the CBD protocol that was used for these measurements involved immediately reducing the voltage to zero following breakdown. As in the forward-biased configuration, each CBD curve was found to be similar regardless of whether or not breakdown had previously been performed on the membrane.

[0216] Fluorescence imaging was performed as described above to confirm the presence of nanopores.

[0217] In comparison to the reverse biased configuration, it was found that an advantage of performing CBD in the forward-biased configuration is that the low breakdown voltage results in slow nanopore formation. This reduces the requirement for stringent feedback conditions to ensure the formation of a single nanopore and allows tuning of the nanopore diameter using active feedback of the applied voltage following breakdown.

Example 5

Bimolecular translocation

[0218] The same type of device as described in Example 1 above was used in this experiment. A single nanopore was created in the membrane by performing CBD in the forward biased configuration. Following breakdown, reservoirs on both sides of the membrane were filled with 3.6M LiCI and the device was left overnight to allow the nanopore to stabilise. Following this, a stable, linear, ionic current was measured through the pore when a voltage was applied between Ag/AgCI electrodes inserted into each reservoir. The potential of the on-chip electrodes and the cis chamber were held at ground while the transmembrane voltage is applied to the trans chamber. The diameter of the nanopore was estimated using the equation above to be 13 nm. [0219] A noise spectrum of the ionic current at an applied voltage of 200mV was obtained. The noise spectrum was comparable to those observed for solid-state nanopores fabricated using other methods. Integrating the normalised noise spectrum between 1-100 Hz to quantify the low-frequency noise using the equation below yielded a value of 6x1 O' ⁴ , which is well below the threshold of 1x1 O' ² required for a nanopore to be usable for single-molecule biosensing applications (Reference 4: M. Waugh et al). where L is a parameter that quantifies the low-frequency noise of the device, I is the mean current, and S(f) is the noise spectrum.

[0220] To detect analyte translocation through the nanopore, the cis chamber was 'spiked' with 1 kbp dsDNA (NoLimits 1000bp DNA, ThermoFisher, SM1671) and the solution was mixed using a pipette resulting in a final DNA concentration of ~5 nM. When a positive voltage was applied to the trans chamber, transient decreases in the ionic current were observed, which are typical of DNA translocation through the nanopore. Closer inspection of the current transients revealed that the translocation events can be classified into two groups: (i) single-level current blockades, and (ii) two-level current blockades. This is typical of DNA translocation through solid-state nanopores with diameters significantly larger than the cross section of dsDNA (~2.4 nm). Single-level and two-level current blockades are indicative of the translocation of dsDNA in an unfolded and folded manner respectively (Reference 5: P. Chen et al). These results confirm that nanopores fabricated via the CBD protocol above can be used for single-molecule biosensing applications.

Example 6

Device geometry and experimental setup

[0221] A schematic of the device geometry is shown in Figure 13(a). The devices are similar to those used in Examples 1 to 5. Each device consisted of a 20 nm thick Si-rich SiN _x membrane (10), where x = 1.14, suspended on a 500 nm thick SiC>2 layer (30) on a 300 pm thick Si substrate (50). Metal electrodes (100) [5/75 nm Cr/Au] were deposited on the membrane surface (10) via photolithography followed by thermal evaporation. Electron beam lithography was then used to pattern a metal nanoconstriction (5/30nm Cr/Au) between two of these electrodes (100b). Unlike the devices in Examples 1 to 5, a thin passivation layer was not deposited over the electrodes (100) for these devices. False colour scanning electron micrographs of the electrode configuration over the suspended region of SiN _x are shown in Figure 13(b). The width at the narrowest part of the nanoconstriction is approximately 200 nm (see Figure 13(b)(ii)).

[0222] Figure 13(c) shows a schematic of the experimental setup used in this example. Similar to Examples 1 to 3, CBD was performed by applying a voltage between an on-chip electrode (100) and an electrolyte solution (200) in contact with the other side of the membrane. A transverse voltage (VTV) was applied across the on-chip electrodes (100b) to drive a current (/TV) through the metal nanoconstriction and produce significant Joule heating at the centre of the constriction. Simultaneous to this, an increasing transmembrane voltage (V™) was applied to the electrolyte solution which drives a transmembrane leakage current (/ _T M) and eventually induces breakdown. Breakdown was performed in the reverse-biased configuration with a positive voltage applied across the nanoconstriction and a negative voltage applied to the electrolyte solution. The reverse- biased configuration was used since, as demonstrated below, the breakdown voltage has a larger temperature dependence than the forward-biased configuration.

CBD temperature dependence

[0223] As indicated in Examples 1 to 4, when CBD is performed by applying a voltage between an on-chip electrode and an electrolyte solution in contact with the other side of the membrane, the mechanism of electrical conduction depends on the direction the electric field is applied. The temperature dependence of breakdown when the voltage is applied between an on-chip electrode and an electrolyte solution in the forward and reverse-biased configurations were measured. Figure 14 shows the breakdown voltage as a function of temperature for the forward and reverse-biased configurations.

[0224] For both electric field configurations, there is an approximately linear decrease in the breakdown voltage with increasing temperature. However, the decrease in breakdown voltage was less pronounced for the forward-biased configuration compared to the reverse-biased configuration. The gradient of the lines of best fit are -0.012 and -0.053 V/°C for the forward and reverse-biased configurations respectively. This difference in the temperature dependence of the breakdown voltage is likely a result of the different conduction mechanisms for these two electric field configurations. For the reverse-biased configuration, conduction is limited by oxidation reactions that must occur at the membrane-electrolyte interface. On the other hand, for the forward-biased configuration, conduction is limited by charge transport through the dielectric. Given the greater temperature dependence of the breakdown voltage for devices in the reverse- biased configuration, this electric field configuration was used in the experiments below. Localised breakdown by Joule heating

[0225] Figure 15 shows a typical CBD measurement where a large current density was passed through the metal nanoconstriction simultaneous to performing CBD. To perform these experiments, the voltage applied across the metal nanoconstriction was quickly ramped to 1V and held at this value (see Figure 15(b)). The voltage across the membrane was then increased in steps of 100mV every 4 s until breakdown was observed (see Figure 15(a)). When breakdown occurs, VTV and V™ were both quickly reduced to zero. Notably, when breakdown occurs, the current through the metal nanoconstriction drops to zero. This indicates that there is damage to the metal nanoconstriction after breakdown.

[0226] To determine the position of the nanopores created by this CBD protocol, fluorescence imaging was performed using the method given in Example 2. The experiment was performed for three different chips. Fluorescence micrographs of the created nanopores are shown in Figure 16 for two different chips. The white dashed box represents the edge of the suspended region of Si N _x , while the solid white lines represent the edges of the electrodes. For each chip, three micrographs are shown representing a time-series of data with a frame before, during, and after the application of a voltage which drives Ca ²⁺ ions through the nanopore. For each device, a single nanopore was formed at the narrowest region of the nanoconstriction.

[0227] These results demonstrate that it is possible to create nanopores that are selfaligned with an on-chip metal nanoconstriction by passing a large current density through the electrodes to locally heat the membrane during CBD.

[0228] As discussed above, the current through the metal nanoconstriction drops to zero when breakdown occurs. This indicates that there is significant damage to the metal nanoconstriction during breakdown. SEM imaging of the devices following CBD was performed to directly observe this damage. For each device, the metal at the narrowest part of the constriction was found to have been removed. Such damage likely results after nanopore formation when the electrolyte solution comes into contact with the metal electrodes. When this occurs, there is temporarily a large voltage between the electrodes and the electrolyte solution, which likely results in electrochemical etching of the metal.

[0229] Damage to the nanoconstriction may be reduced by depositing a thin passivation layer over the electrodes via atomic layer deposition and/or utilising faster feedback conditions to reduce the voltage following breakdown. A similar device geometry with a thin passivation layer over the metal electrodes was used in Examples 1 to 3. References

[0230] The following publications are referenced herein. Each of these publications is incorporated herein by reference.

1. J. K. Rosenstein et al., “Single-molecule bioelectronics”; Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 7(4), 2015, pp. 475-493.

2. Y. Feng et al., “Nanopore-based fourth-generation DNA sequencing technology”; Genomics, Proteomics & Bioinformatics, 13(1), 2015, pp. 4-16.

3. T. Gilboa et al., “Automated, ultra-fast laser-drilling of nanometer scale pores and nanopore arrays in aqueous solutions”; Advanced Functional Materials, 30(18), 2019, 1900642.

4. M. Waugh et al., “Solid-state nanopore fabrication by automated controlled breakdown”; Nature Protocols, 15(1), 2020, pp. 122-143.

5. P. Chen et al., “Probing single DNA molecule transport using fabricated nanopores”; Nano Letters, 4(11), 2004, pp. 2293-2298.